Regulated exocytosis: a novel, widely expressed system

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NATURE CELL BIOLOGY VOL 4 DECEMBER 2002 www.nature.com/naturecellbiology 955

Regulated exocytosis: a novel, widelyexpressed system

Barbara Borgonovo*, Emanuele Cocucci*‡, Gabriella Racchetti*§, Paola Podini*§, Angela Bachi§¶ andJacopo Meldolesi*§#

*Department of Neuroscience, Vita-Salute San Raffaele University and Excellence Centre in Cell Differentiation, via Olgettina 58, 20132 Milan, Italy‡Department of Pharmacology, University of Milan, via Vanvitelli 32, 20133 Milan, Italy

§IRCCS San Raffaele, Dibit, via Olgettina 58, 20132 Milan, Italy¶Department of Cell Pathology, Vita-Salute San Raffaele University and Excellence Centre in Cell Differentiation, via Olgettina 58, 20132 Milan, Italy

#e-mail: [email protected]

Published online: 25 November 2002; DOI: 10.1038/ncb888

Electrophysiological studies in some secretory and non-secretory cells have identified an extensive form of calcium-induced exocytosis that is rapid (hundreds of milliseconds), insensitive to tetanus toxin and distinct from regulatedsecretion. We have now identified a marker of the process, desmoyokin-AHNAK, in a clonal derivative of the neu-ronal cell line, PC12. In resting cells, desmoyokin-AHNAK is localized within the lumen of specific vesicles, butappears on the cell surface during stimulation. Desmoyokin-AHNAK-positive vesicles exist in a variety of cells andtissues and are distinct from the endoplasmic reticulum, Golgi, trans-Golgi, endosomes and lysosomes, and fromGlut4 and constitutive secretion vesicles. They seem to be involved in two models of plasmalemma enlargement: dif-ferentiation and membrane repair. We therefore propose that these vesicles should be called ‘enlargosomes’.

Regulated exocytosis is the process by which, after an appropri-ate stimulus, the membrane of specific organelles fuses withand is incorporated into the plasma membrane of the cell1.

Regulated exocytosis is often associated with regulated secretion, aprocess typical of neurons, endocrine and exocrine cells by whichsecretory granules and vesicles discharge their content under pre-cise temporal and spatial requirements (time constants in neuronsand neurosecretory cells are 1–2 ms and several seconds for clearand dense vesicles, respectively2).

In contrast to neurons, cells that are not specialized for secretionare widely believed to be incompetent for regulated exocytosis.Recently, however, electrophysiological capacitance assays of non-neurosecretory cells (Chinese hamster ovary (CHO) and 3T3fibroblasts) and of a clone of the PC12 phaeochromocytoma cellline specifically defective in neurosecretion (named PC12-27),revealed prompt (hundreds of milliseconds) and extensive(15–30%) surface increases after elevation of cytoplasmic calciumconcentration ([Ca2+]i) induced by flash photolysis of intracellularcaged calcium (refs 3–5). On the basis of extensive evidence, theseresponses can only be due to calcium-regulated exocytosis of smallorganelles (less than 100 nm in diameter4,5). Moreover, in a fractionof the CHO cells analysed, the fusion of a few larger organelles(diameter up to 1.5 µm, possibly secretory lysosomes6) was alsoobserved3. Interestingly, the responses of PC12-27 cells were com-pletely unaffected by tetanus toxin, a classical blocker of synapticand neurosecretory vesicle–granule fusion5. From these results onecan conclude that a new type of exocytosis, fast and regulated butmechanistically distinct from the classical form, operates in at leastthree lines of cultured cells. Evidence of this new process in othercell types, its functional significance and the nature of the exocy-totic vesicles remained to be discovered.

Here we report on studies utilizing a comprehensive cell biolog-ical approach in several PC12 cell clones, competent or defective forneurosecretion, and in a variety of other cell types and tissues. Thenew exocytotic process was found to have a specific protein mark-er present in a population of vesicles distinct from any other cyto-plasmic organelle. In some cell types, in which expression of the

marker is absent or low during growth, an accumulation wasobserved during differentiation.

ResultsIdentification of an exocytotic marker. Our first task was the iden-tification of one or more markers specific for the organelles sus-taining the new calcium-induced exocytotic responses revealed bycapacitance assays5. This was achieved by raising monoclonal anti-bodies against proteins appearing at the surface of PC12-27 cellstreated with the calcium ionophore ionomycin, identified by differ-ential biotinylation as described in Methods.

Among the generated monoclonal antibodies, one (termed Ab)specific for a very-high-molecular-mass protein is expressed by twoneurosecretion-defective PC12 clones, PC12-27 and Trk, and byCHO fibroblasts. In contrast, the antigen was virtually absent inwild-type PC12 cells and in two secretion-competent clones, 16and 38 (ref. 7) (Fig. 1Aa). In two-dimensional (2D) western blotsof immunoprecipitates from PC12-27 cell lysates, various moder-ately acidic Ab-positive spots appeared, which were excised andsequenced by tandem nanoelectrospray mass spectrometry. Allsequences obtained (Fig. 1Ac) were found to be included into thelong (128 amino acids), numerous (more than 30) and characteris-tic repeats of a huge (relative molecular mass ∼700,000 (Mr

~700K)) protein expressed in a variety of cells in multiple species,named both desmoyokin and AHNAK (abbreviated here as dA). Inhumans, the >4,300-amino-acid repeat core of dA is continuouswith both a 251-amino-acid amino-terminal domain and a 1,002-amino-acid carboxy-terminal domain8.

Further studies confirmed the identification of the Ab antigen asdA. First, the purified product (Mr ~200K) of an Escherichia coli-expressed dA construct, z80, including the glutathione S-transferase(GST) tag, several repeats and 80 amino acids of the C-terminaldomain9, was intensely Ab-positive (Fig. 1Ab). Second, mass-spec-trometric analysis of Ab-positive bands appearing in the gels ofGST-containing proteins purified from z80-expressing E. colirevealed in all cases sequences included within dA repeats (not

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NATURE CELL BIOLOGY VOL 4 DECEMBER 2002 www.nature.com/naturecellbiology956

shown). Third, binding of a polyclonal anti-dA antibody (namedKIS), raised against the N-terminal 16-amino-acid polypeptide ofthe repeats10 to the z80 protein (revealed by enzyme-linkedimmunosorbent assay (ELISA)) was competed for by Ab (seeSupplementary Information, Fig. S1). We conclude that the targetsequence of Ab lies in the internal dA repeats, most probably closeto their N-terminal sequence.Cellular distribution of dA. The distribution of dA was investigat-ed by a variety of techniques, first in PC12-27 cells (Figs 1–4) andthen in CHO, HeLa and other cell types (Fig. 4 and SupplementaryInformation, Fig. S2). Confocal microscopy showed stronglabelling over numerous punctae, preferentially distributed in theouter rim of the cytoplasm. The nucleus was consistently negative.In cell lines such a distribution was evident both in sparsely dis-tributed, rapidly growing cells and in thick monolayers.

Under the electron microscope, the Ab immunogold signal wasweak owing to the presence of glutaraldehyde (0.2%, necessary topreserve ultrastructure) combined with formaldehyde in the fixative.

