Secretory Granules (Membrane Capacitance/Microfluorimetry/Video Microscopy/Granule Swelling) L

Proc. Natl. Acad. Sci. USA Vol. 84, pp. 1945-1949, April 1987 Cell Biology

Final steps in exocytosis observed in a cell with giant secretory granules (membrane capacitance/microfluorimetry/video microscopy/granule swelling) L. J. BRECKENRIDGE AND W. ALMERS Department of Physiology and Biophysics SJ-40, University of Washington, Seattle, WA 98195 Communicated by Bertil Hille, December 1, 1986

ABSTRACT Secretion by single mast cells was studied in MATERIALS AND METHODS normal and beige mice, a mutant with grossly enlarged Mast cells were obtained by peritoneal lavage of normal secretory vesicles or granules. During degranulation, the white mice (Swiss-Webster) or beige mice with the Chediak- membrane capacitance increased in steps, as single secretory Higashi defect (bgJ/bgJ strain, The Jackson Laboratory) with vesicles fused with the cell membrane. The average step size a solution containing 140 mM NaCl, 2 mM KCl, 1 mM CaCl2, was 10 times larger in beige than in normal mice, in agreement 2 mM MgCl2, 5 mM glucose, 10 mM Hepes buffer, 45 mM with the different granule sizes measured microscopically in the NaHCO3, 0.4 mM KH2PO4, as well as 300 units of penicillin two preparations. Following individual capacitance steps in and 300 mg of streptomycin per ml. The pH was adjusted to beige mice, individual granules of the appropriate size were 7.2 with NaOH. The cells were placed in an incubator (370C, observed to swell rapidly. Capacitance steps are frequently 5% C02/95% air) for 20-120 min and allowed to settle onto followed by the stepwise loss ofa fluorescent dye loaded into the glass coverslips forming the bottom of experimental cham- vesicles. Stepwise capacitance increases were occasionally in- bers. Experiments were performed at 22-240C in 140 mM termittent before they became permanent, indicating the NaCl/2.5 mM KCl/2 mM CaCl2/5 mM MgCl2/5 mM glu- existence of an early, reversible, and incomplete state of vesicle cose/10 mM Hepes buffer, pH 7.2 (osmolarity, 300 mosM). fusion. During such "capacitance flicker," loss of fluorescent Cells were observed with an inverted microscope (Nikon dye from vesicles did not occur, suggesting that the earliest Diaphot, Zeiss x100/1.30 oil) equipped for epifluorescence aqueous connection between vesicle interior and cell exterior is and voltage-clamped with glass micropipettes in a whole-cell a narrow channel. Our results support the view that the recording mode (5). The composition of the pipette filling reversible formation of such a channel, which we term the solution was 155 mM K glutamate/10 mM NaHepes/5 mM fusion pore, is an early step in exocytosis. MgCl2/4 mM K ITP/1 mM K2EGTA/0.5 mM CaCl2/20 AM GTP[y-S], pH 7.2 (osmolarity, 305 mosM). Pipettes filled with this solution had resistances of 1.5-2.5 MQl in the Chediak-Higashi syndrome is a rare inherited disease char- external solution, and, in the whole-cell mode, established 4- acterized by decreased resistance to bacterial infections and to 10-Mfl connections with the cytoplasm. All reagents were abnormally large granules in leukocytes and mast cells. from Sigma. A lock-in amplifier (6, 7) was used for measuring Analogous diseases occur also in cattle, mink, and mice. In membrane capacitance by superimposing a 320-Hz sinewave mast cells, the giant granules represent enlarged secretory of 44 mV peak-to-peak amplitude onto the -50-mV holding vesicles, because in beige (bgJ/bgJ) mice, a mutant with the potential. The capacitance signal was low-pass filtered with Chediak-Higashi defect, they are readily exocytosed in an RC-circuit of 30-ms time constant, or with a 4-pole Bessel response to Ca ionophores and a secretagogue, compound filter with 100-Hz corner frequency. For fluorescence mea- 48/80 (1). Mast cells ofbeige mice have granules large enough surements, single cells were illuminated with a 20-,um diam- to be easily visible under the light microscope. eter spot of light, and fluorescence was collected through a Exocytosis, or the fusion of cytoplasmic vesicles with the pinhole, mounted in the microscope's image plane, that just cell membrane, is a universal event in eukaryotic cells, but enclosed the area illuminated by the spot. Fluorescence was neither the mechanism of membrane fusion nor the forces measured with a photomultiplier in the photon-counting that drive this event are well understood. In this paper, we mode (model 1140A, Princeton Applied Research, Princeton, combined the study ofthree events in the exocytosis ofsingle NJ). Video recordings (camera model 76, Dage/Maryland granules in the hope that