After proving the potential of mCLING to track endocytic organelles in the relatively large cytoplasmic volume of IHCs, a main concern was its ability to reveal membrane uptake in small synaptic boutons. To test this, mCLING was applied to cultured hippocampal neurons, finding no negative effects on plasma membrane integrity or synaptic vesicle recycling (Figure 3.22). Importantly, mCLING was efficiently taken up by recycling synaptic vesicles, allowing the study of their molecular composition by STED microscopy imaging. Moreover, incubations at low temperature (thereby inhibiting endocytosis) facilitated the distinction between immunostained proteins residing on intracellular organelles, and those sitting on the plasma membrane. These technical advantages were used to better understand aspects of the physiology and organization of conventional synapses, described below.
4.4.1 Molecular differences between spontaneously and actively released synaptic vesicles
In this study we combined mCLING labeling of spontaneously and actively released synaptic vesicles with immunostaining against synaptic- and endosomal-related proteins. The results suggest that these two groups of organelles differ in their molecular composition:
- Spontaneously recycling vesicles presented higher levels of endosomal SNARE proteins, like syntaxin 13 and VAMP4.
- The spontaneously released group presented significantly lower levels of three synaptic vesicle proteins, synaptotagmin 1, synapsin and the SNARE protein VAMP2.
- The two groups had similar levels of other synaptic vesicle proteins, like VGLUT1/2 and synaptophysin.
Taken together, our results indicate that quantitative molecular differences between spontaneously and actively released synaptic vesicles not only reside in SNARE proteins, but also on other protein families (Figure 3.23). Additionally, they indicate that spontaneously released organelles might be more related to constitutive trafficking pathways, being “less vesicular” than their actively released counterparts.
139 Differences in protein make-up between spontaneously and actively released synaptic vesicles were suggested before. Chimeric constructs containing the pH-sensitive protein pHluorin and either VAMP7 or Vti1a, two endosomal SNARE proteins, preferentially located on the spontaneously recycling pool of vesicles (Hua et al., 2011b; Ramirez et al., 2012).
Moreover, the involvement of different synaptic proteins in spontaneous release has been indirectly established upon their mutation or knocking down. Thus, the lower levels of synaptotagmin 1 found here for spontaneously recycling vesicles, would agree with previous reports on the independence of spontaneous release rates from synaptotagmin 1 calcium-sensing activity (Geppert et al., 1994b). On the other hand, the low levels of VAMP2 reported here for spontaneously released vesicles are difficult to reconcile with previous findings indicating a 6-fold reduction in spontaneous release in the absence of this protein (Sara et al., 2005).
Even though spontaneous vesicle release has been related to synaptic development, maintenance and strength, the existence of a physiological role for such events is still debated. In the same context, it is difficult to establish whether the presence of glutamate transporters (VGLUT) and synaptophysin is associated to that theoretical function, or is rather the result of inefficient exclusion of protein components during endocytosis.
Paradoxically, it has been the presence of transporters and neurotransmitter filling, measured through the concomitant miniature post synaptic currents (mEPSC and mIPSC), the standard tool to characterize spontaneous release (Van der Kloot, 1991; McBain and Dingledine, 1992).
4.4.2 Synaptic vesicle proteins stranded on the plasma membrane
mCLING surface labeling of isolated hippocampal neurons was combined with immunostaining against synaptic vesicle-associated proteins, in order to establish the percentage of molecules that remains stranded on the plasma membrane at resting conditions. The evaluated proteins included VGLUT1/2, synaptophysin, synaptotagmin 1, VAMP2, synapsin and Rab3.
Overall, the fraction of proteins present in the plasma membrane ranged between ∼12 and
~22% of the total amount (Figure 3.24). The relatively low variability among these values contrast with a wider range obtained by different groups using protein-pHluorin coupling:
~2% for VGLUT1, ~8% for synaptophysin, ~10-24% for VAMP2 and ~22% for synaptotagmin (Sankaranarayanan and Ryan, 2000; Fernández-Alfonso et al., 2006;
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Granseth et al., 2006; Balaji and Ryan, 2007). Therefore, the mCLING labeling and sample processing protocols used in this study represent a more reliable way to look at protein composition and distribution in synaptic terminals for the following reasons:
- Our method quantifies the endogenously expressed proteins in their native form. In contrast, pHluorin experiments rely on the overexpression of proteins with a large tag at their intravesicular domain (238 aminoacids), likely affecting their targetting, function, clustering and retrieval efficiency from the plasma membrane (Opazo et al., 2010). This is confirmed by similar values for synaptotagmin 1 surface pool of molecules found in this study (~18%) and in a previous report using antibodies against the luminal domain of this protein in resting cells (~19%) (Opazo et al., 2010).
