Neuron, Vol. 14, 689-696, April, 1995, Copyright © 1995 by Cell Press
From Vesicle Docking to Endocytosis:
Intermediate Reactions of Exocytosis
Felix E. Schweizer,* Heinrich Betz,t
and George J. Augustine*
*Department of Neurobiology
Duke University Medical Center
Durham, North Carolina 27710
tDepartment of Neurochemistry
Max Planck Institute for Brain Research
D-60528 Frankfurt
Germany
Neurons have many remarkable tasks to perform. Among
these, the rapid secretion of neurotransmitters from presynaptic terminals is perhaps the most daunting because
it presents numerous biological challenges. First, neurons
must release their transmitters extremely rapidly in response to brief electrical signals. For this reason, neurons
have evolved a form of exocytosis that is tightly regulated
by calcium entry. Second, because of the great spatial
separation between the presynaptic terminals and the
neuronal cell body, neurotransmitter release must be an
autonomous process that is regulated locally, within the
presynaptic terminal. In many neurons, these processes
occur at a specialized presynaptic structure known as the
active zone. Thus, neurons have devised means of targeting and compartmentalizing all of the necessary molecular constituents for neurotransmitter exocytosis at active
zones. As a consequence of this molecular autonomy, the
nerve terminal must also have a means of locally recycling
synaptic vesicles following exocytosis. Indeed, it is now
clear that a sophisticated scheme of membrane and protein recycling underlies neurotransmitter release (Figure
la). In this review, we will focus our attention on recent
progress in elucidating three of the central steps within the
synaptic vesicle life cycle: vesicle docking, vesicle fusion,
and the subsequent endocytosis of vesicular membrane
and proteins.
Mechanisms of Synaptic Vesicle Docking
Docking is a morphological term that developed from the
observation that nerve terminals have regular arrays of
synaptic vesicles juxtaposed just beneath the plasma
membrane at sites where exocytosis occurs (Couteaux
and Pecot-Dechavassine, 1974). Because of their favorable location, it seems likely that these are the vesicles
that undergo exocytosis upon elevation of calcium within
the presynaptic terminal. The short latency between presynaptic excitation and transmitter exocytosis (Katz and
Miledi, 1965) has led to an alternative, functionaldefinition
of docking. According to this definition, docked vesicles
are those that are the first to undergo exocytosis. A third
definition proposed for vesicle docking is molecular and
is based on the notion that docking involves the binding
of vesicular proteins to other proteins on the presynaptic
plasma membrane. It is now common to refer to any such
protein-protein interactions observed in vitro as docking
Review
reactions (Bennett and Scheller, 1994; Rothman, 1994;
Jahn and SLidhof, 1994), although it must be acknowledged that only a subset of all interactions between vesicular and plasma membrane proteins are likely to participate
in vesicle docking. While we can hope that these alternative definitions refer to the same phenomenon, it is more
likely that they describe different steps within a complex
sequence.
Much effort has gone into identifying molecules that
could mediate docking interactions between the vesicle
and plasma membranes. Two soluble proteins, N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (SNAP), are essential for the membrane fusion events underlying Golgi transport (Rothman, 1994).
The search for membrane receptors for these proteins,
called SNAP receptors or SNAREs, led to the identification
of presynaptic proteins known to be components of synaptic vesicle membranes (synaptobrevin) and plasma membranes (syntaxin and 25 kDa synaptosome-associated protein [SNAP-25]). The realization that proteins involved in
Golgi fusion can bind to presynaptic proteins on both vesicular and plasma membranes suggested that SNAREs are
recognition molecules that act, in concert with the cytosolic proteins NSF and SNAP, to mediate synaptic vesicle
docking and/or fusion. The discovery that SNAREs are
also the target of neurotoxins which block transmitter release (Schiavo et al., 1994) lends further credence to such
hypotheses. The few experimental tests of this idea performed thus far in intact synapses suggest that these proteins are involved after synaptic vesicles dock at terminals
(Hunt et al., 1994; DeBello et al., 1995). Thus, the components of the NSF-SNAP-SNARE protein complex are
more likely to be involved in vesicle fusion than in docking.
If the SNAREs do not participate in the primary docking
event, what are the alternative candidates for docking mediators? Several other vesicular proteins have binding
partners on the presynaptic plasma membrane. For example, the calcium-binding vesicle protein synaptotagmin
binds to both syntaxin and neurexin I, the ~-Iatrotoxin receptor (Pevsner et al, 1994; Perin, 1994; H ata et al., 1993;
Bennett et al., 1992). Perhaps the best case can be made
for the vesicular GTP-binding proteins. Vesicle trafficking
is disrupted by nonhydrolyzable GTP analogs (Hess et al.,
1993), and one of these, GDPI3S, inhibits synaptic vesicle
docking (Doroshenko et al., submitted). Synaptic vesicles
possess a number of GTP-binding proteins, including
rab3, which is one member of avery large familyof proteins
involved in vesicular trafficking or fusion (Novick and
Brennwald, 1993). Given the function of other members
of this family, it is plausible that rab3 may be involved in
vesicular docking (Geppert et al., 1994a; Holz et al., 1994);
however, deletion by homologous recombination of the
gene encoding rab3a has only subtle effects on neurotransmitter release, arguing against an obligatory role for
rab3 in synaptic vesicle docking. Nevertheless, during repetitive bouts of exocytosis, neurotransmitter release de-
Neuron
690
,J
Enc'.,k,"}o~o~ls
P~ming
Fusion
~iiiiiiiii,iH"ii!i!i~!
C
b
"Clossical'
d
'kiss a n d run'
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presses more rapidly in nerve terminals deficient in rab3a,
suggesting that this protein could regulate docking events
that take place during intense presynaptic activity (Geppert et al., 1994a).
In addition, a role for lipids in docking cannot be excluded. A phosphatidylinositol lipid transfer protein and
a phosphatidylinositol-4-phosphate 5-kinase stimulate
some steps preceding exocytosis in PC12 cells (Hay and
Martin, 1993; Hay et al., 1995). Interestingly, the calciumbinding vesicle protein synaptotagmin binds to polyphosophoinositides (Fukuda et al., 1994). This suggests that
docking could be mediated by synaptotagmin binding to
phosphatidylinositol phosphates specifically inserted by
transfer proteins at presynaptic active zones. Presynaptic
microinjection of these polyphosophoinositides inhibits
neurotransmitter release (Llinas et al., 1994), although this
effect was suggested to occur at a step downstream of
docking, as defined by physiological criteria. It seems
more likely that these lipids are involved in an ATPdependent priming of vesicles for exocytosis that might
occur after the vesicles have docked (Hay et al., 1995).
In summary, our understanding of the molecular mediators of synaptic vesicle docking is at a very early stage.
At the moment, only GTP-binding proteins, such as rab3,
have been directly implicated in vesicle docking, but additional proteins are certain to be involved.
Regulation of Synaptic Vesicle Fusion
Local Calcium Signals Trigger Vesicle Fusion
When thinking about calcium triggering of transmitter release, one must consider the distance between the sources
of calcium --the voltage-gated calcium channels--and the
calcium receptor proteins that trigger release. In this regard, three geometric arrangements could be relevant
Figure 1. PresynapticCycling of Vesicles
(a) Schematicof the synapticvesiclelife cycle.
Within the nerveterminal,synapticvesiclesare
shownas smallcircles,andtheircontents(neurotransmitter) are shown as shading. These
vesiclesdockat the presynapticplasmamembrane, become primed for exocytosis, and
then, in the presenceof calciumions,fusewith
the plasma membraneto release their contents. Followingfusion,vesicularmembraneis
retrievedvia the processof endocytosis,which
involves coated pits and coated vesicles.
Thesevesicleslosetheir coats(stippled),fuse
with an endosomal compartment, and then
reform into new vesicles (after Heuser and
Reese, 1973).
