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 including organelle transport, chromosome segregation, axonal transport, and signaling pathways. Motor dependent processes are critical for growth, proliferation, and the differentiation of cells and tissues. How motor function is regulated in a developmental context, and the relationship of motor dysfunction to numerous medical problems, including neurodegenerative disease and cancer, is a current focus of research activity. Our work is focused on the microtubule motor cytoplasmic dynein, and the important and unanswered questions regarding how this single motor isoform accomplishes multiple tasks. Our aims will address three mechanisms that potentially regulate dynein targeting and/or activity. (1) First, cytoplasmic dynein contains multiple subunits. The individual subunits or subunit domains could specify where, and to what, dynein is attached. Aim 1 includes biochemical and genetic experiments to address how the dynein light chain and light intermediate chain influence dynein functions. (2) Second, the posttranslational modification of dynein subunits might control dynein subunit activities or binding affinities. We are defining the in vivo sites of phosphorylation on dynein subunits and will study the significance of the target sites. The target sites on the LIC subunit will be mutated to mimic the phosphorylated or unphosphorylated state, and the phenotypes produced by transgenes that express the mutant subunits will be analyzed. (3) In a third mechanism, specific binding partners or effector proteins might mediate the targeting of the dynein motor to specific cargoes or locations. Previous studies have provided evidence that spectrin mediates the attachment of dynactin and dynein to membranes. Our collaborator, Laura Ranum (UMN), recently discovered that Spinocerebellar ataxia type 5 (SCA5), an autosomal dominant neurodegenerative disease, is caused by mutations in 2-III spectrin (SPTBN2). In Drosophila, we have shown that mutant, but not wild type, human ss-III spectrin expressed in neurons causes neurodegeneration and a rough eye phenotype. One goal is to conduct a genome-wide screen to recover modifier loci that identify novel genes in the pathogenic process. A second priority is to determine if mutations in human spectrins, and the corresponding mutations in fly ss spectrin, disrupt axonal vesicular transport in Drosophila. These studies will help to elucidate the molecular underpinnings of SCA5 pathology and neurodegenerative disease. PUBLIC HEALTH RELEVANCE: We are studying the molecular basis of intracellular transport. Our work focuses on the microtubule motor cytoplasmic dynein and the mechanisms by which this motor can accomplish diverse transport tasks. Perturbations in intracellular transport are implicated in human diseases, including neurodegenerative disease, cancer, and birth defects.