Overcrowding in neuronal synapses


Editor's Introduction

Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins

Sometimes we think of the brain as a large biocomputer, taking in information from our senses and sending commands to our body in return. But how does information get from one part of the brain to the other? Neurons pass electrical signals throughout the brain through synapses, but how exactly does that happen? The neuron, and in particular the synaptic terminal, uses a complex set of specialized proteins to create a system of chemical reception and release. Moving beyond the mere identification of the proteins involved, scientists are now using advances in analytics and 3D modeling to create some of the most detailed models of the neuronal synapses to date.  What can these models teach us?

Paper Details

Original title
Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins
Original publication date
Vol. 344 no. 6187 pp. 1023-1028
Issue name


Synaptic vesicle recycling has long served as a model for the general mechanisms of cellular trafficking. We used an integrative approach, combining quantitative immunoblotting and mass spectrometry to determine protein numbers; electron microscopy to measure organelle numbers, sizes, and positions; and super-resolution fluorescence microscopy to localize the proteins. Using these data, we generated a three-dimensional model of an “average” synapse, displaying 300,000 proteins in atomic detail. The copy numbers of proteins involved in the same step of synaptic vesicle recycling correlated closely. In contrast, copy numbers varied over more than three orders of magnitude between steps, from about 150 copies for the endosomal fusion proteins to more than 20,000 for the exocytotic ones.


The quantitative organization of cellular pathways is not well understood. One well-researched membrane trafficking pathway, synaptic vesicle recycling, occupies its own compartment, the synaptic bouton, and can therefore be studied in isolation. It is a relatively simple pathway, comprising only a few steps (13). First, neurotransmitter-filled synaptic vesicles dock to the release site (active zone), are primed for release, and then fuse with the plasma membrane (exocytosis). The vesicle molecules are later sorted and retrieved from the plasma membrane (endocytosis). An additional sorting step in an early endosome (35) may take place before the vesicle refills with neurotransmitter.

To quantify the organization of synaptic vesicle recycling, we first purified synaptic boutons (synaptosomes) from the cellular layers of the cortex and cerebellum of adult rats, using a modified version (6) of a classical brain fractionation protocol (7) (Fig. 1A). The different cellular components were separated by Ficoll density gradients, resulting in a heterogeneous sample, which we first analyzed by electron microscopy. About 58.5% of all organelles were resealed, vesicle-loaded synaptosomes (fig. S1). Most of the remaining organelles, such as mitochondria (~20%) and myelin (8%) (fig. S1), contained few proteins relevant to synaptic vesicle recycling and thus did not bias synaptic protein quantification. The electron microscopy analysis of the synaptosomes also provided their spatial parameters (size, surface, and volume), which are critical in understanding protein concentrations (Fig. 1, B and C).


Fig. 1.  Physical characteristics of the average synaptosome. (A) Schema illustrating the purification of synaptosomes. See the supplementary materials for details. (B) Serial electron micrographs of purified synaptosomes were used to reconstruct entire synapses. The plasma membrane is depicted in light beige, the active zone in red, synaptic vesicles in dark beige, larger organelles in dark gray, and mitochondria in purple. This synaptosome resembles the average physical parameters (C) and was used to model the average presynaptic terminal (Fig. 3). (C) Table listing the average physical parameters of synaptosomes. The values represent mean ± SEM of 65 reconstructions from four independent synaptosome preparations. (D) Quantitative immunoblots of the three synaptic SNARE proteins (SNAP 25, syntaxin 1, and VAMP 2). The lanes on the left represent increasing amounts of the purified protein of interest, forming a standard curve (protein amount versus band intensity). The different synaptosome samples are depicted in the four lanes on the right. (E) Standard curves of the three SNARE proteins obtained from the immunoblots depicted in (D). Linear regression was used to determine the absolute amount of the protein of interest in the synaptosomes.  (F) (Left) The copy numbers for eight major synaptic vesicle proteins, normalized to the number of synaptic vesicles per synaptosome, are compared with the numbers obtained in a previous quantification of synaptic vesicles (8). The red line represents identity. (Middle) The model shows the eight compared proteins in correct copy numbers on an average vesicle. (Right) Correlation between the copy numbers of different vATPase subunits (highlighted in different colors in the vATPase model, above the graph). The immunoblot quantification of the a1 subunit (green; only the transmembrane part is shown) suggests the presence of 742 vATPase complexes per bouton. The copy numbers of the B, C, E, and F subunits (derived from iBAQ mass spectrometry) are plotted against their expected stoichiometries for 742 complexes. The stoichiometry of the different vATPase subunits was obtained from (34). The black line represents identity. All data represent means ± SEM from four independent preparations.

A:  Synaptosome Purification

This figure depicts a general flow chart of the modified brain fractionation technique used to purify and separate synaptosomes from tissue samples.

