Vesicle Tethering on the Surface of Phase-Separated Active Zone Condensates

Tethering of synaptic vesicles (SVs) to the active zone determines synaptic strength, although the molecular basis governing SV tethering is elusive. Here, we discover that small unilamellar vesicles (SUVs) and SVs from rat brains coat on the surface of condensed liquid droplets formed by active zone proteins RIM, RIM-BP, and ELKS via phase separation. Remarkably, SUV-coated RIM/RIM-BP condensates are encapsulated by synapsin/SUV condensates, forming two distinct SUV pools reminiscent of the reserve and tethered SV pools that exist in presynaptic boutons. The SUV-coated RIM/RIM-BP condensates can further cluster Ca2+ channels anchored on membranes. Thus, we reconstitute a presynaptic bouton-like structure mimicking the SV-tethered active zone with its one side attached to the presynaptic membrane and the other side connected to the synapsin-clustered SV condensates. The distinct interaction modes between membraneless protein condensates and membrane-based organelles revealed here have general implications in cellular processes, including vesicular formation and trafficking, organelle biogenesis, and autophagy. [Display omitted] •Synaptic vesicles coat active zone condensate but coacervate with synapsin condensate•SV/synapsin co-condensates encapsulate SV-coated active zone condensates•A presynaptic bouton-like assembly with reserve and tethered SV pools is reconstituted•Membraneless condensates and membrane organelles can interact with distinct modes Each presynaptic bouton contains reserve pool synaptic vesicles (SVs) and a few active zone-tethered SVs. Wu et al. show that SVs coat the surface of protein condensates formed by active zone proteins, such as RIM, RIM-BP, and ELKS, but coacervate with synapsin, forming a multi-phase organization with the docked pool SVs surrounded by the bulk reserve pool SVs as observed in presynaptic boutons.