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• W1995300675 abstract "During recycling of synaptic vesicles (SVs), the retrieval machinery faces the challenge of recapturing SV proteins in a timely and precise manner. The significant dilution factor that would result from equilibration of vesicle proteins with the much larger cell surface would make recapture by diffusional encounter with the endocytic retrieval machinery unlikely. If SV proteins exchanged with counterparts residing at steady state on the cell surface, the dilution problem would be largely avoided. In this scenario, during electrical activity, endocytosis would be driven by the concentration of a pre-existing pool of SVs residing on the axonal or synaptic surface rather than the heavily diluted postfusion vesicular pool. Using both live cell imaging of endogenous synaptotagmin Ia (sytIa) as well as pHluorin-tagged sytIa and VAMP-2, we show here that synaptic vesicle proteins interchange with a large pool on the cell axonal surface whose concentration is ∼10-fold lower than that in SVs. During recycling of synaptic vesicles (SVs), the retrieval machinery faces the challenge of recapturing SV proteins in a timely and precise manner. The significant dilution factor that would result from equilibration of vesicle proteins with the much larger cell surface would make recapture by diffusional encounter with the endocytic retrieval machinery unlikely. If SV proteins exchanged with counterparts residing at steady state on the cell surface, the dilution problem would be largely avoided. In this scenario, during electrical activity, endocytosis would be driven by the concentration of a pre-existing pool of SVs residing on the axonal or synaptic surface rather than the heavily diluted postfusion vesicular pool. Using both live cell imaging of endogenous synaptotagmin Ia (sytIa) as well as pHluorin-tagged sytIa and VAMP-2, we show here that synaptic vesicle proteins interchange with a large pool on the cell axonal surface whose concentration is ∼10-fold lower than that in SVs. Synaptic vesicles (SVs) locally recycle within the presynaptic terminal for multiple rounds of neurotransmitter release (Heuser and Reese, 1973Heuser J.E. Reese T.S. Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction.J. Cell Biol. 1973; 57: 315-344Crossref PubMed Scopus (1583) Google Scholar, Ceccarelli et al., 1973Ceccarelli B. Hurlbut W.P. Mauro A. Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction.J. Cell Biol. 1973; 57: 499-524Crossref PubMed Scopus (620) Google Scholar). The traditional view of protein recycling is that following fusion and merging of vesicles and plasma membrane bilayers (Zenisek et al., 2002Zenisek D. Steyer J.A. Feldman M.E. Almers W. A membrane marker leaves synaptic vesicles in milliseconds after exocytosis in retinal bipolar cells.Neuron. 2002; 35: 1085-1097Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), transmembrane proteins originally in SVs diffuse onto the axonal surface but are efficiently recaptured during endocytosis. SVs contain at least eight known transmembrane proteins with varied relative stoichiometries (Sudhof, 2004Sudhof T.C. The synaptic vesicle cycle.Annu. Rev. Neurosci. 2004; 27: 509-547Crossref PubMed Scopus (1791) Google Scholar, Murthy and De Camilli, 2003Murthy V.N. De Camilli P. Cell biology of the presynaptic terminal.Annu. Rev. Neurosci. 2003; 26: 701-728Crossref PubMed Scopus (270) Google Scholar) and would be expected to be diluted at least 100,000-fold on the axonal surface upon fusion, potentially making recapture based on diffusional encounter with the endocytic machinery unlikely. One possible solution to this problem would be to prevent diffusion of SV proteins out of the site of fusion, as is proposed for “kiss-and-run,” where vesicles are thought to reseal before completely collapsing onto the plasma membrane (Fesce et al., 1994Fesce R. Grohovaz F. Valtorta F. Meldolesi J. Neurotransmitter release: fusion or ‘kiss-and-run’?.Trends Cell Biol. 1994; 4: 1-4Abstract Full Text PDF PubMed Scopus (263) Google Scholar); however the precise conditions under which kiss-and-run retrieval might occur remain to be elucidated (Fernández-Alfonso and Ryan, 2006Fernández-Alfonso T. Ryan T.A. The efficiency of the synaptic vesicle cycle at central nervous system synapses.Trends Cell Biol. 2006; (in press)PubMed Google Scholar) and it does not appear to predominate during high-frequency action potential firing (Fernández-Alfonso and Ryan, 2004Fernández-Alfonso T. Ryan T.A. The kinetics of synaptic vesicle pool depletion at CNS synaptic terminals.Neuron. 