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1
Pankov, Roumen, Edna Cukierman, Ben-Zion Katz, Kazue Matsumoto, Diane C. Lin, Shin Lin, Cornelia Hahn, and Kenneth M. Yamada. "Integrin Dynamics and Matrix Assembly." Journal of Cell Biology 148, no. 5 (March 6, 2000): 1075–90. http://dx.doi.org/10.1083/jcb.148.5.1075.
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Fibronectin matrix assembly is a multistep, integrin-dependent process. To investigate the role of integrin dynamics in fibronectin fibrillogenesis, we developed an antibody-chasing technique for simultaneous tracking of two integrin populations by different antibodies. We established that whereas the vitronectin receptor αvβ3 remains within focal contacts, the fibronectin receptor α5β1 translocates from focal contacts into and along extracellular matrix (ECM) contacts. This escalator-like translocation occurs relative to the focal contacts at 6.5 ± 0.7 μm/h and is independent of cell migration. It is induced by ligation of α5β1 integrins and depends on interactions with a functional actin cytoskeleton and vitronectin receptor ligation. During cell spreading, translocation of ligand-occupied α5β1 integrins away from focal contacts and along bundles of actin filaments generates ECM contacts. Tensin is a primary cytoskeletal component of these ECM contacts, and a novel dominant-negative inhibitor of tensin blocked ECM contact formation, integrin translocation, and fibronectin fibrillogenesis without affecting focal contacts. We propose that translocating α5β1 integrins induce initial fibronectin fibrillogenesis by transmitting cytoskeleton-generated tension to extracellular fibronectin molecules. Blocking this integrin translocation by a variety of treatments prevents the formation of ECM contacts and fibronectin fibrillogenesis. These studies identify a localized, directional, integrin translocation mechanism for matrix assembly.
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Balasubramanian, Lavanya, Abu Ahmed, Chun-Min Lo, James S. K. Sham, and Kay-Pong Yip. "Integrin-mediated mechanotransduction in renal vascular smooth muscle cells: activation of calcium sparks." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 293, no. 4 (October 2007): R1586—R1594. http://dx.doi.org/10.1152/ajpregu.00025.2007.
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Integrins are transmembrane heterodimeric proteins that link extracellular matrix (ECM) to cytoskeleton and have been shown to function as mechanotransducers in nonmuscle cells. Synthetic integrin-binding peptide triggers Ca2+ mobilization and contraction in vascular smooth muscle cells (VSMCs) of rat afferent arteriole, indicating that interactions between the ECM and integrins modulate vascular tone. To examine whether integrins transduce extracellular mechanical stress into intracellular Ca2+ signaling events in VSMCs, unidirectional mechanical force was applied to freshly isolated renal VSMCs through paramagnetic beads coated with fibronectin (natural ligand of α5β1-integrin in VSMCs). Pulling of fibronectin-coated beads with an electromagnet triggered Ca2+ sparks, followed by global Ca2+ mobilization. Paramagnetic beads coated with low-density lipoprotein, whose receptors are not linked to cytoskeleton, were minimally effective in triggering Ca2+ sparks and global Ca2+ mobilization. Preincubation with ryanodine, cytochalasin-D, or colchicine substantially reduced the occurrence of Ca2+ sparks triggered by fibronectin-coated beads. Binding of VSMCs with antibodies specific to the extracellular domains of α5- and β1-integrins triggered Ca2+ sparks simulating the effects of fibronectin-coated beads. Preincubation of microperfused afferent arterioles with ryanodine or integrin-specific binding peptide inhibited pressure-induced myogenic constriction. In conclusion, integrins transduce mechanical force into intracellular Ca2+ signaling events in renal VSMCs. Integrin-mediated mechanotransduction is probably involved in myogenic response of afferent arterioles.
3
Kim, H. J., D. H. Ingbar, and C. A. Henke. "Integrin mediation of type II cell adherence to provisional matrix proteins." American Journal of Physiology-Lung Cellular and Molecular Physiology 271, no. 2 (August 1, 1996): L277—L286. http://dx.doi.org/10.1152/ajplung.1996.271.2.l277.
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Lung injury causes alveolar type I epithelial cell death, basement membrane denudation, and alveolar flooding with serum fibronectin and fibrinogen. For successful restoration of normal architecture, the epithelium must be regenerated from progenitor type II alveolar cells. Using adhesion assays, we examined whether type II alveolar cells adhere to the provisional matrix proteins fibronectin, fibrinogen, and fibrin, and whether integrins mediate this adherence. Rat type II cells adhered to fibronectin, vitronectin, fibrinogen, and fibrin. Synthetic RGD (arginine-glycine-aspartic acid) peptide blocked this adhesion. Flow cytometry and Western analysis indicated that type II cells expressed beta 1- and alpha v beta 3-integrins. Anti-beta 1-and anti-alpha v beta 3-integrin antibodies blocked type II cell adhesion to fibronectin and to fibronectin and fibrinogen, respectively. In summary, type II cells adhered to fibronectin, fibrinogen, and fibrin, and adhesion was partially mediated by integrins. This study provides the first evidence of type II cell adhesion to fibrin gels and vitronectin, beta 1- and alpha v beta 3-integrin mediation of type II cell adhesion, and the presence of the alpha v beta 3-integrin on type II epithelial cells.
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Akimov, Sergey S., Dmitry Krylov, Laurie F. Fleischman, and Alexey M. Belkin. "Tissue Transglutaminase Is an Integrin-Binding Adhesion Coreceptor for Fibronectin." Journal of Cell Biology 148, no. 4 (February 21, 2000): 825–38. http://dx.doi.org/10.1083/jcb.148.4.825.
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The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of β1 and β3 subfamilies, but not with β2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading.
5
Orr, A. Wayne, Mark H. Ginsberg, Sanford J. Shattil, Hans Deckmyn, and Martin A. Schwartz. "Matrix-specific Suppression of Integrin Activation in Shear Stress Signaling." Molecular Biology of the Cell 17, no. 11 (November 2006): 4686–97. http://dx.doi.org/10.1091/mbc.e06-04-0289.
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Atherosclerotic plaque develops at sites of disturbed flow. We previously showed that flow activates endothelial cell integrins, which then bind to the subendothelial extracellular matrix (ECM), and, in cells on fibronectin or fibrinogen, trigger nuclear factor-κB activation. Additionally, fibronectin and fibrinogen are deposited into the subendothelial ECM at atherosclerosis-prone sites at early times. We now show that flow activates ECM-specific signals that establish patterns of integrin dominance. Flow induced α2β1 activation in cells on collagen, but not on fibronectin or fibrinogen. Conversely, α5β1 and αvβ3 are activated on fibronectin and fibrinogen, but not collagen. Failure of these integrins to be activated on nonpermissive ECM is because of active suppression by the integrins that are ligated. Protein kinase A is activated specifically on collagen and suppresses flow-induced αvβ3 activation. Alternatively, protein kinase Cα is activated on fibronectin and mediates α2β1 suppression. Thus, integrins actively cross-inhibit through specific kinase pathways. These mechanisms may determine cellular responses to complex extracellular matrices.
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Wu, C., A. E. Chung, and J. A. McDonald. "A novel role for alpha 3 beta 1 integrins in extracellular matrix assembly." Journal of Cell Science 108, no. 6 (June 1, 1995): 2511–23. http://dx.doi.org/10.1242/jcs.108.6.2511.
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To study the biological role of alpha 3 beta 1 integrins in cell adhesion, migration, and in the deposition of extracellular matrix, we stably expressed the human alpha 3 integrin subunit in the alpha 4, alpha 5 integrin deficient CHO cell line B2. The expression of alpha 3 beta 1 integrins enhanced cell adhesion on entactin (also known as nidogen), but not on fibronectin. Using recombinant GST-fusion proteins that span the entire length of the entactin molecule, we located cell adhesive activity to the G2 domain of entactin. These results suggest that the alpha 3 beta 1 integrin functions as an adhesion receptor interacting with the G2 domain of entactin. On the other hand, the expression of alpha 3 beta 1 integrins did not confer the ability to migrate on entactin. Strikingly, the expression of alpha 3 beta 1 dramatically increased the deposition of entactin and fibronectin into the pericellular matrix. This was accompanied by increased binding activity of the 29 kDa amino-terminal domain of fibronectin. Thus, similar to alpha 5 beta 1 integrins, alpha 3 beta 1 integrins can play an important role in modulating the assembly of pericellular matrices. However, unlike fibronectin deposition supported by alpha 5 beta 1, alpha 3 beta 1 supported fibronectin deposition into pericellular matrix was not inhibited by antibodies binding to the RGD containing cell adhesion domain of fibronectin, demonstrating that the two processes are mechanistically distinct. The role of alpha 3 beta 1 in pericellular matrix assembly potentially implicates this receptor in the assembly and/or recognition of entactin-containing pericellular matrices, an observation consistent with its apparent role in the renal glomerulus of the mammalian kidney.
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Watt, F. M., M. D. Kubler, N. A. Hotchin, L. J. Nicholson, and J. C. Adams. "Regulation of keratinocyte terminal differentiation by integrin-extracellular matrix interactions." Journal of Cell Science 106, no. 1 (September 1, 1993): 175–82. http://dx.doi.org/10.1242/jcs.106.1.175.
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Suspension-induced terminal differentiation of human epidermal keratinocytes can be inhibited by fibronectin through binding to the alpha 5 beta 1 integrin. We have investigated the effect of fibronectin on expression of integrins and proteins of the actin cytoskeleton and have explored the nature of the differentiation stimulus by testing different combinations of anti-integrin monoclonal antibodies or extracellular matrix proteins in the suspension assay. Fibronectin prolonged cell surface expression of beta 1 integrins but did not overcome the inhibition of intracellular transport of integrins that occurs when keratinocytes are placed in suspension. Fibronectin did not prevent the suspension-induced decline in the level of mRNAs encoding the beta 1 integrin subunit, actin, filamin and alpha-actinin; furthermore, the inhibition of terminal differentiation did not depend on the state of assembly of microfilaments or microtubules. Terminal differentiation could be partially inhibited by an adhesion-blocking monoclonal antibody to the beta 1 integrin subunit or by a combination of adhesion blocking antibodies recognising the alpha subunits that associate with beta 1 (alpha 2, alpha 3 and alpha 5). Although laminin and type IV collagen do not inhibit terminal differentiation individually, they were inhibitory when added to cells in combination with a low concentration of fibronectin. We conclude that the proportion of keratinocyte beta 1 integrins occupied by ligand can regulate the initiation of terminal differentiation independently of the state of assembly of the actin cytoskeleton.
