MgCl2/4 mM K ITP/1 mM K2EGTA/0.5 mM CaCl2/20 AM. GTP[y-S], pH 7.2 ... "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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 normal and beige mice, a mutant with grossly enlarged secretory vesicles or granules. During degranulation, the membrane capacitance increased in steps, as single secretory vesicles fused with the cell membrane. The average step size was 10 times larger in beige than in normal mice, in agreement with the different granule sizes measured microscopically in the two preparations. Following individual capacitance steps in beige mice, individual granules of the appropriate size were observed to swell rapidly. Capacitance steps are frequently followed by the stepwise loss of a fluorescent dye loaded into the vesicles. Stepwise capacitance increases were occasionally intermittent before they became permanent, indicating the existence of an early, reversible, and incomplete state of vesicle fusion. During such "capacitance flicker," loss of fluorescent dye from vesicles did not occur, suggesting that the earliest aqueous connection between vesicle interior and cell exterior is a narrow channel. Our results support the view that the reversible formation of such a channel, which we term the fusion pore, is an early step in exocytosis.
MATERIALS AND METHODS Mast cells were obtained by peritoneal lavage of normal white mice (Swiss-Webster) or beige mice with the ChediakHigashi defect (bgJ/bgJ strain, The Jackson Laboratory) with a solution containing 140 mM NaCl, 2 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 5 mM glucose, 10 mM Hepes buffer, 45 mM NaHCO3, 0.4 mM KH2PO4, as well as 300 units of penicillin and 300 mg of streptomycin per ml. The pH was adjusted to 7.2 with NaOH. The cells were placed in an incubator (370C, 5% C02/95% air) for 20-120 min and allowed to settle onto glass coverslips forming the bottom of experimental chambers. Experiments were performed at 22-240C in 140 mM NaCl/2.5 mM KCl/2 mM CaCl2/5 mM MgCl2/5 mM glucose/10 mM Hepes buffer, pH 7.2 (osmolarity, 300 mosM). Cells were observed with an inverted microscope (Nikon Diaphot, Zeiss x100/1.30 oil) equipped for epifluorescence and voltage-clamped with glass micropipettes in a whole-cell recording mode (5). The composition of the pipette filling solution was 155 mM K glutamate/10 mM NaHepes/5 mM 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 external solution, and, in the whole-cell mode, established 4to 10-Mfl connections with the cytoplasm. All reagents were from Sigma. A lock-in amplifier (6, 7) was used for measuring membrane capacitance by superimposing a 320-Hz sinewave of 44 mV peak-to-peak amplitude onto the -50-mV holding potential. The capacitance signal was low-pass filtered with an RC-circuit of 30-ms time constant, or with a 4-pole Bessel filter with 100-Hz corner frequency. For fluorescence measurements, single cells were illuminated with a 20-,um diameter spot of light, and fluorescence was collected through a pinhole, mounted in the microscope's image plane, that just enclosed the area illuminated by the spot. Fluorescence was measured with a photomultiplier in the photon-counting mode (model 1140A, Princeton Applied Research, Princeton, NJ). Video recordings (camera model 76, Dage/Maryland Telecommunications, Michigan City, IN) were stored on a Sony SL 2700 video tape recorder. Fluorescence and capacitance signals were recorded on an FM tape recorder. In some experiments, the capacitance signal was passed through a voltage-to-frequency converter and stored on the audio channel of the video tape recorder. Statistical variation is given as the standard error of the mean, unless indicated otherwise.
