scholarly journals The keratin network of intermediate filaments regulates keratinocyte rigidity sensing and nuclear mechanotransduction

2021 ◽  
Vol 7 (5) ◽  
pp. eabd6187
Author(s):  
Ana C. Laly ◽  
Kristina Sliogeryte ◽  
Oscar J. Pundel ◽  
Rosie Ross ◽  
Michael C. Keeling ◽  
...  
Keyword(s):  
Intermediate Filaments ◽  
Mechanical Response ◽  
Nuclear Lamina ◽  
Fiber Formation ◽  
Cell Stiffness ◽  
Actin Stress Fiber ◽  
Matrix Rigidity ◽  
Rigidity Sensing ◽  
Substrate Rigidity

The keratin network of intermediate filaments provides keratinocytes with essential mechanical strength and resilience, but the contribution to mechanosensing remains poorly understood. Here, we investigated the role of the keratin cytoskeleton in the response to altered matrix rigidity. We found that keratinocytes adapted to increasing matrix stiffness by forming a rigid, interconnected network of keratin bundles, in conjunction with F-actin stress fiber formation and increased cell stiffness. Disruption of keratin stability by overexpression of the dominant keratin 14 mutation R416P inhibited the normal mechanical response to substrate rigidity, reducing F-actin stress fibers and cell stiffness. The R416P mutation also impaired mechanotransduction to the nuclear lamina, which mediated stiffness-dependent chromatin remodeling. By contrast, depletion of the cytolinker plectin had the opposite effect and promoted increased mechanoresponsiveness and up-regulation of lamin A/C. Together, these results demonstrate that the keratin cytoskeleton plays a key role in matrix rigidity sensing and downstream signal transduction.

scholarly journals Mechanisms of sodium fluoride-induced endothelial cell barrier dysfunction: role of MLC phosphorylation

2001 ◽  
Vol 281 (6) ◽  
pp. L1472-L1483 ◽  
Author(s):  
Peiyi Wang ◽  
Alexander D. Verin ◽  
Anna Birukova ◽  
Lydia I. Gilbert-McClain ◽  
Keri Jacobs ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Phosphatase Activity ◽  
Fiber Formation ◽  
Dominant Negative ◽  
Actin Stress Fiber ◽  
Gap Formation ◽  
Barrier Dysfunction ◽  
Transient Activation ◽  
Mlc Phosphorylation

NaF, a potent G protein activator and Ser/Thr phosphatase inhibitor, significantly increased albumin permeability and decreased transcellular electrical resistance (TER), indicating endothelial cell (EC) barrier impairment. EC barrier dysfunction induced by NaF was accompanied by the development of actin stress fibers, intercellular gap formation, and significant time-dependent increases in myosin light chain (MLC) phosphorylation. However, despite rapid, albeit transient, activation of Ca2+/calmodulin-dependent MLC kinase (MLCK), the specific MLCK inhibitor ML-7 failed to affect NaF-induced MLC phosphorylation, actin cytoskeletal rearrangement, and reductions in TER, suggesting a limited role of MLCK in NaF-induced EC activation. In contrast, strategies to reduce Rho (C3 exoenzyme or toxin B) or to inhibit Rho-associated kinase (Y-27632 or dominant/negative RhoK) dramatically reduced MLC phosphorylation and actin stress fiber formation and significantly attenuated NaF-induced EC barrier dysfunction. Consistent with this role for RhoK activity, NaF selectively inhibited myosin-specific phosphatase activity, whereas the total Ser/Thr phosphatase activity remained unchanged. These data strongly suggest that MLC phosphorylation, mediated primarily by RhoK, and not MLCK, participates in NaF-induced EC actin cytoskeletal changes and barrier dysfunction.

scholarly journals P2Y2 receptors regulate osteoblast mechanosensitivity during fluid flow

2014 ◽  
Vol 306 (11) ◽  
pp. C1058-C1067 ◽  
Author(s):  
Joseph Gardinier ◽  
Weidong Yang ◽  
Gregory R. Madden ◽  
Andris Kronbergs ◽  
Vimal Gangadharan ◽  
...  
Keyword(s):  
Mechanical Stimulation ◽  
Fluid Shear Stress ◽  
Rho Kinase ◽  
Fiber Formation ◽  
Cell Stiffness ◽  
Actin Stress Fiber ◽  
Cellular Mechanisms ◽  
Lim Kinase ◽  
Rhoa Gtpase ◽  
Gtpase Activation

