molecular circuitry of endocytosis at nerve...

ANRV389-CB25-07 ARI 4 September 2009 17:25

Molecular Circuitryof Endocytosis atNerve TerminalsJeremy Dittman and Timothy A. RyanDepartment of Biochemistry, Weill Cornell Medical College, New York, NY 10065;email: [email protected], [email protected]

Annu. Rev. Cell Dev. Biol. 2009. 25:133–60

First published online as a Review in Advance onJuly 8, 2009

The Annual Review of Cell and DevelopmentalBiology is online at cellbio.annualreviews.org

This article’s doi:10.1146/annurev.cellbio.042308.113302

Copyright c© 2009 by Annual Reviews.All rights reserved

1081-0706/09/1110-0133$20.00

Key Words

synapse, vesicle cycle, trafficking, clathrin, pHluorin

AbstractPresynaptic terminals are specialized compartments of neurons respon-sible for converting electrical signals into secreted chemicals. This self-renewing process of chemical synaptic transmission is accomplishedby the calcium-triggered fusion of neurotransmitter-containing vesicleswith the plasma membrane and subsequent retrieval and recycling ofvesicle components. Whereas the release of neurotransmitters has beenstudied for over 50 years, the process of synaptic vesicle endocytosis hasremained much more elusive. The advent of imaging techniques suitedto monitor membrane retrieval at presynaptic terminals and the discov-ery of the molecules that orchestrate endocytosis have revolutionizedour understanding of this critical trafficking event.

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Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . 134THE SYNAPSE . . . . . . . . . . . . . . . . . . . . . . 134THE SYNAPTIC VESICLE:

A LOCAL CURRENCY FORINFORMATION FLOW ATTHE SYNAPSE . . . . . . . . . . . . . . . . . . . 135

SYNAPTIC VESICLE RETRIEVALAT NERVE TERMINALS . . . . . . . . 136

THE TIMING OF ENDOCYTOSISAT NERVE TERMINALS:EVIDENCE FOR MULTIPLETIME SCALES OFRETRIEVAL. . . . . . . . . . . . . . . . . . . . . . 137

MODULATION OFENDOCYTOSIS: CALCIUMAND EXOCYTIC LOAD . . . . . . . . . 140

KISS-AND-RUN: ALONG-RUNNING DEBATE. . . . . 141

THE MOLECULAR MACHINERYOF SYNAPTIC VESICLEENDOCYTOSIS. . . . . . . . . . . . . . . . . . 142

A CLATHRIN-MEDIATEDPATHWAY FOR VESICLEREBUILDING. . . . . . . . . . . . . . . . . . . . 143

MECHANISMS FOR SORTING

THE APPROXIMATELY NINECARGO PROTEIN TYPES . . . . . . . 144

CURVING MEMBRANES:INITIATION OF SYNAPTICVESICLE ENDOCYTOSIS . . . . . . . 145

SECONDARY SCAFFOLDINGFOR RECRUITING GENERALENDOCYTIC EFFECTORS . . . . . 149

FINISHING THE JOB:MEMBRANE FISSIONAND COAT DISASSEMBLY . . . . . . 149

CALCIUM SENSORS FORENDOCYTOSIS?. . . . . . . . . . . . . . . . . 151

A ROLE FOR ACTIN DURINGSYNAPTIC VESICLEENDOCYTOSIS?. . . . . . . . . . . . . . . . . 152

REBUILDING SYNAPTICVESICLES: A SINGLE-PASSSORTING AT THE PLASMAMEMBRANE? . . . . . . . . . . . . . . . . . . . . 152

A MOLECULAR PICTUREFOR THE ENDOCYTICMACHINE . . . . . . . . . . . . . . . . . . . . . . . 152

NETWORK PROPERTIES OF THEENDOCYTIC MACHINE . . . . . . . . 153

CONCLUDING REMARKS . . . . . . . . . 154

SV: synaptic vesicle

INTRODUCTION

Chemical communication between cells washarnessed for both distributing and control-ling multicellular behavior early on in thedevelopment of complex life forms. In the ner-vous system, synapses encompass a transduc-tion machine whereby propagating electricalsignals, usually in the form of action potentials,are transformed into chemical messages in theform of packets of neurotransmitters that are se-creted onto a neighboring cell, which in turn aretransduced back into both chemical and electri-cal signals in this cell. The past 60 years havewitnessed an intense scientific effort to under-stand how synapses work, how they form andare controlled, how their function changes over

time scales from milliseconds to months, andhow they are built at the molecular level. In-creasingly, as molecular detail has emerged, ithas overlapped with the recognition that this in-formation is crucial for understanding a varietyof diseased states of neuronal function. Herewe focus our attention on the first half of thesynaptic transmission problem, the cell biologyof the presynaptic nerve terminal and, in partic-ular, the current understanding of endocytosisand synaptic vesicle (SV) recycling.

THE SYNAPSE

The basic structural organization of synapticterminals appears to be relatively well con-served across evolution. Neurotransmitters are

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Fusion

Cargo captureClathrin coat assembly

Membrane curvature

Vesicle budding

Membrane fissionUncoating

Refilling, docking,priming

Cargo mixingLipid mixing

a

b

c d

e

f

Figure 1The synaptic vesicle (SV) cycle. An SV fuses with the plasma membrane (a) and is subsequently rebuiltthrough the assembly of a clathrin-coated pit (b,c) followed by budding (d ), and fission and uncoating of theendocytic coat proteins (e). The vesicle is refilled with neurotransmitter and returned to the vesicle pool forfurther 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 assembledSNARE 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 di-ameter that ranges from ∼30 to 40 nm (de-pending on the species). Typically a few dozento a few hundred SVs are maintained in acluster near the active zone, a dedicated in-tracellular location on the presynaptic plasmamembrane where SVs, upon the arrival of anaction potential stimulus, will fuse with theplasma membrane and release their contentthrough exocytosis (Figure 1). These prefer-ential sites of exocytosis are typically foundjuxtaposed to a postsynaptic specialization ofneurotransmitter-gated ion channels in the re-ceiving cell. As nerve terminals are located

along or at the end of axons very distal tothe cell body and the total number of SVs islimited, ongoing synaptic transmission is main-tained by local recycling of SVs. The mecha-nisms of how vesicles are rebuilt following ex-ocytosis for reuse are the focus of this review.

THE SYNAPTIC VESICLE:A LOCAL CURRENCY FORINFORMATION FLOW ATTHE SYNAPSE

SVs act as key intermediates in convertingelectrical to chemical information at synapses

www.annualreviews.org • Synaptic Vesicle Endocytosis 135

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and represent a currency of information flow inthe nervous system. Because their size is tightlyregulated (see below), it has been possible topurify these organelles to near homogeneity.Such purified vesicles were the starting pointfor identifying SV proteins ( Jahn et al. 1985,Trimble et al. 1988, Perin et al. 1990) and pro-vided the basis for generating the first draft of anSV proteome carried out by the lab of ReinhardJahn (Takamori et al. 2006). SVs are ∼50% pro-tein and 50% lipid, with a 1:1 ratio of phospho-lipid to cholesterol, similar to generic cellularplasma membranes. Following exocytosis, thetransmembrane proteins of SVs become tran-siently incorporated into the plasma membraneand need to be endocytosed to rebuild the SV.Figure 2 illustrates the major transmembraneproteins associated with SVs as well as theiraverage copy number deduced from quantita-tive proteomics. There are roughly nine typesof transmembrane proteins that are specificallyenriched in SVs and of these, only four typeshave been assigned clear-cut functions. These

Neurotransmittertransporter9–14 copies

Synaptotagmin I15 copies

Synaptobrevin 2/ VAMP270 copiesVacuolar ATPase

1–2 copies

Synaptogyrin2 copies

SV22 copies

Synaptophysin30 copies

Scamp1–2 copies

Figure 2The synaptic vesicle (SV) transmembrane proteins. Proteomic analysis ofhighly purified SV preparations allowed estimates of the abundance of each ofthe major transmembrane proteins associated with SVs. The protein copynumbers shown are taken from Takamori et al. (2006).

are (a) the proton pump (vacuolar ATPase),which provides the proton-motive force fordriving neurotransmitter uptake through (b)the vesicular neurotransmitter transporter(Edwards 2007), (c) synaptotagmin I, thecalcium sensor for fast calcium-triggered exo-cytosis (Chapman 2008), and (d ) synaptobrevin2/VAMP2, the vesicle-associated SNARE thatprovides one of the helices required to catalyzemembrane fusion through formation of a four-helix bundle with the specific plasma membraneSNARE proteins syntaxin and SNAP-25 (Rizo& Rosenmund 2008). It is noteworthy that therelative abundance of these different proteinsvaries significantly: Some proteins such asthe proton pump, whose functional role isunequivocal and required for filling vesicleswith transmitter, appear to be expressed atsingle-copy levels. In contrast, synaptophysinis at least one order of magnitude moreabundant but is dispensable, at least in termsof animal viability (McMahon et al. 1996) andbasic synaptic function (Abraham et al. 2006).Presently little is known about variability in thestochiometric relationship of these differentproteins across individual vesicles.

