scholarly journals p190RhoGAP is the convergence point of adhesion signals from α5β1 integrin and syndecan-4

2008 ◽  
Vol 181 (6) ◽  
pp. 1013-1026 ◽  
Author(s):  
Mark D. Bass ◽  
Mark R. Morgan ◽  
Kirsty A. Roach ◽  
Jeffrey Settleman ◽  
Andrew B. Goryachev ◽  
...  
Keyword(s):  
Adhesion Formation ◽  
Focal Adhesions ◽  
Dependent Manner ◽  
Rhoa Activity ◽  
Focal Adhesion Formation ◽  
Convergence Point ◽  
Individual Contributions ◽  
Guanosine Triphosphatase ◽  
Extracellular Matrix Receptors ◽  
Α5β1 Integrin

The fibronectin receptors α5β1 integrin and syndecan-4 cocluster in focal adhesions and coordinate cell migration by making individual contributions to the suppression of RhoA activity during matrix engagement. p190Rho–guanosine triphosphatase–activating protein (GAP) is known to inhibit RhoA during the early stages of cell spreading in an Src-dependent manner. This paper dissects the mechanisms of p190RhoGAP regulation and distinguishes the contributions of α5β1 integrin and syndecan-4. Matrix-induced tyrosine phosphorylation of p190RhoGAP is stimulated solely by engagement of α5β1 integrin and is independent of syndecan-4. Parallel engagement of syndecan-4 causes redistribution of the tyrosine-phosphorylated pool of p190RhoGAP between membrane and cytosolic fractions by a mechanism that requires direct activation of protein kinase C α by syndecan-4. Activation of both pathways is necessary for the efficient regulation of RhoA and, as a consequence, focal adhesion formation. Accordingly, we identify p190RhoGAP as the convergence point for adhesive signals mediated by α5β1 integrin and syndecan-4. This molecular mechanism explains the cooperation between extracellular matrix receptors during cell adhesion.

scholarly journals Pyk2- and Src-Dependent Tyrosine Phosphorylation of PDK1 Regulates Focal Adhesions

2003 ◽  
Vol 23 (22) ◽  
pp. 8019-8029 ◽  
Author(s):  
Yoshihiro Taniyama ◽  
David S. Weber ◽  
Petra Rocic ◽  
Lula Hilenski ◽  
Marjorie L. Akers ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Tyrosine Phosphorylation ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Dependent Manner ◽  
Critical Function ◽  
Focal Adhesion Formation ◽  
Dependent Protein ◽  
And Migration

ABSTRACT 3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a signal integrator that activates the AGC superfamily of serine/threonine kinases. PDK1 is phosphorylated on tyrosine by oxidants, although its regulation by agonists that stimulate G-protein-coupled receptor signaling pathways and the physiological consequences of tyrosine phosphorylation in this setting have not been fully identified. We found that angiotensin II stimulates the tyrosine phosphorylation of PDK1 in vascular smooth muscle in a calcium- and c-Src-dependent manner. The calcium-activated tyrosine kinase Pyk2 acts as a scaffold for Src-dependent phosphorylation of PDK1 on Tyr9, which permits phosphorylation of Tyr373 and -376 by Src. This critical function of Pyk2 is further supported by the observation that Pyk2 and tyrosine-phosphorylated PDK1 colocalize in focal adhesions after angiotensin II stimulation. Importantly, infection of smooth muscle cells with a Tyr9 mutant of PDK1 inhibits angiotensin II-induced tyrosine phosphorylation of paxillin and focal adhesion formation. These observations identify a novel interaction between PDK1 and Pyk2 that regulates the integrity of focal adhesions, which are major compartments for integrating signals for cell growth, apoptosis, and migration.

scholarly journals Calcium-Sensing Receptor Stimulation in Cultured Glomerular Podocytes Induces TRPC6-Dependent Calcium Entry and RhoA Activation

2017 ◽  
Vol 43 (5) ◽  
pp. 1777-1789 ◽  
Author(s):  
Lei Zhang ◽  
Tianrong Ji ◽  
Qin Wang ◽  
Kexin Meng ◽  
Rui Zhang ◽  
...  
Keyword(s):  
Protective Action ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Dependent Manner ◽  
Calcium Sensing Receptor ◽  
Rhoa Activity ◽  
Rhoa Activation ◽  
Calcium Sensing

