Jump to content

User:Marcuse/test

From Wikipedia, the free encyclopedia

BIOLOGICAL SCIENCES / NEUROSCIENCE
Factors regulating the abundance and localization of synaptobrevin in the plasma membrane

Jeremy S. Dittman, and Joshua M. Kaplan[#COR1 *]

Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114

Edited by Pietro V. De Camilli, Yale University School of Medicine, New Haven, CT, and approved June 5, 2006 (received for review January 31, 2006)



After synaptic vesicle fusion, vesicle proteins must be segregatedfrom plasma membrane proteins and recycled to maintain a functionalvesicle pool. We monitored the distribution of synaptobrevin,a vesicle protein required for exocytosis, in Caenorhabditiselegans motor neurons by using a pH-sensitive synaptobrevinGFP fusion protein, synaptopHluorin. We estimated that 30% ofsynaptobrevin was present in the plasma membrane. By using apanel of endocytosis and exocytosis mutants, we found that themajority of surface synaptobrevin derives from fusion of synapticvesicles and that, in steady state, synaptobrevin equilibratesthroughout the axon. The surface synaptobrevin was enrichednear active zones, and its spatial extent was regulated by theclathrin adaptin AP180. These results suggest that there isa plasma membrane reservoir of synaptobrevin that is suppliedby the synaptic vesicle cycle and available for retrieval throughoutthe axon. The size of the reservoir is set by the relative ratesof exo- and endocytosis.

AP180 | endocytosis | pHluorin | synaptic vesicle

Neurotransmitter released at synapses originates from a recyclingpool of synaptic vesicles (SVs) ([#B1 1]–[#B3 3]). Several processesare required for neurotransmitter secretion, including biogenesisof SVs, docking with the plasma membrane, ATP-dependent primingof SVs to make them fusion-competent, calcium-evoked fusion,and endocytic recycling ([#B4 4]–[#B6 6]). The fusion step is believedto be mediated by the SNARE complex, a four-helix coiled-coilstructure consisting of a vesicle SNARE (v-SNARE), synaptobrevin/VAMP,and two target membrane SNARE (t-SNARE) proteins, syntaxin 1and SNAP-25, on the plasma membrane ([#B7 7]–[#B10 10]).

Accurate sorting of SNAREs to vesicle and plasma membranes iscritical for the coordination of SV fusion ([#B11 11], [#B12 12]). The t-SNAREssyntaxin and SNAP-25 are abundant in the plasma membrane butare excluded from recycling SVs ([#B13 13], [#B14 14]), although syntaxinhas been found in some intracellular compartments ([#B13 13], [#B15 15]–[#B17 17]).Several studies have documented a significant fraction of endogenoussynaptobrevin ([#B14 14]) or synaptobrevin GFP ([#B18 18], [#B19 19]) in the plasmamembrane. Surface synaptobrevin could be derived from fusionof SV precursors undergoing anterograde transport via the constitutivesecretory pathway ([#B20 20]–[#B23 23]). Alternatively, surface synaptobrevincould reflect diffusion within the plasma membrane after vesiclefusion ([#B19 19]) or "stranded" vesicles that fail to undergo endocytosis([#B24 24]). Finally, some authors have argued that surface synaptobrevinresults from missorting, particularly in cases in which synaptobrevinis overexpressed ([#B22 22]).

Several questions remain concerning the surface pool of synaptobrevin.Does this pool arise from v-SNAREs that escape retrieval afterexocytosis? Which endocytic pathways regulate this pool of synaptobrevin?Is the spatial distribution of surface synaptobrevin restrictedin some manner? To address these questions, we used synaptopHluorin(SpH), a pH-sensitive variant of GFP fused to the luminal domainof synaptobrevin ([#B25 25]–[#B29 29]), to analyze genes that regulatethe surface pool of v-SNAREs in the nematode Caenorhabditiselegans.


File:Rarrow.gif

Results
[#top File:Uarrow.gifTop
] [#ABS File:Uarrow.gifAbstract
] Results
[#SEC2 File:Darrow.gifDiscussion
] [#SEC3 File:Darrow.gifMethods
] [#ACK File:Darrow.gifAcknowledgements
] [#BIBL File:Darrow.gifReferences
]


To develop an optical reporter for surface synaptobrevin, weexpressed SpH in cholinergic motor neurons. Fluorescence wasobserved both in cell bodies and in axonal processes in theventral and dorsal nerve cords. We used two criteria to determinewhether SpH behaves like endogenously expressed synaptobrevin.First, SpH required the unc-104 KIF1A motor protein for itssynaptic localization, as is the case for endogenous synaptobrevin(data not shown) ([#B30 30], [#B31 31]). Second, pan-neuronal expression ofSpH rescued the locomotion and sensitivity to the cholinesteraseinhibitor aldicarb defects of snb-1 synaptobrevin mutants (datanot shown) ([#B32 32], [#B33 33]). Thus, SpH functionally replaced synaptobrevinat the neuromuscular junction.

SpH is highly pH-sensitive such that fluorescence is expectedto be quenched in the acidic environment of the SV lumen, whereasSpH residing on the plasma membrane will be unquenched, producinga 20-fold increased fluorescence per SpH molecule ([#B25 25]). Individualanimals were dissected, and axonal fluorescence from a 20- to100-µm region of the dorsal nerve cord was focused ontoa photodiode. To determine the surface fraction of axonal SpH,we measured SpH fluorescence changes caused by neutralizingintracellular compartments (with pH 7.4 NH4Cl) and by quenchingsurface SpH (with pH 5.6 Mes) ([#F1 Fig. 1]A and Fig. 6, which ispublished as supporting information on the PNAS web site). Onthe basis of these measurements, we estimate that 30% of axonalSpH resides on the cell surface. This amount was similar tothe surface abundance previously determined in cultured hippocampalneurons ([#B25 25]) and in Torpedo axons ([#B14 14]). The surface fractioncalculation assumes that SVs are acidic, and it may underestimatethe true surface percentage if SVs are closer to a neutral pH,as has been observed in Drosophila terminals ([#B34 34]). However,the ratiometric approach used here gave surface percentage estimatesthat were largely independent of vesicle pH (see SupportingMethods and Fig. 7, which are published as supporting informationon the PNAS web site).


[/cgi/content/full/103/30/11399/F1 File:Zpq0290628380001.gif]
View larger version (23K):
[/cgi/content/full/103/30/11399/F1 [in this window]]
[/cgi/content-nw/full/103/30/11399/F1 [in a new window]]

 Fig. 1. Measuring surface and vesicular SpH in cholinergic motor neurons. (A) A representative experiment in a wild-type animal. Photodiode current measurements from the fluorescent spot were made every 15 sec while the extracellular solution was exchanged as indicated (see Methods). Note that there is a large contribution of background light pooled onto the photodetector (see Supporting Methods for details). (B) Summary of SpH surface ratios in a panel of synaptic mutants: wild-type nuIs122, ric-4 SNAP-25, unc-64 syntaxin, unc-13 Munc13, unc-18 Munc18, dpy-23 µ2 AP2, dyn-1 dynamin, unc-57 endophilin A, and unc-11 AP180. See Methods for the calculation of surface ratios. Data are shown as mean ± SEM. **, P < 0.01 by Student’s t test, compared with wild type.