each may report a different step in Telecommunications, Michigan City, IN) were stored on a the exocytotic Sony SL 2700 video tape recorder. Fluorescence and capac- process. The first is the stepwise increase in itance signals were recorded on an FM tape recorder. In some cell membrane capacitance thought to represent the fusion of experiments, the capacitance signal was passed through a single secretory vesicles with the cell membrane (2), the voltage-to-frequency converter and stored on the audio second is the rapid swelling of secretory granules known to channel of the video tape recorder. Statistical variation is accompany exocytosis (3, 4), and the third is the stepwise given as the standard error of the mean, unless indicated loss of a fluorescent dye loaded selectively into secretory otherwise. vesicles. The main finding is that the stepwise increase in capacitance precedes the other two events. Hence, the RESULTS formation of an electrical connection between the cell exte- Fig. 1 shows bright-field (Left) and fluorescence micrographs rior and the inside of a secretory vesicle is the first among the (Right) of mast cells from normal (A and B) and beige (C-H) three events and cannot be driven by granule swelling. mice. With their hundreds of secretory granules, mast cells of normal mice have a granular appearance (A). By contrast, cells The publication costs of this article were defrayed in part by page charge from beige mice have only some 10-20 grossly enlarged gran- payment. This article must therefore be hereby marked "advertisement" ules; four large and two smaller ones are shown in Fig. 1C, and in accordance with 18 U.S.C. §1734 solely to indicate this fact. several more were located above and below the plane offocus.

Downloaded by guest on September 29, 2021 1945 1946 Cell Biology: Breckenridge and Almers Proc. Natl. Acad. Sci. USA 84 (1987) The fusion of secretory vesicles with the cell membrane increases the cell surface area, and this effect can be assayed by measuring the cell membrane capacitance, Cm (6, 9, 10). In Fig. 2 (A and B), rapid and virtually complete degranulation was induced by allowing GTP[-S], a nonhydrolyzable analog ofGTP, to diffuse out ofthe pipette into the cytoplasm (2). In both cells, degranulation brought a large increase in Cm. In normal mice, Cm increased %3-fold (from 7.1 ± 0.2 pF to 30.3 ± 0.7 pF; n = 31). This increase appears continuous in Fig. 2A, but it can be seen to occur in steps if displayed at c a higher gain (Fig. 2E). A histogram of step sizes is plotted in Fig. 2F; the mean step sizewas 20.3 fF ± 8.1 fF (SD; n = 198). From the mean increase in Cm, an average normal mast cell is calculated to have =1100 vesicles. With the usual Cm of 1 gF/cm2 for biological membranes the average granule surface would be that of a sphere with 0.80-Arm diameter, well within the range observed (0.5-1.0 ,um; see ref. 12). As in similar capacitance measurements on rat mast cells (2), this agreement supports the idea that each ofthe Cm steps in Fig. 2E represents the fusion of a single vesicle with the cell XAL T membrane. In beige mice, degranulation produces a smaller total E increase in Cm (on average, from 5.2 ± 0.3 pF to 8.1 ± 0.4 pF; n = 22), but the steps are so large that they can be seen even at a low gain (Fig. 2B). The histogram (Fig. 2G) shows a wide range of variation, with a possible preponderance of step sizes that are multiples of =50 fF. From the mean values for step size [222 ± 307 fF (SD; n = 418)] and total Cm increase, 13 Cm steps are expected to accompany degranulation in the average cell. The mean Cm step would result from a spherical vesicle of 2.7 A&m diameter. The few but large Cm steps in beige mice are clearly consistent with the presence offew but OW large vesicles. In Fig. 2, exocytosis was monitored also by measuring quinacrine fluorescence. In the normal cell (Fig. 2C), fluorescence diminished continuously, as dye was exocytosed together with other vesicle contents and diffused away into the external solution. Relative to the Cm increase, the fluorescence decline started with a small delay but then FIG. 1. Bright-field (Left) and fluorescence (Right) micrographs progressed with a similar time course. In the cell with giant of mast cells from normal (A and B) and beige (C-H) mice in the granules (Fig. 2D), fluorescence declined in discrete epi- presence of 8 AtM quinacrine. E-G show the same cell; G and H were sodes. A large Cm step occurred at the beginning of each taken after adding a few drops of external solution poisoned with episode, but some steps failed to lead to detectable fluores- compound 48/80 (1 mg/ml). A-D were taken