Tissue was extracted from the brain of rats and then homogenized.

The homogenized material was then fractionated across a Ficoll gradient with a centrifuge.

The gradated density of the Ficoll medium causes the various types of cellular components of the homogenized neural tissue (such as myelin, mitochondria, and synaptosomes) to be stratified into distinct sections of the gradient.

The synaptosomes were then isolated from the gradient for further analysis.

More in depth discussions of the fractionation protocol used may be found in the paper's supplemental material.

B:  Synaptosome Reconstruction with Serial Electron Micrographs

This figure gives a preliminary 3D depiction of one synaptosome.

The reconstruction of these synaptosomes relied on serial electron micrography.

Thereby, 3D models of cellular structures can be obtained by imaging sheets of ~30 nm thickness cut from the sample.

In this reconstruction of the synaptosome, the plasma membrane is shown in a semitransparent beige, the active zone is shown as a large amalgamous red mass, large organelles are shown in grey, mitochondria in purple, and synaptic vesicles in dark beige (green-yellow). 

C:  Physical Characteristics of Synaptosomes

Here we have a rather self-explanatory table giving descriptions of the volume and surface area of synaptosomes as well as the volume of vesicles, number of vesicles observed, inter-vesicle distance, volume of mitochondria, and surface area of the active zone.

Although these numbers are straightforward, they are critical to the development and framing of ultimate results of the paper.

By generating an initial understanding of the general synaptosome architecture the researchers will have a “canvas” on which to place the proteins identified.

Because the ultimate goal of the paper is to determine copy numbers of synaptic proteins, determining the total number of synaptic vesicles present allows researchers to immediately see the percent a given protein represents related to the total aggregate of synaptic proteins. 

D:  Immunoblots of SNAREs

Figure 1D presents the immunoblots generated for the SNARE proteins SNAP 25, syntaxin 1, and VAMP.

The right-hand side of the images shows immunoblot stains for the synaptosomes in their entirety.

On the left of each image, the immunoblots for the desired protein are given.

The thickness of the line is indicative of the quantity of protein present.

In each case, particularly that of VAMP 2, the thickness of the line increases with the concentration of the protein.

E:  Standard Curves of SNAREs.

This figure provides a graphical representation of the findings in Figure 1D.

The relative intensity of each of the synaptic SNAREs is charted against the quantity of purified protein in the sample; these data are given by the scatter-plot style points.

The researchers note that in each case the data present a standard curve.

Linear regressions are added to the graph to demonstrate the standard curvature of the data; these regressions are the three lines.

F:  Copy Numbers of Primary Synaptic Vesicle Proteins

Figure 1F provides data comparing the copy numbers found by Wilhelm et al. with those in previous literature for various synaptic proteins.

The graph on the left shows this comparison for a variety of proteins and the graph on the right presents the comparison specifically for proteins in the vATPase complex.

In the center, a 3D construction of a model synaptic vesicle as a reference for spatial orientation.

The center line of each plot (depicted in red for the general plot and black on the vATPase plot) shows the identity line (i.e.: where the copy number from this study is the same as the copy number from previous literature).

The numbers found by Wilhelm et al. are highly consistent with the literature values.

Before proceeding to investigate the synaptic protein copy numbers, we tested whether the synaptosomes lost a significant proportion of their proteins during the purification procedure. We compared the amounts of 27 soluble proteins and 2 transmembrane proteins in synaptosomes and in undisturbed synapses from brain slices, using fluorescence microscopy (fig. S2, A and B). The large majority of the proteins exhibited no significant changes after synaptosome purification (fig. S2C).

Having verified that the purification procedure maintains the protein composition of the synaptic bouton, we used quantitative immunoblotting to determine the amount of protein of interest per microgram of synaptosomes for 62 synaptic proteins (Fig. 1, D and E). To transform this value into copy numbers per synaptosome, we determined the number of particles in the synaptosome preparation by fluorescence microscopy (~17 million) (fig. S3) and the fraction represented by synaptosomes by electron microscopy (fig. S1, A and B) and by immunostaining for synaptic markers (fig. S1B). Both measurements indicate that ~58% of all particles are synaptosomes, ~9.95 million synaptosomes per microgram.

The results we obtained for all proteins tested are included in table S1. Despite the heterogeneous preparation we started with, our results are very close to synaptic vesicles purified to more than 95% (8), taking into account the known fractions of these proteins on the synaptic plasma membrane (910) (Fig. 1F). We only detected a sizeable difference for synaptic vesicle 2 (SV2) [12 copies per synaptic vesicle in our study, versus 1.7 for (8)]. A more recent study, using an antibody-based approach that is likely to underestimate the copy numbers of abundant synaptic vesicle proteins, found about five SV2 molecules per vesicle (11).

