Sunday. Poster Sessions: Signal Transduction I (46-51)

46

COLONOCYTE DIFFERENTIATON IN SITU IS ACCOMPANIED BY INCREASEDEXPRESSION AND ACTIVATION OF PROTEIN KINASE C (PKC) ISOZYMES. ((G.Veraoysek and J.D.Black)) Roswel Park Cance Institute, Buffalo, NY 14263.

Previous studies in this laboraty have provided evidence for a role of PKC iaozymea in

reguation of post-mitotic eventa in epithelial cells of the mammalan nal intestine (Saxo

et at., J. Cell Biol. 126: 747-764, 1994). To determine whether PKC plays a similar role inepitheial cella of the large intesne clls at diffn stgs of development (obuined bysequential scapng of the colonic epithelial ng) were partitoned io cowlic

particulate fracti and examinod for PKC activity using a nthetic from myclinbasic protein as a A 2-fold incrse PKC actvity wa obsaved as cdls move

from the prolifeation zone into the maurton zone of the colonic crypta Western blotanalysis and immunofluo reacence localization techniques were used to examin theexpression and ssbcellular distibution (i.e. activation sta) of individual PKC isozymes inthe rat colon. Westen blot analysis revealed that six isozymes of PKC (ca, OIL 5, e, ,, and

71) are expressed in olonocytes and that their levels increase along the csypt legh, withthe highest amounts found in the difftiated cels of the epithlil sr. In addition,

higher of activated PKC (i.e. PKC associated with the partculate

subcellular fraction) were obsved in cells of the upper crypt and srface epitheliumrelative to proliferating cdls of the lower crypL _ analysis genrllyshowed increased kvels of PKC iwymes in d tiating and fumctional coloncyt Inaddition, a change in the suboelular distribution of PKC a, OIL, 5, and C indicative ofenzyme activation was observed in the region of the colonic crypt where cels undeogrowth arrest Taken together, these findings support a possible role for PKC in gowthcontrol and/or differentiation of colonic epithehial cells in situ Suppored by NSF grantDCB 8917424 and NIH grant CA 16056.

48

UNIQUE PKC ISOFORMS IN T51B RAT LIVER CELlS AND TER ROLEIN PROLIFERATION AND APOPOTOSIS. ((H. Al-Mexeedi, L.P. Kleie andD.J. Frank)) Department of Patholg and Bioksemity, Faculty of Medicine,University of Ottm, Otawa, Ontario, Canada, KlH 8M5.

We are investigating the regulation of cell proliferation and spoptoa in T51B ratliver celis, a non-neoplastic epitheloid celi line. These cells have proven to be anexcelUent it vro model for celhar hom ti since mogens induce an inmsalburst of cell division followed by apoptos which we are able to monitor by flowtometry. To detmine the role of si trsdon proceases in the reulation

of ceLllar homeostasis we first determined the profil of protein kinase C (PKC)in quiescent T51B cells. PKC-u, -,B, 4, -e and -t were detected in both cytosolicand membrane fractions by immunoblooing. Of thes isoforms, PKC-t was the

most predominant. PKC-g was unique in T51B celis since it appeared as a doublet.Treatment of TS1B cells with okadaic acid indicated that two sbspecies may

rsult from diffrentil phosphorylation of PKC-B in the plasma membrane.Treatment of T51B cells with the umKr promoter, 12-0-r2adecanoyl phorbol 13-acetate (TPA) for 24 hours down-regulated membrane-associated PKC-a, 4 and -

e but was witot effect on PKC-r. In contast, TPA calsed a two fold increasein the level of PKC-, in the membrane. Sinc EGF induces both DNA snthsisand apoptosis in T51B cells, the effect of EGF on the intracelllar lcalizon ofPKC isoforms was investigated by imnunobblting and . EGF(l.5nM) was withot effect on PKC-s, 4, -e mad -r, but caused an increasedtranslocation of PKC4 to the pasma membrane within 8 hours when the cellswere entering S phase. We hypothsize that differential transbcation of PKCisoforms play a rok in cellular homeostasis. (Supported by the geen ofKuwait).

50

b of G aowtk of G UaO by am Ibubitwr otftaPk khec C by a

Feuwhe 1Miw Tinw PrmMwtIMI tia ' Kis Ckimiusa '5 D aigW SWXh O hodb' Do th&yWasb Aataio Sobris Do J key N. Bhus, L IMr W uiaiti2,aer emw, _Dqam.s Gussicsmda malsp 'Dqa-sm of Newrological

SarWy,Caoublbe-Pstyss Md C r,New Yor,NY 10032, 'Dqatsm ofOhedoa -Vlolg, ahe-kigy, CH401 Bud. Swilslcad

PsoR blam C (PKC) physa me alW is igp ecasdtmese pathways dot uafteactin of glrowth lhuI omer raus am d rswosog Vanios inform

ofPKCC e_pe d at big led s mco so -tusm lhey, thafe tha

PKCmit pay a cricel raic i the growth ofh_xeaasoeywcd a tgt

f thethorgy odtu ues.We ch-r-Id two _cgSdaam IlBus(mG-1cd G5-2, _d gSao _u odl

He, GS-I, ot e_mli*adhfrabsk aue at ths

usthoa ceas All tm cog M ms w en h4ghl mes aidd cad misptem I lgl ca

_uinabiseeh _eaIaM' cithgcd ieS W M We bud do dodeivat COP 41251, a pa cad akisee b ioar nEC, _kh tly i bts o gowth

of all of A daint rehed slog C 4270 On dos aodid mm mai The enpacd 12-0 13-am f(Wt A) a poe -us cad m l' aof also tb growtcdl COP 41251 pl- A cmud syasgol ls"it of _wth Catars wit

G0P41251 qrlsmd lathsdo atte at doG2/Mphe o fthde clUcydels da-- ofo dwailswihfsgutd suc Thus =SVm dwt COP 41251 uigM

We bhesgld, daw somori a3miaa ith odorsr,is thet- t

O f

47

pH-REGULATED PROTEIN SYNTHESIS AND PHOSPHORYLATION IN

LEISHMANIA. ((D.Zilberstein and Y.Saar)) Department

of Biology, Technion-Israel Institute of Technology, Haifa

32000, Israel.

During their development, protozoan parasites of the genus

Leishmania are continuously exposed to extreme changes in

temperature and pH, both of which trigger differentiation

of promastigotes to amastigotes in vitro. The aim of our

research was to elucidate the signal transduction pathway

that mediates pH-regulated phosphorylation in L. donovani.

Promastigotes were metabolically labelled using 32-p in

their growth medium at pH 7 at both 26°C and 37C.Subsequently, the pH of these cultures was shifted to pH 5and the parasites were incubated for 10 minutes before

subjecting them to a 2 dimensional gel electrophoresis.

The experiments yielded two patterns of protein

phosphorylation. The first was induced at pH 5, but was

temperature independent and the second pattern appeared

only in cells that were exposed to pH 5 and 37C. A

selected number of phosphoproteins from both groups were

isolated and further analysed. In parallel experiments

we used a polymerase chain reaction to amplify DNA that

codes for the highly conserved catalytic site of protein

kinases. These experiments yielded a 206 bp DNA that

exhibited a high level of homology to the CDC2 gene

family. Two of the amplified DNA were cloned into pQE,

expressed at a high level in E. coli, and the resultant

proteins were purified and specific antibodies were raised

against them, which are used to identify protein kinases.

4,

A YEWST 2 HYBRD SCrEE FCOR PISThNs RACrJNG WIH THESAW. (STI S ACr5IVATED PrIEIN KESES) ((I. Sanchez,M.Kcwlwski,J.Avruch, and J.M. Kyriakis)). Diabetes

Iesearob, Massachusetts General HFpital East, Charlestown,Massadusetta, 02129

The Stress Activated Protein Kinases (SAs) are a subfamilyof the ERR protein (Ser/ttr)kinases and are the dcninant c-Jun

N-teminal kinases activated by cellular stresses, tumornecrosis factor alpha and ILl-B. In order to investigate SAPKfurRLicx and regulatiow esployed the yeast 2 hybrid screen

to idetify polypeptides iich interact with SAPRs. Success-ful interactions are soured in this screen by the transactiv-

ion of a beta-galactosidase reporter gene under the cz:ntrol

of the yeast transcription factor GAL4A. fioe acstxxents of

this System icliue a yeast bait expression plasnid encodingthe full length cdA of SAPX beta fused to the GAL4 1Abinding cdain and the prey library hic comnsists of yeast

ression plasmids fusi proteins of the GAL 4

tranctivation dCgain with hluan T cel cdIs. Eqpression of

the fusion protein specified by the bait plasMnid was ctnfirndby in mot analysis. A scree of 1.8 millio transformants

yielded 100 His+,blue aolaiie. Ilese clones ware susequent-

ly mated with a panel of tester bait strains to rule out any

rsrpcific interacons. ltis test indicated that all except3 of the selected clones specifically interact with the beta

SK isofaz. Finally we obseved an inability of t_ese

clcnses to transactivate in the absence of the beta SAPM baitplasnid. These clnes therefore satisfy all criteria for

protein-pxte interaction in the yeast 2 hybrid screen.

51

INTERACTIONS BETWEEN PROTEIN KINASES IN THEPHEROMONE RESPONSE PATHWAY OF S. CEREVISIAE. ((L.Bardwell, J.G. Cook, B. Cairnsl, E. Chang, D. Ma and J. Thorner))Department of Molecular and Cell Biology, University of Califormia,Berkeley, CA 94720, and IDepartment of Cell Biology, StanfordUniversity School of Medicine, Stanford, CA 94305. (Spon. by S. Linn).

The KSSI, FUS3, STE7 and STElI1gene products are protein kinaseswhich are components of the pheromone response signal transductionpathway of S. cerevisiae. Ksslp and Fus3p are believed to bephosphorylated (and hence activated) directly by Ste7p which is in turnbelieved to be a direct target of StelIp. KSS1and FUS3 are members ofthe MAP kinase/ERK (mitogen activated protein kinase/extracellular-signalregulated kinase) subfamily, while STE7 and STE] I belong to the MEK(MAP-ERKkinase) and MEKK (MEK kinase) subfamilies, respectively. AMEK kinase -> MEK --> MAP kinase cascade is found in many signaltransduction pathways in many (if not most) eukaryotic organisms. Ourstudies are aimed at understanding the role of this evolutionarily conservedmodule in promoting signal transmission, amplification, modulation andintegration. Among other results, we have found that Ste7p, translated in arabbit reticulocyte lysate, forms a stable (Kd < 50 nM) complex with invitro-translated Ksslp or Fus3p. This complex forms between the proteinsin their unactivated states. Moreover, a mutant version of Ksslp, in whichthe loop containing the target residues (T183, Y185) for Ste7p-mediatedphosphorylation has been swapped with the corresponding loop in proteinkinase A, stillassociates tightly with Ste7p. An N-terminal, non-catalyticdomain of Ste7p is sufficient for the Ste7p-Fus3p interaction. Collectively,theseresults suggest that this stable MAPK-MEK complex may function insignal modulation.

9a

Signal Transduction I (52-57). Sunday52

CREB BINDS TO THE 5' FLANKING REGION OF A. NIDULANSCALMODULIN-DEPENDENT PROTEIN KINASE II GENE. ((K.Subbaramaiah, and D. C. Bartelt)) Department of Biological Sciences, St.John's University, Jamaica, NY 11439

A. nidulans calmodulin-dependent multifunctional protein kinase(ACMPK) is a unique protein whose expression is regulated in a cellcycle-dependent fashion at the level of transcription. We have isolatedtwo genomic clones of 3 kb and 6 kb from a X gtl 1 library of A.nidulans DNA. Sequence analysis indicated the presence of multipleintrons which have typical eukaryotic cis-acting elements. One of them isa cAMP response element (cre), a consensus palindromic sequence, 5'-TGACGTCA-3'. Gel shift analysis has shown that purified baculovirusexpressed protein binds to an end labeled PCR generated 200 bp fragmentof 5' genomic DNA containing the cre sequence. This binding iseffectively competed by unlabeled cre oligonucleotide. Similar resultswere obtained when gel retardation assays were performed with HeLa cellnuclear extracts. Earlier studies have shown that CaM kinases I, II, andIV phosphorylate CREB at Ser 133 and that the genes encoding CaMkinases II and IV contain cre sequences in their 5' flanking regions. Ourresults are consistent with these observations and suggest a possibleinvolvement of CREB in the transcriptional regulation of ACMPKexpression. Supported by the American Cancer Society, BE124A.

54

ROLE OF MYOSIN LIGHT CHAIN KINASE IN SHRINKAGE-INDUCEDACTIVATION OF Na+/H+ EXCHANGE IN ASTROCYTES. ((L.D. Shrode', J.D.Klein2, W.C. O'Neil2 andRW. Putniam')) 'Department of Physiology & Biophysics,Wright State University School of Medicine, Dayton, OH 45435; 2Renal Division,Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322.

Primary rat asuroyte exposed to a hyperosmotic solution undergo a Nae-dependent, amiloride-sensitive alkalinization of 0.36 pH unit, indicating that cellshrinkage induces activation of Nae/H+ exchange (NHE). This activation is due toan increase in the affinity of the exchanger for intemal H' and dependent on cellularATP. The pathway of this activation does not involve cAMP, Cae', or PKC (Shrodeet al., Soc. Neurosci. Abst. 19:686, 1993). However, ML-7, an inhibitor of myosinlight chain kinase (MLCK) inhibits the shrinkage-induced activation of NHE in a

dose-dependent fashion with anIC,5 of91 M. Shrinkage-induced activation ofNHEis also inhibited by 500 tsM W-7, 100 pM chlorpromazine and 50 WiMtrifluoperazinne, all inhibitors of calmodulin. Upon cell shrinkage there is an increasein phosphorylation of an 18 kD protein, which is reversed uponretum toisooticsolution. Western immunoblot analysis with amonoclonal antibody against myosinlight chain (MLC) shows thatMLC colocalizes with the 18 kD phosphoprotein andis only present in the Triton-insoluble fraction, indicating that MLC is associatedwith the cytoskeleton astocyt and is phosphorylated upon cell shrinkage. In

swsmary, our data suggest that shrinkage-induced activation of NHE occurs via a

novel pathway involving the activation of calmodulin-dependent MLCK and thephosphorylation of cytoskeletal-associated myosin light chain. We propose a modelwhereby phosphorylation of MLC leads toactivation ofNHE in a process dependenton sts fibers. [Supported by grants from Juvenile Diabetes Foundation (RWP)and NIH (WCO).]

56

THE ROLE OF JUN NUCLEAR KINASE (JNKI) IN IGF-1 RECEPTORSIGNAL TRANSDUCTION. ((B.S.Miller, U.T. Shankavaam, D.T. Kur, andS.A. Rosenzweig)) Department of Cell and Molecular Pharmacology andExperimental Tber cs, Medical University of South Carolina, Chaestn, SC29425.

IGF-I receptor (IGF-IR) activation by its cognate ligand triggers acascade which ultimatelyleadsto proIfr ay cel types.

IGF-lRmdiaed mitogeness appea to occurthough a ra-dependet pathwayinvolving activation of exceular signal-regulatedkin ). We havep viously shownthat IGF-I stimuadon lads to c-Jun o prytin andincreases mActivatorPstein-l (AP-1) DNA acivity. recedicvof JNKl has suggested a direct means of rguladng c-Junp _ aon ad,therefore, AP-1 DNA binding andt tio activ.Inoreto exploretherole of JNK1 in IGF-lR signal tansduction, C4 cells - NIH 3T3 cellsoverexprasn hIGF-IRs (1.6 X 106receptonr/cell) wertanfeceed with JNKlcDNA. JNKItsfcs expre higer levels of JNK1than conols based onimnrsunblottig with JNK1 secific polyclonal antibodies. Whole cel etractswere aayedfor AP-1 bindng activity by gel shiftuay, ERK activity byphosphrylationofmyeln basic protein, and JNK1 actity by phosphoylationofa Glutathone-S-Transferase-Jun 1-79 (GST-Jun) fusion protein. Prlimiayrsults suggest that JNKl overexpreson in C4 cells reduces the UVindhcon ofOST-Junpbwsphoryladon and AP-1 binding activy. IGF-1 simulationlads tosimilar levels of AP-1 DNA binding in contral and JNK1 ranfeced celL BaslERK activity is elevated incedlsoJNK md is incread in eqometo ICF-1. JNKI over ion does not appear to affect IGF-lR sigaltramsduction inC4 el. The role of NKIoveexp n in cels with normallevels of IGF-IRis being furtr exploed. Suppored by JDFIgramt #192230,NIH g -ansBY-06581 (SAR),T32-HIL7260 (UTS) and aPMAF Medical StudtReswArhFeowshbip(BSM).

53

GONDADOatUIREEASI HORMONE(IR AC1VATESMlWOINACrIVA1WPRaOINKWAE(MAP)THROWUHAPIORBaOLESsR SEaSMr PAWAY IN aT31CELLS. ((Srividya Sundresan and J. Larry Jameson)) Departme of Ph_acalogy, and CAawfor Endocrinology, Metabolism and Molclar Medicine, Nordiweslea Univerity MedicalSchool, Chicago, IL-6061 1.

GoRH is a hypothalnic hormone that regulates the synthesis ad reeaa of pituitarygonadotropins, lsteinizisg hormone (LH) and follicle stimulatiag hormone (PSH). GSHactivasm phospholipae C and the protein kinase C pathway, which ha bhae imnpliated inGnRH action. There is evidence of crosstalk between protein kiane C and mioga activWdprotein kinase (MAPK) pathways in other systems, raising the pouibility of steh an event inthe GsRH-gonadotrope system. To investigate a possible rol for MAPK is GaRH daT3-1 cells (derived from a cell line of gonad8ope lineage) were simalaed with GS, d

MAPC activity w exssamined by immunopecipition of the enzyme fo d by a sbsae

phospboylation assay. GnRH indced a rapid nd santsined ctivtion of MAPE, bg

within S minutes after stnulation with mAintenance of maximal ativiq for out mi_Masimal activation by (bRH wan 4- to S- fold in aT3-l cells, ad 2- fold in pin allu ofrat pituitary cells. Treatment with the phoabol eater TPA, likewise caused a 4- to 5- foldinduction of MAPK kinas activity in aT3-1 cells. To detemine wheder de effect ofGnRH was dependent Upon a pheebol enter sensitive pathay, aT3-1 ceb were teadchronicaly with TPA for 24 le to deplt coells of proin kinase C, ad the stiaaadwith cither GeRH orTPA. Pr1etmen with TPA prevented activation of MAPIC by etierGnRH or TPA. The effect of TPA preI atmeat was also exained on the activatio of theglycoprotein hormone a-subunit gene by GoRH or by TPA. Trsient rafectin assays witha constust of the a-subunit promoter linked to a lucifere eper ge, iead thatBtinentwith TPA bcwked TPA- and GnRH- indwed activatio of the a-buat ene.Tefindinp suggest ht OnRH can activate mitogen activaed poti kine (MAPK) in

gonadotrpe cells and that this effect involves a phorbol esaer senitive isofr of proeinkinase C. Fvthrmore, trnscriptional activation of the glyceproteinhbermne a-subunit gene

by GnRH is dependent on a phorbol ester sensitive pathway.

55

RHODOPSIN MUTANTS DISTINGUISH SiTES IMPORTANT FORINTERACTION WITH RHODOPSIN KINASE AND GO. ((W. Sbi S.Osawa, CD. Dickerson and E.R. Weiss)) Departent of Cell Biology adAnatomy, University of North Carolina at Capel Hill, Chapel Hill, NC27599, USA

The cytoplasrnic loops of rhodopsin play important regulatory roles inrhodopsin kdnase activity and tranuducin (Go) activation. To define thespecific regions of interaction between these two proteins and rhodopsin,alanine mutants were generated in the three cytoplasmnic loops of rhodopsinand transiently expressed in HEK-293 cells. All the mutants wereexpressed and bound 1 1-is-rednal nonnally. The level of phosphoryladonof the mutants by rhodopsin kinase and their ability to activate Gt weredetmined. The results suggest that these mutants can be divided into threegroups: those that affect phosphorylation alone, those that affect Gt

activation alone and mutants that are defective both in phosphorylation nd

Gt activation. T62A/V63A/Q64A in Loop I showed a significant inceand R147A/F148A/G149A in Loop II a significant decrease inphosphorylation by rhodopsin kinase, but both activated Gt nornmlly. The

mutants T242A/T243A and V250AJT251A/R252A in Loop m showedreduced ability to couple to Gt, but both displayed nomral levels ofphosphorylation by rhodopsin kinase. In contrast, A233G/A234G/A235Gin Loop m showed severe defects in both phosphorylatdon and in theactivation of Gt. Substitution of asperagines forthese alanines also resultedina severely affected mutant These exeIments suggest differences in theimportance of specific sites for the interaction of rhodopsin with Gt andrhodopsin kinase.

5T

BRADYKININ STIMUIATES TYROSINE PROSPHORYLATION OFp125 FOCAL ADHESION KINASE, PAXILI AND p66 c-ar MASEINHUMAN FIBROBLASTS. ((K-M. Lee ad M.L. Vle))De ofPhmacology and Physiology, Umversity of Chicago, Chicago, L 37.

Bradyinin (BK) mediates its mitogenic effec bybinding to a B2 receptor, amerber of the 7-transmembrane <- reer faily.R itwas sown that BK sfmlae tyro lY via ulnk nw

cani and we reported that tyosne stinted by BKmay play an important role inBK c entry. In thisreport, wecharacterized the mechanism of actvaton of tyrosephospoon andidentified substat that are tyrosn-ph o in respoe toBK. Theincrease in trosine phsophoration of veal btra is lik due to

stimlation of tyrosine nase activitya t modulphosp ase activity. Elevationofint caliumconeta ionlooynortlupganm Iead to an se thffe ltev fph wever, chelation ofCa b BAA-AMmEStedBK-4e protei PlrosememdCZthat OfC 1is not d for an of trosine yion

BK. We d0terned n of PKC to

'on. OfPKC by chrnic treame with PMA 12-mynate 13-aetate- not reduce the alty of BK to inre tphoaphorylation.lhbe resultsdemotadivete w a toactiton of kina.We also report thatp12S ffocal adhsionIdae(FAK)andp inare tyroine kinase uhbstrt u sd by'of anti-s 12 or a nts-

atibodies. BK of p" O7rprelumaly au bosphoWdon. The kiase acty ofpp60 d p12jsvas Wrue d by BK,s jtudged by in vtolnehporp125 u Tnmnesebe,reWts oGpro4,sjcoupled receptors and l

anplayan impon by BKTiwdc as uppnedby NIH pugnGM 41400.

lOa

Sunday. Signal Transduction I (58-63)58

SIGNAL TRAN8DUCTION DURING SPERMATOGZPDUIS((C.W. Walker, M. P. Lener, D. J. Demian, H. Honqust, M.KL Lim, D.F. Daggett . and A.G. Marsh+)) Dept. of Zoology, Univ.of New Hampshire, Durham, NH, Neuroacences, Univ. of N.Carolina, Chapel Hill NC and fCOMB Univ. of Maryland.Baltimore, MD.

In male aimal gonal cells, very little is known about caacadingmolecular eveta that tranaduce mitogenic cues. In the ea ar.Asterlas vulgaris, we do know that the annual initiation ofspermatogonial mitotic divislona Is gated by changing Failphotoperlod and that resulting epr n of the cntgc proto-oncogee is comrlated wIth entry Into 8-phase of the sperma-togonial cel cycle. We have evidence that both steroid- andhrowth f ctor/tyrosine kinase-aed me aresimultaneously involved. Izperimenta were conducted duringthe summer in the eatogenic phase of the gmetogenessusing II ketotestosterone and early September photoperod

as

mitotic stimulants. Redts of experiments were asses bycytolog, Northern hybridizations with an authentic ea star

c-myc probe and Immunochemistry with antibodies todemonstrate whether MAP kinae is phosphorylated on tyrosine.Results indicate that treatments with both non-seaonalphotoperiod and 1 1-ketotestosterone promotemitods in otherwisemitoticaly quiescent sperogoial cells.Our results haveimplications for the potential interaction of these dual mitogenicpathway (steroid and growth factor-based) as pematnormaly occus and for the induction of bioch cal pathwaysby environmental cues. (Supported by NMI [Mol Cytoll, Hatch 353to CWW and SURF to DFD andM1L)

60

BITION OF TYROSINE DEPHOSPHORYLATION LEADS TO INCREAD MAPENASE ACWFrY AND HUMAN BONE CELL PROLIFERA3ION. ((L-W. Wu, H.-ILYoan, DJ. Balink, & KC-H.W. Lau)) Depts. of Med. & Biode., LTus Linda Univer., &I.L. Pettis Menorial VAMC, Lomi Linda, CA 92357.

ide (F), an geft tsed to inms.. dbe oseproaic paents, ha beeshown to meue bone cel polfrato atuM concMtratic u- vitro. Ou p sui. sug-

Sgesed that the milognic action Fis nediJtedby (P-tyr) phospitrylationof ceilulwprondeuim rough iahilbio a P-tyr pbosphate MT . P-tyr& aivato of key nutgenic signaling pr oesi-, asuc MAP kin bosbow. to be _^men for mitogenic rooses. Acordingly, we soug to ted hypotesi

itbion Fm sivity by F would P-tyr & aivity of

MAPK in bsm TE85 osheosavona nib. The P-tyr level o MAP

me.wed foliows: twith mogwc (25-500 pa for venos

l of (upto 24 h), amnisof(cullul pe wemead-P-tyr smbod. Mm P n_pr were sepawed on grdius SDS- E

MAMs (p44~ & the p42~9 wre idetfied with t-MAPE stibody & [mA. MAPIbnds an o by lae demoito-3.MAPrspeciic wtvity wa aayed wilh bsic In study, we foad F

ds-depen into aed cel prlfration (.5fd , Pt laon of te p44&(2-fd but not the p4 & the MAPK specific inhumen TE95with the optima espoose eabdi eff ct at 100 pM lbe dsn ep endas PreasM in

P-tyr vity of MAPK ceered with that te stimiaie in pro-lifaon. The ianeses in P-tyr oxphorlytion ofp44W &the MAP specic activityw epd in tm he increm in eKh p-mana wa grads1 & wcm slative withmaximal inaees (-fold in P-tyr -osphyl & 5-fold in MAP1 adivity) see at 24-hrincubtion widh 100 uM F. e time couses we coneim-Pt with ihibiio of P-tyr

rat activat F at same doses did not

incse cel ro P-tyr phphnlatim& activity of MAPsinb sin fiho-blasIy, sum ywe bave dAmtha iabit of a PTP by F

resuted in stii n of: a) cenl proliferai, b) P-yr ph of MAPK, & c)MAP specific atvity in hue boe cndi. In this iediwhereinenilbitionof P-tyr bu,dte PTFs may r fuisetum for the regletion of cil proliftion ftiugh MAPK.

62

Phosphorylation of a cAMP-specific Phosphodiesterase (HPDEIVB2)by Mitogen Activated Protein Kinase. (J. M. Lenhard, D. Kassel, W.Rocque, L.Hamacher, M. Luther) Glaxo Research Institute, 5 MooreDrive, Research Triangle Park, NC 27709

Cyclic AMP inhibits and ras activates the MAP kinase cascade fortransmitting growth signals. It has been suggested that cAMPsinhibitory effect is a result of PKA activation. PKA is proposed tophosphorylate and block the activity of raf-l, a protein required forsignaling from ras to MAP kinase. Wc report that a cAMP-specific PDE(HPDEIVB2) serves as a substrate for MAP kinase. RecombinantHPDEIVB2 was phosphorylated in Sf9 cells. Deletion of amino acids81-151 and 529-564 had no effect on phosphorylation. Massspectrometry analysis of purified HPDEIVB2 showed thatpbospho71ation occured predominately on se487 and to a lesser extent

on ser4 . Phosphorylation did not occur when these sennes weremutated to alanines. Analysis of the sequence revealed that ser487 andser489 conform to the consensus motifs for phosphotylation bymitogen-activated protein kinase (MAP kinase) and casein kinase ILrespectively. Kinetic experiments showed HPDEIVB2 to be a good invitro substre (Km=63 mM, Vmax=3.0 mmoles/min/mg) for MAPkinase. Using mass speco y and mutational analysis we found thatMAP kinase phosphorylated HPDE1VB2 exclusively at ser487. Theseresults suggest that cAMP-specific phosphodiesterases may contributeto the cross-talk betwoeen the cyclic AMP and MAP kinase signalingpathways.

59

ANTI-GD3-ST1MULATED TYROSINE PHOSPHOPROTEIN EXPRESSION IN GD3.JURKAT CELLS. ((W D Met and V. Der-Minasslan)) Cnrter for Caner and

Transpplination Bology, Chiden's Reseach Insute, Chidren's NatIonMedical Center, Washiton, DC 20010 and Dep et of Pedics, TheGeorge Washton Univty Medical Center, Washington, DC 20010.

The gangNloda GD3 1s xprsa hire human memory T cells, andGD3+ T cell prol_erete hI reponrs to antGD3 antibody; fthr, theymph sts of a mnaorIty of patiet wIh T-eell acute Iymphoblstc leukemib

(T-AL gnglosold' To dYthe0potntil role ot GD3 in sgnSltrnsduction hI GD3* lymPhold cel, we have assesse antl-GD3-inducedtyron phosphop n expreion In GD3' JurLat T-ALL cell. GD3-defclentJurkat lswrei hIcubsted hI GD3-conftining mndia, whkh resulted iniceoporatlon of GD3 in time and dos-epende manner, as hown by

nced binding of the aniGD3 arnibody, R24. GD3* cells wer thn washed,icuad wIh R24, and tyrosn phshoproteins wer detected by

antiph photos1 antibody Immunobkit anarhlys of total te protInssepsted tby SDS-PAGE. The results showed that wIthin 1 minute of R24cubaton, tyros pho of seveal protns, ptcuialy pS-p

and P42-4. incresd, whil long r incubation times resulted in an isoformshft of T cell Lek ksnae, p56 to p60. Incubadon of cell with murine myrnomaIgG3 or incubation of uneatd cell wlth R24 resulted in no changes in tyrosinphosphoprotein phosphorylation. On the other hand, T cell antigen receptoractivation induced by and-CD3 antibody (UCHT-1) rsuld in an altered protein

paStem w mImu that was vey similr to observed

with R24 The result ug tat a vey early evet in GD3-assocltdignaling in T cels hiduced by arnGD3 is actVation of a tymrne kiasecascade, and that thi cascad may be identical to that associated with the T-cel antigen signal transduction patway.

61

60 Hz I G MAGNETIC FIELDS ACTIVATE PROTEIN TYROSINE KINASES ANDPKC IN HUMAN LEUKEMIA CELLS: A POSSIBLE MECHANISM LINKINGLEUKEMIA DEVELOPMENT AND MAGNETIC FIELD EXPOSURE. ((RA. Lubenand F.M. Uckun)) Div. of Biomedical Sciences, Univ. of Califomia, Riverside, CA92521; and Biotherapy Program, University of Minnesota Medical School, Roseville,MN 55113.

Although epidemiological asocation have been found between childhoodleukemia and environmental exposure to low energy electromagnetic fields (EMF),as yet there are no demonstrated mechanisms by which EMF might influenceleukemia development. The current studies test the hypothesis that low energy non-ionizing radiation might alter the acivity of src family PTKs and PKC in humanleukemia cells by pathways simiiar to those of low energy ionizing radiation.Human leukemia B-cell lines were exposed in vitro to a60 Hz sinusoidal, 1.0 Gaussrms magnetic field using a Merritt coil exposure system. Immune complex kinaseassays were done for PTKs and specific peptide phosphorylation assays for PKC.Exposure to the magnetic field produced activation of the lyn and sck PTKs, withpeak activation occurring before 5 min. PTKs are known to be associated with cellsurface receptor (CD40/CD19) modulation of clonogenic leukemia cell growth andapoptosis. Exposure to the field also activated PKC, with a time course slower thanthat of PTKs. PTK inhibitors such as genistein and herbamycin blocked theactivation of PKC, indicating that PKC activation occurs through PTK and PLCy asin the case of other mitogenic stimuli. Activation of PKC by this patrway inleukemia cells is a step in the activation of the immediate-early gene family whichparticipates in cell growth and differentiation. We conclude that60 Hz IG magneticfield exposure produces changesin human leukemia cells which are similar to thoseseen in cells treated with a number of agents which alter cell proliferation anddifferentiation. It has been hypothesized that these changes in signal transductionare a step in the clone-specific development of previously mutagenized B cellprecursor clones into ALL leukemias [Uckun, et al. PNAS 89:9005, 19921. Thesemechanisms may represent a means by which low energy EMF can influence thedevelopment of leukemia in exposed Dersons.

53

THE HUMAN PERIPHERAL CANNABINOID RECEPTOR INHBITS ADENYLATE CYCLASETHROUGH INTERACTION WITH CANNABINOID AGONISTS. ((D.M. SIpet GP. ONl, L FareC. Duesne, M. Gaent Y. Gaeaj, k 1abee, taLl. Gsser nd Klt Metlrs)) Merck Fosst Centre *brTherpeuic Research, llald, Quebec. (SponL by .J. Gamer.)

The hWm perhed cetnod (CB) rceptor has bon doed by everseadhb reacion romhunm RNA end eapressed in orde ow both IWd br*gderledisics end signal aenducon pahways. Recepior blino asays wen perbnid eooyingtheanoekhdole H*N 55212-2 nd using embrnes prered bto rensienl Vestcled COS-

ceb and a sbbi CB expressing CHODK1cd nea. In boh sprssion sysWn, MMN 55212-2spedlc nding wam opIn at pH 7-7.4, moders aitmc by *ebnt and nov&le caon(optenum mM MgCl and 100mM NaC), bht am not sigdc moduabed by GTP)S. The ms dassociation and dssodaion d P N 55212-2 wer rapid and equabim was reacd by 20 min at30'C. Saktion aysin sod t HN 55212-2 specin bidn lo th CB.recpor o1 highallnt wh equumbx bssodaion cnslauls f 2o0 and 3.8 nM (CBW-OS-t and CBrCH0-K1respeie), and sarle, wilt m _m nubers of Unding siles of 25 ad 8 pols pro (CBrCOS-MI and COsCHO-Kl rpectly). Comeition br[HN 55212-2 c badng to te CBsrCaplor wa S_1sectve aid oed tihe baoing mtk order of pobnc or ft moe actileroisoms: HW210 > WN55212-2 - CP-55940 (-) > ALTHC (-) ALTHC (-) > coinidenedaide. The Iged binding rsui show 'ientica prods of PWN55212-2 ie eton wih XtCS2 recepb for both cel ines. The aMy caton and GTP-6 psO suggest t ier th is no

eppererI drenc in CB2 recporWl when ercoetld wkh GTP)6, or that m orpo_pee dreptPl in bo systm ae urcoupled G-protein mectiacldacgnelgAg ocs ugh a smdpopAbof hh al recepbs. The ntioni cion of to C& roptor wi einoi

agot ha ben kesiged t slbe CisCHD-Kl cd CBseceptor ati

StetelyBMACI ibite bgskloin-0sntased inbheidulr cAAMP proucion in a consftdepntm .ner, butdid notc vbsor wse ihac_ie Ca in FURA-2 AMbadCB2-CHOK1 cob. The CBsreprmeilbd inton dofiosninuosd cAM wa oished

by preenet of th cb wth 10 nm of psas toxin (PTX These readl ennebale th ftCBs uncbb onr coupled Io aden4l cy actlly via a PTX-se GTP-bnd1ngPla

Ila

Signal Transduction I (64-69). Sunday64

PROPERTIES OF THE CYCLASE ASSOCIATED PROTEIN IN S.CEREVISL4E. ((N. Freeman, K. Minmr, Z. Chen, A.Weber, and J. Field))Dertm of Pharmacology ad Departqm of Biochemistry and Biophysics,University of Penylvania School of Medicine, 36th St. and Hamilton Walk,Phiadelphia, PA, 19104-6084.

We have been sdyin the cycbse associated protein CAP/SRV2p of S.cerewiae, originally isolated becue it binds yeast cyclase and is required forRAS activadon of cyase in vivo. CAP had previously been shown to beinvolved in a scond fAntion ied by a second set of phenotypes.These phenotypes we upprasedby overexpression of the actin binding proteinprofilin. Inoprcipation experime from yest indicate that a fation ofCAP is associated with actin. Furdemore, purified recombinant CAP inhibitsactin polymerization when measured by failing ball viscometry and byfluorscene of pyrene-labeled acin. The exerim n with pyrene-labeled acwindemonstate a monomer binding activity. These suggest that the second flueionis actin regulation via monwmer binding. CAP proteins in yeast, hydra, C.eleg and mammals are ca d by a proline rich stch in the centr ofthe protein. We have determined that the human and yest proteins both bindSH3 domains via this region. Candidates for in vivo wgets of the yeast proteininclude ABPl and SLAI because they are cytokdeue proteins with SH3domains. Another candi is the CDC2S protein, because it is the RASguanine nucleoide exchaner in yeast. These stuies suggest that CAP

modlates cAMP forntion bybging together adenylyl cyclase, CDC25 andevenally RAS. It may also act in both yeast and mamals to allow SH3proteins to mediate cytoskeletal changes.

66

MONO-ADP-RIBOSYLATION IS A COMPONENT OF THE INSULIN SIGNAWNGPATHWAY IN H411E CELLS. ((P. Zahradka and L Yau)) Division of CardiovascularSciences, St. Bonifsce Geral Hospital Reserch Centre, Department ofPhysiology, University of Manioba, Winnipeg, Manitoba, Canada, R2H 2A6.

ADP-ribosylation is a reversible posttransational modification of proteins whichinvolves the transfer of the ADP-ribose moiety from NAD+ to proteins by specificADP-ri ytse Two distinct cases of ADP-ribosylain reacton havebeen identified in eucaryotic cells: poly-ADP-ribosylation which is aposttransbdtonal dification that occurs only in the nucleus and mono-ADP-ribosylation which is localized in the soluble and microsomal fractions of thecytoplsm. With thed that G proteins and adenylate cyclbse can beADP-nbostd by endogenous transferases, has been of interest to esblish

a direct link between mono-ADP-ribosyltion and specific signalling pathways,Therefore, we have studied th role of mono-ADP-ribosylation in the insulinsignalling pathway in H411E rat hepstoma cells by examining the effect of met-iodobenzylguanidine (MIBG), a novel inhibitor of cellular arginine-specific mono-

ADP-rbosyltransfeae, on the growth simulatory effect of insulin. Insulinstimulation of both rRNA and DNA synthesis in H411E cells was biocked in a dose-dependent manner bythe addition of MIBG but not MIBA(meta-iodobenzylamine),a partial analog of MIBG. Using an in vitro monotransferae asay, addition ofinsulin to H411E ceUls upegulated a microsomal arginine-specific monotransferasactivity over a period of three hours with the peak activity at 30 minutes. Theincrease in activity could be inhibited by the addition of MIBG, but not MIBA. Anin ge activity assay confirmed the activation of a specific monotransfease. Poly-ADP-ribosyltrnsfase asys of the nucwar fraction demonstrated an ctivitywhich was inhibited by 3AB (3-aminobenzamide, a poly-ADP-ribosyltransferaseinhibitor) but not by MIBG. These rksult suggest that a microsomalmonotransferse is a component of the insulin signalling pathway in H411E cells.

68STIMULATION OF PROLFERATION BY N ULIN-LE GROWTH FACTOR-ICORRELATES WrIH PRODUCTION OF 5LIPOXYGENASEMETABOLITES. Mati

Je Inalil Avis, Thma Boyle and James MWshine. Walter Reed Army Inst Res.,Washington, DC 20307-5100, and Biomar and Preento Researach Br., NCI.Behesda, MD 20850.Ths study addreoad questio reated to signal transduction pathways centrd to control of

ceuar gmrwth anddif I as well to cdhular t ty. The cell sstem ued inthis study NCI-H209 muH cedl hlug carcima caultm and the asosm ued to

sasbate cdl growth ilded insnka gwth Ifor IF-) and ga-in reeasig

(GR, while antagmito growth gucag, an antis to the IGF-I re-

coptr (as-IR3), ad a toxn, stop lococcal enteo B (SEB). Insitol(14,i wa genmted up expomr to IGF-I. Pre4acmubon with gucagsora-IR3a_odgad producto ofdts compound by IGF-L IGF-I nd, to agrearex-tegucapo and SEB, incrsd the prduction of uad-d bis andri-phosphates1 compounds ae producd by the sequential actio of A2

and Al indicate that arachidonate mtablism oul als play a role in the obsrved

efficts on groth IGF-I stinsltdprduction of many arhidonate meWboi prisadllyS4rko"cowWramoic Facid (HETE) ad its metebolites products of the 54jpogenapathway). GRP also samlatd 54EE and i4t mtaboli, but its prdon u delayedboWt 2 mmiue (hes and other dat sgge that the GRP growth suimlatoy effc may

be through its simiato of rel of IGF-I). In cooua glucagon alone the

release;d,arachon acid premid IGF-I rle of 5-HE ad SEB, which

toxc to the cells, sui_lod produc of lenkotrees. Addon of 5-rT to seum.

free clue (own in mm d cdel preaon by 40% while inhb-tos of the 5-lipo"genase ptway, AAW6land NDGA, parvnted preaion ts cell

ne ad odhrestablished of mall ce hlg cm L Thoe dn dtt 5-

HEI may play a role a growth (co)factor and sugge mschanim for interfrence isprIfrationof small

cellung cancer.

65

POTENT NEW INHIBITORS OF ADENYLYL CYCLASES POINT TO NEWREGULATORY PATHWAYS. ((R.A.Johnson, L.D6saubry, and I.Shoshani))Dept. of Physiology and Biophysics, SUNY, Stony Brook, NY 11794-8661.

Adenosine 3-phosphates elicit noncompetitive, isozyme-dependent inhibitionof adenylyl cyclases via a site ("Po-site) that is distinct from the catalyticallyactive site. To date, the most potent, naturally occurring 'Po-site ligands havebeen 3AMP and 2'-d3'AMP (IC50s: 9 pM, 1.2 pM, respectively). These can bederived readily from nucleic acids. 3AMP levels varied considerably in tis-sues and roughly reflected the sensitivity of the respective adenylyl cyclases:less 3AMP was found in tissues with the most sensitive enzyme. Cell perme-able "P*-site ligands, 2',5'-dideoxyadenosine (2',5'-ddAdo) and 9-(cyclopentyl)-adenosine (9-CP-Ade) (IC5us: 3 pM, 20 pM, respectivley) can strikingly affectcell function. At concentrations consistent with their inhibition of adenylylcyclase, 2',5'-ddAdo and 9-CP-Ade induced differentiation in ob1771 cells thatwas more rapid and complete than was induced by insulin and triiodothyro-nine. These data suggested that nucleic acid metabolites could influence callfunction via direct inhibition of adenylyl cydases. In exploring this inhibition,we synthesized a series of 2',5-ddAdo-3-phosphates, which gave the follow-ing IC50s for inhibition of Type adenylyl cyclase: 2',5'-dd-3'AMP, 3OOnAt; 2',5'-dd-3'ADP, 30 nM; 2',5'-dd-3'ATP, 10 nM. 2',5'-dd-3'ATP is as potent a regula-tor of adenylyl cyclases as are G-proteins. We are finding also that naturalribo- and 2'-deoxyribo-adenosine homologs potently inhibit this enzyme. Thedata suggest that adenosine 3'-di- and -triphosphates may constitute a partic-ularly important mechanism by which adenylyl cyclases and the cAMP-signal-ing cascade are regulated. The potency and character of these agents sug-gest that they probably regulate other enzymes as well. (NIH: DK-38828)

67Tl FmCT OF THYROID STATUS 01N CARDIAC ARGINIsi-sPzCIFICsOuo-ADP-RIz10aTSYTRANFmRAAS ((I. Igbo and K. Xecitahon))Texas Tech University Health Sciences Center, Lubbock, TX

To determine if thyroid hormone affects arginine-specificmono-ADP-ribosyltransferase activity, adult rats were madeeither hypo- or hyporthyroid or kept euthyroid by i.p.injections of 20mg/kg PTU, 500qg/kg of T,3 or vehicle(alkaline salino) for up to 1S days. Body weight wasmasured daily aa was heart weight at sacrifice. Arginine-apecific mono-ADP-ribosyltransferaso activity and ADP-ribosylated protein in heart membrane preparations were thendetermined. Heart weight/body weight and daily weight gainshowed the status of the animals thyroid. AT 1S days bothhypo- and hyperthyroidim caused 75% inhibition of ensymeactivity a masured by both ADP-ribosylation aay and ADP-ribosyltransferase activity. A difference was seen in therate of onset of inhibition. PTU treatment caued a graduallosa of enzym activity, while T, treatmnt had an imediateonset. In conclusion, thyroid status has a dramatic effecton mono-ADP ribosylation of the heart.

69

REGULATION OF NITRIC OXIDE SYNTHASE BY PLATELET ACTIVATINGFACTOR AND BACTERIAL LIPOPOLYSACHARIDE IN RAT KUPFFER CEULS.((S.B. Mustafa, A.M. Kitten and MS. Olson)) Department of Biochemistry,University of Texas Health Science Center at San Antonio, San Antonio, TX78284. (Spon. by M. S. Olson.)

Bacterial lipopolysacharide (LPS) induces nitric oxide synthase (NOS) inmacrophages.The consequent overproduction of nitric oxide (NO) plays a primaryrole in the pathgenesis of endotoxicshockl Platelet activating factor (PAF) is aproinflammatory mediator, and plasma levels of this Hlpid are elevated inendotoxemna In cultwed rat fiver Kupffer cells (KC) PAY augments LPS- inducedaccumulation of nitrite, a stablemetabolite of NO, ina dose and time-dependentmanner. In parallel with this reaction, PAP and LPS increase protein levels ofNOS. In the absence of extracellular calcium, PAF -and LPS-induced NOproduction is reduced significantly, however following induction, NOS activity iscalcium independent. Treatment ofKC with actinomycinD, cycloheximide anddexamethasone greatly suppresses PAP- and LPS-stimulated nitrite formationdemonstrating that de mow protein synthesisis necessary for the induction ofNOS. Pretreatment ofKC with PAP antagonists WEB 2170 andBN 50739 prior tostimulation by PAP and LPS inhibits NO production. In the presence of differentarginine analogues PAP- and LPS-induced NO formationis significantly lower.Nitrite synthesis by KC stimulated by PAF and LPS is dependent uponextracellular L-arginine and is reduced significantly in the presence of L-lysine.Following formation of NO after PAP and LPS stimulation, guanylate cyclase isactivated resulting in the cellular generation of cGMP. Treatment of KC withcycioheximide inhibits cGMP formation. The present study indicates that NOS isinduced in KC following stimulation by PAF and LPS and this response can beattenuated using PAF receptor antagonists and NOS inhibitors. (Supported bygrantsDK 33538 andThe Robert A. Wekh Foundation to MSO.)

12a

Sunday. Signal Transduction 1 (70)

70ACTIVATION OR SUPPRESSION OF HIV-1 LTR ACTMTY BY THEANTIOXIDANT PYRROUDINE DITHIOCARBAMATE (PDTC) IS PHORBOLESTER DEPENDENT. (K Watanabe, U. Venkatachaam and S. C. Miller) UfeSciences Division, SRI Intemational, Menlo Park, CA 94025.

The intracellular level of reactive oxygen intermediates (ROls) is underdynamic modulation by molecular oxidants and antioxidants. A number oflaboratories have demonstrated a role for redox regulation in the modulationof the NF-kB transcription factor and activation of gene expression directed bythe human immunodeficiency virus long terminal repeat (HIVI-LTR). We haveused the antioxidant pyrrolidine dithiocarbamate (PDTC) to evaluate thepotential mechanism(s) involved in the redox control of signal transductionpathways acting on the HIV1-LTR in the human U937 cell line. Cells wereeectoporaed with a chloramphenicol acetyl transferase (CAT) reporter genedirected by the HIV1-LTR. 24 h postelectroporation, replicate cultures weretreated with increasing doses of TPA to activate HIV1-LTR directed CATprotein. One set of cells were treated with the antioxidant PDTC for 1 h beforethe addition of TPA. Samples were harvested after 24 h and the level of HIVl-LTR-directed CAT protein induced by TPA in the control or PDTC treatedcultures was measured in cell extracts with a CAT ELISA assay. Using thisapproach we have defined conditions that demonstrate that PDTC hasstimulatory and inhibitory activity on the signal transduction pathwaysintegrating at the level of the HIVI-LTR. Moreover, the dose-dependent effectof PDTC to stimulate signal transduction by TPA (p<0.001 by t-test) decreasedwith increasing concentrations of TPA in a dose-dependent manner. Thesignal transduction pathways modulated by intracellular calcium and reactiveoxygen intermediates that integrate NF-k8 activity and HIV1-LTR activation arebeing defined in this model system.

Cell Cycle Controls (71-74)71

GENE EXPRESSION DIVERSITY IN THE G1 AND S PHASES OF THE CELLCYCLE: INVESTIGATION UTILIZING A NOVEL EXPRESSED SEQUENCE TAGPARADIGM.((J.A. Earle-Hughes, M.D. Adams, A. Glodek, C.M. Fraser, and J.C. Venter))Department of Molecular and Cellular Biology, The Institute for GenomicResearch, Gaithersburg, MD 20878.

Expressed sequence tags (EST's) are useful tools in identifying diversityand shifts in expression pattems between different populations of cells.We constructed two directionally cloned cDNA libraries from Jurkat T-cellleukemia cells synchronized in either the Gl or S phase of the cell cycle.Sequencing analysis of 1876 randomly selected clones from Gl and 1550randomly selected clones from S was carried out. This yielded a total of602,648 base pairs of sequence for the Gl phase and 432,503 for the Sphase. Database (GenBank/EMBL) searches yielded exact matches (>95%nucleotide match) to existing sequences for 30-36% of the total EST's, 2-3% were a non-exact (<95% nucleotide match) match, and 6-10% showedhomology to non-human sequence at the protein level. A significantpercentage (46-52%) showed no match to any known database sequence(unknowns). Significant changes in gene expression between the Gl and Sphases of the cell cycle were observed in genes encoding cell cycle regulatedproteins. Differences were also seen in genes involved in the control ofmicrotubule formation and general cell metabolism. However, similarexpression pattems were noted for ribosomal and protein synthesis genes.Preliminary Northern blot studies of several clones confirm expressionpattems observed in EST database analysis.

73REPLICONS MAY BE POISED FOR INITIATION IN MIMOSINE-BLOCKED CHO CELLS. ((J.A. DAnna, J.G. Valdez, S. Peterson, andH.A. Crisman)) Life Sciences Division, Los Alamos National Laboratory,Los Alamos, NM 87545.

