ANNUAL REVIEWS
Further
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Annu. Rev. Physiol. 1990.52:607-624. Downloaded from www.annualreviews.org Access provided by University of Texas Southwestern Medical Center on 01/21/15. For personal use only.
Annu. Rev. Physiol. 1990. 52:607-24 Copyright © 1990 by Annual Reviews Inc. All rights reserved
EXOCYTOSIS W. Almers Department of Physiology and Biophysics, University of Washington, Seattle, Wash ington 98195
KEY WORDS:
fusion pore, patch-clamp, fusion intermediates, membrane capacitance, fusion proteins
INTRODUCTION All eukaryotic cells contain membrane-bounded vesicles that are destined for export, Molecular motors capture these vesicles in the cell interior and carry them to the plasma membrane; this requires metabolic energy. When the vesicle approaches the cell surface to within a few nanometers, it may undergo exocytosis, that is, the membrane surrounding the vesicle may fuse with the plasma membrane. The vesicle membrane then becomes a part of the cell membrane, and any material within the vesicle diffuses into the ex tracellular space. Eukaryotic cells undergo exocytosis either to insert new components into the plasma membrane
or
to export the membrane-imperme
ant substances stored within the vesicles. In constitutive exocytosis, vesicles exocytose approximately as soon as they reach the cell membrane, and no known mechanism controls the fusion event. In stimulated or regulated exocytosis, vesicles gather beneath the cell membrane in clusters and lie there waiting, until a signal reaching the cell membrane causes the appearance of a cytosolic messenger substance. The messenger then causes conformational changes in an unknown receptor protein on either the vesicle or the cell membrane, so that many or most of the vesicles undergo exocytosis in a burst. The regulatory processes vary among different cells; in some (neurons or 2+ eggs), an increase in cytosolic Ca is enough to trigger exocytosis, while in 2+ others (e.g. mast cells; Gomperts, this volume), Ca is merely a cofactor. The mechanisms of fusion also may vary. In this review, however, I make the 607
0066-4278/90/0315-0604$02.00
ALMERS
608
unproven assumption that fusion occurs in the same way in all cells, even when it is coupled to different control mechanisms. One can imagine fusion to be regulated in two ways.
(a) If secretory
vesicles were innately eager to fuse with cell membranes, then regulating fusion would mean preventing it from happening in quiescent cells.
(b)
Annu. Rev. Physiol. 1990.52:607-624. Downloaded from www.annualreviews.org Access provided by University of Texas Southwestern Medical Center on 01/21/15. For personal use only.
Alternatively, the fusion of biological membranes may be energetically diffi cult and require catalysis by specific fusion proteins. These would be con tinuously active in constitutive exocytosis and only occasionally active in regulated exocytosis. There are a number of reasons why the latter is probably correct.
(a) Artificial bilayers made from biological lipids do not readily fuse (56). (b) The
with each other under the conditions found in most living cells
fusion of viral envelopes to host cell membranes is the only fusion process understood at the molecular level, and it requires catalysis by known fusion proteins
(73). (c) In yeast, where secretion is constitutive, several gene (46).
products are required for vesicles to undergo exocytosis
Previous articles have discussed why artificial lipid bilayers are so resistant to fusion
(56), how this resistance may be overcome, and what the findings (7, 20,
with pure lipid bilayers tell us about membrane fusion in living cells
55). The extensive work on pure lipid systems is important and exocytotic fusion clearly does involve the rearrangement of lipids. Nonetheless, it is difficult to disagree with Rand
& Parsegian (55) that "in spite of heroic
efforts, phospholipid bilayer models of fusion do not mimic the cellular processes closely e'1ough to be confident that the cellular mechanism is being probed". The working hypothesis in this review will be that the earliest steps in membrane fusion are due to specific proteins that simultaneously interact with both the plasma and the vesicle membrane
(8, 9, 32, 34, 53, 60, 79).
Although the molecular basis of exocytotic membrane fusion is unknown, promising methods to study exocytosis in a controlled biochemical environ ment have been developed and reviewed [chromaffin cells eggs
(5), sea urchin
(28), Paramecium (51)].
Two Key Properties of Exocytotic Membrane Fusion EXOCYTOSIS CAN BE FAST
After a stimulus reaches a cell, how long does it
take before the first vesicles undergo exocytosis? In the examples listed in Table
1, this delay generally lasts seconds to tens of seconds. Most of this
time is probably spent in generating the cytosolic messenger, in removing cytosolic barrier proteins (e.g. synapsin) or, in some cases, in conveying vesicles from the depth of the cell to the plasma membrane. To appreciate how rapidly exocytotic fusion can occur, it is instructive to consider neurons. In neurons, the vesicles containing the secreted product (i.e. the transmitter) are parked so close to the plasma membrane that they touch, and no chemical
EXOCYTOSIS
Annu. Rev. Physiol. 1990.52:607-624. Downloaded from www.annualreviews.org Access provided by University of Texas Southwestern Medical Center on 01/21/15. For personal use only.
