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Molecular Circuitry
of Endocytosis at
Nerve Terminals
Jeremy Dittman and Timothy A. Ryan
Department of Biochemistry, Weill Cornell Medical College, New York, NY 10065;
email: jed2019@med.cornell.edu, taryan@med.cornell.edu
Annu. Rev. Cell Dev. Biol. 2009. 25:133–60
Key Words
First published online as a Review in Advance on
July 8, 2009
synapse, vesicle cycle, trafficking, clathrin, pHluorin
The Annual Review of Cell and Developmental
Biology is online at cellbio.annualreviews.org
This article’s doi:
10.1146/annurev.cellbio.042308.113302
c 2009 by Annual Reviews.
Copyright All rights reserved
1081-0706/09/1110-0133$20.00
Abstract
Presynaptic terminals are specialized compartments of neurons responsible for converting electrical signals into secreted chemicals. This selfrenewing process of chemical synaptic transmission is accomplished
by the calcium-triggered fusion of neurotransmitter-containing vesicles
with the plasma membrane and subsequent retrieval and recycling of
vesicle components. Whereas the release of neurotransmitters has been
studied for over 50 years, the process of synaptic vesicle endocytosis has
remained much more elusive. The advent of imaging techniques suited
to monitor membrane retrieval at presynaptic terminals and the discovery of the molecules that orchestrate endocytosis have revolutionized
our understanding of this critical trafficking event.
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Contents
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INTRODUCTION . . . . . . . . . . . . . . . . . .
THE SYNAPSE . . . . . . . . . . . . . . . . . . . . . .
THE SYNAPTIC VESICLE:
A LOCAL CURRENCY FOR
INFORMATION FLOW AT
THE SYNAPSE . . . . . . . . . . . . . . . . . . .
SYNAPTIC VESICLE RETRIEVAL
AT NERVE TERMINALS . . . . . . . .
THE TIMING OF ENDOCYTOSIS
AT NERVE TERMINALS:
EVIDENCE FOR MULTIPLE
TIME SCALES OF
RETRIEVAL . . . . . . . . . . . . . . . . . . . . . .
MODULATION OF
ENDOCYTOSIS: CALCIUM
AND EXOCYTIC LOAD . . . . . . . . .
KISS-AND-RUN: A
LONG-RUNNING DEBATE . . . . .
THE MOLECULAR MACHINERY
OF SYNAPTIC VESICLE
ENDOCYTOSIS. . . . . . . . . . . . . . . . . .
A CLATHRIN-MEDIATED
PATHWAY FOR VESICLE
REBUILDING . . . . . . . . . . . . . . . . . . . .
MECHANISMS FOR SORTING
134
134
135
136
137
140
141
142
143
INTRODUCTION
SV: synaptic vesicle
134
Chemical communication between cells was
harnessed for both distributing and controlling multicellular behavior early on in the
development of complex life forms. In the nervous system, synapses encompass a transduction machine whereby propagating electrical
signals, usually in the form of action potentials,
are transformed into chemical messages in the
form of packets of neurotransmitters that are secreted onto a neighboring cell, which in turn are
transduced back into both chemical and electrical signals in this cell. The past 60 years have
witnessed an intense scientific effort to understand how synapses work, how they form and
are controlled, how their function changes over
Dittman
·
Ryan
THE APPROXIMATELY NINE
CARGO PROTEIN TYPES . . . . . . .
CURVING MEMBRANES:
INITIATION OF SYNAPTIC
VESICLE ENDOCYTOSIS . . . . . . .
SECONDARY SCAFFOLDING
FOR RECRUITING GENERAL
ENDOCYTIC EFFECTORS . . . . .
FINISHING THE JOB:
MEMBRANE FISSION
AND COAT DISASSEMBLY . . . . . .
CALCIUM SENSORS FOR
ENDOCYTOSIS? . . . . . . . . . . . . . . . . .
A ROLE FOR ACTIN DURING
SYNAPTIC VESICLE
ENDOCYTOSIS? . . . . . . . . . . . . . . . . .
REBUILDING SYNAPTIC
VESICLES: A SINGLE-PASS
SORTING AT THE PLASMA
MEMBRANE? . . . . . . . . . . . . . . . . . . . .
A MOLECULAR PICTURE
FOR THE ENDOCYTIC
MACHINE . . . . . . . . . . . . . . . . . . . . . . .
NETWORK PROPERTIES OF THE
ENDOCYTIC MACHINE . . . . . . . .
CONCLUDING REMARKS . . . . . . . . .
144
145
149
149
151
152
152
152
153
154
time scales from milliseconds to months, and
how they are built at the molecular level. Increasingly, as molecular detail has emerged, it
has overlapped with the recognition that this information is crucial for understanding a variety
of diseased states of neuronal function. Here
we focus our attention on the first half of the
synaptic transmission problem, the cell biology
of the presynaptic nerve terminal and, in particular, the current understanding of endocytosis
and synaptic vesicle (SV) recycling.
THE SYNAPSE
The basic structural organization of synaptic
terminals appears to be relatively well conserved across evolution. Neurotransmitters are
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Refilling, docking,
priming
Fusion
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a
Cargo mixing
Lipid mixing
f
e
b
c
Cargo capture
Clathrin coat assembly
Membrane curvature
Membrane fission
Uncoating
d
Vesicle budding
Figure 1
The synaptic vesicle (SV) cycle. An SV fuses with the plasma membrane (a) and is subsequently rebuilt
through the assembly of a clathrin-coated pit (b,c) followed by budding (d ), and fission and uncoating of the
endocytic coat proteins (e). The vesicle is refilled with neurotransmitter and returned to the vesicle pool for
further rounds of exocytosis ( f ). Several SV proteins are depicted including synaptobrevin/VAMP ( green),
synaptotagmin ( purple), and the vacuolar ATPase (blue). Syntaxin is shown (red rods), and the assembled
SNARE complex is represented (red-green coils). Other proteins depicted here are clathrin triskelia ( gray),
adaptins (red ellipses), dynamin, and endophilin (red helices).
packaged into small, clear vesicles with a diameter that ranges from ∼30 to 40 nm (depending on the species). Typically a few dozen
to a few hundred SVs are maintained in a
cluster near the active zone, a dedicated intracellular location on the presynaptic plasma
membrane where SVs, upon the arrival of an
action potential stimulus, will fuse with the
plasma membrane and release their content
through exocytosis (Figure 1). These preferential sites of exocytosis are typically found
juxtaposed to a postsynaptic specialization of
neurotransmitter-gated ion channels in the receiving cell. As nerve terminals are located
along or at the end of axons very distal to
the cell body and the total number of SVs is
limited, ongoing synaptic transmission is maintained by local recycling of SVs. The mechanisms of how vesicles are rebuilt following exocytosis for reuse are the focus of this review.
THE SYNAPTIC VESICLE:
A LOCAL CURRENCY FOR
INFORMATION FLOW AT
THE SYNAPSE
SVs act as key intermediates in converting
electrical to chemical information at synapses
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and represent a currency of information flow in
the nervous system. Because their size is tightly
regulated (see below), it has been possible to
purify these organelles to near homogeneity.
Such purified vesicles were the starting point
for identifying SV proteins ( Jahn et al. 1985,
Trimble et al. 1988, Perin et al. 1990) and provided the basis for generating the first draft of an
SV proteome carried out by the lab of Reinhard
Jahn (Takamori et al. 2006). SVs are ∼50% protein and 50% lipid, with a 1:1 ratio of phospholipid to cholesterol, similar to generic cellular
plasma membranes. Following exocytosis, the
transmembrane proteins of SVs become transiently incorporated into the plasma membrane
and need to be endocytosed to rebuild the SV.
Figure 2 illustrates the major transmembrane
proteins associated with SVs as well as their
average copy number deduced from quantitative proteomics. There are roughly nine types
of transmembrane proteins that are specifically
enriched in SVs and of these, only four types
have been assigned clear-cut functions. These
Synaptobrevin 2
/ VAMP2
70 copies
Vacuolar ATPase
1–2 copies
are (a) the proton pump (vacuolar ATPase),
which provides the proton-motive force for
driving neurotransmitter uptake through (b)
the vesicular neurotransmitter transporter
(Edwards 2007), (c) synaptotagmin I, the
calcium sensor for fast calcium-triggered exocytosis (Chapman 2008), and (d ) synaptobrevin
2/VAMP2, the vesicle-associated SNARE that
provides one of the helices required to catalyze
membrane fusion through formation of a fourhelix bundle with the specific plasma membrane
SNARE proteins syntaxin and SNAP-25 (Rizo
& Rosenmund 2008). It is noteworthy that the
relative abundance of these different proteins
varies significantly: Some proteins such as
the proton pump, whose functional role is
unequivocal and required for filling vesicles
with transmitter, appear to be expressed at
single-copy levels. In contrast, synaptophysin
is at least one order of magnitude more
abundant but is dispensable, at least in terms
of animal viability (McMahon et al. 1996) and
basic synaptic function (Abraham et al. 2006).
Presently little is known about variability in the
stochiometric relationship of these different
proteins across individual vesicles.
