499
The synaptic vesicle cycle: exocytosis and endocytosis in Drosophila
and C. elegans
Janet E Richmond* and Kendal S Broadie†
Advances in the study of Drosophila melanogaster and
Caenorhabditis elegans have provided key insights into the
processes of neurotransmission and neuromodulation. Work in
the past year has revealed that Unc-13 and Rab3a-interacting
molecule regulate the conformational state of syntaxin to prime
synaptic vesicle fusion. Analyses of synaptotagmin support its
role as a putative calcium sensor triggering vesicular fusion and
highlight the possible role of SNARE complex oligomerization in
the fusion mechanism. Characterization of endophilin mutants
demonstrates that kiss-and-run endocytosis is a major
component of synaptic vesicle recycling. In neuromodulation,
dcaps mutants provide the first genetic insight into possible
roles of the CAPS protein in mediating dense core vesicle
fusion and modulating synaptic vesicle fusion.
Addresses
*Department of Biological Sciences, University of Illinois,
840 West Taylor Street, Chicago, Illinois 60607, USA
† Department of Biological Sciences, Vanderbilt University,
4270 Medical Research Building III, 465 21st Avenue South,
Nashville, Tennessee 37235-1634, USA;
e-mail: [email protected]
Correspondence: Kendal S Broadie
Current Opinion in Neurobiology 2002, 12:499–507
0959-4388/02/$ — see front matter
© 2002 Elsevier Science Ltd. All rights reserved.
Published online 4 September 2002
Abbreviations
APs
adaptor proteins
awd
abnormal wing discs
CAPS
calcium-activated protein for secretion
DCV
dense-core vesicle
flp
FMRF amide-like protein
FMRF
Phe-Met-Arg-Phe-NH2
GFP
green fluorescent protein
nlp
neuropeptide-like protein
NMJ
neuromuscular junction
NSF
N-ethylmaleimide-sensitive fusion protein
RIM
Rab3a-interacting molecule
SNAP-25 soluble N-ethylmaleimide-sensitive factor attachment
protein 25
SNARE
soluble N-ethylmaleimide-sensitive factor attachment
receptor
SV
synaptic vesicle
Unc
uncoordinated
Introduction
As we describe in our sister review in this journal [1], the
genetic model systems provided by Drosophila melanogaster
and Caenorhabditis elegans offer valuable and exciting
approaches to the study of synapses at the neuromuscular
junction (NMJ). This review highlights the recent
advances in our understanding of synaptic exocytosis and
endocytosis made using genetic approaches in Drosophila
and C. elegans. All of the studies discussed here have been
done at the NMJ, a glutamatergic synapse in flies and a
cholinergic/GABAergic synapse in worms. First, we discuss
the molecular mechanisms regulating presynaptic transmission mechanisms, including synaptic vesicle (SV)
dynamics and the regulation of Ca2+-dependent neurotransmitter release. We consider, in turn, what we have
learned in the past year about vesicular exocytosis, Ca2+sensors and endocytosis in the presynaptic terminal.
Second, we turn to the regulation of dense-core vesicle
(DCV) fusion, which mediates the release of neuromodulators. In particular, we consider the mechanisms that allow
the release of neuropeptides independently of fast-acting,
classical neurotransmitters. The development of synaptic
function during synaptogenesis and the new and/or
unexpected mechanisms of synaptic regulation that have
been uncovered in Drosophila, including dynamic protein
synthesis and degradation mechanisms, form the focus of
our sister review [1].
Exocytosis mechanisms: priming, fusion and
SNARE complex disassembly
Synaptic vesicles dock at active zones where they undergo
a priming step to prepare them for fusion in response to
a calcium signal (Figure 1). During priming, a trimeric
SNARE (soluble N-ethylmaleimide sensitive factor
attachment receptor) complex is formed between the
integral SV protein synaptobrevin, the integral plasma
membrane protein syntaxin and associated SNAP-25
(soluble N-ethylmaleimide sensitive factor attachment
protein 25). The stable assembly of this core complex is
believed to drive membrane fusion and neurotransmitter
release. Following fusion, vesicle components are retrieved
via a process of endocytosis. Several insights into the
molecular mechanisms responsible for these sequential
stages of the exocytosis–endocytosis SV cycle have come
from recent studies in C. elegans and Drosophila.
