Membrane Receptors (509-514). Sunday509

PLATELET ACTIVATING FACTOR PRESYNAPTIC RECEPTOR ACTIVATIONINDUCES GLUTAMIC ACID RELEASE IN THE RETINA, A MECHANISMASSOCIATED WITH RETINAL DAMAGE. ((V.L Marcheselli and N.G. Bazan))Lousiana State University, Eye Center and Neuroscience Center, New Orleans,LA 70112.

PAF (1 Alky -2-acetyl-sn-glycerol--phosphorychlne) a lipid mediator, hasbifunctional acthvty In neuronal tissue. PAF has been shown to be a secondmessenger, Increasing early gene expression, which is mediated by intracellularreceptors (Marcheselli V., and Bazan N., Neurosci. J. 1993., in press).Secondly, PAF utilizes a mechanism which involves membrane receptors whichinduced [3H]glutamic acid release In retinal synaptosomes. PAF, present atalmost undetectable levels, accumulates in neuronal tissue durlng lschemia,hypoxia, and other disorders which involve massive release of excitotoxicneurotransmitters. Ught damage to retnal tissues has been minimized In vivoby pretreatment of animals with a specific PAF antagonist, BN-52021 (Reme C.,et al., Graefe's Arch. Clin. Exp. Ophthalmol. 1992,230;580). A perfusion cellwas mounted with synaptosomes (50 sg of protein) loaded with ['H]glutamicacid, and under conditions of constant flow (Krebs buffer, 6 mVmln), 200 pl of10 nM PAF induces the release of 25% of total rH]glutamic acid taken up.When synaptosomes are treated wfth 1 pM BN-52021, inhibition to controllevels of glutamic acid are measured, with up to 100 nM PAF stimulation. FortymM KCI Induced up to 30% release of [3H]glutamic acid, which is not Inhibitedby the PAF antagonist, BN-52021. This study indicates that a receptormediated mechanism is involved in the release by PAF of glutamic acid inpresynaptic terminals from retina.

511

CELLULAR BUSCEPBILTY TO EXOTOINA. ((D.M. Mucci,J.JForristal, R E. Morris, D. FitzGerald, D.K Strickland and C.B.Saelinger)) NCI, American Red Cross and University of CincinnatiCincinnati OH 45267.

Pseudomons exotoin A (PE) is a three domain tonn that killsmammaLian cells by gaining entry to the cytosol and inhibiting proteinsynthesis. Not all cells are susceptible to the toxin, and resistance couldbe due to ineffective toxin processing, to lack ofa receptor responsible forPE internalization, or to alteration ofreceptor binding capability. Thereceptor for PE has been isolated andis identical to the a2macroglobulin/low density lipoprotein receptor related protein (a2MR1LRP). In additiona 39 kDa receptor associated protein (RAP) blocks both binding andbiological activity of PE for mouse LM fibroblasts. We have examined thecontribution ofa2MR/LRP and ofRAP to cellular susceptibility to toxin.All tissue culture cell lines and mouse tissues which are sensitive to PEhave ct2MR/LRP and minimal amounts ofRAP (e.g. IM fibroblasts,mouse liver). In contrast, cells which exhibit high levels ofresistance (3-4logs) can be divided into two groups: no a2UMRLRP (Hela), or receptorand high levels ofRAP (e.g. Ovcar, PC3). Subcellular fractionationstudies show that RAP and receptor comigrate with plasma membranemarkers, and should be localized to the cell surface. Experimentsutilizing other ligands for LRP, such as lipoprotein lipase, lactoferrin, andactivated a2macroglobulin, show that these ligands minimally reducetoxin binding to TM cels. The results will be discussed in terms of amodel ofRAP regulation of ligand binding to receptor.

510

THE LATERAL MOBILITY OF ALTERNATIVELY SPLICEDISFRMS OF NCAM ((K.A. Jacobsonl ,2 H.P. Weber', S.E. Moore3,B. Yang', P. Doherty3, and F.S. Walsh3)). 'Department of Cell Biology andAnatomy, The University of North Carolina at ha Hill, Chapel Hill, NC.2Lineberger Comprehensive Cancer Center, Chapel ill, NC. and-3Departmentof Expenlmental Pathology, UMDS, Guy's Hospital, London.

Neural cell adhesion molecules (NCAMs) are altematively spliced to form up to30 different isoforms, which, though similar in their extracellular domains,differ in that they are either transmembrane proteins with small or largecytoplasmic domains or are glycosphosphatidylinositol (GPI)-linked to theplasma membrane. When human NCAM isoforms are transfected into NIH313 cells, these cells can serve as a substrate for the outgrowth of neurites fromneuroblasts. We used fluorescence recovery after photobleaching (FRAP) todetermine if the differing membrane anchorage domams affect the NCAM lateraldiffusion in 3T3 cells. Our results showed that there was little difference in thediffusion coefficients (D): (D xI0-10 cm2/s ± SEM for: 120 kD = 24.3±4.2;140 kD = 18.0±3.7; 180 kD = 18.6±3.1) or mobile fraction (MF) (MF ± SEMfor: 120 kD = 57.5±3.3 %; 140 kD = 60.9±4.1 %; 180 kD = 61.4±4.7 %).These results are consistent with the hypothesis that the ectodomain determinesthe lateral diffusivity of some meinbrane proteins, but this measure of lateralmobility of the NCAM isoforms in 3T3 cells did not correlate with growthsignalling potential. The mobility of transfected 120 kD and 140 kD NCAMs inmore biologically relevant cells (PC12 neuroblasts and C2C12 muscle cells)was lower than in 3T3 cells (for 120 kD in PC12 and C2C12, resectively: D=11.4±3.3 and 10.9±1.9; MF = 62.3±5.7 % and 44.3±4.0 %; for 140 kD inPC12 and C2C12 respectively: D = 10.4±1.6 and 4.7±0.5; MF = 55.2±5.0 %and 71.1±4.7%). These results suggest that cis interactions between thetransfected NCAMs and various endogenous components result in reducedlateral mobility when compared with the 3T3 cells. (Supported by GM 41402)

512ALTERATION IN PMN Fcy AND C3bi RECEPTOR EXPRESSION BYNICOTINE, COTININE AND AMMONIA. ((ly. NUMABE, 2M. I. RYDER,1K KIYONOBU and 1K KAMOO)). 1Departent of Periodontology, School ofDentistry at Tokyo, Nippon Dental University, 2Division of Periodontology,University of California, San Francisco.

Clinical studies have shown a correlation between smoking and severaldiseases. Previous studies have demonstrated the effects of whole tobaccosmoke as well as selected components on PMN functions. In this study, theeffects of nicotine, cotinine and ammonium chloride were observed in vitrofor Fcy receptor and C3bi receptor expression. Human peripher PMNswere collected from healthy non smoking subjects and incubated withnicotine (10-2M to 10-6M), cotinine (10-2M to 10-6M) or ammonium chloride(10-2M to 106M) for 45 minutes at 370C. For Fcyreceptor staining, PMNswere incubated with FITC conjugated mouse anti-Fcyreceptor (OK-NK).For C3bi receptor staining, PMNs were first incubated with mouse anti-human monocyte/granulocyte monoclonal antibody (OK-M1), then incubatedwith FITC conjugated goat anti-mouse IgG. Flow cytometric analysis wasperformed on a Ortho Cytoron Absolute ( Ortho Diagnostic Systems K.K.).Significant depressions of Fcy and C3bi receptor staining values wereobserved from 10-2M to 10-6M of nicotine (p<0.05: ANOVA). Depressionsof Fcy and C3bi staining values were observed from 10-2M to 104M ofcotinine and ammonium chloride (p<0.05: ANOVA). llese studiesdemonstrate that nicotine. cotnine and ammnonia inhibit PMN FM and C3bi

receRtor expression which may in turn affect PMN functions such as

513

OXIDANTS ACT AS CHEMOREPELLENTS IN PARAMECIUM BY STIMULATING ANELECTROGENIC PLASMA MEMBRANE REDUCTASE ACTIVITY. ((Frego, LE. andHennessey, T.M.ll Dept. of Biologial Sciences, State Univ. of N.Y. at Buffalo, Buffalo, NY14260.

Plasma membrane associted electron transport ctivities have been shown to affect themembrane potential of many eukaryotic cells, as in chemotaxis of leukocytes. In

Paramcchm membrane potential chanes also regulate the chwmoresponses.Chemoattractants hyperpolarze and cause fast forward swimming while chemorepelientsdepolarize to cause slower and even backwad swimming. Sensory information is integratedin the form of the somatic membrane potential nd behavloral responses are used asbioaays. When single cells ware cored for repellent responses as either a positiveresponse (jrkng or swimming backwards) or no response, a concentration dependentincrease in the % cells responding was seen in cytochrome c, FeCN and nitro bluetetrazolium (NBT). The EC.N values were about 0.4OM, 10,OCM and 2.2uM respectively.These responses were transient, forward swimming resumed within 1 min Use of a 2-waystopcock assay confirmed the repellent nsture of these oxidants. The in yjyg reductaseactivities followed the order of potency in the behavioral asys with cytochrome c beingthe most effective. Cytochrome c caused a conca dependent depolarization thatwaswell correlted with in yby reductase activity (about 4mV/10 unis activity, unit-#M/minhcells). This activity was itself voltage dependent, an excellent correlation was seen betweenthe extent of depolarization caused by ionic stimulation (Ca+ +, Mg+ +, Na+ or K+) andthe % inhibition of in yIX reductase activity (about 15% lnhibitionNlOmV depolarization).Therefore, some drugs may indirectly inhibit this reductase by depolarization. Although bothbody and ciliary membranes contain an NADPH dependent reductese activity, the bodymembrane is sufficient forthistransduction because similar results were seen with decilistedcells. The I viogreductase activities were Inhibied by choroqulne and diphenyllodonlum1DPI) wth ECN values of 1.0mM and 100,uM respectively but side effects impaired thebehavioral assays. The activity showed no pH dependency in the range of pH 5.3 to 8.3.We conclude that compounds that can be reduced by these cells are effective repellents atmicromolar concentrations due to their depolrizing effects. (Supported by NSF Grant BNS89-16228).

514

CELLULAR LOCALIZATION OF HEMOCYANIN INONISCUS ASELLUS. ((E. J. Rappa, G. M. Vemon, and R.Witkus)) Department of Biological Sciences, FordhamUniversity, Bronx, NY 10458 and *Division of Science &Mathematics, Fordham University, New York, NY 10023.

Early in this investigation it was determined thatcommercially purchased anti-keyhole limpet (Megathuracrenulata) hemocyanin cross-reacted with 0. asellushemocyanin. This then allowed the use of the anti-keyholelimpet hemocyanin in subsequent immunologicalexperiments with 0. asellus hemocyanin to locate sites ofhemocyanin. Using Western Blot following SDS/PAGE,hemocyanin was located in the hepatopancreas and indifferent regions of the hindgut. Once this was establishedpost-embedding protein A-gold immunolabeling was usedto locate more specific sites of hemocyanin in the hindgut.Hemocyanin was localized along the length of theplasmalemma of the basal infoldings of the epithelial cells inthe posterior hindgut. This interaction of hemocyanin witha natural biomembrane might be important in oxygenexchange in invertebrates with an open circulatory system incells where there is a great demand for oxygen as in thehindgut.

88a

Sunday. Membrane Receptors (515-520)

515PMA NDUCES A CHANGE N PHENOTYPE OF TE671 CELLS FROMA SMOOTH MUSCLE-TYPE TOWARDS A SKELETAL ORCARDIACMUSCLE-TYPE. ((D. L Mille and J. C. Owicki)) Molecular DevicesCorporation, 4700 Bohannon Drive, Menlo Park, CA 94025

We have previously demonstrated the existence of a muscarinicreceptor inTE671 cells whose activation upon exposure to agonists isdetectable as an enhancement in metabolic activity when measured in aCytosensor° microphyh ometer. Activation of the muscarinic receptorsalso induces cytoplassinc calcium increases in TE671 cells as measuredfluorometrically with intracellular fura-2. Although these cells areknown to express a skletal muscle-type nicodnic receptor as well,activation ofts receptor was not normally measurable either in theCytosensor or with fura-2. Treatmentof these cells with phorbol 12-myn'state 13-acetate (PMA) for several days resulted in a change in thecel morphology, with the cells becomnug much more elongated.Exposure of these "differentiated" cells tonicotinic agonists resulted ina detectable, fast responsein the Cytosensor and a transient fast [Caliincrease. ExtracellularCa was necessary for the [Ca] transient, and KCIdepolarizations of these cells resulted in increases in [Cali. Thus it islikey thata voltage-gated Ca channel is expressed that inundifferentiated cells is not expressed in sufficient number to cause ameasurable change in [Cali. We think it is likely that the Ca transient isresponsible for enihancement of the metabolic activity of these cells suchthat activation of the nAChRis then measurable in the Cytosensor. Weare continuing to investigate both the source of the Ca transient as wellas its metabolic effects.

Supported in part by DARPA contract MDA971-92-C-0005.

517FUNCTIONAL ANALYSIS O TRANSIENTLY TRANSFBCTED RECEPTORS USINGTHE CYTOSENSOR MICROPHYSIOMETER.((HlG. Wads, S.D.K Chan, D. Denne, D.M. Anteniuci KIS. Fok, J. Howison, J.W. Parce,H.M. McConnell* and J.C. Owici)) Molecular Devicea Cop.. Menlo Park, CA 94025 andDeparunent of Cemistry, StnffadUnveesty, Stnod, CA94306.

Cyo micephy moni cellular metbolisin«In r ad has been used

to detct and study bgial responss of cultred cels to affector agents Te metaboLcactivty dat is monitod is dte ra at which cells excrtea protu into heir anvromnment,principay as the caabolic products actic acid sd carbonic acid and also a a result ofNa+/H+ exchange. Roceptor-mediated cell activation by ligands, such a hormones,neuroranau numttr, and grwth factr cma increases in extr luie acidicato ra within

minutes of recepor-lignd bindn. It bs b air wek dtt G-proteinlincod receptors which were stably tnfected into CHO ells can be functionallycharcteized using acidifon ae s thecll rponse o ceptoractivaion (Owickital., PNAS 87:4007-11, 1990). Since inital cha of c-DNA encoding receptormolecules is often acompid using tansiondy tmrnected cells, we have developed twomethods for the functionang of transes usg the Cy ensor. One method uses co-

tansfection of a cell srfacem (Thyl.2) which can be used a a hane to separte thehigh expressing cells from cel Magneic parcles coated with anti-lyl.2were used to accomplish the spdm two days post uanfectn A second method utilizesan Epein-Bar vius (EBV) baed episomal exp s voctrand the human kdney cell line293. This system pennits high eopyex a repiaio and maximiz expressionof the cloned receptor gene after antibioc selection for 4 days. Both of dtese methods resultin cel populto which are predomninantly high expressers and e uitable for analysis inthe Cyosensor. When ml-muscawinic and B2-adrenergic receptors were tansientlytransfected into CHO-kl cell and dopamine Dl receptors were transiently twfocted intointo 293 cells, the Cytosn was able to detect the activation of cellular acidiication ratesby agonists. The responses wer t and could be completely blockedby antagonists. As with stably trfected cells, it wiU be possible to characterinze thepharmcologic propertes of recepto expressed in siently tranfected cells using real-time asments of the activaion ofexualcularadifiation rate,SuWed in pat by Defens Advnes Reseach Projt Agency. CsreaMDA972-92-C-0005.

519

MOLECULAR CLONING AND SEQUENCING AND PROMOTERCHARACIERIZATION OF GOLDFISH ADRENOCEPTOR GENE. ((Y.-LShih, N.-C. Chang, A.-C. Chang and S. J. Lo)) Graduate Institutes ofMicrobiology & Immunology, and Neuroscience, National Yang-Ming MedicalCollege, Taipei, Taiwan, R.O.C.

Although three types of adrenoceptor have been cloned and sequenced inmammals, little is Inown about the structure and oranization of these receptorgenes in lower vertebrates. By using a primer pairs flanking the conservedsequences in the second and sixth transmembrane domain of human a2-adrenergic receptor, a DNA fragment of 834-bp was amplified by polymerasechain reaction (PCR) from the genomic DNA of goldfish cell line. Using thisPCR-amplifled DNA fragment as a probe, we have identified a clone whichcontains a 5.5-Kb, EcoRI-restricted genomic DNA fragment. Sequence analysisrevealed that this gene contains an open reading frame of 1308 base Pairsencoding a putative protein of 436 amino acids. Amino-acid homoorgycomparison to other Ikown adrenoceptors revealed that it exhibits highlyhomology to human or rodent a-2 isoceptors at transmembrane domains,however, the putative 3rd cytoplasmic loop shows extremely little similarity. Aputative promotor sequence TATATAAA was identified and located 383-bpupstream from the first ATG. By using CAT as a reporter gene, we confirmedthe promoter region and a positive regulatory sequence between TATA and thefirst ATG. A polyadenylation signal AATAAA located 143-bp downstream tothe stop codon of the ORF was also identified. Taken together all data, itsupports that the intronless adrenoceptor gene cloned from goldfish is afunctional gene, instead of a pseudogene.

516

ADENOVIRUS E3 PROTEIN DOWN-REGULATES EGF RECEPTORS BY

INCREASING STEADY-STATE CONCENTRATIONS OF RECEPTORS IN

MULTIVESICULAR BODIES WITHOUT INCREASING THE RATE OF RECEPTOR

INTERNALIZATION OR INDUCING INTRINSIC TYROSINE KINASE ACTIVITY.((P.H. Hoffnan and C.R. Carlin)) Department of Physioogy and Biophysics, School of

Medicine, Case Westem Reserve University, Oeveland, Ohio 44106-4970.

We have previously Identified and characterized an lIntegral membrane proteincoded for by the early transcription region 3 (E3) of human group C adenoviruses

that downrulat the EGF receptor (EGFR) (Cell 57:135; J. Biol. Chem.267:13480). Although EGFRs are degraded In adenovirus-infected cells, it has not

been dear whether the E3 protein increse the rate of EGFR Intemalization, causes

sortng to yosomsr, or both. We now show that the rate of EGFR internalization In

adenovirus-Infected cells Is Indistinguishable from the basai Internalization rate for

unoccupid EGFRs endocytosed by a constitutive pathway. Consistent with this

observation, the E3 protein does not Induce EGFR oligomerization or Intrinsic

tyrosine kinase activity, nor does kt affect EGFR affinity for ligand. In addition, Intrinsictyrosine kinase activity Is not required for adenovirus-induced down-regulation, since

kinasedefective EGFRs are degraded to the same extent as wild-type EGFRs, and

EGFR degradation is not blocked by the tyrosine kinase Inhlbitor genistein. Once

intenalized, howver, EGFRs In cells expressing the E3 protein are more likely to

remain sequestered Inside the cell than EGFRs internalzed at steady-state or In cells

Infected with an E3 deletion mutant. interestingly, there is a 3-fold Increase In the

numnber of EGFRs localzed to multivesicular bodies (MVBs) In infected cells

expressing the E3 protein, compared to cells Infected with an E3 deletion mutant.

The MVB is believed to be Important for regulating sorting of receptors that recycle to

the plasma membrane and receptors that are transported to lysosomes. Our data

therefore suggest that adenovirus clears surface EGFRs by regulating receptortrafficking In this organelle, so that relatively more EGFRs Internalzed by a

constitutive pathway at steady-state are transported to iysosomes and degraded.

518

ADENOVIRAL MEDIATED RETROVIRAL TRANSDLICTION INTO RESISTANTCELLS. ((R.M. Adams, M. T. Wang, F.D. Ledley)) Department of Cell Biology,Baylor College of Medicine, Houston, TX, 77054. (Spon. by F.D. Ledley.)

A major determinanit of retroviral host range is the expression of cellularreceptors to cognate viral envelope genes. In cells lackiiig the appropriatereceptor, the amount of retroviral infection is often extremely low (titer < 10o/-6). Adenovirus is known to aid in the transfer and subsequent expression ofnaked DNA and DNA-protein complexes into cells, the supposed mechanism

being solubilization of the endosome. We have found that adenoviralconcentrations of above 10 adenoviral particles,;cell can facilitate thetransduction of cells outside of the infecting retrovirus's host range. Cells inculture were exposed to varying concentrations of adeno- and retrovirus, in the

presence of 8ug/ml polybrene, for 18 hours, followed by washing and

subsequent incubation in normal media for 48 hours. After that time, DNAwas prepared for PCR analysis for the presence of an integrated NEO-R gene, or

the cells were stained for the expression of proviral (beta)galactosidase. Itwas found that in NIH3T3 cells exposed to an N2 vector (packaged in a

xenotropic packaging line, GP+X) at an multiplicity of infection of

approximately 1:1, in the presence of 10 adenoviral particles/cell, the overalllevel of transduction was approximately 2 orders of magnitude higher than thatobserved in the absence of adenovirus, but with the same concentrationi of

retrovirus. Essentially the same results were observed in the human-derivedcell line HeLa exposed to the ecotropic virus, Zen-(beta)-gal, with subsequenthistochemical stain. We believe this transduction is occuring in the absence ofa recognized retroviral receptor, and may represent a novel mechanism forretroviral entry into cells.

520

DO INSULIN/IGF-I AND LOW OXYGEN REGULATE GLUCOSE

METABOLISMIN CANINEMUSCLECULTURESTHROUGHINSULIN/IGF-I RECEPTORS AND GLUT-I? ((N.T. Kuo', S.R. Vadlamudi', R.J. Smith3, K.

Strohl' and R.J. Przybylski')) Departnent of Anatomy' and Medicine2, Case

Westem Reserve University, Cleveland, OH 44106 and Department of Medicine',Harvard University, Boston, MA 02215.

Beagle gluteal skeletal muscle cultures were grown in Dulbecco's DMEM

with 10% calf donor serum in normoxia (21% 0,, 5% CO2, 37°C) until exposureto low oxygen (5% 0,, 5% CO, 37°C). Low oxygen increased the basal level of

2-deoxyglucose uptake by 18%. Additive increases were obtained with insulin,IGF-I and IGF-II equivalent to those obtained in nornoxia suggesting that two

pathways exist. Hormone binding experiments showed low oxygen (19 hr)decreased by 40% the high affinity insulin receptor sites but increased Kd 11.6

nM to 7.6 nM. IGF-I receptors decreased 60%; Kd of 22.1 nM to 6.2 nM. Cross-

linking experiments showed that 12I-insulin and nI-IGF-I bound to 260 and 130

Mr a-subunit proteins. The amount of a-subunit of insulin and IGF-I receptorswere decreased by low oxygen as detacted by cross-linking. Neutralizingantibodies to the insulin and IGF-I receptors cancelled the hormone but not the

low oxygen stimulation of 2-DG uptake. Western blots showed treatment with

low oxygen, insulin, IGF-I and IGF-II increased the amount of GLUT-1 as a 49

kDa protein with no GLUTs 3 or 4 detectable. In sum, low oxygen increases

glucose uptake, decreases insulin/IGF-I receptors but increases receptor affinity

while the GLUT-1 increases in amount. The observations suggest two separatepathways regulate glucose metabolism in skeletal muscle cultures.

89a

Membrane Receptors (521-526). Sunday

521ASSOCIATION OF IP3 RECEPTOR WITH THE PLASMA MEMBRANE.((L. Feng and N. Kraus)) Department of Physiology and Cell Biology,University of Texas Medical School at Houston, Houston, TX 77225.

Studies were carried out to characterize the interaction between inositol 1,4,-5 trisphosphate receptors (IP3R) and the plasma membrane fraction in theliver. Extraction of the membranes with the non-ionic detergents NP-40 andTriton X-100, followed by centrifugation at 100,000 x g, resulted in the

doubling of the IP3R in the pellets while no detectable binding was found in

the supernatants. These data indicate that the detergents did not solubilize thereceptor, and it remained associated with membrane particles, and it is likelyto be associated with the cytoskeleton. The cytoskeletal proteins - actin,ankyrin and spectrin - were identified in the plasma membrane fraction.

However, comparison of the amount of these proteins in different fractions

of the detergent, or otherwise treated plasma membrane fractions, showed no

direct correlation between the presence of any of these proteins in the plasmamembrane fraction and their ability to bind [H]-IP3. This is in contrast to

the brain and T-lymphoma cells in which the IP3R is attached to ankyrin.Thus, the hepatic IP3R, which is different than the brain receptor, might be

attached to the cytoskeleton by anchoring to a different protein. Because of

the plasma membrane localization of the IP3R, it is proposed that it functions

as a Ca2+ influx channel. The release of Ca2W from intracellular stores could

occur through the ryanodine-binding channel, described to be present in the

liver (Feng et al., Cell Calcium 13:79-87, 1992).

523

GESTATIONAL CHANGES IN OXYTOCIN AND ENDOTHELIN-1INDUCED CONTRACTILITY OF PREGNANT RAT MYOMETRIUM.((H. Izumi, N. Tezuka, C. Yallampalli, M. Byam-Smith, R.E. Garfield))Division of Reproductive Sciences, Department of Obstetrics andGynecology, University of Texas Medical Branch, Galveston, TX 77555.

We compared the mechanisms of contraction induced by oxytocin (OT)and endothelin-I (ET-1) on the rat myometrium during gestation.Contractions of longitudinal muscle were measured from rats at mid stage(day 14-16) and late gestation (day 17-19), and during delivery at term.Contractile amplitudes were greater when OT and ET-1 were usedcompared to K+ at the late stage and during delivery and similarlyresponses to OT and ET-1 increased during the progress of gestation.Nifidipine (10 mmol/L) inhibited OT and ET-1 contractions but K+induced contractions were completely abolished. OT and ET-1, but notK+, produced contractions in Ca2+ free solution. In p-escin skinnedmyometrial strips, inositol triphosphate (InsP3) produced contractions byCa2+ release from storage sites and both OT and ET-1 producedcontractions in the presence of GTPyS (1 iM). Oxytocin but not ET-1increased Ca2+ sensitivity for contractile proteins. These results suggestthat the membrane permeability for Ca2+ and the number of receptors forOT and ET-1 increase during the progress of gestation and that both OTand ET-1 produce contractions by activation of voltage-dependent Ca2+channels and production of InsP3 to release Ca2+ from storage sites. Theresults also show that OT, but not ET-1, interaction with receptorsincreases Ca2+ sensitivity for contractile proteins, indicating that theguanine nucleotide protein (G-protein) is linked to the oxytocin receptorbut not to the ET-1 receptor.

525

MOLECULAR CHARACIRITION OF A NOVEL INTERLEUIN-8 RECEPTOR ISOTYPE ((G.N. Prado, KM. Thomas, IL Suzui, GJ. LaRosa, E. Folco, N.C Wilinson, and J. Navarro)) Department of Physiolo andBiophysics, Sealy Center for Moecular Science, Univ. of Texas Medical Branch,Galveston, 1X 77555-0641. (Spon. by D. Carney.)

Interleukin-8 (IL-8) mediates the transendothelial migration of neutrophils tothe site of inflammation. Two ILH8 receptor isotypes have been characterizedon the basis of their pharmacological profile. IL-8 receptor type A binds IL-8and structurally related peptides melanoma growth stimulating activity (MGSA)and neutrophil activating peptide2 (NAP-2)with the following pharmacologicalprofikILe8>- >MGSA>NAP-2, whereas the 11-8 receptor type B profile isIL-8=MGSA>NAP-2. In this study, we isolated a cDNA clone (5Bla) from arabbit neutrophil library encoding a protein corresponding to the superfamilyof G-protein coupled receptors The 5Bla clone encodes a 358 amino acidprotein and exhibits the highest amino acd identity to the human IL-8 receptorB (85%). Tisue distnbution by Northern blot analysis reveals that the 5BlamRNA is expressed preferentially in neutrophils Transfected Chinese hamsterovary (CHO) cells with 5Bla cDNA exhibited specific high affinity I25IL-8binding with a novel pharmacological profile of IL-8>NAP-2> >MGSA. The

corresponding apparent affinities (Ki) for IL-8, NAP-2 and MGSA were 4nM,120nM and 320nM respectively. 11-8induced intracellular calcium mobilizationand deseitization in transfected CHO cells with 5Bla. Sequence analysis ofthe SBla protein with other 11-8 receptor subtypes within the framework oftheir pharmacological profile reveals structural motifs that may correspond tothe ligand binding site of the 11-8 receptor. This work was supported by NIHgrants (AR01810, AR39602) and Tobacco Council for Research.

522EVIDENCE FOR DISTINCT SORTING PATHWAYS FOR THE

THYROTROPIN-RELEASING HORMONE RECEPTOR AND ITSLIGAND. ((C.P. Petrou, and A.H. Tashjian, Jr.)) Dept. Mol. Cell.

Toxicology, Harvard School of Public Health, and Dept. Biol. Chem. and

Mol. Pharmcol., Harvard Medical School, Boston, MA 02115

We have examined the trfficking of the thyrotropin-releasing hormone

receptor (TRHR) and its ligand, after TRH-induced internalizaton in rat

pituitary GH,C, cells. After rapid TRH-induced receptor sequestration, the

cell surface receptor pool was replenished (t6 = 50-70 min). Replenishmentwas insensitive to cycloheximide and was dependent on the duration of

internalization, indicating that the replenished receptors were not newlysynthesized but recycled. The decreasing replenishment of the cell surface

receptor pool with time indicates that, in addition to a receptor recyclingpathway, a TRHR noncycling route exists, which becomes dominant with

increasing intenalization periods. The recycling pathway must branch off

from the noncycling route. The TRHR recycling pathway was distinct from

the ligand cycling pathway; following multiple rounds of internalization, the

percentage of recycled [HJlMeTRH decreased with increasing internalization

time due to accumulation of ligand or its metabolites in a noncycling pathway,but the absolute amount of recycled ligand remained constant after short or

long internalization times. Chloroquine did not affect receptor or ligandrecycling, and dissociation of the TRHR-ligand complex appeared to be

insensitive to low pH. We conclude that after receptor-ligand sequestration,the TRHR and its ligand dissociate via a pH-independent mechanism and are

trafficked into distinct intracellular compartments.

524PHORBOL ESTER MEDIATED STIMULATION OF TRANSCYTOSIS OF THE

POLYMERIC IMMUNGLOBULIN RECEPTOR IN MDCK CELLS INVOLVES

PROTEIN KINASE-C ALPHA TRANSLOCATION. ((M. H. Cardone; Bradley L.Smith*; Daria Mochly-Rosen*; K. E. Mostov))pDepartments of Anatomy and

Biochemistry, University of California, San Francisco, CA 94143-0452. *Department of

Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA 94305-

5332. (Spon. by Kim Topp)

We observed that phorbol myristate acetate (PMA) stimulates transcytosis of the

polymeric immunoglobulin receptor (pIgR) in Madin-Darby canine kidney (MDCK) cells.

Apical release of pre-endocytosed ligand bound to the pIgR (dimeric IgA) can be stimulated2 fold within 7 minutes of addition of PMA. In addition, apical surface delivery of pIgRand cleavage of its ectodomain to secretory component (SC) is also stimulated by PMA.This effect of PMA suggests that protein kinase C (PKC) is involved in the regulation of

pIgR trafficking in MDCK cells. To test this we down regulated PKC activity by pre-treating cells with PMA for 16 hours and observed that transcytosis could no longer bestimulated. Western blots show that the PKC isozymes alpha and to a lesser extentepsilon, are depleted from MDCK cells which have been pre-treated with PMA for 16

hours and that treatmnent of MDCK cells with PMA for 5 minutes causes a dramatic

translocation of the PKC alpha isozyme from the cytosol to the membrane fraction of cell

homogenates. This translocation suUests that the alpha isozyme is involved in PMA

mediated stimulation of transcytosis. We have further demonstrated by isozymetranslocation that IgA binding to pIgR, which stimulates transcytosis, activates PKC-

alpha. The major sites of phosphorylation of pIgR are serines 664 and 726. A mutant

pIgR which has these serines replaced by non-phosphorylatable alanines is stimulated to

transcytose with PMA, suggesting that phosphorylation of pIgR is not required for the

effect of PMA. These results suggest that PMA-mediated stimulation of pIgR transcytosisinvolves the activation of PKC alpha and that IgA binding also activates this PKCisozyme.

526

LATERAL MOBILITY OF COMPLEMENT RECEPTOR 1 (CR1) -

EFFECTS OF THE CYTOPLASMIC RECEPTOR DOMAIN AND OFRECEPTOR CROSS-TALK. ((B. Johanssono, J.P. Paccaud*, W. Reithj, B.Machi, K.E. Magnusson# and J.L Carpentierv)). #Dcpt. of Med. Microbiol.,Univ. of Link6ping, Linklping, Sweden. Depts. of *Morphol. and

iMicrobiol., Univ. Med. Center, Univ. of Geneva, Geneva, Switzerland.

To elucidate the membrane dynamics of complement receptor 1 (CR1), fourCR1 variants with different cytoplasmic domains were expressed in ChineseHamster Ovary (CHO) cells, i.e. wildtype neutrophil CR1 (CRlwt), taillessCR1 (CRltl), a chimera of CR1 and the LDL receptor (CR1LDL) and CR1with a Gly to Tyr mutation in the tail, introducing a NPxY motif (CRltyr).At 37 C, CRltyr was internalized faster than CRlwt, and similar to CR1LDL,even though FxNPxY in CR1LDL is regarded as a more efficient inter-nalization motif than NPxY. Lateral mobility was assessed with fluorescencerecovery after photobleaching. CR1LDL was significantly less mobile (D=7.6x1010cm2/s) than CRltyr (DalOxlOl0cm2/s) and CRlwt (D=12xl-10cm2/s).CRltl was more mobile (D=16xl-10cm2/s) than CRlwt and displayed ahigher intnalizaton rate. This could be explained by an increased likelihoodof CRltl to encounter membrane areas undergoing endocytosis. At 14 C,lateral mobility was similar for all receptor variants. At 37 C, the mobility ofCRItl remained high, while CRlwt, CR1LDL and CRItyr all showeddecreased mobility, possibly because of interaction with cytoplasmic sruc-

tures. In human neutrophils, the lateral mobility of CR1 was significantlylower (D=4.5.10-10cm2/s) than in transfected CHO-cells and also subject toinfluences from other receptor systems; it was found that addition of chemo-tactic peptide (0.1 uM fMLNF) increased the mobility of CR1 (D=7.SxlO-10m2/s), thereby possibly affecting receptor aggregation and intenalization.

90a

Sunday. Membrane Receptors (527-528)

527POST-ENDOCYTOTIC ROUTING OF CD3-£ AND TCR VB8 IN JURKATCELLS. ((K.D. Rigaut, J.E. Scharff, and D.M. Neville, Jr.)) Laboratory ofMolecular Biology, NIMH, Bethesda, MD. 20892

We have constructed immunotoxins directed against the gamma epsilonsubunits of the CD3 complex and the Vg8 region of the T cell receptor (TCR)and tested their toxicity in Jurkat cells, a T cell leukemia line. Consistent withearlier observations, immunotoxins directed by antibodies to CD3 are highlytoxic in Jurkat cells. Surprisingly, immunotoxins constructed using Vg8antibody are only slightly toxic in these cells. We have performed severalexperiments analyzing the intracellular fates of these proteins in attempts tounderstand the differences in the toxicity of these immunoconjugates.Endocytosis assays were performed using iodinated antibodies directedagainst CD3 and Vp8. Both were internalized with identical kinetics and thepercentage of molecules internalized were similar. CD3 and the TCR arereported to be recycling receptors. We performed several experimentsanalyzing the surface expression of these proteins following preincubation ofJurkat cells with monensin, chloroquine and cycloheximide. Preincubationwith cycloheximide had little effect on the level of CD3 and Vp8 expressed onthe cell membrane over a 2 hour period, suggesting that these proteins do notundergo rapid turnover. Monensin reduced the surface expression of CD3and Vg8 to 80% of control with slightly different preincubation times, whereaschloroquine had no effect. When the intracellular localization of CD3 and Vp8was examined using immunofluorescence microscopy, both proteins wereinternalized rapidly into an endosomal/lysosomal compartment. To assesswhether the routing pathways of these molecules diverge, we are examiningthe transport of CD3 and Vg8 simultaneously with real time digitized videomicroscopy. The results of these experiments will be discussed.

