Cell, Vol. 123, 521–533, November 4, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.cell.2005.09.026

Matters ArisingA Slowed Classical PathwayRather Than Kiss-and-Run Mediates Endocytosisat Synapses Lacking Synaptojanin and Endophilin

Dion K. Dickman,1 Jane Anne Horne,2

Ian A. Meinertzhagen,2 and Thomas L. Schwarz1,*1Division of NeuroscienceChildren’s Hospital and Department of NeurobiologyHarvard Medical SchoolBoston, Massachusetts 021152Life Sciences CentreDalhousie UniversityHalifax, Nova Scotia B3H 4J1Canada

Summary

The extent to which a “kiss-and-run” mode of endocy-tosis contributes to synaptic-vesicle recycling re-mains controversial. The only genetic evidence forkiss-and-run at the synapse comes from mutationsin the genes encoding synaptojanin and endophilin,proteins that together function to uncoat vesicles inclassical clathrin-mediated endocytosis. Here wehave characterized the endocytosis that persists innull alleles of Drosophila synaptojanin and endophi-lin. In response to high-frequency stimulation, thesynaptic-vesicle pool can be reversibly depleted inthese mutants. Recovery from this depletion is slowand indicates the persistence of an impaired form ofclassical endocytosis. Steady-state exocytosis ratesreveal that endocytosis saturates in mutant neuro-muscular terminals at 80 vesicles/s, 10%–20% of thewild-type rate. Analyses of quantal size, FM1-43 load-ing, and dynamin function further demonstrate that,even in the absence of synaptojanin or endophilin,vesicles undergo full fusion and re-formation. There-fore, no genetic evidence remains to indicate thatsynaptic vesicles undergo kiss-and-run.

Introduction

To maintain a functional pool of synaptic vesicles in anerve terminal, the rate of endocytosis must balance thatof exocytosis. This is particularly critical during high-fre-quency firing, when the vesicle pool would otherwiserapidly deplete. Indeed, synapses can sustain recyclingrates of at least 1 vesicle/s per active zone in the hippo-campus (Fernandez-Alfonso and Ryan, 2004) and at theDrosophila neuromuscular junction (Delgado et al., 2000).Synaptic endocytosis must also maintain vesicle sizeand ensure sorting of essential proteins to the vesicleeven while operating at rapid rates.

The mechanisms governing synaptic endocytosis arecontroversial: do exocytosing vesicles fully fuse withthe membrane prior to endocytosis, or do they releasetransmitter through a transient fusion pore in a processcalled “kiss-and-run”? The first of these pathways,termed “classical” or “slow” endocytosis, primarily oc-curs outside the active zone and involves the recovery

*Correspondence: thomas.schwarz@childrens.harvard.edu

of components of vesicles that had completely fusedwith the plasma membrane. There is overwhelming evi-dence from genetic, biochemical, electrophysiological,and imaging experiments to support the importance ofthis mechanism in synaptic-vesicle recycling, which ismechanistically akin to endocytosis in other cell typesand involves recruitment of a clathrin coat and subse-quent fission of the vesicle from the plasma membraneby dynamin (Brodin et al., 2000; Heuser and Reese, 1973).Endophilin and synaptojanin then uncoat the vesicle,which is directed back to the functional vesicle pool(Cremona and De Camilli, 2001; Murthy and De Camilli,2003). Genetic and pharmacologic studies have con-firmed the importance of these proteins, and thereforeof classical endocytosis, at synapses in vivo (Guichetet al., 2002; Koenig and Ikeda, 1989; Rikhy et al., 2002;Royle and Lagnado, 2003; Verstreken et al., 2003;Zhang, 2003).

The alternative kiss-and-run model envisions a sim-ple reversal of an exocytotic event, directly at the activezone, in which the vesicle membrane never fully fuseswith the plasma membrane (Kjaerulff et al., 2002; Ryan,2003; Schneider, 2001). Instead, a pore transientlyforms from the vesicle lumen to the extracellular spaceand releases neurotransmitter. The pore then closesand the vesicle returns to the functional vesicle pool tobe refilled with transmitter; no clathrin coat is formedand the protein components need not be sorted fromthe plasma membrane because they never had beenincorporated. While classical and kiss-and-run endocy-tosis are not mutually exclusive mechanisms, the rela-tive importance of each pathway and, indeed, whetherkiss-and-run even occurs at synapses both remain un-resolved issues.

In neurons, the role of kiss-and-run endocytosis bysmall clear synaptic vesicles is highly controversial.Evidence for it has depended primarily on physiologicalmeasurements because no molecules are known withan exclusive role in a kiss-and-run pathway. Phenom-ena that suggest the existence of a fast endocytoticpathway have been observed (Aravanis et al., 2003b;Gandhi and Stevens, 2003; Klingauf et al., 1998; Men-nerick and Matthews, 1996; Staal et al., 2004; Stevensand Williams, 2000; Sun et al., 2002), but, in somecases, the interpretation as kiss-and-run has beencalled into question (Ryan et al., 1996; Sterling andMatthews, 2005; Yamashita et al., 2005; Zenisek et al.,2000, 2002), leading to the suggestion that all synapticendocytosis is classical and dynamin dependent (Del-gado et al., 2000; Fernandez-Alfonso and Ryan, 2004;Koenig and Ikeda, 1983; Ryan et al., 1996; Yamashitaet al., 2005).

The genetic evidence for synaptic kiss-and-run restschiefly on the recent analysis of Drosophila mutants ofendophilin (endo) and synaptojanin (synj) (Verstreken etal., 2002, 2003). Synaptojanin is a polyphosphoinositidephosphatase thought to release the clathrin-adaptorcomplex during uncoating (Cremona et al., 1999). En-dophilin binds to both dynamin and synaptojanin (Ring-stad et al., 1997) and is thought to recruit synaptojanin

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to sites of endocytosis to promote vesicle uncoating(Schuske et al., 2003; Verstreken et al., 2003). AnalyzingDrosophila mutants, Verstreken et al. (2002, 2003) madethe important observation that synaptic transmissionwas sustained even in the face of extended stimulationthat would have exhausted the available vesicle poolhad not a means for the recovery of synaptic vesiclespersisted. Kiss-and-run endocytosis was therefore in-voked as a mechanism to explain the persistence oftransmission at the neuromuscular junction in thesemutants in which classical endocytosis appeared to beabolished (Verstreken et al., 2002).

These mutants have thus figured prominently in thecontroversy surrounding synaptic endocytosis, buttheir interpretation is based on the contention that noclassical endocytosis remained in the mutants to ac-count for the persistence of synaptic transmission.Here, we examine null mutations in synaptojanin andendophilin in order to re-evaluate this conclusion andto clarify their significance for endocytosis. We find thatclassical endocytosis in fact persists, though in an im-paired form, even in the absence of these proteins. Wefind no evidence of or need to invoke a kiss-and-runpathway at the Drosophila neuromuscular junction.

