α1 Integrin cytoplasmic domain is involved in focal adhesion formation via association with intracellular proteins

2001 ◽  
Vol 356 (1) ◽  
pp. 233-240 ◽  
Author(s):  
Klemens LÖSTER ◽  
Dörte VOSSMEYER ◽  
Werner HOFMANN ◽  
Werner REUTTER ◽  
Kerstin DANKER
Keyword(s):  
Signal Transduction ◽  
Focal Adhesion ◽  
Protein Interactions ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Cell Types ◽  
Cytoplasmic Domain ◽  
Integrin Subunit ◽  
Adhesion Receptors ◽  
Focal Adhesion Formation

Integrins are heterodimeric adhesion receptors consisting of α- and β-subunits capable of binding extracellular matrix molecules as well as other adhesion receptors on neighbouring cells. These interactions induce various signal transduction pathways in many cell types, leading to cytoskeletal reorganization, phosphorylation and induction of gene expression. Integrin ligation leads to cytoplasmic protein–protein interactions requiring both integrin cytoplasmic domains, and these domains are initiation points for focal adhesion formation and subsequent signal transduction cascades. In previous studies we have shown that the very short cytoplasmic α1 tail is required for post-ligand events, such as cell spreading as well as actin stress-fibre formation. In the present paper we report that cells lacking the cytoplasmic domain of the α1 integrin subunit are unable to form proper focal adhesions and that phosphorylation on tyrosine residues of focal adhesion components is reduced on α1β1-specific substrates. The α1 cytoplasmic sequence is a specific recognition site for focal adhesion components like paxillin, talin, α-actinin and pp125FAK. It seems to account for α1-specific signalling, since when peptides that mimic the cytoplasmic domain of α1 are transferred into cells, they influence α1β1-specific adhesion, presumably by competing for binding partners. For α1 integrin/protein binding, the conserved Lys-Ile-Gly-Phe-Phe-Lys-Arg motif and, in particular, the two lysine residues, are important.

Control of morphology, cytoskeleton and migration by syndecan-4

1999 ◽  
Vol 112 (20) ◽  
pp. 3421-3431 ◽  
Author(s):  
R.L. Longley ◽  
A. Woods ◽  
A. Fleetwood ◽  
G.J. Cowling ◽  
J.T. Gallagher ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Core Protein ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Cytoplasmic Domain ◽  
Fiber Formation ◽  
Microfilament Bundle ◽  
Focal Adhesion Formation ◽  
And Migration

Syndecan-4 is a widely expressed transmembrane heparan sulfate proteoglycan which localizes to focal adhesions. Previous studies showed that the syndecan-4 cytoplasmic domain can associate with and potentiate the activity of protein kinase C, which is required for focal adhesion formation. To examine further the role of syndecan-4 in cell adhesion, we expressed syndecan-4 cDNA constructs in CHO-K1 cells. Syndecan-2 transfection was used to confirm effects seen were specific for syndecan-4. Cells overexpressing full length syndecan-4 core protein exhibited a more flattened, fibroblastic morphology, with increased focal adhesion formation and decreased cell motility. Expression of a syndecan-4 core protein with either a partial or complete deletion of the cytoplasmic domain or of an antisense construct led to markedly decreased spreading and focal adhesion formation, a more epithelioid morphology, and decreased motility. Overexpression of syndecan-2 changed the adhesive phenotype, but did not markedly alter focal adhesion and microfilament bundle formation. The data suggest that syndecan-4 is a regulator of focal adhesion and stress fiber formation, and influences both morphology and migration.

scholarly journals Interleukin 1-induced calcium signalling in chondrocytes requires focal adhesions

1997 ◽  
Vol 324 (2) ◽  
pp. 653-658 ◽  
Author(s):  
Laura LUO ◽  
Tony CRUZ ◽  
Christopher McCULLOCH
Keyword(s):  
Signal Transduction ◽  
Focal Adhesion ◽  
Cell Attachment ◽  
Interleukin 1 ◽  
Primary Cultures ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Ion Concentration ◽  
Significant Difference ◽  
In Cells