To determine whether the surface SpH pool depended on synaptictransmission, we repeated the measurements in various SV exocytosisand endocytosis mutants. For exocytosis mutants, we examinedSpH fluorescence in mutants lacking the t-SNAREs ric-4 SNAP-25or unc-64 syntaxin 1, as well as in mutants lacking two syntaxin-bindingproteins, unc-13 Munc13 and the Sm protein unc-18 Munc18/nSec1([#B35 35]–[#B39 39]). All four mutants significantly reduced the surfacepool of SpH, with 75% and 94% reductions observed in unc-13and unc-18 mutants, respectively ([#F1 Fig. 1]B). More modest reductionsin SpH surface ratios were observed in t-SNAREs mutants, likelybecause hypomorphic alleles were analyzed, inasmuch as nullalleles cause a lethal phenotype ([#B36 36], [#B40 40]).

The clathrin adaptins unc-11 AP180 and dpy-23 AP2 µ2 andthe endocytic proteins unc-57 endophilin A and dyn-1 dynaminare required for SV endocytosis ([#B41 41]–[#B46 46]). In all four endocyticmutants studied, surface SpH increased significantly (70–160%)([#F1 Fig. 1]B).

To confirm that the surface fraction of SpH was altered in thesemutants, we examined the rate of SpH bleaching in wild-type,unc-18, and unc-11 mutants. A shift toward more quenched SpH(e.g., an exocytosis mutant) would be predicted to slow thebleach rate, whereas the opposite shift should accelerate thebleach rate. This prediction was confirmed experimentally (seeFig. 8, which is published as supporting information on thePNAS web site), thereby providing further evidence that vesiclepH differed appreciably from extracellular pH.

These experiments suggest that a significant fraction of SpHresides on the plasma membrane. This surface pool is suppliedby the exocytosis of SVs and recycled by clathrin-mediated endocytosis.In addition, even if the absolute surface fraction was underestimatedby assuming an acidic SV lumen, the relative shifts in surfaceSpH observed across the panel of synaptic mutants are robust.

Spatial Organization of Vesicular and Surface Synaptobrevin. GFP-tagged synaptobrevin has been widely used as a presynapticmarker, and its enrichment at synapses is thought to correspondto the SV pool. We therefore wondered whether surface synaptobrevinmight also be enriched near synapses. To detect the spatialfeatures of in vivo synaptobrevin distribution, we imaged twodistinct forms of GFP-tagged synaptobrevin in intact animals.First, to analyze the distribution of total (i.e., surface andinternal) synaptobrevin, we imaged an N-terminal GFP-taggedsynaptobrevin (NGFP-SNB) in the motor axons of intact animals.In NGFP-SNB, the GFP is appended to the cytoplasmic domain ofsynaptobrevin; consequently, NGFP-SNB molecules in SVs and thosein the plasma membrane are equally fluorescent. NGFP-SNB fluorescenceis markedly punctate, but we also observed a small amount ofdiffuse axonal fluorescence between puncta, likely representingsurface synaptobrevin ([#F2 Fig. 2]Aa).


[/cgi/content/full/103/30/11399/F2 File:Zpq0290628380002.gif]
View larger version (48K):
[/cgi/content/full/103/30/11399/F2 [in this window]]
[/cgi/content-nw/full/103/30/11399/F2 [in a new window]]

 Fig. 2. Imaging synaptobrevin in intact animals. (Aa) The N-terminal tag places GFP (NGFP-SNB) in the cytoplasm. (Ab) Motor neurons expressing a C-terminal pHluorin-tagged synaptobrevin (SpH) that places the fluorophore in the vesicle lumen. (Scale bars, 5 µm.) (Ba) Expression of SpH in the ventral cord. Neuromuscular junctions are located on the bottom cord (*). (Bb) Expression of UNC-10 RIM1::RFP in the ventral cord. (Bc) Colocalization of SpH (green) and UNC-10 (red). (C) A line scan of SpH in a wild-type animal. Peaks (red arrowheads) and baseline (dashed line) were located by using automated software, as described in Supporting Methods.

We found that 70–90% of SpH fluorescence originates fromthe unquenched surface pool, assuming a vesicle pH between 5.6and 6.3 (see Supporting Methods, Eq. 11); consequently, SpHimages primarily reflect surface synaptobrevin. In wild-typeanimals, SpH fluorescence was moderately punctate, indicatingthat surface SpH was spatially restricted ([#F2 Fig. 2]Ab). TheseSpH puncta colocalized with the presynaptic active zone markerunc-10 Rim1 ([#B47 47], [#B48 48]), suggesting that a local pool of surfaceSpH is sequestered near active zones ([#F2 Fig. 2]B). This surfacecluster of SpH may correspond to a specialized endocytic zonewhere v-SNAREs are recycled to the SV pool, as has been describedin other synapses ([#B49 49]–[#B51 51]).

Total and surface synaptobrevin distributions were measuredin exocytosis (unc-13 Munc13) and endocytosis (unc-11 AP180)mutants by quantitative fluorescence microscopy ([#F2 Fig. 2]C, andsee Methods). In unc-13 mutants, NGFP-SNB punctal fluorescenceincreased 40% ([#F3 Fig. 3]Ab Left). The increased punctal fluorescenceis indicative of an increased SV pool because previous ultrastructuralstudies have shown that unc-13 mutants have a 74% increase inthe number of cholinergic SVs found at neuromuscular junctions([#B35 35]). Decreased SV exocytosis should also cause a reductionin surface synaptobrevin, which was evident in our experiments.The unc-13 mutants had decreased NGFP-SNB axonal fluorescence,and SpH fluorescence was nearly eliminated, particularly inthe axons between puncta ([#F4 Fig. 4]Ab Right). These findings areconsistent with the decreased SpH surface ratio measured indissected unc-13 mutants ([#F1 Fig. 1]B).


[/cgi/content/full/103/30/11399/F3 File:Zpq0290628380003.gif]
View larger version (26K):
[/cgi/content/full/103/30/11399/F3 [in this window]]
[/cgi/content-nw/full/103/30/11399/F3 [in a new window]]

 Fig. 3. Effects of synaptic mutants on synaptobrevin distribution in vivo. (A) Representative image of the dorsal cord in wild-type (a), unc-13 Munc13 (b), and unc-11 AP180 (c) mutant animals. (Left) NGFP-SNB. (Right) SpH. (B) Peak absolute SpH fluorescence across the nine strains: nuIs122, ric-4, unc-64, unc-13, unc-18, dpy-23, dyn-1, unc-57, and unc-11. (C) Axon absolute fluorescence. Individual images are normalized to fluorescent bead standards (see Methods). (D) Plot of percentage change in peak fluorescence vs. percentage change in axon fluorescence for each of the nine strains measured in B and C. A linear regression of the data gave a slope of 0.9, y intercept of 8.2%, and Pearson’s R2 of 0.99 (P < 0.001). All data are normalized to wild type; bars are mean ± SEM. **, P < 0.01 by Student’s t test, compared with wild type. Note that some error bars are too small to be visible on this scale.


