scholarly journals The Mechanical Contribution of Vimentin to Cellular Stress Generation

2018 ◽  
Vol 140 (6) ◽  
Author(s):  
Inge A. E. W. van Loosdregt ◽  
Giulia Weissenberger ◽  
Marc P. F. H. L. van Maris ◽  
Cees W. J. Oomens ◽  
Sandra Loerakker ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Cellular Stress ◽  
Stress Fiber ◽  
Stress Generation ◽  
Stress Fibers ◽  
Adherent Cells ◽  
Wild Type ◽  
Contractile Stress ◽  
Fiber Organization ◽  
Fiber Contraction

Contractile stress generation by adherent cells is largely determined by the interplay of forces within their cytoskeleton. It is known that actin stress fibers, connected to focal adhesions, provide contractile stress generation, while microtubules and intermediate filaments provide cells compressive stiffness. Recent studies have shown the importance of the interplay between the stress fibers and the intermediate filament vimentin. Therefore, the effect of the interplay between the stress fibers and vimentin on stress generation was quantified in this study. We hypothesized that net stress generation comprises the stress fiber contraction combined with the vimentin resistance. We expected an increased net stress in vimentin knockout (VimKO) mouse embryonic fibroblasts (MEFs) compared to their wild-type (vimentin wild-type (VimWT)) counterparts, due to the decreased resistance against stress fiber contractility. To test this, the net stress generation by VimKO and VimWT MEFs was determined using the thin film method combined with sample-specific finite element modeling. Additionally, focal adhesion and stress fiber organization were examined via immunofluorescent staining. Net stress generation of VimKO MEFs was three-fold higher compared to VimWT MEFs. No differences in focal adhesion size or stress fiber organization and orientation were found between the two cell types. This suggests that the increased net stress generation in VimKO MEFs was caused by the absence of the resistance that vimentin provides against stress fiber contraction. Taken together, these data suggest that vimentin resists the stress fiber contractility, as hypothesized, thus indicating the importance of vimentin in regulating cellular stress generation by adherent cells.

scholarly journals Lmna knockout mouse embryonic fibroblasts are less contractile than their wild-type counterparts

2017 ◽  
Vol 9 (8) ◽  
pp. 709-721 ◽  
Author(s):  
I. A. E. W. van Loosdregt ◽  
M. A. F. Kamps ◽  
C. W. J. Oomens ◽  
S. Loerakker ◽  
J. L. V. Broers ◽  
...  
Keyword(s):  
Knockout Mouse ◽  
Stress Fiber ◽  
Stress Generation ◽  
Actin Stress Fiber ◽  
Wild Type ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Contractile Stress ◽  
Fiber Organization

Lmna knockout causes an impaired actin stress fiber organization which results in a fivefold lower contractile stress generation.

Regulation of the RhoA pathway in human endothelial cell spreading on type IV collagen: role of calcium influx

1999 ◽  
Vol 112 (19) ◽  
pp. 3205-3213 ◽  
Author(s):  
L. Masiero ◽  
K.A. Lapidos ◽  
I. Ambudkar ◽  
E.C. Kohn
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Focal Adhesion ◽  
Type Iv Collagen ◽  
Cell Spreading ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Type Iv ◽  
Stress Fiber Formation