SYNAPTIC VESICLE RETRIEVAL
AT NERVE TERMINALS
Synaptotagmin I
15 copies
Synaptogyrin
2 copies
SV2
2 copies
Neurotransmitter
transporter
9–14 copies
Synaptophysin
30 copies
Scamp
1–2 copies
Figure 2
The synaptic vesicle (SV) transmembrane proteins. Proteomic analysis of
highly purified SV preparations allowed estimates of the abundance of each of
the major transmembrane proteins associated with SVs. The protein copy
numbers shown are taken from Takamori et al. (2006).
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Typical CNS synapses contain a limited number of SVs, usually a few hundred or less. As
nerve terminals are typically located great distances from their cell bodies, the biosynthetic
engines of cells, the proteins and lipids that
make up a SV, must be recaptured from the
plasma membrane following exocytosis, refashioned into a SV, and refilled with neurotransmitters (De Camilli et al. 2001). This currency
of synaptic communication is in fact in relatively limited supply. The mechanism of vesicle
retrieval in the SV cycle has been a subject of
intense interest and debate for over 35 years.
The first descriptions of vesicle recycling began
with ultrastructural observations at neuromuscular junctions (NMJ), where uptake of extracellular horse radish peroxidase demonstrated
conclusively the existence of the SV recycling
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pathway (Heuser & Reese 1973). These studies also provided the first evidence that vesicle
retrieval occurred through a clathrin-mediated
pathway. To visualize vesicle recycling at the
EM level, it was necessary to provide a sufficiently rapid and intense stimulus while catching exo- or endocytic events in the act through
rapid freezing. This approach led to the first
kinetic description of endocytosis (Miller &
Heuser 1984) because the time between stimulus and freezing could be varied (Figure 3a,b).
The relatively long time scales observed
(1 min for completion of vesicle endocytosis) fueled speculation that a faster, more direct route
must be operational, and they provided grist for
the idea that endocytosis may occur by reversal of fusion at the active zone, first proposed
by Ceccarelli and colleagues (Ceccarelli et al.
1973); this pathway was later coined kiss-andrun. During the past 10–15 years, efforts to understand the mechanisms of endocytosis of SVs
have been concentrated in two approaches. One
is the examination and discovery of the molecular machinery responsible for endocytosis at
nerve terminals; the other is the biophysical
probing of the time scales and molecular behavior of the endocytic process at the synapse.
THE TIMING OF ENDOCYTOSIS
AT NERVE TERMINALS:
EVIDENCE FOR MULTIPLE
TIME SCALES OF RETRIEVAL
Synaptic endocytosis kinetics measurements
became possible with the advent of new technologies as well as suitable synaptic preparations. Electrical capacitance recordings, which,
in principle, provide direct measurements of
cell surface area, allow one to measure the net
balance of exocytosis and endocytosis. This has
been useful, however, only at select giant synaptic preparations because SVs have a very small
surface area (3000–5000 nm2 ) and sites of fusion are usually quite distant from the cell body,
where the recording electrode makes electrical
contact. Three different types of giant synaptic
preparations have proven useful in this regard.
The first direct real-time measurements of
vesicle retrieval following a burst of exocytosis were made in retinal bipolar cells (von
Gersdorff & Matthews 1994a) and auditory hair
cells (Parsons et al. 1994). Refinement of these
measurements (Beutner et al. 2001, Neves &
Lagnado 1999) revealed that, generally, two
distinct kinetic components of endocytosis are
apparent (with ∼1-s and ∼10-s time constants,
respectively) where the relative proportion of
retrieval via the faster component was sensitive
to calcium influx (Beutner et al. 2001, Neves
et al. 2001). Given that this calcium-driven fast
component was sensitive to the calcium chelator BAPTA (and occurred even when calcium
elevations were spatially confined), the authors
concluded that the faster form was likely occurring near active zones. Similar observations
were made in the giant auditory brainstem calyx
of the Held synapse (Wu et al. 2005).
The appearance of distinct kinetic components leads to the question of how each
participates in regenerating SVs. Interestingly,
the detailed ultrastructural kinetic study by
Heuser and colleagues (Miller & Heuser 1984)
also revealed ultrastructural components with
distinct kinetic signatures following stimulation at the NMJ. Freeze-fracture views of
presynaptic terminal membrane showed that
following stimulation, non-clathrin-coated endocytic vacuole-like structures formed near
the active zone without concentrating cargo
molecules and disappeared within approximately 1 s (Figure 3a,b). In retrospect, it seems
likely that the fast component seen with capacitance measurements could be the same fast
pathway identified by Heuser at the NMJ. Consistent with this interpretation, this component
was shown to be insensitive to clathrin perturbation ( Jockusch et al. 2005). At the calyx of
Held, however, the appearance of fast endocytosis was independent of neurotransmitter release (Yamashita et al. 2005). Pretreatment of
nerve terminals with botulinum toxin blocked
neurotransmitter release without eliminating a
capacitance transient with fast recovery. Thus,
this rapid membrane response may represent a
homeostatic response to acute calcium elevation independent of SV endocytosis.
www.annualreviews.org • Synaptic Vesicle Endocytosis
NMJ: neuromuscular
junction
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a
8
b
Endocytosis profiles (μm–2)
6
5
4
3
2
1
0
0
c
20
40
60
80
100
120
Time (s)
tdwell ~ 0 s
d
tdwell ~ 4 s
0.20
Fraction of events
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7
tdwell ~ 10 s
5
0.10
0.05
tdwell > 22 s
0
0.15
10
15
Time (s)
20
0.00
0.1
5.6
11.1
16.6
22.1
Dwell time (s)
Figure 3
Kinetics of endocytosis from 1984 to 2007 (a) The first measurements of the kinetics of synaptic vesicle (SV) endocytosis were obtained
via pulse-chase freeze fracture experiments at the frog neuromuscular junction (NMJ) by Miller & Heuser (1984). An image that was
obtained by quick freezing an NMJ 30 s after action potential stimulation in 4-AP is shown. The white circles (diameter of 110 nm)
indicate membrane deformations that correspond to clathrin-coated pits. Image adapted courtesy of John Heuser. (b) The histogram of
the accumulation and disappearance of the membrane invaginations obtained by examining freeze-fracture pictures taken at many
different times with respect to the action potential stimulus. Redrawn from Miller & Heuser (1984). (c) Single-vesicle exocytosis and
endocytosis as revealed by following the vesicular glutamate transporter (vGlut1) tagged with pHluorin in a luminal loop. Adapted from
Balaji & Ryan (2007). Upon fusion, pHluorin becomes deprotonated and its fluorescence dequenched. After a variable delay (tdwell ), the
SV protein is endocytosed and the fluorescence becomes quenched as the vesicle lumen reacidifies over a 3- to 4-s time scale. Four
different examples displaying the range of tdwell are shown. (d ) The histogram of tdwell values obtained from approximately 200 events
shows that the timing of endocytosis is dictated by an exponential process (red curve) whose mean time is approximately 13 s.
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Endocytosis
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Exocytosis
V-ATPase
proton pump
Reacidification
pKa ~7.1
Protonated
(quenched)
vGlut-pHluorin
Deprotonated
(fluorescent)
vGlut-pHluorin
Figure 4
pHluorin-based measurements of the vesicle cycle. pHluorin is a modified form of green fluorescent protein
(GFP). GFPs, in general, are excellent proton sensors, as the fluorescence is quenched in an all-or-none
fashion upon protonation, which is well described by a 1-proton binding equilibrium (Haupts et al. 1998).
The pKa of pHluorin is ∼7.1, one log unit more alkaline than GFP and, when resident within the acidic
lumen of a synaptic vesicle, is quenched ∼97% of the time. Redrawn from Sankaranarayanan et al. (2000).
The introduction of optical tracers has
proven particularly powerful for examining endocytosis at small nerve terminals. Pulse-chase
application of the fluorescent amphipathic
molecule FM 1-43 (Betz & Bewick 1992) allowed the first kinetic dissection of endocytosis
in dissociated hippocampal neurons (Ryan et al.
1993, Ryan & Smith 1995). Vesicle retrieval following a large burst of activity indicated that
endocytosis decayed over a 60-s time scale. Although useful for labeling recycling vesicles
pools and measuring pool turnover kinetics,
tracers such as FM 1-43 do not directly provide
real-time endocytosis readouts. The advent of
pHluorins (see Figure 4) provided higherfidelity readout where specific vesicle proteins
fates can be followed. Initial measurements of
VAMP2 endocytosis for relatively large stimuli
showed that the recovery time scale depended
on the amount of exocytosis (Sankaranarayanan
& Ryan 2000). Improvements in these approaches allowed detection of endocytosis following single-action-potential stimuli (Balaji &
Ryan 2007, Granseth et al. 2006), which showed
endocytic recovery occurred with a time constant of approximately 15 s. Analysis of the delay time between exocytosis and endocytosis
for individual vesicles showed that endocytosis times varied stochastically from vesicle to
vesicle, ranging from instantaneous to tens of
seconds with an exponentially distributed dwell
time (Balaji & Ryan 2007), which indicates that
a single rate-limiting stochastic step determines
the time scale of endocytosis.
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MODULATION OF
ENDOCYTOSIS: CALCIUM
AND EXOCYTIC LOAD
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A central goal of research in presynaptic function is to understand the regulation of different
vesicle cycling steps. Prior to assigning specific
molecular roles, one must understand how the
conditions under which the measurements were
performed impact the biophysical observations.