Protein Folding and Assembly (529-532)

528CLONING, FUNCTIONAL EXPRESSION AND TISSUEDISTRIBUTION OF CD 26. (IJ.R. Woska Jr.1, D.D.Jeanfavre1, C.A. Kennedy , J.S. Prendergast3, R.W.Barton2 and B.J. Bormann1)) Departments oflImmunology, 2Pharmacology and 3Pharmaceutics,Boehringer Ingelheim Pharmaceuticals Inc.,Ridgefield, Connecticut 06877

CD 26 (DPPIV), expressed on the surface of T lym-phocytes, is involved in T cell activation and pro-liferation. A full-length cDNA was cloned from YTcells, a T cell lymphoma. CD 26 CHO cell transfec-tants were tested for expression of CD 26 by FACSand Western blotting analysis. CD 26 CHO cellexpression was 40-60% of that seen in the YT cells.Lysates from the transfectants had 20-fold moreDPPIV activity than control cells. This enzymeactivity was inhibited by specific DPP IV inhibitorsand by high concentrations of PMSF and DFP. Cellsurface adenosine deaminase (ADA), which bindsstoichiometrically with CD 26 on YT cells, was notobserved in similar proportions on the CD 26 CHOtransfectants. Therefore, the association of ADA isnot necessary for the peptidase activity of CD 26.The human tissue distribution of CD 26 was analyzedby Northern blotting. High amounts of transcriptwere found in human prostate, kidney and placenta.No transcript was detected in the brain, testes or

colon. Intermediate amounts of transcript was foundin a variety of other tissues.

529

CHARACTERIZATION OF THE FOLDING OF INFLUENZAHEMAGGLUTININ IN ROUGH ENDOPLASMIC RETICULUMDERIVED MICROSOMES. ((D.N. Hebert and A.H. Helenius))Department of Cell Biology, Yale University School of Medicine, NewHaven, CT 06510.

The folding of the viral membrane glycoprotein, influenza hemagglutinin(HA) was characteized in rough endoplasmic reticulum microsomesisolated from dog pancreas. Efficient post-translational folding ofHAoccured in the pH range of 6.7 to 8.7. Below pH 6.7, the stability ofHAwas compromised resulting in the degradation of misfolded HA. HA wasable to fold efficiently (45%) from 15 - 400C. Attpuures above 40OC, increasing amounts of HA was degraded. An inverse linearrelationship was found between teprature and rate of folding between 15and 40 OC with folding half-times of 22.0 and 2.25 min, respectively.Efficient post-translational folding of HA in isolated microsones wasdemonsated in a buffer containing proper redox conditions. IntralumenalATP was required forHA post-translatonal folding. ADP but not AMPinhibited the folding ofHA which resulted in thedegradation of allmonomeric HA while disulfide-linked aggregated HAremained stabile.Inaddition, calcium was not required for p-tranlational folding. Lastly,the lectin-like putative chaperone, calnexin was found to be associated withall folding intmediates and the native form ofHA.

531

ROLE OF N-LINKED OLIGOSACCHARIDES, GLUCOSETRIMMING AND CALNEXIN IN GLYCOPROTEIN FOLDINGAND QUALlTY CONTROL. ((C. Hammond, I. Braakman, and A.Helenius)) Department of Cell Biology, Yale University School ofMedicine, New Haven, CT 06510

Using a pulse chase approach combined with immunoprecipitation,we showed that newly synthesized influenza hemagglutinin (HA)and vesicular stomatitis virus G protein associate transiently duringtheir folding with calnexin, a membrane-bound endoplasmicreticulum chaperone. Inhibitors of N-linkedglycosylation(tunicamycin) and glucosidases I and II(castanospermine and 1-deoxynojirimycin) prevented theassociation, whereas inhibitors of ER a-mannosidases did not.Digestion with jack bean ax-mannosidase showed that the N-linkedside chains of calnexin-bound HA contained at least one terminalglucose residue. Experiments with a temperature sensitive mutant ofVSV G demonstrated that misfolded proteins may also bindcalnexin, but only if they contained appropriately trimmedoligosaccharides. Our results indicated that the binding specificitywas primarily determined by the composition of the N-linkedoligosaccharide side chains with monoglucosylated forms as themost likely binding species. Our data is consistent with a model ofthe ER folding and quality control machinery for glycoproteinswhich includes calnexin as a glycopolypeptide binding lectin,glucosidases I and II as signal modifiers, and UDP-glucose:glycoprotein glucosyltransferse as a folding sensor.

530ASSOCIATION OF FOLDING INTERMEDIATES OFGLYCOPROTEINS WITH CALNEXIN DURING PROTEINMATURATION IN THE ENDOPLASMIC RETICULUM ((W-J. Ou,P.H.Cameron, D.Y.Thomas and J.J.M.Bergeron)) Dept of Anatomyand Cell Biology, McGill University, BRI, Montreal, Canada

Calnexin has previously been identified as a calcium bindingmembrane protein in the endoplasmic reticulum (ER), whichfunctions as a molecular chaperone. The chaperone function ofcalnexin was studied in detail in HepG2 cells. Newly synthesizedglycoproteins including al-antitrypsin, a1-antichymotrypsin, a-

fetoprotein, transferrin, complement 3 and apoBlO0 were foundassociated with calnexin, while albumin was not. Tunicamycinabolished the association, indicating that N-linked glycosylation wasrequired for the association. The interaction of newly synthesizedglycoproteins with calnexin was transient and correlated with theirrates of protein folding and also identified the rank order of transportof glycoproteins from the ER to the Golgi apparatus. An amino acidanalog, azetidine-2-carboxylic acid, previously shown to interferewith protein folding led to a stable association with calnexin andretardation of the ER to Golgi transport. Our results indicate thatcalnexin plays an important role in quality control of glycoproteinfolding in the ER.

532ROLE OF N-LINKED GLYCOSYLATION IN THE SYNTHESIS AND SECRETIONOF HUMAN INTERFERON-y. ((T. Sareneva, J. Pirhonen, K. Cantell, and I. Julkunen))National Public Health Institute, SF-00300 Helsinki, Finland. (Spon. by A. Miettinen)

Interferon-y (IFN-y) is a cytokine that has antiviral, antiproliferative andimmunoregulatory functions. It has two N-linked glycosylation sites and the biologicallyactive form is a dimer. Little information is available concerning the early steps ofsynthesis, dimerization and secretion of IFN-y and the role of glycan residues in these

processes. We modified the human IFN-y gene by PCR for expression in eukaryoticsystems. We also mutated either one or both of the N-linked glycosylation sites bychanging the asparagine residue(s) to glutamine. These constructs were expressed ininsect cells using the baculovirus expression system and expected glycosylated andunglycosylated fonms of IFN-y were obtained. The mutations did not effect the steady-state IFN-ymRNA levels and the translation rates (60 s/molecule) were the same for thewild type and different mutant IFN-y constructs. The overall yields were 10-50 timeslower for the unglycosylated IFN-y construct and 5-10 times lower for the mutantconstruct that lacks the N-terminal sugar chain compared to wild type and the mutantthat lacks the COOH-terminal sugar chain. This may indicate that during the biogenesisof IFN-y the N-terminal sugar residues enhance folding and dimerization of themolecule thus minimizing the misfolding and degradation of the molecules. Secretion,as analysed by pulse-chase experiments, was not affected by the the lack of sugarresidues and secretion half-time was approximately 2.1 hours for all constructs. Theunglycosylated IFN-y showed slightly lower biological activity, but all different formsexisted as dimers and exhibited equal ermal stability. In conclusion, the presence ofNterminal sugar chains in IFN-y are required for high level expression in insect cells.Lack of carbohydrates does not seem to affect the extent of dimerization and subsequenttransport and secretion of recombinant IFN-y.

91a

Protein Folding and Assembly (533-538). Sunday

533

IDENTIFICATION OF A REGION IN BIP THAT ATP

HYDROLYSIS TO POLYPEPTIDE RELEASE

LM. Hendershot)) Dept. of Tumor Cell Biology, Children's

Research Hospital, Memphis, TN 38105 and Dept. Univ.

Tennessee, MemphisTN 38163

Two functional domains have been mapped on the

terminal ATPase domain and a C-terminal polypeptide domain.

The bound polypeptide is released in vitro from BiP ATP

Thus invivo, one role of ATP hydrolysis implied release

bound substrates. In this report, we show that two deletion

(KpnI A444-495 and HCB1A486-516) were

immunoglobulin heavy chain (HC) in transfected failed

release HC in vitro upon Mg2+-ATP addition. Recombinant

proteins, isolated fromE.coli possessed ATPase activities

type protein, demonstrating that their inability to

chains was not due to a defect in their capacity to ATP.

results suggest that this amino acid segment between N-terminal

ATPase domain and C-terminal polypeptide binding BiP serves

to couple ATP hydrolysis to polypeptide release.

as either a transducing or a positioning domain so ATP

hydrolysis can be used to release bound protein.

deletion mutants will behave like ATPase negative mutants transfected

cells, even though they retainfull ATPase activity.

535

PAIRWISE ASSEMBLY OF CD3 CHAINS CONTROLED

CYSTEINE MOTIF.

((F. Letoumeur and S. Even)) Basel Institute

Grenzacherstrasse 487 Postfach CH-4005 Basel,

*Founded and supported by F. Hoffmann-La Roche

The T cell receptor for antigen is composed c/0associated with

4

homodimers and two non

dimers CD3-Sie and CD3-y/e. In order

requirements for pairwise assembly of the CD3

proteins were designed expressing CD3 external

and Tao (CD25) transmembrane and cytoplasmic

study, we show that CD3 EXD are sufficient to

heterodimerization. Within all the EXD

homologous membrane proximal cysteine based

for specific CD3 dimer assembly. A chimeric protein

the CD3-y immunoglobulin-like domain and CD3-e

assembles with CD3-8, overcoming yIS pair inhibition.

swapping partially inhibits 8/c pair assembly.

interactions observed between CD3-y and

the cysteine motif. However this structural is

sufficient to drive complete heterodimerization. CD3

immunoglobulin-like domains must also play

assembly.

537DISRUPTION OF DISULFIDE BOND

HETEROTYPIC AGGREGATION OF NASCENT

((J.T. Sawyer and T. Lukaczyk)) Department

University ofNew York at Buffalo, Buffalo,

To disrupt the early stage of disulfide bond fornation the

secretory pathway of HepG2ceDls we have incubated presence

mM DiT. After a brief pulse with 35S_methionine and 35S_cysteine

SDS extraction and inmunoprecipitation, non-reducing verified that

albumin wassynthesized in its reduced form

experiments using a cell-free translation system

nascent chains might assemble into large heterotypic aggregates. DTT-treatedHepG2 were extracted under non-denaturing albumnuimmunoprecipitates showed a complex polypeptideimmunooprcipitation protocol we determied that serun proteins,

tansferin, plasmiogen, ceruloplsin, alpha-2-macroglobulinand fibrnoggnn were

among the proteins co-immunoprecipitated with

any of these co-iumunoprecipitates with albumin

normal conditions. When any one of these is directy afterD1Ttreatment a complex profile similar to the evident. These

heterotypic "aggregates" are stabilized by non-covalent

we looked at only one,alpha-l-antitrypsin, failed aggregate heterotypically. This

protein is distinguished from the others by the These

data are consistent with a model whereby the fuon of the earliest phase of

disulfide formation is to preventa 'global' heterotypic aggregation of nascent chains

in the ER lumben resulting fr-om the exposure of adhesive domains.

534

OLIGOMERIZATION AND ASSEMBLY OF SEMLIKI

SPIKE PROTEIN MUTANTS. ((W.A.Duffus, P.Levy-Mintz,

M.R.Klimjack, and M.Kielian)) Department of Cell Biology,

Einstein College of Medicine, Bronx, NY 10461.

The El and E2 spike protein subunits of Semliki Forest

form a heterodimeric complex in the RER that is then

the plasma membrane where virusbudding takes place.

interaction is also important duringthe low pH-trig9ered

fusion activity of the virus, since Eland E2 must dissociate

fusion. Using an SFV infectious clone, we have characterized

effects of mutations within the putative fusion peptide El subunit

on dimerization and virus assembly. These mutations previously

demonstrated to block spike protein membrane fusion activity D)

or cause an acid shift in the pH threshold of fusion (G91A).infection of BHK cells at 37,C, both mutants produced

virus proteins indistinguishable from those ofwild type

the assembly of mutant spike proteins into virus was

soluble fragment of El was released into the medium.

infected cells at reduced temperature (28* C) dramatically

El cleavage and permitted assembly of morphologically normal

particles. The dimerization of El and E2 was analyzed G91

mutant using either infected cells or virus particles assembled

Under conditions that preserved the wild type dimer, El

did not co-immunoprecipitate or sediment as a dimer

gradients. These results suggest that the assembly

mutant is due to afterations in the formation or stability El

dimer. Further analysis of the oligomerization and fusion

the mutants is in progress.

536INVOLVEMENT OF CYSTEINE RESIDUES IN SIMIAN

40 VP1OLIGOMERIZATION IN VITRO. ((E Gharakhanian,M.K. Weidman, and A.K. Sajo)) Department of Biological

California State University, Long Beach, CA

The capsid of simian virus 40 (SV40), an icosahedral

virus, is comprised of pentameric capsomeres

intermolecular interactions that stabilize the Vpl

have not been established. This study examines

nature of Vpl-Vpl interactions in capsomere formation

Vpl molecules synthesized in rabbit reticulocyte lysates

assayed for oligomerization via sucrose gradient

followed by immunoprecipitation, and by non-reducing

dodecyl sulfate polyacrylamide gel electrophoresis.

here that Vpl molecules oligomerize in a time concentration

dependent manner in vitro. The oligomers are resistant SDS,

high salt, alkaline pH, and EDTA treatments, but

reduction by dithiothreitol. Thus, Vpl oligomers

via disulfide bonds in vitra SV40 Vpl contains cysteinesany or all of which may be involved in intermolecular

bridge formation. Based on structural and sequence

studies, we have initiated systematic oligo-directed

of Vpl cysteines to serine residues. Current experiments

the ability of these mutants to form stable oligomers vitro.

538

GRP94 BINDING TO IMMUNOGLOBULIN CHAINS

FROM BIP BINDING. ((J.Melnick and Y. Argon))

Immunology, Duke University Medical Center, Durham,

During their transit through the endoplasmic reticulum,

light (L) and heavy (H) chains of immunoglobulins(Ig)two resident ER stress proteins, BiP/GRP78 and

immunoprecipitation of DSP-crosslinked lysates,

GRP94-L binary complexes exist in addition to BiP-L GRP94-BiP-L

complexes. We estimate that GRP94-L complexes

molecules per L chain. In addition, we show that

and BiP to Ig chains can be independently modulated

findings suggest that GRP94 is a polypeptide-binding protein.

ATP causes BiP to dissociate from L and H chains,

not affected. Conversely, depletion of intracellular Al?

BiP but diminished GRP94 association, suggesting

may be ATP-dependent. Phosphorylation also regulates activity:GRP94 is phosphorylated on serine and threonine

phosphorylated GRP94 associates with L chains.

preferentially to mutant L chains whose transport

Using Brefeldin A to arrest a wild-type L chain the

increased association of BiP, but not of GRP94.

retention in the ER is not sufficient for association

GRP94 detects a structural alteration in mutant L

binds transiently to newly-synthesized wild-type L chains, GRP94

association is stable. We propose that GRP94

malfolded immunoglobulin chains, whether

sequence, and may function as an ER scavenger.

92a

Sunday. Protein Folding and Assembly (539-544)

539

THREE MOLECULAR CHAPERONES ASSIST THYROGLOBULIN INTHE ER. ((P.S. Kim and P. Arvan)) Endocrinology, Beth Israel Hospital,Cell and Developmental Biology Program, Harvard Med School, Boston MA

In thyrocytes, molecular chaperones assist thyroglobulin (Tg) mole-cules in early maturation from disulfide-linked aggregates->unfolded mono-mers->folded monomers->dimers (overall tst2 >30min) before export fromthe ER. Since -60 native disulfides are critical to folding, we can decelerateearly folding by brief reduction of live thyrocytes with DTT, to extend theperiod of chaperone involvement. Without DTT treatment at steady-state, co-ppts under native conditions show clear but low Tg associations with calnexin(an ER membrane protein), ERP72, and BiP. Within 10 min of thyrocyteexposure to DTT, nascent Tg is converted to disulfide-linked aggregates witha t1r2 of early folding >2h. These newly-formed aggregates co-ppt ERP72and are quantitatively recovered by anti-calnexin, but association with BiPremains modest. However, during 60-120 min of chase after DTT wash-out,aggregates decline and unfolded monomers increase; calnexin binding fallsand BiP binding rises. Nonreducing SDS-PAGE of co-ppt'd Tg after DTTwashout shows calnexin is bound exclusively to aggregated Tg; BiP bindsthis form only modestly but recognizes better the subsequently formed (un-folded) monomer. Chase beyond 2h shows decreased Tg binding to calnexinand BiP. Since BiP binds non-glycosylated Tg aggregates very well but DTT-induced aggregates only modestly, we examined effect of DTT on chaperonebinding in tunicamycin-treated cells. Calnexin binding of nonglycosylatedTg aggregates went from nearly zero to nearly quantitative, i.e, calnexin doesnot bind any form of N-linked sugars. ERP72 binding also increased afterDTT treatment, while BiP binding (already high) to nonglycosylated Tgremained high after DTT. Indeed, complexes of calnexin-nonglycosylated Tgafter DTT treatment also co-ppt BiP. We conclude that all three of thesechaperone molecules are intimately associated with early folding forms of Tg.

541

DIFFERENTIAL IODINATION OF WILD TYPE AND MUTANTPROINSULINS: IMPLICATIONS FOR THE IN-VIVO MEASUREMENT OFPROTEIN ASSEMBLY. ((T. Kehle, A. Schmitz, M.Maintz, and V.Herzog))Institute for Cell Biology, Ulrich-Haberland-Str. 61a, 53121 Bonn, Germany.(Spon. by W.B. Huttner.)

We have observed that by expressing thyroid peroxidase (TPO) in CHO-cells,proteins can be iodinated during their transport along the secretory pathway. Weapplied this system to study the intracellular iodination of proinsulin, which isknown to form dimers and hexamers, and of a mutant proinsulin bearing an N-glycan in the hexamer forming domain. TPO-CHO cells were cotransfected withproinsulin or the proinsulin mutant, respectively. Electron and light microscopeobservations confirmed that TPO was enzymatically active within the cisternae ofthe rER and that both proinsulin and its glycosylated mutant colocalized to thiscompartment. To our surprise, only the mutant proinsulin was iodinated, whereaswild type proinsulin remained unlabeled. From X-ray data it is known that alltyrosine residues are buried in the hexameric state of insulin or proinsulin, whereasthe tyrosine residues are freely accessible in the monomeric form. We interpret ourresults as an indication that wild type proinsulin was not iodinated because itexisted in hexameric form in the rER. In contrast, in the proinsulin mutant the N-glycan located whithin the hexamer-forming domains inhibited oligomerisationleaving the tyrosine residues accessible to iodination. Since in vitro tyrosineaccessability can be used to detect conformational changes of proteins, ourobservations might be the basis for a new method to determine protein assemblyand conformation in living cells (supported by the Deutsche Forschungs-gemeinschaft, SFB 284 and the Fonds der chemischen Industrie).

543

IN VITRO FIBRINOGIN ASSBMBLY (S. Roy and C0.. Redman))Lindeley F. Kimball Research Institute, New York Blood Center,NY 10021.

Human fibrinogen (340kDa) is a dimer, with each identical

half-molecule composed of 3 different polypeptides (Aar, BP and

y). Previous studies showed that chain assembly occurs in a

step-wise manner and is completed in the BR. To understand

the mechanisms an l vitro transcription/translation systemwas developed. Individual fibrinogen chain cDNAs were

inserted in an expression vector (pYEs2) at the 3 end of T7

pro-oter. Other constructs had 2, or all 3, of the fibrinogencDNA. in tandem, each under the control of T7 promoter.Fibrinogen chain expression and assembly was assayed in a

system coupled to rabbit reticulocyte lysate in the presenceor absence of dog pancreas microsomes. Nicrosomal membranes

were necessary for fibrinogen assembly. In the presence of

oxidized glutathione, but in the absence of membranes,combinations of any 2 chains, or of all 3 chains, yieldedindividual frem chains and some diffuse higher molecular

weight complexes. In the presence of membranea, expressionof Aa and y or BP and y yielded free chains and 2 chain

complexes. Unlike combinations of Aa,y and B$,y expression of

Aa and Bp did not form a distinct 2-chain complex. Expressionof all 3 chains yielded free chains, 2 chain complexes and

higher molecular weight complexes which corresponded to half-

molecules and dimeric fibrinogen. Treatment of this mixturewith plasmin generated digestion products some of which were

similar to plasmin digests of plauma fibrinogen.

540ROLE OF TRANSMEMBRANE DOMAINS IN ASSEMBLY ANDINTRACELLULAR TRANSPORT OF CD8.((P. Cosson and S. Hennecke)) Basel Institute for Immunology*,Grenzacherstrasse 487 Postfach CH-4005 Basel, Switzerland.*Founded and supported by Hoffman-La Roche LTD, Basel

CD8 can be present at the cell surface either as a disulphide-linkedhomodimer of CD8a or as a disulphide-linked heterodimer of CD8a andCD81. We analyzed the assembly and intracellular transport of CD8with particular emphasis on the role of the transmembrane domains(TMD). A chimeric protein made by replacing the TMD of CD8a withthat of the interleukin-2 receptor a chain exhibited reduced ability toform homodimers, while a mutant of Tac containing the CD8a TMDdimerized efficiently. Contrary to CD8a, CD8I expressed alone wasretained in the endoplasmic reticulum (ER). Only a small amount ofCD8 e formed homodimers and these also remained in the ER. A mutantof CD81 that dimerized efficiently was also retained in the ER, thusproving that ER retention of CD8J is not due to its poorhomodimerization. Rather the extracellular domain of CD81 requiresinteraction with that of CD8a to exit the ER. The TMD of CD81 wasalso shown to participate in ER retention by preventing exit ofmonomeric CD8 out of the ER. These findings demonstrate the role ofTMDs in assembly and intracellular transport of the CD8 molecule.

542THE ROLE OF DIMERIZATION IN THE SECRETION OFIMMUNOGLOBULIN LIGHT CHAINS. ((J.L. Dul and Y. Argon)) Dept.of Immunology, Duke University Medical Center, Durham, NC 27710.

The Kc chain of the myeloma MOPC21 is an unusual immunoglobulin (Ig)light (L) chain, in that it fails to form covalent dimers and is not secretedwhen expressed alone. The secretory failure can be overcome by assemblywith a heavy (H) chain. We showed previously that the secretory defect isdue to replacement of the highly conserved Tyr/Phe87 in the variable (V)domain with His. To investigate whether His87 blocks secretion because itaffects L chain dimerization, we examined the ability of MOPC21 x toassemble with various Ig chains in transfected COS-1 cells. We find thatMOPC 21 K chains do not form covalent homodimers in these cells, as isthe case in myeloma cells. Moreover, the revertant K chain containing Tyr87fails to dimerize, but is secreted as well as any wild-type L chain. In doubletransfectants, the revertant and mutant chains also do not assemblecovalently with one another and secretion of the mutant is not restored. Incontrast, a chimera combining a H chain V region with the same CKc regionrescues secretion of the mutant. Although the chimera does not formcovalent dimers with the mutant, it assembles with it non-covalently, asdemonstrated by cross-linking with DSP. Under the same cross-linkingconditions, we are still unable to trap non-covalent homodimers ofMOPC21 K or of the revertant K chain. Apparently, even non-covalentassociation of these L chains is rare. The same is not true of H chains:expressed alone or in combination with L chains, H chains are foundprimarily as covalent oligomers. These data indicate that dimerization per seis not required for L chain secretion, and that L chain monomers that haveachieved a correctly folded conformation are transport competent.

544THE INTERACTION BETWEEN RHODOPSIN AND THECYCLOPHILIN HOMOLOG, NINAA. ((E.K. Baker and C.S.Zuker)) Department of Biology, University of California at SanDiego, La Jolla, CA 92093. (Spon., by N.J. Colley)

In Drosophila, the biogenesis of the major visual pigment,Rhodopsin 1 (Rhl), is dependent on the presence of a visualsystem-specific cyclophilin, ninaA. In flies lacking functionalninaA, Rhl is reduced over one hundred fold, and is retainedwithin the Endoplasmic Reticulum. Because other cyclophilinshave been shown to aid protein folding, in vitro, we havepreviously suggested the model that ninaA is necessary either inthe proper folding and/or transport of Rhl. In order tocharacterize the interaction between ninaA and Rhl we havegenerated transgenic flies expressing a tagged form of Rhlcontaining six histidine residues, and shown that this affinitypurified Rhl is found associated with ninaA. These resultsprovide the first direct biochemical evidence that Rhl andninaA physically interact. Using this assay in conjunction witha collection of genetically-defined alleles of ninaA (Ondek, et.al.JBC, 267(23): 16460-16466, 1992) we have determined that the Cterminal region of ninaA plays a critical role in the interactionwith Rhl.

93a

Protein Folding and Assembly (545-550). Sunday

545ASSEMBLY OF FIBER TRIMERS FROM HUMAN ADENOVIRUS TYPE2. ((B.L. Mulach, J.S. Hong, and J.A. Engler)) Dept. of Biochemistry andMolecular Genetics, University of Alabama at Birmingham, Birmingham, AL35294-0005.

Adenovirus fiber is a trimer composed of three identical subunits. Fibersfrom Ad2 and Ad5 contain the monosaccharide modification of 0-linked N-acetylglucosamine (O-GlcNAc), linked to serine or threonine residues; one rolesuggested for O-GlcNAc has been in the assembly and stabilization ofmultimeric complexes. Point and deletion mutants in the Ad2 fiber gene havebeen made to map the sites on fiber which contain this carbohydratemodification. These mutants, as well as wild type fiber, have been expressed ina recombinant vaccinia virus system. Comparisons of native fiber andrecombinant fiber have been made using Westem blot analysis to show thatthese proteins contain similar amounts of O-GIcNAc, asjudged by reactivity tomonoclonal antibody RL-2, which recognizes O-GlcNAc. Similar experimentshave been performed with fiber mutants in order to compare the sugar contentof wild type and of mutant proteins.Series of deletion mutants of Ad2 fiber were made to study functional

domains for fiber tirmerization and the relationship between sugar modificationand trimerization. Fiber mutants with small N-terminal deletions are defectivein nuclear translocation, but not in trimerization. At least one intemal deletionmutant, 2F-djSph, is able to form trimers, but other internal deletions are not.C-terminal deletion mutants as small as three amino acids were correctiylocalized in the nucleus, but were not able to form trimers.Taken together, Ad2 fiber appears to have at least two functional domains: the

N-terminus for nuclear localization and the C-terminus for trimerization. Thiswork was supported by NIGMS grant GM38234.

547

BIOSYNTHESIS, ASSEMBLY AND SECRETION OF TENASCINHEXAMERS. ((S.D. Redick and J.E. Schwarzbauer)) Department ofMolecular Biology, Princeton University, Princeton, NJ 08544.

Tenascin is ahexameric extaellular matrix molocule hat is expressedduring embryonic development and tumor formation and which hasbeen shown to have anti-adhesive activities. Recently, it has becomeapparent that tenascin is a member of a family of structurally-relatedmodular proteins. Members of this protein family have a uniquetimeric or hexameric swtucre and we have undertaken an examinationof the biosynthesis, assembly and secretion of tenascin hexamers.Cultured U-138 MG human glioma cells secrete tenascin as hexamerscomposed of 320 IDa monomers. These hexamers are secreted intothe cell-conditioned medium rather quickly, appearing after a 30minute labeling. Intracellular tenascin can first be detected following a Sminute pulse labeling. This intracellular tenascin is largely hexawericand the level of intraellular hexamer steadily decreases during asubsequent non-radioactive chase, suggesting that these molecules arebeing seceted into the culture medium. Following a 20 minute chase,the intracellular tenascin begins acquiring endoglycosidase-Hresistance, consistent with its being localized to the Golgi apparatus.The endoglycosidase-H resistant tenascin quicldy disappears from theintracellular fraction, as expected if secretion from the Golgi apparatusis rapid. Similar results were obtained from an examination of thesynthesis and secretion of a recombinant tenascin which encodes anamino-tminal fragment of the molecule. Together, these data suggestthat tenascin hexamer assembly is rapid, consistent with co-tanslational formation of multimers.

549

TRANSMEMBRANE DISPOSITION OF THE ALPHA SUBUNIT OF THENA,K-ATPASE. ((Y. Xie and T. Morimoto)) Department of Cell Biology,New York University School of Medicine, New York, N.Y. 10016.

To ascertain the transmembrane disposition of the a subunit of the Na,K-ATPase, we studied the ER membrane insertion of chimeras consisting oftruncated a polypeptides linked at their C-termini to chloramphenicolacetyl transferase (CAT), a reporter protein, that contains a consensussequence for glycosylation. The chimeras Included aminoterminalportions of the a subunit ending at amino acid residues 121, 167, 313,418, 818, 845, 872, 884, 938, 976 and 008. These sites were chosen onthe basis of a transmembrane disposition inferred from hydropathyanalysis and lie within the various presumptive extra-cellular (121, 313,818, 845, 818, 845 and 938) and cytoplasmic domains (167, 418, 872,884, 976 and 1008) of the a subunit. After transcription of the chimericcDNAs, the mRNA was purified and translated in a rabbit reticulocytelysate In the presence or absence of dog pancreas microsomalmembranes. The products were analyzed by Immunoprecipitation usinganti-dog kidney a subunit antibody and either protein A or Con-ASepharose, followed by Endo H digestion. The results demonstrated thatall the chimeras were glycosylated, except for those in which the a subunitwas truncated at positions 167, 418 and 1008. These results support theexistence of 6 membrane spanning domains wlthin the a subunit, butindicate that amino acid residues 872, 884, and 976 are exposed on theextra-cellular side. Therefore, the first 5 domains have the positionspreviously proposed, but the 6th domain appears to be located betweenamino acid residues 976 and 1008, a region of low hydrophobicity.(Supported by NIH grant GM20277).

546PHOSPHOOLUCOMUTASE SYNThESIE IN RESPONSE TO GLUCOSEDEPRIVATION AND HEAT SHOCK IS UNDEROUCOSYLATED. ((N. Dey, P.Bounelis, TA. Fritz, D.M. Bedwell, and RB. Marchase)) Depts. of CellBiol, Biochem. and Mol. Genet., and Microbiol., Univ. of Alabama atBirmingn, Birmingm, AL 35294.

Phosphoglucomutase (PGM) is the acceptor for UDP-glucose:glycoprotein glucose-l-phosphotransferase (Gic-phosphoransfse) andcontains Gic in a phosphodiester linkage to 0-linked Man. In this study,we have characteied the synthess and glycosylation of PGM by S.cereisiae in response to both glucose depivation and heat shock. PGMsynthesis is inceased in yeast grown to early log phase (OD0W-0.5)following a brief heat shock (370C, 30 min) or when cells re glucosedeprived by growth to an OD6W of 4 as meaured by Western blotanalyses, metabolic labeling with [35Smehonine, and PGM enzymaticactivity. PGM expressed by heat-shocked or glucose-deprved cells wasa) underglucosylated as nmeaued by metabolic labeling with (6-3H]Glc,b) a better acceptor in in vitro Gl-phosphrsrse assays and c) lessnegatvely charged by DEAE chromatography compared to PGMexpCresed by control cells. Both fonns had similar specific activities,suggesdng that the vaiant oligosaccharide expressed during heat shockor glucose deprvation does not directly reglate PGM enymaticfunction. Membrane-associated PGM has been shown to be inactive.The oligosaccharide may participate in this interaction and provide a

means to alter PGM activity and cell metabolism in response to stras.

548THE a AND ,B SUBUNITS OF THE NA,K-ATPASE CAN ASSEMBLEAT THE PLASMA MEMBRANE ((A.W. De Tomaso and R.W. Mercer))Department of Cell Biology and Physiology, Washington University Schoolof Medicine, St. Louis, MO 63110.

We have previously demonstrated that the a and subunits of the rodentNa,K-ATPase are targeted to the plasma membrane when expressedindividually in baculovirus-infected Sf-9 cells. Additionally, the detergent-solubilized a and P polypeptides can be resolved by velocity sedimentationon sucrose gradients into single peaks, suggesting that each subunit is in astable conformation when expressed alone. We then proposed the question:do the two subunits at the plasma membrane retain the ability to assembleinto a functional heterodimer, or is the assembly process restricted to theER? To answer this we have taken advantage of a fortuitous property of thebaculovirus expression system; acidic pH triggers syncitia formation ininfected Sf-9 cells. One of the endogenously expressed viral proteins, gp64,is the baculovirus analog of hemagglutinin. Similar to hemagglutinin, gp64is an acid-triggered fusogenic protein. When Sf-9 cells individually infectedwith either the a or P recombinant baculovirus are plated together at highdensities and subjected to a mild acidic shock, they form large syncitia. Wedemonstrate that in the newly continuous plasma membrane of thesesyncitia the individual Na,K-ATPase a and N subunits assemble into aoiheterodimers as assayed by detergent resistant coimmunoprecipitation.Additionally, the newly assembled heterodimers are functional, as syncitiashow a significant increase in ouabain-sensitive potassium uptake. Thisobservation may be of physiological relevance. It has been proposed(Hundal, et al., JCB 267:5040-5043, 1992) that insulin stimulated Na,K-ATPase activity in rodent muscle is the result of translocation of a and Psubunits to the plasma membrane from two distinct intracellularcompartments, necessitating assembly in a post-ER environment.(Supported by NIH)

550HLPI-I IS INVOLVED IN TARGETING AND ASSEMBLY OFOUTER MEMBRANE PROTEINS IN ESCHERICHL4 COLI. ((A.M.Roy',L. Taylor2,Z. Garcia2,and J. Coleman') 'Louisiana State UniversityMedical Center, New Orleans LA. and 2Louisiana State University -

International Center for Medical Research and Training, San Jos6, CostaRica.

