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 #
5R01GM047607-19
Application #
7794936
Study Section
Intercellular Interactions (ICI)
Program Officer
Flicker, Paula F
Project Start
1991-08-05
Project End
2013-03-31
Budget Start
2010-04-01
Budget End
2011-03-31
Support Year
19
Fiscal Year
2010
Total Cost
$354,552
Indirect Cost
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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Dubois, Fatemeh; Alpha, Kyle; Turner, Christopher E (2017) Paxillin regulates cell polarization and anterograde vesicle trafficking during cell migration. Mol Biol Cell 28:3815-3831
Rashid, Mamunur; Belmont, Judson; Carpenter, David et al. (2017) Neural-specific deletion of the focal adhesion adaptor protein paxillin slows migration speed and delays cortical layer formation. Development 144:4002-4014
Goreczny, G J; Ouderkirk-Pecone, J L; Olson, E C et al. (2017) Hic-5 remodeling of the stromal matrix promotes breast tumor progression. Oncogene 36:2693-2703
Jacob, Andrew E; Amack, Jeffrey D; Turner, Christopher E (2017) Paxillin genes and actomyosin contractility regulate myotome morphogenesis in zebrafish. Dev Biol 425:70-84
Jacob, Andrew E; Turner, Christopher E; Amack, Jeffrey D (2016) Evolution and Expression of Paxillin Genes in Teleost Fish. PLoS One 11:e0165266
Goreczny, Gregory J; Wormer, Duncan B; Turner, Christopher E (2015) A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro. J Vis Exp :e52949
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

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