Dynein is one of the fundamental motor molecules that drive movements in eukaryotic cells. Its microtubule-based activities are essential for many forms of membrane-bound organelle transport, often working in cooperation with the other motor proteins, kinesin and myosin. Specific forms of dynein are required for assembly of the spindle that separates the chromosomes during mitosis, and for chromosome attachment, as well as in mechanisms used to position spindles and orient the sites where the spindle fibers separate after chromosome separation is complete. This project addresses how the dynein motor works, emphasizing regulation and its coordination with opposing motor activities. Dr. Koonce's laboratory has demonstrated a role for dynein as a force-generating anchor of the microtubule array between mitoses (interphase). Overexpression of dynein motor domain fragments causes a dramatic collapse of the microtubule array in the slime mold, Dictyostelium, resulting in robust centrosome movements through the cytoplasm. Microtubules remain attached to and form a trailing "comet tail" as the centrosome moves. Not only dynein, but manipulations to two interacting proteins, Lis-1 and Ndel-1, also produce this phenotype and thus are hypothesized to form a regulatory network that controls dynein movement. The primary objective here tests this hypothesis and seeks to determine how Lis-1 and Ndel-1 regulate the motor. Recent laser microbeam cutting of the comet-like microtubule arrangements demonstrate that their movement results from motors that push on the trailing microtubules. A second objective tests the idea that decreased dynein activity allows kinesin-like motors to push without opposition, and thus interphase microtubule networks are arranged through a balance of both dynein-pulling and kinesin-pushing forces. Finally, in the third objective, an optical trap formed by laser beams will be used on individual molecules to determine the contributions of a lever-arm mechanism to dynein force production, and address how the regulatory machinery may modify this powerstroke. Overall, the project addresses force producing mechanisms that are universal in eukaryotic cells. Dictyostelium offers a multifunctional system in which these forces are exaggerated, and thus highly amenable to study. To date there is very little understanding of how these important proteins are regulated, and if successful, Dr. Koonce's efforts will elucidate a fundamental understanding of how motility is coordinated. Broader Impacts The project will contribute to a fundamental understanding of how multiple dynamic processes are coordinated within cells. In performing the work, graduate students and postgraduate investigators will receive training in state of the art research facilities and be provided significant educational opportunities.
