Proc. Natl. Acad. Sci. USAVol. 92, pp. 8328-8332, August 1995Neurobiology

Rapid endocytosis coupled to exocytosis in adrenal chromaffincells involves Ca2+, GTP, and dynamin but not clathrinCRISTINA R. ARTALEJO*, JOHN R. HENLEYt, MARK A. MCNIVENt, AND H. CLIVE PALFREYt*Department of Neurobiology and Physiology, Northwestern University, Evanston, IL 60208, and Departamento Farmacologia y Terapeutica, Faculdad Medicina,Universidad Autonoma de Madrid, Madrid 28029, Spain; tCentre for Basic Research in Digestive Diseases, Mayo Clinic, Rochester, MN 55905; andtDepartment of Pharmacological and Physiological Sciences, University of Chicago, Chicago, IL 60637

Communicated by Hewson H. Swift, The University of Chicago, Chicago, IL, May 8, 1995

ABSTRACT Rapid endocytosis (RE) occurs immediatelyafter an exocytotic burst in adrenal chromaffin cells. Capac-itance measurements of endocytosis reveal that recovery ofmembrane is a biphasic process that is complete within 20 sec.The ultimate extent of membrane retrieval is precisely con-trolled and capacitance invariably returns to its prestimula-tion value. The mechanism of RE specifically requires intra-cellular Ca2+; Sr2+ and Ba2+ do not substitute, although allthree cations support secretion. Thus the divalent cationreceptors for RE and exocytosis must be distinct molecules.RE is dependent on GTP hydrolysis; it is blocked by GTPremoval or replacement with guanosine 5'-[y-thio]triphos-phate. In the presence of GTP, multiple rounds of secretionfollowed by RE could be elicited from the same cell. RErequires participation of dynamin, a guanine nucleotide bind-ing protein, as revealed by intracellular immunological an-tagonism of this protein. Intact microtubules may be essential,as nocodazole also blocked RE. Whereas anti-dynamin anti-bodies blocked RE, anti-clathrin antibodies did not, suggest-ing that clathrin-coated vesicles are not involved in this formof endocytosis. RE may represent the initial step in the rapidrecycling of secretory granules in the chromaffin cell.

The physiological and molecular basis of secretion has beenstudied in some detail (1), but the nature of the endocytoticprocesses that are coupled to exocytosis is poorly understood.Endocytosis serves the dual function of maintaining cellsurface area constant and retrieving vesicular components forrecycling. In neurons, synaptic vesicle recycling is rapid and itis thought that endocytosed vesicles are recovered virtuallyintact and quickly returned to the releasable pool (2). This isa critical feature of neuronal communication as, withoutrecycling, neural firing would rapidly deplete the pool oftransmitter-containing vesicles ready for release. Whether asimilar phenomenon occurs in other types of secretory cell isunknown, but as these cells may also need to secrete repeti-tively under various conditions, it seems likely that rapidrecycling must be present (3).

Recently, studies on several preparations have suggestedthat rapid endocytotic mechanisms can be assessed by mea-surement of cell capacitance. By this criterion, adrenal chro-maffin cells (4), pituitary melanotrophs (5), and nerve termi-nals (6) rapidly recapture membrane after secretion. Little isknown about the kinetics and regulation of this type ofendocytosis, termed rapid endocytosis (RE) to distinguish itfrom other better-studied phenomena such as receptor-mediated endocytosis (mediated by clathrin-coated vesicles) orvarious kinds of pinocytosis (7). We have conducted a detailedanalysis of RE in bovine adrenal chromaffin cells subjected tophysiological stimulation. We show that it is a kineticallycomplex and highly regulated process that reproducibly follows

each round of secretion. The final amount of membranerecovered seems to be precisely controlled, as most cells returnto their prestimulation capacitance value. RE requires intra-cellular Ca2+ and GTP hydrolysis and we provide directevidence that the guanine nucleotide binding protein (Gprotein) dynamin is intimately involved in the process. Incontrast, our results suggest that RE does not involve aclathrin-coated vesicle-based mechanism.