SYNAPTIC VESICLE RETRIEVALAT NERVE TERMINALS

Typical CNS synapses contain a limited num-ber of SVs, usually a few hundred or less. Asnerve terminals are typically located great dis-tances from their cell bodies, the biosyntheticengines of cells, the proteins and lipids thatmake up a SV, must be recaptured from theplasma membrane following exocytosis, refash-ioned into a SV, and refilled with neurotrans-mitters (De Camilli et al. 2001). This currencyof synaptic communication is in fact in rela-tively limited supply. The mechanism of vesicleretrieval in the SV cycle has been a subject ofintense interest and debate for over 35 years.The first descriptions of vesicle recycling beganwith ultrastructural observations at neuromus-cular junctions (NMJ), where uptake of extra-cellular horse radish peroxidase demonstratedconclusively the existence of the SV recycling

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pathway (Heuser & Reese 1973). These stud-ies also provided the first evidence that vesicleretrieval occurred through a clathrin-mediatedpathway. To visualize vesicle recycling at theEM level, it was necessary to provide a suffi-ciently rapid and intense stimulus while catch-ing exo- or endocytic events in the act throughrapid freezing. This approach led to the firstkinetic description of endocytosis (Miller &Heuser 1984) because the time between stimu-lus and freezing could be varied (Figure 3a,b).The relatively long time scales observed(1 min for completion of vesicle endocytosis) fu-eled speculation that a faster, more direct routemust be operational, and they provided grist forthe idea that endocytosis may occur by rever-sal of fusion at the active zone, first proposedby Ceccarelli and colleagues (Ceccarelli et al.1973); this pathway was later coined kiss-and-run. During the past 10–15 years, efforts to un-derstand the mechanisms of endocytosis of SVshave been concentrated in two approaches. Oneis the examination and discovery of the molec-ular machinery responsible for endocytosis atnerve terminals; the other is the biophysicalprobing of the time scales and molecular be-havior of the endocytic process at the synapse.

THE TIMING OF ENDOCYTOSISAT NERVE TERMINALS:EVIDENCE FOR MULTIPLETIME SCALES OF RETRIEVAL

Synaptic endocytosis kinetics measurementsbecame possible with the advent of new tech-nologies as well as suitable synaptic prepara-tions. Electrical capacitance recordings, which,in principle, provide direct measurements ofcell surface area, allow one to measure the netbalance of exocytosis and endocytosis. This hasbeen useful, however, only at select giant synap-tic preparations because SVs have a very smallsurface area (3000–5000 nm2) and sites of fu-sion are usually quite distant from the cell body,where the recording electrode makes electricalcontact. Three different types of giant synapticpreparations have proven useful in this regard.The first direct real-time measurements of

NMJ: neuromuscularjunction

vesicle retrieval following a burst of exocy-tosis were made in retinal bipolar cells (vonGersdorff & Matthews 1994a) and auditory haircells (Parsons et al. 1994). Refinement of thesemeasurements (Beutner et al. 2001, Neves &Lagnado 1999) revealed that, generally, twodistinct kinetic components of endocytosis areapparent (with ∼1-s and ∼10-s time constants,respectively) where the relative proportion ofretrieval via the faster component was sensitiveto calcium influx (Beutner et al. 2001, Neveset al. 2001). Given that this calcium-driven fastcomponent was sensitive to the calcium chela-tor BAPTA (and occurred even when calciumelevations were spatially confined), the authorsconcluded that the faster form was likely oc-curring near active zones. Similar observationswere made in the giant auditory brainstem calyxof the Held synapse (Wu et al. 2005).

The appearance of distinct kinetic com-ponents leads to the question of how eachparticipates in regenerating SVs. Interestingly,the detailed ultrastructural kinetic study byHeuser and colleagues (Miller & Heuser 1984)also revealed ultrastructural components withdistinct kinetic signatures following stimula-tion at the NMJ. Freeze-fracture views ofpresynaptic terminal membrane showed thatfollowing stimulation, non-clathrin-coated en-docytic vacuole-like structures formed nearthe active zone without concentrating cargomolecules and disappeared within approxi-mately 1 s (Figure 3a,b). In retrospect, it seemslikely that the fast component seen with ca-pacitance measurements could be the same fastpathway identified by Heuser at the NMJ. Con-sistent with this interpretation, this componentwas shown to be insensitive to clathrin pertur-bation ( Jockusch et al. 2005). At the calyx ofHeld, however, the appearance of fast endocy-tosis was independent of neurotransmitter re-lease (Yamashita et al. 2005). Pretreatment ofnerve terminals with botulinum toxin blockedneurotransmitter release without eliminating acapacitance transient with fast recovery. Thus,this rapid membrane response may represent ahomeostatic response to acute calcium eleva-tion independent of SV endocytosis.


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0 5 10 15 20

Time (s)

tdwell ~ 0 s

tdwell ~ 4 s

tdwell ~ 10 s

tdwell > 22 s

Dwell time (s)0.1 5.6 11.1 16.6 22.1

0.00

0.05

0.10

0.15

0.20

Frac

tion

of e

vent

sEn

docy

tosi

s pr

ofile

s (μ

m–2

)

Time (s)

a

c

b

d

0

0 20 40 60 80 100 120

1

2

3

4

5

6

7

8

Figure 3Kinetics of endocytosis from 1984 to 2007 (a) The first measurements of the kinetics of synaptic vesicle (SV) endocytosis were obtainedvia pulse-chase freeze fracture experiments at the frog neuromuscular junction (NMJ) by Miller & Heuser (1984). An image that wasobtained 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 ofthe accumulation and disappearance of the membrane invaginations obtained by examining freeze-fracture pictures taken at manydifferent times with respect to the action potential stimulus. Redrawn from Miller & Heuser (1984). (c) Single-vesicle exocytosis andendocytosis as revealed by following the vesicular glutamate transporter (vGlut1) tagged with pHluorin in a luminal loop. Adapted fromBalaji & Ryan (2007). Upon fusion, pHluorin becomes deprotonated and its fluorescence dequenched. After a variable delay (tdwell ), theSV protein is endocytosed and the fluorescence becomes quenched as the vesicle lumen reacidifies over a 3- to 4-s time scale. Fourdifferent examples displaying the range of tdwell are shown. (d ) The histogram of tdwell values obtained from approximately 200 eventsshows that the timing of endocytosis is dictated by an exponential process (red curve) whose mean time is approximately 13 s.

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Exocytosis

V-ATPaseproton pump

pKa ~7.1

Protonated(quenched)

vGlut-pHluorin

Deprotonated(fluorescent)

vGlut-pHluorin

Endocytosis

Reacidification

Figure 4pHluorin-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-nonefashion 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 acidiclumen of a synaptic vesicle, is quenched ∼97% of the time. Redrawn from Sankaranarayanan et al. (2000).

The introduction of optical tracers hasproven particularly powerful for examining en-docytosis at small nerve terminals. Pulse-chaseapplication of the fluorescent amphipathicmolecule FM 1-43 (Betz & Bewick 1992) al-lowed the first kinetic dissection of endocytosisin dissociated hippocampal neurons (Ryan et al.1993, Ryan & Smith 1995). Vesicle retrieval fol-lowing a large burst of activity indicated thatendocytosis decayed over a 60-s time scale. Al-though useful for labeling recycling vesiclespools and measuring pool turnover kinetics,tracers such as FM 1-43 do not directly providereal-time endocytosis readouts. The advent ofpHluorins (see Figure 4) provided higher-fidelity readout where specific vesicle proteinsfates can be followed. Initial measurements of

VAMP2 endocytosis for relatively large stimulishowed that the recovery time scale dependedon the amount of exocytosis (Sankaranarayanan& Ryan 2000). Improvements in these ap-proaches allowed detection of endocytosis fol-lowing single-action-potential stimuli (Balaji &Ryan 2007, Granseth et al. 2006), which showedendocytic recovery occurred with a time con-stant of approximately 15 s. Analysis of the de-lay time between exocytosis and endocytosisfor individual vesicles showed that endocyto-sis times varied stochastically from vesicle tovesicle, ranging from instantaneous to tens ofseconds with an exponentially distributed dwelltime (Balaji & Ryan 2007), which indicates thata single rate-limiting stochastic step determinesthe time scale of endocytosis.


