Cytoplasmic dynein is a microtubule-based motor which functions in the cell both to drive vesicle motility and to organize the mitotic spindle. Genetic and biochemical studies have suggested that dynactin is a required co-factor for each of these functions. Dynactin is a large complex with a distinctive structure and an unknown cellular mechanism. The studies proposed here have three goals: to define the dynactin complex, to determine its mechanism of function, and to investigate its role in microtubule-based vesicular transport.
In Specific Aim number 1, the molecular characterization of dynactin will be completed, and the biochemical interactions of the subunits of dynactin will be examined.
In Specific Aim number 2, we will examine the effects of dynactin on dynein motor function. Dynactin may serve as a vesicle-bound receptor for dynein, or it may be involved more actively in constraining the parameters of motility, in the adaption of a nonprocessive motor to an intracellular transport function. This hypothesis can be tested by examining the kinetics of the pathway for ATP hydrolysis by cytoplasmic dynein in the absence and in the presence of dynactin. The relative processivity of the cytoplasmic dynein motor will also be examined in a single motor assay.
In Specific Aim number 3, the hypothesis that spectrin mediates the association of cytoplasmic dynein and dynactin with vesicles will be examined using binding studies and peptide competition assays. Evidence from genetic studies in yeast, fungi, Drosophila, and mouse point to the critical nature of the cytoplasmic dynein/dynactin motor complex. However, there is much to learn about the interactions of cytoplasmic dynein and dynactin required to produce vesicular motility along microtubules, and the regulation of this process within the cell. Research over the last several years has led to an increasing awareness of the critical role of the cytoskeleton and its associated proteins in disease. Mutations in both motor molecules and cytoskeletal proteins have been associated with serious and often devastating genetic diseases; motor proteins have also been implicated in the pathogenesis of microbial-induced diseases. Therefore, it is clear that we must continue to improve our understanding of these proteins and how they function both in normal and pathological cellular processes.
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