with a Leitz Dialux cence decline. Large steps not followed by fluorescence microscope (objective, x 100/1.20 water; condenser 1.3 n.a. oil). decline were nearly always observed in this kind of experiment and may represent exocytosis of vesicles that did not The lumen of secretory vesicles in mast cells is known to accumulate dye (as in Fig. 1 C and D). If dye content can be be acidic and to accumulate the fluorescent dye quinacrine, taken as an indicator of pH (8), it follows that intravesicular a weak base with two protonatable groups (8). This dye was acidity is not necessary for exocytosis. Studies on chromaffin present in Fig. 1, and the small fluorescent patches visible in cells have led to similar conclusions (13, 14). the periphery of the normal mast cell (Fig. 1B) almost Sometimes Cm increased in a step and then abruptly certainly represent single vesicles filled with quinacrine; the returned to the original level (arrow in Fig. 2B, see also ref. dark central area marks the location of the nucleus, where 2). Most degranulating cells experience at least one such vesicles are excluded. That fluorescence is localized in the episode. Recalling that Cm steps report only the formation of vesicles is more clearly seen in beige mice (Fig. 1 D and F). an electric connection between the vesicle lumen and the cell However, some vesicles fail to take up dye (arrows in Fig. 1 exterior (see Fig. 6), we conclude that such electric connec- C and D), possibly because their lumen is tion may be reversible and need not represent the (presum- less acidic. ably irreversible) coalescence of secretory vesicles with the Fig. 1 G and H shows the cell of Fig. 1 E and F after partial cell membrane. In Fig. 3A, Cm is seen to "flicker" rapidly degranulation was induced with the potent secretagogue, between two levels before stabilizing at the higher level. compound 48/80. Half of the cell (arrows in Fig. 1 E-H) was Evidently, the electric connection is tenuous at first and swollen and lost its fluorescence as dye diffused out of the becomes permanently established only later. Most of our apparently exocytosed granules. Fluorescence from the re- recordings were made at a slower speed, where flicker maining intact granules, however, remained undiminished. appeared as in Fig. 3B (upper trace); similar episodes were Because quinacrine is exclusively located in secretory vesi- seen in =20% of the degranulating cells. cles, and because it is apparently lost from individual gran- The Cm change in Fig. 3A did not lead to a decline in ules only when they are swollen (a known accompaniment of quinacrine fluorescence (trace not shown), but two other exocytosis), we suggest that the loss of quinacrine fluores- similar events did. In both cases (one of them illustrated in cence may be used as an assay of secretion at the level of Fig. 3B), loss of dye from the granule did not occur until single cells or even single granules. flicker had ceased. However, when Cm steps occurred Downloaded by guest on September 29, 2021 Cell Biology: Breckenridge and Almers Proc. Natl. Acad. Sci. USA 84 (1987) 1947

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01 QF K L -, - -- 0o V T- 5s 02 0.'2 04 0.6 0.8 10 (pF) STEP SIZE (pF) FIG. 2. (A-D) Capacitance (A and B) and fluorescence changes (C and D, in arbitrary units) in single cells from normal (Left) and beige (Right) mice with 8 uM quinacrine added externally. To measure Cm, the tip of a glass micropipette was sealed against the cell. At t = 0, the membrane patch beneath the pipette tip was ruptured by a pulse of suction; this established both electric and diffusional contact between the micropipette and the cytosol. The cell was voltage-clamped, and changes in Cm were followed with a lock-in amplifier. The capacitance changes in A went beyond the linear range of the lock-in amplifier; however, the initial and final values should be correct as they were measured by nulling out the capacitance transient by means of the C, control on the patch-clamp amplifier (11). Vertical lines in D mark the occurrence of capacitance steps. To avoid the degranulation block that tends to result from the illumination ofdye-loaded cells, light was turned on only after patch rupture, so that fluorescence changes during the initial phase of degranulation were often missed. Hence, the dashed lines in C and D are speculative. (E) Cm changes during degranulation of a normal mast cell, shown at high gain. Three steps (horizontal dashed lines) followed each other too rapidly to reproduce well at the playback speed used here. (F and G) Step size histograms in normal (F) and beige (G) mice. (E-G) Without quinacrine.

