Dissociation of LPA-induced cytoskeletal contraction from stress fiber formation by differential localization of RhoA

1997 ◽  
Vol 110 (19) ◽  
pp. 2417-2427 ◽  
Author(s):  
O. Kranenburg ◽  
M. Poland ◽  
M. Gebbink ◽  
L. Oomen ◽  
W.H. Moolenaar
Keyword(s):  
Plasma Membrane ◽  
Tyrosine Kinase ◽  
High Speed ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Dominant Negative ◽  
Cell Periphery ◽  
Src Tyrosine Kinase ◽  
Stress Fiber Formation ◽  
Shape Changes

Addition of lysophosphatidic acid (LPA) to serum-deprived N1E-115 neuronal cells results in rapid f-actin assembly accompanied by neurite retraction and rounding of the cell body due to contraction of the cortical actin cytoskeleton. LPA action is mimicked by activated RhoA, while it is blocked by dominant-negative RhoA (N19RhoA) and the Rho-inactivating C3 toxin. Using immunofluorescence analysis and high speed centrifugation we show that activated RhoA is localized to the plasma membrane. Wild-type RhoA and N19RhoA, however, are mainly cytosolic. We find that LPA-induced shape changes are preceded by translocation of RhoA from the cytosol to the cell periphery. LPA also stimulates translocation of inactive N19RhoA in the absence of ensuing shape changes. When membrane localization of RhoA is prevented by lovastatin, an inhibitor of protein isoprenylation, or by CAAX motif mutation, cytoskeletal contraction is blocked. However, the assembly of f-actin into stress fibers is not affected under these conditions. The effects of both LPA and activated RhoA are blocked by tyrosine kinase inhibitors (herbimycin, genistein, tyrphostin), but not by dominant-negative Src. We conclude that: (1) LPA-induced cytoskeletal contraction, but not stress fiber formation, requires translocation of RhoA from the cytosol to the plasma membrane; (2) translocation of RhoA occurs independently of its activation; and (3), a non-Src tyrosine kinase is involved in RhoA-stimulated contractility.

scholarly journals ADP-Ribosylation Factor 6 Regulates Actin Cytoskeleton Remodeling in Coordination with Rac1 and RhoA

2000 ◽  
Vol 20 (10) ◽  
pp. 3685-3694 ◽  
Author(s):  
Rita L. Boshans ◽  
Stacey Szanto ◽  
Linda van Aelst ◽  
Crislyn D'Souza-Schorey
Keyword(s):  
Cell Surface ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Dominant Negative ◽  
Stress Fibers ◽  
Cortical Actin ◽  
Adp Ribosylation ◽  
Stress Fiber Formation ◽  
Dependent Inhibition ◽  
Adp Ribosylation Factor

ABSTRACT In this study, we have documented an essential role for ADP-ribosylation factor 6 (ARF6) in cell surface remodeling in response to physiological stimulus and in the down regulation of stress fiber formation. We demonstrate that the G-protein-coupled receptor agonist bombesin triggers the redistribution of ARF6- and Rac1-containing endosomal vesicles to the cell surface. This membrane redistribution was accompanied by cortical actin rearrangements and was inhibited by dominant negative ARF6, implying that bombesin is a physiological trigger of ARF6 activation. Furthermore, these studies provide a new model for bombesin-induced Rac1 activation that involves ARF6-regulated endosomal recycling. The bombesin-elicited translocation of vesicular ARF6 was mimicked by activated Gαq and was partially inhibited by expression of RGS2, which down regulates Gq function. This suggests that Gq functions as an upstream regulator of ARF6 activation. The ARF6-induced peripheral cytoskeletal rearrangements were accompanied by a depletion of stress fibers. Moreover, cells expressing activated ARF6 resisted the formation of stress fibers induced by lysophosphatidic acid. We show that the ARF6-dependent inhibition of stress fiber formation was due to an inhibition of RhoA activation and was overcome by expression of a constitutively active RhoA mutant. The latter observations demonstrate that activation of ARF6 down regulates Rho signaling. Our findings underscore the potential roles of ARF6, Rac1, and RhoA in the coordinated regulation of cytoskeletal remodeling.

scholarly journals Involvement of Fyn tyrosine kinase in actin stress fiber formation in fibroblasts

