Neuronal communication relies on precisely maintained synaptic vesicle (SV) clusters, which assemble via liquid-liquid phase separation. This process requires synapsins, the major synaptic phosphoproteins, which are known to bind actin. Reorganization of SVs, synapsins, and actin is a hallmark of synaptic activity, but the molecular details of the interactions between these components remain unclear. Here, we combine in vitro reconstitution with expansion microscopy, super-resolution imaging, and cryo-electron tomography to dissect the roles of SV-synapsin-1 condensates in the organization of the presynaptic actin cytoskeleton. Our results indicate that condensation of synapsin-1 initiates actin polymerization. This process enables SV-synapsin-actin assemblies to facilitate the mesoscale organization of SV clusters along axons, which is similar to the native presynaptic organization observed at both lamprey and mammalian synapses. Understanding the relationship between the actin network and synapsin-synaptic vesicle condensates can help elucidate how coordinated neurotransmission along the axon enables circuit function and behavior.
