Focal adhesions (FAs) are discrete plasma membrane-associated adhesive organelles that play dual roles in cell force transduction and signaling. FAs consist of clustered transmembrane heterodimeric integrin extracellular matrix (ECM) receptors and a large number of cytoplasmic proteins that collectively form thin plaques linking the ECM to actin filament bundles of the cytoskeleton. FAs are complex organelles that can change their composition in response to biochemical or mechanical cues. These compositional differences may underlie the ability of FAs to mediate an array of important cell functions including adhesion, signaling, force transduction, and regulation of the cytoskeleton. These functions contribute to the physiological processes of the immune response, development, and differentiation. However, linking FA composition to FA function has been difficult since there has been no method to isolate intact FAs reproducibly and determine their composition. We report here a new method for isolating FA structures in cultured cells distinct from cytoplasmic, nuclear, and internal membranous organellar components of the cell. We provide protocols for validation of the fractionation by immunofluorescence and immunoblotting, procedures for preparing the isolated FAs for mass spectrometric proteomic analysis, tips on data interpretation and analysis, and an approach for comparing FA composition in cells in which small GTPase signaling is perturbed.
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"Focal adhesion isolation was performed following the protocol described in Kuo et al . ( Kuo et al . , 2012 ) . Briefly , cells were rinsed with PBS and incubated with TEA buffer ( 0 . 2 M triethanolamine , pH 8 . 0 ) for 5 min . To apply hydrodynamic force , the cells were rinsed with PBS for 10 s using a Waterpik dental flosser set at 2 ( Waterpik , Reigate , UK ) . After another wash with PBS , the remaining attached focal adhesions were f"
[Show abstract][Hide abstract] ABSTRACT: Eps8 is an actin regulatory scaffold protein increased in Squamous Cell Carcinoma (SCC) cells. It forms a complex with both Focal Adhesion Kinase (FAK) and c-Src in SCC cells derived from the DMBA/TPA model of skin carcinogenesis. Here, we describe two new roles for Eps8. Firstly, it controls the spatial distribution of active c-Src in a FAK-dependent manner. Specifically, Eps8 participates in, and regulates, a biochemical complex with c-Src and drives c-Src's trafficking to autophagic structures that SCC cells use to cope with high levels of active c-Src when FAK is absent. Secondly, when FAK is expressed in SCC cells, so tethering active c-Src at focal adhesion complexes, Eps8 is also recruited to focal adhesions and is required for FAK-dependent polarization and invasion. Therefore, Eps8 is a critical mediator of Src/FAK-regulated processes; it participates in specific biochemical complexes and promotes actin re-arrangements that determine c-Src's spatial localization and Src/FAK functions in invasive migration.
Journal of Cell Science 10/2014; DOI:10.1242/jcs.157560 · 5.43 Impact Factor
"To further characterize the effects of GEF-H1 in Dex-stimulated FAs, we analyzed the composition and abundance of proteins in FAs from non-silencing and GEF-H1-silencing MSC-3A6 cells (supplementary material Fig. S2A). Serum-starved non-silencing and GEF-H1-silencing MSC-3A6 cells were treated with Dex (0.1 µM, 6 h) and hypotonically shocked to isolate FAs using the FA isolation method (Kuo et al., 2011; Kuo et al., 2012), which has been previously demonstrated to preserve the native FA organization and size. The isolated FA fractions were subject to liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis. "
[Show abstract][Hide abstract] ABSTRACT: Focal adhesions (FAs) undergo maturation culminating in size and composition changes that modulate adhesion, cytoskeleton remodeling and differentiation. While it is well-recognized that stimuli for osteogenesis of mesenchymal stem cells (MSCs) drive FA maturation, actin organization, and stress-fiber polarization, the extent to which FA-mediated signals regulated by the FA protein composition specifies MSC commitment remains largely unknown. Here we demonstrate that, upon dexamethasone (osteogenic induction) treatment, guanine nucleotide exchange factor-H1 (GEF-H1) is significantly enriched in FAs. Perturbation of GEF-H1 inhibits FA formation, anisotropic stress-fiber orientation and MSC osteogenesis in an actomyosin contractility-independent manner. To determine the role of GEF-H1 in MSC osteogenesis, we explore the GEF-H1-modulated FA proteome that reveals non-muscle myosin-II heavy chain-B (NMIIB) as a target of GEF-H1 in FAs. Inhibition of targeting NMIIB into FAs suppresses FA formation, stress-fiber polarization, cell stiffness and osteogenic commitments in MSCs. Our data demonstrate FA signaling in specifying MSC commitment.
Development 08/2014; 127(19). DOI:10.1242/jcs.150227 · 6.46 Impact Factor
"Emerging approaches might include assessing changes in FA and ECM composition, specifically at the cell-surface interface, by employing mass-spectroscopy and proteomic strategies , . As recently shown, FA sites are extremely complex and can consist of hundreds of different proteins which may change in abundance in response to inhibitors and stress , . This flexibility may permit the maintenance of FAs, CCA and adhesion during periods of stress. "
[Show abstract][Hide abstract] ABSTRACT: Integrins, focal adhesions, the cytoskeleton and the extracellular matrix, form a structural continuum between the external and internal environment of the cell and mediate the pathways associated with cellular mechanosensitivity and mechanotransduction. This continuum is important for the onset of muscle tissue generation, as muscle precursor cells (myoblasts) require a mechanical stimulus to initiate myogenesis. The ability to sense a mechanical cue requires an intact cytoskeleton and strong physical contact and adhesion to the microenvironment. Importantly, myoblasts also undergo reorientation, alignment and large scale remodeling of the cytoskeleton when they experience mechanical stretch and compression in muscle tissue. It remains unclear if such dramatic changes in cell architecture also inhibit physical contact and adhesion with the tissue microenvironment that are clearly important to myoblast physiology. In this study, we employed interference reflection microscopy to examine changes in the close physical contact of myoblasts with a substrate during induced remodeling of the cytoarchitecture (de-stabilization of the actin and microtubule cytoskeleton and inhibition of acto-myosin contractility). Our results demonstrate that while each remodeling pathway caused distinct effects on myoblast morphology and sub-cellular structure, we only observed a ∼13% decrease in close physical contact with the substrate, regardless of the pathway inhibited. However, this decrease did not correlate well with changes in cell adhesion strength. On the other hand, there was a close correlation between cell adhesion and β1-integrin expression and the presence of cell-secreted fibronectin, but not with the presence of intact focal adhesions. In this study, we have shown that myoblasts are able to maintain a large degree of physical contact and adhesion to the microenvironment, even during shot periods (<60 min) of large scale remodeling and physiological stress, which is essential to their in-vivo functionality.
PLoS ONE 09/2012; 7(9):e45329. DOI:10.1371/journal.pone.0045329 · 3.23 Impact Factor