Table 1
609
Speed of exocytosis
Cell type
Stimulus
Sea urchin egg
sperm
Neutrophils Mast cells
GTP-,),-S GTP-,),-S antige n 48/80 electrical electrical electrical
Chromaffin cell Frog motor neuron Cat afferent neurons
Delay
Duration
Temperature
(s)
(8)
°C
Method
Reference
40 50 50 60 5 < 0.2 0.0005 0.0002
80 200 200 100 5
15 201 20 1 201 201 201
C C C C C C
30 47
19
psI'
33
psp
42
38
19
36 38 44
13 Delay, time between application of stimulus and first signs of exocytosis; duration, time over which
membrane area increased; C, capacitance measurement; psp, postsynaptic potential. ITemperature given as room temperature.
reaction is required to generate the active cytosolic messenger, Ca2+. Instead, Ca2+ enters the cytosol by crossing the plasma membrane through Ca chan nels. These Ca channels open during an action potential across the plasma membrane, and probably are 10-20 nm away from the vesicles. At the frog motor nerve terminal, the delay between the action potential and exocytosis (as assayed by the post-synaptic potential) is only 0.5 ms at 19°C (33). In mammalian neurons at 38°C, the delay is even less (0.2 ms; 13, 42). In this short time, several events must occur in sequence: first, Ca channels must open; then [Ca2+] near the synaptic vesicles must rise; Ca2+ must bind to its (unknown) receptor; the receptor must change conformation; the molecular rearrangements of membrane fusion must occur; and finally the transmitter must diffuse to its postsynaptic receptor, open ion channels, and produce the postsynaptic response. Even if all of the 0.5 ms synaptic delay in a frog neuromuscular junction were available for the triggering and execution of membrane fusion, it would suffice for at most one enzymatic reaction . Consider the rates of two enzymatic reactions that have been considered to start exocytosis: phosphorylation and dephosphorylation (23, 77). Some placental phosphatases (MW 40,000) have maximal rate constants of up to 45 M-mol of substrate/min and per mg enzyme (67); this translates into one phosphate liberated by one enzyme molecule every 33 ms. One of the fastest known protein kinases, phosphorylase b kinase (MW 1,300,000) will phos phorylate 15 M-mol substrate/min and per mg enzyme (65) at 30°C, or one substrate molecule every 12 ms per catalytic subunit. Both phosphorylation and dephosphorylation are rapid enough to play a role in signal transduction in many secretory cells and, indeed, in many aspects of neuronal secretion. Such reactions, however, are probably too slow to play a role in exocytosis at fast
610
ALMERS
synapses. Judging by the enormous speed of transmitter release, exocytosis is probably triggered by a conformational change in a single macromolecule. EXOCYTOSIS IS TIGHT
The fusion of lipid bilayers involves profound
molecular rearrangements. Yet exocytotic membrane fusion causes damage to
Annu. Rev. Physiol. 1990.52:607-624. Downloaded from www.annualreviews.org Access provided by University of Texas Southwestern Medical Center on 01/21/15. For personal use only.
neither plasma nor vesicle membranes. As far as we know, exocytosis is alway tight; there is no morphologic evidence that exocytosis ever leads to the rupture of vesicles and to the discharge of their contents into the cytosol. The only connection that forms is qetween vesicle lumen and extracellular space, and no leaks form between the vesicle lumen and the cytosol, or between the extracellular space and the cytosol. Lindau & Fernandez (36) have reported an experiment where the electrical conductance between the cytosol and the extracellular space increased by less than 100 pS while about 1000 vesicles underwent exocytosis. Even if none of the vesicles contained open ion channels, each vesicle could have caused a leak of at most 0.1 pS, lOO-fold less than the conductance of a typical ion channel. Clearly the exocytotic mechanism is highly successful in limiting leaks between cytosol and vesicle lumen or between cytosol and extracellular space. This success distinguishes exocytosis from known methods of fusing artificial lipid vesicles with other vesicles or with planar bilayers. It suggests that a highly specific macro molecule must form the first connection between the lumen of an exocytosing vesicle and the cell exterior. The gap junction is a macromolecule that can achieve such a feat because it connects two aqueous compartments selectively without connecting them to a third compartment. Gap junctions do not, however, mediate fusion.
Morphology EARLY STEPS IN EXOCYTOSIS
In thin sections, vesicles caught in the act of
exocytosis are seen to form characteristic omega-figures, which show the vesicles united with the plasma membrane by a narrow neck of membrane. In freeze-fracture, the neck appears as a bump or a dip. The center of the larger bumps can be made into a pit by etching. It must therefore contain aqueous material and represent a pore (called fusion pore in this review) that connects the vesicle lumen with the extracellular space. Even in the same section, pore diameters vary over a wide range. Investigators infer that the pores are narrow at first and later dilate as fusion progresses. The smallest pores seen under the electron microscope are generally about
20 nm in diameter; the preparations examined include posterior pituitary cells (18), mast cells (12), the neuromuscular junction (26), adrenal chromaffin cells (62), and Paramecium (41). In Limulus amoebocytes, an even smaller pore may have been seen. Thin sections of secreting amoebocytes show small areas where the two fusing membranes touch and form a sheet two mem-
Annu. Rev. Physiol. 1990.52:607-624. Downloaded from www.annualreviews.org Access provided by University of Texas Southwestern Medical Center on 01/21/15. For personal use only.