MATERIALS AND METHODSPreparation of Cells and Patch-Clamp. Chromaffin cells

were isolated from calf adrenal medullae, cultured, and patch-clamped as described (8). Capacitance was measured by acomputer program using a phase-tracking technique (9). Se-cretion was elicited by a train of 10 depolarizations, from aholding potential of -90 mV to + 10 mV, each lasting 50 msec;500 msec separated each depolarization. Each depolarizationwas preceded by a 50-msec prepulse to +120 mV to recruitfacilitation Ca2+ channels (8). All experiments were carriedout at room temperature (25°C). The standard patch-pipettesolution contained 110 mM cesium glutamate, 0.1 mMCsEGTA, 40 mM Hepes, 5 mM MgCl2, 2 mM ATP, 0.35 mMGTP, pH 7.2; Mes (40 mM) replaced Hepes in pipettesolutions at pH 6.2. The external solution consisted of 2 mMCaCl2, 150 mM tetraethylammonium chloride, 10 mM Hepes,10 mM glucose, and 1 ,uM tetrodotoxin, pH 7.2. Whereindicated CaCl2 was substituted by equimolar SrCl2 or BaCl2.Fresh preparation of GTP-containing solutions was found tobe essential for reproducible expression of RE.

Preparation and Use of Anti-Dynamin and Anti-ClathrinAntibodies. Affinity-purified polyclonal anti-dynamin peptideantibodies [MC13, against rat brain dynamin-(34-49), andMC63, against rat brain dynamin-(216-241)] were prepared(J.R.H., T. A. Cook, K. Hsiao, and M.A.M., unpublished data).Fab fragments were prepared from affinity-purified IgGs byusing a kit from Pierce. Antibodies were used at the followingfinal concentrations: preimmune IgG, MC13 IgG, and MC63IgG, 1 mg/ml; Fab MC13, 1.7 mg/ml; Fab MC63, 1.6 mg/ml.Two affinity-purified anti-clathrin heavy chain monoclonalIgGs, X19 and X22, were the gift of F. Brodsky (University ofCalifornia, San Francisco) and were used at 2 mg/ml. Allantibodies were dialyzed against pipette solution before use.Immunolabeling of Cells and Extracts. For immunoblot

analysis, cultured chromaffin cells were solubilized directly inSDS sample buffer and electrophoresed on SDS/7.5% poly-acrylamide gels. After transfer to nitrocellulose and blocking[3% (vol/vol) milk in PBS], blots were incubated with MC13or MC63 IgG (0.5 ,tg/ml) and the immune complex wasdetected by enhanced chemiluminescence (Amersham). Forimmunofluorescence, chromaffin cells on coverslips were fixedwith 3% (wt/vol) paraformaldehyde/PBS and permeabilized

Abbreviations: RE, rapid endocytosis; G protein, guanine nucleotidebinding protein; GTP[,yS], guanosine 5'-[y-thio]triphosphate; GDP[,BS],guanosine 5'-[13-thio]diphosphate.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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with 0.1% Triton X-100 in PBS. After blocking (PBS/1%bovine serum albumin), samples were incubated with anti-dynamin IgG (MC13 at 0.5 ,ug/ml) or anti-clathrin IgG (5,tg/ml). Immune reactions were detected with Texas red-conjugated secondary IgGs (Jackson Lab) and visualized byepifluorescence.