The cytokine interleukin 1 (IL-1) is an important mediator of connective-tissue destruction in arthritic joints but the mechanisms by which IL-1 mediates signal transduction in chondrocytes is poorly understood. Previous results have indicated that IL-1 receptors co-localize with focal adhesions [Qwarnstrom, Page, Gillis and Dower (1988) J. Biol. Chem. 263, 8261–8269], discrete adhesive domains of cells that function in cell attachment and possibly in signal transduction. We have determined whether focal adhesions restrict IL-1-induced Ca2+ signalling in primary cultures of bovine chondrocytes. In cells grown for 24 h on fibronectin, the basal intracellular Ca2+ ion concentration ([Ca2+]i) was 100±3 nM. Optimal increases of [Ca2+]i above baseline were induced by 10 nM IL-1 (183±30 nM above baseline). There was no significant difference between cells plated on fibronectin or type II collagen (P > 0.2; 233±90 nM above baseline). Ca2+ transients were significantly decreased by the inclusion of 0.5 mM EGTA in the bathing buffer (74±11 nM above baseline), and 1 μM thapsigargin completely blocked Ca2+ transients. Cells plated on poly-(l-lysine) or suspended cells showed no Ca2+ increases, whereas cells grown on fibronectin exhibited IL-1-induced Ca2+ responses that corresponded temporally to the time-dependent cell spreading after plating on fibronectin. Cells plated on poly-(l-lysine) and incubated with fibronectin-coated beads exhibited vinculin staining in association with the beads. In identical cell preparations, IL-1 induced a 136±39 nM increase of [Ca2+]i above baseline in response to 10 nM IL-1β. There were no IL-1-induced Ca2+ increases when cells on poly-(l-lysine) were incubated with fibronectin-coated beads for only 15 min at 37 °C, in cells maintained for 3 h at 4 °C, in cells incubated with BSA beads for 3 h at 37 °C, or in cells pretreated with cytochalasin D. Labelling of IL-1 receptors with 125I-IL-1β showed 3-fold more specific labelling of focal adhesion complexes in cells incubated with fibronectin-coated beads compared with cells incubated with BSA-coated beads, indicating that IL-1 receptor binding or the number of IL-1 receptors was increased in focal adhesions. These results indicate that, in chondrocytes, IL-1-induced Ca2+ signalling is dependent on focal adhesion formation and that focal adhesions recruit IL-1 receptors by redistribution in the cell membrane.

scholarly journals p130Cas contributes to cellular mechanosensing and force exertion

2018 ◽  
Author(s):  
Hedde van Hoorn ◽  
Dominique M. Donato ◽  
H. Emrah Balcioglu ◽  
Erik H. Danen ◽  
Thomas Schmidt
Keyword(s):  
Focal Adhesion ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Full Length ◽  
Actin Dynamics ◽  
Knock Out ◽  
Focal Adhesion Formation ◽  
Pillar Arrays ◽  
Cellular Machinery ◽  
And Migration

AbstractCell survival, differentiation, and migration are all dependent on the cell’s interaction with its external environment. In addition to chemical cues, cells react to their physical environment, particularly the stiffness of the substrate. In order for cells to react to these elements, they must make use of cellular machinery to signal changes in their microenvironment. One such proposed machinery is the protein p130Cas, which has been shown to regulate focal adhesion turnover, actin dynamics, and cell migration. Here we show that p130Cas localizes to focal adhesions depending on substrate stiffness and subsequently modulates cellular force exertion. We compared on substrates of tunable stiffness knock-out CAS-/-cells to cells re-expressing either the full-length p130Cas or a mutant lacking the focal adhesion targeting domains. On polyacrylamide gels, we observed that p130Cas prevented focal adhesion formation at low stiffness. On structured micro-pillar arrays, p130Cas preferentially localized to sites of force exertion when the apparent Young’s modulus of the substrate was higher than E = 47 kPa. Stiffness-dependent localization of p130Cas coincided with slower, but increased force exertion for the full-length p130Cas. Cas localization to focal adhesions preceded force build-up by three minutes, suggesting a coordinating role for p130Cas in the cellular mechanoresponse. Thus, p130Cas appears to relay mechanosensory information in the cell through its ability to tune force exertion at the focal adhesion.

scholarly journals Activation of Rho-kinase and focal adhesion kinase regulates the organization of stress fibers and focal adhesions in the central part of fibroblasts

