Elsevier

Experimental Cell Research

Volume 314, Issue 4, 15 February 2008, Pages 834-846
Experimental Cell Research

Research Article
Filamin A regulates cell spreading and survival via β1 integrins

https://doi.org/10.1016/j.yexcr.2007.11.022Get rights and content

Abstract

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 β1 integrin function significantly reduced cell spreading and localization of filamin A to cell extensions. Conversely, filamin A-knockdown reduced β1 integrin–collagen binding as measured by 12G10 antibody, suggesting co-dependence between filamin A and β1 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 β1 integrins to mediate cell spreading and prevent apoptosis.

Introduction

Cell spreading and exploration of surface topography are critical processes in cell-mediated immunity, development, wound healing and the interactions of host cells with implanted biomaterials. These processes are dependent in part on subcortical actin remodeling and the formation of cell membrane protrusions in response to soluble signals, and to cues from the extracellular matrix [1], [2]. Cell–matrix signaling involves integrin activation by extracellular ligands, which triggers adhesion formation, actin remodeling and cell spreading [3]. Actin remodeling in response to cell adhesion to extracellular matrices is an elaborate process mediated by complex intracellular signaling pathways involving intracellular Ca2+, small GTPases and a large group of actin-binding proteins.

Actin-binding proteins are crucial for the adhesion of cells to extracellular matrices as they link integrins to the cortical actin cytoskeleton. Several actin-binding proteins including talin, vinculin, paxillin, gelsolin, Arp 2/3 and cortactin have been implicated in cell spreading [4], [5], [6], [7]. Notably, filamin A is an actin-binding protein that cross-links cortical actin filaments into tightly-organized orthogonal networks, a property that may increase the rigidity of cell membranes [8]. Since filamin A strongly influences cellular rheology [9], [10], [11], it may enhance the localized rigidity required for extension of filopodia and lamellipodia. Further, filamin A can inhibit deformation-induced cell depolarization [12] and force-induced apoptosis [13], an important property since cell spreading generates significant membrane-deforming forces during the extension of filopodia and lamellipodia [14], [15].

Despite the well-documented properties of filamin A in regulating cell migration [16], the specific roles of this actin-binding protein in early cell spreading are not well-defined [17], [18]. In addition to cross-linking actin filaments, filamin A can bind more than 30 other proteins [19], including the intracellular domains of β1 and β7 integrins [20], [21], [22]; it also stabilizes cell membranes and reinforces integrin–cytoskeletal interactions [23]. These data suggest that filamin A is an important link between the extracellular matrix and the cytoskeleton and is well-positioned to regulate cell spreading.

Filamin A interacts with calmodulin [24] and calpain [18], [25], two calcium-dependent proteins that participate in actin remodeling and cell spreading. Notably, filamin A also binds to calcium-sensing receptors (CaR) at the cell surface [26], [27], [28], [29], [30], [31]. As intracellular Ca2+ regulation is an important requirement for cell spreading and migration [32], [33], [34], it is conceivable that filamin A governs cell spreading by modulating intracellular [Ca2+].

We tested the hypothesis that filamin A regulates early events in cell spreading through its integrin-binding and calcium-regulating functions. Our data indicate that cortical recruitment of filamin A is strongly associated with cell spreading and exploration of topographically complex substrates, and that filamin A–β1 integrin interactions are critical elements of early steps in cell spreading. However, filamin A and intracellular calcium regulate cell spreading independently.

Section snippets

Reagents

Antibodies against human filamin A (PM6/317), vinculin (FB11), cortactin (4F11) and β1 integrin (JB1A and 12G10) were purchased from Chemicon (Temecula, CA). Anti-actin antibody (AC-15) was purchased from Sigma-Aldrich (Oakville, ON, Canada). The anti-β1 integrin (clone 4B4) antibody was obtained from Beckman-Coulter (Miami, FL). TRITC-phalloidin, fura-2/AM and BAPTA/AM were obtained from Molecular Probes (Eugene, OR). TUNEL reagents and DAPI were purchased from Roche (Mannheim, Germany).

Filamin A localizes to cell extensions of spreading human gingival fibroblasts

Smooth and beaded (0.3 beads/μm2) collagen-coated substrates provided distinctly different topographic features and opportunities for cell spreading (Figs. 1A, E). After 60 min, spreading of human gingival fibroblasts was advanced on the smooth substrate with well-defined cell extensions that included a uniform lamellipodia and isolated spike-like formations resembling long filopodia (Figs. 1B, C). By contrast, spreading and the generation of cell extensions were much more restricted on beaded

Filamin A is required for cell spreading

Filamin A is an important determinant of cell membrane stability and cell locomotion and plays a critical role in cell–matrix signaling pathways [16], [43], [44]. Earlier work on the biochemical properties of filamin identified its role in gelation of cortical actin and in cell motility [9], [45], [46]. Currently its role in early cell spreading has not yet been clearly defined. Our findings indicate that early cell spreading is marked by cortical recruitment of filamin A and that loss of

Acknowledgments

The authors gratefully acknowledge Dr. Yulia Shifrin for performing the filamin A siRNA gene silencing, Mr. Bob Chernecky for the scanning electron micrographs, Mr. Wilson Lee for his assistance with flow cytometry and the late Jaro Sodek for his helpful comments. 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

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