Physiol Rev 92: 1915–1964, 2012
doi:10.1152/physrev.00007.2012
DISTINCT INITIAL SNARE CONFIGURATIONS
UNDERLYING THE DIVERSITY OF EXOCYTOSIS
Haruo Kasai, Noriko Takahashi, and Hiroshi Tokumaru
Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, Graduate School of
Medicine, The University of Tokyo, Tokyo, Japan; and Faculty of Pharmaceutical Sciences at Kagawa, Tokushima
Bunri University, Kagawa, Japan
L
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
INTRODUCTION
EXOCYTOSIS OF SYNAPTIC VESICLES
FUSION OF VESICLES OTHER THAN...
KEY CELLULAR STAGES OF EXOCYTOSIS
PROTEINS REGULATING ULTRAFAST...
MOLECULAR MODELS OF EXOCYTOSIS
KINETIC DETERMINANTS
CONCLUSIONS
1915
1918
1921
1926
1932
1939
1946
1947
I. INTRODUCTION
Secretory vesicles in the cytosol contain a variety of bioactive compounds such as neurotransmitters, hormones, enzymes, and cytokines and also carry membrane lipids and
proteins (212). Fusion of the membrane of these secretory
vesicles with the plasma membrane leads to the opening of
the fusion pore, which connects the inside of the vesicle to
the extracellular space (425). This releases the molecules
contained in the vesicle into the extracellular space and
induces lateral diffusion of molecules in the vesicle membranes into the plasma membrane, a process called exocytosis (476). Fusion of vesicles with the target membrane is
generally well developed in eukaryotic cells and is used for
trafficking various membrane organelles, including the endoplasmic reticulum, Golgi apparatus, lysosomes, and endosomes (283, 534). These membrane fusion events utilize
a family of proteins called soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins.
There is a single vesicle SNARE (v-SNARE), called synaptobrevin or vesicle-associated membrane protein (VAMP),
and two target plasma membrane SNAREs (t-SNAREs),
called syntaxin and SNAP25, which are involved in synaptic exocytosis. When the three proteins are brought together, they form a stable ␣-helical ternary complex, a process that provides the energy for membrane fusion.
Exocytosis occurs either constitutively, without any external stimulus, or is triggered by extracellular signals (212).
The extracellular signals for exocytosis are converted into
intracellular signals via Ca2⫹, cAMP, or protein kinases.
Ca2⫹-dependent exocytosis plays a major role in the release
of neurotransmitters from neurons and in the secretion of
hormones and enzymes from endocrine and exocrine cells.
However, in spite of similar underlying molecular mecha-
0031-9333/12 Copyright © 2012 the American Physiological Society
1915
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Kasai H, Takahashi N, Tokumaru H. Distinct Initial SNARE Configurations Underlying the Diversity of Exocytosis. Physiol Rev 92: 1915–1964, 2012;
doi:10.1152/physrev.00007.2012.—The dynamics of exocytosis are diverse and
have been optimized for the functions of synapses and a wide variety of cell types. For
example, the kinetics of exocytosis varies by more than five orders of magnitude
between ultrafast exocytosis in synaptic vesicles and slow exocytosis in large dense-core vesicles.
However, in all cases, exocytosis is mediated by the same fundamental mechanism, i.e., the
assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. It is often assumed that vesicles need to be docked at the plasma membrane and SNARE
proteins must be preassembled before exocytosis is triggered. However, this model cannot
account for the dynamics of exocytosis recently reported in synapses and other cells. For example,
vesicles undergo exocytosis without prestimulus docking during tonic exocytosis of synaptic vesicles
in the active zone. In addition, epithelial and hematopoietic cells utilize cAMP and kinases to trigger
slow exocytosis of nondocked vesicles. In this review, we summarize the manner in which the
diversity of exocytosis reflects the initial configurations of SNARE assembly, including trans-SNARE,
binary-SNARE, unitary-SNARE, and cis-SNARE configurations. The initial SNARE configurations depend on the particular SNARE subtype (syntaxin, SNAP25, or VAMP), priming proteins (Munc18,
Munc13, CAPS, complexin, or snapin), triggering proteins (synaptotagmins, Doc2, and various
protein kinases), and the submembraneous cytomatrix, and they are the key to determining the
kinetics of subsequent exocytosis. These distinct initial configurations will help us clarify the
common SNARE assembly processes underlying exocytosis and membrane trafficking in eukaryotic cells.
KASAI ET AL.
It is assumed that the ultrafast exocytosis of synaptic vesicles is preceded by the docking of vesicles to the plasma
membrane followed by priming and triggering of exocytosis
(22, 442, 479). This concept is primarily based on ultrafast
(⬍1 ms) exocytosis (FIGURE 1) and is supported by a specific
structure called the active zone. The same concept, however, may not apply to preparations that are devoid of the
active zone. The key unresolved questions are as follows:
1) at which stages of SNARE assembly (initial SNARE configurations) do Ca2⫹ and other triggers come into play, and
2) how does SNARE assembly proceed after the trigger?
New methodologies have been developed to address these
questions, and many mutant animals that lack the key proteins have been generated. Marked progress has been made
since one of the authors of the present review wrote an
article on the same topic in 1999 (301), and it would be
beneficial to reassess the assumptions and terminology, and
to interpret new data. We will review representative studies
on a wide variety of cell types and present a general overview of diverse secretory phenomena, which are normally
Time constants for fusion of single vesicles
Activation energy (kBT)
Time constants
12
14
16
18
21
0.1 ms 1 ms 10 ms 100 ms 1 s
23
25
28
10 s 100 s 1000 s
Synaptic vesicles
Ultrafast exocytosis
Asynchronous exocytosis
Tonic exocytosis
Spontaneous exocytosis
Large dense-core vesicles
Synapses
Chromaffin cells
Islet β cells
PC12 cells
Mast cells
Exocrine cells
Transport vesicles
Kidney duct cells
Gastric parietal cells
Adipocytes
Small vesicles
Chromaffin cells
Islet beta cells
PC12 cells
Mast cells
Proteoliposomes
SNAREs
SNAREs + Munc18
SNAREs in docked vesicles
FIGURE 1. Time constants for the fusion of single vesicles in various preparations. For tonic and spontaneous exocytosis of synaptic vesicles, it is assumed that vesicles are continuously stimulated with cytosolic Ca2⫹.
Activation energies (⌬G) are obtained from the time constant (␶) using: ␶ ⫽ ␶0 exp[⌬G/kBT], where ␶0 ⫽ 10⫺9
s. The kBT unit is the product of the Boltzmann constant and the absolute temperature (353). The prestimulus
docking requirement bar at the bottom of the figure indicates the time constants that are so short that vesicles
must be docked to the plasma membrane before stimulation.
1916
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nisms, the kinetics of exocytosis triggered in secretory cells
by increases in the concentration of cytosolic Ca2⫹ ([Ca2⫹]i)
are highly diverse (FIGURE 1). For example, exocytosis involved in neurotransmission in the brain occurs within 1 ms
of the presynaptic action potential, whereas exocytosis involved in insulin secretion from the islets of Langerhans to
control blood glucose levels can take up to 600 s. Importantly, the same synaptic terminals that show ultrafast exocytosis also show slow exocytosis, i.e., asynchronous and
tonic exocytosis of synaptic vesicles and secretion of peptides
contained in the large dense-core vesicles (FIGURE 1). Thus
understanding exocytoses with diverse kinetic features is essential to synaptic physiology. There are a number of key
bodily processes that involve slow exocytoses. For example,
the diuretic action of vasopressin in the kidney duct cells is
mediated by exocytotic insertion of vesicles that contain
aquaporin into the apical plasma membrane, and the action
of insulin causes exocytotic insertion of vesicles that contain
GLUT4 into the plasma membranes of adipocytes and muscles (TABLE 1).
SNAP25
SNAP23
SNAP23
SNAP23
Syn 3
Syn 3,4
Syn 4
Syn 3
n.d.
VAMP8
VAMP2,7
VAMP2
VAMP7
VAMP8
VAMP2
VAMP2,
VAMP8
VAMP7
VAMP4
n.d.
VAMP3
VAMP7,8
VAMP2
VAMP2,7
VAMP2
VAMP2
VAMP2
VAMP2,4
R-SNARE
c
b
b
b
b
c
a,b,c
n.d.
a
a
a
a
Munc18
U
U
U
U
U
U
C
U
U
U
U
U
U
B, U
B,U,C
T, B, U
T
T
B,U,C
Putative SNARE
Configuration
Insulin:Munc18c
None
cAMP: F-actin(?)
cAMP
antiCD3
Thrombin
Ca2⫹:Syt 6
Ca2⫹:Syt 1,2,9
Ca2⫹:Doc2
Ca2⫹: Syt, 1,2,9,
Syt, 3,5–57,10
Ca2⫹:Syt 1,2,9
Ca2⫹:Syt 1,7
cAMP: SNAP25
Ca2⫹:nd
cAMP: Snapin
Ca2⫹:Syt 1
cAMP: VAMP2
Ca2⫹:Syt7, Doc2
Ca2⫹
Ca2⫹:Syt 4
Ca2⫹:Syt 4
Ca2⫹: Syt 2
IgE:SNAP23
Triggers
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77, 285, 312
281, 485, 509
71, 276
175, 349, 368
260, 482
512, 575
131
43, 162, 182, 275, 680
251, 506
117, 118
315, 623
576, 735
412, 482, 549, 360, 388, 629
214, 215, 386, 458, 459,
604, 612
704
573
703
718
502, 621, 703
Reference Nos.
The four configurations (T, trans-SNARE; B, binary-SNARE; U, unitary-SNARE; C, cis-SNARE) were predicted mainly from the time constants of exocytoses of 0.5–30 ms, 0.03–1 s,
and 1–1,000 s, respectively. Qa-, Qbc-, and R-SNARE are three different subtypes of SNARE. Syn, syntaxin; Syt, synaptotagmin.
T-cells (Immunological
synapse)
Platelets
Spermatozoa (acrosome)
Gastric parietal cells (TV,
H⫹-ATPase)
Kidney duct cells
(Aquaporin2)
Adipocytes, Skeletal muscles
(GLUT4)
Constitutive exocytosis
B cells (IgG), Hela cells
SNAP23
SNAP23
SNAP23
SNAP23
SNAP23
SNAP23
SNAP25
Vtu1b, Stx8
SNAP23
SNAP25
Syn 1
Syn 2
Syn 2, 3
Syn–4
Syn 4
Syn 6
Syn 4
Syn 1
Syn 4,7
Islet ␤ cells (LDCVs:Insulin)
SNAP25
SNAP25
SNAP25
SNAP25
SNAP25
Qbc-SNARE
Syn 7, 11
Syn 2,3
Syn 1
Syn 1
Syn 1
(Spontaneous)
Chromaffin cells (LDCVs)
Exocrine cells
(LDCVs)
Lysosomes
Enlargesomes
Neuronal dendrites
Astrocytes
Basophil, mast cells (LDCVs)
Syn 1
Syn 1
Syn 1
Synaptic vesicles (ultrafast)
(Asynchronous)
(Tonic)
Qa-SNARE
Table 1. Putative SNARE configurations in various preparations
INITIAL SNARE CONFIGURATIONS FOR EXOCYTOSIS
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KASAI ET AL.
dealt with by the separate fields of neuroscience, endocrinology, immunology, diabetology, and digestive, kidney,
and reproductive physiology. Considerable evidence has accumulated that supports the relevance of initial SNARE
configurations to the diversity of exocytosis.
We will first discuss the diversity of exocytoses in neurons and
other cell types (sects. II and III) and give a comprehensive
account of the stages involved in exocytosis (sect. IV). This is
followed by a summary of the major proteins that regulate
exocytosis (sect. V), and a new general framework for exocytosis based on the distinct initial SNARE configurations that
are responsible for its diversity (sects. VI and VII).
Synaptic vesicles in the presynaptic terminals of neurons
undergo exocytosis in four different modes: ultrafast, asynchronous, tonic, and spontaneous. This suggests that the
same types of vesicles show markedly different kinetics depending on the mode of exocytosis. We will review the
kinetics of each mode of exocytosis in turn.
A. Ultrafast Exocytosis
Neurotransmitters are released from synaptic vesicles at the
presynaptic terminal by exocytosis within 1 ms of the arrival of the action potential (22, 442, 479). This ultrafast
exocytosis is found only in the presynaptic terminals of
neurons (FIGS. 1 AND 2A). This short time scale strongly
suggests that the vesicles are already docked to the plasma
membrane when the action potential arrives (FIGURE 3A)
and that SNAREs are preassembled to some degree. Importantly, there is a structural basis for ultrafast exocytosis,
known as the active zone (FIGURE 2A), in which synaptic
vesicles are clustered and some vesicles are docked to the
plasma membrane (83, 473). The active zone comprises an
electron-dense cytoskeletal matrix, referred to as the cytomatrix at the active zone (CAZ), which involves presynaptic
dense projections, actin fibers, ribbons, and grids (152, 287,
572). Most of the molecular components that make up the
active zone have been identified (see sect. V), and some of these
are specific to the active zone, for example, RIM, CAST/ELKS,
bassoon, piccolo, and liprins (242, 249, 572) (sect. V).
Ultrafast exocytosis of synaptic vesicles harnesses the CAZ
proteins in several ways. First, vesicles are localized close to
voltage-gated Ca2⫹ channels such that the vesicles can sense
increases in [Ca2⫹]i close to the open pore of the Ca2⫹
channels before Ca2⫹ is bound by cytosolic Ca2⫹ buffers.
The shortest distance between the vesicles and the Ca2⫹
channels is estimated to be between 30 and 60 nm (410).
This close localization is often called the Ca2⫹ domain, or
Ca2⫹ nanodomain (3, 25, 302, 371, 373, 543), and is supported by CAZ proteins (297). Second, synaptic vesicles are
1918
Vesicles that undergo ultrafast exocytosis upon stimulation
by a single action potential are referred to as vesicles in the
readily releasable pool (RRP), and vesicles on their way
from the site of endocytosis to the active zone are referred to
as vesicles in the recycling pool (232, 527). Those vesicles in
the presynaptic terminal that are not involved in fast recycling or exocytosis are referred to as vesicles in the reserve
or resting pool. The notion of distinct pools of vesicles is
useful when describing the status of the vesicles; however,
these vesicles are not always pooled or segregated in the
cytosol, but rather are scattered and intermingled (142,
232, 527). It should also be noted that the readiness of
synaptic vesicles is also dependent on the states of molecules
in the plasma membrane and the position of vesicles relative
to the membrane. The notion of a vesicle pool is the first
approximation for describing the kinetics of exocytosis.
The number of vesicles in the RRP (N) and the release
probability of each vesicle (p) can be determined. Katz and
del Castillo introduced the binominal statistic to describe
the quantal content of vesicles using N and p (24, 140),
where N correlates with the number of docked and primed
vesicles, and p is dependent on the distance between the
vesicles and the Ca2⫹ channels (410, 678), and on the kinetics of exocytosis after Ca2⫹ binding with fast Ca2⫹ sensors, called synaptotagmins. For small presynaptic boutons
in the cerebral cortex, the release probability per bouton
can be defined. It ranges from 0.1 to 0.9 (60, 410, 431) and
correlates with the expression of CAZ proteins (399). The
number of vesicles in the RRP is estimated to be ⬃10 (527),
the number in the recycling pool to be ⬃20, and the number
in the reserve pool to be ⬃200 (232, 268).
B. Asynchronous Exocytosis
Asynchronous exocytosis often follows ultrafast exocytosis
and lasts for ⬃100 ms after the action potential (479). It is
enhanced when Ca2⫹ is replaced by Sr2⫹ (199). Interestingly, some synapses show asynchronous exocytosis but not
ultrafast exocytosis (45). Although there is a debate about
whether or not asynchronous exocytosis is mediated by
vesicles in the RRP (471, 542), recent studies report that the
vesicles responsible for asynchronous exocytosis are the
same as those that are responsible for ultrafast exocytosis
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II. EXOCYTOSIS OF SYNAPTIC VESICLES
effectively replenished during repetitive stimulation, possibly by actin fibers, presynaptic dense projections, and ribbons at the active zone (545). Silencing of the presynaptic
terminals is induced when CAZ proteins are inactivated
(123, 124, 286). Third, ultrafast exocytosis occurs upon
rapid step increases in [Ca2⫹]i induced by photolysis of a
caged-Ca2⫹ compound (373, 440), suggesting that the exocytotic machinery operates rapidly after binding Ca2⫹. Ultrafast exocytosis has never been observed in preparations
without an active zone, indicating that the active zone is
necessary for ultrafast exocytosis.
INITIAL SNARE CONFIGURATIONS FOR EXOCYTOSIS
A
B
Synapses
Posterior pituitary
PSD
LDCV
SLMV
AZ
Synaptic vesicles
1 µm
C
Adrenal chromaffin cell
D
Islet β cell
M
LDCV
LDCV
Nucleus
FIGURE 2. Electron microscopic images of a synapse and secretory cells. A: synaptic vesicles in the CA1 region
of the rat hippocampus (13). AZ, active zone; PSD, postsynaptic density. [From Applegate and Landfield (13), with
permission from the Society of Neuroscience.] B: a nerve ending in the posterior pituitary gland. Synaptic-like micro
vesicles (SLMV) are clustered beneath the plasma membrane facing the blood vessel, which is surrounded by large
dense-core vesicles (LDCV). [From Klyachko and Jackson (325), with permission from Nature Publishing Group.]
C and D: quick-freeze and freeze-substituted preparations from a bovine adrenal chromaffin cell (C) (494) and a rat
␤ cell (D) (153). Note the absence of the core and halo structures in the LDCVs. M, mitochondrion. [C from Plattner
et al. (494), with permission from Rockefeller University Press; D from Dudek and Boyne (153), with permission
from John Wiley and Sons, Inc.]
(80, 479, 624, 718). It is therefore likely that the vesicles
mediating asynchronous exocytosis are docked to the
plasma membrane in a similar manner to the vesicles in the
RRP (FIGURE 3A). Thus it is suggested that vesicles need to
be docked before stimulation if exocytosis is to be faster
than ⬃100 ms (FIGURE 1). Synaptotagmins confer Ca2⫹
dependence on ultrafast exocytosis, whereas Doc2 may
confer Ca2⫹ dependence on asynchronous exocytosis (718)
(sect. VD).