Priming
Priming likely involves the transition of syntaxin from a
closed to open conformation (Figure 1). The H3 region of
syntaxin required for SNARE complex formation is
occluded in the closed state. Syntaxin adopts a closed
conformation in a complex with uncoordinated (Unc)-18, a
presynaptic protein first cloned from a C. elegans mutant
based on its uncoordinated phenotype [2]. In C. elegans, the
diacylglycerol (DAG)-binding protein Unc-13 has been
shown to displace Unc-18 in vitro [3] and the carboxy
(C)-terminal of Unc-13 is known to interact with syntaxin
in its open state [4]. Thus, Unc-13 could function to
remove Unc-18 and/or stabilize open syntaxin to enable
SNARE complex formation (Table 1). Consistent with this
500
Neuronal and glial cell biology
Figure 1
Model for the role of Unc-13 in synaptic
vesicle priming. Syntaxin adopts a closed
configuration in which part of the α helix
required to interact with synaptobrevin and
SNAP-25 is occluded. In this closed
conformation, syntaxin is unavailable for
SNARE complex formation, a prerequisite for
vesicle priming and fusion competence. The
proposed role of Unc-13 is to stabilize the
open state of syntaxin, thereby facilitating
priming. In support of this model, a
constitutively open form of syntaxin can prime
vesicles in the absence of Unc-13. The role of
the Rab3-interacting protein RIM appears to
be similar to that of Unc-13, because open
syntaxin can also bypass RIM. Possibly, the
arrival of a synaptic vesicle is communicated
to RIM via vesicle-associated Rab3, with
RIM, in turn, activating Unc-13 to interact
with syntaxin.
Rab3
SNAP-25
Synaptobrevin
Unc-13
RIM
LE
Syntaxin
(closed)
Syntaxin
(open)
SNARE
complex
Current Opinion in Neurobiology
hypothesis, Unc-13 knockouts in mice, C. elegans and
Drosophila all result in the loss of evoked synaptic transmission [5–7]. SVs are morphologically docked but fail
to fuse in response to hyperosmotic saline application, a
treatment that induces the fusion of the entire readily
releasable primed vesicle pool. These results suggest that
Unc-13 is required for priming. Richmond et al. [8••]
directly tested the Unc-13 requirement in C. elegans by
engineering a stabilized ‘open’ form of syntaxin and asking
whether transgenic expression of this protein could bypass
the requirement for Unc-13. Overexpression of the ‘open’
syntaxin rescued evoked SV release in unc-13 null mutants
[8••]. Thus, Unc-13 appears to function in a priming mechanism by regulating the conformational state of syntaxin to
control SNARE complex formation (Table 1).
Koushika et al. [9••] recently isolated mutants in the C. elegans
Rab3a-interacting molecule (RIM) gene (unc-10) in a
screen for uncoordinated phenotypes. This mutant was analyzed to provide insights into the normal function of RIM.
RIM interacts with other key synaptic proteins in addition
to Rab3, including Unc-13, synaptotagmin, SNAP-25,
calcium channels, α-liprins, and SH3 domain proteins. In
C. elegans, unc-10 mutants display a 60% reduction in
evoked transmission and reduced spontaneous SV fusion
[9••]; a phenotype much more severe than rab3 mutants
(J Richmond, unpublished data), suggesting that RIM is
more than simply a Rab3 effector. The amino (N)-terminal
zinc finger domain of RIM, which interacts with both
Unc-13 and Rab3, was found to be essential for this RIM
function [9••]. Ultrastructural analysis of unc-10 mutants
showed that SVs were less abundant (reminiscent of rab3
mutants) but that the number of docked vesicles was
normal, suggesting that RIM is not required for vesicle
docking. Given the RIM−Unc-13 interaction, these studies
raised the possibility that RIM may function in SV priming.
As with unc-13 mutants, transgenic expression of a constitutively open syntaxin completely restored evoked release
in unc-10 mutants [9••]. RIM1 knockout mice were subsequently shown to exhibit reduced synaptic transmission,
normal SV docking and defects in synaptic plasticity [10],
consistent with a post-docking role for RIM in exocytosis
(Table 1). Thus, RIM may be a priming factor, possibly
signaling the arrival of a vesicle via a Rab3−RIM interaction,
and facilitating the opening of syntaxin via a RIM−Unc-13
interaction (Figure 1).
