Molecular mechanisms of kinesins that control microtubule and actin polymerization dynamics

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Microtubules and actin filaments form dynamic networks to organize the nucleus and cytoplasm, and to sense and influence cell shape. For example, microtubules grow and shrink to establish the correct size and morphology of the mitotic spindle apparatus for accurate chromosome segregation. Actin filaments are rapidly assembled and bundled together within higher-order structures like the contractile ring to mediate cell division. In both cases, precise temporal and spatial control of their polymeric state is required, but the large differences in their lattice structure and mechanical properties necessitate differential modes of regulation. Our research program is providing a mechanistic understanding of the molecular actions of several different microtubule-depolymerizing kinesins, and a unique kinesin that promotes actin polymerization. We showed that monomers of kinesin-13 family enzymes bind and promote outward bending of tandem tubulin dimers in order to weaken the microtubule lattice and catalyze rapid tubulin detachment from microtubule ends. We also showed that the tubulin-bending force of kinesin-13 does not require energy from ATP hydrolysis. Instead, ATP is hydrolyzed after the kinesin-tubulin complex detaches. Using in vitro biochemical studies, Total Internal Reflection Fluorescence (TIRF) microscopy, X-ray crystallography and small-angle X-ray scattering (SAXS) techniques, we will now determine the biochemical and structural basis for dissociation of the detached kinesin-13-tubulin complex, so that kinesin-13 can catalyze removal of additional tubulins. These same methods will be applied to understand how members of the kinesin-8 and kinesin-14 families employ ATP to catalyze tubulin dissociation from microtubule ends. We will also trap and elucidate the structures of intermediate states of kinesin-8- and kinesin-14-catalyzed microtubule depolymerization by using the molecular strategies that helped us trap the kinesin-13-tubulin complex. To extend this research theme into a new realm for the kinesin field, we will investigate the mechanism by which a unique module in a kinesin-3 motor protein stimulates actin polymerization and bundling. We will use protein truncation studies and chemical biology methods to identify the actin binding interfaces of this kinesin, and then apply X-ray crystallography and electron microscopy (cryo-EM) to determine high-resolution structures of kinesin-3 complexes bound to actin subunits and actin polymers, respectively. These studies will provide a molecular explanation for this unexpected activity of a kinesin, and could guide other hybrid kinesin designs. These studies will ultimately lead to a better mechanistic understanding of how the dimensions of microtubule- and actin-based cellular superstructures are dynamically regulated. Personnel trained by this research program will acquire expertise in biochemical and structural biology research methods that are sought-after by academia and industry.