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At least 19 recordsLinked to original sources
PubMed1990

Actin-membrane interaction in focal adhesions.

Focal adhesions are regions of the plasma membrane where cells in tissue culture adhere strongly to the underlying extracellular matrix, and which at their cytoplasmic face serve to anchor bundles of actin microfilaments. They provide an experimental model for studying the links between the cytoskeleton and the extracellular matrix. Members of the integrin family of extracellular matrix receptors are prominent components, spanning the membrane in focal adhesions, but there is evidence that other membrane components are also needed for these structures to form. A number of proteins are concentrated at the cytoplasmic face of focal adhesions. Recent efforts have sought to determine the links between actin and the integrin cytoplasmic domains. Using in vitro binding assays, two potential bridges between actin and integrin have been identified. One involves talin, which has recently been shown to bind actin directly. The other involves the actin-binding protein, alpha-actinin, which has been found to interact with several integrins. The physiological significance of these two potential bridges between actin and integrin remains to be determined in vivo.

Actinin↗
PubMed1992

Protein kinase C involvement in focal adhesion formation.

Matrix molecules such as fibronectin can promote cell attachment, spreading and focal adhesion formation. Although some interactions of fibronectin with cell surface receptors have now been identified, the consequent activation of intracellular messenger systems by cell/matrix interactions have still to be elucidated. We show here that the kinase inhibitors H7 and HA1004 reduce focal adhesion and stress fiber formation in response to fibronectin in a dose-dependent manner, and that activators of protein kinase C can promote their formation under conditions where they do not normally form. Fibroblasts spread within 1h on substrata composed of fibronectin and formed focal adhesions by 3h, as monitored by interference reflection microscopy (IRM) and by labeling for talin, vinculin and integrin beta 1 subunits. In addition, stress fibers were visible. When cells were allowed to spread for 1h and then treated with kinase inhibitors H7 and HA1004 for 2h, IRM indicated a reduction in focal adhesion formation at concentrations where protein kinase C (PKC) should be inhibited. In contrast, focal adhesions formed normally at concentrations of these inhibitors where cyclic AMP- or cyclic GMP-dependent kinases should be inactivated. Inhibition of PKC, but not that of cyclic AMP- or cyclic GMP-dependent kinases, also prevented the formation of stress fibers and induced a dispersal of talin and vinculin, but not integrin beta 1 subunits, from small condensations present at 1h. Consistent with the reduction in focal adhesion formation when PKC was inhibited, activation of PKC by 30 minutes of treatment with phorbol esters induced focal adhesion formation in cells spread for 3h on substrata composed of the cell-binding (RGD-containing) fragment of fibronectin, while untreated cells or those treated with inactive phorbol esters did not form these structures.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Thrombospondin modulates focal adhesions in endothelial cells.

We examined the effects of thrombospondin (TSP) in the substrate adhesion of bovine aortic endothelial cells. The protein was tested both as a substrate for cell adhesion and as a modulator of the later stages of the cell adhesive process. TSP substrates supported the attachment of some BAE cells, but not cell spreading or the formation of focal adhesion plaques. In contrast, cells seeded on fibrinogen or fibronectin substrates were able to complete the adhesive process, as indicated by the formation of focal adhesion plaques. Incubation of cells in suspension with soluble TSP before or at the time of seeding onto fibronectin substrates resulted in an inhibition of focal adhesion formation. Furthermore, the addition of TSP to fully adherent cells in situ or prespread on fibronectin substrates caused a reduction in the number of cells, which were positive for focal adhesions, although there was no significant effect on cell spreading. In a dose-dependent manner, TSP reduced the number of cells with adhesion plaques to approximately 60% of control levels. The distribution of remaining adhesion plaques in TSP-treated cells was also altered: plaques were primarily limited to the periphery of cells and were not present in the central cell body, as in control cells treated with BSA. The observed effects were specific for TSP and were not observed with platelet factor 4, beta-thromboglobulin, or fibronectin. The TSP-mediated loss of adhesion plaques was neutralized by the addition of heparin, fucoidan, other heparin-binding proteins, and by a monoclonal antibody to the heparin binding domain of TSP, but not by antibodies to the core or carboxy-terminal regions of TSP. The interaction of the heparin-binding domain of TSP with cell-associated heparan sulfate appears to be an important mechanistic component for this activity of TSP. These data indicate that TSP may have a role in destabilizing cell adhesion through prevention of focal adhesion formation and by loss of preformed focal adhesions.

Animals↗
PubMed1990

Paxillin: a new vinculin-binding protein present in focal adhesions.