Vesicular fusion
There is abundant evidence that the SNARE proteins
synaptobrevin, syntaxin and SNAP-25 form a complex
required for vesicular fusion, based in part on knockout
mutations of both syntaxin and synaptobrevin [11].
However, genetic mutations of SNAP-25 had not been
characterized, until Rao et al. [12] recently generated a
temperature-sensitive mutation of SNAP-25 (snap-25ts) in
Drosophila. The mutation disrupts a conserved residue in
the first amphipathic helix of SNAP-25, part of the four
α-helical bundle of the SNARE complex. Although in vitro
SNARE complexes (70kD) were thermally stable in this
mutant, SNARE multimers (~120kD) rapidly dissociated
at 37°C and mutants exhibited impaired SV fusion at 37°C
[12]. Because SNAP-25 contributes two α-helices to the
SNARE complex, a single SNAP-25 may provide one helix
to two different SNARE complexes, thus providing a means
of establishing SNARE complex multimers. Thus, the
snap-25ts phenotype supports a growing body of evidence
The synaptic vesicle cycle Richmond and Broadie
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Table 1
Fly and worm genes involved in endocytosis and exocytosis.
Protein
Fly gene: phenotype
Worm gene: phenotype
Proposed function
CAPS
dcaps: non-autonomous inhibition of latestage SV fusion, block of DCV
exocytosis
dUnc-13: docked SV accumulation,
dramatically decreased vesicle fusion
unc-31: inhibition of cholinergic
transmission
Role in late-stage DCV fusion. Upstream
of indirect modulation of SV fusion
???
unc-10: reduced SV density,
decreased evoked and spontaneous
SV fusion
ric-4: inhibition of cholinergic
transmission
Unc-13
RIM
unc-13: docked SV accumulation,
Required for SV priming via control of
dramatically decreased vesicle fusion syntaxin conformational state
SNAP-25/t-SNARE, snap-25ts: temperature-sensitive allele
membrane
22ºC: increased SV release
associated protein 37ºC: decreased SV release, SNARE
multimer destabilization
Syntaxin
syx: docked SVs accumulate, abolished unc-64: inhibition of cholinergic
SV fusion
transmission
NSF
comatose: temperature-sensitive allele
???
temperature-sensitive block of SV fusion,
SNARE complexes accumulate
Synaptotagmin
syt-1: reduced calcium-dependent
snt-1: reduced cholinergic
evoked release and endocytosis defect transmission and endocytosis defect
SLO-1
slow poke: loss of Ca2+-dependent K+
slo-1: suppressor of unc-64
current
hypomorph, increased duration of
release
Endophilin
Dendo-A: SV depletion, shallow pits,
???
FM1-43 uptake defective, evoked
synaptic depression
Eps-15
???
ehs-1: SV depletion, uncoordinated,
reduced cholinergic transmission
Stoned A/B
stoned: reduced SV density and fusion, unc-41: inhibition of cholinergic
enlarged SVs, reduced synaptotagmin
transmission
at synapse
awd: enhancer of shibire
???
Involved in SV priming, coordinating Rab
3 and Unc-13-dependent functions
Component of core SV fusion machinery,
required for SNARE complex
multimerization
Essential component of core SV fusion
machinery and calcium-channel inhibitor
ATPase required for SNARE complex
disassembly following vesicle fusion
Ca2+-sensor for SV fusion and facilitatory
role in endocytosis
Ca2+-activated K+ channel
regulating SV release duration
Required for clathrin-mediated SV
endocytosis but not ‘kiss-and-run’
transmission
Facilitatory SV endocytosis role, possibly
through a dynamin interaction
Endocytosis; sorting and recycling of
synaptotagmin
Endocytosis: provides GTP for dynamin
activation
Amphiphysin
amphiphysin: flightless, disorganized
???
Role in muscle organization excitation–
muscle T-tubule/sarcoplasmic reticulum
contraction coupling, but not SV
endocytosis
In each case, the NMJ neurotransmission mutant phenotypes are listed for the Drosophila and C. elegans homologs. A putative synaptic
function for each gene product is provided in the last column. DAG, diacylglycerol.
that rings of SNARE complexes linked by SNAP-25 may
be required for efficient vesicle fusion [13].
promise to reveal the complex interactive mechanisms of
the exocytotic process.