In both eukaryotic and prokaryoic cells the protin targetig pathway requr

proteins to cross the membrane in an unfolded state. Proper folding and assembly ofproteins are essntial for survival and optimal growth ofthe cell. Gram negativebacteria have two mnbranes: an imner membrane and an outer manbrne separatedby a periplasmic space. The sequence ofthe gene for HLP-I, hlpA, reval thepresence ofa sigal sequence that dires the protein out ofthe cytoplasm. Electronmicroscopy iunogold labeling localizes the HLP-I protein in the periplasmicspace near the iner mnbrane. Thme et al. (FEBS Lett. 1990; 269:113) proposeHLP-rs involvement in protein scretion, since it can overcome in vitro a non-fuional SecA (a cytoplasmic cmpnat ofthe bacterial socreory machinery).Our in vivo work demonstrates that ovrexpression of HLP-I does not overcome therequiremet for a fimctional SecA. Altdo HLP-I is dispensable for growth underlaboratory conditin, we have evidene to suggest that HLP-I acts as a periplasmicdcape involved in the assembly of outer membrane coponents. A crosslinkintstudy demonstrates an interaction ofHLP-I with oder proteins in vtvo. HLP-I alsointcts with other cellular proteins, one ofthem being dte outer membrane OmpA.HLP-I afficts the assembly ofthe LamB protin imto the outer membrane. Stresfacto such as growth at high temerat , induce HLP-I production fromplasmids contining hlpA, a feaxtre common to chapeones. However, in cont toother heat shock proteins, induction ofHLP-I is slower and maintained for sveralhours during incubation at high temperature.

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Sunday. Protein Folding and Assembly (551-556)

551NATIVE STRUCTURE AND BIOCHEMICAL BEHAVIOR OF A SMALL HEATSHOCK PROTEIN. ((K.W. Osteryoung and E. Vierling)) Department ofBiochemistry, University of Arizona, Tucson, AZ 85721.

In higher plants, the heat shock response is dominated by the induction ofthe small heat shock proteins (sHSPs), which accumulate in the cytosol, ERand chloroplasts. The abundance, diversity and high degree of conservationamong the plant sHSPs suggests they are critical for plant survival at hightemperatures. Recent studies have shown that sHSPs exhibit activities invitro consistent with a molecular chaperone-like function. However,essentially nothing is known concerning the role of the sHSPs in vivo inresponse to heat stress. Using biochemical techniques and transgenic plantapproaches, we are investigating the chloroplast-localized sHSP, HSP21 (21kD), to understand its contribution to the development of thermotolerance inIlants. HSP21 is not detectable in the absence of stress, but accumulates ineat-stressed plants in proportion to temperature. Analysis of soluble

extracts from heat-stressed pea plants by pore exclusion native gelelectrophoresis indicates that in this species HSP21 is present in two highmolecular mass complexes of 200 and 230 kD. In both isolated chloroplastsand intact plants, these soluble complexes become associated with theinsoluble fraction in a time- and temperature-dependent manner. Thetransition to insolubility is reversible; during recovery from stress, insolubleHSP21 redistributes back into the soluble fraction. The soluble and insolubleforms of the protein are not differentially modified based on 2D gel analysis.Assembly of the soluble 200 and 230 kD complexes from the 21 kDmonomer occurs only in chloroplasts isolated from heat-stressed pea plantsand not from control plants, suggesting that HSP21 assembly into higher-order structures requires other heat-inducible factors. However, intransgenic Arabidopsis plants that constitutively overexpress HSP21,assembly of HSP21 into the native complex (300 kD in this species) occursin the absence of heat stress. Further analysis of transgenic plants thateither overexpress or underexpress HSP21 should help to elucidate the invivo function of sHSPs in all organisms.

553IDENTIFICATION OF A NEW HSP70 LIKE GENE IN CHINESE HAMSTERCELLS ((D. Yoon, M. Murawski, and JRI Subjeck))Dept. of Molecular and CellularBiology, Roswell Pas Cancer Institute, Buffalo, NY 14263.

We have screened a cDNA expression library prepared from mRNA obtained fromheat shocked CHO cells with two antibodies prepared against the yeast 104 kDa(generous gift of Dr. S. Lindquist) and mammalian 110 kDa heat shock proteins.Previous studies indicated that immunoprecipitation of HSP10 from CHO cells witheither antibody produced a protein detected by Western blot analysis with the other

antibody. Two cDNA clones were identified as positives with both antibodies.Northern blot analysis indicated that both clones, containing insert sizes of 1 and 3kb, were strongly heat inducible, hybridizing to a message of approximately 3.5 kb.Restriction mapping and sequence analysis indicates that the 1 kb clone is identicalto a Ilkb region at the 3' end ofthe 3 kb clone. Although 85% of the 3kb clone hasbeen sequenced, the initiation site and poly-A tail have not yet been identified.While there was no significant homology(>60%) at the nucleotide level with anyknown sequences, translation of vanous regions in the 5' half of the clone indicatedstng sinilarities with various HSP70s, especially human HSP70. Regions in both5' and 3' halves of the clone also indicated strong sequence simlariies with SeaUrchin sperm receptor (Foltz et. aL Science, 259 1421-1425, 1993). The sequencesexanined to date for these clones bear no homology with yeast HSP104, althoughthey were identified with antibodies prepared against both of the high molecularweight yeast and manmalian HSPs. However,both yeastand mammalian antibodiesappear to exhibita weak cross-reactivity with a 70 kDaprotein which may be HSP70.Present data suggests that this gene encodes a large mammalian HSP. Whether thisis the HSP110 species observed in many mammalian systems and/or a new HSP70species is currendy being examined

555

COFACTOR DEPENDENT UNCOATING OF CLATHRIN BASKETS BY UNCOATINGATPase. ((W. Barouch, K. Prasad, L. Greene, & E. Eisenberg))Lab of Cell Biology, NHLBI, NIH, Bethesda, MD 20892.

Bovine brain uncoating ATPase, a member of the hsc70 familyof heat-shock proteins, uncoats clathrin coated vesicles inan ATP dependent reaction. We previously reported thatclathrin baskets assembled from highly purified clathrin andAP2, the plasma membrane associated assembly protein, cannotbe uncoated by uncoating ATPase and ATP alone, but alsorequires small amounts of a protein cofactor isolated frombovine brain coated vesicles. We have now purified thiscofactor and found that it has a molecular weight of 100 kDa.We also find that, like AP2 baskets, baskets made fromclathrin and AP1i0, the neuronal specific assembly protein,require the 100 kDa cofactor to be uncoated. Using AP1i0-clathrin baskets we find that, in the presence of cofactor,both the uncoating itself, and the ATPase activity associatedwith the uncoating show a rapid initial burst followed by aslow steady state. In the absence of cofactor, neither burstoccurs; there are only very slow background levels ofuncoating and ATPase activity. This demonstrates that thesame cofactor which regulates the uncoating of AP2 basketsalso regulates the uncoating of AP1, baskets and is requiredfor the ATPase activity associated with uncoating. It istherefore likely, that the cofactor dependent regulation ofuncoating by the uncoating ATPase is coimmon to coatedvesicles from all sources and may constitute an importantstep in the endocytosis pathway.

552

HSP-72 MEDIATED STABILIZATION OF PROTEIN SYNTHESIS IN

THERMOTOLERANT CELLS DURING HEAT SHOCK. ((A. De Maio,and S.C. Beck)) Division of Pediatric Surgery, Johns Hopkins UniversitySchool of Medicine, Baltimore, MD 21287.

The synthesis of constitutive cellular proteins is suppressed during moderate

heat shock at 43°C; however, if cells are allowed to recover at 37°C after this

moderate thermal stress for at least 2 hours, they become resistant to the

inhibitory effect of heat shock on protein synthesis. Such tolerance to this

second stress can be correlated with maximal expression of heat shock

proteins, particularly hsp-72. The protection of protein synthesis in

thermotolerant cells was not due to increases of transcription, stabilization of

preexisting messages, or protection from degradation of newly synthesizedpolypeptides. Polysome profiles of thermotolerant and nonthermotolerant cells

incubated at 43°C and control cells incubated at 37°C were obtained. The

polysome pattem of thermotolerant cells under heat shock was identical to the

polysome pattern of control cells maintained at 37°C; this observation is in

contrast to the disruption of polysomes in nontolerant cells incubated at 43°C.Moreover, hsp-72 was found bound to polysomes of thermotolerant cells as

detected by Western blot analysis. We propose that during heat shock the

nascent polypeptides cannot fold correctly and 'precipitate" within the

ribosomes, affecting translational elongation. Binding of hsp-72 to nascent

polypeptides during heat shock may keep these nascent polypeptide chains in

solution maintaining elongation and consequently rescuing translation.

554

PURIFICATION AND CHARACTERIZATION OF EUCARYOTIC TCP-1. ((R.LMartin, C.R. Brown, J.R. Ungappa, V.R. Ungappa, and W.J. Welch))Department of Physiology, University of CalIfornia, San Francisco, Box0854, San Francisco, CA 94143.

Continuing our study of molecular chaperones, we have Investigated thelocalization and function of the putative eucaryotbo cytosollcchaperonin, TCP-1. A polyclonal antibody was raised against TCP-1 to

facilItate these studies. This antibody recognizes a 20S particle(presumably similar In structure to GroEL) that was purffied from bovinebrain. This panicle appears to be homogenos In structure, oonsistingof a single 60 kDA protein. In addition to being found in the cylosol, TCP-1 was found In the nucleus and In the microsomal fraction. UnlIke othermolecular chaperones, e.g., the hsp70 family and mltochondrlal hsp58,which are upreogulated In response to heat shock and other metabolicstressors, TCP-1 was not found to be induced by these stressors. Byuse of varlousIn vivoand In vItroexperiments, the Involvement of TCP-1In polypeptide chain maturation and macromolecular assembly anddlsassembly wHI be presented.

556

THE INTERACTION OF NUCLEOTIDE-FREE hsp70 WITH CLATHRIN AND

PEPTIDES. ((B. Gao, L. Greene, E. Eisenberg)) Lab of Biol.,NHLBI, NIH, Bethesda, MD 20892

The functions of the 70 kDa heat-shock proteins appear to be

regulated by bound nucleotide. We previously developed amethod of preparing functional nucleotide-free bovine brain

uncoating ATPase (hsc70) and in the present study, we usethis nucleotide-free enzyme to investigate the effects of

different bound nucleotides and nucleotide analogues on the

interaction of hsp7O with its protein substrates. We find,first, that clathrin interacts with nucleotide-free enzyme inthe same manner as it interacts with enzyme-ATP; it binds anddissociates very rapidly in contrast to its very slow bindingand dissociation from enzyme-ADP. On the other hand

cytochrome C peptide interacts with nucleotide-free enzyme in

the same manner as with enzyme-ADP; it dissociates veryslowly in contrast to its rapid dissociation from enzyme-ATP.The nucleotide-free enzyme also forms dimers and oligomerslike enzyme-ADP rather than enzyme-ATP. Therefore, the

behavior of the nucleotide-free enzyme appears to depend on

the nature of the substrate with which it is interacting.Second, we find that the nucleotide analogues, AMP-PNP and

dATP, have essentially no effect on the properties of the

nucleotide-free enzyme, either in regard to interaction with

clathrin or peptides, or in regard to polymerization of the

enzyme. Therefore, previous effects observed with these

analogues on the interaction of clathrin with uncoatingATPase were probably due to competitive removal of ADP from

the enzyme rather than to a direct effect of the analogue.

95a

Protein Folding and Assembly (557-558). Sunday557THE EFFECT OF hsp7o POLYMERIZATION ON ITS INTERACTION WITHPEPTIDES AND CLATHRIN. ((B. Gao, E. Eisenberg and L. Greene))Lab of Cell Biol., NHLBI, NIH, Bethesda, MD 20892

Monomeric bovine brain uncoating ATPase (hsc7O) polymerizesinto dimers, and higher order oligomers in the absence ofATP. In this study we investigated the interaction ofpolymerized uncoating ATPase with cytochrome C peptide andwith clathrin. We find that, with both nucleotide-freeenzyme and enzyme with bound ADP, addition of cytochrome Cpeptide shifts the distribution of the enzyme from about 50%polymer to almost all monomer. This formation of monomerdoes not require ATP hydrolysis since it occurs withnucleotide-free enzyme as well as with enzyme with bound ADPor AMP-PNP. Analysis of the binding of peptide by FPLCsuggests that peptide binds only to the monomeric populationof enzyme. Therefore, in contrast to BiP (Blond-Elguindi etal., 1993, J. Biol. Chem. 268, 12730) which appears to bemonomerized by peptides binding directly to dimeric enzyme,our results suggest that peptide monomerizes the uncoatingATPase by reducing the concentration of free monomericenzyme, thereby pulling the monomer-oligomer equilibriumtowards monomer. Preliminary studies indicate that clathrinmay also bind only to uncoating ATPase monomer since withnucleotide-free enzyme we see an initial burst of binding,presumably to monomer, followed by much slower binding whichmay be due to a slow depolymerization of oligomers intomonomers. Our results suggest that both cytochrome C peptideand clathrin may bind to the same general region on theenzyme which is involved in enzyme polymerization.Membrane Domains and Polarity (559-562)

558UNCOUPLING OF PEPTIDE-STIMULATED ATPASE ANDCLATHRIN-UNCOATING ACTIVITY IN DELETION MUTANTOF HSC70. ((M-Y. Tsai and C. Wang)) Institute of MolecularBiology, Academia Sinica, Taipei, Taiwan

Recombinant 60-KD fragment of rat hsc70 has beenoverexpressed in Eschthricliii coli. The recombinant protein isniot capable of disassembling clathrin from coated vesicles.However, the affinity for peptides and the peptide-stimulatedATPase activity of the intact protein are retained in the 60-KDfragment. The dissociation constants of peptide P3a (therecognition sequence of clathrin light chain LCa by hsc70) and S-peptide of ribonuclease for 60-KD protein are 13 and 7 p.M,respectively. The maximal velocities of stimulated ATPaseactivity by peptides P3a anid GT4 are 0.25 and 0.31 nmol/hr/tLg ofproteini, respectively; and, the EC50 values (the concentration ofpeptides brotught about half maximun hydrolysis) for peptidesP3a anid GT4 are 0.56 and 0.30 mM, respectively. These resultsindicate that peptidle-stimulated ATPase activity of hsc70 is notstufficienit for clathriln-Lncoating. We stuggest that other activitiesor cellular components as yet unidentified associated with the C-terminal 10-KD fragment of hsc70 is required for clathrin-uncoating.

559

LOCALIZATION OF A PUTATIVE BASOLATERAL SORTING SIGNAL IN THENEURAL CELL ADHESION MOLECULE, N-CAM. ((AMH. Le Gall, S.K. Powell,A. Rajasekaran. and E. Rodriguez-Boulan))Department of Cell Biology andAnatomy, Cornell University Medical College, NY, NY 10021.

Transmembrane and GPI-anchored isoforms of the neural cell adhesionmolecule, N-CAM, are targeted to opposite domains when transfected intothe polarized epithelial cell line, MDCK. While the GPI anchored form

(ssd) is targeted directly from the TGN to the apical surface, the twotransmembrane forms (sd and Id) are directly delivered to the basolateralsurface (S.K. Powell et al, Nature 353:76, 1991). Because all threeisoforms share a common ectodomain but are differentially targeted,localization must be determined by either regions of membraneassociation and/or the cytoplasmic domain. To address this question. the

cytoplasmic domain of the 140 kD isoform of N-CAM was deleted.Deletion of the cytoplasmic tail resulted in the non-polar steady statedistribution of N-CAM in MDCK as assessed by immunofluorescence andcell surface biotinylation. Membrane targeting assays demonstrated thatthis distribution resulted from the direct targeting of the truncated N-CAM to both surfaces. This suggested that targeting information was notlocalized in the membrane spanning region of N-CAM but in the

cytoplasmic domain. To further characterize the putative basolateralsorting determinant, a nested set of deletion mutants were constructedand analyzed using similar techniques. Analysis of these constructsindicates the presence of a putative basolateral sorting determinant

present 60-78 amino acids from the transmembrane domain of sd-N-CAM.

(Supported by an NSF predoctoral fellowship RCD-9253029 to AHL, and

grants from the Spinal Cord Research Foundation #1152 to SKP, and NIHGM 34107 to ERB).

561

MURINE VASCULAR CELL ADHESION MOLECULE (VCAM-1) PROTEINSENCODED BY ALTERNATIVELY SPLUCED mRNAsARE SELECTIVELYTARGETEDIN POLARIZED EPITHELIAL CELLS ((G. Pirozzi, R. W. Terry, and M. A.Labow)) Molecular Sciences Group, Hoffmann-La Roche Inc. Nutley, NJ07110 (Spons. by D. K. Bums)

VCAM-1 is an immunoglobulin (19) auperfamlly member that is expreaaedin endothelial cells and functons In modating adhesion to a variety ofleukocytes. The predominant VCAM lioform contains seven Ig domainsfollowed by a single tranamembrane region and a short cytoplasmic domain(VCAMTM), while a cytokine inducible isotorm contains only the first threeIg domains and is attached b the cell surface via a glycosyl-phosphatidyl-inositol anchor (VCAMGPl). Both vascular and nonvascular expression ofVCAM has been reported In a variety of normal and patolgical processa,Including a number of human inabmmatby kidney csases where expressionin proximal tubular eplthellal colb was detected. One possible rol of the two

VCAM isoforms Is to allow for domain specific expreslon In polarized celle.In this study, MDCK celis permanenty xpressng both VCAM bohrms wereestablished, and Indiract immunofluoreacence was used to demonstrate thatisoforms with alternative modes of membrane asaociation are targeted todifferent surfaces of polarized epithelial cells. Specifically, VCAMTM isexpressed on the basolateral surface and VCAMGPI on the apical surface.Quantitative domain selective blotinylation was used to demonstrarte that>95% of each isoform was specifically labelled from respective surfacedomains.

560CAVEOLIN-VIP21 AND GLYCOSPHINGOLIPID CLUSTERS IN THETARGETING OF GPI-ANCHORED PROTEINS IN EPITHELIAL CELLS.((Chiara ZurzoloA#,Wouter van't HofM , Gerrit van Meer* and EnriqueRodriguez-BoulanA)) ADept. of Cell Biology and Anatomy, Cornell UniversityMedical College, New York ,#Dept. di Biologia e Patologia Cell. e Molec.,Naples University, Italy *Dept. of Cell Biology Utrecht University, Holland.

GD1-DAF, a model GPI-anchored protein is vectorially targeted to the apicalmembrane of MDCK cells. In FRT cells this protein is directly delivered tothe basolateral domain while in MDCK-ConAr cells it is not sorted. Weanalyzed such variability in the targeting of GPI-anchored proteins in view ofa sorting model that proposes the aggregations of apical transmembrane andGPI-anchored proteins, with glycosphngolipids (GSLs) rafts in the TGN.Although GPI-proteins are not polarized, in the ConAr mutant, the glycolipidglucosylceramide (GlcCer) was sorted to the apical membrane like in MDCKcells. In contrast, GlcCer was basolaterally targeted in FRT cells. In spite ofopposit sorting, and major differences in the GSL composition, both FRT andMDCK cells assembled GSLs into Triton X-100-insoluble complexes withidentical isopycnic densities. In both MDCK and ConAr cells GDI-DAFbecame insoluble in TX100 during transport to the surface and comigratedwith GSLs aggregates on sucrose density gradients. Surprisingly, in FRTcells, GDI-DAF remained compledy soluble and did not associate with GSLscomplexes. The clusteing defect of FRT cells correlated with lack ofexpression of caveolin-VIP21, a protein localized to both the plasmamembrane caveolae and the TGN. This suggests that caveolin may have acrucial role in recruiting GPI-anchored proteins in GSLs complexes. However,since MDCK-ConAr express caveolin and form GSLs cluster , yet missortGPI-anchored proteins, additional factors may be required for the accurateapical sorting of this proteins to the plasma membrane of epithelial cells.

562SORTING SIGNAL AND BASOLATERAL RECYCLING OF TGN-38 INMDCK CELLS. ((Ayyappan K.Rajasekaran, Jeffrey S.Humphrey*, MartinaWagner, Gero Miesenbick **, Andrd Le Bivic#, Juan S.Bonifacino*, EnriqueRodriguez-Boulan))Dept.Cell Biology and Anatomy, Cornell University MedicalCollege, New York.*Cell Biology and Metabolism Branch, National Institute ofHealth, Bethesda, Maryland,*"Cellular Biochemistry and Biophysics Progam,Sloan Kettering Institute, New Yoric, # Facultedes Sciences de Luminy, Marasille,France. (Spon. by A.M.C. Brown)

The Trans Golgi Network (TGN) is the major sorting site for apical andbasolatal plasma membrane proteins in polarized epithelial cells. TGN-38 hasbeen identified as a resident membrane marker protein of this orgmelle. A fusionprotein, composed of the ectodomain of Tac (Interukin 2 recepr a chain) andtransmembrue and cytoplassnic domains of TGN-38 (TGG) localizes precisdy tothe TGN and early endosomes and recycles quite efficiently to the plasmamesnbrane of non polarized cells. The sequence YQRL, contained within the 33amino acid cytoplasmic tail has been identified as the TGN localization signal innon polarized cells; modification or deletion of this signal results inmislocalization to the plasma membrane. We transfected TGO and severalcytoplasmic tail mutants into polarized MDCK cells. As in non-polwized cells,TGG localized to the TGN, defined as a perinuclear network which also stainedwithy- adaptin. A simiar localization was obarved for transfected TGN-38 with aMyc-epitope at the lumenal domain. Transfection of the TGG tail mutantsindicated that YQRL was the TGN-localization signal in polarized MDCK celis.Antibodies against TAC or the myc epitope were taken up selectively from thebasolateral compartnent. Cell surface biotinybtion experiments revealed a fraction

of TGO on the basolaeal membrane. These experiments demonstrate that the

localization signal for TGN-38 in polarized cells is identical to that of nonpolarized cels and that TGN-38 recycles selectively to the basolaseal domain inepithelial cells.

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Sunday. Membrane Domains and Polarity (563-568)563

TRANSCYTOSIS OF APICAL PLASMA MEMBRANE PROTEINS IN THEHEPATOMA-DERIVED WIF-B CELL UNE. ((G. Ihrke, K. Finnegan, and A.L.Hubbard)) Department of Cell Biology & Anatomy, Johns Hopkins University,School of Medicne, Baltimore, MD 21205

We are using WIF-B cells as an in vitro model to study trafficking of plasmamembrane (PM) proteins in polarized hepatocytes. In these cells, apical proteinsare concentrated at bile canalicular-like spaces (BC) between adjacent cells, whichare the functional equivalent to bile canaliculi in viwo (Ihrke et al., manuscriptsubmitted). Hepatocytes differ from other epithelial cells in that they deliver allapical PM proteins that have been studied to the cell surface via the basolateralPM. In living WIF-B cells at 4C, antibodies (abs) bind only to the basolateral PMbut not the apical PM, because tight junctions form a barrier between the twodomains. Abs to the apical PM proteins 5'-nucleotidase, dipeptidyl peptidase IV,amnbopeptidase N and HA4 bind to the basolateral surface of living cells, althoughthe concentration of these PM proteins (except 5'nucleotidase) is below the deectionlimit on fixed cells. When cells were warmed to 37°C for 0-180 min after ab-bindingand washing, then fixed and processed for IMF, increasing amounts of abs weredetected at the apical surface. This was not the case when abs to basolateralproteins were used. We conclude that apical PM proteins are transcytsed from thebasolateral to the apical surface in WIF-B cells, suggesting that newly synthesizedproteins might be transported via the indirect route as in vivo. Transcytosis wasinhibited by drugs that disrupt microtubules (colchicine, nocodazole). Toaccumulate apical PM proteins in the secretory pathway we applied BFA to thecells; 5 pg/ml completely dispersed the Golgi (IMF) and blocked secretion ofproteins (pulse-chase experiments). Protein synthesis was not diminished, butnewly synthesized apical PM proteins seem to accumulate in intracellularcompartments, as they remained in their partially glycosylated form. We arecurrentiy optimizing conditions to follow the trafficking of newwly synthesized PMproteins after release of the BFA induced block (DFG Ih 14/1-2, NIH DK 44375)

565

STIMULATION OF TRANSCYTOSIS BY ELEVATION OFCELLULAR cAMP INMDCK CELLS ((S.H.Hansen and J.E. Casanova)) Pediatric Gastroenterology,Massachusetts Geneal Hospital East, Charlestown, MA 02129

Recent evidence suggests that heterotrimeric G proteins play a role in vesiculartrort processes. Treatment of MDCK cells with cholera toxin (CIX), which activatesGSt, has been shown to enhance apically directed secetion (Pimplikar and Simons,Nature 362;456) and is reported to augment the transcytosis of the polymrcimmunoglobulin receptor (Bomsel and Mostov, Mol. Blol. Cell 3;1317). In agreement withthis latter runding, we have also found that CTX stimulates transcytosis of the pIgR. Todetermine whether this increase is due to a specific action of GSa on the pIgR or is causedby a general increase in membrane flow into the Irncytotic pathway, we have studied theeffect of Ga= activation on the tacytosis of ricin, a toxic lectin which binds to plasmamembane proteins and lipids containing terminal gahctose. Filter-grown MDCK ain II

cells were pretreated with or without CTX (2sgfml) and incubated with 125I-ricin in thebasolteral medium for lh at 37ec. Transytosed ricin was recovered from the apicalmembrane by stripping with 0.lM lactose. Tratment of monolayers with CTX induced a60% increase in ricin transcytosis without affecting monolayer integrity, as determined bymeasurement of transepithelia resistance or permeability to 14C-inulin. In the presence ofthe phosphodistea inhibitor isobutylmethyLxanthine (IBMX), transcytosis of ricinincreased further to >100% of control, suggesting that the effect of CTX was due toincreased cAMP production. Accordingly, forskolin, which activates adenylate cyclasedirectly, was neariy as effective as CTZX in stimulating ricin transcytosis. The effects offorskolin and CTX were not additive. Interestingly, CIX and forskolin also stimulated thetranscytosis of apically intemalized ricin to the basoaei surface, although to a leserextent Pertussis toxin, which inacdvates Gia and Goct, had no effect Overexpression ofthe GSaE subunit in MDCK II cells only increased transcytosis of ricin following treatmentwith CIX. These data combine to suggest that the volume of membrane entering thetrwcytotic pathway can be regulated by cellular levels of cAMP, thus raising thepossibility that tUacytosis in epithdia is influenced by extracellular cues. This hypothesis currently under investigation. Sponsored by a SeniorFellowship of the Danish CancerSociety (S.H.H.) and NTH AB32291 (JE.C.)

567

DIFFERENCES IN GLYCOSPHINGOLIPID AND Na/K-ATPase SORTING INPOLARIZED MDCK EPITHELIAL CELLS: INSIGHTS INTO PROTEINSORTING IN THE TGN. ((1R.W. Mays, 2G. van Meer and IW. J. Nelson))IDept. Mol.& Cel. Physiolog, Stanford Univ. Scb. Med., Stanford, CA 94305,anW 2Dept. Cel Bio, University of Utrecht, The Netherlands.

Sevemi mechanisms have boen prposed for protein sorting to the different membranedomains in polarized MDCK epithelial cells. Sorting of apical membrane proteins isthought to involve the clustering of glycosphingolipids (GSL) and specific proteins(e.g. GPI-anchored proteins) in the trans-Golgi network (TON); as a consequence, itwas proposed that other proteins ae excluded from GSL-rich clusters in the TGN,thby 'sorting" thes proteins to the basal-lateal membane domain. Recent studiesalso indicate that a class of single membrane spanning ligand receptors have anintrinsic sorting signal that specifically sorts them to the basal-lateral pathway. Wehave investigated the targeting and distribudon of GSL and Na/K-ATPase (NKA) intwo different clones of MDCK type II cells. We have found that in clone G cells,fluorescent GSL analop are sorted to the apical plasma membrane domain with arelative polarity of 2:1 compared to the basal-latemimembrane. In this clone, NKA isdiely targeted to the basal-latem membrane within 12 hours of induction of cell-celcontact Clone J cells, on the other hand, do not sort GSL preferentially to eitherplasma membrane domain, and NKA is sorted to both the apical and basal-lateralmembrane domains, but accumulates only in the basal-lateral membrane. Anotherbasal-lteral protein, E-cadhein, is sorted, however, with similar kinetics to only thebasal-lteral membrane in both MDCK clones. Addition of an inhibitor of GSLsynthesis, fumonisin l, d onarates that clone J is insensitive to the drug, but cloneG is hypersensitive; we are currently characzing effects of F9I on GSL and NKAsorting in both cell clones. We suggest that in the presence of GSL clustering andsring in the TGN, NKA is exduded from the apical pathway and becomes sorted withother basl-latemi proteins; without GSL clustering, NKA is included in both sortingpathways.

564THE POLYMERIC IMMUNOGLOBULIN RECEPTOR IS TRANSCYTOSED INCULTURED HIPPOCAMPAL NEURONS. ((E. Ikonen, R.G. Parton, W. Hunziker*,K. Simons, and C.G.Dotti)) Cell Biology Program, European Molecular BiologyLaboratory, D-69012 Heidelberg, Germany and *Institute of Biochemistry, University ofLausanne, CH-1066 Epalinges, Switzerland.

In polarized epithelial cells molecules can be transported across the cell from one plasmamembrane domain to the other in a process known as transcytosis. This mechanism isused to generate cell surface polarity and to transport macromolecules between theluminal and serosal sides of the epithelial layer. The best characterized transcytoticmolecule is the polymeric immunoglobulin receptor (pIgR). It binds the ligand at thebasolateral surface and the receptor-ligand complex is transcytosed to the apical surfacewhere the ligand is released. Our studies have shown that hippocampal neurons mayemploy mechanisms similar to those of epithelial cells to sort proteins to the twoplasma membrane domains. The machinery used for axonal delivery recognizes proteinstargeted apically in epithelia, whereas basolaterally destined proteins are delivered to thedendrites. To explore whether a dendro-axonal transcellular pathway exists in neurons, weexpressed the pIgR in cultured hippocampal neurons with the Semliki Forest virus(SFV) expression system and studied its distribution using immunofluorescencemicroscopy. The majority of the newly synthesized receptor was targeted from the Golgicomplex to the dendrites, with only about 20% of the infected neurons showing axonallocalization. This is consistent with our previous observation that proteins inserted intothe basolateral surface of epithelial MDCK cells are delivered to the dendrites in neurons.Most interestingly, addition of the ligand dIgA resulted in significant redistribution ofthe pIgR, with a 3-fold increase in its axonal localization. Our results suggest that atranscytotic route, analogous to that in epithelia, exists in neurons leading from thesomatodendritic to the axonal domain. Cultured neurons expressing the pIgR offers anexperimental system which should be useful for further characterization of this novelneuronal pathway at the molecular and functional level.

566RECEPTOR-MEDIATED TRANSCYTOSIS OF IgA IN MDCK CELLS IS VIAAPICAL ENDOSOMES. ((G. Apodaca, L. A. Katz, and K. E. Mostov)) Depatment ofAnatomy and Deparment of Biochemiatry and Biophysics, University of Califomia, SanFrancisco, 94143.

Transcytosis is a key membrane tafficking process in polarized cells because it allows forthe exchange of macromolecuks from one cel surface to the opposite one whilemaintaining the barrier funcdon of the epithelia. The best characerized racytotic proteinis the polymeric immunoglobulin receptior (pIgR). The generally accepted model for pIgRtraffic is as follows: the recepor and its ligad are internalized and delivered to a tubulo-vesicular eariy endosoma comparnet shared with other recepts. Here the pIgR and its

ligand are segregated from recycling recepors intom ofendoaomal tubues andpackaged into transcyotic vecies tat direcy fuse with the apical plasma membrane. Amajor finding of our work is that taacytosis of the pIgR in MDCK cels works quitedifferently from this classic mode. When intemalized from the bolatrael surface ofMDCK cells IgA was found to accumulate under the spical plasma membae, and wasaccesible to two apically recycling membrne marr, anti-SC Fab fragments and ricin.Trypsin was found to effectively prevent pIgR molecules from being intemalzed at theapical cell surface. In the pmesce of this protdnase, delivry of basoerally intenalizedIgA to the apical a st was largely dminished and apicaly intenalized ruin hadaccess to this compartnent This result sugess that IgA is delivered from basolatemlenome to apical endosotes and only then to the apical cell surfce. In addition, we fmdthat transfenin, a baolatemd recycling protein, has access to this apical endosomalcompatment, where it is sorted away from the IgA and recycles to the basolateral suface.Therefore, sorting of IgA from basolateral recycling proteins may occur not only inbasolatemi endosomes but in apical endosomes as well. Moreover, fluid phase markers

intenaHzed either apically or basolateraly were excluded from the apical eadosomalatmnt Finmly, this apical cmpamt is concentMed aound the centrwome and

microtubule depolymerization diupts the orgaizatio and function of this compatment

We propose a model is which trncytosis is not a specHized pathway that uses uniquetancytoc vesices, but rather combines portions of pathways used by non-anscytoungmolecules.

568MOLECULAR GENETIC ANALYSES OF A COMPONENT OF THEHEMIDESMOSOME. ((S.B. Hopkinson, J.Asmuth, and J.C.R. Jones)) CMSBiology, Northwestern University Medical School, Chicago, IL 60611.

A l8OkD type II transmembrane protein of the hemidesmosome, an epithelialcell-extracellular matrix attachment device, possesses a cytoplasmic domain of55kD (Hopkinson et al., 1992). We have analyzed sequences that target thisprotein to the hemidesmosome using 8040 cells that assemble hemidesmosomesin vitro. 804G cells were transfected with constructs encoding thetransmembrane portion of human l8OkD and varying lengths of its cytoplasmicdomain. To distinguish mutant protein from wild type 180kD, the transfectedpolypeptide was tagged with the c-myc epitope. Double immunofluorescence oftransfected 804G cells was undertaken using c-myc antibody and humanantibodies against a 230kD component of the hemidesmosome. Sequences at theN-terminus of the l8OkD protein determine both its polarization to the basalaspect of a cell as well as its association with other hemidesmosomal elements.We have extended these observations by transfecting l8OkD constructs into FGcells. These cells express neither the 180 nor the 230kD components of thehemidesmosome but they possess the hemidesmosomal associated integrin n6(.4.In PG cells transfected with the the construct encoding the entire cytoplasmicand trsnsmembrane domains of the I8OkD polypeptide, mutant proteincolocalizes with the tx604 integrin complex. We speculate that the cytoplasmicdomains of the 18OkD polypeptide and those of integrins show specificinteractions. Such interactions may play an important role in assembly of thehemidesmosome and may be involved in matrix-cell signalling phenomena.Supported by NIH.

97a

Membrane Domains and Polarity (569-574). Sunday569

DEVELOPMENT AND MAINTENANCE OF BILE CANALICULI IN COLLAGEN SANDWICHEDHEPATOCYTES. ((E. L. LeCluyse. K. L. Audus, and J. H. Hochman)) INTERxResearch/Merck Research Laboratories and the Departments of Biochemistryand Pharmaceutical Chemistry, University of Kansas. Lawrence. Kansas66047

Elaborate networks of bile canaliculi (BC) are formed by primarycultures of hepatocytes when grown in a collagen sandwich configuration.The initiation and proliferation of the BC network after collagen overlaywere examined by phase, fluorescence, electron, and time-lapse videomicroscopy. De novo repolarization of the plasma membrane was initiatedby the uniform formation of translucent. punctate areas between adjacenthepatocytes. Development of these discrete patches into a contiguous,anastomosing network of BC occurred as a combination of the initiationof new sites and the elongation of previously established sites along thecell borders. Time-lapse video recordings showed BC network formationto be a very dynamic process exhibiting membrane ruffling, channelcontraction and dilation, and vesicle endocytosis. During these initialstages of BC development, there was a marked accumulation of bothmicrofilaments and microtubules at the sites of BC generation. NormalBC network formation was inhibited by both microtubule (colchicine.nocodazole) and microfilament (cytochalasin D. phalloldin) perturbingagents. In cultures where BC network formation was essentially complete.actin microfilaments remained primarily associated with the BCstructures, whereas, intracellular microtubules became more centrallylocated as well as remained associated with the canalicular membranes.Subsequent treatment with mi crotubule or microfilament perturbing agentsresulted in the cessation of normal biliary activity and disruption ofnormal canalicular structure, respectively. These results show thatcollagen sandwiched hepatocytes may be a useful in vitro model forexamining the mechanisms of morphogenesis of the biTiary pole.

