Synaptic vesicle proteins and ATG9A self-organize in distinct vesicle phases within synapsin condensates

ATG9 is the only transmembrane protein of the core autophagy machinery known to be present at presynapses. Here, the authors show that both synaptophysin and ATG9A vesicles assemble into condensates with synapsin but remain segregated from each other. Ectopic expression in fibroblasts of synapsin 1 and synaptophysin is sufficient to generate condensates of vesicles highly reminiscent of synaptic vesicle (SV) clusters and with liquid-like properties. Here we show that unlike synaptophysin, other major integral SV membrane proteins fail to form condensates with synapsin, but co-assemble into the clusters formed by synaptophysin and synapsin in this ectopic expression system. Another vesicle membrane protein, ATG9A, undergoes activity-dependent exo-endocytosis at synapses, raising questions about the relation of ATG9A traffic to the traffic of SVs. We find that both in fibroblasts and in nerve terminals ATG9A does not co-assemble into synaptophysin-positive vesicle condensates but localizes on a distinct class of vesicles that also assembles with synapsin but into a distinct phase. Our findings suggest that ATG9A undergoes differential sorting relative to SV proteins and also point to a dual role of synapsin in controlling clustering at synapses of SVs and ATG9A vesicles.

Automated summary

ATG9 is the sole transmembrane protein found at presynapses in the autophagy machinery. In this study, the authors demonstrate that synaptophysin and ATG9A vesicles form condensates with synapsin but remain separate from each other. Unlike synaptophysin, other integral SV membrane proteins do not form condensates with synapsin but assemble into the clusters formed by the synaptophysin and synapsin proteins. ATG9A localizes to a distinct class of vesicles that also interacts with synapsin but forms a different phase. These findings indicate differential sorting of ATG9A compared to SV proteins and highlight a dual role of synapsin in controlling clustering of SVs and ATG9A vesicles at synapses.