However, muddying this interpretation, the snap-25ts
mutant also exhibits changes in the calcium-sensitivity of
SV fusion, as well as alterations in active zone morphology
[12], suggesting that SNAP-25 may function in several
aspects of synaptic regulation. Multiplicity of function for
synaptic proteins appears to be an increasingly common
finding, as highlighted in a recent site-directed mutagenesis
study targeting the H3 domain of Drosophila syntaxin 1A
[14]. In this study [14], two hydrophobic amino acids
implicated in the inhibitory interaction of syntaxin with
calcium channels and in the formation of a stable SNARE
complex were mutated. The resulting mutants displayed
impaired SNARE complex stability in vitro and virtually
no SV fusion in vivo due to a defect in fusion competence
[14]. Thus, this H3 domain of syntaxin appears to be
involved in two different aspects of fusion: calciumchannel regulation and SNARE complex stabilization.
Targeted structure−function analyses such as these
SNARE complex disassembly
Recruitment of the ATPase N-ethylmaleimide-sensitive
fusion protein (NSF) to SNARE complexes via SNAP
adaptors stimulates SNARE complex disassembly
(Table 1). However, it is unclear when in the SV cycle
SNARE complex disassembly takes place. Two groups
have explored this question using conditional temperaturesensitive mutations in Drosophila NSF (comatose) [15•]. At
the non-permissive temperature, comatose mutants accumulate SNARE complexes, although it is unclear whether
complexes accumulate in the plasma membrane and/or
SVs. By combining comatose with other temperature-sensitive
mutations that block known stages of the SV cycle, these
two groups hoped to uncover where NSF functions to
disassemble SNAREs. With the endocytosis mutant
shibire, which encodes the GTPase dynamin required for
vesicle fission, comatose mutants displayed SV depletion,
suggesting that NSF is not required for vesicle fusion, as
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Neuronal and glial cell biology
Figure 2
(a)
Clathrin
coat
Clathrin
Dynamin
Budding
Fission
Uncoating
Two mechanisms of synaptic vesicle recycling.
In clathrin-mediated endocytosis (a), essential
steps include the recruitment of clathrin and
dynamin to the adaptor protein AP2 which,
along with AP180 and stoned, are linked to
integral vesicle proteins such as
synaptotagmin and synaptobrevin. Following
the formation of a clathrin lattice and
membrane invagination, dynamin participates
in a fission step. Recent analysis of
Drosophila endophilin mutants suggests that
in the absence of clathrin-mediated
endocytosis, a ‘kiss-and-run’ vesicle recycling
pathway persists (b). In this mechanism,
vesicles momentarily fuse with the plasma
membrane to form a fusion pore (kiss) and
then undergo rapid dynamin-dependent
fission (run). The vesicle never collapses into
the plasma membrane.
(b)
Dynamin
Kiss
Run
Current Opinion in Neurobiology
all vesicles were able to fuse and become trapped in the
plasma membrane [15•]. By taking advantage of the lower
restrictive temperature of shibire mutants to inhibit endocytosis before inhibiting NSF, these studies showed that
SNARE complexes did not accumulate, indicating that
NSF disassembly can occur in the plasma membrane
following fusion. When exocytosis was impaired with a
sodium channel temperature-sensitive mutant (paralytic-ts)
in combination with comatose, SNARE complexes did
not accumulate [15•], suggesting that NSF functions after
fusion and before endocytosis by disassembling cisSNARE complexes in the plasma membrane (Table 1).
However, this study does not rule out the possibility that
NSF also acts on SNARE complexes present on SVs. This
is particularly pertinent, given that Drosophila NSF1 has
recently been shown to be enriched in SVs [16].
