Abstract
Synaptic vesicle (SV) recycling enables ongoing transmitter release, even during prolonged activity. SV membrane and proteins are retrieved by ultrafast endocytosis and new SVs are formed from synaptic endosomes (large vesicles—LVs). Many proteins contribute to SV recycling, e.g., endophilin, synaptojanin, dynamin and clathrin, while the site of action of these proteins (at the plasma membrane (PM) vs. at the endosomal membrane) is only partially understood. Here, we investigated the roles of endophilin A (UNC-57), endophilin-related protein (ERP-1, homologous to human endophilin B1) and of clathrin, in SV recycling at the cholinergic neuromuscular junction (NMJ) of C. elegans. erp-1 mutants exhibited reduced transmission and a progressive reduction in optogenetically evoked muscle contraction, indicative of impaired SV recycling. This was confirmed by electrophysiology, where particularly endophilin A (UNC-57), but also endophilin B (ERP-1) mutants exhibited reduced transmission. By optogenetic and electrophysiological analysis, phenotypes in the unc-57; erp-1 double mutant are largely dominated by the unc-57 mutation, arguing for partially redundant functions of endophilins A and B, but also hinting at a back-up mechanism for neuronal endocytosis. By electron microscopy (EM), we observed that unc-57 and erp-1; unc-57 double mutants showed increased numbers of synaptic endosomes of large size, assigning a role for both proteins at the endosome, because endosomal disintegration into new SVs, but not formation of endosomes were hampered. Accordingly, only low amounts of SVs were present. Also erp-1 mutants show reduced SV numbers (but no increase in LVs), thus ERP-1 contributes to SV formation. We analyzed temperature-sensitive mutants of clathrin heavy chain (chc-1), as well as erp-1; chc-1 and unc-57; chc-1 double mutants. SV recycling phenotypes were obvious from optogenetic stimulation experiments. By EM, chc-1 mutants showed formation of numerous and large endosomes, arguing that clathrin, as shown for mammalian synapses, acts at the endosome in formation of new SVs. Without endophilins, clathrin formed endosomes at the PM, while endophilins A and B compensated for the loss of clathrin at the PM, under conditions of high SV turnover.
Highlights
Synaptic transmission is orchestrated by intricate protein machinery (Sudhof, 2013)
Endophilin B Is Encoded by Endophilin-Related Protein in C. elegans In C. elegans, endophilin A and B are encoded by unc-57 and erp-1, respectively
Could ERP-1 be required for formation of bulk endocytosis structures (Figure 1)? We had previously observed in optogenetic stimulation experiments that erp-1 mutants showed a slight reduction in photoevoked ePSCs (Wabnig et al, 2015)
Summary
Synaptic vesicles (SVs) are synthesized in the synaptic terminal, from components (membrane, proteins) delivered from the cell soma (Hannah et al, 1999) These components are thought to be assembled in the synaptic endosome and packaged into uniform vesicles by the clathrin-associated machinery, as shown for mammalian synapses following optogenetic stimulation (Watanabe et al, 2014); see Figure 1A for a model. SVs are filled with neurotransmitter, through the action of the vesicular ATPase and specific, proton-driven neurotransmitter transporters, like the vesicular acetylcholine (ACh) transporter They enter the so-called reserve pool (RP) of vesicles. The process of ultrafast endocytosis retrieves the SV membrane and its protein constituents This form of endocytosis ( called bulk endocytosis) occurs within 50–100 ms after fusion, and at physiological temperature is independent of clathrin (Kittelmann et al, 2013; Watanabe et al, 2013a,b, 2014; Soykan et al, 2017). The precise sequence of events and proteins involved in SV recycling is still under debate
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