Cell migration is essential for normal embryonic development, tissue repair and immune surveillance, but is also a contributing factor in mental retardation, developmental defects, tumor cell invasion and tissue fibrosis. It is a highly dynamic process requiring exquisite spatial and temporal control of cell adhesion to the extracellular matrix (ECM) in coordination with remodeling of the actin cytoskeleton. The Rho family GTPases play a central role in this regulation but the mechanisms controlling the activity of their key regulators, the guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) remain poorly understood. Paxillin is a multi-domain scaffold/adapter protein, which recruits numerous structural and signaling molecules to cell adhesion sites and thereby functions as a central hub in the regulation cell migration.
Aim 1 of this proposal will test the hypothesis that paxillin coordinates the spatial-temporal regulation of Rho family GTPase signaling and focal adhesion dynamics by establishing a local signaling network comprising the ARF GAP PKL/GIT2, the guanine nucleotide exchange factor Vav2 and the tyrosine phosphatase PTP-PEST. Hic-5, a close relative of paxillin is upregulated during epithelial-mesenchymal transition to promote cell migration via Rho-ROCK signaling and is an important regulator of cell motility, as well as patho-physiologic matrix remodeling in myofibroblasts.
In Aim 2, using 2D- and 3D-matrix model systems, we will dissect the mechanism through which Hic-5 controls cell migration and contractility and test the hypothesis that Hic-5 functions both independently and in conjunction with paxillin to regulate these processes. To accomplish these goals, we will suppress endogenous protein expression by RNA interference or express mutant proteins in fibroblasts and utilize confocal fluorescence time-lapse microscopy, combined with Fluorescence Recovery after Photobleaching (FRAP) and Fluorescence Resonance Energy Transfer (FRET) analysis to evaluate cell morphology, polarity and migration as well focal adhesion dynamics and spatial- temporal changes in protein- protein interactions and Rho family GTPase activity. This will be combined with biochemical analysis of changes in intracellular signaling to include GEF activity assays, protein phosphorylation profiling and protein- protein interactions. Completion of these Aims will elucidate the roles of paxillin and Hic-5 and their potential interactions in regulating cell migration vi modulation of the Rho GTPase system.

Public Health Relevance

ll movement is essential for normal processes such as embryonic development and tissue repair but it is also a key factor in cancer progression, tissue fibrosis and several cardiovascular and neurodegenerative disorders. Information gained from the proposed study will contribute to our understanding of how the cell migration machinery is regulated and thereby will potentially identify novel targets for corrective therapies for migration-associated disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM047607-21
Application #
8245845
Study Section
Intercellular Interactions (ICI)
Program Officer
Flicker, Paula F
Project Start
1991-08-05
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2014-03-31
Support Year
21
Fiscal Year
2012
Total Cost
$351,007
Indirect Cost
$127,436
Name
Upstate Medical University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
058889106
City
Syracuse
State
NY
Country
United States
Zip Code
13210
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Deakin, Nicholas O; Turner, Christopher E (2014) Paxillin inhibits HDAC6 to regulate microtubule acetylation, Golgi structure, and polarized migration. J Cell Biol 206:395-413
Wormer, Duncan B; Davis, Kevin A; Henderson, James H et al. (2014) The focal adhesion-localized CdGAP regulates matrix rigidity sensing and durotaxis. PLoS One 9:e91815
Jones, Matthew C; Machida, Kazuya; Mayer, Bruce J et al. (2013) Paxillin kinase linker (PKL) regulates Vav2 signaling during cell spreading and migration. Mol Biol Cell 24:1882-94
Deakin, Nicholas O; Pignatelli, Jeanine; Turner, Christopher E (2012) Diverse roles for the paxillin family of proteins in cancer. Genes Cancer 3:362-70
Wormer, Duncan; Deakin, Nicholas O; Turner, Christopher E (2012) CdGAP regulates cell migration and adhesion dynamics in two-and three-dimensional matrix environments. Cytoskeleton (Hoboken) 69:644-58
Deakin, Nicholas O; Ballestrem, Christoph; Turner, Christopher E (2012) Paxillin and Hic-5 interaction with vinculin is differentially regulated by Rac1 and RhoA. PLoS One 7:e37990
Pignatelli, Jeanine; Tumbarello, David A; Schmidt, Ronald P et al. (2012) Hic-5 promotes invadopodia formation and invasion during TGF-ýý-induced epithelial-mesenchymal transition. J Cell Biol 197:421-37
Pignatelli, Jeanine; LaLonde, Sara E; LaLonde, David P et al. (2012) Actopaxin (α-parvin) phosphorylation is required for matrix degradation and cancer cell invasion. J Biol Chem 287:37309-20
Deakin, Nicholas O; Turner, Christopher E (2011) Distinct roles for paxillin and Hic-5 in regulating breast cancer cell morphology, invasion, and metastasis. Mol Biol Cell 22:327-41

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