In vitro Reconstitution of Kinesin-Driven Vesicle Transport

Abstract

Intracellular vesicle cargos are transported along microtubules by kinesin and dynein motors, towards the cell periphery and cell center, respectively. Individual motors generally have run lengths of several μm, whereas organelles need to travel tens of μm to reach their destinations. Therefore, vesicle cargos usually have multiple copies of motors such that when one or more motors detach from the microtubule, other motors can continue transport. We previously used a 2D lipid bilayer system to measure the microtubule binding kinetics of membrane-bound kinesin-1 motors, from which we predicted long-range transport of 100 nm diameter vesicles required 35 kinesin-1 motors. Our predicted motor number was higher than what was measured on purified neuronal vesicles, thus we aimed to reconstitute kinesin-driven vesicle transport and characterize vesicle motility behaviors in the present study. GFP-labeled kinesin-1 was linked to POPC liposomes through a DNA linker. As the mixing ratio of kinesin-1 and vesicle increased, the vesicle landing density in AMPPNP increased linearly and then reached a plateau. The average vesicle travel distance in ATP increased continuously with the mixing ratio, indicating that multi-motor transport was achieved. Vesicle transport velocity was insensitive to motor number. This system enables reconstitution of vesicle motility with controlled motor densities and can be extended to study bidirectional transport by kinesins and dyneins.