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 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 Caldesmon Inhibits Nonmuscle Cell Contractility and Interferes with the Formation of Focal Adhesions

1999 ◽  
Vol 10 (10) ◽  
pp. 3097-3112 ◽  
Author(s):  
David M. Helfman ◽  
Esther T. Levy ◽  
Christine Berthier ◽  
Michael Shtutman ◽  
Daniel Riveline ◽  
...  
Keyword(s):  
Adhesion Formation ◽  
Focal Adhesions ◽  
Inhibitory Effect ◽  
Ionophore A23187 ◽  
Physiological Regulation ◽  
Cell Contractility ◽  
Inhibitory Function ◽  
Nonmuscle Cell ◽  
Microtubule Disruption ◽  
Dependent Cell

Caldesmon is known to inhibit the ATPase activity of actomyosin in a Ca2+–calmodulin-regulated manner. Although a nonmuscle isoform of caldesmon is widely expressed, its functional role has not yet been elucidated. We studied the effects of nonmuscle caldesmon on cellular contractility, actin cytoskeletal organization, and the formation of focal adhesions in fibroblasts. Transient transfection of nonmuscle caldesmon prevents myosin II-dependent cell contractility and induces a decrease in the number and size of tyrosine-phosphorylated focal adhesions. Expression of caldesmon interferes with Rho A-V14-mediated formation of focal adhesions and stress fibers as well as with formation of focal adhesions induced by microtubule disruption. This inhibitory effect depends on the actin- and myosin-binding regions of caldesmon, because a truncated variant lacking both of these regions is inactive. The effects of caldesmon are blocked by the ionophore A23187, thapsigargin, and membrane depolarization, presumably because of the ability of Ca2+–calmodulin or Ca2+–S100 proteins to antagonize the inhibitory function of caldesmon on actomyosin contraction. These results indicate a role for nonmuscle caldesmon in the physiological regulation of actomyosin contractility and adhesion-dependent signaling and further demonstrate the involvement of contractility in focal adhesion formation.

scholarly journals Intermediate filaments control collective migration by restricting traction forces and sustaining cell-cell contacts

2018 ◽  
Author(s):  
Chiara De Pascalis ◽  
Carlos Pérez-González ◽  
Shailaja Seetharaman ◽  
Batiste Boëda ◽  
Benoit Vianay ◽  
...  
Keyword(s):  
Adherens Junctions ◽  
Cell Monolayer ◽  
Focal Adhesions ◽  
Collective Migration ◽  
Traction Forces ◽  
Mechanical Coupling ◽  
Directed Migration ◽  
Cell Protrusion ◽  
Cell Cell

AbstractMesenchymal cell migration relies on the coordinated regulation of the actin and microtubule networks which participate in polarised cell protrusion, adhesion and contraction. During collective migration, most of the traction forces are generated by the acto-myosin network linked to focal adhesions at the front of leader cells, which transmit these pulling forces to the followers. Here, using an in vitro wound healing assay to induce polarisation and collective directed migration of primary astrocytes, we show that the intermediate filament (IF) network composed of vimentin, GFAP and nestin contributes to directed collective movement by controlling the distribution of forces in the migrating cell monolayer. Together with the cytoskeletal linker plectin, these IFs control the organisation and dynamics of the acto-myosin network, promoting the actin-driven treadmilling of adherens junctions, thereby facilitating the polarisation of leader cells. Independently of their effect on adherens junctions, IFs influence the dynamics and localisation of focal adhesions and limit their mechanical coupling to the acto-myosin network. We thus conclude that IFs promote collective directed migration by restricting the generation of traction forces to the front of leader cells, preventing aberrant tractions in the followers and by contributing to the maintenance of lateral cell-cell interactions.

