R2 - Mechanochemistry of the Cytoplasmic Dynein-Dynactin Motor Complex - Erika L. F. Holzbaur, Ph.D. The molecular motor cytoplasmic dynein is required in mammalian cells for a wide range of cellular functionsincluding vesicle trafficking, Golgi organization, and mitotic spindle assembly. All these functions require an activatorcomplex, dynactin, which binds both to dynein and to microtubules. While cytoplasmic dynein is an essential cellularmotor, its mechanism of force generation has not been explored. We propose to use single motor and optical trapassays to analyze the parameters of force generation by cytoplasmic dynein. We hypothesize that dynactin activelyparticipates in dynein-driven motility, that dynactin is required to increase the efficiency of the dynein motor byconstraining the mechanochemical properties of cytoplasmic dynein to adapt a relatively inefficient and nonprocessivemotor to the cellular environment. We also propose to use structural studies to analyze the cytoplasmic dynein-dynactin complex. We hypothesize that the relative stiffness of dynactin's structure acts to constrain the apparentfloppiness of dynein's power stroke, thus increasing the efficiency of long distance transport in the cell. To test thesehypotheses, we propose: (1) to examine parameters of motility and force generation of cytoplasmic dynein, using singlemotor fluorescent assays and mechanical force measurements in an optical trap; (2) to examine the structure ofcytoplasmic dynein and of the dynein/dynactin complex using AFM and EM, in the presence of ATP or ADP andvanadate (pre-power stroke state), and in the absence of added nucleotide (the apo state); (3) to test the effects ofmutations in cytoplasmic dynein or dynactin on protein structure as well as on parameters of motility and forcegeneration, and (4) to examine the parameters involved in motor-dependent switching of cytoskeletal tracks. Thecorrelation of information from both dynamic and structural studies will allow us to develop and test a model for thegeneration of force by the cytoplasmic dynein and by the dynein-dynactin complex. The observation that mutations inboth cytoplasmic dynein and dynactin cause motor neuron degeneration in mice and in humans highlights theimportance of improving our understanding of dynein structure and function.
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