571

ANALYSIS OF GLYCOSYLPHOSPHATIDYLINOSITOL (GPI-)ANCHORS IN MDCK AND THYROID (FRT) EPITHELIAL CELLS.((Wouter van 't Hof, Anant K. Menon* and Enrique Rodriguez-Boulan)).Department of Cell Biology and Anatomy, Cornell University MedicalCollege, New York, NY 10021, *Laboratory of Molecular Parasitology,The Rockefeller University, New York, NY 10021-6399.

Glycosylphosphatidylinositol (GPI-) anchors act as apical targetingsignals for proteins in MDCK cells (Lisanti et al., 1989, J. Cell Biol. 109:2145-2156). Recently, in a thyroid epithelial (FRT) cell line, the GPI-tailwas found to serve as a basolateral signal (Zurzolo et al., 1993, J. CellBiol. 121: 1031-1039). We analyzed the structure of the GPI-anchors inMDCK and FRT cells, studying the pool of free, non protein-bound GPIfirst. After labeling with 3H-Mannose or 3H-Ethanolamine and TLCanalysis, no differences in the patterns of GPI-anchors were observed.Comparison with well described anchors from Thymoma cells indicatesthat the anchors can contain up to three ethanolamine phosphate residues,attached to the core structure. We are presently testing the structures ofthe protein-bound anchors, by radiolabeling, TCA precipitation, pronasetreatment, isolation of GPI-peptides on octylsepharose columns and TLCanalysis. The biosynthesis of GPI-anchors and the attachment to proteinsoccurs in the ER. Surprisingly, analysis of Influenza and VesicularStomatitis Viruses grown in 3H-Ethanolamine labeled MDCK cellsdetected mature free GPI-anchors in plasma membranes. We are currentlyusing these viruses with the objective of characterizing in detail thecellular location, the sidedness in membranes and the cell surface polarityof GPI-anchors in epithelial cells.

573

ROLES OF THE MEMBRANE-CYTOSKELETON AND CADHERIN-MEDIATED CELL-CELL ADHESION IN GENERATING DIFFERENTDISTRIBUTIONS OF Na+, K+-ATPase IN EPITHELIA. ((J. A. Marrs, E. W.Napoitao', C. Murpby-Erdosh' R. W. Maya, L. F. Relehardt' and W. J. Nelson)).

Department of Moecular and CeDlmlar Physiology, Stanford University, Stanford,CA 94305-5426. 'Department of Physolo, HHMI, University of California,SanFranciseo, CA 94143.0724In simple epitheula, the distibution of ion transporing proteins between the apical or

basal-lateal domains of the plasma membrane is important for determining direcions ofvectorial ion tansport acmss fte epithelium. In the choroid plexus, Na+, K+-ATPase islocalized to the apical plasma membrane; in contrast, Na+, K+-ATPase is localized to thebasl-ltal manbane of most epithelial cells. Previous studies have indicated a role forcadherin mediatd cell-cell adhesion and membne-cyukeleton (anskyrin and fodrin)assembly in regulating Na+, K+-ATPase distribution in absorptive kidney epithelial cells.Confocal immunofluoreacence micscopy reveals that in chicken and rat choroid plexusepithelium, fodrin and ankyrin colocalize with Na+, K+-ATPase at the apical plasmamembrane, but fodrin, ankyrin and adducin also localize at the lateral plasma membranewhere Na', K'-ATPase is absenL Biochemical analysis shows that fodrin, ankyrin andNa+, K+-ATPasea in a high molecular weight complex similar to that found previouslyin kidney epithelial ceUs. Ca++deendet cell adhesion molecules (cadherins) weredetected at lateal membranes of the choroid plexus epithelium and colocalized with adistinct fraction of ankyrin, fodrin and adducin. Cadherins did not colocalize with Na+,K'-ATPase and were absent from the apical membrane. Cadherins were also found in ahigh molecular weight complex with fodrin and ankyrin. We examined proteindistributions in fibroblasts transfected with E-cadherin, the cadherin expressed in mostepithelia, and B-cadherin, a prominent cadherin expressed in the choroid plexusepithelium. The results show that Na+, K+-AlTase and fodrin become concentrated atce-cellcontacts in cells expressing E-cadherin, butnot in celIs expressing B-cadherin,indicating differences in the potential of these cadherins to induce Na+, K'-ATPaseacmulatonat cell-cell contacts. These results provide new insights into the roles of themembrane-cyoskeleon and cadherins in genemting different distributions of the same

protein in simple epithelial cells.

570

MONOCLONAL ANTIBODIES TO THE INTEGRIN 81 SUBUNITINHIBIT EPITHELIAL CELL SURFACE POLARITY REVERSAL.((G.K. Ojakian and R. Schwimmer)) Department ofAnatomy and Cell Biology, SUNY Health ScienceCenter, Brooklyn, NY 11203.

The role of f1 integrins in the regulation ofepithelial cell surface polarity development wasstudied in MDCK cells. Suspension-grown cells formpolarized cysts with apical microvilli and the gp135glycoprotein on the outer membrane. Incubation ofcysts within collagen gel induces reversal(microvilli on lumenal membrane) of cell surfacepolarity (Wang et al., 1990. J. Cell Sci. 95:153).Cyst binding to collagen and laminin was assayed inthe presence of mAbs (AIIB2, AJ2) against theintegrin f1 subunit. These mAbs inhibited bindingsuggesting that integrin a/P heterodimers werepresent on the cyst outer membrane. The a2, a3 anda6 subunits had a non-polarized distribution and thef1 subunit was localized primarily to thebasolateral membrane although low levels were alsodetected on the apical membrane. After incubationin a collagen gel, f1 subunit levels increased onthe outer membrane while microvilli and gp135 levelsdecreased. However, mAb AIIB2 inhibited collagen-mediated microvillar loss and endocytosis of gp135providing evidence that f1 integrins are involved inregulation of epithelial polarity reversal.

572E-CADHERIN INDUCED BASAL-LATERAL Na+, K+-ATPase POLARITY INRETINAL PIGMENT EPITHELIAL CELLS IS ACCOMPANIED BY ARADICAL REORGANIZATION OF THE MEMBRANE-CYTOSKELETON. ((J.A. Marrs, C. Anderson-Fisone, L. R. Nabr, M. C. Jeoug, E. Rodriguez-Boulan' andW. J. Nelson)). Department of Molecular and Cellular Phyaiology, StanfordUniversity, Stanford, CA 94305-5426. "Department of Cell Biology and Anatomy,Cornell University,New York, NY 10021.

Generating polarized distributions of ion transporting proteins is crucial for thefunctions of epithelial cells, and different epithelial cell types can generate oppositedistributions for the same protein depending on physiological needs. Previous studieshave deamnstrated roles for cadherin-mediated cell-cell adhesion and the membrane-cytoskeleton assembly in generating different polarized distributions of Na+, K+-ATPasein different epithelial cell types. While the tissue in vivo has an apical distribution, theretinal pigment epithelium cell line RPE-J has an unpolarized distribution for Na+, K+-ATPase, similar to retinal pigment epithelial cells in primary culture. Transfection ofRPE-J cells with cDNA encoding E-cadherin (RPE-J+LCAM) results in a basal-lateralpolarized distribution of the Na+, K+-ATPase. We have examined the cadherincomplement of the parental and transfected cells. Both cell types express cadherin whichare complexed with the three catenins, suggesting that the different cadherins bindcatenins with similar affinities, and that differences in the catenin binding are notresponsible for the different Na+, K+-ATPase distributions. Fodrin was localized to thelateral plasma membrane in both cell types. However, ankyrin is only detected in theRPE-J+LCAM cells using antibodies raised to the well conserved spectrin bindingdomain. Preliminary PCR experiments suggest different ankyrins are expressed in RPE-Jand RPE-J+LCAM cells respectively. Fially, desmoplakins and desmoglein I were onlylocalized at the plasma membrane by immunofluorscence in RPE-J+LCAM cells butnotin RPE-J cells Together these results show that expression of E-cadherin in these cellsinduces the redistnbution of Na+, Kt-ATPase from an unpolarized distribution to a basal-lateral distribution, perhaps due to expression of a different ankyrin isoform. That E-cadherin also induces expression and assembly ofdemxoomes indicates that it acts as aninducer of specific components of the epithelial phenotype.

574

REINSERTATION OF APICAL SURFACE MEMBRANE PROTEINS FOLLOWINGCELLULAR ATP DEPLETION INDUCED INTERNALIZATION((B.A. Molitoris and R. Dahl)) Univ. Colo. Health Sciences Center and Indiana Univ.Medical Center, Denver, CO and Indianapolis, IN.

ATP depletion in vitro is known to induce smerous morphologic and biochemicalalterations incwlding internalization of surface membrane protems and loss of surac

membane polarity. Alteough r blihntof ssrfacemembmne orgnization is essenlfor normalization of cellular finetion, it is not known whether 'altered' prooeis can bereudlized or if new protein synsaesis is required. To investe these two possibilities areversible model of ATP depletion/repletion in LLCPK, cells was utilized. Cellular ATPwas depleed using 0.1pM Antmycin A in atrated depleted media, and repleted bychanging to physiological media. During the first 2 hr of the ATP recovery phase, cellularATP resumed from <5% to 85% baseline (p<0.01), G-actin recovered frm 48 to 88%baseline (p<0.01), surface ouabainbinding, used to quantify surfaemembrne Na+,K+-ATPase, incrased from 55% to 96% of control vahles (p<0.01). Nether nocodazal

(IOisg/ml) nor cycloheximide (10g/tnl) had any effect on normlization of sufce

membane ouabainbinding. However, reaamentofATP depleted cells during the recoveryperiod with phalloidin (0.61aM) prevented the correction in surface membran ousbainbinding. In additional studies apical membrane proeins were labeled with biotin and texas

red conjugated avidin to directdy visualize in izaion and reinsertion of proteins intothe sufac membrane. Surface protein labeling was biotin dependent a DT sensidve.ATP depletion indced rapid inernalizatonandchumping oflabel which was DTI' resintandlocalized in the cytosol by dital confocal (VayTec) techniques. ATPrepletion resltedin movement of the fluorescently labeled protein back to the srface membrne and thereturn of DIT sensitivity. Taken together, these data indicate intenlized surfacemembrane proteins canbe reutilized during the recovery phase, andreinserdonofNa+,K+-ATPase into the surfae membrane is independent of microtubules and protein synthesisbut requires eorgalization of the actin cytoskeleton.

98a

Sunday. Membrane Domains and Polarity (575-580)

575ULTRASTRUCTURAL LINKS BETWEEN SMOOTHENDOPLASMIC RETICULUM AND BASOLATERALMEMBRANE OF RENAL TUBULES. ((T. Beringer)) Cell Biology& Biophysics, University of Missouri, Kansas City, MO 64108.

Ultrastructural evidence is given to support a long standing theoryimplicating smooth endoplasmic reticulum (SER) with transcellulartransport across rat renal proximal convoluted tubules. Ceriumphosphate impregnated sites in the basolateral membrane (BLM) ofproximal convoluted tubules have been implicated with phosphatetransport (Beringer, J. Cell Biol. 115:467a, 1991). This report revealsan ultrastructural relationship between BLM and SER which may bethe structural basis for transcellular transport of phosphate. Intransmission electron micrographs SER nearly contiguous with theBLM is linked to it by dense structures (5 nm wide, 10 nm long).Tubular invaginations of apical plasmalemma containing endocytoseddextran known to be connected to SER extend towards the BLMapically where their cross-sectioned profiles are superimposed uponcircular electron-dense cerium phosphate domains visible alongtangentially-sectioned BLM in cerium-perfused kidneys. Resultsdescribe structural links between SER and BLM and suggest therelationship of tubular endocytic membranes with cerium phosphateimpregnation sites in BLM to be involved with transcellular phosphatetransport.

577

APICAL ANTIGENS OF THE PROTOZOAN PARASITETOXOPLASMA GONDII. ((N.S. Morrissette and D.S. Roos))Department ofBiology, University ofPennsylvania, Philadelphia,PA, 19104.

Toxoplasma gondii is a protozoan parasite with a highlypolarized morphology. The parasite's specialized apex contains anumber of distinctive structures which define the phylumApicomplexa. We have screened three hybridoma libraries forimmunofluorescent staining to the apical end of Toxoplasma,using as antigen either total protein or detergent-extractedmaterial from two parasite strains. In addition to antibodies topreviously characterized intracellular organelles (rhoptries, etc.),four novel classes of apical antibodies have been identified. (1)Numerous clones which stain the apical quarter of the parasitealso cross-react with host cells in a punctate distribution. Onimmunoblots all ofthese antibodies recognize an identical patternof>10 parasite proteins, and a rapidly migrating host cell antigen.(2) One class of parasite-specific antibodies recognizes a -110 kDprotein and is predominantly apical by IFA, but shows weakerpunctate staining throughout the entire parasite. (3) A singlehybridoma line recognizes a -200 kD antigen at the extremeapical bud of (conoid) parasites. (4) Finally, one class of antibodiesrecognizes both 110 and 200 kD antigens, and appears to stain ina cell-cycle dependent manner -- apical in mature parasites butreorganized to cytoplasmic/perinuclear elements immediatelyfollowing replication.

579

ANKYRING NODE: A NOVEL ISOFORM OF ANKYRIN SELECTIVELYLOCAUZED TO THE PLASMA MEMBRANE OF AXONAL INITIALSEGMENTS AND NODES OF RANVIER IN THE NERVOUS SYSTEM. ((E.Kordeli, S. Lambert and V. Bennett)). Howard Hughes Medical Institute andDepartment of Biochemistry, Duke Univ. Med. Center., Durham, NC 27710.

The initial segment and Node of Ranvier represent specialized domains ofthe axonal membrane responsible for the Initiation and propagation of theneuronal action potential. Membrane depolarization in these domains isachieved by oalized concentrations of Ion channels such as the voltage-dependent sodium channel and the Na+/K+ ATPase. These two proteins areknown to associate with ankyrin, a peripheral proteln believed to act as anadaptor between integral membrane proteins and the spectrin-basedmembrane skeleton. Localized concentrations of other ankyrin bindingproteins such as the cell adhesion molecules LI and ABGP/neurofascin havealso been observed at the initial segments and Node suggesting a role forankyrin and the membrane skeleton in establishing and/or maintaining thesespecialized regions of the axonal membrane. We have recentiy cloned andsequenced cDNA's for a novel ankyrin gene (ankyrin,3) expressed in thenervous system. Ankyrin0 shows strong homology with another ankyrin genefrom the brain, ankyrin3 44OkD, Inciuding an extended 'Wal' domain that mayhave a role in axonal targeting. Specific antibodies raised against theankyrin5 Wail' domain show that in neurons this isoform is localized only to theinitial segment and Node of Ranvier and as such is the first uniquecytoplasmic component of these physiologically important membranedomains. tmatively splied isoforms of ankyrln5 are expressed in othertissues, where hey may also have a rol In the establishment of specializedcell membrane domains.

576LIPOPHOSPHOGLYCAN AND A NOVEL, HYDROPHILIC PROTEINSHOW SIMILAR MEMBRANE DOMAINS ON TlE LEISHMANIASURFACE DURING CAPPING EVENTS. (('P.F.P. Pimenta, 3D.F. Smith,'D. Rangarajan, 2P. Pinto da Silva and 'D.L. Sacks.)) Laboratory of ParasiticDiseases, NIAID1; Structurl Biology Section, NCI1; NIH, U.S.A. andDepartment of Biochemistry, Imperial College of Science, Technology and

Medicine3; London, UX. (Spon. by A. Sher).

During their growth in the sand fly, Leishmania promastigotes undergosequential development from a dividing non-infective stage (procyclic) to amature infective stage (metacyclic). Metacyclogenesis is accompanied bydevelopmental modifications of the Lipophosphoglycan (LPG), which is themajor macromolecule on the cell surface. Conventional immunocytochemistryand freeze-fracture combined with immunogold labeing were used to studyLPG capping and Its association with others cell surface components. Doublelabeling after LPG capping, followed by thin-section showed that antibodiesagainst LPG and Gene B protein (a novel hydrophilic protein associated withthe surface of metacyclic promastigotes) bound to proximal sites on theparasite membrane. This result was confirmed by the capping of Gene Bprotein followed by double labeling. Observation of freeze-fracture replicasrevealed a marked homogeneity in the density and arrangement between theLPG and Gene B protein sites. In contast, the distribution of integralmembrane proteins (IMPs) showed no coneladon between them and the goldlabeling. In conclusion, our results show equivalent membrane domainsbetween LPG and Gene B proein, and suggest tight associadon between these

molecules.

578IDENTiFICATION OF TWO MABS THAT STAIN THE EQUATORIAL REGION OFFIXED AND PERMEABIUZED GUINEA PIG SPERM. ((A. E. Cowan and D. E.Koppel)) Department of Biochemistry, University of Connecticut Health Center,Farmington, CT 06030.

The guinea pig sperm plasma membrane is segregated Into at least four domainsof different composition. Some proteins localized to specffic regions of themembrane are free to diffuse iterally in the membrane, suggesting that barriers todiffusion at the domain boundaries retain proteins within specffic domains. Onesuch domain boundary is located at the equatorial region of the sperm head, anarrow region that separates the anterior and posterior head plasma membranedomains. We have identified two mAbs (ER-1 and ER-2), that specifcally stain theequatorial region o fixed and detergent-extracted cauda epididymal sperm. TheER-1 and ER-2 mAbs show no localization In fixed and extracted testictiar speNm.Staining at the equatoril region first appears In sperm taken from Region of theepididymis, and ilntensifies during epididymal transit In testicular sperm, theplasma membrane of the sperm head is comprised of a single domain thatbecones segregated into two domains only after the sperm has begun to traversethe epididymis, speciflcally in Region II cf the epididymis. Thus, recruitment of ER-1 and ER-2 antigen(s) to the equatorial region occurs Just prior to the segregationof the plasma membrane Into distinct domains. The ER-1 and ER-2 antigen(s) arealso found in developing spermatids. In these cells ER-1 and ER-2 localize to aflamentous network In spermatocytes and round spermatids and to the manchetteIn elongating spermatids, a microtubtie-based structure that surrounds theposterior haif of the nucleus during spermatid elongation. The ER-2 mAbspecifically binds two polypeptides of M, 53 and 47 kD In western blots of testicularcell extracts. These antigens may be components of the spermatid cytoskeletonthat later serve a role In defining the boundary of plasma membrane domains In themature spermatozoan. Supported by NIH GM23585 to DEK

580COMMON SIGNALS CONTROL LDL RECEPTOR SORTING INENDOSOMES AND THEGOLGI COMPLEX OF MDCK CELLS.((K. Matter, J. A. Whitney, E. M. Yamamoto, and I. Mellman))Department of Cell Biology, Yale University School of Medicine, NewHaven, Connecticut 06510 USA.

The generation and maintenance of cell surface polarity in epithelial cellsrequires the continuos sorting of newly synthesized and internalized plasmamembrane proteins. In MDCK cells, basolateral targeting of newlysynthesized membrane proteins involves specific cytoplasmic domaindeterminants. The cytoplasmic domain of LDL receptor bears two tyrosine-containing determinants that can independently target newly synthesizedreceptors from the Golgi to the basolateral plasma membrane ofMDCK cells.We now find that these determinants, localized to the membrane proximal anddistal regions of the receptor's cytoplasmic domain, also serve to controlpolarized sorting in endosomes. Inactivation of the distal determinant reducedthe ability of receptors to return to the basolateral domain followingendocytosis resulting instead in receptor transcytosis from basolateralendosomes to the apical plasma membrane. Similarly, receptors internalizedfrom the apical surface were transported from apical endosomes to thebasolateral surface due to the proximal basolateral targeting determinantThus, receptor recycling in endosomes is directed by the same signals usedfor polarized sorting in the Golgi, indicating that sorting on the endocytic andbiosynthetic pathways involves similar mechanisms. This possibility wasfurther supported by the observation that brefeldin A interfered with sorting,but not transport, in both endosomes and the Golgi complex.

99a

Membrane Domains and Polarity (581-584). Sunday

581MAINTENANCE OF NA,K-ATPASE POLARITY IN HONEYBEEPHOTORECEPTORS BY PHOTORECEPTOR-GLIA INTERACTION.((O. Baumann, and * K. Takeyasu)) Institut fUrZoologie, UniversitMt Regensburg, D-93040 Regens-burg, Germany and * Department of Medical Biochem-istry and Biotechnology Center, The Ohio StateUniversity, Columbus, Ohio 43210.

Arthropod photoreceptors are polarized cells dis-playing distinct surface domains. Using immunocyto-chemical techniques, we have analysed the distri-bution of Na,K-ATPase in honeybee photoreceptorsand the role of glial cells in positioning theNa,K-ATPase on the photoreceptor surface. We demon-strate that the Na,K-ATPase is restricted on thephotoreceptor surface to sites of photoreceptor-glia contact. No junctional structures were ob-served at the borders between Na,K-ATPase-positiveand Na,K-ATPase-negative membrane domains. Remo-deling of photoreceptor-glia contact caused acorresponding redistribution of Na,K-ATPase on thephotoreceptor surface. In isolated photoreceptors,Na,K-ATPase was present on the entire cell surfaceexcept the microvillar membrane. These resultssuggest that adhesion of glial cells to the photo-receptor surface maintains the polarized distribu-tion of Na,K-ATPase in honeybee photoreceptors.Supported by the DFG (Ba 1284/1-1 to O.B.) and theNIH (RO1-GM-44373 to K.T).

583

SIGNALLING MOLECULES AND GPI-LINKED PROTEINSARE CONCENTRATED IN ISOLATED CAVEOLARMEMBRANE DOMAINS. (( M.P. Lisanti, M. Sudol*, Z.-L.Tang and M. Sargiacomo)) The Whitehead Institute forBiomedical Research, MIT, Cambridge MA 02142; and *TheRockefeller University, Laboratory of Molecular Oncology, NewYork, NY 10021

During or after transport to the apical surface of epithelialcells, GPI-linked proteins cluster in cell surface micro-invaginations known as caveolae or plasmalemmal vesicles.Using a recombinant GPI-linked protein and caveolin asmarker proteins, we have devised a rapid, simple, one-stepprocedure for isolating caveolae from cultured MDCK cells.These caveolin-rich membrane domains represent 0.5-0.8 % ofthe plasma membrane and exclude a variety of organelle-specific marker enzymes. In addition to caveolin, we find thatthese isolated caveolae contain a subset of hydrophobic plasmamembrane proteins and cytoplasmically-oriented signallingmolecules. The latter group includes GTP-binding proteins--both small and hetero-trimeric, a non-receptor protein tyrosinekinase (c-Yes) and annexin II, a calcium-dependentphospholipid binding protein with a demostrated role inexocytic fusion events. As both caveolin and annexin II were

originally identified as v-Src substrates in RSV-transformedcells, our results also have implications for understandingtransformation by v-Src.

Cell-to-Cell Interactions I (585-586)

585

ARE THE CADHERINS INVOLVED IN MEDIATING CADMIUM (Cd'4) EMBRYO-TOXICITY IN VITRO? ((B. Chen, and B.F. Hales)) Deparment of Pha &Therapeutlca, McGil Univerity, Montr6al, Qubec, Canada,H3G 1Y6.

Cd2+, a teratogenic and carcinogenic heavy metal, interferes with the functions dcatona such as Zn2+ andCaW. Themechanism(s) underiying the toxicefects of

Cd2+ ae unkinown, butIn LLC-PK, cab there Is evidence t1hat E-cadherin, a Cae+-dependent cell adhesion moloule, is atarget (Prozialeck & Niewenhuis, BBRC181:1118,1991). E- and P-cadherin are expressed during organogenesis. This studydetermined whether these cadherinsareinvolved In medist Cd2+ embryotoxicty.Day 10 rat embryos were pr-incubated 1 hrwithout Cd24,thn cuituredfor 20 hrs in

the absence or presence of 2.5MuM CdCy. Embryce and yolk sacs were collectedand assessedfor Cd+ embryotoxcy, effectson cadherin expressn, and loaliztion

of"Cd'+ binding. Cd'+-treated embryos were growth retarded; their yolk sacs were

thickened and half the dismeter ofcontrols. Northem blot anaysis revealedthat the

concentation of P-cedherin mRNAin the yolk sac, but notin theembryo, weeIncreased 2-fold after exposure to Cd'+; E-cadherin mRNAs were unchanged.

Interestigly, Westem blot analysis showed that therewas a decline in the concentra-

tion of E-cadherin proteinin control yolk sacs with cuiture which wa prevented byCd'+ treatment; E-cadherin protein was increased 2.3-fold alter 20 hrs in Cdc+exposed yolk sacs reative tocontrol. The concentratios of E- or P-cadherin proteins

in Cd+-treated enbryos were not altered; P-cadherin protein In the yolk sacwes not

detectable. Ctol embryo and yolk sac samples were eb andtafred to nitroellulse to Investigte a"Cd binding. "Cd bound to an

unidenfIed 85 Kd proteinin both embryo and yok sacs; binding was attenuated by1 mM Zn2+, but not by1 mM Cs2+. No'laCd binding was detected In the -120 Kdregion, indicing that Cd+ did not bind directly to the cadherins. Thus, Cd2+diferenilly regultes cadherin expression in the yolk sac but not the embryo;however, cadherns are unlikely to be directlyInvolvedin medbting the teratogenicityof Cd2+. Supported by MRC of Canada.

582SURFACE MORPHOLOGY OF LIVING MDCK CELLS BY ATOMICFORCE MICROSCOPY. ((J. H. Hoh and C.-A. Schoenenberger)) M. E. MillerInstitute at the Biocenter, University of Basel, CH4056 Basel, Switzerland.

Here we describe the morphology of the apical surface in an MDCK monolayer,and a ras transformant, by atomic force microscopy (AFM). Normal MDCK cellsgrown on glass form a continuous monolayer of polarized epithelial cells andhave height variations along the apical membrane of a 1-3 microns. The lateralstability conferred by the monolayer, and the small height variation make thesecells a particularly suitable for imaging with the AFM. Cells could be imaged in aphysiological solution for several hours with no noticeable deterioration. Cellboundaries generally appear as ridges that clearly demarcate neighboring cells.Reasonable contrast in the image required forces that deformed the mnembrane onthe order of hundreds of nanometers. In some cases the nucleus could be seen,though apparently only in very thin parts of the monolayer. Two types of protru-sions on the surface could be visualized. Smooth bulges that varied m size from afew hundred nanometers to several microns, which may represent relatively rigidsub-apical structures. Another type of protrusion extended well above the mem-brane and was swept back and forth during the imaging. However, the microvillithat are typically present on the apical surface could not be clearly resolved.Similar morphology was seen on MDCK cells grown on permeable filters, alt-hough nuclei were not visible. Imaging cells on filters resulted in oscillations inthe piezo scanner at normal scan rates. As a result image acquisition time was in-creased to nearly an hour in some cases, making routine use of these filters im-practical. The morphology of the oncogenic transformant is different. When cul-tured on glass these cells grow very flat, and are less than 100 nm thick over largeareas with both extensive processes and rounded edges. Many intracellular struc-tures such as the nucleus, stress fibers and vesicles could be clearly visualized.After imaging, some samples were cultured for an additional 24 hours and cellviability was confirmed by trypan blue exclusion. Cellular dynamics could alsobe observed during imaging over the time scale of hours, further indicating thatthe cells remained viable.

584

THE CYTOPLASMIC DOMAIN OF SYNDECAN-1 IS NOT REQUIREDFOR ASSOCIATION WITH THE DETERGENT-INSOLUBLECYTOSKELETON BUT IS REQUIRED FOR POLARIZEDEXPRESSION IN EPITHELIAL CELLS (M. Jalkanen and H. Miettinen)Turku Centre for Biotechnology, P.O. Box 123, FIN-20521 Turku, Finland.

Cell surface heparan sulfate proteoglycan - syndecan-l binds variousextracellular matrix proteins, is polarized to basolateral surfaces of epithelialcells and has been suggested to interact with the cytoskeleton. To determinethe site on syndecan-l that mediates the interaction with the TX-100insoluble residue, we expressed wild type mouse syndecan-1 and acytoplasmic deletion mutant (tail-less) in CHO and MDCK cells. Weobserved that both the wild type and tail-less syndecan-1 were insoluble inTX-100 at pH 5.0. The insolubility was not affected by increasingtemperature or by cytochalasin D. Removal of the heparan sulfate chainsfrom the ectodomain, however, resulted in complete TX-100 solubility.Syndecan-1 could also be released into the TX-100 soluble fraction byaddition of heparin or heparan sulfate to the extraction buffer. In polarizedMDCK cells the wild type syndecan-1 was almost completely basolateral (>90 %) but tail-less syndecan was equally distributed both to basolateral andapical surfaces. Two other mutants lacking 12 or 24 C-termninal residuesalso failed to polarize in MDCK cells. We conclude that the cytoplasmicdomain of syndecan-l is not responsible for TX-100 insolubility. Instead,our results suggest that TX-100 insolubility is caused by aggregation of thesyndecan-1 molecules with other cellular and/or extracellular molecules viathe heparan sulfate chains. The cytoplasmic domain of syndecan-l andespecially the 12 most distal residues reside information needed for correctcellular localization of syndecan-l in polarized epithelial cells. Supportedby The Academy of Finland.

586

UVOMORULIN LOCALIZATION AND STRUCTURE CHANGES IN MOUSEUTERINE EPITHELIUM DURING THE ESTROUS CYCLE AND EARLYPREGNANCY. ((S.W. Potter, G. Gaza, J.E. Morris)) Department ofZoology, Oregon State University, Corvallis, OR 97331-2914

Uvomorulin (UM), synonymous with e-cadherin, is a major calcium-dependent cell adhesion molecule in adhering junctions of epithelia.Changes in its expression and distribution have been reported duringembryonic development, in cells in culture, and in pathological condi-tions. We investigated UM localization during the normal estrous cycleand early pregnancy, times when cell-cell and cell-matrix associationschange in conjunction with varying hormonal conditions. We examinedfrozen sections of uteri and performed SDS-PAGE of epithelial cell sur-face detergent extracts, using anti-UM for identification. In sections,basal localization of UM increased markedly as uterine epithelium cycledthrough proestrus to estrus, stages at which peak estradiol (E.) levelsstimulate mitosis. In extracts, the 80 kDa UM fragment, relative to the120 kDa intact membrane-spanning UM, was low at diestrus and in-creased through the cycle to peak with a 7-fold increase at metestrus,when epithelial cells are sloughing off. Tissue and extracts from E2-injected immature mice, but not those injected with progesterone alone,showed an estrus-like pattern. At implantation (4.5-5.5 days pregnant),embryonic trophoblast cells attach to and penetrate between epithelialcells; in sections, basal-lateral UM localizations in epithelium decreasedand only UM-containing junctional complexes remained. Just prior toimplantation, at 3.5 days, there was a 3.5-fold peak of the 80 kDa UM(compared to 120 kDa UM). We conclude that changes in UM seenduring the estrous cycle and early pregnancy are under hormonal controland show distribution and degradation consistent with loosening of cellsfrom each other and the matrix. (NIH grant HD 19530)

lOOa

Sunday. Cell-to-Cell Interactions I (587-592)

587INVOLVEMENT OF THE N-CADHERIN/CATENINCOMPLEX IN CARDIAC MYOCYTE INTERACTION ANDMYOFIBRILLOGENESIS. ((A. Peralta Soler,K.R Johnson*,M.J. Wheelock, and KA Knudsen*)) Lankenau MedicalResearch Center, Wynnewood, PA 19096 and 'BiologyDepartment, University of Toledo, Toledo, OH 43606.

N-cadherin is a Ca++-dependent transmembrane cell-celladhesion molecule found in the intercalated disks andextrajunctional sites of cardiomyocytes. Intracellularly,N-cadherin associates with the catenins which are thoughtto anchor the cadherin to the actin cytoskeleton. Additionof specific anti-N-cadherin antibodies to culturedembryonic chick cardiomyocytes inhibits the synchronouscontraction of interacting myocytes. Quantitative electronmicroscopy reveals that anti-N-cadherin significantlydecreases cell-cell junctions, including adherens junctions,gapJunctions, and desmosomes. In addition, the anti-N-cadherin causes myoflbril disarTay and a signiflcantreduction in the cytoplasmic area of the cell occupied bymyofibrils. Together, the data suggest that theN-cadherin/catenin complex plays an important role inpromoting both cell-cell interaction and the formation oforganized myoflbrils in the myocardium.

589SUBSTRATUM AND CELL SHAPE AFFECT EPITHELIAL CADHERINEXPRESSION IN CULTURED ALVEOLAR TYPE II CELLS. ((E.L.Aronsen and J.M. Shannon)) Department of Medicine, National JewishCenter for Immunology and Respiratory Medicine, Denver, CO 80206.(Sponsored by D.R. Voelker.)

In addition to synthesizing and secreting pulmonary surfactant, the typeII alveolar epithelial cell serves as a progenitor for the type I cell. TypeI cells effect gas-exchange in the alveolus and provide a barrier toalveolar flooding. This transition from the cuboidal type II cellmorphology to the highly-attenuated type I cell morphology requireschanges in cell shape and cytoskeletal reorganization. Cadherins are

transmembrane glycoproteins that intracellularly associate with thecytoskeleton and extracellularly mediate cell-cell adhesion. Since cell-cell adhesion between type I cells is required to establish the epithelialbarrier at the air-capillary interface, we hypothesized that the transitionfrom the type II to the type I cell phenotype would be accompanied bychanges in cadherin gene expression. Rat alveolar type II cells were

cultured for 4-8 days on substrata demonstrated previously to promoteeither the maintenance of the type II cell phenotype (EHS matrix) or thedifferentiation of these cells into a type I cell morphology (plasticdishes). Using a cDNA for rat epithelial cadherin (E-cad), we observedthat the transition to a type I cell morphology on plastic wasaccompanied by a significant, time-dependent increase In E-cad mRNAcompared to freshly-isolated cells. In contrast, cells cultured on EHSshowed a small decrease in E-cad mRNA abundance. To directly test theeffects of cell shape on E-cad expression, type II cells were cultured onrat tail collagen gels that were either left attached to the dish or releasedto float free in the medium. E-cad mRNA was increased in the attachedcultures (flattened cells) compared to floating cultures (cuboidal cells).We conclude that Increased E-cad expression correlates with cell shapechanges associated with formation of a barrier epithelium in the rat lung.

591

APPROACHES TOWARDS AN UNDERSTANDING OF MECHANISMAND REGULATION OF E-CADHERIN-MEDIATED ADHESION. ((B.Angres and W.J. Nelson)) Department of Molecular and Cellular Physiology,Stanford University School of Medicine, Stanford, CA 94305.