RESULTS

Kinetics of RE. We investigated exocytosis-endocytosiscoupling in chromaffin cells by patch-clamp recording of cellcapacitance (Cm) (10). Depolarization-activated Ca2+ influxthrough three types of Ca2+ channel (8) leads to stepwiseincreases in Cm that are a direct reflection of secretion in thesecells (ref. 11 and Fig. 1). At the termination of stimulation, Cmrapidly decreased in >90% of all cells examined; a total of 501cells exhibiting RE were studied in the present work. RE couldbe described by two exponentials with Tvalues of -0.3 and -3sec or - 3 sec and 13 sec (Fig. 1 A and B; we refer to thesecomponents as ultrafast, fast-1, and fast-2). The ultrafastcomponent was only seen during a train of depolarizations,never at the end and, when operative, clearly reduced themagnitude of the Cm increase (e.g., Fig. 1A). In a few cases(-9%), Cm returned directly to baseline values, but in mostinstances (-91%), an overshoot indicative of "excess retriev-al" was found, Cm falling below initial values before returningto baseline (Fig. 1). Apart from the ultrafast response, the twokinetic components of RE described here are quantitativelysimilar to observations in melanotrophs (5) and qualitativelysimilar to findings in goldfish neurons (6), the time constantsdiffering in the latter case. These data show that chromaffincells contain a powerful mechanism to rapidly recapturemembrane after exocytosis and are able to "sense" whenoriginal cell surface area has been attained.Repeated depolarizing trains followed by recovery resulted

in successive periods of exocytosis and endocytosis that weresimilar in pattern (Fig. 1C). Tetanization of cells, resulting inmassive secretion, often resulted in failure or a delayed andslow recovery of membrane (data not shown). Under thestimulation conditions used here only -2% of the chromaffingranule population fused with the membrane. Thus it seemslikely that our results approximate the normal physiological

A

'fif- 0.301 secj U2- 6.8 sec

B

behavior of chromaffin cells in the adrenal medulla in responseto intermittent stimulation by the splanchnic nerve.RE Depends on Ca21 Influx; Ba2+ and Sr2+ Do Not

Substitute. Previous work suggested by indirect means thatintracellular Ca2+ may regulate various types of endocytosis (4,12-14). However, the divalent cation specificity of these pro-cesses has never been reported. Secretion in chromaffin cellsis robust if Ba2+ or Sr2+ is substituted for Ca2+ as chargecarrier (15) and it seems likely that a component of theexocytotic machinery recognizes Ca2 , Ba2 , and Sr2 . Incontrast, when RE was examined after a secretory phase withBa2+ or Sr2+ replacing Ca2+ in the external solution, mem-brane retrieval was abolished (Fig. 2). These results suggestthat RE is a Ca2+-dependent process but that neither Ba2+ norSr2+ can bind to the appropriate divalent cation receptor(s).Guanine Nucleotide Turnover Is Necessary for RE. To

evaluate the possible involvement of a G protein in RE, weremoved GTP or introduced the nonhydrolyzable guaninenucleotide guanosine 5'-[y-thio]triphosphate (GTP[-yS]) orthe inhibitor guanosine 5'-[,B-thio]diphosphate (GDP[13S])into cells from the patch-pipette solution (Fig. 3). Such ma-neuvers did not affect evoked Cm increases, indicating that thesecretory mechanism was intact, but RE was completelyblocked. The failure of GTP[yS] to support RE implies thatcontinuous turnover of GTP is important in the process.RE Is Blocked by Anti-Dynamin Antibodies. One G protein

that has been strongly implicated in endocytosis is dynamin(16-19). To test the involvement of dynamin in RE, weintroduced various anti-dynamin antibodies into chromaffincells via the patch pipette (Fig. 4). Although several forms ofdynamin have been identified (e.g., refs. 20 and 21), theantibodies used here would recognize all forms, as they wereraised against regions conserved from Drosophila to humans.Affinity-purified IgG (MC13) had no effect on exocytosis butabolished RE (Fig. 4B; n = 16). Fab fragments of MC13 IgG(Fig. 4C; n = 10), MC63 IgG (n = 22), or its Fab fragments(n = 12) were also inhibitory (Table 1 and data not shown).However, preimmune IgG (Fig. 4D; n = 18), antibodiespreabsorbed with their respective antigenic peptides (n = 20),or boiled immune IgGs (n = 23) were without effect (data notshown). Immunolabeling revealed dynamin to be abundant inchromaffin cells (Fig. 4 E and F).

