Overcrowding in neuronal synapses

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 (1–3). 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 (3–5) 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).

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 (9, 10) (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 (15–17). 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 (4, 6), 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).

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 (23–25). 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.

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 (22, 27, 28).

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 (22, 31–33), 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

www.sciencemag.org/content/344/6187/1023/suppl/DC1

Materials and Methods

Figs. S1 to S7

Tables S1 to S3

Movie S1

References (35–46)

References and Notes

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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.