Single-molecule bouncing at a liquid interface governs presynaptic enrichment

Many cellular processes rely on accumulating highly diffusive cytoplasmic proteins to a specific location within the cell. This spatial enrichment is generally thought to rely on locally immobilizing proteins or slowing diffusion through binding and molecular crowding. Paradoxically, cytoplasmic proteins that are enriched within nerve terminals remain highly diffusive ¹ , raising questions about the mechanisms that govern presynaptic enrichment. Here, using in vivo single-molecule microscopy, we tracked the diffusion of a model cytoplasmic protein, α-Synuclein (α-Syn), and synaptic vesicles (SVs), in motor neuron presynapses of live Drosophila larvae. We demonstrate that α-Syn undergoes rapid and frequent ‘bouncing’ events at a presynaptic interface, locally impeding escape from the presynapse. We measured escape probabilities at the interface, and show that impeding escape, without dramatically slowing diffusion, is sufficient to govern presynaptic enrichment. The interface is liquid-like and coincides with the boundary of the SV cluster, which is a necessary scaffold for the presynaptic enrichment of α-Syn. Our results provide direct evidence of interfacial reflectivity at the interface of liquid-like compartments, and that this phenomenon can govern the subcellular enrichment of proteins and small organelles at the level of individual particles.