However, in 32 ultrathin sections of PC12-27 cells (analysed area110 µm2), more than 30% of the immunogold particles were seenin the 0.5-µm-thick rim below the plasmalemma (Fig. 1D), corre-sponding to less than 10% of the total analysed cytoplasm. This rimis rich in small vesicular profiles ∼60 nm in diameter, most of whichwere labelled (Fig. 1Da–c). The remaining gold particles, scatteredover the rest of the cytoplasm (90% of the area), were randomlydistributed. We conclude that the vesicles are specifically labelledand therefore most probably correspond to the positive punctaerevealed by immunofluorescence.

When resting PC12-27 cells, suspended in 0.32 M sucrose sup-plemented with an antiprotease cocktail, were mildly homogenizedin a cell cracker and fractionated by differential centrifugation, dAwas recovered largely in the particulate fractions (P2 and P3 (seeMethods), enriched in mitochondria and microsomes), whereasthe final supernatant (S, containing the cell cytosol) was consis-tently negative (Fig. 1Ba). With harsher homogenization (with aDounce-type homogenizer), a large part of the P2 labelling moved

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Figure 1 Ab antigen identification and distribution. Aa, Western blots of wild-type PC12 and CHO cells together with four PC12 clones, two (Trk and 27) defec-tive and two (16 and 38) competent for regulated secretion. Notice that no Ab-posi-tive band appears in secretion-competent PC12, whereas in Trk, 27 and CHO amajor band with a very large Mr (>600K) is seen. Ab, Strongly Ab-positive westernblot band of the z80 protein product (GST fused to a dA fragment) purified from E.coli transformed with a vector including the corresponding cDNA construct. Thepreparation purified from E. coli transformed with the empty vector (e.v.) is com-pletely negative. Ac, Mass-spectrometric analysis of peptides obtained from Ab-pos-itive spots, derived from immunoprecipitates and separated by 2D PAGE, revealedin all cases sequences from the internal repeats of dA. B–D, Intracellular distribu-tion of dA, in PC12-27 cells, as revealed by subcellular fractionation (B), confocalimmunocytochemistry (red) (C) and immunogold electron microscopy (D). Whenhomogenates were separated by differential ultracentrifugation, the dA western blotband was recovered primarily in the P2 and P3 particulate fractions, whereas thesoluble fraction (S) was completely negative (Ba). In the P1 fraction, labelling wasmuch less intense, concentrated not only in the dA band but also in a few lower-molecular-mass bands (Ba) generated by the digestion of dA by cytosolic proteas-es. This labelling was due not to nuclei but to large membrane sheets (plasmalem-

ma) as revealed by P1 flotation experiments (not shown). The luminal localization ofdA is shown in Bb and Bc. Exposure of the intact P2 plus P3 combined fraction tothe soluble S fraction had no effect on the dA band (Bb, right lane).Permeabilization of the P2 plus P3 fraction and incubation with LB was also ineffec-tive (Bc, left lane). However, when the fraction was previously permeabilized andthen exposed to S (Bc, right lane) the Ab-positive band was converted into a ladder.Similar results were obtained by using trypsin instead of the S fraction (not shown).The distribution of dA in the lumen of cytoplasmic particles was confirmed by theimmunofluorescence data (C). In PC12-27 cells whose plasma membrane had beenpermeabilized with streptolysin-O, the Ab signal was negative (Cb), whereas withantibodies against cytosolic proteins (such as CAMKII, Ca) the signal was promi-nent. Only when the cells pretreated with streptolysin-O were incubated in DB+, withthe resulting permeabilization of the intracellular membranes, strong Ab-positivepunctae appeared in the cytoplasm, concentrated in the subplasmalemma area(Cc). Scale bars, 10 µm (that in Ca also applies to Cb). Conventional electronmicroscopy revealed that the subplasmalemma rim of PC12-27 cytoplasm con-tained numerous small (50–70 nm in diameter) vesicles (arrows in Da), which in LRWhite sections processed for Ab immunogold labelling often appeared positive(arrows in Db and Dc). Scale bar, 0.2 µm (also applies to Db and Dc).


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to P3 (data not shown). In the heavy P1 fraction, labelling was muchless intense, distributed not only in the high-molecular-mass bandbut also in a few smaller bands. When P1, resuspended in 1.8 Msucrose covered with a 0.3 M cushion, was spun for 60 min at100,000g, most dA floated at the interface (data not shown), showingthat its localization is not in the nucleus but in large, light structures,namely plasma membrane sheets. Taken together, immunocyto-chemical and fractionation data consistently indicate the intracellu-lar distribution of the Ab antigen to be cytoplasmic and particulate.

The luminal distribution of dA emerged consistently from bothimmunofluorescence and the analysis of subcellular fractions. First,when the plasma membrane of living cells was permeabilized withstreptolysin-O before application of antibodies (a condition inwhich proteins in, or exposed to, the cytosol are immunolabelled;Fig. 1Ca), no dA was labelled (Fig. 1Cb). Punctate dA labellingappeared only in cells preincubated in detergent-containing dilu-tion buffer (DB+; Fig. 1Cc). Second, in the isolated P2 and P3 frac-tions resuspended without solubilization in either the S fraction(10 min, 4 °C) or a trypsin solution (10 min, 22 °C), the large dAband remained unaffected (Fig. 1Bb). When the same treatmentswere applied to the fractions resuspended in lysis buffer (LB), thelarge band was converted into a ladder, similar to that observed inthe P1 fraction (Fig. 1Cc). In contrast, when the two combined par-ticulate fractions were resuspended in sodium carbonate (0.5 M,pH 11), about 40% of the dA was released (data not shown), asexpected for a protein lacking transmembrane sequences.The dA-containing vesicles are exocytotic. The results reportedhere refer to stimulated PC12-27 cells. Luminal proteins of vesiclescompetent for regulated exocytosis were expected to appear at thesurface when cells are appropriately stimulated. In vivo immunola-belling results showed that this is so with dA (Fig. 2). At rest, thesurface of PC12-27 cells is almost unlabelled by Ab (Fig. 2a). Incontrast, when stimulation with ionomycin induced an increase in[Ca2+]i more than 5 µM, living cells became rapidly (less than 1min) immunolabelled (Fig. 2b), showing a pattern coinciding with

a lectin surface marker (Fig. 2c). Consistently, a stimulated-versus-resting shift was observed in living PC12-27 cells analysed by fluores-cence-activated cell sorting (FACS) for Ab binding (seeSupplementary Information, Fig. S3D). In contrast, [Ca2+]i increasesof less than 1 µM, induced by ionomycin administered to cells eitherpreloaded with bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetra-aceticacid (BAPTA) (30 min incubation with 50 nM BAPTA acetoxymethylester) or bathed in a calcium-free-EGTA medium, failed to induce anyincrease in surface immunolabelling (Supplementary Information,Fig. S2E–H). The ionomycin-induced surface labelling was long-last-ing, with no appreciable decrease at 30 min after ionophore applica-tion (data not shown). Together with capacitance data5, this resultsuggests that the recycling of exocytotic vesicle membrane occursslowly.

