Journal
EXPERIMENTAL CELL RESEARCH
Volume 314, Issue 4, Pages 834-846Publisher
ELSEVIER INC
DOI: 10.1016/j.yexcr.2007.11.022
Keywords
actin; surface topography; cell guidance
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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. 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. 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 beta 1 integrin function significantly reduced cell spreading and localization of filamin A to cell extensions. Conversely, filamin A-knockdown reduced beta 1 integrin-collagen binding as measured by 12G10 antibody, suggesting co-dependence between filamin A and beta 1 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. We conclude that filamin A integrates with beta 1 integrins to mediate cell spreading and prevent apoptosis. (C) 2007 Elsevier Inc. All rights reserved.
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