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MODULATION OFENDOCYTOSIS: CALCIUMAND EXOCYTIC LOADA central goal of research in presynaptic func-tion is to understand the regulation of differentvesicle cycling steps. Prior to assigning specificmolecular roles, one must understand how theconditions under which the measurements wereperformed impact the biophysical observations.Two main variables associated with driving exo-cytosis at synapses are the extent to which intra-cellular calcium becomes elevated and the totalendocytic burden that ensues from successfulexocytosis. These two variables are closelyentwined as greater elevations in intracellularcalcium generally lead to higher exocytic rates,more material that must be recaptured. Giventhe importance of intracellular calcium insignaling, determining calcium’s role in endo-cytosis is of significant interest. Early attemptsto disentangle calcium elevations from exo-cytosis used α-latrotoxin, a potent secretagogfrom black widow spider venom, to stimulateexocytosis in the absence of extracellular cal-cium (Ceccarelli & Hurlbut 1980). After severalhours of stimulation under these conditions,NMJs became severely depleted of SVs and theplasma membrane developed large enfoldings,indicating that endocytosis was blocked.Ceccarelli & Hurlbut (1980) concluded thatintracellular calcium elevation was necessaryfor SV endocytosis. It was impossible, however,to distinguish the extent to which calciummight simply be modulating rates as opposed toplaying a requisite role in the biochemistry ofendocytosis. A number of different experimentshave shown that, following exocytosis, calciumentry and endocytosis can be decoupled as thepresence of extracellular calcium is no longernecessary for endocytosis to proceed (Gad et al.1998, Ryan et al. 1996) and that bulk calciumlevels typically decay faster than the completionof endocytosis (Wu & Betz 1996). At the otherextreme, persistent photolysis of caged calciumled to complete arrest of endocytosis in giantbipolar terminals so long as bulk cytoplas-mic calcium was greater than approximately

1 μM (von Gersdorff & Matthews 1994b).Although peak intracellular calcium levels nearexocytosis sites transiently reach tens of micro-molar, average sustained intracellular calciumlevels remain well below 1 μM at most nerveterminals following a single-action potential orlow-frequency stimulation. Thus, it is unclear ifsuch inhibition plays a role under physiologicalconditions.

Approaches that allowed direct mea-surements of endocytosis kinetics made itpossible to examine the relationship betweenthe amount of exocytosis and the speed ofendocytosis, as well as how elevations in intra-cellular calcium might modulate this process.Experiments in hippocampal nerve terminalsand at the calyx of Held showed that increasesin exocytosis led to progressively longerendocytosis time scales (Sankaranarayananet al. 2000, Sun et al. 2002), in spite of thefact that endocytosis was accelerated underconditions that led to elevated calcium in thesepreparations (Sankaranarayanan & Ryan 2001,Wu et al. 2005). This slowing of endocytosiswith greater exocytosis may result from anunderlying saturation of the total endocyticcapacity of the synapse. It is notable that suchsaturation behavior is not observed in othersynaptic preparations such as NMJs in mice(Tabares et al. 2007) and flies (Poskanzer et al.2006), suggesting that it is not a fundamentalproperty of endocytosis at nerve terminals.This paradox between acceleration of endocy-tosis due to intracellular calcium versus slowingdue to increased accumulation, and the absenceof either in certain preparations, was recentlyresolved. Taking advantage of single-vesiclesensitivity to monitor endocytosis kinetics sys-tematically over a 40-fold range in exocytosis,Balaji et al. (2008) demonstrated that endocy-tosis recovery follows a single exponential timecourse (15 s) over a wide range of exocytosis butbegins to slow significantly only above a thresh-old accumulation of SVs on the plasma mem-brane. These data imply that until this thresh-old is reached, endocytosis of different vesiclesis likely happening in parallel, each with an

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average time scale of 15 s. Beyond thisthreshold, the synapse can no longer assemblesufficient endocytic machinery to handlegreater endocytic loads, thereby defining theendocytic capacity of the synapse. Importantly,for sufficiently small amounts of exocytosis,the endocytosis time course was insensitive tomanipulations of intracellular calcium. Balajiet al. (2008) observed that the endocytic capac-ity was increased by stimulus conditions thatelevated residual calcium in the nerve terminal.These data thus help reconcile the originalobservations at the frog NMJ as well. Persistentstimulation in the absence of any calcium eleva-tion (as achieved in α-latrotoxin in zero calciumfor prolonged periods) could lead to a situationwhere the amount of exocytosis occurringgreatly exceeds the endocytic capacity, even-tually leading to persistent depletion of vesiclepools. Furthermore, this hypothesis offers anexplanation as to why modulation of endo-cytosis by activity may not be observed: Thedetailed tuning of calcium entry, buffering, andextrusion, along with the specific abundance ofthe necessary endocytic proteins, may be suchthat one never operates in an exocytosis regimewhere the endocytic capacity becomes limiting.For example, at physiological temperatures,the time constant for endocytosis decreases to6 s (Balaji & Ryan 2007) in hippocampal nerveterminals. This increase in clearance timecoupled with the fact that release probabilityis lower at this temperature lead to a situationwhere the endocytic capacity is rarely exceeded.Thus, for three different central nervous sys-tem synapses examined (bipolar terminals, thecalyx of Held, and dissociated hippocampalneurons), a common time scale of 10 to 15 s(at room temperature) appears to prevail in SVrecovery unless significant accumulation of SVcomponents can be driven to the cell surface.

KISS-AND-RUN: ALONG-RUNNING DEBATE

Beginning with the first observations of vesiclerecycling, there has been speculation of arapid recycling pathway whereby the vesicle

maintains its identity and is retrieved intact atthe active zone. This idea was originally fueledby disparity in the time scales of exo- and endo-cytosis observed at the NMJ. Endocytosis tookapproximately 1 min (Miller & Heuser 1984),whereas exocytosis took place on the submil-lisecond time scale. In comparing the time scalefor exo- and endocytosis, however, one shouldconsider the time for the entire recycling vesi-cle pool to fuse with the plasma membrane, notjust a single vesicle. At hippocampal synapsesat 37◦C, exocytosis of the entire recycling poolrequires approximately 20 s for stimulationat 10 Hz (Fernandez-Alfonso & Ryan 2004),which should be compared to the 6-s timeconstant for endocytosis under these condi-tions (Balaji et al. 2008). Thus, it is possiblefor exocytosis to outrun endocytosis, but onlyunder relatively high-frequency stimulation.

The most direct evidence for transient re-versible fusion pores arose from capacitancerecordings of single-vesicle fusion events inchromaffin cells coupled with simultaneousmeasurements of release of catecholamine us-ing carbon fiber amperometry (Albillos et al.1997). In the majority of events, the appear-ance of a small fusion pore was followed byan irreversible expansion and complete loss ofcatecholamine. However, in 5–10% of events,the pore closed without expanding. At con-ventional synapses, this dual approach of ca-pacitance recordings and amperometry has notbeen possible, but single-vesicle fusion eventshave been recorded using capacitance measure-ments alone at the calyx of Held. Wu and col-leagues reported that 17% of events appearedto be transient in nature (He et al. 2006).There are two important caveats in interpretingthese results. First, this approach required ex-amining exocytosis at random locations on thesynaptic surface that were not necessarily activezones. For technical reasons, the events couldbe evoked only using nonphysiological stimuli.Second, the appearance of exocytosis followedsoon after by endocytosis may simply repre-sent a narrow sampling of the select fast eventsof an exponential distribution of time scales aspredicted by a stochastic process (Balaji & Ryan


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Qdot: quantum dot

2007). Verification that such events are insensi-tive to perturbations of elements in the clathrin-mediated pathway will be necessary to assignthem to a distinct endocytosis pathway.

Less direct biophysical methods have alsobeen used to ascertain the presence of kiss-and-run. One approach that has been used exten-sively at hippocampal synapses was to examinethe efficiency of escape of lipophilic tracersfrom SVs during exocytosis. The idea is thata liphophilic dye potentially would dissociatefrom the vesicle lumen membrane too slowly toescape during a kiss-and-run event, but that itmight fully dissociate during a full fusion event.This approach, first introduced more than adecade ago (Ryan et al. 1996), has been used inone variant or another to argue both for (Arava-nis et al. 2003, Harata et al. 2006, Klingauf et al.1998, Pyle et al. 2000, Richards et al. 2005) andagainst (Fernandez-Alfonso & Ryan 2004, Ryanet al. 1996, Zenisek et al. 2002) the presenceof kiss-and-run. Examination of dye loss at thesingle-vesicle level, however, is quite challeng-ing, as one is effectively asking whether lossesin fluorescence are well quantized. A number ofsubtle artifacts can potentially lead to loss of fi-delity of the fluorescence measurements. Chiefamong these is that lipophilic tracers are addedto the outside of the synapse during activity toload recycling vesicles; however, other recy-cling membranes in neighboring glia and post-synaptic dendrites, as well as other presynapticcompartments, can become labeled. Suchspurious labeling can strongly contribute tobackground fluctuations in fluorescence inten-sity. Recent experiments designed to control forsome of these possible complications concludedthat lipophilic dyes destain completely duringaction-potential stimuli (Chen et al. 2008) andsuggest either that kiss-and-run does not occurat these synapses or that one cannot use thesedyes reliably to detect such transient fusions.