Two main variables associated with driving exocytosis at synapses are the extent to which intracellular calcium becomes elevated and the total
endocytic burden that ensues from successful
exocytosis. These two variables are closely
entwined as greater elevations in intracellular
calcium generally lead to higher exocytic rates,
more material that must be recaptured. Given
the importance of intracellular calcium in
signaling, determining calcium’s role in endocytosis is of significant interest. Early attempts
to disentangle calcium elevations from exocytosis used α-latrotoxin, a potent secretagog
from black widow spider venom, to stimulate
exocytosis in the absence of extracellular calcium (Ceccarelli & Hurlbut 1980). After several
hours of stimulation under these conditions,
NMJs became severely depleted of SVs and the
plasma membrane developed large enfoldings,
indicating that endocytosis was blocked.
Ceccarelli & Hurlbut (1980) concluded that
intracellular calcium elevation was necessary
for SV endocytosis. It was impossible, however,
to distinguish the extent to which calcium
might simply be modulating rates as opposed to
playing a requisite role in the biochemistry of
endocytosis. A number of different experiments
have shown that, following exocytosis, calcium
entry and endocytosis can be decoupled as the
presence of extracellular calcium is no longer
necessary for endocytosis to proceed (Gad et al.
1998, Ryan et al. 1996) and that bulk calcium
levels typically decay faster than the completion
of endocytosis (Wu & Betz 1996). At the other
extreme, persistent photolysis of caged calcium
led to complete arrest of endocytosis in giant
bipolar terminals so long as bulk cytoplasmic calcium was greater than approximately
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1 μM (von Gersdorff & Matthews 1994b).
Although peak intracellular calcium levels near
exocytosis sites transiently reach tens of micromolar, average sustained intracellular calcium
levels remain well below 1 μM at most nerve
terminals following a single-action potential or
low-frequency stimulation. Thus, it is unclear if
such inhibition plays a role under physiological
conditions.
Approaches that allowed direct measurements of endocytosis kinetics made it
possible to examine the relationship between
the amount of exocytosis and the speed of
endocytosis, as well as how elevations in intracellular calcium might modulate this process.
Experiments in hippocampal nerve terminals
and at the calyx of Held showed that increases
in exocytosis led to progressively longer
endocytosis time scales (Sankaranarayanan
et al. 2000, Sun et al. 2002), in spite of the
fact that endocytosis was accelerated under
conditions that led to elevated calcium in these
preparations (Sankaranarayanan & Ryan 2001,
Wu et al. 2005). This slowing of endocytosis
with greater exocytosis may result from an
underlying saturation of the total endocytic
capacity of the synapse. It is notable that such
saturation behavior is not observed in other
synaptic preparations such as NMJs in mice
(Tabares et al. 2007) and flies (Poskanzer et al.
2006), suggesting that it is not a fundamental
property of endocytosis at nerve terminals.
This paradox between acceleration of endocytosis due to intracellular calcium versus slowing
due to increased accumulation, and the absence
of either in certain preparations, was recently
resolved. Taking advantage of single-vesicle
sensitivity to monitor endocytosis kinetics systematically over a 40-fold range in exocytosis,
Balaji et al. (2008) demonstrated that endocytosis recovery follows a single exponential time
course (15 s) over a wide range of exocytosis but
begins to slow significantly only above a threshold accumulation of SVs on the plasma membrane. These data imply that until this threshold is reached, endocytosis of different vesicles
is likely happening in parallel, each with an
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average time scale of 15 s. Beyond this
threshold, the synapse can no longer assemble
sufficient endocytic machinery to handle
greater endocytic loads, thereby defining the
endocytic capacity of the synapse. Importantly,
for sufficiently small amounts of exocytosis,
the endocytosis time course was insensitive to
manipulations of intracellular calcium. Balaji
et al. (2008) observed that the endocytic capacity was increased by stimulus conditions that
elevated residual calcium in the nerve terminal.
These data thus help reconcile the original
observations at the frog NMJ as well. Persistent
stimulation in the absence of any calcium elevation (as achieved in α-latrotoxin in zero calcium
for prolonged periods) could lead to a situation
where the amount of exocytosis occurring
greatly exceeds the endocytic capacity, eventually leading to persistent depletion of vesicle
pools. Furthermore, this hypothesis offers an
explanation as to why modulation of endocytosis by activity may not be observed: The
detailed tuning of calcium entry, buffering, and
extrusion, along with the specific abundance of
the necessary endocytic proteins, may be such
that one never operates in an exocytosis regime
where the endocytic capacity becomes limiting.
For example, at physiological temperatures,
the time constant for endocytosis decreases to
6 s (Balaji & Ryan 2007) in hippocampal nerve
terminals. This increase in clearance time
coupled with the fact that release probability
is lower at this temperature lead to a situation
where the endocytic capacity is rarely exceeded.
Thus, for three different central nervous system synapses examined (bipolar terminals, the
calyx of Held, and dissociated hippocampal
neurons), a common time scale of 10 to 15 s
(at room temperature) appears to prevail in SV
recovery unless significant accumulation of SV
components can be driven to the cell surface.
KISS-AND-RUN: A
LONG-RUNNING DEBATE
Beginning with the first observations of vesicle
recycling, there has been speculation of a
rapid recycling pathway whereby the vesicle
maintains its identity and is retrieved intact at
the active zone. This idea was originally fueled
by disparity in the time scales of exo- and endocytosis observed at the NMJ. Endocytosis took
approximately 1 min (Miller & Heuser 1984),
whereas exocytosis took place on the submillisecond time scale. In comparing the time scale
for exo- and endocytosis, however, one should
consider the time for the entire recycling vesicle pool to fuse with the plasma membrane, not
just a single vesicle. At hippocampal synapses
at 37◦ C, exocytosis of the entire recycling pool
requires approximately 20 s for stimulation
at 10 Hz (Fernandez-Alfonso & Ryan 2004),
which should be compared to the 6-s time
constant for endocytosis under these conditions (Balaji et al. 2008). Thus, it is possible
for exocytosis to outrun endocytosis, but only
under relatively high-frequency stimulation.
The most direct evidence for transient reversible fusion pores arose from capacitance
recordings of single-vesicle fusion events in
chromaffin cells coupled with simultaneous
measurements of release of catecholamine using carbon fiber amperometry (Albillos et al.
1997). In the majority of events, the appearance of a small fusion pore was followed by
an irreversible expansion and complete loss of
catecholamine. However, in 5–10% of events,
the pore closed without expanding. At conventional synapses, this dual approach of capacitance recordings and amperometry has not
been possible, but single-vesicle fusion events
have been recorded using capacitance measurements alone at the calyx of Held. Wu and colleagues reported that 17% of events appeared
to be transient in nature (He et al. 2006).
There are two important caveats in interpreting
these results. First, this approach required examining exocytosis at random locations on the
synaptic surface that were not necessarily active
zones. For technical reasons, the events could
be evoked only using nonphysiological stimuli.
Second, the appearance of exocytosis followed
soon after by endocytosis may simply represent a narrow sampling of the select fast events
of an exponential distribution of time scales as
predicted by a stochastic process (Balaji & Ryan
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Qdot: quantum dot
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2007). Verification that such events are insensitive to perturbations of elements in the clathrinmediated pathway will be necessary to assign
them to a distinct endocytosis pathway.
Less direct biophysical methods have also
been used to ascertain the presence of kiss-andrun. One approach that has been used extensively at hippocampal synapses was to examine
the efficiency of escape of lipophilic tracers
from SVs during exocytosis. The idea is that
a liphophilic dye potentially would dissociate
from the vesicle lumen membrane too slowly to
escape during a kiss-and-run event, but that it
might fully dissociate during a full fusion event.
This approach, first introduced more than a
decade ago (Ryan et al. 1996), has been used in
one variant or another to argue both for (Aravanis et al. 2003, Harata et al. 2006, Klingauf et al.
1998, Pyle et al. 2000, Richards et al. 2005) and
against (Fernandez-Alfonso & Ryan 2004, Ryan
et al. 1996, Zenisek et al. 2002) the presence
of kiss-and-run. Examination of dye loss at the
single-vesicle level, however, is quite challenging, as one is effectively asking whether losses
in fluorescence are well quantized. A number of
subtle artifacts can potentially lead to loss of fidelity of the fluorescence measurements. Chief
among these is that lipophilic tracers are added
to the outside of the synapse during activity to
load recycling vesicles; however, other recycling membranes in neighboring glia and postsynaptic dendrites, as well as other presynaptic
compartments, can become labeled. Such
spurious labeling can strongly contribute to
background fluctuations in fluorescence intensity. Recent experiments designed to control for
some of these possible complications concluded
that lipophilic dyes destain completely during
action-potential stimuli (Chen et al. 2008) and
suggest either that kiss-and-run does not occur
at these synapses or that one cannot use these
dyes reliably to detect such transient fusions.
Recently, a novel form of molecular tracer
was introduced for examining the frequency of
full fusion during exocytosis in cultured neurons. In this new technique, a recycling vesicle endocytoses a quantum dot (Qdot) (Zhang
et al. 2007) whose optical properties allow
Dittman
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continuous optical recordings without photobleaching. Because of its large size [hydrodynamic radius of nearly 15 nm (Larson
et al. 2003)], the internalized Qdot escapes
only during full-fusion exocytosis (Zhang et al.