without flicker and a permanent electric connection was from zero. While quinacrine is lost immediately after a stably established rapidly, fluorescence loss began immediately established Cm step, the dye evidently does not readily (Fig. 3C). This was observed in all of five cells without escape from vesicles during flicker. Fluorescence loss was flicker; the average delay between the beginning of the never seen to begin during flicker. fluorescence decline and the preceding step (defined in the In mast cells of beige mice, granules may be observed legend of Fig. 3) was 140 ± 100 ms, at the limit of our time individually to swell during exocytosis (3, 4). In the experiment resolution in these experiments and not significantly different of Figs. 4 and 5, video recordings were made while Cm was recorded as a tone whose pitch varied linearly with Cm. During A the degranulation, individual granules were seen to swell sud- (pF) denly, and this "popping" of granules was accompanied by sudden changes in pitch representing Cm steps. Nearly every Cm 5 step observed in this cell was accompanied by the popping of a B C granule, or by a sudden movement of the cell that presumably (pF) resulted from the popping of a granule. Four granules in this cell, indicated by numbered arrows in the leftmost video frame of Fig. 5, remained in focus throughout the experiment, so that F -,J- - A-1- their diameter could be measured in successive frames and 02 plotted against time (Fig. 4). At different times, each of the 10 s A granules swelled abruptly, and the four episodes of swelling are seen to coincide with Cm steps. FIG. 3. Changes in Cm (in pF) and quinacrine fluorescence (F) in Fig. 5A shows the swelling of the first granule in more degranulating beige mouse mast cells. Experimental conditions are detail. In Fig. SB, the increase in diameter followed the Cm the same as in Fig. 2 B and D. (A) Cm trace only; to improve time step after a delay, but then rapidly went to completion. The resolution, Cm was measured an at with 800-Hz sinusoid and filtered showed no component course 400 Hz. (B and C) Cm (upper trace) and quinacrine fluorescence Cm change slow with the time of granule a (lower trace). Fluorescence is given as a fraction of the value before swelling; evidently, swelling occurred without degranulation. (C) A section of trace D in Fig. 2 (arrow) at expanded change in membrane surface area. Similar results were time scale. The beginning offluorescence decline (2.2 s and 0.1 s after obtained in 11 similar analyses on six cells; both the delay the beginning ofthe Cm changes in B and C, respectively) was defined (see the legend of Fig. 5) and the time required for half the by the intercept, with the initial level of fluorescence, of a straight diameter change varied widely; mean values are 0.44 ± 0.10 line fitted to the section of steepest decline. s for the former (range, 0.20-1.16 s) and 0.91 0.19 s for the Downloaded by guest on September 29, 2021 1948 Cell Biology: Breckenridge and Almers Proc. Natl. Acad Sci. USA 84 (1987)

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1 i 10 s FIG. 4. Traces 1-4, changes in diameterofindividual granules during degranulation ofa beige mouse cell recorded on video tape. The granules and their numbers are indicated in Fig. 5A by arrows. Vesicle diameters were measured on Polaroid prints made of individual frames at x3200 magnification. This was done by using a plastic template with circular holes whose diameters varied in 1/32-inch steps (corresponding to -250 nm) and by determining which of the holes best fit the granule outline. If a granule outline was equally well fitted by two holes differing 1/32 inch in diameter, the granule diameter was taken as halfway between the two hole sizes. Hence, the quantization ofour measurement is in steps of 250/2 = 125 nm. Each granule was measured twice by each of two investigators; the deck of Polaroids was shuffled before each series of measurements, and the investigator did not know, while making the measurement, at which time the video frame was taken. The measurements were highly reproducible, and a 250-nm change in diameter was readily detected. Averages ofthe quadruplicate measurements are plotted. Trace 5, Cm changes in this mast cell; experimental conditions are the same as in Fig. 2D except that no dye was present and Cm was recorded as an FM signal on the audio channel of a video tape recorder and regenerated by a frequency-to-voltage converter during playback. Note that, unlike the initial granule diameter (see text), the amount of swelling is not obviously correlated with the Cm step size.

