The timing and kinetics of neurotransmitter release depend on the relative positioning of clustered Ca²⁺ channels in active zones (AZs) to docked synaptic vesicles (SVs) on presynaptic plasma membranes. However, how AZ might form is not known. Here we show that RIM and RIM-BP, via specific multivalent interactions, form dynamic and condensed assemblies through liquid-liquid phase separation (LLPS). Voltage-gated Ca²⁺ channels (VGCCs), via C-terminal tail-mediated direct binding to both RIM and RIM-BP, can be enriched to the RIM/RIM-BP condensates. We further show that RIM and RIM-BP together with VGCCs form dense clusters on the supported lipid membrane bilayers via phase separation. Therefore, RIMs and RIM-BPs are plausible organizers of the AZ, and the formation of RIM/RIM-BP condensa...[ Read more ]

The timing and kinetics of neurotransmitter release depend on the relative positioning of clustered Ca²⁺ channels in active zones (AZs) to docked synaptic vesicles (SVs) on presynaptic plasma membranes. However, how AZ might form is not known. Here we show that RIM and RIM-BP, via specific multivalent interactions, form dynamic and condensed assemblies through liquid-liquid phase separation (LLPS). Voltage-gated Ca²⁺ channels (VGCCs), via C-terminal tail-mediated direct binding to both RIM and RIM-BP, can be enriched to the RIM/RIM-BP condensates. We further show that RIM and RIM-BP together with VGCCs form dense clusters on the supported lipid membrane bilayers via phase separation. Therefore, RIMs and RIM-BPs are plausible organizers of the AZ, and the formation of RIM/RIM-BP condensates may cluster VGCCs into nano- or micro-domains and position the clustered Ca²⁺ channels with Ca²⁺ sensors on docked vesicles for efficient and precise synaptic transmissions.

Tethering of SVs to the AZ determines synaptic strengths, although the molecular basis governing SV tethering is rather elusive. In this study, we discover that small unilamellar vesicles (SUVs) and SVs purified from rat brains coat on the surface of liquid condensates formed by AZ proteins RIM, RIM-BP, and ELKS. Remarkably, the SUV-coated RIM/RIM-BP condensates are encapsulated by the synapsin/SUV condensates, forming two distinct SUV pools reminiscent of the reserve and tethered SV pools existing in presynaptic boutons. The SUV-coated RIM/RIM-BP condensates can further cluster Ca²⁺ channels anchored on membranes. Thus, we have reconstituted a presynaptic bouton-like structure that mimics SV-tethered AZ 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 might have general implications in other cellular processes including vesicular trafficking, organelle biogenesis, and autophagy.