Background/Aims: Recent studies provided compelling evidence that stimulation of the calcium sensing receptor (CaSR) exerts direct renoprotective action at the glomerular podocyte level. This protective action may be attributed to the RhoA-dependent stabilization of the actin cytoskeleton. However, the underlying mechanisms remain unclear. Methods: In the present study, an immortalized human podocyte cell line was used. Fluo-3 fluorescence was utilized to determine intracellular Ca2+ concentration ([Ca2+]i), and western blotting was used to measure canonical transient receptor potential 6 (TRPC6) protein expression and RhoA activity. Stress fibers were detected by FITC-phalloidin. Results: Activating CaSR with a high extracellular Ca2+ concentration ([Ca2+]o) or R-568 (a type II CaSR agonist) induces an increase in the [Ca2+]i in a dose-dependent manner. This increase in [Ca2+]i is phospholipase C (PLC)-dependent and is smaller in the absence of extracellular Ca2+ than in the presence of 0.5 mM [Ca2+]o. The CaSR activation-induced [Ca2+]i increase is attenuated by the pharmacological blockage of TRPC6 channels or siRNA targeting TRPC6. These data suggest that TRPC6 is involved in CaSR activation-induced Ca2+ influx. Consistent with a previous study, CaSR stimulation results in an increase in RhoA activity. However, the knockdown of TRPC6 significantly abolished the RhoA activity increase induced by CaSR stimulation, suggesting that TRPC6-dependent Ca2+ entry is required for RhoA activation. The activated RhoA is involved in the formation of stress fibers and focal adhesions in response to CaSR stimulation because siRNA targeting RhoA attenuated the increase in the stress fiber mediated by CaSR stimulation. Moreover, this effect of CaSR activation on the formation of stress fibers is also abolished by the knockdown of TRPC6. Conclusion: TRPC6 is involved in the regulation of stress fiber formation and focal adhesions via the RhoA pathway in response to CaSR activation. This may explain the direct protective action of CaSR agonists.

scholarly journals Multiparametric analysis of focal adhesion formation by RNAi-mediated gene knockdown

2009 ◽  
Vol 186 (3) ◽  
pp. 423-436 ◽  
Author(s):  
Sabina E. Winograd-Katz ◽  
Shalev Itzkovitz ◽  
Zvi Kam ◽  
Benjamin Geiger
Keyword(s):  
Cell Adhesion ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Gene Families ◽  
Adaptor Proteins ◽  
Small Interfering Rnas ◽  
Comprehensive Information ◽  
Focal Adhesion Formation ◽  
Multiple Cell ◽  
And Migration

Cell adhesion to the extracellular matrix is mediated by elaborate networks of multiprotein complexes consisting of adhesion receptors, cytoskeletal components, signaling molecules, and diverse adaptor proteins. To explore how specific molecular pathways function in the assembly of focal adhesions (FAs), we performed a high-throughput, high-resolution, microscopy-based screen. We used small interfering RNAs (siRNAs) to target human kinases, phosphatases, and migration- and adhesion-related genes. Multiparametric image analysis of control and of siRNA-treated cells revealed major correlations between distinct morphological FA features. Clustering analysis identified different gene families whose perturbation induced similar effects, some of which uncoupled the interfeature correlations. Based on these findings, we propose a model for the molecular hierarchy of FA formation, and tested its validity by dynamic analysis of FA formation and turnover. This study provides a comprehensive information resource on the molecular regulation of multiple cell adhesion features, and sheds light on signaling mechanisms regulating the formation of integrin adhesions.

scholarly journals Disease-associated keratin mutations reduce traction forces and compromise adhesion and collective migration

2020 ◽  
Vol 133 (14) ◽  
pp. jcs243956 ◽  
Author(s):  
Sachiko Fujiwara ◽  
Shinji Deguchi ◽  
Thomas M. Magin
Keyword(s):  
Molecular Mechanisms ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Missense Mutations ◽  
Collective Migration ◽  
Epidermolysis Bullosa Simplex ◽  
Traction Forces ◽  
Rhoa Activity ◽  
Keratin 5 ◽  
Dependent Cell

ABSTRACTKeratin intermediate filament (IF) proteins constitute the major cytoskeletal components in epithelial cells. Missense mutations in keratin 5 (K5; also known as KRT5) or keratin 14 (K14; also known as KRT14), highly expressed in the basal epidermis, cause the severe skin blistering disease epidermolysis bullosa simplex (EBS). EBS-associated mutations disrupt keratin networks and change keratinocyte mechanics; however, molecular mechanisms by which mutations shape EBS pathology remain incompletely understood. Here, we demonstrate that, in contrast to keratin-deficient keratinocytes, cells expressing K14R125C, a mutation that causes severe EBS, generate lower traction forces, accompanied by immature focal adhesions with an altered cellular distribution. Furthermore, mutant keratinocytes display reduced directionality during collective migration. Notably, RhoA activity is downregulated in human EBS keratinocytes, and Rho activation rescues stiffness-dependent cell–extracellular matrix (ECM) adhesion formation of EBS keratinocytes. Collectively, our results strongly suggest that intact keratin IF networks regulate mechanotransduction through a Rho signaling pathway upstream of cell–ECM adhesion formation and organized cell migration. Our findings provide insights into the underlying pathophysiology of EBS.This article has an associated First Person interview with the first author of the paper.

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.

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