Synchrony of mamalian cells near the G1/S boundary using mimosineinhibits the initiation ofDNA replication. Although this procedure appearsto block cells in late G1, CHOC 400 cells synchronized in this way exhibita marked reduction in the time-dependent transient inhibition of DNAreplication observed in response to ionizing radiation in exponentiallygrowing populations [Larner et al., Mol. Cell. Biol. 14, 1901-1908(1994)]. Curiously, this result is reminiscent of anomalous effects that areobtained when CHO cells pretreated with hydroxyurea are irradiated[Painter and Young, Rad. Res. 64, 648-656 (1975)]. Here we confirmthat blockade of cells with mimosine inhibits DNA synthesis; however, wefind that CHO cells undergo biochemical and stutural changes similar tothose of CHO cells blocked with hydroxyurea, aphidicolin, or other drugsthat do allow the initiation ofDNA synthesis. In both cases, histone HI ispartially depleted in the cell, a large portion of the remaining HI isphosphorylated, there are elevated leveis of cdk2 phosphorylation, and thetreated cultures exhibit unbalanced growth. Thus, whereas CHO cellsblocked with mimosine do not initiate DNA synthesis, their nuclei may bebiochemically and sucturally poised for the initiation ofDNA replication.These results (1) indicate that the restiction point for unbaland growth inCHO ceUs precedes the mimosine block point, and (2) suggest that theevents that regulate the radiation-induced tme-dndent inhibition ofDNAsynthesis precede chromaun structual changes that may be required for theinitiation ofDNA replication. (Supported by the U.S. D.OE).

72

REGULATION OF GI PHASE BY CELL ADHESION IN MOUSEFIBROBLASTS. ((T. Kuzumaki, A. Matsuda, and K. Ishikawa))Department of Biochemistry, Yamagata University School of Medicine,Yamagata 990-23, Japan. (spon. by R.F. Morrison.)

Growth factors and cell adhesion to substratum are essential factors forfacilitating the progression of the fibroblast cell cycle through GI. It is veryimportant to know the precise mechanisms by which cell adhesion regulatesGI phase. We investigated these mechanisms during GO to S phase inmouse BALB/c 3T3 fibroblasts, comparing them with the mechanismregulated by serum. 3H-Thymidine incorporation was measured when cellswere cultured with serum free medium or cultured without cell adhesion(cultured on the dishes coated with polyHEMA) during GO to S phase.Serum was required only in early GI phase for progression into S phase. Incontrast, cell adhesion was clearly needed in late GI phase, especially at theperiod of GI/S transition. Cell adhesion was not required for cells toundergo DNA synthesis once they had passed the G1/S boundary. Northernblot analysis showed that expression of histone H2B and dihydrofolatereductase (DHFR) genes were much suppressed in the cells without celladhesion during late GI and S phase. Further, the transcription factor E2F,which stimulates transcription of DHFR gene, and c-myc genes were alsosignificantly suppressed in the cells without adhesion. These results suggestthat adhesion to substratum plays an important role in regulating theprogression of Gl phase, especially the G1/S transition affecting theexpression of E2F and c-myc genes.

74A UNIQUE G/S CELL CYCLE IN NORMALLY DEVELOPINGMEGAKARYOCYTES. ((Zhengyu Wang, Dimitry Kamen, Ying Zhangand Katya Ravid)) Department of Biochemistry, Boston University Schoolof Medicine, Boston, MA 02118.

During megakaryocyte development the cell stops mitosis and beginsendomitosis, giving rise to a large polyploid megakaryocyte whichfragments into platelets. We have studied the nature of the cell cycle in thisunique cell type, using rat or mouse cultures of primary bone marrow cells.Since megakaryocytes consist only about 0.1% of total bone marrow cells,all studies were done by in-situ staining. DNA synthesis in megakaryocyteswas followed by incorporation of bromodeoxyuridine (BrdU) and wasdetected by immunohistochemistry. These studies revealed that immaturemegakaryocytes (displaying a diameter of 30-50 microns) do not synthesizeDNA continuously, but rather undergo a G/S cell cycle, while the largemature megakaryocytes (>50 microns) cease synthesizing DNA. Cells thatdemonstrate weak staining with BrdU also express the S phase cyclin A, asrevealed by immunostaining. As the megakaryocytes rest in G, theyexpress some of the mitotic cyclin B and the GI cyclin D3. Since these cellsdo not progress through mitosis we suggest that cyclin B, although beingexpressed, is inactivated in this cell type. However, antiseseoligonucleotides designed to suppress cyclin D3 expression, suppressed thedevelopment of megakaryocytes in the bone marrow culture. Amplificationby PCR of cDNA prepared from large mature megakaryocytes, usingprimers for cyclins A or B, yielded cyclin DNA which was barely detectableon agarose gel. We conclude that immature megakaryocytes possess aunique cell cycle and that cyclin D3 plays an important role in normalprogression through this G/S cycle. The cells of high ploidy class stopcycling but continue the process of cytoplasmic maturation.

13a

Cell Cycle Controls (75-80). Sunday

75

MOLECULAR DISSECTION OFA MULT-PARTrTE CEL CYCLEDOMAIN OF H4 GENE TRANSCRIPTION. ((F. Aziz, A. J. vanWijnen, J .B. Lian, J. L. Stein, and G. S. Stein)) Department of CellBiology, University of Massachusetts Medical Center, Worcestr, MA01665.

Cell cycle control of G1/S phase enhancement of histone H4 gene

transcption is regulated by a multipartite protein/DNA interaction domain,(Site II) composed of contiguous and overdappling elements for several

transcription factors. A critical element, known to be a binding site forcomplex HiNF-M, is located in the distal portion of Site HI and contributesto cell cycle regulation. This element is identical to the consensus bindingsequence for interferon regulatory factors 1 and 2. This region also overlaps

with the binding site for the proliferadon-specific factors HiNF P and HiNFD (a multcomponent protein containing the key cell cycle regulators cdc2,cyclin A and an RB-related protein). In this study we have used a series of

factor specific mutations within the Site H domain in conjunction with gel

shift assays to identify the specific nucleotides critical for the binding ofthese factors. Further, we have tested these mutations using transient

transfection assays in HeLa cells, rat osteoblasts, and rat osteosarcoma cells.It was observed that a mutation within the HiNF-M binding site decreasedthe level of expression three-fold. These results observed in vivo are

consistent with the concept that the interaction ofHiNF-M is required for

cell cycle regulation ofH4 gene transcription.

77

A DOMINANT ROLE FOR SPI IN THE EXPRESSION OF A CEL L1CYCLE REGULATED HISTONE H4 GENE. ((M.J. Bimnbaum, AJ. van

Wijnen, KL. Wright, F. Aziz, TJ. Last, B. Frenlde, M.T. Bourke, B.R. Rao,G.S. Stein, and J.L Stein)) Department of Cell Biology, University ofMassachusetts Medical School, Worcester, MA 01655.

Maximal transcription of the cell cycle regulated histone F0108 H4 gene ismediated by two proximal pmoter elements, Sites I and IL While Site IImediates the cell cycle controlled enha nt ofH4 gene transcription at theG1/S phase boundary, maximal levels of tansciption require participation ofSite L By the combined use of the gel mobility shift assay with site-diectedmutaeness, oligonucleodde competition, in vitro expression of t

ptens, and spocific antibody supershifis, we show that Site I is a bi-partitedomain interacting proximally with Spl and distally with members of the ATFfamily of tanscription factrs. Transient gene expression assays in HeLa cellsreveal that a mutation in the Spi binding site, which abolishes Spi binding ingel mobility shift assays, results in a six to ten-fold reduction in reporteractivity as compared to wild-type controls, suggesting that SpI functions invivo at this site. In contrast, mutadon of the asymmeric ATF binding sitealone has no effect on expression. However, in the absence of a functionalSpi binding site, mutation of this ATF site results in a significant decrease inP activity, that this site may indeed function in living cells.

Transactivation studies in Drosophila Schneider cells, which normally lackSpl, show that overexpression of human Spl results in the specific activationof the histonep r via this site. lbus, we show unequivocally that Splplays an important role in enhancing levels of histone H4 gene twanscription.

79

IDENTIFICATION OF MAMMALIAN DNA POLYMERASE P TRANS-DOMINANTMUrANTS BY COMPETiION ANALYSIS INE. COIl.((C.A. Clairmont and lB. Sweasy)), Depatment of Therapeutic Radiology, Yale

University School of Medicine, New Hatn, Cf 06510.

We descbe two mutant DNA polymra e (Pol P) proteins tiate dominantto the wild

type Pot P potein. Pot -2412E prein contains a Y265 to C mno acid altaiom,and the Pol, P-TR protin is a truncat protin contining amin acida 1-154 of the

wildtp Pot P ptin. Usng anwly de-velopedcompeits asy. wedemosaet thatthese two potins are transdominant toDNA polymrase I Ourasmy employs E.co

strain with mutaions in the recA and polA gees (recA718poW12t4) that is unabl to

grow at the restrictive temperatue of 420C on rich medium. However, the

recA718poL4I2U mutant is able to grow on rich medi ut 420C in he pewace ofDNApolymerae P (Sweasy. J.B., and Loeb. LA. J. Biol. Chexs 267: 1407-1410 (1992)).This is most likely due to the ability ofDNA polymerase P to i ase the rate ofjoinigof the Okazla fragments duing DNA replication.We how that recA718poU12 cedlsexpressig the wild type Pol P protin wgethe with eitae the Pol 0-.R or Pod P-2412E

prons ame no lon able to grow on rich medium at 420C to the _me degree thosecells coaning only wild type Pol P. lbose result demm that in dte presence of

Pol P-TR ar Pol 0-2412E. DNA polymerase 0 is unable to complement the

recAl7polA12ts phenotype, suggesng that hes mutant pri interfer with DNApolymrase Ps ability to facilitate the joining of the Okazai fragmnts One possibleexplanatin for this inibition is thatPolP .T ndPdd-24l2Ep may bind to the

DNAin ple of wild type polymera th y the binding sites in an

inactive form. These results stroy sggest tht Pot d Pol p-2412E wedominant to DNA polymerase P This pp ch c be dy effect of kss of

DNApolymerns P activity with respect toDNArpsirep ndr ptcat

76

HISTONE H4 CELL CYCLE ELEMENT BINDING FACTOR HINF-M ISIDENTICAL TO INTERFERON REGULATORY FACTOR 2 (IRF2). ((P.S.Vaughan, A.L. Ramsey-Ewing, A.J. van Wijnen, G.S. Stein andJ.L. Stein)) Department of Cell Biology, University ofMassachusetts Medical Center, Worcester, MA 01655.

The histone H4 gene F0108 is transcriptionally regulatedduring the HeLa cell cycle with a three to five fold increasein transcription during the Gl/S transition. We have definedthe cell cycle element (CCE) required for this activation asan eleven base pair sequence located 20 bases upstream of theTATA box. This site binds a protein factor in gel mobilityshift assays designated histone nuclear factor (HiNF) M. HiNF-M was purified by conventional and DNA affinity chromatographyand determined to be a 48 kilodalton protein by UVcrosslinking and Southwestern blot analysis. Microsequencingof purified HiNF-M revealed it to be identical to interferonregulatory factor 2 (IRF2). IRF2 and the highly relatedprotein IRFI bind a consensus element in the interferon (IFN)P and other IFN inducible genes and the CCE matches the IRFconsensus exactly. In vitro translated IRFI and IRF2specifically bind the CCE in gel shift experiments. It hasbeen demonstrated that IRF2 has oncogenic potential and IRF1has tumor suppressor activity. In order to establish a linkbetween these cell growth related proteins and a cell cycleregulated gene, we are examining the roles of IRF1 and IRF2 intransient transfection experiments with the F0108 gene

78

MOLECULAR CLONING OF THE HUMAN RNA POLYMERASEII LARGEST SUBUNIT GENE RESPONSIBLE FOR INDUCTIONOF SISTER CHROMATID EXCHANGES. ((H. Tsujil, K. Mital, E.

Takahashi2, M. Morimyol, M. Yamauchil, T. Saito', and T. C. James3)) 'NatL

Inst. RadioL Sci., Chiba-shi 263, Japan. 2Ohtsuka Pharmaceutical Co.,

Tokushima-shi 771-01, Japan. 3Fairfield Univ., Fairfield, CT. 06430.

To understand the mechanism of the maintenance of chromosomal stability,

we have isolated many temperature-sensitive (ts) mutants from the Chinesehamster cell line CHO-Ki. One of the mutants, tsTM4, exhibited abnormalinduction of sister chromatid exchanges (SCEs) along with decreased DNA

synthesis in the cells arrested in S phase at the non permissive temperature

(39° C).We have cloned a human gene that complemented the genetic defectof CHO-Kl tsTM4 cells by (a) transfecting human genomic DNA into them,

(b) collecting the human DNA fragments from the secondary transformantsand (c) screening a HeLa cell human genomic DNA library by using these frag-ments. DNA sequence analysis of these fragments revealed that it is the humanRNA polymerase II largest subunit gene, HSRpIILS, with 29 exons spanning31 kbp. Transfection of cloned genomic DNA fragments containing the entirecoding regions of HSRpIILS into CHO-Kl tsTM4 cells rescue their abnormalSCE induction phenotype. The northern analysis shows that the HSRpIILS istranscribed at 390 C and 33.50 C in the transformant. These data strongly sug-gest that a defect in the RpIILS gene is responsible for the abnormal inductionof SCEs in the CHO-Kl tsTM4 mutant. The HSRpIILS gene was mapped to

17p13.1 by fluorescence in situ hybridization

8s

FUNCTION OF BACTERIOPHAGE T4 RNASE H IN DNA

REPLICATION. ((L.J. Hobbs and N.G. Nossal))Laboratory of Molecular and Cellular Biology, NIH,Bethesda, MD 20892.

A vital part of the study of DNA replication isthe coordination of proteins involved in laggingstrand DNA synthesis. T4 RNase H, expressed soonafter infection, removes ribopentamer primers from

nascent DNA chains during synthesis by the T4

replication system in vitro. In this study, RNaseH was purified and characterized using a varietyof 5' and 3' (3P] labelled substrates. T4 RNase H

is a 5' to 3' nuclease that can release RNA from

an RNA/DNA hybrid in primarily 1-3 base fragments,with some products of 4 and 5 nucleotides. It is

also very active on a DNA duplex, somewhat active

on single-stranded DNA (ssDNA) and not active on

ssRNA. 32 Protein (ssDNA binding protein)stimulates the activity of RNase H, causinggreater release of products and larger fragmentsof up to 6 bases. The polymerase accessoryproteins (44/62 and 45 proteins) also moderatelystimulate RNase H together, but not separately.When a downstream DNA primer is present, T4 DNA

polymerase (43 protein) stimulates the RNase H to

cleave larger fragments of up to 20 nucleotides.

It appears that RNase H is not self-regulating.

14a

Sunday. Cell Cycle Controls (81-86)

CEI CYCLECHECKPOINTS: NUCLEARNOT CYTOPLASMCCONTROLOFNUCaEARENVELOPE BREAKDOWN IN SEA URCHIN ZYGOTES ((G. Sluder,

E. Thompson, and F. Miller)) Worester Fdn. Exp. Biol., Shrewsby, MA 01545.

Nudear envelope breakdwn (NEB) and entry into mitosis are dtought oD be driven

by tbe actvato of tie p34-cyclin B lkimse complex or Mitosis Promoing Factor

(MPF). However, when promudear fusion in sea urchin zygotes is blocked with

Colc-mid the female proucetus conastdy undagoe NEB before the male

. ITis ry is not due to regional differea in the time ofMPF

activatin, because closely apposed nacle also break down ro . To

expe this phnomeon we blck the compledo of DNA synthesis eparaely in

male and femalepromcdle by teati eggs or sperm before fertiinti with psonlento form covnt croinks between baspaired strans of DNA. When die female

prmar DNA is croa linked, die male prouae brea down first When the

DNA is cross-linked, male promaclyear breakdown lags female

NED and sometmes doea not ocu even dough the zygote compleles mtsia.

Inactiation of the Colcemid after female NED in such zygotes yields a fucional

spide with die male promuleus loated at one aster. Serial secdon eectr

and m-iroinjection of fluorcent dextran show that the male pmiclear

enveope remains souctualy an fnctdoally intct despile mioDtic levels of MPF

around it. we show tat when psoalen treaftd spermF ci fiue with

feme promacled, die zygote mclei do not break down for more than six his (5-7 normal cell cycla) even dugh HI hist kna activity riss montonicallymitoic or hie leveis. Ie m true for normal zygotes treated with aphidicolin

to block all DNA snthesi. These results show that insea urchin zygotes: 1. NEB

can be undermclear nt cytoplasmic control. 2. Ie target ofthedcekpoint for the

completion of DNA syntesis is within the mdeus and is not activation pathwayforMPF. 3. Mlttcleves of cytoplasmicMPF activity arenot sufficent to drive

NED for amcleus that is under checkpoint ctol.

33

A 1iNETOCHRE PHOSPHOEPITOPE IS A COMPONENT OF A

METAPHASE CELL CYCLE CHECKPONT ((MS. Campbel and GJ.

Gobsky)) DepL of Cell Biology, Univesity of V ia Healft Sciences

Ceter,ChQaritesville, VA 22908.

We haveshawn that a kinecr phosphoepitope is ac nt of a cell

cycle ceckpoint pawaytht mnitos the intarctionof kinedocos with

the sindle. The3F3 m oclonal anti-phohoepitope antibody reopizsa kintoder in mm alia oells dtat is expssed on

diroseone before eirc ne to Usenmaphase plate. Once dromoisomes ae aligned,exrs is lost and cells eter anraphase WhA mnicro-ijectedinto prophse cells, the 3F3n2 Ab causes a concentaon-dqpndeadeLaymthe onset ofaahse InjectedAb ihibit dosphorylataon of the3F3J2 ping npashe. Inirectedcells, thephsphoptrenia exessed at Uhe throuxut the prolongedbtt is finlydeqhosphorylated by Uhe start of anphase.In cells allowed to

progrms nomully to nxaphse and Uxi treated widt miicrotulxale inhibitos,

Use 3F3/2 phosphoepitope rapeas at Use kneochre within 3 mi

Treatnut of cells with irbue inhibitors raeiers the pophoepitopemore resistat tod phospylation by endogmo p as. Immso-

electrn mic py rveals dth Use 3F3/2ep ope is con ed in the

middle layer ofthetrilamina kinethe shuctur Collective, these data

implicate Use 3F3/2epitope in a checkpoint pahwa that govon the etna-phb e to eWhase transiio, and dephoshory on of Use epitope appearsto bereqred foranahase onse. Sported by a grant from the NIGMS.

aTISOASE II A1rIVrTY IS lOUE FOR NEIOflICPEICNtESSICNIN XENOPt LIAEVIS SPERM (CYES- ((M fMorse-Gaio aid M.S.

Risley))D-part2nt of Biological Scienes, Fordham University,

Brxt, NY 10438

Topo II tartt drugs have been used to study the depergnc

of emiosis on topoII activity in cultured spE-tocytes. Met-

aphaa to anaphae progressnwas ihibited by 0.5 and 5pMtniposide (V-26) or 0.5-5AM aclarubicin (ACIA)in s arrested by either drug into dense

es. inhibition of progression tonetaphasewas observed during 6-18 hr incubation in 0.1-5)M VM-26 orAaA. Pachytene pycnotic in 5 or 50qi VM-

26 within 18 hrs and >95% were killed after 6 days in Vt-26.

lie respos of pacytene cells toincubatiin ACLA was

Ravterogmisos: most pahytene cells (60-70%) appeared normalafter 6 days in 0.25-5,M AMA, but 30-40% asamedan inter-

phase-like xorpiDlogy. This atypicln hrlogy was also ob-

served within 18 hrs at tKACA. VM-26 (5)i, 4 hr) irduced

N broeols in 94% of ntocytes aid ias deter-

mired by alkalire single oell gel A (0.5-5.juK) didnot ind ENA breaks, bat caused a dose-deperdentinhibition Of irduction of ENA breaks by V6-26(5M) Tu,

the topoII activity in speantocytes is affected byVU-26AC1A conntrations that block meioticprgressin. Sinceeac

drug inhibits tapo II fuotion by adifferent mucnhanim, the

reslts wxe st that topo II activity isimpotant for pro-

gression frompadyteme throughMII. Pesearchstenr b

IaPEcOductive Hazad in the Workplac, ane, iy andFawironun No 15-93-0669 the March of Dimes.

82DNA TOPOISOMERASE II AND THE 'DECATENATION ADEQUATE?' CELLCYCLE CHECKPOINT. ((DJ. Clarke, J. Gimenez-Abian, A.M. Mullinger, R.T. Johnson& CS. Downes)) CRC DNA Repair Group, Dept. of Zoology, University of Cambridge,Downing St., Cambridge, UK. (Spon. by D. Bray.)DNA topoisomerase II decatenates replicated DNA and is required for chromosomecondensation. We present evidence for a new cycle checkpoint in mammalian cellswhich prevents entry into mitosis until decatenation is adequate for condensation.Standard topo II inhibitors delay cells in G2 but also damage DNA which enforcescycle arrest through the established damage checkpoint. However, ICRF-193 (a topoII inhibitor which does not damage DNA) also blocks progression to mitosis. The'decatenation adequate?' checkpoint is therefore distinct from the damage checkpoint.Typical of checkpoints, ICRF-193-induced arrest is leaky; cells enter mitosis after -4h. Checkpoint-evading drugs, caffeine and kinase/phosphatase inhibitors, force cellsgiven ICRF-193 into mitosis without delay. Condensation, though imperfect, isinduced; thus cells are capable of condensation without topo II activity, but areprevented from starting prophase due to a catenation-sensing mechanism. After shorttimes of ICRF-193 and caffeine treatment, chromosomes (from cells near prophasewhen drugs were added) are quite well condensed. With longer accumulation, lesscondensed material is seen, derived from cells further from prophase when topo II wasinhibited. This time-dependence of condensability indicates progressive decatenatoryactivity in S/G2. In some cells the damage checkpoint is caffeine-insensitive but ICRF-193-induced arrest remains caffeine-sensitive; a component of the damage response isnot part of the decatenation checkpoint. Ataxia telangiectasia group C cells do notdelay in response to damage or ICRF-193 treatment; decatenation and damagecheckpoints have a common element. We suppose the common elements are delayeffector mechanisms, operating downstream of separate damage and catenation-sensors. There is no post-prophase catenation checkpoint. Cells driven into mitosiswith inadequate decatenation (by ICRF-193 and caffeine) attempt anaphaseregardless; cells form a spindle, attempt to separate their catenated chromatids withnormal kinetochore positioning/separation then exit from mitosis.

84PDS1: A NEW GENE INVOLVED IN REGULATION OF ANAPHASE IN YEAST,SACCHAROMYCES CEREVISIAE. ((A. Yamamoto, V. Guacc, and D. Koshland))Department of Embryology, Camegie Institution of Washington, Baltimore,MD 21210

During anaphase duplicated and paired sister chromatids separate andmove along an elongating mitotic spindle to poles of the dividing cell. Yeastmutants deleted for a PDS1 (precocious disassociation of sisters) gene grewslowly and lost chromosomes rapidly at 23 C. They were inviable at 37Capparently because they failed to undergo anaphase but do cytokinesis. Thisresulted in unequal chromosome segregation and accumulation of spindlepole bodies. When pdsl mutants were arrested in S phase at 23XC, theyformed short spindles. When these arrested cells were released from the Sphase block, they completed anaphase even at the 37 C. These resultssuggest that the function of a Pdsl protein for anaphase is executed beforethe anaphase, around the time of bipolar spindle formation. Yeast cells canbe arrested in G2-M phase by nocodazole or by cdc16 or cdc20 mutations.pdsl mutants failed to block dissociation of duplicated sister chromatids innoodazole. Similarly when a pdsl mutation was introduced into cdc16 andcdc2O mutants, sister chromatid separation and spindle elongation were nolonger blocked at the restrictive temperature. The fact that a pdsl mutationallows two differentcdc mutants to complete mitosis, suggests that the Pdslprotein plays a role in regulating the transition from metaphase toanaphase. Consistent with this regulatory role, overexpression of the PDS1gene blocked chromosome separation and spindle elongation but notcytokinesis. Furthermore expression of the PDS1 gene was regulated in acell cycie dependent manner. These results strongly suggest that the Pdslprotein is involved in regulation of the onset of anaphase. Possiblemechanisms of the PDS1 function are discussed.

86IDENTIFICATION OF AMOS-IN ERACTING PROTEIN FROMEXTRACTS OF XENOPUS OOCYTES. ((K.H. Martin and K.I.Swenson)) Department of Cell Biology, Department of MolecularCancer Biology, Duke University Medical Center, Durham, NC27710.

The mos proto-oncogene is a serine/threonine kinase that isnecessary for the meiotic maturation of Xenopus oocytes.Theactivity of mos protein can be assayed experimentally bymicroinjection into Xenopus oocytes, where it triggers theresumption of meiosis, or by the addition to oocyte extracts, whereit induces the activation of MAP kinase. In order to elucidate theevents that occur immediately upstream or downsteam from mos inthe signaling cascade that triggers the biochemical processes leadingto meiosis, we are attempting to identify proteins that interact withmos in a functionally important manner. A functionally active,bacterially-expressed mos fusion protein consisting of maltosebinding protein linked to Xenopus mos (MBP-mos) was used inamodified western blotting assay to probe oocyte extract proteinsseparated by SDS-PAGE andtransferred to nitrocellulose. BoundMBP-mos was detected using a primary antibody to MBP. Resultsfrom this assay showed that MBP-mos bound specifically to oneprotein of 22kD. No binding to this protein was detected in identicalblots probed with MBP protein. Experiments are currentlyunderway to examine the contribution of this protein to mosfunction in oocytes and oocyte extracts.

15a

Cell Cycle Controls (87-92). Sunday

S7isOLATIONOFANOVELANRNKREPEATCONTAINiNG GENE FROM SCEREVISIAE ((D. Lycan1, B. Bolinger1, K. Stafford1 and L Breeden2)) 1.Dept. of Biobgy, Lewis and Clark Collge, Portland OR 97219. 2- FredHutchinson Cancer Research Ctr., Seattle, WA 98104. (Spon. by M.McClellan.)

The ankyrin repeat Is a 33 amino acid motif first Identified as a region ofhomology between two yeast cell cycle regulators (Sw16 of S. cerevislae andCdclO of S. pombe) and two transmembrane proteins with roles in cell fatedetermination (Notch of DrosophIla and Un12 of C. esegans). VarIants of thisrepeat have since been identified in a varlety of proteins from bacteria tohumans, Including transcriptlon factors (MBP, Swi4), regulators oftranscription factors (IkB proteins), a mitochondrial enzyme (glutaminase), acytoskeletal protein (ankyrin). Data from several labs suggest that the functionof the ankyrin repeats In these diverse proteins may be to fadlitateprotein:protein Interactbons. We have identified and sequenced a new repeat-containing gene from S. cerevsiae, YAR1 (yeast ankyrin tepeat protein). YARIIs a small gene, containing a single uninterrupted open reading frame of 627nts. and two noncontiguous ankyrin repeats. YAR1 Is transcribed In both haploidand dipld cellk, and In cells arrested In either GI or S phase of the cell cycle.We have disrupted the chromsomal YAR1 gene In two ways. An insertionaldisruption between the ankyrin repeats of one YAR1 gene In diploids has adominant negative effect upon sporulation. Haterozygous diploids are small,grow slowly and do not sporulate. A truncated polyA+ YAR1 RNA Is detectable inthese diploids In addition to the normal YAR1 RNA. In contrast, a disruptlon thatalso deletes most of YAR1 does not affect sporulation; both heterozygous andhomozygous .yarl diploids sporulate. Haplold Ayarl strains are viable butexhbit a slow growth phenotype. Our workIng hypothesis Is that while theYAR1 gene is not required for sporulation, truncation of YAR1 aiters itsfunction in a way that interferes with sporulation.

89

COMMON MECHANISMS OF NONPROLIFERATION IN HIGH-DENSITY CULTURES, DIFFERENTIATED CELIS AND UNDERTHE INFLUENCE OF HEPARIN.((Y.E.Yegorov,Y.V.Fedorov and I.A.Prudovsky)) Engelhardt Institute of Molecular Biology,Russian Acade-my of Sciences,32 Vavilov str.,ll7984,Moscow,Russia.

Short-term treatment of cels with high-potassium solution inducesDNA synthesis in high density-inhibited cells (Exp.Cell Res. 1992,203,488-490).The same treatment can induce replicative DNA syn-thesis also in some nonproliferating differentiated cells, inclu-ding neurons (Exp.Cell Res.,1993,209,156-l59).We have shown thathepann inhibits cell proliferation preventing cell cycle transi-tion both in G-1 and 0-2 periods. Short-term potassium teatment in-duces DNA synthesis in presence of heparin in inhibitory concen-tradons. Our data indicate the existence of common mechanisms of pro-liferation blockage in cases of high density conditions, differen-tiation and heparin treatment.

91

THE CELL CYCLE ALTERS C/EBP ISOFORM mRNA LEVELS INCOMMA D MAMMARY EPITHELIUM CELLS. ((J. O'Rourke ad J.DeWille)) Ohio State Biochemistry Prog. and Dept. of Vet. Path., The OhioState University, Columbus, OH 43210.

CCAAT enhancer binding proteins (C/EBP) are a family of leucine zipper

trancrition factors implcated in growth control and differentiation. CommaD mammary epithelial cells (CD MEC) were syncronized by sermstarvation and the cell cycle iniated by the addition of serum supplementedmedia. CD MEC expressed C/EBP-beta, C/EBP-delta and CHOPIO, but notC/EBP-alpha. C/EBP-elta mRNA content was maximal during quiesce

and dramatically decrased 1 hour after senm addition coincident with theinduction of early growth respone genes (ie fos, myc). In contrast, C/EBP-beta mRNA content was at basal level during qicence, and moderatlyincreased 1 hour after serum addition. CHOPIO mRNA cotent was elevatedduring quieee and declined gradually to basal level 12 hours after senmaddition. To investigate C/EBP-elta and growth control a stable CD MECcell line expressing a C/EBP-elta antene mRNA (1)2) was produced. In

cell cycl experin, 3H-thymidine uptake peaked at 18 hours in D2 cellscompaed with 20 hours in controls. C-myc and histone IIB mRNA levelswere 20 times greater in quiscent D2 cells compared with controls. In

summary: 1) C/EBP-delta mRNA levels were highest in quiescen CD MEC;2) C/EBP-elta mRNA levels declined rapidly as cells entered the cell cycle;3) proliferation isdites were aberrant in cells expressing C/EBP-deltaantsee mRNA. The data suggest an association between C/EBP-delta and

growth control in CD MEC. (CA 57607).

as

CELLULAR MECHANISMS WHICH CONTROL CELL CYCLEWITHDRAWAL AND SKELETAL MUSCLE DIFFERENTIATION((O. Halevy, B. Novitch, S. X. Skapek, T. Jacks* and A. B. Lassar))Department of Biological Chemistry and Molecular Pharmacology,Harvard Medical School, Boston MA 02115; *MIT Cancer Center,Cambridge, MA.

Skeletal muscle differentiation entails both the activation of muscle-specific gene expression and permanent withdrawal from the cell cycle ofmultinucleated myotubes. The myogenic bHLH regulatory proteins(MyoD family) play a key role in the determination and differentiation ofmuscle cells. Since differentiated myotubes are permanently withdrawalfrom the cel cycle, there must be an integration between myogenic factorfunction and a blockade to cell cycle progression. Using primaryfibroblasts derived from Rb-deficient mouse embryos, we have found thatectopic expression of MyoD in these cells induces skeletal muscledifferentiation in the absence of Rb. Interestingly, differentiated myotubeslacking Rb initiated new DNA synthesis when stimulated with mitogens.Thus Rb is necessary to maintain the absence of DNA synthesis indifferentiated myotubes. Although members of the Rb family and MyoDinteract in vitro, it is still unclear whether direct interaction of MyoD withmembers of the Rb family modulates the activity of Rb in muscle cells.In our poster we wil present biochemical evidence that the myogenicbHLH family may act to indirecdy regulate the activity of the Rb familyduring skeletal muscle differentiation, and thereby secure terminal Goarrest of differentiated myotubes.

90

ALTERATIONS IN CAPACITATIVE CALCIUM ENTRY INTO MOUSEFIBROBLASTS ASSOCIATED WITHSERUM STARVATION AND SV40 LARGE TANTIGEN EXPRESSION ((R. Dyche Mullins*, Molly Peck"5, Teny G. Newcomb***,James W. Jacobberger+, Jesse E. Sisken**)) *Ctr. Biomed. Engr., ***Sch. Biol. Sci.,**Dept. Microbiol. Immun., Univ. of Ky., Lexington, KY, +Dept Genet., Case WesternReserve Univ. Sch. Med., Cleveland OH

In many cell types, depletion of the EP3-sensitive intracelular Ca2+ store activates asignaling pathway, called the capacitative Ca2+entry pathway, that leads to the opening of aplasma membrne Ca2+ channel. We previously found that depletion of the 1P3-sensitiveCa?* store by the intracellular Cae-ATIPase inhibitor, thapsigargin (TG) results in a muchlarger Ca?' influx into SV40-transformed Swiss 3T3 (SV3T3) fibroblasts than intonontranaformed Swiss 3T3 cells. In the present study, we have investigated the relationshipbetween SV40 transformation and capacitative Ca2+ entry. Our previous work was carriedout in 20-24 hour serum-deprived ceUls. We find that, while serum starvation has nosignificant effect on TG-induced Cae entry into SV3T3 cells, serum starvation ofnontransformed Swiss 3T3 cells results in an almost two-fold decease in TO-induced Ca+entry. In addition, we have used SV40 large T antigen-transfected NIH 3T3 cells thatexpress large T antigen at different levela to demonstrate that large T expression alone issufficient to upregulate capacitative calcium entry and that it does so in a dose-dependentmanner. Tbus, there appear to be two mechanisms responsible for the previously observedSV40 transformation-associated increase in capacitative calcium entry: 1) an increaserelated to the difference in proliferative state of the sernm-starved nonral and transformedcells and 2) an increase related to the expreasion of large T antigen. These results raise the

possibility that increased capacitative calcium entry is responsible some of the effects ofSV40 large T antigen on cell cycle regulation. Supported in part by Electric PowerResearch Institute contract RP2965-20.

2

DIFFERENTL EXPRESSION OF CCAAT/ENHANCER BINDING PROTEIN(C/EBP) AND D-TYPE CYCLINS ASSOCIATED WITH THEGLUCOCORTICOID MEDIATED GROWTH CONTROL OF RATHEPATOMA CELLS. ((R.A. Ramos, C.M. Cover, and G.L. Firestone))Department of Molecular and Cell Biology, University of California, Berkeley,CA 94720.

Dexamethasone treatment of BDSI cells, a minimal deviation rat hepatomacell line, causes a GI block in cell cycle progression, whereas, hormonewithdrawal results in the synchronous progression into S phase. In contrast,receptor-positive (EDRI) and receptor-negative (EDR3) glucocorticoid resistantvariants grow asynchronously in the presence of dexamethasone.Dexametasone strongly induced C/EBP transcription factor mRNA and proteinlevels only in the growth suppressible BDS1 cells. The regulated expression ofC/EBP is an early response to glucocorticoids because its transcript levels aremaximally stimulated within one hour of dexamethasone treatment which is wellbefore BDSI cells growth arrest. Neither C/EBP mRNA nor protein weredetected in the glucocorticoid resistant cell lines. In a functional test for C/EBPtranscriptional activity, dexamethasone stimulated alpha 1-acid glycoprotein(al-AGP) promoter activity in growth suppressible BDSI cells transfected withthe CAT reporter gene regulated by the C/EBP- and glucocorticoid-responsiveal-AGP promoter. However, co-transfection of a C/EBP expression vector wasnecessary to observe a reduced level of glucocorticoid responsive al-AGPpromoter activity in the EDR1 glucocorticoid resistant variant. C/EBP functioncorrelates with the growth suppesion of hepatoma cells. In contrast, cyclins Dl

and D2 mRNAs were markedly induced during transit through GI, pealing at 4hours and decreasing by 8 hours after steroid withdrawal; thus, D-type cyclinexpreion correlates with hepatoma cell cycle progression. Curently, the rolesof CJEBP and cyclin Dl in glucocorticoid growth control are being tested bygenetic manipulation of C/EBP and cyclin Dl mRNA and protein levels.Moreover, the C/EBP and cyclin Dl promoters are being examined to preciselydefine the DNA elements involved in the glucocorticoid regulated expression ofthese critical growth controlling genes.

16a

Sunday. Cell Cycle Controls (93-98)

933

SEQUENTIAL CHANGES IN GENES REGULATING STEROL BIOSYNTHESIS

DURING HEPATIC REGENERATION. ((FR Simon, E Sutherland, Fortune, I

Shechter, R Davis, M. Sinensky and A Alexander)) Hepatobiliary Research Center,

VAMC, ERICR, Un. Colorado HSC, Denver, CO 80262

Partial hepatectomy initiates a dynamic series of biochemical and molecular events

resulting in restoration of normal liver size. Hepatic regeneration (HR) can be divided

into 3 stages: pre-replicative (0-12 h), replicative (12-36 h) and mitosis. Although

biochemical studies have shown alterations in sterol metabolism during HR, the

molecular events have not been characterized. Mevalonate, a key intermediate may

be shunted either into isoprenoids or form cholesterol. Isoprenoids are involved in

growth regulation, while cholesterol is necessary for plasma membrane biogenesis.

We examined the hypothesis that HR is associated with changes in gene expression

which might lead to increased mevalonate shunting to isoprenoids during pre-

replication, and increased cholesterol synthesis during replication (12-36 hr). Partial

hepatectomy was performed in rats and compared to sham controls at 4,8,12,24,36 and

48 hrs. Cholesterol was determined by GC and steady state mRNA levels by

densitometry of Northern blots using labeled sequence specific probes for HMGCoA-

R, squalene synthase, 7a-hydroxylase, LDL-R and prenyltransferase. During the

pre-eplicative stage, neither hepatic free cholesterol nor cholesteryl esters (CE) were

significantly changed. However, after 12 hr, CE content was significantly increased

(2 x) indicating cholesterol storage. HMGCoA-R and prenyltransferase mRNA was

rapidly and coordinately increased during pre-replication. But squalene synthaae and

7a-hydroxylase were decreased and LDL-R mRNA was unchanged. In contrast,

squalene synthase, 7a hydroxylase and LDL-receptor mRNA levels were increased

later during the replicative stage. We hypothesize that the hepatocyte coordinately

regulates gene expression forstero1 biosynthesis in order to initially control cell growth

and later plasma membrane biogenesis.

95NDUCTION OF NITRIC OXIDE SYNTHASE GENE EXPRESSION AND

ACT Y BY PA PRIMARY NEONATAL RAT HEPATOCYT ((U.Armatol, M. Menegazi2, C. Guerierol, L. Tetolinl, C. Cardinalet, A. Carcereri

DePrati2, S. Mariotto2, L. Menapacel, and H. Suzuki2)) Institutes of 'Anatomy &

Histology and 2Biochenstry, Unversity of Verona 1-3 7134, Italy

Induciblenitric oide syne (iNOS) is awaked hepatocytes by a variety of

stimuli, including bacterial endotns, inte n dand cytokines. However, the

pathophysiological aim(s) of this idiion remai(s) obsur,and the potential role(s),

if any, played byiNOS in heptcyticprifon and in hePatocacin proc-

esses remain to be adequately assessed. To clarify dte topics furtier, we undertook

the prese work using priny curea of Perooll°purified, normal neonatal rat he-

patocytes attached to polyethylene disk oatig on te top of asenuless MEMme-

dium and treated withth archel tumor promoter phorbol ester 12-0-tetade-canoylpbobol 13-acetate (TPA; 10" M) [1]. Ourresus showthat TPAexerted a

mitogenic action in asignificantfracfion ofthe quient, primary (Go) neonatal hepa-

tocytes. In thesame cells, alredy withmidte first 30 mm ofexpoure,TPA markedlyunduced the mRNA encodng foriNOS.The kvels ofthis specific mRNA peaked at

1-2 hours and doclnod thereafte. No iNOS mRNA levels ocurred the

untrted, paralld primary liver cukura receiving a change of fresh mediunL TPAalso increased the enzymatic activity ofiNOS: an increase of the NO2- + NO3- levels

appeared after 8-h the medium and persisted up to 24-h These remarkable effets of

TPA on both gene expression and enzyme activity of iNOS led credence to the hy-

pothesis that, by mediating the mitogeic action of in hepa-

tocytes,iNOS may be involved in the mechanismsundring the promotion ofliver

chemicalcai s. [1] F. Romano, L. Menapace, U. Armato, Carcinogenesis 9:

2147-2154 (1988).(Work supported by AIRC, Milan; MURST, 40% and 60%funds,

CNR's Target Project on Ageing, and CISMI).

ST

INHIBITION OF HUMANMAM MARY EPITHELIAL CARCINOMACELL PROLIFERATION BY CERES-18. ((H.K. Fatay, N.A. Betz, B.A.

Westhoff and T.C. Johnson)) Center for Basic Cancer Research, Kansas State

University,Manttan, KS 66506-4903.

An interacion among various growth factors, both mitogens and growth in-

hibitors, is aflndmnental requirment for cell growth regulation. Although

positive growth regulators have been studied extensively, studies on negafiveregulators arelimited, We have isolated, and purified to homogeneity, an18

kDa cell regulatory sialoglycopeptide (CeReS-18), obtained from thesurfaces

of intact bovine cerebral cortex cells. CeReS has been shown to inhibit DNA

synthesis and cell proliferation in a wide variety of normal andtransformed

cell types, and a diverse range of species. Most cell types exhibit a similar

sensitivity to the reversible growth inhibitory effects of CeReS-18 at -3 to8 x

I0O M concentrations, while at higherconcentrations CeReS-18 can elicit cy-

totoxicity. The present study was conducted to examine the effects of CeReS-

18 on the prolifiration ofhuman mammary epithelial carcinoma cells. MCF-7

cells which are esten receptor positive, and BT-20 cells which are estrogen

receptor negative, were utilized. Both cell lines were equally cell cycle ar-

rested when trated with an inhibitoryconcntin of CeReS and the inhibi-

tion was completely reversible. When cells were treaed simultaneously with

inhibitor and with mitogens such as, EGF, BFGF, estrogen; IGFI or IGFH, no

alteration of the inhibitory activity of CeReS was observed. FACS analysis of

the inhibited cells revealed that the majority of thes cells were arestedin the

G phase of thecell cycle. CeReS proved to be cytotoxic to both cell lineswhen

it was added at higher than inhibitoryconcentrations. Investigation of

the DNA 'ntegrrty cells klled by the cytotoxic dose of CeReS revealed an

apoptotic death mechanimm as opposed tocell necrosis. In addition; cell ensi-tivity to CeReS increasd up to 10-fold when the culture medium calcium con-

centration was down-shifted from 1.8 mM to 0.18 mM.

94

USE OF TRANSFECTED HEPA 1-6 CELLS TO DISTINGUISH THE ANTI-PROLIFERATIVE AND HEPATOTOXIC EFFECTS OF ACETAMINOPHEN.((CL. Navarro-, S.D. Cohen' and EA Khairallah')), Depts. of Molecularand Cell Biology' & of Pharmacology, Univ. of CT Storrs, CT 06268.

Acetamiinophen (APAP) at high doses is toxic to liver cells both invand in culture. APAP can be activated to its reactive metabolite, N-acetyl-benzoqulnoneimine, via cytochrome p450 and the hepatotozicity isassociated with the covalent binding of this metabolite to specific cellularproteins. Even at 10 mM APAP Hepa 1-6 hepatorma cells do not exhibitcytotoxicity as judged by the release of cytosolic enzymes nor can thecovalent binding of APAP be detected by Western blots using an anti-APAPantibody. However, the proliferation of these cells can be diminished evenat 0.5 mM APAP. Failure to demonstrate cytotoxicity can be explained bythe inability of these cells to express either the p450 isoforms needed toactivate APAP or themajor 58 kDa acetaminophen binding protein (58ABP).The decline in cell growth is concentration dependent with respect toAPAP; higher concentrations result in slower cell division. Hence thiseffect of APAP on these cells must be independant of the activation ofAPAP and the covalent binding seen in the toxicity. Hepa 1-6 cells are

currently being transfected with vectors contaning the cDNA for either orboth the 58ABP and CYP-1A2, one of the p450 isoform that is capable ofactivating APAP. These experiments will enable us to determine whetherthese transfectants will be capable of activating APAP and to shed light onthe importance of the arylation of the 58ABP in distinguishing the anti-proliferative and hepatotoxic effects of APAP. (N.I.H. GM 31460)

96CHANGES IN HEPATIC PLASMA MEMBRANE AND NUCLEAR1,4,5-INOSITOL TRISPHOSPHATE BINDING FOLLOWINGPARTIAL HEPATECTOMY. ((N. Kraus-Ffiedmann and L. Feng))Department of Physiology and Cell Biology, University of Texas MedicalSchool at Houston, P. 0. Box 20708, Houston, TX 77225.

Hepatic parenchymal cells possess two different receptors for 1,4,5-trisphosphate, one isolated with the plasma membrane fraction and anotherisolated with the nuclear fraction (Feng, L. and Kraus-Friedmann, N., Am.J Physiol. 265: C 1588, 1993). Their interaction with antibodies generatedagainst the receptor in the cerebellum indicates that these two receptorproteins are different. The potential involvement of the nuclearIP3receptor in rapid cell proliferation was tested by measuring [3H]-IP3binding, following partial hepatectomy. In nuclear fractions isolated 18hours after the operation, a 33% decline in binding sites was detected. Innuclear fractions isolated 30 hours after the operation, a 60% decline in thebinding sites was detected but, the K. remained unchanged. A 70%decrease in binding sites was also detected in the plasma membranefraction, the Kd forlIP3 increased from21nM to 38nM. These results showthat partial hepatectomy, a state leading to rapid cell proliferation, isassociated with a parallelloss ofIP3 binding sites in the nuclear and plasmamembrane fractions.

98METHIONINE DEPENDENCE. A POSSIBLE GENERAL TUMOR-SPECIFIC THERAPEUTIC TARGET((Guo, ELY., Le, P., Hoffman, R.M)) AntiCancer, Inc 7917 Ostrow Street, SanDiego, CA 92111

The purpose of this study is to determine the frequency of occurrence of thetumor-specific biochemical defect of methionine dependence by comparing theproliferation capacity of 20 different human tumor cellines from the NCIorgan-specific human tumor ceUl line colection and normal fibroblast strains inmethionine-containing medium, in methionine-free, homocysteine-containingmedium and in methionine-free, homocysteine-free medium. Tumor and normalcells were grown in the above culture media and measured for the ability toreduce XIT (2,3-bis (2-methoxy4-Nitro-5-sulfophenyl)-5-l(phenylamino)carbonyll-2H-tetrazolium hydroxide). Al 20 tumor celllines andnormal cell strains grew in the methionine-containing medium. However,approximately one-half of the tumor ceUl lines could not grow in themethionine-free homocysteine-containing medium with the rest growing tovarying degrees. The normal cell strains grew as well in the methionine-free

homocysteine-containing medium as in the methionine-containing medium. TMestriling result was that although the normal cel strains eould grow to someextent and then maintain themseles for two weeks or more in methionine-free,homocysteine-free medium,aUl 20 tumor cel lines died in methionine-free,homocysteine-free medium. All 20 human tumorcellines from 5 differentmajor sites were all methionine-dependent. The results suggest that the newcondition of methionine-free, homocysteine-free medium in vitro hasalowed theconclusion that potentiallyaUl cancers are methionine-dependent. The possiblegeneral occurrence of methionine dependencein cancer allows the possibility oftherapeutic exploitation of the methionine target with agents such as

methioninase which are currently being developed.

17a

Steroid Hormones and Receptors (99-104). Sunday

MORPHOMETRIC ANALYSIS OF ESTROGEN RECEPTORS IN THEOVINE UTERUS DURING EARLY PREGNANCY. ((J. Zheng, D.A. Redmer,L.P. Reynolds)) Dept. of Anim. & Range Sci., North Dakota State Univ.,Fargo, ND 58105.

We have shown that the rate of cell proliferatIon Increases dramatically bydl 8 after mating in the ovine endometrium, primarily within tissue compart-ments near the uterine lumen but remains low in deep glands and myometri-um throughout early pregnancy. In ovariectomized ewes, estradiol Increasesthe rate of cell proliferation in glandular and stromal tissues. To evaluate therole of estrogen in regulating uterine growth durng early pregnancy, 24ewes (n=3/day) were slaughtered on d12 or 14 after estrus (nonpregnant,NP), or on d12, 14, 16, 18, 21 or 24 after mating (pregnant, P). Estrogenreceptors (ER) were immunolocalized In formalin-fixed, paramffin-embeddeduterine tissue sections. Percentage of labeled cells was quantified morpho-metrically for epithelial (Epi), stromal (Str), luminal and deep glandular (LGlaand DGla) and myometrial (Myo) tissues. Positive nuclear staining wasclassified as weak or intense. Weak staining was similar across all days andtissues (49% of cells). Conversely, across all tissues intense staining variedamong days and was similar for P vs NP on d12 and 14 (32 vs 36 and 22vs 21%) but was greater (P<.05) on dl2P (32%) than on d16-24P (11%).Staining was greater (P<.05) for Epi, Str, LGIa, or DGIa on d12 and 14Pthan dl 6, 18, 21, or 24P, but was similar for Myo across all days. Thus, forEpi, LGIa and Str, which are the uterine compartments that exhibit cellproliferation during early pregnancy, ER levels are decreased when thedramatic increase in cell proliferation is occurring. (Supported by NICHD22559 to LPR & DAR).

