Myosins constitute a diverse superfamily of actin-based molecular motors that perform a wide range of fundamental functions, including cytokinesis, cell migration, cell contraction and the intracellular transport and correct positioning of vesicles, organelles and molecules. Recently, it was shown that myosin Va (MVa) is involved in cell migration and is up-regulated in metastatic tumor cells. The steps of invasion and metastasis are responsible for the malignant phenotype of tumor cells, contributing for more than 90% of cancer deaths. Hence, uncovering molecular mechanisms involved in these steps may lead to the development of strategies for cancer treatment or prevention of the disease progression. Cell adhesion is essential to cell motility, proliferation survival and differentiation. It is mediated by integrins and the focal adhesion kinase (FAK) that executes several phosphorylation events that are critical for adhesion, migration and survival. Previous studies in our laboratory showed that MVa directly interacts with FAK and works upstream of FAK to mediate its activation and targeting to the ends of protrusions, at nascent focal adhesion sites, in the leading edge of migrating cells. The aims of the present study are: (i) to map the specific binding sites on MVa and FAK protein structures, using pull down and immunoprecipitation assays; (ii) to image the in vivo dynamics of fluorescently tagged MVa and FAK during the assembly and disassembly of focal adhesions using time-lapse confocal microscopy; (iii) to investigate the involvement of MVa in the process of cell surface expression of integrins in fibroblasts and melanoma cells, using specific antibodies for integrin staining and confocal microscopy and flow cytometry for analysis; (iv) to investigate the involvement of MVa in the global expression of surface molecules using proteomic approaches in which surface proteins are tagged with biotin and captured on streptavidin columns. The proteomic profile of cell surface proteins will be assessed by LC-MS/MS. For these assays, we will use myosin Va-null fibroblasts derived from a Griscelli syndrome patient and same cells rescued by MVa reexpression, as well as human melanoma cells knockdown for MVa by lentiviral-mediated shRNA expression. All cell lines and recombinant DNA tools are already available in our laboratory. These strategies aim to assess the mechanisms underlying MVa and FAK interaction and to shed new light on some of the key events of focal adhesion dynamics, which is central in the control of cell adhesion and migration.
News published in Agência FAPESP Newsletter about the scholarship: