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.
© 2023. The Author(s).