The 68-kD protein (paxillin) is a cytoskeletal component that localizes to the focal adhesions at the ends of actin stress fibers in chicken embryo fibroblasts. It is also present in the focal adhesions of Madin-Darby bovine kidney (MDBK) epithelial cells but is absent, like talin, from the cell-cell adherens junctions of these cells. Paxillin purified from chicken gizzard smooth muscle migrates as a diffuse band on SDS-PAGE gels with a molecular mass of 65-70 kD. It is a protein of multiple isoforms with pIs ranging from 6.31 to 6.85. Using purified paxillin, we have demonstrated a specific interaction in vitro with another focal adhesion protein, vinculin. Cleavage of vinculin with Staphylococcus aureus V8 protease results in the generation of two fragments of approximately 85 and 27 kD. Unlike talin, which binds to the large vinculin fragment, paxillin was found to bind to the small vinculin fragment, which represents the rod domain of the molecule. Together with the previous observation that paxillin is a major substrate of pp60src in Rous sarcoma virus-transformed cells (Glenney, J. R., and L. Zokas. 1989. J. Cell Biol. 108:2401-2408), this interaction with vinculin suggests paxillin may be a key component in the control of focal adhesion organization.

Animals↗
PubMed1989

The role of phosphorylation and limited proteolytic cleavage of talin and vinculin in the disruption of focal adhesion integrity.

Chemical agents which activate specific kinases were employed to disrupt the stress fiber and focal adhesion organization of cells spread on a substratum. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, promoted a rapid loss of stress fibers and focal adhesions from African green monkey kidney (BSC-1) cells. This was paralleled by an increase in the level of talin phosphorylation suggesting that this may play a role in the removal of talin from focal adhesions. Similar morphological changes were produced in the rat embryo fibroblast line (REF 52) by dibutyryl-cAMP, which stimulates protein kinase A. In contrast, however, the phosphorylation of talin was reduced in REF 52 cells when treated with dibutyryl cAMP. In untreated cells we found that the levels of vinculin phosphorylation were very low relative to the levels of talin phosphorylation and did not change following drug treatment in either cell line. Although limited proteolytic cleavage of cytoskeletal proteins represents a potential mechanism for focal adhesion disruption, we observed no proteolysis of talin or vinculin in response to either drug treatment.

Animals↗
PubMed1986

Focal adhesion sites and the removal of substratum-bound fibronectin.

Fibronectin was not removed from the substratum beneath focal adhesion sites when fibroblasts spread in serum-free medium on adsorbed fibronectin substrata, or when fibroblasts spread in serum-containing medium on covalently cross-linked fibronectin substrata. Under these conditions, there was colocalization between 140-kD fibronectin receptors and focal adhesion sites. It was concluded that removal of adsorbed fibronectin from beneath focal adhesion sites was a mechanical process that required serum. The effect of serum was nonspecific since serum could be replaced by equivalent concentrations of serum albumin, ovalbumin, or gamma globulins. Quantitative measurements indicated that the presence of proteins in the incubation medium weakens the interaction of fibronectin with the substratum, thereby allowing the adsorbed protein to be removed from the substratum at sites of high stress. After removing fibronectin from the substratum, cells reorganized this material into patches and fibrils beneath cells, and the reorganized fibronectin colocalized with fibronectin receptors. Some of the patches of fibronectin were phagocytosed. The fibronectin fibrils were observed to be in register with actin filament bundles and sometimes translocated to the upper cell surfaces. It is proposed that removal of fibronectin from beneath focal adhesion sites is an example of how cells can modify their extracellular matrices through contractile activity.

Actins↗
PubMed1992

The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors.

Actin stress fibers are one of the major cytoskeletal structures in fibroblasts and are linked to the plasma membrane at focal adhesions. rho, a ras-related GTP-binding protein, rapidly stimulated stress fiber and focal adhesion formation when microinjected into serum-starved Swiss 3T3 cells. Readdition of serum produced a similar response, detectable within 2 min. This activity was due to a lysophospholipid, most likely lysophosphatidic acid, bound to serum albumin. Other growth factors including PDGF induced actin reorganization initially to form membrane ruffles, and later, after 5 to 10 min, stress fibers. For all growth factors tested the stimulation of focal adhesion and stress fiber assembly was inhibited when endogenous rho function was blocked, whereas membrane ruffling was unaffected. These data imply that rho is essential specifically for the coordinated assembly of focal adhesions and stress fibers induced by growth factors.