2004; 41: 943-953Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Similarly, proteins could be prevented from diffusing beyond sites where endocytosis is likely to occur. However, experiments using GFP-tagged VAMP-2 have shown that synaptic vesicle proteins can diffuse onto axonal surfaces distant from the site of fusion (Sankaranarayanan and Ryan, 2000Sankaranarayanan S. Ryan T.A. Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system.Nat. Cell Biol. 2000; 2: 197-204Crossref PubMed Scopus (325) Google Scholar) and that they can even exchange between boutons (Li and Murthy, 2001Li Z. Murthy V.N. Visualizing postendocytic traffic of synaptic vesicles at hippocampal synapses.Neuron. 2001; 31: 593-605Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), suggesting that no significant barrier to diffusion exists. It is unclear if this represents the behavior of endogenous SV proteins and what impact this has on the potential retrieval mechanisms. Although it has recently been suggested that synaptotagmin Ia (sytIa) resides as clusters on the axonal surface (Willig et al., 2006Willig K.I. Rizzoli S.O. Westphal V. Jahn R. Hell S.W. STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis.Nature. 2006; 440: 935-939Crossref PubMed Scopus (832) Google Scholar), the degree of clustering was only ∼2-fold, which is unlikely to impose significant constraints on lateral diffusion or alleviate the large dilution factor upon exocytosis. Here, using live cell imaging of endogenous sytIa, we demonstrate that sytIa diffuses onto axonal surfaces and exchanges with surface-resident counterparts during recycling. Combining the use of pHluorin-tagged VAMP-2 and sytIa with immunocytochemistry against these proteins, we estimated that their concentration on the axonal surface is ∼10-fold lower than in synaptic vesicles, while the total surface pool is approximately equal in size to that in the entire recycling pool of vesicles. Mixing of SV proteins with this surface reservoir is random, and occurs at both high- and low-frequency stimulation. Our results demonstrate that the molecular identity of SV is not preserved during vesicle recycling and that the large dilution problem is avoided by maintaining a surface complement of at least two important synaptic vesicle proteins. In order to examine the quantitative dynamics of the potential exchange between vesicular and surface pools of SV proteins during recycling, we have taken three complementary approaches. The first makes use of live cell imaging of endogenous sytIa by applying Alexa 488-fluorescently labeled primary antibodies that recognize the lumenal domain of sytIa (α-Stg-488) to dissociated neurons in culture in the absence of any driven synaptic activity. Such staining conditions should allow the preferential visualization of the surface pool of this protein in live cells, as the lumenal domain would be accessible to antibody binding if any of the protein resides on the cell surface. Under these staining conditions, following antibody washout, the fluorescence distribution is mostly uniform along presumed axonal surfaces, with the additional appearance of modest accumulations at presumed presynaptic varicosities (Figure 1A). Following electrical stimulation that recycles the entire releasable pool (600 action potentials [AP] at 20 Hz, Ryan and Smith, 1995Ryan T.A. Smith S.J. Vesicle pool mobilization during action potential firing at hippocampal synapses.Neuron. 