The immunoblot analysis also provided the total mass of each protein per microgram of synaptosome preparation, which could be translated to percentage of the total protein in the preparation. Our quantification of synaptic proteins addressed ~23% of the total protein in the preparation. Because the synaptosomes make ~58% of the preparation, our quantification thus addressed ~40.5% of the total protein in synaptosomes (without presynaptic mitochondria). To test and extend these values, we turned to quantitative mass spectrometry, using a label-free approach, intensity-based absolute quantification (iBAQ) (12). iBAQ estimates the abundance of particular proteins by summing the intensities of all peptides derived from them and then normalizing to the total possible number of peptides. We compared the peptides derived from recombinant synaptic proteins (same as those used for quantitative immunoblotting) from human Universal Protein Standards (UPS2) and finally from synaptosomes, using a hybrid mass spectrometer. iBAQ values were then calculated using MaxQuant (13) and the Andromeda search engine (14), and the amounts of proteins present in synaptosomes were determined by linear regression. The estimates obtained by iBAQ correlated well with the immunoblotting results (fig. S4). The iBAQ approach generated abundance estimates for ~1100 additional proteins in the preparation (see table S2 for a number of well-known proteins relevant to synaptic activity; see table S3 for all other proteins). All quantified proteins (iBAQ and immunoblot analysis) added up to ~88.4% of the protein weight of the entire synaptosome preparation (obtained by summing the percentages indicated in tables S1 to S3).

The members of heteromultimeric complexes, such as the vesicular adenosine triphosphatase (vATPase), were present in the correct (expected) stoichiometries (Fig. 1F), verifying the accuracy of our quantification procedure. The copy numbers of proteins known to be involved in a particular step of synaptic vesicle recycling correlated remarkably well. This observation applied to the exocytotic fusion proteins [SNAREs (fig. S5B), whose abundance was only matched by actin and tubulin (fig. S5M)], to proteins involved in fusion regulation [SNARE-binding or priming proteins (fig. S5C)], to proteins of the clathrin-mediated endocytosis pathway (fig. S5E), to endosomal or constitutive fusion proteins (fig. S5D), to structural vesicle cluster proteins (fig. S5F), to active zone proteins (fig. S5G), to major synaptic vesicle constituents (fig. S5H), or to adhesion proteins (fig. S5I). Proteins involved in membrane trafficking pathways unrelated to synaptic vesicle recycling, such as the exocyst pathway (fig. S5J), were not abundant. There was no correlation between structurally similar proteins, such as those of the Rab or septin families (fig. S5, K and L). Protein copy numbers are high in some steps of the vesicle recycling pathway but much lower in other steps. For example, the exocytotic SNAREs were present in 20,100 to 26,000 copies, despite the fact that one vesicle fusion event requires the formation of only one to three SNARE complexes, which contain one copy of each of the three SNAREs (1517). SNARE-interacting proteins were found at copy numbers of one to several thousands (Munc13a, Munc18a, complexin I, and complexin II) (fig. S5C). In contrast, only ~4000 clathrin molecules and 2300 dynamin molecules were present in the average synapse. Because at least 150 to 180 copies of clathrin are needed for one recycling vesicle (18, 19), the entire clathrin complement of the synapse would be sufficient for the simultaneous endocytosis of only 7% of all vesicles. The dynamin complement of the synapse was only sufficient for 11% of the vesicles, taking into account that at least 52 copies, corresponding to two adjacent dynamin rings, are needed for one pinch-off event (20). Finally, the endosomal SNAREs, which form tetrameric complexes containing one copy of each SNARE (46), were even less abundant (50 to 150 copies) than the endocytotic cofactors.

For some proteins, a strong enrichment in the location where they function may compensate for their low copy numbers. Conversely, abundant proteins may be scattered throughout the synaptic space, which would render their concentrations fairly low at individual sites. To estimate the influence of protein localization, we selected 62 proteins and analyzed them by immunostaining and fluorescence microscopy. We used stimulated emission depletion (STED) (21), a diffraction-unlimited technique, to investigate protein positions with a resolution of ~40 nm (Fig. 2A). To avoid bias owing to possible artifacts connected to the brain homogenization procedure required for generating synaptosomes, we also studied two additional preparations: cultured hippocampal neurons (Fig. 2B) and the levator auris longus neuromuscular junction (Fig. 2C), acutely dissected from adult animals (22).