We have shown that nonvoltage-operated Ca(2+) entry regulates human umbilical vein endothelial cell adhesion, migration, and proliferation on type IV collagen. We now demonstrate a requirement for Ca(2+) influx for activation of the RhoA pathway during endothelial cell spreading on type IV collagen. Reorganization of actin into stress fibers was complete when the cells where fully spread at 90 minutes. No actin organization into stress fibers was seen in endothelial cells plated on type I collagen, indicating a permissive effect of type IV collagen. CAI, a blocker of nonvoltage-operated Ca(2+) channels, prevented development of stress fiber formation in endothelial cells on type IV collagen. This permissive effect was augmented by Ca(2+) influx, as stimulated by 0. 5 microM thapsigargin or 0.1 microM ionomycin, yielding faster development of actin stress fibers. Ca(2+) influx and actin rearrangement in response to thapsigargin and ionomycin were abrogated by CAI. Activated, membrane-bound RhoA is a substrate for C3 exoenzyme which ADP-ribosylates and inactivates RhoA, preventing actin stress fiber formation. Pretreatment of endothelial cells with C3 exoenzyme prevented basal and thapsigargin-augmented stress fiber formation. While regulation of Ca(2+) influx did not alter RhoA translocation, it reduced in vitro ADP-ribosylation of RhoA (P(2)<0. 05), suggesting Ca(2+) influx is needed for RhoA activation during spreading on type IV collagen; no Ca(2+) regulated change in RhoA was seen in HUVECs spreading on type I collagen matrix. Blockade of Ca(2+) influx of HUVEC spread on type IV collagen also reduced tyrosine phosphorylation of p190Rho-GAP and blocked thapsigargin-enhanced binding of p190Rho-GAP to focal adhesion kinase. Thus, Ca(2+) influx is necessary for RhoA activation and for linkage of the RhoA/stress fiber cascade to the focal adhesion/focal adhesion kinase pathway during human umbilical vein endothelial cell spreading on type IV collagen.

Indomethacin Delays Gastric Restitution: Association with the Inhibition of Focal Adhesion Kinase and Tensin Phosphorylation and Reduced Actin Stress Fibers

2002 ◽  
Vol 227 (6) ◽  
pp. 412-424 ◽  
Author(s):  
Imre L. Szabó ◽  
Rama Pai ◽  
Michael K. Jones ◽  
George R. Ehring ◽  
Hirofumi Kawanaka ◽  
...  
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Actin Stress Fiber ◽  
Stress Fiber Formation ◽  
Actin Stress Fiber Formation

Repair of superficial gastric mucosal injury is accomplished by the process of restitution—migration of epithelial cells to restore continuity of the mucosal surface. Actin filaments, focal adhesions, and focal adhesion kinase (FAK) play crucial roles in cell motility essential for restitution. We studied whether epidermal growth factor (EGF) and/or indomethacin (IND) affect cell migration, actin stress fiber formation, and/or phosphorylation of FAK and tensin in wounded gastric monolayers. Human gastric epithelial monolayers (MKN 28 cells) were wounded and treated with either vehicle or 0.5 mM IND for 16 hr followed by EGF. EGF treatment significantly stimulated cell migration and actin stress fiber formation, and increased FAK localization to focal adhesions, and phosphorylation of FAK and tensin, whereas IND inhibited all these at the baseline and EGF-stimulated conditions. IND-induced inhibition of FAK phosphorylation preceded changes in actin polymerization, indicating that actin depolymerization might be the consequence of decreased FAK activity. In in vivo experiments, rats received either vehicle or IND (5 mg/kg i.g.), and 3 min later, they received water or 5% hypertonic NaCl; gastric mucosa was obtained at 1, 4, and 8 hr after injury. Four and 8 hr after hypertonic injury, FAK phosphorylation was induced in gastric mucosa compared with controls. IND pretreatment significantly delayed epithelial restitution in vivo, and reduced FAK phosphorylation and recruitment to adhesion points, as well as actin stress fiber formation in migrating surface epithelial cells. Our study indicates that FAK, tensin, and actin stress fibers are likely mediators of EGF-stimulated cell migration in wounded human gastric monolayers and potential targets for IND-induced inhibition of restitution.

scholarly journals TSC2 modulates actin cytoskeleton and focal adhesion through TSC1-binding domain and the Rac1 GTPase

2004 ◽  
Vol 167 (6) ◽  
pp. 1171-1182 ◽  
Author(s):  
Elena Goncharova ◽  
Dmitry Goncharov ◽  
Daniel Noonan ◽  
Vera P. Krymskaya
Keyword(s):  
Cell Adhesion ◽  
Cell Growth ◽  
Cell Motility ◽  
Focal Adhesion ◽  
Translational Regulation ◽  
Stress Fiber ◽  
Binding Domain ◽  
Stress Fibers ◽  
Actin Dynamics ◽  
Rac1 Gtpase

