The long-term objective of this project is to identify and characterize the forces that drive chromosome movement in meiosis and mitosis. These forces are essential for normal chromosome distribution and arise primarily from microtubule dynamics and microtubule motors. A major goal is to understand the role of microtubule motor proteins in spindle and chromosome dynamics in cell division, including the molecular basis of motor directionality and force generation, and the role of motor directionality and force production in division. The proposed studies focus on Ncd, a minus-end microtubule motor protein of Drosophila. The Ncd motor plays an essential role in spindle assembly and function in meiosis and mitosis. The proposed studies are to examine basic motor properties with the objective of understanding how motors work in the cell. Specific aims of the proposed studies are to 1. Determine the step of nucleotide hydrolysis at which the Ncd stalk/neck rotates. The stalk rotation represents a potential force-producing stroke that drives movement of the motor to the microtubule minus end. We will test the hypothesis that the stalk rotates when the motor binds to microtubules and releases ADP, producing the large displacement we observed in laser trap assays. 2. Define the molecular basis of motor structural changes essential for force generation and transduction. We will test the hypothesis, based on our recent cryoEM results, that switch II helix melting and distortion of the central 2-sheet are essential for motor function by mutating key residues and assaying the mutated motors for ATPase activity and gliding velocity in vitro. Structural analysis of selected mutants will be performed to obtain information regarding the changes the elements undergo. 3. Determine the role of motors in microtubule nucleation in anastral meiosis I spindles and their effects on microtubule dynamics in the spindle. Mature and assembling spindles will be analyzed in live oocytes to determine whether 3tubulin is present and the polarity of microtubules in the anastral meiosis I spindle. Newly designed ncd mutants that affect the ability of the motor to produce force will be examined for their effects on microtubule dynamics in the meiosis I spindle and findings will be correlated with effects of the mutant motors on spindle assembly.
NIH ExPorter
NIH ExPorter