(b-d) Threepossibleformsof endocytosis.The
"classical" view of endocytosis(b) postulates
thatvesiclemembranesinsertedintothe plasma
membraneduringfusionare retrievedthrough
a clathrin-mediatedform of endocytosis.The
"kiss and run" hypothesis(c) states that vesiclesfuseverytransientlywiththe plasmamembrane,andthis processis reversedby pinching
off the vesiclesimmediatelyafterwards.Membranesfrom multiplevesiclesinsertedintothe
plasma membranecan also be taken up by
non-clathrin-mediated"bulk" endocytosis(d).
(Figure 2). The first case occurs if the calcium receptors
are within a few nanometers of calcium channels. When
the calcium channel opens, a highly localized accumulation of calcium results; this calcium signal has been referred to as a calcium domain (Chad and Eckert, 1984)
or, in reference to the spatial dimension involved, a nanodomain (Kasai, 1993). Such nanodomains are too localized and too brief to be measured directly, but theoretical
estimates indicate that, at local calcium receptors, calcium
concentrations could reach tens to hundreds of micromolar (Simon and Llina_s, 1985; Smith and Augustine, 1988;
Roberts et al., 1990; Yamada and Z.ucker, 1992). Another
relevant arrangement occurs when multiple calcium channels are clustered together over a fraction of a micrometer,
as appears to occur at active zones (Roberts et al., 1990;
Robitaille et al., 1990; Smith et al., 1993; Haydon et al.,
1994). When these channels open, a receptor positioned
within a fraction of a micrometer of the cluster will sense
a microdomain of calcium (Kasai, 1993) that can reach
levels of many hundreds of micromolar, due to summation
of calcium coming from multiple calcium channels (Smith
and Augustine, 1988; Roberts et al., 1990; Llin~_s et al.,
1992; Roberts, 1994). Finally, in the case where the calcium channels are far (1 ~.m or more) from the calcium
receptor, calcium must diffuse some distance and will produce radial concentration gradients (Lipscombe et al.,
1988; Hernandez-Cruz et al., 1990). The calcium concentration sensed by such a receptor should be (roughly) in
the low micromolar range. For each of these three cases,
receptors of appropriate affinity and calcium-binding kinetics are needed to transduce the calcium signal into an
appropriate biochemical reaction.
Several lines of evidence now indicate that local calcium
nanodomains are responsible for triggering the rapid exo-
Review: Steps in Exocytosis
691
Nanodom, aJn
Calcium Channel
Microdomain
R a d i a l Shell
Figure 2. Spatial Rangesof CalciumAction
(Top) Once calcium enters through a single calcium channelin the
plasma membrane(PM), it reachesvery high concentrationswithin
a few nanometersof the channel. Calcium receptors(R) within this
nanodomainwill encountercalciumconcentrationsin the rangeof tens
to hundredsof micromolar,and nearbyvesiclescan fusewith minimal
delay. The calcium rise within the nanodomainis very transientand
can onlybe blockedby fast calciumchelatorssuchas BAPTA(calcium
concentrationis indicatedby densityof shading).
(Middle) If calcium channelscluster, their calcium nanodomainswill
overlapand leadto a largecalciumincreaseoverdistancesof a fraction
of a micrometerawayfrom the membrane.Since generationof this
calcium microdomainis dependenton diffusionof calciumawayfrom
the channel,the resultingcalciumsignalis slowerthan in nanodomains
and can be influencedby slower chelators,such as EGTA.