2017 ◽  
Author(s):  
Kazuo Katoh
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Rho Kinase ◽  
Focal Adhesions ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Focal Adhesion Formation ◽  
Specific Regulation

Specific regulation and activation of focal adhesion kinase (FAK) are thought to be important for focal adhesion formation, and activation of Rho-kinase has been suggested to play a role in determining the effects of FAK on the formation of stress fibers and focal adhesions. To clarify the role of FAK in stress fiber formation and focal adhesion organization, we examined the formation of new stress fibers and focal adhesions by activation of Rho-kinase in FAK knockout (FAK–/–) fibroblasts. FAK–/– cells were elliptical in shape, and showed reduced numbers of stress fibers and focal adhesions in the central part of the cells along with large focal adhesions in the peripheral regions. Activation of Rho-kinase in FAK–/– cells transiently increased the actin filaments in the cell center, but these did not form typical thick stress fibers. Moreover, only plaque-like structures as the origins of newly formed focal adhesions were observed in the center of the cell. Furthermore, introduction of an exogenous GFP-labeled FAK gene into FAK–/– cells resulted in increased numbers of stress fibers and focal adhesions in the center of the cells, which showed typical fibroblast morphology. These results indicated that FAK plays an important role in the formation of stress fibers and focal adhesions as well as in regulation of cell shape and morphology with the activation of Rho-kinase.

αvβ3-Integrin antagonists inhibit thrombin-induced proliferation and focal adhesion formation in smooth muscle cells

2003 ◽  
Vol 285 (5) ◽  
pp. C1330-C1338 ◽  
Author(s):  
M. Sajid ◽  
R. Zhao ◽  
A. Pathak ◽  
S. S. Smyth ◽  
G. A. Stouffer
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Focal Adhesion ◽  
Immunofluorescence Microscopy ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Muscle Cells ◽  
Neointimal Formation ◽  
Focal Adhesion Formation ◽  
Integrin Antagonists

αvβ3-Integrin antagonists reduced neointimal formation following vascular injury in eight different animal models. Because α-thrombin contributes to neointimal formation, we examined the hypothesis that αvβ3-integrins influence α-thrombin-induced signaling. Cultured rat aortic smooth muscle cells (RASMC) expressed αvβ3-integrins as demonstrated by immunofluorescence microscopy and fluorescence-activated cell sorting analysis. Proliferative responses to α-thrombin were partially inhibited by anti-β3-integrin monoclonal antibody F11 and by cyclic RGD peptides. Immunofluorescence microscopy showed that α-thrombin stimulated a rapid increase in the formation of focal adhesions as identified by vinculin staining and that this effect was partially inhibited by αvβ3 antagonists. β3-Integrin staining was diffuse in quiescent RASMC and did not concentrate at sites of focal adhesions following thrombin treatment. α-Thrombin elicited a time-dependent increase in activation of c-Jun NH2-terminal kinase-1 (JNK1) and in tyrosine phosphorylation of focal adhesion kinase (FAK). αvβ3-Integrin antagonists partially inhibited increases in JNK1 activity but had no effect on FAK phosphorylation. In SMC isolated from β3-integrin-deficient mice, focal adhesion formation was impaired in response to thrombin but not sphingosine-1-phosphate, a potent activator of Rho. In summary, αvβ3-integrins play an important role in α-thrombin-induced proliferation and focal adhesion formation in RASMC.

pp125FAK tyrosine kinase activity is not required for the assembly of F-actin stress fibres and focal adhesions in cultured mouse aortic smooth muscle cells

1995 ◽  
Vol 108 (6) ◽  
pp. 2381-2391 ◽  
Author(s):  
L. Wilson ◽  
M.J. Carrier ◽  
S. Kellie
Keyword(s):  
Tyrosine Kinase ◽  
Focal Adhesion ◽  
Kinase Activity ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Tyrosine Kinase Activity ◽  
Aortic Smooth Muscle ◽  
Stress Fibre ◽  
Actin Stress Fibre ◽  
Focal Adhesion Formation