Results

Two mutants in synaptojanin (synjLY and synjYN) wereindependently isolated from a forward genetic screenon chromosome 2R using the EGUF-hid method andidentifying defects in photoreceptor transmission (Stow-ers et al., 2002; Stowers and Schwarz, 1999) (see FigureS1 in the Supplemental Data available with this articleonline). There is only a single synaptojanin gene in Dro-sophila, which predicts a protein of 1218 amino acids(Verstreken et al., 2003). We sequenced the entire syn-aptojanin (synj) open reading frame in each allele andfound both to contain an identical C-to-T transition thatcreates a stop codon at the 45th amino acid, before thefirst phosphatase domain, and that is thus likely to bea null mutation. Both alleles failed to complement a chro-mosomal deficiency in this region, Df(2R)x58-7. We usedthe null genotype synjLY/Df(2R)x58-7 in all the experi-ments below. We generated a GFP-synaptojanin trans-gene that could rescue the late-larval lethality of thisgenotype when selectively expressed in the nervoussystem (Figure S1).

Ultrastructure Reveals Depletion of SynapticVesicles in synaptojanin MutantsWhen homozygous for either allele of synj, photorecep-tors differentiated correctly and formed structurallynormal synapses in the lamina. The profiles of photore-ceptor terminals contained synaptic vesicles that weremore clustered than in wild-type (Figure S2), similar tothose seen in endophilin mutant terminals (Fabian-Fineet al., 2003). This close phenotypic similarity to endo-philin provided an obvious parallel between the mutantactions of the two genes, consistent with their knownbiochemical roles.

To characterize the state of the synaptic-vesicle poolat the synapse at which we have studied transmission(see below), we also analyzed the neuromuscular phe-

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otypes of synj and wild-type flies by electron micro-copy (EM; Figure 1). Overall, the mutant varicositiesad significantly fewer synaptic-vesicle profiles (22% ofild-type; p < 0.01; Figures 1A, 1B, and 1E) and feweresicles per area of terminal cross-section (p < 0.02).hese vesicles remained clustered, albeit less tightly

Figure 1B and 1C), and included a subpopulation ofarge vesicle profiles as well as the normal 30–40 nmesicles (inset, Figure 1D), suggesting an alteration inesicle formation or recycling. Indeed, the mean vesicleiameters from synj (35.5 ± 0.0067 nm, mean ± SD, n =09 vesicles) and wild-type (33.6 ± 0.005 nm, n = 314esicles) terminals were significantly different (p <.0005, Kolmogorov-Smirnov test) because of a sub-opulation of large vesicles, apparent in a cumulative-robability histogram (Figure 1E).The observed reduction in the number of vesicles per

erminal was expected from an endocytosis mutant.ome vesicles nevertheless remained, indicating either

hat an endocytotic cycle persisted or that some vesi-les were inert and could not be released. We thereforeurned to detailed analysis of the electrophysiology ofhe neuromuscular junction to clarify the nature of vesi-le cycling in the mutants.

igh-Frequency Stimulation Reversibly Depletesynj and endo Mutant Terminalsespite the paucity of synaptic vesicles in the mutant

erminals, normal-amplitude synaptic responses coulde evoked. We stimulated the motor nerve to muscle 6t 0.2 Hz in 0.6 mM Ca2+ to evoke junctional potentials

EJPs) in synj mutants (12.8 ± 0.8 mV amplitude, mean ±EM, n = 10) that were indistinguishable from controls

12.6 ± 0.6 mV, n = 6). Mutant (42.4 ± 1.4 mV, n = 21)nd control (45.2 ± 1.5 mV, n = 23) EJPs were also indis-inguishable in 10 mM Ca2+, the condition used for de-letion studies below. The properties of spontaneousesicle fusions were also surprisingly unperturbed atynj synapses. Although there was a slight but signifi-ant decrease in mEJP frequency between synj andontrol (1.5 ± 0.18 Hz, n = 11 and 2.2 ± 0.3 Hz, n = 12

n 10 mM Ca2+; p < 0.031, Student’s t test), averageEJP amplitude was unchanged (0.86 ± 0.06 mV, n =

1 and 0.80 ± 0.05 mV, n = 12). The normal quantalontent of synj mutant terminals (14.9 ± 0.7 quanta in.6 mM Ca2+ compared with 15.7 ± 0.7 in controls) andheir nearly normal mEJP frequency indicate that ateast some of the remaining vesicles observed in EM

ust be available and efficiently mobilized for release;t is thus unlikely that many of those detected ultra-tructurally are inert and incapable of exocytosis.oreover, this functional pool of synaptic vesicles muste maintained throughout larval development, suggest-

ng a continuing mechanism for recycling vesicles de-pite the absence of synaptojanin.In a similar electrophysiological analysis of the null

llele endo�4 (Verstreken et al., 2002), the only physio-ogical difference from synj that we found was that

EJP amplitude was increased by 50% in endo mu-ants (1.21 ± 0.17 mV, n = 6; p < 0.006), while mEJPrequency was not significantly different from that ofontrols (2.07 ± 0.53 Hz, n = 6). Thus, evoked responses

n endo mutants are composed of fewer, larger quantal

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Figure 1. Synaptic Vesicles Are Fewer but Clustered in SynapticTerminals Mutant for synj

(A and B) Neuromuscular varicosities from third-instar larvae,shown at the same magnification. By comparison with controls,synj varicosities (B) have fewer synaptic vesicles, which, at somelocations (arrow) close to the T bar ribbon of an active zone, are oflarger diameter. Scale bar: 1 µm.(C) Synaptic sites, as indicated by T bar ribbons (arrowheads), liecloser together in locations not typical of control terminals.(D) Enlarged view of mutant synapse showing residual large vesicleprofiles (arrow) associated with an active-zone T bar ribbon. Scalebar (in [D] for [C] and [D]): 100 nm.(E) The packing densities (left) and spacings (Ra/Re; see Supple-mental Data) of synaptic vesicles in neuromuscular varicosities in-dicate that the reduced populations of synaptic vesicles are moreclustered in mutant terminals than in their corresponding controls.The values of Ra and Re were each significantly higher in the mu-tant (both, p < 0.005), and their ratios were 0.85 in synj and 1.1in wild-type, a difference that was also significant (p < 0.05). Thedistribution of vesicle diameters in synj neuromuscular varicosities(right) is shifted to an increased representation of vesicles >w36nm in diameter (arrow). Above this size, the distributions of diame-ters for synj and wild-type varicosities differed by eye in frequencyhistograms (data not shown), as also revealed by the cumulative-frequency plot of all vesicle sizes. Error bars are mean ± SEM.

events than those of either control or synj terminals.Whereas synaptojanin and endophilin have previouslybeen regarded as functioning jointly in the fission anduncoating steps of endocytosis, the present data sug-gest that endophilin may have a separate functionalongside AP180 in determining vesicle size (Zhang etal., 1998).