101

HSP90 MUTANTS AFFECT THE STABILITY AND LIGAND BINDINGACnIVnlY OF GLUCOCORTCOID APORECEFTO COMPLEXES. ((S.. Bohenand K.R. Yamamoto)) Departmets of Biochemdstry and Biophysis, andPhmcology, University of alfnia, San rancwo, Calfori 94143.048.

lhe recepr (GE) responds tocognte hormonal Igadabyblninto specific DNA squexc and altering the a ctivity of adjacent

promoer gnded,natv eer plexcontalningan

hapg dine, and otherpteins. heGR oe doey ratedrecpt must

intect withlp9O to acquire competenc for signa Uponligandbinding,hsp90 dissocates fm the 4anbund receptor which subIequetiy bbihormone response elements on chrosomes and modulates iption. BY

ning yat for defecthIn rilreceor function we have boltedfouw hsp9O mutun that define at lat two pIenotpc class Afl of the mutnt

remain wmpetent to form cwmple with GR in viw, but bocmia anaysesrevl ateratons in their inctions with GR. A GRE-pedfic hsp90 mutantproduces apoceptor compls that cn bid hmone with hih affinity, but themutant hep9O appeas to dbsoodate ne slowly does wild-type hspi0. In

Contrast, the other hep90 mutuat docae from GR e rapidly than wdtypehsp90 and aporeceptor compks forned by these mutants fail ix vir to bind

hormone with high affinity. he rdative sverity of the alteratons in the

kdnet relme by the hsp9O mutanb comlaes with the severity of the effects onhormone respone. Hsp9 appeas to interact with a broad range of distinct

sigalng proteins in addition to seroid rp, such as elF-2a knle, v-c,

v-Wf and the dioxdn receptor. We peculate that a eneal function of hsp90 maybe to recogize and bind the hictive form of divers signalig acto While

hap90 may play a role in mintaiin these factors in an inactive state, thecharacterization of hspOO mutants clearly indicates that hsp90 binding isinvolved in making receptr cmpetent to repond to lHgnd. We suggest that

hsp90 may be sdminury involved sgnal transducton by othe protein

103

PHOSPHORYLATION OF MEMBRANE-ASSOCIATEDGLUCOCORTICOID RECEPTOR. FURTHER EVIDENCE OF

STRUCTURAL HOMOLOGY WITH THE INTRACELLULAR

GLUCOCORTICOID RECEPTOR. ((B. Gametchu)) Departmet of

Pediatrics, Medical College of Wissonsin, Milwaukee, WI 53226.

We have previously described a novel membrane glucocorticoid receptor(mGR) and correlated the presence of this protein with the lymphocytoliticeffect of glucocorticoids. The only strunal difference that has been noted

to date betwoen the intracellular OR (iGR) and mGR is a molecur size

variation on denatuing polyacrylamide gels (identified by Western blotting)and density grdient analysis with anti-rat OR monoclonal antibodies,BUGR-1 and -2. The mOR is freqiny larger in size and has multiple,large molecular size vrants. We now show that mOE, like iOR, is

phosphorylated. Plas membrane vesicles wer purified from nP_labeledS49W mouse lymphoma cells (superriched for mGR by sequ l

immunoselection techniques) on a sucrose step-gradient Extracted mGRwas conc ed by immuecipitation with BUGR-2 and subjected to I

and 2-D gel analyses. Immunoblotting and antoradiography of theseprepations revealed predominant OR bands of 75, 94, 116 and 150 Kd.The two dimeional analysis showed that most mGR forms focused as a

streak of pepides ranging in chrge from pl 6.6 to 8.5. In smmary, mGRshares yet anoher stctual siilaity with iGR, suggesting thattheprinrymodification resonsible for placement in the ceil membrane is minimal.

1leHSP90 IS PRESENT IN A HETEROMERIC COMPLEX IN THE STEROIDHORMONE RESPONSIVE FUNGUS ACHLYA. (S.A. Brunt', G.H. Perdew2,and J.C. Silver'))'Department of Microbiology and Division of LifeSciences, University of Toronto, Scarborough, Ontario, Canada M 1C 1 A4.2 Department of Food and Nutrition, Purdue University, West Lafayette,Indiana.

Membersof the 9OkDheat shock protein (HSP90)family are constitutivelyexpressed proteins which are upregulated by heat shock, and in certainorganisms during development, and by steroid hormones. HSP90 proteinsare reported to exist in heteromeric complexes with a number of cellularproteins, including kinases, transcription factors and steroid hormonereceptors, as well as other heat shock proteins (HSP70, HSP56). In thefungus Achlys steroid hormones regulate sexual development. Theresponse to hormone is thought to be mediated by a steroid hormonereceptor. Immunoprecipitation of either cellular or in vitro translatedproteins with monoclonal antibodies to HSP9Ofrom Achlya (AC88), mouse(8D3) or rat (2D1 2), showed HSP90 in Achlya can be found in a proteinheterocomplex. Consistently identified in this complex were proteins of11 OkD, 74kD, 64kD, 61 kD, 56kD, 47kD and 23kD. Proteins of similarmolecular weight are observed in vertebrate steroid receptorheterocomplexes. Our results suggest that the association of steroidreceptors with HSP90 in a heteromeric complex with other proteins, likelyextends to this microbial steroid responsive system. (Supported by NSERCCanada)

102MEMBRANE RECEPTORS FOR 1,25(OH)2D3 AND 24,25(OH)2D3ACT THROUGH DIFFERENT SIGNAL TRANSDUCTION PATHWAYSAS DEDUCED BY EFFECTS ON INTESTINAL PHOSPHATETRANSPORT. ((I. Nemere)) Department of Biochemistry,University of California, Riverside, CA 92521,

The steroid hormones 1,25(OH)2D3 and 24,25(OH)2D3enhance 4 Ca transport in the perfused chickduodenal loop through specific basal lateralmembrane (BLM) receptors. The present reportdescribes the effects of vitamin D metabolites on

3P transport. Vascular perfusion with 65 pM1,25(OH)2D3 enhanced transport of 33P within 2 minreaching treated/basal (T/B) ratios of 1.82 + 0.46after 40 min, compared to controls (T/B=l.ll +

0.21). In dose-response studies stimulationoccurred between 33-130 pM 1,25(OH)2D3 but not at6.5 or 650 pM steroid. Lumenal 1,25(OH)2D3 failedto augment 3P transport (T/B=1.05 + 0.11), comparedto vascular hormone (T/B=2.11 + 0.06), suggestingaction through a BLM receptor. Unlike Ca 6.5 nMvascular 24,25(OH)2D3 failed to enhance 33ptransport (T/B=1.0 + 0.27), and suppressed the

effect of 65 pM 1,25(OH)2D3 (T/B=1.18 + 0.21),relative to 1,25(OH)2D3 alone (T/B=1.82 + 0.46).100 nM 25(OH)D3 likewise did not enhance 33P

transport (T/B=0.8 + 0.09), and blocked the effect

of 65 pM 1,25(OH)2D (T/B=0.87 + 0.03). Thus, BLM

receptors activate ioth stimulatory and inhibitorypathways to modulate ion transport.

1"

LEM1, AN ATP-BINDING-CASSETTE TRANSPORTER,SELECTIVELY MODULATES THE BIOLOGICAL EFFICACY OFSTEROID HORMONES. ((A. Kalli S.P. Boben and K.R. Yamamoto))Departments of Pharmacology, and Biochemistry and Biophysics,Univrsity of Califania, San Fancisco, CA 94143- .

The abiLity of the rat glucoccdrtcoid receptor to confer h mon -dependentcriptional t when expressed yeast, enabls the genetic

identifiction of now pr protteim that function in the hormone signaltransductin pathway. We seened for yest mutants that alter the stroid

specificty of the glS i pt and isolated a mutan, lk],that increaaesresponsivxenea todexcx_thsaone andtaconidewithout alteing responivene to a third agonist, deoxycorticosoe.Cloning and of the wild tpe LEMI allele rved a proteinwith puaive mins d sigificant identity to aans es

of the ATP-blading cassette fmily. LEM1 decrases responseness todexamethasoe by pwag this liand frm wild type ces, as uggestedby the ao f wild o

lkns cells. Homone ronsienss of odter semid rece ex ed

wild type and lenl yeastd_monase that LEM1 preferen edxmainobne acenide, one and corco,

but not ogeero, esladio or ticosern Drugs that inhibit the

yeat LEMI activity can also in e the intracellula accumulaon ofdex e in the mouse L929 cells, sugesting the existenc ofmanmaan stes) with LEMl-lke acdvity. Based on thes findings,we propose that transpor lilke LEMI can slectively modulate theintrellular levels of steroid hormones. Putadve manumslian steroid ligandtaotters mae regulate hormone availability, ad thereby hormoneUVng inacl-yespecific manner.

18a

Sunday. Steroid Hormones and Receptors (105-110)

105REiTNOIC ACID IS AN UNEXPECTED DOM[NANT INHIBITOR OFHUMAN BONE CELL DIFFERENnATION. ((Y. Zhou, S. Mohan, T.L.Liidiart, D.J. Baylink, D.D. Strng))Pets VA Med Ctr. and Las LxidaUiv,Loma Lida, CA 92357.

Rainnicaad (RA), the most active metabolite of Vami A, cers profundefcts on proliferationandddfutbn of many cll typesindudg ostooblasts(Obs). In rat ceil models,RA effects nexpresson of the Ob phintype are

developmd tal stag depade.RAinceased preson of the Ob-specific gws

allinep (ALP) and type Iprocdlagm (Col)m preOb stn calls butreduced xpression of sgm in diffa OsH et al., M. Endocrin.6:636, 1992). In humansi, excss Vitamin A causes bone fgility ftures, but

aefcs ofRA n cultured human Obs have not yet boe studiedetiively. Todatine whher RA affcts prolifation and ddifertiatio of huma Obs we

haveexmned actions of all-trans RA prolfatio and xpression of ALP, Col,and osocxlan (OC)in Obsisdated fiom nomal hm bone (NHOB) andinSaOS-2 human cells. 10-- 104 M RA did not significantly affect

prolderation in NHOB or SaOS-2 cells. However, RA markedy reduced basal Coland ALP mRNA expresi in a doseand time depmdat nur to 20-30% ofcotrl n both cell typs. 1,25(0) Vtamn 13 (Vt D) icreased ALP activity,ALP mRNAand OC mRNA levels. RA inhibitedthseinductions by 70% ormore.

RAinibin of Vit D iductions was not due to reduction of Vit D receptor (VDR)expression becauseRAiemsed VDR mRNA levels. These resultssuestthat RAhas a dm atinhibitory effect Vit Dinducion of ALPand OC Sns expressin

m human OBs and seleively inhbits expresion of Mme productswhich compnsethe differntiated OB pheote.

107

INHBITION OF MELATONIN'S AND TAMOXIFENS ACTIONIN MCF-7 CELLS BY MAGNETIC FIELDS. ((J.D. Harland andR.P. Liburdy)) Depamet of Cell & Molecular Biology, LifeSciencs Division, Lawrence Berkeley Laboratory, Berkeley, CA94720.

We have examined whether environmental-level, 60 Hz magneticfields alter the growth of a human breast cancer cell, MCF-7, in thepresence of melatonin or Tamoxifen. We measured MCF-7 cellgrowth over aseven-day exposu period (seoding density: 0.1 x 105cellst35mm plate), using identical incubators and mu-metalchambers enclosing a 4-square Melritt coil system to eliminateendogenous magnetic fields. This approach allowed us to expose

MCF-7 cels simultaneously in matched incubators to highlyuniform magnetic fields at 2 and 12 mGauss(mG). The 2 and 12mGfields had no significant effect ongrowth of unteated MCF-7 cells.In the prsence of melatonin (10 M to 10-IIM) growth inhibitionwas reduced from 20% at 2 mG to <2% at 12mG; p<0.03. The12mG fields reduced the growth inhibition of Tamoxifen at some

pharm ological doses (at 10-7M inhibition fell from 40% at 2mG to6% at 12mG; p<0.001). Tamoxifen acts by binding to the estrogen

receptor, therefore, this receptor may be involved in the fieldintection. These findings provide evidence dt an environmental-level magnetic field can influence the actions of a hormone and a

drug on MCF-7 cell growth.

REGULATIONOFGROWTH FACTOR-INDUCED PROLIFERATIONAND MITOGEN-

ACTIVATED PROTEIN KINASE ACTIVITY BY ESTRADIOL AND ANTI-

ESTROGENS IN ESTROGEN-RESPONSIVETUMOR CELL UNES. ((J.S. Foster, A.

Bukovsky, A. Bragg, M.R. Caudle, and J. Wimaasmnal). Dept. of Obstetrics and

Gynecology, University of Tennesse Medical Center, Knoxville, TN 37920

The effects of estrogen agonist/antagonist administration upon serum and

growth factor-induced proliferation and signal transduction kinrs cascades were

investigated in estrogen-responsive tumor cell lines. Growth of both MCF7

breast cancer cells and BG1 ovarian carcinoma cells wa stimulated by 0.1 to

IOnM estradiol (50-70% increas at 1OnM astradiol) in the presence of serum.

Furthermore growth of MCF7 cells in the presence of 5% fetd calf srum was

inhibited (48% inhibition) by the steroida anti-estrogen ICI 182,780 (0.1 to

1OnM) in the absence of exogenous estradiol. Serum-induced growth of BG1

cells was inhibitable to ICI 182,780 to a lesr extent (24% inhibition). Mitogen-activated kinase (MAPK) activity induced by opidermal growth factor (EGF) in

BG1 cels was enhanced approximately 50% by 24 to 48 hour pretr nt of

cuitures with 0.1 to 1OnM estradiol when masured by immune complex kinase

assay. This effect was rovesibie upon co-treatment with 1 OnM stradiol along

with 1OnM ICI 182,780. Ouantitative immunohistochomical analysis indicasthat estradiol pretrment of BG1 cultures leds to enhanced EGF receptorexpression after 6-24 hour treament with 1 nM estradiol. This may explain in

part the observod increases in kinasm activation. The results suggestinteractions between estradiol action and growth factor signaNing pathways

which may contribute to growth ragulation of strogn responsive cancer clls.

Thus, cross-talk between signalling pathways appears to occur both at the EGF

receptor as well as intracellular kinase cascade stps. AddIitonal studles are

underway to further lucidate the nature of thm Interactions. Supported by

N.I.H.

1I6A NOVEL NUCLEAR FACTOR, HLFP1, MODULATES ER-MEDIATED ESTROGENRESPONSE OF THE HUMAN LACTOFERRIN PROMOTER ACTIVITY. (N.Y.Yang, H. Shigeta and C.T. Teng) Gene Regulation Group,Laboratory of Reproductive and Developmental Toxicology,National Institute of Environmental Health Sciences, NationalInstitutes of Health, Research Triangle Park, NC 27709.

We have previously shown that estrogen-stimulated transcrip-tional activity of human lactoferrin gene in endometriumcarcinoma cells, RL95-2, is mediated through a functionalestrogen response element (ERE) present in the 5' flankingregion of the gene. Upstream from the ERE, a DNA sequence(-418 to -378, FP1) is selectively protected by nuclearprotein of the endometrium (RL95-2) and mamsary gland (HBL100)cells from DNase I digestion. We show that three differentnuclear proteins are involved in binding to the FP1 region(Cl, C2 and C3) and one of them is COUP-TF (C2). From theelectrophoresis mobility shift assay (EKSA), site-directedmutagenesis and DNA methylation interference analysis, we are

able to delineate the position of these proteins that bindFP1. The DNA sequence at the 3' end of FP1, TCAAGGTCATC, isan extended half site of steroid receptor binding element.This DNA sequence matches the consensus element of a newlycharacterized subfamily of nuclear orphan receptors that bindas monomer. Mutation at the Gs in this region abolished theformation of Cl complex and reduced the estrogen responsive-ness of the reporter plasmid containing the intact lactoferrinERE. We isolated a cDNA clone (HLFP1) from the RL95-2expression library and demonstrated that it indeed encodes thebinding protein in Cl complex. These findings demonstratedthat a novel nuclear factor participated in modulatingestrogen response of the human lactoferrin promoter activityby binding to an extended steroid nuclear receptor half-site.

1l5EVIDENCE FOR AROMATASE ACTIVITY AND ITS MODULATION BYGROWTH FACTORS IN A PROSTATE CANCER CELL UNELNCaP.((J.L. Block, B.L. Lokeshwar, and N.L Block)) Departmentof Urology,Unisty of Miami School of Medcne, Miami, FL 33101

Anomalousneabols-ofandrogensisknowntobeinsturnentWinthedevslopm pnnandpcgreslonbottecancer.Thedtencofarmtase-anenzyme that convert androgensinto esogens- in proste c rcoes,has not been sudiceny monstratd , although Its role has been predictedInpa ge dfthe prwtate. We pmvide ioc aandIrmmunoherlcalevWidence for he osdsce of arometasein LNCaP, an androgen responsiveprotat carcnoacll kne. Adose and time-dependatsaturablearomataseactvity wasdemo b in LNCaP cells using a snsv essay to measuete conveslonOf[dB2,1 jtest0ternekinto 3H2O,esto andestradkl.TheKm and Vmax Of arormatese activity in LNCaP clls were 201 nM and 0.76pmrndlVmg, espectiviely. Two well established aromabtse Inhibibrs, 4-mefth d-4-andosnedlone (4-OMA) and 4-hydr-y4-androsterne-l7done(4-OHA), both inhbitd aromatse actviy and polfetion with 1.12

pM and 3.28 FM, epectively. Im loi with an antbody agans

placental aomatse showed a singe species of 55 kDa protein that loalzedto the cYoplasm. Long trm incubation with peptide rowth factors, EGF andTGF-e slgnfcaniyinhbedaromatseacivity (>30%).wWloutataelngmito0cacifvty. Onthe oerhand,incontrastwithobervationsinothertisues, IGF-1(Bu)2cAMP, and protn knase stlators or inhblos did not stirrulate or

inhibltaomase actviy in LNCaP. These reul suggesta potential role ofarma In the development or progression of andogen sense prosatecancer. (WorksupportdbyAlPhaOmegaAlpha ResearCh Award (JLB). NIH-NN Grant No. CA61038(BLL)andAustin L Weeks Endow t Fund (NLB))

110

TISSUE INHIBITOR OF METALLOPROTEINASE I AND PRO-CATHEPSINL FORM A STEROIDOGENESIS STEMULATORY COMPLEX. ((N. Boujrad,

M. Gamier, B.M. Martin, and V. PaPadoPOUs0s)) DePtsrtmen of Cell Biology,

Georgetown University Medical Center, Washington, DC 20007 and #clinicalN rosciences Branch, NIMH, NIH, Bethesda, MD 20892.

Testcular steroidogenesis is under the control of the gonadotopins and locallyproduced unidenfied factors. We isolated from immature rat Sertolh cell

cnditioned media a 70,000 MW protein complex, contining two proteins of28,000 and 38,000 MW, which stmulated Leydig cell steroidogenesis, in a dose-dependent and cAMP- P

manner. Eectroehited 28,000 MW protein

was found to bear the bloactvity whereas the 38,000 MW protein was

responible for the expression of maxmal bioacivity. The 28,000 MW proteinhas an isoclectric point of 6.8, it is glycosylated, containing mosty sialic acid,and its expression is under the control of FSH. Using an anti-28,000 rabbit

antiserum, that we developed, we idefied this protein in the wteulari I fluid and further demonstaed its biological actvity in

studie. N-terminal amnoscid sequence, peptide mapping,and immunologic analyses of the 28,000 protein iicated its identity as the

tissue inhibitor of metalloproteinase-1 (TIMP-l). Furthermore, using RT-PCRand primers based on the lmown mouse TIMP-1 nucleotide sequence we isolated

a 355 bp cDNA frgmen from FSH-simulated Sertoli cell mRNA. This

fragam shared a 92 and 76% sequence homology with the mouse and humanTIMP-i, respectively. Aminoacid sequence and immunologic analyses of the38,000 MW protein showed its identity as the proenzym form of cathepin L,

which was also ientified the interiial fluid. Thusse reults demonsrate that

a rat testis TeMP-ne lp L complex is a potent activator of Leydig cel

steridoeess(Supported by 1H124633 and 11D01031).

19a

Steroid Hormones and Receptors (111-1 13). Sunday

111

ANDROGEN-INDUCED DNA/PROTEIN INTERACTIONS LOCAUZE TO ANDROGENRESPONSE ELEMENTS (ARES) IN INTRON I OF THE 20 kDa AND C3 GENES. ((C.W.Gregory, S.G.A. Power, M.C.W. Avelar, KB. Marschke, J-A Tan, E.M. WiIson, and F.S.French)) The Laboratorie for Reproductive Biology, University of North Carolina, ChapelHill, NC 27599

The 20 kDa protein gene and the C3 subunit gene of prosbtin code for androgenregulatd rat ventral prostet secrtory proteins that contain complex and simple ARES,respectively, wiftin inton 1. 20 kDn protein has sequence homology wfth a cyeine

protease inhibitor and prstetein may functon as a terol cafrier protein. The intron 1 130bp fragment, D2, of the 20 kDa protein gene binds androgen receptor (AR) andglucocorticoid receptor (GR) in gel mobility shift assays. D2 fUnctions as an ARE but nota glucocorbioid response element (GRE) in a tkCAT reporter vector cobansfected intoCV1 cels with an AR expression vector. The 5' (D2-A) and 3' (02-B) halves of D2 alsobind AR, but neither haf fUnctions independently as an ARE in CV1 cells, indicating thatboth the 5' and 3' halves of D2 are required for androgen-dependent transcriptionalenhancer activity. An androgen receptor binding site (ARBS-1), identified in D2-A usingmethylation interfeence, has litfie simiarity to the simple 15 bp partial palindromicconsensus sequence (ARE/GRE). D2 contains one or two additional AR binding sites,

that remain to be localized. The C3 subunit gene contains within a 0.5 kb inbn 1fragment, a strong simple AREIGRE (C3-C) that mediates AR and GR transactivation inthe tkCAT reporter vector. To further daracteze these intron AREs, androgen-dependent protein interacions were analyzed by DNase I footprinting in ventral prostetnudel of intact and 2-day castrate rats. Multiple androgen-dependent protected regionsor hyperensitive sites in the D2 region were revealed by ligation-nediated PCR.Androgen-dependent hyperensitive sites were also observed in the intron 1 C3-C AREregion of the C3 gene. The presence of androgen-dependet footprints in assocatonwith intron I AREs suggests tht AR binding to the AREs induces DNA/protininteractions and chromafn stuctral changes in thene intron regions. These findigprovide addffional evidence that the intron I AREs of the 20 kDa protein and C3 geneshave a role in mediating androgen regulation of gene transcription.

112

ISOLATION OF ANDROGEN REGULATED TRANSCRIPTSFROM PROSTATE STROMA. ((M. J. Gerdes, T. D. Dang, L. McBride,B. Lu, M. Larsen and D. R. Rowley.)) Department of Cell Biology, BaylorCollege of Medicine, Houston, TX 77030.

In development, fetal urogenital sinus mesenchyme is androgen regulated toinduce the growth and differentiation of epithel ui. In the adult prostate gland,stromal-epithelial interactions are necessary for maintenance of androgenregulation and cell differentiation. lTe identification of prostate stroma specificgenes is essential for defining moecular mechanisms central to stronal-epithelialinteractions. Such genes may also be significant to the initiation and progression

of prostatic hyperplasia and carcinoma. In order to identify androgen regulatedgenes, stromal cell lines were developed from fetal rat urogenital sinus (U4F)and adult ventral prostate (PS-i) and characterized in vitro for androgenregulation in chemically defined conditions. Physiological concentrations ofandrogens (5-20 nM testosterone, dihydrotestosterone) stimulated proliferationof U4F and PS-i cells which were relatively unresponsive to glucocorticoids,progesterone, and estradiol. To identify androgen regulated transcripts, PS-icells were cultured in +/- androgen (10 nM DHT, 10 nM T). RNA and cDNAwere prepared and analyzed for regulated expression with differential displayreverse transcription polymerase chain reaction (DDRT-PCR). Replicateanalyses of separate RNA/cDNA preparations to identify consistent androgeninduced transcripts have identified 14 candidate transcripts from the PS-I cellline. Each PCR product was isolated, re-amplified, and cloned into the pCR IITA vector. Expression of 3 of the 14 clones have been examined by Northemanalysis and exhibit androgen induction. Sequence analysis of these clonesreveal no significant match with genes in databases. A cDNA library has beenconstructed from PS-l stromal cells and further efforts are being directedtowards full length cDNA cloning and analysis of in vivo expression pattems.Supported by grants CA58093 and DK45909.

113MODULAON OF ERFATE STROMAL SC4aT1VITY TO HORMONES DY ZMETHELORGANODS IN CULTURE ((C.H. Pgp and D.Kirk)) Palab, Inc., Bothel, WA 98011.

Benip Proattic Hyperplaia (BPH) in thought to be due to an alteatio in dte proatatic-o and our obj ve w to define casaal oal-e l eri )

involved. Decmibed bah in a) o iaolao d nin_tenance of viable epilhelial andarnmal coli lx vo and b) reoWts of a prdimiary bonne senitivity atudy on tonmicela wth ad witdhut pdeial celi preat. BPH nodulea wer dimc_td from rdilproatectomiee, minced ad digdefd wih oollagenaa hy i . An ednichdepithil opoid fraction wa obtined by diffentialad mnintained aaorgaoid by cWuling them over an ago gel (1% w/v) i a SePm-Fro Medium(PFMR4) contaiig inulin, tranferrin ad bovine aeni albumin. Orgaoidarined viabl in cultur for up to at lat 3 nwnths. Individual orgnoida fim thedigeaon wwo allowed to rggrgat ito lger stctu (up to Imn in diaebtr, 2-5days) which won uaed in eperim e ao uctue he baen c ized aundiffeenatd eithli tissaua uingu ltrntrc l and i medods.Mm ceia possesaed mcovili ad ainWe positively for cytlratin 5,6 ad 18 (Cyt)inicatig tht they weo of banal epthdial cel orig Like banl ceis in Ewm, the celawwo alo n tivo for Prostat Spocific Antig (PSA). Th cultud org id howedno prolifative avity (no PCNA atining) with little or no contamiing etronl ceia(vimetin ponaive). In contran, all 8 moalayor tromnl linen from 3 BPH tissu werepotive for vimntin d gtiv e for both PSA rnd Cyt Th defc of dihydro-teto n (DHT) andeadiol-17P (E) onsarouml cdls, with and without 17 da oldorgnoids preoot, wo eluatod for proliferative rponse using the Alanr BlueAay i 24-wel dihes. Stroil celis ao dwed a 2-fold tinulaton by DHT but noreepo_n to idtor EBor to B2 +DHT. In contra, DHT canad a mared inhibition(73%) of aoanl celia in t1ho pomue of BPH orgaoids. Agin, the DHT ffedct wasnetoed bytho proeom of H2 whih had no effect lone. IMm ruts bow thut thetrmn reepon to DHT depnde an tho preance or ab_e of orgaoid ad that B6could abrogte the respoea. (Supported by NIH Grant ROI-DK45870)

G Proteins (114-115).114

G-PROTEIN ALTERATIONS DURING TERMINAL DIFFERENTIATIONOF LENS CELLS. ((M.E. Ireland and L. Mrock)) Department ofAnatomy andCell Biology, Wayne State University School of Medicine, Detroit, MI 48201.

The differentiation of lens epitheial cells into mature fibers occurs throughoutthe life of an organism and is known to be regulated by multiple receptor-mediated events. We examined the toxin-mediated ribosylation of G-proteina-subunitsin these two cell types to detrmine if changes in signal transductionmechanisms occur dunng terminal differentiation in the lens. Pertussis toxin(PT) catalysed the labelling of M, 41 Kd and 39 Kd proteins in isolatedmembranes of both post-mitotic epithelial cells committed to fiber formationand in superficial cells of the lens fiber mass. However, the relative abundanceof the PT substrates differed between these two distinct cells. Epithelial cellsexhibited an equal abundance ofthe PT substrates while the 39 Kd substrate infibers was reduced 50%/o. PT substates were also detected in the oldest, nuclearcells of the fiber mass. Cholera toxin (CT) catalysed labelling was restrictedto a sing 42 Kd protein present in fibers but lacking in epithelial eells. Inaddition, labelling of the CT substrate was observed only after membranes hadbeen pre-incubated with a cytosolic extract These later experiments indicatethe presence of a soluble ADP-ribosylation factor (ARF) in lens fibers. Ourdata show that lens cells during successive stages of their terminal

differentiation exhibit distinct G-protein based signal transducing capabilities.This indicates that dynamic alterations in receptor-regulated events may be

responsible for integrating the orderly progression of events during lens fiberterminal differentiation.

115

MUSCARINIC RECEPTOR COUPLING IN CARDIAC-LIKECELFLS DERIVED FROM EMBRYONIC STEM CELLS LACKING

INBITORY G PROTEIN ALPHA SUBUNITS. ((M.O. Sowell,C. Ye, P.M. Vassilev, S.J. Quinn, and R.M. Mortensen)) Endocrine-Hypertension Division, Brigham and Women's Hospital, Boston, MA02115 (Spon. by M.O. Sowell)

Using the technique of targeted gene inactivation by homologousrecombination, mouse embryonic stem cells were produced that lackone or more of the inhibitory G protein a subunits (cq2, ol3, or thecombination of ci2 and ai3 ). The absence of these ai subunits did notalter growth rate or differentiation of mutant lines into spontaneouslycontracting cardiac-like cells. Carbachol (1-10 produced a

significant decrease in beating rate in wild type (WT) and ai3 null cellswhile a less dramatic slowing was observed with oi2 mutants and cellslacking the combination of ai2 and aic. Analysis of an inwardlyrectifying K+ channel with characteristics of the cardiac muscarnic K+channel was performed using on-cell patch clamp. InWT cellscarbachol produced a large increase in the probability of channelopening while ina2 null cells very little response was seen. Anintermediate phenotype was observed in the ti3 null cells with an

increase in channel activity observed in approximately half the clustersof beating cells.Conclusions: 1) ai2 and cq3 are not essential for EScell growth or for differentiation into beating cardiac-like cells. 2) Themechanism(s) for carbachol's negative inotropic effect involves someai2 independent pathways and does not seem to require oq3. 3) ai2appears to be the pfimary a subunit for muscarinic receptor couplingto the muscarinic K+ channel.

20a

Sunday. G Proteins (1 16-121)

AUGMENTED ADENYLYL CYCLASE ACTIVATION BY CHRONICANTIDEPRESSANT TREATMENT OF C6 GLIOMA CELLS IS DUE TOFACILITATED G PROTEIN COUPLING ((J. Chen* and M. M. Rasenick))Dept. of Physiology & Biophysics, Univ. of Illinois College of Medicine,Chicago, IL 60612-7342.It has been known that chronic antidepressant treaunent results in a significantincrease of Ga-mediated stimulation of adenylyl cyclase in rat cerebral cortexmembrane. However, a down-regulation of 3-adrenergic receptors linkednormally to the stimulation of that enzyme often occurs. In order to

distinguish between these two effects and to determine whether pre- or post-synaptic components of the cell are required. C6 glioma cells were treated withantidepressants. Tricyclic (amitriptyline and desipramine) or atypical(iprindole) antidepressant exposure to C6 cells for 5 days significantlyincreased GppNHp-stimulated adenylyl cyclase activity in membranepreparations in a manner similar to that seen for rat brain membranes after 21day treatment. This effect was drug dose- and exposure time-dependent.Nevertheless, stimulation of adenylyl cyclase by isoproterenol was decreasedafter tbe treatment. By comparison, the antidepressant-induced5-receptordesensitization occurred earlier than the enhancement of GppNHp-activatedadenylyl cyclase, and extensive desensitization of5-receptors by isoproterenoltreatment did not enhance the GppNHp-stimulated adenylyl cyclase activity.These results indicated that the antidepressants have a direct effect on cellsignaling and enhanced GppNHp-stimulated adenylyl cyclase activity was not

correlated with desensitization of 3-adrenergic receptor stimulated adenylylcyclase. Wester Blot using specific antibodies demonstrated that neither theamount of G protein subunits (as. aig, ao and 3) nor their association with theC6 glioma membrane was changed after antidepressant treatment. It issuggested that chronic antidepressant treatment modifies some component ofthe membrane or cytoskeleton which, in tur, increase Gs-induced activationof adenylyl cyclase.

118

ACTTVATION OF RAT LIVER PHOSPHOLIPASE D BY ThESMALLGTP-BINDING PROTEIN RHOA.((KC. Malcolml, M.Symons2, J.H. Extonl)) IHoward Hughes Medical Institute,Dept. Mol. Physiology and Biophysics, Vanderbilt University,Nashville, TN 37232, and 2Onyx Pharmaceuticals, Richmond,CA 94806.

Stimulation of phospholipase D (PLD) byGTPySin rat liverplasma membranes(PM) indicates theinvolvement of GTP-binding(G) proteins. We used rho-GDI, an inhibitor of rho-like small G proteins (as theGST fusion protein, GST-GDI), todetermine the involvement of rho proteins. GST-GDI caused a

dose-dependentinhibition of GTPyS-stimulated PLD activity,as determined by accumulation of phosphatidylethanol (PEth)in the presence of ethanol. Pretreatment of PM with GST-GDIand subsequent washing diminished PEth formation as

compared with control PM. Incubation withGST-GDI causeda rapid and dose-dependent appearance of rhoA, rhoB, racl,and CDC42 in the wash. The extent of appearance of rhoAcorrelated with the extent of inhibition. Reconstitution ofGTPIS-stimulated PLD in GDI-washed PM was achieved withrecombinant, post-translationally modified rhoA, but not rac 1

or CDC42. The response to rhoA was dose-dependent. Theseresults indicate the involvement of rhoA in GTPyS-stimulatedPLD activation in rat liver PM.

120

p140 RAS-GRF (CDC25hb) CONTAINS AN IQ MOTIF AND BINDS

TO CALMODULIN ((R.E. Cheney and M.S. Mooseker)) Biology Dept.

and the Yale Liver Center, Yale University, New Haven, CT 06520

IQ moti& form binding sites for calmodulin or related proteins of the EF-

hand superfamily and are frequently associated with the unusual property of

binding to calmodulin in the absence ofCa". Database searches using a

profile of IQ motifs led us to identify an IQ motif between residues 198-225

of the rat pl40 Ras-GRF. This Ras regulatory protein contains one domain

that is a guanine nucleotide releasing factor (GRF) for Ras and a second

domain that appears to be a GRF for Rac/Rho. Since it is not known what

factors regulate the activity of Ras-GRF, a fusion protein corresponding to

residues 1-470 was constructed and found to bind to calmodulin using a blot

overlay. Interestingly, residues 148-191 of Ras-GRF, which are adjacent to

the IQ motif, are predicted to have a 99% probability of forming a coiled

coil. In addition, 2 PEST regions with PESTFIND scores of 8.1 and 25.8

were identified between residues 792-836; PEST regions in several other

proteins have been associated with cleavage by the Ca++-dependent protease

calpain. These results suggest that the activities of Ras-GRF be

regulated by Ca++and/or calmodulin.

rcrho GR-? ra"t

PESTcoil

117

MICROINJECTION OF Gq SPECIFIC ANTIBODY INHIBITS THE

INCREASE OFCa2+ IN ENDOTHELIN STIMULATED CELLS. ((E.P.LOBO, 0. DUMITRESCU AND HARLAN E. P/ES)), Dept of Anesthesiaand Medicine, University of California San Francisco.The mechanism(s) by which endothelin (ET) activates and stimulates

intracellular Ca2' increase are not totally elucidated. Previous studies suggest

that a pertussis toxin-insensitive GTP-binding protein mediates this

response. In solubilized membranes of CHO cells stably transfected to

overexpress the endothelin receptor (ETA), we have demonstrated coupling

of the Gq to ETA, by co-purification of these two proteins using an ET-1affinity column. To determine whether Gq plays a role in signaling the

release of Ca2' from intracellular stores as well asCa2+ entry from extra

cellular medium, we studied the effects of antibodies to different alpha

subunits on ET- mediated intracellular Ca2' elevation. Antibodies and fura-2 AM were microinjected into transformed CHO cells, and cells were treated

with 5nM ET-l in Ca2' free solution to determine the release ofCa2+ froM

intracellular stores. Ca2' entry was determined by replacing the extracellular

Ca2' free solution with a solution containing Ca2'. IntracellularCa2+ fura-2

AM in an inverted epifluorescence microscope . Both, the release ofCa2+from intracellular stores andCa2+ entry from extracellular space were

inhibited in cells microinjected with antibodies specific to alpha-q subunit

(QL- DuPont NEN). In cells injected with antibodies specific to alpha

common subunit (which does not recognize Gq), Ca2+ responses to ET- 1

were intaCt. These data provide direct evidence that ETA is coupled to Gq,and that release of Ca2' from intracellular stores as well as entry of

extracellularCa2+ is mediated by Gq in thiscell system.

119

EXPRESSION OF ACITIVATED Gsa INHIBITS MYOGENICDIFFERENTIATION IN CaCns CELLS.((C. C. Tsai, J. J. Billadello, andJ. Saffitz)) Departments of Pathology and Medicine, CardiovascularDivision, Washington University School ofMedicine, St. Louis, MO 63110.

Heterotrimericgusnine nucleotide bindingproteins (G.proteins)function in

trnsducing cell surfae receptor generatedsignals across cell membranes to

intracellular effectors. However, recentstudies havelocalized sizeablefractionsoftotal celular heterotimeric G-protein within intracellular andintranucdear loci as wellas in association with the cell membrane-- suggestingtheir involvementin otherfundamental intracellular procsses.Immunocytochemicalstudies have localized significant xa within nuclei ofwild type S49 lymphoma cells.Rimilar nuclear localization ofGsa has beennotedin proliferating, undifferentiated myoblasta but notin post-mitotic,differentiatedcardiac andskeletal myocytes. To explore the potential role ofGoa in the establishment of the muscle phenotype, westudied the effect ofexpression of activated Gao in a well characterized model of myogenicdifferentiation. Westablytransfeted C2C12murinemyoblasts and identifiedmultipleclonal lines (n=24) which expressed activated Gsa,under control ofthesteroid-responsive mouse mammary tumor viruspromoter. Stably tranafectedcells that were not induced to express activatedGsa differentiated normallyinto multinucleated myotubes expressing muscle-specific markers.

Dexamethasone-induced expression of activated Gs. markedly impairedmorphologic and biochemical differentiation into myotubes, as evidenced byformation of 8.3-fold fewer multinucleated myotubes (3.0 + 0.7vs. 24.8 + 2.6,n= 5 random high power fielda) and the accumulation of approximately 3.5-foldless CK activity (98.159 ± 10.116vs. 347.735+ 41.353IU/Ag protein,n-3) in induced cultures relative to the uninduced control plates during a 168hour time course.These data suggest muscle differentiation requiresregulated expression and activity of Gso and are consistent with thepossibility that this G-protein subunit has a novel intra-cellular role inmodulating early events ofthe myogenic differentiationprogram.

121

TLUBULIN-G PROTSIN COMPIEXR)RMATION AND SUBSEQUENT REGULATIONOF SYNAPTIC TRANSMISSION AND CYTOSKELETAL STRUCTURE AND

FUNCTION. ((S. Roychowdhury, A. Mathew and M. M. Rasenick.)) Department of

Physiology & Biophysics, U.Illinois College of Medicine, Chicago.II 60612.

The cytoskeletal protein tubulin canmodulate synaptictruasmission by complexing with

certain G proteins Gs Gi and Gq. Nucleotidetransfer from tubulin to Ga is the key step

of this activation. Recently, it has been observed that the nucleotidetransfer from tubulin

to Gai is followed by hydrolysis and stabilization of bound GDP in the complex.Hydrolysis-resistant GTP analogs elicit asimilar effect. A 2-10 foldincrease in nucleotide

binding is observed when tubulin-[a 32P]AAGTP is incubated with recombinant Gail.The free ysubunits of G proteins diminished the nucleotide binding totubulin suggestinga role of fy as a tubulin-Ga complex terninator which may allowtubulin to release frrimthe complex, rebind GTP and bind to and activate a new Go. The binding sites between

tubulin and Ga appear to involve microtubule polymerization domains, sinceai subunit of

G proteins were demonsaed to inhibit microtubule assembly. Tubulin-G protein

interaction displays several chracteristics of microtubule assembly including activation of

GTPase and partial non-exchangeability of nucleotide bound to tubulin. Thus G proteins,under appropiate conditions may modulate cytoskeletal stucture and function by affecting

tubulin-microtubule system. The antimitotic drug colchicine (0.1FM) had no apparent

effect on tubulin-nucleotide binding or tubulin-Gai induced stabilization of nucleotide

binding. Vinblastine (VBL,l gM), which is known to stabilize GTP binding to tubulin,increased nucleotide binding -4 fold, when incubated in the presence of tubulin-[a32P]GTP. The bound nucleotide was chased completely by mM GTP. However,

incubation of the tubulin-VBL complex with Gai maintained 40% of the [a 32P]GTPbinding even in the presence of I mM GTP. Thus VBL, although, interfering with the

GTP binding properies of tubulin, is unable to affect tusbulin-Gat induced stabilization of

GTP binding, perhaps due to its inability to block the unique interacion between tubulin

and Gail. Tubulin-G protein complex may be involved in modulattion of synaptic

tansmission and modification of cytoskeleal stucture and function. Such protein -protein

interactons may be crucial to control of celhllar signal transduction.

21a

G Proteins (122-125). Sunday

122

CHARACTERIZATION OF p50, A GTP-BINDING PROTEIN SUBSTRATEOF BREFELDIN A-INDUCED ADP-RIBOSYLATION. ((M. Di Girolamo, M.G. Silletta, M. A. De Matteis, A. Braca, A. Colanzi, M. M. Rasenick*, A. Luini,and D. Corda)) Istituto di Ricerche Farmacokgiche Mario Negni, ConsorzioMario Negn Sud, 66030 Santa Maria Imbaro, Italy and *University of Illinois,College of Medicine, Chicago,1L 60680, USA (Spon. by L. Nitsch).

Brefeldin A (BFA), a fungal metabolite with inhibitory activity on membranetransport, induces the mono-ADP-ribosylaffon of two cytosolic proteins of 38and 50 KDa (p50). The 38-KDa substrate has been identified as theglycolytic enzyme glyceraldehyde-3 phosphate dehydrogenase (GAPDH).Here we show that the p50 substrate possesses several properties of a

GTP-binding protein. Thus, 1) GDPPS and GTPyS (0.1 mM) inhibit by -50%its ADP-ribosylation, 2) blot-overlay studies show that a protein comigratingwith ADP-ribosylated p50 is labelled by [a32P]GTP, 3) photoaffinity labellingusing [32P]azidoanilido-GTP (AAGTP) show a comigration between ADP-ribosylated and AAGTP-labelled protein. In bidimensional PAGE the majorAAGTP-labelled spots had identical pi and M.W. of the main degradationproducts of ADP-ribosylated p50, 4) the ADP-ribosylation of p50 was

completely inhibited by brain fy subunit, a behaviour similar to that of the a

subunit of Gs, which is ADP-ribosylated by cholera toxin in the monomericbut not in the frbound form. P50, however, was not ADP-ribosylated by thetoxin. The M.W. of the native protein was determined by gel filtrationchromatography using ADP-ribosylated cytosols from rat brains. The labelledp50 eluted as two main peaks of -180 and -130 kDa and a minor peak of-100 kDa with a tail up to -50 KDa, indicating that p50 could exist as amultimeric complex or could be part of a heterocomplex. Altogheter theseresults suggest that a novel GTP-binding protein could be involved in themechanism of action of BFA.'Supported by A.l.R.C. and CNR (No. 93.01296,93.02378).

124

MECHANO-CHEMICAL OOUPLUNG IN CARDLAC MYOCYTES: INHBMON OFADENYLYL CYCLASE ACTIVITY BY HYPO-OSMOTIC SWELWNG. ((R HL--Dandan and L.L Brunton)) Departments of Pharmacology and Medicine, U.C.SanDiego, La Jolla, CA 92093-0636.

We have studed the effect of hypo-osmotic sweling on cylic AMP (cAMP)metaboism in adult andneonaal rat venriular myocytes. Decreasing omolarlty of

a defined medium by 12.5% to 50% results in graded inhbion (10-40%) ofisoproteriol-nulated cAMP accmulton in myocyles. Swelling cas a

siniar decrease m frskolin-stimuated cAMP accumulation but does not affect

basal and hornone-stimulaed pos ide hydrolysis or celilar ATPcontent. The Inhibitory effect of sweing is readly reversble either by sat additionto restore norMoemolarity or by rlment of the hypotonic medum. Treatment

with pertussi toxin does not reduce the swelling response but abolhes the

inhbitory effect of sweling on cAMP accunulation, suggesting the invement ofthe GI pathway. The response to swelling seems not to involve the reease ofeffectors known to couple to Gi in myocytes: addition of the endctheln receporantgonist BO-123, atropine or adenosine deemmase does not aler the Inhibitory

effect of sweltn on iaproterenol-stimulated cAMP aocumulation. Addknally,condtoned medium from swolln cells, with restored osmlaiy, has no effect on

cAMP accumulation when added to control myocytes. In dsticto to tes

effects, swelling enhacs homone-stiuleed cAMP accumtation in culturedS49 lymphoma cels. We conclude swelln of cardiac myocytes Wtibts cAMP

accumulaton through a mechanim that invs activaton of a pertussis tcsxn-senskive Gi protein. Acivation ofGi by this means may contribute to arenergichypor in hypoxic and ischemic myocardum.

123

PRODUCTION OF CELL LINES STABLY EXPRESSING THERECOMBINANT HUMAN PTH/PTHrP RECEPTOR: EVIDENCE FOIRMULTIPLE SIGNALLING PATHWAYS. ((M. Pines, A.E. Adams, C.Nakamoto, V. Rashti-Behar, S. Stueckle, M. Chorev, M. Rosenblatt, S.Fukayama, A. Tashjian and L.J. Suva.)) Division of Bone and MineralMetabolism, Beth Israel Hospital and Harvard Medical School. I Molecularand Cellular Toxicology, Harvard School of Public Health, Boston, MA02215.

Parathyroid hormone (PTH) is the major hormone responsible for theregulation of extracellular calcium concentration. The initial event in theexpression of PTH bioactivity is binding of the hormone to specific receptors(Rc) located on the surface of target cells in bone and kidney. In this study,we describe the production and characterization of cell lines, derived from thehuman embryonic kidney cell line (HEK-293) and the porcine kidney cell line(LLC-PKI), which stably express different amounts of the recombinant humarPTHlparathyroid hormone-related protein (PTHrP) Rc. Several clones (andthe receptor-lacking parental cell line) were examined for PTH binding, PTH-stimulated cyclic AMP accumulation, PTH-stimulated intracellular calciumand PTH/PTHrP Rc mRNA expression. Receptor-positive clones display aspectrum of PTH-responsiveness that correlates well with Rc number/cell andlevel of Rc mRNA present. In an effort to understand the differences betweenthe biological activity of different fragments of hPTH, we examined the abilityof different regions of hPTH and PTHrP to interact with the cloned Rc. Therewas no apparant interaction of C-terminal peptides with the Rc, although N-terminal peptides showed full bioactivity. The expression of the humanreceptor in a relevant cellular setting should facilitate characterization of theligand-binding properties and physiological signal transduction mechanism ofthe Rc.

125

BOVINE RETINAL ROD TRANSDUCIN STIMULATESTHE ACTIVITY OF A TASTE TISSUEPHOSPHODIESTERASE. ((D.E. Wildman and R.F.Margolskee)) Roche Institute of Molecular Biology, Nutley,NJ 07110.

Soluble cyclic nucleotide phosphodiesterase activities wereextracted from bovine circumvallate papillae and nontastelingual epithelium and subjected to chromatographicseparation on DEAE-Sephacel. A fraction from taste tissuewas obtained which was found to contain a cyclic nucleotidephosphodiesterase activity stimulable by a peptidecorresponding to one of the effector interaction domains ofbovine rod transducin. Aluminum fluoride-activated bovinerod transducin holoprotein also significantly stimulated thetaste tissue phosphodiesterase activity. There was no cyclicnucleotide phosphodiesterase activity in soluble extracts fromnon-taste tissue which exhibited the same enzymatic andchromatographic characteristics as the phosphodiesteraseactivity in the circumvallate extract Thus it appears that thetransducin-stimulable cyclic nucleotide phosphodiesteraseactivity of bovine lingual epithelium is taste tissue-specific.

Apoptosis I (126-127).126

MEMBRANE LIPID PEROXIDATION PRECEDES RADIATION-INDUCEDAPOPTOSIS IN MOLT-4 CELLS. ((D.E. McClain, J.F. Kalinich, and N.Ramakrishnan)) Radiation Biochemistry Department, Armed ForcesRadiobiology Research Institute,Bethesda, MD 20889-5603.

We are investigating mechanisms ofradiation-induced apoptosis usinghuman leukemic MOLT-4 cells as a model system. We have analyzedthe kinetics of DNA fragmentation, morphological changes, andmembrane lipid peroxidation in MOLT-4 cells exposed to differentdoses of ionizing radiation ('tCo gamma, 1-5 Gy, 1 Gy/min). Ughtmicroscopic analysis of irradiated cells indicate that morphologicalchanges characteristic of apoptosis begin to appear 10-12 h afterradiation exposure. DNA fragmentation, the most characteristic featureof apoptosis, also appears after 10-12 h and increases with timepostirradiation. We quantitated the extent of lipid peroxidation inirradiated cells, using a fluorescent fatty acid, cs-parlnaric acid.Peroxidation of this fatty acidresults in a loss ofits fluorescence, whichwas monitored over time. Our measurements show that lipidperoxidatfon begins soon afterradiation exposure, with 20% ofci-parinaric acid oxidized within 30 min postirradiation. Trolox, a peroxylradical scavenger derived from vitamin E, inhibitsradiation-inducedDNAfragmentation and promotesthe proliferative capacity of lrradiatedMOLT-4 cells. These results are consistent with a hypothesis thatperoxidative damage to membranesis a very early lesion that triggersradiation-induced apoptotic death in MOLT-4 clls.

127

CYTOPATHOLOGY OF NUCLEAR EVENTS PRIOR TO SUDDENDEATH (APOPTOSIS) IN NACM-TREATED CHO CELLS. ((T. H.

Dunnebacke and K H. Walen)) Viral and Rickelsial Disease Laboratory, State

of CA Dept of Health Services, 2151 Berkeley Way, Berkieley, CA 94704.

Verebrate cells exposed to the protein NACM (YaegIkria ameba

cytopthogenic material, Dunnebacke and Dixon, 1989, J. Cell Sci.29:391)grow at a normal rate up to 10 generatons before the entire cultureunxrgoessudden death Laiet al (1990, J Cel Biol.1L1:494a) confimed our findingsand added observations ofmargination of the chromntin and laddering of the

cellular DNA in the dying celis (apoptosis). For this cytologic study, we used

Chinese hamsterovaiy (CHO) cells; they have wellchaa izd romosomes

and they have been used in studies of apoplosis Chronmosne evaluaion of

control and NACM-reated cultures showed no differences in the frequencies of

sister chronatid exchange or in the quality of the stains for the first 5 days. Byday 6, in 28% of the mitoic cells in thetreated cultures and in 2% of those in the

contros, the stain one chromatid had a mfotled appe ne, ikhe the

light/dark Halquin patern expcted with the Hoechst 33258 stain (Wolff and

Perry, 1974, Chromosma :341). It is likely thatdaring the first step of

chomosomei cation the of thymidine by 5-booexyuridinewas incomplte, a situation known to occr when the cell cycle has beensloweddown. By 7-8 days, although unchangdin the contols. the miotic activityinthe NACM cultures had ceaseci However,many pair of mirror image teloplase

nuclei were present in adjaent cell They were irnegulaly shaped with

chromos eads proruding as if on during the reformain of the

nuclear membrane had been incomplete. By day 9, all of the cells had shrunk

leaving smll,ghoed cell remnants. In thissystem, the slowing of the mitotic

cedl cycle preceeds byseveral days the morphologic devdlpment of apoptosis.(supported by NSF)

.22a

Sunday. Apoptosis I (128-133)128

MORPHOLOGICAL AND METABOLIC CHARACTERRZATION OFAPOPTOSIS IN NGF-DIFFER 1ATEDPC1 2 CELLS ((J.C. Mills, D. Nelson,M. Erecinska, and R.N. Pittman)) Graduate Group in Cell Biology and Dept ofPharmacology, U. of Pennsylvania, Philadelphia, PA 19104.

