AbstractsBiology & Animal Science

Rho family GTPases are intracellular signal transducers important in cell growth, morphogenesis, motility, cytoskeleton organization, endocytosis and exocytosis. These functions are critical for development, wound healing, and the acquired transformation and metastasis properties of cancer cells. Since gene targeting of Rho GTPases leads to early embryonic lethality, previous studies of Rho GTPase functions were primarily performed by using immortalized cell lines and dominant mutant expression approaches. Those studies, while insightful, are limited by clonality and dominant mutant specificity issues, and by the wide usage of 2D cell culture models on plastic tissue culture dishes that are far from ideal in understanding their physiological functions. Extracellular matrix (ECM) actively participates in normal cell regulation during development and wound healing, and altered cell regulation in the process of tumor progression. Specifically, fibroblasts are a major regulator of ECM interactions. Previous work have suggested that the Rho GTPases Rac and Rho are involved in cell-ECM interactions in both 2D and 3D systems; however, whether the important Rho GTPase member Cdc42 is required in ECM organization has not been studied in this context. The cellular and molecular mechanisms participating in the ECM remodeling in 3D by the Rho GTPases also remain an open question. The central hypothesis of my dissertation is that the Rho GTPase Cdc42 is an essential regulatory signaling molecule involved in maintaining normal ECM interactions and ECM remodeling in a 3D environment. This hypothesis was rigorously tested by utilizing a 3D culture system in conditional Cdc42 gene targeted primary mouse embryonic fibroblast (MEF) cells that better simulates the reciprocal and adaptive interactions that occur between cells and the surrounding matrix in a tissue-like environment. We have found that Cdc42 deficiency leads to global defects in cell-matrix interactions in 3D, as measured by a decrease in collagen gel contraction. This effect is recapitulated by applying a Cdc42 specific small molecule inhibitor to the cells. The matrix contractility defect in Cdc42-/- MEFs is associated with an altered mechanical interaction of the cells with the matrix, as observed by morphologic changes in 3D, and significantly reduced focal adhesion complex formation. The contractility defect of the Cdc42 deficiency cells is also associated with altered ECM and protease regulation required for matrix remodeling, as manifested by an altered fibronectin deposition patterning and a decrease in matrix metalloproteinase 9 (MMP9) expression and activity. Correspondingly, phosphorylation levels of focal adhesion kinase (FAK), paxillin, p21-activated kinase (PAK), cofilin, and Wiskott-Aldrich syndrome protein (WASP) (important focal complex proteins and cytoskeletal regulating proteins) were reduced. Reconstitution of various Cdc42 effector binding mutants into the Cdc42-/- MEFs suggests that the downstream effector of Cdc42, PAK, but not PAR6, may…