Visualization of RIM-BP2's crane-like function in neuronal vesicle transport using FRET

"The last mile" of neuronal vesicles, from being tethered by the active zone filaments to docking at the presynaptic membrane, remains unclear, which limits the deep understanding of synaptic transmission and related physiological changes. Here, we develop two molecular biosensors (BKTS and RKTS) based on fluorescence resonance energy transfer technology according to the structure of RIM-BP2. By detecting the spatial distance between the two ends of the RIM-BP2 and the presynaptic membrane separately, the spatial posture changes in RIM-BP2 are reflected to explore how vesicles are transported to the presynaptic membrane for fusion. In the process of vesicle release, RIM-BP2 in primary cortical neurons and SH-SY5Y cells rotates like a "crane" with amino terminal deviating from the presynaptic membrane while the carboxyl terminal becomes closer. Furthermore, disturbing the microfilament or enhancing cell membrane fluidity inhibits the rotation of RIM-BP2. Through mutating RIM-BP2, we find that actin filaments provide mechanical stress through RIM-BP2 amino terminal, thereby regulating vesicle transport and release. Our work identifies a purely mechanical pathway of vesicle transport, in which microfilaments power the RIM-BP2 to drag vesicles to the presynaptic membrane as a "crane" for further release.