The long-term objective of this grant is to test the hypothesis that corneal epithelial cells have specialized cell-cell and cell-matrix domains that are responsible for stability, differentiation and communication. A specialized basal-lateral domain in the intact embryonic corneal epithelial tissue that contains many actin associated and signaling proteins has been defined. The intracellular protein distribution in this domain is filamentous-actin dependent. The next logical set of experiments involves further characterization of this cell matrix attachment complex and the signal transduction pathways initiated by extracellular matrix that lead to tissue viability.
These specific aims will be addressed with whole avian embryonic epithelia isolated without basal lamina and cultured with extracellular matrix (ECM) molecules in the absence or presence of specific inhibitors or oligonucleotides that block synthesis of specific proteins.
Specific aims : I. Test the hypothesis that zyxin is a nucleating site for ECM stimulated actin bundle assembly. The actin associated protein, zyxin is homologous to an actin nucleating protein and it shuttles from the nucleus to fibroblast focal contacts. Zyxin has a polarized distribution in corneal epithelial sheets that is actin and ECM dependent. The sequence of zyxin and actin reassembly will be determined, then endogenous zyxin will be decreased with antisense oligonucleotides to establish if zyxin acts as a nucleation site for ECM stimulated actin bundle assembly. Zyxin binding proteins will be identified with co-immunoprecipitation, followed by western blot analysis. Zyxin s turnover rate and stability will be determined. II. Test the hypothesis that RhoGTP regulates actin reorganization and cytoplasmic retraction. Rho is a small signaling protein that shuffles between an inactive form (GDP) to an active form (GTP). One of the proteins that participates in changing the active state of Rho has been isolated from the embryonic corneal epithelium (p190RhoGAP). The current project will determine the role of this protein (p190RhoGAP) and other related downstream signaling proteins (ROCK, Dia, MAP kinase and P13 kinase) in extracellular matrix stimulated actin bundle formation. III. Test the hypothesis that epithelial cell-cell and cell-matrix interactions suppresses apoptosis. This model maintains cell-cell attachments while separating the cell-matrix attachment complex. The current project will determine if the stress-inducing signal transduction pathways become active in epithelia isolated without the basal lamina. Further experiments will determine if exogenous ECM can rescue epithelia isolated without the basal lamina or treated with signal transduction inhibitors. In summary, these experiments in our well defined corneal epithelial model will focus on how ECM mediated signal transduction stimulates actin bundle formation and also save the cells from programmed cell death.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY008886-10
Application #
6384626
Study Section
Visual Sciences A Study Section (VISA)
Program Officer
Fisher, Richard S
Project Start
1991-07-01
Project End
2003-03-31
Budget Start
2001-04-01
Budget End
2003-03-31
Support Year
10
Fiscal Year
2001
Total Cost
$232,932
Indirect Cost
Name
Texas A&M University
Department
Other Basic Sciences
Type
Schools of Dentistry
DUNS #
City
College Station
State
TX
Country
United States
Zip Code
77845
Svoboda, Kathy K H; Fischman, Donald A; Gordon, Marion K (2008) Embryonic chick corneal epithelium: a model system for exploring cell-matrix interactions. Dev Dyn 237:2667-75
Svoboda, K H; Gordon, Marion (2008) A tribute to Elizabeth D. Hay, 1927-2007. Dev Dyn 237:2605-6
Kang, P; Svoboda, K K H (2005) Epithelial-mesenchymal transformation during craniofacial development. J Dent Res 84:678-90
Svoboda, Kathy K H; Moessner, Petra; Field, Tamara et al. (2004) ROCK inhibitor (Y27632) increases apoptosis and disrupts the actin cortical mat in embryonic avian corneal epithelium. Dev Dyn 229:579-90
Kang, P; Svoboda, K K H (2003) Nicotine inhibits palatal fusion and modulates nicotinic receptors and the PI-3 kinase pathway in medial edge epithelia. Orthod Craniofac Res 6:129-42
Kang, Pei; Svoboda, Kathy K H (2002) PI-3 kinase activity is required for epithelial-mesenchymal transformation during palate fusion. Dev Dyn 225:316-21
Reenstra, Wende R; Orlow, Daniel L; Svoboda, Kathy K H (2002) ECM-stimulated signaling and actin reorganization in embryonic corneal epithelia are Rho dependent. Invest Ophthalmol Vis Sci 43:3181-9
Watanabe, Michiko; Hitomi, Midori; van der Wee, Kathy et al. (2002) The pros and cons of apoptosis assays for use in the study of cells, tissues, and organs. Microsc Microanal 8:375-91
Svoboda, Kathy Kay Hartford; Reenstra, Wende R (2002) Approaches to studying cellular signaling: a primer for morphologists. Anat Rec 269:123-39
Chu, C L; Reenstra, W R; Orlow, D L et al. (2000) Erk and PI-3 kinase are necessary for collagen binding and actin reorganization in corneal epithelia. Invest Ophthalmol Vis Sci 41:3374-82

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