Molecular Mechanisms of Cytoskeletal Regulators

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DESCRIPTION (provided by applicant): The microtubule cytoskeleton is a dynamic scaffold used to facilitate polarized intracellular transport, cell migration and formation of the mitotic spindle. Microtubules are polymers of a¿-tubulin heterodimers. The microtubule has inherent dynamics regulated by GTP hydrolysis in ¿-tubulin's exchangeable nucleotide site. A host of microtubule associated proteins regulate the polymer's dynamics both spatially and temporally. Defects in microtubule dynamics result in a wide spectrum of diseases including, but not limited to neurodegeneration, spastic paraplegia and aneuploidy. TOG domains are tubulin binding domains found in two conserved protein families that regulate microtubule dynamics, defined by members XMAP215 and CLASP. Across both families, TOG domains are found arrayed; however XMAP215 and CLASP differentially regulate microtubule dynamics, promoting polymerization and pause respectively. The mechanism TOG domains use to bind tubulin and the role arrayed TOG domains play to modulate microtubule dynamics remains to be determined. This proposal develops the hypothesis that arrayed TOG domains provide multiple tubulin/microtubule binding sites and this multivalent architecture coupled with TOG class-specific determinants is central to the mechanism by which XMAP215 and CLASP differentially regulate microtubule dynamics. Three series of experiments examine the structure and function of TOG domains to determine a multi- resolution model for arrayed TOG mechanism. The first objective is to define, at atomic resolution, the structure and unique features of TOG domain classes across the XMAP215 and CLASP protein families using X-ray crystallography. The second objective is to ascertain the tubulin and microtubule binding capacity of individual and arrayed TOG domains and map tubulin binding determinants. This examination will use in vitro tubulin and microtubule binding assays as well as a F"rster resonance energy transfer assay to generate a model of the TOG-tubulin complex. The third objective is to determine the mechanism arrayed TOG domains use to modulate microtubule dynamics in vivo. This study will use live cell fluorescence imaging to monitor microtubule dynamics when the wild-type TOG protein has been depleted and replaced with a fluorescently- labeled truncated, mutant or chimeric construct. The long term objectives of this investigation are to determine at the atomic level, the mechanism arrayed TOG domains employ to modulate microtubule dynamics individually and in concert with other microtubule associated proteins. A fundamental understanding of TOG domain mechanism and how this is utilized in different protein families will enhance our understanding of microtubule dynamics and the role it plays in human health and disease including various neuropathies, ciliopathies, aneuploidy and cancer. PUBLIC HEALTH RELEVANCE: The microtubule cytoskeleton is a dynamic, polarized cellular scaffold that facilitates intracellular transport, ciliogenesis, cell migration and mitosis; all processes that rely on the regulated dynamic instability of the microtubule polymer. Defects in microtubule dynamics manifest in a range of disorders that effect human health including spastic paraplegia, lissencephaly, ciliopathies and aneuploidy; at the same time, microtubule dynamics is a key target for chemotherapeutics as inhibition of microtubule dynamics, and thus mitosis, is a means to prevent the rapid cell division associated with cancer. The proposed research aims to examine the molecular mechanism of TOG domain-containing proteins and the role they play in modulating microtubule dynamics; the research will impact public health by establishing a fundamental mechanistic framework under which healthy microtubule dynamics operates, from which aberrant manifestations can be properly examined and therapeutic strategies developed.