C. Tonic Exocytosis
During long-lasting repetitive stimulation, exocytosis becomes tonic and is no longer time-locked to the action
potential. Tonic exocytosis is more prominent in the
tonic synapses than in the phasic synapses (20, 420) and
displays some characteristics that are distinct from the
asynchronous exocytosis of vesicles. Tonic exocytosis requires a sustained increase in the bulk Ca2⫹ levels (146,
471) rather than the Ca2⫹ domains needed for ultrafast
exocytosis (502). It is independent of Ca2⫹-dependent
activator protein for secretion (CAPS) but dependent on
␣-soluble NSF attachment protein (␣SNAP) (80), and on
VAMP4 instead of VAMP2 (502) (see sect. VA). These
characteristics suggest that tonic exocytosis is dependent
on the vesicles in the recycling pool (502), but not on the
vesicles in the RRP. Exocytosis of nondocked vesicles
during tonic exocytosis has been observed in the active
zone of bipolar terminals (418, 729). Thus, for tonic exocytosis, vesicles are docked after stimulation (FIGURE 3B) and
are continuously resupplied from the recycling pool, with a
diffusion constant 1.5 ⫻ 10⫺2 ␮m2/s (257).
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Blood Vessel
KASAI ET AL.
A
B
Fusion of docked vesicle
Post-stimulus docking & fusion
C
Post-stimulus docking
C
C
C
D
Exocytosis
C
E
F
Sequential exocytosis
Multigranular exocytosis
C
C
C
C
C
FIGURE 3. Schematics depicting various configurations of regulated exocytosis. The red “C” indicates
stimulation, for example by Ca2⫹, which induces the various exocytotic phenomena indicated. A: exocytosis of
a docked vesicle. The stimulus can regulate fusion of the vesicle with the plasma membrane, and the contents
of the vesicle (dark blue) are released. B: exocytosis of a cytosolic vesicle. The stimulus can regulate the
attachment and fusion of the vesicle with the plasma membrane. C: slow exocytosis of a cytosolic vesicle.
Transport or diffusion of the vesicle to the plasma membrane is the major trigger for exocytosis. D: a simplified
notation for all three types of exocytosis. E: sequential exocytosis. F: multigranular exocytosis.
The time taken for one vesicle to undergo exocytosis during
tonic exocytosis (FIGURE 1) is estimated as follows. The tonic
component of exocytosis per second is comparable to the RRP
(219, 471), or ⬃10 vesicles/s/bouton for the small boutons
(433). As ⬃20 vesicles comprise the recycling pool (431), the
rate of exocytosis for a vesicle in the pool is ⬃0.5/s; thus the
average time constant of a vesicle is ⬃2 s during tonic exocytosis (FIGURE 1).
D. Spontaneous Exocytosis
The discovery of miniature endplate potentials led to the
idea of exocytosis (24, 140). Miniature endplate potentials
are thought to represent the spontaneous exocytosis of the
same set of vesicles responsible for ultrafast exocytosis. Despite the simple interpretation that this provides, there are
ample new data suggesting that spontaneous exocytosis involves a set of vesicles that are distinct from those involved
in ultrafast exocytosis (503). First, spontaneous exocytosis
utilizes vesicles in the reserve pool that have VAMP7 (268,
563), VAMP4 (502), and Vps10p-tail-interactor-1a (vti1a)
1920
as v-SNAREs (504). This may account for that fact that spontaneous exocytosis is resistant to the deletion of VAMP2 (139,
268) and to treatment with tetanus toxin (TeNT), which
specifically cleaves VAMP2 (566). In fact, the two pools of
vesicles were differentially labeled in some studies (177,
504, 553), if at all (267, 697). Second, priming proteins
affect spontaneous and evoked exocytosis in different ways
(TABLE 2). For example, complexin and CAPS (sect. V) play
a relatively modest role in spontaneous exocytosis relative
to ultrafast exocytosis. Finally, the exact location of exocytosis in the presynaptic terminal may be different for evoked
and spontaneous exocytosis (19, 552, 728).
Spontaneous exocytosis is constantly stimulated by resting
levels of [Ca2⫹]i (688, 704), and the rate of spontaneous
exocytosis can be estimated from the size of the pool from
which vesicles undergo spontaneous exocytosis (FIGURE 3,
B AND C). With the assumption that spontaneous exocytosis
is induced from a reserve pool comprising more than 100
vesicles in small boutons (268), and the rate of spontaneous
exocytosis is ⬍0.1/s per bouton (432), the average time for
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C
C
INITIAL SNARE CONFIGURATIONS FOR EXOCYTOSIS
Table 2. Contribution of neuronal SNAREs, synaptotagmin1, and priming proteins to the different types of exocytosis
Synaptic Vesicles
Time constant
VAMP2
SNAP25
Munc18a
Munc13-1
RIM
CAPS
Syt1
Complexin
Sync
HTSS
Async
Tonic
Spont
Chromaffin Cells
(Ca2ⴙ-primed)
␤ Cells
Acinar Cells
Mast Cells
1 ms
⫹⫹a
⫹⫹b
⫹⫹c
⫹⫹d
⫹⫹e
⫹⫹f
⫹⫹g
⫹h
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹
⫹⫹
—
⫹
100 ms
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
—
—
10 s
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹
—i
⫹i
⫹
1000 s
⫹
—
⫹⫹
⫹⫹
⫹
⫹f
Inhibitj
ND
30 ms
⫹k
⫹⫹l
⫹⫹m
—⫹n
ND
⫹o
⫹p
⫹q
1s
⫹r
⫹⫹s
⫹t
⫹u
⫹v
⫹w
—x
⫹y
10 s
NDz
—z
—z
ND
ND
ND
NDaa
NDbb
10 s
—cc
—dd
ND
ND
ND
ND
—ee
⫹ff
exocytosis is estimated to be ⬎1,000 s (FIGURE 1). The
differences between tonic and spontaneous exocytosis are
due to differences in [Ca2⫹]i levels (172, 442).
III. FUSION OF VESICLES OTHER THAN
SYNAPTIC VESICLES
For slow exocytosis, which occurs more than 100 ms after
the trigger (FIGURE 1), there is enough time for vesicles to
dock with the plasma membrane and for SNARE to be
assembled after the trigger. In fact, newly docked vesicles in
bipolar terminals undergo exocytosis within 250 ms (729)
and within 100 ms in ␤ cells (310, 590). Such slow exocytosis is mediated by the same types of SNARE that are
utilized for ultrafast exocytosis (TABLE 1), indicating that
neuronal SNAREs are also involved in slow exocytosis. Below, we will discuss the enormously diverse forms of regulated and constitutive exocytosis in a wide variety of preparations.
A. Large Dense-Core Vesicles
in Secretory Cells
Substances secreted from nonneuronal preparations are
condensed in large dense-core vesicles (LDCVs), which undergo regulated exocytosis. The idea of exocytosis and biogenesis of LDCVs was first proposed for pancreatic acinar
cells (476). Unlike synaptic vesicles, LDCVs do not recycle
beneath the membrane but are generated in the Golgi apparatus, where they gradually grow larger in preparation
for massive secretory events. LDCVs in neuroendocrine
cells are relatively small (100 –500 nm) but are larger in
exocrine and hematopoietic cells (1–2 ␮m). LDCVs have an
electron-dense matrix that contains secretogranin and chromogranin (38, 245). In chemically fixed preparations, the
dense cores often shrink, and apparent halo structures are
evident. This shrinkage is very prominent in ␤ cells in the
islets of Langerhans, but electron microscopic investigation
of quick frozen preparations showed that the halo was absent (153, 494) (FIGURE 2, C AND D); this suggests that the
halo is mainly an artifact of chemical fixation. The cytosol
of many secretory cells is filled with LDCVs, suggesting that
their effective mobilization plays a more important role
than the rapid exocytosis of the surface vesicles. Indeed,
compound exocytosis is utilized by certain types of cells for
efficient mobilization of deep vesicles, as will be described in
section IIIB.
Stimulus-secretion coupling of secretory cells was first examined in adrenal chromaffin cells (150, 151), in which
intracellular Ca2⫹ plays a vital role. This discovery led to
the Ca2⫹ hypothesis of neurotransmitter release (311). In
most secretory cells, cytosolic cAMP potentiates Ca2⫹-dependent exocytosis (252, 544, 634). Protein kinase A (PKA)
acts as a trigger in some exocrine cells (182, 556) and epithelial cells (175, 414, 630), although kinase systems other
than PKA are utilized as triggers in pituitary cells (253),
hematopoietic cells (629), and adipocytes (236, 285). When
Ca2⫹ is not a major trigger (FIGURE 3D), exocytosis tends to be
slow, with time constants between 1 s and 10 min (FIGURE 1).
1. Endocrine cells
Endocrine cells secrete hormones into the bloodstream,
whereupon the hormones are extensively diluted. There-
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Sync, ultrafast exocytosis; HTSS, exocytosis induced by hypertonic sucrose solution; Async, asynchronous
exocytosis; Tonic, tonic exocytosis; Spont, spontaneous exocytosis. Proteins necessary for respective exocytosis (⫹⫹); partially required (⫹); not required (—); inhibit exocytosis (inhibit); not intrinsically expressed but
augment exocytosis by their overexpression (—⫹); whose role in exocytosis has not been determined or is
controversial (ND). aRefs. 67, 89, 139, 571, 686; bRefs. 69, 686; cRef. 132; dRefs. 16, 21, 518, 669;
eRef. 141; fRef. 288; gRef. 624; Ref. 704; hRefs. 511, 717; iRef. 80; jRef. 704; kRef. 55; lRef. 606; mRef.
676; nRef. 18; oRefs. 370, 747; pRef. 674; qRef. 86; rRef. 470; sRef. 633; tRefs. 149, 386; uRefs. 339,
588; vRef. 719; wRef. 611; xRefs. 214, 215; yRef. 1; zRefs. 43, 161; aaRef. 162; bbRef. 161; ccRef. 551;
ddRef. 550; eeRef. 412; ffRef. 631.
KASAI ET AL.
A) ADRENAL CHROMAFFIN CELLS. Chromaffin cells develop from
postganglionic sympathetic neurons and are often considered as a model system for rapid neuronal secretion (33).
Secretion of epinephrine by chromaffin cells plays a major
role in the homeostasis of blood circulation when lifethreatening events occur. Exocytosis of LDCVs in adrenal
chromaffin cells occurs with a time constant of ⬃1 s when
depolarized from a resting level of [Ca2⫹]i (321, 675).
Rapid phases of exocytosis occur when cytosolic Ca2⫹ levels are increased for a few minutes (221, 673). After this
“Ca2⫹ priming” process, vesicles undergo more rapid exocytosis, with time constants of 30 ms or less. However, the
time course of exocytosis is still 30 times slower than that of
ultrafast exocytosis that occurs in synaptic vesicles. This
suggests that there are considerable differences in the molecular and cellular bases of the two types of exocytosis (see
sect. V). An intensive quantitative investigation of chromaffin from adult bovine adrenal medulla failed to identify
preferential docking of LDCVs (FIGURE 2B), even when the
chromaffin cells were primed with Ca2⫹ (494), because the
cytosol was already filled with LDCVs. In fact, docking of
LDCVs is detectable in embryonic chromaffin cells, in
which LDCVs are more sparse (134, 135, 676). Importantly, fast exocytosis in Ca2⫹-primed cells is followed by a
slow sustained phase of exocytosis (706, 707), with a time
constant similar to that of slow exocytosis in unprimed
chromaffin cells; this suggests that nondocked vesicles in
chromaffin cells can undergo slow exocytosis (617, 707)
(see sect. VI).
B) PC12 CELLS. The chromaffin cell line PC12 is often used to
investigate the molecular mechanisms underlying exocytosis (26, 32, 36, 99, 120, 185, 205, 305, 323, 346, 391, 499,
591, 622, 625, 656, 657, 744). The kinetics of LDCV exocytosis in PC12 cells are slower (10 s) than in chromaffin
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cells (1 s) and are not increased by Ca2⫹ priming (309, 322).
However, both chromaffin cells and PC12 cells show
intensive compound exocytosis, as described in section
IIIC (FIGURE 3E). Despite the fact that exocytosis is extremely slow in PC12 cells, ultrastructural and biochemical studies using cracked cells often show LDCVs
docked to the plasma membrane (393). This is an example of the dissociation between the morphological docking and fast kinetics of exocytosis (see sect. VIB).
C) ISLET ␤ CELLS. The pancreas, or pancreatic islets, contain
three types of endocrine cell: ␣ cells (which secrete glucagon), ␤ cells (which secrete insulin), and ␦ cells (which secrete somatostatin). An increase in [Ca2⫹]i is the major
trigger for secretion in all three cell types (248, 505, 609,
693, 700). ␤ cells form the major cell mass in the islets, and
insulin secretion is of crucial importance to health and disease as it is the only hormone that facilitates glucose uptake
by muscle and adipocytes, leading to a reduction in blood
glucose levels. Defective insulin secretion is the major etiology of type 2 diabetes mellitus, and stimulation of insulin
secretion is the primary therapeutic target. Insulin secretion
is slow and tonic in general and takes place in two phases.
The first phase of insulin secretion is induced within 5 min
of stimulation and is important for glucose uptake into the
liver. The second phase of insulin secretion is tonic and
gives rise to glucose uptake into muscles and other tissues.
In addition to Ca2⫹, cAMP is an important factor involved
in insulin exocytosis (248, 304, 380, 580). The two phases
of insulin exocytosis are differentially regulated by PKA
(237, 308, 634) and syntaxin (459, 612), and may involve
distinct molecular states (458, 633). It is suggested that
cAMP-guanine nucleotide exchange factor (GEF), Epac,
regulates insulin exocytosis by chronically modulating the
availability of insulin granules (590, 733), but not by
acutely potentiating insulin exocytosis (238, 597, 603).
Importantly, experiments using caged Ca2⫹ compounds in
chromaffin cells (321, 675) and ␤ cells (237, 632, 635) revealed that the speed of exocytosis was slow, even when exocytosis was induced by a rapid increase in [Ca2⫹]i, which induces submillisecond exocytosis in synapses (FIGURE 1). Thus
the exocytotic machinery must be inherently slow. Micromolar increases in [Ca2⫹]i are often required to trigger exocytosis by secretory cells in synapses, and coupling between Ca2⫹ channels and the fusion machinery may occur,
although not as tightly as the coupling involved in synaptic
neurotransmitter release (35, 40, 261, 536). PKA increases
the number of LDCVs that undergo fast exocytosis in both
chromaffin cells (437) and ␤ cells (238, 634).
D) PITUITARY CELLS. Prominent endocrine cells are present in
the anterior and posterior pituitary glands (486). The release of vasopressin and oxytocin from the posterior pituitary
is induced via nerves from the hypothalamus, and the release
behaviors are similar to those observed for LDCV secretion
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fore, the total amount of secretion over a period of minutes
is more important than the millisecond time course of exocytosis from the surface layer of the cytosol. In fact, the
speed of exocytosis is markedly slower in endocrine cells
than the ultrafast exocytosis observed in synaptic vesicles
(FIGURE 1). Moreover, although action potentials trigger
exocytosis of LDCVs in many endocrine cells (49, 319,
700), the temporal correlation between each action potential and exocytosis is never as tight as it is in ultrafast exocytosis (112, 745). Endocrine cells secrete hormones into
the blood vessels, therefore, and exocytosis was predicted to
occur more frequently at plasma membranes facing the
blood vessels (465). However, two-photon imaging experiments show that there is no marked spatial regulation of
exocytosis, and that exocytosis utilizes the entire plasma
membrane (321, 636). In support of this idea, there are no
tight junction structures in endocrine glands, and the intercellular space can act as a pathway for hormonal secretions.
The next section summarizes the specific features of LDCV
exocytosis in different endocrine cell types.
INITIAL SNARE CONFIGURATIONS FOR EXOCYTOSIS
2. Exocrine glands
Exocrine cells secrete digestive enzymes into the gastrointestinal tract. These cells include the acinar cells in the pancreatic, parotid, mandibular, and lacrimal glands. The secretion of enzymes must be slow and tonic to maintain
sufficient concentrations in the extracellular fluids in a regulated manner. Exocrine cells do not express Ca2⫹ channels; rather, Ca2⫹ increases mainly occur via Ca2⫹ release
from intracellular stores (619, 726) and Ca2⫹ influx via the
store-operated Ca2⫹ entry pathways (500).
Secretory granules are localized at the apical region of the
cells, where exocytosis is selectively induced. Even though
the agonist receptors are localized on the basal side of the
cells, Ca2⫹ release is initiated at the apical pole (303), probably due to diffusion of inositol trisphosphate from the
basolateral membrane towards the apical pole (278, 307,
641). The localization of Ca2⫹ mobilization from stores
coincides well with the exocytosis of zymogen granules,
which is apparently advantageous for massive exocytosis
from exocrine glands. The Ca2⫹ increase at the apical pole
is as high as 10 ␮M (279) and is required for the exocytosis
of zymogen granules (443), similar to the situation in synapses and endocrine cells.
There is an electron-lucent layer beneath the apical
plasma membrane in exocrine glands (273), which precludes the attachment of vesicles to the apical plasma
membrane. This electron-lucent layer corresponds with
the apical F-actin layer, toward which the acinar cells
undergo exocytosis (444). In fact, removal of the F-actin
layer by latrunculin facilitates exocytosis (444); thus zymogen granules are not docked to the plasma membrane
prior to stimulation.
3. Hematopoietic cells
Most blood cells, including mast cells, eosinophils, basophiles, and T-cells, have LDCVs that contain various cytokines and enzymes. Exocytosis in blood cells is believed to
depend on cytosolic Ca2⫹ (412); however, other mechanisms also exist (550) (TABLE 1). The secretion of granules is
generally slow in blood cells and secretory granules are
nondocked; however, massive exocytosis can be induced by
compound exocytosis after stimulation (see sect. IIIC).
Exocytosis from blood cells may occur in a pointed manner
toward the target cells. For example, eosinophils are involved in host defense against parasites (84) and are implicated in allergic responses (343). Parasite death is caused by
adherence of eosinophils to the parasite surface, followed
by the targeted release of cytotoxic granule contents by
exocytosis (246). Degranulation in eosinophils occurs via
the formation of degranulation sacs, ensuring the release of
cytotoxic proteins at a high concentration at focal sites
through a single fusion pore (246). It is widely assumed
that, in eosinophils and basophils, compound granules are
formed by homotypic granule-granule fusion inside the cell,
followed by exocytosis and multigranular exocytosis (218).
Ca2⫹ is the trigger for this process in macrophage and dendritic cells (42), whereas kinase signaling is the trigger in
basophils and mast cells (629) and at the immunological
synapses in cytotoxic T-cells (482). Thrombin triggers exocytosis of LDCVs from platelets, which is essential for
thrombosis and is dependent on SNAREs (513).
4. Lysosomal exocytosis
Lysosomes are scattered throughout the cytosol and are
rarely docked to the plasma membrane (376). Exocytosis
from lysosomes is Ca2⫹ dependent (352) and occurs in various cells (447, 506). Lysosomal exocytosis is also used for
ATP release from glial cells (366, 740) and may play a role
in membrane repair (510).