3T3 Cells↗
PubMed1991

Localization of paxillin, a focal adhesion protein, to smooth muscle dense plaques, and the myotendinous and neuromuscular junctions of skeletal muscle.

In this report we have demonstrated that paxillin, a cytoskeletal protein which is present in focal adhesions, localizes in vivo to regions of cell-extracellular matrix interaction which are believed to be analogous to focal adhesions. Specifically, it is enriched in the dense plaques of chicken gizzard smooth muscle tissue and in the myotendinous junctions formed in Xenopus laevis tadpole tail skeletal muscle. In addition, paxillin was identified at the rat diaphragm neuromuscular junction. The distribution of paxillin is thus comparable to that of other focal adhesion proteins, for example, talin and vinculin, in these structures.

Animals↗
PubMed1992

pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions.

Expression of the Rous sarcoma virus-encoded oncoprotein, pp60v-src, subverts the normal regulation of cell growth, which results in oncogenic transformation. This process requires the intrinsic protein-tyrosine kinase activity of pp60v-src and is associated with an increase in tyrosine phosphorylation of a number of cellular proteins, candidate substrates for pp60v-src. We report here the isolation of a cDNA encoding a protein, pp125, that is a major phosphotyrosine-containing protein in untransformed chicken embryo cells and exhibits an increase in phosphotyrosine in pp60v-src-transformed chicken embryo cells. This cDNA encodes a cytoplasmic protein-tyrosine kinase which, based upon its predicted amino acid sequence and structure, is the prototype for an additional family of protein-tyrosine kinases. Immunofluorescence localization experiments show that pp125 is localized to focal adhesions; hence, we suggest the name focal adhesion kinase.

Amino Acid Sequence↗
PubMed1992

Focal adhesion protein-tyrosine kinase phosphorylated in response to cell attachment to fibronectin.

A homology-based cDNA cloning approach was used to identify a widely expressed protein-tyrosine kinase designated as "focal adhesion kinase" (FadK). The entire mouse FadK amino acid sequence was deduced from cDNA clones, revealing a large (119-kDa) non-membrane-spanning protein-tyrosine kinase that lacks Src-homology SH2 and SH3 domains. Immunostaining of BALB/c 3T3 fibroblasts revealed that FadK is concentrated in focal adhesions. FadK is phosphorylated on tyrosine in growing cultures of BALB/c 3T3 cells but contains little or no phosphotyrosine in cells detached by trypsinization. The tyrosine-phosphorylated state is regained within minutes when the cells are replated onto fibronectin. Activation of FadK may be an important early step in intracellular signal transduction pathways triggered in response to cell interactions with the extracellular matrix.

Amino Acid Sequence↗
PubMed1988

Platelet glycoprotein IIb/IIIa complex in cultured cells. Localization in focal adhesion sites in spreading HEL cells.

A panel of mouse monoclonal antibodies (MoAbs) was raised that react with platelet glycoproteins (GP) IIb or IIIa. On immunofluorescence, the MoAbs reacted with 30% to 40% of the human erythroleukemia (HEL) cells. When the HEL cells were induced to spread on fibronectin in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), the MoAbs reacted with the focal adhesion sites. Only some of the GPIIIa MoAbs reacted with cells other than platelets, megakaryocytes, or HEL cells, and these showed focal adhesion sites in cultured human endothelial cells, fibroblasts, and epithelial cells from normal kidney tubules. They did not react, however, with transformed fibroblasts, fibrosarcoma cells, cultured cells from hypernephromas, or cultured human amnion epithelial cells. The results suggest that the platelet-type GPIIb/IIIa complex is only expressed in cells showing an ability to define megakaryoblastic differentiation. Localization of the GPIIb/IIIa complex at the induced focal adhesion sites in HEL cells and localization of the GPIIIa-like molecules in other cells suggest their direct role in the adhesion process and in the actomyocin organization of adherent cells.

Antibodies, Monoclonal↗
PubMed1991

Interleukin 1 beta induces rapid phosphorylation and redistribution of talin: a possible mechanism for modulation of fibroblast focal adhesion.