[/cgi/content/full/103/30/11399/F4 File:Zpq0290628380004.gif]
View larger version (37K):
[/cgi/content/full/103/30/11399/F4 [in this window]]
[/cgi/content-nw/full/103/30/11399/F4 [in a new window]]

 Fig. 4. Loss of tomosyn increases surface synaptobrevin abundance. (A) Representative images of the dorsal cord from the wild-type strain (Upper) and from the tomosyn mutant tomo-1 (Lower). (Scale bar, 5 µm.) (Ba) Absolute peak fluorescence (relative to a fluorescent bead standard, see Methods) for wild type and tomo-1. (Bb) Absolute axonal fluorescence. Data are normalized to wild type; bars are mean ± SEM. **, P < 0.001 by Student’s t test, compared with wild type.

Conversely, disruption of endocytosis in unc-11 mutants significantlyincreased axonal fluorescence of both NGFP-SNB and SpH ([#F3 Fig. 3]Ac),consistent with the increased surface SpH ratio (160%) observedin dissected animals ([#F1 Fig. 1]B). Punctal SpH fluorescence wasalso increased by a similar degree, indicating that perisynapticsurface clusters could persist despite a large increase in totalsurface abundance. Thus, large bidirectional changes in plasmamembrane SpH in intact animals were observed when exocytosisand endocytosis were disrupted, consistent with the pHluorinmeasurements in dissected animals ([#F1 Fig. 1]).

Perisynaptic SpH Is in Equilibrium with Axonal SpH. If SV components are recycled at specialized endocytic zones,we might expect that the recycling of perisynaptic and axonalSpH would be differentially regulated. To test this idea, wequantified perisynaptic and axonal SpH across eight exocyticand endocytic mutants ([#F3 Fig. 3] B and C). Axonal and perisynapticsurface SpH were generally affected equally across the mutantpanel. This correlation was quantified by plotting the relativechanges in peak vs. axonal fluorescence for each mutant ([#F3 Fig. 3]D).Perisynaptic and axonal surface SpH were tightly correlated(R = 0.99), with a regression slope of 0.9, suggesting thatan equilibrium was established between the two surface poolsunder steady-state conditions.

Tomosyn Regulates Surface Synaptobrevin Abundance. In all of the mutants examined thus far, steady-state surfacesynaptobrevin abundance was altered by disruption of vesiclefusion or endocytosis. If the surface abundance of synaptobrevinis activity-dependent, we would expect that mutants that haveincreased rates of vesicle fusion would also have altered surfacesynaptobrevin levels. To test this idea, we imaged tomo-1 mutants,which lack tomosyn, a highly conserved 130-kDa cytoplasmic proteininitially found as a binding partner of syntaxin ([#B52 52], [#B53 53]). Thecarboxyl-terminal SNARE motif of tomosyn forms a core complexwith SNAP-25 and syntaxin and has been proposed to act as acompetitive inhibitor of synaptobrevin. Overexpression of tomosynin neuroendocrine cells inhibits vesicle fusion ([#B53 53], [#B54 54]), andin C. elegans, mutations in the tomosyn gene, tomo-1, enhanceacetylcholine release at the neuromuscular junction ([#B55 55]). Bothpeak and axonal surface SpH increased significantly in tomo-1mutants ([#F4 Fig. 4]). Thus, the surface pool of synaptobrevin canbe augmented by increasing the rate of delivery and by decreasingthe rate of retrieval.

AP180 Regulates the Size of Perisynaptic SpH Clusters. We examined the effects of mutations that disrupt endocytosison the spatial restriction of surface synaptobrevin by measuringthe width of perisynaptic SpH puncta ([#F5 Fig. 5]). The average punctalwidth was File:Ap.gif1 µm in wild-type animals and was not significantlyaltered in dpy-23 AP2, dyn-1 dynamin, or unc-57 endophilin Amutants (all P > 0.05). In unc-11 AP180 mutants, the averageSpH puncta width was increased by 35% ([#F5 Fig. 5] A and B and Fig.9, which is published as supporting information on the PNASweb site), and the entire distribution of punctal widths wasshifted toward larger values ([#F5 Fig. 5]C) (P < 10–10).This change in SpH punctal widths is unlikely to be caused bya change in the geometry of unc-11 AP180 synapses because theSpH puncta are >1 µm wide and are consequently notdiffraction-limited; similar puncta widths were measured withconfocal microscopy (data not shown). We cannot exclude thepossibility that geometric changes contribute to the increasedbrightness of synaptic varicosities; however, prior ultrastructuralstudies ([#B44 44]) did not observe large changes in the geometry ofsynaptic membranes in unc-11 mutants. Transgenic animals expressing50% less SpH displayed identical puncta widths (see Fig. 10,which is published as supporting information on the PNAS website). Finally, internal postendocytic compartments are unlikelyto contribute to increased puncta width because most of theSpH fluorescence in unc-11 mutants could be quenched with acidicsaline (Fig. 11, which is published as supporting informationon the PNAS web site). These results suggest that the increasedSpH puncta widths observed in unc-11 mutants are unlikely tobe caused by changes in SpH abundance or in the distributionof intracellular organelles. Therefore, we propose that AP180may play an important role in restricting a pool of surfacesynaptobrevin near active zones.


[/cgi/content/full/103/30/11399/F5 File:Zpq0290628380005.gif]
View larger version (29K):
[/cgi/content/full/103/30/11399/F5 [in this window]]
[/cgi/content-nw/full/103/30/11399/F5 [in a new window]]

 Fig. 5. Effects of endocytosis mutants on surface synaptobrevin puncta width. (A) Summary of endocytosis mutant effects on SpH punctal width for the following strains (number of animals): wild type, dpy-23 µ2 AP2, dyn-1 dynamin, unc-57 endophilin A, and unc-11 AP180. (B) Assortment of representative puncta from wild-type (Upper) and unc-11 AP180 mutant (Lower) animals. (Scale bar, 1 µm.) (C) Histogram of punctal widths (measured as full width at half maximum, see Methods) for wild-type (black) and unc-11 (red) animals. Widths are binned in 0.2-µm intervals and normalized to the wild-type peak. Data are from 1,672 puncta (90 animals) for wild type and 414 puncta (35 animals) for unc-11. All data are normalized to wild type; bars are mean ± SEM. **, P < 0.01 by Student’s t test, compared with wild type.


