Stimulation of fascin spikes by thrombospondin-1 is mediated by the GTPases Rac and Cdc42

Cell adhesion to extracellular matrix is an important physiological stimulu s for organization of the actin-based cytoskeleton. Adhesion to the matrix glycoprotein thrombospondin-1 (TSP-1) triggers the sustained formation of F -actin microspikes that contain the actin-bundling protein fascin. These st ructures are also implicated in cell migration, which may be an important f unction of TSP-1 in tissue remodelling and wound repair. To further underst and the function of fascin microspikes, we examined whether their assembly is regulated by Rho family GTPases. We report that expression of constituti vely active mutants of Rac or Cdc42 triggered localization of fascin to lam ellipodia, filopodia, and cell edges in fibroblasts or myoblasts, Biochemic al assays demonstrated prolonged activation of Rac and Cdc42 in C2C12 cells adherent to TSP-1 and activation of the downstream kinase p21-activated ki nase (PAK). Expression of dominant-negative Rac or Cdc42 in C2C12 myoblasts blocked spreading and formation of fascin spikes on TSP-1. Spreading and s pike assembly were also blocked by pharmacological inhibition of F-actin tu rnover. Shear-loading of monospecific anti-fascin immunoglobulins, which bl ock the binding of fascin to actin into cytoplasm, strongly inhibited sprea ding, actin cytoskeletal organization and migration on TSP-1 and also affec ted the motility of cells on fibronectin. We conclude that fascin is a crit ical component downstream of Rac and Cdc42 that is needed for actin cytoske letal organization and cell migration responses to thrombospondin-1.