Tuberous sclerosis complex (TSC) 1 and TSC2 are thought to be involved in protein translational regulation and cell growth, and loss of their function is a cause of TSC and lymphangioleiomyomatosis (LAM). However, TSC1 also activates Rho and regulates cell adhesion. We found that TSC2 modulates actin dynamics and cell adhesion and the TSC1-binding domain (TSC2-HBD) is essential for this function of TSC2. Expression of TSC2 or TSC2-HBD in TSC2−/− cells promoted Rac1 activation, inhibition of Rho, stress fiber disassembly, and focal adhesion remodeling. The down-regulation of TSC1 with TSC1 siRNA in TSC2−/− cells activated Rac1 and induced loss of stress fibers. Our data indicate that TSC1 inhibits Rac1 and TSC2 blocks this activity of TSC1. Because TSC1 and TSC2 regulate Rho and Rac1, whose activities are interconnected in a reciprocal fashion, loss of either TSC1 or TSC2 function may result in the deregulation of cell motility and adhesion, which are associated with the pathobiology of TSC and LAM.

scholarly journals ARF1 Mediates Paxillin Recruitment to Focal Adhesions and Potentiates Rho-stimulated Stress Fiber Formation in Intact and Permeabilized Swiss 3T3 Fibroblasts

1998 ◽  
Vol 143 (7) ◽  
pp. 1981-1995 ◽  
Author(s):  
J.C. Norman ◽  
D. Jones ◽  
S.T. Barry ◽  
M.R. Holt ◽  
S. Cockcroft ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
3T3 Fibroblasts ◽  
Stress Fiber Formation ◽  
Swiss 3T3 ◽  
Swiss 3T3 Fibroblasts ◽  
Actin Stress Fibers

Focal adhesion assembly and actin stress fiber formation were studied in serum-starved Swiss 3T3 fibroblasts permeabilized with streptolysin-O. Permeabilization in the presence of GTPγS stimulated rho-dependent formation of stress fibers, and the redistribution of vinculin and paxillin from a perinuclear location to focal adhesions. Addition of GTPγS at 8 min after permeabilization still induced paxillin recruitment to focal adhesion–like structures at the ends of stress fibers, but vinculin remained in the perinuclear region, indicating that the distributions of these two proteins are regulated by different mechanisms. Paxillin recruitment was largely rho-independent, but could be evoked using constitutively active Q71L ADP-ribosylation factor (ARF1), and blocked by NH2-terminally truncated Δ17ARF1. Moreover, leakage of endogenous ARF from cells was coincident with loss of GTPγS- induced redistribution of paxillin to focal adhesions, and the response was recovered by addition of ARF1. The ability of ARF1 to regulate paxillin recruitment to focal adhesions was confirmed by microinjection of Q71LARF1 and Δ17ARF1 into intact cells. Interestingly, these experiments showed that V14RhoA- induced assembly of actin stress fibers was potentiated by Q71LARF1. We conclude that rho and ARF1 activate complimentary pathways that together lead to the formation of paxillin-rich focal adhesions at the ends of prominent actin stress fibers.

scholarly journals Generation of contractile actomyosin bundles depends on mechanosensitive actin filament assembly and disassembly

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Sari Tojkander ◽  
Gergana Gateva ◽  
Amjad Husain ◽  
Ramaswamy Krishnan ◽  
Pekka Lappalainen
Keyword(s):  
Actin Filament ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Physical Mechanisms ◽  
Filament Assembly ◽  
Contractile Stress ◽  
Lateral Fusion