Previous studies of synaptojanin and endophilin re-ported that mutant terminals could not sustain normal-amplitude responses to stimulation at 10 Hz (Verstrekenet al., 2002, 2003). This depletion was interpreted toreflect the full fusion of a population of synaptic vesi-cles with the plasma membrane with the consequencethat these vesicles then become trapped at the mem-brane. The terminals maintained the ability to sustaintransmitter release at a reduced level, which was attrib-uted to a subset of vesicles that could undergo kiss-and-run fusions independently of synaptojanin or en-dophilin function. To examine this model, we stimulatedsynj and endo larvae at 10 Hz for 10 min. In wild-typeterminals, EJP amplitudes initially declined rapidly (τ =3.0 s), probably from depletion of the readily releasablepool, and then declined more slowly (τ = 360 s), to60.2% ± 5.1% of the starting EJP amplitude (mean ±SEM, n = 7; Figures 2A and 2B). EJPs in synj mutantsinitially behaved similarly (τ = 1.5 s), but the secondphase of depression was both more pronounced (de-clining to 19.9% ± 2.0% of initial amplitude) and morerapid (τ = 66 s, n = 11) than in wild-type (Figures 2A and2B). This depletion is somewhat more severe than thatpreviously reported (Verstreken et al., 2003), perhapsbecause synjLY/Df(2R)x58-7 is a null genotype. TheEJPs of endo mutants declined similarly (Figures 2Aand 2B). The total number of quanta released duringthis 10 min period was w350,000 for controls,w100,000 in synj mutants, and w80,000 in endo mu-tants (Figure 2D), far greater than the total vesicle-poolsize for these genotypes, as determined below (Figure2C), and similar to what has been reported (Verstrekenet al., 2003). Therefore, some mechanism of endocyto-sis must persist in these mutants to maintain thissteady-state level of release.

If synj and endo mutations abolish classical endocy-tosis, the vesicle pool should not recover to prestimuluslevels after depletion; those vesicles that had beentransferred to the plasma membrane by full fusion inthe early stages of stimulation would not be recovera-ble. To test this prediction, we stimulated at 10 Hz for10 min and subsequently gave a test pulse every 5 sfor the next 10 min to monitor whether or not the vesiclepool was recovering. Control larvae recovered very rap-idly: within 10 s, responses were 92.2% ± 5.4% of theprestimulus EJP amplitude (n = 5; Figures 2A and 2B).Interestingly, synj neuromuscular junctions could alsorecover, albeit at a substantially slower rate: after 10min, they had attained 67.8% ± 6.8% of the prestimuluslevel (n = 6; Figures 2A and 2B). The recovery in con-trols was best fit by a single-exponential function witha time constant of 4.4 s. A two-exponential functionbest fit the synj recovery curves, with time constants of15.4 s for the initial fast recovery and 582 s for the slowphase of recovery. endo terminals behaved very simi-larly to those in synj larvae during recovery from high-frequency trains (Figures 2A and 2B): a slow kinetic re-

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Figure 2. Depletion and Recovery of the Synaptic-Vesicle Pool at Synapses Lacking Synaptojanin or Endophilin

All error bars are mean ± SEM.(A) Control, synj, and endo mutants were stimulated at 10 Hz for 10 min (until arrow) and subsequently allowed to recover while monitoringthis recovery with stimulation at 0.2 Hz. EJP amplitudes were averaged (binning 2 s of response for each 10 Hz time point), normalized toprestimulus amplitudes, and plotted as a function of time.(B) Representative traces of experiments in (A), with typical individual EJPs shown on a faster timescale, below their corresponding timepoints.(C and D) Determination of the size of the functional synaptic-vesicle pool in control and synj mutant synapses.(C) shits1 and shits1;synjLY/Df(2R)x58-7 mutants were stimulated at 10 Hz at 32°C to deplete the functional synaptic-vesicle pool. EJP ampli-tudes were normalized and averaged as in (A).(D) The total quanta released during 10 Hz stimulation in (A) compared with the total functional synaptic-vesicle pool in (C). During 10 Hzstimulation, 347,000 ± 20,200 synaptic vesicles (mean ± SEM, n = 7) are released from control synapses, 99,600 ± 8,100, from synj (n = 11),and 79,200 ± 14,200 from endo (n = 7). In the absence of recycling, the releasable pool in shi was 49,900 ± 4,600 vesicles (n = 4), while inshi;synj it was 18,500 ± 2,200 (n = 4).

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covery was observed that could be fit with time con-stants of 9.6 s and 421 s. Thus, the recovery of thesynaptic response implies the existence of a persistingendocytotic pathway, albeit one that is slow in the ab-sence of synaptojanin or endophilin. Recovery cannotbe due to recruitment from a reserve pool becausethere is no large reserve of vesicles in the mutant ter-minals. A predominantly kiss-and-run pathway cannotbe reconciled with these observations because vesi-cles that had undergone kiss-and-run fusion shouldhave been available for rerelease within seconds (Pyleet al., 2000; Sara et al., 2002).

The persistence of normal rates of exocytosis at synjneuromuscular junctions despite the apparent deple-tion of vesicles seen by EM suggested that those vesi-cles remaining in the terminals were available for re-lease. For an independent estimate of the number ofvesicles available for exocytosis, we combined synjwith the shibirets1 (shi) mutation, a well-characterizedtemperature-sensitive allele of dynamin that blocks allsynaptic endocytosis at the restrictive temperature(Koenig and Ikeda, 1989). We recorded the postsynap-tic responses of both shi and shi;synj mutants to 10 Hzstimulation at the restrictive temperature and deter-mined the total quanta released as the vesicle pool wasdepleted (Figures 2C and 2D). While control shi larvaereleased w50,000 vesicles before depletion under ourconditions, shi;synj mutants released only w18,000vesicles. Thus, the depletion observed by EM (22% re-maining; Figure 1) was comparable to that determinedphysiologically (36% remaining). Clearly, however, tomaintain responses to 10 Hz stimulation, the remainingvesicles cannot be sequestered in a reserve pool ormade inert by the persistence of a clathrin coat. Thisconclusion is consistent with the observation that theyappear uncoated and remain clustered, probably in thevicinity of the release sites.