EXOCYTOSIS
611
branes thick (50). Such "pedestals" are thought to form as a prelude to the fusion pore, and one of the pedestals was said to contain a small pore of 5 nm diameter. The pores seen in electron micrographs are comparable in diameter to the pores (17) formed by complement (10 nm) or perforin, the toxin of cytotoxic T-Iymphocytes (16 nm). Both complement and perforin pores are assemblies of protein molecules. Whether or not fusion pores include proteins or protein complexes is not clear from electron micrographs. Do the 20 nm pores arise suddenly and without a smaller precursor? Except possibly in amoebocytes, any such precursors are evidently too short-lived to be captured by present morphologic techniques. Unfortunately, it is precisely the early and short-lived forms of the fusion pore that will tell us the most about the mechanism of exocytosis. How does a fusion pore form? What are its molecular constituents? What are its properties at the instant of its forma tion? How does it expand, what is its fate, and ultimately how is it replaced by an apparently seamless neck of lipid bilayer connecting the vesicle to the plasma membrane? Some of these questions must be addressed with methods that can resolve fast events. At neuronal synapses and in some protozoa (such as Paramecium), exocytosis occurs at precisely de termined sites, and one can search for membrane specializations that may be related to exocytosis. In both cell types, freeze-fracture reveals ordered arrays of plasma membrane macromolecules close to sites of exocytosis. In the frog neuromuscular junction, such sites (called active zones) are marked by double rows of intramembrane particles (11), and exocytosis occurs within a few tens of nm of these intramembrane particles. Large and sometimes ordered parti cles in the plasma membrane near active zones also occur at many other synapses, and it is likely that they play a role in exocytosis. In the neuro muscular junction, they cannot be fusion proteins, since exocytosis always occurs near but never exactly above the particles. The particles may represent voltage-sensitive Ca channels (25), but there is no experimental evidence concerning their role. In Tetrahymena (60, 61) and Paramecium (52), each secretory vesicle docks beneath a set of some 10 plasma membrane particles. The particles form a radially symmetric cluster called a rosette that is probably related to exocytosis, since mutants without rosettes cannot exocytose their vesicles (6). In Paramecium, the tip of the secretory vesicle (trichocyst) just beneath the rosette shows an annular-shaped zone enriched with intramembrane particles; whether these particles interact with those of the rosette is unclear. At ftrst the rosette particles were thought to form the fusion pore (60). However, rare images probably representing early stages in membrane fusion (41) show a dispersing rosette, with a 10-20 nm diameter fusion pore forming
SPECIALIZATIONS NEAR EXOCYTOSIS SITES
Annu. Rev. Physiol. 1990.52:607-624. Downloaded from www.annualreviews.org Access provided by University of Texas Southwestern Medical Center on 01/21/15. For personal use only.
612
ALMERS
in the middle. These images suggest that the rosette particles are not at the site of fusion when fusion occurs; hence the particles cannot be fusion proteins. It was later argued that the rosette particles were Ca channels providing the Ca influx thought to be necessary for triggering exocytosis (59). If so, they must be Ca channels of a new type previously unstudied. The voltage-sensitive Ca channels in Paramecium are associated with the cilia and not with the rosette (48), and both deciliated Paramecia and mutants lacking functional voltage sensitive Ca channels are capable of exocytosis (51). In fact, it is unknown how much of the Ca needed for exocytosis comes from the outside in Paramecium. The role of rosettes in exocytosis remains a mystery. In Chlamydomonas (72), an intracellular contractile vacuole gathers low molecular-weight waste products and periodically discharges them into the extracellular environment. This discharge does not represent exocytosis in the strict sense, since the vacuole never fuses with the plasma membrane. In stead, the plasma and vacuole membranes approach each other to within a few nm and enclose between them a periodic array of electron-dense spots. The array of spots corresponds to an array of large intramembrane particles that appears in the cytosol-facing leaflets of both vacuole and plasma membranes. It has been suggested that the particles form aqueous channels connecting the vacuole lumen with the external medium and that the discharge mechanism of the contractile vacuole is an evolutionary precursor of exocytosis (72). Where resting secretory vesicles bulge against the plasma membrane, the narrow cytoplasmic space between vesicle and plasma membrane is some times bridged by narrow filaments [e.g. in mast cells (12) or pancreatic beta cells (49)]. A recent review discusses the possible roles of cytoskeletal . elements in exocytosis (34). Electrophysioiogic Assays .of Exocytosis Electrophysiologic assays allow one to monitor exocytosis at the level of single cells and even single secretory vesicles. At synapses, the postsynaptic potential reports the secretion of transmitter by a nerve terminal. In some synapses, miniature postsynaptic potentials can be recorded and provide, at submillisecond time resolution, an assay for exocytosis at the level of single synaptic vesicles (16). A more recent electrophysiologic assay relies on the fact that each exocytotic event increases the cell surface area. Since all biological membranes have an electrical capacitance of 1 JJ,F/cm2 one may monitor the cell surface area (and hence exocytosis) by measuring the plasma membrane capacitance (21, 30). The method was perfected by Neher & his collaborators (37, 44) and applied to most of the cells in Table 1. When secretion is sufficiently slow (no more than a few vesicles per second), the capacitance (C) can be seen to increase in small steps, each reporting the exocytosis of a single secretory vesicle. Such C steps have been seen in
Annu. Rev. Physiol. 1990.52:607-624. Downloaded from www.annualreviews.org Access provided by University of Texas Southwestern Medical Center on 01/21/15. For personal use only.