Mechanical stimulation of osteoblasts activates many cellular mechanisms including the release of ATP. Binding of ATP to purinergic receptors is key to load-induced osteogenesis. Osteoblasts also respond to fluid shear stress (FSS) with increased actin stress fiber formation (ASFF) that we postulate is in response to activation of the P2Y2 receptor (P2Y2R). Furthermore, we predict that ASFF increases cell stiffness and reduces the sensitivity to further mechanical stimulation. We found that small interfering RNA (siRNA) suppression of P2Y2R attenuated ASFF in response to FSS and ATP treatment. In addition, RhoA GTPase was activated within 15 min after the onset of FSS or ATP treatment and mediated ASFF following P2Y2R activation via the Rho kinase (ROCK)1/LIM kinase 2/cofilin pathway. We also observed that ASFF in response to FSS or ATP treatment increased the cell stiffness and was prevented by knocking down P2Y2R. Finally, we confirmed that the enhanced cell stiffness and ASFF in response to RhoA GTPase activation during FSS drastically reduced the mechanosensitivity of the osteoblasts based on the intracellular Ca2+ concentration ([Ca2+]i) response to consecutive bouts of FSS. These data suggest that osteoblasts can regulate their mechanosensitivity to continued load through P2Y2R activation of the RhoA GTPase signaling cascade, leading to ASFF and increased cell stiffness.

scholarly journals Cell response to substrate rigidity is regulated by active and passive cytoskeletal stress

2020 ◽  
Vol 117 (23) ◽  
pp. 12817-12825 ◽  
Author(s):  
Bryant L. Doss ◽  
Meng Pan ◽  
Mukund Gupta ◽  
Gianluca Grenci ◽  
René-Marc Mège ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Focal Adhesions ◽  
Cell Polarization ◽  
Tumor Formation ◽  
Cell Stiffness ◽  
Matrix Rigidity ◽  
Cytoskeleton Organization ◽  
Local Rigidity ◽  
Rigidity Sensing ◽  
Pillar Arrays

Morphogenesis, tumor formation, and wound healing are regulated by tissue rigidity. Focal adhesion behavior is locally regulated by stiffness; however, how cells globally adapt, detect, and respond to rigidity remains unknown. Here, we studied the interplay between the rheological properties of the cytoskeleton and matrix rigidity. We seeded fibroblasts onto flexible microfabricated pillar arrays with varying stiffness and simultaneously measured the cytoskeleton organization, traction forces, and cell-rigidity responses at both the adhesion and cell scale. Cells adopted a rigidity-dependent phenotype whereby the actin cytoskeleton polarized on stiff substrates but not on soft. We further showed a crucial role of active and passive cross-linkers in rigidity-sensing responses. By reducing myosin II activity or knocking down α-actinin, we found that both promoted cell polarization on soft substrates, whereas α-actinin overexpression prevented polarization on stiff substrates. Atomic force microscopy indentation experiments showed that this polarization response correlated with cell stiffness, whereby cell stiffness decreased when active or passive cross-linking was reduced and softer cells polarized on softer matrices. Theoretical modeling of the actin network as an active gel suggests that adaptation to matrix rigidity is controlled by internal mechanical properties of the cytoskeleton and puts forward a universal scaling between nematic order of the actin cytoskeleton and the substrate-to-cell elastic modulus ratio. Altogether, our study demonstrates the implication of cell-scale mechanosensing through the internal stress within the actomyosin cytoskeleton and its coupling with local rigidity sensing at focal adhesions in the regulation of cell shape changes and polarity.

Regulation of reactive oxygen species-induced endothelial cell-cell and cell-matrix contacts by focal adhesion kinase and adherens junction proteins

2005 ◽  
Vol 289 (6) ◽  
pp. L999-L1010 ◽  
Author(s):  
Peter V. Usatyuk ◽  
Viswanathan Natarajan
Keyword(s):  
Cell Adhesion ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Barrier Function ◽  
Fiber Formation ◽  
Actin Stress Fiber ◽  
Cell Matrix ◽  
Cell Cell

Oxidants, generated by activated neutrophils, have been implicated in the pathophysiology of vascular disorders and lung injury; however, mechanisms of oxidant-mediated endothelial barrier dysfunction are unclear. Here, we have investigated the role of focal adhesion kinase (FAK) in regulating hydrogen peroxide (H2O2)-mediated tyrosine phosphorylation of intercellular adhesion proteins and barrier function in endothelium. Treatment of bovine pulmonary artery endothelial cells (BPAECs) with H2O2 increased tyrosine phosphorylation of FAK, paxillin, β-catenin, and vascular endothelial (VE)-cadherin and decreased transendothelial electrical resistance (TER), an index of cell-cell adhesion and/or cell-matrix adhesion. To study the role of FAK in H2O2-induced TER changes, BPAECs were transfected with vector or FAK wild-type or FAK-related non-kinase (FRNK) plasmids. Overexpression of FRNK reduced FAK expression and attenuated H2O2-mediated tyrosine phosphorylation of FAK, paxillin, β-catenin, and VE-cadherin and cell-cell adhesion. Additionally, FRNK prevented H2O2-induced distribution of FAK, paxillin, β-catenin, or VE-cadherin toward focal adhesions and cell-cell adhesions but not actin stress fiber formation. These results suggest that activation of FAK by H2O2 is an important event in oxidant-mediated VE barrier function regulated by cell-cell and cell-matrix contacts.