The mechanisms of neural apoptosis were studied an NGF-differentiatedsubclone of thePC12 cell line. Longterm time-lapse videomicroscopy revealed thatapoptosis (as induced by withdrawal of NGF) is characteized by dramaticalterations in cellular morphology that include dynamic extrusion and retaction ofvery large pseudopod-hike pouches.Siice these protrusions seem to be formed bythe 'streaming" ofnonmal cytoplasmic constituents, including, often, the ceflnucleus, it appears that apoptosis involves substantial solation of the cytoskeletalmeshwork. At times, a pouch will grow as large as the cell body, giving thecell

a bipolar, symmetrical shape, which is reminiscent of a mitotic cell at the beginningof cytokinesis. Such substantial cytoplasmic activity suggests that the cell mighthe expending large amounts of energy. Hence, basic metabolic rate, glycolyticactivity, macromolecular synthesis, and concentration of a variety of nucleotideswere determined in order to study changes in overall metabolic state during death.The results suggest that alterations in metabolism occur only very late in the deathprocess as most paameters are very similar in dying and control populations. Anunexpected finding from the metabolic studies which were conducted on dyingpopulations of cells was the marked asynchrony of death. Tumecourses of viabilityloss tended to follow a declining exponential curve rather than the expectedsigmoid, suggesting, surprisingly, that the rate of death (when expressed as a

fraction of viable cells at each point) is a constant.

130

EVIDENCE THAT INTRACELLULAR PLASMINOGEN ACTIVATOREIN IBITOR TYPE 2 (PAI-2) DIN ITS A PROTEASE INVOLVED IN CELL

DEATH. L .L.Dickinson, K. Donnan, M.L. Linn, A. Suhrbier, T.M.Antalis)) Queensland Institute of Medical Research, Brisbane,Australia 4027

Many cellular stimuli trigger cell death as a means of controllingtissue homeostasis. There is increasing evidence for theinvolvement of proteases in several cellular death pathways. PAI-2 is a serine protease inhibitor which has been found to play aprotective role in tumor necrosis factor-a (TNF) mediated celldeath [Kumar et al (1991) J. Biol. Chem. 266, 20960-20964]. We haveexplored the mechanism by which PAI-2 confers protection sad

investigated the protective role of PAI-2 cell death initiated byother agents. HeLa cells, stably transfected with PAI-2 cDNA, weretested for their sensitivity to TNF-induced cytolysis compared withwild-type cells, and cells expressing antisense PAI-2, vector aloneor a CAT control plasmid. PAI-2 expression was demonstrated toprotect from TNF-mediated cytolysis in a dose dependent manner.PAI-2 expression did not protect from cell death induced throughdamage by UV or ionizing radiation, but was protective againstlysis by certain viruses. The type of cell death blocked by PAI-2involves programmed cell death, or apoptosis. The serine protease,urokinase-type plasminogen activator (uPA), which is the onlyknown substrate of PAI-2, was demonstrated not to be involved inthe protective mechanism. Mutagenesis of the PAI-2 serpin activesite residue demonstrated that this amino acid was important forprotection. The results suggest that PAI-2 inhibits cell death bytargeting an, as yet, unidentified intracellular protease.

CORRELATIONS BETWEEN DNA FRAGMENTAION AND CHANGES IN NUCLEAR MORPHOLOGY

DURING DEXAETASONE INDUtJED APOPTOSIS IN IMMATURE THYMOCYTES. M. Sikorska.E. Carsn , VHl-.^va r N. Chalv2 D.L. Brown3, and P.R. Walker ,*.Institute for Biological Sciences, National Research Council of Canada,

Ottawa, ON, KtA OR6, 5Department of Biology, Carleton University, Ottawa, ON,KlS 5B6, 3Department of Biology, University of Ottawa, Ottawa, ON, KlN 6N5.

Apoptosis was induced in rat thyaocytes by 1 jM Dexaaethasone (Dex) treatmentcells were analysed at several times points for up to 6 hr after the

treatment. DNA fragmentation was analysed using pulsed field gel electro-

phoresis and visualised by light microscopy after labeling with biotinylated

dUTP (TUNEL technique). Changes in nuclear morphology were assessed by imuno-fluorescence and confocal microscopy. Nuclear envelope protein(s) were

with P12 mAb, nuclear lasina with lasin B specific Ab 119D5-F1,matrix protein with Nu1A specific A-204 mAb and chromosome binding

protein(s) with P1 mAb. High molecular weight DNA fragments (50 kbp) could be

detected after 1 hr of Dex treatment and they preceded internucleosomal DNAcleavage. In situ labeling of DNA breaks revealed two patterns of staining:

bright rim around the highly condensed chromatin mass of small apoptotic

and diffuse nuclear staining of large and apparently morphologically

cells. IWhile P12 staining remained largely unaffected, there

of lamin B in apoptotic cells with collapsed chromatin. This was

accompanied by rearrangement of nuclear matrix and chromosome binding proteins.

Confocal microscopy of double labeled cells revealed that DNA breaks preceded

degradation of the lasina which represented the earliest of the changes innuclear morphology.

129

Mn-INUCED APOPTOSIS IS PRECEDED BY PHOSPHORYLATION OF A23kDA PROTEIN Di DIFEREMNTATINGPCP 2 CEL. ((K.E. Larsen, J. Roth,

andJIM Aleta)) Department of Pharmacolg, SUNY at Buffalo, Buffalo, NY 14214.

Two major issues of int in the field of neuronal are neurite

formation and cell death Neiher are lunderstood, although both are cucial to

neuronal deelpment Our aboratory is interested in the role that proteinphosphorylati pla these events To this end, we ntili PC2 cells, which are a

well model of sympathetic neurons that eatend neurites when exposed to

nerve growth factor (NGF). We have also recenty character a variant cell line(PC12m,) that acquires a neuronal phenotype with neurite extension when exposed to

eithe NGF or mangnese choride (Mn). Treatment of PC12 cells with NGF andMn or Mn alone for 1 or more days not only promotes neurite outgrowth but alsoresuls in the selective phosphorylatio of a23 kDa prtein. In addition to iuing

differentiation and phosphoryon, Mnalso leads to programmed ce death asdetermined by counting cel nuclei and by the presence ofcharacteric apoptoticcondensed chromatin following stainin with the nuclear stain, Hoechst 33342, In theparental cel line, PC12p, Mn administration merely promotes flatening of thecels;sneurites are not obseivd unlesa the cells are exposed to NGF at the same time. In thiscase, neurites form more rapidly with NGF alone comitan treatment of

PCI4 cells also results in the phosphorylation of a 23 kDa protein and subsequentapoptosis, whereas NGF alone does not promote p hr or apoptosis. Mn, on

the ohder hand, can induce apopsosis but camot enhance phosphoryato Although it

is hatth de phosphoryation of tih 23 kDa protein is not correlated with neuritefonrato it may be related to endogenous survival/death program. Initial experimentsusing the protein syte inhibitor, anisomycin, indicate that this agent is capable ofprotectng agains Mn-induced apoptosis and diminishing the Mn-inducedphosphoylation of the 23 kDa protein. We thereore cncude that thedifferentitneuronal phenotype inreases the of both cellnes to exhibit enhanced

phosphorAyation of a23 kDa protein and toinitiae a unique cell death program.

131

CHARACTERIZATION OF ENDONUCLEASE ACTMITY FROM PC2 2

PHEOCHROMOCYTOMA CELLS.((D. Ferrari, M. Vilalba and F. Di Virgliol)intiu of Gera Pathology. University of Fwrra, Frrara, IMy.

A dinctive feare of apoptosisis dogradation of cellar DNA intonudl.osom.-size fragenet a consequence of activation of putative

enouceamse. LUtti informationis avaabl on the nature, reguLtionand tracu of the enxmes invoived. Previous studies

suggest Vt a type-11 Mndnuclease could be acvated durig apoptosis,butrecenrporrts on the hdependency of DNA degradation triggeredby many s ili thu hypotei. We haveinvesti th presence, regulion localzation of endonuclacivt in fctio isolted from culures of PC12 cels. Our resultssugt tt it ispo abe toisolt twoendonuclas activitis that differfor ionic requirement: 1) at pH 5 we meaured an activity that was Ca2+

Mg+jep_ndent 2) at pH 7.6 an actvity that was Ca2+independentbut Mgr dependent. Both activities wer inhbtd by Zn2+. Endonucldactivity was high inuc er factons nd vary low in nuclar extracts(soluble nuclear frcti). Hower, we al found activity in themitochondria and microsomal but not cytosolic frations. In fact,

aactivity corretd best with the mncrosomal marker glucose-

6-phoaphae.thdu we think that the enzyme(s) might be assocated tomicrosomal membranes and nuclear nveope, whis presence in othercelulr fctons might be due to microsomal contamination. We alsofoundthat plasmid DNA wa digesd independentiy of type or molculrweight. Thes rest suggstthat PC12 clls express twoCa+independnt endonudeaswavies that can be serated according to pHoptium and Mg2 dependency.

133

E)CRACHROMOSOMAL CIRCULAR DNAS AND NATIVECHROMOSOMES ARE DEGRADED TO 50KB DNA FRAGMENTSDURING CIS-PLATINUMINDUCED APOPTOSIS INTHE MULTIDRUGRESISTANT EPIDERMOID CELL UNE BVI. ((P.V. chen1 A. M.

Sanchez5 , and J. T. Baft2)) DI p ofC Biolgy andAnaomy n 2 bI of Radiaion Oncology, School of Medicine,liedcal Coe Ger , Augu, Ga. 30912. (Sponsor M.J. Mulroy)

extrachromoa omal circular DNAs ae tequenily detected in

human canes ad often habor amplified ges, such ancogeres, that

cotrt to malignn prhgreson. In addion, a variety of culured cal

Ines slected for dru reaistance conota circulr DNAs harborin ampd

drug resistance genes. For exanple, the . mullidrug

resistant human cal KB-VI harborsa0 copies Of the

mutdrug rsisac gene (M1) on cirl DNAs,ap750

and 1500 kobas in siz. To geinhsght ito th stuctra organkitonand biolo of circular DNAs, tis sudyiv ad tW fate Of the circalrDNAs in lB-VI during apoptois, a mode of call death cactize bybreakdownofdchroomoal DNA, whlie th nuclear nmambne remainital interesthily, aftr cispiatininduced apoptosis, hiHohular-weht DNA fbagnt of 50 kb reutd frm th b nof DR-1-containing cdrcular DNAs, wl as from th nie .Bes releae of 50 kp DNA fragments is believed to result frm tereem of loop domains of DNA inohed in the hIgher-order packn ofDNA, tis result provides ston evienc that the padng of

_~achromoamal DNAs (epsms anddotble minutchromomes) Is

simiar tat nrativ chomosom

23a

Apoptosis I (134-139). Sunday

134

ACTIVITY AND ISOENZYMATIC SPECTIRUM OF NUCLEARENDONUCLEASES IN ISCHEMIZED RABBIT MYOCARDIUM.((LG.Lebedeva, S.S.Alexandrova, I.D.Kirsanova, SJavadov, and I.I.Votrin)) Institute of Biological and Medical Chemistry, Russian Academyof Medical Sciences, Moscow 119832, Russia (Spon. by A.G.Basnakian.)

Ischemia of tissue often leads to apoptosis due to the activation ofCa/Mg-dependent endonuclease (CME). Using perfused rabbit bearts,changes of the activity and isoenzymatic spectrum of the CME family inmyocardial cell nuclei were studied. CME activity in nuclear extractsmeasured by plasmid incision assay was found to be increased after 40min ischemia induced by break in the perfusion. Further extension of theischemia led to the decrease of the activity. Reperfusion after 40 minischemia did not stop the activation of CME. Interestingly, that diabetesmyocardium expressed initially higher level of CME activity in nuclearextracts, and following perfusion was associated with the decrease of theCME activity. To determine the CME isoenzymatic spectrum in theextracts, the electrophoresis of proteins in SDS-polyacrylamide gel withthe subsequent detection of endonucleases in the gel was carried out.Extracts from control animals contained 5 isoenzymes of molecularweights from 15 to 130 kDa. Under ischemia, increased activity of theCME was associated with the increase of the number of isoforms up toeleven from 14 to 170 kDa, but it had still 5 isoforms when ischemia was

combined with diabetes. We interpret the increase ofCME activity as theevidence of apoptosis in myocardium.

136

OVEREXPRESSION OF HUMAN BCL-2 PROTEIN IN CHO CELLSEXTENDS THEIR SURVIVAL AND GROWTH IN SERUM FREEMEDIA. ((P. Reddy, D. Zhu, and J. Thomas)) Immunex Corporation,51 University Street, Seattle, WA 98101. (Spon. by K. Grabstein.)

Overexpression of the Bcl-2 protein in factor dependent lymphoid,myeloid and neuronal cell lines blocks apotosis and promotessurvival but not growth of these cells in the absence of growth factor.We used the pCDNA3 vector to express human Bcl-2 in CHO DXB-11 cells expressing tumor necrosis factor receptor (TNFR) andstudied the effects of Bcl-2 on cell viability, growth and recombinantprotein expression in serum free media in the presence andabsence of insulin like growth factor (IGF-1). We found that the Bcl-2 clones exhibit greater cell viability and grow to higher celldensities than the parental cell line in the presence or absence ofIGF-1 over a 10 day culture period. A corresponding increase involumetric TNFR expression in Bcl-2 clones was observed althoughthe amount of TNFR per cell was similar to the parental cells. Thehalf maximal response of Bcl-2 clones to IGF-1 was lowered by 10fold compared to the parental cells. Hence Bc1-2 protein in CHOcells confers a survival and growth advantage leading to anincrease in total viable cell days and a concomitant increase inprotein production in serum free media. Bcl-2 may act bydecreasing IGF-1 or other growth factor requirements of CHO cells.

138

THE EXPRESSION OF BCL-2 IN HL-60 CELLS IS NOT DOWN-REGULATED DURING APOPTOSIS INDUCTION BYCYCLOHEXIMIDE. ((Z.Y. Han, D. Chatterjee and J.H. Wyche)).Division of Biology and Medicine, Brown University, Providence, RI02912. ((J. Early and P. Pantazis)) The Stehlin Foundation for CancerResearch and St. Joseph Hospital Cancer Laboratory, Houston, TX 77003.(Sponsored by J.H. Wyche.)

Treatment of HL-60 cells with cycloheximide (CHX) for short (2.5 hours)time periods induced apoptosis. Flow cytometry analyses demonstred thisis not a cell cycle-specific phenomenon. The induction of apoptosis in HL-60 cells by CHX suggests that under normal conditions, apoptosis fatorsare constitutively expressed. However, the activity of apoptosis factorsmay be inhibited by labile apoptosis suppressors The oncogene bcl-2 hasemerged as a critical gene that may regulate apoptosis in many cell types.

Western blot analyses indicated that treatment of HL-60 cells with 50 ugmlcycloheximide for up to 10 hours did not significantly alter the cellular levelof bcl-2. This indicates that bcl-2 is very stable and by itself not likely toprevent HL-60 cells from undergoing apoptosis. Fur ratment ofHL-60 cells with a differentiation-inducing agent, dimethylsulfoxide(DMSO), for 4-5 days induced spontaneous apoptosis. Prior to theoccurrence of apoptosis in DMSO-treated cells, bcl-2 levels were notsignificantly altered. This further suggests that apoptosis in HL-60 cellsmay be regulated by other factors such as bax (Oltvai et al., Cell, 74: 609-619,1993). We also characterized a mutant, HCW-2, derived from the HL-60 cell line that is resistant to apoptosis induction by CHX. Our studiesthus far indicate that these mutant cells no longer express some putativeapoptosis factors. This suggests thatHCW-2 mutant cells should provide auseful system for potentially identifying putadve apoptosis factors.

135

MOLECULAR CLONING OF CELL DEATH GENES FROMINSECT MUSCLE. ((D. Sun*, R. Ziegler$, C. E Milligan+, S.Fahrbach#, and L M. Schwartz*+)) *Molculalr and Cellular BiologyProgram, +Dept. of Biology, University of Massachusetts, Amhe, MA01003; $ Dept. of Biochemistry, University of Arizona, Tucson, AR85721; #Dept. of Entomology, University of Illinois, Urbana, IL 61801.

The intersegmental muscles (ISMs) of the tobacco hawkmothMAaniscasexta die during the 30 hour period after the emergence of the adult mothThe ability of the ISMs to die requires new gene expression. Using a

differential hybridization cloning strategy, we have screened an ISM cDNAlibrary to isolate genes that are upregulated coincident with the commitmentof the cells to die. Four such genes were isolated. The mRNAs of thesegenes increase dramatically when the ISMs become committed todegenerate. Sequence analysis revealed that one of the genes showsgreater than 60% identity to a protein from human hippocampus. Thefunction of this gene is unlnown. Another identified gene was shown toencode Apolipoprotein HII a lipid transfer molecule found in fat body andhemolymph. Both the mRNA and protein levels of Apolipoproteinmincrease with death. The role of this gene product in cell death is ofparticular interest considering recent evidence m ing the presenceof a specific human stncture homolog of Apolipoprotein l,

Apolipoprotein E4, found to be associated with Alzheimee's Disase.

137CHO CELLS AND CELL DATEL ((C.lA.g, K.Mrray, J HlHkmaa, XGaIIa A.J.Dkka))-o Mlury Dlidon, 2205, SdeoolfBlgIa SdensMancester Umlversty, Oxford Road, Manchester M13 95P, U.K

Recenty, there has been a slow but inesod atton on the possible inportance ofaptosis for animal cell Several rport have idat that myelnahybriDma and cdes die by apoptosis. Chines Hamster Ovary (CHO) ods a

further inusaly important in. CHO duk cels are usd widely with ampliable

exesso syms for producto of reconbinant proteins. We shwn that in batch

culture, CHO cdels e logical and biochical chags cbhrtistic ofappotc cdls when enter the death phae. Thes imlude nulear condensation and

fmation of apeptotic bodies and I markers such as DNA -mntGenes whi reglate apoptois avebeen identied andone of them, the bcl-2 gene hs

been cloned from the t(14,18) tanaocation brakpoint found in folicular B-coUlYnmhoma of Bcl-2 has been down to prvet apoposs seal

sysm. We have ivesited the likely bnefiil effbc of of the bcl-2pse CO cel since geneaton of cel lnes with iimpoved sesvial abiity will have

immsne imions in Hmn bcl-2 driv by the glutamine sntha

e syem d by Celtch hs been transfcted into CHO cdls. Bothwestrn and northr blo deected the presence of the _dhentic size Bc:-2 (26KDa)and it is epressedup to 2-4% of the total cela prems. Two Bcl-2 prodcing loneand tw conl clones (tanafcted by co l vector only) wre ed in this study. Wecxamined the effect of Bcl-2 on the uvival of cels i batch cultu and the resisacto atuospoine which isa potet inde of ap_pa. Vable cols thi Study we

aseedby the of ypan blue. Bcl-2 does cell growth

rate and it is unble to proct cdls fom su which trigger aopposi. Th, in CHOcels, Bd-2 i st suffwicient to pevent apoposs the condition of study.

139

FUNCTIONAL ANALYSIS OF Wt p53 IN TWO P53 NULLCELL LINES.((LKM Leffet, D. Sharp, J. F. Boylan)), DuPont MerckPharmaceutical Company, Experimental Station, P.O. Box80400, Wilmington, DE 19880.

The p53 tumor suppressor protein is a cell cycle regulatorthat appears to be involved in the Gl/S phase checkpoint.Normal p53 detects DNA damage and holds the cel at theG1/S cell cyde boundary until the damage is repaired.Mutated p53 containing cells continuously cycle perpetuating

and generating additional mutations some of which providethe transformed cell with a unique growth advantage drivingtumor cell progression. We have used two human p53 nullcell lines; H1299 (derived from a large cell lung carcinoma)and PC3 (derived from a prostate carcoma) to examine the

function of exogesly expressed p53 on the cell cycle. Wedeveloped several cell lines that contain Wt-p53 under thecontrol of a dexamethasone inducible extrachromosomalepession vector. Although both H1299 and PC3 cells stablyexpress inducdble p53 functional protein, these lines respondvery differently to p53 induction. Our results suggest thatthe effects of Wt-p53 on the cell cycle and apoptosis dependon the recipient cell type.

24a

Sunday. Apoptosis I (140-145)140

APOPTOSIS SIGNALS DELIVERED THROUGH THE T CELLRECEPTOR OF A T CELL HYBRID REQUIRE THEIMMEDIATE-EARLY GENE NUR77. ((Z-G Liu, S.W. Smith,KA. Mclaughlin, LM. Schwartz, and B.A. Osborne)) Program inMolcular and Cellular Biology, University of Massachusetts, Amherst, Ma01003. (Spon. by CE. Milligan.)

of the T cell receptor on immatr thymic T cells induces deathby apoptosis. While several lines of evidence indicate that apoptosis

requires de novogene expression, little is known about the molecularpathways that mediate this response. Here we identify nur77, a zinc fingertrAnscription factor, that is expressed in response to TCRe ngagement in

immature T and T cell hybrds.Antisense inhibition nur77expression prevents apoptosis in TCR stimulated cells. While nur77 also isexpressed in response to mitogens, tAnscription is regulated by 5 upstreamelements that are distint for those used for induction of apoptosis. Inaddition, polyadenylation is only observed on nur77 trancripts found incondemned cells. These data support role fornur77 in cell death that mayhe distinct from that of activation.

142

INHIBITION OF PROTEIN GERANYLGERANYLATION RESULTS INCELL DEATH BYAPOPTOSIS. ((7.Y. Han, WA. Anderson*, andJRWyche)). Division of Biology and Medicine, Brown University,Providence, RI 02912 and *Biology Department, Howard University,Washington, DC 20059. (Sponsored by Z. Han.)

Treatment of cells with lovastatin results in the inhibition of isoprenoid andcholesterol production. When OVIN cells, which are derived from a rat

metastatic ovarian tumor cell line were cultured in medium containinglovasttin, cellular and biochemical changes were observed. After treatmentwith 10 uM lovastatin for 20-22 hours, most cells assumed a rounded up

appearance. Thereafter cell bodies began blebbing and cells were foundfloating in the medium. At the end of a 48-hour treatment, almost all cellswere dead. The appearance of blebbing cell bodies suggested thatlovastatin-treated OVIN cells might be undergoing apoptosis. Gelelectrophoresis of DNA isolated from these cels showed typical apoptoticdegradation of DNA. This induction of cell morphology changes andapoptosis hy lovasttain treatment was not blocked by cycloheximide,indicating that the activation of apoptosis in these cells does not require theexpression of new gene products. Addition of farnesol, squalene,lanosterol, LDL-cholesterol, dolichol, coenzyme Q or isopentenyladenine tothe culture medium failed to prevent induction of OVIN cell apoptosis bylovastatin. However, apoptosis was blocked by the addition of mevalonicacid or geranylgeranylpyrophosphate to the culture medium. Lovastatin-treated OVlN cells accumulated several species of unprenylated proteinsprior to apoptosis. Among them, at least three were found to requiregeranylgeranylation under normal conditions.These results indicate thatapoptosis in lovastatin-treated OVIN cells was due to a lack of adequateamount of geranylgeranylated proteins.

144

Acetaldehyde-induced cell death: an internal signal for the elevation of

apoptosis. ((G.D. Crawford, Dahl R., Simon, F.R., Mapoles, J.E.))Hepatobiiary Center, University of Colorado Health Science Center, Denver,CO 80262.

We have previously reported a CHO cell line which expresses a transfectedmurine-liver alcohol dehydrogenase (ADH) and constituitively expresses a

low activity of acetaldehyde dehydrogenase. This allows chronic exposure to

in situ generated acetaldeyde (AcH) to be easily controlled by supplementingthe culture with low levels of ethanol. The basal level of apoptotic cell death

common to transformed cells was elevated by acetaldehyde in both a dose-

dependent and cumulative manner. The level of apoptotic cell death was

unchanged from untreated controls when the synthesis of AcH from ethanol

by ADH was inhibited with 4-methylpyrazole. DNA fragmentation was

quantified as a measure of apoptosis, and this was confirmed by presence of

regular DNA ladders and condensed chromatin. When a potent apoptoticagent, beauvericin, was applied, the extent of apoptotic cell death was

increased by prior chronic exposure to AcH. These data suggest that AcHacts to elevate alow frequency stochastic event which results in apoptosis, as

well as to potentiate the degree of apoptosis generated by more potent

triggers. This mechanism of apoptosis-induction is in contradistinction to the

more commonly discussed examples of thymocyte interaction with

glucoonrticoid which require external signals to initiate the apoptoticprogram.

141

MULTISTEP CONTROL OF PROGRAMMED CELL DEATH IN CUL-TURED FIBROBLASTS. ((E. Wang, D. Uu, & S. Pandey)) The Bloom-field Ctr. for Research in Aging, Lady Davis Institute, Jewish General Hos-pital & Dep't of Medicine, McGill University, Montreal,Quebec, Canada

The active suicidal event of programmed cell death or apoptosis helps ridorganisms of cells that are extra or no longer needed; genes protecting cellsfrom or committing cells to apoptosis are increasingly being characterized.We find that early cell cycle genes are involvedin the initial events ofapoptosis: during the first 3-6 hours after serum deprivation of mouse 3T3fibroblasts, before the detection of oligonucleosome ladders, up-regulatedexpression of c-fos, c-myc,cjun, cdc2 and RB phosphorylation are seen, aswell as the disappearance of statin, a nonproliferation-specific protein.Rescue from eventual death is possible by adding serum back to the cultureup to 12 hours after serum deprivation, when DNA fragmentation becomesnoticeable; this point of no return to living is marked biochemically by theappearance of a terminin protein (Tp30) of 30 kda, which remains at highlevels till the final death at 24 hours. Tp3O is a product of a precursor of90 kda (Tp9O), found only in growing and quiescent fibroblasts; a 60 kda(Tp60) form is found in their senescent counterparts. Tp9O proteolysis toTp3O seems to occur at the apoptosis-commitment stage, for which Tp3Ois a marker, Tp3O presence is also associated with cell death events inrodent tissues undergoing involution. Senescent human fibroblasts resistapoptosis; this failure to die is related to a lack of Tp3O. Our resultssuggest that the molecular events dictating apoptosis may be separated intotwo stages: initiation, marked by the activation of some early cell cyclegenes and statin disappearance, which cascades into committment, markedby the appearance of Tp3O from activated Tp9O proteolysis. The tandemorchestration of these two events may regulate successful apoptotic death.

143

EFFICIENT INHIBITION OF SODI IN PCl2 CELLS BY ANTISENSEOLIGONUCLEOTIDES LINKED TO ANTENNAPEDIA PEPTIDES. ((C.M.Troy, D. Derossi, A. Prochiantz, andM.L. Shelanski)) DepL of Pathology,Columbia Univ., New York, NY 10032 and Ecole Normale Superieure, Paris,France.

We have previously shown that the down-regulation of SODI in PCl2 cells byantisense oligonucleotides (ASODI) results in apoptoticceUl death (Troy andShelanski PNAS 91:6384-6387. 1994). However, the lability of theoligonucleotides in serum neessitat working in srum-free medium and at highASODI concenatiraon. In the current studies we have coupled ASODI by S-Sbond to a 16 aminoacid peptde homologous to the third helix of the drosophilaantennapedia homeodomain (pAntp-ASODI). pAntp peptide has beendemonstrated to be tasported through the membrane and to accumulate inneuronal nuclei (Derossi et aL JBC 269:10444-10450, 1994), where the S-Sbond is cleaved, releasing the oligonucleotide. WhenPC12 cells are incubatedwith pAntp-ASODI for 5 hours in 15% serum there is 67% inhibition of theSODI activity. More than 60% of thettrated cells dle in 24 hours, a resultsimilar to that obtained with conventional delivery of ASODI in serum-freemedium, but requiring 3 nMratherthan 8 iM concentrations of oligonucleotide.The antennapedia delivery system is a powerful method of delivering antisenseoligonucleotides and has a number of advantages over conventionaloligonucleotides: 1) it can be used in serum-containing media; 2) it requires muchlower concentrations of antisense oligonucleotide (nM instead of IM range); 3)unlike other modifications of oligonucleotides to increase stability, theoligonucleotide itself is unmodified and therefore will interact weU with theappropriae mRNA; 4) it shouldbe effectivein prinary neuronal cultures, as wellasincelllines; 5) it shouldbe effective for inWiostudies.

145

MYCOPHENOUC ACID, AN INHIBITOR OF INOSINE MONOPHOSPHATEDEHYDROGENASE, INDUCES ACCUMULATION OF NEUTRAL UPID INMONOCYTIC CELL UNES AND APOPTOSIS IN LYMPHOID CELL UNES. ((R.G.

Cohn, A. Mirkovich, E. Eugui, P. Burton, B. Dunlap,J.P. Caullield)) Syntex

Discovery Research, Palo Alto, CA 94304. (Spon. by S. Porter).

Mycophenolic acid (MPA) inhibits inosine monophosphate dehydrogenase, an

essential enzyme in de novo guanosine synthes, and is the active constituent of a

potent immunosuppresveagent, myphe mofetil. We have exarrmned the

effects of MPA on 4 cel Ines in vitro. Incubation of the mnoytic cal InesU937and THP-1 with I piM MPA for 72 hr indurce anincreae in the neutral lipid content

of hese cab as measured by nie red furesc and thin layer chromnatography.MPA caused an increase in nile red fluorescence of 66% in U937 cell and 129% in

THP-1 cab compared to vehide treated controla. Both Epid dropets and

tiacy1golycerola were alao increased in MPA-teaed cab. An increase in

triaCglyceroI folowing 72 hr incubafon with 1 iJM MPA was also observed in the

lymphoid cal Ines CCRF and MOLT-4. However, the most draatic effect of MPA

on the Iymphoid nes w the inducion of apoptosis as seen by nuclear

wmrphology. In pren expemn MOLT-4 colla showed adoseincrease in apopItois baween 0.1 to 1 pM MPA. After 48 hr incubation,approximately 10% of ceo in untreated cultures were apototic, as were 50% of

cb with 0.1 pM MPA and 90% of celb treated with 0.5 lIMMPA. Guaeine addiion up to 8 hr folowing initiation of MPA-Atnent inhibitedapoptala in MOLT-4 cab; when added after 12 hr thee wee rninsma inhbibiton.These stuIles suggest that MPA Induces apoptoWsl In rapidly prolfersting lymphoid

Ines and alersld metaboram in both mnocytic and lymphid cal nes. Thesefindings maey ba important in theimi induced by MPA in vivo.

25a

Apoptosis I (146-147). Sunday146

CELL CYCLE DEPENDENCY OF TNFa EFFECTS ON TF-1 CELLS: A ROLE FORTHE TNFR II IN MEDIATING APOPTOSIS ((R. C. Kuo, *D.J. MacEwan, and G. T.Baxter)) Moecular Deoicee Corp., Sunnyvale, CA and 'Dept. ofB i, Univ. ofGlsgow, GWagow, Scotand UK

Diverse physiological responses in a number of different cell types have beenattributed to tumor necrolis factor alpha (TNFa). At the cellular level, TNFa has beenshown to modulate cell prolieration, dlfferentiatlon, and apoptosis. Two cell surfacereceptors, the P TNFR I and p80 TNFR II, have been identified for TNFa. Theprecise signaling mechanisms of TNFx by these receptors have not beendetermined. In this study we demonstrate that TNFa induces either apoptosis orprolNeration depend on the state of cell cycle progession in the TF-1 cell line. The

1 cell line, originally isoted ftmm a human eiythroleukemia patient, is CD34+ andexhibis chacterisics of a myeloid sbm cell lire. cells require a growth factorsuch as GM-CSF or IL-3 for survival and proliferation. Immunoblot analysis indicatesthat TF-1 cells expres both the TNFR and TNFR II forms of the TNFa receptor. Initialexperiments using a Cytosensor microphys1ometer to continuously monitor cellularmetabolic activity suggest that these cells respond to TNFa in two dramaticallydifferent ways. Induction of apoptosis only occurs with cells in log growth (mitoticallyactve), while quiescent cells respond by stimulation of cellular proliferation. The fimecourse and concentration deendence of TNFa Induced apoptosis was confirmed byboth Hoechst staining and fragmentation. Proliferation was measured using acolorimetric cellular proliferation assay. To determine the involvement of TNFR It insignaling for apoptosis, we used a neutralizing anti-TNFR 11 monoclonal antibody.Hoechst stain anaysis indiates a greater than 80 percent inhibition of TNFamediated apoptosla, thus demonstrating a role for TNFR II in the TNFa-dependentapoptotic signaling mechanism.

Partial support by ARPA, Contract MDA972-92-C-0005.

147

TUMOR NECROSIS FACTOR ALPHA-MEDIATED CYTOTOXICI1TYAND DNA FRAGMENTATION ARE INDUCED BY ACTINOMYCIN-D IN BOVINE MAMMARY ACINAR CELLS. ((S.H. Kim, Y.S. Moon,W.L. Keller and C.S. Park)) Dept. of Animal and Range Sciences, NorthDakota State University, Fargo, 58105

Tumor necrosis factor (TNF)-a is thought to modulate apoptosis innormal cells. The objective of this study was to determine if TNF-a caninduce apoptosis in mammary acinar cells. Mammary acinar cells wereisolated from mammary tissues of lactating cows. Acinar cells were platedon collagen gel in Dulbecco's minimum essential medium supplementedwith 5% fetal bovine serum and incubated at 37 'C. Cells were assigned toeither a TNF-a(10-gM) only treatment group or a TNF-a(10-8M)/actinomycin D(5 ng/mL) group. Cells were also exposed to variableconcentrations of actinomycin D(0, 5, and 10 ng/mL) in the presence ofTNF-a(10-BM). DNA fragmentation is a hallmark of apoptosis in mosttypes of cells. DNA fragmentation of the TNF- (10-8M)/actinomycin D(5ng/mL) group was increased by 70% at d 3. DNA synthesis in the TNF-a(10-4M)/actinomycin D(5 ng/mL) group was reduced by d 3 comparedwith d 1. TNF-)(10-8M) alone did not significantly reduce DNA synthesisover the three day period. Cells treated with TNF-a(l0-8M)/actinomycinD(5 ng/mL) had significantiy reduced levels of DNA synthesis at d 3compared with cells treated with TNF-a(l0-8M)/actinomycin D(0 or 10ng/mL). These results suggest that TNF-a is cytotoxic to normal cellswhen they are exposed to actinomycin D. TNF-a together withactinomycin D appear to function as inducers of DNA fragmentation innormal bovine mammary acinar cells.

Muscle and Muscle-Associated Proteins (148-151).148

TRANSGENIC MICE EXPRESSING A DYSTROPHIN GENEWITH A DELETION IN THE ACTIN-BINDING DOMAINPROVIDE A MODEL FOR BECKER MUSCULAR DYSTROPHY((K. Corradol, P.L. Mills2, J. Wrightl, J.S. Chamberlain2, K Wangl)).Department of Chemistry and Biochemistry, University of Texasl, Austin,TX 78759; Department of Human Genetics, University of Michigan MedicalSchool2, Ann Arbor, MI 48109.

Duchenne (DMD) and Becker (BMD) muscular dystrophies are allelic X-linked recessive disorders caused by defective expression of the proteindystrophin. The N-terminal 240 amino acids (domain I) of dystrophin sharessequence identity with the actin binding domain of members of the spectrinfamily of F-actin binding proteins. We are investigating the interactionsbetween actin and dystrophin utilizing a combination of in vitro (Corrado etal, FEBS 344, 1994) and in vivo techniques. Using the dystrophin deficientmdx mouse as an animal model for DMD, we have created transgenic mdxmice that express a dystrophin molecule carrying an in-frame deletion withinthe actin-binding domain. Based on histological and immunofluorescencemicroscopy analysis, our data suggest that a wild-type level of expression ofthe deleted protein is able to correct most, but not all, of the histologicaldefects associated with mdx mice and display a mild "Becker-like"phenotype. These results suggest that the relatively severe Becker phenotypeobserved in human patients that have deletions within domain I is due to acombination of the observed low levels of the truncated protein and loss of animportant functional domain. To further our understanding of the function ofthis domain at the cellular level, we are examining the effects of this deletionon the interactions between dystrophin and various cytoskeletal proteins at thesarcolemma as well as at myofibrillar locations. This transgenic line will beuseful as an animal model for Becker muscular dystrophy.

150

EXTRAOCULAR MUSCLE IN THE MDX MOUSE: ABSENCE OF PATHOLOGYCORRELATES WITH SUPEROXIDE DISMUTASE ACTIVITY. ((R.J. Ragusa andJ.D. Porter)) Depetret of Anatomy and Neurobology, Univerty of KentuckyColege of Medidne, Lexdngton, KY, 40536-0064.

In the mdx mouse, extaocular muscle (EOM) is protected from the deogeneratlveregeneratve cyce that occurs In Umb muscle. The objectives of this study wero toquantify this differntial repons, and to examine the hypohsis EOM may be

proected from fre dt cell damuge. Whle 80% to 90% of the fibersin the mdx Umb are replacd by genee fibers by po aal day 90 (90), ourelectron mkosop*analysis, revealed that iIto of damage and egerneration(agocytc cell iNfratIon, acIae myoblast, centro-nuceat flbes) are not

present hi the rotatory EOMLs. In addlIton, as in the ontol strain, exprdon ofembyonic myosin heav chain protein occur only In the orbital awr of the adultmdx mouse. Therefore, the absence of thi isofomi at ypicsl sites furtr supportsthe finding that sinicat fibertumover is not ocmuri in these muscles. Also,evaion of the overall myosin heav chain expresio pettem, combined with otnormal nmophological prfi suggests tha none of the six unique fibertypes presentIn the EOMs appea to be prefentially afected by the pahologics processespresent in the mdx mouse. In contras to this, howewr, the retractor bulbi muscles,which contain fibertypes itr de betwon llmb and EOM types, do undero anilid _la luev- W- I W-jW cycle which results in approxImately 30%replacement with oegeneative fibes by P120. Alhough them s)

responsibi for conferrng protection to the rotatomy EOMs hav not beendetermdned, our preliminary studles usin a colorimetrIc assay for superoxidedbrutase have indIkod th the acivity/m protein of this onzyme may be

grea In thee musce as compared to the llmb muscls. This

finding, twfore, suggests that a potential mochanism for protecion fromdegeneatIon in mdx EOM may involve a more efficInt removal of free radicals.Supported by NIH EYO0L4 and Reserch to Prvent Blndne.

149DY3TOOPHINTRANSFECION LOW INRACELIILARCALCIUM ANDCALCIUM LEAK CHANNELACIIVlTY IN MDXMYOTUBES ((G.C. McCarter, W.F. DenetdawJr, P. Reddy, ad RLAteinliardt)DepO of Molecular and Cell Biology, Univ. of Calfrnia,

Berkeley, CA 94720

The lack of dystrophin in muscle cells from Duchenne mculardystrophy patients and the mdx mouse is assoclated with risedhth bar free calium ([Ca+2Di and a hier open probatblty (PO) of

the calcium leak channeL We have recently shown that rnul ([ClS+2DIand leak channel activity is rered in myotubes from transgenc mdxmice epreng dystrophin (Denetdlaw etaL, 1994, MoL BiaL CAl4930.Here, we have tanfcted primary myotube cultures frommdx mdie witha plasmid containing the dystrophin gene and _meaued their ([Ca+21)and leak channel Po. The plasmid, pRSVDy (Asadi et aL., 1991, Nature352;815),contained the Rous sarcona virus promoterand washnrodluedat the myoblast stage by lipofection. The presence of dystrphhn wconfirmed by immunohlstochemlsty. The Intraceluhrfree calchmwdetermined with Pura-PE3: tansfected mdx 62i3 nM,n-30 mdz 77t3nM, n=22; and normul 51 t 2 nM, n=19. The leak channel openprobabilities were also lower for the transcted myotubes 0.0185 t 01,n-16, versus 0.0810 .04, n-19 foruntan d mdx. The Pb for norulmyotube has previously been reported to range from 0.008 to 0G.Conductance and voltage-dependence were not altered by the

We have thus shown that adult dyr can hae

ncrmalcalhcm regulation restored ix vitro by hntrouctof aDNAcnstru containing the dystophin gene.

151

CALPAIN ACTIVITY AND DISTRIBUTION ARE MODIFED DURINGNECROSIS AND REGENERATION OF DYSTROPHIC MUSCLE ((M.J.Spencer, D.E. Croall, J.G. Tidball)) DepL Physiological Science, UCLA,Los Angeles, CA 90024

Death of dystrophin-deficient muscle purportedly results fromincreases in intracellular [Cal that causes the activalion of calpins. We tstedwhethercpains play a role in this process by assaying for changes incalpaactivity, concentration and distribution during the peak period of muscle celldeath in mdx mice (4 wk) and in completely regenerated, adult mdx muscle.Calpain isoforms were fractionated and then analyzed using antisera thai bindthe propeptides present only on the procalpains. Our findings show that atpeak necrosis, -capain becomes highly activated, although its concentationis unchanged. At this same stage, m-calpain concentration is increased,although its activation level is unchanged. During regeneration, It-calpainactivity and m-calpain concentration return to control values. Northernanalysis demonstrated that the increase in [calpain] is not from increasedtranscription, because neither calpain mRNA differed in concentration fromcontrols. Immunohistochemistry shows that in necrotic fibers, calpain'sdistribution is altered from normal fibers. However, during regeneration,calpain concentration is elevated at the cell membrane, although its activityreturns to normal. These observations agree with current hypotheses thatcalpain can be deactivated and retained at the membrane. The findingsreported here show that changes in the concentration, activation anddistribution of calpains over the course of mdx dystrophy support its role in

both the degenerative and regenerative aspects of mdx dystrophy. Supportedby the NIH and MDA.

26a

Sunday. Muscle and Muscle-Associated Proteins (152-157)152

VISUALIZAION OF DYWSTRP IMUSCJZ FBBlY EVANS BLUE-VITALSTA8IN IN THE MDX MOUSE. (CL Matuda and A.N1sh1kawa¶A

of l of Tokyo. K oma Tokyo JapanOf Phyaloio SaitamLa Medical School

Saitama350-044 Japa

To st the dystrophic fber distrtio In the akeletal muscle of the mdx

moue. It necesaay to prepare many erial aectios of muscle tisaue andreconstitute three-dimensional images of degenerated fibers by time-

ming way. And to study m muscle fibe biochemicaly, itwas not practical to isolate DN A and proteins from degeneratingmuscle fibers from thin sections. We have developed a new method tovisu dysr skeletal musle fies in the mdx moue by Evans blue-vital stainine. One egLoghody weigt of Evan blue dislved In PS

Injected into tal vein. The moue was sacrificed 3 hr after the injectio. It

found that blue-colored bers wre vle i n many skeletal muscles of

the mdx mouse. However, such blue-coloed fiber was not found in the B10control mouse. Fluorescent icr ic ob tn with 0-activation

revealed that Evans blue stained fibers can be visualied by reddishnluoesceee. Hematoxyinosin stang of the same section showed that

the Evans blu-stained muscle fbes are either hypercontracted fiber or

opaque and e This method alowed to count the number

o fdegenerat in fibers more antitively and to exCise and

intact fibers to study biochemically. For example, genomic DNAIsolated frm these blue-colored and non-colored muscle fibers of the ame

musle tue of the md moue andwere Into an agarose gel

DNA ladders with 200kb unit size were seen In the DNA of blue-coloreddegenerating fibers, however, such ladder was not found in the DNA of un

stained intact fibers. Theresul t suggesta that active cel death or apoptosimightcci In the dystrophic muslefSis in the mdz mose.Evans ble vital staining method will be useful not only to study the procesof muscle degnenertion but also to evaluate the theraputic effect of myoblattrnsfer and tnsfer in the mdx moie.

154

TRUNCATED DESMIN EXPRESSION AND ISOMETRICFORCE MEASUREMENT IN ISOLATED QUAIL SKELETALMYOTUBES. ((H.S.Feng, Z.H.Yang, K.R.Yu, T.H,jIkata,H.Holtzero and H.-Sweeney)) Departments of Physiologyand Cell & Developmental Biology, University ofPennsylvania School of Medicine, Philadelphia, PA19104. (Spon. by H.L.Sweeney)We have esablished a newsysem to exanine the structumal and contracilepropetiesof muscle proteinshn cultured avian myotubse. Skeaal myob ats frompecoralendceof quad embrysweer cultured over mati-gel costed sides A g

d hyocyesw m transfected with cDNA ercoding aruncated desren in which pertof the rod doman had been removed. (1) Polyconl anti-emin and monoclonal

anti-fast myosin wwer used to stain the myotubes d day 6, 9and12 cultures. 50-70% d the myotubcureweer tmraneced and dsplayed enormous numbes of

2-3pm desmin+ spheroids rsthe than numerous long desmin+ IFs. (2) The caDswere perieabinxed and a single myctbe mourted with 'T-c1ip' between a post

and a forceb trbnducer messurln iomatric force. Theerfire wast- mwunmtedon the stage of a mics equiped wih Nomarskd optics for observaton of

ptterm and d_terfination of sarcomee ength. Images d contractions

were recored on vio tope for dynami obseraon of sarcomem stabilty. Each

myotube mau then checked byi mnuno-staining after mechanical study. Themeasured forces (per cross-sectional area) for control and mock transfectionwbeswere 2to 4 imes higher thann mydotubes in whichdeesrm n IFs were

disrupted by expresion of truncateddeemin. Additiorally, the stabiliy of thelore striation patrnwm dminished byesression oftnrcateddesmin. Thus

intactdesminIFs may play a role in providing mechanical stability for forced gm inskletlal muscleceas.

153

THE BINDING DOMAINS AND MEMBRANE TOPOLOGY OF TRIADIN INSKELETAL MUSCLE TRIAD JUNCTION. ((H. Fan and A.H. Caswell)) Dept. ofCellular and MoleculaPharmaology, University of Miami School of Medicim, Mimi,FL 33136

Tniadin, an intnic protein of the juntion, binds to the dihy ropyridinercepto(DHPPr) an the unction footproden (1FF) western blot overlay expriments.

inteaction between triadin/DEPr andtrtr din /JFP suggests that riadin may berplnsible; for maintainig the junion archtectu and possibly for mediating

sigal tns In order to lix the iteracng domains wihin thetriadicprotein,fusion pepides wer synthesized using an expression system pGag whi

inludes a protenknl e A phosphosylaionsit e. Pepdes which have been expressed

are DHPr6w,,,, encodin DHPr al subunit amino acds 664-799, triadin (1-

49), triadin 2 (68-389), 2a (68-278), tiadin 3 (370-706), tiadin 3a (370-562),

triadin 3b (551-706), 3bl (551-672) andtriadin 3b2 (673-706) (the numbers inbrackets corrspond to the amio acid sequene of triadin). mAb AE.8 coains a

cytoplasmic eptope sinceimmunoelectron microscopy of intacttriadic vesile usingthis

antibody reveals prficles adjacet to the protuding junctional foet. It binds to

t iadin 2 and 2a specifically. A second nAb against tiadin, GE 4.90, also has a

cytoplamic epitope, since it inhibits CaP reles from SR. It binds totriadin 3, 3b and3b2 indcating that the C-temina 34mio a cids are ltdin the cytosol.mosphoryll DH[Prnr binds totriadinin thein tat vecle under non-reducing and

reducing conditions.It binds to fusion peptides,ttradin 2, 2a, 3, 3b and3bl bet

not tiadin and triadi 3b2. Similar patter ofbindin g to triadin are using the

iodinated JFP aa probe. Direct binding between DHPr/or DHPrft,,, and JFP were not

see. Based on tee findings, proposethat tiadin contain an extensive cytoplaicdomain which binds to theJFP and to the region of theDH[Pr which is considered

ritical fr signal trsmission.

155

CAPACITY OF AN EMBRYONIC ISOFORM OF MYOSIN HEAVYCHAIN (MHC) TO FUNCTIONALLY SUBSTII UTERORENDOGENOUS MHCS IN DROSOPHILA MUSCLE. ((L. Wells, N.Hess, V. L. Collier, K. A. Edwards, andS.I. Bernstein)) MolecularBiologyrIntitute, San Dicgo State Univcrsity, San Diego, CA 92182.

Drosophila melawgaster has a sie musle MHC gene The vaiousisoforn s needed fordifferen muscle funtions ar geserated by alternativeRNA splicing. To study the iprae of alterative isoforms, hine offlies wansfo with a gene encoding only an embiryonc-typeisofomm Flies with the transgene were then mated with a myosin-null flyline, creating a viable fly with only a single myosin isofom.phenotypic effects of intchanging isofoen sethen stioudied in severalmuscle types. On aphysiologicalevdel, these flies are coipletely flightess,have difficulty mating, have reduced jumping ability, and are sluggish.Polarized light microscopy revealed aprogessive loss of organization in theindiret flight muscles, correspondg to a total loss offunction.Legmuscles, on the other hand, look essentially normal, conesponding torelavely no al waldngabiliy. Electnm icroscopy revealed severe

ultUaCturaldefec inthe indict flight muscle; but only minordeefectsinthe TDT orjump muscle. In thesehighlyspeciaized muscles an isoformswitchresultsin loss of muscle functo Leg muscles, which contract at a

nmer rate,fution fairly well with theemnbryonicisofom Nofunctional or izational were detocted inlarvalmuscl,which is thought to contract slowly. Since the isoform used for this studywas embryonic, that it functioned well intransformed larvae is notsurprising. We concludethat isofonn ype does havefunctionalsignificance, especially in highlyspecalizedmuscles.

156 15?

IMMUNOFLUORESCENT CONFOCAL ANALYSIS OFTROPOMYOSIN IN DEVELOPING HEARTS OF NORMALAND CARDIAC MUTANT AXOLOTLS, AMBYSTOMAMEXICANUM. ((Sheb 1 L.Frac and LayF.I )Deqtntof Anaomy dCell Biold, SUNY Health Science Center, 750

Es Adam Stree, Syracue, NY 13210.

