Structure and Function of Cytoplasmic Dynein

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DESCRIPTION (provided by applicant): Molecular motor proteins function in a multitude of intracellular transport processes that include the organization and transport of organelles, chromosome segregation, axonal transport, and signaling. Motor dependent processes are critical for the growth, proliferation, and differentiation of cells and tissues. How motor function is regulated during development, and the relationship of motor dysfunction to numerous medical problems including neurodegenerative disease, congenital chromosomal syndromes, and birth defects is a current focus of research activity. Our work is focused on the microtubule motor cytoplasmic dynein, and how this single motor isoform accomplishes multiple tasks. How is dynein targeted to specific cargoes and/or cellular locations? Our aims will address three non-exclusive mechanisms that potentially contribute to dynein targeting. (1) Cytoplasmic dynein contains multiple subunits. The individual subunits or subunit domains could specify where, and to what cargo, dynein is attached. To test this hypothesis we will ask whether domains within the light intermediate and the intermediate chain polypeptides confer specific functions. Mutagenesis and molecular genetic approaches will be used to disrupt domain function and the mutant phenotypes will be characterized. (2) The posttranslational modification of dynein subunits might control whether subunits are competent to bind a cargo with high affinity. A collaboration with Dr. John Yates (Scripps Research Institute) will define the sites of phosphorylation on subunits within the dynein complex using a mass spectrometry approach. Subsequently, the phosphorylation sites identified will be mutated to mimic the phosphorylated or unphosphorylated state of the respective subunit. The phenotypes produced by transgenes that express the mutant subunits will be analyzed to reveal the functional significance of dynein phosphoregulation. (3) Specific binding partners or "effector" proteins might mediate the targeting of the dynein motor to specific cargoes or locations. We will pursue the functional analysis of candidate interacting proteins identified in the previous period and will continue with secondary tests on other interacting loci.