2007). Zhang et al. (2007) found that stimulusdependent unloading of Qdots followed a
slower time course than an FM dye loaded into
the same synapses, suggesting that some fusion
events release FM dye but not Qdots. A significant caveat with this approach comes from the
large size of Qdots relative to the vesicles that
contain them. If the Qdot interacts with the
vesicle lumen and stabilizes the curvature of a
fusing vesicle, perhaps an otherwise rare partialfusion event could be made more probable.
Investigations into the molecules that underlie endocytosis have yielded little evidence
in support of kiss-and-run mechanisms to date
(see below). Furthermore, the observation that
vesicle components exchange with counterparts
resident on the cell surface during endocytosis over a wide range of stimulus conditions
(Fernandez-Alfonso et al. 2006, Wienisch &
Klingauf 2006) indicates that vesicles typically
lose some of their components following exocytosis. Finally, detailed measurements of the
time between exocytosis and endocytosis for
individual vesicles showed that, although vesicles do occasionally endocytose quickly, the frequency of such events is well predicted by a
stochastic (single-exponential) process with a
mean endocytic time of 15 s (Balaji & Ryan
2007). Taken together, the biophysical evidence
weighs largely against significant vesicle recycling via a kiss-and-run mechanism. Furthermore, as discussed below, most experiments
aimed at explicitly targeting clathrin or associated machinery at the synapse support a major role for the classical endocytic pathway in
vesicle retrieval.
THE MOLECULAR MACHINERY
OF SYNAPTIC VESICLE
ENDOCYTOSIS
Molecular and genetic insights over the past
25 years from a variety of model systems have
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culminated in a formidable list of potential proteins and lipids involved in endocytosis. Many
of the proteins share common binding partners and exhibit overlapping functions within
the endocytosis machinery. This property of interconnectivity may be important in explaining
an emerging dilemma in the field of SV endocytosis: Gene knockouts and RNA interference
knockdown of endocytic proteins generally do
not eliminate SV endocytosis and, in many
cases, have remarkably subtle effects (Di Paolo
et al. 2002, Ferguson et al. 2007, Gu et al. 2008,
Sato et al. 2009). This situation starkly contrasts
with SV exocytosis, where removal of any one
of a small collection of essential proteins eliminates SV fusion (Südhof 2004). If altered SV
endocytosis is observed after a particular endocytic protein is removed, there are three possible considerations for the residual endocytosis.
First, this perturbation may uncover a second,
independent pathway with distinct underlying
molecular mechanisms. Second, the measured
endocytosis is identical to the wild-type process except that a functional paralog has taken
the place of the deleted molecule, although it
may not function with the same efficacy. Third,
the impaired endocytosis proceeds via the identical molecular pathway but with lower efficacy
because the deleted molecule played a contributory rather than obligatory role in the process.
In this case, the network of endocytic proteins
is robust to deletions of individual members.
Consistent with the third scenario, experiments to date have demonstrated that no single protein appears to be absolutely essential
to the process of SV endocytosis. Perhaps particular components of the synaptic endocytic
network play a larger or smaller role in different animal phyla, but all the components are
generally found in presynaptic specializations of
metazoa. Furthermore, these components show
a high degree of conservation, both at the sequence level and in terms of interactions with
other proteins and phospholipids (Lloyd et al.
2000). In the following section of this review, we
introduce 15 proteins/protein complexes that
compose the SV endocytic network (Figures 6
and 7). These proteins are separated into three
layers of the network on the basis of their function, binding interactions, and protein domain
content. At the core of the endocytic network is
the capacity to gather up cargo into a local patch
of plasma membrane and deform this patch into
a separate compartment destined for internalization. A second layer of proteins acts to stabilize the core proteins while recruiting additional effector molecules that both catalyze and
terminate the process. These effectors make up
the third layer, and they function in numerous
cellular trafficking processes in addition to SV
endocytosis.
A CLATHRIN-MEDIATED
PATHWAY FOR VESICLE
REBUILDING
The first details of the molecular basis of SV
recycling arose with the pioneering studies of
Heuser & Reese (1973), who noted that endocytosis following stimulation appeared to occur via the newly discovered clathrin coats.
Since these early discoveries, progress in our
understanding of the molecular basis of SV recycling has benefited from a number of approaches in different model systems. Although
the relevance of the finding that clathrin-coated
pits could be found at synapses originally was
debated, the evidence that this pathway predominates as the route of vesicle recovery has
become overwhelmingly strong in the past
10 years.
Three types of experiments have been performed, all of which support a major role for
the clathrin reuptake pathway. First, microinjections of peptides or antibodies that interfere
with different steps of endocytosis were followed by electron microscopy and functional
assays. Second, genetic ablations of endocytic
proteins were followed by kinetic assays and
electron microscopy. Third, the effects of photoinactivation of clathrin in Drosophila NMJs
were followed by functional and ultrastructural
assays. At synapses such as in the lamprey giant
reticulospinal axons, injections of peptides and
antibodies designed to interrupt dynamin function (see below), followed by activity-driven
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a
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*
200 nm
b
Clathrin-coated pits
SVs
SVs
Plasma
membrane
Figure 5
The accumulation of clathrin-coated pits at nerve terminals during activity in
the absence of dynamin-1. Strong evidence for synaptic vesicle (SV)
endocytosis normally proceeding through a clathrin-coated pit pathway has
been obtained from a number of experiments in which molecular perturbations
led to trapping vesicle endocytosis at an intermediate stage. Here, an example
of the ultrastructure of a hippocampal synapse derived from a dynamin-1
knockout mouse taken from Ferguson et al. (2007) is shown. (a) Single section
of a tomogram taken from a synapse where spontaneous activity led to the
development of profound invaginations studded with clathrin-coated profiles.
The small arrows indicate the presence of interconnected clathrin-coated buds,
the asterisk shows an evagination of an adjacent cell into this nerve terminal,
and the large arrow shows a small cluster of heterogeneously sized synaptic
vesicles. (b) 3D tomographic reconstruction shows that these clathrin-coated
profiles were associated with plasma membrane invagination.
vGlut: vesicular
glutamate transporter
144
exocytosis, led to accumulation of clathrincoated buds (Shupliakov et al. 1997). Similar accumulations were observed in hippocampal neurons when the abundant brain-specific
isoform, dynamin-1, was genetically ablated
(Figure 5). Additionally, peptide injections designed to interfere with clathrin assembly led
to dramatic slowing of the kinetics of endocytosis ( Jockusch et al. 2005) as well as to activitydependent interruption of exocytosis (Gad et al.
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2000; Morgan et al. 2000, 2003), consistent with
a depletion of available vesicles due to a block in
endocytosis. At hippocampal synapses, the kinetics of endocytosis can be dramatically slowed
when clathrin is removed by siRNA (Granseth
et al. 2006). Finally, photoinactivation of fluorescently labeled clathrin, which offers the
most temporally precise molecular interruption, showed that acutely inactivating clathrin’s
inability to assemble in Drosophila NMJs undergoing exocytosis leads to the formation of
large dead-end vacuoles (Heerssen et al. 2008,
Kasprowicz et al. 2008) and the loss of any further vesicle recycling. Thus, data using several
distinct approaches and examining multiple different types of synapses all argue that clathrin
assembly normally plays a critical role in SV
endocytosis.
MECHANISMS FOR SORTING
THE APPROXIMATELY NINE
CARGO PROTEIN TYPES
An important unsolved question in SV recycling is, how is each of the nine different types
of SV transmembrane proteins resorted into
SVs following exocytosis? As retrieval occurs
through clathrin-mediated endocytosis, the
expectation is that known mechanisms for
receptor-mediated endocytosis will be manifest
here. For instance, perhaps there are interactions of sorting motifs in vesicle protein cytoplasmic tails with the plasma membrane adaptor
protein complex AP2. AP2, a heterotetrameric
complex that additionally binds clathrin, is
thought to coordinate clathrin assembly with
cargo recognition during endocytosis (Keen
1987). At present, only a single putative sorting
motif has been functionally identified in SV
proteins. Mutation of a dileucine-like motif in
the vesicular glutamate transporter (vGlut1)
slows vGlut1 internalization at nerve terminals
(Voglmaier et al. 2006). Synaptotagmin has
also been identified as a binding partner to
AP2 (Grass et al. 2004, Haucke & De Camilli
1999, Haucke & Krauss 2002, Haucke et al.
2000, Zhang et al. 1994). In the absence
of Synaptotagmin 1 ( Jorgensen et al. 1995,
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Nicholson-Tomishima & Ryan 2004, Reist
et al. 1998), or following either photoinactivation (Poskanzer et al. 2003) or mutation in
residues in its C2B domain (Poskanzer et al.
2006), endocytosis of SVs is impaired. Surprisingly, deletion of the AP2 μ2 subunit fails to
eliminate cargo retrieval and SV recycling in
both Caenorhabditis elegans (Gu et al. 2008) and
dissociated hippocampal neurons (Kim & Ryan
2009). In the absence of μ2 , however, there
was a significant loss of SVs and endocytosis
kinetics were measurably slower, implying that
AP2 normally does play a role at synapses.
These results imply that additional sorting
mechanisms must be operating and open up the
possibility that AP2 serves a more subtle role
in SV endocytosis than originally anticipated.