- Immunostaining levels are similar among neurons cultured in the same coverslip.
Transfection efficiency is, however, a more variable parameter that could affect the quantification of protein distributions.
- More accurate quatifications can be obtained with high-resolution imaging, in contrast to confocal imaging performed in pHluorin analysis.
- mCLING labeling and immunostaining are easier and more reproducible methods, enabling the study of several proteins in parallel.
- In our study we also analysed cytoplasmic proteins that transiently associate to synaptic vesicles before vesicle release (Rab3 and synapsin). This would have been impossible with pHluorin coupling, since it can only be applied to integral membrane proteins.
Among our results, the abundance of Rab3 (~22%) and synapsin (~13%) on the membrane is surprising, considering their disengagement from the vesicular membrane after exocytosis is accomplished (Fischer von Mollard et al., 1991; Tarelli et al., 1992).
Nevertheless, synapsin molecules have been seen before on the plasma membrane by immuno-electron microscopy (Tarelli et al., 1992; Pieribone et al., 1995). This fraction, however, has been difficult to determine due to the difficulties imposed by immuno-EM.
Similarly, a fraction of Rab3A molecules was also found on the plasma membrane of small rat brain synapses (Mizoguchi et al., 1990), as well as in the rat NMJ (Mizoguchi et al., 1992).
The results presented here suggest that a relatively large fraction of synaptic vesicle proteins stays on the plasma membrane. It is not clear, however, if these molecules remain together as a vesicular patch, as suggested by STED-microscopy studies (Willig et al., 2006;
141 Opazo et al., 2010), or if they diffuse and intermix with plasma membrane resident proteins (Sankaranarayanan and Ryan, 2000; Li and Murthy, 2001; Wienisch and Klingauf, 2006).
The latter hypothesis, however, is supported by studies using pHluorin expression, which has been shown to impair protein-protein interactions and clustering (Opazo et al., 2010). In the first case, the retrieval of synaptic vesicles as a unit would be facilitated, possibly making vesicle replenishment faster. In the second case, mechanisms of protein organization and clustering would be required either before vesicle retrieval, or by sorting through an endosome (Hoopmann et al., 2010), with expected delays in the recycling processes. The similar percentages found here for all proteins could suggest that their levels on the plasma membrane resemble the stoichiometry of an average synaptic vesicle, supporting their role as a readily retrievable pool of molecules that is eventually endocytosed as a reformed synaptic vesicle.
4.4.3 Differences in protein clustering between SNAP-25 and Syntaxin 1
In our last approach to hippocampal neurons we studied how the two membrane SNARE proteins, SNAP-25 and syntaxin 1, organize in intracellular organelles and on the plasma membrane. For this, mCLING surface labeling and immunostaining were combined in hippocampal neurons, in the same way as for the previous section. We found that, on average, the size of SNAP-25 clusters on the axolemma was similar to their counterparts from intracellular organelles (Figure 3.25B). In contrast, syntaxin 1 clusters found on the plasma membrane were larger and brighter than the syntaxin 1 staining from organelles (Figure 3.25C), suggesting that this protein forms relatively large molecular assemblies (Sieber et al., 2007; Bar-On et al., 2012) on the plasma membrane, but not on organelles.
Syntaxin 1 clustering depends on homophilic interactions between SNARE motifs, as well as on electrostatic interactions with membrane phosphoinositides (Sieber et al., 2006, 2007;
Khuong et al., 2013). Hence, it has been suggested that differences in phosphoinositide composition across organellar and plasma membranes could determine cluster size (Khuong et al., 2013), and therefore explain the results obtained here. SNAP-25 forms less dense and larger clusters than syntaxin 1, with no dependence on its SNARE motifs. Due to its palmitoyl anchoring to the membrane, SNAP-25 diffuses more easily than Syntaxin 1, which contains a C-terminal transmembrane domain, suggesting a less tight control of its cluster size (Halemani et al., 2010; Bar-On et al., 2012). Another important player determining cluster size on intracellular organelles could be cholesterol, as its effect has
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been shown before on plasma membranes (Chamberlain et al., 2001; Lang et al., 2001).
Overall, the results of this study are in line with the complex mechanisms by which syntaxin 1 forms molecular assemblies, compared to the apparently simple membrane localization of SNAP-25. Further experiments using mCLING labeling could help to study clustering factors for these and other proteins by mutation in their structures or changes in membrane lipid composition.