File:Rarrow.gif

Discussion
[#top File:Uarrow.gifTop
] [#ABS File:Uarrow.gifAbstract
] [#SEC1 File:Uarrow.gifResults
] Discussion
[#SEC3 File:Darrow.gifMethods
] [#ACK File:Darrow.gifAcknowledgements
] [#BIBL File:Darrow.gifReferences
]


Our results lead to four primary conclusions. First, there isa sizeable pool of synaptobrevin on the plasma membrane of cholinergicmotor axons in C. elegans. This surface synaptobrevin is derivedfrom the SV pool, and it clusters near active zones in a mannerregulated by AP180. Second, the surface pool of synaptobrevinis largely recycled by clathrin- and dynamin-dependent endocytosis.Third, perisynaptic surface synaptobrevin is in equilibriumwith nonsynaptic axonal surface synaptobrevin. Fourth, increasingthe rate of secretion (in tomo-1 mutants) results in a net increaseof the surface pool.

Sources of Plasma Membrane Synaptobrevin. Synaptobrevin has been observed in the plasma membrane in multipleneuronal cell types by using immunofluorescence, electron microscopy,biochemical fractionation, and GFP fusion proteins ([#B14 14], [#B18 18],[#B19 19], [#B56 56]). In principle, there are two likely sources for thisplasma membrane pool: (i) diffusion from fused SVs and (ii)exocytosis of SV precursors undergoing anterograde transportto nerve terminals. Our results favor the former hypothesis.For surface synaptobrevin to accumulate from SVs, some fractionof vesicular protein must escape retrieval during endocytosisand diffuse away from perisynaptic endocytic zones, as has beenobserved in cultured hippocampal neurons ([#B19 19], [#B56 56]). Surface synaptobrevinwas nearly eliminated in mutants lacking UNC-13 and UNC-18,proteins that are critical for SV exocytosis at active zonesbut are not required for the constitutive secretion pathway.Enhanced synaptic acetylcholine secretion in tomo-1 tomosynmutants increased the surface abundance of synaptobrevin, furthercorroborating the synaptic origins of this surface component.Thus, the surface pool of synaptobrevin derives largely fromSV fusion at nerve terminals.

Routes of Synaptobrevin Retrieval. Several endocytic pathways have been proposed to mediate v-SNARErecapture. On a time scale of 1–30 sec, SV endocytosisis thought to occur at specialized endocytic zones that neighboractive zones. The perisynaptic enrichment of surface synaptobrevinobserved here could reflect the spatial extent of these endocyticzones. On slower time scales, constitutive endocytosis and pinocytosisof nonsynaptic membrane will capture this axonal synaptobrevinand, therefore, will likely contribute to recycling throughoutthe axon ([#B50 50], [#B57 57], [#B58 58]). We observed a tight correlation betweenthe amounts of SpH in perisynaptic surface clusters vs. nonsynapticaxonal SpH over a 5-fold change in surface SpH concentrationunder steady-state conditions. This correlation is probablythe result of equilibration between the two compartments. Inasmuchas we observed parallel effects on surface synaptobrevin bothlocally (perisynaptic membrane) and globally (axonal membrane)at steady state, all of these endocytic routes are likely tocontribute to regulation of the surface synaptobrevin pool.The sorting of plasma membrane synaptobrevin to SVs dependson a targeting sequence found in its cytoplasmic domain ([#B22 22]).This sorting signal can act autonomously to direct synaptobrevinto SVs, regardless of the synaptobrevin’s initial location([#B20 20], [#B21 21], [#B59 59], [#B60 60]). These studies suggest that nonsynaptic synaptobrevincan be efficiently sorted into SVs, thereby providing a meansof coupling surface abundance to replenishment of the vesiclepool and enhancing the recycling efficiency of the vesicle cycle.

The equilibration of surface synaptobrevin across the axon,as observed here, is consistent with several prior studies ([#B19 19],[#B56 56]). Allersma et al. ([#B61 61]) reported that in <1 sec after vesiclefusion, GFP-tagged synaptobrevin equilibrated over a 1-µm2region of membrane. Rapid forms of endocytosis such as "kiss-and-run"events may limit lateral diffusion of synaptobrevin, whereascomplete collapse of the vesicle membrane followed by clathrin-mediatedendocytosis could result in relatively greater lateral diffusion.Rapid endocytic events occurring as soon as 1 sec after exocytosishave been reported at a mammalian central synapse ([#B62 62]). On thistime scale, endogenous synaptobrevin would be expected to diffuseFile:Ap.gif3 µm, assuming a diffusion coefficient of 2.5 µm2/sec([#B56 56], [#B63 63]). However, after more intense periods of activity, recyclingtimes slowed to >20 sec, theoretically allowing a synaptobrevinmolecule to diffuse >10–15 µm from its releasesite. Because three to four synapses are found per 10 µmin the worm nerve cords, these results support our conclusionthat surface synaptobrevin derived from SV exocytosis rapidlyequilibrates throughout the axon.

Maintenance of a Surface v-SNARE Pool. The steady-state abundance of plasma membrane synaptobrevinresults from a balance between the rate of insertion by meansof SV fusion and removal by endocytosis. In hippocampal neurons,increasing the rate of exocytosis (beyond a threshold levelof 2 Hz at room temperature) overwhelmed retrieval mechanismsand resulted in a parallel increase in surface synaptobrevin([#B64 64]). We observed a similar phenomenon (a 30–35% increasein surface SpH) when exocytosis was increased in mutants lackingthe inhibitory SNARE tomosyn. Because all mutants tested inthis study affected the ratio of surface-to-vesicular synaptobrevin,we conclude that SV exocytosis is not inextricably coupled toendocytosis. Taken together, these results suggest that thesurface pool of synaptobrevin is determined by the rates ofSV exocytosis and endocytosis, and consequently correlates withrecent synaptic activity.

Role of AP180 in Recycling of Surface Synaptobrevin. AP180 localizes to presynaptic endocytic zones and is thoughtto regulate local curvature of the lipid bilayer during assemblyof the clathrin lattice ([#B43 43], [#B65 65], [#B66 66]). Loss of AP180 resultedin a 35% widening of perisynaptic surface SpH clusters. Nonetet al. ([#B44 44]) reported that AP180 mutants selectively disruptedretrieval of synaptobrevin from the plasma membrane, suggestinga role for AP180 as a synaptobrevin chaperone. AP180 also regulatessynaptic localization of synaptotagmin and cysteine-string protein,two additional SV-associated proteins ([#B43 43]). Our observationsprovide evidence that AP180 functions to concentrate surfacesynaptobrevin near active zones. However, AP180 is not essentialfor maintenance of a steady-state gradient; we still detectedlocalized SpH peaks in unc-11 AP180 mutants.