A leading candidate adaptor for dedicated
SV protein sorting is stonin, first identified
as a temperature-sensitive paralytic mutant in
Drosophila (where it is named Stoned ) more
than 35 years ago (Grigliatti et al. 1973, Kelly
1983). Stonin 2, which contains a μ2 homology domain (Figure 6), interacts genetically
(Fergestad & Broadie 2001, Phillips et al. 2000)
and biochemically ( Jung et al. 2007, Walther
et al. 2004) with synaptotagmin. Removal of
Stoned B in Drosophila NMJs leads to defects in
endocytosis (Stimson et al. 2001) and, when coexpressed with synaptotagmin in nonneuronal
cells, it facilitates synaptotagmin retrieval (Diril
et al. 2006).
Evidence for defects in cargo sorting in SV
endocytosis has also emerged from examination of mutations in proteins associated with
clathrin assembly. In particular, deletion of the
monomeric adaptor protein AP180 led to mislocalization of VAMP2 but not synaptotagmin or synaptogyrin (Nonet et al. 1999, Zhang
et al. 1998), suggesting a specific role in sorting
VAMP2 during endocytosis. In general, these
clathrin-associated sorting proteins usually associate with both AP2 and clathrin for proper
function (Brett & Traub 2006). The fact that
endocytosis and protein sorting can occur without AP2 suggests that redundant mechanisms
that link sorting proteins, cargo, and coated pits
must be operational.
One proposed scenario for efficiently recapturing SV proteins is that they remain stably associated after exocytosis and directly nucleate clathrin-coat formation. Although this
may occur for certain SV proteins, this is not
the case for VAMP2 or synaptotagmin. At hippocampal nerve terminals, these proteins diffuse onto the axonal surface (Li & Murthy
2001, Sankaranarayanan & Ryan 2000), and
they exchange with surface counterparts during endocytosis (Fernandez-Alfonso et al. 2006,
Wienisch & Klingauf 2006). Currently, the issue of how SV proteins are retargeted into SVs
during endocytosis remains largely unresolved.
PIP2 : phosphoinositol-(4,5)-bisphosphate
CURVING MEMBRANES:
INITIATION OF SYNAPTIC
VESICLE ENDOCYTOSIS
In addition to recollecting and sorting SV proteins, the endocytosis machinery must locally
curve the plasma membrane and eventually
pinch off 3000 to 5000 nm2 of bilayer in the
form of a 30- to 40-nm-diameter vesicle. This
process can be broken into two parts: deformation of membrane into a vesicular or tubular
shape and scission of the stalk connecting this
structure to the plasma membrane.
The process of membrane deformation begins with the organization of endocytic coat
proteins on the inner leaflet of the synaptic
membrane. This assembly is thought to be
driven in part by the generation of phosphoinositol-(4,5)-bis-phosphate (PIP2 ), which recruits several endocytic proteins to the plasma
membrane including AP2, epsin, dynamin,
endophilin, amphiphysin, and AP180. At
synapses, activity-dependent PIP2 synthesis
is largely carried out by phospho-inositol-5kinase Iγ (Di Paolo et al. 2004), which presumably drives rapid recruitment of endocytic
factors during repetitive exocytosis. In addition to providing a scaffold for concentrating
appropriate cargo proteins destined for endocytosis, endocytic coat proteins are thought
to regulate the area destined for internalization in a manner that leads to a precise SV
size.
www.annualreviews.org • Synaptic Vesicle Endocytosis
145
ANTH
N
NPF
AP2/Clath
Positive
feedback
+
Avidity
+ + + +
SH3
C
C
C
MHD
C
+ + + +
+
• Cargo binding
• Curvature
SHD
Dynamic properties
N-BAR
Amphiphysin
N
AP2/Clath
ENTH
AP180
N
Epsin
N
NPF
NPF
Stonin 2
NPF
AP2/Clath
Clathrin
NPF
EH
EH
EH
EH
N-BAR
F-BAR
EH
EH
Sac1p
N
Stonin 1
N
ANTH
EVH1
CALM
N
N-WASP
N
PH
EH
GBD/CRIB
5' p’ase
Synaptojanin
N GTPase
Dynamin
N
Syndapin
N
Endophilin
N
Eps15
N
Intersectin 1L
N
Intersectin 1S
NPF
Primary scaffold
Primary effectors
AP2/Clath
SH3
GED
NPF
AP2/Clath
NPF
Core module
C
PRD
C
C-C
SHD
PRD
PRD
SH3
C-C
C-C
MHD
C
SH3
SH3
C
VCA
C
BARS
SH3
SH3
NPF
AP2
NPF
AP2/Clath
C
C
C
SH3
SH3
PH
+ + + +
DH
C
C2
C
+ + + +
+
+ + + +
+
Secondary effectors
SH3
SH3
PRDs
SH3
SH3
Secondary scaffolds
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AP2/Clath
Dittman
NPF
146
AP2/Clath
GDP + Pi
GTP
C-C
SH3
EH
C-C
PRD
Protein-protein interactions
F-BAR
C2
ENTH
N-BAR
PH
ANTH
Lipid-binding domains
GTPase
5' p’ase
PI(4)P + Pi
Sac1p
PIP2
Enzymatic domains
Domain properties
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On the basis of biochemical, structural,
and genetic evidence, several protein domains
have been implicated in the curving of membranes. ENTH (epsin N-terminal homology)
domains found at the amino termini of epsin,
AP180, and CALM are phospholipid-binding
modules (Hurley 2006, Itoh & De Camilli
2006). ENTH domains interact preferentially
with PIP2 -containing phospholipids and tubulate liposomes in vitro as well as plasma membranes of living cells when overexpressed (Ford
et al. 2002, Itoh et al. 2001). Structural studies of ENTH domains suggest that an Nterminal amphipathic alpha helix inserts into
the lipid bilayer upon binding to PIP2 (Itoh
et al. 2001). The canonical example of an
ENTH domain protein at the synapse is epsin.
Thought to function as a clathrin adaptor, epsin
binds clathrin heavy chain and AP2. These
associations position epsin as part of the endocytic core module. Epsin contains several
NPF (asparagine-proline-phenylalanine) motifs near its carboxy terminus that interact with
Eps15 homology (EH) domain modules found
in the secondary scaffolds Eps15 and intersectin/Dap160 (Figure 6). Epsin also contains
ubiquitin-interacting motifs, which may function in the recognition of ubiquitinated cargo
proteins. In yeast, expression of the ENTH
domain is sufficient to rescue viability of the
Ent1/2 double mutant (the two yeast epsin orthologs), suggesting that this domain has an
essential role irrespective of its adaptor functions (Wendland et al. 1999). Under some conditions, epsin and clathrin triskelia, but not
clathrin alone, can invaginate lipid monolayers
(Ford et al. 2002). Disruption of epsin function by antibody binding in the lamprey giant synapse decreased SV numbers and led to
an accumulation of large coated pits following stimulation ( Jakobsson et al. 2008). These
data suggested that SV endocytosis was trapped
at an early stage in the absence of functional
epsin. In contrast, loss of the Drosophila epsin
ortholog Liquid Facets did not appear to impair SV endocytosis, although synapse architecture was significantly affected (Bao et al.
2008). Epsin is also found in other cellular
compartments, suggesting that it may have a
general role in clathrin-mediated endocytosis
(Horvath et al. 2007).
AP180 and CALM share an ENTH-like
module at their amino termini along with
clathrin and AP2-interacting domains within
their unstructured carboxy termini. However,
interaction of these proteins with PIP2 is distinct from epsin’s ENTH domain interaction
and the N-terminal domain contains additional alpha helices (Itoh & De Camilli 2006).
These biochemical and structural differences
led investigators to call these ANTH (AP180
N-terminal homology) domains. ANTH domains do not appear to deform lipids on
their own, but they may influence curvature indirectly through interactions with other
membrane-binding proteins. AP180 is predominantly found at presynaptic terminals,
whereas CALM is ubiquitous and also possesses
multiple NPF motifs (which are not found
in AP180) near its carboxy terminus (Itoh &
De Camilli 2006). C. elegans and Drosophila
each have a single AP180/CALM ortholog
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Figure 6
Categories of endocytosis proteins. Schematic diagrams of protein domain organization are shown for the core module (AP2, clathrin,
stonin 2, epsin, AP180, and amphiphysin), secondary scaffolds (intersectin and Eps15), and secondary effectors (endophilin, syndapin,
dynamin, synaptojanin, N-WASP, CALM, and stonin 1). The domain abbreviations are as follows: PH, pleckstrin homology domain;
ANTH, AP180 N-terminal homology domain; ENTH, epsin N-terminal homology domain; BAR, bin amphiphysin rvs; EH, eps15
homology domain; PRD, proline-rich domain; C-C, coiled-coil domain. NPF represents any of five possible motifs that interact with
EH: NPF, WW, FW, SWG, and H(ST)F. AP2/Clath represents two AP2-binding motifs [WXXF and DP(WF)] and three clathrinbinding motifs [D(LI)(LFQ), L(FWY)X(FWY)(DE), and PWXXW]. The diagram depicts these as one localized domain, but these
motifs are actually distributed throughout the polypeptide. Two isoforms of intersectin are shown: long and short. Dynamic properties
are depicted as either positive feedback (polymerization, enzymatic catalysis) or avidity (multiple binding sites linked
together).