Chediak-Higashi syndrome is a rare inherited disease characterized by decreased resistance to bacterial infections and abnormally large granules in leukocytes and mast cells. Analogous diseases occur also in cattle, mink, and mice. In mast cells, the giant granules represent enlarged secretory vesicles, because in beige (bgJ/bgJ) mice, a mutant with the Chediak-Higashi defect, they are readily exocytosed in response to Ca ionophores and a secretagogue, compound 48/80 (1). Mast cells ofbeige mice have granules large enough to be easily visible under the light microscope. Exocytosis, or the fusion of cytoplasmic vesicles with the cell membrane, is a universal event in eukaryotic cells, but neither the mechanism of membrane fusion nor the forces that drive this event are well understood. In this paper, we combined the study of three events in the exocytosis of single granules in the hope that each may report a different step in the exocytotic process. The first is the stepwise increase in cell membrane capacitance thought to represent the fusion of single secretory vesicles with the cell membrane (2), the second is the rapid swelling of secretory granules known to accompany exocytosis (3, 4), and the third is the stepwise loss of a fluorescent dye loaded selectively into secretory vesicles. The main finding is that the stepwise increase in capacitance precedes the other two events. Hence, the formation of an electrical connection between the cell exterior and the inside of a secretory vesicle is the first among the three events and cannot be driven by granule swelling.
RESULTS Fig. 1 shows bright-field (Left) and fluorescence micrographs (Right) of mast cells from normal (A and B) and beige (C-H) mice. With their hundreds of secretory granules, mast cells of normal mice have a granular appearance (A). By contrast, cells from beige mice have only some 10-20 grossly enlarged granules; four large and two smaller ones are shown in Fig. 1C, and several more were located above and below the plane of focus.
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.
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Proc. Natl. Acad. Sci. USA 84 (1987)
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FIG. 1. Bright-field (Left) and fluorescence (Right) micrographs of mast cells from normal (A and B) and beige (C-H) mice in the presence of 8 AtM quinacrine. E-G show the same cell; G and H were taken after adding a few drops of external solution poisoned with compound 48/80 (1 mg/ml). A-D were taken with a Leitz Dialux microscope (objective, x 100/1.20 water; condenser 1.3 n.a. oil). The lumen of secretory vesicles in mast cells is known to be acidic and to accumulate the fluorescent dye quinacrine, a weak base with two protonatable groups (8). This dye was present in Fig. 1, and the small fluorescent patches visible in the periphery of the normal mast cell (Fig. 1B) almost certainly represent single vesicles filled with quinacrine; the dark central area marks the location of the nucleus, where vesicles are excluded. That fluorescence is localized in the vesicles is more clearly seen in beige mice (Fig. 1 D and F). However, some vesicles fail to take up dye (arrows in Fig. 1 C and D), possibly because their lumen is less acidic. Fig. 1 G and H shows the cell of Fig. 1 E and F after partial degranulation was induced with the potent secretagogue, compound 48/80. Half of the cell (arrows in Fig. 1 E-H) was swollen and lost its fluorescence as dye diffused out of the apparently exocytosed granules. Fluorescence from the remaining intact granules, however, remained undiminished. Because quinacrine is exclusively located in secretory vesicles, and because it is apparently lost from individual granules only when they are swollen (a known accompaniment of exocytosis), we suggest that the loss of quinacrine fluorescence may be used as an assay of secretion at the level of single cells or even single granules.