8
Sottile, J., D. C. Hocking, and K. J. Langenbach. "Fibronectin polymerization stimulates cell growth by RGD-dependent and -independent mechanisms." Journal of Cell Science 113, no. 23 (December 1, 2000): 4287–99. http://dx.doi.org/10.1242/jcs.113.23.4287.
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Many aspects of cell behavior are regulated by cell-extracellular matrix interactions, including cell migration and cell growth. We previously showed that the addition of soluble fibronectin to collagen-adherent fibronectin-null cells enhances cell growth. This growth-promoting effect of fibronectin depended upon the deposition of fibronectin into the extracellular matrix; occupancy and clustering of fibronectin-binding integrins was not sufficient to trigger enhanced cell growth. To determine whether the binding of integrins to fibronectin's RGD site is required for fibronectin-enhanced cell growth, the ability of fibronectin lacking the integrin-binding RGD site (FN(Delta)RGD) to promote cell growth was tested. FN(Delta)RGD promoted cell growth when used as an adhesive substrate or when added in solution to collagen-adherent fibronectin-null cells. Addition of FN(Delta)RGD to collagen-adherent fibronectin-null cells resulted in a 1.6-1.8x increase in cell growth in comparison with cells grown in the absence of fibronectin. The growth-promoting effects of FN(Delta)RGD and wild-type fibronectin were blocked by inhibitors of fibronectin polymerization, including the anti-fibronectin antibody, L8. In addition, FN(Delta)RGD-induced cell growth was completely inhibited by the addition of heparin, and was partially blocked by either heparitinase-treatment or by addition of recombinant fibronectin heparin-binding domain. Heparin and heparitinase-treatment also partially blocked the growth-promoting effects of wild-type fibronectin, as well as the deposition of wild-type fibronectin into the extracellular matrix. These data suggest that cell surface heparan-sulfate proteoglycans contribute to the growth-promoting effects of FN(Delta)RGD and wild-type fibronectin. Addition of heparin, treatment with heparitinase, or incubation with monoclonal antibody L8 all inhibited the formation of short linear FN(Delta)RGD fibrils on the cell surface. Inhibitory (beta)1 integrin antibodies had no effect on FN(Delta)RGD fibril formation, FN(Delta)RGD-induced cell growth, or cell adhesion on FN(Delta)RGD-coated substrates. These data suggest that fibronectin fibril formation can promote cell growth by a novel mechanism that is independent of RGD-integrin binding, and that involves cell surface proteoglycans.
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Guan, J. L., J. E. Trevithick, and R. O. Hynes. "Fibronectin/integrin interaction induces tyrosine phosphorylation of a 120-kDa protein." Cell Regulation 2, no. 11 (November 1991): 951–64. http://dx.doi.org/10.1091/mbc.2.11.951.
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We describe a 120-kDa protein (pp120) that is phosphorylated on tyrosine in cells attached to fibronectin-coated surfaces. The protein appears to be located in focal contacts where it codistributes with beta 1 integrins. pp120 is distinct from the beta 1 subunit of integrins and from vinculin and alpha-actinin. pp120 is rapidly dephosphorylated in cells suspended by trypsinization but becomes rapidly phosphorylated in cells attaching and spreading on fibronectin. Attachment of cells to RGD-containing peptides, polylysine, or concanavalin A is not sufficient to induce phosphorylation of pp120. The 120-kDa cell-binding domain of fibronectin can induce some phosphorylation of pp120, but further phosphorylation occurs in the presence also of the heparin-binding domain of fibronectin. Phosphorylation of pp120 precedes, but is correlated with, subsequent cell spreading. Phosphorylation of pp120 can also be triggered by attachment of cells to anti-integrin antibodies, and this requires the cytoplasmic domain of the integrin beta 1 subunit. Thus interaction of beta 1 integrins with extracellular ligands (fibronectin or antibodies) triggers phosphorylation of an intracellular 120-kDa protein, pp120, that may be involved in the responses of cells to attachment.
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TANI, Patricia H., Joseph C. LOFTUS, and Ron D. BOWDITCH. "In vitro selection of fibronectin gain-of-function mutations." Biochemical Journal 365, no. 1 (July 1, 2002): 287–94. http://dx.doi.org/10.1042/bj20020067.
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Directed protein evolution, which employs a combination of random mutagenesis, phage display, and in vitro selection, was used to identify second-site suppressors of the fibronectin (Fn) cell binding domain mutation Asp1495Ala (RGA). The mutations in the Fn 9th (3fn9) and 10th (3fn10) type III repeats obtained after selection on purified integrins αIIbβ3(D119Y) and α5β1 are reported. The 3fn9–10(D1495A) phage with substitution mutations at Asp1418, which is located within the linker region between 3fn9 and 3fn10, enhanced binding to the integrins αIIbβ3 and α5β1, but not αvβ3. The substitution mutations identified at residue Asp1418 were introduced into the native recombinant 3fn9–10 sequence and found to augment binding to αIIbβ3, demonstrating that the observed gain-of-function phenotype was independent of the multivalent character of the phage. These results support the following conclusions. First, regions of Fn in addition to the RGD loop are in close proximity to αIIbβ3 and α5β1 and are capable of participating in the binding to these integrins. Secondly, the conformational relationship between the 3fn9 and 3fn10 modules may be an important factor in the binding of Fn to these two integrins. Thirdly, other altered properties of Fn-integrin interactions, such as integrin specificity, may also be selected. This is the first description of Fn mutations that augment binding to integrins. The ability to select for particular phenotypes in vitro and the subsequent characterization of these mutations should further our understanding of the molecular details involved in the association of integrins and their ligands. Additionally, these higher-affinity 3fn9–10 ligands provide a starting point for further in vitro evolution and engineering of integrin-specific modules.
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Triantafilou, Kathy, Martha Triantafilou, Yoshikazu Takada, and Nelson Fernandez. "Human Parechovirus 1 Utilizes Integrins αvβ3 and αvβ1 as Receptors." Journal of Virology 74, no. 13 (July 1, 2000): 5856–62. http://dx.doi.org/10.1128/jvi.74.13.5856-5862.2000.
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ABSTRACT Human parechovirus 1 (HPEV1) displays an arginine-glycine-aspartic acid (RGD) motif in the VP1 capsid protein, suggesting integrins as candidate receptors for HPEV1. A panel of monoclonal antibodies (MAbs) specific for integrins αvβ3, αvβ1, and αvβ5, which have the ability to recognize the RGD motif, and also a MAb specific for integrin α2β1, an integrin that does not recognize the RGD motif, were tested on A549 cells. Our results showed that integrin αv-specific MAb reduced infectivity by 85%. To specify which αv integrins the virus utilizes, we tested MAbs specific to integrins αvβ3 and αvβ1 which reduced infectivity significantly, while a MAb specific for integrin αvβ5, as well as the MAb specific for α2β1, showed no reduction. When a combination of MAbs specific for integrins αvβ3 and αvβ1 were used, virus infectivity was almost completely inhibited; this shows that integrins αvβ3 and αvβ1 are utilized by the virus. We therefore proceeded to test whether αv integrins' natural ligands fibronectin and vitronectin had an effect on HPEV1 infectivity. We found that vitronectin reduced significantly HPEV1 infectivity, whereas a combination of vitronectin and fibronectin abolished infection. To verify the use of integrins αvβ3 and αvβ1 as HPEV1 receptors, CHO cells transfected and expressing either integrin αvβ3 or integrin αvβ1 were used. It was shown that the virus could successfully infect these cells. However, in immunoprecipitation experiments using HPEV1 virions and allowing the virus to bind to solubilized A549 cell extract, we isolated and confirmed by Western blotting the αvβ3 heterodimer. In conclusion, we found that HPEV1 utilises both integrin αvβ3 and αvβ1 as receptors; however, in cells that express both integrins, HPEV1 may preferentially bind integrin αvβ3.
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Testaz, S., M. Delannet, and J. Duband. "Adhesion and migration of avian neural crest cells on fibronectin require the cooperating activities of multiple integrins of the (beta)1 and (beta)3 families." Journal of Cell Science 112, no. 24 (December 15, 1999): 4715–28. http://dx.doi.org/10.1242/jcs.112.24.4715.
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Based on genetic, functional and histological studies, the extracellular matrix molecule fibronectin has been proposed to play a key role in the migration of neural crest cells in the vertebrate embryo. In the present study, we have analyzed in vitro the repertoire and function of integrin receptors involved in the adhesive and locomotory responses of avian truncal neural crest cells to fibronectin. Immunoprecipitation experiments showed that neural crest cells express multiple integrins, namely (alpha)3(beta)1, (alpha)4(beta)1, (alpha)5(beta)1, (alpha)8(beta)1, (alpha)v(beta)1, (alpha)v(beta)3 and a (beta)8 integrin, as potential fibronectin receptors, and flow cytometry analyses revealed no major heterogeneity among the cell population for expression of integrin subunits. In addition, the integrin repertoire expressed by neural crest cells was found not to change dramatically during migration. At the cellular level, only (alpha)v(beta)1 and (alpha)v(beta)3 were concentrated in focal adhesion sites in connection with the actin microfilaments, whereas the other integrins were predominantly diffuse over the cell surface. In inhibition assays with function-perturbing antibodies, it appeared that complete abolition of cell spreading and migration could be achieved only by blocking multiple integrins of the (beta)1 and (beta)3 families, suggesting possible functional compensations between different integrins. In addition, these studies provided evidence for functional partitioning of integrins in cell adhesion and migration. While spreading was essentially mediated by (alpha)v(beta)1 and (alpha)8(beta)1, migration involved primarily (alpha)4(beta)1, (alpha)v(beta)3 and (alpha)8(beta)1 and, more indirectly, (alpha)3(beta)1. (alpha)5(beta)1 and the (beta)8 integrin were not found to play any major role in either adhesion or migration. Finally, consistent with the results of inhibition experiments, recruitment of (alpha)4(beta)1 and (alpha)v(beta)3, individually or in combination using antibodies or recombinant VCAM-1 and PECAM-1 molecules as a substratum, was required for migration but was not sufficient to produce migration of the cell population as efficiently as with fibronectin. In conclusion, our study indicates that neural crest cells express a multiplicity of fibronectin-binding integrins and suggests that dispersion of the cell population requires cooperation between distinct integrins regulating different events of cell adhesion, locomotion and, possibly, proliferation and survival.
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Aplin, J. D., A. Sattar, and A. P. Mould. "Variant choriocarcinoma (BeWo) cells that differ in adhesion and migration on fibronectin display conserved patterns of integrin expression." Journal of Cell Science 103, no. 2 (October 1, 1992): 435–44. http://dx.doi.org/10.1242/jcs.103.2.435.
