Cell locomotion is critical to development, wound healing and tissue remodeling, and a central problem during metastasis in cancer. Thus, it is important to gain an understanding of cell motility at the basic level. The cytoskeletal polymers, microtubules and filamentous actin (f-actin) are required for cell motility. Polarized f-actin assembly and movement is thought to drive cell motility, while microtubules are believed to spatially direct sites of f-actin assembly. There is evidence that microtubules may achieve this by activation of cellular signaling via small GTPases of the Rho family. However, the organization and dynamics of microtubules and how they are affected by actin is not thoroughly characterized, and the molecules involved in mediating their interactions during cell motility are unknown. I approach these problems at 3 levels: First, by examining microtubules and f-actin dynamics in living cells using multimode microscopy, by exploring with both microscopy and biochemistry microtubule/f-actin interactions in a cellular extract model systems, and finally by examining the biochemical interactions of purified components of the motile machinery in vitro. The combination of unique microscopy methods and molecular biochemistry is a novel means of carefully characterizing cellular phenomenon and then going directly to the test tube test hypotheses about the molecular mechanisms underlying the phenomena. In this proposal, I describe three specific aims to shed light on our understanding of microtubule/f- actin interactions in cell motility. 1. To develop new microscopy methods for the simultaneous visualization and analysis of f-actin and microtubule dynamics and to carefully analyze microtubule/actin interactions in living migrating cells. 2. To biochemically and microscopically probe the molecular mechanism dynamic microtubule/actin interactions in Xenopus egg extracts. 3. To biochemically analyze microtubule mediation of Rho family signaling and study the mechanism by which growing microtubules activate Rac1 during cell motility. The results of these studies will greatly enhance our understanding of the mechanisms and mechanics of cell locomotion and will provide new methods for analyzing protein dynamics in living cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061804-04
Application #
6653907
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Deatherage, James F
Project Start
2000-09-01
Project End
2005-08-31
Budget Start
2003-09-01
Budget End
2004-08-31
Support Year
4
Fiscal Year
2003
Total Cost
$261,343
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Schneider, Ian C; Hays, Cristen K; Waterman, Clare M (2009) Epidermal growth factor-induced contraction regulates paxillin phosphorylation to temporally separate traction generation from de-adhesion. Mol Biol Cell 20:3155-67
Kroboth, Karin; Newton, Ian P; Kita, Katsuhiro et al. (2007) Lack of adenomatous polyposis coli protein correlates with a decrease in cell migration and overall changes in microtubule stability. Mol Biol Cell 18:910-8
Prigozhina, Natalie L; Waterman-Storer, Clare M (2006) Decreased polarity and increased random motility in PtK1 epithelial cells correlate with inhibition of endosomal recycling. J Cell Sci 119:3571-82
Kita, Katsuhiro; Wittmann, Torsten; Nathke, Inke S et al. (2006) Adenomatous polyposis coli on microtubule plus ends in cell extensions can promote microtubule net growth with or without EB1. Mol Biol Cell 17:2331-45
Wittmann, Torsten; Waterman-Storer, Clare M (2005) Spatial regulation of CLASP affinity for microtubules by Rac1 and GSK3beta in migrating epithelial cells. J Cell Biol 169:929-39
Torreano, Paul J; Waterman-Storer, Clare M; Cohan, Christopher S (2005) The effects of collapsing factors on F-actin content and microtubule distribution of Helisoma growth cones. Cell Motil Cytoskeleton 60:166-79
Prigozhina, Natalie L; Waterman-Storer, Clare M (2004) Protein kinase D-mediated anterograde membrane trafficking is required for fibroblast motility. Curr Biol 14:88-98
Wittmann, Torsten; Bokoch, Gary M; Waterman-Storer, Clare M (2003) Regulation of leading edge microtubule and actin dynamics downstream of Rac1. J Cell Biol 161:845-51
Rodriguez, Olga C; Schaefer, Andrew W; Mandato, Craig A et al. (2003) Conserved microtubule-actin interactions in cell movement and morphogenesis. Nat Cell Biol 5:599-609
Ponti, A; Vallotton, P; Salmon, W C et al. (2003) Computational analysis of F-actin turnover in cortical actin meshworks using fluorescent speckle microscopy. Biophys J 84:3336-52

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