Attempts to immunolabel patch-clamped PC12-27 cells wereunsuccessful. In contrast, experiments of caged-calcium photolysis,the technique employed in capacitance experiments5 to induce fast[Ca2+]i jumps, were quite revealing. Sparse monolayers, previouslyloaded with nitrophenyl EGTA (NP-EGTA), were flashed with axenon arc lamp, transferred to ice, processed for Ab immunofluo-rescence and finally fixed. Single flashed cells showed promptswelling, as expected for an exocytotic burst. Concomitantly, a dAsignal appeared at the surface, a much stronger one than thatinduced by ionomycin (compare Fig. 2d with Fig. 2b). Non-flashedcells, in which [Ca2+]i remained at the resting level, failed to showany increase in surface signal. When the images of flashed cells weremathematically deconvoluted, the labelling appeared punctate, asexpected after exocytosis of membrane-bound vesicles (Fig. 2e; seealso Supplementary Information, Fig. S3). The photolysis-inducedsurface transfer of dA was unaffected by tetanus toxin; however, itwas active in PC12-27 cells, as revealed by the cleavage of a target,cellubrevin (data not shown).

Exocytotic transfer of dA to the PC12-27 plasma membrane wasalso confirmed by subcellular fractionation and by the surfacebiotinylation of dA. For the former, the dA patterns of stimulatedand resting cells were similar in P2 and P3 (large bands) and S (notrace of dA) (Fig. 2h). In contrast, the plasma membrane labellingof P1 was much more prominent and was distributed as a ladder ofbands resembling that observed after the proteolysis of solubilizedP2–P3 (compare Fig. 2h with Fig. 1Bc). The result is not surprisingbecause plasma membrane sheets are not sealed, and surface pro-teins therefore remain in direct contact with cytosolic proteases.For surface biotinylation of dA, resting and ionomycin-stimulatedPC12-27 cells were cooled, biotinylated on the surface and thenlysed. Immunoprecipitates were probed with either Ab or strepta-vidin, to reveal total and surface-exposed dA, respectively.Stimulation induced a large (almost fivefold; Fig. 2g) increase inthe surface-exposed band, with no change in the total pool(Fig. 2f).Unique nature of the new exocytotic vesicles. Confocal analysis ofPC12-27 cells doubly labelled with Ab together with antibodies oragents specific for various cytoplasmic organelles failed to revealany consistent overlap. In particular, the distribution of the dA-positive punctae in the proximity of the plasmalemma did notsuperimpose the endoplasmic reticulum network, revealed by cal-reticulin (Fig. 3a), the Golgi complex and trans-Golgi networkstained with mannosidase II and tgn38, respectively (Fig. 3b, c),early and recycling endosomes (EEA1 and transferrin receptor,Fig. 3d, e), late endosomes, multivesicular bodies and lysosomes(lamp1 (ref. 11) and Lucifer yellow; Fig. 3f, g), the insulin-sensi-tive Glut4 vesicles (insulin-regulated aminopeptidase (IRAP)12;Fig. 3h), and vesicles of constitutive secretion (labelled by thetransfected green fluorescent protein (GFP)–chromogranin B chi-maera13 that in PC12-27 cells, in contrast to wild-type PC12, issecreted via the constitutive pathway14; Fig. 3i). Finally, dA-marked vesicles exclude DAMP (β-(2,4-dinitroanilino)-3´-amino-N-methyldipropylamine) and are therefore not acidic (datanot shown).

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Figure 2 Surface distribution of dA in stimulated PC12-27 cells. In intactPC12-27 cells, surface immunofluorescence labelling with Ab was almost impercep-tible at rest (a) and rose considerably after treatment with ionomycin in calcium-containing medium (b). In ionomycin-treated cells (c), the surface distribution of dA(green) coincided largely with that of the plasmalemma-directed lectin fromLycopersicon esculentum (red). The appearance of dA on the surface in ionomycin-treated PC12-27 cells was also supported by surface biotinylation, showing analmost fivefold increase in the surface signal with respect to resting cells (right andleft bands in g) with no change in the total dA pool (corresponding bands in f), andby subcellular fractionation, showing a large increase of the signal in the plasmamembrane sheets of the P1 fraction (compare the western blots of h with those ofresting cells in Fig. 1Ba). When stimulation was not by ionomycin but by photolysisof caged calcium, PC12-27 cells promptly increased in volume and the surfaceappearance of dA was extensive (d). When analysed by deconvolution, the dAlabelling of photolysed cells was found to consist of numerous small dots (e), asexpected for an exocytosis-generated signal. Scale bars, 10 µm (that in b alsoapplies to a, and that in e also applies to c and d).


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Expression and functions of the new vesicles. The expression andsurface translocation of dA after stimulation occur not only inPC12-27 cells but also in other lines: CHO (Fig. 4Bb–d), Rin, HeLa(Fig. 4A, Ba) and others (see Supplementary Information, Fig. S2).

Similarly, various primary cultures (dendritic cells and neurons;Fig. 4Ca–c) are dA-positive. In various organs of the rat, expressionis widespread but not ubiquitous (see Fig. 4A). In adult rat tissues,dA is present in various cell types. In the pancreas, acinar cells arelabelled moderately, whereas a minor component of islets, distinctfrom insulin and glucagon-secreting cells, is strongly labelled (Fig.4Cd and Supplementary Information, Fig. S2D).

In the same cell type, dA expression can change during differen-tiation. The positivity of human dendritic cells was reinforced bytreatment with tumour necrosis factor-α or lipopolysaccharide15

(Fig. 4Ca, b). More interestingly, dA-negative wild-type and clonalPC12 accumulate the marker beginning at 6 h after the application ofnerve growth factor (NGF) (Fig. 4Da, b). In these cells, dA-positivevesicles are distinct from other organelles, in particular dense gran-ules (chromogranin B; data not shown) and clear vesicles (synapto-physin; Fig. 4Dc). The distinction between exocytotic organelles ofthe classical and new systems was confirmed in differentiated wild-type PC12 cells treated with tetanus toxin, stimulated with iono-mycin and then surface-labelled for both synaptotagmin I and dA. Infact, the appearance of synaptotagmin I decreased considerably (−40%), whereas that of dA was unchanged, confirming the insensi-tivity of the new system to toxin (Fig. 4E). In fully differentiated cellsof both wild-type PC12 and PC12-27 cells, dA-positive vesicles accu-mulate in neurites, especially at growth cones (Fig. 4Dd). Exocytosisof new vesicles, although distributed over the whole plasmalemma,therefore seems to be more concentrated at sites where processes ofmembrane expansion take place.

To investigate the possible involvement of dA-positive vesicles inplasmalemma wound healing16,17, we exposed living PC12-27 cells tovarious injuries (including scraping and micropipette puncturing)followed, after 1 min at 22 °C for resealing, by Ab labelling. Theresults showed surface dA appearance in most wounded cells (datanot shown). After electroporation, which induces many small holesin the plasmalemma, cells were all surface-positive (Fig. 4Fa, b)except for a few cells that were probably dead before immunola-belling which showed intense positivity for the extracellular mark-er tetramethylrhodamine β-isothiocyanate (TRITC)–dextran. Inparallel experiments, resealed PC12-27 cells, doubly labelled for dAand the lysosome marker lamp1, showed strong surface labellingfor the first but not for the second antibody (Fig. 4Fc), whichremained distributed in punctae throughout the cytoplasm(Fig. 4Fd).