(Bottom) If calcium receptorsare far from the calciumchannels,they
will sensea slowrise in calciumas this ion diffusesradiallythroughout
the cell.
cytosis that occurs at active zones. Rapid jumps in calcium
concentration show that calcium concentrations comparable to those produced within micro- or nanodomains are
necessary to produce physiological rates of secretion
(Chow et al., 1994; Heidelberger et al., 1994). The slowly
binding calcium buffer EGTA does not block transmitter
release, while a rapidly binding homolog, BAPTA, efficiently blocks release (Adler et a11991; von Gersdorff and
Matthews, 1994a). Based on the kinetics of calcium binding by EGTA, these data indicate that the calcium receptor
which triggers release must bind calcium within tens of
nanometers of the calcium channel, i.e., within the nanodomain (Neher, 1986; Stern, 1992). To position the calcium receptor within the nanodomains, there must be
some molecular coupling between the exocytotic apparatus and presynaptic calcium channels. Syntaxin, a plasma
membrane SNARE that binds to both calcium channels
and vesicle proteins, may serve this purpose (Yoshida et
al., 1992).
The more widespread presynaptic calcium signal resulting from gradual diffusion of calcium away from nano-
domains may also regulate transmitter release. It has long
been thought that a form of presynaptic plasticity known
as facilitation is due to the residual presence of calcium
(Katz and Miledi, 1968; Rahamimoff, 1968), and diffuse
calcium signals may also be responsible for producing
other forms of plasticity, such as augmentation and posttetanic potentiation (Erulkar and Rahamimoff, 1978; Zengel
and Magleby, 1981; Delaney et al., 1989; Swandulla et
al., 1991; Kamiya and Zucker, 1994; Regehr et al., 1994).
Consistent with these notions, the ability of calcium entry
to promote exocytosis is enhanced by elevation of basal
calcium levels (Charlton et al., 1982) or by activation of
calcium-regulated kinases (Nichols et al., 1990; Llin~,s et
al., 1991). In endocrine cells, diffuse calcium signals also
seem to regulate mobilization of newvesicles into a releasable pool (Sch&fer et al., 1987; Thomas et al., 1993; Neher
and Zucker, 1994). Such calcium signals can also trigger
exocytosis in these cells (Knight and Baker, 1982), although they very rapidly release a small fraction of vesicles
(Horrigan and Bookman, 1994) perhaps requiring calcium
concentrations as high as those reached in micro- or nanodomains (Heinemann et al., 1994). A specific molecular
mechanism for calcium regulation of synaptic vesicle mobilization has been proposed by Greengard et al. (1993),
who studied the phosphorylation of the vesicle-associated
protein synapsin I by the type II calcium/calmodulin-dependent protein kinase. In vitro experiments and microinjection studies suggest that synapsin I acts as a calcium- and
phosphorylation-sensitive anchor connecting synaptic
vesicles to the presynaptic cytoskeleton (Greengard et al.,
1993). This mechanism has recently been questioned by
experiments in which the synapsin I gene has been deleted by homologous recombination, with little effect on
neurotransmitter release (Rosahl et al., 1993). Ectopic expression or overexpression of synapsin I also has only
limited morphological or physiological effects (Geppert et
al, 1994b; Lu et al., 1992). However, direct methods of
measuring vesicle dynamics and addressing the possible
roles of compensatory proteins, such as synapsin II, will
be needed before experiments will allow us to understand
the role of synapsins in transmitter release.
Molecular Mechanisms of Synaptic Vesicle Fusion
An ever increasing amount of experimental data supports
the concept that synaptotagmin plays a prominent role in
calcium-regulated neurotransmitter release, perhaps serving as the calcium receptor that triggers synaptic vesicle
fusion. Early work established that synaptotagmin both
binds calcium and participates in transmitter release (summarized in Popov and Poo, 1993; DeBello et al., 1993).
Microinjection experiments documented that this protein
participates at a step between vesicle docking and fusion
(Bommert et al., 1993), precisely where the calcium receptor is thought to act. Genetic experimentation in Drosophila and mice provides more direct evidence in support of
a role for synaptotagmin as a calcium receptor regulating
exocytosis. First, transgenic mice defective in synaptotagmin I have greatly impaired calcium-dependent neurotransmitter release (Geppert et al., 1994c). This lesion is
Neuron
692
selective for the most rapid component of calcium-dependent release, with a slower calcium-dependent component
and spontaneous transmitter release being little affected.