EXOCYTOSIS
613
adrenal chromaffin cells (44), mast cells (19), pancreatic acinar cells (39), neutrophils (47), and pituitary lactotrophs (40). C steps as small as 2 tF can be resolved, which correspond to vesicles of 0.2 JLm2 surface area or 250 nm diameter (44, 47). Exocytosis of single synaptic vesicles containing a fast neurotransmitter (typically 50 nm in diameter) is still beyond the reach of this assay, but C changes due to the release of many synaptic vesicles can be detected (22). The method can also be used for monitoring episodes of endocytosis (2). Mast cells have been useful for studying single exocytotic events because their secretory vesicles are large. Particularly convenient are mast cells from beige mice (strain C57BL/6J-bgj/bgj),which have a genetic defect resembling Chediak-Higashi syndrome in humans. Because of this defect,mast cells and other granulocytes of beige mice are unable to limit the size of their secretory vesicles. Instead of having some thousand vesicles of about 0.8 JLm diameter, mast cells of beige mice have only 10-40 giant vesicles of 1-5 /Lm diameter. They secrete readily when stimulated by mast cell secretagogues (54) or intracellular GTP-,),-S (8, 78) and individual vesicles can be seen under the light microscope while they exocytose (15). Nowhere are the electric signals associated with the exocytosis of single vesicles as large as in mast cells of beige mice. Time-Resolved Exocytosis of Single Vesicles
DO OSMOTIC FORCES DRIVE MEMBRANE FUSION? Pure lipid bilayers can be fused to each other by placing them under mechanical stress. Secretory vesicles often swell as cells secrete,which suggests that swelling stretches the vesicle membrane and thereby causes membrane fusion [for a review of this hypothesis see (20)]. Mast cells of beige mice provide an opportunity to test this idea, because one can watch individual vesicles swell while one measures the capacitance (e) to monitor fusion. Two independent studies (1,8,78,79) have shown that a step increase in C always precedes swelling. In these studies, the e step clearly represented fusion of the vesicle because when the vesicle's diameter was measured under the light microscope, the surface area calculated agreed well with the amplitUde of the e step. Swelling followed the step with a mean delay of 0.4 s (8). Even in osmotically shrunk vesicles, fusion preceded swelling; hence even vesicles with a slack membrane can fuse. Work on chromaffin cells provides additional evidence that vesicle membranes can fuse without being stretched (27). Swelling of the vesicle may have an important role in hastening extrusion of the contents from the vesicle cavity; the mechanism of swelling has been reviewed (Verdugo, this volume). It is likely, however, that vesicle swelling is a consequence rather than a cause of membrane fusion. In the search for early events in membrane fusion one must look elsewhere.
614
ALMERS
REVERSIBLE INTERMEDIATES IN MEMBRANE FUSION
It is widely believed
that exocytosis is irreversible and that the subsequent retrieval of the exocy tosed membrane occurs by a different mechanism. In agreement with this view, a plot of mast cell capacitance (i.e. mast cell surface) against time
Annu. Rev. Physiol. 1990.52:607-624. Downloaded from www.annualreviews.org Access provided by University of Texas Southwestern Medical Center on 01/21/15. For personal use only.
usually looks like a staircase, with each step building upon the previous one
(9, 19). Occasionally, however, one obtains recordings as in Figure l a, where C increases in a step, but then fluctuates for a second and once even returns to baseline. Since the step increase in C reports the formation of an electric connection between the exterior and the lumen of the exocytosing vesicle (the fusion pore), the experiment shows that this connection can form and break repeatedly. Only later does the step become stably established, which sug gests that the vesicle has fused completely. Figure
1a is reminiscent of a
single-channel recording. Just as the sudden appearance and disappearance of current through the single ion channel represents the sudden opening and closing of that channel, so do the flickering fluctuations in
C represent the
opening and closing of the fusion pore.
(19), capacitance flicker as in Figure l a has (1, 19) and from beige mice (4, 8, 9, 78). A flickering vesicle filled with a First seen by Fernandez et al
now been seen by several other groups in mast cells both from normal rats
fluorescent dye will not readily release this dye, hence a flickering fusion pore must be narrow
(8). The electrical conductance of a flickering pore (0.7-4 nS (4, 9) is consistent with a 4 to 10 nm diameter pore of 150
in giant vesicles)
nm length (9). Unfortunately, we do not know why some vesicles flicker, and most do not. Flicker suggests that a narrow pore is an early intermediate in exocytosis. Because flicker is rare, one imagines that the intermediate is usually short lived and only occasionally survives for seconds. Indeed, even vesicles that do not flicker form transient pores of high electrical resistance. If one looks at
C steps with high enough time resolution, one can often see that C grows gradually to a final value (Figure l b). The time course reflects the increasing conductance of the dilating fusion pore (Figure
Ie). Unfortunately, the initial
phase of dilation is too rapid even for time-resolved capacitance recordings. Hence the method misses early steps in the formation of the pore.
THE EARLIEST FUSION PORE
The secretory vesicles of mast and chromaffin
cells actively accumulate biogenic amines, and this active uptake is powered by a pH gradient across the vesicle membrane
(31). The gradient is main
tained by an electrogenic H+ pump that generates a lumen-positive potential across the vesicle membrane
(9, 58). As soon as the pore opens, the plasma
and vesicle membrane become electrically connected. The difference between plasma and vesicle membrane potentials then drives an electric discharge through the fusion pore that adjusts the charge on the vesicle membrane
EXOCYTOSIS
615
A (pF) 0.1
1-��-"""""'"..;
Annu. Rev. Physiol. 1990.52:607-624. Downloaded from www.annualreviews.org Access provided by University of Texas Southwestern Medical Center on 01/21/15. For personal use only.