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 of GTP, to diffuse out of the 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 a higher gain (Fig. 2E). A histogram of step sizes is plotted in Fig. 2F; the mean step size was 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 of the Cm steps in Fig. 2E represents the fusion of a single vesicle with the cell membrane. In beige mice, degranulation produces a smaller total 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 of few but 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 progressed with a similar time course. In the cell with giant granules (Fig. 2D), fluorescence declined in discrete episodes. A large Cm step occurred at the beginning of each episode, but some steps failed to lead to detectable fluorescence decline. Large steps not followed by fluorescence decline were nearly always observed in this kind of experiment and may represent exocytosis of vesicles that did not accumulate dye (as in Fig. 1 C and D). If dye content can be taken as an indicator of pH (8), it follows that intravesicular acidity is not necessary for exocytosis. Studies on chromaffin cells have led to similar conclusions (13, 14). Sometimes Cm increased in a step and then abruptly returned to the original level (arrow in Fig. 2B, see also ref. 2). Most degranulating cells experience at least one such episode. Recalling that Cm steps report only the formation of an electric connection between the vesicle lumen and the cell exterior (see Fig. 6), we conclude that such electric connection may be reversible and need not represent the (presumably irreversible) coalescence of secretory vesicles with the cell membrane. In Fig. 3A, Cm is seen to "flicker" rapidly between two levels before stabilizing at the higher level. Evidently, the electric connection is tenuous at first and becomes permanently established only later. Most of our recordings were made at a slower speed, where flicker appeared as in Fig. 3B (upper trace); similar episodes were seen in =20% of the degranulating cells. The Cm change in Fig. 3A did not lead to a decline in quinacrine fluorescence (trace not shown), but two other similar events did. In both cases (one of them illustrated in Fig. 3B), loss of dye from the granule did not occur until flicker had ceased. However, when Cm steps occurred
Biology: Breckenridge and Almers
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Proc. Natl. Acad. Sci. USA 84 (1987)
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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 of dye-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 established rapidly, fluorescence loss began immediately (Fig. 3C). This was observed in all of five cells without flicker; the average delay between the beginning of the fluorescence decline and the preceding step (defined in the legend of Fig. 3) was 140 100 ms, at the limit of our time resolution in these experiments and not significantly different ±
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FIG. 3. Changes in Cm (in pF) and quinacrine fluorescence (F) in degranulating beige mouse mast cells. Experimental conditions are the same as in Fig. 2 B and D. (A) Cm trace only; to improve time resolution, Cm was measured with an 800-Hz sinusoid and filtered at 400 Hz. (B and C) Cm (upper trace) and quinacrine fluorescence (lower trace). Fluorescence is given as a fraction of the value before degranulation. (C) A section of trace D in Fig. 2 (arrow) at expanded time scale. The beginning offluorescence decline (2.2 s and 0.1 s after the beginning of the Cm changes in B and C, respectively) was defined by the intercept, with the initial level of fluorescence, of a straight line fitted to the section of steepest decline.
from zero. While quinacrine is lost immediately after a stably established Cm step, the dye evidently does not readily escape from vesicles during flicker. Fluorescence loss was never seen to begin during flicker. In mast cells of beige mice, granules may be observed individually to swell during exocytosis (3, 4). In the experiment of Figs. 4 and 5, video recordings were made while Cm was recorded as a tone whose pitch varied linearly with Cm. During the degranulation, individual granules were seen to swell suddenly, and this "popping" of granules was accompanied by sudden changes in pitch representing Cm steps. Nearly every Cm step observed in this cell was accompanied by the popping of a granule, or by a sudden movement of the cell that presumably 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 their diameter could be measured in successive frames and plotted against time (Fig. 4). At different times, each of the granules swelled abruptly, and the four episodes of swelling are seen to coincide with Cm steps. Fig. 5A shows the swelling of the first granule in more detail. In Fig. SB, the increase in diameter followed the Cm step after a delay, but then rapidly went to completion. The Cm change showed no slow component with the time course of granule swelling; evidently, swelling occurred without a change in membrane surface area. Similar results were obtained in 11 similar analyses on six cells; both the delay (see the legend of Fig. 5) and the time required for half the diameter change varied widely; mean values are 0.44 0.10 s for the former (range, 0.20-1.16 s) and 0.91 0.19 s for the ±
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Proc. Natl. Acad Sci. USA 84 (1987)
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FIG. 4. Traces 1-4, changes in diameter of individual 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 x 3200 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 of our 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 diameter was 38% ± 3%. Similar granule swelling followed the Cm increase also when the tonicity of the cytosol was approximately doubled by adding 300 mM sucrose or stachyose to the pipette solution (not shown). If the vesicle membrane is normally under stress, a hypertonic cytosol should have relieved the 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 have been detected. In experiments as in Fig. 5, 12 granules looked approximately round; their surfaces were calculated from the diameters measured before swelling (mean, 3.1 ± 0.2 ,um) by assuming a spherical shape and were plotted against the amplitude of the Cm step (mean, 376 ± 36 if) preceding the swelling of that granule (plot not shown). The two variables
were strongly correlated; the chance of this correlation being fortuitous (correlation coefficient, 0.872) is
