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.

Agency
National Institute of Health (NIH)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM038499-26
Application #
8726986
Study Section
No Study Section (in-house review) (NSS)
Program Officer
Gindhart, Joseph G
Project Start
Project End
Budget Start
Budget End
Support Year
26
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Pharmacology
Type
Schools of Medicine
DUNS #
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Hendershott, Melissa C; Vale, Ronald D (2014) Regulation of microtubule minus-end dynamics by CAMSAPs and Patronin. Proc Natl Acad Sci U S A 111:5860-5
Sirajuddin, Minhajuddin; Rice, Luke M; Vale, Ronald D (2014) Regulation of microtubule motors by tubulin isotypes and post-translational modifications. Nat Cell Biol 16:335-44
Hui, Enfu; Vale, Ronald D (2014) In vitro membrane reconstitution of the T-cell receptor proximal signaling network. Nat Struct Mol Biol 21:133-42
Schroeder, Courtney M; Ostrem, Jonathan M L; Hertz, Nicholas T et al. (2014) A Ras-like domain in the light intermediate chain bridges the dynein motor to a cargo-binding region. Elife 3:e03351
Petry, Sabine; Groen, Aaron C; Ishihara, Keisuke et al. (2013) Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2. Cell 152:768-77
Singhvi, Aakanksha; Teuliere, Jerome; Talavera, Karla et al. (2011) The Arf GAP CNT-2 regulates the apoptotic fate in C. elegans asymmetric neuroblast divisions. Curr Biol 21:948-54
Goodwin, Sarah S; Vale, Ronald D (2010) Patronin regulates the microtubule network by protecting microtubule minus ends. Cell 143:263-74
Trammell, Matthew A; Mahoney, Nicole M; Agard, David A et al. (2008) Mob4 plays a role in spindle focusing in Drosophila S2 cells. J Cell Sci 121:1284-92
Yildiz, Ahmet; Tomishige, Michio; Gennerich, Arne et al. (2008) Intramolecular strain coordinates kinesin stepping behavior along microtubules. Cell 134:1030-41
Goshima, Gohta; Wollman, Roy; Goodwin, Sarah S et al. (2007) Genes required for mitotic spindle assembly in Drosophila S2 cells. Science 316:417-21

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