1995; 14: 983-989Abstract Full Text PDF PubMed Scopus (241) Google Scholar), the original diffuse distribution becomes much more punctate (Figure 1B). Subsequent stimulation in the presence of FM 4-64 indicates that these sites of stimulus-driven sytIa accumulation correspond to functional presynaptic terminals (Figure 1C). Quantification of sytIa signal before and after stimulation at FM-positive sites indicates an average fold increase in fluorescence of 1.63 ± 0.13 (n = 4). Measurements of sytIa signals along the diffuse axonal regions decrease to 0.60 ± 0.04 (n = 3). These results indicate that a population of sytIa exists on synaptic and axonal surfaces that redistributes significantly away from axons and toward nerve terminals during vesicle recycling, consistent with the idea that part of this surface pool is internalized and exchanges with what were originally vesicle-resident sytIa proteins. To further test this hypothesis, we asked whether additional rounds of labeling of the cell surface after stimulation lead to additional surface staining. We used the same antibody but labeled it with a different fluorophore, Alexa 546 (α-Stg-546). When applied after labeling with α-Stg-488 (Figure 1D), but prior to any electrical stimulation, only weak labeling was achieved (Figure 1E), indicating that the initial α-Stg-488 labeling saturated the available binding sites. In contrast, when an additional round of α-Stg-546 labeling was applied after stimulation, which led to clustering of the original surface labeling as in Figure 1B (Figure 1F), the staining was much greater (Figure 1G, “3rd labeling”). On average the increase in surface labeling achieved in the third round after stimulation compared to the second round prior to stimulation was 2.5- ± 0.22-fold (n = 3). We attribute this large increase in labeling to the appearance of copies of sytIa that were originally resident in SVs, but became resident on the surface following SV recycling. Taken together these experiments strongly suggest that endogenous sytIa in SV exchanges with surface sytIa during recycling. A second approach was to make use of secondary immunofluorescence detection of endogenous sytIa, taking advantage of the fact that a judicious choice of labeling, fixation, permeabilization, and stimulation conditions could be used to determine how much sytIa is on the surface compared to the recycling vesicle pool and how much interchange between these pools takes place during vesicle turnover. Application of anti-sytIa antibodies to live cells followed by fixation leads to the appearance of a surface distribution (Figure 2Ai) similar to that seen in live cells (Figure 1A), which in this case is detected using fluorescently labeled secondary antibodies (2ry) applied after fixation. We verified that the primary antibody concentration used led to near saturation of available binding sites (Figure 2C). This saturable staining, as well as the absence of any significant signal when only 2ry antibodies were applied (Figure 2Aiii) or when a primary antibody against the cytoplasmic tail was used (Figure 2D), provides good evidence that the staining is specific in nature. As with live cell imaging, in addition to a diffuse fluorescence we also detected fluorescence puncta which we interpret as arising from sytIa on the surface of presynaptic boutons. We restricted further quantitative analysis to these regions. To determine the amount of surface staining compared to that in all recycling vesicles within boutons, we compared the amount of staining obtained when applied to nerve terminals at rest (as in Figure 2Ai) with that obtained when the primary antibody is present during intense electrical stimulation (Figure 2Bi) such that the recycling pool of sytIa, which we refer to as the total recycling pool (TRP), was labeled to near saturation (Figure 2C). The relative amount of surface versus vesicular labeling in the stimulated cells can be determined in two ways. 2ry antibodies applied prior to permeabilization result in 23% ± 7% of the labeling obtained when applied after permeabilization. Application of unlabeled 2ry antibody prior to permeabilization can be used to block all surface epitopes (Figure 2Aii). Comparison of staining with and without surface blockade indicates that the surface sytIa staining comprises 34% ± 6% of that in the TRP. Taken together, these two estimates of the relative amount of sytIa on the surface compared to the vesicular pool indicates that it is ∼30%. Since only approximately half of all vesicles in the terminals participate in recycling (Li et al., 2005Li Z. Burrone J. Tyler W.J. Hartman K.N. Albeanu D.F. Murthy V.N. Synaptic vesicle recycling studied in transgenic mice expressing synaptopHluorin.Proc. Natl. Acad. Sci. USA. 2005; 102: 6131-6136Crossref PubMed Scopus (124) Google Scholar, Poskanzer and Davis, 2004Poskanzer K.E. Davis G.W. Mobilization and fusion of a non-recycling pool of synaptic vesicles under conditions of endocytic blockade.Neuropharmacology. 