Recently, a novel form of molecular tracerwas introduced for examining the frequency offull fusion during exocytosis in cultured neu-rons. In this new technique, a recycling vesi-cle endocytoses a quantum dot (Qdot) (Zhanget al. 2007) whose optical properties allow

continuous optical recordings without pho-tobleaching. Because of its large size [hy-drodynamic radius of nearly 15 nm (Larsonet al. 2003)], the internalized Qdot escapesonly during full-fusion exocytosis (Zhang et al.2007). Zhang et al. (2007) found that stimulus-dependent unloading of Qdots followed aslower time course than an FM dye loaded intothe same synapses, suggesting that some fusionevents release FM dye but not Qdots. A signif-icant caveat with this approach comes from thelarge size of Qdots relative to the vesicles thatcontain them. If the Qdot interacts with thevesicle lumen and stabilizes the curvature of afusing vesicle, perhaps an otherwise rare partial-fusion event could be made more probable.

Investigations into the molecules that un-derlie endocytosis have yielded little evidencein support of kiss-and-run mechanisms to date(see below). Furthermore, the observation thatvesicle components exchange with counterpartsresident on the cell surface during endocyto-sis over a wide range of stimulus conditions(Fernandez-Alfonso et al. 2006, Wienisch &Klingauf 2006) indicates that vesicles typicallylose some of their components following exo-cytosis. Finally, detailed measurements of thetime between exocytosis and endocytosis forindividual vesicles showed that, although vesi-cles do occasionally endocytose quickly, the fre-quency of such events is well predicted by astochastic (single-exponential) process with amean endocytic time of 15 s (Balaji & Ryan2007). Taken together, the biophysical evidenceweighs largely against significant vesicle recy-cling via a kiss-and-run mechanism. Further-more, as discussed below, most experimentsaimed at explicitly targeting clathrin or asso-ciated machinery at the synapse support a ma-jor role for the classical endocytic pathway invesicle retrieval.

THE MOLECULAR MACHINERYOF SYNAPTIC VESICLEENDOCYTOSIS

Molecular and genetic insights over the past25 years from a variety of model systems have

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culminated in a formidable list of potential pro-teins and lipids involved in endocytosis. Manyof the proteins share common binding part-ners and exhibit overlapping functions withinthe endocytosis machinery. This property of in-terconnectivity may be important in explainingan emerging dilemma in the field of SV endo-cytosis: Gene knockouts and RNA interferenceknockdown of endocytic proteins generally donot eliminate SV endocytosis and, in manycases, have remarkably subtle effects (Di Paoloet al. 2002, Ferguson et al. 2007, Gu et al. 2008,Sato et al. 2009). This situation starkly contrastswith SV exocytosis, where removal of any oneof a small collection of essential proteins elim-inates SV fusion (Sudhof 2004). If altered SVendocytosis is observed after a particular endo-cytic protein is removed, there are three possi-ble considerations for the residual endocytosis.First, this perturbation may uncover a second,independent pathway with distinct underlyingmolecular mechanisms. Second, the measuredendocytosis is identical to the wild-type pro-cess except that a functional paralog has takenthe place of the deleted molecule, although itmay not function with the same efficacy. Third,the impaired endocytosis proceeds via the iden-tical molecular pathway but with lower efficacybecause the deleted molecule played a contrib-utory rather than obligatory role in the process.In this case, the network of endocytic proteinsis robust to deletions of individual members.

Consistent with the third scenario, experi-ments to date have demonstrated that no sin-gle protein appears to be absolutely essentialto the process of SV endocytosis. Perhaps par-ticular components of the synaptic endocyticnetwork play a larger or smaller role in differ-ent animal phyla, but all the components aregenerally found in presynaptic specializations ofmetazoa. Furthermore, these components showa high degree of conservation, both at the se-quence level and in terms of interactions withother proteins and phospholipids (Lloyd et al.2000). In the following section of this review, weintroduce 15 proteins/protein complexes thatcompose the SV endocytic network (Figures 6and 7). These proteins are separated into three

layers of the network on the basis of their func-tion, binding interactions, and protein domaincontent. At the core of the endocytic network isthe capacity to gather up cargo into a local patchof plasma membrane and deform this patch intoa separate compartment destined for internal-ization. A second layer of proteins acts to sta-bilize the core proteins while recruiting addi-tional effector molecules that both catalyze andterminate the process. These effectors make upthe third layer, and they function in numerouscellular trafficking processes in addition to SVendocytosis.

A CLATHRIN-MEDIATEDPATHWAY FOR VESICLEREBUILDING

The first details of the molecular basis of SVrecycling arose with the pioneering studies ofHeuser & Reese (1973), who noted that endo-cytosis following stimulation appeared to oc-cur via the newly discovered clathrin coats.Since these early discoveries, progress in ourunderstanding of the molecular basis of SV re-cycling has benefited from a number of ap-proaches in different model systems. Althoughthe relevance of the finding that clathrin-coatedpits could be found at synapses originally wasdebated, the evidence that this pathway pre-dominates as the route of vesicle recovery hasbecome overwhelmingly strong in the past10 years.

Three types of experiments have been per-formed, all of which support a major role forthe clathrin reuptake pathway. First, microin-jections of peptides or antibodies that interferewith different steps of endocytosis were fol-lowed by electron microscopy and functionalassays. Second, genetic ablations of endocyticproteins were followed by kinetic assays andelectron microscopy. Third, the effects of pho-toinactivation of clathrin in Drosophila NMJswere followed by functional and ultrastructuralassays. At synapses such as in the lamprey giantreticulospinal axons, injections of peptides andantibodies designed to interrupt dynamin func-tion (see below), followed by activity-driven


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200 nm

a

b

*

Clathrin-coated pits

SVsSVs

Plasmamembrane

Figure 5The accumulation of clathrin-coated pits at nerve terminals during activity inthe absence of dynamin-1. Strong evidence for synaptic vesicle (SV)endocytosis normally proceeding through a clathrin-coated pit pathway hasbeen obtained from a number of experiments in which molecular perturbationsled to trapping vesicle endocytosis at an intermediate stage. Here, an exampleof the ultrastructure of a hippocampal synapse derived from a dynamin-1knockout mouse taken from Ferguson et al. (2007) is shown. (a) Single sectionof a tomogram taken from a synapse where spontaneous activity led to thedevelopment 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 synapticvesicles. (b) 3D tomographic reconstruction shows that these clathrin-coatedprofiles were associated with plasma membrane invagination.

vGlut: vesicularglutamate transporter

exocytosis, led to accumulation of clathrin-coated buds (Shupliakov et al. 1997). Simi-lar accumulations were observed in hippocam-pal neurons when the abundant brain-specificisoform, dynamin-1, was genetically ablated(Figure 5). Additionally, peptide injections de-signed to interfere with clathrin assembly ledto dramatic slowing of the kinetics of endocy-tosis ( Jockusch et al. 2005) as well as to activity-dependent interruption of exocytosis (Gad et al.

2000; Morgan et al. 2000, 2003), consistent witha depletion of available vesicles due to a block inendocytosis. At hippocampal synapses, the ki-netics of endocytosis can be dramatically slowedwhen clathrin is removed by siRNA (Gransethet al. 2006). Finally, photoinactivation of flu-orescently labeled clathrin, which offers themost temporally precise molecular interrup-tion, showed that acutely inactivating clathrin’sinability to assemble in Drosophila NMJs un-dergoing exocytosis leads to the formation oflarge dead-end vacuoles (Heerssen et al. 2008,Kasprowicz et al. 2008) and the loss of any fur-ther vesicle recycling. Thus, data using severaldistinct approaches and examining multiple dif-ferent types of synapses all argue that clathrinassembly normally plays a critical role in SVendocytosis.