DiscussionThe principal tool of our work has been Ab, a monoclonal antibodyagainst dA, the very-high-molecular-mass protein that appears atthe surface of PC12-27 cells after stimulation by ionomycin or, evenmore, after photolysis of a calcium caged compound. The identifi-cation of dA was based on multiple criteria: peptide sequencing;specific Ab labelling of a protein in E. coli transformed with a dAconstruct; and competition of Ab with KIS10, another antibodyagainst dA. By the use of Ab, dA was shown to be localized withinsmall cytoplasmic vesicles that, after appropriate increases in[Ca2+]i, undergo exocytotic fusion. After cell stimulation, no dA isrecovered in the incubation medium (data not shown). Because theprotein seems to be devoid of transmembrane sequences, its per-sistence and the ensuing slow recycling to the cytoplasm is mostprobably dependent on specific interaction(s) with other vesiclecomponent(s). These results nicely complement previous dataobtained by capacitance assays3–5. dA is therefore the first cell bio-logical marker of a regulated exocytosis system that was previouslyknown only on the basis of electrophysiology.

The vesicular localization of dA was unexpected because theprotein, cloned almost 10 years ago, had been reported at many otherintracellular sites: associated with desmosomes and particles in theGolgi area, within the nucleus and/or the cytosol, attached to the plas-malemma and in transit to and from the above compartments8,10,18–20.

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Figure 3 The intracellular distribution of dA is unique. The intracellular distribu-tion of dA does not coincide with that of any known cytoplasmic organelles. In thisfigure the left panels (red) show dA, the middle panels (green) the markers of theorganelles specified below, revealed by antibodies against specific markers (a–f, h),a dye (g) and a transfected secretory-protein–GFP construct (i). The right panelsshow the merged images. a, Endoplasmic reticulum (calreticulin); b, Golgi complex(mannosidase II); c, trans-Golgi network (tgn38); d, early endosomes (EEA1); e, recy-cling endosomes (transferrin receptor); f, g, late endosomes and lysosomes (lamp1and Lucifer yellow), respectively; h, Glut4 vesicles (IRAP); I, constitutive secretion(GFP–chromogranin B). Scale bar, 10 µm (applies to all panels).


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However, part of the previous data were obtained by cell-freeexperiments in which dA, isolated from detergent-lysed cells, wasincubated with other proteins or DNA21–23, whereas others were

based on the expression of truncated constructs19,22,23. Additional,extensive studies were conducted with the parallel use of polyclon-al antibodies raised against dA peptides8,10,18,20. One such antibody,

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Figure 4 Expression, intracellular distribution and exocytosis of dA in cellsand tissues; effects of differentiation and plasma membrane lesion. A, Abwestern blots of cells and adult rat tissues. Sr, adrenal gland; Br, brain; Cbl, cere-bellum; Hrt, heart; Mus, skeletal muscle; Pcrs, pancreas; Kdn, kidney. B, dA imm-munolabelling (red) of resting HeLa (a) and CHO (b) cells permeabilized with DB+,and intact CHO cells, surface-labelled before (c) and after (d) stimulation with iono-mycin. C, dA immunolabelling (red) of cells permeabilized with DB+. a, b, Humandendritic cells, before (a) and after (b) differentiation by lipopolysaccharide, docu-mented also by the appearance of the specific antigen, B7.1 (green). c, Rat hip-pocampal neuron in culture. d, Rat pancreas, showing low dA labelling (red) of aci-nar cells, intense labelling of an unidentified cell type within and around the islet,and negativity of most islet cells positive for the secretory vesicle protein synapto-physin (green). Da–c, DB+-permeabilized wild-type PC12 cells, competent for regu-lated secretion, immunolabelled with Ab (red) before (a) and after (b, c) NGF-induced differentiation. Notice the appearance in the latter of dA punctae that are,however, distinct from the conventional secretory vesicles because they do notcoincide with the punctae positive for their specific markers, synaptophysin (green,c) and chromogranin B (not shown). d, NGF-differentiated PC12-27 cells immunola-

belled for dA after permeabilization with DB+ buffer. E, Wild-type PC12 differentiatedwith NGF and stimulated with ionomycin: surface labelling with both Ab (red) andanti-synaptotagmin I antibody (green). The cells in a had received no pretreatment,whereas those in b had been pretreated with tetanus toxin (TeNT). c, Quantitativedeconvolution analyses, expressed as arbitrary fluorescence units and shown asmeans ± s.d., performed in a total of 30 pretreated and 30 non-pretreated cells.STG, synaptotagmin. Fa, b, PC12-27 cells electroporated in the presence of TRITC-dextran (red), then resealed and surface-immunolabelled with Ab (green). Most cellsappear surface-positive for dA, often without co-labelling with TRITC. In contrast, thecells strongly positive for the latter are often dA-negative and might already havebeen dead before immunolabelling. c, Surface immunolabelling of electroporatedPC12-27 cells for dA (green) and the lysosomal membrane marker lamp1 (red). Thelatter is not evident at the cell surface. d, As in c, except that cells were permeabi-lized in DB+ before immunolabelling. Lamp1-positive punctae (red) are numerouswithin the cytoplasm and do not coincide in distribution with dA (green), which ismostly at the surface and in the subplasmalemma rim of the cytoplasm. Scalebars, 10 µm (that in Ba also applies to Bb–d and E; that in Ca also applies to Cb,Dc and F; that in Cc also applies to Cd, Da, Db and Dd).


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KIS, when employed for immunofluorescence (as described inMethods), failed to induce any specific staining even in dA-positivecells. In contrast, when KIS was used for western blotting, theresults were fully consistent with those with Ab, in terms of boththe cell expression and subcellular distribution of dA. In contrast,immunocytochemical results obtained with another antibody,FEN10, were completely inconsistent. In agreement with previousresults8,18,20, we found strong labelling of nuclei (never labelled byAb) accompanied by weak decoration of cytoplasmic punctae dis-tinct from those labelled by Ab. Moreover, labelling with FEN wasnot restricted to Ab-positive cells but extended to others, apparent-ly Ab- and KIS-negative, such as the undifferentiated wild-typePC12 cells employed in our laboratory. We therefore consider thatFEN (but not KIS) is specific for, or at least cross-reacts with,another antigen distinct from dA.

The existence of non-classical routes of exocytosis, needed forthe surface transfer of important proteins (such as receptors, chan-nels and transporters24–28), and distinct from both regulated secre-tion and constitutive cycling to and from the plasma membrane,has emerged during the past several years. However, the possibilitythat dA-specific exocytosis corresponds to one or more of the aboveprocesses seems unlikely because of their distinct properties,including slower time constants and smaller cumulative membraneareas. The new system also seems to be distinct from secretory lyso-somes, that is, exocytotic organelles similar to the acidic granules ofblood leukocytes29, which have been described in some but not allcells6,29. dA vesicles are much smaller than lysosomes, are not acidicand are negative for lysosomal markers. Finally, dA-marked vesiclesare distinct from clear and dense vesicles of neurosecretory cells. Infact, within wild-type PC12 cells treated with NGF, the two familiesof organelles exhibit distinct intracellular localizations, and theirexocytosis is differentially affected by tetanus toxin: inhibition ofneurosecretion and no change in dA surface translocation, respec-tively. The co-expression of distinct families of regulated exocytot-ic organelles might occur in many cell types. Capacitance assays inmast cells and pancreatic acinar cells have revealed exocytosis ofsmall vesicles together with that of typical large granules30,31, where-as in chromaffin cells classical neurosecretion is accompanied by aminor, calcium-dependent exocytotic process, unaffected byclostridial neurotoxins32.