We are interested in the role of linkage of E-cadherin to the cytoskeleton,cytoskeletal organization and second messenger pathways to elucidatemechanism and regulation of E-cadherin-mediated cell-cell adhesion. Toexamine regulation of cell-cell adhesion and the cadherin-mediatedphenomenon of cell compaction, we have taken two approaches. In the firstapproach, compaction is analyzed in a cell aggregation assay. Mousefibroblasts (L-cells), which show no typical cadherin-like adhesion, were

transfected with full length E-cadherin resulting in a change from a non-

compacting fibroblastic phenotype into a compacting epithelial phenotype.Thetransfection of L-cells with E-cadherin truncated in the cytoplasmic domain,which results in loss of E-cadherin binding to catenins and the cytoskeleton,does not lead to a compacting cell phenotype, demonstrating the importance ofthe intracellular domain in cell compaction. We show that E-cadherin-mediatedcompaction can be inhibited by buffering intracellular Ca2+ with BAPTA (10-20i±M) indicating a role of [Ca2+]i in functional interactions between E-cadherin and the cytoskeleton. In the second approach, E-cadherin-mediatedadhesion is quantitated in a centrifugal-force-based adhesion assay (McClay et

al. (1981) Proc. Natd. Acad. Sci. USA 78, 4975-4979) using MDCK epithelialcells. The extracellular domain of E-cadherin was purified from dogkidney andused as a solid-phase substrate to study E-cadherin-mediate7d adhe-sion.Preliminary results suggest that adhesion is dependent on metabolic activity ofthe cell. Chelation of intracellular Ca2+, however, did not abolish or decreaseadhesion under the applied conditions. These results suggest that cadherin-mediated adhesion and compaction are regulated differently.

588EFFECT OF AD5 ElA 12S ON THE EXPRESSION OF UVOMORULIN, INIMMORTAUZED AND TRANSFORMED CELL LINES. ((S. Gopalakrishnan andM. P. Quinlan)). Department of Microbiology and Immunology, University ofTennessee, Memphis, TN 38163.

Adenovirus is a DNA tumor virus that infects quiescent epithelial cells.The Adenovirus ElA gene induces epithelial cell DNA synthesis,proliferation and immortalzaton of primary Baby Rat Kidney (BRK) epithelalcells. Immortalized epithelial cells retain expression of epithelial cellcharacteristics such as morphology, expression of cytokeratins andattachment to extracellular matrix. The expression of uvomorulin, a Ca++ -

dependent, intercellular adhesion molecule, in mock and 12S infectedBRKs was analyzed. At early times after plating, primary BRK epithelial celsexpress uvomorulin at the intercellular junctions. With time in culture thereis a decrease in uvomorulin expression. Infection of BRK cells with the12S virus results in increased expression of uvomorulin, with a maximum at4 days postinfection, as detected by immunofluorescence. Epithelial cellsestablished by 12S continue to express uvomorulin. In contrast toimmortalization, BRK cells trensfected with an activated RAS gene andEIA give rise to transformed cells. Progression along the transformationpathway is modulated by the second exon of ElA. These transformedcells have altered intracellular distnbution of uvomorulin. Preliminary resultsfrom RT-PCR experiments confirm the above observations. Thus, AD5EIA 12S may act as a differentiating factor that maintains the epithelialphenotype of cells by increasing uvomorulin expression.

590CONTROL OF EXPRESSION AND FUNCTION OF THE EPITHELIAL CELLADHESION MOLECULE E-CADHERIN. (a. Behrens, 0. Lowrick, G. Hennig,J. Htilsken, and W. Birchmeier)) Max-Delbrf3ck-Centrum, D-13122 Berlin-Buch,Germany.

The cell-cell adhesion molecule E-cadherin is expressed in epithelial cells anddownregulated in many poorly differentiated carcinomas, which leads to highermotility and invasiveness of the cells. Here we demonstrate by mutational analysisthat the E-cadherin promoter is composed of positive and negative regulatoryelements, the combination of which generates epithelial specificity. A GC-richregion (between -58 and -25) and a CCAAT-box (at -65) exhibit positiveregulatory activity in both epithelial and nonepithelial cells. In contrast, thepreviously identified palindromic element E-Pal (between -86 to -75) has a dualfunctional role: in epithelial cell it weakly promotes transcription, whereas innonepithelial cells it suppresses promoter activity, apparently by inhibiting thefunction of the GC-rich region and the CCAAT-box. We show further that helix-loop-helix transcription factors are candidates for binding to E-Pal, and that theGC-rich region interacts with transcription factor AP-2 or closely related proteins.We have shown previously that the protein tyrosine kinase pp60v-src inducesdrastic disturbances of intercellular adhesion of epithelial cells and concommitandtyrosine phosphorylation of E-cadherin and of the associated protein I-catenin. Wehave now isolated the human B-catenin cDNA and found that the protein ishomologous to armadillo of Drosophila (69% amino acid identity), and iscomposed of 13 repeats of 40 amino acids which are flanked by nonrepeated NH2-and COOH-termini. In order to identify the functional domains of B-catenin, wehave prepared deletion mutants of the protein and tested these for interaction withE-cadherin in transient transfection assays. Our results suggest that the repeats areessential for binding to E-cadherin, whereas the nonrepeated N- and C-terminaldomains are dispensable and may serve other functions, e.g. in signal transfer.

592DYNAMIC EXCHANGE OF CELL SURFACE AND INTRACELLULAR POOLS OFE-CADHERIN ((K.A. Siemers, E. Shore, W. J. Nelson)) Department of Molecular &Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5426

E-cadherin is an important protein in the regulation of cell-cell adhesion and thedevelopment of epithelial cell polarity. We present evidence that there is both a surfaceand intracellular pool of E-cadherin in MDCK cells. We have characterized these twopools by detecting protein with four independent methods: cell surface biotinylation,trypsinization,iodination, and immunoflorescence. We find that upon induction of cell-cell contact there is a five fold increase in the cell surface level of E-cadherin. Thisincrease in cell surface levels of E-cadherin occurs on the (basal-)lateral membrane andnot at the apical membrane. Recruitment of this surface pool occurs from a pre-existingor intracellular pool, rather than a newly synthesized pool Treatment withcyclohexamide, under conditions which >95% of protein synthesis is inhibited, does notprevent rcruitment of E-cadherin from the intracellular pool to the cell surface pool. Inaddition, using cell surface iodination of E-cadherin, we find internalized protein isdirectly degraded and does not recycle through the intracellular pool. We were also ableto separate and examine both pools of E-cadherin by employing a technique whichselectively precipitates biotinylated E-cadherin from Triton X-100 soluble and insolublesamples, and allows immunoprecipitation of the non-biotinylated, intracellular pool. Ourdata indicatethat 50% of the E-cadherin is distributed in the intracellular pool and 50% isin the cellsurface pool. The distribution of the intracellular and cell surface pool of E-cadherin was examined by immunoflorescence microscopy. Results demonstate two verydifferent staining patterns. The intracellular pool appears as large vesicular structures inthe cytoplasm of the cell, where as the surface pool is characterized by punctate staining.These results show thatthere is a dynamic regulation of E-cadherin levels at cell-cellcontacts by recruitment of protein from an intracellular pool. This regulation mayprovide a mechanism forrapid response to cell interactions in the fornation of strong

cell-cellcontacts.

lOla

Cell-to-Cell Interactions (593-598). Sunday593FLOW-INDUCED DISAGGREGATION OF HUMAN BREASTCANCER CELL AGGREGATES CORRELATES WITH E-CADHERINEXPRESSION. ((S.W. Byers, C.L. Sommers and A. Tozeren))Department of Cell Biology and the Lombardi Cancer Research Center,Georgetown University Medical Center, Washington D.C. 20007 andDepartment of Mechanical Engineering, The Catholic University ofAmerica, Washington D.C. 20064

Defects in the expression or function of E-cadherin are common ininvasive, metastatic carcinomas. We have now used a laminar flow assay tocompare the response of aggregates of normal breast epithelial cells(NBEC), and breast carcinoma cells (BCC) to forces imposed by laminarflow. E-cadherin negative invasive BCCs do form cell aggregates in thepresence of calcium, but these are significantly more likely than E-cadherinpositive cell aggregates to disaggregate in response to low shear forces suchas those found in a lymphatic vessel or venule. E-cadherin positive NBECsand E-cadherin positive BCC aggregates could not be disaggregated evenwhen exposed to shear forces in excess of those found in arteries.Transfection of E-cadherin did not alter the response of invasive tumorcells to laminar flow. However, the absence of a triton soluble pool of E-cadherin in these cells points to a defect in E-cadherin interaction with thecytoskeleton. In contrast, E-cadherin transfected L-cells acquired calcium-dependent cell-cell adhesion and had disaggregation properties similar tothose of E-cadherin positive NBECs. Our results show that E-cadherinnegative cells, or cells in which it is inefficiently linked to thecytoskeleton, are far more likely than E-cadherin positive cells to detachfrom a tumor mass in response to lymphatic or venous flow. Since aprimary route of dissemination of BCCs is to the local lymph nodes theseresults point to a novel mechanism whereby defects in cell-cell adhesioncould lead to BCC dissemination.

595E-CADHERIN CHANGES CELL-CELL BUT NOT CELL-SUBSTRATESPECIALIZATIONS WHEN IT INDUCES MESENCHYMAL-EPITHELIALTRANSFORMATION (MET). ((C.R. Vanderburg and E.D. Hay)) Departmentof Cell Biology, Harvard Medical School, Boston, MA. 02115

Six day old embryonic chick corneal fibroblasts were cotransfected withE-cadherin (Ecad) and Neol cDNA using an impact mediated transfectiontechnique. After selection, Ecad positive cells form stratified aggregates ofepithelioid cells by 3 wks in culture. These cells express Ecad anddesmoplakin on lateral surfaces (Vanderburg and Hay, 1992. Molec. Biol.Cell., 3:5a). Here we extend our findings, showing by TEM that thesestratified epithelioid cells form zonulae adherentes, tight junctions, and truedesmosomes. They also upregulate connexins, keratin, and otherdesmosome associated proteins. To further evaluate the ability of Ecad topromote epithelial substrate interactions, we used TEM andimmunofluorescence/confocal microscopy to follow production of basallamina by transfected fibroblasts cultured on glass, collagen gels, or

denuded corneal stromas containing living fibroblasts. Prior to and afterEcad transfection, corneal fibroblasts synthesize laminin, type IV collagenand Na K*ATPase. After culturing on living stromas, fibrillogranular materialpolymerizes under the epithelial basal surface. A true basal lamina doesnot form. Both transfected and untransfected fibroblasts cultured on

collagen downregulate collagen IV and laminin dramatically. In transfectedcells on all substrates, Na'K'ATPase localizes to cell-cell contacts, but notapical or basal surfaces. We conclude that expression of Ecad onlypromotes epithelial structures involved in cell-cell interaction. Apical-basalpolarity, basal lamina formation and ECM synthesis appear to be controlledseparately during MET.

597

CHARACTERIZATION OF ADHERENS JUNCTIONS INFIBROBLASTS. ((#K. R. Johnson, *K. A. Knudsen, *A. P. Soler, and#M. J. Wheelock)) #Department of Biology, University of Toledo,Toledo, OH 43606 and*Lankenau Medical Research Center,Wynnewood, PA 19096

Classical adherens junctions are made up of a transmembraneprotein (a member of the cadherin family) and cytoplasmic proteinsthat link the cadhenn to the cytoskeleton. The cadherin molecule isdirectly associated with three proteins termed "catenins" which arethen associated in some way with actin filaments (presumably viaproteins such as vinculin and a-actinin). Using bacterially derivedfusion proteins we produced monoclonal antibodies against a- and pcatenins and plakoglobin (rcatenin) having molecular weights of102, 92 and 80 kD respectively. Using these antibodies, we showthat cultured normal human and chicken fibroblasts havecadherin/catenin complexes similar to but not identical to complexesfound in cellswith classical adherens junctions. The cadherin foundin fibroblast junctions Is N-cadherin and various subsets of thecatenins are associatedwith N-cadherin in these cells.Immunofluorescence with either anti-N-cadherin or anti-cateninantibodies show a localization at regions of cell-cell contact andalong actin filaments. Ultrastructural analysis demonstrates that bothhuman and chicken fibroblasts have membranous structures very

much like epithelial cell adherens junctions. Supported by NIH CA44464 and AR37945, The American Cancer Society, The AmericanHeart Association and The W.W. Smith Charitable Trust.

594CLONING OF HUMAN CADHERIN ASSOCIATED PROTEINa-(E)CATENIN SHOWS A SECOND ISOFORM SIMILAR TO META-VINCULIN. ((P. Kebriaei, J.S. Morrow, and D.L. Rimm)) Dept. ofPathology, Yale University School of Medicine, New Haven, CT 06510

A 102 kD protein called a-catenin (or CAP102) is associated with thecytoplasmic domain of cadherin molecules and is required for theirhomotypic cell-cell adhesive activity. The mouse a-catenin clone showssequence homology to vinculin in three domains. We have used PCR withprimers based on the mouse sequence to obtain >20 human cDNA clones.Sequencing of 3463 bp reveals a 2724 bp open reading frame coding for a 907 aaprotein with 97% identity to mouse a-catenin. Northern blots reveal messagesof 3900 and 4100 bp in multiple epithelial and non-epithelial tissue types.Within the molecule, three regions show homology to vinculin in the 25 - 40%range. An alternative splice form of vinculin has been described which codesfor a 68 aa insert (by splicing in exon 19) in the C-terminal region of themolecule called 'meta-vinculin'. We have identified an a-catenin clonethat also appears to contain a similar type insert of 24 aa located in ananalogous region of the protein. The location of the putative alternative splicecorresponds exactly with an insert seen in CAP-R, a human a-Ncateninhomologue, but shows no sequence homology. Transcripts of both formsappear to be present in a range of human tissues as judged by RT-PCR. Weconclude that the epithelial isoform of a-catenin has an inserted sequenceisoform analogous to the meta-vinculin splice form.

596BLOCKING THE FUNCTION OF E-CADHERIN TOGETHER WITH P-CADHERIN INHIBITS CALCIUM-INDUCED REORGANIZATION OFJUNCTIONAL COMPONENTS IN CULTURED HUMAN KERATINO-CYTES. ((#J. E. Lewis, *P. J. Jensen, and #M. J. Wheelock))#Department of Biology, University of Toledo, Toledo, OH 43606 and*Department of Dermatology, University of Pennsylvania,Philadelphia, PA 19104

Human keratinocytes grown in 30 sM calcium display minimal cell-cell interactions. However, elevation of calcium to 1 mM induces theformation of adherens junctions and desmosomes. Within 3-6 daysafter elevation of calcium to 1 mM, the cells stratify to form amultilayered structure that resembles skin in vivo. Humankeratinocytes express two members of the cadherin family ofadhesion molecules, E-cadherin and P-cadherin. E-cadherin isexpressed throughout the stratified culture while P-cadherin isrestricted to the basal layer. We have previously shown that functionblocking antibodies to E-cadherin delay the formation of adhesivestructures and disrupt the formation of a stratified culture. Antibodiesto E-cadherin, however, do not prevent the adhesive structures fromeventually forming and do not prevent stratification. In the presentstudy we show that antibodies to P-cadhern alone have minimaleffect on the ability of the cells to form junctional complexes butantibodies to both cadherins, when added together, preventformation of normal junctions. We are currently investigating theeffect of blocking both cadherns on the ability of these cells to stratifyin culture. Supported by NIH CA44464 & AR39674.

598ESTRADIOL STIMULATES E-CADHERIN mRNA LEVELS INMCF-7 CELLS. ((R. Jednak, C.D. MacCalman, and O.W.Blaschuk)) Department of Surgery, McGill University, Montreal,Quebec, Canada.

E-cadherin (E-cad) is an integral membrane glycoprotein whichmediates calcium-dependent, epithelial cell adhesion. The factors thatregulateE-cad levels in mammalian cells are imperfectly known. Wehave previously shown that 17f-estradiol (E2) stimulates cadherinlevels in rat granulosa cells (Develop. Biol. 136: 564). Based on thisobservation, we have proposed that steroids are key regulators ofcadherin levels. In order to test this hypothesis, we have investigatedthe ability of steroids to regulateE-cad mRNA levels in the humanbreast cancer cell line, MCF-7. The MCF-7 cells contain estrogen,androgen, and progesterone receptors (Sem. Oncology 19: 286). E2(30nM) was found to be a potent stimulator of E-cad mRNA levels inMCF-7 cells. This steroid significantly stimulated E-cad mRNAlevels in these cells within 90minutes after it was added to the culturemedium. Furthermore,tamoxifen (a competitive inhibitor of estrogen)blocked the effect of E2 on theE-cad mRNA levels in the MCF-7cells. These results support our hypothesis that steroids are capable ofregulating cadherin levels in mammalian cells.Supported by the Medical Research Council of Canada.

102a

Sunday. Cell-to-Cell Interactions (599-603)

599TYROSINE PHOSPHATASE INHIBITORS NCREASE TIGHTJUNCTIONAL PERMEABILITY N STRAIN I MDCK CELLS (G.M.Staddon, C. Smales and L.L. Rubin)) Eisai London ResearchLaboratories, University College London, London WC1E 6BT, U.K.

Pervanadate, a tyrosine phosphatase inhibitor, increased the tyrosinephosphorylation of adherens junction components in MDCK cells,resulting in junctional disassembly (Volberg et al. (1992) EMBO J. 11,1733-1742). However, the integrity of tight junctions was apparentlyunaffected, at least as determined by the staining of ZO-1, a tightjunction-associated protein. In contrast, we find that pervanadatecauses a rapid opening of tight junctions in filter-grown Strain IMDCK cells, as measured by decreased transcellular electricalresistance (TER). Phenylarsine oxide (PAO), another tyrosinephosphatase inhibitor, also caused a rapid decrease in TER, but notas substantially as that observed with pervanadate. Immuno-fluorescence staining of fixed cells revealed an increase in phospho-tyrosine at the intercellular borders after both pervanadate and PAOtreatment. However, PAO did not give rise to the diffuse cytoplasmicstaining observed with pervanadate. Western blotting demonstratedincreased tyrosine phosphorylation of many proteins in response topervanadate in contrast to the phosphorylation of oniy a few bandsin response to PAO. These findings suggest that the tyrosinephosphorylation of a limited number of proteins may directly, or

indirectly, control the permeability of intercellular tight junctions.

601

ROLE OF INTEGRINS IN SPERM-EGG BINDING AND FUSION.((E.A.C. Almeida, P.G. Calarcov, A.E. Sutherland§, D.G. Mylesi, P.Prymakoffl, and J.M. White')) *Departments of Pharmacology andBiochemistry and Biophysics, and vDepartment of Anatomy and§Department of Stomatology, University of California San Francisco,CA 94143, and ¶University of Connecticut Health Center, Farmington,CT 06030. (Spon. by S.I. Waters)

Binding and fusion of sperm and eggs are critical events inmammalian fertilization. We previously showed that PH30, anintegral protein of the guinea pig sperm plasma membrane: 1) isinvolved in sperm-egg fusion, 2) contains an integrin binding domain,and 3) contains a putative membrane fusion domain. Thesecharacteristics led to the hypothesis that PH30 plays roles in bothgamete adhesion, via an egg surface integrin, and gamete fusion. Inthis study we tested the involvement of egg surface integrins inbinding and fusion of murine sperm and eggs. We assayed sperm-egg binding by video phase contrast microscopy and fusion by both anovel fluorescent lipid mixing assay and by a fluorescent cytoplasmiccontent mixing assay. A rabbit polyclonal antiserum recognizing theintegrins a31l, ax51, a601l, and avJ3 inhibited sperm binding tozona-free eggs by 80%, from 14+/-2 to 3+/-1 sperm bound per egg.Despite inhibiting binding, the antibody had no effect on fusion per se.On average 1.5 +/-0.5 sperm fused per egg in both antibody treatedand control samples. These results indicate that an egg surfaceintegrin(s) is involved in fertilization, and that it is involved in spermbinding to the egg plasma membrane. Current efforts should identifywhich specific integrin(s) act as sperm ligand(s).

600MOLECULAR CLONING OF A NOVEL DESMOSOME-ASSOCIATED PROTEIN.((P.OUYANG and*S.Sugrue)) Dept. Anatomy, Chang Gung MedicalCollege, Taiwan, ROC and *Dept. Cell Biology, Harvard MedicalSchool, Boston, MA 02115

The desmosome serves as an important adhesion structureinvolved not only in integration of adjacent cells but alsofunctioning as anchorage point for cytoplasmic intermediatefilaments. We have identified a protein, termed 08L, of Mr=140,000, associated with mature desmosome of epithelium, andcDNA clones encoding for the carboxyl two thirds of the total08L protein were identified (JCB 118:1477-88 and NBC 3:292a).Here we confirm that these cDNA clones represent genuine 08Land the extension of open reading frame to the 5' end has beenaccomplished with 5'-RACE. To verify that 08L had been cloned,we carried out a series of experiments involving expression ofcDNA-encoded proteins and imnunoblotting using original 08Lantibody as well as the generation of new antibodies directedagainst fusion proteins. Western blots of PCR-generated fusionproteins revealed that 08L epitope was located near to thecarboxyl extreme of the total 08L protein. The antisera, frommouse inoculated with fusion protein, immunostained the 140 kD08L on Western blot and stained the lateral cell surfacesconsistent with 08L staining pattern. We have performed RACEutilizing gene-specific primers to obtain primary structureinformation of 5' end of 08L. An additional 600 bp fragment was

identified. We are now in the process of analyzing the 5' endof 08L primary structure and this information will add up toour understanding of the function of 08L in the stabilizationof epithelial adhesion.

602

Ga PROTEINS ARE INVOLVED IN THE REGULATION OFPHAGOCYTOSIS AND SEXUAL CELL FUSION INDICTYOSTELIUM DISCOIDEUM. ((D.D. Browning, K.E. Lewis,and D.H. O'Day)) Department of Zoology, University ofToronto, Erindale College, Mississauga, Ontario, Canada L5L1C6.

We have used a novel phase of sexual development inDictyostelium discoideum to investigate the role of GTP-binding proteins in 2 separate events: sexual cell-fusion andcannibalistic phagocytosis. Phagocytosis of vegetativeamoebae zygote giant cells was inhibited by the G-proteinagonist GDPOS whereas sexual cell-fusion was slightlyaugmented. In contrast, receptor independent activation ofGa-proteins using aluminum fluoride and GTPyS wasapproximately twice as effective at inhibiting cell-fusion asphagocytosis. Using both I and 2-dimensional SDS-PAGE andwestern blotting with 2 different immune sera, we demonstratethat GaS is unique to phagocytes in this organism.Measurement of cAMP levels intracellularly using the isotopedilution assay, suggests that adenylate cyclase may be theclassical effector enzyme and is important in the regulation ofphagocytosis.

603

ROLE OF PROTEIN KINASE C IN THE REGULATION OF THEINTRACELLULAR pH AND GAP JUNCTION PERMEABILITY.((I.V. Budunoval, L.A. Mittelman2, and J.Miloszewska 3)) ILaboratory ofCarcinogen Screening Methods, National Cancer Center, Moscow, Russia;2Department of Physiology and Pharmacology, Sackler Medical School,Tel-Aviv University, Tel-Aviv, Israel; 3 Department ofTumor Biology,M. Sklodowska-Cffie Institute, Cancer Center, Warsaw, Poland(Spon. by R.R. Hewitt)

In protein kinase C (PKC)-depleted cells the function of many membrane-associated proteins which are substrates for this enzyme (including Na+/H+antiporter, gap junctional proteins, etc.) could be expected to change due to thedecrease of the extent of their phosphorylation. Using monolayer cultures ofhamster fibroblasts DM15 we studied the effects of PKC depletion on suchparameters as the level of gap junctional intercellular communication,intracellular pH (pHi) and the sensitivity of cells to the effects of tumorpromoters. Teatment ofDM15 cells with 12-0-tetradecanoylphorbol-13-acetate(TPA) for 24 hr induces strong decrease of PKC activity especially in themembrane fraction. In PKC-depleted cells the level of dye-coupling is 30-40%higher in comparison with the untreated control cells. However the level ofphosphorylation of Cx43, the main gap junctional protein for fibroblasts, doesnot decrease in TPA-pretreated cells. PKC-depleted cells are resistant to theuncoupling effect of such tumor promoters as meserein, teleocidin and Ca2+ionophore A23187; and are as sensitive as control cells to the effect ofDDT andnigericin. PKC down-regulation does not affect pHi in DM15 cells. The effectof tumor promoters teleocidin and A23187 which usually induce cytoplasmalkalinization is different in the PKC-depleted cells. Thus these data show thatPKC plays the role in pHi and gap junctional permeability regulation mainly as

the mediator for the effects of agonists and Ca2+.

103a

Chloroplasts and Mitochondria (604-609). Sunday

604PLASTID GENOME REPLICATION AND CONFORMATION. ((A.M.Nerozzi and A.W. Coleman)) Division of Biology and Medicine, BrownUniversity, Providence, RI 02912.

The structure of the individual molecule of the plastid genome is now wellknown in many algae and plants. Single plastids can contain up to 200copies of the plastid DNA (ptDNA). However, the arrangement of thesemolecules within the plastid "chromosome' and the role of this structure inthe successful replication and segregation of the plastid genome duringplastid and cell division has yet to be unravelled. We have examined thisrelationship in the chromophyte alga Ochromonas danmca using a variety oftechniques. 0. danica is particular useful for such work because the ptDNAlies in a relatively large and easily recognizable ring nucleoid just within theplastid envelopes. Microspectrofluorometry of DAPI-stained ring nucleoidshas revealed that the plastid genome complement per ring nucleoid isroughly correlated with plastid size and that the ring nucleoid increases insize in both length and thickness. Immunocytochemical detection of sites ofincorporation of 5-bromodeoxyuridine into the ring nucleoids of individualcells has revealed that ptDNA synthesis is active at 3-5 discrete sites spacedalong the ring structure. The extent of labelling increases with the length ofthe pulse. Plastid DNA synthesis occurs throughout GlI/S/G2 and thereforeis under separate control from nuclear DNA synthesis. In multiplastidiccells the ptDNA synthesis pattern is similar for all plastids in all stages ofthe cell cycle. Treatment of cells with the DNA gyrase inhibitor nalidixicacid results in the accumulation of densities along the ring nucleoid whichare strongly correlated with sites of ptDNA synthesis. Pulsed field gelelectrophoresis of total DNA from cells lysed and digested in agarose blocksfollowed by hybridization with a ptDNA-specific probe has revealed thepresence of multimeric ptDNA molecules. We are using these approachestogether to elucidate the structure of the plastid nucleoid.

606

AN APPARENT COUPLING BETWEEN CELL SIZE CONTROLAND CHLOROPLAST GENOME INHERITANCE IN THEUNICELLULAR ALGA CHLAMYDOMONAS. ((E.V. Armbrust,A. Ibrahim, and U. W. Goodenough )) Department of Biology, Box1229, Washington University, St. Louis, MO 63130.

A common feature of eukaryotes is that organelle genomes areinherited almost exclusively from the female parent Uniparentalinheritance of chloroplast traits has been examined most extensivleyin the haploid unicellular green alga, Chlamydomonas reinhardtii.Approximately 1-2 hr after zygote fonnation, the chloroplast DNAfrom the mr parent is selectively destroyed, presumably due to theaction of a zygote-specific nuclease (Armbrust et al., Cell 74, 1993);thus meiotic progeny generally inherit chloroplast traits from theMt+ parent only. The destruction of the mr chloroplast DNA isinhibited and hence biparental inheritane is enhanced when themt+-linled mutation mat-3 is carried by the mt+ parent (Gillham etal, Genet. 115:677, 1987). Since the mat-3 phenotype is rapidlysuppressed (Gillham), we backcrossed a suppressed mat-3 strain andrecovered progeny displaying the biparental trait Interestingly,these progeny proved to be remarkably small (-2.5 jim instead of 10pum in diameter), reminiscent of the wee] mutant described forfission yeast (Russel & Nurse, Cell 49:559, 1987 ). By TEM, all ofthe organeLes are proportionately reduced in size. The size defecthas now been recovered from each of the 3 original mat-3 strains, issuppressed over time, and is displayed only by mt+ progeny thatcarry mat-3, suggesting that mat-3 somehow influences both cellsize and organelle inheritance.

608

FREEZE-ETCH MORPHOLOGY OF CYTOPLASMIC MALE STERILEAND FERTILE PETUNIA MITOCHONDRIA. ((C. A. Conley, M. V.Parthasarathy, A. G. Cobb, and M. R. Hanson)) Section of Plant Biology andSection of Genetics, Cornell University, Ithaca, NY 14853

Plant mitochondria carry out many and more diverse functions than animalmitochondria. Although mammalian mitochondrial mutations are common,only two classes of non-lethal plant mitochondrial defects are known. One ofthese, cytoplasmic male sterility (CMS), causes pollen abortion in otherwisemorphologically normal plants. A physiological phenotype correlating withPetunia CMS involves differential partitioning of electron flow through thecytochrome oxidase and alternative oxidase pathways. We have recentlyreported tissue-specific localization of a number of mitochondrial proteinsinvolved with aspects of energy production, as well as a CMS-associatedprotein. In Petunia CMS-associated mitochondria a novel region of DNA,named pcf, codes for a protein found at high levels only in sterile plants. ThePcf protein has a localization pattern very similar to that of the mitochondrialatpase subunit A (AtpA), somewhat similar to the localization of the altemativeoxidase (AoA), and quite different than the localization of cytochrome oxidasesubunit II (CoxII). In order to determine possible associations between theseproteins at the molecular level, we are performing freeze-etch studies of isolatedmitochondria and cells from fertile and male-sterile plants, using previouslycharacterized antibodies as probes for molecular localization. This informationshould lead to a greater understanding of the interactions between plantmitochondrial energy pathways.

605THE POLYPROTEIN PRECURSOR TO A EUGLENA THYLAKOIDPROTEIN, LHCPII, IS TRANSPORTED FROM THE ER TO THECHLOROPLAST PRIOR TO POLYPROTEIN PROCESSING. ((S.Chidananda and S. D. Schwartzbach)) School of Biological Sciences,University of Nebraska, Lincoln, NE 68588. (Spon. by G. Veomett)

The precursor to the Eualena light harvesting chlorophyll a/b binding proteinof photosystem 11 (pLHCPII) is a polyprotein composed of 8 mature LHCPllscovalently linked by a decapeptide. pLHCPII is synthesized on membranebound polysomes and appears to be transported to the ER and Golgiapparatus prior to chloroplast localization. To confirm the transport pathwayand identify the intracellular site for processing pLHCPII into 8 LHCPlls, cellswere pulse labeled with 35S-sulfate, organelles separated on sucrosegradients, (p)LHCPlls immunoprecipitated from gradient fractions andpLHCPlls and LHCPlls separated on SDS gels. Microsomal, chloroplastand mitochondrial fractions were free of contamination by other organellesas determined by marker enzyme distributions. After a 10 min pulse, theunprocessed pLHCPII polyprotein was found in microsomes but not inchloroplasts. LHCPII was undetectable after a 10 min pulse consistent withthe 20 min half time for pLHCPII processing. When pulse labeled cellswere chased for 20 or 40 min with unlabeled sulfate, pLHCPII was foundin microsomes and chloroplasts. LHCPII was found in chloroplasts butnever in microsomes. Localization of newly synthesized pLHCPII tomicrosomes and the appearance of pLHCPII in chloroplasts coincident withthe processing of pLHCPII to LHCPII indicates that pLHCPII is transportedas a polyprotein from the ER to the chloroplast; the site of proteolyticprocessing of pLHCPII to produce 8 LHCPIIs. (NSF-MCB-9118721)

607NADH REGULATES THE PERMEABILITYOF THEMITOCHONDRIAL OUTER MEMBRANE BY CONTROLLING THEGATINGOF VDAC CHANNELS. ((A.C. Lee, M. Zizi, and M.Colombini)) Dept. Zoology, Univ. Maryland, College Park, MD 20742(Spon. by M.D. Goode)

Energy production by glycolysis and oxidative phosphorylation ishighly regulated and includes reciprocal inhibition through the Pasteurand Crabtree effects. The hydrogen carrying cofactor, NADH, mayparticipate in this "cross-talk" by controlling the permeability of themitochondrial outer membrane through the regulation of a majortransport pathway, the voltage gated channels called VDAC (alsoreferred to as mitochondrial porin). We present two independentlines of evidence: pure VDAC channels reconstituted intophospholipid membranes and isolated intact mitochondria. In thereconstituted system, the voltage dependence of human HVDAC1, S.cerevisiae VDAC, and N. crassa VDAC is doubled specifically bymicromolar amounts of NADH and not by millimolar amounts ofNAD+, ATP, ADP, cAMP, or cGMP. Using mitochondria with intact(99%) outer membrane, ADP permeability is reduced 6 fold by theaddition of micromolar quantities of NADH. Damaging the outermembrane eliminated the NADH effect. The KD in both cases is very

similar, about 50 AM. These results add to the growing evidence thatthe mitochondrial outer membrane plays a critical regulatory role inenergy metabolism. (Supported ONR grant N00014-90-J-1024)

609

COUPLING OF MITOCHONDRIAL METABOLISM AND MITOCHONDRIALTRANSLATION IN THE RAT HEART ((E. E. McKee, and A. T. Bentley)), SouthBend Center for Medical Education, Dept. of Biochem. and Mol. Biol., IndianaUniv. Sch. of Med., and Dept. of Chem./Bioch., Notre Dame, IN 46556.

The biogenesis of the mitochondrial inner membrane requires the coordination oftwo genomic systems: the nuclear cytoplasmic system, responsible for the majorityof inner membrane proteins; and the mitochondrial system, responsible for 13essential peptides of 4 inner membrane enzyme complexes. It is well known thatthe cellular content of the inner membrane is adjusted to meet the energy demandsof the cell; but the mechanisms are urnknown. The goal of the present work is tounderstand the relationship between mitochondrial translation and metabolism. Inthis study, mitochondrial translation, the inner membrane electrochemical gradient,and the matrix and medium levels of adenine and guanine nucleotides were deter-mined in isolated heart mitochondria under a variety of oxidizing and non-oxidizingconditions. Thus far our data have demonstrated the following. I) Mitochondrialtranslation is regulated by the adenine energy charge of the system which is inequilibrium with the guanine energy charge of the system. 2) Mitochondrialtranslation prefers the addition of adenine nucleotide as ADP rather than ATP (1.5-2fold increase at 4 mM) even though the same energy charge is maintained by bothafter several minutes of incubation. This does not appear to be due to an inhibitorycontaminant of ATP. 3) Mitochondrial translation is nearly completely inhibited innon-oxidizing mitochondria, and in mitochondria in which the membrane potentialhas been disniptred, even under conditions in which the medium and matrix adenineand guanine energy charges are maintained at a high level. These results suggest thehypothesis that a membrane potential may be required for the insertion of mito-chondrial translation products, and if insertion is blocked, translation is inhibited.

104a

Sunday. Chloroplasts and Mitochondria (610-615)

610

CORRELATION BETWEEN ADENINE NUCLEOTIDE CONTENT ANDSUCCINATE OXIDATION RATES OF MITOCHONDRIA ISOLATEDFROM RAT LIVERS PERFUSED WITH GLUCAGON. ((P.V. Blair))Department of Biochemistry and Molecular Biology, Indiana UniversitySchool of Medicine, Indianapolis, Indiana 46202-5122

The adenine nucleotide (AdN) content of rat liver mitochondria (RLM) canbe varied by treatment of perfused rat livers with different levels of glucagon.The change in mitochondrial AdN content produces concomitant changes inADP-stimulated and uncoupler-stimulated oxidation of succinate. Mitochondriaisolated from untreated, perfused rat livers have an AdN content of 12.1 0.6nmoles/mg RLM protein, an ADP-stimulated succinate oxidation rate of 165±16 ngatoms oxygen/min x mg RLM protein and an uncoupler-stimulated rateof 205 t19. At a glucagon induced intermediate AdN content of 16.2t1.4nmoles/mg RLM protein the succinate oxidation rates are 199 ±14 with ADPand 246±14 with uncoupler. The highest average mitochondrial content ofAdN induced by glucagon was 23.8t 1.4 nmoles/mg RLM protein and was

accompanied by increased succinate oxidation rates of 313±16 and 36222 withADP and uncoupler, respectively. The regression and correlation coefficientsindicate over 90% of the variation in succinate oxidation rates can beexplained by a linear dependence on the mitochondrial AdN content within thephysiological range of 12 to 24 nmoles AdN per mg RLM protein. It is alsoevident that intramitochondrial AdN plays a dominant role in uncoupler-stimulated respiration. (Supported by US-PHS Grant DK 13939 and the GraceM. Showalter Trust).