MECHANISMS FOR SORTINGTHE APPROXIMATELY NINECARGO PROTEIN TYPES

An important unsolved question in SV recy-cling is, how is each of the nine different typesof SV transmembrane proteins resorted intoSVs following exocytosis? As retrieval occursthrough clathrin-mediated endocytosis, theexpectation is that known mechanisms forreceptor-mediated endocytosis will be manifesthere. For instance, perhaps there are interac-tions of sorting motifs in vesicle protein cyto-plasmic tails with the plasma membrane adaptorprotein complex AP2. AP2, a heterotetramericcomplex that additionally binds clathrin, isthought to coordinate clathrin assembly withcargo recognition during endocytosis (Keen1987). At present, only a single putative sortingmotif has been functionally identified in SVproteins. Mutation of a dileucine-like motif inthe vesicular glutamate transporter (vGlut1)slows vGlut1 internalization at nerve terminals(Voglmaier et al. 2006). Synaptotagmin hasalso been identified as a binding partner toAP2 (Grass et al. 2004, Haucke & De Camilli1999, Haucke & Krauss 2002, Haucke et al.2000, Zhang et al. 1994). In the absenceof Synaptotagmin 1 ( Jorgensen et al. 1995,

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Nicholson-Tomishima & Ryan 2004, Reistet al. 1998), or following either photoinacti-vation (Poskanzer et al. 2003) or mutation inresidues in its C2B domain (Poskanzer et al.2006), endocytosis of SVs is impaired. Surpris-ingly, deletion of the AP2 μ2 subunit fails toeliminate cargo retrieval and SV recycling inboth Caenorhabditis elegans (Gu et al. 2008) anddissociated hippocampal neurons (Kim & Ryan2009). In the absence of μ2, however, therewas a significant loss of SVs and endocytosiskinetics were measurably slower, implying thatAP2 normally does play a role at synapses.These results imply that additional sortingmechanisms must be operating and open up thepossibility that AP2 serves a more subtle rolein SV endocytosis than originally anticipated.

A leading candidate adaptor for dedicatedSV protein sorting is stonin, first identifiedas a temperature-sensitive paralytic mutant inDrosophila (where it is named Stoned ) morethan 35 years ago (Grigliatti et al. 1973, Kelly1983). Stonin 2, which contains a μ2 homol-ogy domain (Figure 6), interacts genetically(Fergestad & Broadie 2001, Phillips et al. 2000)and biochemically ( Jung et al. 2007, Waltheret al. 2004) with synaptotagmin. Removal ofStoned B in Drosophila NMJs leads to defects inendocytosis (Stimson et al. 2001) and, when co-expressed with synaptotagmin in nonneuronalcells, it facilitates synaptotagmin retrieval (Dirilet al. 2006).

Evidence for defects in cargo sorting in SVendocytosis has also emerged from examina-tion of mutations in proteins associated withclathrin assembly. In particular, deletion of themonomeric adaptor protein AP180 led to mis-localization of VAMP2 but not synaptotag-min or synaptogyrin (Nonet et al. 1999, Zhanget al. 1998), suggesting a specific role in sortingVAMP2 during endocytosis. In general, theseclathrin-associated sorting proteins usually as-sociate with both AP2 and clathrin for properfunction (Brett & Traub 2006). The fact thatendocytosis and protein sorting can occur with-out AP2 suggests that redundant mechanismsthat link sorting proteins, cargo, and coated pitsmust be operational.

PIP2: phospho-inositol-(4,5)-bis-phosphate

One proposed scenario for efficiently re-capturing SV proteins is that they remain sta-bly associated after exocytosis and directly nu-cleate clathrin-coat formation. Although thismay occur for certain SV proteins, this is notthe case for VAMP2 or synaptotagmin. At hip-pocampal nerve terminals, these proteins dif-fuse onto the axonal surface (Li & Murthy2001, Sankaranarayanan & Ryan 2000), andthey exchange with surface counterparts dur-ing endocytosis (Fernandez-Alfonso et al. 2006,Wienisch & Klingauf 2006). Currently, the is-sue of how SV proteins are retargeted into SVsduring endocytosis remains largely unresolved.

CURVING MEMBRANES:INITIATION OF SYNAPTICVESICLE ENDOCYTOSIS

In addition to recollecting and sorting SV pro-teins, the endocytosis machinery must locallycurve the plasma membrane and eventuallypinch off 3000 to 5000 nm2 of bilayer in theform of a 30- to 40-nm-diameter vesicle. Thisprocess can be broken into two parts: deforma-tion of membrane into a vesicular or tubularshape and scission of the stalk connecting thisstructure to the plasma membrane.

The process of membrane deformation be-gins with the organization of endocytic coatproteins on the inner leaflet of the synapticmembrane. This assembly is thought to bedriven in part by the generation of phospho-inositol-(4,5)-bis-phosphate (PIP2), which re-cruits several endocytic proteins to the plasmamembrane including AP2, epsin, dynamin,endophilin, amphiphysin, and AP180. Atsynapses, activity-dependent PIP2 synthesisis largely carried out by phospho-inositol-5-kinase Iγ (Di Paolo et al. 2004), which pre-sumably drives rapid recruitment of endocyticfactors during repetitive exocytosis. In addi-tion to providing a scaffold for concentratingappropriate cargo proteins destined for endo-cytosis, endocytic coat proteins are thoughtto regulate the area destined for internaliza-tion in a manner that leads to a precise SVsize.


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On the basis of biochemical, structural,and genetic evidence, several protein domainshave been implicated in the curving of mem-branes. ENTH (epsin N-terminal homology)domains found at the amino termini of epsin,AP180, and CALM are phospholipid-bindingmodules (Hurley 2006, Itoh & De Camilli2006). ENTH domains interact preferentiallywith PIP2-containing phospholipids and tubu-late liposomes in vitro as well as plasma mem-branes of living cells when overexpressed (Fordet al. 2002, Itoh et al. 2001). Structural stud-ies of ENTH domains suggest that an N-terminal amphipathic alpha helix inserts intothe lipid bilayer upon binding to PIP2 (Itohet al. 2001). The canonical example of anENTH domain protein at the synapse is epsin.Thought to function as a clathrin adaptor, epsinbinds clathrin heavy chain and AP2. Theseassociations position epsin as part of the en-docytic core module. Epsin contains severalNPF (asparagine-proline-phenylalanine) mo-tifs near its carboxy terminus that interact withEps15 homology (EH) domain modules foundin the secondary scaffolds Eps15 and inter-sectin/Dap160 (Figure 6). Epsin also containsubiquitin-interacting motifs, which may func-tion in the recognition of ubiquitinated cargoproteins. In yeast, expression of the ENTHdomain is sufficient to rescue viability of theEnt1/2 double mutant (the two yeast epsin or-thologs), suggesting that this domain has anessential role irrespective of its adaptor func-tions (Wendland et al. 1999). Under some con-ditions, epsin and clathrin triskelia, but notclathrin alone, can invaginate lipid monolayers

(Ford et al. 2002). Disruption of epsin func-tion by antibody binding in the lamprey gi-ant synapse decreased SV numbers and led toan accumulation of large coated pits follow-ing stimulation ( Jakobsson et al. 2008). Thesedata suggested that SV endocytosis was trappedat an early stage in the absence of functionalepsin. In contrast, loss of the Drosophila epsinortholog Liquid Facets did not appear to im-pair SV endocytosis, although synapse archi-tecture was significantly affected (Bao et al.2008). Epsin is also found in other cellularcompartments, suggesting that it may have ageneral role in clathrin-mediated endocytosis(Horvath et al. 2007).

AP180 and CALM share an ENTH-likemodule at their amino termini along withclathrin and AP2-interacting domains withintheir unstructured carboxy termini. However,interaction of these proteins with PIP2 is dis-tinct from epsin’s ENTH domain interactionand the N-terminal domain contains addi-tional alpha helices (Itoh & De Camilli 2006).These biochemical and structural differencesled investigators to call these ANTH (AP180N-terminal homology) domains. ANTH do-mains do not appear to deform lipids ontheir own, but they may influence curva-ture indirectly through interactions with othermembrane-binding proteins. AP180 is pre-dominantly found at presynaptic terminals,whereas CALM is ubiquitous and also possessesmultiple NPF motifs (which are not foundin AP180) near its carboxy terminus (Itoh &De Camilli 2006). C. elegans and Drosophilaeach have a single AP180/CALM ortholog

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 6Categories 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, eps15homology domain; PRD, proline-rich domain; C-C, coiled-coil domain. NPF represents any of five possible motifs that interact withEH: NPF, WW, FW, SWG, and H(ST)F. AP2/Clath represents two AP2-binding motifs [WXXF and DP(WF)] and three clathrin-binding motifs [D(LI)(LFQ), L(FWY)X(FWY)(DE), and PWXXW]. The diagram depicts these as one localized domain, but thesemotifs are actually distributed throughout the polypeptide. Two isoforms of intersectin are shown: long and short. Dynamic propertiesare depicted as either positive feedback (polymerization, enzymatic catalysis) or avidity (multiple binding sites linkedtogether).


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that presumably fulfills the duties of both pro-teins. In both of these model systems, loss ofAP180/CALM substantially broadened the dis-tribution of SV sizes and increased the mean di-ameter by over 30% (Nonet et al. 1999, Zhanget al. 1998). In addition, AP180 and CALMregulate the plasma membrane abundance ofsynaptobrevin (Dittman & Kaplan 2006, Harelet al. 2008, Nonet et al. 1999, Zhang et al.1998), suggesting that ANTH domain pro-teins play a role in both cargo recruitment andsize of the endocytic area destined to undergoendocytosis.