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Starting from the BeWo choriocarcinoma cell line, two stable variant cell lines (epi and lc) were isolated. Epi cells displayed an epithelioid colony morphology while lc were fibroblastoid. lc cells attached and spread on fibronectin-coated surfaces at significantly lower density of fibronectin than epi or the parent cell line. lc also migrated more efficiently to fibronectin in a trans-filter assay than either epi or parent cells. Integrin expression by the cell lines was investigated by flow cytometry and immunoprecipitation from surface-labelled cells with a panel of subunit-specific antibodies. Integrins alpha 2 beta 1, alpha 5 beta 1, alpha v beta 1 and alpha 6 beta 4 were detected in each case, and levels of expression were identical in the two variant lines. Anti-functional antibodies were used to probe the role of integrins in fibronectin- and vitronectin-mediated adhesion. Complete inhibition of adhesion to fibronectin was observed with anti-beta 1 antibody, and partial inhibition with anti-alpha 5, suggesting that integrin alpha 5 beta 1 is mainly responsible for the interaction. Adhesion to vitronectin was inhibitable using anti-alpha v and anti-beta 1 antibodies, suggesting that integrin alpha v beta 1 is active in these cells as a vitronectin receptor. There was a correlation between the altered morphology of the variant cells and alterations in the distribution of integrin alpha 6 beta 4 and laminin in monolayer cultures. The results support the idea that fibronectin may mediate the migratory behaviour of extravillous trophoblast in vivo. Switch to a more migratory phenotype may be mediated by the selective activation of integrins and altered interaction with basement membrane.
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Kim, H. J., C. A. Henke, S. K. Savik, and D. H. Ingbar. "Integrin mediation of alveolar epithelial cell migration on fibronectin and type I collagen." American Journal of Physiology-Lung Cellular and Molecular Physiology 273, no. 1 (July 1, 1997): L134—L141. http://dx.doi.org/10.1152/ajplung.1997.273.1.l134.
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Acute lung injury leads to type I alveolar epithelial cell (AEC) death, denudation of the alveolar basement membrane, and formation of an alveolar provisional matrix from fibronectin, fibrinogen, and type I collagen. The provisional matrix provides a scaffold for alveolar repair. To restore normal lung architecture, surviving type II AECs must reepithelialize denuded alveoli. We examined whether AECs migrate on provisional matrix proteins and whether integrins mediate this migration using a Boyden chemotaxis chamber. Cultured AECs migrated on fibronectin-coated filters by haptotaxis (defined as movement on a solid-phase substrate) more than one type I collagen-coated filters, and they did not migrate on fibrinogen-coated filters. Soluble fibronectin augmented migration on type I collagen-coated filters, but not on fibronectin-coated filters. Anti-alpha v beta 3-integrin monoclonal antibody (MAb) inhibited migration on substrate-bound fibronectin by 62-77%, whereas anti-beta 1-integrin MAb inhibited migration by 48%. Anti-alpha 2-integrin MAb almost completely inhibited migration on substrate-bound type I collagen, but not on fibronectin. The novel findings in this study are as follows: 1) AECs migrate by haptotaxis more effectively on substrate-bound fibronectin than on type I collagen; 2) alpha v beta 3- and beta 1-integrins partially mediate AEC haptotaxis on fibronectin; and 3) the alpha 2 beta 1-integrin mediates AEC migration on type I collagen. These results support the importance of type II cell migration on provisional matrix proteins during repair of lung injury.
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Boyd, Nikhat D., Bosco M. C. Chan, and Nils O. Petersen. "β1 integrins are distributed in adhesion structures with fibronectin and caveolin and in coated pits." Biochemistry and Cell Biology 81, no. 5 (October 1, 2003): 335–48. http://dx.doi.org/10.1139/o03-063.
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Integrins are found in adhesion structures, which link the extracelullar matrix to cytoskeletal proteins. Here, we attempt to further define the distribution of β1 integrins in the context of their association with matrix proteins and other cell surface molecules relevant to the endocytic process. We find that β1 integrins colocalize with fibronectin in fibrillar adhesion structures. A fraction of caveolin is also organized along these adhesion structures. The extracellular matrix protein laminin is not concentrated in these structures. The α4β1 integrin exhibits a distinct distribution from other β1 integrins after cells have adhered for 1 h to extracellular matrix proteins but is localized in adhesion structures after 24 h of adhesion. There are differences between the fibronectin receptors: α5β1 integrins colocalize with adaptor protein-2 in coated pits, while α4β1 integrins do not. This parallels our earlier observation that of the two laminin receptors, α1β1 and α6β1, only αaβ1 integrins colocalize with adaptor protein-2 in coated pits. Calcium chelation or inhibition of mitogen-activated protein kinase kinase, protein kinase C, or src did not affect localization of α1β1 and α5β1 integrins in coated pits. Likewise, the integrity of coated-pit structures or adhesion structures is not required for integrin and adaptor protein-2 colocalization. This suggests a robust and possibly constitutive interaction between these integrins and coated pits.Key words: adhesion, endocytosis, extracellular matrix, microscopy, confocal, signalling.
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Klass, C. M., J. R. Couchman, and A. Woods. "Control of extracellular matrix assembly by syndecan-2 proteoglycan." Journal of Cell Science 113, no. 3 (February 1, 2000): 493–506. http://dx.doi.org/10.1242/jcs.113.3.493.
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Extracellular matrix (ECM) deposition and organization is maintained by transmembrane signaling and integrins play major roles. We now show that a second transmembrane component, syndecan-2 heparan sulfate proteoglycan, is pivotal in matrix assembly. Chinese Hamster Ovary (CHO) cells were stably transfected with full length (S2) or truncated syndecan-2 lacking the C-terminal 14 amino acids of the cytoplasmic domain (S2deltaS). No differences in the amount of matrix assembly were noted with S2 cells, but those expressing S2deltaS could not assemble laminin or fibronectin into a fibrillar matrix. The loss of matrix formation was not caused by a failure to synthesize or externalize ECM components as determined by metabolic labeling or due to differences in surface expression of alpha5 or beta1 integrin. The matrix assembly defect was at the cell surface, since S2deltaS cells also lost the ability to rearrange laminin or fibronectin substrates into fibrils and to bind exogenous fibronectin. Transfection of activated alphaIIbalphaLdeltabeta3 integrin into alpha(5)-deficient CHO B2 cells resulted in reestablishment of the previously lost fibronectin matrix. However, cotransfection of this cell line with S2deltaS could override the presence of activated integrins. These results suggest a regulatory role for syndecan-2 in matrix assembly, along with previously suggested roles for activated integrins.
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Mould, A. Paul, Ewa J. Koper, Adam Byron, Grit Zahn, and Martin J. Humphries. "Mapping the ligand-binding pocket of integrin α5β1 using a gain-of-function approach." Biochemical Journal 424, no. 2 (November 11, 2009): 179–89. http://dx.doi.org/10.1042/bj20090992.
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Integrin α5β1 is a key receptor for the extracellular matrix protein fibronectin. Antagonists of human integrin α5β1 have therapeutic potential as anti-angiogenic agents in cancer and diseases of the eye. However, the structure of the integrin is unsolved and the atomic basis of fibronectin and antagonist binding by integrin α5β1 is poorly understood. In the present study, we demonstrate that zebrafish α5β1 integrins do not interact with human fibronectin or the human α5β1 antagonists JSM6427 and cyclic peptide CRRETAWAC. Zebrafish α5β1 integrins do bind zebrafish fibronectin-1, and mutagenesis of residues on the upper surface and side of the zebrafish α5 subunit β-propeller domain shows that these residues are important for the recognition of the Arg-Gly-Asp (RGD) motif and the synergy sequence [Pro-His-Ser-Arg-Asn (PHSRN)] in fibronectin. Using a gain-of-function analysis involving swapping regions of the zebrafish integrin α5 subunit with the corresponding regions of human α5 we show that blades 1–4 of the β-propeller are required for human fibronectin recognition, suggesting that fibronectin binding involves a broad interface on the side and upper face of the β-propeller domain. We find that the loop connecting blades 2 and 3 of the β-propeller, the D3–A3 loop, contains residues critical for antagonist recognition, with a minor role played by residues in neighbouring loops. A new homology model of human integrin α5β1 supports an important function for D3–A3 loop residues Trp157 and Ala158 in the binding of antagonists. These results will aid the development of reagents that block integrin α5β1 functions in vivo.
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Huang, Jing, Lance C. Bridges, and Judith M. White. "Selective Modulation of Integrin-mediated Cell Migration by Distinct ADAM Family Members." Molecular Biology of the Cell 16, no. 10 (October 2005): 4982–91. http://dx.doi.org/10.1091/mbc.e05-03-0258.
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A disintegrin and a metalloprotease (ADAM) family members have been implicated in many biological processes. Although it is recognized that recombinant ADAM disintegrin domains can interact with integrins, little is known about ADAM-integrin interactions in cellular context. Here, we tested whether ADAMs can selectively regulate integrin-mediated cell migration. ADAMs were expressed in Chinese hamster ovary cells that express defined integrins (α4β1, α5β1, or both), and cell migration on full-length fibronectin or on its α4β1 or α5β1 binding fragments was studied. We found that ADAMs inhibit integrin-mediated cell migration in patterns dictated by the integrin binding profiles of their isolated disintegrin domains. ADAM12 inhibited cell migration mediated by the α4β1 but not the α5β1 integrin. ADAM17 had the reciprocal effect; it inhibited α5β1- but not α4β1-mediated cell migration. ADAM19 and ADAM33 inhibited migration mediated by both α4β1 and α5β1 integrins. A point mutation in the ADAM12 disintegrin loop partially reduced the inhibitory effect of ADAM12 on cell migration on the α4β1 binding fragment of fibronectin, whereas mutations that block metalloprotease activity had no effect. Our results indicate that distinct ADAMs can modulate cell migration mediated by specific integrins in a pattern dictated, at least in part, by their disintegrin domains.
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Eshghi, Shawdee, Mariette G. Vogelezang, Richard O. Hynes, Linda G. Griffith, and Harvey F. Lodish. "α4β1 integrin and erythropoietin mediate temporally distinct steps in erythropoiesis: integrins in red cell development." Journal of Cell Biology 177, no. 5 (June 4, 2007): 871–80. http://dx.doi.org/10.1083/jcb.200702080.