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that presumably fulfills the duties of both proteins. In both of these model systems, loss of
AP180/CALM substantially broadened the distribution of SV sizes and increased the mean diameter by over 30% (Nonet et al. 1999, Zhang
et al. 1998). In addition, AP180 and CALM
regulate the plasma membrane abundance of
synaptobrevin (Dittman & Kaplan 2006, Harel
et al. 2008, Nonet et al. 1999, Zhang et al.
1998), suggesting that ANTH domain proteins play a role in both cargo recruitment and
size of the endocytic area destined to undergo
endocytosis.
Another membrane binding module employed during SV endocytosis is the BAR (Bin,
amphiphysin, Rvs) domain (Figure 6). BAR
domains found in endophilin, amphiphysin,
syndapin, and sorting nexin 9, among others,
bind phospholipids as crescent-shaped dimers,
and they are thought to generate or stabilize highly curved regions of the membrane
(Gallop & McMahon 2005, Itoh & De Camilli
2006, Ren et al. 2006). In vitro, these domains can tubulate liposomes and, in some
cases, appear to bind preferentially to particular liposome sizes, suggesting that the BAR
domain can sense lipid curvature (Gallop &
McMahon 2005). Amphiphysin and endophilin
share amino-terminal domains that form amphipathic helices capable of penetrating the cytoplasmic bilayer leaflet and perturbing lipid
packing, particularly in the presence of acidic
phospholipids. Thus, their N-terminal helix
and BAR motif are together termed N-BAR
domains. Both amphiphysin and endophilin
contain carboxy-terminal SH3 domains, which
are thought to interact with the proline-rich
domains of dynamin and synaptojanin (Itoh
& De Camilli 2006). The combination of
N-BAR and SH3 domains makes these proteins ideally suited for the dual task of deforming membranes and recruiting other endocytic
molecules to the curved region. Amphiphysin
additionally has clathrin- and AP2-binding motifs, linking it to the core endocytic machinery.
The mouse knockout of amphiphysin I displayed a modest decrease in recycling SV pool
size as well as a slower recycling rate (Di Paolo
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et al. 2002). Fly mutants lacking amphiphysin
did not appear to have significant transmission
defects at the NMJ, and nervous system function is largely intact (Razzaq et al. 2001, Zelhof
et al. 2001). Similarly, neither deletion nor RNA
interference knockdown of the worm ortholog
AMPH-1 impacts nervous system function
appreciably ( J. Kaplan and J. Bai, personal communication). However, loss of endophilin significantly impairs synaptic transmission in both
flies and worms (Rikhy et al. 2002, Schuske
et al. 2003, Verstreken et al. 2002). The SV pool
is decreased, whereas SV diameter and quantal size are increased. Ultrastructural similarities between endophilin and synaptojanin mutants, such as accumulations of clathrin-coated
pits and vesicles, queues of vesicles far from
the active zone, and large cisternae, support
the hypothesis that endophilin and synaptojanin act together at a similar stage during endocytosis (Schuske et al. 2003, Verstreken et al.
2003).
Syndapin, a member of another class of BAR
domain known as F-BAR (formerly known as
extended FCH) domain proteins, may play a
role during SV endocytosis, particularly during
prolonged or intense stimulation (Andersson
et al. 2008). Also known as PACSIN, syndapin possesses an N-terminal F-BAR domain,
several NPF motifs, and a C-terminal SH3
domain, which interacts with dynamin and
N-WASP (Anggono & Robinson 2007, Kessels
& Qualmann 2004). Syndapin binds to EH
domain proteins via its NPF motifs and participates in trafficking through the recycling
endosome pathway (Braun et al. 2005). Fly syndapin mutants failed to display any significant
defects in synaptic transmission or SV endocytosis (Kumar et al. 2009). However, antibodies
against syndapin caused enhanced synaptic depression and large VAMP2-containing cisternae after sustained stimulation when injected
into lamprey giant synapses (Andersson et al.
2008). Because no effect was observed at low
stimulus frequencies, syndapin may be part of
a secondary pathway for retrieving SV components through endocytic intermediates following prolonged bouts of activity.
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SECONDARY SCAFFOLDING
FOR RECRUITING GENERAL
ENDOCYTIC EFFECTORS
The proteins described thus far can be
organized either into a core endocytic module (clathrin, AP2, AP180, stonin 2, epsin,
amphiphysin) or into secondary effectors
(endophilin and synaptojanin; others mentioned below) based on their interactions (or
lack thereof ) with vesicle cargo molecules or
cargo-binding proteins. Two synaptic proteins
bridge this gap between the core module and
secondary effectors, acting as recruiters and
organizers during SV endocytosis. Eps15 and
intersectin/Dap160 are binding partners and
each has highly conserved domain structures
coupling the endocytic coat to secondary
effectors (Montesinos et al. 2005). Eps15 is a
110-kDa protein containing three N-terminal
EH domains, a central coiled-coil domain, and
clathrin/AP2 binding motifs near its carboxy
terminus (Morgan et al. 2003, Salcini et al.
2001). The EH domains mediate interactions
with NPF motifs in epsin, stonins, AP180, and
synaptojanin, whereas the coiled coil interacts with intersectin (Kelly & Phillips 2005,
Morgan et al. 2003). Loss of Eps15 in C. elegans
results in decreased SV number, particularly at
elevated temperatures (Salcini et al. 2001). The
Eps15 mutant ehs-1(ok146) genetically interacts
with the worm temperature-sensitive dynamin
mutant dyn-1(ky51) such that locomotion is
severely impaired in the double mutant even at
permissive temperatures (Salcini et al. 2001).
Eps15 also appears to enhance AP180-mediated
clathrin assembly in vitro, suggesting that it
may have an early role in building clathrin pits
(Morgan et al. 2003). The loss of Eps15 in
Drosophila causes substantial decrease in synaptic intersectin, dynamin, stonin, synaptotagmin
I, α adaptin, and endophilin (Koh et al. 2007).
Intersectin/Dap160 is a synaptic protein
containing two N-terminal EH domains, a central coiled-coil domain, and four to five SH3 domains (depending on the species) at its carboxy
terminus (Koh et al. 2004, Marie et al. 2004).
A long isoform in vertebrates also contains
Dbl homology domain, a pleckstrin homology
domain, and a C2 domain after the SH3 repeat. This module can act as a guanine nucleotide exchange factor for Cdc42, regulating
actin dynamics through Cdc42 and N-WASP
(Hussain et al. 2001). Interestingly, Drosophila
and C. elegans intersectin orthologs do not contain this module. The EH domains interact
with epsin; the coiled coil binds Eps15; and the
SH3 domains bind to dynamin, synaptojanin,
and N-WASP (Marie et al. 2004, Montesinos
et al. 2005). The loss of intersectin in the fly results in diminished SV endocytosis; decreased
synaptic levels of AP180, endophilin, synaptojanin, and dynamin; and increased synaptic
depression during prolonged stimulation (Koh
et al. 2004, Marie et al. 2004). Eps15 intersectin double mutants exhibit synaptic depression and decreased FM1-43 uptake to a similar degree as either single mutant (Koh et al.
2007). Microinjection of the intersectin SH3
domain in lamprey giant synapses inhibits SV
recycling and traps a large number of coated pits
in the periactive zone (Evergren et al. 2007).
Furthermore, intersectin appears to negatively
regulate recruitment of dynamin to periactive
zones at this synapse. In C. elegans, ITSN-1
localizes to the periactive zone in NMJs, and
loss of ITSN-1 reveals a substantial increase in
large, irregular vesicular structures as well as
a decrease in spontaneous transmitter release
(Rose et al. 2007, Wang et al. 2008).
FINISHING THE JOB:
MEMBRANE FISSION
AND COAT DISASSEMBLY
The final step of endocytosis involves severing
the endocytic bud from the plasma membrane.
In vitro studies have shown that when artificial
membrane necks are narrowed to sufficiently
small diameter, thermal fluctuations result
in a collapse of the neck, presumably the
starting point for fission (Bashkirov et al. 2008,
Israelachvili 1992). Dynamin, a mechanochemical GTPase, was originally identified as the
gene product of the Drosophila shibire mutant,
a temperature-sensitive paralytic that becomes
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Clathrin/AP2 binding motif
SH3–PRD interaction
EH–NPF interaction
Other protein interactions
PI(4,5)P2
Phospholipid
binding
Cytoskeleton
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Second scaffold
Scaffold
?
Core module
Clathrin
?
Cargo
selector
Membrane
binding
Secondary scaffold
Secondary effectors
Eps15, Intersectin
Stonin 1, CALM
Syndapin
AP2, stonin 2, AP180
Dynamin
N-WASP
Epsin, amphiphysin
Synaptojanin
Endophilin
?