WITHDRAWN inyoii anjor tof the din filamets in anized

cadiac Fle during con tcion

iswell-d1oumned. Recdy, in has been implcd in aciz welL_hndic ge cmuitantaxod,

he-na that fail Dbes due to alackof ornied myofibri Thse

isag aoaconcorn zantreductioin amountof to si in mutnmt hear

cllsk Cofocal was used tDexamin t date-I Io- tinsoftropo duing myofibi essin normal

he ver dt coofdevelopme fm de hea-ben-s (35) dtroughthe advancedemitycic ste (40) and Inmyofibrils form

of Op omyo in mutan

agesN ounalheaiu at stag 35 nascentmyofibrilsiocainlalond thecon At later stages, a bas t apicalognizatinwoWeu m"yocardtwn, That is, nascent

myoibrils we fod s new the basl surfae ofcells while larg,m myofibrils e fo arthespical (or lu_inal) surfesofcell By

sta 40, norul' d filed with cablesof In

_tt beam tr-qm7ispretat vsylow levels and in an

Suftligbn at csT538, and40. cm s werenot w2k18 nd he, t ata de, op site n sa geHL314adHL-37702 and anAHA Grant).

27a

Muscle and Muscle-Associated Proteins (158-163). Sunday

158

LOCAIIZATIONAND QTATATONOFMYOFIBRIUIARPROTEINS BY CONFOCALMOCROSCOPY INDEVELAPING HEARTS

OFNORMALANDCARDIAC MUTANTMEA CAN AXOWLS.

((Robert Zajdel andLnyyF Lmanski)) Department of AnDMYnddCellBiology, SUNY Health Science Center, Syracuse, NY 13210.

The cardiac letha mutation in axolodis results in an absence of normalmyofibril formation. Mutant embryos can suvive fora lmited tim withoutbeating bearts. Bichemalepements have sugsted that tiopomyosi is

greay reduced in mutant hea and that the other myofibrinlareprotereducedorexpressedin an fashion. The preaentimmunolt uceantitative tudy exmines whole mounts of normal and

mutant heas duringde with otcal sconing using laserscning confocal Three-die ing of

s

seve

myofibriluteins have conannealack of normaly-orgnized myofibrdlsin mutant hearts as wel as a drstic reducton of tpomyosin in the mutants.The stining for -actin, myosin and -actinin are also somewhat reduced inmutant hearts and there is essentially no normal myofibril orgion atanyof the stages examined. Stage 35 mutant hearts have actin,opomyosin, a-actinin and myosin expressed both at the periphery andtthroghout thecytoplas.The amou nt ofst aning for al proteins at stageshows a slightreduction compard to normaL As development proges to 39 and41, mutant heats show drastic reductions in tropomyosin staining as well as

significantreductions in sining fortheotherproteins. The proteins are

localized primarily in amorphous aeasin the mutantcesU To verifyqualitative mphological observations ofthe staining levls in developinghearts, we are curny employingquant ttin prm m in conjunctionwith the confocal micrsc ps. by NIH Grants HL32184 and

H L 37702 andan AHAa LFLF)

160

A CATALYTIC COMPONENT OF THE LOBSTER MUSCLEPROTEASOME THAT DEGRADES MYOFIBRILLAR PROTEINS. ((DL.Mykles)) Department of Biology, Colorado State University, Fort Collins,CO 80523.

The proteasome is a multicatalytic proteinase that hydrolyzes peptide andprotein substrates. As the catalytic center of the 26S protease, it mediates theATP-dependent degradation of ubiquitinated proteins. The complex has atleast five peptidase activities; these are the trypsin-like, chymotrypsin-like,peptidylglutamyl peptide hydrolase (PGPH), small neutral amino acid-preferring, and banched chain amino acid-preferring (BrAAP) activities. Theproteolytic activity of the lobter prote is activated by heating Im mi at 60C; this heat-activated form is highly stable and degrades myosin,

tropomyosin, and troponin at room temperature (Arch. Biochem. Biophys.288: 543-551). Using troponin as a model substrate, in conjunction withcations or protease inhibitors, the BrAAP activity was identified as a

component involved in the hydrolysis of myofibrillar proteins. Of the fiveactivities, only the BrAAP activity was stimulated by heat-treatment. In

addition, both the BrAAP and proteolytic activities were inhibited by 3,4-dichloroisocoumarin, chymostatin, N-ethylmaleimide, and Mg2,, hot wereunaffected by Li+, Ieupeptin, or PMSF. Finally, the BrAAP activity was

competitivelyihhibited by toponin, while the proteolytic activity was inhibitedby the BrAAP substrate. In contrast, the trypsin-like and PGPH activitieswere completely inhibited by leupeptin and Li+, respectively, and thechymotrypsin-like activity was not inhibited by troponin. These resultssuggest that the BrAAP component mediates the initial cleavages ofendogenous protein, andthus may constitute therate-limiting step in thecomplete hydrolysis of myofibrillar proteins by the ATP/ubiquitin-dependentproteolytic pathway. Supported by NSF (DCB-8919315 andIBN-9218337).

162

CALDESMON PHOSPHORYLATION, BUT NOT MYOSIN LIGHTCHAIN PHOSPHORYLATION, OCCURS DURING CALCIUM-INDEPENDENT PHORBOL ESTER-INDUCED SMOOTH MUSCLECONTRACTION ((Thlrockmorton, D.C., Brophy, C., Packer, C.S., Isales,C., Rasmussen, H.)) Institute for Molecular Medicine and Genetics,Medical College of Georgia, and the Augusta VAMC, Augusta, GA 30912.

The role of protein kinase C (PKC) in sustained vascular smooth musclecontraction is controversial. We investigated the contraction of bovinecarotid artery smooth muscle (BCASM) stimulated by phorbol 12,13-dibutyrate (PDBu) under Ca2+-free conditions. Caldesmon and myosinlight chain (MLC)' phosphorylation were also measured. BCASM,incubated in Ca2+-free buffer containing the extracellular Ca2+ chelatorEGTA and the intracellular Ca2+ chelator BAPTA, had an intracellularCa2+ concentration of <lOnM (determined using FURA-2). PDBu inducedBCASM contractions of similar magnitude in the presence or absence ofCa2+. While PDBu increased caldesmon phosphorylation after 60 minutesin the presence or absence of Ca2 +, PDBu did not increase MLCphosphorylation over basal levels in the absence of Ca2+. Western blottingof BCASM using PKC isoform-specific antibodies demonstrated thepresence of two "Ca2+-independent" PKC isoforms: epsilon and zeta.Under Ca2+-free conditions, PDBu induces BCASM contraction andcaldesmon phosphorylation without increases in MELC phosphorylation.

Ca2+-independent isoforms of PKC may mediate PDBu-inducedcontractions through a kinase cascade which involves caldesmonphosphorylation.

159

PROTEIN SYNTHESIS DURING HEART DEVELOPMENT INNORMAL AND CARDIAC MUTANT AXOLOTLS. ((N. Erginel-Unltlns, DX,Dube andL.F. Lemanski)) Department of Anaomy and CellBiology, SUNY Health Science Center, 750 East Adams Street Syracuse,New York 13210.

Recessive mutant c in axoiots causes a failure of the hearts of affectedembxyos to funcion. mutt het (c) lackorgnizedsa

myofibril present study was underakmn to determine he overal

patter of in vivo protein synthesis and subsequent ofthenewiy synhsinzd proei fora 24 ho ourpeiodin norm(+/+or+k) and

cardiac mutant(c/c) axolot heart at various stages of developmenAdditionaldy, selectedcytoskeeibmyoflbril proteins were analyzed indetil forthei rs ynthesis dUinghear t develpmen Forsuc h analyses,thehearts were radiolabeld with [35S-mewnine for24 hours and subjectd to

SDS-PAGE analyses and au y. Quantitative densisomeiicanalyses of the bands showthateven though the overall protein syntheticpatterns are simia normal and mutant heart tis a general redction inthe synthes ofthe proteins in mutant hearts is observed even atthe earlierstages of development (stages 35 and 38). Most interestingly, actin issynthsized in normal amounts in mutant heat at stage 35, whiletroo snsynthesis is significanly reduced att hs stage. Synthess and

accumulatio fmostofthe proteins including ropomyoinare s ignifi cantyreduced in mutant hear ater stage s (staes 41- 42). l iscoa oborates

earlierbi ocheicalandand sties. (NIH grants

HL-32184, HL-37702 and an AHA grant)

161

A CALPAIN-LIKE CALCIUM-DEPENDENT PROTEINASE FROMLOBSTER SKELETAL MUSCLE. ((J.R. Beyette and D.L. Mykles))Department of Biology, Colorado State University, Fort Collins, CO 80523.

A variety ofCa2+-dependent proteias cal calpains have been identfiedvertebrate tissues. The m- and It-calpains of this class of proteinases are

present in all tissues surveyed, and the p94 calpain occurs only in skeletalmuscle. In the lobster, Homarus americanus, four calcium-dependentproteinase (CDP) activities have been identified from skeletal muscle. Thes

enzymes may function in the breakdown of proteins during normal clawmuscle atrophy that occurs during molting, since the CDPs degrademyofibrillar proteins to acid-soluble products. We have characterized themajor activity, called CDP Hb, with espe to autolytic activatio and Westerblot analysis. The CDP Ub is a 195-kDa homodimer of 95-kDa subunits.Like the vertebrate calpains, the CDPIIb is a cysteine proteinase thatundergoes aCa2+-dependent autolysis of the N-terminus. Autolysis requires at

leat ImMCa2+ and initillly reslts in two proteolytic fragmnts of 80 and 85kDa. Autolysis is intramolecular, since the appearnce of autolyic products is

independent of enzyme or substrate concentration. Native CDPIIb requiresapproximately 2.2 mMCa2+ for substrate hydrolysis; autolysis lowers the

Ca2+ requirement to approximately 1.7 mM. TheCa2+ concentraton requiredfor autolysis closely follows thatrequWed forproteolytic activity ofnativeCDP

IIb, suggesting thatautolysis is required for enzyme activity. Polyclonalantibodies to vertebratem-, ii-, and p94 calpains do not cross-react with CDPlb;similarly, a polyclonal antibody to CDPll b does not cross-react withm-,ti-, or p94 calpains, showing that there are few,if any, epitopes sharedbetween the CDPIIb and the calpains. Although both lobster CDPIb andvertebrate calpains share many biochemical properties, ourrsults suggest thatthe lobster enzymerepresents a group of Ca2U-dependent proteinases distinctfrom calpains. Supported by NSF (DCB-8919315 and IBN-9218337).

163

INFLUENCE OF HYPOXIA ON POST-NATAL ADAPTATION OF PORCINEPULMONARY ARTERIES: CHANGES IN MEDIAL SMOOTH MUSCLE CELLS((S M Hall, M Gorenflo, J Reader, T Faulwner and S G Haworth))Instiute of Child Health, London. WC1 N 1EH UK (Spon. by CAKing)In newboms,chronic hypoia causes persistentpulmonary hypertenson andchtnge

in pulmonay artery (PA) smoothmuscl (SM)cal cytoskelal proteins. Pigeb were

exosed to coic hypobaric hypoxia (HP)(380kPa) for 3 days, eihr from birth, 3or 14 days old and compared with newbor anImals and aged mathed controls (CT).The dsrbution of cytolkeletal proteins was studled by inmmunobluoraacant labengof te wal of small estic and muscular arteries and of medial SM cs dspersedfrom main PAs. Immunolabellingd CT and HP elaic and muscular PAs showe SMspcilic a-stWin and nmosin heavy chain (MHC) in al 3 and 6day old arImals,embyonic SM-MHC was not found in sini mwsculararteries at any age, and desminappeared only in 6 and 17 day HP. In cos dspered from CT and HP 3 day oldanials contractile protein containing filamenrts (SM-a.actin, SM-MHC, embryonic

specifc MHC, a-actinin) formwd strss fibres but formed striated shets IncOas fromolder animals. Coes from CT and HP anlais, ofaN ages, oonprsed 3 phenotypes,elongated fusiform, small and rge epthelod, all blled with SMa-actin and SM-MHC. Following hypoxia there was no change In the pattern staining but inteiyof actin and myosin staining increased. Using SDS-PAGE the proportion of MHCisoforms (SM1/SM2/non-muscle) was determined in CT extra-pulmonary,andperipheral intrapulronary elastic arteres. At birth no non-muscle MHC was detected

in main PAs and the SM1/SM2 ratio hI Coomasle blue stained gels was the sameas the adult (3&8/2). In smal elastic vessae there was an alteration In proporton of

SM11SLnon-muscle isoforms betwen birtwM days (43M3(27) and adult (55/3314).Thes results demonstrate a spectrum SMC cytoskletal difetiation In difntsegnment of pulmonary artery In the newborn period. The cad dispersion studies

demonstrate heterogeneiky of caN populations in a give arteria segment.

28a

Sunday. Muscle and Muscle-Associated Proteins (164-166)

164

MUSCLE STRUCTLRAL VARIATIONS WITH HEIGI{T. ((M. Prado

Fgura, E. Buz and G. RamlreO)) Ilnst. Invesfigacl6n Bo micaCONICET/UNS, 8000 Bahla Banca Argern 2Dept. B gla UMS,3inst. Nac. Enfeiedades Neopisicas, Llma 34, Pert.

ml studie carfed out under conditions of low oxygen tension

have demonstrated enzyme enrkhMet at the respiratory chain. Wehave anayzed utrastructural changes In skeletal muwsce -daphrag-from rats taken to the lnst. de Boogla Adina at Morococs (Peru)altude: 4.540 hts above sa level, tar acc_imatlzaton. Animabwere classied hto gups: I) cotrols, 1) acute hypoxia (hours

perma ) and I) c c hypoxda (one to sx monwt pera c).

Musco fgmeets from thesegrops wee fid p

accordrig to classical nmtodoogy for te observon by TEM. Fromthe microgrph ana we can hfer that a) at acute hypoa there

exists an e at mohondria crse wtich preset a h1gherelectron densl b) at chn hypox mitochondia are reased In

number, size and wth numerous cste in a crcar shope. We

iAsalzed different mJchon l biogenes stges. We can concudetopogrphic ultrsuctural aro tae pisce hI setal musle

aster acute and chronic hypoIa; these dch es bin much moreinaI hn chronic hypoxIa. This apper to ha a con,eIatlon wIth

miochondral enzyme ncrease pmduced by b l methods.

165

NOVEL ISOFORM EXPRESSIONS OF MOUSE CARDIAC TROPONIN T:cDNA CLONING AND CHARACIERIZATION OF THE ALTERNATIVELYSPLICED VARIABLE REGIONS. ((J.-P. Jin, J. Wang, M.E. Resek, and J.Zhang)) Department of Medical Biochemistry, University of Calgary, Calgary,Alberta, Canada T2N 4N1

Troponin T (TnT) is the tropomyosin-binding subunit of the vertebrate striatedmuscle troponin complex and participates in the Ca2+-regulation of contraction.There are different genes encode the cardiac and skeletal muscle TnTs and theexpression of multiple isoforms from either gene is regulated throughdevelopmental and/or muscle type-specific alternative mRNA splicing. Thehypervariable N-terminal region and the C-terminal exon 16/17 splicing ofskeletal muscle TnT have been documented and a cardiac TnT isoformswitching is observed during both avian and mammalian heart development.However, the biological significance of these isoform expressions and functionof the variable regions in TnT molecule have not been established. We haveisolated over 60 independent cDNA clones from a XZapII library constructedfrom mouse cardiac mRNA. DNA sequencing has revealed the full lengthsequences of the major embryonic and adult cardiac TnT isoforms as well asseveral novel isoforms. We have summarized the splicing patterns of exon 12that is also found in rat cardiac TnT gene and its presence may guide themolecular anatomy ofTnT for structure-function relationships. A novel exon inthe N-terminal variable region has been identified in the mouse cardiac TnTsequences. The statistically large number ofcDNA clones sequenced providesdata to evaluate the relationships among splicing of the two variable regions inthe cardiac TnT sequence. The cDNA cloning results are funther compared withthe protein expression pattern during mouse heart development. Genomicanalysis of the mouse cardiac TnT gene is underway to establish a system tostudy the biological significance of TnT isoform expression. (Supported in partby a grant from the Margaret Gunn Endowment for Animal Research.)

166

ANALYSIS OF MUSCLE-SPECIFC ENHANCER TRAP LINES INDROSOPHILA MELANOGASTER. ((M. Mardahl, N. Drain, S. LBemstein, G. L. Harris)) Molecular Biology Institute, San Diego StateUniversity, San Diego, CA 92182.

P-element-mediated enhancer detcion in Drosophila melanogaster wasused to identify genes that are expressed specifically in muscle. Thistechnique utilizes a reporter gene contained within a tanposable elementthat has inserted at random in the genome. Transcriptional regulatoryelements located near the site of insertion will influence the expression ofthe reporter gene in a tissue specific or developmental manner. Twoenhance trap lines that exhibited muscle-specfic staining of the lac-Zreporter gene product in cryo-secdoned adult flies were used in this study.Both lines exclusively express the reporter gene in the indirct flightmuscle. The position of the P-element insertions have been mapped onpolytene chromosomes by in situ hybridization. One line mapped to 22A,an area not previously noted to contain a muscle-specific gene, and theother maps to 66DE, close to the paramyosin gene location. Severalmutants for each enhancer line that have been generated by P-elementexcision are homozygous lethal indicating that these genes may be requiredfor viability. A genomiic clone obtained from the 22A line was used as aprobe to detect a 1.7 kbRNA transcipt on a developmental northem.Three cDNA clones resulting from a library screen were 96% homologousto the gene encoding enolase. The genomic upstream regions are beingsequenced to uncover the presence of muscle-specific enhancers to explainthe muscle specificity of the reporter gene. The other enahancer trap line at66DE is being used to isolate paramyosin mutants by insertionalmutagenesis due to by the local hops of the mobilized P-element.(Supported by Amerin Heart Association- California Affiliate)

Kinesin (167-168)167

mRNA EXPRESSION OF KIFIA, KIF2, KIF3A, KIF3B, KIF5, ANDCYTOPLASMIC DYNEIN DURING AXONAL REGENERATION.((R. Takemura-2, T. Nakata', Y. Okada', Y. Sekine', H. Yamazaki', Z. Zhang',and N. Hirokawa'))'Department of Anatomy and Cell Biology, School ofMedicine, University of Tokyo,Tokyol 13; 2Okinaka Memorial Institute forMedical Research, Tokyo 105, Japan.

Mouse neuronal cells express multiple members of kinesin family proteinsand cytoplasmic dynein, which are microtubule-dependent motors involvedin axonal transport of various organelles. To elucidate the role of individualmotor proteins during regeneration, we examined the gene expression ofthese proteins in mouse dorsal root ganglion cells after sciatic nerve crush.The level of mRNA expression was examined by Northern hybridization.Seven to 14daysafteraxotomy, mRNA forclass II p-tubulin, and neurofilamentL protein showed typical changes previously known in regenerating rat lumbersensory neurons, suggesting that axonal sprouting was succesfully occuring.At these stages, levels of mRNA for kifla, kif2, kif3a, kif3b, and kif5 were

reduced to 50-80% of control. In contrast, mRNA for cytoplasmic dyneinwas slightly increased, up to 140%. The results suggest that the expression ofmembers of kinesin family proteins is slightly down regulated in a coordinatedfashion in murine lumber sensory neurons during regeneration. In addition,the results suggest that the expression of cytoplasmic dynein is slightly up

regulated, and partially support the previous suggestions that retrogradetansport plays important roles in regeneration as means to probe the progress

of regeneration.

168

POINT MUTATION OF ATP BINDING MOTIF GENERATEDRIGOR-KINESIN, WHICH SELECTIVIELY BLOCKSANTEROGRADE LYSOSOME MEMBRANE TRANSPORT INVIVO ((T.Nakata and N.Hirokawa)) Department. of Anatomyand Cell Biology, School of Medicine, University of Tokyo, Tokyo,1 3, Japan.

Point mutations were introduced in the ATP binding motif ofkinesin heavy chain. These mutants and wild-type kinesin cDNAwas introduced into fibroblasts. Wild-type kinesin accumulated atthe tips of the long processes, while some mutants tightly boundto microtubules in the center of the cells, indicating that thesemutants are rigor type. We analyzed vesicular tr.ansports usingthese mutants. The stationary localization of membrane organelleswas not severely affected in these cells. Retrograde transport fromGolgi to endoplasmic reticulum, and lysosome dispersion are bothmicrotubule-dependent, plus-end directed movements. The latterwas selectively blocked in the rigor mutant cells. The microtubulesminus-end directed lysosome motion was not affected. The resultsindicated a selective blockade of membrane transport by thedominant effect of rigor-type kinesin mutants in the cells.

29a

Kinesin (169-174). Sunday

CLONING A CDNA ENCODING A NEW NEURAL-SPECIFICMEMBER OF THE MOUSE KINESIN SUPERFAMILY ((Z Yang andL.S.B. Goldstein)) Howard Houghes Medical Institute, Division of Cellularand Molecular Medicine, Department of Pharmacology, UCSD, La Jolla,CA 92093-0683. (Spon. byH.J. Matthies)

Proteins of the kinesin superfamily define a class of microtubule-dependentmotors that are involved in cell division and intracellular transport. On thebasis of the conserved amino acid sequences shared by eachkinesin-likemotor in a putative motor domain, several primers were designed. Whentheseprimers were used for PCR, several DNA fragments with the expectedsize were amplifiedfrom a mouse brain cDNAlibrary. The DNAfragmentswere cloned and subjected to sequence analysis. While several differentclones representing known mouse kinesin superfamily members wereisolated, several distinct clones representing new members of thissuperfamily were found. One PCR fragment was analyzed further byNorthern analysis and found to be expressed mainly in brain. A 4 kbcDNA corresponding to this fragment was isolated, analyzed, and found tocontain an open reading frame of 794 amino acids. Sequence analysisindicates that theamino acid sequence is most similar to members of theKif3-KLP68D family of kinesin-like proteins from mouse and Drosophila,respectively. Since these proteins are thought to play roles in the nervoussystem, we suggest that this cDNA encodes a new neural-specific memberof themouse kinesin superfamily.

lYl

IDENTIICATION OF HUMAN UNC-104-RELATED GENES. ((A.J.Otsuka and W. Klinke)) Department of Biological Sciences, 4120IllinoisState University, Normal,IL 61790-4120.

The product of the unc-104 gene is akinesin-related protein that is requiredfor axonal transport of synaptic vesicles in the nematode, Caenorhabdtiselegans. We hypothesize that similar proteins may be present in humans andserve similar functions. In order to identify human unc-104-related genes, theEntrez nucleic acid sequence database (release 10, June 1994) was searchedwith the MacVector computer program (Kodak) using the C elegansunc-104cDNA sequence. The highest matching score was obtained with an

unidentified open reading frame (ORF) from the male myeloblast cell-line,KG-1 [Nomura, N. et al. (unpublished), Entrez document ID 452516].Unlike kinesin or kinesin-related proteins which show strong sequence

similarities only in the motor domain (-300 aa), this ORF shows brokenregions of similarity over the amino terminal half (-800 aa) of the UNC-104protein. The ORF is not equivalent in sequence to the mouse unc-104-relatedKiflb product, which also has hyphenated regions of similarity to the aminoterminal portion of unc-104. We are continuing to analyze the human brainunc-104-related HHCMC63 cDNA clone previously identified by J. CraigVenter's group [Adams, M. D. etal., Nature, 355, 362-634 (1992)].

173

THE KLP2 GENE OF SCHIZOSACCHARONYCES PONBE ENCODES A MEMBER OFTHE KAR3 SUBFAMILY OF KINESIN-LIKE PROTEINS. ((C.L. Troxell andJ.R. McIntosh)) Department of Cellular, Molecular andDevelopmental Biology, Univeristy of Colorado, Campus Box 347,Boulder, CO 80309-0347.

We are using Schlzosaccaromyces pombe as a model system inwhich to examine the mitotic functions of novel kinesin-likeproteins (KLPs). We have used a PCR strategy to isolategenomic clones encoding KLPs from S. pombe. Two genes, SpoKLPI(-PKL1, A. Pideux and Z. Cande, 1993, Mol. Biol. Cell 4:243a)and SpoKLP2, were identified and mapped to the left arm ofchroosome 1; they encode proteins belonging to the subfamilyof motors which includes KAR3p, NCD and KLPAp (O'Connell etal., J. Cell Biology 120: 153). Members of this subclass havethe kinesin motor domain at the COOH terminus, rather that atthe NH, terminus, as in other members of the kinesinsuperfamily. The nucleotide sequence of SpoKLP2 predicts a

protein of 833 residues and molecular weight 92,580, withoverall similarities to KLPAp (59%), KAR3p (58%)and NCD (55%).The ore highly conserved motor domain of SpoKLP2 sharesgreater similarity (76%)) to that of KLPAp over a length of 407residues, about 69% similarity to that of KAR3p over 372residues and a relatively low 43% similarity to that of NCDover 284 residues. Experiments are in progress to determinethe effects deleting the SpoKLP2 gene, to localize KLP2p inliving and fixed cells, and to,.characterize the biochemicalproperties of the bacterially expressed protein. This work was

supported in part by NSF (C.L.T.) and NIH (J.R.M).

170

GENETIC ANALYSIS OF DROSOPHILA KINESIN LIGHT CHAINFUNCTION. ((J. G. Gindhart, Jr.1. C. Desai2, K. Zinn2, and L. S.B. Goldsteinl)) 1 Howard Hughes Medical Institute, UCSDSchool of Medicine, La Jolla, CA and 2Califomia Institute ofTechnology, Pasadena, CA.

Native kinesin is a heterotetramer composed of twohaavychains and two light chains. Whereas the heavy chains are

responsible for the microtubule binding and ATP-dependenttranslocation activities of kinesin, the functions of the light chainare unknown. Current models propose that the kinesin lightchain regulates or mediates the tethering of thekinesin heavychain motor domain to organelles, or that the kinesin light chainregulates kinesin heavy chain motor activity. Todistinguishamong these and other models, a mutational analysis of theDrosophila kinesin light chain (kk) gene has been initiated. kicmutants were generated by mobilizing a P-element insertionlocated in the same cytological interval askkc, then screening forinsertion events in or near klc using Southern blots and PCR.One P-element insert was mapped toIntron ofkkc. Thephenotypic analysis of defects associated with this insert, as wellas deletions created byits imprecise excision, will helpdetermine the in vivo function of the kinesin light chain.

172

TOWARDS EXPRESSION OF THE UNC-104 KINESIN-RELATEDPROTEIN FROM CAENORPJ4BDDSTIELEGANS. ((V. Anupunpisit andA-J. Otsuka)) Department of Biological Sciences, Illinois State University,Normal,IL 61790.

Kinesins are a family of microtubule-based motor proteins involved in theintracellular transport of vesicles and organelles alongmicrotubules. Thenematode CaenorhabdIs elegans is a free-living animal that provides an

excellent system for the study of neural development. The behavioral andcellular phenotypes ofwucoordnated-104 (unc-104) mutants are consistentwith the identification of theunc-104 product as akinesin-related proteinused for proper axonal transport of synaptic vesicles. To demonstrate thatthe unc-104 protein is a microtubule-based motor, construction of an unc-

104 gene expression system using Escherichia coli has been undertaken.Using the polymerase chain reaction, a 2.4 kb DNA fragment that includesthewuc-104 motor domain and a portion of the rod domain has beengenerated. Although several approaches were used, it has not been possibleto clone this DNA fragment. However, attempts are underway to clone a

subfragment containing only the motor domain of the unc-104 product(amino acids residues 1-468). If unc-104 expression is successful, bacterialextracts expressing the unc-104 protein fragment will be tested formicrotubule-based motility.

174

STRUCTURAL AND FUNCTIONAL ANALYSIS OFKINECTIN"Yu,H., Kumar,J.,Nicchitta,C.V.,Toyoshima,l. and Sheetz,M.P."Duke University Medical Center

Kinectin has been identified as a kinesin-associated membrane protein inchick brain microsomes. A kinectin cDNA of 5.3 kb has been isolated froman embryoric chick brain cDNA library by immunoscreening with a panel ofmonoclonal anti-kinectin antibodies. The cDNA contained an open readingframe encoding a protein of 156 kDa. Sequence analysis suggested that theN-terminal 30 residues might function as a membrane integration signal andthe rest of the molecule capable of forming alpha-helical coiled coils (likelyto be dimers). Cell free in vitro translation, menbrane translocation andassociation studies indicated that the translation product is a membraneassociated protein with major portion of the molecule on the cytosolic sideof the membrane. The expressed protein is also capable of binding to kinesinin blot binding assay consistent with all the characteristics of native kinectin.One monodonal antibody against the cytoplasmic portion of kinectin couldinhibit 90% of the plus-end directed vesicle motility in vitro even as Fabfragment. This inhibition of in vitro kinesin driven veside motilitycorresponds to similar inhibition of the kinesin binding to vesicles. These dataand the structual features of kinectin all indicated that kinectin is an essentialmembrane anchor for kinesin in the functional motile complex.

30a

Sunday. Kinesin (175-180)

175

ISOLATION OF PROTEIN PHOSPHATASE ACTIVITY IN MT-

AFFINITY PURIFIED MOTOR FRACTIONS. (Q.M. McIlvain Jr.', L.

Lindesmith2, Y. Argon' and M.P. Sheetz2)) Depts. of 'Immunology

and 2Cell Biology, Duke University Medical Center, Durham, NC

27710Identification of the protein phosphatase involved in the 2-3 fold

increase in kinesin motor activity (see McIlvain et al. 1994. JBC.

269(29):19176-19182) would greatly enhance our understanding of

the mechanism of microtubule (MT) dependent motor regulation.

Using a MT-affinity/ATP release protocol, we demonstrated that

both kinase and phosphatase activities are retained in the semi-

purified motor fractions of mouse T-cell lymphoma (Th) and hu-

man promyelocytic leukemia (HL-60) cells. Treatment of the ATP

eluate with 125 nM okadaic acid (OKA), resulted in the same 2-3 fold

activation of kinesin motor activity as previously reported for OKA

treated cytosol (see above ref.). Additionally, maximal inhibition of

the phosphatase was observed at an okadaic acid concentration of

500 nM, indicating that the novel phosphatase is a member of the

PP1 phosphatase family. The phosphatase was further purified on

an FPLC sizing column and a molecular weight of approximately

120 kD was determined. Two peaks of activity were recovered from

the column, one at 120 kD and a second smaller peak at 36kD, which

is consistent with the size of the PP1 catalytic subunit. These results

suggest that the regulatory components involved in MT-dependent

motor activity are present in the motor fraction.

1I7

A KINESIN-LIKE PROTEIN IS ASSOCIATED WITH MICROTUBULES

DWIDING ARABIDOPSIS CELLS AND POLLEN TUBES OF

TRADESCANTIA. ((B.Liu, and B.A. Palevitz)) Department of Botany,

University of Georgia, Athen, GA 30602.

A polyconal antibody was raised against two peptides of themnicrotubule(Mt)-binding domain of an Arabidopsis kinesin derived from a published

sequence (katA;Mitsui et al., 1993. MGG. 238362). Monospecific antibody

was prepared against the peptides covalently bound to Imnmobilon-AV

membrane. The monospecific antibody recognizes a polypeptide with

molecular mass of approximately 120 kDa on SDS-PAGE with Arabidopsis

seedling extracts.The punfied antibody was used to localize theIdnesin-

like protein (KLP) in Arabidopsis suspension cells. KLP appears to

specifically associated with the mitotic apparatus and phragmoplast.

metaphase cells, KLP is present on kinetochore Mts adjacent to the

kintochores, but appears to be absent near the poles. Antibody labeling

striking on Mts that branch from thekinetochore fibers and cross

interzone. By late anaphase, KLP is restricted to the interzone region.

During early phragmoplast development, KLP is broadly localized

the forming cell plate. At later stages, it is present in the phragmoplast

either side of the cell plate. In Tradescantia pollen tubes, the antibody

stains themitotic apparatus of the generative cell but not the prominent

in the vegetative cytoplasm. Our results show that KLP(s) is present

dividing cells of Arabidopsis and oher species andmay play roles

uchmtosome separation and phragmoplast organization. Furher

biochemical and functional analyses ofKLP is undenray. Supported

USDA-NRICGP grant 93-37304-9142.

119

PKL1: A KINESIN-LIKE PREOTEI FROM SCHIZOSACCHAROMYCES

POMBE. ((AL. Pidoux and W.Z. Cande)) Department of Molecular

CeCl Biology, University of California, Berkeley, CA 94720.

Kinesin-like microtubule motor proteins are thought to contribute

the spindle and the cytoplasm. PKL1 is a 96kD kinesin-related

was isolated from anS.pombe cDNA library using peptide

LAGSE and anti-HIPYR) specific for the motor domains of

proteins. putative motor domnai of PKLi is at the C-terminus and

degree Of homology is with the KAR3/NCD subfamily. non-essential

gene, butdissptpion causes an inreased sensitivity to mictuble-depolynerisingdrugs. PKLI-disrupted srains are able to mate and sporulate,

PICL is not required for keryogamy. Disruption of PKL1 partally suppresses

temperature sensitivity associated with a mutation inCUC7, another e-related protein. This observation is similar onkesin S. cereviase and Aspergillus and may support the idea that miaintenance

spindle structure requires balancing forcea. The PKLI protei localsssmitotc spindle and when overexpresaed it is readily detectable cytoplass cmicrotubules as well. PKL1 behaves as a motor protein bacterially

xpressed fuiion proteins bind microtubules, show ncrtubue-stiimulatedactivity and bundle nmcrotubues in vitro an ATP-dependent manner. Genetic

screens are currently underway to identify genes which are synthetically

with PCLi. We believe thatPKLL may function as a motor the

(eg anaphase A, spindle integrity) and/or cytoplasm (eg nuclear

though its precise role is not yet clear.

176

SUPPRESSION OF KINESIN EXPRESSION BY ANTISENSE OUGONUCLEOTIDEINHIBITS INSUUN SECRETION FROM CULTURED MOUSE PANCREATIC BETACELLS ((Y.X Meng and R.D. Balzon)) Department d Structural and CellularBiology, University of South Alabama College of Medicine, Mobile, AL 36688

Diabetes is a severe diseaseFt is characterized by chronic hyperglycemia anddsturbances ofmetabolsm. In most cases, insulin defikcncy is the direct reasonfor hyperglycemia in diabetes patients. Alhough insulin has been extensivelystudied over the past several years, the molecular and cellular mechanism of

insulin secretion from pareatic beta cells is not completely understood. The betacell microtubule cytoskeleton has been shown to be an imponrant regulator of

insulin secretion. In this study the microtubule-Iependent ATPase kinesin hasbeen proposed to functio as a translocator in beta cells that ansports thesecretory vesicbs along microtubuletrack from deepwithin the cytoplasm, whereinsulin is synthesized and processed, to the surface of the beta cells uponsecretory stimuation. To test this hypothesis directly, the heavy chain of a mousepancretic beta cell kinesin (mKHC) was cloned and sequenced and then mKHCexpression was suppressed by a complementary antisenseor " Incubation of cultured monolayerpancreatic beta cellswith the anti-mKHC oigomer decreased insulin secretion by 50%, whereas acontrol oligomer didnot significantly affect the insulin secretion. Thus, the effectsof the anti-mKHC oigonmerexibited sequence specifity. In addWion, the inhibitionofthe insulin secrebon induced by theanfisense tea wasnot the result of

cel damage due to toxic effects of oligonucleotides because the inhibitory effectscould be reversed bywashingoutthe olgonuclotide. Thesefindings indicatethatpa eatic beta cell kinesin plays an important role in insulin secretion.

178

KINESIN IS LOCALIZD TO ENDOSOMAL AND SECRETORYMEMBRANES IN ACINAR CELLS. ((S.F. Hamm-Alvarezl, J. Prietol, J.P.Gierowl, T. Yangl, A. Bekmnezianl, R.A. Walker2, and A.K. Mircheffl))tDepartments of Pharmaceutical Sciences, Physiology and Biophysics, and Celland Neurobiology, Univ. of Southem Califomia, Los Angeles CA 90033 and2Department of Biology, Virginia Tech, Blacksburg VA 24061.

We have examined the role of microtubule-dependent vesicle transport inmembrane trafficking in rabbit lacrimal gland acinar cells. Video microscopy ofisolated acinar cells reveals numerous small vesicles (.500 nm diameter) thatmove in a directed manner. The frequency of these vesicle movements is inhibitedby nocodazole to a level <5% of that seen in control cells. Membranes fromacinarcells fractionated over sorbitol density gradients followed by phase-partitioningyield membrane fractions derived from distinct membrane compartments. Theassociation of the microtubule-dependent motor, kinesin, with these membranecomparatents was examined by Westem blotting with kinesin monoclonalantibodies DK410-2.1 and DK410-4.1. Kinesin enrichment was detected inmembranes with high levels of a-glucosidase, an endoplasmic reticulum marker,and in membranes with high levels of J-hexosaminidase, an marker associatedwith lysosomal and trans-Golgi membranes and secretory vesicles. Significantassociation of kinesin with endosomalmembranes containing endocytosedhorseradish peroxidase and endogenous acid phosphatase and Na,K-ATPase was

also observed. The inhibition of vesicle transport by nocodazole and theenrichment of kinesin in endosomal and secretorymembranes suggests thatmicrotubule-dependent vesicle transport is a major component of membranetrafficking in the lacrimaa gland. Supported by NIH grants CA- 63387 and EY-05801.

Distribution of solublekinesin in rapidly frozen and substituted squidaxoplasm ((J. E. Moreira, and T. S. Reese)). Laboratory of Neurobiology,

Bethesda MD 20892

Kmnesm ,the putative motor for anterogade organelle movements

in squids axons, is thought to exist in vesicle-bound and soluble pools in the

squd giant axon. The functon of the free kinesins remains unclear. Thespatial distributon and loose associaton of the free and bound kinesins isan example of a difficult clas of problem for immunocytochemicallocalizatn since chemicalfixation would be expected toredisuibute the

ents He we presenta protocolofferingasolutiontothisproblem Freshy dissected squid axons were extuded and the axoplamwas immediately frozen on a cold copper block, freeze-substituted in 0.1 %

uranylaceet in acetone at-80°CC infilted with LowicrylK11 M at

-, C, and polymeried by UV, iniutiay at-70° C Electron micimmmunolabeing of thinsections with apolyclonal antibody directed againstthe headregion of squid kiinesi heavy chain followed by protein A-goldshowed gold grais on both organell es and cytosol while paralel

expermimnt with thepre inmune serum showed only scattered gold

particles. The antigens reacting with the heavy chain antibody arewidelydistributed in the cytoplasm but are concentrated (after correcting for thesmall bacgkgound) 62 fold around vesicles, 26 fold around ERcisternae, 12fold aound mirnotubules and 5 fold aound mitochondria. These results

suggest that the preset protocol is both sensitive and specific.Microtubules appear to bindkInesin, or related proteins. The majoity ofthe axopl asmic kinesin is on organelles, thoughap a fifthappears to free in axoplasm.

31a

Kinesin (181-186). Sunday181

ONE AXON: MANY MOTORS. ((V. Muresan, D. Winter, T. Abramson, andB.J. Schnapp)) Department of Cell Biology, Harvard Medical School, Boston,MA 02115. (Spon. by H. Green).

An affinity purified, polyclonal antibody to squid linesin heavy chain (KHC)motor domain blocks 45% of the plus-end directed vesicle transport in KI-extracted vesicle fractions from squid axoplasm, but < 10% of a highly purifiedplus-end vesicle fraction, suggesting that other motor proteins, in addition toKHC, might function as vesicle motors in squid axons. To determine whetherother kinesin-related proteins are present in squid axoplasm, we performedWestem blots using peptide antibodies to conserved epitopes of the kinesin motordomain (Sawin et al., 1992, J. Cell Sci., 101: 303). At least ten polypeptides,which are not proteolytic fragments of classical KHC, were labeled onimmunoblots of homogenized squid axoplasm. In addition to KHC, fivepolypeptides (200 kD, 92 kD, 78 kD, 36 kD, and 23 kD) which stained with thepeptide antibodies co-sedimented with and were released from microtubules in anucleotide-dependent manner. Only KHC and the 92 kD polypeptide were

present at significant concentration in purified plus-end vesicle-microtubulecomplexes. These results suggested that neurons might be a rich source of novelmotor proteins for cytoplasmic transport, and motivated us to screen a ratforebrain cDNA library with the peptide antibody. We isolated 130 cDNA clonesfrom this library screen. Partial sequences from a small subset of the clonesindicated - 80% were kinesin-related proteins, including KHC, KIF3A (Aizawaet al., 1992, J. Cell Biol., 119: 1287), and a novel kinesin-related protein,rbKRPl. Antibodies to a fusion protein made downstream of the motor domainof rbKRPl stain a single - 90 kD polypeptide on Western blots of squidaxoplasmic cytosol. Although preliminary, these results suggest that neurons,even a single axon, contain multiple kinesin-related proteins, and at least some ofthese are conserved between mammals and squid. These motors might beinvolved in fast or slow axonal transport

183

THE MITOSIS-SPECIFIC KINESIN-LIKE PROTEIN KLP61F OFDROSOPHILA MELANOGASTER IS AN ESSENTIAL COMPONENTOF THE MlTOTIC SPINDLE. (N.R. Bartonl, AJ. Pereira2, and L.S.B.Goldstein1, 1Howard Hughes Medical Institute, Div. of Cell. & Molec.Med., Dept. of Pharn., U.C. San Diego, La Jolla, Ca 92093-0683 and2Dept. of Cell & Develop. Biology, Harvard Univ., Cambridge, Ma.02138)

Classical genetic analysis has shown that the kinesin-like proteinKLP61F is essential for Drosophila development Mutations in theKLP61F gene result in the failure ofduplicated centrosoms to separate.This phenotypic behavior is similar to the results obtined from loss offunction analysis of other members of the bimC class of kinesin-likeproteins. We have begun the enzymatic and biochemical characteaization ofboth recombinant and native KLP61F and find that its moility andmicrotubule binding chtractristics appear to be most similar to those of thebimC family members EgS and KRP130,an apparent EgS homologue inDrosophila. In addition, we generated KLP61F-specific polyclonalantisera and perfonned in situ localization of KLP61F during the variousstages of mitosis in Drosophila syncitial blastoderm embryos. The resultsshow that KLP61F is not restricted to the centrosome but is founddistributed both at the spindle poles and along the length of the mitoicspindle fibers. These results indicate that KLP61F may have a functionbeyond its essential role in centrosome separation. Additionally, we usedthe KLP6lF-specific antisera to demonstrate that KLP61F and KRP130 are

immunologically distinct This result, in combination with the observationthat KLP61F activity is essential for development, confrms the existence ofmultiple non-redundant bimC family members in Drosophila.

185

KIF2 IS A NEW ANTEROGRADE MICROTUBULE BASED MOTOR WHICH

TRANSPORTS MEMBRANOUS ORGANELLES DISTINCT FROM THOSE CARRIED BY

KHC AND KIF3A/B Y. Noda, R. Soto-Yoshitake, S. Kondo, M.Nangaku, N. Hirokawa. Department of Anatoey and Cell Biology,Faculty of Medicine, University of Tokyo, Japan.

We have previously cloned five different sesbers of kinesin

superfamily proteins (KIFs) in the souse brain (JCB 119:1287-

1296, 1992) and demonstrated one of them, KIF3A as an anterogrademotor (JCB 125:1095-1107, 1994). We have now characterized

another unique axonal transport motor, KIF2 having its motor

domain in the center of the molecules. Recombinant KIF2 exists as

a dimer and moves microtubules plus end directedly at the

velocity of 0.47+0.11 us/sec, which is two thirds of kinesin's.

Immunological examination showed that native KIF2 is abundant in

developing axons and that it accumulated in the proximal regionof the ligated nerves after twenty hours' ligation. KIF2 exists

without light chain in soluble form. KIF2's associated vesicles

immunoprecipitated by anti-KIF2 antibody, are different from

those carried by existing motors such as kinesin and KIF3A. They

are also dintinct from synaptic vesicles although KIF2 is

accumulated in so-called synaptic vesicle fractions and embryonalgrowth cone particles. These results strongly suggested KIF2

function as a novel anterograde motor specialized for a certain

group of small vesicles in fast axonal transport.

182INTERACTON OF MOTOR PROTEINS WITH ANTEROGRADE ANDRETROGRADE VESICLE POPULATIONS FROM SQUID AXOPLASM.((V. Muresan, CM. Vines, T.S. Reese*, and BJ. Schnapp)) Department of CellBiology, Harvard Medical School, Boston, MA 02115 and *Laboratory ofNeurobiology, NIH, Bethesda, MD 20892.

There is good evidence that kinesin and cytoplasmic dynein are motors for,respectively, anterograde (i.e. toward microtubule plus-ends) and retrogradevesicle transport in the squid axon. We are interested in elucidating themechanisms which cause anterograde and retrograde vesicles to be transported in

the correct direction along microtubules, i.e. how do vesicles seleet the motorprotein with the correct polarity, given the large soluble pools of both plus-endand minus-end motors? We previously separated anterograde and retrogradevesicle populations by nucleotide-dependent binding Qf vesicles to microtubules(Godek & Schnapp, Mol. Biol. Cell, 3: 172a). Results from in vitro motilityassays led us to propose a model wherein 1) cytoplasmic dynein, but not kinesin,interacts with all vesicles via a nonspecific association with the lipid bilayer, and2) only organelles programmed to move in the anterograde direction interact withkinesin. We proposed that the kinesin-driven movement overcomes nonspecifi-cally bound dynein because kinesin has a shorter dwell time off the microtubule.To test this model, we have used Western blotting to determine the distribution ofkinesin and cytoplasmic dynein on anterograde and retrograde vesicles.Preliminary results support the model: kinesin co-purifies with the anterogradevesicles, not the retrograde vesicles, while both populations of vesicles co-purifywith cytoplasmic dynein after incubation with cytosol. As a further test of thismodel, we have made a function blocking antibody to the motor domain ofkinesin heavy chain, and we are asking whether purified anterograde vesicles,which normally move exclusively to the plus-ends of microtubules in the

presence of cytosol containing active cytoplasmic dynein, will reverse theirdirection if the vesicle-bound kinesin is inactivated with the antibody.

184

A ROMOTETRAMERIC KESIN-RELATED MOTORPROTEIN LSOLATED FROM DROSOPHILA EMBRYOS.((D.G. Cole*, W.M. Saxtona, KB. Sheehana, and J.M. Scholey*))Section Of Molecular and Cellular Biology, Univesity of CaliforniA,Davis, CA 95616. aD t of Bio Is,Isttute for Moecular andCellar Biology, Univeri, B nton, Indiana 47405

We have used pan-lknesinPeptide antibodies (Cole et al., J. CellSc., 101, 291-301 (1992); Sawn et ot., J. Cell Sd. 101, 303-313(1992)) to identify ad isolate kinedn-rdated proteins (KRPs) frmDrosophia _°lu8ogaster embyonic cyosol These KRPs cediinted

with tobule Ws) poymeried d hmesiedvwthAMPPNP,ad oem of them, X 1RP30, wu furtb urifed fom ATP eluas of the

MTs (Cole etal., J. Bid. Af

dust KRP13o hs a StoWkes radius of 16.2 mr ndra vaue

7.6S, we estmatd the radve olecular mas of KRP130 to be 490-kDa.

Beus no detctable found to copurify with dt 130-

kDa subunit, we co13ciuemat RP3O a ric comple

comosed of four 130-ka polypqde& sibunis KRPU3o displas aus-end di rcted mntr acivity of moving sing s at

0Q04:k i.01 pmhc. 130-kDa tod for reactivity with

In andRdyla ant s specific forvaious member of thekinesin Re indicae th the KRPL3O sbunit is rlated toXenopus EgS (Sawin et al., Naure 359, 540-543 (1992)), a nmmber ofthe binC subfamily of kinesins Therefore, KRP3 tobe the first

Drosophila ICRP, and the first member of the biC subfamily in anyorganis, tobe ied frm naie tisseas afuntonal mutmer motor

compx Stuctual s are in progres Supported by grants frotNIH, ACS, and AHA.

186KIF3B FORMS A HETERODIMER WITH KIF3A AND WORKS AS A NEWMICROTUBULE-BASED ANTEROGRADE MOTOR OF MMBRANE ORGANELE

TRANSPORT. ( (H. Yamazaki, T. Nakata, Y. Okada and N. Hirokawa))Department of Anatomy and Cell Biology, Faculty of Medicine,

University of Tokyo, Tokyo 113, Japan. (Spon. by T. Nagano)

We cloned a new member of murine brain kinesin super family,

KIF3B, and found that its sequence is highly homologous but

not identical to KIF3A. KIF3B is localized in various organtissues and developing neurons, and accumulates with

anterogradely moving membrane organelles after ligation of

nerve axons. Immunoprecipitation assay revealed that KIF3B

forms a complex with KIF3A and three other associated

polypeptides, named as the Kinesin superfamily Associated

Protein 3 (KAP3). In vitro reconstruction assay showed that

KIF3A and KIF3B directly bind with each other. The recombinant

KIF3A/B demonstrated plus-end-directed microtubule sliding

activity. Subcellular fractionation of brain homogenates showed

a large amount of the native KIF3 complex to be associated

with membrane fractions other than synaptic vesicles. In

addition, we found that the composition of IAP3 in the brain

is different in the soluble and membrane-bound forms, and

also differs between the brain and testis. Our findings

suggest that KIF3A/B is a unique heterodimric microtubule-based

anterograde translocator for m-mhranousorganelles. and that

MAP3 may determine membrane association and functional diversity

of KIF3 in vivo.

32a

Sunday. Kinesin (187-192)

167

KIPlA IS A UNIQUE IO IC KINSIN-LI MTOR THE FASTAXONAL TRANSPR. ((Y. Okada, H. Yamasaki, Y. Sekine and N.Hirokawa)) Department of Anatomy and C0ll Biology, School ofMedicine, University of Tokyo, Tokyo, 113, JAPAN.

We have cloned and sequenced the cDNA of KIF1A, a kinesinsuperfamily protein in murine brain. It was composed of 1695amino acids with N terminal motor domain, short a-helical stalkdomain and large tail domin at its C-terminal. No otherkinesin superfamily proteins already reported showedsignificant similarity in the tail domain, except nematodeunclO4. Unc104 showed 71.0% similarity throughout themolecule, suggesting that KIFlA is the murine homologue ofunc104. To elucidate the in vivo function of KIFlA, we have

purified and characterized recombinant KIFlA protein.Unexpectedly, KIFlA behaved as a monomer in a sucrose velocitygradient centrifugation and appeared as a globular protein bylow angle rotary-shadowing 3N. It showed a plus-end directedmicrotubule-dependent motor activity. Its velocity was

1.2,uml, twice as fast as conventional kinesin, and wellcoincided with the velocity of the anterograde vesicle movementin the axon. Furthermore, anti-KIPlA antibodies stained axons

and synaptic regions in vivo, and also stained intensively atthe proximal ends of nerves even after lhr ligation, suggestinstrongly that KIFlA is a new motor for anterograde fast axonaltransport.