Fig. 2.  Presynaptic protein organization. (A) Protein organization in synaptosomes. The scheme indicates an overview of the preparation. AZ, active zone; ves, synaptic vesicles. Purified synaptosomes were immunostained in parallel for the protein of interest, VAMP 2 (red, STED resolution), for an active zone marker, bassoon (blue, confocal resolution), and for a vesicle marker, synaptophysin (green, confocal resolution). The fourth panel shows the relative spatial distribution of VAMP 2 as obtained from average images (several hundred synapses from two independent experiments; see the supplementary materials for further details). The putative outline of the synapse is indicated by the white line, the active zone by the black circle; the relative spatial abundance is color-coded (see color bar). Scale bars are 500 nm (image panels) and 200 nm (fourth panel). The last two panels on the right are density distributions for two additional presynaptic proteins, amphiphysin and syntaxin 16. Scale bar is 200 nm. (B) Protein organization in hippocampal cultures. Details as in (A). Scale bars are 2 μm and 200 nm, respectively. (C) Protein organization in the mouse neuromuscular junction. Instead of immunostaining for bassoon, the active zone position was obtained by labeling postsynaptic acetylcholine receptors with bungarotoxin. All other details as in (A). Scale bars are 2 μm and 500 nm, respectively. Imaging data for all the other proteins are provided in fig. S6. (D) Different spatial parameters were measured for each of the 62 proteins we imaged, as indicated by the labeling of the rows. Parameter values were normalized to the maximum (100%). All values are indicated according to the color scale (right). The proteins are grouped according to functional categories: active zone proteins (bassoon, piccolo, and RIM1), synaptic vesicle proteins (synaptophysin, VGlut 1/2, VAMP 2, VAMP 1, SV2 A/B, synapsin I/II, and synaptogyrin 1), calcium sensor proteins (synaptotagmin 2, synaptotagmin 1, synaptotagmin 7, doc 2A/B, and calmodulin), SNARE cofactors (CSP, Munc13a, Munc18a, NSF, α-SNAP, and complexin 1/2), small guanosine triphosphatases (GTPases) (Rab3, Rab5, and Rab7), disease-related proteins (α/β-synuclein, APP, and β-secretase), mitochondrial proteins (VDAC), endocytosis proteins (AP-2 mu2, SGIP1, synaptojanin, epsin 1, clathrin heavy chain, clathrin light chain, dynamin 1,2,3, endophilin I,II,III, amphiphysin, Hsc70, intersectin 1, PIPK Iγ, AP 180, and syndapin 1), endosomal SNAREs (syntaxin 13, syntaxin 16, syntaxin 7, syntaxin 6, Vti1a, and VAMP4), plasma membrane SNAREs (syntaxin 1, SNAP 23, SNAP 25, and SNAP 29), general secretory proteins (CAPS, SCAMP 1, SGTα, and vATPase a1), calcium buffer proteins (calbindin, calretinin, and parvalbumin), and cytoskeletal proteins (actin, septin 5, and tubulin).       

A:  Organization of Synaptic Proteins in the Synaptosome

Figure 2A provides an overview of the immunostaining that was used to mark desired proteins. For each protein, a tissue preparation was stained for the protein of interest itself, VAMP 2 in red, bassoon in blue (to mark the active zone), and synaptophysin in green (to mark the vesicles). The fourth, fifth, and sixth panels respectively show the average distribution of VAMP 2, Amphiphysin, and Syntaxin 16 in the synaptosome. For reference, the synaptosome is artificially outlined in white in the fourth panel.

B:  Organization of Synaptic Proteins in Hippocampal Cultures. 

Figure 2B shows the same general form of preparation depicted in Figure 2A. As opposed to the purified and isolated adult rat synaptosomes used for the data in 2A, the 2B preparation used cultures from the hippocampus of postnatal animals. This secondary data using hippocampal neurons, as well as a tertiary set of data shown in Figure 2C, serve as references of comparison to ensure that the findings from the purified cultures of 2A were not the result of artifacts of the purification procedure and homogenization process.

C:  Organization of Synaptic Proteins in the Neuromuscular Junction.

 Figure 2C, as in Figure 2B and 2A, shows the general scheme of the immunostaining technique used by the paper and the protein organization of VAMP 2, Amphiphysin, and Syntaxin 16. Here, the data were obtained from the levator auris longus neuromuscular junction of the mouse. 

D:  Spatial Parameters of Synaptic Proteins. 

Perhaps one of the more seemingly complicated figures of the paper, Figure 2D shows the relative distribution (abundance) of the proteins studied against a variety of spatial parameters. The spatial parameters are given as the row headings of the table and the proteins, separated into functional groups, are given as the columns. The colors are "normalized to the maximum." This means that the protein (or proteins) with the highest value for a given parameter will have the darkest red color. The color of each other cell in that row will indicate that protein’s value for the given spatial parameter as a percentage of the maximum. For example: Suppose the maximum value obtained for a given parameter is 10 and suppose that we are coloring the cell for a protein that has a value of 5 for that parameter. In this case our protein would have a value of 50% as 5 is 50% of 10 and the cell would therefore be green.