Further information about dA-positive vesicles was obtained bydual-labelling experiments with Ab and antibodies specific forother cytoplasmic organelles. Consistent with their exocytotic rolewas the observation that dA-positive vesicles are concentrated inthe cytoplasmic rim below the plasmalemma, an area from whichother organelles (endoplasmic reticulum, Golgi and trans-Golgielements) are, in contrast, largely excluded. With regard to endo-somes at various stages of maturation, and also multivesicular bod-ies and lysosomes, dA vesicles were distinguished both by localiza-tion and by pH (dA-marked organelles are not acidic). In addition,Glut4 and constitutive secretion vesicles do not coincide with dAvesicles. We suggest that the novel, rapid exocytosis system is sus-tained by a new class of molecularly distinct organelles.

The spectrum of cells and tissues rich in dA is broad, rangingfrom the brain to the exocrine and endocrine glands, from viscerato lymphoid organs. However, within adult rat tissues its expressionis not ubiquitous and even in single cell types dA levels can be vari-able. Wild-type PC12 cells, and also antigen-presenting dendriticcells and neuroblastoma10 cells, although devoid of or poor in dAwhen undifferentiated, begin to accumulate the marker shortlyafter exposure to the differentiating conditions, and transfer it tothe surface when stimulated. Expression of the new system mighttherefore not be a stable property of specific cell types, but could beacquired (or increased) transiently at some stages.

The differentiation-induced expression of dA vesicles, togetherwith the slow post-exocytotic recycling of their membranes, sug-gests their involvement in cell surface enlargement, a function thatin neurite extension has already been assigned to a still-unidentified

vesicle system33. In non-nerve cells, membrane additions for surfaceexpansion, although necessary for key processes such as phagocy-tosis and locomotion34,35, have not yet been widely investigated.

Wound healing is a ubiquitous process performed by exocytosedmembranes16,36. So far, the involvement of various types oforganelle has been claimed: specific large granules in oocytes16,36,secretory lysosomes in NRK cells17, endosomes in 3T3 fibroblasts37

and synaptotagmin-positive vesicles in wild-type PC12 cells38.Other healing organelles are expected to exist6. In PC12-27 cellswounded by various treatments, dA was always seen to appear onthe surface, whereas the lysosomal–endosomal marker lamp1 wasnot detected. Taken together, the present and previous results seemto suggest that healing is due to the exocytosis of not merely onebut several types of organelle competent for rapid exocytosis,whose expression can vary in different types of cell.

In conclusion, the list of cytoplasmic organelles needs to beexpanded to include a new type of vesicle competent for rapid, cal-cium-dependent exocytosis. This mechanism of surface plasticityseems to operate in a variety of cell types. Although the informa-tion, especially that relating to the functional role, is not yet com-plete, it is clear that fusion of these vesicles induces an enlargementof the plasmalemma. We therefore propose that they be called‘enlargosomes’.

MethodsMaterialsThe enhanced chemiluminescence detection system, protein G– and protein A–Sepharose agarose

beads, 2D strips, IPGphor system and pGEX-4T-1 vector were from Amersham Biosciences (Uppsala,

Sweden); the bicinchoninic acid protein assay reagent, sulpho-N-hydroxysuccinimide (NHS)-LC-

biotin, sulpho-NHS-SS-biotin and streptavidin–agarose beads were from Pierce (Rockford, IL); Lucifer

yellow was from Molecular Probes (Leiden, The Netherlands); the TRITC-labelled Lycopersicon escu-

lentum lectin, streptolysin-O and TRITC-labelled dextran were from Sigma (St Louis, MO); and NGF

was from Alamone labs (Jerusalem, Israel).

The purified tetanus toxin was a gift from C. Montecucco. The CgB–GFP plasmid13 and the pGEX-

4T-1-z80 vector containing the AHNAK fragment9 were kindly provided by H. H. Gerdes and F.

Sköldberg, respectively.

The following antibodies were obtained as gifts: rabbit polyclonal anti-AHNAK KIS and FEN pep-

tides from E. Shtivelman; rabbit polyclonal anti-mannosidase II from M. Farquhar; rabbit polyclonal

anti-EEA1 from A. D’Arrigo; rabbit polyclonal anti-CaMKII and anti-synaptophysin from F. Valtorta;

rabbit polyclonal anti-IRAP from P. Pilch; mouse-monoclonal IgG1 anti-lamp1 from I. Mellman; rab-

bit polyclonal anti-synaptotagmin I (luminal epitope) from A. Malgaroli; and anti-chromogranin B

from P. Rosa.

The following antibodies were from commercial sources: rabbit polyclonal anti-calreticulin and

mouse monoclonal IgG1 anti-TGN38 from Affinity BioReagents (Golden, CO); mouse monoclonal

IgG1 anti-transferrin receptor, goat anti-mouse IgGγ, IgG(H+L) and IgM (all conjugated with alkaline

phosphatase) from Zymed (San Francisco, CA); mouse monoclonal anti-human B7.1 from BD

PharMingen (San Diego, CA); peroxidase-conjugated goat anti-mouse and goat anti-rabbit antibodies

from Bio-Rad (Hercules, CA); fluorescein isothiocyanate-conjugated and rhodamine-conjugated goat

anti-mouse and goat anti-rabbit antibodies, and colloidal gold particles (12 nm) coated with anti-

mouse IgG from Jackson Laboratories (West Grove, PA); and goat anti-mouse IgG subclasses from

Southern Biotechnology Associated (Birmingham, AL).

The composition of lysis buffer (LB) was 50 mM HEPES pH 7.5, 150 mM NaCl, 15 mM MgCl2, 1

mM EGTA, 10% glycerol and 1% Triton X-100, and that of dilution buffer (DB+) was PBS plus 1%

BSA, 0.3% Triton X-100 and 20% goat serum. Dilution buffer without Triton is indicated as DB−. Tris-

buffered saline (TBS) contained 200 mM NaCl, 0.5% Tween 20 and 50 mM Tris, pH 7.4. Cytomix

buffer contained 120 mM KCl, 0.15 mM CaCl2, 10 mM potassium phosphate, 2 mM EGTA, 5 mM

MgCl2 and 25 mM HEPES, pH 7.6.

Cell culturesAll cells were grown at 37 °C in a humidified 5% CO2 atmosphere, using media supplemented with 2

mM L-glutamine and 100 U ml−1 penicillin, streptomycin (Biowhittaker, Verviers, Belgium). In particu-

lar, PC12 wild-type and cell clones were grown in Dulbecco’s modified Eagle’s medium supplemented

with 10% horse serum (Euroclone, Wetherby, UK) and 5% fetal clone III serum (Hyclone, Logan, UT);

hybridoma cells in Iscove’s MDM medium with 10% fetal clone I serum, 5% macrophage conditioned

medium (MCM) and 50 µM β-mercaptoethanol. PC12 differentiation was induced by NGF39.

Cultured rat hippocampal neurons and human blood-derived dendritic cells were generously provided

by A. Bergamaschi and C. Paolucci (San Raffaele Institute, Milan, Italy), respectively.

Isolation of exocytosed membrane proteinsPC12-27 cell suspensions, washed twice in PBS at 4 °C, were resuspended and incubated at 10 °C for

30 min in the presence of 2 mg ml−1 sulpho-NHS-LC-biotin (Pierce). After three washes in chilled PBS

containing 1% BSA (Boehringer Mannheim, Germany), they were incubated at 37 °C for 5 min in PBS

with 15 mM CaCl2 (PBS+) supplemented with 5 µM ionomycin to stimulate exocytosis, then chilled

again on ice and exposed for 30 min to 2 mg ml−1 sulpho-NHS-SS-biotin40. After two washes in PBS

supplemented with 1% BSA, and two washes in PBS, cells were solubilized in LB. Nuclei were removed


articles


by low-speed centrifugation at 4 °C (10 min). Cell lysate supernatants were then incubated for 1 h

(gentle mixing) with equal volumes of streptavidin–agarose beads (Pierce) at 4 °C, then washed three

times in PBS. The biotinylated plasma membrane proteins bound to the beads were then incubated for

30 min in PBS containing 50 mM β-mercaptoethanol at 4 °C, and then centrifuged. The supernatant,

containing proteins that appeared at the surface during the exocytotic event, was recovered and used to

immunize mice.