latter (range, 0.30-1.82 s). The mean increase in granule were strongly correlated; the chance ofthis correlation being diameter was 38% ± 3%. fortuitous (correlation coefficient, 0.872) is <0.001. The Similar granule swelling followed the Cm increase also regression line through the origin had a slope of 1.17 ILF/cm2, when the tonicity of the cytosol was approximately doubled well in line with values found for other biological membranes. by adding 300 mM sucrose or stachyose to the pipette These results strongly support the view that the Cm steps solution (not shown). If the vesicle membrane is normally accompanied the exocytosis of the granules that were sub- under stress, a hypertonic cytosol should have relieved the sequently observed to swell. stress by causing shrinkage. In none of six exocytosing granules was there evidence of swelling in the 1 s preceding the Cm step, even though an 8% change in diameter would DISCUSSION have been detected. Ourresults show that mast cells ofbeige mice, with theirgiant In experiments as in Fig. 5, 12 granules looked approxi- granules, offer uniquely favorable conditions for studying mately round; their surfaces were calculated from the diam- exocytosis at the level of single vesicles. Our main finding is eters measured before swelling (mean, 3.1 ± 0.2 ,um) by that, among three events associated with the exocytosis of assuming a spherical shape and were plotted against the single vesicles-stepwise increases in Cm, quantized release amplitude of the Cm step (mean, 376 ± 36 if) preceding the of a fluorescent dye, and granule swelling-the step change swelling of that granule (plot not shown). The two variables in Cm precedes the two others.

Ad FIG. 5. (A) Swelling of granule 1 as seen in video frames taken around the time of the second Cm step in Fig. 4. (B) Superimposed time courses of granule diameter (a and o) and Cm (solid line). Measurement of granule diameter as in Fig. 4; the sequence of circles was obtained from the sequence of video frames in A. The Cm step was used to trigger a square pulse, which in turn grounded the video input to the cassette 49 recorder for =30 ms. The scrambled video frame recorded during that time contained no useful image of 354 Z the cell but marked the occurrence ofthe C. step on the ; video track. The recording ofthe Cm increase in B was 0 limited in its time resolution by an RC filter of 30 ms 4.7 time constant. Error bars reflect the reproducibility of K I G measuring the granule diameter on a given Polaroid o Q print; none are given if <4 nm. The delay of granule e swelling is defined as the time between the beginning of the Cm step and the intersection with the baseline of a 4.5 straight line fitted to the sequence of dots showing the 0.5 s steepest increase. Downloaded by guest on September 29, 2021 Cell Biology: Breckenridge and Almers Proc. Natl. Acad. Sci. USA 84 (1987) 1949 Fig. 6 shows a sequence of events that would be consistent with planar lipid bilayers (21), and that hypertonic solutions with our observations. The vesicle (A) first binds to the cell tend to inhibit exocytosis (20). However, it has been difficult membrane via specific sites (B). Virus infection is an example to tell whether swelling and other morphologic changes in of how this may be accomplished with a single specific granules (such as the dissembly of crystalline structures seen membrane protein (15), which could be located in either the in quiescent granules; see ref. 22) occurs before or after the vesicle or the cell membrane. In other cases, membrane formation of fusion pores, or other, similar fusion contracts proteins in both vesicle and cell membrane may be involved that are too small to be readily visible microscopically. In (e.g., mucocyst discharge in Tetrahymena; see ref. 16). Upon mast cells, granule swelling clearly follows the opening of binding, cell and vesicle membranes come in close apposition fusion pores, even when possibly preexisting mechanical (17, 18). Next, an aqueous channel forms (C), connecting the stress on the vesicle membrane is relieved by an osmotic vesicle interior to the cell exterior much as a gap junction gradient. Hence, mechanical stress on the vesicle membrane connects the cytoplasm of two adjacent cells. Both electric cannot be the driving force for membrane fusion during current and small solutes may pass through this channel, exocytosis. The same may be true in other secretory cells. which we term the "fusion pore." Since the fusion pore Recent findings suggest, for example, that osmotic