We analyzed the proteins of interest in relation to the positions of the release site (identified by marking active zone proteins) and of the vesicle cluster (visualized by staining for the protein that is most strongly enriched in purified synaptic vesicles, synaptophysin) (8). We averaged single synapses by overlapping their active zones and rotating the images until reaching the best possible alignment of the vesicle cluster and of the protein of interest. This procedure provided an overview of the relative spatial distribution of each protein. Overall, many of the protein distributions were similar (Fig. 2, A to C, and fig. S6). Active zone proteins were mostly confined to the active zone areas. Most of the other proteins could be found throughout the synaptic boutons [albeit they were enriched to different levels in areas such as the active zone or the vesicle cluster (Fig. 2D); see fig. S7, A to H, for a more detailed analysis of differences between the proteins]. These observations are consistent with the presence of most of the proteins on purified synaptic vesicles (8) and with the fact that the synaptic vesicle cluster occupies much of the synaptic bouton volume (Fig. 1B). Thus, for the majority of proteins, localization does not appear to compensate for low copy numbers.

Although the imaging parameters measured above did not pinpoint actual positions within the synapse, they allowed us to make broad estimates for the organization of each protein (fig. S7I). We used the data to generate a three-dimensional (3D) model containing 60 proteins placed within a typical synaptic volume (obtained from an individual electron microscopy reconstruction whose parameters were close to synaptosome averages) (Fig. 3). The proteins were modeled in atomic detail, according to their known molecular structures, and were placed in the synaptic space according to the information provided by the STED images and the literature (Fig. 2 and fig. S6). For example, the SNARE molecules syntaxin 1 and SNAP 25 are shown in clusters with a specific organization (2325). The hippocampal culture images (Fig. 2B) were used to obtain an additional set of data, the correlation of protein amounts with synapse size [judged from the amount of vesicles (26) (fig. S6)]. The copy numbers of some proteins increase linearly with synapse size; others, including most endocytotic proteins, follow an exponential curve, which implies that small synapses contain proportionally larger amounts of these proteins than large synapses.


Fig. 3.  A 3D model of synaptic architecture. (A) A section through the synaptic bouton, indicating 60 proteins. The proteins are shown in the copy numbers indicated in tables S1 and S2 and in positions determined according to the imaging data (Fig. 2 and fig. S6) and to the literature (see fig. S6 for details). (B) High-zoom view of the active zone area. (C) High-zoom view of one vesicle within the vesicle cluster. (D) High-zoom view of a section of the plasma membrane in the vicinity of the active zone. Clusters of syntaxin (yellow) and SNAP 25 (red) are visible, as well as a recently fused synaptic vesicle (top). The graphical legend indicates the different proteins (right). Displayed synaptic vesicles have a diameter of 42 nm.

A:  Cross Sectional Depiction of the Synaptic Bouton. 

After determining the copy numbers of the proteins investigated, the researchers constructed a 3D model of an average synaptosome. In Figure 3A, we see a cross sectional view of the synaptosome in it’s entirety. For a video look, check out the video of the model linked in the additional science links section at the bottom of the page. The researchers note that despite the synaptic proteins technically representing only a minority percentage of the synaptosome’s total volume, the spatial configuration of the proteins within the synaptic terminal makes the space feel crowded none the less. One may also notice that vesicles are often clustered rather than uniformly distributed throughout the terminal. A legend of the protein’s depicted (which include 60 of the 62 proteins initially studied) is provided in the bottom right hand side of the Figure.

B:  Close up of the Active Zone. 
Figure 3B simply shows a close up section of the active zone area. Notice that the relative abundances of synaptic proteins is slightly different than in other parts of the synaptosome, reminding us that the functional class of a protein impacts it’s location within the cell. 
C:  Close up of a Synaptic Vesicle. 

Figure 1C shows a close up of a single synaptic vesicle. Much as the relative abundances of synaptic proteins has a characteristic makeup near the active zone, so too do the vesicles have a particular set of proteins. 

D:  Close up of Plasma Membrane Near the Active Zone. 

Figure 1D provides a 3D model of a portion of the plasma membrane near the active zone. The large collection of red molecules are SNAP 25, a critical protein for the exocytosis pathway. The yellow cluster is composed of syntaxin 1, a protein that, like SNAP 25, helps to facilitate vesicle fusion. Above the syntaxin cluster, we can see [a] the remainders of a synaptic vesicle that has recently fused to the membrane.  

We used the modeled volumes of the proteins to calculate the fraction of the synaptic volume that they occupy. This value, ~7% of the synaptosome volume (excluding mitochondria), is comparable to the space occupied by the synaptic vesicles (~6%, derived from the electron microscopy measurements). These low values could lead to the impression that the synaptic volume is not densely populated by proteinaceous structures. However, the 3D model suggests that the synaptic space is rather crowded, especially inside the vesicle cluster and at the active zone (Fig. 3, A to C, and movie S1).