189

MULMPLE KINESINLIK TRANSCRPM IN THE RETINAL PIGMENTEDEPrIiELUM ((LALUungerand BL Bunide)) Departmnt-ofMoleculsrand COUioloy,UnlverltyofCamcfnils, Berkey,C* 947M)

It is now appaet tht kines1n4ke protein (KLPs) cmprise a large mult-gnufamiy ofmotm poteis In Drowpd elee dfferent

have boen kdified, whers five KLPs have bee fund in he mouse bran andfour in Xeuopus oocyte. We have used a degenerate pim reverse

aiptaepona reation (PCR) cloning str to kIntfy andisolate putative kIndns family nmbers eprsd in etinal pmned

epihum (IM), feshy ioated hrm the sriped bs, Moroc Mxtl Sine

adult fish RIE conta a sngle posetitotic cel type, this approach wuld avoidmitotic or mulotic knesi sad Idntfy only toe _odated withd_ifentatedeptlulial functions, Sx classes of kiesn famly mnu bers P6)identified, and regions th motor domains we dond ad

sequenued, A homol search at theamno add lewd sugg that thefish counterpat of the k1zuin hevy chsin, whreas PLP2-6 appear to be nodspecaes Nortanblotanuydirvealed dtt flue ELPs w abundanly in

mural retina and intact retna-IF, but eqxesion leb were deWctble in theisoated RF only after long term expoe, suggsig a low abundance FEYnums in cells. PFLP showed different pattns of exprsion in a

varity of tssues examined, with the KHC homwoog PILFP-l being most wideyexprsed, whethe mot abundanmeniber, iaP-2, sowed irongexpron in

thetina and in bra rhus, we have ison that six diffesut ILPe can be

PCI amplfied from the poetdmtotic celb of the fish RPI We gget tht tuePICLPs may participate in known RPE transpot pross such vectal

and lyoom and re cy

pn rb NHgas EO id EY063994 .

191

OBSERVATION OF KNESIN MOTILrTY ON CHEMICALLY CROSS-LINKED MICROTUBULES. ((D.C. Tur, C. Chang, S.L Brandow, andD.B. Murphyt)) Center for Biomolecular Science and Engineering, Code6930, Naval Rcsearch labratoy, Washington, D.C 20375-5348, andtDeprtmnt of Cell Biology, Johns Hopkins University Medical School,Baltimore, M.D. 21205

Microtubules that have been chemically cross-linked were investigated as

substrates for dinesin motility. Atomic force microscopy was used toevaluate gross structral changes in the microtubules while kinesin beadstudies were used to evaluate activity. Microtubules were fonned in vitrofrom chicken brain tubulin and taxol stabilized. Following dilution to aworidng concentradon of 0.6 pM, the microtubules were chemically cross-

linked in a buffer containing 0.05% gluteraldehyde (5 mM) or 1.5 tM EGS(ethylene glycolbis[succinimidylsuccinate]) for 60 seconds. The reactionwas quenched with addition of TRIS buffer and the treated microtubuleswere then immobilized on DEAE-dextran coated coverslips. AFM ofthese microtubules indicate that both cross-linking treatments are

sufficient to stabilize the gross strucure of microtubules during air-drying.

They also remained stable after a challenge with distilled water. In thepresence of ATP, movement of kinesin coated beads was observed on theboth sets of cross-linked microtubules. The rate of movement along thesecross-linked microtubules was at least 50% of the bead velocity observedon untreated, native microtubules. Applications of this work may include

multiple use substrates for kinesin motility studies and rugged microtubulesubsttsfor affinity chromatography of kinesin. Supported by the Officeof Naval Research.

eg8KIF1B: A NEW MONOMERIC ANTEROGRADE MOTOR FORMITOCHONDRIAL TRANSPORT. ((M. Nangaku, R. Sato-Yoshitake,Y. Okada, Y. Noda, R. Takemura, H. Yamazaki, and N. Hirokawa))Departnent of Cell Bioogy, School of Medicine, University of Tokyo,Tokyo, 113. (Spon. by T. Saito)

In order to understand the mechanism of organel;e transports in

the cell, we cloned, sequenced, and characterized a new kinesin-

related motor protein, KIFI B. KIFl B works as a monomer, having a

microtubule plus-end directed motility. lmmunocytochemically, KIF1B

was colocalized with mitochondria in vivo. Furthermore, a subcellular

fractionation study showed that KIFlB was concentrated in the

mitochondrial fracton, and purified KIF1B could bansport mitchondria

along microtubules in vitra These data strongly suggested that

KIFIB works as a unique monomeric motor for anterograde

mHtochondrial transport.

1iKINESIN STRUCTURE, MICROTUBULE STRUCTURE, ANDKINESIN-MICROTUBULE INTERACTIONS. ((Y. H. Song,M. Thormihien, A. Marx, E.-M. Mandelkow, E. Mandelkow))Max-Planck-Unit for Struct. Molec. Biol., Hamburg, Germany.

Microtubules can be decorated with kinesin heads periodically and with astoichiometry of one head per tubulin dimer, with kinesin binding tobeta-tubulin (Song & Mandelkow, PNAS 90:1671, 1993). This interactioncan be used for studying the stucture of tubulin-bound kinesin by imagereconstruction; however, it can also be used to highlight aspects ofmicrotubule structure that were hitherto not visible because of the lowcontrast between alpha and beta tubulin. The image reconstruction showsthat kinesin is bound asymmetrically: If one views a microtubule with theplus end up and from the outside, the ldnesin head is on the right sideof a protofilament, reaching over to the next protofilament. Depending onthe cutoff representation the shape is reminiscent of a pear or aboomerang. The specificity of interaction provides a basis forinvestigating the subunit arrangement and surface lattice ofmicrotubules, especially of flagellar outer doublet and central pairmicrotubules, both of which have a OBO-lattice which implies somediscontinuity in the wall. One unexpected finding concerns the polarity:Microtubule plus and minus ends can be distinguished by kinesin labeling.This shows that the plus end has a crown of alpha tubulin, the minus endhas a crown of beta tubulin. This is consistent with the idea that betatubulin interacts with gamma tubulin at the microtubule organizing center(Oaldey, TICB 2:1, 1992). Since the exchangeable GTP is located atthe plus end (Mitchison, Science 261:1044, 1993) this means that even theterminal beta-tubulin-GTP is already buried within the microtubule,rAthern an stickdng out at the end. - Supported by DFG.

192A NEW KINESIN-LIKE PROTEIN (IKpl) LOCALIZED TO A SINGLEMICROTUBULE OF THE CHLAMYDOMONAS FLAGELLUM. ((M. Ber-stein, P.L. Beech, and J.L. Rosenbaum)) Department of Biology, Yale University, NewHaven, CT 06520.

We have begun studies on motor proteins that could be responsible for dynein-independent motilities of Chlamydomones flagella. Using RT- PCR, we isolated partialclones for five kinesin-like proteins (klps) from Chiampdomonas. The mRNAs for threeof these ldps are upregulated during fiagelar regeneration, indicating that they encodeflagilar ldpe. One full lngth cDNA, KLP1, has been cloned and sequenced KLPIencodes an 83 kDa protein that contains an anino-terminal motor domain which isapproximately 40% identical to the motor domains of other klpe. The C-terminal por-tion of the Klpl protein is unique. A Klpi specific antibody was raised and used as

a probe for KIpl in fractions of isolated flagella and in situ. Immunofluorescence ofwhole cells indicated that Klpl was present in both the flagella and cell body. In wildtype flagella, Klpl was bound tightly to the axoneme; immunogold labeling of wildtype axonemal whole mounts showed that KIpi was restrkted to one of the two centralpair microtubules at the core of the axoneme. Klpl failed to ocalize to the flagellaof mutants ladcng central pair microtubules, but was present in mutant flagella frompfl6 cells, which contain an unstable Cl microtubulk, indicating that Klpl was boundto the C2 central pair microtubuk. Localization of Klpi to the C2 microtubule was

confirmed by immunogold labeling of negatively stained and thin sectioned axonemes.

These finding suggest that Klpl may play a role in rotation or twisting of the cen-

tral pair microtubules. We have also detected KlpI immunoreactive proteins in ciliafrom other non-vertebrates and in mouse teste. Currently, we are asying the in vitromotility and biochemical properties of KIpi protein that has been expressed in bacteriaor that has been salt-extracted from axoneme.

33a

Kinesin (193-197). Sunday

193THE ISOMETRIC FORCE OF A SINGLE KINESIN MOLECULE((Chris. M. Coppin*, Jeff T. Finer#, Jim A. Spudich#, and Ronald D. Vale*))Departments of of *Pharmacology and Biochemistry, University of California,San Francisco, CA 94143, The Marine Biological Laboratory, Woods Hole, MA02543 #Departments of Biochemistry and Developmental Biology, BeckmanCenter, Stanford University School of Medicine, Stanford, CA 94305.

The displacements and isometric forces produced by a single kinesin moleculetranslocating along a stationary axoneme were measured using the optical trapfeedback apparatus of Finer et. aL (Finer, J. T. et aL Nature 368: 113-119,1994). As a kinesin-bound bead moved up the force gradient of the optical trap,it usually exhibited discrete displacements of approximately 8 nm, similar tothose described by Svoboda et aL (Svoboda, K. et aL Nature 365: 721-727,1993). Frequently, the kinesin could move the bead against forces of about 3-5pN before it released from the microtubule and returned to the trap center. Thesteps were separated by pauses ranging in duration from a few milliseconds tomore than two seconds. A statistical analysis of all the observed pauses is underway. When the position of the bead was clamped by feedback control of theoptical trap's position, kinesin consistently achieved somewhat greater maximalforces than during translocation of the bead. Unlike myosin, which exhibitssingle force transients, kinesin reached its maximal force in a discontinuousfashion. At 20 uM ATP, the maximal force was often sustained for long periodslasting up to several seconds, but the force transient was sometimes abruptlyterminated much sooner. This apparatus is currently being used to furtherdefine the characteristics of kinesin's force-generating and elastic components.

195

ncd MOTOR DOMAIN ATPase. ((T. Shimizul 2, E. Sablin3, R.Fletterick3, R. Vale1.3, and E. Taylor1'4)) 'Marine Biol. Lab., WoodsHole, MA 02543, 2Nati. Inst. Biosci. Human-Tech., Ibaraki, Japan, 3UnivCalif. San Francisco, CA 94143, and 4Univ. Chicago, 60637 IL

ncd is a kinesin-related protein from Drosophila that moves in theopposite direction along microtubules to kinesin. To learn more aboutthe nod mechanism, nod motor domain (R335-K700) was expressed in

E. coli and its enzymatic characteristics were studied. The purified nodmotor domain exhibited unusual UV spectrum with a broad peakaround 272-275 nm. Upon incubation with radioactive ATP, 3H atadenine but not 32P at i-phosphate was incorporated in the protein,indicating it bound probably ADP but not ATP. Thus, like kinesin,nucleotide binding to ncd motor domain is tight, aithough there is anequilibrium between the protein and fee nucleotide. A fluorescent ATPanalogue, mantATP, was also incorporated in the protein, probably asmantADP. Concomitantly, the fluorescence intensity increased at 0.005s-1, which probably reflects the release of ADP from the protein. Uke-wise, the fluorescent intensity of the nod motor domain having boundmantADP decreased upon mixng with ATP; the rate was acceleratedmore than 1000 fold to 3 s-1 by the presence of excess microtubules.The profile of the rate vs microtubule concentration was almost super-imposable with that of mantATPase vs microtubule concentration.These characteristics are very similar to those of kinesin.

194TE INESIN HEAD DOMAIN 1401 IS A DM R. ((JJ.

Carreiae, S.P. Gilxet$, M.L. Mot nd .A. JOhen) *Dopt. ofBiochemistry, Univ. of .M. Medical Ceter, Jacko, S. 39216 andtDept. ofdMolcu and Cel Bidoa, Pm. State Univ., UniversityPY}, PA. 16802.

The bacterial expressed had domain of ,kinein K401, binds and

hydrolymes ATP and p r micrtabue motility. Theinterpretation of m ot niengi and ATP hydrol-d rate

kAowledge of the state of asembly of the head domain. It

ha routindy been tat head doman are momorn ad thatthe tnia mediated by the stk rgion. We have directlymeasudthe stakte of assembly of the lhd domai costrt

K401 by tionveloty and eqili methods i a OptimaXLA anlytical ultracentrifug. Betwe 1 and 20 M 1K401 MW45,079, is predminantly a di wit a sedm tatioaeetS20,w, of 4.9 s. M ar ht msmen establish thediaso&iation constant for imesation at 0.1 pM, butfitting reqIie the inlsdi a hi

-arm The extent of

oafATP ADP, or AMPPNP. The saalle cstrut 131,MW 37,825, is a monomer (maured MW = 37,795 * 726) with a

cOiaeet, S20,,, Of 2.9 s. Thus, the dimerisatondomai is either between amino acid resdue 342 and 401, or stroyaffected by the removal of thi reon Thee reuts have impotant

implcationa the of ATP depet motity aa.[Supported by GM41117 (J.J.C.), BIRE-216150 (JJ.C.) and GM26726

196KINESIN DISSOCIATION FROM THE MICROTUBULE OCCURSAFIER ATP HYDROLYSIS. ((S.P. Gilbert', M.R. Webbt, M. Brunet, &K.A. Johnson*)) Department of Biochemistry & Molecular Biology,Pennsylvania State University, University Park, PA 16802 USA andNational Institute for Medical Research, Mill Hill, London, UKt.

quench flow and stopped flow medtods have been used to define aminimal mechanism for the microtubule-kinesin ATPase using theDrosophila kinesin motor domain (K401) expressed in Escherichia coli(Gilbert & Johnson, 1993, Biochemistry 32, 4677; Bioch=eistry, 1994,33, 1951). The results indicate that ATP binds the M'K complex rapidly(I., = 2 FM-IS-s, k., = 200 s-' ) but weakly with an apparentK = -100

IAM. ATP hydrolysis occurs at the active site at 100 s-I which is ntlyfaster than steady sate turnover at 20 s-1, showing that the rate limiting stepoccurs after ATP hydrolysis. K401 dissociation from the MoK complexwas measured direcdy and at saturating ATP concentrations is 13 s-1,documenting that kinesin crossbridge detachment from the microtubuleoccurs after ATP hydrolysis. Measurements of Pi release during the firstturover determined the rate constant to be 13 s-1. The kinetics of kinesindissociation and Pi release can be explained by a sequential mechanism inwhich the first step is slow and rate limiting followed by the fast secondstep. K'ADP binding to the microtubule is microtubule concentrationdependent (20 pM-is-1) and followed by fast ADP product release. These

results reveal that kinesin in contrast to myosin spends most of it ATPasecycle bound to the microtubule, and the mechanism de ed accounts forthe ability of a single kinesin molecule to promote translocation along amiotubule atmaximmnvelocity. Supported by NH GM 26726 to KAJ.

1973 DIMENSIONAL STRUCTURE OF THE KINESIN HEAD-MICROTUBULECOMPLEX BY CRYOELECTRON MICROSCOPY((M. Wkkewa", T.Ishkawat, M. Alsuot, T. Nakat, Y. Fujlyoshit, T.Wakabayashilt and N.Hirokawa*))*Departnent of Anatomy and Cell Blogy, School of Medicineand tDeparment of Physics, Schol of Sie,nce,Unhverskty of Tokyo. 7-3-1Hongo Bunkyo-ku Tokyo 113 Japan *Protein Engneering ResearchInsitue Osaka Japan

Kinesin is an ATP-drlven meter on a mrebule (MT), which may take partInthe organella Iasperb in cells. Its motor domain structure, and how It bindsto mikrotubules ar hiporbat informatlos to know the mechanism ofmechno-ebemlcal coupling MNctbues consist otfa- and 0-tub_nbeteredimers fbming mainly 13 wr 14 proto_flaments From dectromicroscopc and X-ry fiber diffaton studies, the tuun monmers pess12-nm helical pitch and a 4-nm logitdinal pitch along a proto In

term of the arrangement of beterodime In a mlrotubue, recentt udes udingspefc binin of a kisnsi head to a tn al revesied the helicklptch tobe 12 nm, althongh having a 8-nm lnitu pitch. Ti dirncy suW

a discontinuity should exsts. Here we studied the arrangementofh edimeina mkcotul by exmini the srfame strctur of kinei head decorated-mkcrotuls ing qulck-freeze,deep-etchelectren miroscopy, then Dund thatthe ihecl discotnutied are aligned parale to the protofilaments, formlng a

"seam ins.' In addition, we alsoeond two adjolni seam lies, caleod 'dobiesosm Mues,' previously clsieled a mixed lttice stcture vice as a B tice

on. Thn we performed threedimnsol cto of a recombina_tkinesin head-mlcrotuhbucmplex cryo-eiectrn microwcopy md Imag

analysi bad on thi microtubule modeL revealed the feture of inesin

hed a the head bind on the ridge of mkrotubmi, polnting outside by the tallike structure.

34a

Sunday. Cilia and Flagella (198-203)198

MICROTUBULE SLIDING IN CILIA DURING AVOIDANCERESPONSES OF CEIL ((M.E.J. Holwill)) Department ofPhysics, Kings College, London WC2R 2LS, U.K. (Spon. by P.Satir)

Avoidance responses observed in many ciiates and flagellatesare a direct result of changed beat patterns in the propulsive ciliaor flagella. The changes can involve a reorientation of theorganelle, modifications to the bend symmetry or shape, reversalof the direction of bend propagation, transient cessation ofmotion or a combination of these. The responses indicate that thecell is able to control the beat patterns of its propulsiveorganelles. From analysis of the bend shapes, the patterns ofmicrotubule sliding within a cilium can be determined. In cellssuch as Crithidia oncopelti, which exhibit wave reversal, thedirections of relative microtubule sliding in the axoneme reverseduring the avoidance response. Since active microtubule slidingis probably unidirectional, wave reversal could be achieved byswitching activity from the set of doublets on one side of theaxoneme to the diametrically-opposed set; such a mechanismrepresents a development of the switch-point hypothesis proposedby Satir (1985, Mod. Cell Biol. 4:1) for normal ciliarymovement. For organisms such as Phytophthora zoospores,where one flagellum stops beating during cell reorientation,sliding in both halves of the axoneme is switched offsimultaneously. A mechanism which switches sliding, withappropriate phasing, among microtubule groups can explain otherpatterns of avoidance response.

200

INHIBITION OF MOTILE, MECHANOSENSITIVE CILIA USINGCo2+. ((B.T.Lowe, S.L.Tamm))Boston Univ.Harine ProgramWoods Hole, MA 02543We are investigating how motile, pacemaker cilia ofthe ctenophore statocyst are excited or inhibited bymechanical deflection of the statolith depending onthe sign of geotaxis. Using a horizontal microscopewith a vertically rotating stage, we measured beatfrequencies (BF) of upper and lower cilia ofdissected adult statocysts and intact larva. The BFwere reversibly decreased in 79% of adult statocystsand 85% of larva using 5-20mM Co2+. The BF of excitedvs inhibited cilia (regardless of geotactic sign) were

24:3, 2:2, 30:2 Hz for seawater (SW), Co2+, and SW,respectively. Using micromanipulation,isolatedclusters of cells bearing groups of cohesive pacemakercilia show directional mechanosensitivity. Differentgroups of cilia exhibited different signs ofdirectional sensitivity. In 10-20mM Co2+, the ciliaresponse to deflection was reversibly decreased. TheBF of excitation vs inhibition were 8:0, 0:0, and 4:0Hz for SW, Co2+, and SW ,respectively. Data indicatethat the cellular mechanism for directionalsensitivity may not require continuous input from thenerve-net. The inhibitory effect of Co2+, a calciumchannel blocker, indicates that Ca2+ channels may beinvolved in the mechanotransduction process of thesedirectionally sensitive cilia.

202

REGULATION OF FLAGELLAR LENGTH IN CHL4MYDOMONAS. ((J.Lappin and W.L. Dentler)) Department of Physiology and Cell Biology,University of Kansas, Lawrence, KS 6045.

To study signals that regulate flagellar microtubule (MT) assembly, we areusing insertional mutagenesis to generate Chianydomonas mutants that cannotassemble flagellar MTs and are examining existing mutants that cannot maintainnormal flagellar length. In synchronized cultures, one of the latter mutants,pfl8,grws normal lOpm long flagela that gradually shorten to 2-5nm within 8 hrs.Wild type (WI = cc124) cells cultured identically maintain 10-12jm longflagella throughout the day. Flagellar length inpfl8 is regulated by a clock. ifpfl8 is deflagellated at various times during the day, flagella regenerate to theshort lengths found on control (shortening)pfl8 cells. Althoughpfl8 cells havea small pool of flagellarprotein and canmot regenerate flagella in the pfesence ofcycloheximide (CX), flagellar shortening cannot be due to a depletion of a'critical protein pool": if 25 mM LiCI is added to cells with fully grown or

shortening flagella, MT disassembly is arrested, and some flagella grow anadditional 1-2pm even in the presence of CX. LiC does not alter the cell cycle-dependent shortening, since flagella rapidly shorten to the control (short) lengthsafter LiCl removal. The LiQeffect is specific: 25mM NaCIor KCI do notinduce flagellar growth. The target for LiCI is not known but it may involvecAMP-dependent signal transduction. 1 mM IBMX (with or without db-cAMP)induces flagellar shortening in WI' Chlamydonmonas cells and accelerates thenormal shortening seen withpfl8 ceUs. Flagelar shortening induced by db-cAMEP and IBMX occurs at a slower rate inpfl8 than in Wr cells. LiCl partiallyattenuates IBMX-induced shortening. Both IBMX- and normal pfl8 flagelarshortening are inhibited by H-8, an inhibitor of cAMP-stimulated protein kinaseA activity. These results support a role for cell cycle-dependent signaltrasduction pathways in flagelar MT growth control. Sudies of flagelarmutants that cannot assemble or maintain specific MTs may reveal defects in

this signaling process as well as in structunal defects in MTs and MT-associatedproteins.

1BS

CHARACTERIZATION OF A pl70 ANTIGEN ON THE SURFACE OF THETIPS OF THE SOMATIC CILIA. ((RF. Ali, M.S. Aihara, R.D. Allen and A.K.Fok)), Pacific Biomedical Research Center and Department of Microbiology,University of Hawaii, Honolulu, HI 96822. (Spon. R.G. Kleinfeld).

On the surface of Paranecium the somatic cilia are responsible for motilitywhile the oral cilia play a role in phagosome formation. We have recentlyobtained a mAb to a hydrophilic p170. This protein was located on theextracellular surface as it was readily labeled in live and in fixed butnonpermeabilized cells. It was located on the surface of the tips of the somatic butnot the oral cilia. In some cells the labeled tips formed a bright halo around theunlabeled cell body and in others, only the cilia tips on one side of the cells or thetips of the cilia at the posterior and anterior ends were labeled. In frozen thinsections this antigen wvas located at the very distal end of the somatic cilia and nooral cilia was labeled. In differential centrifugation, this antigen was found in thelysosomal and microsomal pellets. When live cells were incubated at 120 C tolower the rate of protein synthesis, this antigen appeared to be sloughed off, as a210 kD protein that reacted with the mAb was recovered from the spent culturemedium. This antigen did not appear to be an immobilization antigen whose M,

ranges from 250 to 300 kD and was also distinct from the p190 resident plasmamembrane antigen previously reported (Allen et al., J. Cell Sci. 101:449, 1992).The function of this antigen is not known at this time, but it may be involved insome sensing function in this ciliate. (Supported by NSF, MARC and MBRSgrants.)

201

SIMULTANEOUS MEASUREMENT OF CILIARY ACIIVITY ANDINTRACELLULAR CALCIUM BY HIGH-SPEED VIDEO MICROSCOPY((Michael J. Sanderson and Elen R. Dirksen)) Dept of Physiol. Univ. of Mass.Med. Center, Worcester, MA 01655 and Dept. Anat. & CeUl Biol. UCLA Sch. ofMed., University of California, LA, CA, 90024-1763

An understanding of the regulation of ciliary activity requires a correlation ofchanges in ciliary beat frequency with changes in intracellular messengers.Quandfication of ciliary activity is achieved by the analysis of the variation in thelight intensity of a microscope image of chiary movement. Becse the fame rateof NTSC video is inadequate to ch rize the cillary beat cycle, we havedeveloped a high-speed video technique that utilizes an inexpensive CCD camerathat has been modified to record up to 240 images per sec. This is achieved byreseeting the vertical synchronization multiple times during a normal video field.Although image size is reduced with increased frame rate, the video signalproduced is compatible with conventional recording devices. Data is extracted fromthe recorded video images by digital processing and this allows for thesimultaneous analysis of multiple cells and for the analysis of ciliary metachrony.Simultaneous measremts of [Ca2+1i are achieved by incorporating the videocamera into the optical train of a fluorescence microscope. By manipulating thelight wavelengths used for fura-2 excitation and phase'contrast illumination, therespective microscope images can be directed to the appropriate video camera withthe aid of a specific dichroic mirror. The use of this CCD camera with an imageintensifier also offers the prospect of high-speed Ca2+ imaging.Supported by NIH HL 49288 (MJS), the Smokeless Tobacco Research Council.,Inc, and the Tobacco Related Disease Research Program of UC (ERD).

203

STRONTIUM CAN SUBSTITUTE FOR CALCIUM IN THE CHLAMYDOMAONASREINHARDTII FLAGELLAR REGENERATION SYSTEM ((J.H.Evans J.LnSmith, andLR.Kelr)) Depaqtmt dBIogkcl Sclense The Flordd State Unlvrsty,Talahassee, FL32308.

When bfegelate green alga re cis stimuiated by pHshock, Quwamoby ata. have ound a + h/x routed ho dcwnels located hi

the ceO body. This Ca2+ hluxdrects excision of flagellafrom the cellbody and ladsthe coordnthe expression of gene enodIg flagear proteins. Outgrowth of new

flagela is measuable wIthin mIn after excision, and is cmplee hi -2 hours. Theprocesses of flagelar excision gene Idxction, and ougroh are Ighty coupledtemraly.butmay be unc ld by hiterri wIh the C bmn dtecell,suggetn that fselar excision, fgellar hiduction, and flagellar outgrowth are

eslNta_y separable processesthat areld by a common requdrwrnt forCab. To hIsestga uherh eCa sorqeen of thepH shock pon, we

need a msa by which to qumy the Ca-

hti after slon. C2Ca'+hasbeenused to atudyCa' hikux laoing the cIsion response, but isnot feasible for our usehi sudying the gene Indudon response to pH shock for a variety of reasons. Wehave been unucessftl in our atempts to Ca2+ hdcator dyes as wel. We

report here tatSr doll the same repon from cel atrpH shock as doesCa2+ md at no other metal ion we tested1aellceda smkarcellar sponse. Sr2+requIes a higher extrsellular contralton than does Ca+ for a full flagellarexcision repo efolown pH shock but the gene hxduxton respone I

disthigulshable from produced by C&2+ and flagarou hi Sr2+sallrto hi CaN- Our aim is to dermine t feasblt of using Sr2+ as a

of in ourfuture sdudIs, (Work su by NSF grart 90208 to

LRK)

35a

Cilia and Flagella (204-209). Sunday

204

CALCIUM INFLUX AND FLAGELLAR GENE INDUCTION IN THE CHLAMVDOMONASREINHARDTlI FLAGELLAR REGENERATION SYSTEM. ((J.H.Evana and L.R.Kelber))Departmt of BIologicScbnncs TheFblod Slate University, Talaaswee, FL 32306

Stimulation of the bflagellate green alga Chlsnydomonas rniif by pH shockproduces s that drect aer excision from the cell body, the coordinateInduction of genes encodingfibgp proteins, and flagelar outgrowth. The procsesof flagellar excison, flagsllr gene induction, and flagelar outgrowth are tightly coupledtemsporlly, wIh induction and otrwth beginning soon after excsIon. We haverported flagelar exlsion, flagel gee e inductIon, and flagelar outgrowth are

rentaly sparable processes, lnked by common Ca2- requireoent, and thatflagearexcialon and outgrowth are not required forthe gene inductIon response. Itre,we report furtr studies on roled Ca2+ in the induction response to pH shock.Wlld-type cell pH shodwd Ina low Ca2+medium (<IOIM Ca24 fall to excise theirflagela and exhibi a gene inductionresponse of lower magniude and different kicnetcsfrom that of cells shoccd Ina high Ca2qmeidum. In the mutant fe-i, which does notexcisefaglla In resporne to pH shock, gene induction resnse are aimilar to

t thseobsrved Inwhi ype when ta-i is shocsd in lowand high C media. Faellarexcislon, therefore, Is not required forthe high typeinductin response. BecauseQuambyd aL have reportedthat aninflu of extacllarCa routedthroughLa3+senstve channelsoccurs folowing pH shock, we pH shocked cells ina hghCa2+medium withLa3+ and found a low

tp inductionresponse. AddingLa3s at times after pH shockresut a graded, highCa2+ typeinduction responseaithowugh the ces have excised flagela tothe same extent, suggestng that themagnitude ofthe highCa2+ typeinduction response Is indpendent of the extent offlagellar excsion but is dependent onCa+ Infhlw Cells shocked in high Ca2+ and in thepresenoe of Cd2+ excisetheir flagela andinduce their genes normally,but do notexhibit flagellaroutgrowth, suggestingthat outgrowth plays no or a small role Inthe highCa2+hductionresponse. We suggest the excision, induction,and outgmwthrespone to pH shockare medited bydistnt pathways which sharea common Ca2+influx. (This work supported by NSFgrant 9020856 to LRK)

206

MOLECULAR GENETIC ANALYSIS OF CHLAMYDOMONAS REINHMRD71IFLAGELLAR COMPONENTS BY INSERTIONAL MUTAGENESIS. ((A.Koutoulis, GJ. Pazour, C.G. Wilkerson, and G. B. Witman)) Worcester Foundationfor Experimental Biology, Shrewsbury, MA 01545.

Non-homologousinsertional mutagenesis frequently results in the tagging of thedisrupted gene by the inserted DNA, so that the mutated gene can be readily cloned.We transformed a nitl mutant of C. reinhardtl with a plasmid containing the nitratereductase gene(AMT) attached to the pUC1 19 vector, and then screened the resulting7TI transformants for a slow swimming phenotype, which is characteritic ofmutants with defects in the outer dynein arm (termed oda mutants). From 2978colonies screened, 24independently isolated cell lines with reduced swimming speedswere obtained. Southern blot analysis using cloned DNA encoding the a, andydynein heavy chains (DHCs) and the 69 and 78 kDa (IC78) intermediate chains ofouter arm dynein revealed that the gene forIC78 was disrupted in8 of these celllines, andthat the gene encoding the P DHC was disrupted in two others. Electronmicroscopy of the other 14 mutants revealed that 3 completely lack outer arms whilean additional 4 lack some butnot alloftheouterarms. Inonemutantthedyneinarms appear normal but the central microtubulesare twisted around each other alongthe axoneme. The remaining 6 mutants are still being analyzed. The motility defectof at least one of the oda mutants co-segregates with the NIT1 phenotype and theinserted DNA, suggesting that the defective gene is tagged. Inasmuch as theSouthern blot analysis did not detect a disruption of any of the 5 cloned dynein genes

in this mutant, wecrossed the mutant to those previously known oda mutantsrepresenting genes that have not yet been doned, and tested for complementation instable diploids. The new oda strain complemented all of the tested strains except foroda3, suggesting that it has a disruption in the oda3 gene. We are in the process ofcloning the defective gene using the inserted pUC1 19 sequence as a tag.

208

MOLECULAR DISSECTION OF THE CENTRAL APPARATUS INCHLAAYDOMONAS FLAGELLA. ((Elizabeth F. Smith and Paul A.Lefebvre)) Department of Genetics and Cell Biology. University ofMinnesota St. Paul, MN 55108.

Genetic, structural, biochemical and functional studies of flagellar motilityhave indicated that the central apparatus may regulate flagellar beating.However, characterization of specific central apparatus components has beenlimited. To begin charcterizing these components, we used transformation as

a gene disruption technique to obtain central apparatus mutants. Nit- cellswere trnsformed with the wild type Chlamydomonas nitrate reductue geneand selcted as colonies on nitrate media. Central appratus defective mutantswere identified by immotility and electron microscopy. Among the centralapparatus mutants obtained, two represnt previously identified loci. Usingdiploid complementation tests we determined that a mutant lacking theentire central apparatus (AIO) is an allele of pf20, and a mutant lacking theCl tubule, is an allele of pfl6 (D2). Using dikaryon rescue and reversionanalysis techniques, Dutcher et al. (1984), determined that the gene product

of pf16 was a S7kd axonemal structural protein. A genomic sequence

fragment flnking the insertion site was cloned into pUCI19, and then usedas a hybridization probe to screen a genomic library. Overlapping clones

averaging 15kb in length were obtained which, when transformed into D2and pfl6 cells, complemented their structural and motility defects. Thesmallest subclone which complemented the mutant upon transformation was

4kb. We used this subclone as a probe on northern blots of total RNA fromwild type cells. The probe recognized a 2kb transcript consistent with a

flagellar structural protein of S7kd. This probe was then used to screen a

cDNA library from which we obtained a 2kb clone. We are currentlysequencing this clone to begin analyzing possible functions of the pf16 geneproduct.

205

IDENTIFICATION OF AN HSP70 PROTEIN IN THE EUKARYOTICFLAGELLUM ((Karl A. Johnson and Melissa A. Bloch)) Department of Biology,HaverfordCollege, Haverford PA 19041.

Heat Shock Proteins (HSPs), originally identified as proteins universally expressed in

response to cellular stress, also function in protein folding, targeting and assembly.As part of an investigation into the mechanism(s) of flagellar assembly in the green

alga Chlamydomonas we became interested in whether HSPs might play a role in

chaperoning this process. Using two different monoclonal antibodies (clones 3a3 and

7.10) that recognize different, but highly conserved, epitopes shared by HSP70 fam-

ily members from evolutionarily diverse organisms, a prominent70kDa polypeptideis detected on immunoblots of total Chlamydomonas cell protein. The amount ofthis protein increased two-fold following beat shock at40°C, demonstrating that themonoclonals indeed recognize Chlamydomonas HSP70(s). Interestingly, these mono-

clonals cross-reacted with a 70 kDa polypeptide on immunoblots of Chlaomydomonsflagellar protein. Treatment of flagella with the nonionic detergent NP-40 releasedroughly 40% of this flagellar HSP70 with the detergent-soluble membrane and matrix

protein fraction, while the remaining 60% was tightly associated with the detergent-insoluble axonemes. A biochemical characteristic of HSP70 family members is release

from macromolecular complexes in the presence of ATP. Treatment of axonemes with

10mM ATP released a significant fraction of the previously axonemal-bound flagellarHSP70. This release was specific for ATP and did not occur with either10mM AMP or

10mM AMP-PNP, a non-hydrolyzable ATP analog. These data showimmunologicallyand biochemically for the first time that an HSP70 family member is present in the

Chlamydomonas flagellum; a flagellar HSP70 may play a role in targeting axonemal

proteins into the flagellar compartment and in chaperoning the process of axonemalassembly.

207

ISOLATION AND CLARCIERZATION OFABNORMALFLAGEILAR-NUMBER MUTANTS IN IAMYDOMONAS.((W.-C. Wu, L.-W. Tam, and C. Silflow)) Dept. of Genetics and Cell Biology,Univ. of Minnesota, St Paul, MN 55108.

Wild-tpe cells in the green alga Chlanydomnasreind w n/have two

flagella. Two mutants with abefrant flagellar number wer generated byinserdonal mutagenesis and desigated 12A10 and 31E3. Aplasmidcontaining the nitrae reductase strutual gene (NIlT) wastransfomedinto nitlP cels andNirtransmants were screened for an abnormal

numberof flagella. For both mutants, half of the cellslack flagella and 20-30% of the cells have only one flagellum. Biflagellae cls in thepopulation show an abnormal tumbling motility. The cosegregation of the

flagellar-numberpocype with theNW phenotype idat that thedefects maybecausedbyplasmid integra Ajunction fragment adjacentto the planid intgrto site in12A10 DNA was cloned and used to

scen a lambda genomiclibry. Nlne ovelappig ckxnes wer obtand

which covered aregion of 26kb.Ibeplas idinseon events caused a

deletionwithinthis genomic regionin each of the two mutants. Whentran into muta cells, each of the ne clones conplented thedects in 31E3 but only parally comemented the12A10phenotype;normal flagdlarnumber was rcoverd but not the wild-type swimmingpattern. Theresults suggest thata gene involvedin the control of flagellrnumber is located in a 6kb region common tothese clones. We curntdyare usingfrgments from this 6kb region to screen for cDNAs deivedfrom this region. RFLP analysis was used to map the mutations to linkagegroup IX.

209

ISOLATION AND CHARACTERIZATION OF CHLAMYDOMONASRElNHARDTll FLAGELALES MUTANTS. ((C D. Amundsen and P. A.

Lfebvre))D t of Geneics andCdlBiCeolo Uoiverity ofMNnesota,St. Paul, MN 55108.

We have used insertonal mutagenesis to generate twenty four flagellalesstins. The mutaons were generated by sor a plasmidcon gthe nie reductae (NlT1)pge intoa Ch kneyomaas stain with a detonin NITl. Transformants wae screened for the absence of flagella Whenwildype ametes induced to shedidrcal wals, each c wal canbe

to have a pair of suct collars) through which the flagella

extend. The suctu the cell wllsoftreeofourntnt

stains. Theothcr twenty onetris have have obvous flagellar colas ontheir cell walls. We are perfo ng; n oe nC ments usingan

anti-acetylated--tubulin n l antibody (Piperno, G., andM T. Fulle,1985, J. Cell Biol, 101:2085-2094) to check for abnomal roodet micr tubuleassembly. We are each flglless stain toa swimming strain to

check for tght linkage betwee the fgelals phenotype and the rcmarker (NrTl) used in the tnsfrmaton. Souther Blot analysis is beingconducted on those straim that have demotrd linha between theflagellaless and NIT+ phenotps, in prpatio for

genes.

36a

Sunday. Cilia and Flagella (210-211)210MEASUREMENT OF OOCYTE CUMULUS COMPLEX (OCC) TRANSPORTRATES Si VITRO. ((M. Knoll, S. Huang, N. Dressen, and P. Talbot)) Departmentd Biology, University of California, Riveride, CA. 92521.

Furional assays for measuring OCC transport rates in y&m are needed. Wehave developed a method for measurng the rate of OCC pick up by the hamsterkfundbtum and for correlatn OCC trasport rates to CBF in the same sample.We have used this method to examne the effects of culture fluid vicosity on OCCtransport rates hI32. Perfusion dcane were made by plcing a holding pietteand inlet and oulet pWtes in organ culture dIsmes. Hamster hVundbula weredissected frorn oviducts and stablized by drawing them part way into a holdingpipete. OCC colected from maure follicles and stained with methylene blue wereplaced by micropette on the surface of an infundbdum sway from the ostium.The time required for the OCC to move to the ostium along a defined path wasdetermined. The path length was measured and the OCC tranwsport rate calculated.OCC transport rate in control medium was measured along one pathway on 19irfundibula and averaged 41.3* 9.5 mm/sec. In general, rates remaied constant

over the 15 minutes required to collect the deta For ndividual Infundibula, thecoeficiens of variatlon for rates along a particuar path were always lass t.; 5%.In a second expemNent, rates aong three paths on indiviul WundibuLh warecompared and found to vary in some experiments with the path chosen. In a thirdexperinent, the method was used to assess the effects of visoosity on OCCtransport rate. lnfunxxula were exposed sequentilly to control medium, controlmedium containing 1, 5, or 10% Ficoll, and control medium alone. OCC transportrates decreased as Fool conentib eased. When 10% Ficoll was washedout with control medium, OCC transport rates retumed to control values within 10minutes of washout. This method wil be usefl for studying piccup of the OCC bythe infundibulum, and data obtakied wfth the method indicate that viscositykifences transport rate of the OCC.

211THE GAS PHASE OF MAINSTREAM (MS) CIGARETTE SMOKE CAUSESPERSISTENT INHIBITION OF OOCYTE CUMULUS COMPLEX (OCC)TRANSPORT RATES IN VITRO. ((M. Knoll and P. Talbot)) Department ofBiology, University of Califomia, Riverside, CA. 92521.

Eplmiologal studies idicate that women srokers are at inceased risk forectopic pregnancies. We have previusly shown ectopic pregnancies mayicrease in smokers due to inhibiion of oviducal dcsay beat frequency (CBF). Inthis study, we used a new tehique to quantitate the effect of MS gas phasesmoke solXions on the rate at which the OCC is picked up by the infundibnium of

the oviduct h vir. Smoke sotions containig levels of nicotine and cyanide foundin the serum of human smokers were made as descrbed previously (Mole BKd Cell4: 273a, 1993). Infundrbula, which have cilia on their outer surface, were rermovedfrom female hamnsters and placed in organ culture dishes modified for OCCtransport masureets and perusion. OCC were transported over the surface of

infundbla at a rate of 48±10 mm/sec in control medium; rates did not change

during 15 minutes of observation. When gas phase MS smoke solufions wereperfWused into the charrber, OCC tiansport rates dropped signifficantly to an averageof 8±0.8 mrrasec. In smoke solution, rates abo dereased during the incubationinterval. When smnoke soluion was replced with control medium, OCC transportrates averaged 3*0.4 mnmsec. In washout medium, transport rates generallydereased slihtly during the 15 minute inubation interval. CBF, measured in thesame samples, averaged 8.9 Hz in control medium, 6.1 Hz in smnoke solution, and6.0 Hz in the washout medium. These data show that solutions of gas phase MSsmoke can inhibit OCC transport hi vm that this effect is persistent even whensmoke sdlution is removed, and that inhibition of OCC btansport Is related to factorsin addition to CBF. These observations suggest that persistent inhbition of OCCtransport rate by factors in MS gas phase smoke could cause ectopic pregrnacy in

smokers.

Centrosomes and Kinetochores (212-215)212

IDENTIFICATION OF A CELL CYCLE DEPENDENTCENTORSOMAL 8SKD ANTIGEN BY HUMAN AUTOIMMUNESERUM. ((MJ. Pacios, E. Stillwell, HB. Shu, N. Fertig, T. Mdspgr,andHLC Joshi)) Emory University, Atlanta, GA 30322.

A very useful approach to investigate the molecular nature of thecentrosome has been the use of autoantibodies spontaneously clicitedin a human disease condition, characterized as CREST syndrome(Calcinosis, Raynaud's Phenomenon, Esophageal Dysmotility,Schlerodactyly and Telangiectasia). Here, we describe a serum thatidentifies centrosome throughout the mammlian cell cycle in humanHela cells. In addtion, as cells enter the M-phase, the antibody stainingappears throughout the cytoplasm particularly emiched in the mitoticspindle and ch ns. This cytoplasmic staining persists throughanaphase and telophase, and it becomes restricted to the centrosome inG1. Immunoblot analysis shows that the serum contains antibodies toprimarily two polypeptides of 85KD and lOOKD. The 85KD reactiveantibody, when affinity purified from the blots, retains all thecharacteristics of the serum staining. Confocal images show a tight ringaround each of the centriolar cylinders. We conclude that the 85 KDantigen defines a novel class of centrosomal polypeptides that becomesassociated with other spindle structures during mitosis.

214

ANALYSIS OF CENTROSOMAL COMPONENTS WITH MONOCLONALANTIBODES. ((T. Otal, J. VogeP2, G. Peng2, R. Palazzo2, R. Kuriyama'))IDeparnt of Cell Biology & Neuroaatomy, University of Minnsota,Minneapolis, MN, 2Deparunent of Physioogy & Cell Biology, University ofKansas University, Lwrence, KA. (Spon. by V. Iwanij.)

Monoclonal antibodies were raised in order to probe for molecular components

specific to the entrosome. Highl purified centrosomes, composed of 14 maorbands on SDS-PAGE, were prepared from activeed oocytes of the surf clam,Spird soldisbra by diffential centirfugtion and sucrose density gradiaetcesrifugaton. Indivihal excised bands as well as the whole centrosomalfraction were used for mnmiaion of mice. Hybridmna atants were

screened by indirect immanofluorescence staining of isolated Sprulacentrososnes and whole CHO cells on coverlips. Twenty six cones wereobtaie and their specfic ai&ges were ientified using inmunobWots.Seveneen cle were able to label cenosones in only Sirua. Among dte

nine clones, si (SS4 to SS7, SSG3C, SSDl4D) were positive in both

sa sd CHO cels, and throe cone (SS1 to SS3) stained CHO centoone

exclusively. While small Centriole-like dots were revealed by SS1 and SS3,almost all CHO-positive antibodiS recognized per materia-lilkeaWates. Besides centrosomes, four clones (SS2, SS4, SS6, SS7) were alsocro-reactive with inermediate filament-lik fibers in mlin cells. Sincemobalur species identified by te anibodies were disnct from each odtr,differ types of rediae filanin-lilke protins appear to be inchded inthe tucture specfic m

(Supported by CR and NH.

213

A 50 kDa VIMENTIN-RELATED PROTEIN ASSOCIATED WITH THECENTROSOME AND INTERMEDIATE FILAMENTS IN CULTURED CELLS.((A.L. Khodjakov * #, E. Bibikova*, O.B. Solovyanova**, and E.S.Nadezhdina ** #)) *Biological Department, Moscow StateUniversity, Moscow; **A.N. Belozersky Institute for Physico-chemical Biology, Moscow State University; #Institute of ProteinResearch, Pushchino, Russia.

Monoclonal antibody SN-3C8 (Mab 3C8) was raised against apartially purified fraction of centrioles from calf spleen. Mab 3C8immunostains the mitotic spindle, the centrosome and a subset ofinterphase microtubules (mts) in cultured cells. The percent ofinterphase mts positive for 3C8 staining varied from the majority inL929 and CHO-KI cells to a minor subset in PKE cells, while no 3C8positive mts were detected in mouse embryo (MEF) or humanskin(HSF) fibroblasts. Mab 3C8 was used to screen a CHO-K I Unizapexpression library (Stratagene), and a single positive clone wasisolated. Polyclonal antibodies raised against the fusion proteinencoded by the cDNA immunostain the centrosome in all cell linestested. In addition, it stained intermediate filaments in CHO-KI andL929 cells, but not in MEF, HSF, CVI or PKE cells. On protein blots,the antibody stained a single band having an apparent molecularmass of 50 kDa present in total protein and the triton X-100insoluble fraction of CHO-K1 and L929 cells, but did not recognizeany protein in total protein from HSF, CVI cells or in calf brainproteins. Partial sequence analysis of the cDNA showed a highdegree of similarity to hamster vimentin. Experiments areunderway to determine the full sequence and function of thisprotein, and its relationship to the mt-associated Mab 3C8 antigen.Supported in part by ISF Grant# M4AOOO.

215

MOLECULAR CLONING OF cDNA ENCODING A NOVELCENTROSOMAL PROTEIN. ((S. Komesli and K. Gull)) School ofBiological Sciences, 2.502, Stopford Building, University ofManchester, Manchester, M13 9PT, United Kingdoom.

Isolated and purified lamb thymus centrosomes were used as theimmunogen to raise polyclonal antibodies against centriolar proteins.Using this approach we have produced a panel of rat polyclonalantibodies against centriolar proteins. One of these sera, CLT050,raised against native centrosomes extracted with 0.5 M NaCl beforeinjection recognises centrosomes by immunofluorescence onmammalian cells. It also detects several bands of 207, 170, 128, 77 and37 KD in immunoblots of centrosomal preparations. We have used thisserum as a probe to screen Xgtl expression libraries of Swiss 3T3(mouse fibroblast) to clone cDNAs encoding centrosomal proteins. Wehave obtained 3 different positive clones. One of them, a 365 bpfragment, has been sequenced. The analysis of the predicted aminoacid sequence suggest that this polypeptide is part of a novel protein.We have cloned and expressed this fragment in Ecoli as a fusionprotein. The fusion protein has been purified and used to raise ratpolyclonal antisera. Immunofluorescence of mammalian cells usingthese antisera recognises specifically the centrosomal region. Theseresults represent an initial identification of a novel protein of themammalian centrosome.

37a

Centrosomes and Kinetochores (216-221). Sunday216

SUCCESSIVE ASSEMBLY OF TWO PROTEINS ONTO THECENTROSOME COORDINATED WITH SPECIFIC EVENTS DURINGMrIOSIS. Oegema, W.F. Marshall, J.W.BJdat and B.M. Alberta)),Department of Biochemistry and Biophysics, UCSF, SF CA 94143-0448.

CP190 (Centrosomal Protein of 190 kDa, originally called DMAP 190)was previously identified using a combination of microtubule affinitychromatography and immunocytology. Antibodies made to this proteinstain the centrosome during part of the cell cycle in Drosophila embryos;nuclear staining during interphase is also apparent. Immunoaffinitychromatography experiments using affinity purified anti-CP190

antibodies identified a group of interacting proteins. One of these is CP60(previously called DMAP 60). Antibodies to CP60 also stain thecentrosome and nucleus. We have characterized the cell cycle dependentlocaizations of CP60 and 190 in greater detail 3D microscopy wa donein live embryos injected with fluoreseently labeled 6-His fusion proteinsderived from CP60 and CP190. In late interphase, both CP 190 and 60 arefound in the nucleus. Labeled CP190 and 60 have each been coinvectedwith a fluorescently labeled 40,000 MW dextran, which is excluded fromnuclei, to determine when their transition from the nucleus to the

centrosome occurs relative to nuclear envelope breakdown. Recruitmentof CP190 to centrosomes appears to occur immediately following nuclearenvelope breakdown; CP60 remains in the nucleus until it assmbles ontocentrosomes at the metaphase to anaphase transition. We have also

stained fixed embryos from the syncytial nuclear cycles 10-14 for y-tubulin, CP60, CP190 and DNA simultaneously. Combining the datafrom the fixed and live experiments, we have constructed a detailedpicture of the localization of these proteins throughout the nuclear cycleduring these divisions. We have also characterized the nuclearlocalizations of these two proteins in 3D at high resolution. CP60 and

CP190 appear to form fibrous networks within the nucleus which do notcolocalize with each other or with DNA.