The majority of interleukin 1 (IL-1) receptors in human fibroblasts has been shown to be localized at focal adhesions. This study describes rapid alterations caused by IL-1 beta/IL-1-receptor interaction at these sites. Fibroblast monolayers, incubated with IL-1 beta and prepared for electron microscopy, showed successive loss of cell-substratum contact and fewer and less-pronounced processes. Immunocytochemistry revealed loss and redistribution of the talin staining initially observed after 5-15 min of IL-1 beta incubation. Similarly, the cytoskeleton showed a decrease in staining and a disorganization starting from 15 to 30 min after IL-1 addition, whereas extracellular fibronectin appeared largely unaffected. Prelabeling with [32P]phosphate showed a 2- to 3-fold increase in the level of talin phosphorylation, peaking at 15 min. Phospho amino acid analyses revealed a higher level of serine and threonine phosphorylation. The data suggest that the action of IL-1 beta on fibroblasts may be partially mediated by direct phosphorylation of talin via activation of a protein serine/threonine kinase, leading to changes in transmembrane linkage proteins and the cytoskeleton. Such alterations at focal adhesions may provide a mechanism by which IL-1 can rapidly modulate cell-matrix interactions during inflammation and wound healing.

Cell Adhesion↗
PubMed1991

Focal adhesion integrity is downregulated by the alternatively spliced domain of human tenascin.

Tenascin, together with thrombospondin and SPARC, form a family of matrix proteins that, when added to bovine aortic endothelial cells, caused a dose-dependent reduction in the number of focal adhesion-positive cells to approximately 50% of albumin-treated controls. For tenascin, a maximum response was obtained with 20-60 micrograms/ml of protein. The reduction in focal adhesions in tenascin-treated spread cells was observed 10 min after addition of the adhesion modulator, reached the maximum by 45 min, and persisted for at least 4 h in the continued presence of tenascin. This effect was fully reversible, was independent of de novo protein synthesis, and was neutralized by a polyclonal antibody to tenascin. Monoclonal antibodies to specific domains of tenascin (mAbs 81C6 and 127) were used to localize the active site to the alternatively spliced segment of tenascin. Furthermore, a recombinant protein corresponding to the alternatively spliced segment (fibronectin type III domains 6-12) was expressed in Escherichia coli and was active in causing loss of focal adhesions, whereas a recombinant form of a domain (domain 3) containing the RGD sequence had no activity. Chondroitin-6-sulfate effectively neutralized tenascin activity, whereas dermatan sulfate and chondroitin-4-sulfate were less active and heparan sulfate and heparin were essentially inactive. Studies suggest that galactosaminoglycans neutralize tenascin activity through interactions with cell surface molecules. Overall, our results demonstrate that tenascin, thrombospondin, and SPARC, acting as soluble ligands, are able to provoke the loss of focal adhesions in well-spread endothelial cells.

Actins↗
PubMed2025

The downregulation of ubiquitin-specific peptidase 2 indicates a poor prognosis and promotes the progression of gastric cancer through focal adhesion and ECM pathway signaling.

Gastric cancer ranks among the most prevalent forms of cancer worldwide. Recent rapid advancements in diagnostic methods, neoadjuvant or adjuvant therapies, and surgical procedures have significantly improved survival rates for patients with gastric cancer. Nonetheless, these benefits have not yet reached the majority of individuals affected. Previous research has indicated that USP2, a component of the ubiquitin system, plays a crucial role in reshaping the proteome and enhancing the prognosis of diseases. However, the current understanding of USP2 expression and the associated pathways in gastric cancer remains unclear. The differential expression of USP2 was examined in pan-cancer, with a particular focus on its expression in gastric cancer cells and patients. Additionally, the impact of USP2 on the proliferation, migration, and apoptosis of gastric cancer cells was explored via CCK8, transwell, and invasion assays. RNA sequencing was employed to investigate pathways associated with USP2, and RT-qPCR and western blotting were utilized to confirm the expression of related pathway genes and proteins. The prognostic value of a model derived from USP2 expression was assessed and validated. USP2 expression was significantly reduced in gastric cancer cells and patient samples (p&#x2009;<&#x2009;0.05). Patients with low USP2 expression are primarily associated with genetic variations, neoantigen loads, microsatellite instability (MSI) scores, and immune cell infiltration (p&#x2009;<&#x2009;0.05). The overexpression of USP2 suppresses proliferation, migration, and cell cycle progression while enhancing apoptosis in GC cells. Concurrently, we identified 865 genes whose expression was downregulated. KEGG and GSEA enrichment analyses revealed significant suppression of the focal adhesion and ECM receptor interaction pathways following USP2 overexpression. A genomic model derived from USP2 was constructed and validated for its reliability in predicting patient prognosis. The expression of USP2 was positively correlated with sensitivity to small-molecule drugs, including entinostat, SB590885, and PF-562,271. USP2 acts as a negative regulator of gastric cancer progression. Consequently, USP2 has the potential to be utilized as a therapeutic target to improve the clinical prognosis and survival rates of patients.

