Cell adhesion and spreading are regulated by complex interactions between the cytoskeleton, matrix adhesion receptors and extracellular matrix proteins, but the molecular determinants of these interactions in early events in cell spreading are not defined. I found that the actin-binding proteins cortactin, vinculin and filamin A were enriched in the earliest formed extensions of HEK-293 cells spreading on collagen. Knockdown of filamin A by short hairpin RNA reduced spreading and the number of cell extensions. Antibody blockade of collagen binding sites on ß1 integrin reduced (p<0.05) cell spreading and the localization of filamin A at cell extensions. Knockdown of filamin A reduced ß1 integrin occupancy by collagen as measured by 12G10 antibody, suggesting a functional co-dependence of filamin A and ß1 integrin. Based on mass spectrometry screening of potential filamin A interacting proteins I examined the interaction of filamin A with the intermediate filament protein vimentin. Filamin A and vimentin-expressing cells were well-spread on collagen and exhibited numerous cell extensions enriched with filamin A and vimentin. By contrast, knockdown of filamin A or vimentin inhibited spreading, cell adhesion, cell surface ß1 integrin expression and ß1 integrin activation. Knockdown of filamin A reduced vimentin phosphorylation and blocked recruitment of vimentin to cell extensions while knockdown of filamin A and/or vimentin inhibited the formation of cell extensions. Inhibition of cell spreading, vimentin phosphorylation and ß1 integrin surface expression and activation were all phenocopied in cells treated with the protein kinase C inhibitor bisindolylmaleimide; cell spreading was also reduced by siRNA knockdown of protein kinase Cє. By immunoprecipitation of cell lysates and by pull-down assays using purified proteins I found an association between filamin A and vimentin. Filamin A also associated with protein kinase Cє, which was enriched in cell extensions. In vitro pull-down assays using deletional mutants of purified filamin A showed that both vimentin and protein kinase Cє bound to a region of filamin A that included repeats 1-8. Reconstitution of filamin A-deficient cells with full-length filamin A or filamin A repeats 1-8 restored cell spreading, vimentin phosphorylation and the cell surface expression of ß1 integrins. I conclude that interactions of filamin A with vimentin and protein kinase Cє may be important for the trafficking and activation of ß1 integrins and cell spreading on collagen.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/29970 |
| Date | 15 September 2011 |
| Creators | Kim, Hugh |
| Contributors | McCulloch, Christopher |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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