Adhesion and morphogenesis of many non-muscle cells are guided by contractile actomyosin bundles called ventral stress fibers. While it is well established that stress fibers are mechanosensitive structures, physical mechanisms by which they assemble, align, and mature have remained elusive. Here we show that arcs, which serve as precursors for ventral stress fibers, undergo lateral fusion during their centripetal flow to form thick actomyosin bundles that apply tension to focal adhesions at their ends. Importantly, this myosin II-derived force inhibits vectorial actin polymerization at focal adhesions through AMPK-mediated phosphorylation of VASP, and thereby halts stress fiber elongation and ensures their proper contractility. Stress fiber maturation additionally requires ADF/cofilin-mediated disassembly of non-contractile stress fibers, whereas contractile fibers are protected from severing. Taken together, these data reveal that myosin-derived tension precisely controls both actin filament assembly and disassembly to ensure generation and proper alignment of contractile stress fibers in migrating cells.

Modeling of Stress Fiber Organization in Cyclically Stretched Cells

2007 ◽  
Author(s):  
Zhensong Wei ◽  
Vikram S. Deshpande ◽  
Robert M. McMeeking ◽  
Anthony G. Evans
Keyword(s):  
Stress Fiber ◽  
Stress Fibers ◽  
Parameter Study ◽  
Transverse Strain ◽  
Fiber Alignment ◽  
Cyclic Stretching ◽  
Cycling Frequency ◽  
Strain Magnitude ◽  
Distribution Of Stress ◽  
Fiber Organization

Numerical simulations that incorporate a bio-chemo-mechanical model for the contractility of the cytoskeleton have been used to rationalize the following observations. Uniaxial cyclic stretching of cells causes stress fibers to align perpendicular to the stretch direction, with degree of alignment dependent on the stretch strain magnitude, as well as the frequency and the transverse strain. Conversely, equibiaxial cyclic stretching induces a uniform distribution of stress fiber orientations. Demonstrations that the model successfully predicts the alignments found experimentally are followed by a parameter study to investigate the influence of the straining frequency and the transverse contraction of the substrate. The primary predictions are as follows. The fiber alignment increases with increasing cycling frequency. Transverse contraction of the substrate causes the stress fibers to organize into two symmetrical orientations with respect to the primary stretch direction.

scholarly journals Biomechanical regulation of focal adhesion and invadopodia formation

2020 ◽  
Vol 133 (20) ◽  
pp. jcs244848
Author(s):  
Or-Yam Revach ◽  
Inna Grosheva ◽  
Benjamin Geiger
Keyword(s):  
Focal Adhesion ◽  
Actin Polymerization ◽  
Protein Complexes ◽  
Focal Adhesions ◽  
Stress Fibers ◽  
Mechanical Forces ◽  
Molecular Machinery ◽  
Contractile Stress ◽  
Functionally Diverse ◽  
Invadopodia Formation

ABSTRACTIntegrin adhesions are a structurally and functionally diverse family of transmembrane, multi-protein complexes that link the intracellular cytoskeleton to the extracellular matrix (ECM). The different members of this family, including focal adhesions (FAs), focal complexes, fibrillar adhesions, podosomes and invadopodia, contain many shared scaffolding and signaling ‘adhesome’ components, as well as distinct molecules that perform specific functions, unique to each adhesion form. In this Hypothesis, we address the pivotal roles of mechanical forces, generated by local actin polymerization or actomyosin-based contractility, in the formation, maturation and functionality of two members of the integrin adhesions family, namely FAs and invadopodia, which display distinct structures and functional properties. FAs are robust and stable ECM contacts, associated with contractile stress fibers, while invadopodia are invasive adhesions that degrade the underlying matrix and penetrate into it. We discuss here the mechanisms, whereby these two types of adhesion utilize a similar molecular machinery to drive very different – often opposing cellular activities, and hypothesize that early stages of FAs and invadopodia assembly use similar biomechanical principles, whereas maturation of the two structures, and their ‘adhesive’ and ‘invasive’ functionalities require distinct sources of biomechanical reinforcement.

scholarly journals Phospholipase D Activity Is Required for Actin Stress Fiber Formation in Fibroblasts

