Mechanistic Analysis of Microtubule Based Motors

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The overarching goal is to gain critical insights into the fundamentals of kinesin motor structure and function and to extrapolate this understanding to the inner workings of the cell. Kinesin superfamily members share a common catalytic domain yet participate in a wide range of cellular functions including intracellular transport, mitosis and meiosis, regulation of microtubule dynamics for remodeling of the cytoskeleton, and generation of cell polarity. It is now recognized that sequence differences modify the mechanochemistry, microtubule interactions, and the response to force, each of which is critical for the specific physiological function. The goal of this proposal is to establish the mechanistic and structural features shared by kinesin- 14 Kar3Cik1, Kar3Vik1, and Ned and at the same time to reveal unique features that result in functional specificity. Members of the kinesin-14 subfamily are the only kinesins known to promote microtubule minus- end-directed force generation and to use an ATP-promoted powerstroke mechanism. In contrast, members of kinesin-1, 2, 5, and 7 subfamilies generate microtubule plus-end-directed force, and these molecular motors are processive. Conventional kinesin-1, kinesin-5 Eg5, and kinesin-7 CENP-E generate dimeric motors from the same gene product, yet the functional catalytic dimer for kinesin-2 KIF3AB and KIF3AC arises from two different gene products. Therefore, what is the selective advantage of heterodimeric catalytic enzymes for in vivo function, how is head-head communication established to modulate interactions with the microtubule lattice and/or microtubule end, and what mechanisms regulate the interplay of processivity and response to force? The research proposed evaluates heterodimeric Kar3Cik1 and Kar3Vik1 in comparison to homodimeric Ned, and heterodimeric Kinesin-2 KIFAB and KIFAC in comparison to other processive homodimeric kinesins. Experimental approaches include presteady-state kinetics methodologies, single molecule and ensemble fluorescence microscopy, optical trapping to determine the force-dependent motility properties, X-ray crystallography, cryo-electron microscopy and tomography, and computational modeling. This comprehensive analysis will provide new insights to understand the mechanochemistry that underlies structure-function relationships required for cellular organization and physiological function. RELEVANCE (See instructions): The overall goal is to understand the mechanochemistry of kinesin motors that underlies their ability to promote intracellular transport, generation of cell polarity, and remodeling of the microtubule cytoskeleton for cell division, cell differentiation, and morphogenesis during human development. Defects in kinesins have been linked to diverse pathologies including cancer, ciliopathies, neuropathies, and birth defects.