Changes in mEJP Amplitude after TetanusWe noted that the amplitude of the mEJPs increased insynj mutants after the 10 Hz stimulation in Figure 2. Wetherefore quantified mEJP amplitude before the tetanus(“pre” conditions), for 2 min immediately after the 10min tetanus (“post” conditions), and again after 10 minof rest posttetanus (“post+10” conditions) in control,synj, and endo larvae (Figure 3). The mEJP amplitudedistribution in controls decreased slightly after the teta-nus and partially recovered to a prestimulus distributionafter 10 min (Figures 3A and 3D). This may be due todesensitization of the postsynaptic glutamate recep-tors or decreased filling of the synaptic vesicles withneurotransmitter but was not investigated further. Incontrast, although the amplitude distribution of synjmEJPs was similar to that in controls before the stimu-lus, synj amplitudes were larger right after the stimulusand even larger after 10 min rest (Figures 3B and 3E).This change was chiefly due to the appearance of asubpopulation of particularly large mEJPs. The mea-sured increase may be an underestimate if the pro-cesses that caused the decrease in amplitude at wild-type synapses are also occurring at these synapses.Regardless, the alterations in mEJP amplitude may bea correlate of the subpopulation of large vesicles seen

Figure 3. mEJP Amplitude Changes after a Tetanus

(A) Representative traces of mEJP recordings from control syn-apses in prestimulus time points (controlpre) and 10 min after thetetanus (controlpost+10).(B and C) Similar representative traces for synj (B) and endo (C).(D) Cumulative-probability histograms showing mEJP amplitudedistributions in control synapses during prestimulus conditions(controlpre, n = 3158), immediately following the tetanus (controlpost,n = 418), and 10 min following the tetanus (controlpost+10, n = 1030).All curves are significantly different (Kolmogorov-Smirnov [KS] test,p < 0.001).(E) Similar histograms for synj during prestimulus conditions (synj-

pre, n = 1935) compared with controlpre (not significantly different;KS test, p > 0.05), synjpost (n = 823), and synjpost+10 (n = 429). Bothsynjpost and synjpost+10 distributions are significantly different fromsynjpre and controlpre (KS test, p < 0.001).(F) Similar histograms of endo mutant (endopre, n = 2275) distribu-tions relative to controlpre and endopost+10 (n = 1492). All are signifi-cantly different from each other (KS test, p < 0.001). Events from 2min of recordings from each of at least 5 animals were pooled foreach genotype and condition.

by EM (Figure 1), and, thus, while we cannot exclude apostsynaptic mechanism, the change in mEJPs is mostlikely to have arisen presynaptically. When endo mEJPamplitudes were measured in similar conditions, ampli-tude distributions actually became smaller after thetetanus (Figures 3C and 3F). Thus, the pathway main-taining the uniformity of synaptic-vesicle size is com-promised by the absence of synaptojanin or endophilin,particularly when endocytosis must be rapid. Because

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Figure 4. Loading and Unloading FM1-43 Dye in Cycling and Re-Forming Synaptic Vesicles

All error bars are mean ± SEM.(A–C) Synapses on muscles 6 and 7 loaded with FM1-43 in control, synj, and endo larvae. Animals were dissected and stimulated with 90mM K+ and 2 mM Ca2+ for 10 min in the presence of FM1-43 dye to load recycling synaptic vesicles, then washed in 5 mM K+, 0 mM Ca2+

saline for 12 min, and finally imaged. Inset: individual labeled boutons of each genotype.(D) Control larvae failed to load dye when the nerve cord was cut, and the dye was instead applied in 5 mM K+, 2 mM Ca2+.(E–G) Synaptic terminals were unloaded following an additional 5 min incubation in 90 mM K+ without dye.(H) Loading was quantified (n = 7 for wt, n = 8 for synj, n = 7 for endo, and n = 5 for wt controls; see Experimental Procedures).(I–K) Control, synj, and endo synapses were loaded in a manner similar to that in (A)–(C) using endogenous activity from the nerve cord during10 min incubation in 5 mM K+, 2 mM Ca2+ and dye.(L) Synapses with a severed nerve were not competent to load in the same conditions.

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fective in loading dye even in mutant synapses. With

(M–O) These loaded synapses were unloaded by 5 min incubation in 90 mM K+.(P) Loading was quantified (n = 5 for all genotypes).(Q–S) Depletion/re-formation loading in shi (shits1) (R) and shi;synj (shits1;synjLY/Df(2R)x58-7) (S) mutant terminals. Larvae were dissected andequilibrated to 34°C in 5 mM K+, 2 mM Ca2+ saline, then depleted of synaptic vesicles with a 5 min incubation in 90 mM K+, 2 mM Ca2+, andwere finally allowed to re-form synaptic vesicles for 15 min at 22°C in 5 mM K+, 2 mM Ca2+ saline. FM1-43 was not loaded when larvae weretreated similarly but the dye was removed before the 15 min recovery phase (Q).(T) Loading was quantified (n = 9 in shi, 11 in shi;synj, and 4 in shi controls).Scale bar: 10 µm for (A)–(G), 8 µm for (I)–(O), and 2 µm for (Q)–(S). Images were acquired with identical settings within each experimentalgroup: (A–G), (I–O), (Q–S). All mutant loading was above background (p < 0.05).

stages. However, both shi and shi;synj terminals were

a kiss-and-run mechanism preserves the integrity andtherefore the size of a vesicle, the changed amplitudesare not easily explained if the sustained release pro-ceeds by kiss-and-run.

FM1-43 Dye Loading of Synaptic Vesiclesin endophilin and synaptojanin MutantsIn several systems, anomalies in the loading and un-loading of lipophilic dyes have served as evidence of akiss-and-run pathway (Aravanis et al., 2003a; Cochillaet al., 1999; Klingauf et al., 1998). At the Drosophilaneuromuscular junction, endo null mutants have beenreported to be incapable of taking up FM1-43 dye, andit was therefore concluded that the recycling of vesiclesduring extended stimulation was accomplished by akiss-and-run mechanism in which a proteinaceous fu-sion pore was a barrier to dye loading (Verstreken etal., 2002). However, the small size of the vesicle poolin individual synj and endo boutons might alternativelyaccount for difficulties in observing dye loading, and sowe re-examined this issue.

Control larval preparations (Figure 4A) could be loadedefficiently with a 10 min exposure to FM1-43 in 90 mMK+, 2 mM Ca2+ followed by a 12 min wash in 5 mM K+,0 mM Ca2+ saline (Kuromi and Kidokoro, 1998). Wewere also able to load dye in synj, endo1, and endo�4

terminals using this protocol (Figures 4B, 4C, and 4Hand data not shown). The degree of loading was greatlyreduced, however, as expected from slowed endocyto-sis and the reduced number of vesicles observed byEM in mutant terminals, a number which was probablyfurther reduced by the depolarizing conditions em-ployed to load dye. Similar results were obtained byloading in 60 mM K+, 2 mM Ca2+ (data not shown). Thatthe dye loading in both wild-type and mutant larvaerepresented a releasable pool of synaptic vesiclesrather than background staining or uptake into anothercompartment was confirmed by observing that dyecould subsequently be unloaded by a further 5 min in-cubation in 90 mM K+, 2 mM Ca2+ (Figures 4E–4G). Thespecificity of the loading was also confirmed by thefinding that dye was not loaded into control terminalsin 5 mM K+ saline provided that the nerve had beensevered from the nerve cord to prevent the activationof the terminals by action potentials arising spontane-ously in the nerve cord (Figure 4D).