Calcium sensors at the synapse
Although the SNARE complex constitutes the essential fusion
machinery of the SV, it is unclear how fusion is triggered by
calcium. Synaptotagmin has been proposed as the major calcium
sensor that mediates fusion, yet the precise mechanism of
its fusogenic action is unknown. Synaptotagmin is an integral
vesicle protein that interacts directly with the SNARE complex
and it contains a large cytoplasmic region that binds calcium via
two C2 domains (C2A and C2B). In an elegant study, Littleton
et al. [17••] explored the role of the C2B calcium-binding
domains of Drosophila synaptotagmin I. This study showed that
synaptotagmin facilitates SNARE complex formation and that
calcium acts via synaptotagmin to promote SNARE complex
dimerization [17••]. A mutant that deletes the entire C2B
region disrupts synaptotagmin-dependent SV endocytosis,
but a single point mutation within C2B selectively inhibits
calcium-dependent exocytosis at a post-docking step [17••].
Analyses of this Drosophila mutant demonstrated that both calcium-dependent synaptotagmin oligomerization and SNARE
complex assembly were disrupted. These data suggest a model
in which calcium-dependent conformational changes in C2B
cause synaptotagmin oligomerization and trigger fusion via
the assembly and clustering of SNARE complexes [17••]. In
another study, overexpression of engineered mutations targeting a polylysine motif of the Drosophila synaptotagmin I C2B
domain yielded a similar reduction in evoked release, although
detailed analyses of synaptotagmin oligomerization and
SNARE assembly were not undertaken [18].
Calcium influx undoubtedly influences neurotransmitter
release via several other synaptic calcium sensors. For
The synaptic vesicle cycle Richmond and Broadie
example, recent evidence suggests that one form of
plasticity, augmentation, is mediated by calcium-dependent
phospholipase C activation which may target Unc-13 and
cause an increase in the size of the readily releasable pool
[19,20]. In Drosophila, the ATPase HSC-70 has been
implicated along with cysteine string protein in a mechanism
that regulates the calcium-sensitivity of release [21]. Finally,
in C. elegans, a link has been established between the
calcium-activated potassium channel SLO-1 and the
regulation of release [22]. slo-1 mutants, isolated in a screen
for suppressors of syntaxin mutant hypomorphs, exhibited
greatly increased quantal content attributable to a longer
duration of release in the absence of SLO-1-mediated
repolarization. Clearly, a complete understanding of
transmission will require an integrated understanding of
interactions between multiple calcium sensors.
Endocytosis mechanisms: kiss-and-run and
protein sorting
Vesicle recycling is essential for sustained synaptic
transmission. Clathrin-mediated endocytosis is generally
thought to be the major pathway for this vesicle retrieval.
Adaptor proteins (APs) including the AP2 complex and
AP180 recruit a clathrin lattice to the plasma membrane,
which induces membrane invaginations that are pinched
off in a dynamin-dependent reaction (Figure 2). Several
proteins have been implicated in endocytosis, on the basis
of their interactions with these established components of
the endocytotic machinery. Using both forward and reverse
genetics, several of these candidates have recently been
targeted in Drosophila and C. elegans.
Endophilin
Endophilin is an SH3-domain-containing protein that
exhibits lysophosphatidic acid acyl transferase activity and
interacts with both dynamin and synaptojanin [23].
Consistent with previous in vitro studies, Drosophila
endophilin mutants abolish clathrin-mediated endocytosis,
as revealed by the absence of FM1-43 dye-uptake, vesicle
protein redistribution, arrested shallow pit formation and
severe depletion of SVs throughout the presynaptic terminal in these mutants [23,24•]. A deeply surprising finding
in these endophilin mutants was that evoked transmission
amplitude approximated that of wild-type controls and
could be sustained at 20% of normal levels, even during
high frequency stimulation [24•]. Given the very small SV
pool in endophilin mutants, these data argue for the prominence of a second, very rapid vesicle retrieval pathway that
does not require complete fusion and recovery (i.e. kissand-run recycling; Figure 2). The kiss-and-run mechanism
of transient and reversible pore formation presumably also
requires dynamin, because shibire mutants at the non-permissive temperature completely lack vesicle release and
recycling in Drosophila [24•]. This observation is consistent
with dynamin antibody perturbation experiments that
eliminate kiss-and-run events detected in chromaffin cells
[25]. Now that an apparent kiss-and-run mode of vesicle
recycling can be isolated and studied in the Drosophila
503
endophilin mutant, it will be interesting to determine which
other proteins are required for this elusive, but apparently
central vesicle recycling pathway.