FEBS Letters ◽  
2007 ◽  
Vol 581 (27) ◽  
pp. 5227-5233 ◽  
Author(s):  
Dan Xu ◽  
Hiroko Kishi ◽  
Hozumi Kawamichi ◽  
Katsuko Kajiya ◽  
Yuichi Takada ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Actin Stress Fiber ◽  
Stress Fiber Formation ◽  
Actin Stress Fiber Formation ◽  
Fyn Tyrosine Kinase

scholarly journals Bartonella effector protein C mediates actin stress fiber formation via recruitment of GEF-H1 to the plasma membrane

2020 ◽  
Author(s):  
Simon Marlaire ◽  
Christoph Dehio
Keyword(s):  
Plasma Membrane ◽  
Stress Fiber ◽  
Effector Proteins ◽  
Fiber Formation ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Actin Stress Fiber ◽  
Type Iv ◽  
Stress Fiber Formation ◽  
Actin Stress Fiber Formation

AbstractBartonellae are Gram-negative facultative-intracellular pathogens that use a type-IV-secretion system (T4SS) to translocate a cocktail of Bartonella effector proteins (Beps) into host cells to modulate diverse cellular functions. BepC was initially reported to act in concert with BepF in triggering major actin cytoskeletal rearrangements that result in the internalization of a large bacterial aggregate by the so-called ‘invasome’. Later, infection studies with bepC deletion mutants and ectopic expression of BepC have implicated this effector in triggering an actin-dependent cell contractility phenotype characterized by fragmentation of migrating cells due to deficient rear detachment at the trailing edge, and BepE was shown to counterbalance this remarkable phenotype. However, the molecular mechanism of how BepC triggers cytoskeletal changes and the host factors involved remained elusive. Using infection assays, we show here that T4SS-mediated transfer of BepC is sufficient to trigger stress fiber formation in non-migrating epithelial cells and additionally cell fragmentation in migrating endothelial cells. Interactomic analysis revealed binding of BepC to a complex of the Rho guanine nucleotide exchange factor GEF-H1 and the serine/threonine-protein kinase MRCKα. Knock-out cell lines revealed that only GEF-H1 is required for mediating BepC-triggered stress fiber formation and inhibitor studies implicated activation of the RhoA/ROCK pathway downstream of GEF-H1. Ectopic co-expression of tagged versions of GEF-H1 and BepC truncations revealed that the C-terminal ‘Bep intracellular delivery’ (BID) domain facilitated anchorage of BepC to the plasma membrane, whereas the N-terminal ‘filamentation induced by cAMP’ (FIC) domain facilitated binding of GEF-H1. While FIC domains typically mediate post-translational modifications, most prominently AMPylation, a mutant with quadruple amino acid exchanges in the putative active site indicated that the BepC FIC domain acts in a non-catalytic manner to activate GEF-H1. Our data support a model in which BepC activates the RhoA/ROCK pathway by re-localization of GEF-H1 from microtubules to the plasma membrane.Author SummaryA wide variety of bacterial pathogens evolved numerous virulence factors to subvert cellular processes in support of a successful infection process. Likewise, bacteria of the genus Bartonella translocate a cocktail of effector proteins (Beps) via a type-IV-secretion system into infected cells in order to interfere with host signaling processes involved in cytoskeletal dynamics, apoptosis control, and innate immune responses. In this study, we demonstrate that BepC triggers actin stress fiber formation and a linked cell fragmentation phenotype resulting from distortion of rear-end retraction during cell migration. The ability of BepC to induce actin stress fiber formation is directly associated with its ability to bind GEF-H1, an activator of the RhoA pathway that is sequestered in an inactive state when bound to microtubules, but becomes activated upon release to the cytoplasm. Our findings suggest that BepC is anchored via its BID domain to the plasma membrane where it recruits GEF-H1 via its FIC domain, eventually activating the RhoA/ROCK signaling pathway and leading to stress fiber formation.

scholarly journals Involvement of an SHP-2-Rho Small G Protein Pathway in Hepatocyte Growth Factor/Scatter Factor–induced Cell Scattering