2004; 47: 714-723Crossref PubMed Scopus (21) Google Scholar; our unpublished data), this suggests that the surface fraction of sytIa accounts for ∼15% of total sytIa at synapses, in good agreement with previous estimates obtained using VAMP-2 fused to pHluorin (Sankaranarayanan et al., 2000Sankaranarayanan S. De Angelis D. Rothman J.E. Ryan T.A. The use of pHluorins for optical measurements of presynaptic activity.Biophys. J. 2000; 79: 2199-2208Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). We then determined the extent to which sytIa in synaptic vesicles could interchange with sytIa on the surface during exo-endocytosis. First, we asked what fraction of sytIa residing on the surface that is labeled with anti-sytIa antibodies becomes protected from staining 2ry antibody due to activity-dependent internalization. Comparison of the fluorescence intensity in boutons where the surface is labeled in resting neurons (as in Figure 2Ai) with ones that have been labeled with primary antibody, stimulated to achieve maximal vesicle cycling, and only with labeled 2ry antibody after endocytosis has potentially internalized some of the primary antibody (without permeabilization) indicates that 48% ± 5% (n = 3) of the surface sytIa is internalized during extensive vesicle cycling. Second, following labeling of surface sytIa and antibody washout, synapses were stimulated to achieve maximal vesicle pool turnover and fixed. Any remaining surface antibodies were blocked by addition of blocking 2ry antibodies prior to permeabilization. Probing with staining 2ry antibody at this point reflects the total amount of surface label that has become protected from antibody block by activity-induced internalization (Figure 2Biii). This was compared to the amount of labeling achieved by leaving the anti-sytIa antibody present during the stimulus, and then blocking the surface prior to permeabilization to obtain a measure of the releasable pool (RP) (Figure 2Bii). 57% of sytIa in the RP is replaced by surface sytIa (ratio of iii/ii in bar graph). Under resting conditions, 21% of the RP appears to be replaced (iv/ii), likely due to spontaneous events occurring during the incubation with primary antibody (Sara et al., 2005Sara Y. Virmani T. Deak F. Liu X. Kavalali E.T. An isolated pool of vesicles recycles at rest and drives spontaneous neurotransmission.Neuron. 2005; 45: 563-573Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). Taken together, these results indicate that there is a pool of endogenous sytIa residing on the axonal and synaptic surface, accounting for ∼30% of the TRP at presynaptic boutons, which is taken up into vesicles following exo-endocytosis. Our third approach was to use synapto-pHluorin (spH), a pH-sensitive form of GFP (pKa ∼7.1) fused to the lumenal domain of VAMP-2, to better quantify the size of the axonal and vesicle pools of SV proteins and to study the dynamics and conditions under which they would mix. SpH fluorescence is quenched when exposed to the SV lumen pH (∼5.5), but undergoes an ∼20-fold increase following fusion and exposure to the extracellular pH. In a resting bouton, a large fraction of the signal arises from a portion of spH residing on the synaptic surface (Sankaranarayanan and Ryan, 2000Sankaranarayanan S. Ryan T.A. Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system.Nat. Cell Biol. 2000; 2: 197-204Crossref PubMed Scopus (325) Google Scholar). Although it is possible that overexpression of VAMP-2 might lead to abnormal surface accumulation of spH, we have performed calibrations based on fluorescence intensity and estimated the number of spH molecules per vesicle to be at most two (see Figure S1 in the Supplemental Data). To determine if this resting surface distribution was the VAMP-2 equivalent of the dynamic reservoir of endogenous sytIa described above, we photobleached the surface pool of spH of an entire neuron (see Experimental Procedures) and tested whether it would mix with nonbleached spH (in SVs) after exo-endocytosis of all recycling vesicles. Figure 3A shows spH fluorescence arising from bouton and interbouton areas of a single neuron. The initial fluorescence in the interbouton area arises almost exclusively from surface spH while that in the bouton area (F0) arises from the surface with a small contribution from the intracellular SV pool. Depletion of the releasable pool onto the surface (driven by firing 720 APs at 30 Hz) causes an increase in fluorescence (ΔF1) measured at boutons that reflects the size of the RP (this signal is reproducible in successive control stimulus rounds; Fernández-Alfonso and Ryan, 2004Fernández-Alfonso T. Ryan T.A. The kinetics of synaptic vesicle pool depletion at CNS synaptic terminals.Neuron. 2004; 41: 943-953Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). A simultaneous interbouton signal increase indicates axonal spreading of spH. Analysis of the distribution of endogenous VAMP-2 in bouton and interbouton areas in stimulated versus nonstimulated cells shows that endogenous VAMP-2 also redistributes during stimulation (Figure S2). Following the stimulus, the fluorescence returns to F0 after synaptic vesicle endocytosis, indicating that the size of the surface pool remains unaltered after a round of vesicle cycling. Photobleaching of surface spH is reflected in the magnitude of the change in the resting fluorescence (F0– F0bl). The degree to which surface and internal pools of spH intermix during recycling of SVs can be determined by measuring the resting fluorescence value (F0bl∗) following additional AP firing. If internal and surface spH do not intermix during vesicle recycling then fluorescence levels should return to (F0bl) after additional stimulation. In contrast, we found that F0bl rises significantly after an additional round of stimulation to the new level (F0bl∗) (Figure 3A, middle panel) that corresponds to a surface replacement of 44% ± 7% (n = 9 neurons). This calculation assumes that only surface spH was bleached and represents a slight underestimate of the degree of replacement (see Experimental Procedures). Thus a significant fraction of spH previously in vesicles now resides on the surface. This level of stimulation appears to equilibrate the two pools, as both the magnitude of the subsequent activity induced fluorescence change (ΔF2) and the new resting levels achieved after stimulation (F0bl∗) remain unchanged through two additional rounds of stimulation (Figure 3A, right panel). The replacement of bleached spH with unbleached spH is also evident in the interbouton region (Figure 3A, lower traces). We estimated the degree of fluorescence replacement of unbleached spH in vesicles by bleached spH in the RP during extensive vesicle recycling. Comparison of the size of the AP-induced fluorescence increase before and after bleaching and re-equilibration (i.e., ΔF1 and ΔF2 in Figure 3A) indicates that 52% ± 6% (n = 5) of spH in vesicles is replaced by that on surface after exo-endocytosis of the RP. Similar results were found using pHluoro-tagmin, a construct consisting of synaptotagmin fused to pHluorin in its lumenal domain (Figure S3). Following bleaching of the surface pool and exocytosis of the unbleached pool of spH, the kinetics of endocytosis should also reflect mixing of bleached and unbleached spH. If bleached and unbleached molecules are endocytosed at equal rates then the net rate of unbleached spH endocytosis (and hence the fluorescence decay rate) will decrease in proportion to the amount of mixing of these pools. Measurements of the kinetics of fluorescence decay indicate that the effective rate of spH endocytosis is reduced ∼2-fold immediately following bleaching, but is restored once the pools have equilibrated (see Table S1 in the Supplemental Data). Taken together, these experiments demonstrate that spH in vesicles replaces ∼50% of spH on the plasma membrane when the entire releasable pool turns over. Conversely, spH on surface replaces ∼50% of that in recycling vesicles under the same conditions. Thus, surface- and vesicle-resident pools of recycling spH are comparable in size, and they randomly mix after depletion of SVs onto the surface, such that the probability of endocytosis of any given spH is at most 0.5. We next performed experiments to determine the extent of interchange between surface and vesicular pools of spH with shorter AP trains and at lower frequencies. 