We also could load FM1-43 by taking advantage ofthe presence of spontaneous electrical activity in prep-arations in which the nerve cord remained attached tothe neuromuscular junction. These conditions, whichmore closely resemble physiological stimuli, were ef-

an intact nerve, in 2 mM Ca2+ and 5 mM KCl, controlterminals became brightly fluorescent after 10 min in-cubation in FM1-43; synj and endo terminals were alsoloaded, although again at a reduced level (Figures 4I–4K and 4P). The loaded terminals could then be un-loaded of dye by 5 min stimulation in high K+ (Figures4M–4O). By contrast, loading did not occur in 5 mM KClwhen the nerve was severed from the ventral nervecord (Figure 5L). Similar results were obtained with themore hydrophobic dye FM1-84, using either this proto-col or K+ depolarization (data not shown). Thus, syn-apses lacking synaptojanin or endophilin are still com-petent to load and release lipophilic dyes.

The ability to load dye in synj and endo removes thebasis for invoking a narrow, transient fusion pore thatselectively excludes the dye but does not in and ofitself require that the endocytosis be mediated by aclassical pathway; some models of kiss-and-run endo-cytosis may be compatible with dye uptake and release(Aravanis et al., 2003a; Klingauf et al., 1998). The persis-tence of a classical endocytotic pathway, even in theabsence of synaptojanin, could be tested, however, bydepleting the terminals of synaptic vesicles and thendetermining if they were capable of re-forming. Ter-minals can be reversibly depleted of vesicles by manip-ulating the temperature-sensitive shi mutant, in which,at the restrictive temperature, clathrin-coated endocy-totic vesicles can no longer be severed from the plasmamembrane (Koenig and Ikeda, 1989). When stimulatedat the nonpermissive temperature, shi terminals looseall of their synaptic vesicles and contain some profiles,not at active zones, of membrane arrested in the pro-cess of endocytosis (Koenig and Ikeda, 1989). By EM,we have determined that shi;synj double mutants arealso depleted of vesicles by stimulation at nonpermis-sive temperatures (data not shown). We therefore ex-amined dye loading in shi and shi;synj double mutants.If classical endocytosis were to occur, we reasonedthat a double-mutant terminal could be depleted ofsynaptic vesicles at the nonpermissive temperature (asin Figure 2C) and then loaded with dye upon restorationof dynamin function and release from endocytoticblockade at the permissive temperature. To test thispossibility, we used a protocol in which high K+ at 34°Cwas used to deplete the vesicle pool, and then re-for-mation of vesicles was assayed by incubating the lar-vae in 5 mM K+, 2 mM Ca2+ at 22°C for 15 min in thepresence of dye (Kuromi and Kidokoro, 1998). In thispoststimulus loading phase, vesicles that had beentrapped on the plasma membrane would re-form andincorporate dye. We could not assay shi;endo mutantsbecause they did not survive to third-instar larval

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Figure 5. Stimulation Frequency Determines Steady-State Transmission in Wild-Type, synj, and endo Synapses

All error bars are mean ± SEM.(A) 1 Hz stimulation for 10 min in control (n = 4), synj (n = 6), and endo (n = 4) synapses. For each graph, 2 s of responses was averaged ateach time point, normalized to initial EJP amplitudes, and plotted as a function of time.(B) A comparison of 5 and 10 Hz stimulation for 5 min in control (n = 4 and 5), synj (n = 7 and 4), and endo (n = 6 and 6) terminals.(C) Sequential stimulation at 1 Hz for 2 min, 7 Hz for 2 min, 10 Hz for 3 min, and 20 Hz for 3 min in control (n = 6) and synj (n = 7) terminals.(D) Sequential stimulation at 7 Hz for 3 min, 20 Hz for 3 min, 7 Hz for 5 min, and 0.2 Hz for 5 min in synj (n = 6) and endo (n = 5). Note thatEJP amplitudes are dependent on the stimulation frequency and can recover from high-frequency trains, as seen in Figure 2, to reach thesteady-state amplitude characteristic of that frequency.(E) Steady-state EJP amplitude at each frequency for wild-type, synj, and endo terminals. The data in (A)–(D) were fit with the equation f(t) =A1e−t/τ

1 + A2e−t/τ2 + B to determine the normalized steady-state amplitude, B, at each frequency.

(F) Quanta released per second during steady states at 1 Hz, 5 Hz, 7 Hz, 10 Hz, and 20 Hz in control, synj, and endo terminals. Quanta werecalculated by dividing the EJP amplitudes at steady state by the mEJP amplitude and introducing a correction for nonlinear summation(Martin, 1955).

able to load dye under these conditions (Figures 4R–4T). As expected, this loading was dependent on thepresence of FM1-43 dye after the shi blockade was re-lieved (Figure 4Q). Thus, synaptic vesicles incorporatedinto the membrane because of a block in dynamin func-tion were able to re-form into the vesicle pool once thatblock was removed; this depletion/re-formation experi-

di

SAAb

ment therefore provided direct evidence for a classical,

ynamin-dependent endocytotic pathway that persistedn the absence of synaptojanin.

timulus Frequency Determines the Steady-Statemplitude of the EJPs presented above, a slow endocytotic process cane demonstrated in synj and endo mutants. If this pro-

cess accounts for the sustained release observed in

Matters Arising529

Figure 2, then the sustained phase would represent asteady state in which exocytotic and endocytotic ratesare balanced. The level of transmission in such a steadystate would not be fixed by the size of a specializedpool of kiss-and-run vesicles but rather would be de-pendent on the rate of stimulation. Thus, examining thedependency of sustained release on stimulus fre-quency could provide a critical test of our hypothesisthat a slow process sustains transmission and couldalso serve to measure the rates of endocytosis atthese synapses.

We therefore examined the responses of mutant andwild-type synapses to stimulation at different fre-quencies. In control larvae, upon stimulation at 1 Hz for10 min, EJP amplitude declined slightly in the first 20 sof stimulation and thereafter was maintained at a nearlyconstant level that remained at 85.2% ± 7.8% of theinitial amplitude (n = 4; Figure 5A). synj and endo larvaehad similar initial levels of depression, followed by amodest but steady decrease in amplitude to 67.8% ±4.0% and 61.0% ± 2.8% of the starting EJP amplitude(n = 6 and 4; Figure 5A). Thus, the synaptic-vesicle poolwas maintained at near wild-type levels under pro-longed low-frequency stimulation even in the absenceof synaptojanin or endophilin function; at 1 Hz, mutantendocytotic rates were sufficient to counterbalancenearly wild-type rates of exocytosis.

