Focal contacts are the main structural specializations of the cell membrane and of cortical actin cytoskeleton responsible for the adhesion of cultured cells to the substratum; however, little is known about the cytoskeletal mechanisms controlling their development. Preliminary data, obtained by the group in Moscow, with microtubule-depolymerizing drugs resulted in the hypothesis that the microtubule system may control the organization of focal contacts via the regulation of the state of the actin cortex. In turn, the contractile forces developed by the cortex which act on the cytoskeleton-attached membrane proteins of the focal contacts are affected. The goal of this project is to study the role of microtubular system in the development of focal contacts.
Specific aims i nclude the analysis of the alterations of structure and dynamics of focal contacts in cultured fibroblasts after interventions specifically changing the structure and function of microtubular system. These include exposure of cells to the drugs depolymerizing or stabilizing microtubules as well as injection of antibody to microtubule motor protein, kinesin. Two complementary technologies will be used to assess the state of focal contacts: interference-reflection microscopy to reveal these contacts in living cells and immunocytochemical methods to identify proteins specifically localized in the focal contacts and for other cytoskeletal proteins. To test the alterations of contractile forces in the cortex induced by microtubule-specific agents and to correlate these alterations with those of focal contacts the methods of force measurements developed in the Chapel Hill laboratory will be used. The planned experiments will lead to better understanding of the mechanisms of cytoskeletal control of the formation of cell-matrix adhesions. The project will explore the nature of alterations in the cytoskeleton-membrane-matrix relationships responsible for pathological adhesion and motility of neoplastic cells.