Figure 7
Synaptic vesicle endocytic network. The proteins that compose the endocytic machinery at the synapse are
classified in two ways: location in the network (color) and functionality (shape). Blue proteins make up the core
module, red proteins are secondary scaffolding, and green proteins are secondary effectors. Proteins found
exclusively at the synapse are shown in bold type. Scaffolds are hatched lines, cargo binders are triangles,
membrane benders are crescents, and secondary effectors are diamonds. Interactions between proteins are
depicted as arrow connectors for clathrin/AP2 binding, SH3 to PRD binding, Eps15 homology domain to
NPF binding, and other protein interactions (see figure key for arrow types). Membrane interactions are
shown as downward gray arrows, and the PIP2 phosphatase synaptojanin is shown interacting with PIP2 (red
arrow). Endophilin ( purple) has no known interactions with either the core module or the secondary scaffold
and is therefore distinguished from the other secondary effectors.
devoid of SVs during activity at the nonpermissive temperature (Koenig & Ikeda 1989). This
protein, which is recruited to the endocytic
bud neck, is the minimal essential fission
machinery in vitro, provided the membranes
undergoing fission are under tension (Roux
et al. 2006). Dynamin interacts with a number
of SH3-containing proteins such as endophilin,
amphiphysin, syndapin, and intersectin via
its proline-rich domain (Figures 6 and 7).
Together with other BAR domain proteins, dynamin also appears to coordinate the shaping
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of endocytic buds (Itoh et al. 2005). The
evidence for dynamin’s role in endocytosis at
the synapse is very strong as many dominantnegative strategies targeting dynamin block SV
endocytosis (Koenig & Ikeda 1999, Newton
et al. 2006, Yamashita et al. 2005). Surprisingly, genetic ablation of dynamin-1, which
encodes ∼85% of brain dynamin, resulted in
only partial defects in SV endocytosis. Mice
lacking dynamin-1 survive about two weeks
(Ferguson et al. 2007). Under these conditions,
endocytosis appeared to be completely arrested
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only during intense activity, presumably owing
to the elevation of intracellular calcium. However, soon after cessation of action potential
firing, endocytosis resumed with remarkably
normal kinetics. Although synapses lacking
dynamin-1 in mouse still contain a small
amount of dynamin-3 and even less dynamin-2
(Ferguson et al. 2007), one possible conclusion
from this work is that the fission step in
endocytosis is not catalyzed by the action of
dynamin alone, but it is simply accelerated by
this protein.
Once the endocytic vesicle is severed from
the plasma membrane, the newly sculpted
vesicle must shed all the machinery that was
used in rebuilding prior to reuse. Clathrin uncoating occurs via the concerted action of the
ATPase Hsc70 and auxilin, which helps recruit
this enzyme to the vesicle (Eisenberg & Greene
2007). One important mystery is the identity
of the molecular trigger to initiate uncoating,
as it would be energetically costly and perhaps detrimental to launch this process prior
to fission. A mechanistic clue has arisen from
genetic ablation studies of synaptojanin: The
loss of this lipid phosphatase leads to a pronounced accumulation of clathrin-coated vesicles at nerve terminals as well as a delay in the
availability of newly endocytosed vesicles for
additional rounds of exocytosis (Cremona et al.
1999, Mani et al. 2007, Schuske et al. 2003,
Verstreken et al. 2003). Synaptojanin is stabilized at endocytic buds through multiple interactions including binding to the SH3 domains
of intersectin and endophilin and the EH domains of intersectin and Eps15, as well as direct binding to clathrin and AP2 (Haffner et al.
1997, Marie et al. 2004, Montesinos et al. 2005,
Schuske et al. 2003, Verstreken et al. 2003). The
5 phosphatase domain at the amino terminus
of synaptojanin rapidly dephosphorylates PIP2.
This depletion of PIP2 decreases the phospholipid binding affinity of the core module and
BAR proteins, simultaneously destabilizing the
coat assembly. Another possible coupling between membrane fission and synaptojanin arises
from the theoretical consideration that local depletion of PIP2 selectively on the vesicle bud
causes a transient gradient of PIP2 between
the bud and the plasma membrane to which it
is connected. If this gradient contributes to a
lipid phase separation between these compartments, the interfacial line between the neck and
bud tends to be minimized, spontaneously constricting the neck (Liu et al. 2006). Regardless of
the details of fission and uncoating, these processes are quite rapid as evidenced by the difficulty of capturing a coated vesicle, even when
rapid freeze EM is used.
CALCIUM SENSORS FOR
ENDOCYTOSIS?
As described, presynaptic calcium affects numerous aspects of SV endocytosis. However,
unlike SV exocytosis, in which a single protein (synaptotagmin 1) accounts for much of the
calcium dependence of SV fusion (Chapman
2008), it is not clear where calcium acts during endocytosis. Nearly every endocytic protein
interacts with the membrane or directly binds
to a lipid-binding protein. Because elevated
calcium can alter the energetics of these membrane interactions, it is plausible that presynaptic calcium functions to change the efficiency of endocytosis through general effects on
protein-lipid interactions rather than through a
dedicated calcium sensor. However, it is worth
noting that synaptotagmin 1 interacts with the
core module and its C2 domains may affect
this interaction, at least in regimes where calcium is highly elevated (Diril et al. 2006, Jung
et al. 2007, Mohrmann et al. 2008, Walther
et al. 2001). Multiple lines of evidence support a role for synaptotagmin in endocytosis.
Another candidate mechanism for calcium in
endocytosis is through the dephosphins, endocytic proteins such as dynamin, synaptojanin,
and amphiphysin, that are dephosphorylated
in a calcium-dependent manner by the phosphatase calcineurin (Cousin & Robinson 2001).
The significance of these dephosphorylation
events has not been fully delineated, although
dephosphorylation of dynamin promotes its
interaction with syndapin (Anggono et al.
2006). Dephosphins seem to play a greater role
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during conditions that lead to bulk-endocytosis
(Clayton et al. 2007), which may happen only
rarely under physiological stimuli.
A ROLE FOR ACTIN DURING
SYNAPTIC VESICLE
ENDOCYTOSIS?
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The regulation of actin polymerization is a
vital part of membrane remodeling and intracellular trafficking, and multiple endocytic proteins mentioned above have a direct impact
on actin and the cytoskeleton. For example,
the secondary effectors dynamin, syndapin, and
N-WASP, as well as intersectin, all connect directly or indirectly to proteins that control actin
polymerization (Hussain et al. 2001, Kessels
& Qualmann 2004, Koh et al. 2004, Miki &
Takenawa 2003, Shin et al. 2007). In yeast,
actin plays an essential role in endocytosis
(Kaksonen et al. 2006). In vitro membrane scission by dynamin can utilize actin as an anchor to
transduce its force on tubules into longitudinal tension (Roux et al. 2006). However,
actin does not appear to be important for
SV endocytosis per se in hippocampal neurons (Sankaranarayanan et al. 2003), but
it may contribute as a secondary scaffold
(Figure 7). Interestingly, actin seems to have
a more pronounced role in the endocytosis of SVs at a giant synapse (Brodin &
Shupliakov 2006, Shupliakov et al. 2002).
Perhaps the cytoskeletal connections to endocytosis depend on the geometry and size of a
synaptic terminal, particularly because regulation of the cytoskeleton is likely to be a major
determinant of synapse size.
REBUILDING SYNAPTIC
VESICLES: A SINGLE-PASS
SORTING AT THE PLASMA
MEMBRANE?
One of the assumptions we have made in
discussing vesicle recycling is that the sorting
of SV proteins occurs in a single endocytic
step, as opposed to having an intermediate
organelle after endocytosis at the plasma
membrane makes passage through a recycling
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endosome for further cargo sorting (Koenig
& Ikeda 1996). The existence of such a recycling endosome remains a formal possibility,
although very few experiments have provided
definitive evidence for any functional significance. In the earliest studies of Heuser &
Reese (1973), they identified a nonvesicular
horse radish peroxidase–positive compartment
that appeared only transiently following
stimulation. More recent experiments in which
serial-sectioning electron microscopy was
performed demonstrated that, during intense
stimulation, deep invaginations of the plasma
membrane often form, which can easily be
confused with internal organelles in any single
section (e.g., see Figure 5), but, in fact, remain
topologically connected to the cell surface
(Ferguson et al. 2007). Nonetheless, an endosomal intermediate may play a role under some
stimulus conditions, particularly following
intensive stimulation, when bulk endocytosis
has been reported to operate at a number of
synapses (Clayton et al. 2008, Holt et al. 2003).
A MOLECULAR PICTURE FOR
THE ENDOCYTIC MACHINE
Ideally, the molecules described here can
be mapped to the phenomenology of SV
endocytosis on the basis of their intermolecular
interactions, genetics, structure, and cell
biological roles in other forms of endocytosis.
A host of studies published over the past
decade have made apparent a rough version
of this molecular map and the dynamics of the
endocytic machinery. The core module couples
nucleation of an endocytic coat with cargo
recruitment. Polymerization of clathrin and
membrane interactions with ENTH and
N-BAR domains rapidly commence the
process of curving the lipid bilayer at the
site of endocytosis. Furthermore, assembly
of the core may increase the avidity of cargo
binding because a multiplicity of binding sites
will be brought into a small region of the
membrane. In this scenario, cargo capture,
scaffold assembly, and membrane curvature are
highly coupled coordinated events.
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The N-BARs endophilin and amphiphysin
contribute to membrane bending and may
initiate an additional positive-feedback cycle
of binding to curved membrane and selfassembly, driving further membrane curvature.