Immunization and hybridoma productionTwo 4-week-old female Balb-C mice were injected twice (with a 2 wk interval) in the footpad with the

antigen pool diluted in PBS and emulsified in Freund’s adjuvant (the first time complete, the second

incomplete). At 16 days after the first injection, animals were first bled (to perform preliminary analy-

ses of sera with ELISA and western blots of wild-type PC12 and PC12-27 cells) and then given a third

boost of antigen in PBS. The animals were killed 3 d later. Lymphocyte fusion with P3X63Ag8 myelo-

ma cells was performed as recommended41. Fused cells were centrifuged and resuspended in hybrido-

ma complete medium, seeded in 96-well plates (105 cells per well), and selected with HAT (hypoxan-

thine, aminopterin, thymidine) medium.

Antibody purification and screeningPC12-27 cells (104) were seeded on microtitre plates precoated with 10 µg ml−1 poly-(L-lysine) in PBS,

stimulated with 5 µM ionomycin in PBS+ for 5 min, then centrifuged and fixed with 4% paraformalde-

hyde. After blocking for 1 h with PBS plus 1% BSA, the wells were incubated with hybridoma super-

natants for 2 h at 37 °C, washed, and incubated with 1 µg ml−1 goat anti-mouse IgG or IgM, both con-

jugated with alkaline phosphatase. Hydrolysis of p-nitrophenyl phosphate was measured with an

ELISA reader filtered at 405 nm. Positive hybridomas were tested three times, then cloned by limiting

dilution to obtain monoclonal cultures. Antibodies were purified either on hydroxyapatite columns

(Bio-Rad), or by affinity chromatography on protein G cartridges (Amersham Biosciences) in accor-

dance with the manufacturer’s instructions.

Western blottingWashed cells were solubilized in ice-cold LB, then quickly centrifuged at 20,000g for 5 min to sediment

nuclei, which were discarded. Adult rat tissues were homogenized in 0.32 M sucrose, 5 mM HEPES pH

7.4 supplemented with a protease inhibitor mixture (Sigma), with a Dounce hand homogenizer.

Protein concentration was determined with the bicinchoninic acid assay. Fixed amounts of protein

were separated by SDS–polyacrylamide gel electrophoresis, then transferred to nitrocellulose filters

that were processed at 22 °C. Filters were first blocked for 1 h with 5% non-fat dry milk in TBS, then

incubated for 3 h with the primary antibody diluted in PBS plus 3% BSA, washed five times for 10 min

in TBS, incubated for 1 h with the appropriate peroxidase-conjugated secondary antibody (1 µg ml−1),

washed five times for 10 min in TBS and once in PBS, and developed photographically by chemilumi-

nescence.

Subcellular fractionation and protein digestionPBS-washed preparations of PC12-27 cells, resting and pretreated with ionomycin (5 µM, 5 min) were

suspended at 4 °C in a mixture containing 0.32 M sucrose, 5 mM HEPES pH 7.4, and protease

inhibitor cocktail. After gentle homogenization by 20 passages in a cell cracker (from EMBL; clearance

12 µm), the suspensions were centrifuged for 10 min at 1,000g to obtain P1, which was solubilized in

LB and cleared by centrifugation at 20,000g for 5 min to sediment nuclear debris. Alternatively, P1 was

resuspended in 1.8 M sucrose, transferred to a centrifuge tube and covered with 0.3 M sucrose. After

centrifugation at 100,00g for 1 h, the floating band, containing plasma membrane sheets, was recov-

ered at the interface, diluted to 0.3 M sucrose, sedimented by centrifugation and analysed. The super-

natants of the first, low-speed centrifugation were further centrifuged in sequence, first at 10,000g for

10 min to obtain the P2 pellet, then at 100,000g for 1 h to obtain the P3 pellet and the final super-

natant, S. All fractions were analysed by western blotting.

For digestion experiments in vitro, mixtures of the P2 and P3 fractions were first incubated for

10 min with either trypsin (10 µg ml−1, 22 °C) or S (4 °C), with or without LB containing 0.2% Triton

X-100 (final concentration), and then analysed as above.

Immunofluorescence and immunogoldCells plated on poly-(L-lysine)-coated coverslips were fixed for 20 min on ice in 4% formaldehyde dis-

solved in PBS pH 7.4, quenched with 0.1 M glycine, washed in DB+ and transferred to 22 °C. After

exposure for 2 h to the primary antibodies in DB+ they were washed extensively in PBS, exposed for

2 h to fluorescein isothiocyanate-conjugated or TRITC-conjugated goat anti-mouse or anti-rabbit sec-

ondary antibodies, incubated for 1 h in DB+, washed, mounted, and finally analysed in a laser scanning

confocal microscope (Bio-Rad MRC 1024).

For plasma membrane permeabilization, cells plated on coverslips were incubated on ice with 0.5 U

ml−1 streptolysin-O in PBS, then warmed (5 min at 37 °C) to induce pore formation. After extensive

washing with PBS, the monolayers were either incubated with the primary antibody diluted in DB−

(30 min on ice) and then fixed and quenched, or directly fixed, quenched and then exposed to the pri-

mary antibody in DB+. After being washed and exposed to the second antibody in the same buffers, the

monolayers were processed as above.

For surface immunolabelling, monolayers of living cells, covered with PBS+ and kept at 22 °C, were

treated with ionomycin (5 µM, 5 min) alone or after loading with BAPTA (BAPTA-AM, 50 µM,

15 min). Additional monolayers were processed while covered with calcium-free PBS supplemented

with 1 mM EGTA. After transfer to 4 °C they were incubated (30 min on ice) with the primary anti-

bodies diluted in DB−, then washed, fixed in formaldehyde for 20 min, quenched with glycine, exposed

to the second antibody in DB+ and processed as above. In parallel experiments, suspensions of PC12-

27 cells, at rest or treated with ionomycin, as indicated previously, were quickly chilled and incubated

at 4 °C with Ab diluted in DB−. After washing, the fluorescent surface staining of the population was

evaluated by FACS analysis.

For combined surface labelling with Ab and the TRITC-labelled Lycopersicon esculentum lectin, the

latter was included in the primary antibody solution. After incubation in ice for 30 min and washing,

the monolayers were fixed and processed as described in the preceding paragraph.

For caged-calcium experiments, cells plated on poly-(L-lysine)-coated coverslips and loaded for

45 min with 10 µg ml−1 NP-EGTA-AM compound (Molecular Probes) were flashed with an ultraviolet

xenon lamp to induce exocytosis42. Experiments and imaging were carried out in the laborarory of M.