Focal adhesion formation by F9 embryonal carcinoma cells after vinculin gene disruption

1995 ◽  
Vol 108 (6) ◽  
pp. 2253-2260 ◽  
Author(s):  
T. Volberg ◽  
B. Geiger ◽  
Z. Kam ◽  
R. Pankov ◽  
I. Simcha ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Embryonal Carcinoma ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Fluorescent Labeling ◽  
Carcinoma Cells ◽  
Fluorescence Labeling ◽  
Embryonal Carcinoma Cells ◽  
F9 Cells ◽  
F9 Embryonal Carcinoma Cells

The assembly of focal adhesions was investigated in F9 embryonal carcinoma cells in which the expression of vinculin was eliminated by a targeted disruption of the vinculin gene. Vinculin-deficient F9 cells were capable of adhering to fibronectin-coated surfaces, though they displayed a reduced spreading compared to the parental cells. Transmission electron microscopy as well as interference reflection microscopy of live cells showed that vinculin-null F9 cells formed focal adhesions that were indistinguishable from those of the control cells. Fluorescent labeling for actin, talin, alpha-actinin, paxillin and phosphotyrosinated components indicated that the organization of all these focal contact-associated components was essentially identical in the vinculin-containing and vinculin-null cells. However, quantitative, digitized microscopy indicated that the intensity of fluorescence labeling in focal adhesions for alpha-actinin, talin and paxillin was significantly higher in cells lacking vinculin. The results suggest that there are multiple molecular mechanisms for the formation of focal adhesions in the absence of vinculin.

scholarly journals Intermediate filaments control collective migration by restricting traction forces and sustaining cell–cell contacts

2018 ◽  
Vol 217 (9) ◽  
pp. 3031-3044 ◽  
Author(s):  
Chiara De Pascalis ◽  
Carlos Pérez-González ◽  
Shailaja Seetharaman ◽  
Batiste Boëda ◽  
Benoit Vianay ◽  
...  
Keyword(s):  
Adherens Junctions ◽  
Cell Monolayer ◽  
Focal Adhesions ◽  
Collective Migration ◽  
Traction Forces ◽  
Mechanical Coupling ◽  
Directed Migration ◽  
Cell Protrusion ◽  
Cell Cell

Mesenchymal cell migration relies on the coordinated regulation of the actin and microtubule networks that participate in polarized cell protrusion, adhesion, and contraction. During collective migration, most of the traction forces are generated by the acto-myosin network linked to focal adhesions at the front of leader cells, which transmit these pulling forces to the followers. Here, using an in vitro wound healing assay to induce polarization and collective directed migration of primary astrocytes, we show that the intermediate filament (IF) network composed of vimentin, glial fibrillary acidic protein, and nestin contributes to directed collective movement by controlling the distribution of forces in the migrating cell monolayer. Together with the cytoskeletal linker plectin, these IFs control the organization and dynamics of the acto-myosin network, promoting the actin-driven treadmilling of adherens junctions, thereby facilitating the polarization of leader cells. Independently of their effect on adherens junctions, IFs influence the dynamics and localization of focal adhesions and limit their mechanical coupling to the acto-myosin network. We thus conclude that IFs promote collective directed migration in astrocytes by restricting the generation of traction forces to the front of leader cells, preventing aberrant tractions in the followers, and by contributing to the maintenance of lateral cell–cell interactions.

scholarly journals Vinculin controls focal adhesion formation by direct interactions with talin and actin

2007 ◽  
Vol 179 (5) ◽  
pp. 1043-1057 ◽  
Author(s):  
Jonathan D. Humphries ◽  
Pengbo Wang ◽  
Charles Streuli ◽  
Benny Geiger ◽  
Martin J. Humphries ◽  
...  
Keyword(s):  
Binding Sites ◽  
Molecular Mechanisms ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Direct Interaction ◽  
Functional Link ◽  
Binding Partners ◽  
Potential Binding ◽  
Focal Adhesion Formation ◽  
Potential Binding Site