218

RECONSTITUTlION OF THE HUMAN SPERM CENTROSOMEIN VITRO.((S S. Zoran,, CSimerlf, P. Sdhoff, T. Steams5,J.Ssalbury,and Gerald Schatten))* University of Wisconsin, Madisonk, WI, Stsnford

Univerty, Stanforfd, CA, Mayo Clnink Rochester, MN

During fertilizAtion, the sperm centrosome attracts egg proteins asit is reconstituted into a replicating,fully functional centrosomecapable of nucleating sperm astral microtubules. This process

was explored in vitro using human sperm and Xenopus egg

exetacts. Centrin was found human sperm, as one or two dots,at the base of the sperm head,and its presence was not altered byexposure to egg extract. Theseresults have been confirmed byWestern blotting.-gTubulmn was not detectable human sperm.Some sperm displayed i-tubulin after a 'priming' step involvingdisulfidebond reduction or SDS-PAGE and Western blotting.All sperm exhibited y-tubulin staining after 'priming' andexposure toextract. Thecentrosome became phosphorylated afterexposure to egg extract, as detected by MPM-2. Sperm astral MTformation within these extracts was observed live usingXRhodamine-tubulin. These results demonstrate the presence ofcentrin and suggest some residual paternalT-tubulin may befound in human spermatozoa. Upon incubation in extract,maternal y-tubulin will bind to the human sperm centrosome inpreparation for its MT nucleating role. Supported by the NIH.

220

SUBCELLULAR DISTRIBUTION AND FUNCTIONAL ORGANIZATIONOF MIP-90 IN CULTURED CELLS. (M. Gonzlez1.3, L Tapia1.2, V.Cambiazo1 and R.B. Macdonil-2)1Lab. Cell. & Mol. Biol, ICC, 2Univ. ofChileand 3Cathollc University, Casilla 70111, Santiago 7, CHILE.

Our researchinterestis toinvestgate in vivo functions ofMip-90, anovel microtubule-interacting protein isolated from HeLa celW on thebasis ofitsinteraction with C-terminal tubulin peptides affinity columns.Immunological and biochemical studies showed that Mip-90 bearsstructural features which are not shared with known neuronal MAPs.Double immunofluorescences using an specfic anti-Mip-90 antibodyrevealed an striking co-localization of this protein with interphasemicrotubuies (Mt). Nocodazol-treqted cells showed significant changesin Mip-90 distIbution as correlated to disuption of the Mt network. Inthe drug-treated cells, Mip-90 remained associated to an stableperinuear subse of Mt's, suggesting its involvement In stabilization ofthe Mt structure. Within this context, it Is Interesting that Mip-90 hasbeen found In other celkular structures, including the midbody of HeLacels and axonal processes In primary neuronal cultures, that containstable subsets of Mt* Moreover, Mip-90 co-iocalized with Mt bundleswith a perdnucear diibuton in taxol-treated ceils In mitotic celis, Lip-90 was identifbid In association with centrosomes suggesting its in vivoregulatory role In Mt assembly, In agreement with the findings thatpurifid Mip-90 Induced tubuin assembly In a concentration dependentfashion. The stdies suggest th Lip-90 is a new Mt-interacting proteinthat nay play a roe En modulating different aspects of Mit dynamics, and

their Interactons with other elements of the cytostructure. (Support:;CouncNl forTobwwc Research, USA. Fondecyt, DTI and TWAS).

217

STRUCTURE AND BIOCHEMISTRY OF THE MICROTUBULE-NUCLEATING SITEOF THE CENTROSOME ((M.Moritz, *M. Braunfeld, *D.A. Agard, andB.M. Alberts)) Department of Biochemistry & Biophysics, *HowardHughes Medical Institute, UCSF, San Francisco, CA 94143-0448.

Using centrosomes isolated from early Drosophila embryos, weare exploring the structure and biochemistry of the microtubule(nT) nucleating site. We have developed a biochemical assay

for centrosomal proteins involved in MT nucleation. This assayinvolves the in vitro complementation of centrosomes that havebeen rendered nonfunctional for MT nucleation by treatment withIM KCI. These nonfunctional centrosomes can be complemented invitro by a 100,000 x g supernatant of an extract prepared from0-2 hr embryos. We are currently fractionating this extract byFPLC to identify proteins required for complementation. We arealso using electron tomography to reconstruct three-dimensionalimages af the isolated centrosomes with reqrown asters. Recon-structions to 55 Angstroms resolution have allowed us to identi-fy minus ends of MTs within the nucleating material of thecentrosome. No large structures are apparent at the minus ends.We are attempting to localize known centrosomal proteins withrespect to the minus ends of MTs usinq inmmunoqold labeling.

219IN VITRO FUNCTIONAL ANALYSIS OF CENTROSOME-NUCLEUSINTERACTIONS ((Sigrid Rcinsch and Eric Karsenti)) EMBL,Meyerhofstrasse 1. Postfach 10.2209, D69012 Hcidclberg Germany

In many cells centrioles sre associated with the nuclearenvelope, In others cells, for example epithelia, centrioles areassociated with the apical surface of the cell. During fcrtilizationin most vertebrate embryos. the egg lacks centrioles which arethen provided by the sperm. Through interactions between thefemale pronucleus and the sperm microtubule aster, the twopronuclei are brought together. In each of these cases, themechanism by which the centrioles interact with either thenucleus or plasma membrane are not understood. We are seekingto develop an in vitro system to study the interaction ofcentrioles and nuclei, and have preliminary evidence thatinterphase extracts made from Xenopus eggs are a good candidatesystem. These extracts support the decondensation andmaturation of exogenous dembranated sperm and have abundantspontaneous microtubule assembly both in the presence andabsence of exogenous sperm nuclei. When centrosomes areadded to these extracts, spontaneous assembly is substantiallyinhibited, and centrosomes cluster and form large microtubuleasters in the presence of cytochalasin. Centrosomes appear tointeract minimally with the exogenous sperm nuclearmembrane but affect the maturation process. In contrast,centrosomes will cluster around added HeLa nuclei but only inthe presence of cytochalasin. Experiments in progress examineother factors that influence the interaction - includingnucleotides, microtubules, and microtubule-associated proteins.

221

TH PENCRCE LCE NKE NTA MCBfNROSCM ISNOTTHE MAJORMICROTUBULE ORGANINGCENTER IN SERTOU CELLS ((A.W.Vogl, Weistand D.C. Pfelffer)) Depatment of Analomyand tBloSences Imaging Facty,The UnIverty of British Columbia, Vanoouver, B.C. Canada, V6T1Z3.In this study weInvestigate the perdnuclear cntrbileontanlng centrosomeas a MTOC In Sertoll cells. Fragments or dsscted pieces of fixedsemhiferous epitheltium of rat testis were fluoresoently labelled for tubulnand actin. The three-mensional pattem of micotubules In Seroi cels wasdetermined usin data coleced with a confocal nIcroscopeand alyzed usinthe NIH-Image program. The detaile ar of microtubus around,and the number of microtubule ends assocded with, centrosomes weredetermined from composite projeti construcsed from serial thin sections.The nudeating potential of perinudear centrosomes was determined byperfusing testes for 6 hrs with 10 pghdvi nzo and then for up to 3 hrswith oonto buffer prior to fixation and analysis by confca and standardfluorescence microscopy. Microtubuls are not organized around a focalperncear ste and few icrotuu ends are assodated wih the centrsomeIn normal tissue. Moreover, in celi recovering from nocodazole treatment,microtububs first appear apical (peripheral) regions. Our data indicatethat the centriol-containing perinuciear centrosome Is not a significantMTOC In Sertoll calls. Rather, microtububs are nucleated In peripheralregions and projct basally. Based on the observatIons that mxotubuesappear to cuir the nucleus, rIntermedIate filaments are conceontrated arondthe nucleus, microtubules project Into the perlnuclear intermediate filamentnetwork, and micotubuls intermediate Uaments are often coailgned, we

suggest that microtubules are captured at the base of Fe call by theperinuclear Intermediate filament-rich cytoskeleton. Our nucleation-capture model may be appiicab-e to other epeilal systeme. Supported by agrant frm the B.C.H.R.F.

38a

Sunday. Centrosomes and Kinetochores (222-227)

222

PURIFICATION OF G2- and M-PHASE CENTROSOMES FROM

OOCYTES OF SPISULA SOUDISSIMA ((JX Vogel ad R.E. Palazzo))Departm of Physiology and Cell Biology, Universit of Kansas, Lawec KS 66045.

Oocytes of Spiuna a arrested at the G2/M bder, late prophas, of mnioss I. Nohave been detected is oocyte before activato P eogenetic

avauion with KCI Induces GVBD, cntosome maturati and the completiofmems.Lysates prepard frm unatvatedoocyse cm be induced to urgo centosomema_sados

in vitro, indicated by centriole assembly and duplication, correlated with increased

microtbule nucleation potetial (Plazzo, et aL Scienco 256:219-221 1992). We have

extnded tese stuesn dfoud that unactivated as cota a partie wtch contmIytnbulin and sa ceats few -irsetubules We sugges that this p tcle the pc a rewhich is indued to underg matiai when oocyts or lysases activated. In orde to

characteiz cntosome frm vars stWaes of the coil cycle we prified pro_ntrosomesfrom umactiwvated oocytes (LI-cetosomes) and cenosomes from oocyt acwvated for 4mm. with Ka (A4-cenuoomes) (Pazzo, at aL ZooL ScL 5:603411 1988) by liner

sucrose density gradient centrifugaptio Grdient fracto were aasyed for centrosomecontent by rcstut twublin muder defed condmi and analyzed by polarzdlight microscopy and immofltresec mirscopy. Centsesedimented at 56.4%(U-cestosomes) ad 582% A-centrosomes) is gaden Both U- and M-

controsomes sedimented at -59% sucrse in discondinu preparative radients.Immunofluoresene revealed that both U- and A4-entrosomes conained ytubulin, butU-centDSOMeS nucleated fewer microtabues tha A4-controsomes. This differenc innucleation potil was asociaed with 1) a quatitatve difference is protin pacentosom (4 pgL-centrosome compared to 5.9 pg/A4-centrosome), 2) -5 fod kss ytsbulin is U-mosoms late to A4-centrosomes, ad 3) an icr is MP-2 epiopecontent A4-centrosomes.In cocusod we have purnfied cent me frm two phae the ceol cydce, G2- adM-phas of meiosis Futher, we have deermined that dtes orgaeies ar distic baed on (1)differnc in miombulc nucleaon potental (2) prtein coet ad (3) post-tasadonlmodificanon of centosomal proteis. The rles of these pri in centme functo ae

currenty under stdy.

224

CENIROSOMALPOCUS OF GAMMA TUBULIN IS NOT REQUIRDFORMIOSIS IN DROSOPHILA ((P. G. Wison ANDM. T. Fuller))D URmn OF , Stanfod University School ofMedicine, Stanfor CA 94305

Gamma tubun is a conserved memberofthe tbulin i that iscated at miottbul pnmn8ceDtLCs1ofg mmatubulm function

results in a virtual an oanialatubule rgania. Muions itb (a) menein Doophila

eveal mma buin function doesnotrequireorganstInataspindlesin testis, ovaries, ad larval of mutas are

sp.depolo ladc a wel defined focus of gamni hbuin.

,cehow diffs massof mmtbn.

TLDoli inofDMAP190O to dtect ce somnsemi miotic cdii

Cmtuomes wem detectedin i cdlswtaw bh _od t

WtWulin, but these foci wem oba rved inpp rm later tagesofmitos Dep ofthelacfofiantx mitoticcells, alleks of 1maefuwdblyex anddonot _mtedefet in Lme cTeshofeshsye and

Qeare moresensvtoeto lo of Cma fu thnmilotic division m_uta ae mWale d feme si

Wehaw ideifie uti_ve muo inthetw doestht ecodettthuHinisofirmainNkosophila. apenandipaba of tuun cDNAs on polyte omes revedsth

Ws des not enoodegabmmatuln M e, thep otpep_ dby

loss of Bris fnco om the yepratd by of mm

tubulin functi Our cts thee ia olf mtuuinin

ssemb!y doanoz i a focus P themost iing m ofour ta mitod mdcU diviso inanimal cdls appently donote a definedc m

226ANALYSIS OF KINErOCHORE ASSEMBLY AND MICROTUBULE DYNAMICS

IN CLARIFED XENOUIS EGG EXTRACIS. ((A. Dessi, H. Deacon L Wordenan,ad TJ. Mitchison)) Depaptme of Pharmacology, Universty of Califonia, SaFrncso, CA 94143.(sromosome seg aion invemtebres occurs by a precise control of Adromosome-

micrtubule inweai which are mediated by the kinetche. In ore to undestad

ts pocess at a biocmical levl we h been anlyzig khrlochm assembly inmiotic Xenopu egg extra MNtodc dcomat asembled in crude Xenopus exucashows discrete ocaizato of a ceatomere aociated kiesin (CAKI) to poisestructures Antiosdy which stas kmeohrsin fsog dce culture cell. We

have expoitod this loc1imdon to analyz kieicore ssembly in daified Xenopu egg

ext which exibt reproducsble rcho easmby. Two-fold diludon of

ecisl resul in Sig csI wish good morhly. Only a blw peceage of

tbese cimomosom show CARI ali bat preincobios of te nuclei in

unMltd extact pior to diatio results in a drmIIc ices in th nember of

dromosome with CAKI localization result suggest that kinetchore

asyed by localzation of CAK1 is dilution sesitidve. We ar also isdagmsom asembled in excts to deterine ff these mewill bind

bules ad if the micrule bnding region cincides w the rgio for CAK1

Aalysis of mictbule assembly in crude Xenops egg extas showed that

nm ovemore apidly mitoicetata bes exua soew a

fold h4beg cat phe rate ie cpha extra. We have loked at micro8muledynancs in Xentpus egg extac clardd by centrifgato usngvideoa banced DIC

ad we vefound o lrdlif d extrac very st e Bycoamping diludtos of clarified extr we haw fotd Xt the dcricon does notsuover the cdl cycle diffme in microbol dynanics but oPpeP- to elimine acounsisntive mchanim fo increseag mcrotubul trver. We we aimptig to aid

bawkd-fMPrP ntfd c fm the cheifiration domise whe is respnsibe forthehigh

tr veofmir in vivo.

223

IDENTIFICATION OF INTINSIC DBIER AND PURIFICATION OFOVEREXPESSED MONOMERIC FORMS OF 7-TUBULIN IN SF9CEL ((A. Vasailev', M. Kimble', C.D. Silflow, M. LaVoie2, R.

Kuriyanal)) IDepris of Cell Biolo & Neuanuwony University ofMinnesota, Minneapolis, MN, of Gentics & Cell Biolo,

Univenity of Minota, St Paul, MN.

Calamydosmw cDNA encoding the fulllength, 468 smin acid sequence of

-y-uulin wa expresed in inmct S19 cell using de baculov expression

sysem. the induced 52 kDa y-bulin ws recovered in

tie aftrceifugation of Sf9 coll lysts at 18,000 for 15 min.

Wben dcell sernatant wa analyzd by FPLC on a Superde 200 sizing

cohlsn C(aadonns-y-sul separatd ito two mjor peak. lMc la

peak contained a onnomeric form of y-sbulin which inseracts with the

Superdex colus in a dqpendes mnmer. lit lanin peak, with an

qppaat molea mas of 900 kDa, aorresponded to a moleua henn

complex, ad TCPI dcyerin releaed folded ubuln polpeptide from thecomplex in the presence of MSATP. lit relasd -tbulin monomers werecapable ofbin tmtoio Wo ad biochemical q ties of active

furth puified usn a coonmbinaton of size exclusion and ionexcha cohumn chromaogrpyl.iT nou SO cce --suxin miraedfaste than Cslaydomnas tubuln wih an appaest molcula mas of 49

kDa on gl. Whle ov rssd 7--bin was prest in amonomerc form, a tsul from S9 ad HeA colb exists u adsn, suggesting posbit tai 7-tubuln could form a heteoduner with

hito unbno molecule(s). (Supported by CTR and NIH).

225

ROLE OFGAMMA-TUBULIN IN MIIDSIS-SPECIFIC ACTIVATION OFTHE S. POMEBSPINDLE POL BODY. ((IL Masuda, and T. Shibata))Laboratory of Cellular ad Molecl Biolog, Tle Insdtute of Physical andatmical Reseach (RIKEN), Wako, Saiuma 351-01, Japan.

lit ability of the S. pvmbe pindle pole body (SPB) to nuleate

microtubles T(ffs) is activad at mosis, whera k is inactivased duringTo study emolecular mechanism of miosis-pecific SPBwe have used an in vitroassay capable ofcosarting inephaw

SPBs into a miotic sta. MT nwleats acivity ofSPBs in pereabilized inerphase & pombeoell is acivated by incubtion inXenOptS mitotcexuat SPB acivaton reqires actvity of proein

other thancdc2. Inurpase SPBs are preincubated with antibodye ed against S. prnbe gamma-tubulin and then activated in Xenopusmittic extcs cannot nuclae MTLu mitic SPBs

with te antibody cannot nuTbe fL antbody blockingafbothproessesissuppresep byadditi of a C-uminalgamma-tubulinfagment expresed in colL iTbese sus sugettbatmma-tubulin ispartoflMTnucleang complex atthc SPB to interact with alpha-bea tubulin

Addition of gmma-tubulin o is N-tminal fragment toXcopus mitodc exracts inhibits SPB actvation Affini hrmtgphy Of

Xenopus mitoic extacts with the N-teminal fragment shows that SPBactidvaon requie both fraction bound and unbound to the firgmen SPBacdivation does not requir Xenopusgamma-tubulin results suggstthat miosis-specif modfiation ofthe N-terminal domain ofSamma-tubulinis required for tng the immatu MTnuceting complex into thematr complex, and that two factors present in the extracts, a protein kinasand a protein modifying gammat n, d two SPB aomponenta,akinasetarget and gamma-ubulin, are involved in thc SPB

22?

CENTROMERE (PREKNETOCHORE) ASSOCIATION WrH THE NUCLEAR

MATIX AND CHROMOSOME SCAFFOLD. ((D. He and BRBrikley))Dqssrtmentof Cei Bidogy, Baylor CoUrse ofMedicie, H,osX,lX 77030.

Previous sudie in our labortwy hav shown pr_ne c

protei ric DNA comple in he isrphawe nuc ) undo c

auuctw m dulxt_qkaio pudoni in a a comaisner li pr study ows tht tl movene and stuctr

rearangm m of pr occur in imsate associatin with the nuclear

Ix nuclearma core filaent systm examind in Hel, CaSk, MCF-

7 and Ixda n*ac cells. RavAl of 75% of te nuclearp ad 95% of dteDNA had Litte effet on stang intsity, distribution ad nizatiof

stained with baiman from _dera CRESI paents. Aftr 0.25

M (NH4)2SO4 extacton, reIdual DNA coloalized with CREST stand dob,gesig at edo CEN-DNA/pti complex may sere to ancbor prlneochores to thenwucla matrix. Using resines setim ofH eIa cells, d was used to

diretl visualize the amociatio of CREST astigens with coe filuamn network of

the ncer sti Core fiasnss appeared as a relatvely uniform netwrk of 9-13onu filawmet associated with nucleolar rennants and smalle -ose represantinpilfnhe CREST labeledthassociated masses

fiha themsev lM lbabded w a asme stan, stuc

and distributio as tdio in te itc, usextacted .sln. P bands of approxi-satey S0 and 140 IcD, prnwmbly CENP-B ad C, we idenified on SDS by

imm_noblnig pin with CREST antsera CENP-A and a portio of dte

tga a ing to the nucla -a woe a o detected on meesphase crm

scaffold.

39a

Centrosomes and Kinetochores (228-233). Sunday

228

CENP-A CONTAINS A HISTONEH33 HOMOLOGY DOMAIN THAT IS REQUIREDFOR TARGETING TO THE CENTROMERE. ((KF. Sullivan)) Departdmt ofCollBiology, The Scripps Research Institute, La Jolla, CA 92037. (Spon. by M. Yeagew

The centrmere Is a differentiated chromosomal domain that contains uniquelyorgnized chomatin required formitotl chrmosome segregation. To study the

stuctur and assembly of the contmere, we have coned cDNAs encodingmammalian hiAtone-lke oentromwe autoantien CENP-A. A patl bovineCENP-A cDNA waisolated by RT-PCR. using dg ate lonu d primedesied from paptide sequence data. This was used toisolt ahomo fulllngth human cDNA. The human CENP-A cDNA encodes a 140as polyedewith two domains based on sequence honmolgy a uniqueN-terminal domain of 48resdues and a 92 resIdue C-terminal domain thatshars60%identity wih histoneH3. In transfected animal cels, the human cDNA direted the synthesis of a 17 kdpolypeptide reactive with human ant ntromere sera that co-migrates with HoLacell CENP-A. We have studied the targeng of CENP-A to cordtmeres byconstruction of anopitope taggeddeivative and shown that CENP-Aisofficentylocalized to centromeres In transfected human and other mammaliancels. Thelocaion of thecentrmeri tareti signal within CENP-A was examined byconstuction of chimic CENP-Ahlstone 113 genes. Surprisingly, the C-tminalhistone fol domain of CENP-A wasn cessyan sufficient for centromefrtarget rather than the uniqueN-terminus. By co-expession of CENP-A withCENP-B, the aiphold DNA bindig centmereautoantigen, we show that the

localization of CENP-Ais not specied by CENP-B. These da clearlyindicatethat theassembly of the centromereis driven, In part, by the specif n poationof a novel core histone protein. They furher suggest thattawgeting of CENP-A tocoentromeresis specified ptmaly byinteractions at the level oftm nudiosome,raising the possibility of nucleosome-specf DNA sequence reonition.

Supported by NIH ROi GM3908M

230

CHARACTERIZATION OF THE KINETOCHORE PROTEINCENP-F: MOLECULAR CLONING, LOCALIZATION ANDEXPRESSION.((H. Liao# A, B. Winkfein*, G, Mack*, J.B. Rattner*, and T.J. Yene))Cell Biology Graduate Group, University of Pennsylvania,Philadelphia, PA. 19104,*Department of Medical Biochemistry,University of Calgary, Calgary, AB, Canada T2N 4N1, Fox ChaseCancer Center, Philadelphia, PA. 19111.

CENP-F is a novellinetochore protein that was previously identifiedby an autoimmune serum that recognized a.ca 400kDa chromosomeprotein (RattnereLaL, Cell Motility and CytosAkeCton 26: 214-226,1993). Using this serum, we have cloned a 10.1 Ikb human cDNA thatencodes CENP-F. Analysis of its DNA sequence reveals that itencodes a 377 kDa protein that does not appear to be homologous toany known proteins. Immunoflourescence staining reveals thatCENP-F accumulates in the nucleus from S to G2 phases of the cellcycle. Kinetochore staining is first detected at prophase and thislocalization persists until metaphase. At anaphase, CENP-F isrelocalized fromkinetochores to the equatorial region of the spindle.By telophase, CENP-F is localized at the intracellular bridge.Expression studies show that CENP-F is synthesized throughout thecell-cycle but its steady-state level reaches a peak during G2 and M.Pulse-chase experiments reveal that CENP-F turnoverrate is increasedtenfold when cells progress through mitosis. Amongst the chromosomepassenger family, CENP-F appears to bind to kinetochores the earliest.CENP-F may be one of the first proteins to initiate the formation andmaturation of the trilaminar plates.

232CIN2 AND CIN4 FUNCTION AS A MODULATOR OF KINETO-CHORE /MICROTUBULE ACTIVTY. ((SONG, X-Y., SHAH, J., andBLOOM, K.S.)) Dept. of Biology, University of North Carolina at Chapel Hill,N.C. 27599-3280.

We have used three phenotypes to isolate mutations in kinetochore/ micro-tubule function from S. cerevisae These phenotypes include transcriptionalreadthrough the centromere (Perier and Carbon, 1992), suppression of dicentricchromosome breakage (Doheny et aL, 1993) and supersensitivity to a micro-tubule poison, benomyL One mutant has been identified that exhibits all threephenotypes. The gene complementing the mutation, CIN4, encodes a proteinof 191 amino acids with three motifs characteristic of G-proteins. Interestingly,the amino acid change in the cin4 mutant is outside the conserved motifs. CIN4as well as CINI and 2 were originally identified as chromosome instability andbenomyl sensitive mutants (Hoyt et aL, 1990; Stearns et al., 1990). Null mu-

tations of cin2 suppress breakge of both dicentric plasmids and chromosomes,cin4 null mutants suppress only dicentric plasmid breakage and cinl null mu-

tants fail to suppress both. Other benomyl sensitive mutants, such as tubl-1and mad2-1 also fail to suppress dicentric breskage. The benomyl sensitivity

and suppression of dicentric breakage characteristic of the cin2 and cin4 mu-

tants indicates these gene products may play a functional role in kinetochore/microtubule interactions. Since these genes are dispensible, we propose thatCIN2 and CIN4 modulate chromosome/microtubule attachment.Perier and Carbon, Genetics 132:39; Doheny et aL, Cell 73:761; Hoyt et aL,MCB 10.223; Stearns et aL, Genetics 124:251.

229

CENP-E-ASSOCIATED KROUIUBULE MOTO1R LSOlATED FROM

CELl IN PROMETAPHASFM_TAPHASE IS MINUS-END

DIRECTE. ((DLA Throwerl, MA. Jordanl, B.T. Scharr2, Ti. Yen3,andL Wilsol)) Dpatentof BioogicalSc nces, Univesity of

California Santa Barbara, CA 931061, Cell Biolog Gradum Group,

Univasity of Pennsylvania2, and FoxCbase Cance Centr Philadelphia,PA 191113.We haveprtally purified andcharacterizedanmimns end-directed

nuicrtbue motorfom extasof HeLa cellsblc edinprom e N byvinblast. Cnileexpeimnt evidence

suggests that the motormay beCENP-Eits which has been predicted to

be amicrotubc e motr basedon squence honologylooinesin Thracdvity is immuno- with an atdibody specific forCENP-E (HX-1). The motoracvit o-purifies with CENP-E pr thrghmic pStubule-f caon, gel filtraon, and sucrose densitgradientcentrifugation. MDtoracr is differentfiomdynen rrh nbased uponmicoubegiding rates (0.24 psa at2° C) andnucm oie andinhibitorscificities. Furth, theo activity is clearly ntduc todyneinas itis Otinhiedbyadynein-specific antbody (Bob) orby treatment with

vanadt andUVli t Suceoedetysity -tion and gelIfilratoindicat thatbtenve mol ar ofthmo in is approximely874,000 and that itisamx0nm) thatistwice the esmtlengtho Th rts ngly sugt thatCENP-E nayact as am dusnddieced microubul mo tr durinfle RJR 11- F f adtosis Suppored y Amcan &ncerSoiq ty DP43 and USPHS grantZ4M4477.

231

UBQUMN-CONJUGATjIG ENZYMES SUPPRESS ACONDITIONAL-LETHAL MUTATIONOPNDC10, A GENEENCODENG A KINETOC RE

PR0HIN INS. CERISIAE. ((OL Kopdi. GJ. Beagle, KK Scdlr, nd T.C. Hsbfer)) Dep_wts ofBiochemistry, Mdlecl dCelll Biology, Conell

University, Iha NY 14853.

Toid eifn ew p n in ved skneochs funti we eboth

asecond-sh andur o acondition al-

lea of NDCIO. NdclOpiskmBIo be copoem of te CBF3comnple. nking dheceatIo'n the spinlae icacotubul (Lxchner, J..

audJ. Cuba.. 1991. CdL 64:717-725 md Hyum, A., etat. 1992. Nae. 359.533-536. We be previously isoldu aIkolefNDC10 (ndcO-2) by scring a

collection forde ina spindle rnnationmd/ocromosome Seg Aeaon usigmt-- Hb i mmonfiu esa ce.m Atdeo a -

pnumsve mpare adclO-2 cdt forms appamsty mdalspindle, butdmagsphae DNA fals toproply sewgrga and smains in the modheceL. Thi

neicldivision producea man cell amd cell with a 2C DNAcom ent.

we ideatfied dre litae of spontanous aPrmssars adc)0-2.Two naeb opshavebeen doedaud paraly ced. One grwpcosins ofmatons in UBC6 ad the ote in UBC7,both peviouslycba(Sommer, T., and S. Jeuwach. 1993. Natme 365:176-179 d ingnsmsmJ.,etaL1993. N_e361:369-371). BothUbc6pandUbnp reubqui-caJugatngenzymes, md te iscvidene th Ubc6p md Ubc7p hyil iniect eachothe (Chen, P., eta. Cell 74: 357-369). These asq are alel spocific. In

uddion, we abo isolatd 12 supgsnuos a 2-t om yes

geomuic lwy. Only owe hs beo eqondmd isidenl toUWC6.Twrestingly, diutig the UBC6 ne alsoaqpees them*0-2 mutati. Both

theaecoad-lemif d ovesxpressn aqipmnmr, sor suggestarole forthed qulim-moesiAed psuaelideg mni ochcre fo cmL

233

MPS2 FUNCTIONBS REQUIRED FOR M1TOTIC SPINDLE FORMATIONIN SACCHAROMYCFS CEREVIAE ((Antonio Mnral, Willow Dres-

chdrl, Susn Miler2, BreckByers2, and Mark Wlneyl)); IDepartment of Mo-leulas, Celluas, and Developmenal Biolgy, Univeity of Colorado,Boul-der, Colondo;21 tm ofGenetka, University of Washington, Seattle,Washigton-

Itis cuclalto theeukaotic cel cycle that the centrosomeundego precisedupllcaton to genme thetwo pol ofthe mitotlc spinde. Inthe buddinyeast Sd y enrooma fuitons are provided by thespInde pole body (B). Our laboratory hs isoated a specific mutant,m,2-1 on ar spindle 2-1), whh, at thenonp shre temperature,prevents properSPB duption and leads to the formation ofamonopolarspindle phsadefective SPB (Winy, et al, 1991,JCB, 114:745).TheMPS2 gene s predktdtoencodea271ainoacddpolypeptldethat nmycontain coied-coi doma, as well as a higly hydophob region that

might se to span a Seque data of theAPS2 promoter re-

gion indicate that MPS2 bt c n les may be regulated by the cellcyde.Another a spindle nutation, ndcl-1 (nudear division cycle 1-1),causes a phenoypethat b very milar to the one caisedby mpe2-1. are

currently thepossible itainbetweh APS2 and NDC1.

are also (1) _ gc tatn ta that mayucover other s2m utarnta 2) completlngtheid ofthe pqu2-1 mutantleso

(3)atemtingto ocalizetheMpa2prote Inwhol cel saswel asexacts,

and (4)det g whetherMPS2 Is esential foryest ceRvabilty by ex-

amninghephteno oftcellwhoeeMPS2gae havebeencompletelyde-leted

40a

Sunday. Centrosomes and Kinetochores (234-237)234

DYNAMICS OF SPINDLE POLE BODY BEHAVIOR INSACCHAROMYCES CE1RIEISIAR OBSERVED IN VIVO. ((J. A. Ehana,B. J. Schnapp*, and P. A. Silver)) Department of Biological Chemistry andMolecular Pharmacology, Dana-Farber Cancer Institute, and 'Departmentof Call Biology Harvard University Medical School, Boston, MA 02115.(Spon. by B. J. Schnapp)

The spindle pole body (SPB) of is the buding

yeast's analog of the mammalian centrosome. The progress of SPBmigration can be directly correlated to the progress of the cell cycle.Investgations of mitosi, and of SPB function and behavior in yeast havebeen limited by the lark of a method to visualize SPBs in lving cells. Byutilizing the gn-fluoresnt protein fused to a SPB-asaociated protein,NUF2p, we have observed by flu nce micr the dynamics ofSPBmigration in living yeast cells. We show that NUF2-GFP is associatedwith SPB's by co-immunolcalization with a known SPB-aaociated antigenand we have observed the rate, directionality, and pattern of SPBmovement directly in vivo by time-lapse digital microscopy. During afraction of the cell cycle, the SPB's appear to be relatively quiescent. Theonset of the period of spindle pole segegation begins with an increase inmovements of the pair of SPB's within the mother cell. Initially the spindle(as defined by the positions of the SPB's) is randomly oriented, butpreceding its motion into the bud, it aligns along the mother-bud axis. TheSPB's then separate with a velocity of approsimately 0.1-0.3 imsecond.Furthermore, we have oberved that one SPB can migrate properly evenwhen the other is immobile. Finally, we show that once SPB separation is

complete, the entire spindle moves as a unit along the mother-bud axisThus, we demonstrate that it is feasible to study the movements ofintracellular components in structurally-intct living cells. The ability totrack the dynamics of SPB's in living cells creates unique experimentalopportunities for investigations ofmicrotbule organizing centers and theirroles in mitosis

238EFFECTS OF MBC ON SPINDLE POLE ORGANIZATION IN DIVIDINGHUMAN GRANULOSA CELLS ((A.Can, S.Messinger, D.F.Albertini))Department of Anatomy and Cell Biology, Tufts University School ofMedidne, Boston, MA 02111.

MBC (Carbendazim) is known as a potent fungicide which shows itseffect by antimitotic activity. The predse mechanism of its antimitotic effecton cell cyde and related subcellular structures remains unknown. Thepresent study was designed to assess if MBC has an antimitotic effect oncultured human cells and whether this effect is due to any impairment onmitotic spindle formation and function. For this purpose human ovariangranulosa cells were treated with MBC (3OpM) for 12 hours. Samnpes wereanalyzed using triple fluorescence labeling with antibodies against total andacetylated tubuin, centoomal proteins and DNA. Results showed that i)MBC blocks mitotic cell cyde progression with a seven fold increase asquantified by mitotic cell index, ii) mitotic arrest is achieved without anygross disturbance of microtubule (MT) organization, iii) MBC treated cells

give rise to the formation of multipolar spindles of two distinct type; one

possesses cen boomal material, acetylated kinetochore MTs and astral MTswhereas the second does not possess cenbtsomal material and astral MTsbut does contain acetylated MTs, iv) mitotic arrested cell display unequalpartitioning of chr to spindle poles. Taken together, these resultssuggest that MBC as an aneuploidy inducing antimitotic agent interferesprimarily with cenbtsome behavior and secondarily with spindle formation,astral and kinetochore microtbule organization which leads to unequalmovement of chmosom and arrest in mitosis. (Supported byUSEPA/Center for Environmental Management, Tufts University)Intracellular Movement (238-239)

235POLYPEPTEDE SP 83 IS A COMPONENT OFTHE STABLE PROTOFILAMEWbOF FLAGELLAR MICROTUBULES AND IS ALSO LOCALIZED IN BASALBODIES ((E.H. Hincholiffe & R.W. Linck)) Department of Cell Biology andNaromasemy, Univey of Mliuaese Mineal MN 55455

Axonemal doublet microtubules can be fractionated into detergentresistant protofilament (pf) ribbons which are composed of tubulin,tektins A. B, and C, and 77- and 83-kDa polypeptides. Anti-tektinantibodies have previously been shown to label basal bodies andcentrioles in echinoderms, molluscs and mammals (Steffen and Linck1994, J. Cell Sci. 107: 2095-2105). The 83 kDa protein (Sp 83) from seaurchin S. purpuratu flagellar microtubules was SDS-PAGE/blot-purified and used to raise a polyclonal antibody which is specificfor flagellar Sp 83 on western blots. Sarkosyl fractionation andsubsequent immunoblot analysis of axonemes demonstrates that Sp83 is associated wholly with the detergent-stable fraction.Furthermore, immuno-TEM shows that Sp 83 is distributed along thelength of the pf-ribbon. Immunofluorescence of splayed sperm tailsshows anti-Sp 83 labeling of all nine outer doublets; interestingly itstains the basal body as well. To examine the possibility that Sp 83 isa centriolar component we investigated its presence in sea urchinegg cytoplasm. Sea urchin oocytes contain in their cytoplasm all thenecessary polypeptides to build at least 16 pairs of embryoniccentrioles (Sluder et al 1990, J. Cell Biol. 110: 2025-2032). An 83-kDapolypeptide recognized by anti-Sp 83 was found in egg cytoplasm byimmuno-affinity chromatography and immunoblot anlysis. Thecellular redistribution of this protein following fertilization iscurrently under study. This work is supported by NIGMS GM-35648and NSF-RTG DIR-9113444.

237

MICROTUIBULE ORGANIZATION CENTERS IN MAMMALIANRETINE ((U. Wolfir1n2 ad J.L. Salisbuy1)) lDept. of Biochmiy andMolecular Biolgy. Mayo Cliic Foundaon, R MN 55905;2ZooobiIntitut Univesitlt Karbruhe (T.H.), Kob r. 13, D-612iKasnruheGmay.

he neuronal retina in vaubrtes is a hily ordeed and active neurona network

with wel defined cellar ayer. Most dynamic process. in ceds or tissues areasocated with their qct kl ements In

neurons, nuicretubulen are at ci liated cels such asvertebrate photoeceptor ced (PRCs). the basl bodies ae myor microiubkr,l-lztonF-F(foas). _S_0fiaronc dshw tg

an ess_til cowpon of MTOCs for microube fonmation, is localized in the

ce.trosonws of cells of al layer of imeronal rein aMd at die basal bodies of

dt PRCs in bovne as well huna Hower, a positive an-gammnatubulinreacto wa also observed with he cdiay roedets orngiatg at te basal bodis of

dte PRCs. Adcivati of MTOCs in sectoned retina using cytoplasic extract of

XaKupus e reveals td naicr ub zato is resticted to

rnon- and basal bodis. No mi buie forma is inuoed at t gsm-tubulin roeets. In adion, te a atin expertnents rv

differences in tde abilit to induce the of micrctles betwenr u sm md bal bodie. Despe the smm b _f anti-gan_na-tubuin s_aini only a few mico were py ad th basalbodie in PRCs, whi in cosr Ilage micretnubakars we at the

r oso1es ofc of edher layes,j min ntly in gangon cells. TIis ates

a darift "molecu ar itio of both maor MTOC of the cel d basal bodyconple md te crosome. Support by dte DFG (Wo 548/1-1) and NIH(HD29366).

238

PIGM GRANULE TRANSPORT IN ISOLATED RETINALPIGMENTED EPiTHELIAL (RPE) CMLLS DOES NOT REQUIREMICROTUBULES, BUT IS BLOCKED BY CYTOCIALASIN.((C King-Sth, P. Paz and B. Burside)) D afMolculandCell Biology, Uni of Califri, Brkey,94720

RPE of teleost contain .Mrmbrsne-bound ani pignotga

which dispers into loe; apical proc or teintothe celbody,d m a112 - .TO At npos in RPE we

ha dv preparaions of toalprocesse extended away from the a

seollate video

apregate nstt rate,but dip e saa fas In

dpigumen grnanles in apical -atcesse arwe not statie,butdm'ctrfp mdSM--MLPeiu

RPEdtna co-aws had t i t k _ eWupwbkdd byf f i wie & lpr,odoWleof_ ssRPE ti IepX by Ig

oells with lOpM o Deete&pa omicrotubules from cells beoth

igmetoa to

10f1 oD veribly blocked net ag

i atd cells, but dicited rapd "htdin mOeIenta ofnd

pinent granules in bh di Our gest dttin RPE n n,mcro--bules may perform a c is reqired in

isolated cdls wher apical projectios are atded to the subst Thes

observatonisi that mntorasiswdtrnsp a s notrequired for pigmnt gule nlation R , pit ue

transpartapea to be pyblynmor.(Suppotdby 5S ad )

239

MOTILITY OF MICROINJECTED NATIVE AND FOREIGNPARTICLES IN THE SQUID GIANT AXON. ((M. Terasald*, A.Schmidek*, JA. Galbraitht, PE. Gallant*, and T.S. Reese')) MarineBiological Labs, Woods Hole, MA 02543, 'Lab of Neurobiology,NINDS, NIK Behesda, MD 20892, tDept of Bioenginering, UC SanDig, LA Jolla, CA 92093.

In der to ivestidgate transport of cytsolic components of axoplasm,native and forcip fl particles were microinjected into freshlydistd squid gat axons on the stage ofa confocal micpe (BioRad).At low magnification, the fluorescence associated with rhodamine

phaldidn-i rabbit muscle actin filaments moved anterogradely.Short pieces of rhodamine conjugated, taxol-stabilized cow brainmhrobl al moved aneogely. Athigermasificai individualmim ;bul fiugms made stozy, neo moen at an avof 1 to 6 pwnmin When Tex red conjugates of soluble axopa c

prtins we injected, flu aegates moved anterogradely at acompable Tbes resutTs suggest dacertain naiv coenpoent theaxoplasm can be movedan y inp le form by a commonaxoplasic mech which to be defined. It is possible that

movements are related to the mcanim in axoplasm reported tomove vaous anionic ptculate in crab axons (Adams and Bray, Nature303:718). In squid axons, anionic latex polystyre beads ranging in size

from 10 mJ to 500 em in diaumer moved nterogradely in linear tract,while no movement was observed using beads lager than 500 em.Lae

anionic flu ntly labeled dexts (500 kD to 4000 kD) also moveddeyin tracts, while a neutral dextran (500i), and smaller

aniomc dexrans (10.70kD) diffud miforny in all dicions.

41a

Intracellular Movement (240-245). Sunday240

ACIIN DYNAMICS IN THE SEA URCHIN EGG. ((M. Terasaki))Marine Biologic:al Labs, Woods Hole, MA 02543, and Lab ofNeurobiology, NINDS, NI, Bethesda, MD 20892. (Spon. by L.A. Jaffe)

Sea urchin eggs (L. pics) wae injected with rhodamine (Rh) phalloidin(final 1.2-2 and ob ved byconfocalLabeld flaments orbuds (- 2-10 pm) underwent shifking motions aswell as ans at about 8 pm/minL CytochalasinB (1 caused the

oir f the fi indicating that this phalloidin concentaiondoes not irrevesibly stabiliz actin filamns in the egg. Atfrlization, thefilets became immobile dming the Ca wave, then resmed motion andbecame fAgmented tD shrter lengths. Lengtning filamen were observedatthe fertilizaon cone and at the cell cortex. At the dmt ofpronuclearmigration, filaments at the cone release and follow the sperm astr andpronuclus. Cortical filam ts also relase and travel in the geneal directionof the spem astr. The Rh phalloiin injected eggs undergo apparnynonalceavage; peliinary rm did not deecta stridng change in

te cleavage furrow. Since Rh phaloidin can alter actin filament assemblyand disembly in vitro, it is not lcnown to what degree the labeled filamentsand theirbehavorare normal. In Spportof the valdity of theseobservation, actin in the frdizadon cone and the lengtheing of corticalfiamens have previously been obsered by elern microscopy,cytochalasin causes disassembly, and early development appears normaLPossible candidates for the substrat for acdnmo are the ER and/ormiciotubule

242

PARTICLES MOVE ALONG ACTIN FILAMENTS IN NERVE GROWTH CONES.((L.L. Evans and P.C. Bridgman)) Dept. of Anatomy and Neurbiobgy,Washington University School d Medicine St. Louis, MO 63110.

Organele movement alng actin filaments has been demonstrated in dissociatedsquid axoplsm (Kurznetsov at al., Nature 356:722-725), but has not been shownto occur in neurons. We used video-enhanced differential interference

contrast (DIC) miroscopy to observe particle transport in cutured superiorcenical ganglon growth cones. To determine I this particle movemert oocursalong actin, we then used fluorescnce nmirosopy to correlate this movementwith nicrotubuile and actin distributions in the same growth cones. Particles,ranging In size from <0.5-1.5prn, moved In the growth cone pehey along linear

structures (termed transport bundles) at an average rate of 150nmlsec. ThelIklood d observig particle movement inceased after treating cells with a lowconcentration of cytochalasin, which had no detectable effect on the transportbundles. This treatment presumably decreased the density of the periphealcytoplasm, thereby alowing particls easier access to the perphery. Particlemovements were sometime halting, as when a particle encountered obstacissuch as bundle intersections or other particles occupying the same bundle; onencounterig obstacles, particles often stopped and reversed direction. Particlsfrequently moved into the growth cone periphery from the thick, central region dthe growth cone, where organele traffic (pre ably microtubule-bsed) is rapidand abundant. Occasinaly partiles also appeared to form from andVor fuse withthe lading edge of the growth cone, indicating that they may participate inrecycling of materials required for extension. After fixation of samples viewed byDIC, cells were labeled with rhodami phalloidin and an antibody to tubuin. Theobserved trnaport bundls cobocalzed with actin, but not microtubules. The ratesof partkie movement, and the association of moving particles with actin filamentbundles, suggest that myoeis partiipate in the transport of organeles (or othermaterais) in living neurons.

244

MICROTUBULEDEPENDENT ORGANELLE MOTILiTY IS LIMITEDBY ATP METABOLISM IN CELL EXTRACrS. ((K. Fang, G. Nakhuda,B. Berry, and D.B. Murphy)) Dept Cell Biology and Anatomy, JohnsHopkins Medical School, Baltimore, MD 21205.

Acanthameba contains significant amounts of kinesin and dynein. Lowspeed extacts, pr red by homonizaon in 10 cell pellet volumes of 15mM imidazole, pH 7.5, 1 mM MgCl2, 2 mM EGTA, and 0.6 M mannitol andcentifugation at 300 x g for 5 min, usually exhibit robust motility oforganeLes on microtubues (Mrs) in vitro, but in some prpations themotility is coderably reduced. In inactive prearons, organelies bind butdo not move on MTs, although some movements can be induced by additionof 1 mM Mg-ATP. To determine if ATP was the limiting factor, we measuredthe concenation ofATP in amebae and in extacts using a firefly luciferaseassay capable of detectng 1 nM ATP. Intact amebae contain 250 pM ATP;however, following lysis and homogenizaton the halftime for decay and theequilibrium concentration (detamined by fitting an exponential) are 5 min and2pM, respctively. This is tme regardless of whether 1 mM ATP is added tothe lysis bufer. Kinein-coated latex beads move in the presence of 1 pMATP, but no movement occurs at <15 pM ATP with 1 mM ADP, indiaingthat ADP is inhibitory at high concentrations. Pully half of the ATP-consuming activity in extrc is contained in the mitochondria. Althoughmitochondria exhibit MTdepeadent motility in vivo and associate with kinesinas detmined by immuofluno ence, isolated mitochondria do not moveefficienty on MTs in vitro. Calculations show that the rate ofATP depletionwithin a 1 urm radius of a mitochondion matches the rte of diffusion ofATPin the dum, sugengAgain tiat ATP concentradon may be a limitingfactor in motility. These observations indicate that ognele

motility in vitro is strongly affected by nuclotdde hydrolysis.

241

ACTIN-BASED M0lLITY OF ISOLATED AXOPLASMIC ORGANELLES.((JA DeGorgis, T.S. Roese, and E.L. Bearer.)) MBL at Woods Hole, MA02543, Brown University, Providence, R.I., 02912, and NINDS, NIH,Bethesda MD 20893.

A 235 kDa myosin is the pedinant myosin in K-washed ognellefacti from the squid giant axon as tested by Wester blottng with a pan-myosin andbody. By inmu ochemistry, this myosin is on the surfacesof KI-washd organelles and, thus, is proposed to be a motor for movementof organees along actn flmets (Beareet aL 1993). Here,wc amine

the motlity and other acdvides of the KI-washed andles KI-washedfrom squid axoplsam wee isolated by bigh speed centrifugadon

over a sucrose density adient. To detrmine whet the myosin on these

org< es was funcidonal, we measured theacd n Mg-ATPaseactivit of each facdon of the sucrose gradent andc d the reslts afternomizing fortotal protein content. The ognelle fon contained thehighest specific acdvity. We then tested whetherKI-washed oranmes bindactinflaments byinubatingoraneles with acdn plumes nueated off theends ofacsomal processes, and observing the interacdons of the oranelleswith actin by video and election microscopy. In the absence ofATP, actnfilaments wee heavily decorted with organelles, while in the presence ofAT, vimrally no orgnelles were found stuck to the filaments Actn-basedmodlity of theseorpnees was tested in the video assay-hey movedvigoroudy in only one direction (1.114 +/- .03 um/sec) on actin filametThus, KI-washed organelks retain vigorous ATPas and motility actvitywhich is atibutable to the 235 kDa myosin on their faces. Supported byCIM Grant #3192, and NIH GM47368.

243

AXONAL TRANSPORT OF MITOCHONDRIA ON MICROTUBULES AND F-ACTININ VIVO. ((R.L Morris & P.J. Hollenbe)) Dqe of Neurobiolog and Program inBbbgia & Bbmedicl Sdences, Harvard Medcal School, Bosion MA 02115.

Mitochondria in axons move bidirectionally at fast transport raes consistent withther mot being microtubule (MT)-based, but ev that organeles can moveaong scUn microfilaments (MFs) In vitro has rased questons about which

flmet suppout cgql movement hi vivo. To identify which flmentstm(s) mnilochoncia can utlie hI vMv, we have ten two appo ahes. First, wegrewchlck synqalheic neurons the presence of eIer (i) cylchasinE to create neurs which laked MFs, or (li) noodazole or vinblastine to produceneuxle whIch cd MTs. M itochorKa movedbronay at noma velocintalong the ere of neua which contaned MTs but lacke MFs. In oortradilochoiKriacid notevn enrneur i Ich had ways lacked MTs but cont

MFs. In a sond approch, we tated neuroe culures with cqtokel-

etal drugs o disrpt eiter MTs or MFs in an which already contained nmchon-drlaalong their lengths. Before drug treatment, mchondriamoved bldrectlonalyat a ide rae ofveies lo poduce net anterogede movemt In cylochalasi-treated cells, which contain MTs but lack MFs. average m lochondra velocthxxas in both die,os allog notbof decrsd. In vn0sieraecel., lacitng MTs but containn essentialy norfnal ieels of MFs, mochondla

contined lo move bidlraclondy. eiraveragevaoly was reduced and wa shimlin the anterograde and rtogradedIect but fte mlit nra spent 3-fold as

much Ime ov n the reograde as in the anIteograe diorecin, resuing in netrtograde trnsport Thus, neurltes grown without lack mle because,in fe absence of MTs, their movements on acUn are primariy directed tod the

celi body. Ourdataindicatehatvesbrate mo n acan move on both MTs andMFs hi vft, andqune dal suggest at rapid m ments are MT-based whieat least a fracon of slow milochondria movements are actin-based.

245

MICROTUBULE-DEPENDENT ORGANELLE MOTlLITY INACANTHAMOEBA. ((O. Baumann, B. Kristo, K.D. Fang, and D.B.Murphy)) Dept Cell Biology & Anatomy, Johns Hopkins Medical School,Baltimore, MD

Kinesin- and dynein-dependent organelle motility along microtubules (MTs)is robust in amoeba extracs, and Linesin bas becn biochemically puified fmrmthis cell type, indicatng amoebae as an ideal model system for studies ofMT-dependent organelle transport The identities of the organelles exhibitingmovement are as yet undetmined, although we previously reported thatmitochondria appear to be associated with kinesin and dynein as detemin&t

by immunofluorscence microscopy. However, isolated mitochondria movepoorly in vitro, whereas organelles from microsomal fractions exhibit activemovements. We therefore examined organelle motility in vivo by video-enhanced DIC microscopy and report the following observations: (1) Inspiny, relatively buoyant amoebae, mitochondria and small (<1 pm) vesicularorganelles exhibit frequent movements over distances of several micmetsand at a speed of -2 pm/sec. Organelle motlity is inhibited by nocodazoletretm (1-10p, indicating that it occurs along MTs. (2) MTs could bedirectly visualizd in flatned amoebae. MT are highly dynamic and emantein the form of an aster from a MTOC next to the nucleus and are associatedwith foci at the plasma membane. Nocodazole tatment elimints the MTs.(3) In flattened amoebae, bidirectional movement of vesicles, and in rarecases, mitochondria, occurred along the path of MTs. (4) The amount of invivo organelle movement depends strongly on the conditions of cell cultureand is most active in well ated cultures in rapid log phase growth

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Sunday. Intracellular Movement (246-251)

246

MICROTUBULE-BASED VESICLE TRANSPORT IN EXTRACFS FROMDICrYOSIEUUM DISCOIDEUM ((N. Pollock, E. de Hostos, J. Niclas, and R. D.Vale)) Department of Pharmacology, University of California San Francsco, SanFrancsco, CA 94122.

