bodies blocked RE, anti-clathrin antibodies did not, suggest- ing that clathrin-coated ... analysis of RE in bovine adrenal chromaffin cells subjected to physiological ... (J.R.H., T. A. Cook, K. Hsiao, and M.A.M., unpublished data). Fab fragments ...
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 8328-8332, August 1995 Neurobiology
Rapid endocytosis coupled to exocytosis in adrenal chromaffin cells involves Ca2+, GTP, and dynamin but not clathrin CRISTINA 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; and tDepartment 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 immediately after an exocytotic burst in adrenal chromaffin cells. Capacitance measurements of endocytosis reveal that recovery of membrane is a biphasic process that is complete within 20 sec. The ultimate extent of membrane retrieval is precisely controlled and capacitance invariably returns to its prestimulation value. The mechanism of RE specifically requires intracellular Ca2+; Sr2+ and Ba2+ do not substitute, although all three cations support secretion. Thus the divalent cation receptors for RE and exocytosis must be distinct molecules. RE is dependent on GTP hydrolysis; it is blocked by GTP removal or replacement with guanosine 5'-[y-thio]triphosphate. In the presence of GTP, multiple rounds of secretion followed by RE could be elicited from the same cell. RE requires participation of dynamin, a guanine nucleotide binding protein, as revealed by intracellular immunological antagonism of this protein. Intact microtubules may be essential, as nocodazole also blocked RE. Whereas anti-dynamin antibodies blocked RE, anti-clathrin antibodies did not, suggesting that clathrin-coated vesicles are not involved in this form of endocytosis. RE may represent the initial step in the rapid recycling of secretory granules in the chromaffin cell.
each round of secretion. The final amount of membrane recovered seems to be precisely controlled, as most cells return to their prestimulation capacitance value. RE requires intracellular Ca2+ and GTP hydrolysis and we provide direct evidence that the guanine nucleotide binding protein (G protein) dynamin is intimately involved in the process. In contrast, our results suggest that RE does not involve a clathrin-coated vesicle-based mechanism.
MATERIALS AND METHODS
The physiological and molecular basis of secretion has been studied in some detail (1), but the nature of the endocytotic processes that are coupled to exocytosis is poorly understood. Endocytosis serves the dual function of maintaining cell surface area constant and retrieving vesicular components for recycling. In neurons, synaptic vesicle recycling is rapid and it is thought that endocytosed vesicles are recovered virtually intact and quickly returned to the releasable pool (2). This is a critical feature of neuronal communication as, without recycling, neural firing would rapidly deplete the pool of transmitter-containing vesicles ready for release. Whether a similar phenomenon occurs in other types of secretory cell is unknown, but as these cells may also need to secrete repetitively under various conditions, it seems likely that rapid recycling must be present (3). Recently, studies on several preparations have suggested that rapid endocytotic mechanisms can be assessed by measurement of cell capacitance. By this criterion, adrenal chromaffin cells (4), pituitary melanotrophs (5), and nerve terminals (6) rapidly recapture membrane after secretion. Little is known about the kinetics and regulation of this type of endocytosis, termed rapid endocytosis (RE) to distinguish it from other better-studied phenomena such as receptormediated endocytosis (mediated by clathrin-coated vesicles) or various kinds of pinocytosis (7). We have conducted a detailed analysis of RE in bovine adrenal chromaffin cells subjected to physiological stimulation. We show that it is a kinetically complex and highly regulated process that reproducibly follows