Rupnik at the European Neuroscience Institute, Göttingen. Where indicated, the same experiments

were performed after a 24-h preincubation of the cells with tetanus toxin (100 nM). The latter condi-

tions were chosen on the basis of the results of preliminary experiments showing in PC12-27 cells a

decrease (about −40%) in a protein target of the toxin, cellubrevin, as revealed by western blotting, and

in wild-type PC12 cells a decreased appearance to the surface of synaptotagmin I, after 5 min stimula-

tion with either ionomycin or 50 mM KCl, as revealed by immunofluorescence. The surface appear-

ance of both dA and synaptotagmin I was investigated concomitantly by dual surface labelling in cells

expressing both classical and new exocytotic systems, namely NGF-differentiated wild-type PC12 cells,

treated or not for 24 h with tetanus toxin (100 nM) and stimulated with ionomycin as specified above.

Quantification of the results was made after deconvolution analysis (see below).

Optical sections taken every 150 nm with a wide-field microscope were deconvoluted with the

softWoRx Deconvolve software (Applied Precision) on the Delta Vision System, to remove the blur in

fluorescence and to perform three-dimensional reconstruction of the images.

For electron microscopy, PC12-27 cells were fixed in 4% formaldehyde plus 0.2% glutaraldehyde

and then processed in parallel for conventional Epon embedding, for LR White resin embedding, and

for freezing. Ultrathin sections of Epon samples were stained; ultrathin sections in LR White and

cryosections were exposed to Ab and then labelled with immunogold43. Controls were ultrathin sec-

tions of wild-type PC12 cells processed as above and of PC12-27 cells exposed to immunogold without

previous treatment with Ab.

Immunoprecipitation and protein biotinylationLysates from PC12-27 cells (1 ml in LB), precleared by the addition of 30 µl of protein G–Sepharose

beads, were incubated overnight with Ab at 4 °C, with gentle mixing, after which 50 µl of protein

G–Sepharose beads was added. After incubation for 2 h at 4 °C, the beads were washed three times in

LB and twice in 10 mM Tris-HCl pH 7.4. Bound proteins were analysed by 2D PAGE staining and

western blotting.

In specific experiments, cell suspensions at rest or after treatment with ionomycin for 5 min were

transferred to ice and labelled with cell-impermeant sulpho-NHS-SS-biotin. After cell lysis in LB, the

preparations were immunoprecipitated with Ab as mentioned above, and the proteins bound to the beads

were analysed by western blotting probed with either Ab, to evaluate the total amount of antigen immuno-

precipitated, or horseradish peroxidase-conjugated avidin, to estimate the surface-biotinylated antigen.

Two-dimensional PAGE, protein expression in E. coli and peptide sequencingTwo-dimensional PAGE analyses of Ab-immunoprecipitated proteins were performed by the IPGphor

method (Amersham Biosciences). After separation in the second dimension, the proteins were either

revealed with silver staining or transferred to nitrocellulose filters. The spots recognized by Ab were

excised, digested in gel with pepsin, processed for mass-spectrometric analysis and sequenced as

described in ref. 44.

The BL21 E. coli, transformed with pGEX-4T-1 empty vector or with the vector containing the z80

construct coding for an AHNAK fragment fused to GST, were induced with isopropyl β-D-thiogalacto-

side as indicated elsewhere9. The GST fusion proteins produced were recovered from crude bacterial

lysates by glutathione–Sepharose 4B resin (Amersham Biosciences) in accordance with the manufac-

turer’s instructions, and labelled in western blots with Ab. Various GST–z80 fragments recognized by

Ab were excised from gels and analysed by mass spectrometry44.

Membrane lesionsMicropipette puncturing and scrape wounding were performed as described previously17.

The protocol for electroporation45 was as follows. PC12-27 cells were suspended in cytomix buffer

supplemented with 2 mM ATP and 5 mM glutathione, in the presence of dextran-TRITC (1 mg ml−1).

Suspensions were electroshocked at 400 V and 125 µF in a 2-cm cuvette placed in a Bio-Rad Gene

Pulser apparatus equipped with a capacitance extender, then transferred to ice. After washing, surface

exposure of the antigen was detected in resealed cells by incubation of coverslips first with Ab with or

without anti-lamp1 antibody (30 min on ice), followed by washing, centrifugation onto slides, fixation

and processing as mentioned above for surface immunolabelling. In parallel experiments the resealed

cells were fixed and permeabilized before exposure to Ab and anti-lamp1, to reveal the intracellular

distribution of the two antigens.

RECEIVED 26 NOVEMBER 2001; REVISED 29 JULY 2002; ACCEPTED 24 OCTOBER 2002; PUBLISHED 25 NOVEMBER 2002.

1. Jahn, R. & Südhof, T. C. Membrane fusion and exocytosis. Annu. Rev. Biochem. 68, 863–911 (1999).

2. Kasai, H. Comparative biology of Ca2+-dependent exocytosis: implications of kinetic diversity for

secretion function. Trends Neurosci. 22, 88–93 (1999).

3. Coorssen, J. R., Schmitt, H. & Almers, W. Ca2+ triggers massive exocytosis in Chinese hamster ovary

cells. EMBO J. 15, 3787–3791 (1996).

4. Ninomiya, Y., Kishimoto, T., Miyashita, Y. & Kasai, H. Ca2+-dependent exocytotic pathways in

Chinese hamster ovary fibroblasts revealed by a caged-Ca2+ compound. J. Biol. Chem. 271,

17751–17754 (1996).

5. Kasai, H. et al. Multiple and diverse exocytosis in wild-type and defective PC12 cells. Proc. Natl

Acad. Sci. USA 96, 945–949 (1999).

6. Andrews, N. W. Regulated secretion of conventional lysosomes. Trends Cell. Biol. 10, 316–321

(2000).

7. Clementi, E., Racchetti, G., Zacchetti, D., Panzeri, M. C. & Meldolesi, J. Differential expression of

markers and activities in a group of PC12 nerve cell clones. Eur. J. Neurosci. 4, 944–953 (1992).

8. Sussman, J., Stokoe, D., Ossina, N. & Shtivelmann, E. Protein kinase B phosphorylates AHNAK and

regulates its subcellular localization. J. Cell Biol. 154, 1019–1030 (2001).

9. Sköldberg, F. et al. Identification of AHNAK as a novel autoantigen in systemic lupus erythemato-

sus. Biochem. Biophys. Res. Commun. 291, 951–958 (2002).

10. Shtivelman, E. & Bishop, J. M. The human gene AHNAK encodes a large phosphoprotein located

primarily in the nucleus. J. Cell Biol. 120, 625–630 (1993).


articles


11. Piper, R. C. & Luzio, J. P. Late endosomes: sorting and partitioning in multivesicular bodies. Traffic

2, 612–621 (2001).

12. Thoidis, G. & Kandrov, K. V. A Glut4-vesicle marker protein, insulin-responsive aminopeptidase, is

localized in a novel vesicular compartment in PC12 cells. Traffic 2, 577–587 (2001).

13. Kaether, C., Salm, T., Glombik, M., Almers, W. & Gerdes, H. H. Targeting of green fluorescent pro-

tein to neuroendocrine secretory granules: a new tool for real time studies of regulated protein

secretion. Eur. J. Cell Biol. 74, 133–142 (1997).

14. Malosio, M. L. et al. Neurosecretory cells without neurosecretion: evidence of an independently

regulated trait of the cell phenotype. J. Physiol. (Lond.) 520, 43–52 (1999).