B. Compound Exocytosis
One prominent feature of exocytosis of LDCVs is compound exocytosis, which is defined as exocytosis of more
than one vesicle at the same time (492). The advantage of
compound exocytosis is the effective mobilization of deep
vesicles within the cytosol without the need for transportation to the target plasma membrane, thereby avoiding deformation of cellular structures. This is particularly important for LDCVs, as their large size makes diffusion and
transport to the proper release sites in the plasma membrane more difficult. Therefore, compound exocytosis provides the most efficient method of mobilizing LDCVs stored
deep within the cytosol. There are two forms of compound
exocytosis: sequential and multigranule.
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from nerve endings (see sect. IIIC), although LDCVs seldom
dock to the membrane (FIGURE 2B). An interesting characteristic of the posterior pituitary gland is the presence of small
synaptic vesicles with unknown functions (FIGURE 2B)
(325). The anterior pituitary gland comprises cells that secrete six different hormones: coricotrophs secrete adrenocorticotropic hormone, somatotrophs secrete growth hormone, thyrotrophs secrete thyroid stimulating hormone,
lactotrophs secrete prolactin, and gonadotrophs secrete luteinizing hormone and follicle stimulating hormone. Exocytosis of all of these LDCVs is triggered by increases in
[Ca2⫹]i, which are mediated by Ca2⫹ channels in corticotroph and somatotroph cells, and by Ca2⫹ release from
intracellular Ca2⫹ stores within thyrotroph, lactroph, and
gonadotroph cells (618), probably in much the same way as
in exocrine cells. In addition to Ca2⫹, PKC is proposed to
act as a trigger of exocytosis in gonadotroph cells (253).
Compound exocytosis is observed in cells that utilize Ca2⫹
release from intracellular stores (116).
KASAI ET AL.
1. Sequential exocytosis
In 1965, structural studies predicted sequential exocytosis in
pancreatic acinar cells (273), and the dynamics were identified
using two-photon excitation imaging (443) (FIGURE 3E). Interestingly, maintenance of the omega-shaped structure of
already-fused granules requires an actin coating (444); otherwise, sequential exocytosis results in vacuoles, akin to
those found in acute pancreatitis (444, 507). Thus exocrine
cells are well prepared for the sequential progression of
exocytosis from deep within the cells, maintaining defenses
against the harsh environment in the gastrointestinal tract.
Interestingly, although actin coating of the granules slows
the fusion between the granules, it does not block it (444),
suggesting that the granules are not docked to each other or
to the plasma membrane, and that the submembrane actin
cytoskeleton can dynamically regulate exocytosis.
Two-photon imaging of clusters of chromaffin cells shows
that sequential exocytosis results in vacuoles akin to those
observed in latrunculin-treated acinar cells, even without
latrunculin treatment (321). Such vacuolar structures were
seen in earlier studies but were thought to represent endocytotic processes (33). Compound exocytosis can mobilize
more than 50% of granules stored in the cytosol. Although
compound exocytosis has been observed at the basal surface of cells facing a glass surface using total internal reflection fluorescence (TIRF) imaging (7), it is particularly
prominent in tissue preparations in which the extracellular
space is faced by an intact extracellular matrix, which
blocks diffusion of gels within the granules. After sequential
exocytosis, the gel inside the granules swells and forms expanding luminal structures, which bring the vacuolar membrane closer to the cytosolic vesicles and facilitate the progression of exocytosis. Thus mechanical pressure appears to
facilitate exocytosis in chromaffin cells.
Studies of exocytosis in acinar and chromaffin cells show
that the progression of exocytosis is mostly sequential, and
that the speed of fusion of granules in the superficial layer is
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The fusion pore in primary vesicles plays a pivotal role in
sequential exocytosis because all subsequent secretion is
mediated through the same initial fusion pore on the original plasma membrane. Indeed, ultrastructural analysis of
chromaffin cells shows that the vesicles and plasma membrane are bridged by fine strands, which are preserved even
after exocytosis and may represent annexins (438). Similar
structures are also found in the anterior pituitary gland, in
which sequential exocytosis also occurs (581). In addition,
submembraneous F-actin plays a role in stabilizing both the
fusion pore and sequential exocytosis in chromaffin cells
(321).
2. Multigranular exocytosis
In multigranule exocytosis, vesicles fuse within the cytosol
to form the multigranular vesicles that later undergo exocytosis (FIGURE 3F). Typical examples of multigranular exocytosis in hematopoietic cells were given in the previous
section. It seems that both multigranular and sequential
exocytosis are used by many cells, including mast cells (8),
lactotroph cells (116), gastolic parietal cells (175), ␤ cells
(262), and synapses (23, 244, 398). Multigranular exocytosis has the advantage of secreting a great number of messengers at the same time; however, unlike sequential exocytosis, the amounts cannot be controlled as precisely. Multigranular exocytosis is a prominent feature of exocytosis
from blood cells. Multigranular (multivesicular) exocytosis
may also be utilized during synaptic neurotransmitter release; for example, botulinum toxin treatment increases the
frequency of giant miniature excitatory postsynaptic potentials (498, 566), suggesting that multivesicular exocytosis
may be involved in some pathological states at the synapses.
C. Large Dense-Core Vesicles in Synapses
Presynaptic terminals contain synaptic vesicles, and LDCVs and
electron microscopic analyses show that most of the LDCVs are
nondocked to the plasma membrane (FIGURE 2B) (76, 152, 201,
539, 560, 610). Exocytosis of synaptic LDCVs requires repetitive stimulation (583, 692); thus the molecular basis of
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In sequential exocytosis, vesicles fuse with the plasma membrane (preserving their omega-shaped structure to some extent) where they become the target for further exocytosis of
deep vesicles within the cytosol (FIGURE 3E). Sequential exocytosis is a highly sophisticated three-dimensional phenomenon, the quantification of which requires a two-photon microscope. The prevalence of sequential exocytosis
may, therefore, be underestimated. Sequential exocytosis
has been observed in exocrine acinar cells (224, 443, 444),
adrenal chromaffin cells (7, 190, 321), PC12 cells (322,
734), ␤ cells (464, 632), pituitary lactotrophs (116), human
neuroendocrine Bon cells (652), the nasal gland (469), eosinophils (218), and mast cells (8, 213), and even in bipolar
synapses (398). Sequential exocytosis is particularly prominent in exocrine acinar cells and chromaffin cells, as described below.
akin to that of vesicles in the deep cellular layers (321, 444).
This suggests that the vesicles in the deep layers have a
“fusion readiness” comparable to that of superficial vesicles. Granules are not docked to other granules in the cytosol, supporting the notion that the secretory granules are
also not docked to the plasma membrane before stimulation. Moreover, the strictly sequential nature of exocytosis
suggests that a key factor must diffuse from vesicle membranes that have already undergone exocytosis to assist exocytosis of vesicles in the deeper cell layers. In fact, diffusion
of t-SNAREs into the fused vesicles has been found in chromaffin cells (321), ␤ cells (632), and exocrine cells (493).
Thus sequential exocytosis suggests that vesicles are nondocked (see sect. VI).
INITIAL SNARE CONFIGURATIONS FOR EXOCYTOSIS
LDCV exocytosis in presynaptic terminals is similar to that
in endocrine cells. The 20-ms delay in the exocytosis of
LDCVs (75) may correspond to the crash fusion events observed in adrenal chromaffin cells (7). Exocytosis of LDCVs is
mainly limited by the number of mobile granules and their
slow rate of diffusion (81); the mobility of LDCVs is also Ca2⫹
dependent (583).
D. Small Vesicles in Nonneuronal Cells
Small vesicle exocytosis is usually characterized using imaging techniques. Small vesicles in PC12 cells selectively
contain acetylcholine (ACh), and their exocytosis was visualized by labeling the vesicle with a vesicular ACh transporter labeled with pHluorin (64). Exocytosis of small vesicles from PC12 cells and islet ␤ cells was also studied using
two-photon imaging, which revealed that the time constants for exocytosis induced by Ca2⫹ uncaging were 0.8
and 0.3 s for PC12 cells and islet ␤ cells, respectively (238,
367). These time constants correlate well with the fast component of increases in membrane capacitance (309, 635).
The fast component of the capacitance increase in ␤ cells is
augmented by cAMP but is resistant to Rp-cAMP (515),
and is congruent with the facilitation of small vesicle exocytosis by Epac (238, 544), which regulates small vesicle
exocytosis (0.3 s) in ␤ cells more rapidly than PKA regulation of LDCVs (4.5 s) (238). Despite the abundance of
Ca2⫹-dependent exocytosis of small vesicles in these cells,
small vesicles are found nondocked to the plasma membrane before stimulation (305, 494); therefore, they may
undergo poststimulus docking (FIGURE 3, B AND C) (see
sect. IVA).
E. Regulated Membrane Recycling Systems
1. H⫹-ATPase trafficking in gastric parietal cells
Regulated membrane recycling has been proposed as the
major mechanism underlying histamine-induced acid secretion from gastric parietal cells, in which H⫹-ATPases in endosomal compartments of tubulovesicles (TV) are brought to
the apical secretory surface by exocytosis (175). The involvement of SNAREs in this process was recently confirmed
using TeNT (300, 349). This recycling is induced by histamine and H2 receptors, which activate a heterotrimeric G
protein Gs, resulting in an increase in cytosolic cAMP levels
(175, 253, 368). The signal is then relayed to PKA. The TV
membranes are mutually fused, apparently undergoing
compound exocytosis, to reorganize the membrane structure. Reorganization of the TVs is dependent on actin fibers,
suggesting that cAMP regulates poststimulus docking of
TVs to the plasma membrane (FIGURE 3C).
2. Aquaporin trafficking in kidney-collecting ducts
Another prominent example of the recycling of transport
vesicles can be found in kidney collecting duct cells, where
vasopressin raises cytosolic cAMP levels, which results in
the exocytosis of vesicles that contain aquaporin-2 into the
collecting duct membrane in a PKA-dependent manner.
This underlies the major diuretic action of vasopressin (71,
554). This form of exocytosis requires SNAREs, as it is
blocked by TeNT (202) and botulinum toxins A, B, and C
(501); it is also highly dependent on the subcortical actin
layer, and the actions of cAMP may be mediated by depolymerization of the cortical actin layer by inhibition of Rho
small GTPases (324, 450). Vesicles containing aquaporin-2
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Ca2⫹-dependent exocytosis of small vesicles has been demonstrated in various cells other than synaptic terminals and
may be mediated by specific types of vesicles that, like synaptic vesicles, can be labeled with synaptophysin. These
vesicles are called “synaptic-like microvesicles” (SLMV;
Refs. 290, 439)(FIGURE 2) or “enlargesomes” (117, 118,
305) (TABLE 1). Exocytosis of small vesicles has been studied using capacitance measurements in the cell-attached
patch configuration, where single exocytotic events are seen
as stepwise increases in the membrane capacitance of neuroblastoma NG108 –15 cells (137), pituitary nerve endings
(325), pancreatic ␤ cells (379), or PC12 cells (743). The
estimated size of the small vesicles ranges from 40 to 100
nm, which is consistent with ultrastructural observations
(305, 494) and imaging studies (238, 367). Ca2⫹-dependent
exocytosis of small vesicles is also found in fibroblasts and
muscles, where it may play a role in membrane repair (404,
614). Small vesicle exocytosis in chromaffin cells (706) and
PC12 cells (305) is resistant to TeNT but is blocked by
TeNT in fibroblasts (587). Enlargesomes may share a common ancestor with synaptic vesicles (537, 587) and appear
to be utilized by cells for growth, differentiation, migration,
and wound healing (117, 118, 238).
Amperometry studies show that the rapid increase in capacitance happens more quickly than LDCV exocytotic events
in chromaffin cells (221, 309, 428, 448, 457), PC12 cells
(448), ␤ cells (304, 635), CHO cells (447), and mast cells
(320) (FIGURE 1). The most likely reason for the dissociation between the capacitance and amperometric signals in
these cells is the presence of small vesicle exocytosis. The
dissociation is minimized under Ca2⫹-priming conditions,
in which the basal Ca2⫹ level is increased to more than 0.3
␮M over 3 min (221, 434), although this procedure should
not exclude the contribution of small vesicles. It is also
reported that the amperometric measurement was affected
by a diffusional delay of unknown nature (221), which
might reflect sequential exocytosis (321) or the monoamine
content of small vesicles. In addition, membrane capacitance measurements record both exocytosis and endocytosis, which are difficult to separate (367). Hence, the kinetic
features of LDCV exocytosis from endocrine cells would be
better investigated using imaging approaches, which are
able to unambiguously distinguish the exocytosis of LDCVs
and small vesicles (41, 87, 261, 306, 321, 367, 479).
KASAI ET AL.
undergo spontaneous exocytosis, akin to synaptic vesicles.
Similar to TV vesicles, zymogen granules in parotid acinar
cells express aquaporin and undergo exocytosis induced by
cAMP during stimulation with isoproterenol (182, 556).
3. GLUT4 trafficking in adipocytes and
skeletal muscles
4. Trafficking of glutamate receptors
in neuronal dendrites
Activity-dependent trafficking of glutamate receptors in
postsynaptic dendrites is involved in the synaptic plasticity
of glutamatergic synapses (315). Vesicles carrying glutamate receptors are found in the cytosol and undergo Ca2⫹dependent exocytosis (384, 481). Thus docking and fusion
of these vesicles must be induced in a Ca2⫹-dependent manner to enable rapid activity-dependent changes in synaptic
functions. Since exocytosis is Ca2⫹ dependent, these vesicles may fall into the same class as enlargesomes (117).
F. Constitutive Exocytosis
Constitutive exocytosis occurs without any particular external stimulation and carries membrane lipids and membrane proteins to the plasma membrane, which is necessary
for cell survival (212). TIRF imaging of constitutive exocytosis reveals that it occurs in a manner similar to regulated
exocytosis (569). Constitutive exocytosis is mediated by
SNAREs (485) (TABLE 1), as is the case for regulated exocytosis, and some secretory cells appear to utilize the same
set of SNAREs as are utilized for constitutive exocytosis
(TABLE 1, see sect. VIC). Thus SNARE-dependent exocytosis can proceed without the need for a specific trigger, and
exocytosis can be regulated at any stage during transport,
docking, and fusion of vesicles (FIGURE 3, A–C).
G. Fusion of SNARE-Bearing
Proteoliposomes
The idea of fusion assay systems that use liposomes bearing
SNAREs was first introduced in 1998 (690), when dequenching of membrane fluorescent tracers was used to
monitor lipid mixing, which is indicative of membrane
hemifusion. Such hemifusion was associated with full fusion content mixing in some studies (445, 665, 682). Lipo-
1926
Recently, a single vesicle fusion system was developed in
which lipid mixing and content mixing can be simultaneously studied in vesicles that are linked by SNAREs (341).
The fusion is extremely rapid (⬍250 ms), possibly due to
the selection of vesicles that were already docked with each
other. The rapid fusion events, coupled with a complete
reconstituted system, may allow the study of ultrafast
Ca2⫹-dependent fusion in the near future.
IV. KEY CELLULAR STAGES
OF EXOCYTOSIS
In this section, we explain some of the key notions of exocytosis that are necessary for understanding the diversity of
SNARE-dependent exocytoses. In addition, the concepts of
docking, priming, and triggering need some clarification;
therefore, we also summarize what is known regarding the
intermediate steps involved in membrane fusion and the
effects of lipid composition.
A. Docking of Vesicles
1. Definition of docking
The vesicle membrane must make physical contact with the
plasma membrane for membrane fusion to occur at any
time before or after stimulation (402, 671). For ultrafast
exocytosis, such physical or molecular contact is likely
formed before stimulation, and the word docking is often
used to describe the conceptual state of vesicles before stimulation. Docking, however, can simply mean contact between vesicles and the plasma membrane irrespective of the
timing of the stimulation. These two meanings of docking
are often used interchangeably, giving the erroneous impression that docking of the vesicles needs to precede the
triggering of exocytosis. To avoid this confusion, we use the
terms prestimulus and poststimulus docking in the following sections.
2. Prestimulus docking
Prestimulus docking has been studied in neurons and secretory
cells using electron microscopy, where vesicles are often
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The actions of insulin promote the uptake of glucose by
skeletal muscle and adipocytes via exocytosis of vesicles
containing a highly efficient glucose transporter, GLUT4
(174, 236), which is impaired in type 2 diabetes. Exocytosis
of GLUT4 is SNARE-dependent (77). The vesicles are
stored in the cytoplasm; therefore, insulin stimulation triggers the docking and fusion of GLUT4-containing vesicles
to the plasma membrane (30, 285, 708).
some fusion is both constitutive and slow (10 –30 min)
(258), even when the system is preconditioned with a small
COOH-terminal fragment (residues 49 –96) of VAMP2
(496). Liposome fusion can be induced in a Ca2⫹-dependent manner in the presence of a soluble synaptotagmin1
fragment (661) or full-length synaptotagmin1 (347, 685),
but the fusion is still slow (1–10 min). A cell-based assay
using “flipped” SNAREs reported that SNAREs can promote content exchange (265), but the kinetics are slow (10
min), even when synaptotagmin and complexin are included in the fusion assay. These model systems may explain the slow exocytosis observed in secretory cells, at least
with respect to the time course (FIGURE 1).
INITIAL SNARE CONFIGURATIONS FOR EXOCYTOSIS
Prestimulus docking is also found in cells that show only
slow exocytosis, such as PC12 cells, in which docked and
nondocked granules undergo exocytosis over the same time
course (317). Moreover, prestimulus docking does not hasten insulin exocytosis in ␤ cells (310, 655, 719). If prestimulus docking does not hasten exocytosis in slow secretory
cells, then why are vesicles found tightly docked to the
plasma membrane in PC12, chromaffin, and pituitary cells?
One idea is that prestimulus docking assists sequential exocytosis (see sect. IIIB) by stabilizing the fusion pore of the
primary exocytotic vesicles (FIGURE 3E) (321, 322, 632).
Such docking is mediated by annexins (306, 438, 581),
which can tightly link vesicles with the plasma membrane.
Another mechanism for docking or tethering vesicles to the
plasma membranes involves Rab, a small GTPase, and
other tethering proteins (68, 72, 272, 725). Such loose molecular contacts may play an important role in directing
vesicles to their proper targets, because SNARE interactions
are too promiscuous (32, 558, 714) to explain the specificity
of vesicle trafficking.
