Filamin A regulates cell spreading and survival via b1 integrins

Objectives: Cell spreading and exploration of topographically complex substrates require tightly-regulated interactions between extracellular matrix receptors and the cytoskeleton, but the molecular determinants of these interactions are not defined. Methods: We examined whether the actin-binding proteins cortactin, vinculin and filamin A are involved in the formation of the earliest extensions of cells spreading over collagen or poly-L-lysine-coated smooth and beaded substrates. Results: Spreading of human gingival fibroblasts was substantially reduced on beaded or poly-L-lysine-coated substrates. Filamin A, vinculin and cortactin were found in cell extensions on smooth collagen. HEK-293 cells also spread rapidly on smooth collagen and formed numerous cell extensions enriched with filamin A. Knockdown of filamin A in HEK-293 cells by short hairpin RNA reduced spreading and the number of cell extensions. Blocking b1 integrin function significantly reduced cell spreading and localization of filamin A to cell extensions. Conversely, filamin A-knockdown reduced b1 integrin-collagen binding as measured by 12G10 antibody, suggesting co-dependence between filamin A and b1 integrin functions. TUNEL staining showed higher percentages of apoptosis after filamin A-knockdown in spreading cells. Chelation of [Ca²⁺]_i with BAPTA/AM reduced spreading of wild-type and filamin A-knockdown cells, however wild-type cells showed recruitment of filamin A to the subcortex, indicating independent roles of filamin A and [Ca²⁺]_i in cell spreading. Conclusions: We conclude that filamin A integrates with b1 integrins to mediate cell spreading and prevent apoptosis.

This study was supported by Canadian Institutes of Health Research operating (MGP-37783), Group and Research Resource grants to C.A. McCulloch. H. Kim acknowledges fellowship support from the Canadian Arthritis Network, the University of Toronto Harron Fund and the CIHR Cell Signals Program (STP-53877).