Spatio-temporal dynamics of Rac1, PI 3 -kinase, and the cytoskeleton during cadherin -mediated cell -cell adhesion

Cell-cell adhesion is a fundamental aspect of life as a multi-cellular organism, providing both essential structural support to tissues and permitting the compartmentalization of distinct physiologic spaces. Aberrations in cell-cell adhesion result in pathologic dysfunction and loss of homeostasis. As such, an understanding of the mechanisms by which cell-cell adhesions are established is important to our understanding of physiology and disease.;In epithelial tissues, molecules in the cadherin family are primary mediators of cell-cell adhesion. In this thesis, I have studied events that occur downstream of interaction between E-cadherin molecules on adjacent cells. Establishment of cadherin-dependent adhesion is regulated by the action of Rho-family small GTPase signaling molecules, but the precise role of these signaling proteins is poorly understood. Using timelapse microscopy and quantitative image analysis, I have demonstrated that cell-cell contact in the MDCK model epithelial cell system coincides with a spatio-temporal reorganization of plasma membrane Rac1 (a Rho GTPase family member) and a focusing of lamellipodia from non-contacting to contacting surfaces. Within cell-cell contacts, Rac1 and lamellipodia transiently concentrate at newest sites, but decrease at older, stabilized sites. Products of PI 3-kinase activity also accumulate dynamically at contacts, but are not essential for either initiation or development of cell-cell adhesion. These results define a role for Rac1 in regulating the rates of initiation and strengthening of cell-cell adhesion.;Cadherin-dependent adhesion is also associated with extensive actin cytoskeleton reorganization at cell-cell contact sites, but the function of this cortical actin restructuring has remained unclear. Observational studies suggest that cortical actin is under tension at the margins of developing contacts. Using timelapse microscopy and immunofluorescence with probes specific for activated myosin, I have also studied the dynamics and role of actin-myosin contractility during cell-cell adhesion. I show that a contractile actin-myosin ring is formed between contacting MDCK epithelial cells, and Ser19-phosphorylated myosin is enriched at margins of cell-cell contacts. By applying inhibitors of myosin activation, we demonstrate that actin-myosin tension generated at these sites is important for contact lengthening. These results substantiate a proposed novel role for purse-string actin-myosin contractility in development of cell-cell adhesion.*.;*This dissertation includes a CD that is compound (contains both a paper copy and a CD as part of the dissertation). The CD requires the following application: QuickTime Movie Player.