An extracellular-matrix-specific GEE GAP interaction regulates Rho GTPase crosstalk for 3D collagen migration

Rho-family GTPases govern distinct types of cell migration on different extracellular matrix proteins in tissue culture or three-dimensional (3D) matrices1-3. We searched for mechanisms selectively regulating 3D cell migration in different matrix environments4'5 and discovered a form of Cdc42-RhoA crosstalk governing cell migration through a specific pair of GTPase activator and inhibitor molecules. We first identified pPix, a guanine nucleotide exchange factor (GEF), as a specific regulator of migration in 3D collagen using an affinity-precipitation-based GEF screen. Knockdown of PPix specifically blocks cell migration in fibrillar collagen microenvironments, leading to hyperactive cellular protrusion accompanied by increased collagen matrix contraction. Live FRET imaging and RNAi knockdown linked this I3Pix knockdown phenotype to loss of polarized Cdc42 but not Racl activity, accompanied by enhanced, de-localized RhoA activity. Mechanistically, collagen phospho-regulates pPix, leading to its association with srGAP1, a GTPase-activating protein (GAP), needed to suppress RhoA activity. Our results reveal a matrix-specific pathway controlling migration involving a GEF-GAP interaction of f3Pix with srGAP1 that is critical for maintaining suppressive crosstalk between Cdc42 and RhoA during 3D collagen migration.