We then compared the steady-state responses ofsynj and endo mutants to stimulation at 1, 5, 7, 10, and20 Hz, either stimulating a given synapse for 5 min atone frequency (Figure 5B) or sequentially stimulating asingle synapse at several frequencies (Figure 5C). Atfrequencies greater than 1 Hz, the amplitude of the re-sponses decreased and reached a steady-state levelafter approximately 3 min. The decline could be fit bythe double exponential f(t) = A1e−t/τ

1 + A2e−t/τ2 + B,

where B represents the steady-state level attained.These steady-state levels are tabulated in Figure 5E.The amplitude of the steady state was dependent onthe frequency of stimulation. Moreover, the decline wasreversible. When a preparation was stimulated sequen-tially at 7 Hz, 20 Hz, and again at 7 Hz, the amplitudeof the EJP recovered to a steady-state level character-istic of 7 Hz regardless of the intervening lower re-sponses during 20 Hz stimulation. Thus, the steadystate in which exocytosis and endocytosis are bal-anced is frequency dependent and reversible.

During these steady states, the number of availablevesicles must be maintained despite their rapid turn-over; the rate of replenishment by endocytosis conse-quently must equal the rate of exocytosis. Therefore,from the steady-state EJP amplitudes, we calculatedthe number of quanta that must be recycled per secondto sustain the steady state in wild-type, synj, and endoterminals at 1, 5, 7, 10, and 20 Hz stimulation fre-quencies (Figure 5F). In wild-type synapses, the rate ofendocytosis increased as the rate of stimulation in-creased, indicating that the endocytotic pathway isnormally capable of sustaining endocytosis at rates ofat least 360 vesicles/s, consistent with what othershave measured (Delgado et al., 2000). In contrast, insynj and endo preparations, endocytotic rates in-creased as stimulation increased from 1 to 5 Hz butwere saturated by approximately 7 Hz stimulation.

From 7 to 20 Hz, the mutant synapses consistently re-

cycled approximately 80 vesicles/s at the steady state(Figure 5F). Thus the responses of synj and endo syn-apses to repetitive stimulation can be described as aconsequence of a substantial slowing of the maximalrate of the classical pathway for endocytosis.

Discussion

Loss of Synaptojanin or Endophilin Impairs butDoes Not Abolish the Classical Endocytotic PathwayOur studies at the neuromuscular junction have uncov-ered a slow endocytotic pathway that persists in theabsence of synaptojanin or endophilin function. Fromthe evidence summarized below, we conclude that thispathway has the properties of a classical clathrin- anddynamin-mediated mechanism. Moreover, we find noevidence for a kiss-and-run mechanism and no need toinvoke such a mechanism to explain the physiology ofthe mutant terminals.

As noted by others (Verstreken et al., 2002; Ver-streken et al., 2003), the existence of a means of recycl-ing vesicles is clear from the ability of the synapse tosustain a normal EJP amplitude under conditions of lowactivity and to sustain release even at high frequencies,albeit with reduced amplitudes, for extended periods.That this recovery mechanism consists of a slow, clas-sical pathway is indicated by three lines of evidence. (1)Synaptic transmission can recover to initial amplitudeswhen released from tetanus. Were only kiss-and-runmechanisms available to recover vesicles, it would notbe possible for those that had fully fused to be restoredto the functional vesicle pool. The depleted pool wouldtherefore give rise to a reduction in EJP amplitude untilthe much slower process of vesicle biogenesis and ax-onal transport could restore those vesicles that hadbeen lost. That pathway cannot contribute significantlyin our experiments because the motor neuron cell bod-ies are no longer attached to the axons and becausethere is no measurable recovery in shi at the nonper-missive temperature (Figure 2). The recovery of EJPamplitude in synj and endo, however, takes place witha time constant of hundreds of seconds (Figure 2). Thistime course is far too slow for kiss-and-run, which ishypothesized to require milliseconds or a few secondsat most for the fusion pore to open and close and inwhich vesicles should rapidly be available for rerelease(Aravanis et al., 2003b; Gandhi and Stevens, 2003; Klin-gauf et al., 1998; Sara et al., 2002). (2) The mEJP ampli-tude increased after kinetically challenging conditionsin synj terminals (Figure 3); this observation stronglysuggests that the vesicles re-form by a classical endo-cytotic pathway, albeit one that is defective in its abilityto preserve vesicle size. Alterations in vesicle size, indi-cated both by EM and by mEJP amplitudes, probablyreflect alterations in the ability of clathrin and AP180 toshape the nascent vesicle (Zhang et al., 1998). In con-trast, a kiss-and-run mechanism in which vesicle integ-rity is maintained during transmitter release does notoffer an explanation for the stimulation-induced changesin quantal size at both synj and endo mutant synapses.(3) That transmitter release proceeds by full-fusionevents in both endo and synj mutants is also indicatedby the persistence of FM1-43 loading and unloading in

a variety of protocols. The lipophilic dye was incorpo-

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Figure 6. A Slow Classical Endocytotic Path-way Can Explain the Endocytotic Defects insynj and endo Mutants

Under conditions of low activity (left panels),a functional synaptic-vesicle pool and anEJP with normal amplitude can be main-tained in both wild-type and mutant ter-minals. High-frequency stimulation in-creases the rate of exocytosis (center andright panels), but, in wild-type terminals, en-docytotic rates increase to maintain a func-tional vesicle pool of nearly prestimulus size.In synj or endo terminals, however, endocy-tosis rates cannot adequately increase.While exocytotic rates are greater than en-docytotic (center panel), the releasable poolis depleted, and the amplitude of the EJPdeclines. A new steady state is reachedonce the depletion of this pool is sufficientto reduce the rate of exocytosis to a levelthat is equal to the endocytotic rate (rightpanels). The thickness of the arrows repre-sents the rates (vesicles/s) at which vesiclesare cycled between intracellular and plasma-membrane pools.

rated into recycling vesicles during periods of stimula-tion and could be unloaded in a stimulation-dependentway (Figure 4). Most notably, re-formation of synapticvesicles by a classical pathway was directly demon-strated by FM1-43 loading into synaptic vesicles thathad been trapped on the plasma membrane with theshibire mutant (Koenig and Ikeda, 1989) and then al-lowed to re-form. Thus, the recovery of the releasablepool is coupled to the endocytosis of membranes thathad fused with the cell surface. Therefore, data fromthe recovery of EJP amplitudes, changes in mEJP am-plitudes, and dye loading independently demonstratethe persistence of a slow, classical pathway for endo-cytosis in both synj and endo.

