? The cytoplasmic dynein and dynactin motor complex generates force to provide movement for a vast array of cellular functions. Long-range movements are especially important in the axonal processes of neurons where dynein and dynactin move retrograde cargo over distances ranging from microns to meters. Genetic lesions in components of the dynein and dynactin motor machinery alter axonal transport and can result in severe neuronal disorders. The ability of dynein to take multiple steps without dissociating from the microtubule, called processivity, is enhanced by dynactin. For this processivity enhancement to take place, dynactin must be able to bind microtubules. The goal of this proposal is to elucidate the mechanism by which the dynactin/microtubule interaction increases cytoplasmic dynein processivity. Our hypothesis is that processivity enhancement occurs because dynactin acts as a molecular tether between dynein, cargo, and the microtubule cytoskeleton. As a molecular tether, dynactin prevents the complete dissociation of the motor/cargo complex from the microtubule and biases the rebinding of dynein to the microtubule. ? ? The first specific aim of this proposal is to characterize the interactions that occur between dynactin and microtubules with the goal of testing the validity of the dynactin tethering model and discriminating between possible mechanisms of action. This will be done by A) determining what portions of dynactin play a role in microtubule binding and characterizing the apparent binding strengths of the interactions, B) analyzing the single-molecule behavior of the dynactin/microtubule interaction, and C) characterizing the role of the dynactin/microtubule interaction in processive motility. The second specific aim is to elucidate regulatory mechanisms of the dynactin/microtubule interaction. This will be done by A) examining dynactin molecular heterogeneity and B) examining lis1 function. ? ? The wide range of human health problems that may be caused or compounded through the aberrant function of the cytoplasmic dynein and dynactin motor complex raises the possibility that dynein and dynactin components may be useful targets for therapeutic intervention. However, we require a better understanding of how dynein-based motility occurs and the role that dynein and dynactin may play in these disorders before intervention can be attempted. ? ?
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