C

I I I I

lOOsecIlI

FIG. 1. Kinetics of RE in chromaffin cells. Changes in Cm in adrenal chromaffin cells during exo- and endocytosis. Secretion was initiated bya train of depolarizations. After exocytosis was complete (rising phase), the Cm trace declined, reflecting endocytosis (dashed line is baseline).Breaks in Cm records correspond to application of test depolarizations. (A and B) RE has three possible kinetic components termed ultrafast (uf),fast-1 (fl), and fast-2 (f2) with Tvalues of 0.28 + 0.01, 3.40 ± 0.09, and 12.92 ± 0.64 sec (mean ± SEM; n = 202), respectively (significantly different;P < 0.0001); cell inA displayed ultrafast kinetics between depolarizations 2 and 3 (arrow) and fast-2 kinetics on termination of secretion; the cellin B displayed fast-1 and fast-2 kinetics. In any single round of endocytosis, cells exhibited two, but never all three kinetic components. Best fitexponentials derived with the Simplex algorithm are superimposed on the traces as continuous lines. (Insets) Slower time base (calibration 80 msec)to show excess retrieval. (C) Successive cycles of exocytosis and endocytosis during 20 min of continuous recording. Four sequential trains ofdepolarization similar to those described above were applied; 4 min separated each train.

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FIG. 2. RE is Ca2+-dependent. Ca2+ (A), Sr2+ (B), or Ba2+ (C) wasused in different cells as charge carriers, secretion was elicited, and Cmwas recorded as in Fig. 1. Each cation supported secretion equally wellas measured by total Cm in,crease (for means, see Table 1) but RE wasabolished in the presence of Ba2+ and Sr2+. (Insets) Currents elicitedby the first depolarization.

RE Does Not Involve Clathrin-Coated Vesicles. In view of itspostulated role in synaptic vesicle recycling (22, 23), we directlytested whether clathrin might be involved in RE. Two anti-clathrin heavy chain monoclonal antibodies (ref. 24; 2 mg/ml)were introduced into chromaffin cells via the patch pipette(Fig. 5A) but were without effect on RE, suggesting thatclathrin is not involved in these processes. The same antibodieswere used in parallel immunolabeling studies to ensure theycould recognize clathrin heavy chain in bovine chromaffin cells(Fig. 5B). Moreover, when microinjected into chromaffin cells,these antibodies (at 2 mg/ml) inhibited transferrin uptake by33.5 ± 2.2% (X19, n = 18) and 36.7 ± 2.7% (X22; n = 10)compared to preimmune IgG (n = 22), indicating that they arecapable of interfering with receptor-mediated endocytosis, asshown in other cells (25).The initial phase of receptor-mediated endocytosis is gen-

erally considered to require K+, to be independent of micro-tubules, and to be susceptible to lowered cytoplasmic pH (7).We tested the effects of each of the above conditions on RE.(i) All experiments were performed with a pipette solutionlacking K+, thus it is evident that RE does not require thiscation. (ii) RE was blocked by preincubation of chromaffincells with nocodazole, suggesting that microtubule integrity isessential for RE (Fig. SC, trace a). The effects of nocodazolewere completely reversible; during washout, cells consistently(n = 14) exhibited "capacitance flicker," probably due to theflickering of a fission pore prior to complete endocytosis (Fig.5C, trace b). As RE recovered fully, capacitance flicker waslost, suggesting that it may be due to incomplete reformationof polymerized microtubules. Nocodazole had no effect onexocytosis, in agreement with earlier data suggesting thatmicrotubule integrity is not required for regulated secretion ofcatecholamines in these cells (26). (iii) Cytoplasmic acidifica-tion completely blocks clathrin-mediated endocytosis in othersystems (7, 27). However, lowering pipette pH from 7.2 to 6.2had no effect on the extent or rate of RE (Fig. SD; cf. ref. 5).We also found that antibodies against the small G proteinrab5a, implicated in the early stages of receptor-mediatedendocytosis (28), failed to affect RE (n = 9). Thus, by severalcriteria, it is unlikely that RE in chromaffin cells involvesclathrin-coated vesicles.