Eps15
A reverse genetic approach has also been applied to study
the function of an Eps15 homologue in C. elegans (Table 1).
Eps15 is the prototype of a conserved EH-domaincontaining protein family that is implicated in many
intracellular vesicular sorting pathways, including endocytosis. To better understand the function of Eps15 in
endocytosis, a targeted deletion of the worm Eps15
homologue (ehs-1) was isolated by the C. elegans Knockout
Consortium and studied by Salcini et al. [26•]. The ehs-1
mutants appear normal at 18°C, but show a locomotory
defect at higher temperatures. Similar phenotypes were
observed by RNA-mediated interference of EHS-1 synthesis and by the transgenic expression of a truncated
EHS-1 protein that acted as a dominant-negative [26•].
ehs-1 mutants were resistant to acetylcholinesterase
inhibitors; this demonstrated that EHS-1 perturbations
affecting worm locomotion at raised temperatures were
due to reduced presynaptic neurotransmission, consistent
with the immuno-localization of EHS-1 within the
neuropil. Ultrastructural analysis of ehs-1 mutants at restrictive temperatures revealed SV depletion, a hallmark for an
endocytosis requirement [26•]. A role for EHS-1 in endocytosis was further supported by genetic interactions
between ehs-1 and dynamin mutants; double mutants
display a subviable, severely uncoordinated phenotype.
EHS-1 was subsequently shown to directly interact with
dynamin [26•]. Because ehs-1 mutants only exhibit defects
at elevated temperatures, these results establish that
EHS-1 is not an essential component of the endocytotic
machinery, such as dynamin, but rather appears to act as an
accessory protein.
Stoned
Despite a growing appreciation of kiss-and-run transmission, clathrin-mediated endocytosis is clearly an important
retrieval mechanism following complete vesicle fusion.
This process is finely tuned to generate vesicles of a
precise dimension with the appropriate complement of
proteins and lipids, by recognition molecules (adaptins)
that link vesicle-derived proteins in the membrane to the
clathrin scaffold. For example, AP180 is responsible for the
retrieval of synaptobrevin [27] and the adaptin AP2 complex recruits synaptotagmin [28]. Recently, a pair of novel
Drosophila proteins, stoned A and B (STNA and STNB),
derived from dicistronic mRNA, have also been implicated
in synaptotagmin recycling [29]. STNA and STNB both
bind synaptotagmin, and contain several domains typical
of adaptor proteins (Table 1). Both stoned and synaptotagmin
mutants exhibit SV depletion and abnormally sized
vesicles, indicative of similar roles in SV endocytosis. In
two recent papers, the endocytotic defect in stoned mutants
was confirmed in FM1-43 dye-uptake studies [29,30].
STNA and STNB were both shown to be colocalized
504
Neuronal and glial cell biology
with the endocytotic lattice; stoned mutants exhibited
abnormal compartmentalization of endocytosed vesicles
[31•]. Most strikingly, overexpression of synaptotagmin
suppressed the stoned phenotype and restored normal
endocytosis dye-uptake, whereas shibire mutants exacerbated the endocytosis defect [31•]. Together these new
findings strengthen the argument that stoned proteins are
involved in recycling of synaptotagmin, and thus
impact both endocytosis and exocytosis in which synaptotagmin participates.
Dynamin
Dynamin is the GTPase required for separation of vesicles
from the plasma membrane during all forms of endocytosis
(Figure 2). Dynamin–GTP appears rate limiting for endocytosis and increasing the GTP-bound activated form of
dynamin has been shown to enhance rates of endocytosis
[32]. Thus, it is likely that this critical step of the vesicle
cycle is under tight regulation. To uncover proteins
involved in the regulation of dynamin function, Krishnan
et al. [33] undertook a forward genetic screen in Drosophila,
looking for enhancers of the temperature-induced paralysis
of the dynamin mutant shibire. This screen uncovered
three hypomorphic alleles of abnormal wing discs (awd), a
Drosophila ortholog of the human tumor suppressor gene
nm23. The awd gene encodes a nucleoside diphosphate
kinase that appears to act as an unconventional guanosine
nucleotide exchange factor to provide the GTP required
for dynamin function [33] (Table 1).
Amphiphysin
Although the molecular machinery that evolved to
subserve synaptic release appears to be highly conserved,
there do appear to be exceptions. This is highlighted by
the recent analysis of Drosophila amphiphysin mutants [34].
