The overall aims are to understand the molecular basis of cell matrix adhesion and its effects on cell structure, behavior and differentiation. Work will concentrate on the extracellular matrix proteins, fibronectins, their cell surface receptors, particularly integrins and syndecans, and the cytoplasmic proteins (both cytoskeletal and signal transduction) whose organization and function are affected by fibronectin-integrin interactions to produce the cellular responses (adhesion, spreading, motility, cytoskeletal organization, proliferation, survival, gene expression) affected by cell-matrix interactions. l. Investigate the functions of specific modules/domains of fibronectins (FNs), including their three alternatively spliced segments. These studies will use recombinant expressed FN fragments with defined complements of domains and alternatively spliced segments and will concentrate on cell types known to be exposed in vivo to FN isoforms including all spliced segments. 2. Investigate the differing functions of fibronectin-receptor integrins (alpha5beta1, alpha4beta1, alpha3Abeta1, alpha3Bbeta1, some alphaV integrins) using recombinant FNs with/without specific domains/binding sites and cells lacking specific integrins (derived from mutant mouse strains). In particular, mesenchymal cells, myoblasts and keratinocytes lacking various FN receptor integrin subunits (alpha3, alpha4, alpha5), will be analyzed for cell adhesion, spreading and migration, cytoskeletal organization, proliferation, survival and specific differentiation.
The aim will be to identify cell types with differing responses depending on the sets of FN-integrin interactions to which they are subjected. 3. Investigate signal transduction events arising from FN-integrin interactions. This will be initiated with (a) human keratinocytes adherent to defined recombinant FNs and (b) murine mesenchymal cells differing in their complement of integrin receptors that bind to FN. The work will be extended to one or two cell systems established in specific aim 2. 4. Investigate the interactions of the cytoplasmic domains of different FN receptor integrins (alpha3A, alpha3B, alpha4, alpha5, alphaV) using a variety of methods. 5. Investigate the contributions of syndecans (and possibly other membrane proteoglycans) to cellular responses to fibronectins. Cell lines differing in syndecan expression will be studied for their responses to FN and will be analyzed for signalling and protein-protein interactions, as above. 6. Investigate the interactions of talin, a major cytoskeletal associate of integrins, using fusion proteins, yeast two-hybrid system and other approaches. These studies should illuminate how the complex responses of cells are controlled by their interactions with the extracellular matrix. Such interactions play important roles in many aspects of normal physiology and in pathologies as diverse as wound healing, cancer, thrombosis, fibrosis and inflammation.
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