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

Area of Science:

• Neuroscience
• Cell Biology
• Biophysics

Background:

• The final steps of neuronal vesicle transport to the presynaptic membrane are not fully understood.
• This knowledge gap limits understanding of synaptic transmission and associated physiological processes.

Purpose of the Study:

• To elucidate the mechanism of vesicle transport to the presynaptic membrane.
• To investigate the role of RIM-Binding Protein 2 (RIM-BP2) in vesicle docking and fusion.

Main Methods:

• Development of two Förster Resonance Energy Transfer (FRET)-based molecular biosensors (BKTS and RKTS) targeting RIM-BP2.
• Detection of spatial distance changes between RIM-BP2 ends and the presynaptic membrane in primary cortical neurons and SH-SY5Y cells.
• Investigation of the effects of microfilament disruption and altered cell membrane fluidity on RIM-BP2 dynamics.
• Functional analysis through RIM-BP2 mutation studies.

Main Results:

• RIM-BP2 exhibits a "crane-like" rotation during vesicle release, with its amino terminus moving away from and carboxyl terminus towards the presynaptic membrane.
• Inhibition of microfilaments or increased membrane fluidity disrupts RIM-BP2 rotation.
• Actin filaments exert mechanical stress through the RIM-BP2 amino terminus, regulating vesicle transport and release.

Conclusions:

• A novel, purely mechanical pathway for vesicle transport is identified.
• Microfilaments, via RIM-BP2 acting as a "crane," actively drive vesicle transport to the presynaptic membrane for release.
• This mechanism provides new insights into synaptic transmission regulation.