Flightless I is a focal adhesion-associated actin-capping protein that regulates cell migration

Matrix Dynamics Group, University of Toronto, Toronto, Ontario, Canada.
The FASEB Journal (Impact Factor: 5.04). 05/2012; 26(8):3260-72. DOI: 10.1096/fj.11-202051
Source: PubMed


The role of adhesion-associated actin-binding proteins in cell migration is not well defined. In mouse fibroblasts we screened for focal adhesion-associated proteins that were isolated with collagen-coated beads and detected by tandem mass spectrometry. We identified flightless I (FliI) as an actin-binding protein in focal adhesion fractions, which was verified by immunoblotting. By confocal microscopy most FliI was distributed throughout the cytosol and in focal adhesions. By sedimentation assays and in vitro binding assays, we found that FliI associates with actin filaments and actin monomers. Assays using purified proteins showed that FliI inhibits actin polymerization and caps but does not sever actin filaments. Cells with FliI knockdown or cells overexpressing FliI migrated more or less rapidly, respectively, than wild-type controls. Compared with controls, cells with FliI knockdown were less adherent than wild-type cells, exhibited reduced numbers of focal adhesions containing activated β1 integrins and vinculin, and exhibited increased incorporation of actin monomers into nascent filaments at focal adhesions. These data indicate that FliI regulates cell migration through its localization to focal adhesions and its ability to cap actin filaments, which collectively affect focal adhesion maturation.

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    • "Pull-down assays have been described (Mohammad et al., 2012). Briefly, recombinant FliI proteins bound to Sepharose beads (GST- GLD 1–6, GST-GLD 2–6, or GST-LRR) were incubated with purified myosin rods (from A.B.). "
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    ABSTRACT: We examined the role of actin capping protein flightlessI (FliI) in collagen remodeling by mouse fibroblasts. FliI overexpressing cells exhibited reduced spreading on collagen but formed elongated protrusions, which stained for myosin10 and fascin, and penetrated pores of collagen-coated membranes. Inhibition of Cdc42 blocked formation of cell protrusions. In FliI knockdown cells, transfection with constitutively active Cdc42 did not enable protrusion formation. FliI overexpressing cells displayed increased uptake and degradation of exogenous collagen and strongly compacted collagen fibrils, which was blocked by blebbistatin. Mass spectrometry analysis of FliI immunoprecipitates showed that FliI associated with non-muscle myosin IIA (NMMIIA), which was confirmed by immunoprecipitation. GFP-FliI colocalized with NMMIIA at cell protrusions. Purified FliI containing gelsolin-like domains (GLD) 1-6 capped actin filaments efficiently whereas FliI GLD 2-6 did not. Binding assays showed strong interaction of purified FliI protein (GLD 1-6) with the rod domain of NMMIIA (kD = 0.146 μM) whereas FliI GLD 2-6 showed lower binding affinity (kD = 0.8584 μM). Cells expressing FliI GLD 2-6 exhibited fewer cell extensions, did not colocalize with NMMIIA and showed reduced collagen uptake compared with cells expressing FliI GLD 1-6. We conclude that FliI interacts with NMMIIA to promote cell extension formation that enables collagen remodeling in fibroblasts. © 2015 by The American Society for Cell Biology.
    Preview · Article · Apr 2015 · Molecular biology of the cell
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    • "Cells with reduced levels of gelsolin migrate slower, while a reduction in Flii levels leads to an increase in migration.5 Both fibroblasts and keratinocytes, cells typically found in wounds, that have less Flii migrate faster in vitro and in vivo and vice versa.7,10 The impaired wound healing seen in mice overexpressing Flii could in part be due to the ability of Flii to inhibit cell adhesion and migration. "
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    ABSTRACT: Intracellular Flightless I (Flii), a gelsolin family member, has been found to have roles modulating actin regulation, transcriptional regulation and inflammation. In vivo Flii can regulate wound healing responses. We have recently shown that a pool of Flii is secreted by fibroblasts and macrophages, cells typically found in wounds, and its secretion can be upregulated upon wounding. We show that secreted Flii can bind to the bacterial cell wall component lipopolysaccharide and has the potential to regulate inflammation. We now show that secreted Flii is present in both acute and chronic wound fluid.
    Full-text · Article · Nov 2012 · Communicative & integrative biology
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    ABSTRACT: Gelsolin superfamily members are Ca(2+) -dependent, multi-domain regulators of the actin cytoskeleton. Calcium binding activates gelsolin by inducing molecular gymnastics (large-scale conformational changes) that expose actin interaction surfaces by releasing a series of latches. A specialized tail latch has distinguished gelsolin within the superfamily. Active gelsolin exhibits actin filament severing and capping, and actin monomer sequestering activities. Here, we analyze a combination of sequence, structural, biophysical and biochemical data to assess whether the molecular plasticity, regulation and actin-related properties of gelsolin are also present in other superfamily members. We conclude that all members of the superfamily will be able to transition between a compact conformation and a more open form, and that most of these open forms will interact with actin. Supervillin, which lacks the severing domain 1 and the F-actin binding-site on domain 2, is the clear exception. Eight calcium-binding sites are absolutely conserved in gelsolin, adseverin, advillin and villin, and compromised to increasing degrees in CapG, villin-like protein, supervillin and flightless I. Advillin, villin and supervillin each contain a potential tail latch, which is absent from CapG, adseverin and flightless I, and ambiguous in villin-like protein. Thus, calcium regulation will vary across the superfamily. Potential novel isoforms of the superfamily suggest complex regulation at the gene, transcript and protein levels. We review animal, clinical and cellular data that illuminate how the regulation of molecular flexibility in gelsolin-like proteins permits cells to exploit the force generated from actin polymerization to drive processes such as cell movement in health and disease.
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