Functional Significance of Surface v-SNAREs. We propose that the plasma membrane pool of synaptobrevin isan integral component of the SV cycle. Several results are consistentwith this idea. First, we and others have shown that a significantfraction of total synaptobrevin is found at the cell surface([#B56 56]). Further, the surface pool of synaptobrevin is derivedfrom SV exocytosis and is equilibrated throughout the axon.In cultured rodent neurons, synaptobrevin released from onesynapse can be recycled at a neighboring synapse to replenishits vesicle pool ([#B19 19]). In these examples, surface abundanceof synaptobrevin is regulated by synaptic activity, and theplasma membrane pool provides a source of v-SNAREs during recoveryfrom bouts of exocytosis ([#B67 67]). Taken together, these resultssuggest that surface synaptobrevin is an important componentof the SV cycle. Further experiments will be necessary to determinewhether the abundance of surface synaptobrevin regulates synapticefficacy.


File:Rarrow.gif

Methods
[#top File:Uarrow.gifTop
] [#ABS File:Uarrow.gifAbstract
] [#SEC1 File:Uarrow.gifResults
] [#SEC2 File:Uarrow.gifDiscussion
] Methods
[#ACK File:Darrow.gifAcknowledgements
] [#BIBL File:Darrow.gifReferences
]


Strains and Plasmids. Strain maintenance and genetic manipulation were performed asdescribed in ref. [#B68 68]. Animals were cultivated at 20°C onagar nematode growth media seeded with HB101 bacteria. Strainsused in this work are listed in Supporting Methods. Strain KP#557encodes a pHluorin-tagged worm synaptobrevin (SNB-1) in whichsuperecliptic pHluorin was inserted at the C terminus expressedunder the snb-1 promoter. KP#558 is the same SNB-1::pHluorinconstruct expressed under the acr-2 promoter. KP#704 encodesa GFP-tagged SNB-1 in which GFP was inserted at the N terminus(NGFP-SNB) expressed under the acr-2 promoter. KP#930 encodesunc-10 cDNA tagged with a tandem repeat of monomeric red fluorescentprotein (mRFP) expressed under the acr-2 promoter.

In Vivo Microscopy and Image Analysis. GFP and pHluorin-expressing animals were mounted on agarosepads and viewed on a [/cgi/redirect-inline?ad=Zeiss Zeiss] Axiovert microscope with an OlympusPlanApo 100x NA 1.4 objective, as described in ref. [#B69 69]. Imageswere captured with a Hamamatsu Photonics ORCA digital camera,and line scans were analyzed with custom software in IGOR Pro(WaveMetrics, Lake Oswego, OR). Images of 500-nm fluorescein-conjugatedbeads (Molecular Probes) were captured during each imaging sessionto provide a fluorescence standard for comparing absolute fluorescencelevels between animals. Background signal (charge-coupled devicedark current and slide autofluorescence) was subtracted beforeanalysis. Automated line scan analysis is described in SupportingMethods.

Worm Dissection and Fluorometric Microscopy. Saline solution recipes are listed in Supporting Methods. SpH-expressinganimals were glued to Sylgard-coated coverslips (Dow-Corning),dissected, and placed in a perfusion chamber for gravity-fedsuperfusion of extracellular saline solutions. A 20- to 100-µmregion of the dorsal nerve cord was excited under epifluorescenceillumination by a xenon arc lamp, and the emission fluorescencewas focused onto a custom-built photodiode to establish thebaseline fluorescence value. The photodiode current was sampledfor 30 msec every 15 sec, digitized at 10 kHz (National Instruments,Austin, TX), digitally filtered, and analyzed with custom softwareusing IGOR Pro (WaveMetrics). For most experiments, the applicationof NH4Cl followed by Mes pH 5.6 was repeated at least once andthe results were averaged. In some experiments, the order ofapplication was reversed, with similar results. NH4Cl and Messolutions were applied until a stable level of fluorescencewas observed (at least 5 min). Derivation of the surface fractioncalculations is provided in Supporting Methods.


File:Rarrow.gif

Acknowledgements
[#top File:Uarrow.gifTop
] [#ABS File:Uarrow.gifAbstract
] [#SEC1 File:Uarrow.gifResults
] [#SEC2 File:Uarrow.gifDiscussion
] [#SEC3 File:Uarrow.gifMethods
] Acknowledgements
[#BIBL File:Darrow.gifReferences
]


We thank G. Garriga (University of California, Berkeley, CA)and the C. elegans Genetic Stock Center for strains, J. Rothman(Columbia University, New York, NY) for reagents, J. Madisonfor advice, J. Bai and S. Cotman for assistance with the confocalimaging, and members of the Kaplan laboratory for comments onthis manuscript. This work was supported by National Institutesof Health Research Grant GM54728 (to J.M.K.) and by a DamonRunyon postdoctoral fellowship (to J.S.D.).


File:Rarrow.gif

Footnotes


Abbreviations: SV, synaptic vesicle; v-SNARE, vesicle SNARE; t-SNARE, target membrane SNARE; SpH, synaptopHluorin; NGFP-SNB, N-terminal GFP-tagged synaptobrevin.

  • To whom correspondence should be addressed at: Department of Molecular Biology, Massachusetts General Hospital, Simches Research Building, Seventh Floor, 185 Cambridge Street, Boston, MA 02114. E-mail: kaplan{at}molbio.mgh.harvard.edu

Freely available online through the PNAS open access option.

Author contributions: J.S.D. and J.M.K. designed research; J.S.D.performed research; J.S.D. contributed new reagents/analytictools; J.S.D. analyzed data; and J.S.D. and J.M.K. wrote thepaper.

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

© 2006 by [/misc/terms.shtml The National Academy of Sciences] of the USA


File:Rarrow.gif

References
[#top File:Uarrow.gifTop
] [#ABS File:Uarrow.gifAbstract
] [#SEC1 File:Uarrow.gifResults
] [#SEC2 File:Uarrow.gifDiscussion
] [#SEC3 File:Uarrow.gifMethods
] [#ACK File:Uarrow.gifAcknowledgements
] References


