Allosteric Role of Dynein Light Chains in Dynein Assembly and Regulation

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Cytoplasmic dynein is a microtubule-based molecular motor that uses the energy derived from ATP hydrolysis to move and transport cellular cargo towards the minus ends of microtubules. This transport is essential in several aspects of cell behavior including cell migration and division, chromosome segregation, and vesicle trafficking. The dynein heavy chain subunits bind to microtubules and contain the ATP sites that provide the motive force, while the light and intermediate chains are thought to bind to cargo and regulate the activity of the motor. Recent data from the Barbar lab have suggested that the role of light chain LC8 is to facilitate dimerization of the natively disordered N-terminal domain of dynein intermediate chain, rather than to primarily function as a cargo adaptor, as commonly thought. Light chain Tctex-1, which is a structural homolog of LC8 and binds the intermediate chain at a site contiguous to the LC8 binding site, is likely to have a similar role as LC8. The research project will test the hypothesis that the tandem roles of light chains LC8 and Tctex-1 are to promote dynein assembly and the interaction with the cargo adaptor dynactin, and not to act as cargo adaptors themselves. Using a combination of biophysical approaches including nuclear magnetic resonance spectroscopy (NMR), isothermal titration calorimetry and X-ray crystallography, the Barbar lab will elucidate the mechanisms of three important outcomes of the interactions of both light chains to dynein intermediate chain: 1) how they contribute to formation of a tight dynein assembly, 2) how they promote dimerization of the intermediate chain, and 3) how they may promote a tighter interaction of the intermediate chain with p150Glued subunit of dynactin. The essential roles of these light chains will be tested in vivo in the lab of Professor Tom Hays, a collaborator on this project. The studies should provide novel insights into the structural biology and thermodynamics of dynein motor function, and will complement the cell biological approaches that predominate in the field of intracellular transport. Since these studies provide the first structural insights into the role of the disorder-to-order transition in dynein assembly and its association with dynactin, they allow the opportunity to develop a tractable system to probe the role of intrinsic disorder in the assembly of large macromolecular complexes. Broader Impacts: The PI has a strong record of research contributions with undergraduate students, and of mentoring minorities and students with disabilities. The PI is taking a leading role on her campus to introduce molecular biophysics to the research community by providing access to instrumentation and training workshops. Furthermore, the PI will take an active role to promote basic science among high school students by providing opportunities for research lab experience, and by developing instructional material and a hands-on module in the Saturday Academy program. The latter is a highly successful program at Oregon State University for outreach for high school students in which biochemistry and biophysics have not yet been represented.