Intersectin and endophilin condensates prime synaptic vesicles for release site replenishment.

Following synaptic vesicle fusion, vacated release sites are replenished immediately by new vesicles for subsequent neurotransmission. These replacement vesicles are assumed to be located near release sites and used by chance. Here we find in mouse hippocampal excitatory synapses that replacement vesicles are clustered near the active zone where release sites reside by intersectin-1. Specifically, intersectin-1 forms dynamic molecular condensates with endophilin A1 and sequesters vesicles around this region. In the absence of intersectin-1, fewer vesicles cluster within 20 nm of the plasma membrane, and consequently vacated sites cannot be replenished rapidly, leading to synaptic depression. Mutations in intersectin-1 that disrupt endophilin A1 binding result in similar phenotypes. In the absence of endophilin A1, intersectin-1 is mislocalized, and this replacement pool of vesicles cannot be accessed, suggesting that endophilin A1 is needed to mobilize these vesicles. Thus, our work describes the replacement zone within a synapse, where replacement vesicles are stored for replenishment of the release site.