Another membrane binding module em-ployed during SV endocytosis is the BAR (Bin,amphiphysin, Rvs) domain (Figure 6). BARdomains found in endophilin, amphiphysin,syndapin, and sorting nexin 9, among others,bind phospholipids as crescent-shaped dimers,and they are thought to generate or stabi-lize highly curved regions of the membrane(Gallop & McMahon 2005, Itoh & De Camilli2006, Ren et al. 2006). In vitro, these do-mains can tubulate liposomes and, in somecases, appear to bind preferentially to partic-ular liposome sizes, suggesting that the BARdomain can sense lipid curvature (Gallop &McMahon 2005). Amphiphysin and endophilinshare amino-terminal domains that form am-phipathic helices capable of penetrating the cy-toplasmic bilayer leaflet and perturbing lipidpacking, particularly in the presence of acidicphospholipids. Thus, their N-terminal helixand BAR motif are together termed N-BARdomains. Both amphiphysin and endophilincontain carboxy-terminal SH3 domains, whichare thought to interact with the proline-richdomains of dynamin and synaptojanin (Itoh& De Camilli 2006). The combination ofN-BAR and SH3 domains makes these pro-teins ideally suited for the dual task of deform-ing membranes and recruiting other endocyticmolecules to the curved region. Amphiphysinadditionally has clathrin- and AP2-binding mo-tifs, linking it to the core endocytic machinery.

The mouse knockout of amphiphysin I dis-played a modest decrease in recycling SV poolsize as well as a slower recycling rate (Di Paolo

et al. 2002). Fly mutants lacking amphiphysindid not appear to have significant transmissiondefects at the NMJ, and nervous system func-tion is largely intact (Razzaq et al. 2001, Zelhofet al. 2001). Similarly, neither deletion nor RNAinterference knockdown of the worm orthologAMPH-1 impacts nervous system functionappreciably ( J. Kaplan and J. Bai, personal com-munication). However, loss of endophilin sig-nificantly impairs synaptic transmission in bothflies and worms (Rikhy et al. 2002, Schuskeet al. 2003, Verstreken et al. 2002). The SV poolis decreased, whereas SV diameter and quan-tal size are increased. Ultrastructural similari-ties between endophilin and synaptojanin mu-tants, such as accumulations of clathrin-coatedpits and vesicles, queues of vesicles far fromthe active zone, and large cisternae, supportthe hypothesis that endophilin and synapto-janin act together at a similar stage during en-docytosis (Schuske et al. 2003, Verstreken et al.2003).

Syndapin, a member of another class of BARdomain known as F-BAR (formerly known asextended FCH) domain proteins, may play arole during SV endocytosis, particularly duringprolonged or intense stimulation (Anderssonet al. 2008). Also known as PACSIN, syn-dapin possesses an N-terminal F-BAR domain,several NPF motifs, and a C-terminal SH3domain, which interacts with dynamin andN-WASP (Anggono & Robinson 2007, Kessels& Qualmann 2004). Syndapin binds to EHdomain proteins via its NPF motifs and par-ticipates in trafficking through the recyclingendosome pathway (Braun et al. 2005). Fly syn-dapin mutants failed to display any significantdefects in synaptic transmission or SV endocy-tosis (Kumar et al. 2009). However, antibodiesagainst syndapin caused enhanced synaptic de-pression and large VAMP2-containing cister-nae after sustained stimulation when injectedinto lamprey giant synapses (Andersson et al.2008). Because no effect was observed at lowstimulus frequencies, syndapin may be part ofa secondary pathway for retrieving SV compo-nents through endocytic intermediates follow-ing prolonged bouts of activity.

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SECONDARY SCAFFOLDINGFOR RECRUITING GENERALENDOCYTIC EFFECTORS

The proteins described thus far can beorganized either into a core endocytic mod-ule (clathrin, AP2, AP180, stonin 2, epsin,amphiphysin) or into secondary effectors(endophilin and synaptojanin; others men-tioned below) based on their interactions (orlack thereof ) with vesicle cargo molecules orcargo-binding proteins. Two synaptic proteinsbridge this gap between the core module andsecondary effectors, acting as recruiters andorganizers during SV endocytosis. Eps15 andintersectin/Dap160 are binding partners andeach has highly conserved domain structurescoupling the endocytic coat to secondaryeffectors (Montesinos et al. 2005). Eps15 is a110-kDa protein containing three N-terminalEH domains, a central coiled-coil domain, andclathrin/AP2 binding motifs near its carboxyterminus (Morgan et al. 2003, Salcini et al.2001). The EH domains mediate interactionswith NPF motifs in epsin, stonins, AP180, andsynaptojanin, whereas the coiled coil inter-acts with intersectin (Kelly & Phillips 2005,Morgan et al. 2003). Loss of Eps15 in C. elegansresults in decreased SV number, particularly atelevated temperatures (Salcini et al. 2001). TheEps15 mutant ehs-1(ok146) genetically interactswith the worm temperature-sensitive dynaminmutant dyn-1(ky51) such that locomotion isseverely impaired in the double mutant even atpermissive temperatures (Salcini et al. 2001).Eps15 also appears to enhance AP180-mediatedclathrin assembly in vitro, suggesting that itmay have an early role in building clathrin pits(Morgan et al. 2003). The loss of Eps15 inDrosophila causes substantial decrease in synap-tic intersectin, dynamin, stonin, synaptotagminI, α adaptin, and endophilin (Koh et al. 2007).

Intersectin/Dap160 is a synaptic proteincontaining two N-terminal EH domains, a cen-tral coiled-coil domain, and four to five SH3 do-mains (depending on the species) at its carboxyterminus (Koh et al. 2004, Marie et al. 2004).A long isoform in vertebrates also contains

Dbl homology domain, a pleckstrin homologydomain, and a C2 domain after the SH3 re-peat. This module can act as a guanine nu-cleotide exchange factor for Cdc42, regulatingactin dynamics through Cdc42 and N-WASP(Hussain et al. 2001). Interestingly, Drosophilaand C. elegans intersectin orthologs do not con-tain this module. The EH domains interactwith epsin; the coiled coil binds Eps15; and theSH3 domains bind to dynamin, synaptojanin,and N-WASP (Marie et al. 2004, Montesinoset al. 2005). The loss of intersectin in the fly re-sults in diminished SV endocytosis; decreasedsynaptic levels of AP180, endophilin, synap-tojanin, and dynamin; and increased synapticdepression during prolonged stimulation (Kohet al. 2004, Marie et al. 2004). Eps15 inter-sectin double mutants exhibit synaptic depres-sion and decreased FM1-43 uptake to a simi-lar degree as either single mutant (Koh et al.2007). Microinjection of the intersectin SH3domain in lamprey giant synapses inhibits SVrecycling and traps a large number of coated pitsin the periactive zone (Evergren et al. 2007).Furthermore, intersectin appears to negativelyregulate recruitment of dynamin to periactivezones at this synapse. In C. elegans, ITSN-1localizes to the periactive zone in NMJs, andloss of ITSN-1 reveals a substantial increase inlarge, irregular vesicular structures as well asa decrease in spontaneous transmitter release(Rose et al. 2007, Wang et al. 2008).

FINISHING THE JOB:MEMBRANE FISSIONAND COAT DISASSEMBLYThe final step of endocytosis involves severingthe endocytic bud from the plasma membrane.In vitro studies have shown that when artificialmembrane necks are narrowed to sufficientlysmall diameter, thermal fluctuations resultin a collapse of the neck, presumably thestarting point for fission (Bashkirov et al. 2008,Israelachvili 1992). Dynamin, a mechanochem-ical GTPase, was originally identified as thegene product of the Drosophila shibire mutant,a temperature-sensitive paralytic that becomes


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Core module Secondary scaffold Secondary effectors

Scaffold

Second scaffold

Cargoselector

Clathrin

Cytoskeleton

Stonin 1, CALM

Dynamin

Synaptojanin Endophilin

N-WASP

Syndapin

Epsin, amphiphysin

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AP2, stonin 2, AP180

?