0.5
s
B 0.1 pF
[
� - - - - - - - - - - - - - - - - - - - - - - - -
100
\I)
� lu '-'
Further
Quick links to online content
Annu. Rev. Physiol. 1990.52:607-624. Downloaded from www.annualreviews.org Access provided by University of Texas Southwestern Medical Center on 01/21/15. For personal use only.
Annu. Rev. Physiol. 1990. 52:607-24 Copyright © 1990 by Annual Reviews Inc. All rights reserved
EXOCYTOSIS W. Almers Department of Physiology and Biophysics, University of Washington, Seattle, Wash ington 98195
KEY WORDS:
fusion pore, patch-clamp, fusion intermediates, membrane capacitance, fusion proteins
INTRODUCTION All eukaryotic cells contain membrane-bounded vesicles that are destined for export, Molecular motors capture these vesicles in the cell interior and carry them to the plasma membrane; this requires metabolic energy. When the vesicle approaches the cell surface to within a few nanometers, it may undergo exocytosis, that is, the membrane surrounding the vesicle may fuse with the plasma membrane. The vesicle membrane then becomes a part of the cell membrane, and any material within the vesicle diffuses into the ex tracellular space. Eukaryotic cells undergo exocytosis either to insert new components into the plasma membrane
or
to export the membrane-imperme
ant substances stored within the vesicles. In constitutive exocytosis, vesicles exocytose approximately as soon as they reach the cell membrane, and no known mechanism controls the fusion event. In stimulated or regulated exocytosis, vesicles gather beneath the cell membrane in clusters and lie there waiting, until a signal reaching the cell membrane causes the appearance of a cytosolic messenger substance. The messenger then causes conformational changes in an unknown receptor protein on either the vesicle or the cell membrane, so that many or most of the vesicles undergo exocytosis in a burst. The regulatory processes vary among different cells; in some (neurons or 2+ eggs), an increase in cytosolic Ca is enough to trigger exocytosis, while in 2+ others (e.g. mast cells; Gomperts, this volume), Ca is merely a cofactor. The mechanisms of fusion also may vary. In this review, however, I make the 607
0066-4278/90/0315-0604$02.00
ALMERS
608
unproven assumption that fusion occurs in the same way in all cells, even when it is coupled to different control mechanisms. One can imagine fusion to be regulated in two ways.
(a) If secretory
vesicles were innately eager to fuse with cell membranes, then regulating fusion would mean preventing it from happening in quiescent cells.
(b)
Annu. Rev. Physiol. 1990.52:607-624. Downloaded from www.annualreviews.org Access provided by University of Texas Southwestern Medical Center on 01/21/15. For personal use only.
Alternatively, the fusion of biological membranes may be energetically diffi cult and require catalysis by specific fusion proteins. These would be con tinuously active in constitutive exocytosis and only occasionally active in regulated exocytosis. There are a number of reasons why the latter is probably correct.
(a) Artificial bilayers made from biological lipids do not readily fuse (56). (b) The
with each other under the conditions found in most living cells
fusion of viral envelopes to host cell membranes is the only fusion process understood at the molecular level, and it requires catalysis by known fusion proteins
(73). (c) In yeast, where secretion is constitutive, several gene (46).
products are required for vesicles to undergo exocytosis
Previous articles have discussed why artificial lipid bilayers are so resistant to fusion
(56), how this resistance may be overcome, and what the findings (7, 20,
with pure lipid bilayers tell us about membrane fusion in living cells
55). The extensive work on pure lipid systems is important and exocytotic fusion clearly does involve the rearrangement of lipids. Nonetheless, it is difficult to disagree with Rand
& Parsegian (55) that "in spite of heroic
efforts, phospholipid bilayer models of fusion do not mimic the cellular processes closely e'1ough to be confident that the cellular mechanism is being probed". The working hypothesis in this review will be that the earliest steps in membrane fusion are due to specific proteins that simultaneously interact with both the plasma and the vesicle membrane
(8, 9, 32, 34, 53, 60, 79).
Although the molecular basis of exocytotic membrane fusion is unknown, promising methods to study exocytosis in a controlled biochemical environ ment have been developed and reviewed [chromaffin cells eggs
(5), sea urchin
(28), Paramecium (51)].
Two Key Properties of Exocytotic Membrane Fusion EXOCYTOSIS CAN BE FAST
After a stimulus reaches a cell, how long does it
take before the first vesicles undergo exocytosis? In the examples listed in Table
1, this delay generally lasts seconds to tens of seconds. Most of this
time is probably spent in generating the cytosolic messenger, in removing cytosolic barrier proteins (e.g. synapsin) or, in some cases, in conveying vesicles from the depth of the cell to the plasma membrane. To appreciate how rapidly exocytotic fusion can occur, it is instructive to consider neurons. In neurons, the vesicles containing the secreted product (i.e. the transmitter) are parked so close to the plasma membrane that they touch, and no chemical
EXOCYTOSIS
Annu. Rev. Physiol. 1990.52:607-624. Downloaded from www.annualreviews.org Access provided by University of Texas Southwestern Medical Center on 01/21/15. For personal use only.