Modeling Exocytosis and Endocytosisin Mutant TerminalsWe propose that endocytosis at the Drosophila neuro-muscular junction is mediated by a single classicalpathway and have presented evidence that the rate atwhich this pathway operates is slowed but not abol-ished by the loss of either synaptojanin or endophilin.A special pool of kiss-and-run synaptic vesicles is notinvoked to explain the persistence of transmitter re-lease in these mutants, even under prolonged high-fre-quency stimulation. Rather, the rate of vesicle releasefrom synj and endo terminals reflects a steady-state re-lationship in which the rate of endocytosis balancesthat of exocytosis (Figure 6). Under conditions of lowactivity, wild-type and mutant terminals have compara-ble pools of readily releasable vesicles despite theoverall reduction in vesicle numbers in synj and endoboutons. Mutant terminals therefore have EJP ampli-tudes comparable to those of controls. However, underconditions of high-frequency stimulation, a wild-typeterminal can maintain a large synaptic-vesicle pool,while rates of endocytosis at mutant terminals areslower. Indeed, we have calculated (Figure 5F) that en-docytosis in mutant neuromuscular junctions saturatesat 80 vesicles/s. With high rates of exocytosis and in-

saqiIdsbtqwo

DtTwpsaetntpspsaptaehsarncd

ufficient compensatory rates of endocytosis, the avail-ble pool of synaptic vesicles declines, and, in conse-uence, the quantal content decreases until exocytosis

s sufficiently reduced to produce a new steady state.n this steady state, the rate of exocytosis from the re-uced pool matches its rate of restoration by endocyto-is. This model explains the observation that the num-er of quanta released per stimulus during prolongedrains varies with the stimulus frequency: at higher fre-uencies, fewer vesicles per stimulus can be releasedithout surpassing the limiting rate of vesicles per sec-nd that can be restored by endocytosis.

oes a Kiss-and-Run Mechanism Exist Alongsidehe Conventional Endocytotic Pathway?he persistence of a slow, classical endocytotic path-ay in both synj and endo synapses is sufficient to ex-lain the ability of these terminals to sustain transmis-ion. But might a kiss-and-run pathway be present inddition? The possibility of kiss-and-run is difficult toxclude by genetic means because there are no pro-eins known to be selectively required for this mecha-ism. However, one line of evidence strongly suggestshat endocytosis proceeds entirely by the conventionalathway at the fly neuromuscular junction: sustainedtimulation in shi mutants leads to a rapid and com-lete loss of transmission, in contrast to what is ob-erved in synj and endo null mutants. Thus, dynamin isbsolutely required for the maintenance of the vesicleool. This protein is known to play an essential role inhe fission step of clathrin-mediated endocytosis botht nerve terminals and in nonneuronal cells (De Camillit al., 1995; Koenig and Ikeda, 1989). Kiss-and-run,owever, because it does not involve full membrane fu-ion but merely reverses the opening of a protein-ceous fusion pore, has no need of a membrane fissioneaction and thus should have no requirement for dy-amin. Large peptidergic vesicles may present a spe-ial case in which a fusion reaction is followed by aynamin-dependent fission reaction without complete

Matters Arising531

discharge of all the vesicular contents (Holroyd et al.,2002), a process sometimes included among kiss-and-run models. However, there is no evidence for dynaminfunction in the rapid, reversible pore opening that wehave considered here as kiss-and-run.

To invoke kiss-and-run at this synapse would there-fore require invoking a function for dynamin outside itsestablished biochemical properties as a GTPase thatassembles into an oligomer on invaginating mem-branes and catalyzes fission (De Camilli et al., 1995).Such a role seems unlikely. Kiss-and-run endocytosishas been estimated to operate within milliseconds(Gandhi and Stevens, 2003; Klyachko and Jackson,2002; Stevens and Williams, 2000), and dynamin wouldbe unlikely to assemble on a vesicle, oligomerize, andcomplete its enzymatic reaction within this time frame.Moreover, dynamin is predominantly localized to a re-gion away from active zones (Estes et al., 1996; Marieet al., 2004; Roos and Kelly, 1999). We have confirmedthat dynamin colocalizes with clathrin and other endo-cytotic proteins but not with the active-zone markernc82 (Figure S3); by contrast, kiss-and-run vesicleswould necessarily recycle at the active zone. Thus theabsolute dependence of transmission on dynamin is in-consistent with a significant kiss-and-run pathway atthe Drosophila neuromuscular junction.

Implications for the Mechanismsof Synaptic-Vesicle EndocytosisThe significance of synaptojanin and endophilin to therecycling pathway can be considered in the context ofthe remarkable efficiency of endocytosis at nerve ter-minals. We determined the maximum rate in wild-typeto be >360 vesicles/s per synapse, corresponding to atleast 0.65 vesicles/s per active zone. Previous studies,under different ionic conditions, have estimated ratesof 1–2 vesicles/s per active zone at this synapse (Del-gado et al., 2000); these estimates are consistent withmeasurements of up to 1 vesicle/s at vertebrate activezones (Fernandez-Alfonso and Ryan, 2004), suggestingthat maximal rates of endocytosis appear to be con-served across species.

In contrast, synj and endo mutant terminals maxi-mally recycle 5- to 10-fold fewer vesicles/s and thusmay resemble nonneuronal cells, which are also esti-mated to operate at rates at least 10-fold slower thanobserved at synapses (Gaidarov et al., 1999). Whilemany proteins necessary for constitutive clathrin-medi-ated endocytosis, such as dynamin, the AP2 complex,and clathrin itself, are expressed in most and perhapsall cell types, other proteins and splice variants are par-ticularly enriched in neurons, including endophilin, syn-aptojanin, synaptotagmin, AP180, and dap160/intersec-tin. Indeed, we and others have found that the onlyessential function of these proteins is in the nervoussystem (DiAntonio and Schwarz, 1994; Marie et al.,2004; Verstreken et al., 2002). We therefore hypothesizethat this latter set is not essential for the formation, fis-sion, or uncoating steps of endocytosis but rather ispresent to optimize the efficiency of these processes innerve terminals. Consistent with this hypothesis, previ-ous studies have shown that synaptic transmissionalso persists, albeit in compromised form, in synapto-

tagmin, AP180, and dap160 mutants (Koh et al., 2004;Marie et al., 2004; Nicholson-Tomishima and Ryan,2004; Poskanzer et al., 2003; Schwarz, 2004; Zhang etal., 1998), as shown here for synaptojanin and endophi-lin. Thus, classical endocytosis probably persists at areduced level in synaptic terminals lacking any of theseproteins individually. In contrast, all endocytosis seemsto be abolished in synapses lacking the ubiquitous coreproteins dynamin, clathrin, or AP2 (Gonzalez-Gaitanand Jackle, 1997; Koenig and Ikeda, 1989; Morgan etal., 2000).