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Erythropoietin (Epo) is essential for the terminal proliferation and differentiation of erythroid progenitor cells. Fibronectin is an important part of the erythroid niche, but its precise role in erythropoiesis is unknown. By culturing fetal liver erythroid progenitors, we show that fibronectin and Epo regulate erythroid proliferation in temporally distinct steps: an early Epo-dependent phase is followed by a fibronectin-dependent phase. In each phase, Epo and fibronectin promote expansion by preventing apoptosis partly through bcl-xL. We show that α4, α5, and β1 are the principal integrins expressed on erythroid progenitors; their down-regulation during erythropoiesis parallels the loss of cell adhesion to fibronectin. Culturing erythroid progenitors on recombinant fibronectin fragments revealed that only substrates that engage α4β1-integrin support normal proliferation. Collectively, these data suggest a two-phase model for growth factor and extracellular matrix regulation of erythropoiesis, with an early Epo-dependent, integrin-independent phase followed by an Epo-independent, α4β1-integrin–dependent phase.
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Lomas, Amanda C., Kieran T. Mellody, Lyle J. Freeman, Daniel V. Bax, C. Adrian Shuttleworth, and Cay M. Kielty. "Fibulin-5 binds human smooth-muscle cells through α5β1 and α4β1 integrins, but does not support receptor activation." Biochemical Journal 405, no. 3 (July 13, 2007): 417–28. http://dx.doi.org/10.1042/bj20070400.
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Fibulin-5, an extracellular matrix glycoprotein expressed in elastin-rich tissues, regulates vascular cell behaviour and elastic fibre deposition. Recombinant full-length human fibulin-5 supported primary human aortic SMC (smooth-muscle cell) attachment through α5β1 and α4β1 integrins. Cells on fibulin-5 spread poorly and displayed prominent membrane ruffles but no stress fibres or focal adhesions, unlike cells on fibronectin that also binds these integrins. Cell migration and proliferation were significantly lower on fibulin-5 than on fibronectin. Treatment of cells on fibulin-5 with a β1 integrin-activating antibody induced stress fibres, increased attachment, migration and proliferation, and stimulated signalling of epidermal growth factor receptor and platelet-derived growth factor receptors α and β. Fibulin-5 also modulated fibronectin-mediated cell spreading and morphology. We have thus identified the β1 integrins on primary SMCs that fibulin-5 interacts with, and have shown that failure of fibulin-5 to activate these receptors limits cell spreading, migration and proliferation.
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Tomaselli, K. J., C. H. Damsky, and L. F. Reichardt. "Purification and characterization of mammalian integrins expressed by a rat neuronal cell line (PC12): evidence that they function as alpha/beta heterodimeric receptors for laminin and type IV collagen." Journal of Cell Biology 107, no. 3 (September 1, 1988): 1241–52. http://dx.doi.org/10.1083/jcb.107.3.1241.
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Cells of the rat neuronal line, PC12, adhere well to substrates coated with laminin and type IV collagen, but attach poorly to fibronectin. Adhesion and neurite extension in response to these extracellular matrix proteins are inhibited by Fab fragments of an antiserum (anti-ECMR) that recognizes PC12 cell surface integrin subunits of Mr 120,000, 140,000, and 180,000 (Tomaselli, K. J., C. H. Damsky, and L. F. Reichardt. 1987. J. Cell Biol. 105:2347-2358). Here we extend our study of integrin structure and function in PC12 cells using integrin subunit-specific antibodies prepared against synthetic peptides corresponding to the cytoplasmic domains of the human integrin beta 1 and the fibronectin receptor alpha (alpha FN) subunits. Anti-integrin beta 1 immunoprecipitated a 120-kD beta 1 subunit and two noncovalently associated integrin alpha subunits of 140 and 180 kD from detergent extracts of surface-labeled PC12 cells. Immunodepletion studies using anti-integrin beta 1 demonstrated that these two putative alpha/beta heterodimers are identical to those recognized by the adhesion-perturbing ECMR antiserum. Anti-alpha FN immunoprecipitated fibronectin receptor heterodimers in human and rat fibroblastic cells, but not in PC12 cells. Thus, low levels of expression of the integrin alpha FN subunit can explain the poor attachment of PC12 cells to FN. The PC12 cell integrins were purified using a combination of lectin and ECMR antibody affinity chromatography. The purified integrins: (a) completely neutralize the ability of the anti-ECMR serum to inhibit PC12 cell adhesion to laminin and collagen IV; (b) have hydrodynamic properties that are very similar to those of previously characterized integrin alpha/beta heterodimeric receptors for ECM proteins; and (c) can be incorporated into phosphatidylcholine vesicles that then bind specifically to substrates coated with laminin or collagen IV but not fibronectin. Thus, the ligand-binding specificity of the liposomes containing the purified PC12 integrins closely parallels the substrate-binding preference of intact PC12 cells. These results demonstrate that mammalian integrins purified from a neuronal cell line can, when incorporated into lipid vesicles, function as receptors for laminin and type IV collagen.
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Grenz, H., S. Carbonetto, and S. L. Goodman. "Alpha 3 beta 1 integrin is moved into focal contacts in kidney mesangial cells." Journal of Cell Science 105, no. 3 (July 1, 1993): 739–51. http://dx.doi.org/10.1242/jcs.105.3.739.
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The movement of integrins into focal adhesive structures accompanies cell attachment to extracellular matrix. The kinetics of incorporation of integrins into focal contacts was studied during attachment to matrix of mesangial cells of the kidney glomerulus. On collagen, fibronectin, laminin and vitronectin, the number and intensity of talin-focal contacts increased with time. Talin-containing focal contacts were present in mesangial cells within 2 h of plating and in control cells (HT1080 and Rugli) within 1 h. Integrin alpha-chains colocalized with talin, dependent on the matrix substrate. The attachment, spreading and organization of integrin into focal contacts was not affected when endogenous protein synthesis was suppressed with cycloheximide. In Rugli, alpha 1 beta 1 organized into focal contacts on collagen and laminin, while in HT1080 alpha 2 beta 1 organized on collagen type I, alpha 5 beta 1 on fibronectin, alpha 6 beta 1 on laminin, and alpha 3 beta 1 and alpha 4 beta 1 were diffusely distributed on all substrates. These distributions mirrored the usage and expression patterns previously established for integrins in these cells and was as predicted from the literature. In mesangial cells, however, alpha 3 beta 1 was also organized into prominent focal contact arrays on collagen, fibronectin, EHS and human placental laminins, but not on vitronectin, while alpha 6 beta 1 was not organized. Initial attachment and spreading of mesangial cells was absolutely dependent on divalent cations. Mg2+ and Mn2+ supported attachment on all substrates, while Ca2+ stimulated attachment on laminin (E8), fibronectin and vitronectin. The data suggest that the functional integrins on mesangial cells include alpha 1 beta 1 (on collagen and laminin) alpha 2 beta 1 (on collagen), alpha 5 beta 1 (on fibronectin) and alpha V beta 3 (on vitronectin). However, mesangial cells do not use alpha 6 beta 1 on laminin, and the data support a role for alpha 3 beta 1 as putative receptor for fibronectin, collagen and laminin.
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Marques, M. Rubia, D. Hajjar, K. Gomes Franchini, A. Sigari Moriscot, and M. Fagundes Santos. "Mandibular Appliance Modulates Condylar Growth through Integrins." Journal of Dental Research 87, no. 2 (February 2008): 153–58. http://dx.doi.org/10.1177/154405910808700210.
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Functional orthopedic therapy corrects growth discrepancies between the maxilla and mandible, possibly through postural changes in the musculature and modulation of the mandibular condylar cartilage growth. Using Wistar rats, we tested the hypothesis that chondrocytes respond to forces generated by a mandibular propulsor appliance by changes in gene expression, and that integrins are important mediators in this response. Immunohistochemical analyses demonstrated that the use of the appliance for different periods of time modulated the expression of fibronectin, α5 and αv integrin subunits, as well as cell proliferation in the cartilage. In vitro, cyclic distension of condylar cartilage-derived cells increased fibronectin mRNA, as well as Insulin-like Growth Factor-I and II mRNA and cell proliferation. A peptide containing the Arginine-Glycine-Asparagine sequence (RGD), the main cell-binding sequence in fibronectin, blocked almost all these effects, confirming that force itself modulates the growth of the rat condylar cartilage, and that RGD-binding integrins participate in mechanotransduction.
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Pasqualini, R., E. Koivunen, and E. Ruoslahti. "A peptide isolated from phage display libraries is a structural and functional mimic of an RGD-binding site on integrins." Journal of Cell Biology 130, no. 5 (September 1, 1995): 1189–96. http://dx.doi.org/10.1083/jcb.130.5.1189.
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Many integrins recognize short RGD-containing amino acid sequences and such peptide sequences can be identified from phage libraries by panning with an integrin. Here, in a reverse strategy, we have used such libraries to isolate minimal receptor sequences that bind to fibronectin and RGD-containing fibronectin fragments in affinity panning. A predominant cyclic motif, *CWDDG/LWLC*, was obtained (the asterisks denote a potential disulfide bond). Studies using the purified phage and the corresponding synthetic cyclic peptides showed that *CWDDGWLC*-expressing phage binds specifically to fibronectin and to fibronectin fragments containing the RGD sequence. The binding did not require divalent cations and was inhibited by both RGD and *CWDDGWLC*-containing synthetic peptides. Conversely, RGD-expressing phage attached specifically to immobilized *CWDDGWLC*-peptide and the binding could be blocked by the respective synthetic peptides in solution. Moreover, fibronectin bound to a *CWDDGWLC*-peptide affinity column, and could be eluted with an RGD-containing peptide. The *CWDDGWLC*-peptide inhibited RGD-dependent cell attachment to fibronectin and vitronectin, but not to collagen. A region of the beta subunit of RGD-binding integrins that has been previously demonstrated to be involved in ligand binding includes a polypeptide stretch, KDDLW (in beta 3) similar to WDDG/LWL. Synthetic peptides corresponding to this region in beta 3 were found to bind RGD-displaying phage and conversion of its two aspartic residues into alanines greatly reduced the RGD binding. Polyclonal antibodies raised against the *CWDDGWLC*-peptide recognized beta 1 and beta 3 in immunoblots. These data indicate that the *CWDDGWLC*-peptide is a functional mimic of ligand binding sites of RGD-directed integrins, and that the structurally similar site in the integrin beta subunit is a binding site for RGD.
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Danen, Erik H. J., and Kenneth M. Yamada. "Fibronectin, integrins, and growth control." Journal of Cellular Physiology 189, no. 1 (2001): 1–13. http://dx.doi.org/10.1002/jcp.1137.
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Praetorius, H. A., J. Praetorius, S. Nielsen, J. Frokiaer, and K. R. Spring. "β1-Integrins in the primary cilium of MDCK cells potentiate fibronectin-induced Ca2+ signaling." American Journal of Physiology-Renal Physiology 287, no. 5 (November 2004): F969—F978. http://dx.doi.org/10.1152/ajprenal.00096.2004.