2000 ◽  
Vol 11 (8) ◽  
pp. 2565-2575 ◽  
Author(s):  
Atsuko Kodama ◽  
Takashi Matozaki ◽  
Atsunori Fukuhara ◽  
Mitsuhiro Kikyo ◽  
Masamitsu Ichihashi ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Dominant Negative ◽  
Stress Fibers ◽  
Dominant Negative Mutant ◽  
Stress Fiber Formation ◽  
Cell Scattering ◽  
Hepatocyte Growth

Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering through the tyrosine kinase–type HGF/SF receptor c-Met. We have previously shown that Rho small G protein (Rho) is involved in the HGF/SF-induced scattering of Madin-Darby canine kidney (MDCK) cells by regulating at least the assembly and disassembly of stress fibers and focal adhesions, but it remains unknown how c-Met regulates Rho activity. We have found here a novel signaling pathway of c-Met consisting of SHP-2-Rho that regulates the assembly and disassembly of stress fibers and focal adhesions in MDCK cells. SHP-2 is a protein-tyrosine phosphatase that contains src homology-2 domains. Expression of a dominant negative mutant of SHP-2 (SHP-2-C/S) markedly increased the formation of stress fibers and focal adhesions in MDCK cells and inhibited their scattering. C3, a Clostridium botulinum ADP-ribosyltransferase, and Y-27632, a specific inhibitor for ROCK, reversed the stimulatory effect of SHP-2-C/S on stress fiber formation and the inhibitory effect on cell scattering. Vav2 is a GDP/GTP exchange protein for Rho. Expression of a dominant negative mutant of Vav2 blocked the stimulatory effect of SHP-2-C/S on stress fiber formation. Conversely, expression of mutants of Vav2 that increased stress fiber formation inhibited HGF/SF-induced cell scattering. These results indicate that SHP-2 physiologically modulates the activity of Rho to form stress fibers and focal adhesions and thereby regulates HGF/SF-induced cell scattering. In addition, Vav2 may be involved in the SHP-2-Rho pathway.

scholarly journals Bartonella effector protein C mediates actin stress fiber formation via recruitment of GEF-H1 to the plasma membrane

2021 ◽  
Vol 17 (1) ◽  
pp. e1008548
Author(s):  
Simon Marlaire ◽  
Christoph Dehio
Keyword(s):  
Plasma Membrane ◽  
Ectopic Expression ◽  
Stress Fiber ◽  
Effector Proteins ◽  
Fiber Formation ◽  
Host Cells ◽  
Actin Stress Fiber ◽  
Nucleotide Exchange ◽  
Cell Contractility ◽  
Stress Fiber Formation

Bartonellae are Gram-negative facultative-intracellular pathogens that use a type-IV-secretion system (T4SS) to translocate a cocktail of Bartonella effector proteins (Beps) into host cells to modulate diverse cellular functions. BepC was initially reported to act in concert with BepF in triggering major actin cytoskeletal rearrangements that result in the internalization of a large bacterial aggregate by the so-called ‘invasome’. Later, infection studies with bepC deletion mutants and ectopic expression of BepC have implicated this effector in triggering an actin-dependent cell contractility phenotype characterized by fragmentation of migrating cells due to deficient rear detachment at the trailing edge, and BepE was shown to counterbalance this remarkable phenotype. However, the molecular mechanism of how BepC triggers cytoskeletal changes and the host factors involved remained elusive. Using infection assays, we show here that T4SS-mediated transfer of BepC is sufficient to trigger stress fiber formation in non-migrating epithelial cells and additionally cell fragmentation in migrating endothelial cells. Interactomic analysis revealed binding of BepC to a complex of the Rho guanine nucleotide exchange factor GEF-H1 and the serine/threonine-protein kinase MRCKα. Knock-out cell lines revealed that only GEF-H1 is required for mediating BepC-triggered stress fiber formation and inhibitor studies implicated activation of the RhoA/ROCK pathway downstream of GEF-H1. Ectopic co-expression of tagged versions of GEF-H1 and BepC truncations revealed that the C-terminal ‘Bep intracellular delivery’ (BID) domain facilitated anchorage of BepC to the plasma membrane, whereas the N-terminal ‘filamentation induced by cAMP’ (FIC) domain facilitated binding of GEF-H1. While FIC domains typically mediate post-translational modifications, most prominently AMPylation, a mutant with quadruple amino acid exchanges in the putative active site indicated that the BepC FIC domain acts in a non-catalytic manner to activate GEF-H1. Our data support a model in which BepC activates the RhoA/ROCK pathway by re-localization of GEF-H1 from microtubules to the plasma membrane.

