The extracellular matrix (ECM) plays a critical role in cellular decision-making. It serves as a framework for tissue architecture and it stimulates cell growth, migration, and a host of other cell processes. Transmission of ECM signals into cells is carried out by integrin receptors. These receptors link the ECM to the actin cytoskeleton and activate a number of different signaling pathways. To understand how cells coordinate extracellular signals with intracellular processes requires insights into the organization of the ECMas well as information about the signaling molecules that are activated by ECM-integrin interactions. We are applying a combined approach to this problem using mammalian cell culture and the nematode Caenorhabditis elegans. The proposed experiments make use of the distinct and complementary advantages provided by these two systems. In HT1080 human fibrosarcoma cells, Src family kinases appear to play an essential role in fibronectin (FN) matrix assembly and matrix stability. We will determine the timing of Src-action during assembly, its effect on matrix turnover, and its role in formation of matrix assembly sites at the cell surface. Biochemical analysis of detergent-insoluble matrix, microscopic analysis of FN fibril organization, and addition of inhibitors and mutant proteins will be used for these experiments. Matrix turnover decreases cell adhesion as it promotes migration. Therefore, we will examine the effects of matrix assembly on cell migration rate using in vitro cell migration assays. With C. elegans, which has only 2integrin receptors, genetic analyses to identify major signals downstream of ECM-integrin interactions in vivo will be performed. We are able to rescue beta integrin (pat-3) null animals with genes mutated in conserved beta integrin cytoplasmic tyrosine residues. Rescued nematodes are viable and fertile, but show defects in gonad morphogenesis, a process dependent on integrin-mediated cell movements. To identify additional functional sequences within the beta tail, rescued nematode lines with other tail mutations will be analyzed for defects in gonad formation and other integrin-related phenotypes. Nematode lines will also be used in RNA interference and EMS mutagenesis screens to isolate novel genes that act in pathways downstream of integrins. Results from these aims will provide molecular and genetic information about the role of ECM-integrin interactions in regulating cell migration and matrix assembly during tissue morphogenesis.
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