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Because β1-integrin is involved in sensing of fluid flow rate in endothelial cells, a function that in Madin-Darby canine kidney (MDCK) cells is confined to the primary cilium, we hypothesized β1-integrin to be an important part of the primary ciliary mechanosensory apparatus in MDCK cells. We observed that β1-integrin, α3-integrin, and perhaps α5-integrin were localized to the primary cilium of MDCK cells by combining lectin and immunofluorescence confocal microscopy. β1-Integrin was also colocalized with tubulin to the primary cilia of the rat renal collecting ducts, as well as to the cilia of proximal tubules and thick ascending limbs. Immunogold-electron microscopy confirmed the presence of β1-integrin on primary cilia of MDCK cells and rat collecting ducts. Intracellular Ca2+ levels, monitored by fluorescence microscopy on fluo 4-loaded MDCK cells, significantly increased on addition of fibronectin, a β1-integrin ligand, to mature MDCK cells with an IC50 of 0.02 mg/l. In immature, nonciliated cells or in deciliated mature cells, the IC50 was 0.40 mg/l. Blocking the fibronectin-binding sites of β1-integrin with RGD peptide prevented the Ca2+ signal. Cross-linking of β1-integrins by Sambucus nigra agglutinin produced a Ca2+ response similar to the addition of fibronectin. Furthermore, the fibronectin-induced response was not dependent on flow or a flow-induced Ca2+ response. Finally, the flow-induced Ca2+ response was not prevented by the fibronectin-induced signal. Although β1-integrin on the primary cilium greatly potentiates the fibronectin-induced Ca2+ signaling in MDCK cells, the flow-dependent Ca2+ signal is not mediated through activation of β1-integrin.
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Regen, C. M., and A. F. Horwitz. "Dynamics of beta 1 integrin-mediated adhesive contacts in motile fibroblasts." Journal of Cell Biology 119, no. 5 (December 1, 1992): 1347–59. http://dx.doi.org/10.1083/jcb.119.5.1347.
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Motile chick skeletal fibroblasts adhere to a laminin substrate by means of clustered beta 1 integrins. These integrin "macroaggregates" are similar to classic focal contacts but do not appear dark under interference-reflection microscopy. They contain alpha 5 integrin and are associated with extracellular fibronectin. To study their behavior during cell movement, time-lapse, low-light video microscopy was used to image integrins on living cells tagged with a fluorescent anti-beta 1 integrin antibody. Integrin macroaggregates remain fixed with respect to the substratum, despite the fact that they fluctuate in size, density, and shape over a period of minutes. Upon detachment of the cell rear, as much as 85% of the beta 1 integrin density of a macroaggregate remains behind on the substrate, along with both alpha 5 integrin and fibronectin. Release of the cell rear does not involve cleavage of the beta 1 integrin cytoplasmic domain from the remainder of the protein. These results indicate that cell motility does not require regulated detachment of integrin receptors from the substrate. On the other hand, cytoskeletal components and a variable fraction of the integrins are carried forward with the cell during detachment, suggesting that some type of cortical disassembly process does occur. Integrin macroaggregate structures are not recycled intact after detachment of the cell rear from the substrate. They do not persist on the cell surface, nor can they be seen to be engulfed by vesicles; yet, some of the individual integrins that make up these macroaggregates are eventually transported forward by both vesicular and cell-surface routes. Antibody-tagged integrins accumulate in dense patches at the lateral edges and dorsal surface of the cell, and move forward on the cell surface. The tagged integrins also enter cytoplasmic vesicles, which move forward within the cytoplasm. Macroaggregates generally form and grow at the cell front; however, application of fluorescent antibody causes integrins to disappear from the leading edge. Therefore, it has not been possible to directly visualize the recycling of the forward moving tagged integrins into new macroaggregates at the cell front. Surprisingly, under these conditions cells move normally despite the absence of any delivery of tagged integrin to the leading edge, indicating that recycling of integrins to the lamella is not required for apparently normal motility.
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Wu, Xin, Zhe Sun, Andrea Foskett, Jerome P. Trzeciakowski, Gerald A. Meininger, and Mariappan Muthuchamy. "Cardiomyocyte contractile status is associated with differences in fibronectin and integrin interactions." American Journal of Physiology-Heart and Circulatory Physiology 298, no. 6 (June 2010): H2071—H2081. http://dx.doi.org/10.1152/ajpheart.01156.2009.
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Integrins link the extracellular matrix (ECM) with the intracellular cytoskeleton and other cell adhesion-associated signaling proteins to function as mechanotransducers. However, direct quantitative measurements of the cardiomyocyte mechanical state and its relationship to the interactions between specific ECM proteins and integrins are lacking. The purpose of this study was to characterize the interactions between the ECM protein fibronectin (FN) and integrins in cardiomyocytes and to test the hypothesis that these interactions would vary during contraction and relaxation states in cardiomyocytes. Using atomic force microscopy, we quantified the unbinding force (adhesion force) and adhesion probability between integrins and FN and correlated these measurements with the contractile state as indexed by cell stiffness on freshly isolated mouse cardiomyocytes. Experiments were performed in normal physiological (control), high-K+ (tonically contracted), or low-Ca2+ (fully relaxed) solutions. Under control conditions, the initial peak of adhesion force between FN and myocyte α3β1- and/or α5β1-integrins was 39.6 ± 1.3 pN. The binding specificity between FN and α3β1- and α5β1-integrins was verified by using monoclonal antibodies against α3-, α5-, α3 + α5-, or β1-integrin subunits, which inhibited binding by 48%, 65%, 70%, or 75%, respectively. Cytochalasin D or 2,3-butanedione monoxime (BDM), to disrupt the actin cytoskeleton or block myofilament function, respectively, significantly decreased the cell stiffness; however, the adhesion force and binding probability were not altered. Tonic contraction with high-K+ solution increased total cell adhesion (1.2-fold) and cell stiffness (27.5-fold) compared with fully relaxed cells with low-Ca2+ solution. However, it could be partially prevented by high-K+ bath solution containing BDM, which suppresses contraction by inhibiting the actin-myosin interactions. Thus, our results demonstrate that integrin binding to FN is modulated by the contractile state of cardiac myocytes.
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Behrendtsen, O., C. M. Alexander, and Z. Werb. "Cooperative interactions between extracellular matrix, integrins and parathyroid hormone-related peptide regulate parietal endoderm differentiation in mouse embryos." Development 121, no. 12 (December 1, 1995): 4137–48. http://dx.doi.org/10.1242/dev.121.12.4137.
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The outgrowth of parietal endoderm (PE) cells from precursor endodermal cells is one of the first differentiation events that occur in mouse embryos. We have analyzed the molecular determinants of this process by placing isolated inner cell masses (ICMs) on defined extracellular matrix substrata in microdrop cultures. Differentiation and outgrowth of PE required a fibronectin substratum. Laminin supported the adhesion and outgrowth of visceral endoderm (VE) and actively suppressed the differentiation of PE in mixtures of fibronectin and laminin. Collagen type IV, gelatin, vitronectin or entactin supported little or no endodermal outgrowth. Trophectoderm (TE) cells have been implied to be important in PE induction in vivo. We found that recombination of ICMs in culture with TE cells, or with medium conditioned by TE cells, greatly increased the differentiation of PE. TE cells stimulated PE outgrowth on substrata other than fibronectin. One cytokine secreted by trophoblast and endodermal cells, parathyroid hormone-related peptide (PTHrP), was critical for outgrowth on any substratum. A function-perturbing antibody to PTHrP reduced the number of PE cells, whereas the addition of PTHrP increased that number. Furthermore, addition of PTHrP changed the substratum requirements for outgrowth, making laminin, vitronectin and low concentrations of fibronectin permissive for PE outgrowth. Immunostaining with anti-integrin antibodies showed that fully differentiated PE cells outgrowing on fibronectin expressed alpha 5, alpha 6 and alpha v beta 3 integrins. However, analysis of outgrowths in the presence of function-perturbing antibodies to alpha 5, alpha 6 and alpha v beta 3 integrins showed that these integrins directed PE outgrowth only on fibronectin, laminin and vitronectin substrata, respectively. We have shown that there is a cooperative interplay of extracellular matrix, integrins and PTHrP that modulates PE outgrowth.
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Zhang, Z., AO Morla, K. Vuori, JS Bauer, RL Juliano, and E. Ruoslahti. "The alpha v beta 1 integrin functions as a fibronectin receptor but does not support fibronectin matrix assembly and cell migration on fibronectin." Journal of Cell Biology 122, no. 1 (July 1, 1993): 235–42. http://dx.doi.org/10.1083/jcb.122.1.235.
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The fibronectin receptor, alpha 5 beta 1, has been shown to be required for fibronectin matrix assembly and plays an important role in cell migration on fibronectin. However, it is not clear whether other fibronectin binding integrins can take the place of alpha 5 beta 1 during matrix assembly and cell migration. To test this, we expressed the human alpha v subunit in the CHO cell line CHO-B2 that lacks the alpha 5 subunit. We found that the human alpha v combined with CHO cell beta 1 to form the integrin alpha v beta 1. Cells that expressed alpha v beta 1 attached to and spread well on fibronectin-coated dishes, but did so less well on vitronectin-coated dishes. This, along with other data, indicated that alpha v beta 1 functions as a fibronectin receptor in CHO-B2 cells. The alpha v beta 1-expressing cells failed to produce a fibronectin matrix or to migrate on fibronectin, although the same cells transfected with alpha 5 do produce a matrix and migrate on fibronectin. The affinity of the alpha v beta 1-expressing cells for fibronectin was fourfold lower than that of the alpha 5 beta 1-expressing cells. In addition, alpha v beta 1 was distributed diffusely throughout the cell surface, whereas alpha 5 beta 1 was localized to focal adhesions when cells were seeded onto fibronectin-coated surfaces. Thus, of the two fibronectin receptors, alpha v beta 1 and alpha 5 beta 1, only alpha 5 beta 1 supports fibronectin matrix assembly and promotes cell migration on fibronectin in the CHO-B2 cells. Possible reasons for this difference in the activities of alpha v beta 1 and alpha 5 beta 1 include the lower affinity of alpha v beta 1 for fibronectin and the failure of this integrin to localize in adhesion plaques on a fibronectin substrate. These results show that two integrins with similar ligand specificities and cell attachment functions may be quite different in their ability to support fibronectin matrix assembly and cell motility on fibronectin.
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Bunni, Marlene A., Inga I. Kramarenko, Linda Walker, John R. Raymond, and Maria N. Garnovskaya. "Role of integrins in angiotensin II-induced proliferation of vascular smooth muscle cells." American Journal of Physiology-Cell Physiology 300, no. 3 (March 2011): C647—C656. http://dx.doi.org/10.1152/ajpcell.00179.2010.
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Angiotensin II (AII) binds to G protein-coupled receptor AT1 and stimulates extracellular signal-regulated kinase (ERK), leading to vascular smooth muscle cells (VSMC) proliferation. Proliferation of mammalian cells is tightly regulated by adhesion to the extracellular matrix, which occurs via integrins. To study cross-talk between G protein-coupled receptor- and integrin-induced signaling, we hypothesized that integrins are involved in AII-induced proliferation of VSMC. Using Oligo GEArray and quantitative RT-PCR, we established that messages for α1-, α5-, αV-, and β1-integrins are predominant in VSMC. VSMC were cultured on plastic dishes or on plates coated with either extracellular matrix or poly-d-lysine (which promotes electrostatic cell attachment independent of integrins). AII significantly induced proliferation in VSMC grown on collagen I or fibronectin, and this effect was blocked by the ERK inhibitor PD-98059, suggesting that AII-induced proliferation requires ERK activity. VSMC grown on collagen I or on fibronectin demonstrated approximately three- and approximately sixfold increases in ERK phosphorylation after stimulation with 100 nM AII, respectively, whereas VSMC grown on poly-d-lysine demonstrated no significant ERK activation, supporting the importance of integrin-mediated adhesion. AII-induced ERK activation was reduced by >65% by synthetic peptides containing an RGD (arginine-glycine-aspartic acid) sequence that inhibit α5β1-integrin, and by ∼60% by the KTS (lysine-threonine-serine)-containing peptides specific for integrin-α1β1. Furthermore, neutralizing antibody against β1-integrin and silencing of α1, α5, and β1 expression by transfecting VSMC with short interfering RNAs resulted in decreased AII-induced ERK activation. This work demonstrates roles for specific integrins (most likely α5β1 and α1β1) in AII-induced proliferation of VSMC.
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Li, Jing, and Timothy A. Springer. "Energy landscape differences among integrins establish the framework for understanding activation." Journal of Cell Biology 217, no. 1 (November 9, 2017): 397–412. http://dx.doi.org/10.1083/jcb.201701169.
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Why do integrins differ in basal activity, and how does affinity for soluble ligand correlate with cellular adhesiveness? We show that basal conformational equilibrium set points for integrin α4β1 are cell type specific and differ from integrin α5β1 when the two integrins are coexpressed on the same cell. Although α4β1 is easier to activate, its high-affinity state binds vascular cell adhesion molecule and fibronectin 100- to 1,000-fold more weakly than α5β1 binds fibronectin. Furthermore, the difference in affinity between the high- and low-affinity states is more compressed in α4β1 (600- to 800-fold) than in α5β1 (4,000- to 6,000-fold). α4β1 basal conformational equilibria differ among three cell types, define affinity for soluble ligand and readiness for priming, and may reflect differences in interactions with intracellular adaptors but do not predict cellular adhesiveness for immobilized ligand. The measurements here provide a necessary framework for understanding integrin activation in intact cells, including activation of integrin adhesiveness by application of tensile force by the cytoskeleton, across ligand–integrin–adaptor complexes.
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von Wichert, Götz, Guoying Jiang, Ana Kostic, Kurt De Vos, Jan Sap, and Michael P. Sheetz. "RPTP-α acts as a transducer of mechanical force on αv/β3-integrin–cytoskeleton linkages." Journal of Cell Biology 161, no. 1 (April 7, 2003): 143–53. http://dx.doi.org/10.1083/jcb.200211061.
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Cell motility on ECM critically depends on the cellular response to force from the matrix. We find that force-dependent reinforcement of αv/β3-integrin–mediated cell–matrix connections requires the receptor-like tyrosine phosphatase α (RPTPα). RPTPα colocalizes with αv-integrins at the leading edge during early spreading, and coimmunoprecipitates with αv-integrins during spreading on fibronectin and vitronectin. RPTPα-dependent activation of Src family kinases, in particular activation of Fyn, is required for the force-dependent formation of focal complexes and strengthening of αv/β3-integrin–cytoskeleton connections during the initial phase of ECM contact. These observations indicate that Src family kinases have distinct functions during adhesion site assembly, and that RPTPα is an early component in force-dependent signal transduction pathways leading to the assembly of focal complexes on both fibronectin and vitronectin.
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Mould, A. Paul, Susan E. Craig, Sarah K. Byron, Martin J. Humphries, and Thomas A. Jowitt. "Disruption of integrin–fibronectin complexes by allosteric but not ligand-mimetic inhibitors." Biochemical Journal 464, no. 3 (December 5, 2014): 301–13. http://dx.doi.org/10.1042/bj20141047.
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Integrins are protein receptors on cells, and inhibiting these receptors is a strategy for treating many diseases. We show that commonly used integrin inhibitors are unable to dissociate pre-formed integrin–ligand complexes, whereas inhibitors that function allosterically are able to do so.
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Wu, C., A. J. Fields, B. A. Kapteijn, and J. A. McDonald. "The role of alpha 4 beta 1 integrin in cell motility and fibronectin matrix assembly." Journal of Cell Science 108, no. 2 (February 1, 1995): 821–29. http://dx.doi.org/10.1242/jcs.108.2.821.
Full textAbstract:
The alpha 4 beta 1 integrin has been suggested to play important roles in embryogenesis and pathogenesis of many diseases which involve both cell adhesion and cell migration. Previous studies using anti-alpha 4 beta 1 antibodies and fibronectin (Fn) fragments have suggested that alpha 4 beta 1 integrins may be involved in cell motility on Fn and vascular cell adhesion molecule-1 (VCAM-1). However, the cells used in these studies also express other Fn integrin receptors including alpha 5 beta 1 integrin, which is known to function in cell motility on Fn. To test whether alpha 4 beta 1 integrins mediate cell motility on Fn and VCAM-1 in the absence of alpha 5 beta 1 integrin, we expressed human alpha 4 integrin in a Chinese hamster ovary (CHO) cell line that is deficient in alpha 5 beta 1 integrin (CHO B2). The parental alpha 5 deficient CHO B2 cells were unable to adhere, spread or migrate on Fn, nor could they assemble a fibrillar Fn matrix. Expression of alpha 4 beta 1 integrin in the CHO B2 cells enabled the cells to adhere, spread and migrate on Fn and on VCAM-1 but not to assemble a fibrillar Fn matrix. The cellular processes mediated by the interaction of alpha 4 beta 1 with Fn or VCAM-1 were inhibited by the CS1 peptide derived from the major alpha 4 beta 1 binding site on Fn. These findings demonstrate that alpha 4 beta 1 integrins not only function as cell adhesion receptors but also as cell motility receptors for Fn and VCAM-1 independent of alpha 5 beta 1. Moreover, they reveal important functional differences between Fn binding integrins. The alpha 4-positive, alpha 5-negative CHO cells described in this report will be useful tools in studying the mechanism of molecular signalling during integrin mediated cellular processes.
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Thibault, Gaétan, Marie-Josée Lacombe, Lynn M. Schnapp, Alexandre Lacasse, Fatiha Bouzeghrane, and Geneviève Lapalme. "Upregulation of α8β1-integrin in cardiac fibroblast by angiotensin II and transforming growth factor-β1." American Journal of Physiology-Cell Physiology 281, no. 5 (November 1, 2001): C1457—C1467. http://dx.doi.org/10.1152/ajpcell.2001.281.5.c1457.
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Using a novel pharmacological tool with125I-echistatin to detect integrins on the cell, we have observed that cardiac fibroblasts harbor five different RGD-binding integrins: α8β1, α3β1, α5β1, αvβ1, and αvβ3. Stimulation of cardiac fibroblasts by angiotensin II (ANG II) or transforming growth factor-β1 (TGF-β1) resulted in an increase of protein and heightening by 50% of the receptor density of α8β1-integrin. The effect of ANG II was blocked by an AT1, but not an AT2, receptor antagonist, or by an anti-TGF-β1 antibody. ANG II and TGF-β1 increased fibronectin secretion, smooth muscle α-actin synthesis, and formation of actin stress fibers and enhanced attachment of fibroblasts to a fibronectin matrix. The α8- and β1-subunits were colocalized by immunocytochemistry with vinculin or β3-integrin at focal adhesion sites. These results indicate that α8β1-integrin is an abundant integrin on rat cardiac fibroblasts. Its positive modulation by ANG II and TGF-β1 in a myofibroblast-like phenotype suggests the involvement of α8β1-integrin in extracellular matrix protein deposition and cardiac fibroblast adhesion.
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Venstrom, K., and L. Reichardt. "Beta 8 integrins mediate interactions of chick sensory neurons with laminin-1, collagen IV, and fibronectin." Molecular Biology of the Cell 6, no. 4 (April 1995): 419–31. http://dx.doi.org/10.1091/mbc.6.4.419.
Full textAbstract:
Integrins are major receptors used by cells to interact with extracellular matrices. In this paper, we identify the first ligands for the beta 8 family of integrins, presenting evidence that integrin heterodimers containing the beta 8 subunit mediate interactions of chick sensory neurons with laminin-1, collagen IV, and fibronectin. A polyclonal antibody, anti-beta 8-Ex, was prepared to a bacterial fusion protein expressing an extracellular portion of the chicken beta 8 subunit. In nonreducing conditions, this antibody immunoprecipitated from surface-labeled embryonic dorsal root ganglia neurons a M(r) 100 k protein, the expected M(r) of the beta 8 subunit, and putative alpha subunit(s) of M(r) 120 k. Affinity-purified anti-beta 8-Ex strongly inhibited sensory neurite outgrowth on laminin-1, collagen IV, and fibronectin-coated substrata. Binding sites were identified in a heat-resistant domain in laminin-1 and in the carboxyl terminal, 40-kDa fibronectin fragment. On substrates coated with the carboxyl terminal fragment of fibronectin, antibodies to beta 1 and beta 8 were only partially effective alone, but were completely effective in combination, at inhibiting neurite outgrowth. Results thus indicate that the integrin beta 8 subunit in association with one or more alpha subunits forms an important set of extracellular matrix receptors on sensory neurons.
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Meijne, A. M., D. M. Casey, C. A. Feltkamp, and E. Roos. "Immuno-EM localization of the beta 1 integrin subunit in wet-cleaved fibronectin-adherent fibroblasts." Journal of Cell Science 107, no. 5 (May 1, 1994): 1229–39. http://dx.doi.org/10.1242/jcs.107.5.1229.
Full textAbstract:
Using immuno-EM, we have studied the distribution of the beta 1 integrin subunit in chicken embryo fibroblasts allowed to adhere and spread for 3 hours on a fibronectin-coated surface in serum-free medium. The cells were wet-cleaved, which removed most of the cell body, yielding ventral plasma membranes with little, and sometimes virtually no, associated cytoskeleton. The beta 1 integrin subunit was detected with antibodies against the cytoplasmic domain. In immune fluorescence, it colocalized with adhesion plaques, in a punctate staining pattern, and often seemed to be at the periphery of the plaque. By immuno-EM, beta 1 was in fact found in discrete clusters, not throughout the plaque. In deep-cleaved cells from which virtually all cytoskeleton was removed, clusters could often be seen to be located on fibronectin fibrils. Furthermore, beta 1 was present in clusters at the cell margins, and isolated or in small groups at the very edge of the cell. When fibronectin synthesis, and consequently fibril formation, was inhibited by cycloheximide, large adhesion plaque-like structures were formed at the cell margin. This phenotype was reversed by addition of soluble fibronectin, which was incorporated into fibrils. As in normal plaques, talin and vinculin were present, the plasma membrane was very close (10-20 nm) to the substratum and the fibronectin layer underneath was removed. These plaques did contain beta 1 integrins but they were not in clusters. These observations indicate that the talin-vinculin network of an adhesion plaque is normally anchored to the substratum at discrete beta 1 integrin clusters that may be located on fibronectin fibrils, and that elsewhere the plaque is not necessarily attached to the substratum by interaction of integrins with matrix proteins. In the absence of fibronectin fibrils, adhesion plaque-like structures can be formed, but these are aberrant in size, location and fine structure.
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Penberthy, T. W., Y. Jiang, F. W. Luscinskas, and D. T. Graves. "MCP-1-stimulated monocytes preferentially utilize beta 2-integrins to migrate on laminin and fibronectin." American Journal of Physiology-Cell Physiology 269, no. 1 (July 1, 1995): C60—C68. http://dx.doi.org/10.1152/ajpcell.1995.269.1.c60.
Full textAbstract:
Recruitment of monocytes to inflammatory sites involves a series of sequential attachments and detachments to extracellular matrix proteins in response to a chemoattractant gradient. In this study we compared the migration of human peripheral blood monocytes on different extracellular matrix proteins in response to monocyte chemoattractant protein-1 (MCP-1) and N-formylmethionyl-leucyl-phenylalanine. Monocytes migrated more effectively on laminin compared with other extracellular matrix proteins. In contrast, this preference was not observed with neutrophils, suggesting that the monocytes and neutrophils may have differences in their migration on extracellular matrix proteins. To study this further, function-blocking monoclonal antibodies were used to examine mechanistically whether beta 1- and beta 2-integrins were involved in monocyte migration on fibronectin or laminin in response to MCP-1. Monocyte migration on both laminin and fibronectin was blocked 100% (P < 0.05) by intact monoclonal antibody, F(ab') fragments, and F(ab')2 fragments to beta 2-integrins. We also determined that antibodies to beta 2-integrins block monocyte migration that has already been initiated. In contrast, antibody to the beta 1-integrins inhibited monocyte migration by approximately 40% (P < 0.05). Thus monocytes that express both beta 1- and beta 2-integrins require utilization of beta 2-integrins in migration on extracellular matrix proteins. The results also suggest that beta 1-integrins facilitate monocyte migration but that monocyte migration is not absolutely dependent on the interaction of beta 1-integrins with extracellular matrix proteins. In contrast, neutrophil migration is beta 2-integrin dependent and is not facilitated by beta 1-integrins.
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Deryugina, E. I., and M. A. Bourdon. "Tenascin mediates human glioma cell migration and modulates cell migration on fibronectin." Journal of Cell Science 109, no. 3 (March 1, 1996): 643–52. http://dx.doi.org/10.1242/jcs.109.3.643.
Full textAbstract:
The role of tenascin in mediating tumor cell migration was studied using two cell migration models. In migration/invasion Transwell assays U251.3 glioma cells rapidly migrated through the 8 mu m pore size membranes onto tenascin- and fibronectin-coated surfaces. In this assay the number of cells migrating onto tenascin was 52.2 +/- 9.6% greater than on fibronectin within 4 hours. To assess cell migration rates and cell morphology, U251.3 migration was examined in a two-dimension spheroid outgrowth assay. The radial distance migrated by U251.3 cells from tumor spheroids was found to be 53.8 +/- 4.9% greater on tenascin than on fibronectin. Cells migrating on tenascin display a very motile appearance, while cells migrating on fibronectin spread and maintain close intercellular contacts. Cell migration in the presence of integrin blocking antibodies demonstrated that migration on tenascin and fibronectin is mediated by distinct integrins, alpha2beta1 and alphavbeta5/alphavbeta3, respectively. Since tenascin is coexpressed in malignant tumor matrices with fibronectin, we assessed the effects of tenascin on U251.3 cell migration mediated by fibronectin. Tenascin was found to provide a positive effect on fibronectin-mediated migration by altering cell morphology and enhancing cell motility. These effects of tenascin on fibronectin-mediated cell migration were inhibited by blocking beta1 and alpha2beta1 integrins. The results suggest that tenascin may play a significant role in promoting tumor cell migration and invasiveness by modulating cell responses to normal matrix components.
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Martel, V., L. Vignoud, S. Dupe, P. Frachet, M. R. Block, and C. Albiges-Rizo. "Talin controls the exit of the integrin alpha 5 beta 1 from an early compartment of the secretory pathway." Journal of Cell Science 113, no. 11 (June 1, 2000): 1951–61. http://dx.doi.org/10.1242/jcs.113.11.1951.
Full textAbstract:
Talin is a major cytosolic protein that links the intracellular domains of beta1 and beta3 integrins to the cytoskeleton. It is required for focal adhesion assembly. However, its downregulation not only slows down cell spreading and organization of focal adhesions but also impairs the maturation of some beta1 integrins, including the fibronectin receptor alpha5beta1. To investigate this, we characterized the beta1 integrin synthesized in cells expressing talin anti-sense RNA (AT22 cells). We identified a large intracellular pool of beta1 integrins that is abnormally accumulated in an earlier compartment of the secretory pathway. In this report, we show that in talin-deficient AT22 cells, the aberrant glycosylation of integrin receptors is accompanied by a delay in the export of the integrin alpha5beta1. In normal cells, talin was found associated with beta1 integrins in an enriched membrane fraction containing Golgi and endoplasmic reticulum. Finally, microinjection of anti-talin antibodies resulted in accumulation of the integrins within the cells. These data strongly suggest that talin plays a specific role in the export of newly synthesized integrins. We propose that talin binding to the integrin may disclose a diphenylalanine export signal, which is present in the membrane-proximal GFFKR motif conserved in all integrin alpha chains.
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Hangan-Steinman, Dolores, Wai-chi Ho, Priti Shenoy, Bosco MC Chan, and Vincent L. Morris. "Differences in phosphatase modulation of α4 β1 and α5 β1 integrin-mediated adhesion and migration of B16F1 cells." Biochemistry and Cell Biology 77, no. 5 (October 1, 1999): 409–20. http://dx.doi.org/10.1139/o99-050.
Full textAbstract:
It is well established that a biphasic relationship exists between the adhesive strength of β1 integrins and their ability to mediate cell movement. Thus, cell movement increases progressively with adhesive strength, but beyond a certain point of optimal interaction, cell movement is reduced with further increases in adhesive function. The interplay between the various kinase and phosphatase activities provides the balance in β1 integrin-mediated cell adhesion and migration. In the present study, the significance of protein tyrosine phosphatases (PTP) and ser/thr protein phosphatases (PP) in α4β1 and α5β1 integrin-mediated mouse melanoma B16F1 cell anchorage and migration on fibronectin was characterized using phosphatase inhibitors. At low fibronectin concentration, α5β1 functioned as the predominant receptor for cell movement; a role for α4β1 in B16F1 cell migration increased progressively with fibronectin concentration. Treatment of B16F1 cells with PTP inhibitors, sodium orthovanadate (Na3VO4) and phenylarsine oxide (PAO), or PP-1/2A inhibitor, okadaic acid (OA), abolished cell movement. Inhibition of cell movement by PAO and OA was associated by a reduction in the adhesive strength of α4β1 and α5β1. In contrast, treatment of B16F1 cells with Na3VO4 resulted in selective stimulation of the adhesive function of α5β1, but not α4β1. Therefore, our results demonstrate that (i) both PTP and PP-1/2A have roles in cell movement, (ii) modulation of cell movement by PTP and PP-1/2A may involve either a stimulation or reduction of β1 integrin adhesive strength, and (iii) distinct phosphatase-mediated signaling pathways for differential regulation of the various β1 integrins exist. Key words: phosphatases, integrins, cell movement, cell adhesion.
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Danen, Erik H. J., Jacco van Rheenen, Willeke Franken, Stephan Huveneers, Petra Sonneveld, Kees Jalink, and Arnoud Sonnenberg. "Integrins control motile strategy through a Rho–cofilin pathway." Journal of Cell Biology 169, no. 3 (May 2, 2005): 515–26. http://dx.doi.org/10.1083/jcb.200412081.
Full textAbstract:
During wound healing, angiogenesis, and tumor invasion, cells often change their expression profiles of fibronectin-binding integrins. Here, we show that β1 integrins promote random migration, whereas β3 integrins promote persistent migration in the same epithelial cell background. Adhesion to fibronectin by αvβ3 supports extensive actin cytoskeletal reorganization through the actin-severing protein cofilin, resulting in a single broad lamellipod with static cell–matrix adhesions at the leading edge. Adhesion by α5β1 instead leads to the phosphorylation/inactivation of cofilin, and these cells fail to polarize their cytoskeleton but extend thin protrusions containing highly dynamic cell–matrix adhesions in multiple directions. The activity of the small GTPase RhoA is particularly high in cells adhering by α5β1, and inhibition of Rho signaling causes a switch from a β1- to a β3-associated mode of migration, whereas increased Rho activity has the opposite effect. Thus, alterations in integrin expression profiles allow cells to modulate several critical aspects of the motile machinery through Rho GTPases.
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Mangale, Sachin S., and K. V. R. Reddy. "Expression pattern of integrins and their ligands in mouse feto-maternal tissues during pregnancy." Reproduction, Fertility and Development 19, no. 3 (2007): 452. http://dx.doi.org/10.1071/rd06143.