More direct implication of synaptotagmin in transducing
presynaptic calcium accumulation into exocytosis comes
from characterization of point mutations in the synaptotagmin gene of Drosophila. Certain mutations cause a change
in the cooperative triggering of transmitter release by calcium ions, altering the stoichiometdc relationship between
extracellular calcium concentration and rate of evoked release (Littleton et al., 1994). Although it is not yet known
what these mutations do to the calcium-binding properties
of synaptotagmin, their effects on the calcium sensitivity
of transmitter release provide the most compelling evidence yet that synaptotagmin serves as the calcium receptor that triggers exocytosis. Whether synaptotagmin acts
as a calcium-regulated promoter of vesicle fusion or a calcium-sensitive barrier to fusion is not yet resolved (Popov
and Poo, 1993; DeBello et al., 1993). Deletion of the synaptotagmin gene does not completely abolish evoked release (Broadie et al., 1994; Geppert et al., 1994c), indicating that alternative calcium receptors are involved in
synaptic transmission (DeBello et al., 1993; Geppert et
al., 1994c), at least under conditions in which synaptotagmin is lost. This residual release has a calcium dependence indistinguishable from wild type, suggesting a
synaptotagmin-like molecule as the alternate calcium receptor. Possible candidates include isoforms of synaptotagmin (UIIrich et al., 1994), rabphilin (Shirataki et al.,
1993), Doc2 (Orita et al., 1995), and other proteins capable
of binding calcium with low affinity (for references, see
Kasai, 1993).
Evidence that a NSF-SNAP-SNARE complex is directly
involved in transmitter release has come from recent microinjection experiments. Two neurotoxins that cleave the
vesicle SNARE, synaptobrevin, inhibit neurotransmitter release at the squid giant synapse, while injection of a cytoplasmic domain of synaptobrevin leads to a reversible inhibition of neurotransmission (Hunt et al., 1994). Terminals
injected with neurotoxin had an increase in the number
of docked synaptic vesicles, suggesting that synaptobrevin is involved in transmitter release at a step downstream of docking. Disruption of SNAP function also perturbs transmitter release: presynaptic injection of SNAP
enhances transmitter release, while SNAP-derived peptides block release and cause accumulation of docked
vesicles (DeBello et al., 1995). Introduction of SNAP into
permeabilized chromaffin cells also enhances exocytosis
of catecholamines (Morgan and Burgoyne, 1995). These
findings suggest that SNAP is involved in neurotransmitter
release and that its concentration at release sites is rate
limiting. In summary, all data are consistent with the idea
that synaptobrevin and SNAP are important for neurotransmitter release, although comparable data are not yet
available for NSF and other SNAREs.
The above-mentioned results indicate that components
of the NSF-SNAP-SNARE complex are important for
steps that intervene between vesicle docking and fusion.
Because synaptotagmin also seems to be involved in
steps that intervene between vesicle docking and fusion
(Bommert et al., 1993), it is important to determine the
functional relationship between the NSF-SNAP-SNARE
complex and synaptotagmin. Biochemical data indicate a
competitive binding relationship between SNAP and synaptotagmin: synaptotagmin is displaced from the NSFSNAP-SNARE complex when SNAP is added in increasing amounts (SSIIner et al., 1993). These data have been
interpreted to mean that SNAP works downstream from
synaptotagmin, with the endogenous ATPase activity of
NSF (Whiteheart and Kubalek, 1995) perhaps providing
the energy for vesicle fusion. The numerous binding and
hydrolysis steps required by this model imply a relatively
long delay between elevation of calcium and triggering of
exocytosis, although it is possible to imagine circumstances in which a brief calcium signal could trigger rapid
fusion of a small fraction of available vesicles despite an
intrinsically slow fusion mechanism (Almers, 1994). Because calcium binds within tens of microseconds of entering the nerve terminal (Adler et al., 1991), and exocytosis
quickly follows a step elevation in presynaptic calcium concentration (Heidelberger et al., 1994), the fusion process
apparently is rapid. Thus, it seems probable that the NSFSNAP-SNARE complex works before synaptotagmin,
priming synaptic vesicles by conferring fusion competence upon them (O'Connor et al., 1994), while synaptotagmin follows to act as a rapid trigger for exocytosis as soon
as calcium enters the nerve terminal. In summary, a rich
cascade of reactions involving multiple proteins appears
to occur between the docking and fusion of synaptic vesicles. Defining these reactions and understanding their
sequences within the cascade will be essential for comprehending the forces that underlie vesicle fusion (Zimmerberg et al., 1993).
Endocytosis
Electron microscopy studies have established that vesicular membrane incorporated into the plasma membrane
during exocytosis is retrieved from the nerve terminal via
a slower process of endocytosis (Heuser and Reese, 1973;
Ceccarelli et al., 1973). However, the intracellular pathways employed in endocytosis and the speed at which
endocytosis takes place are still topics of debate.
The "classical" view of the pathway of vesicular endocytosis is that it is initiated by the formation of coated pits on
the presynaptic plasma membrane. These pits somehow
identify and scavenge vesicular membrane, which is then
taken into the presynaptic cytoplasm when coated vesicles are formed (see Figure lb). There is good evidence
that this pathway is employed in nerve terminals (Heuser
and Reese, 1973; Koenig and Ikeda, 1983). An alternative
pathway for endocytosis is that vesicles fusing with the
plasma membrane could immediately be retrieved via a
reversal of exocytosis (see Figure lc). Although there
is little evidence for this form of "kiss and run" exocytosis
at nerve terminals, it remains a popular idea (Ceccarelli
et al., 1973; Fesce et al., 1994). A rapidly reversible form
of endocytosis is often observed in mast cells (Monck and
Fernandez, 1994); this could serve as a model for understanding the possible importance of kiss and run endocytosis in presynaptic terminals. A third mechanism for
Review: Steps in Exocytosis
693
endocytosis has been observed in nerve terminals (Miller an('] Heuser, 1984) and endocrine cells (Neher and
Marty, 1982; Rosenboom and Lindau, 1994; Thomas
et al., 1994) that have undergone extensive exocytosis.
Under these conditions, bulk retrieval of presynaptic membrane occurs via uncoated invaginations (see Figure ld).
The relative importance of these mechanisms in the
normal life cycle of the synaptic vesicle has yet to be
evaluated.
The time course of retrieval of vesicular membrane is
even more perplexing. Most early electron microscopy
suggested that endocytosis is a process that takes place
over a period of minutes (Heuser and Reese, 1973). However, experiments employing more dynamic measurement methods indicate that nerve terminal endocytosis
can be more rapid (Miller and Heuser, 1984). Studies
based on the uptake of the fluorescent membrane dye
FM1-43 suggest that an entire cycle of exocytosis, endocytosis, and reformation of fusion-competent synaptic vesicles takes on the order of one minute, with endocytotic
half-times estimated at less than 30 seconds (Betz and
Bewick, 1993; Ryan et al., 1993; Ryan and Smith, 1995).
Capacitance measurements made from nerve terminals
suggest an even more rapid time course, with the most
rapid phase of endocytosis taking place within a couple
of seconds (von Gersdorff and Matthews, 1994a). These
kinetic discrepancies could result from differing contributions of the various pathways for endocytosis (see Figures
l b - l d ) , particularly if these pathways are important over
different time courses and under varying stimulation conditions. A priority for future work is to determine the speeds
and relative importance of the endocytotic mechanisms
shown in Figure 1.