Preparation of Cells and Patch-Clamp. Chromaffin cells were isolated from calf adrenal medullae, cultured, and patchclamped as described (8). Capacitance was measured by a computer program using a phase-tracking technique (9). Secretion was elicited by a train of 10 depolarizations, from a holding potential of -90 mV to + 10 mV, each lasting 50 msec; 500 msec separated each depolarization. Each depolarization was preceded by a 50-msec prepulse to +120 mV to recruit facilitation Ca2+ channels (8). All experiments were carried out at room temperature (25°C). The standard patch-pipette solution contained 110 mM cesium glutamate, 0.1 mM CsEGTA, 40 mM Hepes, 5 mM MgCl2, 2 mM ATP, 0.35 mM GTP, pH 7.2; Mes (40 mM) replaced Hepes in pipette solutions at pH 6.2. The external solution consisted of 2 mM CaCl2, 150 mM tetraethylammonium chloride, 10 mM Hepes, 10 mM glucose, and 1 ,uM tetrodotoxin, pH 7.2. Where indicated CaCl2 was substituted by equimolar SrCl2 or BaCl2. Fresh preparation of GTP-containing solutions was found to be essential for reproducible expression of RE. Preparation and Use of Anti-Dynamin and Anti-Clathrin Antibodies. Affinity-purified polyclonal anti-dynamin peptide antibodies [MC13, against rat brain dynamin-(34-49), and MC63, 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 by using a kit from Pierce. Antibodies were used at the following final concentrations: preimmune IgG, MC13 IgG, and MC63 IgG, 1 mg/ml; Fab MC13, 1.7 mg/ml; Fab MC63, 1.6 mg/ml. Two affinity-purified anti-clathrin heavy chain monoclonal IgGs, X19 and X22, were the gift of F. Brodsky (University of California, San Francisco) and were used at 2 mg/ml. All antibodies were dialyzed against pipette solution before use. Immunolabeling of Cells and Extracts. For immunoblot analysis, cultured chromaffin cells were solubilized directly in SDS sample buffer and electrophoresed on SDS/7.5% polyacrylamide gels. After transfer to nitrocellulose and blocking [3% (vol/vol) milk in PBS], blots were incubated with MC13 or MC63 IgG (0.5 ,tg/ml) and the immune complex was detected by enhanced chemiluminescence (Amersham). For immunofluorescence, chromaffin cells on coverslips were fixed with 3% (wt/vol) paraformaldehyde/PBS and permeabilized
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: RE, rapid endocytosis; G protein, guanine nucleotide binding protein; GTP[,yS], guanosine 5'-[y-thio]triphosphate; GDP[,BS], guanosine 5'-[13-thio]diphosphate. 8328
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with 0.1% Triton X-100 in PBS. After blocking (PBS/1% bovine serum albumin), samples were incubated with antidynamin IgG (MC13 at 0.5 ,ug/ml) or anti-clathrin IgG (5 ,tg/ml). Immune reactions were detected with Texas redconjugated secondary IgGs (Jackson Lab) and visualized by epifluorescence.
RESULTS Kinetics of RE. We investigated exocytosis-endocytosis coupling in chromaffin cells by patch-clamp recording of cell capacitance (Cm) (10). Depolarization-activated Ca2+ influx through three types of Ca2+ channel (8) leads to stepwise increases in Cm that are a direct reflection of secretion in these cells (ref. 11 and Fig. 1). At the termination of stimulation, Cm rapidly decreased in >90% of all cells examined; a total of 501 cells exhibiting RE were studied in the present work. RE could be described by two exponentials with Tvalues of -0.3 and -3 sec or 3 sec and 13 sec (Fig. 1 A and B; we refer to these components as ultrafast, fast-1, and fast-2). The ultrafast component was only seen during a train of depolarizations, never at the end and, when operative, clearly reduced the magnitude of the Cm increase (e.g., Fig. 1A). In a few cases (-9%), Cm returned directly to baseline values, but in most instances (-91%), an overshoot indicative of "excess retrieval" was found, Cm falling below initial values before returning to baseline (Fig. 1). Apart from the ultrafast response, the two kinetic components of RE described here are quantitatively similar to observations in melanotrophs (5) and qualitatively similar to findings in goldfish neurons (6), the time constants differing in the latter case. These data show that chromaffin cells contain a powerful mechanism to rapidly recapture membrane after exocytosis and are able to "sense" when original cell surface area has been attained. Repeated depolarizing trains followed by recovery resulted in successive periods of exocytosis and endocytosis that were similar in pattern (Fig. 1C). Tetanization of cells, resulting in massive secretion, often resulted in failure or a delayed and slow recovery of membrane (data not shown). Under the stimulation conditions used here only -2% of the chromaffin granule population fused with the membrane. Thus it seems likely that our results approximate the normal physiological -
A