Amphiphysin has direct binding interactions
with clathrin and AP2 (placing it in the core
module), whereas endophilin has no known
interaction with these or other proteins in
the core module (qualifying endophilin as a
secondary effector). The secondary scaffold
molecules Eps15 and intersectin/Dap160 bind
to the core module and provide additional
binding sites for recruiting the secondary
effector molecules: dynamin, synaptojanin,
and N-WASP. Both dynamin and synaptojanin
convey amplification of a sort within the endocytic complex. Dynamin self-assembles and
can utilize some of the energy released by polymerizing to deform membranes. Synaptojanin
contains a phospholipase enzymatic domain,
and thus a small number of synaptojanins
would be expected to deplete PIP2 rapidly
from the membrane. These effectors terminate
the endocytic process by irreversibly severing
the membrane connection and releasing the
endocytic coat with the help of Hsc70 and
auxilin. The AP180 paralog CALM, the stonin
2 paralog stonin 1, and the F-BAR syndapin
may also contribute to SV endocytosis, but
their specific requirements are not well defined.
We therefore include these components as
potential secondary effectors (Figures 6 and 7).
NETWORK PROPERTIES OF THE
ENDOCYTIC MACHINE
There are several advantages to considering the
endocytic proteins as a network (Alon 2007,
Hintze & Adami 2008, Schmid & McMahon
2007). First, fault-tolerant networks exhibit
graceful degradation, because the failure of one
component has little effect on the overall performance. Evidence to date suggests that SV
endocytosis continues after the removal of any
one protein. Second, weak interactions can be
transiently strengthened and amplified by positive feedback within the network. Thus, the
binding of cargo; polymerization of the core
module; and membrane bending by N-BARs,
epsins, and dynamin all collaborate to create
a fast, high-fidelity endocytic process. Third,
modularity within the network allows the basic
endocytic machinery to be utilized in many separate types of trafficking processes within a neuron. For instance, most of the SV endocytosis
machinery is likely to be involved in postsynaptic receptor trafficking, intracellular trafficking
between organelles, and membrane remodeling during synapse development (Bushlin et al.
2008, Gong & De Camilli 2008, Itoh & De
Camilli 2006, Koh et al. 2007, Montesinos et al.
2005). Another consequence of a densely connected network is that the removal of one protein can have repercussions on the stability of
many others. For instance, in fly Eps15 mutant synapses, many of the core proteins have
significantly decreased abundance along with
the Eps15-binding partner intersectin/Dap160
(Koh et al. 2004, 2007). This interdependence
of protein stability emphasizes the importance
of surveying the abundance of all endocytic proteins following a molecular deletion.
Parsing the endocytic proteins into three
layers (Figure 7) is not absolute in that members of the core module and the N-BARs also
assist in recruiting secondary effectors (Schuske
et al. 2003, Takei et al. 1999, Verstreken et al.
2003), whereas Eps15 and intersectin also help
stabilize the core module (Koh et al. 2007,
Morgan et al. 2003). The relative impact of removing particular proteins does not necessarily
correspond to the location within this endocytic
network. For instance, the core module proteins AP2, clathrin, and amphiphysin all have a
relatively small effect on SV endocytosis when
removed individually (Di Paolo et al. 2002, Gu
et al. 2008, Sato et al. 2009). These observations would be predicted in a redundant network such as this, because secondary scaffolds
and effectors functionally compensate for the
loss of these core proteins. Perhaps double deletions that specifically target redundant function
may have a more dramatic effect on SV endocytosis. For example, removal of clathrin and
intersectin simultaneously would be predicted
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to impact the scaffolding function of the network. Concomitant removal of ENTH- and
BAR-domain proteins may eliminate the ability
to deform the membrane.
CONCLUDING REMARKS
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In the half century following Katz’s quantum
hypothesis at the nerve terminal (Del Castillo
& Katz 1954), advances in molecular, imaging,
and physiological techniques have conspired
to produce a fairly detailed understanding of
the SV cycle. The process of manufacturing,
trafficking, and recycling SVs involves the
coordinated efforts of hundreds of proteins
discovered over the past few decades. As the list
of molecular players grows, so does the need
to synthesize and organize this collection. As
with many aspects of modern cell biology, the
detailed properties of SV endocytosis may be
emergent properties of a network. One of the
impediments to understanding this emergence
is that we still lack in-depth information about
the precise location of most of these molecules
during various stages of the SV cycle. Quantitative studies of the dynamic interactions, as well
as application of new super-resolution techniques, should provide a rich new level of detail
to understand the functioning of the endocytic
network.
SUMMARY POINTS
1. Clathrin-mediated endocytosis accounts for the majority of SV endocytosis under normal conditions, whereas bulk endocytosis may contribute following high-frequency
stimulation.
2. SV proteins are endocytosed stochastically such that their dwell time on the plasma
membrane obeys an exponential distribution with a 6-s time constant at 37◦ C (13-s time
constant at room temperature).
3. The proteins of endocytosis can be organized in a simple hierarchical network based on
protein interactions, domains, and genetic ablation studies.
4. No single component of the SV endocytosis machinery appears to be essential: The
network of endocytic proteins appears robust to these molecular perturbations.
DISCLOSURE STATEMENT
The authors are not aware of any biases that might be perceived as affecting the objectivity of this
review.
ACKNOWLEDGMENTS
The authors apologize in advance to all the investigators whose research could not be appropriately
cited owing to space limitations. We thank members of the Ryan and Dittman labs for useful
discussions and suggestions.
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Contents
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Chromosome Odds and Ends
Joseph G. Gall p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Small RNAs and Their Roles in Plant Development
Xuemei Chen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p21
Annual Review
of Cell and
Developmental
Biology
Volume 25, 2009
From Progenitors to Differentiated Cells in the Vertebrate Retina
Michalis Agathocleous and William A. Harris p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p45
Mechanisms of Lipid Transport Involved in Organelle Biogenesis
in Plant Cells
Christoph Benning p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p71
Innovations in Teaching Undergraduate Biology
and Why We Need Them
William B. Wood p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p93
Membrane Traffic within the Golgi Apparatus
Benjamin S. Glick and Akihiko Nakano p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 113
Molecular Circuitry of Endocytosis at Nerve Terminals
Jeremy Dittman and Timothy A. Ryan p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 133
Many Paths to Synaptic Specificity
Joshua R. Sanes and Masahito Yamagata p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 161
Mechanisms of Growth and Homeostasis in the Drosophila Wing
Ricardo M. Neto-Silva, Brent S. Wells, and Laura A. Johnston p p p p p p p p p p p p p p p p p p p p p p p p p 197
Vertebrate Endoderm Development and Organ Formation
Aaron M. Zorn and James M. Wells p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 221
Signaling in Adult Neurogenesis
Hoonkyo Suh, Wei Deng, and Fred H. Gage p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 253
Vernalization: Winter and the Timing of Flowering in Plants
Dong-Hwan Kim, Mark R. Doyle, Sibum Sung, and Richard M. Amasino p p p p p p p p p p p p 277
Quantitative Time-Lapse Fluorescence Microscopy in Single Cells
Dale Muzzey and Alexander van Oudenaarden p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 301
Mechanisms Shaping the Membranes of Cellular Organelles
Yoko Shibata, Junjie Hu, Michael M. Kozlov, and Tom A. Rapoport p p p p p p p p p p p p p p p p p p p p 329
The Biogenesis and Function of PIWI Proteins and piRNAs: Progress
and Prospect
Travis Thomson and Haifan Lin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 355
vii
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Mechanisms of Stem Cell Self-Renewal
Shenghui He, Daisuke Nakada, and Sean J. Morrison p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 377
Collective Cell Migration
Pernille Rørth p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 407
Hox Genes and Segmentation of the Hindbrain and Axial Skeleton
Tara Alexander, Christof Nolte, and Robb Krumlauf p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 431
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Gonad Morphogenesis in Vertebrates: Divergent Means to a
Convergent End
Tony DeFalco and Blanche Capel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 457
From Mouse Egg to Mouse Embryo: Polarities, Axes, and Tissues
Martin H. Johnson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 483
Conflicting Views on the Membrane Fusion Machinery and the Fusion
Pore
Jakob B. Sørensen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 513
Coordination of Lipid Metabolism in Membrane Biogenesis
Axel Nohturfft and Shao Chong Zhang p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 539
Navigating ECM Barriers at the Invasive Front: The Cancer
Cell–Stroma Interface
R. Grant Rowe and Stephen J. Weiss p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 567
The Molecular Basis of Organ Formation: Insights from the
C. elegans Foregut
Susan E. Mango p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 597
Genetic Control of Bone Formation
Gerard Karsenty, Henry M. Kronenberg, and Carmine Settembre p p p p p p p p p p p p p p p p p p p p p p 629
Listeria monocytogenes Membrane Trafficking and Lifestyle:
The Exception or the Rule?
Javier Pizarro-Cerdá and Pascale Cossart p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 649
Asymmetric Cell Divisions and Asymmetric Cell Fates
Shahragim Tajbakhsh, Pierre Rocheteau, and Isabelle Le Roux p p p p p p p p p p p p p p p p p p p p p p p p p p p 671
Indexes
Cumulative Index of Contributing Authors, Volumes 21–25 p p p p p p p p p p p p p p p p p p p p p p p p p p p 701
Cumulative Index of Chapter Titles, Volumes 21–25 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 704
Errata
An online log of corrections to Annual Review of Cell and Developmental Biology articles
may be found at http://cellbio.annualreviews.org/errata.shtml
viii
Contents