3. Poststimulus docking
Most vesicles stored in the cytosol are nondocked to the
plasma membrane in synapses and secretory cells. Indeed,
most synaptic vesicles are nondocked in the presynaptic
terminals (like LDCVs in synapses) in endocrine and exocrine cells. The same is true for small vesicles in nonsynaptic
preparations, such as transport vesicles in gastric parietal
cells and kidney duct cells, as described in section III. Live
imaging has directly demonstrated that Ca2⫹ can induce
poststimulus docking of vesicles for exocytosis, referred to
as a “crash fusion event,” in adrenal chromaffin cells (7,
671), islet ␤ cells (310, 459, 460, 590), PC12 cells (317,
555), and mast cells (31). Such crash fusion events are
found even in synaptic vesicles, where they are involved in
the tonic exocytosis of ribbon synapses (257, 418, 729).
Because TIRF imaging reaches at least 40 nm from the
plasma membrane, and SNARE complexes are only 12 nm
in length, it is unlikely that ternary-SNARE complexes form
before stimulation during crash fusion events (353). Crash
fusion events are dominant during insulin exocytosis from ␤
cells (310, 590), where the rates of granule arrival at, and
departure from, the submembrane space are two orders of
magnitude higher than the release rates. Such dynamics may
determine the rate of exocytosis (239).
4. Submembranous actin layers and exocytosis
We can infer whether pre- or poststimulus docking is involved in exocytosis from the dependence of exocytosis on
submembraneous actin fibers. If vesicles are already docked
before stimulation, removal of the actin layer should not
affect exocytosis. Indeed, latrunculin A treatment, which
disrupts actin fibers (122), does not affect ultrafast exocytosis from the active zone (426, 545), but it augments spontaneous exocytosis (268, 426). These results are consistent
with the idea that ultrafast exocytosis is mediated by
docked vesicles and spontaneous exocytosis occurs from
nondocked vesicles in the reserve pool (268, 503). Similarly,
disruption of subcortical actin is reported to augment exocytosis from adrenal chromaffin cells (653), ␤ cells (647),
exocrine acinar cells (444), gastric parietal cells (175), and
kidney duct cells (71, 450). Deletion of Munc18a reduces
morphological docking of LDCVs in embryonic adrenal
chromaffin cells (676) because Munc18a removes the subcortical actin layers (133, 651). Treatment with latrunculin
A restores morphological docking, but not exocytosis, suggesting that Munc18a removes the subcortical actin layer
for docking in addition to priming SNARE assembly (69,
135, 270, 571, 686).
Prestimulus docking is a morphological concept and is absolutely necessary only for ultrafast exocytosis (FIGURE 1);
however, it neither necessarily accompanies tight SNARE
assembly nor hastens exocytosis. Thus we should better
specify the initial states of the SNARE assembly, rather than
the “docking” of vesicles, wherever possible.
B. Priming of Exocytosis
Priming of exocytosis generally pertains to the molecular
and cellular processes that facilitate stimulus-induced fusion reactions. Priming in permeabilized chromaffin cells
(259) and PC12 cells (393) requires Mg-ATP. Priming is
often assumed to follow vesicle docking in these preparations and in the active zone (446, 451); however, priming
can precede docking. For instance, ATP-dependent disassembly of SNAREs by N-ethylmaleimide-sensitive fusion
protein (NSF) is necessary for exocytosis (336, 392, 705),
and such priming reactions precede the docking of vesicles.
In addition, there is a specific priming process called Ca2⫹
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docked to the site of exocytosis in resting samples (FIGURE 2)
(133). Prestimulus docking is considered necessary for fast
secretory cells and synapses (699). In fact, the number of
docked vesicles in the synapse correlates with the number of
vesicles in the RRP (568). SNAREs and Munc18 are the
epicenter of exocytosis (see sect. V), and both might be
involved in prestimulus docking. In fact, docking of LDCVs
in embryonic chromaffin cells was reduced by the deletion
of SNAP25 (135), Munc18a (676), or cleavage of syntaxin
with BoNTC (134). Consistent with this, docking of synaptic vesicles in the synapses of nematode mutants studied
under high-pressure electron microscopy was impaired
when syntaxin or Munc18a was deleted (222, 223, 691).
However, in mammalian (69, 270, 571, 670, 686) and fly
(67, 577) synapses, SNAREs and Munc18a are dispensable
for docking, indicating that prestimulus docking is more
dependent on CAZ proteins, such as RIM (297, 330) and
Munc13 (595). Therefore, prestimulus docking does not
specify the state of SNAREs.
KASAI ET AL.
priming (673, 677), which is induced by moderate increases
in [Ca2⫹]i and markedly hastens exocytosis, presumably by
inducing prestimulus docking and assembly of SNARE
complexes in synapses and chromaffin cells (442). Priming
can even be positional, as the vesicles need to be brought
close to the Ca2⫹ channels to trigger ultrafast exocytosis
(442, 678).
C. Triggering of Exocytosis
1. Physiological triggering
SNARE-dependent exocytosis can occur constitutively without stimulation, meaning that exocytosis can be regulated at
any stage from transport, SNARE assembly, and fusion.
Once such regulation, or “clamp,” is removed, exocytotic
reactions may proceed to fusion. Exocytosis can be triggered by an increase in [Ca2⫹]i (479) or cAMP (630), activation of kinases (253, 285, 629), or even changes in membrane voltage (731, 732). Such stimulation can be very
rapid and transient, or repetitive and tonic, lasting for minutes or hours, for example, tonic exocytosis of synaptic
vesicles and insulin exocytosis. Exocytosis can be regulated
at multiple stages (597, 634).
2. Ca2⫹-dependent triggering steps
Synaptotagmins and Doc2 are Ca2⫹ sensors for Ca2⫹-dependent exocytosis (see sect. VD). Synaptotagmins sense
increases in [Ca2⫹]i caused by influxes of Ca2⫹ through
Ca2⫹ channels located close to the site of exocytosis (Ca2⫹
domain) for ultrafast exocytosis (see FIGURE 8). Exocytosis
evoked by a single action potential, however, is rather exceptional, and Ca2⫹-dependent exocytosis is usually induced by an increase in [Ca2⫹]i, which occurs diffusely over
space and time. This applies even to synaptic neurotransmitter release, particularly for tonic and spontaneous exocytosis. In endocrine and exocrine cells, increases in [Ca2⫹]i
can be slowly achieved by Ca2⫹ release from intracellular
Ca2⫹ stores. In these cases, Ca2⫹ induces poststimulus
1928
D. Membrane Dynamics
The free energy required for the fusion of a vesicle to the
membrane has been theoretically estimated as 40 –120 kBT,
where 1 kBT ⫽ 4 ⫻ 1021 J, the product of the Boltzmann
constant and the absolute temperature (119, 338). This is
the same order of magnitude as the energy supplied by the
hydrolysis of ATP (38 kBT) and the reduction in the free
energy of SNAREs after zippering (35 kBT) (353). Thus the
fusion reaction and SNARE assembly have comparable energy barriers, consistent with the SNARE hypothesis of exocytosis.
Membrane fusion proceeds via the following three steps:
1) initial contact, 2) formation of hemifusion intermediates,
and 3) opening of the fusion pore (FIGURE 4) (119, 332).
Molecular dynamic simulations suggest that the process of
hemifusion and fusion pore opening are very rapid after
initiation (181, 452). In contrast, forming the initial contact
may take a long time, and the narrow fusion pore can be
stable over seconds. It is likely that fusion is arrested before
initial contact, at least for slow exocytosis, and possibly for
all forms of exocytosis.
1. The initial step of membrane fusion
The two membranes must make physical contact for membrane fusion to occur. In biological fusion reactions, such
contact is actively guided by tethering molecules in the active zone (725), including small G proteins (Rabs) (281),
myosin V, microtubules, and F-actin (594). This is to ensure
the selectivity of the fusion events. Conversely, the contact
zone is crowded with membrane-associated proteins, such
as the subcortical actin meshwork (337), membrane integral proteins, and adaptor proteins, which cover 50% of the
surface of the plasma membrane (102). In principle, the
intimate contact involved in fusion requires the opening of
protein-depleted patches in the opposing membranes.
These proteinaceous obstacles are dynamic, allowing access
to vesicles for only a short period of time (138), and these
dynamics can regulate the kinetics of exocytosis. In the
active zone, CAZs may promote the initial contact.
Examples of initial contact formation are found in many
biological tissues, including exocrine acinar cells, in which
exocytosis is induced toward the cortical F-actin layer
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Many priming molecules that regulate SNAREs have been
identified (80, 392), including Munc18, Munc13, CAPS,
complexin, and snapin (sect. V). Thus priming can be defined as a molecular process that facilitates exocytosis by
regulating SNAREs. It should be noted that, since SNARE
assembly may occur after stimulation in slow secretory cells
(sect. VI), the same molecule used for priming of ultrafast
exocytosis may be utilized after stimulation for slower exocytosis (see FIGURE 8). For instance, although CAPS is
normally assumed to be involved in prestimulus SNARE
assembly in PC12 cells, it also facilitates Ca2⫹-dependent
SNARE assembly after stimulation (317). Many other examples are presented in section VI. Thus priming proteins
regulate exocytosis and SNARE assembly both before and
after the triggering of exocytosis.
docking and, therefore, acts upstream of SNARE assembly
(633), as is the case for other triggers such as cAMP. Thus
triggering is achieved by resumption of SNARE assembly,
which is halted at various stages depending on the cell type
(see sect. VI). Thus exocytotic vesicles experience the following stages (FIGURE 8): 1) SNARE disassembly and partial
SNARE assembly, 2) triggering, 3) poststimulus SNARE assembly, and 4) the fusion reaction that occurs in parallel with
the final SNARE assembly.
INITIAL SNARE CONFIGURATIONS FOR EXOCYTOSIS
A
End-cup formation
D
Small vesicle
B
E
Hemi-fusion
Large vesicle
C
Fusion pore
F
Membrane tension
within a few seconds of stimulation (444). The rate of exocytosis in secretory cells may be regulated by the frequency
of vesicles approaching the plasma membrane (138, 239).
Interestingly, vesicle movements are increased before fusion
(6, 138), suggesting that lipidic contact may be made immediately before exocytosis. The initial contact between
vesicles with the plasma membrane may be induced 40 –100
ms before exocytosis (7, 239, 310, 317, 555, 590). Thus the
transport of vesicles and the submembraneous cytomatrix
are major factors determining the dynamics of exocytosis.
The shortest distance between the two membranes would
be 2–3 nm for a lipid bilayer at full hydration (598), and the
initial step of membrane fusion may be point-like local contact between the two membranes (FIGURE 4A) (159, 598)
from which the hemifusion stalk intermediate (FIGURE 4B)
is formed. The early metastable and transition states are
mostly inferred from molecular dynamics simulations, as
the transitional state is too fast to directly observe. The
prestalk fusion intermediate may be initiated from only one
lipid molecule, whose two hydrocarbon chains are inserted
into the opposing membranes (598). Such reactions must be
facilitated by the buckling of the plasma membrane and end
cap formation (FIGURE 4A) (402) by proteins such as
SNAREs, synaptotagmin (269, 387, 582), or Doc2 (208)
(see sect. V). The end cap, or membrane dimple, was reported in ultrastructural studies of secretory cells (425) and
TIRF microscopy (10).
In ultrafast exocytosis of docked synaptic vesicles, the osmotic pressure generated by a hypertonic sucrose solution
(HTSS) can induce exocytosis (67, 532). Positive pressure
also enhances exocytosis from chromaffin cells (321). The
positive pressure forces the vesicles closer to the plasma
membrane, which might facilitate initial contact formation
of the two lipid bilayers (179).
Another factor that may affect contact formation is the
curvature of the vesicle membranes. Small vesicles with a
greater curvature can more readily form a contact with the
plasma membrane (FIGURE 4, D AND E). This may explain
why the rate of exocytosis of larger vesicles is slower than
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FIGURE 4. Schematics depicting membrane dynamics during exocytosis. A: end-cap formation. Fusion is
initiated by contact between the vesicle and the plasma membrane. B: a stalklike hemifusion intermediate.
C: the fusion pore. Fusion pore opening is facilitated by an inverse cone-shaped lipid in the outer leaflet of the
membrane (red arrowhead) or a cone-shaped lipid in the inner leaflet (blue arrowhead). D and E: preferential
contact by smaller vesicles due to reduced steric hindrance and a larger membrane curvature. F: membrane
tension assisting the opening of the fusion pore. Smaller vesicles may generate a larger tension to facilitate the
opening of the fusion pore.
KASAI ET AL.
that of the small vesicles (FIGURE 1). Although this discrepancy in the rate of exocytosis may be due to the different
types of SNAREs expressed, exocytosis of small vesicles in
cells lacking neuronal SNAREs is as fast as the exocytosis of
LDCVs that have neuronal SNAREs (⬍1 s), suggesting that
the size of the vesicles contributes to the kinetics of exocytosis.
After the initial physical contact between the two membranes, a local lipidic connection between the proximal
(contacting) monolayers of the fusing membranes is formed
from a pointlike contact (FIGURE 4A) (331). This hemifusion intermediate is called the stalk intermediate and is currently thought to adequately describe the transitional stage
of membrane fusion (103, 280). Molecular dynamic simulations have predicted similar or lower energy barriers (377,
452, 598). The stalk intermediate can rapidly lead to either
opening of the fusion pore or to stable hemifusion diaphragms (402).
In contrast, many lines of evidence suggest that the fusion
pore is mostly, if not entirely, lipidic. First, the semi-stable
nature of the fusion pore has been demonstrated using pure
lipid bilayers (94). Second, there is considerable transfer of
lipid bilayers during flickering of the fusion pore (424),
before the opening of a 1.4-nm fusion pore (636), and before diffusion of vesicle proteins (640). Finally, exocytosis
depends critically on the composition of membrane lipids,
in the same manner as predicted by lipid structure (see sect.
IVF). This is difficult to reconcile with a purely proteinaceous model.
The hemifusion diaphragms can be fixed and their ultrastructures confirmed (375, 727). Such structures are detected in protein-free bilayers (100, 106, 381, 716),
SNARE-incorporated lipid bilayers (2), and biological
membranes (350, 413, 602, 701). Vesicles in the active zone
might already be hemifused (727). The fusion pore can
rapidly open from the stalk intermediate; however, the
hemifusion diaphragm may be a dead-end structure if no
tension is applied. In fact, a recent single vesicle imaging
study directly demonstrated that hemifusion of some vesicles does not proceed to full fusion (341, 701).
2. How does the fusion pore open?
E. Fusion Pores
Opening of the fusion pore that connects the vesicle cavity
with the extracellular space is a critical step in exocytosis,
and it is induced from the stalk intermediates (FIGURE 4B).
The early stage of fusion pore opening was identified in
ultrastructural studies (463) and later revealed to be semistable using membrane capacitance measurements, which
enable rapid and quantitative measurement of fusion pore
sizes between 0.2 and 2 nm (357, 425, 441). The fusion
pore normally rapidly dilates to induce irreversible full fusion for complete secretion and insertion of membrane lipids and proteins, whereas it shuts again and becomes semistable before undergoing full fusion.
1. The lipidic nature of the fusion pore
The semi-stable nature of the fusion pore was initially interpreted as suggesting that the fusion pore was made of gap
junction-like proteins spanning the two membranes (65,
160, 280). The V0 membrane sector of the V-ATPase is one
such candidate in yeast vacuolar fusion (489, 663) and in
1930
It is theoretically predicted that fusion pore opening and
growth consume energies of each ⬃40 kBT (119, 338),
which is larger than the formation of hemifusion intermediates (104). In addition, fusion pore opening and growth
depends on membrane tension (FIGURE 4F). Such tension
may be supplied by the elastic stress of the curved plasma
membrane (FIGURE 4D) (402) and/or by the elastic stress on
the membranes of curved vesicles (FIGURE 4F) (424). In
small vesicles, the membrane tension can be large, which
facilitates the opening of the fusion pore. This provides
another possible explanation as to how the size of vesicles
may affect the kinetics of exocytosis (FIGURE 1). All three
steps (FIGURE 4, A–C) of membrane fusion are facilitated by
increasing membrane curvature and tension.
If the energy supplied by the proteins exceeds that of membrane fusion, the fusion pore will rapidly dilate and reach
full fusion (636). If the two energies are similar, the fusion
pore can be closed after the initial opening, or can flicker
open and closed. These behaviors have been observed using
membrane capacitance measurements (171, 247, 291,
441), amperometry (5, 746), and fluorescence imaging
(321, 636). Fusion pore flickering has also been reported in
proteoliposomes (722). Such transient opening may be sufficient for neurotransmitter release, and a short recycling
pathway has been postulated (736). Short and narrow fusion pore opening, however, is not sufficient to release large
molecule hormones (37, 636) or supply membrane proteins. Thus transient opening of a fusion pore is considered
a fusion failure for hormonal secretion. The major population of vesicles in adrenal chromaffin cells (322) and pancreatic ␤ cells (380, 638) undergo full fusion, particularly in
intact preparations, whereas kiss-and-run exocytosis is
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2. Hemifusion intermediates
Drosophila synapses (250). It has, however, since been revealed that the role of V0 ATPase may be indirect (405,
695), possibly by helping SNAREs to open the fusion pore
(160). Another candidate was the oligomerized syntaxin
transmembrane domain (225, 227). It needs to be explained,
however, how a tightly oligomerized transmembrane domain
(TMD) can reversibly disassemble again to dilate the fusion
pore. Also, the protein spanning the other bilayer has not been
identified for the V0-ATPase or syntaxin TMD.
INITIAL SNARE CONFIGURATIONS FOR EXOCYTOSIS
more abundant in dissociated culture preparations in which
exocytosis was measured at the membrane facing the coverslip (659). Since full collapse of vesicles into the plasma
membrane occurs rapidly (1 s) (321), their exocytosis may
result in water secretion (658).
F. Lipid Composition
The kinetics of exocytosis are profoundly affected by lipid
composition, which exert either direct physical effects on
membrane dynamics or indirect effects on the membrane
proteins.
1. Direct effects on membrane structure
The plasma membrane is composed of various phospholipids and cholesterol, the concentrations of which reach molar levels and physically affect exocytosis. The composition
of membrane lipids varies in different cells, different subcellular domains, and in each leaflet of the bilayer. Synaptic
phospholipids are enriched in unsaturated fatty acids, such
as arachidonic acid, and comprise 10% phosophatidylcholine (PC), 20% phosphatidylserine (PS), 60% phosphatidylethanolamine (PE), 5% phosphatidylinositol (PI), and
15% sphyingomyelin (SM) (130). Each lipid can be characterized quantitatively by the curvature of the monolayer
that it spontaneously forms and can be classified into one of
three groups according to the shape of that monolayer
(749). Some membrane lipids are flat, such as PC and SM.