Dicyosteiumn dicoidum, a unicellular eukaryote tractable by both biochemicaland genetic memns, has been a productive system for the study of molecularmotos We have now made extracts from Dictyostdium which support highlevels of microtubule-based vesicle transport in vitro. Cultures of the axenically-grown strain AX2 were lysed by forced passage through a 03 im filter, and acrude mentrane fraction and a membrane-free high speed supernatant weresubsequently prepared. ATP-dependent movement of vesides along bovinebrain microtubules was stimulated by combination with the high-speedsupenatant. The directionality of movement was investigated by assayingvescle movement on bovine brin microtubules that had been nucleated off ofsea urchin axonemes. Vesicles moved in both directions along microtubules withrates rntging from 0.7-1.1 lm/s. Preliminary results suggest that vesicles in theendocytic pathway labelled by fluid phase uptake of a fluorescent dye alsonoved along microtubules. This in vitro assay may prove useful for dissectingthe components involved in microtubule-based organelle transport.

248

UNCOATNG OF HERPES SIMPLEX VIRUS - 1 AND TRANSPORTOF THE VIRAL CAPSIDS TO THE NUCLEAR ENVELOPE. ((B.Sodeik and A. Helenius)) Department of Cell Biology, Yale UniversitySchool of Medicine, New Haven, Cr 06520-8002

After binding to the host celL the membrane envelope of herpes simplexvirus 1 (HSV-1) fuses with the plasma membrane, thereby delivering thevial capsid covered by the tegument proteins into the cytoplasm In orderto determine the subcellular fate of the incoming viral capsids, we infectedVero and HeLa cells with HSV-l(;F) in the presence of cycloh de, andanalyzed them by SDS-PAGE, immunofluorescence and imunoelecton

microscopy. Polyclonal sea made against heavy, DNA containing capsids(anti-HC and against the major capsid protein VP5 did not detectextracellular virions bound to the host cell at 40C. After warming the cellsto 370C, there was a time dependent increase in punctate cytoplasmiclabeling, suggesting the removal of tegument proteins and the exposure ofcapsid epitopes. At 2 to 4 h post infection the majority of the labeledstructures, which most likely represent single viral capsids, hadconcentrated around the nucleus. Double-labelling experiments, usinganti-tubulin antibodies, showed that the majority of those capsids notlocalid to the nucleus wee co-localzd with microtubules and oftenaccumulated at the microtubue orgniing center (MDiC). Transport ofthe vial capsids to the nucleus was reduced by taxol and cytochalasin andalmost completely inhibited by nocodazole, colchicine, and vinblastine.Our data suggest that after HSV-1 fusion at the plasma membrane andremoval of the tegument proteins, the viral capsids bind to microtubuelesand are transported byh a minus-end d motor along microtubulesfrom the cell periphery to the MTOC and subsequendy to the nuclearpores

250

NUDF, A NUCLEAR MIGRATION GENE IN ASPERGILLUS NIDULANS, ISSIMILAR TO THE HUMAN LIS-1 GENE REQUIRED FOR NEURONALMIGRATION((X Xiang, A. H. Osmass*, S. A. Osmani*, M. Xin, and N. R. Morris)) UMDNJRobert Wood Johnson Medical School, Department of Pharmacology, 675 HoesLane, Piscataway, NJ 08854* Weis Center for Research, Geisinger Clinic, Danvile, PA 17822

Nuclear migration plays an important role in the growth and development ofmany organisms. In the filamentous fungus Aspergillus nidulans, nuclearmigration occurs during vegetative growth, as well as during conidiation(asexual spore formation) and ascosporogenesis (sexual spore formation). Itis known that the nuclear migration process in A. nidulais is microtubuledependent. We have identified several genes in which temperature sensitivemutations affect nuclear migration is A. nidulans. The audA gene encodes a

homolog of cytoplasmic dynein that may act as a motor for the nuclearmigrtion process. NudC encodes a 23 kD protein with no obvious homologyto any protein with a known function. During the cloning of ,sudC, a plasmidclone that partially suppresaes the mudC3 mutation was isolated. Thisplasmid clone has been identified as the audF gene. The NUDP protein leveldeclines in the sudC3 as well as the amdF mutants grown at restrictivetemperature. NsdF encodes a protein with P-transducin-like repeats mostsimilar to the Miller-Dieker lissencephaly gene (LIS-1) in human. Patientswith the Miller-Dieker syndrome show defects in neuronal migration anddie young. These respilts suggest that the sudF and nsdC gene productsinteract in some way and may participate in the regulation of the nuclearmigration process; and the LIS-1 gene in human may regulate neuronal

migration by regulating the nuclear migration process in migrating neurons.

247COLCHICINE INHIBITS TRANSCYTOSIS IN M-CELLS OFMOUSE PEYER'S PATCHES (A. Kracke and H. Bartels)Laboratory of Cell Biology and ElectronMicroscopy, D-30625 Hannover, Germany

N-cells transport soluble and particulateantigens across the dome-epithelium of Peyer'spatches (PP) by transcytosis. To investigate therole of microtubules in the vesicular traffic ofN-cells, native ferritin (NF) was injected intoisolated ileal loops containing PP two hoursafter Balb/c mice had received an intraperitone-al dose of colchicine (1.25mg/100g body weight)in 0.9% NaCl (n=6) or NaCl (n-5; control). PPwere fixed in glutaraldehyde and processed forthin section electron microscopy two hours afterexposure to NF. In controls, NF was present insmall vesicles, multivesicular bodies and thepocket-like invagination of the basal plasmamembrane of ca 60% of the M-cells. In contrast,only 16% of the M-cells in the colchicine-treated animals possessed NF-containing vesi-cles which were restricted to the most apicalcytoplasm. The results indicate that transcyto-sis in M-cells is microtubule-dependent.

249AN EXTRAGENIC SUPPRESSOR OF NUCLEAR MIGRATIONMUTATIONS CONFERS SENSITIVITY TO COLD ANDBENOMYL. ((D. A. Willins and N. R. Morris)) Dept. ofPharmacology, UMDNJ - Robert Wood Johnson MedicalSchool, Piscataway, NJ 08854

In the filamentous fungus AspergiUus niduikns, sporegermination is followed by nuclear division and migration ofnuclei outwards along the growing multinuclear germ tube.The nud (nclear distribution) mutants have a temperature-sensitive defect in-growth and nuclear migration. Severalkey participants in nuclear migration have been identified:microtubules, the nudA product (cytoplasmic dynein heavychain), and the nudF product (a G protein n-subunit-likeprotein). In order to identify additional participants in theprocess, we isolated extragenic suppressors of a nudFmutation. One nudF suppressor also suppresses the growthdefect of several other nud mutations, including nudAl,nudC3, and nudG8. This suppressor (designated snfA forsuppressor of nude confers sensitivity to cold and benomyl,both microtubule-destabilizing conditions. SrJA maps tochromosome VIII and is tightly linked to tubA, which encodesthe major Aspergilus a-tubulin. Together, the resultssuggest that a tubA mutation can suppress the growth defectof several nuclear migration mutations.

251IDENTIFICATION AND CHARACTERIZATION OFEXTRAGENIC SUPPRESSORS OF nudC, A GENE REQUIREDFOR NUCLEAR MIGRATION IN Aspergillus nidus . (( Ya-HuiChiu and N. Ronald Morris )) Department of Pharmacology,UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ08854

Nuclear migrtion is essential for proper growth, development andcellular function in both higher and lower eukaryotes. We haveidentified four genes which are involved in nuclear movement in A.nidulans. A temperature-sensitive mutation in the nudC geneprevents the microtubule-dependent movement of nuclei atrestrictive temperature. The nudC gene encodes a protein of 23 kDwith no obvious homology to any protein whose function is known.However, it does exhibit strong sequence similarity to expressedsequences of C.ekgans, Drosophila and rat, suggesting that it is aconserved protein. We also found that the homologous clone ofDrosophila can complement the nudC3 mutation in A. nidsdw. Tostudy the biological function of nudC, we isolated extragenicsuppressors of nudC to identify proteins that interact with the nudCprotein. Snc (Suppressor of wudC ) A-I represent 9 linkage groups.All available diploids, which are homozygous for the ts- nudC3mutation and beterozygous for the suppressor mutation, are ts+,indicating that they are dominant suppressors. Three cosmidlibraries have been constructed for SncA, B, C mutations. Byransforming theAt nidslds nudC3 stain with these libraries, one

clone from the SscB library has been shown to suppress the nudC3mutation and its sequencing is in progress.

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252"SELF-CENTERING" ACTIVITY OF CYTOPLASM.((V.I.Rodionov andG.G.Borisy)) Laboratory of Molecular Biology, University of Wisconsin,Madison, WI 53706.

Most eukaryotic cells have a prominent cell center with a centrosome whichserves as a nucleation site for radially arranged microtubules (MTs).However, evidence exists that establishment of the cell center does not stricflydepend on the eentrosome. Cytoplasmic fragments of fish melanophores,within several hours after severing, have been reported to establish a newfocus for the aggregation of pigment granules (McNiven and Porter, 1988, J.Cell Biol. 106:1593). In the prent work, we generated fragments of blacktetra melanophores microsurgically and examined their reorganization bycorrelated time-lapse video and immunofluorescence microscopy. Theposition of fragment centers was assayed (i) by analysis of the arrangement ofMTs as revealed by staining with -tubulin antibody; (ii) by location of thepigment aggregate induced by adrenalin treatment; and (iii) by antibodystaining for y-tubulin which has been associated with MT minus ends.Fragments showed several remariable properties: (i) a new center becameestablished quicldy after severing (15 min), consistent with the time scale ofMT turnover (tli2= 5 min); (ii) center formation apparently depended onmotor activity and pigment aggregation as microtubules did not becomeradially reanranged in fragments with dispersed melanosomes; (iii) centerformation depended on MT dynamics as determined by taxol inhibition; and(iv) centers lacked any detectable focus of y-tubulin whereas intact cellsshowed a prominent y-tubulin spot at the cell center. We conclude thatmelanophore cytoplasm is endowed with a "self-centering" activityindependent of the centrosome and y-tubulin. We present a two-step modelfor formation and positioning of a new center involving motor activity andMT dynamnics. Supported by NIH grantGM 25062 (GGB).Cytoskeleton-Membrane Interactions: Structure (253-256).

253CO-LOCALIZATION OF ANKYRIN AND Na+-Ca2+ EXCHANGEPROTEIN IN DEVELOPING RABBIT MYOCYTES ((Fuhua Chen, VictoriaY. Shin, Kenneth D. Philipson and Joy S. Frank)). University of California,Los Angeles, Los Angeles, CA 90024.

Ankyrin is a peripheral membrane protein which links integral membraneproteins to other elements of the cytoskeleton. Previous studies haveindicated that ankyrin is associated with Ca2+ transport proteins such as theDHP receptor and the Na+-Ca2+ exchanger (Na-Ca-X). In adult myocardialcells, the Na-Ca-X is abundant in the T tubular (T-T) membrane. Linkage ofthe exchanger to ankyrin could result in the initial placement of the exchangerin T-T membrane. Accordingly, indirect immunofluorescent studies wereperformed to localize ankyrin and the Na-Ca-X in isolated myocytes fromneonate (5 day old), which lack T-T, as well as juvenile (2 week old) andadult rabbit hearts. Cells were incubated with a polycolonal antibody (Ab)to ankyrin followed by FITC-labeled secondary Ab and a monoclonal Ab toNa-Ca-X followed by Texas Red-labeled secondary Ab. At 5 days of age,ankyrin labeling was mainly present at the Z-disc, while the Na-Ca-X wasonly present on the peripheral sarcolemma. In adult cells, co-localization ofthese two proteins occurred. At 2 weeks of age, the Na-Ca-X was expressedin partially developed T-T as was ankyrin. These results show that ankyrinis present at the Z disc before T-T form in the immature cells. Ankyrin isthus available to immobilize the Na-Ca-X during development of T-T. Thechange in distribution of the Na-Ca-X may reflect different interactions withthe cytoskeleton at various development stages.

255TRANSPOSON INSERTION MUTATIONS OF THE UNC-44 ANKYRINGENE IN C. ELLEGANS ((P. Boontralpoontawee and A. J. Otsuka))Department ofBiological Sciences, Illinois State University, Normal, IL61790.

Variou studies have shown that mutations in the Caenorhabits elegansunc-44 gene are associated with abnormal axonal outgrowth and guidance.OurDNA sequence data and Northern blot analysis demonstrate that the unc-44 encodes several ankyrin-related proteins. Ankyrins represent anintracellla protein family that mediate the interaction between cytoskeletaland tansembrane proteins. In analogy to the vertebrate ankyrins, theputative UNC-44 protein is composed of4 basic stuctura domains: anankyrin repeat domain, a linker domain, a spectrin-binding domain, and a C-terminal domain. Northern blot anablsis revealed that there are at least 5 unc-44t ript in the wild-type C. ekgns, ranging from 3 to 14 kb. Northemblot anaysis of3 mutants revealed that the 14-kb message is the onlytranucript affected in common. Therefore, it is hypothesized that the 14-kbtrscript is required for proper axonal outgrowth and guidance. Onetransposon insertion mutation ofunc-44 has been mapped to the ankyrinrepeat domain and five DNA insertions to the C-terminal domain. Twospontaneous uc-44 reversions analbzed are due to Tcl excisions resulting inwild-type DNA sequence, the other two are due to a 3-bp deletion and a 3-bpinsertion, rectively. Surprisgly, ww-44(rhl042) and st200 reverntscontain a second transposon inserted at a downstream exonrmtron boundarywhich, hypothetically, may alter the unc-44 splicing pattern.

254THE N-TERMINAL 79 AMINO ACIDS OF AEl (BAND 3) AREESSENTIAL FOR HIGH AFFINITY BINDING TO ANKYRIN((Y. Ding and R. R. Kopito)) Department of Biological Sciences,Stanford University, Stanford, CA 94305-5020, USA

The AEI gene is expressed in erythroid cells and in intercalated cellsof the distal nephron. In erythrocytes the AE1 gene product, band 3,functions both as a plasma membrane anion exchanger and, via high affinityinteraction with ankyrin, as an anchor for the spectrin cytoskeleton. Thelatter activity is associated with the NH2-terminal cytoplasmicallydisposed-420 amino acid domain of band 3. The principal form of kidneyAE1 (kAE1) lacks the 5' exons which encode amino acids 1-79 of theerythrocyte protein. kAE1 is located in the basolateral membrane of acid-secreting (a-type) intercalated cells. It has been proposed that interaction ofkAE1 with the spectrin cytoskeleton contributes to its polarized distribution.In this study, we tested the ability of kAE1 and band 3 to bind erythrocyteand brain ankyrins. Binding was assessed by co-immunoprecipitation ofdetergent-solubilized recombinant anion exchangers with radioiodinatedfragments of ANK1 (erythrocyte ankyrin or ANKR) and ANK2 (brain ankyrinor ANKB) containing the 33-amino acid repeat band 3-binding domain.Erythrocyte AE1 (band 3) associated with the 43 kD ANK1 fragmentbiphasically with high affinity (Kd=10-40 nM) and low affinity (Kd = 100-150nM) sites. Erythroid AE1 associated with 82 kD ANK2 fragment with at least10 fold lower affinity. In contrast, we could detect no significant associationof kAE1 with either ANK1 or ANK2 (Kd< 400 nM). We conciude that theN1-2-terminal 79 amino acids of AEI are essential for its high affinity andspecific binding to erythrocyte (ANK1) ankyrin. (Supported by postdoctoralfellowship 93-65 from the American Heart Association to Y.D. and by NIHGrant GM38543 to R.R.K.).

256DOMAIN STRUCTURE AND MEMBRANE ASSEMBLY OF DROSOPHIlAANKYRIN. ((RIL Dubreuil and M. Yau)) Committee on Cell Physiology,University of Chicago, Chicago, IL 60637.

We previously identified a 170 kD ankyrin-like protein in Drosophila. We now

report cDNA sequence analysis of the Drosophila ankyrin coding region. Thesequence encodes a 170 kD protein that is 53% identical to mammalian brainAnk2. The regions of greatest similarity to mammalian ankyrins are the amino

terminal repeat domain, that is asociated with membrane binding activity, andthe central speczin-binding domain. The carbowxy terminal domain offly ankyrinis shorter than known mammalian sequences in this region and exhibits onlylimited sequenee milarity to other ankyrins. Sequence differenees in thisdomain alone are sufficient to explain the difference in size between fly andmammalian ankyrins (170 vs >200 kD). The sequence conservation of themembrane- and spectrin-binding domains provides further support for a role ofDrosophila ankyrin as a membrane-cytokeleton connector, analogous to its rolein mammals. Mutations of a spectrin are lethal in Drowphila, althoughembryonic and early larval development seem only mildly affected. In addition,a marker of eell polarity (the sodium pump) was not detectably altered in a

spectrin mutants, challenging the proposed role of the membrane skeleton indevelopment of cell polaibty. We used an antibody agaunst the carboxy terminalregion of fly ankyrin to study its fate in spectrin mutants. We previously

showed that ankyrin and 0 spectrin continue to accumulate in a spectrinmutants. We now show that the accumulated ankyrin is present at the plasmamembrane of several cell types ofboth mutant and wild type first instar larvae.The independent assembly of ankyrin in a spectrin mutants raises thepossibility of partial membrane skeleton function in the absence of a spectrin.Genetic studies of other membrane skeleton components such as ankyrin willbe important to further evaluate the role of the membrane skeleton duringdevelopment.

Intracellular Movement (252). Sunday

Sunday. Cytoskeleton-Membrane Interactions: Structure (257-262)257

MINIMAL ANKYRIN-BINDING DOMAIN ON Na,K-ATPase. ((P.Devarajan, A. Dorfman, J.S. Morrow)). Department of Pediatricsand Pathology, Yale University, New Haven, CT 06520. tspabyJMi.)

Interactions between ankyrin and Na,K-ATPase are crucial to themaintenance of epithelial cell polarity. We have shown that thisinteraction is mediated by the 150-residue second cytoplasmicdomain of Na,K-ATPase a subunit (PNAS 1994, 91:2965-9). Forthis study, we used PCR to construct a series of a-Na,K-ATPasedeletions, in order to identify the minimal ankyrin-binding domain.The deleton mutants were expressed in bacteria, purified with abioaffinity column, and tested for binding to both red cell ankyrinas well as to an ankyrin isoform in Madin-Darby Canine Kidneycells, using an in vito solution binding assay. A 25-residuepeptide, corresponding to codons 140-165 on a-Na,K-ATPase,was found to retain much of the specific binding to both ankyrinisoforms. This active sequence is highly conserved within allknown a-Na,K-ATPase isoforms and species, but is absent fromall other known ankyrin-binding proteins. This suggests that theinteraction of ankyrin with membrane proteins may involvecomplex tertiary structural requirements not deducible from theprimary sequence, and that a singular binding site on Na,K-ATPases for ankyrin isoforms has evolved by a uniqueevolutionary pathway. The impact of this interacton on themaintenance of Na,K-ATPase polarity in epithelial cells is beinginvestigated.

259ANK-3 ASSOCIATES W1TH LATE LYSOSOMES IN MOUSE MACROP-AGES ((T. C. Hoock*,L L Peters*, and S. E. Lux*)). Div. of Med. Sci., Program in Biol. and Biomed. Science,Harvard Univ., Boston, M& *Div. ofHematol./Oncol,ChildrenYs Hosp., Boston,

nyrins have been charactenzed as a fanily of plasma membrane-associated proteinsthat link irtegral membrane ionhannels to the underlying membrane skeleton. There

are now three members of this farily: ery cyte ankyrin Nk-l brain ankyrin Ank-2)and the recery identfied epithelia/axonal ankynn WMk-3) (Peters et al., Blood 80:272a,1992). To further caatenize the expression and localization of Ank-3 we conductedimmnunofluorescence, westem and northern blottng experiments in mIacr)phages. Usingantibodies directed against the reguatory dorain, we show hat Ank-3 surrounds phasedense intracellular vescles throughout the nmaorphage cytoplasm. These vesides are

phase dense lte somes (PDllJ based on two cnteria: I they are acidic in nature,

deterrmined by acridine orange stairing, and 21 they are late in the endosomal pathway,revealed by Ank-3 antibody colocalizing with fluorescence from endocytosed, FITCtagged dextrans, only alter a 24 hour chase penod. Westem blot analsis shows twoisoforms of Ank-3 in macrophages: 100 and 70 kD. Based on high stringency northernblot analysis using domain specific probes, two messages of approximately 3.3 and 4.8kb are made, both are varants that lack the Nerninal 89 kD repeat domain. Anantibody directed against the predicted N-terrinus of this truncated form of ankynn alsostains PDLL while an antibody directed aganst an alternativy spiced exon that liesbetween the 89 kD and spectnn binding domarins fails to localize to PDLL Macrophagesused in these experiments were differentiated ix vifo from bone marrow using L-cell

conditioned media, and the growth rate or the cells inversely correlated with theappearance of PDLL In contast, neither stainng nor Ank-3 mlNA were detectable inthe immortal J774e macrophage cell line. In condusion, Ank-3 is exressed inmacrophages and witn these cells appears to be associated with the intracellularmembranes or PDLL This Is the first demonstaon o the presence of an ankynin on this

intracellular membrane and suggests that the varnants oAnk-3 missing the N-terminal 89kD repeat domain play a role in the stability or physiology of PDLL

261

MAPPING THE INTERACTION BETWEEN a-ACTININ AND THECYTOPLASMIC DOMAIN OF 1 INTEGRIN. ((A.P. Gilmore, C. A. Otey#,D. R Critchley*, and I Burridge)). Dept. Cell Biol. and Anat, UNC at ChapelHill, Chapel Hill, NC; #Dept. Cell Biol., Univ. Virginia, Charlottesville,VA;*Dept Biochem., Univ. Leicester, Leicester, UK

Focal adhesions are regions where the actin cytoskeleton is anchored to thecell membrane via transmembrane adhesion molecules of the integrin family.The assembly of these structures is important for both cell adhesion andadhesion-mediated signal transduction. We have investigated the role of theactin bundling protein a-actinin in linking actin microfilaments to thecytoplasmic domain of . It has previously been shown that the 1 integrincytoplasmic domain binds to a-actinin via its central spectrin-like repeats(Otey et al., 1990). We have used bacterially expressed GST/ct-actinin fusionproteins in a solid phase assay to test the ability of individual repeats to interactwith the P1 peptide. These results suggested that all four of the repeatscontained sequences which allowed binding to the peptide. These experimentswere initially carried out using a-actinin repeats which were incorrectlyphased. We have recently correctly defined the phasing of the four spectrin-like repeats in a-actinin (Gilmore et al., in press), and these also all bind to the0 1 peptide in vitro. To investigate any correspondence between in vitrobinding to the peptide and possible binding to A1 integrin in vivo wemicroinjected the fusion proteins into REF-52 cells and localized the injectedprotein by fluorescence microscopy. Localization to focal adhesions onlyoccurred with a fusion protein containing all four repeats in tandem. The fourrepeats are required for dimerization in native a-actinin, and this dimerizationmay be required for it to interact with native P1 integrin. We are currentlyinvestigating this by testing the ability of the repeat fusion proteins to bind tointac purified integrins. Supported by an EMBO fellowship (APG) and NIHgrants.

258

MECHANSM FOR THE GENERATION OF BINDIIYG SITE DIVERSIr ON THE

MEMBRANE BINDING DOMAIN OF ANKYRIN. ((P. Michaely1 and V.

Bennett1,2)) Departments of Biochemistry1 and Cell Biology2, HowardHughes Medical Institute, Duke University Medical Center, Durham, NC27710

Ankyrin is involved In immobilizing several membrane proteins atfunctionally important regions of the plasma membrane. At the node ofRanvier, for example, ankyrin is believed to contact the voltage-gated Nachannel, Na/Ca exchanger, and NatK ATPase. Other neuronal binding sitesfor ankyrin include members of the diversely iocalized Ll/neurofascinfamily of cell adhesion molecules. The sites for all of these targets onankyrin appear to require the N-terminal 'membrane binding' domain.This region Is dominated by a tandem array of 24 ankyrin repeats whichare organized into four foding domains of six repeats each. The foldingdomain organization Is surprising since more than four independentmembrane targets have been identified. How this diversity of sites isgenerated by the four repeat domains was examined using a panel of repealdomain constructs and two ankyrin binding membrane proteins; theCVHCO3 anion exchanger and rat neurofascin (ABGP186). Using directbinding assays, the CVHCO3 exchanger was shown to require both repeatdomains three and four for high affinity interaction with ankyrin.Neurofascin required primarily repeat domain two aithough slightlyhigher affinity was seen with a construct containing both repeat domainstwo and three. An additional site for neurofascin was seen with aconstruct containing both repeat domains three and four. Diversity ofinteractions with ankyrin thus appears to be achieved through acombinatorial mechanism wherein Individual domains can act eitherindependently or in unison with other repeat domains to form sites.

260

IT1ERACTION OFTHE CYTOPLASMIC DOMAIN OF ICAM-2 W1TH a-

ACTININ ((L. Heiska, O.-M. Mykklnen, C. Kantor, C.G. Gahmberg, and

0. Carpen)) Deparanents of Pathology and Biochemistry, University of

Helsinkd, Helsinki, Finland

ICAM-2 is a cell surface adhesion molecule that functions as a counter-

receptor for LFA-1 (CD1 la/CD18). It is mainly expressed on endothelial cells

and cells of lymphoid origin. We have studied the intracellular interactions of

ICAM-2 by synthesizing a peptide encompassing the 26 amino acid cyto-plasmic domain of ICAM-2 and testing the ability of various proteins to

interact with the immobilized peptide. We found that purified a-actinin, an

actin-binding cytoskeletal protein, specifically bound to the cytoplasmicdomain peptide. No interaction was seen with ca-actinin and peptides of

similar length containing sequences from the exctracellular domain of ICAM-2

or from other irrelevant proteins. The interaction could be competed with

excess cold cz-actinin, it was not dependent on divalent cations and occurred

in the presence of IM NaCl. Denaturation of a-actinin, on the other hand,totally abolished binding. A further indication of a direct interaction between

ICAM-2 and a-actinin was obtained with the yeast two hybrid/imteraction trapsystem. Yeast cells co-transfected with the bait vector/ICAM-2 cytoplasmicdomain sequence and the pray vector/a-actinin plasmid demonstrated an

ability to grow on leu-selection plates and formed blue colonies on X-Gal

plates. The results, together with previous studies demonstrating an

interaction between a-actinin and ICAM-1, PI-integrins and CD18 emphasizethe role of a-actinin as a linker between cell surface adhesion molecules and

actin-containing cytoskeleton.

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AVIAN ZYXIN LOCALIZES TO ADHESION PLAQUES IN RAT EMBRYOFIBROBLAST CELLS. ((D.A. Nix and M.C. Beckerle )) Department ofBiology, University of Utah, Salt Lake City, Ur 84112.

Zyxin is a low abundance phosphoprotein that is localized at adhesionplaques and the terminal portions of actin stress fibers. The protein is 542aain length and exhibits an unusual proline-rich N-terminal domain (aa 1-348)and three tandemly arrayed protein binding interfaces called LIM domains (aa349-542). In order to determine which domain of zyxin contributes toadhesion plaque targeting, we exprssed various portions of avian zyxin in arat embryo fibroblast cell line (REF-52). Specifically we microinjectedeukaryotic expression constructs containing zyxsn cDNA sequences and usedindirect immunoflourescence to visualize both the expressed avian sequencesas well as endogenous rat zyxin in single cells. The expression and sub-cellular localization of avian zyxin sequences were detected by an avian-specific anti-zyxin antibody or by epitope-tagging the avian zyxin sequences.A pan-zyxin antibody was used to detect the total complement of zyxinpresent within each cell. Using this strategy, we determined that full lengthavian zyxin localizes to adhesion plaques of REF-52 cells in a manner that isindistinguishable from the pattem of endogenous zyxin. This illustrates thatthe sequences that target zyxin to focal contacts as well as the correspondingdocking sequences are evolutionarily conserved. Deletion of 244aa from theN-terminus of zyxin fails to affect the protein's ability to localize to adhesionplaques. However, removal of the entire proline rich domain (aa 1-348)essentially eliminates adhesion plaque localization. Likewise, successivedeletion of each C-terminal LIM domain produces a graded reduction in thepercentage of transfected cells displaying adhesion plaque localization andresults in an increased cytosolic distribution of the avian protein.Identification of the region of zyxin that targets it to adhesion plaques shouldallow us to develop reagents that specifically perturb zyxin localization andfunction in vivo.

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Cytoskeleton-Membrane Interactions: Structure (263-268). Sunday

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THE FOCAL ADHESION PROTEINS ZYXIN AND VINCULIN ARELOCALIZED AT BASAL LATERAL MEMBRANES IN EMBRYONICAVIAN CORNEAL E1PTHELIA. ((jlirawuthiworavong, KLKH. Svoboda))Det of Anatomy and Neurobiology, Bostn Univ. Sch. of Med., Bost MA.

Zyxin and vinculin are components of focal adhesions. It has also beenshown that zyincolocalizes with a-actinin at cell-cell adhns juncto of

cultured pigmented retinal epithelial cells (Crawford ct aL, 1992. J.C.B.116:1381-93). The 6-day embryonic Avian corneal epithelia, composed oftwo layers, can be isolated as a sheet of cells with (+) or without (-) basallamina (BL). Epithelia isolated -BL have F-actin localized in basal cellcytoplasmic blebs. These blebs respond to ECM molecules by reorgnizingF-actin into a filamenious netwodr, the actin cortical mat (ACM). Epithelia

isolated -BL and cultured with laminin (LM) and cytochalasin D (CD),contain diruped F-acdn distibuted ino aggregates. We asked: What is theintracellula distribution of zyxin and vinculin in embryonic Avian corneal

Dozyxin andvincuincolocaliz withF-actinmcytoplasmic blebs,in LM induced reorganized ACM, or CD induced actin apregates? Inepithelia isolated +BL, zyxin and vinculin were loalizd to discrete areas inbasal lateral mbranes. InLM tred -BL epiti, zyxin and vinculn hada definite polarized distrbution along lateral membranes in apical and basalregions. At the level of the ACM, zyxin and vinculin were distibuted nearlateral cell membranes at the ends of actin filaments. In CD teated cells,zyxinremaiedpunctate dtrgh the epitheli. We eport for the first timethat zyxin has a polarized indicating focal adhesions are located

on lateral cell membranes in this whole epithelial tissue. Our whole tissuemodel can be used to elucidate the role and function of zyxin and odter actin-associatedprotins during ECM stinmulatedrse anizaton

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AN av INTEGRIN ASSOCIATED PR07EIN COMPLEX LOCALIES TOTHE Z-LINE OF SKELETALMUSCLE AND CONTAINS THE ENZYMEGAPDH. ((ILA. McDonald, J. Muschler, ML Lakonishok, A.F. Horwitz))Deuarent of Cell Biology, University of Mlinois at Urbanaa pnpaign,Urbana, IL 61801.

Integrins are mane adhesion molecules that link the extracellulrmatrix to the cytoeleton Previou s dies of thec yeetal molcules withwhich integrins interact have focussed on adheion plaques in fibroblasHowever we find that different intgrins localiz in distinct junctional regionson skdell muscl. For example, the c7 integin subunit localizes in the

myotendious junction, the av integrin subunit in the costanee, and the 05

intin subunit in the adhesion plaqu. Taken together tis argues strongly fornovelcyosetal-ingrin ling To ideiy novel integrin binding proinswe produced MAbs agat molecues which co-purified with integrin fromadult tissues. Thbis apprah poduced a MAb, P4B2, which _in ies acomplex of proteins migrting atmoleclarwet pimnarily of 37,27 and 18kD from both adultchickn brain and Aeleal nmscle. By westen blot analysiste P4B2 antigen contanms av integrin and 81 integrin, but not 015, oa6 or o7integrin subunits. Intnal anino acid quence analysis of the mpjor protinsidentified the 37 kDpotein as gly dahye-3-phoatdeydrogenae(GAPDH). The oth pepronsa to be noveL The P4B2 antibody stains

pronnntiyinneural tissue and localizes at the Z-lin in striated muscle. Both81 integrin and GAPDH have previously benr ported to localize to the Z-line.A MAb directed against the cxv intgrin also co-locaizes in the Z-band ofmuscle and purifies somc of these molecules, e.g., GAPDH. Tbeseobsevations point to a novel integrin juntonal structe.

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ASSOCIATION OF DYSTROPHIN WITH THE SARCOLEMMA ANDCYTOSKELETON IN CARDIAC AND SKELETAL MUSCLE.((T.J. Byers', D.R. Swartz", K.S. Sobieck')) 'Department ofPhysiology & Biophysics and 'Department of Anatomy, IndianaUniversity School of Medicine, Indianapolis, IN 46202.

In purified sarcolemma vesicles, dystrophin associates tightly witha complex of membrane proteins and glycoproteins (conditions suchas 2 M KI or pH 11 are necessary to disrupt this interaction), andbinds to the Triton X-1 00-stable cytoskeleton. We have exploredthe biochemical partitioning of dystrophin into fractions extractedfrom whole tissue and crude microsomes. From whole tissue, 40 -

60% of dystrophin was extracted with 1 % Triton X-1 00, and asimilar amount was extracted in low ionic strength buffer at pH 9.0,37eC. Lesser quantities were extracted from microsomes underthese conditions and there were quantitative differences betweencardiac and skeletal muscle tissues. These data indicate a pool ofdystrophin in whole muscle that is not tightly associated with theglycoprotein complex. In addition, a significant pool of dystrophinremains tightly associated with the myofibrillar cytoskeleton (- 40%)after detergent extraction. By immunofluorescence microscopy thisdystrophin is present on a subset of myofibrils, and appears on oneface of the myofibril surface. Distinct pools of dystrophin withdifferent solubility properties may indicate differences in dystrophinstructure and/or differeeices in interactions with other proteins.

2"CHANGESINTHECYTOSKELETON-ADHESIONCOMPLEXDEFINEDISTINCT STAGES IN THE DIFFERENTIATION OF LENS FIBERS.((D.C.Beebe, JD. Potts and S. Bassnett)) Department of Anatomy and CcllBiology, USUHS, Bethesda, MD, 20814-4799, USA.

Lens fiber cells have a prominent cytoskeletal-adhesion complex Previousstudies have identified several components of this complex, including N-cadherin, a-actinin, spectrin, ankyrin and F-actin. We used immuno-cytochcmistry and western blots to define the organization of the adherenscomplex during lens fiber differentiation in chicken embryos. N-cadherin, atransmembrane cell-cell adhesion protein, was present on the lateralmembranes of elongating coricl fiber cells. N-cadherin staining disappearedabruptly as the fiber cells completed elongation. A similar pattern ofexpression was seen using an antibody to band 4.1, a molecule that links

trananembrane proteins to the cyto&keleton. Viulin and pailin, which arefound in cell-cel and cell-substrate adhesion complexes, were not detectedon the lateral membranes of elongating fibers However, once fibers reachedtheir maximum length, vinculin and paxillin staining appeared along thelateral membranes. The appearance of vinculin and paxiln coincided withthe loss of staining for N-cadherin and band 4.1. Western blots ofmicrodissected lens slices confirmed the distribution of these antigens in fibercells at different stages of differentiation. These studies reveal a majorreorganization of the cytoskeletal-adhesion complexes in lens fibers as theseceDls reach maturity. Increased expression of vinculin and paxillin appears tobe a late event in fiber cell maturation.Supported by NIH EY04853 (DCB) and EY09852 (SB).

266A POSSIBLE ROLE OF INTEGRIN a6p1 AT THE CONTRACTIONFORCE TRASMITTING SITES OF CARDIOMYOCYTES.((K.Imanaka-Yoshida, *M.Enomoto-lwamoto, T.Yoshida, *F.Suzuki,and T.Sakakura)) Department of Pathology, School of Medicine,Mie University, Mie 514, *Department of Biochemistry, Faculty ofDentistry, Osaka University, Osaka 565, JAPAN.

Recently, we have reported that vinculin and talin form attachmentcomplex at costamere of cardiomyocyte, which transmitscontraction forces from myofibrils to the extracellular matrix. Thepurpose of this paper was to determine if integrin, another majoradhesion protein, is involved in the adhesion complex. Wecultured adult rat cardiomyocytes on laminin coated coverslips. Onthe 8th day of cuiture, the coverslips were incubated with a non-

penetrating cross-linker Bis(sulfosuccinimidyl)-suberate, thenextracted with 0.1% SDS, to separate only the integrin moleculesinvolved in substrate adhesion. The slides were fixed and stainedwith either one of anti-a3, a6, av, or p1 integrin antibodies. In thearea previously occupied by cardiomyocytes were, a6 and p1

showed striated distribution whose periodicity coincides with Z-lines, which is characteristic of costameric attachment. Whereas,a3 was distributed as tiny dots and av demonstrated arrow headssimilar to adhesion plaque of fibroblasts. These findings suggest

that integrin a6p1 is involved in the attachment of cardiomyocytesto extracellular matrix at costamere and could mediatetransmission of contraction force.

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SITE DIRECTED MUTAGENESIS OF THE 1tCALPAIN CLEAVAGE SITE IN alISPECTRIN. ((Paul R. Stabach, Susan Glantz-Tuschman, and Jon S.Morrow)) Yale University School of Medicine, Dept. of Pathology, NewHaven, CT 06510. (Spon. by J. Morrow.)

The ceavage of proteins by calcium activated neutral proteases such as{I-Calpain is both ubiquitous and hliyh specific. While the precise roleof these enzymes remains enigmantic, they are beleved to play animportant role in mediating cytoskeletal changes during synapticremodeling, and may be excessively activated in cells subjected toschenic stress. One inmortant target of p-calpain action is the al (non-erythroid spectrin or fodrin). In vitro and In vivo, j-calpain cleavesthis subunit of spectrin betwen tyr104 wa gy105Of the 11th repetitveunit of aln spectrin (Harris et al., JBC 263:15754, 1988). Thiscleavage is highly specific, despite the occurance of presumablysusceptible X-tyr and X-arg sequences at last 35 times In anI spectrin.In order to better understand the site specificity of 1i-calpain forspectrin, and as a first step to preparing transgenic mice with p-calpainresistant spectrin, site directed mutatgensis has been used to preparerecombinant alI spectrin with 20 dfferent amino acid substitutins at the-2 position Each of these were evaluated for their susceptibility to -

calpain using in vitro ckavage ssays. The rplacemet of the wt valine atthe -2 position with glycine, proline, and arginine abolishedsusceptibility. Several other amino acids yieded reduced susceptibility,aithough overal a surprising range of substution were well tolerated at

this position without substaintive loss of cleavabity. These studiessuggest that secondary and tertiary structural factors resident inproteins, and difficult to evaluate in synthetic peptides, may be theprimary deterrnnants of -calpain specificity.

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Sunday. Cytoskeleton-Membrane Interactions: Structure (269-274)269

ACTIVATION OF CALPAIN MODULATES ACTIN,-ASD CYTOSKELETON INGASTRIC PARIETAL CELLS. ((X Yao, S. Olha, A. Thlbodeau and J.G. Forte,Dept d Molcular and Ceal Blgy, Univ. dCaforia. Berkey, CA 94720)).

Gasbt ezrin, a putave protein, has been inplbated in te

8 k1 wHvgu1W mmrnremnodeling d gaatric parlet cel. Our prevkouswork has establahed an ezrin-calpain refaonahip (Am. J. Physl. 265:C36,1993). To tet the effect da rise in intracellular Ca2+ (Ca2+)# on the acn-ezrItion, we carried out NP-40 extraction folowing the elevation d [Ca2+Jiin thepreeec of capain inhbitor I (Cl). The aaodation d ezrin with actin-baaedcytoekeeton was evaluated by iruc d NP-40 extracted glands andWeatem analyaia of NP-40 extracted fractions. Elevation of [Ca2+,withoutpretreatment with Cl led to hydrolyai d ezrin and liberation of eznn from actin-baaedcytosketn. Pretreatment of Cl (50 ngfml) minimized the proteolysb but not thediociation d ezrin from the actin cytoskeleton. Parall double imnunotainingahowed that ezrin co-resides with F-actin mainly in carliculJi and apcal plasmanmebrane of parietal cells, in the control group, as reported previously (EMBO J.10.2363, 1991). Diffuse cytoplasmic staining for ezrin became apparent in Clpretreaed ginds whle fuzzy F-actn localization was seen. lonoryn aloe causedthe bss of ezrin and F-actin signals within par col whie F-actin steining at theapice of other epithelial cellkwas unaffected. To exanine wheter elevated [Ca2+1cauase at of actin filament (e.g., via acdin-evering protein), ordepolymerization of actin filemet (e.g., oes of actin-filament stabllzer), we carriedout DNase assay folowing NP-40 extrin. Actition of calpain, achieved with 2IM lonomycin disrupts acn-bae cyok on reflected by Increaed contnt of

mn (G) and short fametoua (F) actin in NP-40 soluble fiwton whie Clprotects actin cytoskeleton from fragemenaton and depolyization. Theincrease of aectin, G plus F, content in NP-40 soluble fction corres with the lssof ezrin onomycin-treated ginds. Currenty, we are testing whetherfr and final despmrzeton of actin filament is due to the loss of actinfilament stabilizer. (Support by NIH grant DK10141)

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]IDENTIFICATION OF THREONINE-346 AS A PUTATIVE IN VIVOPHOSPHORYLATION SITE ON BRAIN B SPECTRIN. ((R.K. Sihag)) McLeanHospital, Havard Medical School, Belmont, MA 02178.

Brain spectrin (Fodrin) is a major constituent of neuronal membrane skeleton. In

Wwo only the 275 kDa B-subunit of the 560 kDa aS heterodimer of brain spectrinis known to be phosphorylated. To idenify the sites of phosphorylation, Triton-insoluble cytoskeleton preparat from mouse spinal cord were phospborylatedIn Wro by endogenous cytoskdeton - asociad or purified protein kinases. Two-dimensional e maps of a vro pbosporylated n-spectrn wercompared with that of in vwi phosphorylated, n-spectrin. As many as 9phosphorylatable sites were identfied. Phosphoamino acid analysis showed thatboth srine and thronine residues were phosphorylated in Wvo. PbosphopeptideS, one of the two major in Wvo phosphorylaed peptides which was pbosphorylatdby a cytoskeleton associated second mesenger-independent protein kinae, wasisolated. The at-chymotryptic digest of nP-labeled -spectrin was separated byHPLC on a C,, reverse-phase column. The fraction contining phosphopeptide S2was identified by two-dimensional phosplopeptide mapping of the 'P-labeledpeptides. The 2P-labeled peptide S2 was further purified on a C, reverse-phasecolumn. The purified peptide yielded molecular mass of 1264 Da by lasr-

desorption mass spectrometery. By automated Edman degradation we haveidentified the peptide S2 sequence (V Q Q Q L Q A F NT Y) on the B, repea ofthe non-erythoid spectrin. These results show that Thr-346 may be a majorputative Ln Wo phosphorylationsiteon B-spectrin. PhosphorylationofThr-346 mayplay a role in regulating the binding of synapsin-I to brain spectrin. Supported byNSF and AHAF.

273

ISOLATION OF THE CYrOSKELETAL PROTEIN MOESIN FROM XENOPS LAEVWS((Armstrong, N., Thorn, J., and Kay, B.K)) University of North Carolina,Chapel Hill, North Carolina, 27599-3280

Interactions between the actin cytoskeleton and the plasma membrane arethought to play an important role during several aspects of developmentincluding cell migration, changes in cell shape, and establishment ofspecialized membrane domains. To better understand how the actincytoskeleton and the plasma membrane interact during development, we havebegun an examination the ERM family of proteins (ezrin, moesin, andradixin) in developing Xenamus laevis embryos. These proteins are thoughtto Knk actin and the plasma membrane and play an important role inregulating cell adhesion. Western blots of Xeou embryos stained using a

polyclonal antibody specific for sea urchin moesin indicate the presence ofthree ERM like proteins with MW of approximately 54, 57, and 63 kDa. The63 kDa protein is expressed at a constant level from oocyte stage IVthroughout early devebpment. The 54 and 57 kDa proteins are notexpressed in the embryo prior to tailbud stage but are expressed at very highlevels in the tadpole. Examination of adult frogs revealed that these threeproteins are expressed in different tissues. The 63kDa protein is expressedin both the kidney and intestine while the 54 kd protein is expressed in theintestine and the kings. The 57 kDa protein was not observed in any of thetissues examined. Using primers corresponding to highly conserved regionsof the amino-terminus of ezrin, partial clones of ERM-like proteins weregenerated from a Xeo sua oocyte cDNA library by PCR. Full length cbneswere subsequently obtained by rescreening the library using these partialclnes as a probe. Through this method we have obtained a full length cloneencoding a 421 amino acid protein. Sequence comparison indicates that thisprotein is 90% identical to human moesin at the amino acid level.Correspondingly we refer to this protein as Xenopus moesin.

270

HUMAN RECOMBINANT a-CATENIN BINDS TO SPECTRIN. ((C.R.Lombardo, D.L. Rimm, E. Koslov, and J.S. Morrow)) Yale UniversitySchool of Medicine, Dept. of Pathology, New Haven, CT 06510.

A central feature of the E-cadherin based cell-cell adhesion machinery isa cytoplasmic complex composed of E-cadherin, a-catenin, and 0-catenin.Spectrin and actin also co-localize with E-cadherin and the catenins veryearly during nascent cell-cell contact in cultured Madin Darby CanineKidney Cells. These findings suggest a direct interaction between thenonerythroid spectrin-actin cytoskeleton and the cell adhesion complex.Using recombinant a-catenin, a novel interaction with both erythroid andnon-erythroid spectrin has been detected in vitro, using both co-precipitation assays and quantitatively by the technique of surfaceplasmon resonance (BlAcorem). Both erythrocyte (aIPI) and non-erythroid (aI$II) spectrin bound a-catenin with Kd values of 4 to 6 JIM.a-catenin also bound to both subunits of spectrin. The N-terminal 228amino acids of a-catenin comprised the spectrin binding domain; this isthe same region responsible for its binding to F-actin (see abstract byRimm et al). These biochemical studies demonstrate a novel functionalinteraction of spectrin with a component of the cell-adhesion complex,and may account for the co-localization of these molecules at points ofcell-cell contact.

272

MOESIN AND OTHER ACTIN-BINDING PROTEINS IN THENEUTROPHIL MEMBRANE SKELETON. ((K.N. Pestonjamasp1, C.P.Strassell, H. Furthmayr2, W.M. Nauseefl.3, E.J. Lunal)) lWorcesterFoundation for Experimental Biology, Shrewsbury, MA 01545.2Department of Pathology, Stanford University, Stanford, CA 94305.3University of Iowa College of Medicine, Iowa City, IA 52242.

F-actin columns and blot overlays with [125]1-labeled rabbit muscle F-actinwe used to identify actin-binding proteins (ABPs) in membranes frombovine neutrophils (PMNs). One group of ABPs (17-, 25-, 43-, and 50-kDa)resists extraction with sodium carbonate, is solubilized with 3% octylglucoside(OG), and binds tightly to an F-actin affinity matrix. Other ABPs, includingprominent 68- and 72-kDa proteins, are insoluble in OG but bind to [125]I-labeled F-actin in blot overlays. This binding can be competed with unlabeledF-actin, but not with poly-aspartic acid or taxol-stabilized microtubules. Also,no binding is seen with [125]I-labeled G-actin or heat-denatured actin. The68-kDa polypeptide appears to be membrane-bound moesin. This protein co-puifies with plasma membrane-enriched fractions from both bovine andhuman PMNs and, along with a number of surface-biotinylated proteins,resists extraction with CHAPS or 3-6% OG. After extraction of membraneswith 1% Triton X-100, 5 mM EDTA, 250 mM NaCl, 25 mM Tris, pH 7.5,the 68-kDa ABP can be immunoprecipitated with anti-moesin antibodies andco-migrates on SDS-PAGE with moesin. Similarly, the less abundant 72-kDaABP co-immunoprecipitates and co-migrates with ezrin, suggesting that bothmoesin and ezrin bind directly to [125]1-actin in blot overlays. In immuno-fluorescence, moesin localizes at the plasma membrane of bovine PMNs,consistent with a role for moesin-like proteins in the PMN membrane skeleton.

274

EZRIN SELF-ASSOCIATION INVOLVES HEAD-TO-TAIL BINDING OF AMINO-AND CARBOXY-TERMINAL DOMAINS. ((R. Gary and A. Bretscher))Section of Biochemistry, Molecular and Cell Biology, Comell University,Ithaca, NY 14853.

Ezrin is a membrane-cytoskeletal linking protein which is concentratedin actn-rich cell surface structures. It partially cofractionates with thedetergent-insoluble cytoskeleton, and has recently been shown (Tsukitaet al., JCB 126:391, 1994) to bind to a transmembrane extracellularmatrix receptor. Both monomeric and oligomeric forms of ezrin arepresent in tissue extracts, and the ability of ezrin to self-associate invitro has been demonstrated using a blot overlay binding assay (Gary andBretscher, PNAS 90:10846, 1993). The remarkable specificity of thisassociation and the likelihood that it is regulated in vivo suggest that itmay be functionally important. To better understand the moleculararchitecture of the ezrin-ezrin complex, we used truncated forms ofezrin to map the domains responsible for this binding activity. Weproduced a large collection of truncated ezrin fusion proteins which wereresolved by SDS-PAGE, transferred to a blot, and probed withblotinylated full-length ezrin. All fusion proteins containing the 107residue C-terminus of ezrin (P479-L585) displayed full probe-bindingactivity, whereas E555-L585 was inactive. All fusion proteins with C-terminal deletions were incapable of binding the probe. When truncatedezrin fusion proteins were biotinylated and used in solution as probes,only those containing the N-terminus (P1-1368) were active. Theseresults demonstrate that the N-terminal 'head' domain of ezrin bindsintermolecularly to a C-terminal 'tail'. Furthermore, the N-terminaldomain is irreversibly inactivated by SDS-PAGE whereas the C-terminaldomain is not

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