Role of protein kinase G in barrier-protective effects of cGMP in human pulmonary artery endothelial cells

2006 ◽  
Vol 290 (5) ◽  
pp. L919-L930 ◽  
Author(s):  
Aigul Moldobaeva ◽  
Laura E. Welsh-Servinsky ◽  
Larissa A. Shimoda ◽  
R. Scott Stephens ◽  
Alexander D. Verin ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Pulmonary Artery ◽  
Recombinant Adenovirus ◽  
Fiber Formation ◽  
Independent Effect ◽  
Actin Stress Fiber ◽  
Barrier Dysfunction ◽  
Human Pulmonary Artery

Increases in endothelial cGMP prevent oxidant-mediated endothelial barrier dysfunction, but the downstream mechanisms remain unclear. To determine the role of cGMP-dependent protein kinase (PKG)I, human pulmonary artery endothelial cells (HPAEC) lacking PKGI expression were infected with a recombinant adenovirus encoding PKGIβ (Ad.PKG) and compared with uninfected and control-infected (Ad.βgal) HPAEC. Transendothelial electrical resistance (TER), an index of permeability, was measured after H2O2 (250 μM) exposure with or without pretreatment with 8-(4-chlorophenylthio)guanosine 3′,5′-cyclic monophosphate (CPT-cGMP). HPAEC infected with Ad.PKG, but not Ad.βgal, expressed PKGI protein and demonstrated Ser239 and Ser157 phosphorylation of vasodilator-stimulated phosphoprotein after treatment with CPT-cGMP. Adenoviral infection decreased basal permeability equally in Ad.PKG- and Ad.βgal-infected HPAEC compared with uninfected cells. Treatment with CPT-cGMP (100 μM) caused a PKGI-independent decrease in permeability (8.2 ± 0.6%). In all three groups, H2O2 (250 μM) caused a similar ∼35% increase in permeability associated with increased actin stress fiber formation, intercellular gaps, loss of membrane VE-cadherin, and increased intracellular Ca2+ concentration ([Ca2+]i). In uninfected and Ad.βgal-infected HPAEC, pretreatment with CPT-cGMP (100 μM) partially blocked the increased permeability induced by H2O2. In Ad.PKG-infected HPAEC, CPT-cGMP (50 μM) prevented the H2O2-induced TER decrease, cytoskeletal rearrangement, and loss of junctional VE-cadherin. CPT-cGMP attenuated the peak [Ca2+]i caused by H2O2 similarly (23%) in Ad.βgal- and Ad.PKG-infected HPAEC, indicating a PKGI-independent effect. These data suggest that cGMP decreased HPAEC basal permeability by a PKGI-independent process, whereas the ability of cGMP to prevent H2O2-induced barrier dysfunction was predominantly mediated by PKGI through a Ca2+-independent mechanism.

scholarly journals Cell-type-specific mechanical response and myosin dynamics during retinal lens development in Drosophila

2020 ◽  
Vol 31 (13) ◽  
pp. 1355-1369
Author(s):  
Laura Blackie ◽  
Rhian F. Walther ◽  
Michael F. Staddon ◽  
Shiladitya Banerjee ◽  
Franck Pichaud
Keyword(s):  
Epithelial Cell ◽  
Mechanical Response ◽  
Cell Types ◽  
Cell Stiffness ◽  
Force Transmission ◽  
Cell Type ◽  
Lens Development ◽  
Retinal Cells ◽  
Cell Type Specific

We describe and study the role of the contractile Myosin cytoskeleton in the different epithelial cell types that make up the fly retina. We find that medial contractile Myosin meshworks control the apical geometry and area of retinal cells. Our work also suggests that force transmission within a group of cells depends on cell stiffness.