Most are cone-shaped, such as PE, cholesterol, and unsaturated free fatty acids, and generate a negative curvature of
the membrane monolayer (FIGURE 4C) (188). Finally, lysophosphatidylcholine (LPC), which is generated by phospholipase A2 (PLA2), and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], which is generated by phosphorylation
of PI, are inverse-cone-shaped and impart a positive curva-
One valuable example is the effect of snake presynaptic
phosphlipase A2 neurotoxins (SPANs), which cause exhaustive exocytosis and paralyze neuromuscular junctions
(525). This action appears to operate downstream of
SNARE complex formation (526). The toxin hydrolyzes PC
into LPC and fatty acids (408, 526). Inverse-cone-shaped
LPCs remain confined to the external leaflet of the presynaptic membrane, whereas fatty acids have a high rate of
transbilayer movement and will partition between the two
membrane leaflets. The cone-shaped fatty acids in the inner
leaflet of the plasma membrane facilitate the formation of
hemifusion intermediates, whereas the inverted cone-shaped
LPCs in the outer leaflet of the plasma membrane promote
opening of the fusion pore (FIGURE 4C). These effects of
SPANs can be fully mimicked by the extracellular application of LPC and free fatty acids (FFA) (525). This illustrates
that a change in lipid composition can have serious consequences in terms of synaptic exocytosis.
2. Lipid effects on membrane proteins
Many effects of membrane lipids on SNAREs and synaptotagmins have been reported (130, 695). Some of these are
listed below.
Phophoinositides such as PIP2 profoundly modulate membrane dynamics, including signal transduction, the membrane cytoskeleton, dynamin, and ion channels (143). Thus
PIP2 may affect exocytosis indirectly via the cytoskeleton
(50). PIP2 increases the speed of synaptotagmin binding to
the plasma membrane (28, 565), and binds to CAPS (374),
Mint (462), and rabphilin3 (114), which might result in
stabilization of the fusion pore (200). PIP2 also influences
both the spatial and functional properties of syntaxin at the
plasma membrane (430, 666).
Synaptic vesicles are enriched with cholesterol (30 – 40% of
membrane lipids), which is known to modify the TMD of
VAMP to form a parallel dimer rather than a scissor-like
conformation and, consequently, to facilitate lipid mixing
(649). Moreover, cholesterol serves as a strong stimulus for
hemifusion but acts as a mild stimulus for pore opening and
expansion during SNARE-mediated fusion (93). Cholesterol enhances spontaneous exocytosis, but suppresses
evoked exocytosis in synapses (687).
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The stability of the fusion pore depends on a variety of
factors other than membrane tension. The lifetime of the
fusion pore is affected by its cargo (416, 417). Full fusion is
favored by stronger stimulation in some preparations (517,
561, 736), while kiss-and-run events are favored by stronger stimulation in the other preparations (4). Exocytosis of
LDCVs containing a brain-derived neurotrophic factor
(BDNF) is full fusion in dendrites and kiss-and-run fusion in
axons (397). SNAREs, and many SNARE-interacting proteins, including Munc18a (173, 292), synaptotagmins (579,
742), Doc2 (178), SNAP25 (163, 436), VAMP2 (66, 316),
syntaxin (227), SNARE complex (226), and complexin (9,
17), affect fusion pore properties (see sect. V). This supports
the idea that SNARE-related proteins impinge on the opening of the fusion pore. It is not always easy for fusion proteins to supply a tension that is sufficient to readily overcome the energy required for fusion pore opening and dilation (589), and the lifetime of the fusion pore may
contribute to the time courses of secretion (415).
ture to the membrane (FIGURE 4C). The curvature of lipids
affects the two membrane leaflets in an opposing manner
(FIGURE 4C). For example, abundant PEs in the inner leaflet
facilitate negative curvature and fusion (101, 329), whereas
those in the outer leaflet prevent negative curvature and
fusion. Conversely, inverse-cone-shaped lipids in the inner
leaflet hinder negative curvature and fusion (411) and exocytosis (105), whereas those in the external leaflet promote
exocytosis.
KASAI ET AL.
t-SNAREs are clustered within cholesterol-rich domains in
the plasma membranes of secretory cells (92, 344). Depletion of cholesterol disperses this clustering and reduces exocytosis (344). Interestingly, depletion of cholesterol increases the rate and onset of sequential exocytosis in ␤ cells
due to diffusion of t-SNAREs (632).
PS regulates synaptotagmin-plasma membrane interactions, which facilitates opening of the fusion pore but prevents its dilation (741).
Unsaturated fatty acids, such as arachidonic acid, act on
syntaxin in its closed conformation and facilitate exocytosis
(127, 128, 351).
Membrane lipids have a direct effect on ion channels, which
are involved in triggering exocytosis. For example, PIP2
modulates K⫹ channels and arachidonic acids modulate
Ca2⫹ channels (180).
V. PROTEINS REGULATING
ULTRAFAST EXOCYTOSIS
In this section, we describe the molecular bases of ultrafast
exocytosis. Exocytosis is completely abolished by deletion
of SNAREs, NSF, Munc18, and Munc13, and partially impaired by deletion of complexin, CAPS, and snapin. The
same molecules are utilized for slower exocytosis, but they
may be used at different stages of the process, as described
in section VI. Specific CAZ proteins are required for the
organization of exocytosis in the active zone, but not for
exocytosis itself.
The individual SNARE motifs are largely unstructured in
solution, but when four SNARE motifs are mixed, they
assemble into a stable ␣-helical ternary complex. The crystal structure of the SNARE complex reveals a parallel fourhelix bundle with all COOH termini exposed on one side
(628) (FIGURE 5B). Four charged residues from individual
SNARE proteins form ionic interactions at the center of the
complex (called zero layer); one arginine (R) from VAMP2,
one glutamine (Qa) from syntaxin, and two glutamines (Qb
and Qc) from SNAP25. Therefore, SNAREs are classified
into four groups: R-, Qa-, Qb-, and Qc-SNAREs (165).
Assembly of the SNARE complex proceeds from the NH2terminal to the COOH-terminal transmembrane region in a
zipper-like fashion and induces membrane fusion. It is not
known how far the zippering is completed before ultrafast
exocytosis is triggered, but the resulting SNARE complex in
trans (trans-SNARE complex) is expected to bring vesicles
and the plasma membranes into close apposition for ultrafast exocytosis (283) (FIGURE 8). The SNARE complex is
remarkably stable; therefore, the energy derived from its
formation can be used to overcome energy barriers for
membrane fusion (229). This energy can be passed to the
TMDs to locally disrupt lipid membranes (613).
A. SNAREs
B. Munc18
SNAREs are the minimal machinery required for membrane
fusion (356, 533, 620, 690) and are the targets of the clostridial neurotoxins that cause tetanus and botulism (51, 52, 358,
564). These neurotoxins specifically cleave SNAREs and
completely block ultrafast exocytosis of synaptic vesicles
(89, 270, 282, 546). Loss of SNARE proteins results in a
complete loss of Ca2⫹-evoked ultrafast exocytosis in Drosophila (67, 139, 577), C. elegans (540), and mice (571,
686). Injection of the syntaxin SNARE motif causes strong
inhibition of ultrafast exocytosis in the squid giant synapse
(456). SNAREs are associated with the plasma membrane
voltage-gated Ca2⫹ channels responsible for the Ca2⫹ influx that triggers the exocytosis of synaptic vesicles (44,
541, 723).
Sec1/Munc18 (SM) proteins are essential for intracellular
membrane trafficking (233, 264, 455). Deletion of Munc18a,
a major neuronal SM protein in mammals, completely
blocks evoked and spontaneous synaptic transmission
(670). The binding partner of SM proteins is syntaxin1
(FIGURE 5, C AND D) (192, 193, 234, 491). There are three
distinct modes of Munc18a binding with syntaxin1 (82).
First, Munc18a binds to closed syntaxin1. The horseshoe-shaped cavity of Munc18 –1 binds to closed syntaxin1 (FIGURES 5D AND 8). Munc18a may clamp syntaxin1 to block nonspecific SNARE complex assembly during intracellular trafficking by shielding syntaxin1 from
intracellular SNARE partners (407). Munc18 may act as a
chaperone for syntaxin (193, 235, 490) because when
Munc18a is deleted, syntaxin expression is reduced by 70%
(78, 650, 691). Second, Munc18a binds with the NH2terminal sequence of syntaxin (59, 156, 484, 508). An open
SNAREs are characterized by a conserved heptad repeat of ⬃60
residues called a SNARE motif. VAMP2 contains one SNARE
1932
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Sphingosine facilitates SNARE complex assembly and activates synaptic vesicle exocytosis (129). This effect may be
due to the interaction between VAMP and sphingosine in
the vesicle membrane and the increased size of the RRP.
motif adjacent to the COOH-terminal TMD (FIGURE 5A). Syntaxin contains a large NH2-terminal region that consists of
three autonomously folded ␣-helical bundles (Habc domain), an unfolded linker, a SNARE motif (also called a H3
domain), and a COOH-terminal TMD (FIGURE 5, A AND B).
The Habc domain binds to the SNARE motif intramolecularly
to form a closed conformation, which is stabilized by Munc18
(155) (FIGURE 8). SNAP25 possesses two SNARE motifs
separated by a short linker containing palmitoylated cysteines, which are responsible for membrane anchoring
(538) (FIGURE 5A).
INITIAL SNARE CONFIGURATIONS FOR EXOCYTOSIS
A
Mouse syntaxin1A
N-pep
Qa SNARE (H3 domain)
Habc domain
TMR
Mouse SNAP25B
Qb SNARE
Qc SNARE
Mouse VAMP2
R SNARE
TMR
B
syntaxin 1A
(191-256)
SNAP25 (140-203)
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syntaxin 1A
(28-144)
C
N
SNAP25 (7-83)
VAMP2 (28-89)
C
Rat Munc18-1
Domain 1
Domain 2
Domain 3
Domain 2
D
Domain 2
(135-245)
(480-592)
Domain 1
(4-134)
Domain 3
(246-479)
N-pep
(2-15)
Habc domain
H3 domain
FIGURE 5. A: the primary structures of syntaxin1A, SNAP25b, and VAMP2. The number of residues is
indicated above each diagram. Cysteine residues predicted to be palmitoylated are labeled with a “C”. N-pep,
N-peptide; TMR, transmembrane region; SNARE, SNARE motif. B: the crystal structure of a SNARE complex
[Protein data bank (PDB) ID 1SFC, ID 1BR0] (170, 628). The connection between the Habc domain and the
H3 domain was drawn by hand. C: the primary structure of rat Munc18a. The N-pep, Habc domain, and H3
domain are from syntaxin 1A. D: the crystal structure of Munc18a and syntaxin1A (PDB ID 3C98) (82).
mutant of syntaxin (syntaxin LE mutant; L165A, E166A),
which disrupts the closed conformation, can interact with
Munc18a, whereas syntaxin lacking the NH2-terminal sequence cannot and fails to assemble a SNARE complex (523).
Thus the binding of Munc18a to the NH2-terminal region of
syntaxin is critical during SNARE assembly as it allows the
transition of syntaxin1 from the inactive to the active conformation (FIGURE 8). Finally, Munc18a directly interacts with
the ternary-SNARE complex (154, 318, 586) (FIGURE 8) to
facilitate the regulation of SNARE functions.
C. NSF and SNAP
A tight SNARE complex (cis-SNARE complex) remains in
the plasma membrane after exocytosis and needs to be disassembled and recycled to enable another round of fusion
events. Disassembly of the SNARE complex is mediated by
the NSF (Sec18p in yeast), and the interaction between the
SNARE complex and NSF requires SNAPs (Sec17p in
yeast). NSF is a soluble homo-hexameric ATPase, which
contains an NH2-terminal domain and two ATP-binding
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KASAI ET AL.
domains. Although overall sequence similarity between
SNARE subtypes is limited, all SNARE complexes are disassembled by ␣SNAP and NSF (12, 385, 475, 601). The
extreme stability of the SNARE complex means that NSF
has to use energy derived from ATP hydrolysis to disassemble it (FIGURE 8). Three SNAPs are recruited from the cytoplasm to every one SNARE complex in the membrane, and
the resulting SNAP/SNARE complex recruits a NSF hexamer, leading to the disassembly of the SNARE complex
(189, 230, 231, 256, 601).
D. Synaptotagmins and Other Ca2ⴙ Sensors
Synaptotagmin1 is enriched in synaptic vesicles and is the
major Ca2⫹ sensor for ultrafast exocytosis (22, 95, 144,
328, 364, 453, 621, 724), but not for asynchronous exocytosis (194). Presynaptic microinjection of synaptotagmin
peptides or anti-synaptotagmin antibodies inhibits synaptic
transmission in squid (53, 187, 419).
Asynchronous (194, 365, 449) and spontaneous (364, 704)
exocytoses are augmented in neurons lacking synaptotagmin1. This suggests that asynchronous and spontaneous
exocytoses are mediated by Ca2⫹ sensors other than synaptotagmin1. Attractive candidates for a slow Ca2⫹ sensor are
the double C2 domain (Doc2) proteins (467, 468). Doc2
proteins are soluble proteins, which are enriched in synapses. Doc2 has three isoforms, Doc2␣, Doc2␤, and Doc2␥
(186, 547). Like synaptotagmins, these proteins contain C2
domains but have relatively high affinity for Ca2⫹ (478).
Knocking out Doc2 in cultured hippocampal neurons results in normal evoked responses, but reduces spontaneous
release frequency by 50% (208). Doc2 may be a Ca2⫹ sensor for asynchronous exocytosis (718) and may also be
involved in spontaneous exocytosis (209, 478). Synaptotagmin1 acts as a Ca2⫹ sensor for spontaneous exocytosis,
although its deletion increases spontaneous exocytosis as it
also clamps a more sensitive Ca2⫹ sensor (704).
E. Complexin
Synaptotagmin1 contains two tandem C2 domains (C2A
and C2B), which are homologous to the regulatory C2 region of protein kinase C. The crystal structure of the two C2
domains shows that they have a very similar ␤-sandwich
structure (FIGURE 6B) (169, 191, 626, 627). Both domains
bind Ca2⫹ via loops located at the top of the ␤-sandwich.
Five acidic amino acid residues on the two loops form three
Ca2⫹-binding sites on the C2A domain and two Ca2⫹-binding sites on the C2B domain (169, 584). Both C2 domains
bind phospholipids in a Ca2⫹-dependent manner (488,
621). Synaptotagmin1 interacts with other synaptotagmins
(70, 186, 487, 702), including syntaxin1 (29, 396), SNAP25
(29, 567, 739), VAMP2 (184), a syntaxin1/SNAP25 binary
complex (29, 520, 660), and the SNARE complex (29, 57,
125, 739). It has been noted that bacterially expressed synaptotagmin1 shows nonspecific binding in vitro (662).
Complexins/synaphins are soluble proteins with a molecular mass of 19 –20 kDa, which are expressed in the brain
(277, 403, 637). Deletion of complexin results in a 70%
reduction of ultrafast exocytosis (254, 271, 295, 389, 400,
511, 710 –712, 717) and facilitates spontaneous exocytosis
(295, 400, 717). This is akin to the effects of synaptotagmin1 (194) (TABLE 2), except that it can be overcome by an
increase in external Ca2⫹ concentration. Complexin does
not bind Ca2⫹ but directly associates with synaptotagmin1
(643). Complexin binds with the assembled SNARE complex at the central helix with nanomolar affinity (644), and
this binding is necessary for complexin function (400, 644,
711). The crystal structure of the complexin/SNARE complex shows that the central helix of complexin binds to the
surfaces of VAMP2 and syntaxin in an anti-parallel fashion
(58, 97) (FIGURE 6C). The NH2-terminal complexin region
is more important for ultrafast exocytosis than the COOHterminal region (295, 711). The NH2 terminus of complexin also binds to the COOH terminus of the SNARE
complex to facilitate ultrafast exocytosis (400, 711) and
priming of synaptic vesicles (717).
Ca2⫹ binding to the C2B domain is more important for
transmitter release than Ca2⫹ binding to the C2A domain.
Disrupting Ca2⫹-binding sites on the C2B domain has a
dramatic effect on ultrafast exocytosis (382, 449), whereas
disruption of Ca2⫹-binding sites on the C2A domain has a
In addition to these mechanisms, complexin inhibits liposome or cell-cell fusion by clamping full zippering of the
trans-SNARE complex (195, 562, 639). In fact, the accessory helix (residues 26 to 47) of complexin (FIGURE 6D) can
interact with the membrane-proximal region of the SNAREs
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In mammals, SNAPs form a small protein family with three
isoforms, termed ␣, ␤, and ␥SNAP, in which ␣SNAP is the
most abundant and ubiquitous isoform (115). Neither deletion of ␤SNAP nor a reduction in ␣SNAP levels affects nerve
cell viability, differentiation, or function (80); however, constitutive deletion of ␣SNAP in mice leads to embryonic lethality (91, 259). ␣SNAP/NSF acts at the priming stage during
synaptic transmission (313, 363, 578). Mutations in NSF
block synaptic transmission due to accumulation of syntaxin
and SNARE complexes in synaptic vesicles (362, 363). Microinjection of NSF peptides that inhibit the ATPase activity of
NSF also inhibited synaptic transmission, and did so in a few
seconds before ultrafast exocytosis (336).
small effect (167, 615). Ca2⫹ bound to the C2B domain of
synaptotagmin1 promotes binding between two membranes due to its highly positive electrostatic potential. The
positively charged sites on the C2B domain directly interact
with the negatively charged phospholipid head groups, and
may result in local membrane bending for initiation or acceleration of membrane fusion (14, 387).
INITIAL SNARE CONFIGURATIONS FOR EXOCYTOSIS
A
C 2A
TMR
C2 B
Mouse synaptotagmin-1
(1-421)
C2A
C2 B
Mouse Doc2
(1-405)
B
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C2B domain
(273-408)
C2A domain
(142-264)
C
NT
Accessory
helix
Central
helix
CT
Mouse Complexin I
(1-134)
D
Cplx (26-72)
N
C
SN25N (10-81)
C
N
VAMP2 (28-92)
SN25C (139-204)
Syx H3 (191-256)
FIGURE 6. A: the primary structures of mouse synaptotagmin1 and Doc2. The number of residues is
indicated above each diagram. TMR, transmembrane region; MID, Munc13–1 interacting domain. B: the
crystal structure of the C2 domains [Protein data bank (PDB) ID 1BYN, ID 1K5W] (169, 584). The connection
between the C2A domain and the C2B domain was drawn by hand. The spheres represent Ca2⫹. C: the
primary structure of mouse complexin I. NT, NH2-terminal region; CT, COOH-terminal region. D: the crystal
structure of a SNARE complex and complexin (PDB ID 1KIL) (97).
to prevent their zippering (196) and may cross-link SNAREs
into a zig-zag array (294, 334, 354). The complexin clamp
may be competitively relieved by Ca2⫹-bound synaptotagmin1, thereby inducing fast Ca2⫹-evoked membrane fusion
(400, 639, 717).
F. Munc13
Munc13 proteins are large soluble proteins that are necessary for ultrafast exocytosis (16, 21, 518, 669). Munc13
proteins contain multiple domains including C2 domains
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KASAI ET AL.
(C2A, C2B, and C2C), a RIM-binding domain (RBD), a
calmodulin-binding domain (CBD), a C1 domain (C1), and
Munc homology domains (MHD1 and MHD2) (FIGURE 7).
The RBD may take part in the Munc13/RIM interaction to
recruit Munc13 to the active zone (11), and the CBD and
C2 domains may be involved in Ca2⫹-dependent short-term
plasticity (293). The C1 domain of Munc13 proteins binds
diacylglycerol and mediates the action of phorbol esters
during exocytosis (46, 390, 516).
C 2A
CBD
C1
The Munc13 family comprises brain-specific Munc13–1,
Munc13–2, and Munc13–3 and nonneuronal Munc13– 4.
Munc13–1 is utilized in phasic synapses in which the RRP is
full, and Munc13–2 is utilized in tonic synapses, in which the
RRP is gradually refilled after repetitive stimulation (531). A
splice variant of Munc13–2, ubMunc13–2, and Munc13– 4
are ubiquitously expressed (327) and may be necessary for
exocytosis of vesicles in nonneuronal cells (251, 513).
G. CAPS
CAPS was originally isolated from brain cytosol as a factor
required for Ca2⫹-triggered LDCV exocytosis (679). Deletion of CAPS reduces ultrafast exocytosis by 70% by reducing the RRP (288, 514), suggesting that CAPS is also in-
C2B
MHD1
C 2C
MHD2
Munc13-1 (1-1735)
MUN domain
RIM-binding domain
C2
PH
MHD1
CAPS1 (1-1355)
Snapin (1-136)
CC
N-terminal propeller
C-terminal propeller
R-SNARE
Tomosyn (1-1152)
WD40 blades
ZF
PDZ
C2 A
C2B
RIM1 (1-1462)
CC
CC
CC
CC
IWA
Cast1 (1-957)
ZF
ZF
CC
CC
CC
Bassoon (1-3942)
ZF
ZF
CC
CC
CC
PDZ
C2
C2
Piccolo (1-5165)
FIGURE 7. The primary structures of priming and CAZ proteins. The number of residues is indicated above
each diagram. From top to bottom: mouse Munc13–1, CAPS1, snapin, tomosyn, RIM1, CAST1, bassoon, and
piccolo. C2A, C2A domain; CBD, a calmodulin-binding domain; C1, C1 domain; MHD, Munc homology domain;
C2C, C2C domain; C2; C2 domain; PH, PH domain; CC, a predicted coiled-coil domain; SNARE, VAMP-like
SNARE motif; ZF, zinc-finger domain; PDZ, PDZ domain.
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The MUN domain (residues 859 –1531) partially rescues
release in Munc13 knockout mice (39), which is further
augmented by a domain encompassing the C2C domain
(383). Ultrafast exocytosis in a C. elegans UNC13 null mutant was partially rescued by overexpression of the open
syntaxin1 LE mutant (518), indicating that UNC13 facilitates the open configuration of syntaxin. Such action may
be mediated by the MUN domain, which can bind the NH2
terminus of syntaxin (47), and to heterodimeric and heterotrimeric SNARE complexes (211, 378, 694). The MUN
domain also interacts with the NH2-terminal domain of
Doc2 (466) and mediates Ca2⫹-dependent recruitment of
vesicles to the target membrane synergistically with diacylglycerol (207, 251).
INITIAL SNARE CONFIGURATIONS FOR EXOCYTOSIS
volved in ultrafast exocytosis. CAPS is an evolutionarily
conserved multidomain protein, for example, Caenorhabditis elegans has a single CAPS isoform (UNC-31), and
vertebrates have two closely related isoforms (CAPS1 and
CAPS2). The pleckstrin homology (PH) domain interacts
with PIP2 (206). The CAPS protein exhibits sequence
similarity to the MUN domain of Munc13–1 (47, 327)
(FIGURE 7), which is suggested to be a tethering factor (355).
CAPS binds independently to each of the three SNARE proteins (126) and markedly accelerates SNARE-dependent liposome fusion in vitro by promoting transSNARE complex assembly.
H. Snapin
I. Tomosyn
Tomosyn is a 130-kDa cytosolic protein identified as a
syntaxin-binding protein in rat brain cytosol (183). Tomosyn is a negative regulator of exocytosis because its
overexpression causes a significant reduction in exocytosis (27) and its deletion causes enhanced neurotransmitter release (158, 548) through increased synaptic vesicle
priming (198, 203, 204, 401). Tomosyn comprises two
domains: a large conserved NH2-terminal domain and a
COOH-terminal SNARE motif, which is homologous to
the VAMP2 SNARE motif (395) (FIGURE 7). The COOHterminal SNARE motif can form a ternary-SNARE complex with syntaxin and SNAP25 in the same manner as
the neuronal SNARE complex (240, 495). Thus tomosyn
can compete with VAMP2 for assembly with binarySNAREs. The crystal structure of Sro7p, a yeast homolog
of tomosyn, reveals that the NH2-terminal 14 WD40
repeats fold into two seven-bladed ␤-propeller domains
(241) (FIGURE 7), which directly bind to synaptotagmin1
J. Other CAZ Proteins
1. RIM
RIM (Rab3-interacting molecule) is a binding partner for
Rab3 (683), a highly abundant protein in synaptic vesicles. Deletion of RIM strongly reduces presynaptic Ca2⫹
channel density and the size of the RRP (228, 297). RIM
is a large soluble protein that contains various domains
including a zinc-finger, PDZ, and C2 (FIGURE 7). RIM1
also binds to several other proteins, including cAMP-GEFII, a
cAMP sensor (474), RIM-binding proteins (RIM-BPs) (684),
Munc13–1 (48), synaptotagmin, SNAP25, N-type Ca2⫹
channels (121, 297), and alpha-liprins (570) to form a
protein scaffold in the presynaptic nerve terminal. This suggests that RIM tethers N- and P/Q-type Ca2⫹ channels to
presynaptic active zones via a direct PDZ domain-mediated
interaction and acts during vesicle priming by interacting
with Munc13. RIM may also facilitate SNARE assembly by
reversing autoinhibitory homodimerization of Munc13
(141).
2. CAST
CAZ-associated structural protein (CAST) directly or indirectly binds CAZ proteins to form a large molecular complex at the active zone (249, 461) (FIGURE 7). CAST also
binds to RIM1 and, indirectly, to Munc13–1 to form a
ternary complex. The last three amino acids (I, W, and A)
are critical for binding to the PDZ domain of RIM1. Bassoon, another CAZ protein, is also associated with this
ternary complex. Thus there exists a network of proteinprotein interactions between CAZ proteins (461). Deletion
of CAST selectively affects the exocytosis of inhibitory synapses by reducing the size of the RRP (296).
3. Bassoon
Bassoon is a large protein containing two NH2-terminal
zinc-finger domains, several coiled-coil domains, and a
stretch of polyglutamines at its COOH terminus (646)
(FIGURE 7). The zinc-finger domains bind to the prenylated Rab acceptor protein-1 (PRA1) (79, 394), a molecule that interacts with rab3A and VAMP/synaptobrevin
II in vitro (166). Bassoon is localized at the CAZ of
excitatory and inhibitory synapses (519, 646) as well as
at the base of retinal ribbon synapses (61). In the retinas
of bassoon knockout mice, the presynaptic ribbons are
not anchored to the plasma membrane and are free-floating (145). The interaction between bassoon and RIBEYE,
a ribbon-specific protein, is required for the formation of
photoreceptor ribbon synapses (645).
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Snapin was identified as a SNAP25-binding protein and
is expressed in both neuronal and nonneuronal cells (85,
274) (FIGURE 7). Microinjection of a SNAP25-binding
site peptide derived from snapin inhibits synaptic transmission of cultured superior cervical ganglion neurons
(274). The SNAP25-binding site peptide of snapin also
blocks the interaction between synaptotagmin1 and the
SNARE complex. Deletion of snapin reduces excitatory
postsynaptic currents (EPSC) by 70% and slows EPSC
kinetics (477). Deletion of snapin also leads to a marked
reduction in the amount of synaptotagmin1/SNARE
complex in the mouse brain (477). These results suggest
that snapin stabilizes the coupling between synaptotagmin1 and the SNARE complex during Ca2⫹-triggered
ultrafast exocytosis at central nerve terminals. The synaptotagmin1/SNARE complex, which also contains snapin, does not include complexin (477), suggesting that
snapin and complexin exclusively regulate the synaptotagmin1/SNARE complex.
in a Ca2⫹-dependent manner and inhibit Ca2⫹-dependent exocytosis (713).
KASAI ET AL.
clustering without directly affecting neurotransmitter release (429).
4. Piccolo
Piccolo is a 500-kDa protein containing zinc-finger domains. It is structurally related to bassoon, but it also contains PDZ and C2 domains (90, 166) (FIGURE 7). The piccolo zinc-finger domains directly interact with PRA1 (79,
166, 394). Deletion of piccolo and bassoon impairs vesicle
Cis-SNARE
Thus 10 different proteins are required for ultrafast exocytosis. They may appear redundant if they were only utilized
for ultrafast exocytosis; in reality, however, they support
different forms of exocytosis, even in the active zone, and
Unitary-SNARE
Binary-SNARE
Trans-SNARE
Cis-SNARE
Vesicle
Munc18
SNAP25
SNAREs
Syntaxin
NSF
Munc13
ATP
ADP+Pi
Tomosyn
CAPS
RIM
PIP2
Complexin
Snapin
Synaptotagmins, Doc2
CAZ
Ca2+
Ca2+
Ca2+
Trans-SNARE configuration (T)
1 ms – 30 ms
Cytomatrix
Binary-SNARE configuration (B)
30 ms – 1 s
Ca2+, cAMP, phosphorylation, etc.
Unitary-SNARE configuration (U)
1 s – 100 s
20 kBT
Reaction coordinate
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VAMP
INITIAL SNARE CONFIGURATIONS FOR EXOCYTOSIS
pleiotropic exocytoses in a variety of other preparations
(see sect. II), as described in the next section.
VI. MOLECULAR MODELS OF EXOCYTOSIS
A. Different trans-SNARE Configurations
1. Energetics of SNARE-mediated ultrafast
exocytosis of synaptic vesicles
The entire process of ultrafast exocytosis is depicted by the
energy profile of a SNARE complex in FIGURE 8. Hydrolysis
of ATP generates 38 kBT of energy and induces disassembly
of the SNARE complex, whose zippering produces at least
⬃35 kBT of energy (353). This is less than the 43 kBT of
energy needed for hemifusion of pure lipid bilayers (333);
the formation and expansion of the fusion pore requires
even more energy (119). Thus hydrolysis of multiple ATPs
and SNAREs likely contributes to the exocytosis of single
vesicles.
The two forms of the ternary-SNARE complex, cis- and
trans-, are depicted in FIGURE 8. Disassembly of cis-SNARE
by NSF generates a “unitary-SNARE” state, in which each
SNARE is not bound to other SNAREs. Subsequent assembly of t-SNAREs (syntaxin and SNAP25) generates a “binary-SNARE” complex, and then a v-SNARE (VAMP2)
assembles with the binary complex to form a ternary transSNARE complex connecting the vesicle with the plasma
membrane. Priming processes represent a progression from
the cis-SNARE state to the trans-SNARE state, which fa-
If the trans-SNARE complex represents the initial state that
triggers exocytosis, it needs to be protected from further
SNARE assembly and fusion. Although synaptotagmin1
and complexin may have a clamping role (108, 195, 197,
341, 562, 639, 661, 717), the accumulation of vesicles at
the active zone is unaffected in mice lacking synaptotagmin1 or complexin, suggesting that these molecules alone
may not explain the arrest of fusion. It is also possible that
SNAREs (341, 685) and CAZ proteins may act structurally
as the fusion clamp. Also, the heptagonal CAZ grid encasing the vesicles at the active zone (152) may sterically hinder
membrane fusion. Such barriers can readily be overcome by
alteration of the lipid composition (525, 654) (see sect. IVF)
or osmolarity (see below).
Exocytosis from the trans-SNARE state can be triggered by
Ca2⫹ sensors, such as synaptotagmin1 and Doc2. Accounting for a fusion rate of 1 ms, the last energy barrier for the
trans-SNARE configuration for ultrafast exocytosis in the
presence of Ca2⫹ activation is 14 kBT (FIGURE 8, transSNARE configuration). Importantly, exocytosis in the active zone can be induced by a HTSS (80, 532), even in mice
lacking synaptotagamin1 (704) or complexin (511). This
suggests that the trans-SNARE complex takes a particular
organization for ultrafast exocytosis (217) such that HTSS
mimics the action of synaptotagmin1 and complexin. HTSS
may shrink the cytosol (532) and force the plasma membrane closer to the vesicles, facilitating the initial contact
between the two membranes. This may be the role of Ca2⫹dependent binding of synaptotagmin1 and Doc2 on the
membrane phospholipids (208, 269). The mechanical pressure may be utilized for synaptic functions, as is the case for
chromaffin cells (321). Importantly, Munc18a, Munc13, and
CAPS are necessary for HTSS-induced exocytosis (TABLE 2),
FIGURE 8. The energy profile of SNARE proteins in the cytosol, where they exist in three distinct states: trans-SNARE, binary-SNARE, and
unitary-SNARE. Top panel shows a schematic illustration of SNARE and Munc18 during exocytosis of a vesicle. Blue circles represent vesicles.
Munc18 catalyzes the transition from the unitary-SNARE to the cis-SNARE. The free energy profiles of a set of SNARE proteins and associated
molecules are predicted from the time constants of transitions between the states as described in the text and are depicted in the bottom three
panels. The gray trace in each panel depicts the default profile for that configuration. The black and red traces show the energy profiles in resting
and stimulated cells, respectively. In the trans-SNARE configuration in the active zone, Munc13 assists in the formation of the binary complex,
and RIM, CAPS, and PIP2 promote formation of the trans-SNARE complex. Fusion is prevented by CAZ, SNAREs, synaptotagmins, and
complexin, whereas it is triggered by Ca2⫹ binding with synaptotagmins and Doc2. In the binary-SNARE configuration, exocytosis is triggered
from the binary-SNARE state. Cytomatrix and tomosyn prohibit the formation of the trans-SNARE complex, whereas CAPS and PIP2 promote it
after stimulation. Ca2⫹ reduces the energy barrier for the trans-SNARE and fusion steps. In the unitary-SNARE configuration, exocytosis is
initiated from unassembled SNAREs. The motility of the vesicles in the cytomatrix and assembly of unitary-SNAREs are regulated. Triggers
promote vesicle motility and SNARE assembly, which is assisted by priming proteins, such as Munc13, CAPS, and snapin. Distinct initial SNARE
configurations can naturally account for the enormous kinetic diversity (1 ms to 100 s) of exocytosis.
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The neuronal SNAREs (syntaxin-1, SNAP25, and VAMP2)
assemble to form a ternary complex that bridges the vesicle
and the plasma membrane at some point during exocytosis.
To produce sufficient force for membrane fusion, the transSNARE assembly cannot be as tight as the cis-SNARE assembly. Indeed, the trans-SNARE complex can be reversibly disassembled (432, 707). The trans-SNARE state may
be the initial configuration required for ultrafast exocytosis
in the active zone and fast exocytosis in neuroendocrine
cells.
vors the membrane fusion reaction. From the length of time
taken to form the RRP (10 –20 s), we estimate that the
activation energy of the unitary and binary states is ⬃20
kBT (80, 532) (FIGURE 1). Likewise, the activation energy of
exocytosis is predicted to be 20 kBT for vesicles in the RRP
at resting levels of [Ca2⫹]i, which undergo exocytosis with a
time constant of ⬃10 s (373). These resting transitions in
SNARE states are depicted as gray lines in FIGURE 8.
KASAI ET AL.
indicating that they are involved in the formation of tight
SNARE assemblies for ultrafast exocytosis. Ultrafast exocytosis shows only one kinetic component when induced by
the uncaging of caged Ca2⫹ in the calyx of Held (678),
whereas it shows two kinetic components when induced by
depolarization, reflecting the heterogeneous distribution of
synaptic vesicles relative to Ca2⫹ channels. Such heterogeneous distribution of vesicles, however, may not exist in
small boutons or inhibitory synapses (543).
terminal mutation of SNAP25 (608), and deletion of synaptotagmin1 (674) and was slowed by a R233Q mutation
in synaptotagmin1 (605), replacement of SNAP25a with
SNAP25b (607), and replacement of the SNAP25b COOHterminal linker with SNAP23 (436). The amplitude of the
fast component, but not the slow component, of the capacitance increases was regulated by PKA-mediated phosphorylation of SNAP25 (437). These results suggest the existence of two distinct trans-SNARE states in Ca2⫹-primed
chromaffin cells.
2. Different trans-SNARE states
By conforming to slow kinetics, exocytosis in chromaffin
cells does not require synaptotagmin1 (674) and Munc13–1
(TABLE 2). The time constant for the capacitance increases
was limited to 30 ms and did not decrease upon overexpression of CAPS and Munc13–1 (369). To date, HTSS has not
been reported to induce exocytosis in endocrine cells (54,
427). Replacement of synaptotagmin1 with synaptotagmin2 slows exocytosis (434), whereas it hastens the ultrafast exocytosis in synapses (703). Thus the trans-SNARE
states in Ca2⫹-primed chromaffin cells have molecular
bases distinct from those in ultrafast exocytosis.
Two distinct trans-SNARE states have been distinguished
in Ca2⫹-primed chromaffin cells (706), in which the increases in the membrane capacitance induced by large increases in [Ca2⫹]i show two kinetic components, with time
constants of 30 and 300 ms. Rigorous kinetic analyses indicated that the two components could be ascribed to two
distinct trans-SNARE complexes: tight and loose. These
states were interconvertible in a Ca2⫹-independent manner,
with a time constant of 4 s (675). The fast trans-SNARE
state was selectively eliminated by BoNTA (706), COOH-
1940
The amplitude of both components of the capacitance increase was reduced by TeNT, BoNTC, -D, and -E (706);
depletion of PIP2 (421); complexin (86); CAPS (370); snapin (642); NH2-terminal mutation of SNAP25 (608); and
expression of tomosyn (720, 721) and was augmented by
PKC-dependent phosphorylation of SNAP25 (435). These
delineate the common priming steps for the two transSNARE states, which bear some similarity with those of
ultrafast exocytosis.
