Assembly of synaptic active zones requires phase separation of scaffold molecules

The components of active zones at neuronal synapses are well known, but the processes underlying the assembly of these structures are less so; here, a role for liquid-liquid phase separation of scaffold proteins is identified. The formation of synapses during neuronal development is essential for establishing neural circuits and a nervous system(1). Every presynapse builds a core 'active zone' structure, where ion channels cluster and synaptic vesicles release their neurotransmitters(2). Although the composition of active zones is well characterized(2,3), it is unclear how active-zone proteins assemble together and recruit the machinery required for vesicle release during development. Here we find that the core active-zone scaffold proteins SYD-2 (also known as liprin-alpha) and ELKS-1 undergo phase separation during an early stage of synapse development, and later mature into a solid structure. We directly test the in vivo function of phase separation by using mutant SYD-2 and ELKS-1 proteins that specifically lack this activity. These mutant proteins remain enriched at synapses in Caenorhabditis elegans, but show defects in active-zone assembly and synapse function. The defects are rescued by introducing a phase-separation motif from an unrelated protein. In vitro, we reconstitute the SYD-2 and ELKS-1 liquid-phase scaffold, and find that it is competent to bind and incorporate downstream active-zone components. We find that the fluidity of SYD-2 and ELKS-1 condensates is essential for efficient mixing and incorporation of active-zone components. These data reveal that a developmental liquid phase of scaffold molecules is essential for the assembly of the synaptic active zone, before maturation into a stable final structure.