There is evidence that the time course of endocytosis
is regulated by calcium. Early work showed that nerve
terminals treated with a-latrotoxin, a toxin that triggers
massive exocytosis of synaptic vesicles, do not undergo
endocytosis if bathed in calcium-free media (Ceccarelli
and Hurlbut, 1980). It has therefore been proposed that an
elevation of calcium concentration within the presynaptic
terminal is a necessary trigger for endocytosis. Some work
in endocrine cells is consistent with this notion (Neher and
Zucker, 1994), although it has also been reported that
neuronal endocytosis is unaffected by removal of external
calcium (Ryan et al., 1993). A recent report indicates that
the situation may be still more complex: elevation of intracellular calcium concentration actually slows the rate of
endocytosis in certain nerve terminals (von Gersdorff and
Matthews, 1994b). It may be that endocytosis requires
some intermediate level of intracellular calcium, and prolonged exposure to calcium-free solutions could lower the
internal calcium concentration below the threshold for supporting endocytosis. Thus, it appears that intracellular calcium regulates endocytosis, exocytosis, and vesicle docking. Such intricate regulation could underlie some of the
variations in the reported speed of endocytosis.
Our understanding of the molecules responsible for endocytosis in nerve terminals is still at an early stage, but
is advancing rapidly. The role of coated pits and coated
vesicles in the classical pathway (see Figure 1) implicates
@
I
~3a
~
ClGhrin
SNARE
SNAP
NSF
SynapTolagmin
Ca
Figure 3. Tentative Assignmentof Proteins to Specific Steps in the
Vesicle Life Cycle
clathrin and other coat proteins, as well as the clathrin
adaptor-complex proteins (adaptins), which regulate coat
formation (Robinson, 1994). It is thought that these proteins are essential for the early formation of coated pits.
Like synaptotagmin, adaptins bind to phosphoinositides
(Ye et al., 1995), and synaptotagmin binds to clathrin adaptor-complex proteins (Zhang et al., 1994); this could allow
synaptotagmin to be a multifunctional protein that regulates
endocytosis as well as exocytosis (Jorgensen et al., unpublished data), and some of the complex effects of calcium
upon endocytosis might be mediated via synaptotagmin.
Another protein essential for endocytosis is dynamin, a
GTP-binding protein associated with coated pits and
coated vesicles (Herskovits et al., 1993). A mutation of
the dynamin gene in Drosophila leads to an impairment of
endocytosis in nerve terminals (Koenig and Ikeda, 1983).
Because this mutation stops the budding of coated pits
to form coated vesicles, it appears that dynamin is involved
in the formation of coated vesicles. Consistent with this
hypothesis, treatment of nerve terminals with a nonhydrolyzable analog of GTP produces tubular membrane invaginations coated by dynamin, suggesting that the GTP analog freezes dynamin in the midst of budding off coated
vesicles (Takei et al., 1995). Dynamin can self-assemble
into ring-like structures (Hinshaw and Schmid, 1995);
these may serve to constrict the neck of the coated pit
and thereby cause the transition from coated pit to coated
vesicle (Takei et al., 1995). Dynamin is also phosphorylated by protein kinase C and dephosphorylated by calcineurin, two calcium-sensitive enzymes (Liu et al., 1994).
Thus, changes in the state of phosphorylation of dynamin
offer still more possible mechanisms for calcium regulation of endocytosis.
Conclusion
In summary, the combination of cell biological, molecular
biological, and physiological methods has led to many new
insights into the partial reactions that make up the life cycle
of synaptic vesicles. Figure 3 summarizes our tentative
assignment of several proteins to stages of this life cycle,
Neuron
694
but it is likely that m a n y o t h e r p r o t e i n s will s o o n be a d d e d
to this picture. T h e n e x t c h a l l e n g e will be to identify the
specific roles o f t h e s e p r o t e i n s in this e x q u i s i t e l y r e g u l a t e d
s e q u e n c e of events.
Acknowledgments
We thank all members of our laboratories for stimulating discussions
and other colleagues for sharing results prior to publication. This work
was supported by National Institutes of Health grant NS 21624 to
G. J. A. and by grants from Deutsche Forschungsgemeinschaft (LeibnizProgramm) and Fends der chemischen Industrie to H. B.
March 20, 1995
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