The observed increase in phosphotyrosine content of focal adhesion-associated proteins, in response to integrin engagement, indicates a role for integrin-regulatable tyrosine kinase(s) in cytoskeletal re-organisation. The tyrosine kinase pp125FAK, by virtue of its focal adhesion localisation in fibroblasts, represents a prime candidate to perform this function. We have investigated whether pp125FAK performs a similar function in mouse aortic smooth muscle cells (MASMC). MASMC cultured for 16 hours exhibit F-actin stress fibres and focal adhesions. We have shown that vinculin, pp125FAK and tyrosine-phosphorylated proteins are localised in focal adhesions during this time period. MASMC, under these culture conditions exhibit elevated pp125FAK tyrosine kinase activity, as measured by an increased autophosphorylation potential. We investigated the development of F-actin stress fibres and focal adhesions in MASMC in response to adherence to fibronectin, conditions shown to promote cytoskeletal reorganisation in fibroblasts. Within 30 minutes, MASMC exhibited well-developed F-actin stress fibres and prominent focal adhesions which immunostained intensely for vinculin, pp125FAK and phosphotyrosine. Adherence to fibronectin has been reported to activate pp125FAK tyrosine kinase in fibroblasts, leading to the proposal that pp125FAK plays a critical role in focal adhesion formation. Therefore pp125FAK activation, in response to adherence to fibronectin, was investigated in MASMC. Anti-phosphotyrosine immunoblotting and in vitro kinase assays of MASMC lysates have revealed that, under conditions which promote focal adhesion formation, pp125FAK remains inactive. Since overnight cultures of MASMC exhibited elevated pp125FAK tyrosine kinase activity, we investigated whether these cells deposit their own combination of extracellular matrix (ECM) molecules and/or secrete factors into their conditioned medium which are capable of activating pp125FAK tyrosine kinase. Our results indicate that MASMC-elaborated ECM, but not their conditioned medium, supported pp125FAK tyrosine kinase activation. Furthermore, MASMC exposed to MASMC-ECM displayed a poorly defined F-actin stress fibre network and rudimentary focal adhesions. Thus we have demonstrated the existence of two adhesion-mediated situations in MASMC; one in which fibronectin promotes cytoskeletal reorganisation in the absence of pp125FAK tyrosine kinase activity and the other in which cells adhering to MASMC-ECM display elevated pp125FAK tyrosine kinase activity in association with an impaired ability to promote F-actin stress fibre and focal adhesion formation. These results indicate that in MASMC, pp125FAK tyrosine kinase activity is not involved in F-actin stress fibre assembly and focal adhesion formation.

Smooth muscle cell rigidity and extracellular matrix organization influence endothelial cell spreading and adhesion formation in coculture

2007 ◽  
Vol 293 (3) ◽  
pp. H1978-H1986 ◽  
Author(s):  
Charles S. Wallace ◽  
Sophie A. Strike ◽  
George A. Truskey
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Endothelial Cell ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Tissue Engineered Blood Vessels ◽  
Focal Adhesion Formation ◽  
Fibrillar Adhesion

Efforts to develop functional tissue-engineered blood vessels have focused on improving the strength and mechanical properties of the vessel wall, while the functional status of the endothelium within these vessels has received less attention. Endothelial cell (EC) function is influenced by interactions between its basal surface and the underlying extracellular matrix. In this study, we utilized a coculture model of a tissue-engineered blood vessel to evaluate EC attachment, spreading, and adhesion formation to the extracellular matrix on the surface of quiescent smooth muscle cells (SMCs). ECs attached to and spread on SMCs primarily through the α5β1-integrin complex, whereas ECs used either α5β1- or αvβ3-integrin to spread on fibronectin (FN) adsorbed to plastic. ECs in coculture lacked focal adhesions, but EC α5β1-integrin bound to fibrillar FN on the SMC surface, promoting rapid fibrillar adhesion formation. As assessed by both Western blot analysis and quantitative real-time RT-PCR, coculture suppressed the expression of focal adhesion proteins and mRNA, whereas tensin protein and mRNA expression were elevated. When attached to polyacrylamide gels with similar elastic moduli as SMCs, focal adhesion formation and the rate of cell spreading increased relative to ECs in coculture. Thus, the elastic properties are only one factor contributing to EC spreading and focal adhesion formation in coculture. The results suggest that the softness of the SMCs and the fibrillar organization of FN inhibit focal adhesions and reduce cell spreading while promoting fibrillar adhesion formation. These changes in the type of adhesions may alter EC signaling pathways in tissue-engineered blood vessels.