Focal adhesions (FAs) regulate cell migration. Vinculin, with its many potential binding partners, can interconnect signals in FAs. Despite the well-characterized structure of vinculin, the molecular mechanisms underlying its action have remained unclear. Here, using vinculin mutants, we separate the vinculin head and tail regions into distinct functional domains. We show that the vinculin head regulates integrin dynamics and clustering and the tail regulates the link to the mechanotransduction force machinery. The expression of vinculin constructs with unmasked binding sites in the head and tail regions induces dramatic FA growth, which is mediated by their direct interaction with talin. This interaction leads to clustering of activated integrin and an increase in integrin residency time in FAs. Surprisingly, paxillin recruitment, induced by active vinculin constructs, occurs independently of its potential binding site in the vinculin tail. The vinculin tail, however, is responsible for the functional link of FAs to the actin cytoskeleton. We propose a new model that explains how vinculin orchestrates FAs.

scholarly journals Superior Alignment of Human iPSC-Osteoblasts Associated with Focal Adhesion Formation Stimulated by Oriented Collagen Scaffold

2021 ◽  
Vol 22 (12) ◽  
pp. 6232
Author(s):  
Ryosuke Ozasa ◽  
Aira Matsugaki ◽  
Tadaaki Matsuzaka ◽  
Takuya Ishimoto ◽  
Hui-Suk Yun ◽  
...  
Keyword(s):  
Bone Tissue ◽  
Structural Organization ◽  
Critical Role ◽  
Bone Matrix ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Collagen Scaffold ◽  
Patient Specific ◽  
Specific Cell ◽  
Adhesion Behavior

Human-induced pluripotent stem cells (hiPSCs) can be applied in patient-specific cell therapy to regenerate lost tissue or organ function. Anisotropic control of the structural organization in the newly generated bone matrix is pivotal for functional reconstruction during bone tissue regeneration. Recently, we revealed that hiPSC-derived osteoblasts (hiPSC-Obs) exhibit preferential alignment and organize in highly ordered bone matrices along a bone-mimetic collagen scaffold, indicating their critical role in regulating the unidirectional cellular arrangement, as well as the structural organization of regenerated bone tissue. However, it remains unclear how hiPSCs exhibit the cell properties required for oriented tissue construction. The present study aimed to characterize the properties of hiPSCs-Obs and those of their focal adhesions (FAs), which mediate the structural relationship between cells and the matrix. Our in vitro anisotropic cell culture system revealed the superior adhesion behavior of hiPSC-Obs, which exhibited accelerated cell proliferation and better cell alignment along the collagen axis compared to normal human osteoblasts. Notably, the oriented collagen scaffold stimulated FA formation along the scaffold collagen orientation. This is the first report of the superior cell adhesion behavior of hiPSC-Obs associated with the promotion of FA assembly along an anisotropic scaffold. These findings suggest a promising role for hiPSCs in enabling anisotropic bone microstructural regeneration.

scholarly journals Novel keratin 5 mutation in a family with epidermolysis bullosa simplex

2015 ◽  
Vol 10 (6) ◽  
pp. 2432-2436
Author(s):  
JIAJIA GAO ◽  
XUEBIN WANG ◽  
FANG ZHENG ◽  
SUFANG DONG ◽  
XUEPING QIU
Keyword(s):  
Epidermolysis Bullosa ◽  
Epidermolysis Bullosa Simplex ◽  
Keratin 5

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 Mutation analysis of the entire keratin 5 and 14 genes in patients with epidermolysis bullosa simplex and identification of novel mutations

2003 ◽  
Vol 21 (4) ◽  
pp. 447-447 ◽  
Author(s):  
Petra H.L. Schuilenga-Hut ◽  
Pieter v.d. Vlies ◽  
Marcel F. Jonkman ◽  
Esm� Waanders ◽  
Charles H.C.M. Buys ◽  
...  
Keyword(s):  
Mutation Analysis ◽  
Epidermolysis Bullosa ◽  
Epidermolysis Bullosa Simplex ◽  
Novel Mutations ◽  
Keratin 5
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