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Membranebinding

SH3–PRD interaction

EH–NPF interaction

Clathrin/AP2 binding motif

Other protein interactionsPhospholipid

bindingPI(4,5)P

2

Figure 7Synaptic vesicle endocytic network. The proteins that compose the endocytic machinery at the synapse areclassified in two ways: location in the network (color) and functionality (shape). Blue proteins make up the coremodule, red proteins are secondary scaffolding, and green proteins are secondary effectors. Proteins foundexclusively 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 aredepicted as arrow connectors for clathrin/AP2 binding, SH3 to PRD binding, Eps15 homology domain toNPF binding, and other protein interactions (see figure key for arrow types). Membrane interactions areshown as downward gray arrows, and the PIP2 phosphatase synaptojanin is shown interacting with PIP2 (redarrow). Endophilin ( purple) has no known interactions with either the core module or the secondary scaffoldand is therefore distinguished from the other secondary effectors.

devoid of SVs during activity at the nonpermis-sive temperature (Koenig & Ikeda 1989). Thisprotein, which is recruited to the endocyticbud neck, is the minimal essential fissionmachinery in vitro, provided the membranesundergoing fission are under tension (Rouxet al. 2006). Dynamin interacts with a numberof SH3-containing proteins such as endophilin,amphiphysin, syndapin, and intersectin viaits proline-rich domain (Figures 6 and 7).Together with other BAR domain proteins, dy-namin also appears to coordinate the shaping

of endocytic buds (Itoh et al. 2005). Theevidence for dynamin’s role in endocytosis atthe synapse is very strong as many dominant-negative strategies targeting dynamin block SVendocytosis (Koenig & Ikeda 1999, Newtonet al. 2006, Yamashita et al. 2005). Surpris-ingly, genetic ablation of dynamin-1, whichencodes ∼85% of brain dynamin, resulted inonly partial defects in SV endocytosis. Micelacking 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 owingto the elevation of intracellular calcium. How-ever, soon after cessation of action potentialfiring, endocytosis resumed with remarkablynormal kinetics. Although synapses lackingdynamin-1 in mouse still contain a smallamount of dynamin-3 and even less dynamin-2(Ferguson et al. 2007), one possible conclusionfrom this work is that the fission step inendocytosis is not catalyzed by the action ofdynamin alone, but it is simply accelerated bythis protein.

Once the endocytic vesicle is severed fromthe plasma membrane, the newly sculptedvesicle must shed all the machinery that wasused in rebuilding prior to reuse. Clathrin un-coating occurs via the concerted action of theATPase Hsc70 and auxilin, which helps recruitthis enzyme to the vesicle (Eisenberg & Greene2007). One important mystery is the identityof the molecular trigger to initiate uncoating,as it would be energetically costly and per-haps detrimental to launch this process priorto fission. A mechanistic clue has arisen fromgenetic ablation studies of synaptojanin: Theloss of this lipid phosphatase leads to a pro-nounced accumulation of clathrin-coated vesi-cles at nerve terminals as well as a delay in theavailability of newly endocytosed vesicles foradditional rounds of exocytosis (Cremona et al.1999, Mani et al. 2007, Schuske et al. 2003,Verstreken et al. 2003). Synaptojanin is stabi-lized at endocytic buds through multiple inter-actions including binding to the SH3 domainsof intersectin and endophilin and the EH do-mains of intersectin and Eps15, as well as di-rect 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). The5′ phosphatase domain at the amino terminusof synaptojanin rapidly dephosphorylates PIP2.This depletion of PIP2 decreases the phospho-lipid binding affinity of the core module andBAR proteins, simultaneously destabilizing thecoat assembly. Another possible coupling be-tween membrane fission and synaptojanin arisesfrom the theoretical consideration that local de-pletion of PIP2 selectively on the vesicle bud

causes a transient gradient of PIP2 betweenthe bud and the plasma membrane to which itis connected. If this gradient contributes to alipid phase separation between these compart-ments, the interfacial line between the neck andbud tends to be minimized, spontaneously con-stricting the neck (Liu et al. 2006). Regardless ofthe details of fission and uncoating, these pro-cesses are quite rapid as evidenced by the diffi-culty of capturing a coated vesicle, even whenrapid freeze EM is used.

CALCIUM SENSORS FORENDOCYTOSIS?

As described, presynaptic calcium affects nu-merous aspects of SV endocytosis. However,unlike SV exocytosis, in which a single pro-tein (synaptotagmin 1) accounts for much of thecalcium dependence of SV fusion (Chapman2008), it is not clear where calcium acts dur-ing endocytosis. Nearly every endocytic proteininteracts with the membrane or directly bindsto a lipid-binding protein. Because elevatedcalcium can alter the energetics of these mem-brane interactions, it is plausible that presy-naptic calcium functions to change the effi-ciency of endocytosis through general effects onprotein-lipid interactions rather than through adedicated calcium sensor. However, it is worthnoting that synaptotagmin 1 interacts with thecore module and its C2 domains may affectthis interaction, at least in regimes where cal-cium is highly elevated (Diril et al. 2006, Junget al. 2007, Mohrmann et al. 2008, Waltheret al. 2001). Multiple lines of evidence sup-port a role for synaptotagmin in endocytosis.Another candidate mechanism for calcium inendocytosis is through the dephosphins, endo-cytic proteins such as dynamin, synaptojanin,and amphiphysin, that are dephosphorylatedin a calcium-dependent manner by the phos-phatase calcineurin (Cousin & Robinson 2001).The significance of these dephosphorylationevents has not been fully delineated, althoughdephosphorylation of dynamin promotes itsinteraction 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 onlyrarely under physiological stimuli.

A ROLE FOR ACTIN DURINGSYNAPTIC VESICLEENDOCYTOSIS?

The regulation of actin polymerization is avital part of membrane remodeling and intra-cellular trafficking, and multiple endocytic pro-teins mentioned above have a direct impacton actin and the cytoskeleton. For example,the secondary effectors dynamin, syndapin, andN-WASP, as well as intersectin, all connect di-rectly or indirectly to proteins that control actinpolymerization (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 scis-sion by dynamin can utilize actin as an anchor totransduce its force on tubules into longitu-dinal tension (Roux et al. 2006). However,actin does not appear to be important forSV endocytosis per se in hippocampal neu-rons (Sankaranarayanan et al. 2003), butit may contribute as a secondary scaffold(Figure 7). Interestingly, actin seems to havea more pronounced role in the endocyto-sis of SVs at a giant synapse (Brodin &Shupliakov 2006, Shupliakov et al. 2002).Perhaps the cytoskeletal connections to endo-cytosis depend on the geometry and size of asynaptic terminal, particularly because regula-tion of the cytoskeleton is likely to be a majordeterminant of synapse size.

REBUILDING SYNAPTICVESICLES: A SINGLE-PASSSORTING AT THE PLASMAMEMBRANE?

One of the assumptions we have made indiscussing vesicle recycling is that the sortingof SV proteins occurs in a single endocyticstep, as opposed to having an intermediateorganelle after endocytosis at the plasmamembrane makes passage through a recycling

endosome for further cargo sorting (Koenig& Ikeda 1996). The existence of such a recy-cling endosome remains a formal possibility,although very few experiments have provideddefinitive evidence for any functional signif-icance. In the earliest studies of Heuser &Reese (1973), they identified a nonvesicularhorse radish peroxidase–positive compartmentthat appeared only transiently followingstimulation. More recent experiments in whichserial-sectioning electron microscopy wasperformed demonstrated that, during intensestimulation, deep invaginations of the plasmamembrane often form, which can easily beconfused with internal organelles in any singlesection (e.g., see Figure 5), but, in fact, remaintopologically connected to the cell surface(Ferguson et al. 2007). Nonetheless, an endo-somal intermediate may play a role under somestimulus conditions, particularly followingintensive stimulation, when bulk endocytosishas been reported to operate at a number ofsynapses (Clayton et al. 2008, Holt et al. 2003).

A MOLECULAR PICTURE FORTHE ENDOCYTIC MACHINE

Ideally, the molecules described here canbe mapped to the phenomenology of SVendocytosis on the basis of their intermolecularinteractions, genetics, structure, and cellbiological roles in other forms of endocytosis.A host of studies published over the pastdecade have made apparent a rough versionof this molecular map and the dynamics of theendocytic machinery. The core module couplesnucleation of an endocytic coat with cargorecruitment. Polymerization of clathrin andmembrane interactions with ENTH andN-BAR domains rapidly commence theprocess of curving the lipid bilayer at thesite of endocytosis. Furthermore, assemblyof the core may increase the avidity of cargobinding because a multiplicity of binding siteswill be brought into a small region of themembrane. In this scenario, cargo capture,scaffold assembly, and membrane curvature arehighly coupled coordinated events.

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The N-BARs endophilin and amphiphysincontribute to membrane bending and mayinitiate an additional positive-feedback cycleof binding to curved membrane and self-assembly, driving further membrane curvature.Amphiphysin has direct binding interactionswith clathrin and AP2 (placing it in the coremodule), whereas endophilin has no knowninteraction with these or other proteins inthe core module (qualifying endophilin as asecondary effector). The secondary scaffoldmolecules Eps15 and intersectin/Dap160 bindto the core module and provide additionalbinding sites for recruiting the secondaryeffector molecules: dynamin, synaptojanin,and N-WASP. Both dynamin and synaptojaninconvey amplification of a sort within the en-docytic complex. Dynamin self-assembles andcan utilize some of the energy released by poly-merizing to deform membranes. Synaptojanincontains a phospholipase enzymatic domain,and thus a small number of synaptojaninswould be expected to deplete PIP2 rapidlyfrom the membrane. These effectors terminatethe endocytic process by irreversibly severingthe membrane connection and releasing theendocytic coat with the help of Hsc70 andauxilin. The AP180 paralog CALM, the stonin2 paralog stonin 1, and the F-BAR syndapinmay also contribute to SV endocytosis, buttheir specific requirements are not well defined.We therefore include these components aspotential secondary effectors (Figures 6 and 7).