DISCUSSIONThe present results demonstrate that RE is the major

pathway for membrane retrieval under physiologically relevant

200 PAL15 msec

FIG. 3. Guanine nucleotide turnover is necessary for RE. Cm wasmonitored continuously after establishment of whole-cell patch con-figuration with different intrapipette solutions. (A) No GTP in pipette.(B) GDP[,BS]-(350 ,M). (C) GTP[yS] (350 ALM). Secretion was elicitedas described in Fig. 1 at the times indicated above the capacitancetrace. The Ca2+ current elicited by the first test depolarization isplotted below the Cm trace. Replacement of GTP with GDP[13S] (B)or GTP[-yS] (C) blocked endocytosis without reducing exocytosis. Theinitial endocytosis remains intact in A presumably because sufficientcellular GTP remains at this time to support one round of membranerecovery. It also remains intact in B, probably because GDP[,BS]cannot gain access to binding sites on G proteins until bound GTP hasbeen hydrolyzed. In C we surmise that GTP[,yS] can displace boundGTP and, hence, no endocytosis is possible. The absence of GTP or

presence of GDP[13S] does not modify chromaffin-cell Ca2+ currentswhen measured as peak current; however, GTP[-yS] did reduce Ca2+current (due to a reduction in components other than the facilitationcurrent, data not shown). Neither the extent nor the rate of secretionwas markedly affected by these conditions (see Table 1).

conditions in chromaffin cells. RE potentially performs twocritical functions: enabling the cell to maintain a constantsurface area while simultaneously preserving the integrity ofthe secretory granule membrane in the form of a recapturedvesicle. The fate of rapidly endocytosed vesicles is not knownbut may involve direct recycling of granules back to thesecretory pool. In neurons RE may be the first step in the rapidrecycling of synaptic vesicles. While there is less evidence forthis phenomenon in nonneuronal cells, the fact that RE isfound in both chromaffin cells and melanotrophs suggests thatdirect recycling may exist in diverse types of secretory cells.Indeed, experiments using an entirely different approach toours indicate that rapid recycling of endocytosed membranedoes occur after secretion in chromaffin cells (29).We show that RE is a kinetically complex process with

several phases. The rate of membrane retrieval can be de-scribed by three time constants, only two of which wereobserved together in any single round of endocytosis. Ofparticular interest is the ultrafast process that occurred duringbut never after an exocytotic burst. The existence of such amechanism means that there is not necessarily a lag betweenexocytosis and endocytosis, as has been claimed to occur inother systems (5). In addition, most cells displayed "excessretrieval" indicative of the recovery of surplus membrane.Nevertheless, capacitance then recovered back to baseline,suggesting the existence of some compensatory mechanismable to sense cell surface area. RE is kinetically distinct fromeither coated-vesicle-mediated or fluid-phase endocytosis,processes that undergo delayed activation after chromaffincells are induced to massively secrete (30, 31). Under the latter

AI Calcium

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A Control Cell B Anti-dynamin IgG C Anti-dynamin Fab4 min 22mn