This probably places constraints on both organelle and protein diffusion. The high copy numbers of exocytosis-related proteins may have evolved as a mechanism to cope with these constraints, to ensure the high speed of neurotransmitter release. In contrast, endocytosis can take place for many tens of seconds after exocytosis. This allows endocytosis to proceed with proportionally lower numbers of cofactor proteins. In principle, the synaptic boutons could increase the speed of endocytosis by accumulating larger amounts of endocytotic proteins. This, however, would result in an even greater congestion of the synaptic space, which presumably might perturb synaptic function. A simpler solution for the problem of balancing rapid release with slow vesicle retrieval appears to have been to maintain a large enough reservoir of vesicles (222728).

Our data reveal a correlation between the copy numbers of proteins involved in the same steps of synaptic vesicle recycling. The mechanisms behind this correlation are unclear. A simple hypothesis would be that such proteins either are produced together or are transported to the synapse together. However, these proteins have different lifetimes (29) and are transported from the neuronal cell body on different precursors (30). One possible explanation, at least for the soluble cofactor proteins, is that the synaptic vesicle cluster regulates their number. The vesicles are known to bind to and buffer such proteins (223133), thereby retaining in the synapse only a defined number of cofactors. Such mechanisms do not apply, however, to transmembrane proteins, whose regulation remains to be determined.

Supplementary Materials


Materials and Methods

Figs. S1 to S7

Tables S1 to S3

Movie S1

References (3546)

References and Notes

  1. V. Haucke, E. Neher, S. J. Sigrist, Protein scaffolds in the coupling of synaptic exocytosis and endocytosis. Nat. Rev. Neurosci. 12, 127–138 (2011).

  2. R. Jahn, D. Fasshauer, Molecular machines governing exocytosis of synaptic vesicles. Nature 490, 201–207 (2012).

  3. T. C. Südhof, The synaptic vesicle cycle. Annu. Rev. Neurosci. 27, 509–547 (2004).

  4. P. Hoopmann, A. Punge, S. V. Barysch, V. Westphal, J. Bückers, F. Opazo, I. Bethani, M. A. Lauterbach, S. W. Hell, S. O. Rizzoli, Endosomal sorting of readily releasable synaptic vesicles. Proc. Natl. Acad. Sci. U.S.A. 107, 19055–19060 (2010).

  5. V. Uytterhoeven, S. Kuenen, J. Kasprowicz, K. Miskiewicz, P. Verstreken, Loss of skywalker reveals synaptic endosomes as sorting stations for synaptic vesicle proteins. Cell 145, 117–132 (2011).

  6. S. O. Rizzoli, I. Bethani, D. Zwilling, D. Wenzel, T. J. Siddiqui, D. Brandhorst, R. Jahn, Evidence for early endosome-like fusion of recently endocytosed synaptic vesicles. Traffic 7, 1163–1176 (2006).

  7. D. G. Nicholls, T. S. Sihra, Synaptosomes possess an exocytotic pool of glutamate. Nature 321, 772–773 (1986).

  8. S. Takamori, M. Holt, K. Stenius, E. A. Lemke, M. Grønborg, D. Riedel, H. Urlaub, S. Schenck, B. Brügger, P. Ringler, S. A. Müller, B. Rammner, F. Gräter, J. S. Hub, B. L. De Groot, G. Mieskes, Y. Moriyama, J. Klingauf, H. Grubmüller, J. Heuser, F. Wieland, R. Jahn, Molecular anatomy of a trafficking organelle. Cell 127, 831–846 (2006).

  9. F. Opazo, A. Punge, J. Bückers, P. Hoopmann, L. Kastrup, S. W. Hell, S. O. Rizzoli, Limited intermixing of synaptic vesicle components upon vesicle recycling. Traffic 11, 800–812 (2010).

  10. M. Darna, I. Schmutz, K. Richter, S. V. Yelamanchili, G. Pendyala, M. Höltje, U. Albrecht, G. Ahnert-Hilger, Time of day-dependent sorting of the vesicular glutamate transporter to the plasma membrane. J. Biol. Chem. 284, 4300–4307 (2009).

  11. S. A. Mutch, P. Kensel-Hammes, J. C. Gadd, B. S. Fujimoto, R. W. Allen, P. G. Schiro, R. M. Lorenz, C. L. Kuyper, J. S. Kuo, S. M. Bajjalieh, D. T. Chiu, Protein quantification at the single vesicle level reveals that a subset of synaptic vesicle proteins are trafficked with high precision. J. Neurosci. 31, 1461–1470 (2011).