swelling establishes an electric connection between vesicle lumen and is not required for exocytosis of adrenal chromaffin granules cell exterior, the vesicle membrane now contributes to the (23). Cm. A step increase in Cm is expected to accompany the opening, and a step decrease to accompany the closing, of a We thank Dr. Erwin Neher for providing us with circuit boards for fusion pore. Hence, fusion pores may cause Cm flicker (Fig. the lock-in amplifier. This work was supported by Grant AM-17803 3 A and B) by opening and closing repeatedly before a wider from the National Institutes of Health. and more permanent connection is established. We do not know whether the lining of the fusion pore is proteinaceous 1. Poon, K. C., Liu, P. I. & Spicer, S. S. (1981) Am. J. Pathol. or lipidic, but the fusion pore must be narrow because it 104, 142-149. retards or prevents the escape of quinacrine. In Limulus 2. Fernandez, J. M., Neher, E. & Gomperts, B. D. (1984) Nature amoebocytes undergoing exocytosis, a narrow channel was (London) 312, 453-455. occasionally observed to bridge vesicle and cell membranes 3. Curran, M. J., Brodwick, M. S. & Edwards, C. (1984) Bio- in regions of close proximity (18); it may correspond to the phys. J. 45, 170a (abstr.). fusion pore inferred here from electrical measurements. 4. Curran, M. J. & Brodwick, M. S. (1984) Biophys. J. 45, 170a When the vesicle irrevocably commits itself to fusion with (abstr.). the cell membrane (D), the fusion pore is replaced by a wider 5. Hamill, 0. P., Marty, A., Neher, E., Sakmann, B. & Sig- opening, and the vesicle matrix worth, F. J. (1981) Pflugers Arch. 391, 85-100. (presumably identical with 6. Neher, E. & Marty, A. (1982) Proc. Natl. Acad. Sci. USA 79, the granule observed under the microscope) swells and 6712-6716. extrudes itself from the vesicle cavity. In amoebocytes, the 7. Almers, W. & Neher, E. (1987) J. Physiol. (London), in press. morphologic changes that accompany matrix swelling begin 8. Johnson, R. G., Carty, S. E., Fingerhood, B. J. & Scarpa, A. near the pore (18), suggesting that the swelling, here and in (1980) FEBS Lett. 120, 75-79. amoebocytes, is induced by the entry or release of small 9. Jaffe, L. A., Hagiwara, S. & Kado, R. T. (1978) Dev. Biol. 67, solutes fusion pores. 243-248. through Conceivably, the swelling ofthe 10. Gillespie, J. I. (1979) Proc. R. Soc. London Ser. B 206, matrix in mast cells drives the dilation of the fusion pore. 293-306. However, the swelling cannot be accompanied by significant 11. Marty, A. & Neher, E. (1983) in Single Channel Recording, stretching of the vesicle membrane, because in experiments eds. Sakmann, B. & Neher, E. (Plenum, New York), pp. as in Fig. SB, the increase in Cm has no component with a time 107-122. 12. Chi, E. Y. & Lagunoff, D. (1975) J. Histochem. Cytochem. 23, course similar to that of granule swelling. 117-122. Swelling and related morphologic changes in granules have 13. Holz, R. W., Senter, R. A. & Sharp, R. R. (1983) J. Biol. been observed to accompany exocytosis in so many cell Chem. 258, 7506-7513. types that they have been considered to be necessary 14. Knight, D. E. & Baker, P. F. (1985) J. Membr. Biol. 83, preludes to membrane fusion. it 147-156. Specifically, has been 15. White, J., Kielian, M. & Helenius, A. (1983) Q. Rev. Biophys. suggested that granule swelling drives fusion by mechanically 16, 151-195. stretching the vesicle membrane (19, 20). This view is 16. Satir, B., Schooley, C. & Satir, P. (1973) J. Cell Biol. 56, reinforced by the finding that osmotic gradients in the 153-176. appropriate direction can induce the fusion of lipid vesicles 17. Lawson, D., Raff, M. C., Gomperts, B., Fewtrell, C. & Gilula, N. B. (1977) J. Cell Biol. 72, 242-259. 18. Ornberg, R. L. & Reese, T. S. (1981) J. Cell Biol. 90, 40-54. A _ B _ C A 19. Zimmerberg, J. & Whitaker, M. (1985) Nature (London) 315, 581-584. 20. Finkelstein, A., Zimmerberg, J. & Cohen, F. S. (1986) Annu. Rev. Physiol. 48, 163-174. 21. Cohen, F. S., Akabas, M. H. & Finkelstein, A. (1982) Science FIG. 6. Hypothetical sequence of events during exocytosis of 217, 458-460. mast cell secretory vesicles. Stippled area represents the "granule" 22. Caulfield, J. P., Lewis, R. A., Hein, A. & Austen, K. F. observed under the microscope. In D, the granule is no longer (1980) J. Cell Biol. 85, 299-311. entirely surrounded by membrane and, hence, can swell without 23. Holz, R. W. & Senter, R. A. (1986) J. Neurochem. 46, 1835- stretching the vesicle membrane. 1842. Downloaded by guest on September 29, 2021