Table 1
609
Speed of exocytosis
Cell type
Stimulus
Sea urchin egg
sperm
Neutrophils Mast cells
GTP-,),-S GTP-,),-S antige n 48/80 electrical electrical electrical
Chromaffin cell Frog motor neuron Cat afferent neurons
Delay
Duration
Temperature
(s)
(8)
°C
Method
Reference
40 50 50 60 5 < 0.2 0.0005 0.0002
80 200 200 100 5
15 201 20 1 201 201 201
C C C C C C
30 47
19
psI'
33
psp
42
38
19
36 38 44
13 Delay, time between application of stimulus and first signs of exocytosis; duration, time over which
membrane area increased; C, capacitance measurement; psp, postsynaptic potential. ITemperature given as room temperature.
reaction is required to generate the active cytosolic messenger, Ca2+. Instead, Ca2+ enters the cytosol by crossing the plasma membrane through Ca chan nels. These Ca channels open during an action potential across the plasma membrane, and probably are 10-20 nm away from the vesicles. At the frog motor nerve terminal, the delay between the action potential and exocytosis (as assayed by the post-synaptic potential) is only 0.5 ms at 19°C (33). In mammalian neurons at 38°C, the delay is even less (0.2 ms; 13, 42). In this short time, several events must occur in sequence: first, Ca channels must open; then [Ca2+] near the synaptic vesicles must rise; Ca2+ must bind to its (unknown) receptor; the receptor must change conformation; the molecular rearrangements of membrane fusion must occur; and finally the transmitter must diffuse to its postsynaptic receptor, open ion channels, and produce the postsynaptic response. Even if all of the 0.5 ms synaptic delay in a frog neuromuscular junction were available for the triggering and execution of membrane fusion, it would suffice for at most one enzymatic reaction . Consider the rates of two enzymatic reactions that have been considered to start exocytosis: phosphorylation and dephosphorylation (23, 77). Some placental phosphatases (MW 40,000) have maximal rate constants of up to 45 M-mol of substrate/min and per mg enzyme (67); this translates into one phosphate liberated by one enzyme molecule every 33 ms. One of the fastest known protein kinases, phosphorylase b kinase (MW 1,300,000) will phos phorylate 15 M-mol substrate/min and per mg enzyme (65) at 30°C, or one substrate molecule every 12 ms per catalytic subunit. Both phosphorylation and dephosphorylation are rapid enough to play a role in signal transduction in many secretory cells and, indeed, in many aspects of neuronal secretion. Such reactions, however, are probably too slow to play a role in exocytosis at fast
610
ALMERS
synapses. Judging by the enormous speed of transmitter release, exocytosis is probably triggered by a conformational change in a single macromolecule. EXOCYTOSIS IS TIGHT
The fusion of lipid bilayers involves profound
molecular rearrangements. Yet exocytotic membrane fusion causes damage to
Annu. Rev. Physiol. 1990.52:607-624. Downloaded from www.annualreviews.org Access provided by University of Texas Southwestern Medical Center on 01/21/15. For personal use only.
neither plasma nor vesicle membranes. As far as we know, exocytosis is alway tight; there is no morphologic evidence that exocytosis ever leads to the rupture of vesicles and to the discharge of their contents into the cytosol. The only connection that forms is qetween vesicle lumen and extracellular space, and no leaks form between the vesicle lumen and the cytosol, or between the extracellular space and the cytosol. Lindau & Fernandez (36) have reported an experiment where the electrical conductance between the cytosol and the extracellular space increased by less than 100 pS while about 1000 vesicles underwent exocytosis. Even if none of the vesicles contained open ion channels, each vesicle could have caused a leak of at most 0.1 pS, lOO-fold less than the conductance of a typical ion channel. Clearly the exocytotic mechanism is highly successful in limiting leaks between cytosol and vesicle lumen or between cytosol and extracellular space. This success distinguishes exocytosis from known methods of fusing artificial lipid vesicles with other vesicles or with planar bilayers. It suggests that a highly specific macro molecule must form the first connection between the lumen of an exocytosing vesicle and the cell exterior. The gap junction is a macromolecule that can achieve such a feat because it connects two aqueous compartments selectively without connecting them to a third compartment. Gap junctions do not, however, mediate fusion.
Morphology EARLY STEPS IN EXOCYTOSIS
In thin sections, vesicles caught in the act of
exocytosis are seen to form characteristic omega-figures, which show the vesicles united with the plasma membrane by a narrow neck of membrane. In freeze-fracture, the neck appears as a bump or a dip. The center of the larger bumps can be made into a pit by etching. It must therefore contain aqueous material and represent a pore (called fusion pore in this review) that connects the vesicle lumen with the extracellular space. Even in the same section, pore diameters vary over a wide range. Investigators infer that the pores are narrow at first and later dilate as fusion progresses. The smallest pores seen under the electron microscope are generally about
20 nm in diameter; the preparations examined include posterior pituitary cells (18), mast cells (12), the neuromuscular junction (26), adrenal chromaffin cells (62), and Paramecium (41). In Limulus amoebocytes, an even smaller pore may have been seen. Thin sections of secreting amoebocytes show small areas where the two fusing membranes touch and form a sheet two mem-
Annu. Rev. Physiol. 1990.52:607-624. Downloaded from www.annualreviews.org Access provided by University of Texas Southwestern Medical Center on 01/21/15. For personal use only.