15. Paolucci, P., Rovere, P., De Nadai, C., Manfredi, A. A. & Clementi, E. Nitric oxide inhibits the tumor

necrosis factor alpha-regulated endocytosis of human dendritic cells in a cyclic GMP-dependent

way. J. Biol. Chem. 275, 19638–19644 (2000).

16. McNeil, P. L. & Terasaki, M. Coping with the inevitable: how cells repair a torn surface membrane.

Nature Cell Biol. 3, E124–E129 (2001).

17. Reddy, A., Caler, E. V. & Andrews, N. W. Plasma membrane repair is mediated by Ca2+-regulated

exocytosis of lysosomes. Cell 106, 157–169 (2001).

18. Hashimoto, T. et al. Desmoyokin, a 280 kDa keratinocyte plasma membrane-associated protein, is

homologous to the protein encoded by human gene AHNAK. J. Cell Sci. 105, 275–286 (1993).

19. Nie, Z., Ning, W., Amagai, M. & Hashimoto, T. C-terminus of desmoyokin-AHNAK protein is

responsible for its translocation between the nucleus and cytoplasm. J. Invest. Dermatol. 114,

1044–1049 (2000).

20. Hashimoto, T., Gamou, S., Shimizu, N., Kitajima, Y. & Nishikawa, T. Regulation of translocation of

the desmoyokin-AHNAK protein to the plasma membrane in keratinocytes by protein kinase C.

Exp. Cell Res. 217, 258–266 (1995).

21. Haase, H. et al. Signalling from β-adrenoreceptor to L-type calcium channel: identification of a

novel cardiac protein kinase A target possessing similarities to AHNAK. FASEB J. 13, 2161–2172

(1999).

22. Sekiya, F., Bae, Y. S., Jhon, D. Y., Hwang, S. C. & Rhee, S. G. AHNAK, a protein that binds and activates

phospholipase C-γ1 in the presence of arachidonic acid. J. Biol. Chem. 274, 13900–13907 (1999).

23. Gentil, B. J. et al. The giant protein AHNAK is a specific target for the calcium- and zinc-binding

S100B protein. J. Biol. Chem. 276, 23253–23261 (2001).

24. Lu, W. et al. Activation of synaptic NMDA receptors induces membrane insertion of new AMPA

receptors and LTP in cultured hippocampal neurons. Neuron 29, 243–254 (2001).

25. Ward, D. T., Hammond, T. G. & Harris, H. W. Modulation of vasopressin-elicited water transport

by trafficking of aquaporin2-containing vesicles. Annu. Rev. Physiol. 61, 683–697 (1999).

26. Whitehead, J. P. et al. The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells. J.

Biol. Chem. 276, 27816–27824 (2001).

27. Simpson, F., Whitehead, J. P. & James, D. E. GLUT4—at the cross roads between membrane traf-

ficking and signal transduction. Traffic 2, 2–11 (2001).

28. Banerjee, A., Shih, T., Alexander, E. A. & Schwartz, J. H. SNARE proteins regulate H+-ATPase redis-

tribution to the apical membrane in rat renal inner medullary collecting duct cells. J. Biol. Chem.

274, 26518–26522 (1999).

29. Blott, E. J. & Griffiths, G. M. Secretory lysosomes. Nature Rev. Mol. Cell Biol. 3, 122–131 (2002).

30. Almers, W. & Neher, E. Gradual and stepwise changes in the membrane capacitance of rat peri-

toneal mast cells. J. Physiol. (Lond.) 386, 205–217 (1987).

31. Thomas, P. et al. Two types of exocytosis observed in single, rat pancreatic acinar cells. J. Physiol.

(Lond.) (in the press).

32. Xu, T., Binz, T., Niemann, H. & Neher, E. Multiple kinetic components of exocytosis distinguished

by neurotoxin sensitivity. Nature Neurosci. 1, 192–200 (1998).

33. Coco, S. et al. Subcellular localization of tetanus neurotoxin-insensitive vesicle-associated mem-

brane protein (VAMP)-VAMP7 in neuronal cells: evidence for a novel membrane compartment. J.

Neurosci. 19, 9803–9812 (1999).

34. Hackam, D. J. et al. v-SNARE-dependent secretion is required for phagocytosis. Proc. Natl Acad. Sci.

USA 95, 11691–11696 (1998).

35. Mellman, I. Quo vadis: polarized membrane recycling in motility and phagocytosis. J. Cell Biol. 49,

529–530 (2000).

36. Steinhardt, R. A., Bi, G. & Alderton, J. M. Cell membrane resealing by vesicular mechanism similar

to neurotransmitter release. Science 263, 390–393 (1994).

37. Togo, T., Alderton, J. M., Bi, G. O. & Steinhardt, R. A. The mechanism of facilitated cell membrane

resealing. J. Cell Sci. 112, 719–731 (1999).

38. Detrait, E. R. et al. Plasmalemmal repair of severed neurites of PC12 cells requires Ca2+ and synap-

totagmin. J. Neurosci. Res. 62, 566–573 (2000).

39. Leoni, C. et al. Neurite extension occurs in the absence of regulated exocytosis in PC12 subclones.

Mol. Biol. Cell 10, 2919–2931 (1999).

40. Schmidt, A., Hannah, M. J. & Huttner, W. B. Synaptic-like microvesicles of neuroendocrine cells

originate from a novel compartment that is continuous with the plasma membrane and devoid of

transferrin receptor. J. Cell Biol. 137, 445–458 (1997).

41. Galfré, G., Howe, S. C., Milstein, C., Butcher, G. W. & Howard, J. C. Antibodies to major histocom-

patibility antigens produced by hybrid cell lines. Nature 266, 550–552 (1977).

42. Rupnik, M. et al. Rapid regulated dense-core vesicle exocytosis requires the CAPS protein. Proc.

Natl Acad. Sci. USA 97, 5627–5632 (2000).

43. Gatti, G., Podini, P. & Meldolesi, J. Overexpression of calsequestrin in L6 myoblasts: formation of

endoplasmic reticulum subdomains and their evolution into discrete vacuoles where aggregates of

the protein are specifically accumulated. Mol. Biol. Cell 8, 1789–1803 (1997).

44. Wilm, M. et al. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray

mass spectrometry. Nature 379, 466–469 (1996).

45. Van den Hoff, M. J. B., Moorman, A. F. M. & Lamers, W. H. Electroporation in ‘intracellular’ buffer

increases cell survival. Nucleic Acids Res. 20, 2902 (1992).

ACKNOWLEDGEMENTS

We thank H. Kasai and his laboratory staff for their pioneering collaboration and advice; E. Shtivelman

and M. Rupnik for generous support; M. Matteoli and G.P. Schiavo for advice, A. Lorusso for partici-

pating in some experiments; C. Paolucci for FACS analysis, R. Barsacchi for performing the [Ca2+]i

measurements; and D. Dunlap for the critical revision of the text. Imaging experiments were per-

formed within Alembic (Advanced Light and Electron Microscopy Bio-Imaging Center), San Raffaele

Scientific Institute. This work, supported by grants from Telethon, the European Union

(QLG1–02233), CNR (Ag. 2000 and the CNS degenerative diseases), the Cofin Italian University

System (2001.053389-033) and the Armenise–Harvard Foundation, was performed in the framework

of the Italian MIUR Center of Excellence in Physiopathology of Cell Differentiation.

Correspondence and requests for material should be addressed to J.M.

COMPETING FINANCIAL INTERESTS

The authors declare that they have no competing financial interests.


Regulated exocytosis: a novel, widely expressed system

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