NETWORK PROPERTIES OF THEENDOCYTIC MACHINE

There are several advantages to considering theendocytic proteins as a network (Alon 2007,Hintze & Adami 2008, Schmid & McMahon2007). First, fault-tolerant networks exhibitgraceful degradation, because the failure of onecomponent has little effect on the overall per-formance. Evidence to date suggests that SVendocytosis continues after the removal of anyone protein. Second, weak interactions can betransiently strengthened and amplified by pos-itive feedback within the network. Thus, the

binding of cargo; polymerization of the coremodule; and membrane bending by N-BARs,epsins, and dynamin all collaborate to createa fast, high-fidelity endocytic process. Third,modularity within the network allows the basicendocytic machinery to be utilized in many sep-arate types of trafficking processes within a neu-ron. For instance, most of the SV endocytosismachinery is likely to be involved in postsynap-tic receptor trafficking, intracellular traffickingbetween organelles, and membrane remodel-ing during synapse development (Bushlin et al.2008, Gong & De Camilli 2008, Itoh & DeCamilli 2006, Koh et al. 2007, Montesinos et al.2005). Another consequence of a densely con-nected network is that the removal of one pro-tein can have repercussions on the stability ofmany others. For instance, in fly Eps15 mu-tant synapses, many of the core proteins havesignificantly decreased abundance along withthe Eps15-binding partner intersectin/Dap160(Koh et al. 2004, 2007). This interdependenceof protein stability emphasizes the importanceof surveying the abundance of all endocytic pro-teins following a molecular deletion.

Parsing the endocytic proteins into threelayers (Figure 7) is not absolute in that mem-bers of the core module and the N-BARs alsoassist in recruiting secondary effectors (Schuskeet al. 2003, Takei et al. 1999, Verstreken et al.2003), whereas Eps15 and intersectin also helpstabilize the core module (Koh et al. 2007,Morgan et al. 2003). The relative impact of re-moving particular proteins does not necessarilycorrespond to the location within this endocyticnetwork. For instance, the core module pro-teins AP2, clathrin, and amphiphysin all have arelatively small effect on SV endocytosis whenremoved individually (Di Paolo et al. 2002, Guet al. 2008, Sato et al. 2009). These observa-tions would be predicted in a redundant net-work such as this, because secondary scaffoldsand effectors functionally compensate for theloss of these core proteins. Perhaps double dele-tions that specifically target redundant functionmay have a more dramatic effect on SV endo-cytosis. For example, removal of clathrin andintersectin simultaneously would be predicted


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to impact the scaffolding function of the net-work. Concomitant removal of ENTH- andBAR-domain proteins may eliminate the abilityto deform the membrane.

CONCLUDING REMARKS

In the half century following Katz’s quantumhypothesis at the nerve terminal (Del Castillo& Katz 1954), advances in molecular, imaging,and physiological techniques have conspiredto produce a fairly detailed understanding ofthe SV cycle. The process of manufacturing,trafficking, and recycling SVs involves thecoordinated efforts of hundreds of proteins

discovered over the past few decades. As the listof molecular players grows, so does the needto synthesize and organize this collection. Aswith many aspects of modern cell biology, thedetailed properties of SV endocytosis may beemergent properties of a network. One of theimpediments to understanding this emergenceis that we still lack in-depth information aboutthe precise location of most of these moleculesduring various stages of the SV cycle. Quantita-tive studies of the dynamic interactions, as wellas application of new super-resolution tech-niques, should provide a rich new level of detailto understand the functioning of the endocyticnetwork.

SUMMARY POINTS

1. Clathrin-mediated endocytosis accounts for the majority of SV endocytosis under nor-mal conditions, whereas bulk endocytosis may contribute following high-frequencystimulation.

2. SV proteins are endocytosed stochastically such that their dwell time on the plasmamembrane obeys an exponential distribution with a 6-s time constant at 37◦C (13-s timeconstant at room temperature).

3. The proteins of endocytosis can be organized in a simple hierarchical network based onprotein interactions, domains, and genetic ablation studies.

4. No single component of the SV endocytosis machinery appears to be essential: Thenetwork 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 thisreview.

ACKNOWLEDGMENTS

The authors apologize in advance to all the investigators whose research could not be appropriatelycited owing to space limitations. We thank members of the Ryan and Dittman labs for usefuldiscussions and suggestions.

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AR389-FM ARI 14 September 2009 14:58

Annual Reviewof Cell andDevelopmentalBiology

Volume 25, 2009

ContentsChromosome Odds and Ends

Joseph G. Gall � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Small RNAs and Their Roles in Plant DevelopmentXuemei Chen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �21

From Progenitors to Differentiated Cells in the Vertebrate RetinaMichalis Agathocleous and William A. Harris � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �45

Mechanisms of Lipid Transport Involved in Organelle Biogenesisin Plant CellsChristoph Benning � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �71

Innovations in Teaching Undergraduate Biologyand Why We Need ThemWilliam B. Wood � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �93

Membrane Traffic within the Golgi ApparatusBenjamin S. Glick and Akihiko Nakano � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 113

Molecular Circuitry of Endocytosis at Nerve TerminalsJeremy Dittman and Timothy A. Ryan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 133

Many Paths to Synaptic SpecificityJoshua R. Sanes and Masahito Yamagata � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 161

Mechanisms of Growth and Homeostasis in the Drosophila WingRicardo M. Neto-Silva, Brent S. Wells, and Laura A. Johnston � � � � � � � � � � � � � � � � � � � � � � � � � 197

Vertebrate Endoderm Development and Organ FormationAaron M. Zorn and James M. Wells � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 221

Signaling in Adult NeurogenesisHoonkyo Suh, Wei Deng, and Fred H. Gage � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 253

Vernalization: Winter and the Timing of Flowering in PlantsDong-Hwan Kim, Mark R. Doyle, Sibum Sung, and Richard M. Amasino � � � � � � � � � � � � 277

Quantitative Time-Lapse Fluorescence Microscopy in Single CellsDale Muzzey and Alexander van Oudenaarden � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 301

Mechanisms Shaping the Membranes of Cellular OrganellesYoko Shibata, Junjie Hu, Michael M. Kozlov, and Tom A. Rapoport � � � � � � � � � � � � � � � � � � � � 329

The Biogenesis and Function of PIWI Proteins and piRNAs: Progressand ProspectTravis Thomson and Haifan Lin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 355

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Mechanisms of Stem Cell Self-RenewalShenghui He, Daisuke Nakada, and Sean J. Morrison � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 377

Collective Cell MigrationPernille Rørth � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 407

Hox Genes and Segmentation of the Hindbrain and Axial SkeletonTara Alexander, Christof Nolte, and Robb Krumlauf � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 431

Gonad Morphogenesis in Vertebrates: Divergent Means to aConvergent EndTony DeFalco and Blanche Capel � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 457

From Mouse Egg to Mouse Embryo: Polarities, Axes, and TissuesMartin H. Johnson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 483

Conflicting Views on the Membrane Fusion Machinery and the FusionPoreJakob B. Sørensen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 513

Coordination of Lipid Metabolism in Membrane BiogenesisAxel Nohturfft and Shao Chong Zhang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 539

Navigating ECM Barriers at the Invasive Front: The CancerCell–Stroma InterfaceR. Grant Rowe and Stephen J. Weiss � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 567

The Molecular Basis of Organ Formation: Insights from theC. elegans ForegutSusan E. Mango � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 597

Genetic Control of Bone FormationGerard Karsenty, Henry M. Kronenberg, and Carmine Settembre � � � � � � � � � � � � � � � � � � � � � � 629

Listeria monocytogenes Membrane Trafficking and Lifestyle:The Exception or the Rule?Javier Pizarro-Cerda and Pascale Cossart � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 649

Asymmetric Cell Divisions and Asymmetric Cell FatesShahragim Tajbakhsh, Pierre Rocheteau, and Isabelle Le Roux � � � � � � � � � � � � � � � � � � � � � � � � � � � 671

Indexes

Cumulative Index of Contributing Authors, Volumes 21–25 � � � � � � � � � � � � � � � � � � � � � � � � � � � 701

Cumulative Index of Chapter Titles, Volumes 21–25 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 704

Errata

An online log of corrections to Annual Review of Cell and Developmental Biology articlesmay be found at http://cellbio.annualreviews.org/errata.shtml

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