scholarly journals Enhanced focal adhesion assembly reflects increased mechanosensation and mechanotransduction at maternal–conceptus interface and uterine wall during ovine pregnancy

Reproduction ◽  
2009 ◽  
Vol 137 (3) ◽  
pp. 567-582 ◽  
Author(s):  
Robert C Burghardt ◽  
James R Burghardt ◽  
James D Taylor ◽  
Adele T Reeder ◽  
Bar T Nguen ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Mechanical Stretch ◽  
Uterine Wall ◽  
Placental Development ◽  
Focal Adhesion Formation ◽  
Tensile Forces ◽  
Tight Connection

The integrity of the fetal–maternal interface is critical for proper fetal nourishment during pregnancy. Integrins are important adhesion molecules present at the interface during implantation; however,in vivoevidence for integrin activation and focal adhesion formation at the maternal–conceptus interface is limited. We hypothesized that focal adhesion assembly in uterine luminal epithelium (LE) and conceptus trophectoderm (Tr) results from integrin binding of extracellular matrix (ECM) at this interface to provide increased tensile forces and signaling to coordinate utero-placental development. An ovine model of unilateral pregnancy was used to evaluate mechanotransduction events leading to focal adhesion assembly at the maternal–conceptus interface and within the uterine wall. Animals were hysterectomized on days 40, 80, or 120 of pregnancy, and uteri immunostained for integrins (ITGAV, ITGA4, ITGA5, ITGB1, ITGB3, and ITGB5), ECM proteins (SPP1, LGALS15, fibronectin (FN), and vitronectin (VTN)), cytoskeletal molecules (ACTN and TLN1), and a signal generator (PTK2). Focal adhesion assembly in myometrium and stroma was also studied to provide a frame of reference for mechanical stretch of the uterine wall. Large focal adhesions containing aggregates of ITGAV, ITGA4, ITGA5, ITGB1, ITGB5, ACTN, and PTK2 were detected in interplacentomal uterine LE and Tr of gravid but not non-gravid uterine horns and increased during pregnancy. SPP1 and LGALS15, but not FN or VTN, were present along LE and Tr interfaces in both uterine horns. These data support the idea that focal adhesion assembly at the maternal–conceptus interface reflects adaptation to increasing forces caused by the growing fetus. Cooperative binding of multiple integrins to SPP1 deposited at the maternal–conceptus interface forms an adhesive mosaic to maintain a tight connection between uterine and placental surfaces along regions of epitheliochorial placentation in sheep.

scholarly journals Activation of Rho-kinase and focal adhesion kinase regulates the organization of stress fibers and focal adhesions in the central part of fibroblasts

2017 ◽  
Author(s):  
Kazuo Katoh
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Rho Kinase ◽  
Focal Adhesions ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Focal Adhesion Formation ◽  
Specific Regulation

Specific regulation and activation of focal adhesion kinase (FAK) are thought to be important for focal adhesion formation, and activation of Rho-kinase has been suggested to play a role in determining the effects of FAK on the formation of stress fibers and focal adhesions. To clarify the role of FAK in stress fiber formation and focal adhesion organization, we examined the formation of new stress fibers and focal adhesions by activation of Rho-kinase in FAK knockout (FAK–/–) fibroblasts. FAK–/– cells were elliptical in shape, and showed reduced numbers of stress fibers and focal adhesions in the central part of the cells along with large focal adhesions in the peripheral regions. Activation of Rho-kinase in FAK–/– cells transiently increased the actin filaments in the cell center, but these did not form typical thick stress fibers. Moreover, only plaque-like structures as the origins of newly formed focal adhesions were observed in the center of the cell. Furthermore, introduction of an exogenous GFP-labeled FAK gene into FAK–/– cells resulted in increased numbers of stress fibers and focal adhesions in the center of the cells, which showed typical fibroblast morphology. These results indicated that FAK plays an important role in the formation of stress fibers and focal adhesions as well as in regulation of cell shape and morphology with the activation of Rho-kinase.

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