Surface SV2A-Syt1 nanoclusters act as a sequestration hub that limits dynamin-1 recruitment and targeting to recycling synaptic vesicles
Following exocytosis, the recapture of plasma membrane-stranded vesicular proteins into recycling synaptic vesicles (SVs) is essential for sustaining neurotransmission. Surface clustering of vesicular proteins has been postulated as a pre-assembly mechanism for endocytosis - ensuring high-fidelity retrieval. Here, we used single-molecule imaging to examine the nanoclustering of synaptotagmin-1 (Syt1) and synaptic vesicle protein 2A (SV2A) in hippocampal neurons. Syt1 forms surface nanoclusters through interaction of its C2B domain with SV2A, that are sensitive to mutations in this domain (Syt1K326A/K328A) and knocking down SV2A. SV2A co-cluster with Syt1 and blocking SV2As cognate interaction with Syt1 (SV2AT84A) also decreased SV2A clustering. Surprisingly, impairing SV2A-Syt1 nanoclustering enhanced plasma membrane recruitment of key endocytic protein dynamin-1, leading to accelerated Syt1 endocytosis, altered intracellular sorting and decreased trafficking of Syt1 to Rab5-positive endocytic compartments. SV2A-Syt1 surface nanoclusters therefore negatively regulate the rate of their own re-entry into recycling SVs by controlling the recruitment of the endocytic machinery.