612

MITOCHONDRIAL TOXICITY OF DIDEOXYNUCLEOSIDES ANDTHEIR DERIVATIVES ((3.S. Modica-Napolitano and B.T. Brunelli)) TuftsUniversity, Department of Biology, Medford, MA 02155

Several dideoxynucleosides and their derivatives are currently being testedfor their potential as more potent, less toxic antiretroviral alternatives toAZT in the treatment of AIDS. Since recent evidence suggests that alimiting toxic side effect of long term AZT therapy (mitochondrialmyopathy) may be the result of tissue specific inhibition of mitochondrialrespiration (Modica-Napolitano, J; BBRC 194:170-177, 1993), we

investigated the possibility that other dideoxynucleosides and theirderivatives may similarly affect mitochondria by measuring the effect ofthese compounds on mitochondrial bioenergetic function. Our results showthat like AZT, the pyrimidine dideoxynucleosides DDU (2',3'-dideoxyuridine) and DDT (2',3'-dideoxythymidine) and dideoxynucleosidederivative AZU (3'-azido 3'--deoxyuridine) inhibited both succinate-linkedrespiration and, to a lesser extent, NADH-linked respiration in mitochondriaisolated from rat skeletal muscle. However, over the range of concentrationstested (0-5 mg/ml) DDC (2',3'-dideoxycytidine) had no effect on either ofthese measures of mitochondrial function. The testing of additionalcompounds is ongoing, as is an analysis of structure/function relationships.These data may prove useful in predicting and/or preventing limitingtoxicities of dideoxynucleosides and their derivatives in the treatment ofAIDS.

614

MESSENGER RNA PROCESSING: C-TO-U EDMNG IN VASCULAR PLANTMITOCHONDRIA. ((G.L Sper-Whitis, A.L Russell and J.C. Vaughn))Department of Zoology, Miami University, Oxford, OH 45056.

Editing of mRNAs in the mitochondria of "higher" plants (Angio-sperms and Gymnosperms) is a novel form of RNA processing by C-to-U transitions. The mechanism conserves amino acid residues thatperform critical functions, and has not yet been observed in either"lower" vascular or non-vascular plants. To begin determination ofthe phylogenetic origin of this phenomenon, total genomic DNA wasisolated from every major branch of the vascular plant tree. PCRamplification gave a 600 to 750 nucleotide long segment of the mito-chondrial genes for cytochrome c oxidase subunits I and II (=d andraW). The PCR products were cloned into the pCRII vector and then

sequenced. Alignment with homologous gene sequences of diverseplant species having known RNA editing sites was used to infer nineedits in Ginks9 (Gymnosperm) and three in Nephroleis (Pteropsid,fern) rg gene fragments. Although no edits were found in the

corresponding md fragments from Pailotum (Psilopsid) or Ly:

nmi=m (Lycopsid), eight sites of C-to-U transitions were observed inthe C gene fragment from Psilotnm. In every case, the predicted

edit would confer the selective advantage of conserving the aminoacid residue at a site where divergence has not yet been observed inother plant species nor for many non-plant species. These resultsnow extend to a primitive vascular plant (1falJQm) the previousobservations that there are preferred editing sites. The plant mito-chondrial editing machinery is shown for the first time to extend to

one of the deepest branches of vascular plant phylogeny, an obser-vation which raises the question of its origin.

611RAT LIVER MITOCHONDRIAL PROCESSING PEPTIDASE (MPP): a- AND,6- SUBUNITS ARE REQUIRED FOR ACTIVITY. ((P. Rysavy, V. Saavedra-Alanis, and F. Kalousek)) Department of Genetics, Yale UniversitySchool of Medicine, New Haven, CT 06510

The mitochondrial processing peptidase, alone or together with themitochondrial intermediate peptidase (MIP), is responsible for maturationof most precursor proteins after their translocation across mitochondrialmembranes. The enzyme, isolated and purified from rat livermitochondria, consists of two non-identical subunits like those from N.crassa and S. cerevisiae. In contrast to the Neurosoora enzyme, whosesubunits can reversibly dissociate from each other, rat MPP forms astable heterodimer of a- (55kDa) and B- (52kDa) subunit. To determinethe role of the subunits in the catalytic activity, we expressed themature sequence of each of subunit individually in E.coli, first as fusionproteins (with the maltose binding protein) and more recently as freesubunits. In all cases, we generated mainly insoluble and inactivesubunits. We therefore used a reconstitution protocol, which consistedof denaturation in urea, followed by renaturation, dilution, and dialysisunder reducing conditions, to attempt to recover enzymatic activity.Individual subunits subjected to this procedure did not produce anyactivity. Active MPP was recovered only when both a- and ,8-MPPsubunits were present. Reconstituted recombinant MPP resembled thenative rat liver enzyme as judged by its molecular weight, its inhibitionby EDTA, and its ability to process a variety of mitochondrialprescursors appropriately to either an intermediate or mature form.Thus, both subunits of rat MPP are required to form active, nativeenzyme. Supported by NIH grant DK 09527.

613EXPRESSION OF NUCLEAR GENES IN ISOLATED HUMAN MITOCHONDRIA.((Marie A. D'Agostino)) Genetic Ventures, Inc., Basic ResearchProgram, ' conshohocken, PA 19428.

Although the human mitochondrial genome codes for 13 polypep-tides of the oxidative phosphorylation (oxphos) chain, the pre-ponderance of oxphos subunits are coded for by nuclear genes

and certain fatal encephalomyopathies with mitochondrial (mt)dysfunction may be the result of nuclear DNA (nDNA) mutations.

In this study, restriction fragments containing normal nuc-

lear oxphos subunit coding sequences were cloned in E. coli,PCR-amplified, and purified by elution from agarose gels. nDNA-mtDNA recombinants were then constructed and transfected intorespective populations of human mitochondria, Each populationwas then examined in vitro for the synthesis of RNA transcripts(by hybridgation analysis) and mitochondrial translation pro-

ducts (by S-labelling). In each case, successfully trans-fected mitochondria demonstrated the presence of both an mRNAtranscript and polypeptide translation product appropriate tothe inserted nDNA sequence. This first (to our knowledge) de-monstration of the insertion and expression of nuclear genes inisolated human mitochondria may provide an experimental stra-tegy both for the understanding of mitochondrial genetic func-tions as well as a possible genetic therapy model for mitochon-drial diseases caused by nuclear gene mutations. Supported byGenetic Ventures, Inc.

615

INHIBITION OF MITOCHONDRIAL DNA POLYMERASE-y BYPHOSPHORYLATED ZIDOVUDINE (AZT): INSIGHTS INTO AZTSIDE EFFECTS. ((W. Lewis, J. F. Simpson and R. R. Meyer))Department of Pathology, University of Cincinnati College ofMedicine, Cincinnati OH 45267-0529.

Zidovudine (azidothymidine, AZT) inhibits human immuno-deficiency virus replication and reduces AIDS severity. Limitingside effects of AZT include mitochondrial skeletal and cardiacmyopathies. Evidence suggests that pharmacologically-activeAZT triphosphate (AZTTP) plays a role in myopathy pathogenesis.This study identified AZTTP as an inhibitor of mitochondrial DNApolymerase-y in vitro. At comparable pharmacologicconcentrations, unphosphorylated and monophosphorylated AZTderivatives (AZT and AZTMP) showed little inhibition of DNApolymerase-y. AZTTP's inhibition kinetics were defined usingpurified bovine cardiac mitochondrial DNA polymerase-y andAZTTP in vitro. The K for dTTP was 0.8 ± 0.3 gM. AZTTPincubation with DNA poIymerase-y in viftro yielded mixed inhibitionkinetics. A competitive K- of 1.8 ± 0.2 FM and a non-competitive Ki' of 6.8 ± 13t M were found. Both Ki and Ki'were strikingly higher than that for reverse transcriptase but lowerthan that for cellular DNA polymerases. At 200 pM AZTMP, 15%inhibition of DNA polymerase-y occurred. Similarly, at 1000 aMAZT, DNA polymerase-y was inhibited approximately 15%.Biochemical findings suggest that inhibition of mitochondrial DNApolymerase-y by pharmacologic concentrations of AZTTP may beintegral to the pathogenesis of AZT-induced myopathy.

105a

Peroxysomes (616-621). Sunday

616CHARACTERIZATION OF A HUMAN PEROXISOME PROLIFERATOR-ACTIVATED RECEPITOR. ((P.J. Andre and G.M. Small)) Department ofCell Biology/Anatomy, Mount Sinai Medical Center, New York, NY 10029.

A wide range of structurally diverse compounds, including hypolipidemicdrugs, herbicides and industrial plasticizers cause peroxisome proliferation,induction of peroxisomal enzymes and eventual formation of hepatocellulartumors in rodents. The recent identification of a mouse nuclear receptor,termed theperoxisomeproliferator-activated receptor(mPPAR), suggeststhatthese compounds stimulate the transcription of genes encoding peroxisomalproteins by interacting with this member of the steroid hormone receptorsuperfamily. We have cloned a human homolog of this receptor (hPPAR)from a HepG2 cDNA library. The DNA bears 85% identity with the rathomolog (rPPAR) and 70.5% identity with NUCI, a member of the PPARreceptor group isolated from a human osteosarcoma cell library. The

deduced amino acid sequence of hPPAR is 92%and 62% identical to that forrPPAR and NUCI respectively. Northern analysis with an hPPAR proberevealed that the mRNA is expressed as a large transcript, >er than 9 kb.We found the highest level of hPPAR expression in human skeletal muscle,and lower levels in liver, kidney, heart, pancreas and mammary gland. Thisresult suggests that the expression of hPPAR differs from mPPAR in both thesize of the transcript and that it is most abundant in muscle with lowexpression in liver. These differences may help account for species specificresponses to peroxisome proliferators.

618PEROXISOMAL MALATE DEHYDROGENASE IS CRMTICALFOR FAITY ACID B-OXIDATION IN YEAST PEROXISOMES.((N. Singhi, CV. Roermund, Y. Elgersmat, H.F. Tabak-c and RJAWanders*)) Case Westem Reserve University School of Medicine,Cleveland, OH 44106. Departments of *Pediatrics and@Biochemistry, University Hospital Amsterdam, The Netherlands.

In the yeastS caeeviae, three distinct isozymes of malatedehydrogenase are localized in mitochondria, cytosol andperoxisomes. While the mitochondrial and cytosolic isozymesfunction in exchange of reducing equivalents across the mitochondrialmembrane, the function of peroxisomal isozyme is not clear. If itfunctions in re-oxidation of intra-peroxisomal NADH, its absenceshould affect peroxisomal fatty acid B-oidation in a significantmanner. To test this hypothesis, we disrupted the yeast peroxisomalmalate dehydrogenase gene (mdh3 ) by one step gene replacement.The resulting mutants grew on glucose, glycerol and acetate as solecarbon sources, but not on oleate. WhenB-oxidation of 14C octanoateand palmitate was measured in living cells, the production of labeledCO2 and acetyl CoA was -4-5 fold lower in the mutants, despitewild-type levels of two B-oxidation enzymes in homogenates: acyl CoAoxidase and trifunctional protein. These results imply that MDH3plays a critical role in peroxisomal B-oxidation, probably in theputative malate/aspartate shuttle. Furthermore, these results implythat the yeast peroxisomal membrane is not permeable to NADH.

620

PER3, A GENEREQUIRED FORPEROXISOME BIOGENESIS IN THEMETHYLOTROPIIC YEAST PICHIA PASTORIS. ((H. Liu, M. Venhuis,*X. Tan, and J. M. Cregg)) Department of Chemistry, Biochemistry,and Molecular Biology, Oregon Graduate Institute of Science &Technology, Portland, OR 97291, Laboratory for ElectronMicroscopy, University of Groningen, Haren, The Netherlands.(Spon. by R. Rachubinski)

We have initiated molecular genetic studies on the peroxisomeusing Pichia pastoris as a model system. This yeast was selectedbecause peroxisomes are required for its growth on eithermethanol or oleic acid--easily observed phenotypes--and becausemethods for its classical- and molecular-genetic manipulation are

well developed. By functional complementation of one of our

peroxisome-deficient mutants (per3), we have cloned a DNAfhagment containing the P. pastoris PER3 gene. DMA sequencingof the complementing region revealed a single open readingframe of 1,827 base pairs (bp) capable of encoding a protein of 609amino acids (-67 kDa). Methanol-induced cells in which 1,326 bpof the coding region were deleted contained no peroxisomes upon

electron microscopic examination. PER3 produces a conadtudvebut methanol-inducible transcript of approximately 2.0 kb. Thepredicted amino acid sequence showed no significant similarity to

other reported proteins nor to consensus sequence motifs thatwere examined. The three carboxy-terminal amino acids of Per3p

are Ala-Lys-Leu, a common targeting signal for peroxisomalmatrix enzymes; therefore, Per3p may be a novel constituent ofthe peroxisomal matrix.

617

CRARACTzRIzATION OF 'LIOGT' PDIOzISOmS IN FIDROBLASTS FROMPATIEN'F WITH SELLWEMM SYNDROU. ((M. Forstner, D. RLeglanegg,I. Sal-hofer and O. Ioefler)) University of Graz, Institute ofPathology, Graz, A-8036, Austria

Zellwger syndrome (ZS) is the most severe peroxisomaldisorder, characterLied by a complete loss of peroisomalfunctions supposedly due to the absence of orphologicallydetectable peroxisomes (POs). Upon dnasity gradientcentrifugation of fibroblast homogenates from a patient with ZSwe found catalase not in the cytosol but associated withparticles of intermediate density which are also present in

control fibroblasts together with the usual 'heavy, Pos.Ieunofluorescence with an antibody against catalase rzeeals a

punctate pattern, indicating a particle bound nature of theenzym, in dependence of the fixation method used. The use ofmthanol/acetone at -20'C leaves almost all of the particlesintact whereas fixation with formaldehyde/Triton X-100 leads tothe loss of these structures. Imeuno electron microscopy alsoreveals the loss of structures when using detergens in thefixative. 3lectron microscopy of catalase positive subcellularfractions shows catalase associated with structures thatresemble in shape and size the isolated POn from control

fibroblasts. Western blot analysis of subcellular fractionsshows that catalase, the 70 kD mmbrane protein and the 44 kD

thiolase precursor protein co-sedimet at the same densLty,whereas the peroxisomal bifunctional enzym or ia vitroexpressed firefly lucLferase cannot be detected in thesefractions. This gLves rise to the assumption that proteias thatutilize the SKL topogenic signal for sorting to POs cannot beimported into the preexlsting peroxisomal structures in skinfibroblasts from this ZS patient. In accordance to thedescription of light, precursor POs ia donsLty gradLents fromrat liver, we assume that these particles are precursor POs that

cannot be processed in ZS fibroblasts to become functLonal POs.

619I3ONOCYTOCEEMICAL LOCALIZATION OF URATE OXIDASF INFROG LIVER AND KIDNEY. ((N. Usuda, S. Hayashi, S.Fujiwara, T. Noguchi, J.K. Reddy, K. Alvares, M.S. Rao,and A.V. Yeldandi )) Department of Pathology,Northwestern University Medical School, Chicago, IL60611, and Department of Biochemistry, Kyushu DentalCollege, Kokura, Kitakyushu 803, Japan.

Earlier differential and density gradientcentrifugation studies have established the presence ofurate degrading enzymes, urate oxidase, allantoinaseand allantoicase in hepatic peroxisomes of amphibia andfish. In order to confirm this subcellular distributionof urate degrading enzymes directly it is necessary tovisualize their presence using immunocytochemicalapproaches. This report deals with the localization ofurate oxidase in frog liver and kidney. Urate oxidasewas detected in both liver and kidney of frogs andtadpoles by immunoblot analysis. Subcellularlocalization of urate oxidase was ascertained byprotein A-gold immunocytochemical staining of LowicrylK4M-embedded tissue. Peroxisomes in frog liverparenchymal cells and kidney proximal tubularepithelium contained a dense sub-crystalloid core whichis found to be the exclusive site of urate oxidaselocalization. These results demonstrate that, unlikerat kidney peroxisomes which lack urate oxidase,peroxisomes of frog kidney contain this enzyme.

621

ISOLATION AND CHARACTERIZATION OF PER6, A GENEREQUIRED FORPEROXISOME BIOGENESIS IN THE YEAST PICHIA PASTORIS.((J. M. Cregg, W. Xic, M. Veenhuis,* and X. Tan)) Department ofChemistry, Biochemistry, and Molecular Biology, Oregon GraduateInstitute of Science & Technology, Portland, OR 97291, *Laboratoryfor Electron Microscopy, University of Groningen, Haren, TheNetherlands.

A DNA fragment containing the PER6 gene of the methylotrophicyeast Pichia pastoris was cloned by functional complementation ofa per6 mutant of the yeast, one of a number of peroxisome-deficient mutants isolated in this laboratory. The DNA sequencerevealed a 1,383 base pair open reading frame which potentiallyencodes a 461 amino acid protein of -51 kDa. The gene istranscribed into a message of 1.4 kilobases that is constitutivelyexpressed but induced several fold in cells growing on methanol.A mutant strain was constructed in which a large portion of theopen reading frame was deleted and was used to geneticallydemonstrate that the cloned gene was the same as that which wasdefective in the originally isolated per6 mutant. Upon electronmicroscopic observation, methanol-induced celis of the per6-deletion strain had no peroxisomes. The predicted amino acidsequence of the PER6 product revealed several features of whichthe most interesting was a significant regional similarity to theproduct of PAF-I, a gene known to be defective in some patientswith Zellweger syndrome (a lethal human genctic disease causedby peroxisome deficiency).

106a

Sunday. Peroxysomes (622-626)

622ISOLATION AND ANALYSIS OF PER8, A GENE REQUIRED FORPEROXISOME BIOGENESIS IN THE YEAST HANSENULA POLYMORPHA.((X. Tan, M. Veenhuis,* and J. M. Cregg)) Department of Chemistry,Biochemistry, and Molecular Biology, Oregon Graduate Institute ofScience & Technology, Portland, OR 97291, *Laboratory forElectron Microscopy, University of Groningen, Haren, TheNetherlands. (Spon. by C. Enns)

The methylotroph Hansenula polymorpha is an attractive modelorganism for studies on peroxisome biogenesis becauseperoxisomes are required for growth on methanol and becausemethods for genetic manipulation are well developed. Peroxisome-deficient (per) mutants have been isolated and are being utilized toclone the affected genes from a genomic DNA library byfunctional complementation. We have isolated an H. polymorphaDNA fragment that complements a per8 strain. The DNA sequenceof the complementing region revealed a single long open readingframe of 885 base pairs capable of encoding a protein of 295 aminoacids (-33 kDa). Northern blots revealed a message that wasconstitutively expressed but induced several fold upon shifting ofH. polymorpha cells to methanol medium. The predicted aminoacid sequence of PER8 (Per8p) harbors a region of approximately38 amino acids in which 8 cysteines are grouped in two zinc-finger-like motifs. Per8p showed significant similarity to anumber of other putative zinc-finger proteins (e.g., RADI 8), butonly in this specific region. To identify the subcellular location ofPer8p, antibodies have been raised to Per8p expressed as a fusionprotein in E. coli. These antibodies are being affinity purified foruse in biochemical and immunocytological experiments.

624HETEROLOGOUS SORTING TO THE PEROXISOME IN S. CEREVISIAE:THE PTS1 TRIPEPTIDE IS SUFFICIENT FOR PEROXISOMAL TARGETING.((J.A. McNew and J.M. Goodman)) Department of Pharmacology, TheUniversity of Texas Southwestem Medical Center, Dallas, TX 75235.

The peroxisomal targeting sequence (PTS1) has been shown to function in avariety of species induding yeast. However, recent evidence has indicated thatthe PTS1 motif is necessary but may not be sufficient in S. cerevisiae. Toanswer this question directly we expressed a chimeric protein ending in thePTS tripeptide AKL. This construct consisted of the bacterial enzymechloramphenicol acetyltransferase (CAT) appended with the carboxy-terminaltwelve amino acids of PMP20, a peripheral peroxisomal membrane proteinfrom the methylotrophic yeast Candida boidinii. CAT-PMP20 has been shownpreviously to sort to the peroxisome in animal cells (EMBO J. 9:85 1990). Wefound that this molecule sorted CAT to the peroxisome in S. cerevisiae, suchthat approximately 50% of expressed protein was in the organelle asdetermined by western blotting of cell fractions. Further chimera wereconstructed to determine the effects of sequences immediately upstream of theAKL. When the tripeptide was placed directly behind CAT, the sortingefficiency was 50%, as before. However, placing a spacer of nine glycineresidues before AKL increased sorting efficiency to about 90%. We concludethat the PTS1 tripeptide aione is sufficient (in the context of CAT) for efficientperoxisomal targeting in S. cerevsiae.

623EXPRESSION OF TRUNCATED FORMS OF THE RAT URATE OXIDASEIN INSECT CELLS USING BACULOVIRUS. ((R. Chu, N. Usuda,K. Alvares, J. K. Reddy, M.S. Rao, and A.V. Yeldandi))Department of Pathology, Northwestern UniversityMedical School, Chicago, IL 60611.

Urate oxidase forms a crystalloid core in peroxisomesof rat and most other mammals that catabolize uric acidto allantoin. When a full-length rat urate oxidase cDNAwas expressed insect cells it formed large peroxisomalcrystalloid core-like structures. The present studydeals with the expression of four truncated urateoxidase cDNAs using the baculovirus insect cell systemto elucidate the structural determinants. Constructsencoding 244 aa in the C-terminal region, or 223 aa inthe N-terminal region produced an insoluble proteinwithout a clear crystalloid substructure. The C-terminus 244 aa fragment, which contains theperoxisomal targeting signal, was transported intocytoplasmic vacuoles, whereas the N-terminus 223 aapeptide without the targeting signal appeared in thecytoplasm as insoluble non-crystalloid aggregates. Theproducts of two smaller truncated cDNAs coding for N-terminal 80 aa or C-terminal 59 aa, however were notdetected in the cell as insoluble aggregates. None ofthe truncated urate oxidase proteins showed enzymeactivity. These studies show the truncated largerurate oxidase peptides though remain as insoluble andeasily purifiable aggregates, they loose the enzymeactivity, as well as, the crystalloid configuration.

625CHARACTERIZATION OF PEROXISOME BIOGENESIS MUTANTS:IMPLICATION OF THREE PEROXISOME PROTEIN PACKAGING PATHWAYS.((J. W. Zhang, C. Luckey, Y. Han and P. B. Lazarow)) Departent of Cal Biolgyand Anatomy, Box 1007, Mount Sinai School of Medicine, Fifth Avenue & 100thStreet, New York, NY 10029.

We have devised a poative selection procedure to identify peroxisomebkogenea (peb) mutants In Sachromyces c.revlsiae. With this method, wehave isolated five complementation groups of peb mutants. One mutant fromeach oomplementation group was further characterized. A gentle col fraction-ation method using digitonin was developed for the mutant analysis. Thepresence of peroxiaomes in mutant cells was analyzed by elctron morosopy.The subeelkilar bocalation of threp proteins, catalase, thiolaae andacyl-CoA oxldase, was determined by imrnunoflluorescenc, eWctron micoecopiccytochemistry, immuno.electron microacopy and the new cell fractionationmethod. One mutant, peb3-1, appeara to express reduced amounts ofperoxisomal proteins. Two d the mutants, peb2-1 and peb4-1, lack recognizableperoxisomes, although they might contain peroxisome membrane ghosts likethose found In Zellweger syndrome. In these two mutants, all three peroxaomalproteins arefund in the cytoac. Two additional mutants are partially defectIve inpackaging peroxisome proteins and show striking intracellular clustering ofnormal-looking peroxisomes. One of them, pebl-1, is defective in packagingthiolase. The other one, peb6s1, is defective In cataiase packaging. Both of themare normal in acyl-CoA oxidae packaging. These data suggest that threeperoxisome protein packaging pathways, or three branches of a pathway, arepresent in yeast celI. These branches are represented by catalae, thioiase andacyl-CoA oxsdae.

626

FURTHER EVIDENCE FOR INVOLVEMENT OF A PROTON-MOTIVEFORCE IN IMPORT OF PROTEINS INTO PEROXISOMES. ((M. Sohaskey,K. Campbell and J.M. Goodman)) Department of Pharmacology, TheUniversity of Texas Southwestem Medical Center, Dallas, TX 75235

Alcohol oxidase is an abundant peroxisomai protein in the methylotrophicyeast Candida boidinii. It is synthesized in the cytoplasm and is transportedto peroxisomes where it assembles into octamers, the active form of theenzyme. Several years ago we reported that proton ionophores such asCCCP prevented import and octamerization (Cell 48:165, 1987), suggestingthat a peroxisomal proton-motive force is esseritial for protein import intoperoxisomes. However, it is now known from studies in vitro that ATP is arequirement for import. To determine whether CCCP is inhibitingperoxisomal assembly only indirectly by acting on mitochondria and therebylowering ATP concentrations, the simultaneous effects of CCCP on adeninenucleotide concentrations and alcohol oxidase assembly was determined. Asimple and rapid method for extraction of nucleotides from yeast wasdeveloped for this purpose. Addition of 25 ILM CCCP to intact cells led toalmost complete inhibiton of assembly of alcohol oxidase yet lowered ATPlevels only 50%. To determine whether a 50% decrease in ATP wassufficient to inhibit assembly, cells were treated with antimycin A, a drugwhich is highly specific for its inhibition of mitochondrial electron transport.Antimycin at 5 pg/mI depressed ATP concentrations more than 80% yet hadonly a slight effect on assembly of alcohol oxidase. We conclude thatlowering ATP concentrations cannot account for the disruptive effect ofproton ionophores on peroxisomal import. The simplest iriterpretation of ourdata is that a peroxisomal proton-motive force is responsible for import ofproteins irito peroxisomes in vivo, at least in yeast.

107a

Endocrine and Exocrine Glands (627-632). Sunday

627PANCREASTATIN STIMULATES SECRETION OF A CHONDROITINSULFATE PROTEOGLYCAN FROM NEONATAL RAT ATRIALCARDIOMYOCYTES. ((S.S. Pence and S.-U. Gorr)), Dept. Biological andBiophysical Sciences, University of Louisville Health Sciences Center,Louisville, KY 40292. (Spon. by M.M. Jumblatt)

Sulfated proteoglycans (PG) are secreted by most cell types and act incell adhesion and protein binding. In endocrine cells, PG can be secretedby either a regulated or constitutive secretory pathway. In order todetermine if sulfated proteoglycans are secreted by the regulated pathwayin atrial cardiomyocytes, neonatal rat cardiomyocytes were prepared from1-3 day old rats. Cells were labeled with Na2"5SO4 and then chased in thepresence or absence of 100 nM endothelin or pancreastatin. Aliquots ofthe labeling and chase media were analyzed by SDS-PAGE. The cellssecreted a high molecular weight sulfated molecule that was digested bychondroitinase ABC but not heparinase. Continuous labeling experimentsindicated that the PG required more than 2 h for release, consistent withnon-constitutive release. Endothelin-1, a known stimulator of atrialnatriuretic factor secretion, stimulated PG secretion about 100%. Theseresults indicate that rat atrial cardiomyocytes express a chondroitin sulfateproteoglycan that is secreted via a regulated secretory pathway. Atrialsecretory granules contain chromogranin A that plays a role in regulationof hormone secretion in a variety of endocrine cells. The chromogranin-derived peptide pancreastatin (100 nM) stimulated secretion of thesulfated proteoglycan by about 50%. These data suggest thatchromogranin A can play a role in regulation of atrial secretion. Supportedby a Grant-in-Aid from the American Heart Association, Kentucky Affiliateand NIH grant T35 DE071 71.

629THE INHIBMON OF ATRIAL NATRIURETIC FACTOR RELEASE BYMELANOCYTE STIMULATING HORMONE.IA. Bower and R. Mino)) Mount St. Mary's College, Los Angeles, CA90049

This study was designed to investigate possible effects between atrialnatriuretic factor (ANF) and melanocyte stimulating hormone (a-MSH). MSHis produced and secreted by the intermediate pituitary. It has beensuggested to function as a physiological natriuretic agent in man, but howit brings this action about is not well understood. ANF was originally identi-fied in cardiomyocytes and shown to have natriuretic, diuretic, andvasorelaxant properties. It was of Interest, therefore, to examine whetherthe natriuretic action of MSH is produced through the atrial natriuretichormone of the heart. Sprague-Dawley rats were used. Animals werelightly anesthetized using carbon dioxide gas. A thoracic incision was madeand the right atrial lobe excised. The tissue was rinsed, cleansed of fat,gently minced, and divided into equal amounts for control and experimentalincubations. Cells were incubated for a total of 120 minutes in Tyrode'ssalt solution with or without a-MSH 1 0.-1 04M. Endothelin (10.-104M)was used to establish stimulation of ANF release. Fractions were collectedevery 30 minutes for analysis by reversed phase high performance liquidchromatography. Results suggest an inhibition of ANF release by a-MSHand a stimulation of ANF release byendothelin that is blocked by a-MSH.Supported by NIH-NIGMS- IGMO 8232)

631

IMMUNOCYTOCHEMICAL LOCALIZATION OF TRANSFORMING GROWTHFACTOR ALPHA DURING TESTICULAR DEVELOPMENT OF BOARS. ((J.C.Gillespie, L.A. Jaeger, L Johnson, and T.H. Welsh)) Depts. of AnimalScience and Vet. Anat., Texas A&M Univ., College Station, TX 77843.

How various growth factors coordinate development of testicular cellsessential for gametogenesis and sterokdogenesis Is unclear. Temporalaspects of the proliferation of Leydig (LC) and Sertoil (SC) cells and thepresence of transforming growth factor alpha (TGFc) were determind bythe immunocytochemical and the morphometric analyses of the porcinetestes (from boars of 6 litters; littermates assigned at birth to 1 of5 age

groups; Age>1 70d-pubertal; Age<l 70d-prepubert). Weight of the testesand epkidymide as wel as Leydig ce number Increased with agp. The %of TGFa immuno-positive LC was consistently high through developmentThe % TGFa immuno-positive SC increased with age. The intensity ofstaining for TGFa in the LC declined whereas it increased In the SCthroughout puberty. Temporal changes In appearance anddistribution oftesticular TGFa may influence develpment of the male reproductivesystem

Age Testes Epidkymkdes #LC/testis % LC % SC(days) Wt. (g) Wt. (g) (109) staining staining

14 5.2 .9 1.6± .1 .3 .04 99 < 528 6.6 .8 2.1 .2 .3± .01 98 4056 14.7± 1.5 6.1 1.1 1.7± -.1 90 65

112 115.0±11.4 34.6±2.1 15.6±1.8 95 85170 360.2±t23.1 68.8 4.9 20.2± 1.6 95 90(Supported by 1993 NPPC grant and 1992 HLSR grnt )

628EFFECTS OF ENDOTHELIN 1,2 & 3 ON THE SECRETION OFATRIAL NATRIURETIC PEPTIDE FROM SINGLE CELLS OF THERAT RIGHT AND LEFT ATRIA ((T.L.Rice and H.A. Miller 1II))Department of Biological Sciences, East Tennessee State University,Johnson City, TN 37601.

Atrial Natriuretic Peptide (ANP) is released from the heart andregulates blood pressure and volume. Several hormones havebeen shown to influence ANP release, among these endothelins.Endothelin is released from endothelial cells in response to stretchand has been implicated as the mediator in the volume-inducedrelease of ANP. We have developed a reverse hemolytic plaqueassay that enables us to monitor the release of ANP from singleatrial cardiocytes. Using this assay we determined the effect ofendothelin 1,2, and 3 on the proportion of cardiocytes that releaseANP from each rat atrial chamber. Individual cardiocytes weredissociated and incubated with varying concentrations of endothelinfor 18 hrs and then the proportions of secreting cells weredetermined by a 2 hr plaque assay. Exposing right atrial cells toendothelin 1(1V M ) resulted in a 131% increase in the proportionof ANP secreting cardiocytes. Endothelin 1 did not significantlyincrease the ANP secreting population in the left atrium. However,when endothelin 2 & 3 were tested, the proportion of ANP secretingcells from both chambers was increased by more than 75%. Theseresults imply a discordance between the ANP secreting cells of theright and left atria in their response to endothelin 1 but not 2 or 3.

630

PARATHYROID CELLS CONTAIN A MEMBRANE-ASSOCIATED FORM OFDARPP-32. ((L.M. Matovcik, J.F. Schaefer, F.S. Gorelick, P. Greengard and B.K.Kinder)) Depts. of Surgery, Medicine, Cell Biology and Cellular and MolecularNeuroscience, Veterans Affairs Medical Center, West Haven, CT 06516, Yale Univ., NewHaven, CT 06510, and The Rockefeller Univ., New York, NY 10021.

Primary cultures of bovine parathyroid cells secrete PTH in response to low extracellularCa+2 or high intracellular cAMP levels; conversely, secretion is inhibited by raising the

[Ca+2]. Intracellular [Ca+2] directly reflects that outside the parathyroid cell. Themechanism by which Ca+2 and cAMP act with opposing but coordinated effects on PTHsecretion has not been determined. In the rat caudate putamen, stimulatory dopaminergicinput increases cAMP and activates cAMP-dependent protein kinase, whichphosphorylates a soluble, cytoplasmic protein, DARPP-32 (dopamine and cAMPregulatedghosphoprotein of 32 kDa). Inhibitory glutamatergic input increases Ca42 andactivates protein phosphatase-2B, which dephosphorylates DARPP-32. DARPP-32 is aprotein phosphatase-1 inhibitor, which has been characterized in the central nervoussystem, where it appears to play a role in coordinating the intracellular effects of calciumand cAMP. We have described a novel form of DARPP-32 in the parathyroid gland,which, in contrast to the soluble brain form, is tightly associated with intracellularmembranes. DARPP-32 is observed in small vesicles distributed throughout thecytoplasm of the parathyroid chief cell when examined by confocal immunofluorescencemicroscopy. Immunoblot analysis of bovine parathyroid homogenate with a monoclonalantibody to DARPP-32 demonstrates a single immunorcactive protein with an apparent Mrof 32 kDa. The majority of parathyroid DARPP-32 is present in a particulate fractiondefined by differential cell fractionation. It remains particulate aftertreaunent with 1.0 MNaCI or 0.1 M Na2CO3. Protease digestion studies indicate that DARPP-32 is present onthe cytoplasmic face of the membrane. When isolated bovine parathyroid cells labeledwith RS-[5-3H(N)lmevalonolactone are subjected to immunoprecipitation with polyclonalanti-DARPP-32, a predominant 32 kDa protein is observed, indicating that parathyroidDARPP-32 is isoprenylated. This modification of DARPP-32 may be responsible in partfor the association of DARPP-32 with intracellular membranes.