Full textAbstract:
The role of integrins, the cell-surface glycoproteins involved in various cellular functions, is well documented. However, information about their role and expression profile during pregnancy is still scant. In the present study, the expression of the integrin subunits β3, α6 and α5, along with their ligands vitronectin, osteopontin, laminin and fibronectin, was investigated in mouse uterus during different stages of pregnancy, namely 6.5, 8.5 and 13.5 days post coitus (d.p.c.) by immunohistochemical localisation. Integrins β3, α6 and α5 and the extracellular matrix molecules vitronectin and osteopontin exhibited dynamic spatiotemporal changes in their expression pattern in gestational endometrium, whereas fibronectin and laminin demonstrated more-or-less ubiquitous expression. The inter-implantation sites showed localisation of most of these molecules predominantly in the luminal epithelium, whereas their expression was negligible in the stroma. The present study explores the possible role and relevance of the spatiotemporal expression of integrins and their ligands in endometrial/decidual function and the maintenance of pregnancy.
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Pinon, Perrine, Jenita Pärssinen, Patricia Vazquez, Michael Bachmann, Rolle Rahikainen, Marie-Claude Jacquier, Latifeh Azizi, et al. "Talin-bound NPLY motif recruits integrin-signaling adapters to regulate cell spreading and mechanosensing." Journal of Cell Biology 205, no. 2 (April 28, 2014): 265–81. http://dx.doi.org/10.1083/jcb.201308136.
Full textAbstract:
Integrin-dependent cell adhesion and spreading are critical for morphogenesis, tissue regeneration, and immune defense but also tumor growth. However, the mechanisms that induce integrin-mediated cell spreading and provide mechanosensing on different extracellular matrix conditions are not fully understood. By expressing β3-GFP-integrins with enhanced talin-binding affinity, we experimentally uncoupled integrin activation, clustering, and substrate binding from its function in cell spreading. Mutational analysis revealed Tyr747, located in the first cytoplasmic NPLY747 motif, to induce spreading and paxillin adapter recruitment to substrate- and talin-bound integrins. In addition, integrin-mediated spreading, but not focal adhesion localization, was affected by mutating adjacent sequence motifs known to be involved in kindlin binding. On soft, spreading-repellent fibronectin substrates, high-affinity talin-binding integrins formed adhesions, but normal spreading was only possible with integrins competent to recruit the signaling adapter protein paxillin. This proposes that integrin-dependent cell–matrix adhesion and cell spreading are independently controlled, offering new therapeutic strategies to modify cell behavior in normal and pathological conditions.
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Féral, Chloé C., Andries Zijlstra, Eugene Tkachenko, Gerald Prager, Margaret L. Gardel, Marina Slepak, and Mark H. Ginsberg. "CD98hc (SLC3A2) participates in fibronectin matrix assembly by mediating integrin signaling." Journal of Cell Biology 178, no. 4 (August 6, 2007): 701–11. http://dx.doi.org/10.1083/jcb.200705090.
Full textAbstract:
Integrin-dependent assembly of the fibronectin (Fn) matrix plays a central role in vertebrate development. We identify CD98hc, a membrane protein, as an important component of the matrix assembly machinery both in vitro and in vivo. CD98hc was not required for biosynthesis of cellular Fn or the maintenance of the repertoire or affinity of cellular Fn binding integrins, which are important contributors to Fn assembly. Instead, CD98hc was involved in the cell's ability to exert force on the matrix and did so by dint of its capacity to interact with integrins to support downstream signals that lead to activation of RhoA small GTPase. Thus, we identify CD98hc as a membrane protein that enables matrix assembly and establish that it functions by interacting with integrins to support RhoA-driven contractility. CD98hc expression can vary widely; our data show that these variations in CD98hc expression can control the capacity of cells to assemble an Fn matrix, a process important in development, wound healing, and tumorigenesis.
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Artym, Vira V., and Howard R. Petty. "Molecular Proximity of Kv1.3 Voltage-gated Potassium Channels and β1-Integrins on the Plasma Membrane of Melanoma Cells." Journal of General Physiology 120, no. 1 (June 10, 2002): 29–37. http://dx.doi.org/10.1085/jgp.20028607.
Full textAbstract:
Tumor cell membranes have multiple components that participate in the process of metastasis. The present study investigates the physical association of β1-integrins and Kv1.3 voltage-gated potassium channels in melanoma cell membranes using resonance energy transfer (RET) techniques. RET between donor-labeled anti–β1-integrin and acceptor-labeled anti-Kv1.3 channels was detected on LOX cells adherent to glass and fibronectin-coated coverslips. However, RET was not observed on LOX cells in suspension, indicating that molecular proximity of these membrane molecules is adherence-related. Several K+ channel blockers, including tetraethylammonium, 4-aminopyridine, and verapamil, inhibited RET between β1-integrins and Kv1.3 channels. However, the irrelevant K+ channel blocker apamin had no effect on RET between β1-integrins and Kv1.3 channels. Based on these findings, we speculate that the lateral association of Kv1.3 channels with β1-integrins contributes to the regulation of integrin function and that channel blockers might affect tumor cell behavior by influencing the assembly of supramolecular structures containing integrins.
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Schick, P. K., C. M. Wojenski, X. He, J. Walker, C. Marcinkiewicz, and S. Niewiarowski. "Integrins Involved in the Adhesion of Megakaryocytes to Fibronectin and Fibrinogen." Blood 92, no. 8 (October 15, 1998): 2650–56. http://dx.doi.org/10.1182/blood.v92.8.2650.
Full textAbstract:
Abstract We studied integrins involved in the adhesion of resting and activated megakaryocytes (MK) to fibronectin (FN) and fibrinogen (FGN). Guinea pig MK were isolated and in some experiments were activated by thrombin. MK adhering to FN or FGN coated on coverslips were quantitated by a computerized image analysis program. The binding of soluble human FN to MK was detected by Western blotting. Anti-integrin antibodies, disintegrins, and cyclic RGD peptides were used to identify integrins involved in the adhesion of MK to FN or FGN. Resting MK adhered to coverslips with immobilized FN. The adhesion of MK to FN was primarily inhibited by an anti-5 antibody and EMF-10, a distintegrin highly specific for 5β1. However, the adhesion of MK to FN was not blocked by agents that inhibit ΙΙbβ3, vβ3 or 4β1. A β1 activating antibody increased the number of MK bound to FN due to the activation of 5β1. The binding of soluble FN was also primarily inhibited by agents that block 5β1. Resting MK did not adhere to FGN. However, MK activated by thrombin did adhere to FGN. This binding was mediated by ΙΙbβ3, because binding was inhibited by bitistatin, a disintegrin, and a cyclic RGD peptide that are known to block this integrin. The binding of thrombin-activated MK to FN was mediated by both 5β1 and ΙΙbβ3 based on the additive effect of agents that inhibit these integrins. The study indicates that resting MK bind to FN but not to FGN and that 5β1 is the major integrin involved in the binding of MK to FN. Activated MK bind to FGN primarily by IIbβ3. However, the binding of activated MK to FN is due to both 5β1 and IIbβ3. The demonstration that 5β1 and that IIbβ3 are involved in MK adhesion indicates that these integrins may have a role in MK maturation and platelet production. © 1998 by The American Society of Hematology.
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Schick, P. K., C. M. Wojenski, X. He, J. Walker, C. Marcinkiewicz, and S. Niewiarowski. "Integrins Involved in the Adhesion of Megakaryocytes to Fibronectin and Fibrinogen." Blood 92, no. 8 (October 15, 1998): 2650–56. http://dx.doi.org/10.1182/blood.v92.8.2650.420k37_2650_2656.
Full textAbstract:
We studied integrins involved in the adhesion of resting and activated megakaryocytes (MK) to fibronectin (FN) and fibrinogen (FGN). Guinea pig MK were isolated and in some experiments were activated by thrombin. MK adhering to FN or FGN coated on coverslips were quantitated by a computerized image analysis program. The binding of soluble human FN to MK was detected by Western blotting. Anti-integrin antibodies, disintegrins, and cyclic RGD peptides were used to identify integrins involved in the adhesion of MK to FN or FGN. Resting MK adhered to coverslips with immobilized FN. The adhesion of MK to FN was primarily inhibited by an anti-5 antibody and EMF-10, a distintegrin highly specific for 5β1. However, the adhesion of MK to FN was not blocked by agents that inhibit ΙΙbβ3, vβ3 or 4β1. A β1 activating antibody increased the number of MK bound to FN due to the activation of 5β1. The binding of soluble FN was also primarily inhibited by agents that block 5β1. Resting MK did not adhere to FGN. However, MK activated by thrombin did adhere to FGN. This binding was mediated by ΙΙbβ3, because binding was inhibited by bitistatin, a disintegrin, and a cyclic RGD peptide that are known to block this integrin. The binding of thrombin-activated MK to FN was mediated by both 5β1 and ΙΙbβ3 based on the additive effect of agents that inhibit these integrins. The study indicates that resting MK bind to FN but not to FGN and that 5β1 is the major integrin involved in the binding of MK to FN. Activated MK bind to FGN primarily by IIbβ3. However, the binding of activated MK to FN is due to both 5β1 and IIbβ3. The demonstration that 5β1 and that IIbβ3 are involved in MK adhesion indicates that these integrins may have a role in MK maturation and platelet production. © 1998 by The American Society of Hematology.
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Pierini, Lynda M., Moira A. Lawson, Robert J. Eddy, Bill Hendey, and Frederick R. Maxfield. "Oriented endocytic recycling of α5β1 in motile neutrophils." Blood 95, no. 8 (April 15, 2000): 2471–80. http://dx.doi.org/10.1182/blood.v95.8.2471.
Full textAbstract:
Abstract During cell migration, integrin attachments to the substratum provide the means to generate the traction and force necessary to achieve locomotion. Once the cell has moved over these attachments, however, it is equally important that integrins detach from the substratum. The fate of integrins after detachment may include release from the cell, lateral diffusion across the cell surface, or endocytosis and redelivery to the cell surface. Polymorphonuclear neutrophils (PMNs) become stuck on the extracellular matrix proteins fibronectin and vitronectin when their intracellular free calcium concentration ([Ca++]i) is buffered. Taking advantage of this feature of PMN migration, we investigated the fate of integrins to differentiate among various models of migration. We demonstrate that 5β1, one of the fibronectin-binding integrins, is responsible for immobilization of [Ca++]i-buffered PMNs on fibronectin. We find that 5 and β1 are in endocytic vesicles in PMNs and that 5 colocalizes with a marker for an endocytic recycling compartment. When [Ca++]i is buffered, 5 and β1 become concentrated in clusters in the rear of the adherent cells, suggesting that [Ca++]i transients are required for 5β1 detachment from the substratum. Inhibition of 5β1 detachment by buffering [Ca++]i results in the depletion of 5 from both endocytic vesicles and the recycling compartment, providing compelling evidence that integrins are normally recycled by way of endocytosis and intracellular trafficking during cell migration. This model is further refined by our demonstration that the endocytic recycling compartment reorients to retain its localization just behind the leading lamella as PMNs migrate, indicating that membrane recycling during neutrophil migration has directionality.