There are two vertebrate amphiphysin proteins that are
brain-enriched, although an amphiphysin 2 splice variant is
found in skeletal muscle. Amphiphysins have N-terminal
BAR domains involved in dimerization and lipid interactions; a central region that binds clathrin, the AP2 subunit
α-adaptin, and endophilin; and a C-terminal SH3 domain
that binds dynamin and synaptojanin. Experiments using
dominant-negative overexpression of amphiphysin domains
have been shown to block endocytosis in several systems.
It is therefore surprising that, in Drosophila, the only identified amphiphysin gene is enriched in muscle and exhibits
no presynaptic defects attributable to disruption of endocytosis
[34]. In light of the fact that Drosophila amphiphysin lacks
the clathrin-binding motif this is perhaps understandable.
Instead, Drosophila amphiphysin mutants exhibit a pronounced
locomotory defect that appears due to a disorganized muscle
T-tubule/sarcoplasmic reticulum system [34]. Thus, the
proposed endocytic function of vertebrate amphiphysin
appears to be less conserved than that of, for example,
clathrin and dynamin (Table 1). In Drosophila, alternative
proteins such as DAP-160, which binds several endocytic
proteins, may fulfil the dynamin-recruiting role proposed
for vertebrate amphiphysin.
Neuromodulation: dense core vesicles and
neuropeptides
Neuropeptides play critical roles in synaptic signaling.
Unlike classical neurotransmitters, however, neuropeptides
are packaged into dense core vesicles (DCV), which are
trafficked and triggered to fuse with the plasma
membrane using molecular mechanisms distinct from
those used in rapid SV neurotransmission. Neuropeptides
are thought to modulate synaptic function primarily
through G-protein-coupled receptors, but they also
mediate rapid communication through direct gating of ion
channels. The sheer abundance of neuropeptides and
their receptors is daunting, and perhaps explains the slow
progress in our understanding of this vital arena of
synaptic biology.
Neuropeptide function
Recent work in Drosophila and C. elegans has begun to chart
the size of this field by determining the number of putative neuropeptides and neuropeptide receptors encoded in
these genomes. The most complete picture emerges from
C. elegans analyses that predict: at least 130 genes for
neuropeptide receptors [35]; 92 neuropeptide-encoding
genes; 37 insulin-related genes; 32 neuropeptide-like
protein (nlp) genes; and 23 Phe-Met-Arg-Phe-NH2 (FMRF)
amide-like protein (flp) genes [36–38]. Many of the nlp
genes encode proproteins, which are processed to produce
more than 150 putative neuropeptides. Expression studies
show that these neuropeptides are found almost exclusively
in neurons [36].. Thus, as in other systems, neuropeptides
represent far and away the most abundant signaling
molecules in the invertebrate nervous system.
Prior to 2001, mutant analyses of neuropeptide function in
Drosophila and C. elegans were limited to behavioral studies.
For example, studies showed that C. elegans lacking flp-1
displayed uncoordinated movement and hyperactivity [39]
and Drosophila lacking pigment dispersing factor peptide
in pacemaker neurons showed a loss of circadian locomotor
rhythms [40]. This type of study confirms the significance
of neuropeptide signaling, but gives little insight into the
mechanisms by which neuropeptides are released or
mediate their activity at synapses.
The first direct electrophysiological studies addressing
neuropeptide function have recently been carried out in
C. elegans; they examine the pharynx musculature to study
the functions of a subset of the FMRF-like peptides [41].
Three peptides operate at physiological concentrations: one
increases pharyngeal action potential frequency, similarly to
the action of serotonin; two inhibit the pharynx, similarly to
the action of octopamine. Among these three FMRF-like
peptides, two are expressed only in extrapharyngeal neurons
and modulate the activity of the pharynx only indirectly,
whereas one is expressed in a neuron with direct synaptic
contact on the pharynx and mediates regulation directly
[41]. These results represent the first functional information
on the action of neuropeptides in C. elegans.