Importantly, the kinetics of exocytosis varies according to
the subtype of Ca2⫹ sensor in the synapses and secretory
cells. In synapses, synaptic currents are fastest with synaptotagmin2, and slower with synaptotagmin1 and synaptotagmin9 (703). Importantly, although synaptotagmin3,
-5, -6, -, and -10 do not rescue ultrafast exocytosis, they can
evoke slow or tonic exocytosis upon repetitive stimulation
(703). In agreement with the results in synapses, deletion of
synaptotagmin1 and -7 impair fast and slow capacitance
increases, respectively, in Ca2⫹-primed chromaffin cells
(573). The difference between synaptotagmin1 and synaptotagmin7 arises, in part, from subtle sequence changes
(709). Cochlear hair cells utilize otofelin together with specific SNAREs as a fast Ca2⫹ sensor (289, 454, 535). Asynchronous exocytosis may be mediated by Doc2 and is suppressed by synaptotagmin1 and complexin (TABLE 1) (717).
Interestingly, the kinetics of exocytosis are markedly altered
by the linkers between the SNARE motif and the TMDs of
VAMP (66, 136, 217, 316, 406) and syntaxin (668), and by
the linker between the two SNARE motives in SNAP25 (69,
436). Thus the linker regions outside the SNARE motif play
an important role in the kinetics of membrane fusion mediated by SNAREs, in line with the idea that SNARE assembly can be induced up to the transmembrane regions (613).
3. How many SNARE complexes are required
for exocytosis?
Proteoliposome studies show that a single trans-SNARE
complex is capable of inducing exocytosis (56, 665),
whereas other studies report that 3 (148) or between 5 and
11 (299) are required. Smaller numbers might reduce the
fusion rate (299). In living cells, at least two SNARE complexes are involved in synaptic transmission (596), and
three to five SNARE complexes are involved in exocytosis in
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Compared with ultrafast exocytosis in the active zone, exocytosis in adrenal chromaffin cells is slower. Membrane
capacitance measurements indicate that the fastest time
constant in Ca2⫹-primed adrenal chromaffin cells is 30 ms
(673, 675). Nevertheless, it was suggested that SNAREs in
adrenal chromaffin cells are in the trans-state because rapid
capacitance increases were resistant to botulinum neurotoxins (BoNTs) (706) and an antibody against free SNARE
(707), neither of which act on assembled SNAREs. The slow
kinetics of LDCV exocytosis from Ca2⫹-primed chromaffin
cells may be partly due to the large size of the vesicles; 500 nm
compared with 40 nm in the synapse (FIGURE 3, D AND E).
Moreover, CAZ is absent in chromaffin cells, and exocytotic vesicles contact with an ordinary submembraneous
cytomatrix. The distance between chromaffin granules and
the plasma membrane in the active zone may be functionally larger than for synaptic vesicles. As a result, the transSNARE complex is looser than that formed for ultrafast
exocytosis. Ca2⫹ priming may help to remove part of the
cytomatrix to facilitate trans-SNARE complex formation
and speed up exocytosis; otherwise, exocytosis is slow in
chromaffin cells, with a time constant of ⬃1 s.
INITIAL SNARE CONFIGURATIONS FOR EXOCYTOSIS
PC12 cells (227, 266) and Ca2⫹-primed chromaffin cells
(423). Oligomers of SNAREs purified from the brain comprise three to five SNARE complexes (110, 521). The number of SNAREs is not a simple determinant of the kinetics of
exocytosis, given that two or three SNAREs might be
enough for fast exocytosis. Consistently, the amplitude, but
not kinetics, of exocytosis are affected by BoNTC and
TeNT (546). The number of SNAREs may affect the stability of the fusion pore (589). It is also possible that release
probability is regulated by the number of VAMP2 molecules, given the fact that each synaptic vesicle has 70 of
them (638).
4. SNARE oligomerization
There are several potential mechanisms for the oligomerization of SNAREs. Oligomerization of SNAREs may be induced by TMD (227, 255, 335, 342), SNARE motifs (409,
593), or complexin (644). Recent liposome experiments
demonstrate that SNAREs form zig zag-shaped oligomers
with the help of complexin (294, 334, 354). It remains to be
seen whether such oligomers and large changes in supramolecular structures can be utilized for ultrafast exocytosis.
Alternatively, the WD40 domains of tomosyn induce oligomerization of SNAREs and inhibit synaptic transmission
(548). Deletion of tomosyn reduces SNARE oligomerization by 30%, and oligomerization of SNAREs may play an
inhibitory role. Another possibility is that Ca2⫹-induced
oligomerization of synaptotagmin may induce synaptic vesicle fusion via assembly and oligomerization of SNARE
complexes (96, 361), either by direct binding (111, 702) or
by indirect binding mediated by complexin (643).
Unlike SNAP25 and SNAP23 in animal cells, Qb-SNARE
and Qc-SNARE are separate molecules in plants (359). It is
possible that the two Q-SNAREs are linked in animal cells
to promote the efficiency of membrane fusion events for
more rapid activity of animal cells relative to plant cells.
The linker region may just bring the two SNARE motives of
SNAP25 closer to allow assembly, but it may also have
another role. The oligomerization of SNAREs can be
achieved by cross-linking (or domain swapping) of SNARE
complexes through two distinct SNARE domains (Qb and
Qc) in two SNAP25 molecules. The existence of such a
biochemical state was postulated in early studies (164, 340,
Taken together, many studies show that there are many
diverse trans-SNARE configurations, even for neuronal
SNAREs (syntaxin1, SNAP25, and VAMP2), in terms of
1) the tightness of trans-SNARE complex, 2) the kinetics of
poststimulus SNARE assembly, 3) the number of transSNARE complexes for each exocytosis event, 4) oligomerization, and 5) domain swapping, all of which depend on
priming proteins and sensors. Also, the initial conformation
of lipid bilayers (FIGURE 4) may be different depending on
the trans-SNARE configurations.
B. Non-trans-SNARE Configurations
In the previous section we described the molecular mechanisms of exocytosis induced within 100 ms after stimulation, where prior formation of a trans-SNARE complex
seems a reasonable assumption. If exocytosis is slower,
however, such assumptions are no longer valid, and there
are many lines of evidence for other initial SNARE configurations. The trans-SNARE configuration, however, has
been adopted as the sole model of regulated exocytosis in
most studies, and no other possibilities have been systematically explored. Therefore, we will first summarize the
reasons for considering non-trans-SNARE configurations and then provide some examples and mechanisms
in detail.
1. Rationale for non-trans-SNARE configurations
1) There is no direct proof or quantitative demonstration
of prestimulus trans-SNARE complex formation for exocytosis, even in the active zone. The key structural prob-
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Oligomerization of SNARE complexes is found in many
preparations. Star-shaped oligomerized SNAREs have been
purified from the brain (521). They have also been formed
in an aqueous solution (164). Ring-shaped SNARE complex oligomers have been detected by atomic force microscopy (AFM) and electron microscopy when full-length
SNARE was expressed in liposomes (109). Such oligomers
could be disassembled by NSF (284). These SNARE complexes were all cis-SNARE. Also, electron paramagnetic
resonance (EPR) analysis demonstrated the formation of
SNARE oligomers preceding hemifusion (375).
497, 644) but has met with several criticisms. First, SNARE
coiled-coil structures can be formed without a SNAP25
linker region (627). Second, a single molecular Förster resonance energy transfer (FRET) study showed that the occurrence of domain swapping is infrequent at very low
(⬍100 pM) molecular concentrations (694). Third, in chromaffin cells, SNAP25 with two mutations in distinct SNARE
motifs was effective on the capacitance increases only when
both mutations were in the same SNAP25 molecule (423).
Finally, isolated SNARE domains of SNAP25 can rescue
exocytosis of PC12 cells (98, 558) and proteoliposomes
(480). These arguments indicate that domain swapping is
not necessary to form the SNARE complex, may need
higher concentrations of SNAP25 (340), and may be specifically utilized for ultrafast exocytosis. If the SNAP25 domains are swapped, the SNAREs are placed in close proximity to the fusion pore, which may boost efficient formation of hemifusion and fusion pore opening. Domain
swapping may be assisted by the fact that the Qb-SNARE
motif of SNAP25 first binds with syntaxin, leaving the QcSNARE motif free (524, 694). The domain swapping model
must, therefore, be directly examined in the active zone in
the future.
KASAI ET AL.
lem is related to how tight SNAREs are assembled so that
the remaining SNARE assembly can induce ultrafast fusion.
3) TIRF and confocal experiments have shown that vesicles
are nondocked to the plasma membrane before stimulation
in synaptic vesicles in ribbon-type synapses in bipolar terminals (257, 418, 729) and LDCVs in secretory cells (7,
310, 317, 555, 590, 671) (see sect. IVA), indicating that
SNAREs are not assembled. In secretory cells, small vesicles
are nondocked but undergo fast Ca2⫹-dependent exocytosis (see sect.IID).
4) Some vesicles in secretory cells, such as PC12 and ␤ cells,
show tight morphological docking, but the docking does
not affect (126, 655, 719), or even slow, exocytosis (310)
(sect. IIIA). This suggests that SNAREs are not preassembled in the docked vesicles.
5) Sequential exocytosis is found in a variety of secretory
cells and neurons (sect. IIIB) and is best explained by the
assembly of v-SNARE with t-SNAREs, which are supplied
from the plasma membrane into the fused vesicle membrane
(see sect. VID).
6) Stimulus-induced assembly of SNAREs during exocytosis has been demonstrated both biochemically (43, 603,
698) and using FRET imaging (633) (see sect. VID).
7) The tonic (80) and spontaneous exocytosis (268) of synaptic vesicles likely involves nonprimed and nondocked vesicles (see sect. VIB2).
8) SNAREs for cAMP-dependent slow exocytosis have been
identified in many cells (630) (TABLE 1; sect. IIIE), in which
vesicles are apparently nondocked to the membrane prior to
stimulation.
9) Constitutive exocytosis also utilizes SNAREs (485) (TABLE 1),
indicating that nondocked vesicles can undergo fusion
without any stimulation, and that any step in between maturation, transport, tethering, docking, and fusion can be
used to control exocytosis (see sect. IIIF).
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2. Tonic exocytosis of synaptic vesicles
Tonic exocytosis is induced by repetitive action potentials
in the active zone and is not time-locked to each action
potential as is the case for slow exocytosis in endocrine
cells. The rate of tonic release is determined by the availability of unitary-SNAREs through ␣SNAP and ␤SNAP
(80). Thus, during tonic exocytosis, vesicles reside within
the recycling pool (557). This involves the disassembly of
cis-SNAREs, transport of vesicles to the active zone, and
assembly of trans-SNARE complexes. In fact, nondocked
vesicles undergo exocytosis during tonic stimulation underneath the ribbon in bipolar terminals (418, 730).
3. Spontaneous exocytosis of synaptic vesicles
Spontaneous exocytosis involves vesicles in the recycling
and reserve pools, which are characterized by VAMP7
(268) and vti1a as a Q-SNARE (504). Interestingly, spontaneous exocytosis shows markedly different molecular
characteristics to ultrafast exocytosis. Spontaneous exocytosis is resistant to knockout of VAMP2, SNAP25, RIM,
and CAPS and is enhanced by knockout of synaptotamin1
(TABLE 2). It is also inhibited by deletion of Doc2 (208,
478). Mutation of the linker region in VAMP2 by insertion
of 12 or 24 residues between the SNARE motif and transmembrane region affected ultrafast exocytosis, but not
spontaneous exocytosis (136). Cholesterol has opposite effects on ultrafast and spontaneous exocytosis (687). These
results indicate that molecular events downstream of Ca2⫹
stimulation of vesicles in the reserve pool are very different
from those involved in Ca2⫹ stimulation of vesicles in the
RRP. The finding that the actin depolymerizing agent latrunculin A increased spontaneous exocytosis but did not
affect ultrafast exocytosis (268, 426) suggests that the vesicles responsible for spontaneous exocytosis are nondocked
to the plasma membrane (see sect. IVA4). Thus spontaneous exocytosis does not seem to selectively utilize transSNARE configurations, unlike ultrafast exocytosis.
4. Slow exocytosis in secretory cells
Although relatively fast exocytosis can be induced in Ca2⫹primed chromaffin cells, sustained exocytoses follow a fast
exocytotic burst (673, 706). This is thought to be mediated
by vesicles with unassembled SNAREs. Ca2⫹-induced
SNARE assembly before exocytosis has been directly demonstrated in ␤ cells (633).
Ca2⫹-dependent exocytosis is slower in secretory cells with
nonneuronal SNAREs and synaptotagmins than in those
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2) There is huge kinetic diversity in exocytosis (1 ms to 1,000 s),
even though the same set of SNARE proteins is used by neurons
and neuroendocrine preparations (FIGURE 1, TABLE 1). Such diversity can be naturally explained if initial configurations
other than trans-SNARE states are considered (FIGURE 8).
Otherwise, vesicles for slow exocytosis must be bound to
the membrane by trans-SNARE complexes for an unnecessarily long time before exocytosis, which renders mobilization of vesicles stored in the cytosol extremely inefficient.
Hence, trans-SNARE configurations may be physiologically relevant only for ultrafast or fast exocytosis with time
constants shorter than 100 ms.
10) Proteoliposome experiments show that SNAREs are
sufficient for slow exocytosis (689) and that the ratelimiting step for fusion can be a Ca2⫹-dependent docking
step (599) (see sect. VIC). Fast exocytosis can be induced
only when docked vesicles are selected as the initial state
(341).
INITIAL SNARE CONFIGURATIONS FOR EXOCYTOSIS
In epithelial and hematopoietic cells, exocytosis is not triggered by Ca2⫹, and vesicles are rarely attached to the
plasma membrane. Exocytosis from these cells is mainly
mediated by nonneuronal SNAREs (TABLE 1). For instance,
exocytotic insertion of aquaporin in kidney epithelial cells is
triggered by cAMP and mediated by syntaxin4, SNAP23,
and VAMP2 or -7 (TI-VAMP) (276). Gastric parietal cells
utilize cAMP to trigger H⫹-ATPase insertion, which is mediated by SNAP25, syntaxin3, and VAMP2 (368). Mast
cells utilize syntaxin4, SNAP23 (550), VAMP7 and -8 (endobrevin) (551), and synaptotagmin2 (412), and phosphorylation of SNAP23 plays an important role in triggering
exocytosis induced by IgE (629). The immunological synapses of T-cells utilize syntaxin7 and VAMP7 (260, 482).
Exocytosis of vesicles containing GLUT4 in adipocytes and
skeletal muscles is mediated by syntaxin4 and SNAP23, and
triggered by tyrosine phosphorylation of Munc18c by insulin receptors (15, 285), and the docking step is regulated by
PI3-kinase (30). A syntaxin3/SNAP23/VAMP8/Munc18b
complex is utilized for regulated exocytosis in exocrine cells
and platelets and for constitutive exocytosis. These exocytoses are not mediated by the trans-SNARE configuration:
exocytosis is slow, vesicles are not docked before stimulation, and exocytosis is often potentiated by removal of subcortical actin layers (see sect. IVA).
4. Different non-trans-SNARE configurations
If exocytosis is initiated by non-trans-SNARE configurations, there are at least three possible initial states for exocytosis: binary-SNARE, unitary-SNARE, and cis-SNARE
(FIGURE 8). It is not easy to identify which of these states is
used for the initial configuration, and more than one configuration may be utilized for a particular type of exocytosis. To support their existence, however, we will scrutinize
possible mechanisms involving each of the three non-transSNARE configurations in the next section. Slow exocytosis provides us with valuable examples to aid our understanding of the processes of SNARE assembly because it
proceeds gradually after it is triggered, unlike the ultrafast exocytosis.
C. Binary-SNARE Configurations
The binary t-SNARE acceptor complex of the two plasma
membrane SNAREs, syntaxin and SNAP25, has been proposed as an intermediate stage of SNARE assembly (520,
522, 524). Further assembly of VAMP2 to form a transSNARE complex is the next stage of the fusion reaction
(FIGURE 8). We will describe existing lines of evidence that
the binary-SNARE state can act as the initial configuration
for exocytosis in many secretory phenomena. Such binarySNARE configurations are particularly suited for mobilizing a large pool of vesicles, which are stored in the relatively
superficial layers of the cytosol.
1. Biochemistry of the binary configuration
Binary-SNARE states are formed when the Munc18a/syntaxin1 complex binds to SNAP25, a process catalyzed by
Munc13 (378) (FIGURE 8). A binary-SNARE complex can
efficiently induce trans-SNARE complex formation upon
increases in [Ca2⫹]i because synaptotagmins bind with the
binary-SNARE complex (29, 520, 660), as well as with
membrane phospholipids (488, 621), in a Ca2⫹-dependent
manner. Such binding has a micromolar requirement for
[Ca2⫹]i, in accordance with the [Ca2⫹]i requirement of exocytosis in synapses and endocrine (635, 674) and exocrine
cells (443). The binding of synaptotagmins with the plasma
membrane supplies VAMPs within the vesicles to the binary
complex in the plasma membrane. VAMP2 can rapidly assemble with the binary complex (496).
Proteoliposome experiments indicate that Ca2⫹ can stimulate docking of vesicles (722) by synaptotagmin (113, 347,
599) and can induce full fusion in 30 ms (682) (FIGURE 3C).
A binary-SNARE configuration can act as the initial configuration for Ca2⫹-dependent fusion events in proteolipose,
because t-SNAREs frequently form the binary complex in
proteoliposomes (496), and because binary-SNARE states
are stabilized by many priming proteins, including complexin, synaptotagmin, Munc13, and Munc18 (694). A
new liposome assay system using a microfluidic device and
low ionic strength solutions revealed that synaptotagmin
can act as a distance regulator to control the assembly of the
binary t-SNARE complex and VAMP in a Ca2⫹-dependent
manner (667). This suggests that binary configurations account for exocytosis with time constants between 30 ms and
1 s (FIGURE 1, TABLE 1).
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with neuronal SNAREs (283). TABLE 1 lists the SNAREs
and synaptotagmins that have been identified in immunohistochemical or knockout experiments. Of particular note,
islet ␤ cells utilize neuronal SNAREs, but not synaptotagmins 1, 2, 7, or 9 (214, 215). Insulin vesicles are nondocked
before exocytosis (310, 590) and therefore utilize nontrans-SNARE configurations (633). Nondocked vesicle exocytosis is generally found in other cells that utilize nonneuronal SNAREs and synaptotagmins. For example, postsynaptic dendritic exocytosis utilizes syntaxin4, SNAP23, and
synaptotagmin4 (315, 623). Synaptotagmin7 and -10 are
utilized for insulin-like growth factor and glucagon secretion in the brain (88) and ␣ cells in the islets (216), respectively. Exocrine acinar cells utilize syntaxin2, SNAP23, and
VAMP2 for primary exocytosis of zymogen granule exocytosis (43). Glial exocytosis may be mediated by lysosomes
(352, 740) utilizing syntaxin1, SNAP23, and VAMP3 (cellubrevin) (576) and is regulated by synatotgamin4 (735).