EXOCYTOSIS
611
branes thick (50). Such "pedestals" are thought to form as a prelude to the fusion pore, and one of the pedestals was said to contain a small pore of 5 nm diameter. The pores seen in electron micrographs are comparable in diameter to the pores (17) formed by complement (10 nm) or perforin, the toxin of cytotoxic T-Iymphocytes (16 nm). Both complement and perforin pores are assemblies of protein molecules. Whether or not fusion pores include proteins or protein complexes is not clear from electron micrographs. Do the 20 nm pores arise suddenly and without a smaller precursor? Except possibly in amoebocytes, any such precursors are evidently too short-lived to be captured by present morphologic techniques. Unfortunately, it is precisely the early and short-lived forms of the fusion pore that will tell us the most about the mechanism of exocytosis. How does a fusion pore form? What are its molecular constituents? What are its properties at the instant of its forma tion? How does it expand, what is its fate, and ultimately how is it replaced by an apparently seamless neck of lipid bilayer connecting the vesicle to the plasma membrane? Some of these questions must be addressed with methods that can resolve fast events. At neuronal synapses and in some protozoa (such as Paramecium), exocytosis occurs at precisely de termined sites, and one can search for membrane specializations that may be related to exocytosis. In both cell types, freeze-fracture reveals ordered arrays of plasma membrane macromolecules close to sites of exocytosis. In the frog neuromuscular junction, such sites (called active zones) are marked by double rows of intramembrane particles (11), and exocytosis occurs within a few tens of nm of these intramembrane particles. Large and sometimes ordered parti cles in the plasma membrane near active zones also occur at many other synapses, and it is likely that they play a role in exocytosis. In the neuro muscular junction, they cannot be fusion proteins, since exocytosis always occurs near but never exactly above the particles. The particles may represent voltage-sensitive Ca channels (25), but there is no experimental evidence concerning their role. In Tetrahymena (60, 61) and Paramecium (52), each secretory vesicle docks beneath a set of some 10 plasma membrane particles. The particles form a radially symmetric cluster called a rosette that is probably related to exocytosis, since mutants without rosettes cannot exocytose their vesicles (6). In Paramecium, the tip of the secretory vesicle (trichocyst) just beneath the rosette shows an annular-shaped zone enriched with intramembrane particles; whether these particles interact with those of the rosette is unclear. At ftrst the rosette particles were thought to form the fusion pore (60). However, rare images probably representing early stages in membrane fusion (41) show a dispersing rosette, with a 10-20 nm diameter fusion pore forming
SPECIALIZATIONS NEAR EXOCYTOSIS SITES
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612
ALMERS
in the middle. These images suggest that the rosette particles are not at the site of fusion when fusion occurs; hence the particles cannot be fusion proteins. It was later argued that the rosette particles were Ca channels providing the Ca influx thought to be necessary for triggering exocytosis (59). If so, they must be Ca channels of a new type previously unstudied. The voltage-sensitive Ca channels in Paramecium are associated with the cilia and not with the rosette (48), and both deciliated Paramecia and mutants lacking functional voltage sensitive Ca channels are capable of exocytosis (51). In fact, it is unknown how much of the Ca needed for exocytosis comes from the outside in Paramecium. The role of rosettes in exocytosis remains a mystery. In Chlamydomonas (72), an intracellular contractile vacuole gathers low molecular-weight waste products and periodically discharges them into the extracellular environment. This discharge does not represent exocytosis in the strict sense, since the vacuole never fuses with the plasma membrane. In stead, the plasma and vacuole membranes approach each other to within a few nm and enclose between them a periodic array of electron-dense spots. The array of spots corresponds to an array of large intramembrane particles that appears in the cytosol-facing leaflets of both vacuole and plasma membranes. It has been suggested that the particles form aqueous channels connecting the vacuole lumen with the external medium and that the discharge mechanism of the contractile vacuole is an evolutionary precursor of exocytosis (72). Where resting secretory vesicles bulge against the plasma membrane, the narrow cytoplasmic space between vesicle and plasma membrane is some times bridged by narrow filaments [e.g. in mast cells (12) or pancreatic beta cells (49)]. A recent review discusses the possible roles of cytoskeletal . elements in exocytosis (34). Electrophysioiogic Assays .of Exocytosis Electrophysiologic assays allow one to monitor exocytosis at the level of single cells and even single secretory vesicles. At synapses, the postsynaptic potential reports the secretion of transmitter by a nerve terminal. In some synapses, miniature postsynaptic potentials can be recorded and provide, at submillisecond time resolution, an assay for exocytosis at the level of single synaptic vesicles (16). A more recent electrophysiologic assay relies on the fact that each exocytotic event increases the cell surface area. Since all biological membranes have an electrical capacitance of 1 JJ,F/cm2 one may monitor the cell surface area (and hence exocytosis) by measuring the plasma membrane capacitance (21, 30). The method was perfected by Neher & his collaborators (37, 44) and applied to most of the cells in Table 1. When secretion is sufficiently slow (no more than a few vesicles per second), the capacitance (C) can be seen to increase in small steps, each reporting the exocytosis of a single secretory vesicle. Such C steps have been seen in
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EXOCYTOSIS