120 AP stimuli at 10 Hz led to a surface spH replacement of 23% ± 4% (n = 3) (Figure 3B). Lowering the stimulus frequency to 1 Hz (120 AP), where normally little net accumulation of spH is observable (Fernández-Alfonso and Ryan, 2004Fernández-Alfonso T. Ryan T.A. The kinetics of synaptic vesicle pool depletion at CNS synaptic terminals.Neuron. 2004; 41: 943-953Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar; Figure 3C, left panel) resulted in 12% ± 4% (n = 7) replacement (Figure 3C) and is accompanied by clear axonal dispersion (Figure 3C, middle lower panel). Comparisons of the degree of replacement with the fraction of the RP fusing with the membrane for various stimulus lengths and frequencies used (estimated using alkaline trapping, see Experimental Procedures) indicate a linear relationship that extrapolates to the origin on the graph with a slope of 0.5 (Figure 4A). These data support a model that upon fusion with the plasma membrane, spH mixes into a pool equal in size to the entire RP, and that endocytosis draws randomly from this mixed pool over a wide range of stimulus conditions. We used VAMP-2 and sytIa as model SV proteins to show that during endocytosis, synaptic vesicles reform their complement of transmembrane proteins by making use of a large axonal and synaptic surface reservoir of these proteins. Analysis of all our data leads to the following conclusions regarding the distribution of sytIa in the TRP: 50% of this protein is found in recycling vesicles, 20% in the surface of boutons, and 30% in the surface of interbouton regions (Figure 4B). From simple geometrical considerations, assuming that the diameter of the bouton and the axons are 0.2 μm and 0.6 μm, respectively, and the interbouton length is ∼3 μm, and that on average there are 50 vesicles recycling per bouton (each with a diameter of 40 nm), our results would predict that VAMP-2 and sytIa are approximately ten times more concentrated in vesicles compared to the surface. Since there is no barrier to lateral diffusion of SV proteins following fusion (at least for the stimuli used here, Figure 1, Figure 3 and Figure S2; Sankaranarayanan and Ryan, 2000Sankaranarayanan S. Ryan T.A. Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system.Nat. Cell Biol. 2000; 2: 197-204Crossref PubMed Scopus (325) Google Scholar, Sankaranarayanan et al., 2000Sankaranarayanan S. De Angelis D. Rothman J.E. Ryan T.A. The use of pHluorins for optical measurements of presynaptic activity.Biophys. J. 2000; 79: 2199-2208Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, Li and Murthy, 2001Li Z. Murthy V.N. Visualizing postendocytic traffic of synaptic vesicles at hippocampal synapses.Neuron. 2001; 31: 593-605Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), a large reservoir alleviates the challenge of recovering SV proteins following fusion by reducing the dilution factor on the axonal surface from ∼105 to ∼10. Although the original qualitative studies using anti-sytIa labeling of SV recycling failed to detect a surface pool of sytIa (Matteoli et al., 1992Matteoli M. Takei K. Perin M.S. Sudhof T.C. De Camilli P. Exo-endocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons.J. Cell Biol. 1992; 117: 849-861Crossref PubMed Scopus (280) Google Scholar), recent quantitative analyses documented the presence of such a surface pool (Willig et al., 2006Willig K.I. Rizzoli S.O. Westphal V. Jahn R. Hell S.W. STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis.Nature. 2006; 440: 935-939Crossref PubMed Scopus (832) Google Scholar), in agreement with our studies. In addition, ultrastructural experiments have described the presence of several synaptic proteins (including VAMP-2 and sytIa) on the surface of resting synapses (Taubenblatt et al., 1999Taubenblatt P. Dedieu J.C. Gulik-Krzywicki T. Morel N. VAMP (synaptobrevin) is present in the plasma membrane of nerve terminals.J. Cell Sci. 1999; 112: 3559-3567Crossref PubMed Google Scholar, Morel et al., 2003Morel N. Dedieu J.C. Philippe J.M. Specific sorting of the a1 isoform of the V-H+ATPase a subunit to nerve terminals where it associates with both synaptic vesicles and the presynaptic plasma membrane.J. Cell Sci. 2003; 116: 4751-4762Crossref PubMed Scopus (69) Google Scholar). The study by Willig et al., 2006Willig K.I. Rizzoli S.O. Westphal V. Jahn R. Hell S.W. STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis.Nature. 