Henkin, Gilead Phillip; (2022) Self-organization of microtubule and mixed-motor networks in vitro. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The core activity of cell division is the transmission of a full set of genes from a parent cell to its progeny. The error-free accomplishment of this process is tightly controlled and relies on robust assembly of a dynamic microtubule structure, the spindle apparatus. Crosslinking motors with different structures and directionalities determine the architecture and dynamics of the spindle, consisting of simultaneous contractile and extensile phases. Generally, it is not understood why some motor-filament systems are contractile and some extensile, and moreover, it is not clear how both types of network simultaneously exist in the spindle. Recent work has illuminated underlying principles of spindle assembly by rebuilding them from the bottom up. Accordingly, we explore the organization of microtubule networks with contractile and/or extensile properties by biochemical reconstitution of crosslinking motor activities. We examine networks of growing microtubules organized by HSET and KIF11, mitotic kinesins with different structures and different directionalities, which in cells promote pole-formation and poleward flux, respectively. We find that while they can each contract microtubules nucleated from low concentrations of tubulin, their activities diverge in high concentrations of tubulin – HSET tends to strongly contract networks, whereas KIF11 can drive turbulent extensile bundles. In combination, we find that although HSET drives network contraction when dominant, it also assists the opposing KIF11 to generate extensile networks, suggestive of multiple roles for HSET in spindles. We do not observe coexistence of simultaneous extensile and contractile phases. The emergent phenomena can be explained by the different crosslinking structures of the motors, and not by the simple rules that determine organization by single motor species. Lastly, seeking mechanisms that recapitulate stronger minus-end motors and poleward flux that promote bipolarity of biological spindles, we study the behavior of oligomeric HSET constructs. While these oligomeric HSETs did not successfully driving pole-formation in the presence of KIF11, we demonstrate that they can target the depolymerase kif2A to minus end poles and subsequently promote progressive contractile activity.

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