632THAPSIGARGIN (THAP) INCREASES CYTOSOLIC CALCIUM([Ca'i-)ANDINHIBITS SECRETION OF PARATHYROID HORMONE (Pl H) FROMBOVINE PARATHYROID CELLS. ((T.R. Femeandi and R.R. MacGregor))Department of Anatomy and Cell Biology, University of Kansas Medical Center,Kansas City, KS 66160-7400.

The secretion of PTH isnegatively controUed by extracellular [Cal ((Ca.d).Both[Ca*J6 and protein kinase C have been prposed to be cellular trnusducersof [Ca!l,. To examine the specificrole of [Can,, in bovine parathyroid cells,we utilized THAP, a sesquiterpene lactone thatraises [Cai., THAPinhlibtsthe Ca"-ATPawe of the endoplasmic reticulum, but does not alter levels ofinositol phosphates oractivity of protein kinase C. The effects of THAP on[Ca]',, were examined using the fluorescent Ca! indicator Indo-l;PIrHsecretion was monitored by radioimmunoassay of media At 2.5 mM [CwL.,1 pM THAP raised[Cai,, but had no effect on FF11 secretion. At 0.5 to0.7 mM[Cai,,, 1 pM THAP increased [Ca'i,1 to the level observed when[Cai,]. is 1.2 to 1.5 mM, and inhibited FMH secretion to a degreecommensurate with those[Ca!]n. Significantinhibition ofPFFH secretionoccurred by 15 minutes of treatment, and continued for 18 hours. Inhibidon ofsecretion by either 2.5 mM [Ca"], or 0.7 mM [car; + I pM THAP wasparially overcome by treatment of cells with 3-methyladenine, an inhibitor ofautophagosome formation in hepatocytes. SDS-PAGE analysis of parathyroidproteins after incubations with [5HJleucine showed that both 2.5 mM [Ca"],.and 0.7 mM [Ca'lI, + 1 pM THAP decrasedincorporation of [3H]leucine intocellular parathyroid proteins by 10-20%. These data lead us to conclude that

[Cani,, is a major component of the 2nd messenger system for the inhibition of

PFFH secretion by [Ca],a.

108a

Sunday. Endocrine and Exocrine Glands (633-638)

633AUTOPROTEOLYTIC ACTIVATION OF THE PROHORMONE CONVERTASE,mPC1. ((L. J. Goodman and C. M. Gorman)) Department of Cell Genetics,Genentech, Inc., 460 Pt. San Bruno Blvd., South San Francisco, CA 94080.

In this study the activation of the mouse prohormone convertase, mPC1 was

characterized. Transient expression of native mPC1 in the human embryonickidney cell line, HE1 293, results in the production of an amino-terminallyprocessed, 81 kDa protein which undergoes further carboxyl-terminal truncation toyield a 66 kDa protein. We investigated the possibility that these cleavages

occured by an autocatalytic mechanism Expression and N-terminal sequencing ofmutant proteins altered in their putative catalytic domain (Ser382 to Ala or

His208 to Ala) demositrated that the amino-terminal pro-region of PCI isprocessed by an autocatalytic mechanism. In contrast, an unidentified protease isinvolved in the production of the 66 kDa protein. Furthermore, these resultssuggest that autocatalysis may be the result of an intramolecular reaction, sinceprocessing of the active-site mutant to the 81 kDa form could not be complementedby the overexpression of active furin or PC1. Pulse-chase kinetics and Brefeldin A(BFA) experiments verified that proteolysis occured in the Endoplasmic Reticulumprior to reaching the Golgi apparatus. To determine if cleavage of the pro-regionis required for activation of PC1, a mutant was expressed containing an alteredamino-terminal deavage site (ArgllO to Ala), which resulted in the production ofa90 kDa uncleaved form. Coexpression of wild-type PC1 or the deavage-sitemutant with the substrate relaxin, demonstrated that the pro-region is requiredfor the processing of prorelaxin to mature relaxin, suggesting that autoproteolysisis required for PC1 activity.

635

ALTERATIONS IN COLLAGEN GENE EXPRESSION ANDDEPOSITION IN VENTRICULAR MYOCARDIUM OFTHYROIDECTOMIZED RATS. ((LE. Klein, M. Eghbali)) YaleUniversity School of Medicine, New Haven, CT 06510

Previouslywe demonstrated that cardiac fibroblasts, collagen producingcells in the heart, are targets for the regulatory effects of L-thyroxin. Toestablish the regulatory role of thyroid hormones on myocardial collagenmatrix, we examined collagen gene expression and deposition in thehearts of Sprague Dawley rats after surgical thyroidectomy. Depletionof thyroid hormone was monitored by radioimmunoassay andfluorescence analysis ofT3 and T4, respectively. Northern analysis ofventricular RNA from thyroidectomized rats showed a 160% (P<0.01)increase for Proa I (I) collagen mRNA compared to that in normal age-

matched rat heart At 12 weeks following thyroidectomy, collagenmatrix of the heart, as examined byimmunofluoresent lightmicroscopyof frozen heart sections, showed alteratons of type I collagen fibers inventricular myocardium of thyroidectomized rats compared to age-

matched normal or sham operated rat heart. These findings indicatethat collagen matrix of the heart is subject to regulation by thyroidhormones and that their normal production may be necessary formaintaining the integrity of collagen matrix.

63T

RU 486 AS AN ANTIGLUCOCORTICOID: REVERSED DEXAMETHASONE (DEX)INHIBMON OF ADRENOCORTICOTROPIN (ACTH) SECRETION IN CULTUREDBOVINE AND PORCINE CORTICOTROPHS. ((S.T. Willard, J.A. Carroll, P.G.Harms and T.H. Welsh, Jr.)) Dept. Animal Science, Texas A&M University,Colege Station, TX 77843-2471.

The ability of RU 486 (mifepristone, courtesy of RousselUclaf) to negateDE)(s Inhibition of ACTH secretion in vitro was studied. On day 5 ofprimary culture, bovine (steers) and porcine (boars) cortkcotrophs were

challenged for 3 h and 4 h, respectively, with DMEM alone (Control), .1sM cortlcotropln-releasing hormone (CRH), 1j vasopressin (VP), 1 pM

of a VP analog l[DeamIno'(D-3-(PyrIdyl) AlS,Arg8)-VP]; VPB}, .1 and1 pM DEX,.1 and 1 pM RU 486, or various cobinations Medium contentof ACTH was determined by radionmmunoassay. In bovine cortlcotrophs,the stimulatory effects of CRH, VP and VPB were reduced (P < .0001)59%, 80% and 72%, respectively, by co-trestment with DEX In porcinecorttcotrophs, the stinuastory effect of CRH was reduced (P< .0001) 66%by co-treatment with DEX. Relative to Control, co-treatment withDEX+RU 486 did not Incresse (P> .1) ACTH secretion. However, co-

addition of RU 486 to CRH+DEX, negated (P<.OOO1) DEXsinhibitory effecton CRH-induced ACTH secretion by bovine (54%) and porcine (42%)corticotrophs. Similarly, RU 486 negated (P<.0001) by 90% DEX'sinhibition of VP- and VPB-Induced ACTH secretion. Thee data denmostrtethat relfepristone, RU 486, can act as a potent antiglucocorticoid in vitro

due to it's ability to block DEXs Inhibitory actions on both proteinkinase A(CRH) and protein kinase C (VP) mediated secretion of ACTH by

coticotrophs#[Supported by TAMU Facuity Mini-Grant 186515S and USDA CRG 85-

CRCR-1-1 823].

634

a-SPECIFIC ANTI-CLUSTERIN ANTIBODY: DEVELOPMENT ANDCHARACTERIZATION. ((M.L. Sanders', J.A. Farris', A.R. Strauch2, D.N.Foster', and B.G. Crabo')) Department of Animal Science, University ofMinnesota, St. Paul, MN 55101' and Department of Cell Biology,Neurobiology and Anatomy, Ohio State University, Columbus, OH 432102.

Clusterin is expressed at significant levels, and possibly several isoforms,in the mammalian reproductive system. However, it's role and specificfunction in the male reproductive system remain unclear. We generated ahighly conserved 15 amino acid alpha-subunit specific peptide (homologousfor human, pig, rat and mouse) for polyclonal antibody production. Theresulting pa4 was compared with existing anti-ram and anti-humanmonoclonal and anti-mouse anti-clusterin polyclonal antibodies. pa4recognizes reduced immunoprecipitated products from boar rete testis fluid,cultured porcine testicular cell lysates and porcine and transfected murinetissue culture supernatant at M, - 34kDa. Both monoclonal antibodies reveala 35kDa band in reduced samples. pa4 does not cross-react strongly witheither of the monoclonal immunoprecipitated products, nor vice-versa. Inunreduced samples, pa4 precipitates an -83 kDa band, while themonoclonals produce a larger -97 kDa band. However, the anti-mousepolyclonal antibody cross-reacts with all precipitated products under bothconditions, at the apparent above-mentioned relative molecular weights. Weconclude that our po4 antibody is specific for the alpha subunit of one formof clusterin, while the monoclonal antibodies recognize a higher molecularweight isoform of clusterin. Future studies will target the functionality androle each subunit and various isoforms play in the male porcine reproductivetract. (Supported by USDA grant #9102466.)

636

CADMIUM CHLORIDE UNIQUELY AFFECTS ADRENAL CELLCHOLESTEROL METABOLISM DURING UNSTIMULATEDSTEROIDOGENESIS. ((O. Mgbonyebi, C. Smothers, and J. Mrotek))Physiology, Meharry Medical College, Nashville, TN 37208.

Cadmium chloride (CdCl2) affects unstimulated and stimulated Y-1 mouseadrenal tumor cell steroid secretion differently. We examined unstimulatedY-1 cells incubated with or without 45 ug CdC12/ml in combination with orwithout exogenously added 20-hydroxycholesterol (200HC), 22-hydroxy-cholesterol (220HC), 25-hydroxycholesterol (250HC), pregnenolone (PEG),or progesterone (POG). These steroids increase 20-dihydroxyprogesterone(20DHP) secretion, bypass rate-limited cholesterol transport steps and maybypass sites that CdCI inhibits. Under unstimulated conditions the 25, 20,or 220HC hydroxyls Facilitate plasma membrane, cytoplasm, and outer andinner mitochondrial membrane solubility, diffusion and/or transport, bypass-ing the rate-limited step(s). CdCl significantly reduced unstimulated and250HC-induced 2ODHP secretion 2N%. Unstimulated 200HC, 220HC, PEG,or POG synthesis into 2ODHP was unaffected by CdC12. CAMP acceleratescytoplasmic cholesterol transport to and through the mitochondrialmembranes before cholesterol conversion to pregnenolone by side-chaincleavage enzymes occurs on the inner membrane matrix face. Stimulation-inhibiting CdC12-plasma membrane interactions were significantly bypassedusing dbcAMP. Thus, CdCl2 inhibited endogenous cholesterol and 250HC,but not dbcAMP-stimulated cholesterol, 20 or 220HC, PEG or POG, con-versions to 2ODHP. Unstimulated cholesterol and cAMP-regulatedmitochondrial cholesterol may be independently controlled; 250HC may be auseful tool to examine this phenomenon. (AFOSR: F49620; NIH: IK14-HLO2482 & S06GM08037; NSF: RR03032 & HRD9106096. OPM & CTS:NIGMS Pre-doctoral Fellows. OPM: 1992 Porter Physiology Fellow.)

638

SPACE FLIGHT ALTERS CATECHOLAMINE-SYNTHESIZING ENZYMEEXPRESSION AND ACTIVITY IN THE RAT ADRENAL MEDULLA((P.I. Lelkes, E. Ramos, D. chick, J. Liu and B.R. Unsworth )) Lab. Cell Biol.,Univ Wisc. Med School and Dept. Cell Biol., Marquette Univ, Milwaukee, WI.

The effects of prolonged exposure to microgravity during space flight on theneuwoendocrine system are not well understood. Using adrenals from 6 rats flown for8 days on space-shuttle mission SST54 and from 6 matched, grounded controls, wecompared the total catecholamine (CA) contents and the expression and enzymaticactivities of two of the catecholamlnesynthesizing enzymes, tyrosine hydroxylase(TH) and phenylethanolamine-N-methyltransferase (PNMT). Total catecholamineswere measured by HPLC with elctrochemical detection, TH and PNMT activitiesassayed radioenzymatically.The level of expression of the steady state mRNA levelsof TH was analyzed by comparing the intensities of the PCR products to that of ahousekeeping gene GAPDH. Changes inthe expression were assessed from alterationsin the ratio of TH/GAPDH. Immmunoreactivity was assessed by indirectimmunoperoxidase staining of paraffin sections of formaldehyde-fixed glands. In spiteof significant fluctuations between individual animals, the data obtained to dateindicate: 1.The space flight did not change PNMT activity, however it significantlydecreased TH activity (m35%). Also, we found a similar decrease in the TH mRNAlevels as well as in the total catecholamine contents. As assessed byimmunoperoxidase staining, the amount of immunoreactive PNMT was unchanged,however, paradoxically, there seemed to be more TH present after space flight thanin the controls. Since thespace-animals were recovered between 5.5 -12.5 hrs afterlanding, thusm=nimiing the possibllity of stress-induced short term effects, wehypothesize that exposure to microgravity during space flight might alter both theexpression, enzymatic activity and also the turn-over of TH.

109a

Endocrine and Exocrine Glands (639-643). Sunday

639GABAA-RECEPTORS IN NEUROENDOCRINE GASTROPANCREATIC CELLS.((G. von Blankenfeld1), H. Kettenmann1), J Turner2) G. Ahnert-Hilger3) and 8Wiedenmann3)))1) Max-DelbrOck-Center for Molecular Medicine, Robert-RSasle-Str. 10, D13122 Berlin-Buch 2) Resarch Laboratories of Schering AG, D-13342 Berlin 3) KUnikurrSteglitz, Free University of Beroin, Dept. of Gastroenterology, Hindenburgdamm 30, D-1220(Berlin, FRG (Spon. by B. Wiedenmann)

The pancreatic epithelial cell line AR42J synthesizes vesicles similar to small synaptkvesicles of neurons. In additon, AR42J cells contain considerable amounts of thineurotransmitter -taminobutyric acid (GABA) (Rosewicz et al., 1992, Eur J Cell Biol, 59, 80)Recently, AR42J cets have been shown to take up, store and specifically release GABS(Ahnert-Hilor and Widenmann, 1992, FEBS Letters, 314, 41). Assuming that endogeneoutGABA may act in neurendocrine cells as an autocrine modulator, we looked for functionsGABA-receptors on AR42J cells.Using the patch-clamp technique, GABA-activated Cl currents were recorded, whictresemble classical GABAA-receptors. Furthermore, GABA-activated currents wersmimicked by muscimol and blocked by bicuculline or enhanced by diazepam. Like irimmature neurons or glial cells, GABA triggered an increase in cytosolic Ca++. Thiunderlying mechanism is most likely a Cr-afflux, which induces a membrane dapolarizatiorand a resulting activation of voitage-gated Ca++- channels. The functional studies warssupported by immunocytochemical analyses using antibodies against GABAA-receptor a-

A-, and -tsubunits. The expression of GABAA-receptor subunits in AR42J calls was too lo%for immunocytochamical detdction. However, a pronounced immunoreaction with anthodiesagainst the al and a2-subunits, as well as the P2- and the y2-subunit was obtained irseveral gastropancreatic normal and neoplastic gastrin-producing cells as well as In p-callsInterestingly, these cells contained also considerable amounts of endoganeous GABA.The data suggest an autocrine mechanism of GABA in various neuroendocrine catls of thsgastropancreatic system.

641MORPHOLOGIC AND PHYSIOLOGIC COiRELATES OF SECRETION IN CUL-TURED LACRIMAL GLAND ACINI. ((R.L os, T.Y. Filids, and M.M. CrIpps))Departments of Anatomy and Physiology. Louana State University MedicalCenter, New Orlam, LA 70119.

The objectIe of th audas Is to develop a prkmay culture syatem for lacrimalgland acini and to detemrtinthe stuctural and physiolgIcalo IarimrtIs of culturedsecr~toy.seilatothoir Invvou Lacrimal gand acifrom adult mab NowZ whlte rabbis wer culured for 3 days In suppementd DME/F12 medlumon Matrigdl-coated call culture int Cultured acni wer reponsIve tochoilnergic stImulatIon by 1O0uM carbachol with a 4X Increm In total secreteprotein during 30 min. This increas In secreted protein was blocked by themuscarinic antagonist atrophn (10uM). TranwnIsion electron microscopicexamination of the cultured calls demonstrated that after three days In culture theacinl wero attached to the culture subtatum and retaIed many In vivoulrasuctural charctrIs includings k cdlar apical-baa polalty, apIcal juconacomplexes, distinctive lumons, and wel organized secretory organelles. Acinarcalls In unrtimulated culturs contained numorous socrotory granules, while acinarcells In carbachol-treated cultures were largely dograrlated. These studiesdemontrabt the fesblllty of culturing functIonal socretory hlrmal acini that may beused In physiologk and cel signaling stdis (Supported by NIH NEI grantEYT .8)

640

AGONIST-INDUCED ALTERATIONS IN PROTEIN KINASE C(PKC) ISOFORMS IN DISPERSED CHIEF CELLS FROM GUINEAPIG STOMACH. ((R.D.Raffaniello and J-P.Raufman))Department of Medicine, SUNY-Health Science Center,Brooklyn, NY 11203.

Previously, we have demonstrated that carbachol andPMA cause an increase in PKC activity and stimulatepepsinogen secretion from gastric chief cells(Gastroenterology 104:A848, 1993). In the presentstudy, we used antibodies to PKC isoforms to examineagonist-induced alterations in PKC in dispersedchief cells from guinea pig stomach. Immunoblottingrevealed the presence of a conventional (a) and anovel (C) PKC isoform in chief cell extracts. PMA(100 nM) and carbachol (10 mM) induced a 154% and53% increase, respectively, in membrane-associatedaPKC. Although 10 pM carbachol was maximal forstimulation of pepsinogen secretion, this concentra-tion did not cause an increase in membrane-associat-ed aPKC. PMA or carbachol did not cause an increasein membrane-associated CPKC at any concentrationtested. Whereas a 4-hr incubation with PMA (300 nM)caused a 53% down-regulation of aPKC, CPKC levelswere not altered. These results indicate that, ingastric chief cells, (1) aPKC and CPKC responddifferently to carbachol and PMA; and (2) Anincrease in membrane-associated PKC isoforms is notrequired for carbachol-induced pepsinogen secretion.

2

DERIVATION OF SUBLINES FROM AN INSULINOMA CELL LINE WHICHMAINTAIN CORRECT GLUCOSE RESPONSIVENESS. ((David Knack, DeborahM. For, Megan E. Laurenoe, Sarah Eckroth, Shimon Efrat', Orion D. Hegre))Department of Diabetes, CytoTherapeutics, Inc., Providence, RI 02906'Department of Phamcol, A Eintein Collag of Medicin, Bronx, NY.

Trnaganic mioe bearing the SV40 large T antigen gene driven by the rat insuOnpraooter develop spontaneous insuinomas 3.4 montha after birth. Insuinomasfrom thes gic ki have ben usd by a number of laboratories toeeablleh a vaiety of insuin releadng B-c lin including the rnin, nit, and BTCli. While a number of thee i, in their early paages, heve demonstratedinsulin releb in response to glucoso dh simnler to that obsved In intactWaft, the response ch.racteristics have tended to deterorate over time. Thepolyconal B TC 6 line was generated from a RIPTag Instinorm. The cell linemantains correct gko reponse dcaacteistis until approKtdely pasge 25.Over 50 suiine have bean generated from pasage 18-22 B TC8 cells using avariety of cloning and FACS techniques. Characterization of these sublinesindicate a wide heterogeneity among sublines for insulin secetory levels,glucose response cheracteriatics and growth rate. One subline SB1,demonstrated haf maximal insulin rsponse at 8mM glucose, with bal insulinreease levels being maintained up to about 2 mM glucose at passage 38(approximately 1 yr in vitro). Side by aide peifusion expeiments with intactmouse lelets. demonstrated remarkabe simiarity between the SB1 line and intacties, with both demonstrating a bipasic insulin rel eas in response to 16mMglucose with reduced and prolonged second pha". A smaller, transientrespns to 8 mM glucose was also observed. These data indicate corretlyreted B cell can be bolated and maintained from an inkial haterogenousB-cal parent population that does not othwise retain stable glucose responsedcaacteristic in vio.

DEVELOPMENT OF AN EMBRYONIC CHICK ORGAN CULTURE SYSTEM FORFUNCTIONAL STUDIES ON CHOLINERGIC REGULATION OF PANCREATICSECRETIONS. ((A. D. Johnson)) Department of BiologicalSciences, University of Alabama, Huntsville, AL 35899

Carbohydrate metabolism is dependent on cholinergic regulationof pancreatic exocrine release. Organ cultures were preparedusing the whole panceas from 18 day-old embryonic chicks toexamine the effects of the synthetic parasympathomimeticagents, carbamylcholine chloride (carbachol) and bethanecholchloride, on the stimulatory effects of amylase release over a

4-24 hour incubation period. Bethanechol and carbacholconcentrations of <.05-5Oml stimulated (P<.05) amylase releaseduring the 4-24 hour incubation period when compared to controlcultures. Bethanechol had a higher percent stimulation (345Z)for total amylase release when compared to 2401 for carbachol.Lactate dehydrogenase activity and histological examinationrevealed funictional changes in the pancreatic tissue after 12hours of incubation. The organ culture system describedseems to be a functional in vitro model to test cholinergicagents for short term incubation periods.

1lOa

Sunday. MethodsI (644-649)

644

MICROWAVE FIXATION IMPROVES ANTIGENICITY OF AGLUTARALDEHYDE SENSITIVE ANTIGEN WHILEPRESERVING MORPHOLOGY. ((M.C. Jamurt, C.D. Faraco', L.O.Lunardi2, and C. Oliver))'U; Federal do Parana, Curitiba, PR, Brazil,2Fac. Med Ribeirao Preto-USP, Sao Paulo, Brazil,3 Office of NavalResearch, Arlington, VA, USA.

Microwave fixation for electron microscopy has been used to preserve

antigenicity (Login and Dvorak, Histochem. J. 20:373, 1988). Here, we

examine the ability of microwave fixation to preserve the antigenicity ofa mast cell specific ganglioside (Oliver, et al., J. Cell Biol. 116:635,1992) on rat bone marrow derived mast cells. RBL-2H3 cellsconventionally fixed with glutaraldehyde concentrations as low as 0.1%,

failed to react with a mAb (AA4) against the ganglioside. Strongstaining of mast cell plasma membranes was seen when bone marrowwas initially fixed with 2% formaldehyde and then refixed in 2%glutaraldehyde - 2% formaldehyde following immunostaining.However, the ultrastructural preservation of the cells was poor.

Antigenicity and morphological detail were both preserved when bonemarrow was fixed in 0.05% glutaraldehyde 2% formaldehyde for 4seconds in a 750 watt microwave oven. Using this method, mast cellsin various stages of maturation as well as cells that did not containgranules were immunoreactive. The agranular cells were tentativelyidentified as tissue mast cell precursors. No other cells from the bonemarrow were reactive. This method should prove useful with otherantigens that are sensitive to glutaraldehyde fixation.

646

A COMBINED SCANNING PROBE AND LIGHT MICROSCOPE FORLIVING CELL STUDIES. ((A. Stemmer)) Marine Biological Laboratory,Woods Hole, MA 02543.

It is becoming increasingly likely that the architectural dynamics in the cellinterior is governed by the distribution and activity of structures at the surfaceof the cell, e.g. receptors, ion channels and, at times, organizing centers, whichare expected to guide the spatial organization of macromolecular assembliesand organelles in the cytoplasm. This guidance, effected through mechanicaland/or biochemical links, could account for a long sought physical basis forthe various forms of cell polarity observed during directed cell movement, celldivision and differentiation. To study such interactions in vivo, a hybridmicroscope has been developed that allows the visualization of functionalstructures at the surface of living cells at a resolution reaching moleculardimensions, and their correlation with the three-dimensional architecturaldynamics within the active cell. The new microscope is based on a scanningprobe microscope mounted coaxially with a high-resolution video-enhancedinverted light microscope for simultaneous observation of the same specimenin both modes. The scanning probe microscope is designed to accommodateforce, near-field optical, or ionic probes to obtain a multitude of topographicand spectroscopic data from the surface at nanometer resolution, while thelight microscope equipped with high NA optics provides for time-lapsedoptical sections that generate three-dimensional maps of intracellularstructures at sub-micrometer resolution. (Supported through a Swiss NationalScience Foundation Fellowship and research grants R37 GM31617-12 fromNIH and MCB-8908169 from NSF (both to Dr. ShinyaInoue)).

648

NEW POLARIZED LIGHTMICROSCOPE FOR FAST ANDORIENTATION INDEPENDENT MEASUREMENT OFBIREFRINGENT FINE STRUCT1URE IN LIVING CELLS.((Rudolf Oldenbourg and Guang Mei))Marine Biological Laboratory, Woods Hole MA 02543, USA.

The polarized light microscope has the unique potential tomeasure submicroscopic molecular arrangements dynamically andnon-destructively in living cells and other specimens.

We are developing a new kind of polarized light microscopewhich combines speed and high resolution in its measurement ofthe specimen anisotropy, irrespective of its orientation. The designof the new pol-scope is based on the traditional polarized lightmicroscope replacing the conventional compensator by electro-optical devices. A video camera and computer assisted imageanalysis provide measurements of specimen anisotropy in rapidsuccession for all points of the image comprising the field of view.

The new instrument has the ability to display sequentially thespatial distribution, and rapidly measure the temporal changes in:birefringence due to intra-molecular anisotropy or fine-structuralform; and (with some modification to the basic scheme) polarizedfluorescence in molecular moiety selectively labeled with reporterdyes, and dichroism exhibited by naturally occurring chromo-phores. The instrument development is supported by the NationalInstitutes of Health grant R01 GM49210 awarded to RO.

645COMPUTER RECONSTRUCTION OF GRAFTS FROM SERIALLYSECTIONED RAT BRAIN. ((R.V. Blystone, G.A. Mickleyt, and P.A.Mason*)) Department of Biology, Trinity University, San Antonio, TX78212, tArmstrong Laboratory, Radiofrequency Radiation Division, BrooksAFB, TX 78235, and*Operational Technologies, San Antonio, TX 78229.

Three dimensional views of microanatomical structures are difficult tovisualize from serially sectioned tissues. We have developed a computertechnique for the rapidreconstruction of serially sectioned brain grafts. Thegustatory neocortex or amygdala areas of mature malerats were injectedthrough 26gauge canulae with selected brain tissue from 18-day ratfetuses.Five or more months post-injection, the adultrats were formalin perfused and6 pm serial coronal sections were taken through Bregma +1.2 or -2.3 regions(after Paxinos & Watson, 1986). Every tenth section was stained with Luxolfast blue and cresyl violet acetate. These stained sections were viewed withan Olympus Vanox microscope and the resultant images digitized using theImage -1 system. The TIFF images were fine-aligned with NIH Image 1.50software and enhanced with Adobe Photoshop 2.5 software. The files wereconverted to HDF format using the Spyglass Data Transform Utility. Thesequential tenth sections were then three-dimensionally rendered into avolume using Spyglass Dicer 2.0. This software allows the rotation of therendered volume and its reslicing. Care was taken to bring the x, y, and z

pixel planes into proper dimensional agreement. The total time needed tobuild each graft reconstruction beginning with image capture was twodays.Reconstructed grafts were directly compaired by computer pastingrenderingsinto their proper position on a computer reconstructed 3-D stereotaxic atlasmodeled after Paxinos & Watson, 1986. These procedures provided arapidand cost effective means to dimensionally visualize an initial 2-D brain dataset. Supported by the U.S. Air Force (JOCAS 77570185) and the Air ForceOffice of Scientific Research Summer Research Program.

647

OPTIMIZED EPIPOLARIZED-LIGHT MICROSCOPE VISUALIZATION OFIMMUNOGOLD-SILVER STAINING OF HEPATIC PEPCK ANTIGENICSITES. ((K. Gao, B.F. Giffin, R.E. Morris, E.L. Cardell, and R.R. Cardell))Department of Anatomy and Cell Biology, University of Cincinnati College ofMedicine, Cincinnati, OH 45267.

We have used the immunogold-silver staining method to localize antibodies tophosphoenolpyruvate carboxykinase in 10 pm cryosections of 4%paraformaldehyde perfusion-fixed normal male rat liver. The resolution and thesensitivity of detection wereimproved by epipolarization microscopy of 0.5 pmsemithin plastic sections prepared from immunogold-silver enhanced 10 pmthick cryosections. When epipolarized illumination was combined withtransmitted light, the image of antigenic sites, produced by epipolarized light,and the tissue morphology, delineated by transmitted light, were demonstratedsimultaneously. In order to optimize the conditions for high resolution, a IOOXoil immersion objective lens with adjustable diaphragm was used with variousintensity settings for both light sources. Our observations indicated that if theintensity of the transmitted light was too high, the visibility of the gold-coredsilver grains by epipolarized illumination was decreased; while if the intensityof epipolarization was too strong, halos appeared around the gold-cored silverparticles. By adjusting the aperture in the objective lens and neutral densityfilters in the transmitted light pathway to balance the intensities of transmittedand epipolarized lights, the best image was gained which showed the maximumnumber of antigenic sites with properly balanced delineation of the tissueprofile.

649

A SENSiIVE AND STABLE METAL-ENHANCED DAB SUBSTRATE pVRIMMUNOHISTOCIEMICAL ANDIMMUNOBLOTIlN APPIICATIONS.((LK. Hines, KCD. Feather, and D.C. Kienk)) Rescuch and Deveopment, PieChemical Company, Rockford, IL 61105. (Spon. By C. R. Clark.)

3,3-diaminobenzidine (DAB) isa trditonal subsme for die develome ofhorsadish perxidae (HR may sytems. A common method so improv ansi-

tivity has been lo addbeavy metals (AdmsL 1981. J. Histo. Cyto., 2,229). Thismethod is useful butohn has problems sooiaed with including decesd solu-bility, Unwaned pripiton andiead bwkgound coow. We bave devlopeda metal-enhaced DAB subsa soluion which utilizes cobaltchrde ad nickelchloide in a optmized fomlion wi a sta peoxide buffer. This subsume

wa anaysedby westn bkytn to dcetabiotinylated protein with HRPconjugat-

ed trptvidin nd in isumunistocheit by stainin forbwman chorionicgnoin I

pacenal tisu The prcipos which resu fm using theimoved sm duk bron-blak cdo with very litt background

develomt Thi makes th subse ideal for immmobkotg ad inmunohisto-chemical applio In a dotbktanlysi, senitivity (as determied by the tower

detctability imit) wth is new substae wu Icead 50 fold over traditional

DAB subsa and 35 fold over other metl enhanc methods. Th simple twocomponent stem is sable for least6 mot and the dilutd woring substrtesolu is stable atroom mpeatu forup to 6 hours whtho affecting the signalto noie rato d whouta y pecipitae fonning. In concludon, we find that theaddion of both nckel and cobal the DAB combined with optimization of buffers

in a s ior staining solution, not achievable by traditiona proocols of sub-s me doe

llla

Methods I (650-655). Sunday650

DESIGN AND USE OF AN ATOMIC FORCE MICROSCOPE (AlM) FORBIOLOGICAL IMAGING (( Silvio P. Marchese-Ragona)) TopoMetrix,5403, Betsy Ross Drive, Santa Clara, CA 95054-1162 USA

The Atomic Force Microscope (AFM) is becoming an increasinglyimportant tool for biological sciences primarily due to the ability toscan specimens in liquids under physiological conditions. To extendthe usefulness of the instrument we have combined the AFM with aninverted optical microscope. This unique combination allowsaccurate positioning of the scanning tip to within less than 1.0 um ofthe sample. The AFM will scan an area of 150 um by 150 um with a

10 um z-range in a droplet of liquid as small as 50 ul. The ability toaccurately position the tip over the sample allows data to becollected in one pass thereby reducing the possibility of damage tothe sample that could occur from multiple scans. To further optimizethe data available from the single pass imaging method, we collectfive different signals in both the forward and reverse scans, makingten channels of data in all. As biological test specimens we havebeen using polytene chromosomes isolated from Drosophila salivarygland. AFM analysis of the inter-band area of the chromosome showsbundles of fibers running parallel to one another along the axis of thechromosome. We are currently attempting to obtain higherresolution information using different specimen preparations andsharper tips.

652OPTIMIZATION OF NORTHERNWSOUTHERN ANALYSISUSING CHEMILUMINESCENCE: ACHIEVING SENSITIVITYEQUAL TO RADIOACTIVITY ((H. Xbao and M. Parls)) United StatesBiochemical Corporation, 26101 Miles Road, Cleveland, OH 44128 (Spon. byM. Snider)

We have optimized the condItIons necessary for relable detectIon of Northemand Southem blots using chemiluminescence to provide results equal insensitMty to radloactive detecdon. Successful chemiluminescent detection hasbeen achieved by increasing the biotln-labeling efficiency of DNA probes andopfImizing the conditions for nucleic acid hybridIzation. Probes are labeled withblotin using the random prImed lbeling method In which Blotin-14-dCTP Isreadily incorporated by Exonuchae-free Kienow into newly synthesizedDNA. The probes are subsequentiy hybridized to target RNAADNAimmobilized on positively charged nylon membrane. Hybridization isperformed using an aqueous buffer containing a high concentration of SDS(7%). Use of this buffer results In stronger hybridl zation signals than resultsobtained using standard formarnide buffer. After the probe is bound to thetarget, it is detected by binding streptavidin-alkaline phosphatase to the biotin.A chemiuminescent substrate for alkaline phosphatase, Lumi-Phos 530, isapplied to the membrane and the sealed blot is exposed to X-ray fim. Typicalexposure times are short (30 min -4 hrs) unlike the longer exposures (days)required to detect radio-active blots. By using chemniluminescence, we areable to detect single copy genes in as lIKtle as 0.5 ILg of genomic DNA onSouthem blots. We are equally successful in Northem analyis, exemplifiedby the detection of GAPDHmRNA In just 0.5 pg of total RNA exacted fromHeLa cells. Lower copy numbers ofmRNA have also been successfullydetected. Examples include the detection of c-sis and p53. mRNAs in totalRNA extracted from the A172 human gNoblastoma cell line. The level ofsensitivity is the same as that observed using f32P]-labeled probes for bothNorthern and Southem analysis.

654

MICRO DIRECT DETECTION PCR: A ONE-STEP METHOD OF TYPINGMHC CLASSII DRB1 GENE ALLELES. ((H.H. Chan, D. Chia, P.I. Teusaki, andA.J. Acalinovich)) Tissue Typing Lab., Departmt of Surgey, UCLA School ofMedicine, L.A., CA 90024.

Rcently,methods such as RFLP, oligo-probe hybridizton, and sequence-speific-primer PCR(SSP-PCR) have boen applied to HLA allele typig. Most of thetechniques reqire laborintensive and/or time consuming steps for sigl detecton.

We have steamlined HLA DNA typing through a single-step PCR/detection method

perforned atmicro levelin one vessel-a 72 wells Tersali micro-tray.PCR reactions were performed in a 72-well Terasali try using 1.5 ul primer pairmix and 1.5 ul DNA. Annealing was perfonmed at 63' Cand [Mg++] was adjusted to

obtain the maximm signal/noise ratio. After 30 cycles ofamplification, 2 ulethidium homodimer was added, thende sials were detcted bymeasurngfluorescenceemission energy at 617 em witi a One LambdaMicroscope Scamer.Using 21 pairs of SSP, we were able to typethe fllowing alleles of DRB1: 01, 15,16, 03, 04, 11, 12, 13, 14, 07, 08, 09, and 10.Todem the accuracy of themethod, we obtained two sets of previouslytyped

DNA sanples: 50 RFLP typed samples had 100 % accuracyand another 50consensus DNAs were highly correlatd with results from diffent detectionmethdsperfomed in 30 laboratories. Also, 2 DNA samples weretested 5 times to show thereproducibility. The coefficient of variation of the positive wells were between 10.6and 20%.Wehave demntrtd a simple, fast methiod for MHC gene typing can also be

applied tosttidylage scalesc of speific genetic defects.

