The microtubule cytoskeleton plays an essential role in cell shape, migration, and division. One of the major functions of the microtubule cytoskeleton is to facilitate transport of organelle and macromolecules to particular destinations in the cell. Transport can be mediated by two strategies. First, ATP hydrolyzing motor proteins can carry cargoes along the surface of the microtubule. Second, certain proteins (+TIP proteins) can selectively track along the growing tip of a microtubule as it extends to the cell cortex and can bind to and deliver certain cargoes (e.g. signaling molecules). Our goals are to understand motor-protein cargo recognition, regulation of motor proteins, and the mechanism by which +TIP interact with microtubule growing ends. In general, we wish to dissect the mechanisms of these proteins using a variety of techniques including x-ray crystallography, electron microscopy, biochemical approaches, in vitro reconstitution assays, and cell biological approaches in living cells. In this grant, we propose the following aims. 1) We wish to determine how a particular subset of mRNAs are selected for transport by motor proteins in yeast. In particular, we wish to solve an atomic structure for a minimal element of such mRNAs complexed with the proteins that are involved in the transport pathway. 2) We have solved crystal structures of several +TIP domains and developed a model suggesting that these proteins function as 'polymerization chaperones' that deliver oligomeric tubulin to the growing end of the microtubules. We propose to better define how these proteins interact with tubulin and develop functional assays to garner support for this model. 3) We will study activators of the dynein motor protein, in particular testing the notion that they affect dynein motor activity. We have also identified a new protein that may regulate dynein at kinetochores, and we will pursue further studies of this protein. 4) We will investigate new ATPases that we believe may modulate the dynamics of microtubules. This work has several potential medical applications. First, the +TIP proteins are essential for microtubule function in mitosis and in cell migration, and their selective inhibition may be useful in cancer chemotherapy (by inhibiting the spindle) or in inflammatory disease (by blocking cell migration). Our work on dynein regulators is likely to be important for understanding the spindle checkpoint, a topic of great interest in cancer since modulation of the checkpoint may enhance cancer cell death after chemotherapy.
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