Thus nonneuronal SNAREs and synaptotagmins may be
optimized for Ca2⫹-dependent non-trans-SNARE configurations.
KASAI ET AL.
2. Possible examples of the binary configuration in
synapses and endocrine cells
D. Unitary-SNARE Configurations
Unitary-, or free-SNAREs, are a highly reactive form of
SNARE complex (FIGURE 8) and are an obvious candidate
for the initial SNARE state. We call them unitary-SNAREs
to avoid the possible misunderstanding that such SNAREs
are completely free and not bound to other molecules, such
as Munc18. Such a configuration is considered to be the
default in plant biology (359). The unitary-SNARE configuration has a great advantage in inducing slow and compound exocytosis for intensive mobilization of vesicles from
deep within the cytosol.
3. How is the binary-SNARE configuration clamped?
1. Biochemistry of unitary configurations
Several potential mechanisms are considered, which may
prevent the binary complex from formation of transSNARE complexes in the resting state (FIGURE 8).
Unassembled SNAREs can be generated from ternary- and
binary-SNARE complexes (243), oligomerized SNAREs
(256, 456, 472, 483), and syntaxin/SNAP25/tomosin hetrotetramers (240, 548). Thus unitary-SNAREs are dynamically maintained and may be generated shortly before exocytosis is triggered (336). Unassembled syntaxin can be
chaperoned by Munc18a, which bundle SNARE motifs and
Habc for stabilization (715).
1) Vesicles cannot form trans-SNARE complexes if the plasma
membrane region for exocytosis is already occupied with vesicles in the RRP. This is the case for nondocked vesicles in the
active zone and in Ca2⫹-primed chromaffin cells (673, 706). In
many other preparations, however, vesicles do not seem to
form trans-SNARE complexes, even though the membrane
region is available (see sects. III and IV).
2) A major barrier to the binary complex forming the transSNARE complex in the resting state may simply be due to
the low incidence of the collision of vesicles with the binary
complex in the plasma membrane. SNARE-mediated fusion
events occur slowly, even in proteoliposomes in which diffusion of liposomes is unlimited (496, 667, 689), unless the
vesicles are already sited close to each other (341, 682). In
the cytosol of living cells, vesicle movement is retarded by
the cytoskeleton and organelles, and the approach of vesicles to the plasma membrane is restricted by the submembraneous cytomatrix. Even though the vesicles reach the
plasma membrane, the v-SNARE may not collide with the
binary-SNARE complexes (138, 239) as their residence
time is short. In tonic synapses, Munc13–2 allows the formation of a trans-SNARE complex only after repetitive
stimulation (531).
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The vesicle membrane protein, cysteine-string protein (CSP),
chaperones SNAP25, helps proper folding of SNAP25 for
SNARE formation, and prevents ubiquitylation and proteasomal degradation of SNAP25 (585). In fact, knockout of
CSP␣ in neurons reduces SNAP25 levels and induces synaptic degeneration (168). In ␤ cells, CSP is expressed in
insulin granules, and overexpression suppresses insulin
exocytosis (73). Thus unitary-SNAREs are bound to their
respective chaperones and are protected from SNARE
assembly.
In spite of the clamping role of Munc18a, proteoliposome
experiments demonstrate that Munc18a facilitates the formation of the binary-SNARE and trans-SNARE complexes
(378, 528, 586). Munc13 induces dissociation of Munc18
from closed syntaxin and the formation of binary- and ternary-SNARE complexes (378, 518). Unsaturated fatty acids act on syntaxin in its closed conformation and facilitate
exocytosis (see sect. IVF2). If vesicles contact with the plasma
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TIRF images of endocrine cells show that clusters of syntaxins and SNAP25 often overlap to form binary complexes
(157), but not ternary complexes (524). In ␤ cells, a FRET
probe of SNAP25 identified a high FRET region, in which
binary-SNAREs were formed, and exocytosis was faster
than in a low FRET region. Crash fusion events in ␤ cells
occur within 40 ms after docking of vesicles to the plasma
membrane (7, 310, 590, 651), suggesting a binary configuration based on rapid time courses (FIGURE 8). The rapid
(10 –100 ms) exocytosis of nondocked LDCVs in synapses
(74) also fits with the idea of a binary configuration. Graded
ribbon-type synapses in bipolar terminals also display exocytosis of nondocked vesicles during tonic stimulation (418,
730). Such sustained vesicular release at high rates is also
observed in mossy fiber terminals in the cerebellar granule
layer (557), and may be generally applicable to tonic exocytosis in synapses (80). Binary-SNAREs might act as release sites, the formation of which is facilitated by endocytosis (263). In ␤ cells and bipolar terminals, it has been
proposed that exocytosis is limited by the diffusion of vesicles (239, 257). Thus the binary-SNARE configuration appears to be utilized for various forms of Ca2⫹-dependent
exocytosis (TABLE 1).
3) The binary state is specifically stabilized by soluble
VAMP-like proteins, such as tomosyn (183) and amisyn
(559), which can bind to binary complexes and stabilize
them. Their binding can be competitively removed by
VAMP, which leads to exocytosis. Thus these proteins control the speed of exocytosis in the binary mode to avoid
exhaustion of vesicles. In fact, overexpression of tomosyn
causes a significant reduction in exocytosis in PC12 cells
(120, 240), chromaffin cells (721), adipocytes (696), ␤ cells
(107, 738), and neurons (27). Likewise, VAMPs with the
longin domain (LD), such as VAMP7, will be less reactive
with the binary acceptor complex (672).
INITIAL SNARE CONFIGURATIONS FOR EXOCYTOSIS
membrane after stimulation, VAMPs and synaptotagmins introduce intimate contacts with syntaxins and SNAP25, and
facilitate SNARE assembly (108, 748) (FIGURE 8).
2. Possible examples of unitary configurations in
endocrine cells and synapses
Ca2⫹-dependent assembly of t-SNAREs was demonstrated
in ␤ cells in the islets using a FRET probe. There are two
regions in the plasma membrane of ␤ cells in which
SNAREs are unitary or binary (633). Exocytosis from the
unitary-SNARE region is slow and is associated with an
increase in FRET levels, indicating that SNAP25 is assembled with syntaxin, and that the unitary-SNARE configuration is utilized for the slow phase of insulin exocytosis.
TIRF imaging of endocrine cells often shows that vesicles
are docked for a short time after stimulation (⬍3 s), but
before fusion events in ␤ cells (310, 590) and PC12 cells
(555), consistent with FRET results indicating that fusion is
induced 3 s after SNARE assembly in the unitary-SNARE
configuration (633).
Sequential exocytosis is most simply explained by the unitary-SNARE configuration. The spread of sequential exocytosis into the cytosol may be best explained by the diffusion of SNAP23 or SNAP25, as syntaxins are integral membrane proteins and may have more difficultly passing
through the narrow fusion pores. In fact, redistribution of
SNAP25 and SNAP23 has been demonstrated in endocrine
cells (322, 632) and mast cells (213). Syntaxin may also be
a diffusive factor, particularly when the fusion pore is
widely open, as has been reported in exocrine cells (493).
Exocrine acinar cells express syntaxin3 and VAMP8 for
granule-granule fusion during sequential exocytosis (43,
224), and the unitary-SNARE configuration may be utilized
during multigranular exocytosis. In neutrophils, SNAP23 is
expressed in secretory vesicles (592), perhaps providing a
reason for the abundance of multigranular exocytosis
(218), in which vesicle-vesicle fusion precedes exocytosis.
It is proposed that the sustained phase of exocytosis observed
after an increase in [Ca2⫹]i is mediated by unitary-SNAREs in
chromaffin cells (706) and in ␤ cells (633). Sustained exocytosis is considered to represent assembly of SNAREs from the
unitary states, as it is blocked by BoNTs and antibodies (705–
707). The sustained phase is augmented by Munc13–1 (18,
cAMP-dependent exocytosis is likely mediated by unitarySNARE configurations (TABLE 1). This is because exocytosis must spontaneously proceed from unitary-SNAREs,
with constitutive exocytosis mediated by syntaxin4/SNAP23/
VAMP8/Munc18b (485) and slow exocytosis induced by
Ca2⫹, cAMP, and kinases. The long time constants of cAMPdependent exocytosis (between 1 and 1,000 s; FIGURE 1 AND
TABLE 1) can be explained by the time required for the
unitary-SNAREs to be assembled and induce exocytosis
(FIGURE 8). SNAP23 can induce only slow exocytosis (⬎1 s)
(607); therefore, SNAP23 may not allow a stable binarySNARE state (176). The slow time course of spontaneous
exocytosis in the synapse (1,000 s) suggests that it is at least
partly mediated by the unitary configuration (FIGURE 8) or
even the cis-SNARE configuration.
3. How is the unitary-SNARE configuration clamped?
There are several possible mechanisms for preventing the
unitary-SNARE configuration from undergoing further
SNARE assembly in the resting state (FIGURE 8).
1) The availability of unitary-SNAREs is regulated. For example, tyrosine phosphorylation of Munc18c, which insulates syntaxin (235, 378, 737), is the major trigger of exocytosis of vesicles containing GLUT4 (15, 285). Likewise,
phosphorylation of SNAP23 is a key factor regulating exocytosis in mast cells (629). Exocytosis in salivary glands is
triggered by cytosolic cAMP and phosphorylation of
VAMP2 by PKA (182).
2) SNARE assembly itself may be regulated. Glucose-induced insulin exocytosis is potently regulated by PKA in a
manner that is dependent on snapin (603). Snapin may
allow SNARE assembly only when it is phosphorylated by
PKA in ␤ cells. Snapin can associate with SNAP23 and is
able to form a ternary complex with SNAP23 and syntaxin4
(85), which may be involved in various forms of slow exocytosis.
3) Access of the vesicles to the plasma membrane may be a
limiting factor for binary-SNARE complex formation. Vesicle proteins such as synaptotagmins, Doc2, and VAMP2
bind to t-SNAREs (184, 314, 567) and may facilitate formation of the binary- and trans-SNARE states (326). Such
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t-SNAREs are found unassembled (unitary), but clustered
and reactive, in the plasma membranes of PC12 cells (36,
220, 344, 345, 524), where syntaxin1 is likely associated
with Munc18a (600). There are abundant t-SNAREs surrounding the docked vesicles in PC12 cells (36), and they
are dynamically diffusing (326). Thus SNAREs are unassembled, even though vesicles are docked in PC12 cells,
which may account for the long latency of exocytosis (10 s)
in these cells (FIGURE 1). Poststimulus assembly of SNAREs
has been reported in ␤ cells of the islets (603).
747) and Munc13–2 in chromaffin cells (747) and in ␤ cells
(298), suggesting that the binary-SNARE assembly is facilitated by Munc13 in a Ca2⫹-dependent manner (FIGURE 8).
The sustained phase is also facilitated by CAPS (369, 611)
and PIP2 (421), suggesting that CAPS also facilitates
SNARE assembly after the Ca2⫹ trigger (FIGURE 8). The
sustained phase is greater with SNAP23 than with SNAP25
(436, 607), consistent with the idea that SNAP23 is involved in the unitary-SNARE configuration.
KASAI ET AL.
access is limited until cAMP-dependent removal of the submembranous cytomatrix has occurred, including the subcortical actin layer (324, 450).
E. Cis-SNARE Configurations
1. Sperm acrosome exocytosis
There is tremendous diversity in the initial assembly states
of neuronal SNAREs. This critically affects the time constants of exocytosis. The more tightly formed the SNARE
complex is, the sooner exocytosis occurs after stimulation.
Neuronal SNAREs are able to form the trans-SNARE state
before stimulation only in the active zone, but this is not
their sole mode of action, as nondocked vesicles may also be
involved (see sect. VI). The unitary- and binary-SNARE
modes may be widely utilized for slower exocytosis in synapses and secretory cells. The initial SNARE configurations
appear to be the major factor governing the kinetics of
exocytosis and are determined by the factors B–G below.
These factors may also affect the kinetics of exocytosis by
regulating poststimulus SNARE assembly.
B. Subtypes of SNAREs and
Priming Proteins
Neuronal SNAREs mediate rapid exocytosis, whereas nonneuronal SNAREs, such as VAMP4, syntaxin2, and SNAP23,
mediate slower exocytosis and are associated with ubiquitous priming proteins such as Munc18b and Munc13– 4. In
preparations with nonneuronal SNAREs, vesicles are nondocked and the trans-SNARE complex is not formed before
stimulation. SNARE linker regions may also affect the fusion processes directly (sect. VI). The number of SNARE
complexes may regulate the probability of exocytosis.
C. Ca2ⴙ-Sensor Subtypes
2. Other possible cases
Even if cells utilize neuronal SNAREs, the speed of exocytosis is dependent on the type of Ca2⫹ sensor used: from
fastest to slowest, the speed of the synaptotagmins is 2 ⬎1
⬎9 ⬎7 ⬎6 (703). Doc2 causes slower exocytosis than synaptotagmin1. It is thus likely that the triggering proteins
also regulate the initial configurations of SNAREs (sect. VI).
There are abundant cis-SNAREs in synapses (521) and
yeast vacuoles (664), and they may be used as a reservoir of
SNAREs that can be used on demand. Indeed, NSF and
␣SNAP act 1–5 s before fast exocytosis in synapses (336), and
SNAREs are recycled during tonic release (80). Tonic release is
dependent on ␣SNAP, suggesting that cis-SNAREs are also
utilized during tonic release (80). The action of NSF may be
regulated by rab3 (131), which is required for recycling
(348) and used for priming and biogenesis of LDCVs in
chromaffin cells (34, 574, 705). Cytosolic Ca2⫹ promotes
short-term plasticity (422) and recovery from depletion of
RRP (147, 616, 681) in various synapses, partly by activating cisSNAREs.
The localization of vesicles relative to the Ca2⫹ channels
profoundly affects the kinetics of exocytosis (sect. II). This
localization is regulated by SNAREs, and by priming and
triggering proteins.
VII. KINETIC DETERMINANTS
E. Submembraneous Cytomatrix
We have described the major factors affecting the kinetic
diversity of exocytosis in various secretory vesicles in neurons and secretory cells. We can now summarize the major
determinants of the time constants of exocytosis after stimulation based on all the data described in this review.
The cytomatrix at the active zone (CAZ) allows transSNARE configurations. In non-trans-SNARE configurations, the diffusion properties of vesicles and the distance
between the vesicles and the plasma membrane also play a
major role in the kinetics of exocytosis. The submembra-
1946
D. Vesicle Distance From the Ca2ⴙ Channels
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The acrosome of sperm cells undergoes exocytosis within a
few minutes after contact with the zona pellucida of an
oocyte, which is a crucial step for fertilization. The initial
trigger for acrosomal exocytosis is Ca2⫹ entry into the cytosol (131). Then, Rab3 is activated and triggers NSF/
␣SNAP-dependent disassembly of cis-SNARE (372) (which
is composed of syntaxin1A, SNAP25, and VAMP2) in both
the acrosome and the plasma membrane (648). The resulting disassembled SNAREs reassemble to form transSNARE complexes between the acrosome and the plasma
membranes. Ca2⫹ entry is followed by Ca2⫹ release from
the acrosome, which in turn induces fusion of acrosome
with the plasma membrane in a synaptotagmin6-dependent
manner (131). ␣SNAP also sequesters syntaxin and blocks
trans-SNARE conformation (529). cAMP/Epac is necessary
for the entire process to work (63) via rap1 and rab3b (62).
An anti-complexin antibody blocked formation of the
trans-SNARE complex, and subsequent exocytosis was
blocked by excess complexin (530). This series of events
during acrosomal exocytosis proceeds in much the same
way as other SNARE-dependent exocytoses, except that
exocytosis is triggered from the cis-SNARE state.
A. The Initial SNARE Configurations
INITIAL SNARE CONFIGURATIONS FOR EXOCYTOSIS
nous cytomatrix directly regulates exocytosis after triggering (see sect. VI).
ACKNOWLEDGMENTS
We thank G. J. Augustine and M. Kozlov for helpful discussion.
F. Vesicle Size
G. Lipid Composition
The lipid composition of both the vesicles and plasma membranes profoundly affects the fusion readiness of vesicles by
affecting both membrane fusion and the formation of
SNARE complexes (524) (sect. IV).
VIII. CONCLUSIONS
We have described the molecular and cellular mechanisms
of SNARE-dependent exocytosis that underlie the diverse
experimental results shown for synapses, endocrine, exocrine, epithelial, hematopoietic, muscle cells, and model
systems. This diversity can be mostly ascribed to distinct
initial SNARE configurations, although one must admit
that the molecular mechanisms underlying the clamping
and triggering of SNARE assembly have not been fully elucidated. The diversity of initial configurations will provide
us with valuable opportunities to scrutinize the individual
steps of the common SNARE assembly processes for exocytosis and membrane trafficking. As such, experimental
determination of initial SNARE states holds the key to understanding exocytosis, even though it is technically demanding. Direct FRET measurement of SNARE organization in the active zone and in secretory cells would go a long
way to achieving this goal. If various molecules are incorporated in the assay, single molecular FRET analyses will be
informative in identifying the precise molecular interactions
between molecules. Single vesicle liposome assays may provide an opportunity to study ultrafast exocytosis in vitro, as
the molecular composition can be controlled. These in vitro
methodologies have a tendency to give weighted views of
the actual in vivo phenomena. Super-resolution imaging,
such as PALM, STED, and RESOLFT (210), will help to
characterize high-order molecular structures and vesicle dynamics. Two-photon FRET/fluorescence lifetime imaging
will yield key information from intact tissues. Armed with
these new tools and ideas, we believe that a better understanding of exocytosis will be achieved in the next decade.
GRANTS
This work was supported by Grant-in-Aid for Specially Promoted Area 2000009 (to H. Kasai), the Global COE Program (Integrative Life Science based on the study of Biosignaling Mechanisms to H. Kasai), and the Strategic Research
Program for Brain Sciences (Bioinformatics for Brain Sciences to H. Kasai) from the Ministry of Education, Culture,
Sports, Science, and Technology (MEXT). This work was
also supported by a Research Grant from the Human Frontier
Science Program and CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Agency
(JST) (to H. Kasai).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the authors.
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