Kinesin vesicular transport through 2D and 3D microtubule intersections.

Abstract

Exocytic vesicular cargoes, driven by kinesin motor ensembles, reach their destination by navigating the cell's 3D multi-intersectional microtubule (MT) cytoskeleton. Using reductionist approaches, we investigate how kinesin-1 motor ensembles maneuver cargo through MT-MT intersections in vitro. 350-nm fluid-like liposomes, a biologically relevant cargo that allows surface diffusion of motors, were incubated with truncated kinesin-1 (K543) at varying stoichiometries. Kinesin-cargo complexes (5-20 motors per liposome) have track-limited run lengths, implying multiple simultaneously engaged motors. When kinesin-liposome complexes encounter an obstructing, intersecting MT in 2D, liposomes with fewer motors (i.e., 5) prefer going straight (56%) to turning (33%), yet liposomes with more motors (i.e., 10-20) go straight (48%) and turn (47%) equally. Interestingly, liposome pause frequency and duration at intersections scale with motor number, indicating motor teams engaged with each of the intersecting MTs enter a tug-of-war. With less motors the tug-of-war outcome favors going straight because less free motors can engage the intersecting MT. Whereas with more motors, the tug-of-war is between teams with equivalent motor number, leading to no directional preference. To better model the cells’ MT cytoskeleton, 3D intersections were created by suspending MTs between 0.7 and 3 micron pedestal beads. Super-resolution fluorescence microscopy allowed the spatial relationship between liposomes and intersecting MTs to be precisely defined. While approaching the intersection, liposomes can move in a helical trajectory that tracks the in vitro MT protofilament supertwist (5 microns). Upon reaching the intersection, liposomes pause and deform the intersecting MTs as competing motor teams undergo a tug-of-war. After the tug-of-war resolves, liposomes transported by 10 kinesins prefer going straight (58%) to turning (29%), which differs from the 2D intersection outcomes. In silico modeling of both 2D and 3D intersection outcomes will provide insight into the underlying biophysical mechanism(s) governing kinesin-cargo transport in vivo.