2001 ◽  
Vol 21 (12) ◽  
pp. 4055-4066 ◽  
Author(s):  
Yoonseok Kam ◽  
John H. Exton
Keyword(s):  
Actin Cytoskeleton ◽  
Phospholipase D ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Dominant Negative Mutant ◽  
Actin Stress Fiber ◽  
Wild Type ◽  
Stress Fiber Formation ◽  
Significant Difference

ABSTRACT Phospholipase D (PLD) is a ubiquitously expressed enzyme of ill-defined function. In order to explore its cellular actions, we inactivated the rat PLD1 (rPLD1) isozyme by tagging its C terminus with a V5 epitope (rPLD1-V5). This was stably expressed in Rat-2 fibroblasts to see if it acted as a dominant-negative mutant for PLD activity. Three clones that expressed rPLD1-V5 were selected (Rat2V16, Rat2V25, and Rat2V29). Another clone (Rat2V20) that lost expression of rPLD1-V5 was also obtained. In the three clones expressing rPLD1-V5, PLD activity stimulated by phorbol myristate acetate (PMA) or lysophosphatidic acid (LPA) was reduced by ∼50%, while the PLD activity of Rat2V20 cells was normal. Changes in the actin cytoskeleton in response to LPA or PMA were examined in these clones. All three clones expressing rPLD1-V5 failed to form actin stress fibers after treatment with LPA. However, Rat2V20 cells formed stress fibers in response to LPA to the same extent as wild-type Rat-2 cells. In contrast, there was no significant change in membrane ruffling induced by PMA in the cells expressing rPLD1-V5. Since Rho is an activator both of rPLD1 and stress fiber formation, the activation of Rho was monitored in wild-type Rat-2 cells and Rat2V25 cells, but no significant difference was detected. The phosphorylation of vimentin mediated by Rho-kinase was also intact in Rat2V25 cells. Rat2V25 cells also showed normal vinculin-containing focal adhesions. However, the translocation of α-actinin to the cytoplasm and to the detergent-insoluble fraction in Rat2V25 cells was reduced. These results indicate that PLD activity is required for LPA-induced rearrangement of the actin cytoskeleton to form stress fibers and that PLD might be involved in the cross-linking of actin filaments mediated by α-actinin.

Simulation of the Coupling of Cell Contractility and Focal Adhesion Formation

2007 ◽  
Author(s):  
Amit Pathak ◽  
Vikram S. Deshpande ◽  
Robert M. McMeeking ◽  
Anthony G. Evans
Keyword(s):  
Focal Adhesion ◽  
Adhesion Formation ◽  
High Stress ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Cell Contractility ◽  
Focal Adhesion Formation ◽  
Pattern Shape ◽  
Stress Dependent

The remodeling of the cytoskeleton and focal adhesion distributions for cells on substrates with micro-patterned ligand patches is investigated using a bio-chemo-mechanical model. All the cells have approximately the same area and we investigate the effect of ligand pattern shape on the cytoskeletal arrangements and focal adhesion distributions. The model for the cytoskeleton accounts for the dynamic rearrangement of the actin/myosin stress fibers and entails the highly non-linear interactions between signaling, the kinetics of tension-dependent stress-fiber formation/dissolution and stress dependent contractility. This model is coupled with a focal adhesion (FA) model that accounts for the mechano-sensitivity of the adhesions from thermodynamic considerations. This coupled stress fiber and focal adhesion model is shown to capture a variety of key experimental observations including: (i) the formation of high stress fiber and focal adhesion concentrations at the periphery of circular and triangular, convex–shaped ligand patterns; (ii) the development of high focal adhesion concentrations along the edges of the V, T, Y and U shaped concave ligand patterns; and (iii) the formation of highly aligned stress fibers along the un-adhered edges of cells on the concave ligand patterns. When appropriately calibrated, the model also accurately predicts the radii of curvature of the un-adhered edges of cells on the concave-shaped ligand patterns.

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