Mechanisms Underlying the Nucleation and Kinesin-Driven Assembly of Microtubule Rings

Abstract

Active self-assembly processes rely on the conversion of chemical energy into mechanical work to overcome the limitations associated with diffusion-driven, passive self-assembly. For example, the transport of biotinylated microtubule filaments by surface-adsorbed kinesin motor proteins results in the actively assembly of bundles, rings, and spools in the presence of streptavidin. Observing the nucleation and assembly of these structures has been hindered by issues including controlled buffer exchange and photo-oxidative damage from fluorescent excitation. To address these issues, we developed a custom PDMS microfluidic device to characterize the earliest events involved in active assembly of microtubule rings and spools. Three distinct nucleation mechanisms were observed: pinning, collisions, and induced curvature. Pinning occurred when the leading tip of a microtubule encounters and stalls at an inactive motor. Experiments and numerical energy minimizations suggest that the diameter of ring/spools formed by pinning is strongly dependent on the surface density of kinesin motors. Collision-induced nucleation occurred when three (or more) microtubules simultaneous collide, forming a closed triangle that further evolved into a ring/spool. Collision accounted for the majority of nucleation events when photodamage was mitigated with our microfluidic device. The third nucleation mechanism, induced-curvature, was only observed at long-time scale, and attributed to a frozen-in curvature due to the binding of streptavidin-coated quantum dots to microtubules. We further showed that nucleation mechanisms affected both the diameter and rotation direction of the assembled rings/spools. Collectively, our studies provide fundamental insights on active assembly processes, which may be applied for regulating the morphology and functional properties of the resulting structures.Sandia National Laboratories is a multi-mission laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.