It is therefore likely that synaptojanin and endophilinin neurons have been superimposed upon a basicmechanism of constitutive endocytosis to acceleratethe synaptic-vesicle cycle. In the absence of synapto-janin and endophilin, the core machinery of classicalendocytosis has now been shown to remain functionalat the synapse, permitting a slower vesicle cycle andwith no evidence of or need to invoke kiss-and-run.

Experimental Procedures

Drosophila Stocks and GeneticsBriefly, the EGUF-hid screen was based on engineering heterozy-gous flies whose eyes were homozygous for a single EMS-muta-genized chromosome arm (Stowers and Schwarz, 1999). Wescreened such flies for defects in photoreceptor synaptic transmis-sion by behavioral and electrophysiological assays (phototaxis andelectroretinogram, ERG). From 350,000 flies screened (correspond-ing to 87,500 chromosomes), 1,836 flies failed to phototax in the F1

generation and were kept. From these, we established 72 mutantstocks with a defective ERG; 35 showed no response to light, while37 stocks representing at least 11 complementation groups, includ-ing synj, lacked “on/off-transients” but retained the sustained com-ponent of the ERG.

ElectrophysiologyMutant larvae were separated from heterozygous siblings raised at25°C (Loewen et al., 2001). Third-instar larvae were dissected in 0Ca2+ HL-3 and then bathed in modified HL-3 saline (in mM): NaCl70, KCl 5, MgCl2 10, NaHCO3 10, CaCl2 10, sucrose 115, trehelose5, HEPES 5 (pH 7.2). Current-clamp recordings were performed onmuscle 6 in abdominal segments A2 or A3 (Atwood et al., 1993),and severed ventral nerves were stimulated with suction elec-trodes. Recording electrodes (10–20 M�) were filled with 3 M KCl.Data were analyzed only from recordings with resting potentials>60 mV (average resting potentials: control −64.5 ± 0.993 mV, synj−65.9 ± 1.08 mV, endo −68.3 ± 1.74 mV) that did not deviate bymore than 10 mV during the protocol. Data sets were rejected inwhich the stimulated nerve did not function throughout the record-ing, as determined by abrupt drops in EJP amplitude. Data werecollected using a Digidata and Axopatch 200B amplifier. pClamp 8by Axon Instruments, Microsoft Excel, MatLab 7, and miniAnalysisby Synaptosoft were used for data analysis. Quantal content wasdetermined by dividing the amplitude of the EJP by the averagemini amplitude (before stimulation) and correcting for nonlinearsummation (Martin, 1955). The change in mEJP amplitude that ac-companies high-frequency stimulation was not taken into accountin the analysis of exocytotic and endocytotic rates because wecould not determine how much of this change had occurred at anygiven point in the stimulation and because the effects were notlarge enough to substantially alter the calculated rates.

Electron MicroscopySynapses at neuromuscular junctions on larval muscles 6 and 7(Atwood et al., 1993) were analyzed from digital EM images of shortseries of 55 nm sections through individual varicosities from filletedlarvae (either wild-type or synj; three larvae each). Data were com-pared between 14 varicosities from 3 synjLY/Df(2R)x58-7 larvae and7 varicosities from 2 yw;FRT42D larvae. Values analyzed were

Cell532

means of mean values from one to four varicosities per larva. Ana-lyzing vesicle sizes, we found that they were evenly distributed inall terminals of each genotype as judged by eye in frequency histo-grams of vesicle diameters. Thus, variation in vesicle size is intro-duced mostly by the vesicle diameters themselves, not from theterminals (e.g., through physiological differences). Therefore, wesought differences in the mean vesicle diameters by pooling valuesfor all the terminals of a given genotype.

FM1-43 ExperimentsProtocols were modified from those of Kuromi and Kidokoro, 1998.Larvae were dissected in a dish in 0 Ca2+ modified standard saline(Jan and Jan, 1976) and then transferred to normal saline (in mM):NaCl 130, KCl 5, MgCl2 2, CaCl2 2, sucrose 36, HEPES 5 (pH 7.2)or high-K+ saline: NaCl 45, KCl 90, MgCl2 2, CaCl2 2, sucrose 36,HEPES 5 as specified.Activity-Dependent LoadingLarvae were dissected in 0 Ca2+ saline, washed three times, andplaced on a shaker in a dish containing high-KCl solution with 10�M FM1-43 (Molecular Probes, Eugene, Oregon) for 10 min. Larvaewere then washed three times quickly in 0 Ca2+ saline and washed12 min further in 0 Ca2+ saline before imaging synapses on a ZeissLSM 510 confocal microscope with a 63/1.4× water-immersionlens. Z stack images of 14–18 sections 0.3 �m apart were pro-cessed with LSM software.Intact LoadingLarvae dissected as described above but without severing thenerve cord were incubated in 2 mM Ca2+ normal saline for 10 minin the presence of 10 �M FM1-43 and washed and imaged as de-scribed.Depletion/Re-Formation Loadingshits1 or shits1;synjLY/Df(2R)x58-7 larvae were dissected and incu-bated in prewarmed normal solution with 10 µM FM1-43 at 34°Cfor 5 min. Solutions were then changed, and larvae in prewarmed60 mM KCl solution with FM1-43 were incubated at 34°C for 5 minto deplete the vesicle pool. Larvae were then washed three timesquickly in normal saline at 22°C with FM1-43 and incubated for 15min at 22°C before washing and imaging.QuantificationImages were quantified using Metamorph 6.3 (Molecular Devices,Downingtown, Pennsylvania) with a looping program that analyzedthe average pixel intensity per section per synapse, subtractingbackground pixel intensity on the muscle and adding the values ofeach section.

Supplemental DataSupplemental Data include Supplemental References and three fig-ures and can be found with this article online at http://www.cell.com/cgi/content/full/123/3/521/DC1/.

Acknowledgments

We thank Wade Regehr for comments on the manuscript. We alsothank H. Bellen, M. Ramaswami, T. Orr-Weaver, E. Buchner, A. Hof-bauer, and the Bloomington Drosophila Stock Center for reagentsand fly strains and U. Eden for advice on endocytosis models. Thiswork was supported by NIH grants NS-41062 to T.L.S. and EY-03592 to I.A.M. and by the MRRC Imaging Core at Children’s Hos-pital. D.K.D. was supported by a Howard Hughes Medical InstitutePredoctoral Fellowship.

Received: April 21, 2005Revised: July 27, 2005Accepted: September 20, 2005Published: November 3, 2005

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