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FIG. 4. RE is blocked by anti-dynamin antibodies. Continuous Cm recordings after formation of whole-cell configuration from a cell loadedwith the following components: (A) Control-no addition. (B) Affinity-purified polyclonal anti-dynamin IgG (MC13). (C) Fab fragments of MC13.(D) Preimmune IgG. The times of depolarization trains (vertical bars) are indicated above each trace; generally >18 min was allowed between thefirst and second trains for antibodies to diffuse into the cell. Initial rates of endocytosis after the first exocytotic burst were not significantly differentfrom control (for means, see Table 1) reflecting the fact that insufficient antibody has diffused into the cell at this time to affect RE. (E) Immunoblotof total chromaffin cell protein (100 ,ug; lanes 1 and 3) and rat brain synaptosomal protein (20 ,tg; lanes 2 and 4) reacted with anti-dynamin IgGsMC13 and MC63. Note that either antibody reacts with the prominent 105-kDa dynamin band in both samples (arrow). Staining was absent ifantibodies were preincubated with a 100-fold excess of their respective immunogenic peptide (in lane 3, the asterisk indicates a nonspecific bandthat was not competed out by the peptide). (F) Immunofluorescent detection (MC13 IgG) of dynamin in chromaffin cells. (Upper) Differentialinterference contrast image. (Lower) Fluorescence. (Bar = 10 ,um.)

circumstance, granule markers appear on the cell surface andmay mix with other constituents before being endocytosed,possibly by a coated-vesicle pathway involving clathrin. Fluid-phase endocytosis is much slower than RE (T 3.7 min; ref.

31) and does not require Ca2+ or GTP (32). Several additionalpieces of evidence presented here suggest that RE does notinvolve a clathrin-based mechanism in chromaffin cells. Mostimportantly, anti-clathrin antibodies fail to affect RE but doinhibit receptor-mediated endocytosis in these cells.Our results show that the RE mechanism involves intracel-

lular Ca2+, GTP hydrolysis, dynamin, and microtubules. RE is

Ca2+-dependent and we show that it cannot be supported byBa2+ or Sr2 . We speculate that Ca2+ may be the critical linkthat ensures tight coupling between RE and the precedingsecretory event. As with exocytosis, the nature of the Ca2+receptor(s) involved in endocytosis is unknown, but as exocy-

tosis can occur with either Ba2+ or Sr2+, it is likely that thedivalent cation receptors for exocytosis and RE are distinctmolecules. RE requires GTP hydrolysis and a critical G proteinregulating the process appears to be dynamin. This protein wasoriginally discovered as a microtubule-binding protein (33)and microtubules markedly stimulate the GTPase activity of

Table 1. Statistical analysis of the molecular requirements for RE in chromaffin cells

Maximum rate Maximum MembraneTotal Maximum rate endocytosis rate retrieved

Peak current, capacitance exocytosis, ultrafast, endocytosis- withinCondition pA change, fF fF/sec fF/sec fast, fF/sec 20 sec, %

Control (n = 202) -797.4 ± 46.9 861.1 ± 70.6 869.1 ± 54.4 1017.1 ± 126.8 91.7 ± 5 100.4 ± 1.20Barium (n = 28) -1383.4 ± 74 1099.5 ± 102.9 430.6 ± 48.7 0Strontium (n = 20) -1273.1 ± 83.1 912.8 ± 107.1 567.7 ± 62.1 0No GTP (n = 33) -731.7 ± 48.9 970 ± 109.2 888.1 ± 84.6 0.87 ± 0.40GTP[yS] (n = 13) -386.7 ± 26.7 744.2 ± 97.8 982.9 ± 122.1 - 0GDP[P3S] (n = 16) -761.16 ± 38.3 946.6 ± 108 856.7 ± 68 0.66 ± 0.32Anti-dynamin IgG (MC63) (n = 22) -725.7 ± 46.1 812.6 ± 107.7 718.3 ± 59.6 - 0.90 ± 0.44Anti-dynamin IgG (MC13) (n = 16) -691.8 ± 31.1 831.1 ± 72.1 774.2 ± 53.3 - 1.08 ± 0.68Anti-clathrin IgG (X19) (n = 13) -748.4 ± 59 822 ± 116 839.9 ± 67.4 936.3 ± 157 102.3 ± 8.7 102.3 ± 3.36Anti-clathrin IgG (X22) (n = 10) -793.3 ± 58.2 811.4 ± 71 889.1 ± 80 1034.1 ± 92 92.8 ± 9.7 105.4 ± 3.42Nocodazole (n = 21) -789.2 ± 55.1 796.8 ± 84.1 920.1 ± 80.4 - 0pH 6.2 (n = 15) -659.3 ± 28.7 799.5 ± 68.5 872.8 ± 55.7 1233.7 ± 129.7 86.6 ± 7.3 106.9 ± 4.06