The synaptic vesicle cycle Richmond and Broadie
Comparable studies in Drosophila have not yet been
reported. Instead, the first functional assay has attempted
to harness optical techniques to visualize a green fluorescent protein (GFP)-tagged rat atrial natriuretic factor
peptide [42•]. Because no similar peptide exists in flies,
this approach is limited to monitoring the trafficking and
release of an ectopic peptide. At the larval NMJ, approximately 50% of the fluorescence associated with this tagged
peptide was released following prolonged depolarization
(15 min high [K+]) [42•], suggesting that functional assays
in this system may be possible. Native neuropeptides
identified at the Drosophila NMJ include proctolin,
insulin-like neuropeptide and pituitary adenylate cyclaseactivating peptide (PACAP) [43,44].
The role of CAPS in dense-core vesicle exocytosis
The only protein proposed to play a restricted role in DCV
exocytosis is calcium-activated protein for secretion
(CAPS) [45]. Mammalian CAPS binds Ca2+ and membrane
phospholipids and is proposed to mediate a late post-docking stage in a DCV-specific exocytotic pathway [46]. At
present, CAPS represents the only known way to putatively
dissect apart SV and DCV mechanisms at the synapse.
For this reason, the Drosophila ortholog (dCAPS; 59% identity, 91% similarity to mammalian CAPS) was targeted by
reverse genetic mutagenesis and the mutants characterized
at the NMJ [47••]. Null dcaps mutants are late embryoniclethal and display severe movement defects, showing that
the gene is essential for survival. Surprisingly, given the
reported DCV specificity of CAPS in mammals, dcaps
mutants display a 50% decrease in glutamatergic transmission
at the NMJ and docked SVs accumulate at presynaptic
active zones [47••]. However, transgenic replacement of
CAPS in these synaptic terminals fails to rescue these
defects, indicating an indirect CAPS requirement in other
cells. Indeed, it was recently found that rat CAPS expression
in a small, defined set of peptidergic neurons is sufficient
to rescue both the lethality and the NMJ defects in dcaps
mutants (R Renden and K Broadie, unpublished observations). Thus, CAPS is required in these peptidergic
neurons to indirectly regulate SV fusion at the glutamatergic
NMJ, presumably by controlling the release of a hormonelike modulator (Table 1).
In dcaps mutants, DCVs accumulate over three-fold in
synaptic terminals [47••], suggesting that CAPS plays an
essential role in DCV exocytosis. This accumulation appears
specific, because dUnc-13, an essential effector of SV fusion,
does not display a block in DCV fusion [47••]. Although
similar studies of the C. elegans CAPS ortholog (Unc-31)
have not been reported, the gross phenotypes of dcaps and
unc-31 mutants suggest it performs a similar function to
CAPS in the two organisms. Impaired cholinergic function
at the unc-31 mutant NMJ is suggested by the increased
resistance to acetylcholinesterase inhibitors, which may be a
secondary consequence caused by impaired DCV release in
neuromodulatory cells [48]. The hiatus in these promising
505
studies is the lack of a suitable functional assay for DCV
exocytosis in either Drosophila or C. elegans. Approaches
being explored now in Drosophila and C. elegans include
fluorescent peptide monitoring (similarly to [42•]) and
development of direct electro-physiological assays including
capacitance and amperometry measurements. Only with
these tools will it be possible to elucidate the role of CAPS
in DCV-specific exocytosis pathways.
Conclusions and future directions
So what have flies and worms taught us during the last year
about endocytosis and exocytosis? In neurotransmission,
an intriguing mechanism emerged in which Unc-13 and
RIM act together to regulate the conformational state of
syntaxin to prime SV for fusion. Clearly, SV priming is
closely regulated by multiple pathways, including both
calcium and diacylglycerol, which impinge on Unc-13
function. Perhaps most surprising were the analyses of
Drosophila endophilin mutants, which revealed that kissand-run exocytosis is apparently a major component of
neurotransmission.. As new mutants affecting endocytosis
emerge, we are able to refine our understanding of this
complex process. Similarly, our understanding of the
process of DCV release and the mechanisms of peptide
action have been furthered by mutant perturbation
analysis in these tractable genetic organisms.
Acknowledgements
We particularly thank Robbie Weimer in Erik Jorgensen’s lab for assistance
with the figures. We are grateful to members of the Broadie lab for
critical discussions.
References and recommended reading
Papers of particular interest, published within the annual period of review,
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• of special interest
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