The role of Rho GTPase in cell stiffness and cisplatin resistance in ovarian cancer cells

2014 ◽  
Vol 6 (6) ◽  
pp. 611-617 ◽  
Author(s):  
Shivani Sharma ◽  
Chintda Santiskulvong ◽  
Jianyu Rao ◽  
James K. Gimzewski ◽  
Oliver Dorigo
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Cisplatin Resistance ◽  
Rho Gtpase ◽  
Cell Stiffness ◽  
Ovarian Cancer Cells ◽  
Actin Stress Fiber ◽  
Direct Role ◽  
Actin Remodeling

Measurements of cell stiffness, IC50 and cellular actin stress fiber organization reveal a direct role of Rho mediated actin remodeling mechanism in cisplatin resistance of ovarian cancer cells.

scholarly journals Regulation of vimentin intermediate filaments in endothelial cells by hypoxia

2010 ◽  
Vol 299 (2) ◽  
pp. C363-C373 ◽  
Author(s):  
Tiegang Liu ◽  
Oscar E. Guevara ◽  
Rod R. Warburton ◽  
Nicholas S. Hill ◽  
Matthias Gaestel ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Intermediate Filaments ◽  
Endothelial Permeability ◽  
Biomechanical Properties ◽  
P38 Map Kinase ◽  
Fiber Formation ◽  
Endothelial Barrier ◽  
Intermediate Filament Protein ◽  
Actin Stress Fiber ◽  
Hsp27 Phosphorylation

Hypoxia triggers responses in endothelial cells that play roles in many conditions including high-altitude pulmonary edema and tumor angiogenesis. Signaling pathways activated by hypoxia modify cytoskeletal and contractile proteins and alter the biomechanical properties of endothelial cells. Intermediate filaments are major components of the cytoskeleton whose contribution to endothelial physiology is not well understood. We have previously shown that hypoxia-activated signaling in endothelial cells alters their contractility and adhesiveness. We have also linked p38-MAP kinase signaling pathway leading to HSP27 phosphorylation and increased actin stress fiber formation to endothelial barrier augmentation. We now show that vimentin, a major intermediate filament protein in endothelial cells, is regulated by hypoxia. Our results indicate that exposure of endothelial cells to hypoxia causes vimentin filament networks to initially redistribute perinuclearly. However, by 1 hour hypoxia these networks reform and appear more continuous across cells than under normoxia. Hypoxia also causes transient changes in vimentin phosphorylation, and activation of PAK1, a kinase that regulates vimentin filament assembly. In addition, exposure to 1 hour hypoxia increases the ratio of insoluble/soluble vimentin. Overexpression of phosphomimicking mutant HSP27 (pmHSP27) causes changes in vimentin distribution that are similar to those observed in hypoxic cells. Knocking-down HSP27 destroys the vimentin filamentous network, and disrupting vimentin filaments with acrylamide increases endothelial permeability. Both hypoxia- and pmHSP27 overexpression-induced changes are reversed by inhibition of phosphatase activity. In conclusion hypoxia causes redistribution of vimentin to a more insoluble and extensive filamentous network that could play a role in endothelial barrier stabilization. Vimentin redistribution appears to be mediated through altering the phosphorylation of the protein and its interaction with HSP27.

scholarly journals EpCAM promotes endosomal modulation of the cortical RhoA zone for epithelial organization

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Cécile Gaston ◽  
Simon De Beco ◽  
Bryant Doss ◽  
Meng Pan ◽  
Estelle Gauquelin ◽  
...  
Keyword(s):  
Cell Shape ◽  
Specific Protein ◽  
Cell Polarization ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Endosomal Trafficking ◽  
Transient Zone ◽  
And Behavior ◽  
Fine Tune

AbstractAt the basis of cell shape and behavior, the organization of actomyosin and its ability to generate forces are widely studied. However, the precise regulation of this contractile network in space and time is unclear. Here, we study the role of the epithelial-specific protein EpCAM, a contractility modulator, in cell shape and motility. We show that EpCAM is required for stress fiber generation and front-rear polarity acquisition at the single cell level. In fact, EpCAM participates in the remodeling of a transient zone of active RhoA at the cortex of spreading epithelial cells. EpCAM and RhoA route together through the Rab35/EHD1 fast recycling pathway. This endosomal pathway spatially organizes GTP-RhoA to fine tune the activity of actomyosin resulting in polarized cell shape and development of intracellular stiffness and traction forces. Impairment of GTP-RhoA endosomal trafficking either by silencing EpCAM or by expressing Rab35/EHD1 mutants prevents proper myosin-II activity, stress fiber formation and ultimately cell polarization. Collectively, this work shows that the coupling between co-trafficking of EpCAM and RhoA, and actomyosin rearrangement is pivotal for cell spreading, and advances our understanding of how biochemical and mechanical properties promote cell plasticity.

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