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behavior of chromaffin cells in the adrenal medulla in response to intermittent stimulation by the splanchnic nerve. RE Depends on Ca21 Influx; Ba2+ and Sr2+ Do Not Substitute. Previous work suggested by indirect means that intracellular Ca2+ may regulate various types of endocytosis (4, 12-14). However, the divalent cation specificity of these processes has never been reported. Secretion in chromaffin cells is robust if Ba2+ or Sr2+ is substituted for Ca2+ as charge carrier (15) and it seems likely that a component of the exocytotic machinery recognizes Ca2 , Ba2 , and Sr2 . In contrast, when RE was examined after a secretory phase with Ba2+ or Sr2+ replacing Ca2+ in the external solution, membrane retrieval was abolished (Fig. 2). These results suggest that RE is a Ca2+-dependent process but that neither Ba2+ nor Sr2+ 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, we removed GTP or introduced the nonhydrolyzable guanine nucleotide guanosine 5'-[y-thio]triphosphate (GTP[-yS]) or the inhibitor guanosine 5'-[,B-thio]diphosphate (GDP[13S]) into cells from the patch-pipette solution (Fig. 3). Such maneuvers did not affect evoked Cm increases, indicating that the secretory mechanism was intact, but RE was completely blocked. The failure of GTP[yS] to support RE implies that continuous 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, we introduced various anti-dynamin antibodies into chromaffin cells via the patch pipette (Fig. 4). Although several forms of dynamin have been identified (e.g., refs. 20 and 21), the antibodies used here would recognize all forms, as they were raised against regions conserved from Drosophila to humans. Affinity-purified IgG (MC13) had no effect on exocytosis but abolished 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), antibodies preabsorbed with their respective antigenic peptides (n = 20), or boiled immune IgGs (n = 23) were without effect (data not shown). Immunolabeling revealed dynamin to be abundant in chromaffin cells (Fig. 4 E and F).
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FIG. 1. Kinetics of RE in chromaffin cells. Changes in Cm in adrenal chromaffin cells during exo- and endocytosis. Secretion was initiated by a 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 in A displayed ultrafast kinetics between depolarizations 2 and 3 (arrow) and fast-2 kinetics on termination of secretion; the cell in B displayed fast-1 and fast-2 kinetics. In any single round of endocytosis, cells exhibited two, but never all three kinetic components. Best fit exponentials 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 of depolarization 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) was used in different cells as charge carriers, secretion was elicited, and Cm was recorded as in Fig. 1. Each cation supported secretion equally well as measured by total Cm in,crease (for means, see Table 1) but RE was abolished in the presence of Ba2+ and Sr2+. (Insets) Currents elicited by the first depolarization.
RE Does Not Involve Clathrin-Coated Vesicles. In view of its postulated role in synaptic vesicle recycling (22, 23), we directly tested whether clathrin might be involved in RE. Two anticlathrin 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 that clathrin is not involved in these processes. The same antibodies were used in parallel immunolabeling studies to ensure they could 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 by 33.5 ± 2.2% (X19, n = 18) and 36.7 ± 2.7% (X22; n = 10) compared to preimmune IgG (n = 22), indicating that they are capable of interfering with receptor-mediated endocytosis, as shown in other cells (25). The initial phase of receptor-mediated endocytosis is generally considered to require K+, to be independent of microtubules, 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 solution lacking K+, thus it is evident that RE does not require this cation. (ii) RE was blocked by preincubation of chromaffin cells with nocodazole, suggesting that microtubule integrity is essential for RE (Fig. SC, trace a). The effects of nocodazole were completely reversible; during washout, cells consistently (n = 14) exhibited "capacitance flicker," probably due to the flickering of a fission pore prior to complete endocytosis (Fig. 5C, trace b). As RE recovered fully, capacitance flicker was lost, suggesting that it may be due to incomplete reformation of polymerized microtubules. Nocodazole had no effect on exocytosis, in agreement with earlier data suggesting that microtubule integrity is not required for regulated secretion of catecholamines in these cells (26). (iii) Cytoplasmic acidification completely blocks clathrin-mediated endocytosis in other systems (7, 27). However, lowering pipette pH from 7.2 to 6.2 had no effect on the extent or rate of RE (Fig. SD; cf. ref. 5). We also found that antibodies against the small G protein rab5a, implicated in the early stages of receptor-mediated endocytosis (28), failed to affect RE (n = 9). Thus, by several criteria, it is unlikely that RE in chromaffin cells involves clathrin-coated vesicles.