2006; 440: 935-939Crossref PubMed Scopus (832) Google Scholar indicated that sytIa exists in clusters on the plasma membrane and vesicles, and it was concluded that the protein is not likely to disperse onto the axonal surface during recycling. However, it is important to note that (1) their data suggest a very small degree of clustering (∼1–3 sytIa), which is unlikely to impede diffusional exchange during recycling, and (2) they used secondary immunodetection of sytIa which itself could lead to clustering of the protein, especially as the concentration of primary antibody was not shown to be saturating the sytIa binding sites. Thus we do not think their data is fundamentally incompatible with our results, but it would imply that the difference in concentration between the surface and vesicles would be slightly lower than estimated above. Although pHluorin-tagged proteins provide a wealth of real-time, quantitative dynamic information, it is unclear to what extent either the addition of the GFP moiety or the ensuing overexpression perturbs the normal dynamics and cellular distribution of the target (Pennuto et al., 2003Pennuto M. Bonanomi D. Benfenati F. Valtorta F. Synaptophysin I controls the targeting of VAMP2/synaptobrevin II to synaptic vesicles.Mol. Biol. Cell. 2003; 14: 4909-4919Crossref PubMed Scopus (91) Google Scholar). Several observations indicate that we are not disrupting the distribution of spH between surface and vesicles in our experiments. First, a surface pool of endogenous sytIa equal in size to that measured with spH was detected using live and fixed cell imaging approaches (Figure 1, Figure 2). Although this approach made use of bivalent antibodies that may result in dimerization of sytIa, this was likely avoided as we worked under saturating binding conditions (Figure 2C), and thus, each epitope would be expected to be occupied by a different antibody. Second, our data are consistent with other reports of surface-residing SV proteins (see above). Third, we previously showed that the surface fraction is independent of the amount of spH expression (Sankaranarayanan et al., 2000Sankaranarayanan S. De Angelis D. Rothman J.E. Ryan T.A. The use of pHluorins for optical measurements of presynaptic activity.Biophys. J. 2000; 79: 2199-2208Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). Fourth, spH is only expressed in trace amounts compared to endogenous VAMP-2 (Figure S1). These observations strongly support the notion that the dynamic behavior of spH reflects that of endogenous VAMP-2. Our photobleaching experiments allowed us to measure spH dispersion along the surface of axons following fusion even at stimulation frequencies low enough (1 Hz) to prevent significant accumulation of the probe onto the axonal surface (Figure 3). Our data are difficult to reconcile, however, with recent results that implied that no mixing occurred between surface and vesicle pools of spH during recycling (Gandhi and Stevens, 2003Gandhi S.P. Stevens C.F. Three modes of synaptic vesicular recycling revealed by single-vesicle imaging.Nature. 2003; 423: 607-613Crossref PubMed Scopus (362) Google Scholar). Two possible explanations for this discrepancy come to mind. First, the fast disappearance of spH signal following fusion in some events in the Gandhi and Stevens work could have resulted not from endocytosis and reacidification, but from lateral diffusion of the protein away from the synaptic bouton. Second, it is possible that mixing was avoided under the very low-frequency conditions used in that" @default.
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• W1995300675 title "Synaptic Vesicles Interchange Their Membrane Proteins with a Large Surface Reservoir during Recycling" @default.
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