Control of SNARE-driven vesicle fusion by synapsin condensates on freestanding membranes

Single-molecule observations of membrane protein dynamics have long been challenging due to the limitations of traditional model membrane systems. Here, we developed a simple method to prepare freestanding lipid bilayers (FLBs) for studying protein diffusion and clustering. An array of large-area, protein-embedded membrane patches were stably formed on an electron microscopy grid for high-resolution fluorescence imaging. Applying this technique to SNARE-driven vesicle fusion, the core mechanism for synaptic vesicle release, we dissected vesicle docking and membrane fusion activities through diffusion-based analysis at the single-particle level, revealing distinct intermediate species on membranes. Upon adding complexin and synapsin, well-established regulators of the synaptic vesicle cycle, we observed their unique effects on the membrane-bound populations. Notably, synapsin condensates not only clustered VAMP2-containing vesicles but also engaged closely with the target membrane, releasing the vesicles for immediate fusion upon dispersal. This suggests a more direct role for synapsin in vesicle release than previously assumed. The FLB platform enables the study of diverse protein–membrane activities, with future applications in membrane deformation and tension.