Humans↗
PubMed1990

Adhesions, focal sclerosis, protein crescents, and capsular lesions in membranous nephropathy.

All biopsies of membranous glomerulopathy collected between 1965 and 1988 (n = 198) were examined for the presence of adhesions, focal sclerosis, protein crescents, capsular drops, and other capsular lesions. These were observed with a frequency related to the number of glomeruli in the biopsy and the number of sections examined. A peculiar interrelation between these lesions was found. In the 63 cases with sufficient clinical follow-up, the capsular lesions were associated with a lower incidence of remission of the disease, and with more proteinuria and a higher serum creatinine at the last follow-up. It is hypothesized that focal detachment of podocytes may result in the formation of a protein crescent if a plasma-like filtrate is 'injected' between the epithelium of Bowman's capsule and its basement membrane. The detachment of the epithelium may lead to focal sclerosis and the formation of adhesions. The capsular drop is thought to be a final stage of inspissation of the protein crescent.

Adult↗
PubMed1990

Distinct functions for integrins alpha 3 beta 1 in focal adhesions and alpha 6 beta 4/bullous pemphigoid antigen in a new stable anchoring contact (SAC) of keratinocytes: relation to hemidesmosomes.

Basal cells of stratified epidermis are anchored to the basement membrane zone (BMZ) of skin via hemidesmosomes. We previously identified integrin alpha 3 beta 1, in focal adhesions (FAs), of cultured human keratinocytes (HFKs) as a mediator of HFK adhesion to secreted BMZ-like extracellular matrix (ECM; Carter, W.G., E.A. Wayner, T.S. Bouchard, and P. Kaur. 1990. J. Cell Biol. 110: 1387-1404). Here, we have examined the relation of integrins alpha 6 beta 4 and alpha 3 beta 1, to bullous pemphigoid antigen (BPA), a component of hemidesmosomes. We conclude that alpha 6 beta 4 in HFKs localizes in a new stable anchoring contact (SAC) that cooperates with alpha 3 beta 1-FAs to mediate adhesion to ECM, based on the following. (a) Comparison of secreted ECM, with exogenous laminin, fibronectin and collagen identified ECM as the preferred ligand for HFK adhesion and spreading and for formation of both alpha 6 beta 4-SACs and alpha 3 beta 1-FAs. (b) Inhibition of HFK adhesion with combined anti-alpha 3 beta 1 (P1B5) and anti-alpha 6 beta 4 (GoH3) antibodies indicated that both receptors were functional in adhesion to ECM while alpha 3 beta 1 played a dominant role in spreading. (c) alpha 6 beta 4 colocalized with BPA in SACs that were proximal to but excluded from FAs. Both alpha 6 beta 4-SACs and alpha 3 beta 1-FAs were in contact with the adhesion surface as indicated by antibody exclusion and interference reflection microscopy. (d) In contrast to alpha 3 beta 1-FAs, alpha 6 beta 4-SACs were present only in nonmotile cells, not associated with stress fibers, and were relatively stable to detergents and urea, suggesting a nonmotile, or anchoring function for SACs and motility functions for alpha 3 beta 1-FAs. (e) alpha 6 beta 4 formed a detergent-insoluble complex with exogenous ECM in an affinity isolation procedure, confirming the ability of an unidentified ECM ligand to interact with alpha 6 beta 4. (f) We suggest that alpha 6 beta 4/BPA-SACs in culture restrict migration of HFKs on ECM while alpha 3 beta 1-FAs form dynamic adhesions in spreading and migrating cells. alpha 6 beta 4/BPA-SACs in culture bear functional and compositional similarities to hemidesmosomes in skin.

Autoantigens↗
PubMed1991

Identification of cytoskeletal, focal adhesion, and cell adhesion proteins in growth cone particles isolated from developing chick brain.

Growth cones are intimately involved in determining the direction and extent of neurite elongation during development. They are able to monitor their environment and respond to it by undergoing directed motility. We have isolated a fraction enriched in growth cone particles from embryonic chick brain. Assayed by immunoblots, this fraction is enriched in GAP-43, and contains the cytoskeletal proteins actin, myosin II, neurofilament protein, tubulin, kinesin, and dynamin. All of the major components of focal adhesions are also present: alpha-actinin, vinculin, talin, and integrin. In addition to integrin, we also identify the cell adhesion molecules A-CAM, L1, fibronectin, and laminin in these particles. This preparation of isolated growth cone particles may be a useful model system for studying growth cone adhesion and motility.

Actinin↗