613
adrenal chromaffin cells (44), mast cells (19), pancreatic acinar cells (39), neutrophils (47), and pituitary lactotrophs (40). C steps as small as 2 tF can be resolved, which correspond to vesicles of 0.2 JLm2 surface area or 250 nm diameter (44, 47). Exocytosis of single synaptic vesicles containing a fast neurotransmitter (typically 50 nm in diameter) is still beyond the reach of this assay, but C changes due to the release of many synaptic vesicles can be detected (22). The method can also be used for monitoring episodes of endocytosis (2). Mast cells have been useful for studying single exocytotic events because their secretory vesicles are large. Particularly convenient are mast cells from beige mice (strain C57BL/6J-bgj/bgj),which have a genetic defect resembling Chediak-Higashi syndrome in humans. Because of this defect,mast cells and other granulocytes of beige mice are unable to limit the size of their secretory vesicles. Instead of having some thousand vesicles of about 0.8 JLm diameter, mast cells of beige mice have only 10-40 giant vesicles of 1-5 /Lm diameter. They secrete readily when stimulated by mast cell secretagogues (54) or intracellular GTP-,),-S (8, 78) and individual vesicles can be seen under the light microscope while they exocytose (15). Nowhere are the electric signals associated with the exocytosis of single vesicles as large as in mast cells of beige mice. Time-Resolved Exocytosis of Single Vesicles
DO OSMOTIC FORCES DRIVE MEMBRANE FUSION? Pure lipid bilayers can be fused to each other by placing them under mechanical stress. Secretory vesicles often swell as cells secrete,which suggests that swelling stretches the vesicle membrane and thereby causes membrane fusion [for a review of this hypothesis see (20)]. Mast cells of beige mice provide an opportunity to test this idea, because one can watch individual vesicles swell while one measures the capacitance (e) to monitor fusion. Two independent studies (1,8,78,79) have shown that a step increase in C always precedes swelling. In these studies, the e step clearly represented fusion of the vesicle because when the vesicle's diameter was measured under the light microscope, the surface area calculated agreed well with the amplitUde of the e step. Swelling followed the step with a mean delay of 0.4 s (8). Even in osmotically shrunk vesicles, fusion preceded swelling; hence even vesicles with a slack membrane can fuse. Work on chromaffin cells provides additional evidence that vesicle membranes can fuse without being stretched (27). Swelling of the vesicle may have an important role in hastening extrusion of the contents from the vesicle cavity; the mechanism of swelling has been reviewed (Verdugo, this volume). It is likely, however, that vesicle swelling is a consequence rather than a cause of membrane fusion. In the search for early events in membrane fusion one must look elsewhere.
614
ALMERS
REVERSIBLE INTERMEDIATES IN MEMBRANE FUSION
It is widely believed
that exocytosis is irreversible and that the subsequent retrieval of the exocy tosed membrane occurs by a different mechanism. In agreement with this view, a plot of mast cell capacitance (i.e. mast cell surface) against time
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usually looks like a staircase, with each step building upon the previous one
(9, 19). Occasionally, however, one obtains recordings as in Figure l a, where C increases in a step, but then fluctuates for a second and once even returns to baseline. Since the step increase in C reports the formation of an electric connection between the exterior and the lumen of the exocytosing vesicle (the fusion pore), the experiment shows that this connection can form and break repeatedly. Only later does the step become stably established, which sug gests that the vesicle has fused completely. Figure
1a is reminiscent of a
single-channel recording. Just as the sudden appearance and disappearance of current through the single ion channel represents the sudden opening and closing of that channel, so do the flickering fluctuations in
C represent the
opening and closing of the fusion pore.
(19), capacitance flicker as in Figure l a has (1, 19) and from beige mice (4, 8, 9, 78). A flickering vesicle filled with a First seen by Fernandez et al
now been seen by several other groups in mast cells both from normal rats
fluorescent dye will not readily release this dye, hence a flickering fusion pore must be narrow
(8). The electrical conductance of a flickering pore (0.7-4 nS (4, 9) is consistent with a 4 to 10 nm diameter pore of 150
in giant vesicles)
nm length (9). Unfortunately, we do not know why some vesicles flicker, and most do not. Flicker suggests that a narrow pore is an early intermediate in exocytosis. Because flicker is rare, one imagines that the intermediate is usually short lived and only occasionally survives for seconds. Indeed, even vesicles that do not flicker form transient pores of high electrical resistance. If one looks at
C steps with high enough time resolution, one can often see that C grows gradually to a final value (Figure l b). The time course reflects the increasing conductance of the dilating fusion pore (Figure
Ie). Unfortunately, the initial
phase of dilation is too rapid even for time-resolved capacitance recordings. Hence the method misses early steps in the formation of the pore.
THE EARLIEST FUSION PORE
The secretory vesicles of mast and chromaffin
cells actively accumulate biogenic amines, and this active uptake is powered by a pH gradient across the vesicle membrane
(31). The gradient is main
tained by an electrogenic H+ pump that generates a lumen-positive potential across the vesicle membrane
(9, 58). As soon as the pore opens, the plasma
and vesicle membrane become electrically connected. The difference between plasma and vesicle membrane potentials then drives an electric discharge through the fusion pore that adjusts the charge on the vesicle membrane
EXOCYTOSIS
615
A (pF) 0.1
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