  1. Betz, W. J. & Angleson, J. K. (1998) Annu. Rev. Physiol. 60, 347–363.[/cgi/external_ref?access_num=10.1146/annurev.physiol.60.1.347&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000072713900018&link_type=ISI [ISI]][/cgi/external_ref?access_num=9558468&link_type=MED [Medline]]
  2. Schneggenburger, R., Sakaba, T. & Neher, E. (2002) Trends Neurosci. 25, 206–212.[/cgi/external_ref?access_num=10.1016/S0166-2236(02)02139-2&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000174512200011&link_type=ISI [ISI]][/cgi/external_ref?access_num=11998689&link_type=MED [Medline]]
  3. von Gersdorff, H. & Matthews, G. (1999) Annu. Rev. Physiol. 61, 725–752.[/cgi/external_ref?access_num=10.1146/annurev.physiol.61.1.725&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000079229000030&link_type=ISI [ISI]][/cgi/external_ref?access_num=10099708&link_type=MED [Medline]]
  4. Calakos, N. & Scheller, R. H. (1996) Physiol. Rev. 76, 1–29.[/cgi/ijlink?linkType=ABST&journalCode=physrev&resid=76/1/1 [Abstract/Free Full Text]]
  5. Jahn, R. & Südhof, T. C. (1999) Annu. Rev. Biochem. 68, 863–911.[/cgi/external_ref?access_num=10.1146/annurev.biochem.68.1.863&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000082693200027&link_type=ISI [ISI]][/cgi/external_ref?access_num=10872468&link_type=MED [Medline]]
  6. Murthy, V. N. & De Camilli, P. (2003) Annu. Rev. Neurosci. 26, 701–728.[/cgi/external_ref?access_num=10.1146/annurev.neuro.26.041002.131445&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000185093200024&link_type=ISI [ISI]][/cgi/external_ref?access_num=14527272&link_type=MED [Medline]]
  7. Sollner, T. & Rothman, J. E. (1994) Trends Neurosci. 17, 344–348.[/cgi/external_ref?access_num=10.1016/0166-2236(94)90178-3&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=A1994NY34400012&link_type=ISI [ISI]][/cgi/external_ref?access_num=7526506&link_type=MED [Medline]]
  8. Rothman, J. E. & Warren, G. (1994) Curr. Biol. 4, 220–233.[/cgi/external_ref?access_num=10.1016/S0960-9822(00)00051-8&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=A1994NC83700004&link_type=ISI [ISI]][/cgi/external_ref?access_num=7922327&link_type=MED [Medline]]
  9. Fasshauer, D., Sutton, R. B., Brunger, A. T. & Jahn, R. (1998) Proc. Natl. Acad. Sci. USA 95, 15781–15786.[/cgi/ijlink?linkType=ABST&journalCode=pnas&resid=95/26/15781 [Abstract/Free Full Text]]
  10. Sutton, R. B., Fasshauer, D., Jahn, R. & Brunger, A. T. (1998) Nature 395, 347–353.[/cgi/external_ref?access_num=10.1038/26412&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000076083800043&link_type=ISI [ISI]][/cgi/external_ref?access_num=9759724&link_type=MED [Medline]]
  11. Jahn, R., Lang, T. & Südhof, T. C. (2003) Cell 112, 519–533.[/cgi/external_ref?access_num=10.1016/S0092-8674(03)00112-0&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000181252600010&link_type=ISI [ISI]][/cgi/external_ref?access_num=12600315&link_type=MED [Medline]]
  12. Südhof, T. C. (2004) Annu. Rev. Neurosci. 27, 509–547.[/cgi/external_ref?access_num=10.1146/annurev.neuro.26.041002.131412&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000223246300019&link_type=ISI [ISI]][/cgi/external_ref?access_num=15217342&link_type=MED [Medline]]
  13. Mitchell, S. J. & Ryan, T. A. (2004) J. Neurosci. 24, 4884–4888.[/cgi/ijlink?linkType=ABST&journalCode=jneuro&resid=24/20/4884 [Abstract/Free Full Text]]
  14. Taubenblatt, P., Dedieu, J. C., Gulik-Krzywicki, T. & Morel, N. (1999) J. Cell Sci. 112, 3559–3567 Pt 20.[/cgi/ijlink?linkType=ABST&journalCode=joces&resid=112/20/3559 [Abstract]]
  15. Salem, N., Faundez, V., Horng, J. T. & Kelly, R. B. (1998) Nat. Neurosci. 1, 551–556.[/cgi/external_ref?access_num=10.1038/2787&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000077437100009&link_type=ISI [ISI]][/cgi/external_ref?access_num=10196561&link_type=MED [Medline]]
  16. Otto, H., Hanson, P. I. & Jahn, R. (1997) Proc. Natl. Acad. Sci. USA 94, 6197–6201.[/cgi/ijlink?linkType=ABST&journalCode=pnas&resid=94/12/6197 [Abstract/Free Full Text]]
  17. Kretzschmar, S., Volknandt, W. & Zimmermann, H. (1996) Neurosci. Res. 26, 141–148.[/cgi/external_ref?access_num=10.1016/S0168-0102(96)01086-3&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=A1996VU69700005&link_type=ISI [ISI]][/cgi/external_ref?access_num=8953576&link_type=MED [Medline]]
  18. Ahmari, S. E., Buchanan, J. & Smith, S. J. (2000) Nat. Neurosci. 3, 445–451.[/cgi/external_ref?access_num=10.1038/74814&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000086752000012&link_type=ISI [ISI]][/cgi/external_ref?access_num=10769383&link_type=MED [Medline]]
  19. Li, Z. & Murthy, V. N. (2001) Neuron 31, 593–605.[/cgi/external_ref?access_num=10.1016/S0896-6273(01)00398-1&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000170759200013&link_type=ISI [ISI]][/cgi/external_ref?access_num=11545718&link_type=MED [Medline]]
  20. Sampo, B., Kaech, S., Kunz, S. & Banker, G. (2003) Neuron 37, 611–624.[/cgi/external_ref?access_num=10.1016/S0896-6273(03)00058-8&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000181136500010&link_type=ISI [ISI]][/cgi/external_ref?access_num=12597859&link_type=MED [Medline]]
  21. Grote, E., Hao, J. C., Bennett, M. K. & Kelly, R. B. (1995) Cell 81, 581–589.[/cgi/external_ref?access_num=10.1016/0092-8674(95)90079-9&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=A1995QZ71000015&link_type=ISI [ISI]][/cgi/external_ref?access_num=7758112&link_type=MED [Medline]]
  22. Grote, E. & Kelly, R. B. (1996) J. Cell Biol. 132, 537–547.[/cgi/ijlink?linkType=ABST&journalCode=jcb&resid=132/4/537 [Abstract]]