DISCUSSION The present results demonstrate that RE is the major pathway for membrane retrieval under physiologically relevant
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FIG. 3. Guanine nucleotide turnover is necessary for RE. Cm was monitored continuously after establishment of whole-cell patch configuration with different intrapipette solutions. (A) No GTP in pipette. (B) GDP[,BS]-(350 ,M). (C) GTP[yS] (350 ALM). Secretion was elicited as described in Fig. 1 at the times indicated above the capacitance trace. The Ca2+ current elicited by the first test depolarization is plotted below the Cm trace. Replacement of GTP with GDP[13S] (B) or GTP[-yS] (C) blocked endocytosis without reducing exocytosis. The initial endocytosis remains intact in A presumably because sufficient cellular GTP remains at this time to support one round of membrane recovery. It also remains intact in B, probably because GDP[,BS] cannot gain access to binding sites on G proteins until bound GTP has been hydrolyzed. In C we surmise that GTP[,yS] can displace bound GTP and, hence, no endocytosis is possible. The absence of GTP or presence of GDP[13S] does not modify chromaffin-cell Ca2+ currents when measured as peak current; however, GTP[-yS] did reduce Ca2+ current (due to a reduction in components other than the facilitation current, data not shown). Neither the extent nor the rate of secretion was markedly affected by these conditions (see Table 1).
conditions in chromaffin cells. RE potentially performs two critical functions: enabling the cell to maintain a constant surface area while simultaneously preserving the integrity of the secretory granule membrane in the form of a recaptured vesicle. The fate of rapidly endocytosed vesicles is not known but may involve direct recycling of granules back to the secretory pool. In neurons RE may be the first step in the rapid recycling of synaptic vesicles. While there is less evidence for this phenomenon in nonneuronal cells, the fact that RE is found in both chromaffin cells and melanotrophs suggests that direct recycling may exist in diverse types of secretory cells. Indeed, experiments using an entirely different approach to ours indicate that rapid recycling of endocytosed membrane does 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 described by three time constants, only two of which were observed together in any single round of endocytosis. Of particular interest is the ultrafast process that occurred during but never after an exocytotic burst. The existence of such a mechanism means that there is not necessarily a lag between exocytosis and endocytosis, as has been claimed to occur in other systems (5). In addition, most cells displayed "excess retrieval" indicative of the recovery of surplus membrane. Nevertheless, capacitance then recovered back to baseline, suggesting the existence of some compensatory mechanism able to sense cell surface area. RE is kinetically distinct from either coated-vesicle-mediated or fluid-phase endocytosis, processes that undergo delayed activation after chromaffin cells are induced to massively secrete (30, 31). Under the latter
Neurobiology: Artalejo et al. A
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Proc. Natl. Acad. Sci. USA 92 (1995)
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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 loaded with 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 the first and second trains for antibodies to diffuse into the cell. Initial rates of endocytosis after the first exocytotic burst were not significantly different from control (for means, see Table 1) reflecting the fact that insufficient antibody has diffused into the cell at this time to affect RE. (E) Immunoblot of 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 IgGs MC13 and MC63. Note that either antibody reacts with the prominent 105-kDa dynamin band in both samples (arrow). Staining was absent if antibodies were preincubated with a 100-fold excess of their respective immunogenic peptide (in lane 3, the asterisk indicates a nonspecific band that was not competed out by the peptide). (F) Immunofluorescent detection (MC13 IgG) of dynamin in chromaffin cells. (Upper) Differential interference contrast image. (Lower) Fluorescence. (Bar = 10 ,um.)