Rate of the exocytotic burst was measured as the initial rate of Cm increase divided by the peak amplitude. The rate of Cm decline was obtainedby measuring the steepest downward slope of the Cm trace divided by the amplitude of the decline. Peak amplitudes and rates of Cm were eachdivided by the initial capacitance and then multiplied by the average initial capacitance of the cell to normalize data.

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8332 Neurobiology: Artalejo et al.

A Anti-clathrin heavy chain IgG( X19 ) C

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al I pH 7.2 a2

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bi I pH 6.2 b2 I

B 5 fF IB _ \ 1^ ~~~~~ ~~~~~~~~~~~~~40sec40 sec

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FIG. 5. RE is independent of clathrin and clathrin-coated vesicles. (A) Continuous Cm records after formation of whole-cell configuration froma cell loaded with affinity-purified anti-clathrin heavy chain monoclonal IgG (X19; 2 mg/ml); no effect on exocytosis or RE was found. (B)Immunofluorescence of cultured chromaffin cells stained with anti-clathrin IgG (X19; 5 ,tg/ml). (Bar = 10 ,u&m.) (C) Functional microtubules arerequired for complete endocytosis: Traces: a, nocodazole pretreatment (10 ,uM; 2 h) blocks endocytosis but has no effect on Ca2+ currents orexocytosis (see Table 1); b, capacitance flicker 2 h after nocodazole washout (in this case prolonged fluctuations of 30, 40, 50, 55, and 60 fF; 15-30granules). (D) Acidification of the cytosol does not influence RE. Traces: al and a2, internal pH 7.2; bl and b2, internal pH 6.2. At internal pH6.2, cells still respond with ultrafast (al and bl) and fast (a2 and b2) kinetic components of endocytosis (for statistics, see Table 1).

dynamin (34). Although the relationship between dynamin andmicrotubules in vivo is debatable (18, 19, 35), our results areconsistent with the notion that a dynamin-microtubule inter-action drives membrane recapture, as microtubule disruptionby nocodazole blocked RE.Dynamin is also implicated in receptor-mediated endocyto-

sis (18, 19), a process distinct from RE as discussed above.Morphological studies of Drosophila shibire mutants, in whichdynamin is defective, reveal long tubulovesicular structuresemanating from the plasma membrane (36) and recent micro-scopic data from fibroblasts suggest that dynamin is critical forthe pinching-off of vesicles from coated pits (37). It seemspossible that dynamin plays a similar physical function in RE:it may be responsible for constricting the fission pore and/oractively retracting the invaginating membrane from the cellsurface. In this regard, it is interesting to recall that allendocytosis appears to be blocked in shibire flies (36). Curi-ously, the early stages of receptor-mediated endocytosis arenot affected by microtubule depolymerization (7); thus, dy-namin may interact with targets other than microtubules tomodulate this process, e.g., Grb2 (38). Identification of thecomponents interacting with both dynamin and Ca2+ will beessential to a full understanding of RE.

We thank Drs. F. Brodsky and A. Wandinger-Ness for their gifts ofanti-clathrin and anti-rab5a antibodies, respectively, and Drs. T. F. J.Martin, T. L. Steck, and D. E. Clapham for comments on themanuscript. This work was supported by grants from the Sloanfoundation and the National Institutes of Health.

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