  23. Tsukita, S. & Ishikawa, H. (1980) J. Cell Biol. 84, 513–530.[/cgi/ijlink?linkType=ABST&journalCode=jcb&resid=84/3/513 [Abstract]]
  24. Gandhi, S. P. & Stevens, C. F. (2003) Nature 423, 607–613.[/cgi/external_ref?access_num=10.1038/nature01677&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000183301200032&link_type=ISI [ISI]][/cgi/external_ref?access_num=12789331&link_type=MED [Medline]]
  25. Sankaranarayanan, S., De Angelis, D., Rothman, J. E. & Ryan, T. A. (2000) Biophys. J. 79, 2199–2208.[/cgi/ijlink?linkType=ABST&journalCode=biophysj&resid=79/4/2199 [Abstract/Free Full Text]]
  26. Miesenbock, G., De Angelis, D. A. & Rothman, J. E. (1998) Nature 394, 192–195.[/cgi/external_ref?access_num=10.1038/28190&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000074705900057&link_type=ISI [ISI]][/cgi/external_ref?access_num=9671304&link_type=MED [Medline]]
  27. Samuel, A. D., Silva, R. A. & Murthy, V. N. (2003) J. Neurosci. 23, 373–376.[/cgi/ijlink?linkType=ABST&journalCode=jneuro&resid=23/2/373 [Abstract/Free Full Text]]
  28. Poskanzer, K. E., Marek, K. W., Sweeney, S. T. & Davis, G. W. (2003) Nature 426, 559–563.[/cgi/external_ref?access_num=10.1038/nature02184&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000186944300039&link_type=ISI [ISI]][/cgi/external_ref?access_num=14634669&link_type=MED [Medline]]
  29. Li, Z., Burrone, J., Tyler, W. J., Hartman, K. N., Albeanu, D. F. & Murthy, V. N. (2005) Proc. Natl. Acad. Sci. USA 102, 6131–6136.[/cgi/ijlink?linkType=ABST&journalCode=pnas&resid=102/17/6131 [Abstract/Free Full Text]]
  30. Hall, D. H. & Hedgecock, E. M. (1991) Cell 65, 837–847.[/cgi/external_ref?access_num=10.1016/0092-8674(91)90391-B&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=A1991FP51600014&link_type=ISI [ISI]][/cgi/external_ref?access_num=1710172&link_type=MED [Medline]]
  31. Yonekawa, Y., Harada, A., Okada, Y., Funakoshi, T., Kanai, Y., Takei, Y., Terada, S., Noda, T. & Hirokawa, N. (1998) J. Cell Biol. 141, 431–441.[/cgi/ijlink?linkType=ABST&journalCode=jcb&resid=141/2/431 [Abstract/Free Full Text]]
  32. Miller, K. G., Alfonso, A., Nguyen, M., Crowell, J. A., Johnson, C. D. & Rand, J. B. (1996) Proc. Natl. Acad. Sci. USA 93, 12593–12598.[/cgi/ijlink?linkType=ABST&journalCode=pnas&resid=93/22/12593 [Abstract/Free Full Text]]
  33. Rand, J. & Nonet, M. (1997) in C. elegans II eds. Riddle, D., Blumenthal, T., Meyer, B.& Priess, J. (Cold Spring Harbor Lab. Press, Woodbury, NY), pp. 1049–1052.
  34. Sturman, D. A., Shakiryanova, D., Hewes, R. S., Deitcher, D. L. & Levitan, E. S. (2006) Biophys. J. 90, L45–L47.[/cgi/ijlink?linkType=ABST&journalCode=biophysj&resid=90/6/L45 [Abstract/Free Full Text]]
  35. Richmond, J. E., Davis, W. S. & Jorgensen, E. M. (1999) Nat. Neurosci. 2, 959–964.[/cgi/external_ref?access_num=10.1038/14755&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000083883300011&link_type=ISI [ISI]][/cgi/external_ref?access_num=10526333&link_type=MED [Medline]]
  36. Saifee, O., Wei, L. & Nonet, M. L. (1998) Mol. Biol. Cell 9, 1235–1252.[/cgi/ijlink?linkType=ABST&journalCode=molbiolcell&resid=9/6/1235 [Abstract/Free Full Text]]
  37. Augustin, I., Rosenmund, C., Südhof, T. C. & Brose, N. (1999) Nature 400, 457–461.[/cgi/external_ref?access_num=10.1038/22768&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000081715000052&link_type=ISI [ISI]][/cgi/external_ref?access_num=10440375&link_type=MED [Medline]]
  38. Weimer, R. M. & Richmond, J. E. (2005) Curr. Top. Dev. Biol. 65, 83–113.[/cgi/external_ref?access_num=000228295600003&link_type=ISI [ISI]][/cgi/external_ref?access_num=15642380&link_type=MED [Medline]]
  39. Verhage, M., Maia, A. S., Plomp, J. J., Brussaard, A. B., Heeroma, J. H., Vermeer, H., Toonen, R. F., Hammer, R. E., van den Berg, T. K. & Missler, M., et al. (2000) Science 287, 864–869.[/cgi/ijlink?linkType=ABST&journalCode=sci&resid=287/5454/864 [Abstract/Free Full Text]]
  40. Nonet, M. L., Saifee, O., Zhao, H., Rand, J. B. & Wei, L. (1998) J. Neurosci. 18, 70–80.[/cgi/ijlink?linkType=ABST&journalCode=jneuro&resid=18/1/70 [Abstract/Free Full Text]]
  41. Takei, K., Mundigl, O., Daniell, L. & De Camilli, P. (1996) J. Cell Biol. 133, 1237–1250.[/cgi/ijlink?linkType=ABST&journalCode=jcb&resid=133/6/1237 [Abstract]]
  42. Zhang, B., Koh, Y. H., Beckstead, R. B., Budnik, V., Ganetzky, B. & Bellen, H. J. (1998) Neuron 21, 1465–1475.[/cgi/external_ref?access_num=10.1016/S0896-6273(00)80664-9&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000077910600027&link_type=ISI [ISI]][/cgi/external_ref?access_num=9883738&link_type=MED [Medline]]
  43. Bao, H., Daniels, R. W., Macleod, G. T., Charlton, M. P., Atwood, H. L. & Zhang, B. (2005) J. Neurophysiol. 94, 1888–1903.[/cgi/ijlink?linkType=ABST&journalCode=jn&resid=94/3/1888 [Abstract/Free Full Text]]
  44. Nonet, M. L., Holgado, A. M., Brewer, F., Serpe, C. J., Norbeck, B. A., Holleran, J., Wei, L., Hartwieg, E., Jorgensen, E. M. & Alfonso, A. (1999) Mol. Biol. Cell 10, 2343–2360.[/cgi/ijlink?linkType=ABST&journalCode=molbiolcell&resid=10/7/2343 [Abstract/Free Full Text]]
  45. Schuske, K. R., Richmond, J. E., Matthies, D. S., Davis, W. S., Runz, S., Rube, D. A., van der Bliek, A. M. & Jorgensen, E. M. (2003) Neuron 40, 749–762.[/cgi/external_ref?access_num=10.1016/S0896-6273(03)00667-6&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000186651200010&link_type=ISI [ISI]][/cgi/external_ref?access_num=14622579&link_type=MED [Medline]]
  46. Harris, T. W., Schuske, K. & Jorgensen, E. M. (2001) Traffic 2, 597–605.[/cgi/external_ref?access_num=10.1034/j.1600-0854.2001.002009597.x&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000170866200002&link_type=ISI [ISI]][/cgi/external_ref?access_num=11555413&link_type=MED [Medline]]
