Fibroblasts grown in a floating 3D collagen matrix deposit and remodel the surrounding ECM through mechanical interactions between the cells and the ECM and through secreted proteases (2,8). Contraction of the matrix is a visible readout of cell-matrix interactions

Extracellular matrix (ECM) actively participates in normal cell regulation and in the process of tumor progression. The Rho GTPase Cdc42 has been shown to regulate cell-ECM interaction in conventional two-dimensional culture conditions by using dominant mutants of Cdc42 in immortalized cell lines that may introduce non-specific effects. Here we employ three-dimensional culture systems for conditional gene targeted primary mouse embryonic fibroblasts (MEFs) that better simulate the reciprocal and adaptive interactions between cells and surrounding matrix, to define the role of Cdc42 signaling pathways in ECM organization. Cdc42 deficiency leads to a defect in global cell-matrix interactions reflected by a decrease in collagen gel contraction. The defect is associated with an altered cell-matrix interaction that is evident by morphologic changes and reduced focal adhesion complex formation. The matrix defect is also associated with a reduction in synthesis and activation of MMP9 and altered fibronectin deposition patterning. Cdc42 mutant rescue experiment found that downstream of Cdc42, PAK, but not Par6 or WASP, may be involved in regulating collagen gel contraction and fibronectin organization. Thus, in addition to the previously implicated roles in intracellular regulation of actin organization, proliferation and vesicle trafficking, Cdc42 is essential in ECM remodeling in 3-dimensions.