  12. B. Schwanhäusser, D. Busse, N. Li, G. Dittmar, J. Schuchhardt, J. Wolf, W. Chen, M. Selbach, Global quantification of mammalian gene expression control. Nature 473, 337–342 (2011).

  13. J. Cox, M. Mann, MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008). 

  14. J. Cox, N. Neuhauser, A. Michalski, R. A. Scheltema, J. V. Olsen, M. Mann, Andromeda: A peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).

  15. R. Mohrmann, H. de Wit, M. Verhage, E. Neher, J. B. Sørensen, Fast vesicle fusion in living cells requires at least three SNARE complexes. Science 330, 502–505 (2010).

  16. R. Sinha, S. Ahmed, R. Jahn, J. Klingauf, Two synaptobrevin molecules are sufficient for vesicle fusion in central nervous system synapses. Proc. Natl. Acad. Sci. U.S.A. 108, 14318–14323 (2011).

  17. G. van den Bogaart, M. G. Holt, G. Bunt, D. Riedel, F. S. Wouters, R. Jahn, One SNARE complex is sufficient for membrane fusion. Nat. Struct. Mol. Biol. 17, 358–364 (2010).

  18. Y. Cheng, W. Boll, T. Kirchhausen, S. C. Harrison, T. Walz, Cryo-electron tomography of clathrin-coated vesicles: Structural implications for coat assembly. J. Mol. Biol. 365, 892–899 (2007).

  19. H. T. McMahon, E. Boucrot, Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nat. Rev. Mol. Cell Biol. 12, 517–533 (2011).

  20. V. Shnyrova, P. V. Bashkirov, S. A. Akimov, T. J. Pucadyil, J. Zimmerberg, S. L. Schmid, V. A. Frolov, Geometric catalysis of membrane fission driven by flexible dynamin rings. Science 339, 1433–1436 (2013).

  21. K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, S. W. Hell, STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440, 935–939 (2006).

  22. Denker, I. Bethani, K. Kröhnert, C. Körber, H. Horstmann, B. G. Wilhelm, S. V. Barysch, T. Kuner, E. Neher, S. O. Rizzoli, A small pool of vesicles maintains synaptic activity in vivo. Proc. Natl. Acad. Sci. U.S.A. 108, 17177–17182 (2011).

  23. D. Bar-On, S. Wolter, S. van de Linde, M. Heilemann, G. Nudelman, E. Nachliel, M. Gutman, M. Sauer, U. Ashery, Super-resolution imaging reveals the internal architecture of nano-sized syntaxin clusters. J. Biol. Chem. 287, 27158–27167 (2012).

  24. J. J. Sieber, K. I. Willig, R. Heintzmann, S. W. Hell, T. Lang, The SNARE motif is essential for the formation of syntaxin clusters in the plasma membrane. Biophys. J. 90, 2843–2851 (2006).

  25. J. J. Sieber, K. I. Willig, C. Kutzner, C. Gerding-Reimers, B. Harke, G. Donnert, B. Rammner, C. Eggeling, S. W. Hell, H. Grubmüller, T. Lang, Anatomy and dynamics of a supramolecular membrane protein cluster. Science 317, 1072–1076 (2007).

  26. V. N. Murthy, T. Schikorski, C. F. Stevens, Y. Zhu, Inactivity produces increases in neurotransmitter release and synapse size. Neuron 32, 673–682 (2001).

  27. V. Marra, J. J. Burden, J. R. Thorpe, I. T. Smith, S. L. Smith, M. Häusser, T. Branco, K. Staras, A preferentially segregated recycling vesicle pool of limited size supports neurotransmission in native central synapses. Neuron 76, 579–589 (2012).

  28. T. Rose, P. Schoenenberger, K. Jezek, T. G. Oertner, Developmental refinement of vesicle cycling at Schaffer collateral synapses. Neuron 77, 1109–1121 (2013).

  29. L. D. Cohen, R. Zuchman, O. Sorokina, A. Müller, D. C. Dieterich, J. D. Armstrong, T. Ziv, N. E. Ziv, Metabolic turnover of synaptic proteins: Kinetics, interdependencies and implications for synaptic maintenance. PLOS ONE 8, e63191 (2013).

  30. D. Bonanomi, F. Benfenati, F. Valtorta, Protein sorting in the synaptic vesicle life cycle. Prog. Neurobiol. 80, 177–217 (2006).

  31. Denker, K. Kröhnert, J. Bückers, E. Neher, S. O. Rizzoli, The reserve pool of synaptic vesicles acts as a buffer for proteins involved in synaptic vesicle recycling. Proc. Natl. Acad. Sci. U.S.A. 108, 17183–17188 (2011).

  32. O. Shupliakov, The synaptic vesicle cluster: A source of endocytic proteins during neurotransmitter release.

  33. S. O. Rizzoli, Synaptic vesicle recycling: Steps and principles. EMBO J. 33, 788–822 (2014).

  34. N. Kitagawa, H. Mazon, A. J. R. Heck, S. Wilkens, Stoichiometry of the peripheral stalk subunits E and G of yeast V1-ATPase determined by mass spectrometry. J. Biol. Chem. 283, 3329–3337 (2008).

  35. G. Fischer von Mollard, B. Stahl, A. Khokhlatchev, T. C. Südhof, R. Jahn, Rab3C is a synaptic vesicle protein that dissociates from synaptic vesicles after stimulation of exocytosis. J. Biol. Chem. 269, 10971–10974 (1994).

  36. D. Angaut-Petit, J. Molgo, A. L. Connold, L. Faille, The levator auris longus muscle of the mouse: A convenient preparation for studies of short- and long-term presynaptic effects of drugs or toxins. Neurosci. Lett. 82, 83–88 (1987).

  37. H. Schägger, Tricine-SDS-PAGE. Nat. Protoc. 1, 16–22 (2006).

  38. Bethani, T. Lang, U. Geumann, J. J. Sieber, R. Jahn, S. O. Rizzoli, The specificity of SNARE pairing in biological membranes is mediated by both proof-reading and spatial segregation. EMBO J. 26, 3981–3992 (2007).

  39. Pechstein, O. Shupliakov, V. Haucke, Intersectin 1: A versatile actor in the synaptic vesicle cycle. Biochem. Soc. Trans. 38, 181–186 (2010).

  40. L. von Kleist, W. Stahlschmidt, H. Bulut, K. Gromova, D. Puchkov, M. J. Robertson, K. A. MacGregor, N. Tomilin, A. Pechstein, N. Chau, M. Chircop, J. Sakoff, J. P. von Kries, W. Saenger, H. G. Kräusslich, O. Shupliakov, P. J. Robinson, A. McCluskey, V. Haucke, Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition. Cell 146, 471–484 (2011).

  41. Vervaeke, A. Lorincz, P. Gleeson, M. Farinella, Z. Nusser, R. A. Silver, Rapid desynchronization of an electrically coupled interneuron network with sparse excitatory synaptic input. Neuron 67, 435–451 (2010).

  42. Rappsilber, M. Mann, Analysis of the topology of protein complexes using cross-linking and mass spectrometry. Cold Spring Harbor Protocols 2007, pdb.prot4594 (2007).

  43. J. V. Olsen, L. M. de Godoy, G. Li, B. Macek, P. Mortensen, R. Pesch, A. Makarov, O. Lange, S. Horning, M. Mann, Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol. Cell. Proteomics 4, 2010–2021 (2005).

  44. W. Huang, B. T. Sherman, R. A. Lempicki, Bioinformatics enrichment tools: Paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1–13 (2009).

  45. W. Huang, B. T. Sherman, R. A. Lempicki, Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

  46. T. Staudt, M. C. Lang, R. Medda, J. Engelhardt, S. W. Hell, 2,2′-thiodiethanol: A new water soluble mounting medium for high resolution optical microscopy. Microsc. Res. Tech. 70, 1–9 (2007).

Acknowledgments: We thank the following collaborators for providing purified proteins and antibodies: R. Jahn, C. Griesinger, B. Shwaller, and A. Roux. We thank H. Martens for technical help and support, B. Rizzoli for helpful comments on the manuscript, and T. Sargeant for providing the carve source code for creation of the voltage-dependent anion channel (VDAC)-based mitochondrial membrane cut-outs. B.G.W. was supported by a Boehringer Ingelheim Fonds PhD Fellowship. S.T. was supported by an Excellence Stipend of the Göttingen Graduate School for Neurosciences, Biophysics, and Molecular Biosciences (GGNB). The work was supported by grants to S.O.R. from the European Research Council (FP7 NANOMAP and ERC-2013-CoG NeuroMolAnatomy) and from the Deutsche Forschungsgemeinschaft (DFG) Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, as well as from DFG grants RI 1967 2/1, RI 1967 3/1, and SFB 889/A5. We acknowledge support by the DFG to V.H. (Exc-257-Neurocure and SFB 958/A01), H.U. (SFB 889), and M.K. (SFB 958/A11). Author contributions: B.G.W. prepared the synaptosomes and performed all immunoblotting experiments. K.K. performed the electron microscopy imaging and all neuromuscular junction imaging. C.S. performed the hippocampal culture imaging. S.T. performed the synaptosome imaging. B.R. generated the synapse model. S.J.K., G.A.C., and M.K. participated in the biochemistry experiments. S.M. and H.U. designed and performed all mass spectrometry experiments. S.O.R., B.G.W., and V.H. designed the experiments. All authors analyzed the data and contributed to writing the manuscript.