651CHARACTERIZATION OF A TRIPLE BAND FILTER FOR MULTI-WAVELENGTH EPIFLUORESCENCE VIDEO MICROSCOPY.((R.J.Lowy)) Physiology Dept., AFRRI, Bethesda, MD 20889.

Interference filters with multiband pass regions(MBPR) are now available to allow simultaneous or

sequential examination of staining patterns ofmultiple fluorescent probes. The filter set examinedhas broad band excitation (ex) and emission (em)regions at 360ex/460em, 490ex/520em and 560ex/610emand was used with Cascade Blue (CB), Bodipy (BD) andTexas Red (TR). Images of polychromatic beads showedsub-pixel registration for all MBPR as would beexpected, as neither the dichroic mirror nor theemission filter is moved between wavelengths. Thedegree of spectral separation or "cross-talk" (Xtalk)between MBPR regions was assessed using albumin-dyeconjugates at concentrations between 0.010 and 10mg/ml. Small volumes were examined using a digitallyenhanced video microscopy system. Xtalk was evaluatedfor each dye by sequential exposure to all 3 ex wave-lengths and normalizing the em intensities to thevalue at the optimum ex wavelength. Most dye-excita-tion combinations showed Xtalk of 10% or less. BDXtalk at 360 nm was higher, 25-40%, showed a complexconcentration dependence, but was relatively constantat high concentrations. Corrections should bepossible in some circumstances allowing concurrenttriple dye localization and quantitation usingdigital imaging methods.

653RNA-PCR-IN SITU: A NOVEL APPROACH TO DETECT CYTOKINEGENE EXPRESSION IN NORMAL HUMAN PERIPHERAL BLOODMONOCYTES (PBMC). ((Kumar NM, Nair MPN, Schwartz SA,Lwebuga-Mukasa JS.)) Lung Biology Research Program, Divisions ofPulmonary and Critical Care, Allergy, and Immunology, Dept. of Medicine,SUNY/AB School of Med, Buffalo General Hospital, Buffalo NY 14203

RNA-Polymerase Chain Reaction (PCR) is useful for selective detectionof cytokdne gene expression in inflammatory responses and disease states.On the other hand in situ hybridization permits the localization of targetgenes in specific cell types in a mixture of cells or tissues. We hypothesizedthat since many cytokines are expressed and affect cell functions at verylow concentrations, the combinatson of the two techniques may provide apowerful tool for studying cytokine gene expression. Lipopolysaccharide(LPS) and ethanol are known to induce tumor necrosis factor (T'NF) andcolony stimulating factor (GM-CSF) gene expression in normallymphocytes. We examined the effect of LPS and ethanol on the expressionof these cytokine genes using RNA-PCR-in situ hybridization. PBMC werecultured in LPS (lOug/ml), 0.3% (v/v) ethanol or the combination of LPSand ethanol for 8 hours. Treated and untreated cells were fixed in 4%paraformaldehyde and stored in 70% ethanol at 4C and divided into twosets. One set was subjected to reverse transcription followed by PCR andin situ hybridization with randomly primed PI labeled cDNA probesspecific for TNF and GM-CSF. The second set was subjected only toconventional in situ hybridization with the same probes. The signals forboth genes were barely detectable in cells subjected to conventional in situhybridization. In contrast, the signals were dramatically enhanced in thecells that were subjected to RNA-PCR prior to in situ hybridization. Weconclude that RNA-PCR-in situ technique may provide a useful tool fordetection and localization of rare genes in tissue and mixed cell populations.

655SYNTHESIS OF RNA PROBES BY THE DIRECT IN Vr7RO TRANSCRIPTIONOF PCR-GENERATED DNA TEMPLATES. ((R. Urrutia, 'B. Kachar, and M.A.McNiven)) Center for Basic Research in Digestive Diseases, Mayo Clinic, RochesterMN 55905 and 'Me Laboratory of Cellular Biology, National Institute on Deafnessand Other Communication Disorders, National Institutes of Health Bethesda MD20891. (Spon. by G.J. Gores.)

We report a novel plasmid-free method for the generation of RNA probes basedupon the in vitro transcription of PCR-generated DNA templates carrying theT7 andT3 RNA polymerase promoters. In the first step of this method, primers aredesigned which contain sequences from the gene of interest linked to the promoterregions for17 andT3 RNA polymerases. PCR-ampliflcation using these primers isthen done to generate a template which contains one of these promoter sequences ateither end. Subsequendy, a labelled RNA probe is generated by in vitro transcriptionof this template with either 17 or T3 polymerase in the presence of digoxigeninuridine triphosphate (DIG-UTP). Tbe major advantages of this technique are that:1) the probes can be generated using unpurified DNA templates directly amplifiedby PCR from a tissue-specific cDNA pool without the need of DNA cloning, plasmidpurification and linearization, 2) strand-specific RNA probes can be synthesizedquickly, allowing the ability to choose between either the sense or antisense strandfor a particular application. Tbis is a major advantage with respectto PCR-labeleddouble stranded DNA probes, and 3) because the RNA polymerases synthesize RNAin excess with respect to the DNA template, this method can generate large amountsof high quality RNA probes. We have successfully applied this technique to theidentification of kinesin-encoding sequences by Southern blot and library screening.This method may find a significant application for the identification of genes byhybridization experiments because it is fast, efficient, and easily performed.

112a

Sunday. Methods I (656-661)

656DETECTION OF INTRACELLULAR HIV-1 PROVIRAL DNASEQUENCES BY STRAND DISPLACEMENTAMPLIFICATION AND I SITU HYBRIDIZATION. ((K.L.Lohman*, N. OstrerovaS T. Ward* M. Janszen*, V.C.Maino*, R. Singert and J.M. Mathyst)) Becton DickinsonImmunocytometry Systems, San Jose, CA 95131, tDepartmentof Cell Biology, University of Massachusetts Medical Center,Worcester, MA 01655.

The ability to detect and enumerate HIV infected cellsrepresents an important index for determining HIV-1 viral loadin AIDS patients. Identification of HIV-1 proviral DNA andmRNA sequences in HIV-1 infected cells and tissues using inAita hybridization techniques combined with nucleic acidamplification protocols (e.g. PCR), has been recently describedby a number of investiptors Here we describe the use ofStrand Displacement Amplification (SDA), an isothermic gene

amplification methodology, to amplify a region of the HIV-1gag mRNA and proviral DNA gene sequences in both HIV-1infected tissues (J.M. Mathys in preparation) and cellsuspensions. SDA methodology was adapted for the rapiddetection of intracellular HIV target sequences by flowcytometry or image cytometry using fluoresceinatedoligonucleotide probes or light microscopy employing substratedeveloped alkaline phosphatase conjugated oligonucleotideswith specificity for the HIV-1 gag region sequences.

658IN SITU HYBRIDIZATION TO HIV-1 RNA IN GEL MICRO-DROPS ((J. Abebe*, K.G. Lazzari*, R.H. Singer*, B.R. Goguen*, D.W.Bradley*)) *Dept. of Cell Biology, Univ. Mass. Medical Center, Worchester,MA; *One Cell Systems, Inc., 100 Inman St., Cambridge,MA 02139.

We report a method of performing fluorescence in situ hybridization utilizingGel Microdrop Technology (1). The 8E5/LAV cell line (2) was chosen as a

model system to detect HIV infected cells because they produce HIV-1 RNAbut do not produce infectious virus. Single 8E5/LAV cells were encapsulated inmicropdrops by mixing with molton CelGel V (One Cell Systems, Inc.),applying a shear force with a CellSyslO0 maker (One Cell Systems, Inc.),cooling the emulsion, and separating the microdrops from the oil phase.Encapsulated cells were hybridized to a mixture of biotinylated HIV probes (3),stained with FITC-conjugated avidin, and visualized with epifluorescencemicroscopy. The 8E5/LAV cells probed with HIV DNA fluoresced brightiy.The gel microdrops did not interfere with the hybridization, staining, or

detection procedures. As a control, NS-1 cells (a myeloma mouse cell line withno HIV RNA (4)) were processed concurrently; they were barely visible underfluorescence microscopy. Encapsulated 8E5/LAV cells treatd with RNAsebefore hybridization were also dimmer, indicating that the probe was binding toHIV RNA. Replacement of a 4% paraformaldehyde fixation step with a 0.05%Triton X100 permeabilization step substantially improved the signal to

background ratio. Gel microdrops can be analysed by flow cytometry and mayhelp preserve structural integrity. These results demonstrate that gel microdropsare compatible with in situ hybridization procedures.

(1) Supported by NIH SBIR Phase I grant # 1R43 AI32329-01.(2) J. Exp. Med. 164: 280, 1986.(3) Developed by RHS through NIH funds (grant #HB67022).(4) Eur. J. Immunol. 6: 511, 1976; J. Mol. Biol. 90: 691, 1974.

660THE EFFECTIVENESS OF A MODIFIZD MTT PROLIFERATION ASSAY FOR

COMPARING GROWTH RATES OF BOR II AMPHIBIAN CELLS. ((M.A. Delgadoand J. Wesson)) Department of Biology, Southeast Missouri StateUniversity, Cape Girardeau, MO 63701. (Spon. by Walt W. Lilly.)

The MTT proliferation assay is designed for use in 96-11microplates, so that absorbance due to the blue formazan dye at

570 nm wavelength can be measured in a microplate reader.Background absorbance due to cellular debris is accounted for by

subtracting absorbance at 690 nm from the absorbance at 570 nsOur microplate reader lacks the necessary 690 nm filter, so we

developed a modified MTT assay which utilizes contrifugation to

reduce background absorbance due to cellular debris. We

typically conduct growth experiments using cells plated in 24-

well culture plates, so we compared results of our assay using

96-well plates verses 24-well plates. The lower mean absorbanceand standard errors of the means obtained using our modifiedassay indicate that the procedure effectively reduces background

absorbance thereby reducing the variation within our data.Measuremnts of absorbance in 24-well plates were too variableto be useful, probably due to incompatibility of the 24-wellplates with the positioning and optics of the microplate reader.Future use of this assay to compare proliferation of various

subclones of the Bor II amphibian cell line is based on theassumption that differences in assay results are directly

proportional to differences in cell numbers, and not due to

clonal differences in metabolism of MTT. In order to test thisassumption, proliferation rates of C4 and R6 clones were

measured using our modified MTT assay and by directly counting

cells with a hemacytometer. The growth curves generated by

these two methods were parallel to each other, indicating that

differences in NTT results do directly reflect differences in

cell numbers. Therefore, the assay can be used to compare thegrowth rates of these subclones.

657NEW APPROACH FOR QUANTITATIVE PCR AMPUFICATION OF

HIV-1 DNA AND RNA SPECIES

((C.C. Novick("'), G.E. Novick0l, L. Resnick(", R.J. Herrera°t))(0Department of Biological Sciences, Florida International University, Miami, Fl,

USA. (nRetrovirology Laboratories, Mount Sinai Medical Center, Miami Beach,Fl, USA. (Spon. by C.Bigger)

Quantitative PCR amplification of is one of the main challenges of today's

biotechnology.A novel approach based on the concept of competitive PCR using internal

controls was developed to detect and quantify HIV-1 DNA and RNA species

from clinical samples.A recombinant full length HIV-1 DNA was created and a full length RNA

transcript was in vitro transcribed from it. Both contain a distinct feature that

allows to discriminate between recombinant and endogenous HIV-1 DNA and

RNA when they are coamplified in the same reaction tube. A radioactive PCRreaction is carried out with a known number of molecules of intemal standard

and unknown number of molecules of patient template; the original amount of

sample template can be calculated.The way in which full length recombinant HIV-1 DNA and RNA were

designed, the optimization of the coamplification and the sensitivity and

accuracy of the detection and quantitation of the radioactive signal of the

amplified product, make this quantitative PCR approach an invaluable tool for

the detection and quantitation of the viral load of a particular HIV-1 infected

sample.This research was supported by U.S.Public Health Service Grant RR08205 to

R.J.H.

659

THE SIMULTANEOUS ISOLATION OF RNA, DNA AND PROTEINS

FOR GENE EXPRESSION ANALYSIS. ((K. Mackey and P.Chomczynski)) University of Cincinnati, College of Medicine, andMolecular Research Center, Inc., Cincinnati, OH 45212.

We describe a new method for gene expression studies that allows thesimultaneous isolation of RNA, DNA and proteins from the same cell or

tissue sample. This is an improved version of the single-step liquid-phaseseparation method developed initially for isolating total RNA (AnalBiochem 162: 156. 1987). RNA isolation can be completed in 1 h, andDNA and proteins in 3 h. The simultaneously isolated RNA and DNA can

be used for Northern or Southern blotting, and for RT-PCR or PCR,respectively. Proteins can be analyzed by Western blotting. In addition,the complete recovery of DNA concomitant with RNA and protein isolationmakes it possible to normalize the results of gene expression studies per

DNA content instead of the more variable total RNA, protein content, or

tissue weight. The use of this method is exemplified by the simultaneous

isolation of RNA, DNA and proteins from rat pituitary cells and mammarygland. The detection of nondegraded growth hormone (GH) mRNA and a-lactalbumin mRNA in Northern bloting, EcoR1 and Pstl fragments of theGH and a-lactalbumin genes in Southern blotting, and GH and a-

lactalbumin in Western blotting illustrated the effectiveness of this method.The isolated RNA was used for amplification of various mRNA fragmentsby RT-PCR. No DNA contamination was detected in these rcactions.

661

TRANSFORMATION OP A DOMINANT SELECTABLE MARKERINTO CHLAMYDOMONAS REINHARDTII. (Q.A.E. Nelson and

P.A. Lefebvre)) Department of Genetics and Cell Biology,

University of Minnesota, St. Paul, MN 55108

We have constructed a dominant selectable marker gene for use in

nuclear transformation of all C. reinhardtii strains. Transform-

ation was previously limited to specific mutant strains for which

the auxotrophic marker genes had been cloned because of the

failure of heterologous genes to be expressed in C. reinhardtii. The

CRYl-1 mutation alters the C. reinhardtii S14 ribosomal protein

gene, conferring resistance to the eukaryotic translational

inhibitors cryptopleurine and emetine. We have determined the

site of the CRYl-1 mutation to be a single missense mutation in the

last codon for the S14 protein. The plasmid used for successful

transformation contained the CRYl-1 coding region fused to the C.

reinhardtii RUBISCO small subunit 2 promoter to produce a high

level of CRYl-1 expresion. The chimeric construct produced high

resistance to cryptopleurine and emetine in transformed cells, both

by direct selection and in cotransformation with a second marker

gene.

113aI I

Film Session 1 (662-667). Sunday

662DYNAMICS OF TRACTION FORCES, CYTOSKELETAL STRUCTURE, AND FOCAL

CONTACTS IN SERUM-DEPRIVED FIBROBLASTS. ((K. Burton & D1. Taylor)).

Center for Light Mioscope Imaging, Carnegie Mellon University, Pitsburgh. PA, 15213.

We have correlated positions and movements ofcytoskelet structure, myosin II, focal

contacts, and traction forces in living Swiss 3T3 fibroblass Traction forces were monitored

using a modification of the method intoduced by Harris (Science 208:177,1980) in which

cells are cultured on transparent silicone. In order tomonitor forces applied by relatively weak

colls, including fibroblasts deprived of serum, we have employed polysiloxanes which are

significantly more compliant than those reported heretofore. We are able to control the

compliance of these silicon sheets appropie to a wide range of traction forces. These

substrata also possess improved optical properties permitting high quality interference

reflection microscopy (IRM). Several methods of force calibration are being explored.

Cytoskeletal structure was monitored using three modes of timelapse microscopy for each

cell. Images were acquired using IRM to determine ceUl-substrnm contacts and Nomarski

DIC to visualize general ceUular strucure and distortions in the substrtm. T'hc distribution

of myosin was determined using fluorscence microscopy. Since precise comparison of

stuctural dynamics and traction forces required measurmts overtime, fluorescence signals

from livingcells wer acquired from adwdamine-myosin analog which had incorporated into

cytoskeletal strucures following microinjection of cells cultured on the silicone substrata.

Images using each mode of microscopy were separated by about 20 sec., timepoints by 5 min.

Resolution of cytoskeletal stucture wasimproved by inducing cells to flatten by culturing

in mediaat low concentration ofseum (0.2%) for48 hours before observation. Under these

conditions, the number of stres fibersincreases and many are observed to truansportroughlyperpendicular to their long axes at speeds of 10-20 pm/hr (Giuliano and Taylor, Cell Mot. &

Cyto. 16:14,1990). We have tested the hypothesis that termini of stress fibers, focal

contacts, and sites of force application move together. Wefind no evidence that sites of

appLscation oftraction forces, which correlate strongly with focal contacts,show movements

to thoseoftaunsported fibers. Manystress fibers are observed to terminate in

focal contacts, but these do notexhibittrnsort Traction forces are observed to be directed

axialy along fibers terminating in contacts. These results suggest thatstur fibertransport

requires one or both ends of the fibers to be unrestrained by mechanical connections to the

substratum, thus allowinglateral movanent in response to some force within the cell.

664

MECHANISM OF GLIDING MOTILITY IN THE

CYANOBACTERIUM SPIRULINA SUBSALSA.

((A.T. Tabor and N. S. Allen)) Department of Biology, Wake Forest

University, Winston-Salem, NC 27109, and Marine Biological Laboratory,

Woods Hole, MA 02543

The mechanism of the gliding motility of bacteria and cyanobacteria has

remained elusive. S. subsalra 's translational motility (0.8-1.2 pmsec-1) was

invariably acoopanie by both sini revolution arndthe longitudinal axis

of the filament ad synchronous secretion of mucilage. Spirulinatrichomes

appe segmented,and microaphere binding occurs, in a ounterhelical manner

over ten segments. This binding pattem is found to correlate spatially and

temporally with the pattern of secreted single and multiple pass tracks, the

pattern of substrate adhesionareas, side-to-side osdllations or "waves ocaurring

during locomotion, andnewly discovered putative mucilage extrusion

structures. These paired periodic particles occur in a counterhelical manner

within thefilament near theinner cncave regions of the helical cells and are in

a position to provide a highly directional secetion of mucilage. The location

of these partides during locomotion is such that they could produce theproper

rotationa torque to propel the bacteia forward or backward. A video tape will

demonstrate thes phenomena. A computer-genrated 3-D conceptual model

was constnscted to further undand themechanism of motility.Supported by Howard Hugha Medical Resarch Foundation Award, Department

of Biology (WFU, AT) and Archie Funds, (WFU, NSA).

666

THE CENTR TL TRANSPORT OF MICROTUBULES IN MOTILE

CELLS. ((A.Mikhailov and G G Gundemen))) Dqeamets of Patholoy and

Anatotm & Cell Biology, Colunbia Univest, New York, NY.

The centripedl transport of cell srface components (e.g., beads, particles, aggretedmemrane astigens)and actin-ased flmentoussruuctures within motile cells is thought

to reflct anactin-dependent process that is essential for cell locomotion. Other

cytkeletalsuctur have not been reported to undergo centripetal transport We

proide evidencefor the ekstce of the centripetaltransport of snicroebules(MlTs)motile NRK fibrblat To detect Ivis in living cells, we microisnectedrhodamine-labeled tubslin, allowedit to incorporae into all the Ts (1-2 hr.) and then

imagd theM Ts under lowkvel ilhainaion with an ISIT camera.Images were recorded

evy -10 onoptcl disc and digitallyenhanced to imprve contra. We observed a

distinct bted ofMbTs, those orientd paalle to the leangedge of the cell, to move

backwardswith respct to the kading dge with pect to the substratum. The rate

of rarwardm oveent of indidualMTs was fairly constant over time and in

om 24 cells avep d 0.63*0.37 pntin. The rate of MT apt

sinilr to therate of of fluorcent beads on the sufac of NRK cells

measrd under the sanme conditions (0.21*0.24 pns min)and to therates of centripetal

transpoftreported fbr othersruct inamelae of fibrblasts (0.2-3 pm/nmin). During

theit MT ends appeared to go and shink, suggeting that the MTs were

still dynamic. At 0.2 pM cytochalasn D (CD)centripetl transport ofMTs was not

bowd,although mecmane ruffling wa inhibited, at higher ounoentratln of CD we

could not observe MTs due to contation of the cell We also observedon number of

oceaslon th e ntrpeta tranpor of pinocytotic vesicles; these vesicdes form ed

near the lang edge and than movedrear rd enmeshed in a group of conbipetlymn MTs. Our reults dhw that other non-tn baed sucur within the cytoplam

of motile cells undroeecntripeal transport, and rai the posbility that the ceonpettansport Of MTs is involved in vesicle tranor Supportd by NIH grant GM42026

ACS CB-34B.

663SLOW CELLULAR DYNAMICS OF NORMAL AND TRANSFORMEDMDCK CELLS BY ATOMIC FORCE MICROSCOPY. ((C.-A. Schoenenbergerand J. H. Hoh)) M. E. MUiller Institute at the Biocenter, University of Basel,CH4056 Basel, Switzerland.

We have examined slow cellular dynamics in an epithelial monolayer formed byMDCK cells, and an MDCK ras transfornant, cultured on glass cover slips. Anatomic force microscope (AFM) was used to image cells under conditions atwhich they remain viable, and time lapse movies representing on the order of 60seconds real time per frame were produced. In normal MDCK cells two types ofprotrusions in the apical membrane exhibit dynamic behavior. Structures thatcreate bulges in the apical plasma membrane form transiently over the time scaleof minutes to lgs of minutes. These bulges probably represent structures belowthe plasma membrane, although their identity is not known. Another class ofprotrusion from the apical membrane extends well above the surface. Thesestructures appear first as bulges, extend above the membrane and appear to de-tach. Based on this behavior we propose that this represents a secretory event. Anoncogenic transformant derived from MDCK cells by retroviral infection growsvery flat on glass. This allows the plasma membrane to deform around a numberof intracellular structures during imaging with the AFM and for the direct moni-toring of their movement. Two morphological variants behave differently duringimaging. Some cells have processes that extend away from the cell body, withstress fibers that stretch along the process. In response to perturbation by theAFM tip, these process withdraw. As few as 2-3 scans (256 lines per scan) can beenough to induce the movement. During the withdrawal, movement of the stressfibers can be clearly seen. Transformed cells with rounded edges have filamentsrunning parallel to the edge of the cell. In the flatregions of these cells both ante-rograde and retrogradetransport of intracellular particles can be seen. In additionwe have observed two types of wave like movements through the cell, one movesradially out from center of the cell while another moves circularly around the pe-riphery of the cell.

665VISUALIZATION OF CALCIUM TRANSIENTS CONTROLLINGCILIARY MOTILITY. S.L. Tamm and M. Terasald*. BUMP, MBL,Woods Hole, MA 02543 and*Laboratory of Neurobiology, NINDS, NIH,Bethesda, MD 20892.

Depolarizing stimuli activate voltage sensitive-Ca channels locatedsomewhere in the ciliary or flagellar membrane, resulting in a transient Cainflux that triggers specific changes in axonemalmotility (i.e. beat reversal,arrest, wave asymmetry, etc). We visualized sites ofCaenty and hencedistribution of Ca channels along ciiarymembranes by injecting Ca Greendextran into ctenophore eggs (Mn mionsis). Eggs developed normally in 1-2days into free-swimminglarvae with their cells and 100 gm long ciliarycomb plates loaded with the indicator. Electrical stimulation causedsynaptically-driven reversed beating of larval comb plates, correlated with arapid rise of fluorescence emission along the entire length of immobilizedcilia and in the cell bodies. Fluorescence rises within 17 msec and to asimilar relative extent from base to tip of the cilia, Ca must therefore enterthrough channels distributed along the entire length of the ciliary membranes.Ca Green emission then declines more slowly to resting level (1-2s) at asimilar rate from base totip, indicating that Ca is pumped out along the entrecilia. Cell body emission peaks slightly after their cilia and decays moreslowly. The location of Ca channels along thediliary membranes suggeststhat the Ca targets controlling motility are distributed along the length of theaxonemes. Visualization of Ca transients in cilia for the first time is due tothe large size and early development of ciliary comb plates in ctenophores,and the use of dextran-conjugated fluorescent Ca indicators formicroinjection. Supported by NEI grant GM 45557.

667

FORMATION OF ENDOPLASMIC RETICULUM-LIKE MEMBRANE NETWORKSOCCURS BY MICROTUBULE GROWTH IN INTERPHASE XENOPUS EXTRACTS.((C. M. Waterman-Storer, C. Zhu, D. Frank,K W. Murray and E.D. Salmon)) MarneBiological Laboratory, Woods Hole, MA (Spon. by T. Shirrizu)

The formationand maintenance of thelace-like network of membrane tubules whichcomprise the endoplasmic reticulum (ER)in cells has been demonstrated to be amicrotubule (MT)-dependent process. In vitro assays have been developed in whichER-Ike networks of membranous tubules form on coversqa bound MTs incrude cellextracts. Microtububased motor proteins have beenimplicatedin the formation ofthese membrane networks in vtro. Here, we demonstrate thatin additlon to motorprotesi activiy, the process of MT growth and shrinkage maycontribute signNficantlyto the fomiation of ER-like networks Inknterphase-aested Xenopus egg extracts.By using video enhanced DIC microscopy, we have observed In high speedsupematants that as a growing MT endimpinged on a membranous vesile in theextract, a thin tubularprocess was pulled from the vesile. These tubular processesoften became stretched out over100 gm as they were dragged through the cytosol at20 30pm/min by theirattachment solely at the growing MT end. Over time, asextending membrane processes contacted oneanother, they fused and a tubularmembrane reticukim formedIn the extract. In additon, the membranous tubulesremained attached to MT ends as the MT switched from the growth to the shrinkingphase of dynamic instabil4 Thisindicates that the membrane tubules could retaintheirattachmernt to MT ends during both additon and removal of tubulin subunits.The nature ofthe attachment was not resolvable,althoughit appeared to be robust.When a membrane tubule became stuck distal fromits attachment to the MT end, theMT became bowed asit continued to elongateunder the tenslon of the tubuleattached toRs growing end. To rule out the possibilty that membrane tubules wereattached to the ti of a transbocating MT, as opposed to a growing one, we haveobserved asirrilar rate of formation of membrane networks in extractscontaining5mM AMP-PNP. This level of non-hydrolyzable ATP analog has been shown toInhibit MT motor actIvtyin Interphase Xenopus extracts. These resultsindicate thatthe phenomenon of MT dynamic instabiliky is Important in the formation of the ER invivo. (Supported by NIH GM31 136-14 to the MBL Physiology Course)

114a

Sunday. Film Session 1 (668-669)

668FORMATION OF FILOPODIA IN CELL-S INFECTED BY LISTERIA((J.W.Sanger, F.T. Ashton, F. Dold, B. Mittal, D. Nanavati and J.M.Sanger))Dept. of Cell and Developmental Biology, Univ. Penn. Sch. Med., Phila., PA.

The motility of the pathogenic bacterium, Listeriac monocvtogenes insidePtK2 cells occurs initially within the normal boundaries of the host cell, butafter longer times of infection, the Listeria induce the formation of filopodialprojections of the plasma membrane. Actin polymerization takes place at thebacterial surface creating a 'tail' that is necessary for the forward movementof Listeria in the cytoplasm, and for the formation, maintenance and motilityof the filopodia. With time, the length of the filopodium exceeds the lengthof the actin bundle. When this occurs, the membranous connection to the cellbecomes very thin and the filopodium exhibits serpentine undulations. Ifcytochalasin is added to these cells, the movements of the filopodia stop andthe bacteria are pulled back into the cell by the retraction of the filopodialmembranes. Removal of the drug leads to the reformation of the filopodia.Exposure of infected cells to agents that affect the cytoskeleton (DMSO,nocodazole) leads to an increase in the number of filopodia, suggesting thatweakening the cell cortex facilitates deformations of the cell surface byListeria. When the 53 kD proteolytic fragment of alpha-actinin, responsiblefor the binding of alpha-actinin to integrin, is injected into cells, Listeria losetheir ability to form tails and filopodia. Injection of the fragment into cellswith filopodia leads to the loss of the filopodia. These experiments support amodel in which intact alpha-actinin molecules are needed for (1) the cross-linking of actin filaments into stable bundles (i.e., tails) and for (2) theinteraction with integrins in the cell membranes to form filopodia. Researchsupported by an award from the National Research Initiative CompetitiveGrants Program of the USDA (Agreement number 92-37204-7926).

6693D ANALYSIS OF CELL MOVEMENT DURING NORMAL ANDMYOSIN-II-NULL MORPHOGENESIS IN DICTYOSTELIUM. (U.G.McNally and K. Doolittle)) Department of Biology and Institute forBiomedical Computing, Washington University, St. Louis, MO 63130

We are using 3-D computational optical-ectioning microscopy tovisualize and quantify the 3-D cell trajectories of both normal (Ax2)and myosin-II-null celis in Dictyostelium mounds in the process oftip formation. Mounds containing a subset of fluorescently taggedcells are viewed at 1.3pm resolution in x,y and z, and 60 such 3-Dimages are collected at 2 minute intervals. 3-D movies ofmorphogenesis generated from these time-lapse data and 3Dtrajectories obtained by tracking individual cells both show that Ax2cells exhibit a complex assortment of motile behaviors in themound. Some cells jiggle in place, others appear to follow eitherlinear or spiral trajectories, some reverse their directions and othersconvert from one motile behavior to another. These results suggestthat a number of different, potentially competing cell-guidancemechanisms are operative in the mound. In contrast, in myosin-II-null mounds, nearly all of the cells jiggle in place. This radicalreduction of motility in myosin-II-null cells in the mound comparedto their locomotion on a substrate suggests that myosin-II is far morecritical for motion within a tissue mass than on a substrate.

Monday. Symposium IV: Probing Nuclear Organization: Structural, Genetic, and Cytological Approaches (670)670THE ROLE OF THE TELOMERE IN CHROMOSOME STABILITY INYEAST. ((V.A. Zakian and L.L. Sandell)) Fred Hutchinson CancerResearch Center, Seattle, WA 98104.

Telomeres, the ends of eukaryotic chromosomes, are thought to berequired for the stable maintenance of linear chromosomes. Chromosomesin Saccharomvces cerevisiae end in -300 bps of the heterogeneous repeatC1_,3A. To determine directly the contribution of telomeric DNA tochromosome stability, strains were constructed in which the terminal tractof C1_-3A DNA could be eliminated from one end of an authentic, butdispensable, chromosome inyvivo. The effects of telomere elimination werestudied in a wild type strain, in a strain lacking the major pathway forrecombinational repair of double strand breaks (nm= cells), and in a strainlacking the major system for cell cycle arrest in response to DNA damage(raa2 cells). In wild type cells, elimination of a single telomere caused aRADM-mediated cell cycle arrest, indicating that one critical function oftelomeres is to help cells distinguish intact chromosomes from damagedDNA. In all strains examined, loss of a telomere also resulted in very highrates of chromosome loss, demonstrating that telomeres are also essentialfor chromosome stability. Broken chromosomes could acquire a newtelomere either by recombinational repair or by de novo telomereformation. However, many wild type cells recovered from the RAD9 arrestwithout repairing the broken chromosome, replicating and segregating thedamaged chromosome for as many as 10 cell divisions before its eventualloss. These experiments demonstrate that even in cells thought to haveefficient mechanisms to detect and repair DNA damage, loss of a singletelomere results in chromosome loss in many cells. These data haveimportant implications for the generation of genetic instability during agingand neoplasia in human cells.

Sunday. Symposium V: Cell Biology of the Extracellular Matrix (671-672)671

EXTRACELLULAR MATRIX DIRECTS TISSUE-SPECIFIC GENES:IMPLICATIONS FOR DEVELOPMENT AND BREAST CANCER.((M.J. Bissell)) Lawrence Berkeley Laboratory, Berkeley, CA 94720

Throughout development, the differentiated phenotype is heavily influencedby dynamic and reciprocal interactions between the cells and the surroundingmicroenvironment. Over the last 15 years, we have begun to systematicallyelucidate the elements of this microenvironment in the mouse mammary gland.We have established that the extracellular matrix (ECM) in general, and thebasement membrane in particular, play a significant role in regulating theexpression of milk protein genes both in culture and in vivo. The followingpicture has emerged: a) The regulation is transcriptional; b) There are novelenhancers that respond to ECM; c) The enhancers are tissue-specific, but alsofunction with heterologous promoters; d) The signals are transmitted throughintegrins, and they are responsive to changes in cell shape; e) The ECMcomponent that is most important for such regulation is laminin; f) Thelaminin domain that is responsible has been identified; g) Utilizing transgenicanimals that express the activated form of the matrix degradingmetalloproteinase, stromelysin, we have shown that remodelling of thebasement membrane is crucial both for branching morphogenesis andexpression of milk proteins. We have begun to apply to human cells theknowledge gained from the mouse model and we have succeeded inestablishing a simple yet informative three-dimensional assay fordistinguishing nonnal from malignant human breast cells. This distinction hasbeen difficult to obtain in conventional cultures. We have applied this versatiletechnique to defsne the function of suppressor genes such as NM23. We arenow in a position to define meaningful markers for normal and malignant cellsand to analyze the basis for the dramatic differences in cellular behaviourobserved in our assay system. Futhermore, we have applied this versatiletechnique to study the effect of the expression of tumor suppressor genes inthese cells.

672ARCHITECTURAL DESIGN IN THE ECM: THE ROLE OF FIBRILLIN GENESAND MICROFIBRILS. ((L.Y. Sakai, G.M. Corson, N.L. Charbonneau,S.C. Chalberg, and D.R. Keene)) Department of Biochemistry andMolecular Biology, Oregon Health Sciences University, andShriners Hospital for Crippled Children, PorLland, Oregon.

We first isolated and characterized fibrillin (Sakai, et al.,J. Cell Biol. 103: 2499-2509, 1986) and hypothesized thatTibFIMin molecules assemble into extracellular microfibrils.In order to understand hov fibrillin assembles intomicrofibrils, we have studied the unpolymerized monomer,obtained from cell culture medium (Sakai, et al., J. Biol.Chem. 266: 14763-14770, 1991). We have clonied and sequencedlTFiillin cDNA (Maslen, et al., Nature 352: 334-337, 1991;Corson, et al., Genomics 17: 476-484,1993) to identifystructural domains vhich may play important functional roles.We nov also knov that mutations in the fibrillin gene lead to ahuman heritable disorder of connective tissue, the Marfansyndrome, which is characterized by many variable phenotypicmanifestations. Most of the mutations identified so far occuras single base changes resulting in the substitution of anincorrect amino acid in a single epidermal grovth factor-likerepeat, of the calcium binding type. There are 43 of thesemotifs tandemly repeated in the FBN1 sequence. Mutations havealso been found in an unusual motif containing 8 cysteineswhich is also present in transforming growth factor 01 bindingprotein. The function of this unusual 8-cysteine repeat isunknown. These results implicate fibrillin microfibrils, andperhaps certain structural domains within fibrillin molecules,in processes important to the growth and development ofskeletal elements and blood vessels.

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