  47. Koushika, S. P., Richmond, J. E., Hadwiger, G., Weimer, R. M., Jorgensen, E. M. & Nonet, M. L. (2001) Nat. Neurosci. 4, 997–1005.[/cgi/external_ref?access_num=10.1038/nn732&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000171325800013&link_type=ISI [ISI]][/cgi/external_ref?access_num=11559854&link_type=MED [Medline]]
  48. Betz, A., Thakur, P., Junge, H. J., Ashery, U., Rhee, J. S., Scheuss, V., Rosenmund, C., Rettig, J. & Brose, N. (2001) Neuron 30, 183–196.[/cgi/external_ref?access_num=10.1016/S0896-6273(01)00272-0&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000168412900020&link_type=ISI [ISI]][/cgi/external_ref?access_num=11343654&link_type=MED [Medline]]
  49. Roos, J. & Kelly, R. B. (1999) Curr. Biol. 9, 1411–1414.[/cgi/external_ref?access_num=10.1016/S0960-9822(00)80087-1&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000088977300020&link_type=ISI [ISI]][/cgi/external_ref?access_num=10607569&link_type=MED [Medline]]
  50. Teng, H. & Wilkinson, R. S. (2000) J. Neurosci. 20, 7986–7993.[/cgi/ijlink?linkType=ABST&journalCode=jneuro&resid=20/21/7986 [Abstract/Free Full Text]]
  51. Teng, H., Cole, J. C., Roberts, R. L. & Wilkinson, R. S. (1999) J. Neurosci. 19, 4855–4866.[/cgi/ijlink?linkType=ABST&journalCode=jneuro&resid=19/12/4855 [Abstract/Free Full Text]]
  52. Pobbati, A. V., Razeto, A., Boddener, M., Becker, S. & Fasshauer, D. (2004) J. Biol. Chem. 279, 47192–47200.[/cgi/ijlink?linkType=ABST&journalCode=jbc&resid=279/45/47192 [Abstract/Free Full Text]]
  53. Hatsuzawa, K., Lang, T., Fasshauer, D., Bruns, D. & Jahn, R. (2003) J. Biol. Chem. 278, 31159–31166.[/cgi/ijlink?linkType=ABST&journalCode=jbc&resid=278/33/31159 [Abstract/Free Full Text]]
  54. Yizhar, O., Matti, U., Melamed, R., Hagalili, Y., Bruns, D., Rettig, J. & Ashery, U. (2004) Proc. Natl. Acad. Sci. USA 101, 2578–2583.[/cgi/ijlink?linkType=ABST&journalCode=pnas&resid=101/8/2578 [Abstract/Free Full Text]]
  55. Dybbs, M., Ngai, J. & Kaplan, J. M. (2005) PLoS Genet. 1, 6–16.[/cgi/external_ref?access_num=10.1371/journal.pgen.0010006&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=16103915&link_type=MED [Medline]]
  56. Sankaranarayanan, S. & Ryan, T. A. (2000) Nat. Cell Biol. 2, 197–204.[/cgi/external_ref?access_num=10.1038/35008615&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000086397000012&link_type=ISI [ISI]][/cgi/external_ref?access_num=10783237&link_type=MED [Medline]]
  57. Holt, M., Cooke, A., Wu, M. M. & Lagnado, L. (2003) J. Neurosci. 23, 1329–1339.[/cgi/ijlink?linkType=ABST&journalCode=jneuro&resid=23/4/1329 [Abstract/Free Full Text]]
  58. Meshul, C. K. & Pappas, G. D. (1984) Brain Res. 290, 1–18.[/cgi/external_ref?access_num=10.1016/0006-8993(84)90730-3&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=A1984SB41300001&link_type=ISI [ISI]][/cgi/external_ref?access_num=6607088&link_type=MED [Medline]]
  59. West, A. E., Neve, R. L. & Buckley, K. M. (1997) J. Cell Biol. 139, 917–927.[/cgi/ijlink?linkType=ABST&journalCode=jcb&resid=139/4/917 [Abstract/Free Full Text]]
  60. Martinez-Arca, S., Arold, S., Rudge, R., Laroche, F. & Galli, T. (2004) Traffic 5, 371–382.[/cgi/external_ref?access_num=10.1111/j.1398-9219.2004.00180.x&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000220838200006&link_type=ISI [ISI]][/cgi/external_ref?access_num=15086786&link_type=MED [Medline]]
  61. Allersma, M. W., Wang, L., Axelrod, D. & Holz, R. W. (2004) Mol. Biol. Cell 15, 4658–4668.[/cgi/ijlink?linkType=ABST&journalCode=molbiolcell&resid=15/10/4658 [Abstract/Free Full Text]]
  62. Wu, W., Xu, J., Wu, X. S. & Wu, L. G. (2005) J. Neurosci. 25, 11676–11683.[/cgi/ijlink?linkType=ABST&journalCode=jneuro&resid=25/50/11676 [Abstract/Free Full Text]]
  63. Bacia, K., Schuette, C. G., Kahya, N., Jahn, R. & Schwille, P. (2004) J. Biol. Chem. 279, 37951–37955.[/cgi/ijlink?linkType=ABST&journalCode=jbc&resid=279/36/37951 [Abstract/Free Full Text]]
  64. Fernandez-Alfonso, T. & Ryan, T. A. (2004) Neuron 41, 943–953.[/cgi/external_ref?access_num=10.1016/S0896-6273(04)00113-8&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000221458300013&link_type=ISI [ISI]][/cgi/external_ref?access_num=15046726&link_type=MED [Medline]]
  65. Yao, P. J., Petralia, R. S., Bushlin, I., Wang, Y. & Furukawa, K. (2005) J. Comp. Neurol. 481, 58–69.[/cgi/external_ref?access_num=10.1002/cne.20362&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000225517600005&link_type=ISI [ISI]][/cgi/external_ref?access_num=15558718&link_type=MED [Medline]]
  66. McMahon, H. T. (1999) Curr. Biol. 9, R332–R335.[/cgi/external_ref?access_num=10.1016/S0960-9822(99)80206-1&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000080232900009&link_type=ISI [ISI]][/cgi/external_ref?access_num=10330371&link_type=MED [Medline]]
  67. Kaneda, Y., Grote, E., Bonzelius, F., Desnos, C., Herman, G., Clift-O’Grady, L. & Kelly, R. B. (1995) Cold Spring Harbor Symp. Quant. Biol. 60, 379–387.
  68. Brenner, S. (1974) Genetics 77, 71–94.[/cgi/ijlink?linkType=ABST&journalCode=genetics&resid=77/1/71 [Abstract]]
  69. Burbea, M., Dreier, L., Dittman, J. S., Grunwald, M. E. & Kaplan, J. M. (2002) Neuron 35, 107–120.[/cgi/external_ref?access_num=10.1016/S0896-6273(02)00749-3&link_type=DOI [CrossRef]][/cgi/external_ref?access_num=000176601800014&link_type=ISI [ISI]][/cgi/external_ref?access_num=12123612&link_type=MED [Medline]]





Nicoll R, Schmitz D (2005). "Synaptic plasticity at hippocampal mossy fibre synapses". Nat Rev Neurosci. 6 (11): 863–76. PMID 16261180.

Retrieved from "https://en.wikipedia.org/w/index.php?title=User:Marcuse/test&oldid=853427820"