circumstance, granule markers appear on the cell surface and mix with other constituents before being endocytosed, possibly by a coated-vesicle pathway involving clathrin. Fluidmay
phase endocytosis
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31) and does not require Ca2+ or GTP (32). Several additional pieces of evidence presented here suggest that RE does not involve a clathrin-based mechanism in chromaffin cells. Most importantly, anti-clathrin antibodies fail to affect RE but do inhibit receptor-mediated endocytosis in these cells. Our results show that the RE mechanism involves intracellular Ca2+, GTP hydrolysis, dynamin, and microtubules. RE is
Ca2+-dependent and we show that it cannot be supported by Ba2+ or Sr2 We speculate that Ca2+ may be the critical link that ensures tight coupling between RE and the preceding secretory event. As with exocytosis, the nature of the Ca2+ receptor(s) involved in endocytosis is unknown, but as exocytosis can occur with either Ba2+ or Sr2+, it is likely that the divalent cation receptors for exocytosis and RE are distinct molecules. RE requires GTP hydrolysis and a critical G protein regulating the process appears to be dynamin. This protein was originally 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
Membrane Maximum rate Maximum rate retrieved Maximum rate endocytosis within endocytosisultrafast, Peak current, exocytosis, capacitance 20 sec, % fast, fF/sec pA fF/sec Condition fF/sec change, fF 1017.1 ± 126.8 91.7 ± 5 100.4 ± 1.20 -797.4 ± 46.9 861.1 ± 70.6 869.1 ± 54.4 Control (n = 202) 0 Barium (n = 28) -1383.4 ± 74 1099.5 ± 102.9 430.6 ± 48.7 0 912.8 ± 107.1 567.7 ± 62.1 Strontium (n = 20) -1273.1 ± 83.1 0.87 ± 0.40 970 ± 109.2 888.1 ± 84.6 No GTP (n = 33) -731.7 ± 48.9 0 744.2 ± 97.8 -386.7 ± 26.7 982.9 ± 122.1 GTP[yS] (n = 13) = ± ± ± 68 0.66 ± 0.32 -761.16 38.3 946.6 108 856.7 (n 16) GDP[P3S] 0.90 ± 0.44 -725.7 ± 46.1 812.6 ± 107.7 718.3 ± 59.6 Anti-dynamin IgG (MC63) (n = 22) 1.08 ± 0.68 831.1 ± 72.1 774.2 ± 53.3 -691.8 ± 31.1 Anti-dynamin IgG (MC13) (n = 16) 936.3 ± 157 102.3 ± 8.7 102.3 ± 3.36 -748.4 ± 59 822 ± 116 839.9 ± 67.4 Anti-clathrin IgG (X19) (n = 13) 105.4 ± 3.42 92.8 ± 9.7 1034.1 ± 92 -793.3 ± 58.2 811.4 ± 71 889.1 ± 80 Anti-clathrin IgG (X22) (n = 10) 0 796.8 ± 84.1 920.1 ± 80.4 Nocodazole (n = 21) -789.2 ± 55.1 86.6 ± 7.3 106.9 ± 4.06 1233.7 ± 129.7 -659.3 ± 28.7 799.5 ± 68.5 872.8 ± 55.7 pH 6.2 (n = 15) 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 obtained by measuring the steepest downward slope of the Cm trace divided by the amplitude of the decline. Peak amplitudes and rates of Cm were each divided by the initial capacitance and then multiplied by the average initial capacitance of the cell to normalize data. Total
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Neurobiology: Artalejo et al. A Anti-clathrin heavy chain IgG( X19 ) C 5 min
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