A Small Fibronectin-mimicking Protein from Bacteria Induces Cell Spreading and Focal Adhesion Formation

Abstract

Eukaryotic cell adhesion is a fundamental process in tissue development, homeostasis, and disease and is mediated by specific interactions of cell surface receptors with extracellular matrix (ECM) 2 proteins (1-5). The ECM is a meshwork of fibrillar and nonfibrillar components assembled into complex structures such as basement membranes. The latter provide a scaffold for cell adhesion, spreading, and migration. ECM regulates numerous cell functions by activating multiple signaling pathways at the adhesion sites. ECMs, composed of collagens, laminins, and other glycoproteins such as fibronectin (FN), serve as substrates for different adhesion molecules including the integrin family of transmembrane receptors. The assembly of ECM components into functional supramolecular modules is highly regulated (3-7). FN matrix assembly alone is a dynamic cell-driven process in which the soluble FN molecules assemble into insoluble fibrillar polymeric ECM structures (8).FN and integrin receptors play crucial roles in a variety of morphogenetic processes, which are regulated by processes termed outside-in and inside-out signaling cascades (3-5). Deregulation of integrin and FN functions associates with disease development including chronic inflammation, heart failure, cancer, and metastasis (7, 9 -11). The outside-in signaling triggered by ligation of integrin receptors with FN and other ECM components results in the reorganization of cytoskeletal and signaling molecules into complexes of more than 90 proteins (9 -13). This occurs by synergistic processes dependent on integrin aggregation and occupancy, as well as tyrosine phosphorylation. Integrins also cooperate with growth factor receptors such as epidermal growth factor receptor (EGFR) to enhance signaling (14).FN consists of multiple domains (classified types I-III) that show binding specificities for specific cell membrane receptors, collagen, fibrin, and heparin. FN alone is sufficient to induce highly efficient spreading of many mammalian cell types including fibroblast and epithelial cells in vitro. An important functional unit of FN is its RGD tripeptide motif, which acts in

Effects of exogenous ganglioside GM1 on different stages of cell spreading studied by directly quantifying spreading rate

“…The problem is that cells start spreading very early on upon attachment and that almost all cells spread out within 1-3 h as previously reported (Tamura et al, 1998;Tegtmeyer et al, 2010). In our study, more than 60% of the HUVECs changed from a spherical state into a spread state within 0.5 h and more than 90% of the HUVECs within 2 h (Figures 2A, 3, 4 and 5A).…”

Helicobacter pylori controls NLRP3 expression by regulating hsa-miR-223-3p and IL-10 in cultured and primary human immune cells

“…Then the whole-cell extracts were pre-cleared with Protein-G sepharose beads (Amersham Pharmacia, Munich, Germany) for 2 h at 4 C and beads were removed by centrifugation at 2400 g for 5 min at 4 C. The Abs against human NLRP3 (Santa Cruz Biotech, Santa Cruz, CA, USA) were added to pre-cleared lysate and incubated for 16 h at 4 C in a rotatory shaker at low speed. The immune complexes were precipitated using protein-G sepharose beads by incubating for 4 h at 4 C and centrifugation at 2400 g for 5 min at 4 C. 39,40 The protein-G sepharose bead-bound immune complexes were washed three times with PBS (pH 7.4) and then added 2 Â SDS lysis buffer and boiled for 5 min before using for SDS-PAGE and Western blot analysis. A portion of the remaining lysate mixed with equal amount of 2 Â SDS lysis buffer and boiled for 5 min, which is used for detection of loading control GAPDH protein.…”

Helicobacter pylori-Induced Disruption of Monolayer Permeability and Proinflammatory Cytokine Secretion in Polarized Human Gastric Epithelial Cells

“…It is the direct contact of the T4SS with the epithelial cell membrane, however, that is believed to be primarily responsible for induction of the secretion of interleukin-8 (IL-8) and other host immune response events (14)(15)(16)(17). Epithelial ␣5␤1 integrin has been implicated as one of the host receptors for this activity (18)(19)(20)(21). Expression of these integrins, however, is limited to the basolateral membranes.…”