scholarly journals Active Rho is localized to podosomes induced by oncogenic Src and is required for their assembly and function

2004 ◽  
Vol 166 (3) ◽  
pp. 317-323 ◽  
Author(s):  
Rebecca L. Berdeaux ◽  
Begoña Díaz ◽  
Lomi Kim ◽  
G. Steven Martin
Keyword(s):  
Specific Inhibitor ◽  
Stress Fiber ◽  
Small Gtpase ◽  
Fiber Formation ◽  
Dominant Negative ◽  
Stress Fiber Formation ◽  
Transformed Fibroblasts ◽  
C3 Exoenzyme ◽  
And Function

Transformation of fibroblasts by oncogenic Src causes disruption of actin stress fibers and formation of invasive adhesions called podosomes. Because the small GTPase Rho stimulates stress fiber formation, Rho inactivation by Src has been thought to be necessary for stress fiber disruption. However, we show here that Rho[GTP] levels do not decrease after transformation by activated Src. Inactivation of Rho in Src-transformed fibroblasts by dominant negative RhoA or the Rho-specific inhibitor C3 exoenzyme disrupted podosome structure as judged by localization of podosome components F-actin, cortactin, and Fish. Inhibition of Rho strongly inhibited Src-induced proteolytic degradation of the extracellular matrix. Furthermore, development of an in situ Rho[GTP] affinity assay allowed us to detect endogenous Rho[GTP] at podosomes, where it colocalized with F-actin, cortactin, and Fish. Therefore, Rho is not globally inactivated in Src-transformed fibroblasts, but is necessary for the assembly and function of structures implicated in tumor cell invasion.

Regulation of Osteoblast-Like Cell Behaviors on Hydroxyapatite by Electrical Polarization

2007 ◽  
Vol 361-363 ◽  
pp. 1055-1058 ◽  
Author(s):  
Miho Nakamura ◽  
Akiko Nagai ◽  
Natalie Ohashi ◽  
Yumi Tanaka ◽  
Yasutaka Sekijima ◽  
...  
Keyword(s):  
Cell Movement ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Wound Healing Assay ◽  
Cell Behaviors ◽  
Stress Fiber Formation ◽  
Cytoskeleton Reorganization ◽  
Osteoblast Adhesion ◽  
As Stress

The osteoblast adhesion to the substrates are recognized to play a fundamental role in osteoconduction process. The purpose of this study was to evaluate the in vitro behavior of osteoblasts cultured on polarized hydroxyapatite (HA), having the enhanced osteobonding abilities. Osteoblast-like cells were seeded onto the polarized HA and investigated the adhesion and motility. The polarization had no effects on the percentage of the number of the spreaded cells against all the adhered cells, but had significant effects on the elongation of adhered cells from fluorescent observation and on the cell motility showed by the wound healing assay. The charges induced on the HA surface accelerated the cytoskeleton reorganization of the adhered cells cultured on HA specimens. The acceleration was emerged as the cells shape, actin filament pattern such as stress fiber formation, and the prolongation of the cell movement distances.

scholarly journals Novel Roles for α3β1 Integrin as a Regulator of Cytoskeletal Assembly and as a Trans-dominant Inhibitor of Integrin Receptor Function in Mouse Keratinocytes

1998 ◽  
Vol 142 (5) ◽  
pp. 1357-1369 ◽  
Author(s):  
Kairbaan M. Hodivala-Dilke ◽  
C. Michael DiPersio ◽  
Jordan A. Kreidberg ◽  
Richard O. Hynes
Keyword(s):  
Stress Fiber ◽  
Fiber Formation ◽  
The Other ◽  
Collagen Type Iv ◽  
Focal Contact ◽  
Actin Organization ◽  
Α3β1 Integrin ◽  
Stress Fiber Formation ◽  
Associated Proteins ◽  
Mouse Keratinocytes

Previously we found that α3β1 integrin–deficient neonatal mice develop micro-blisters at the epidermal–dermal junction. These micro-blisters were associated with poor basement membrane organization. In the present study we have investigated the effect of α3β1-deficiency on other keratinocyte integrins, actin-associated proteins and F-actin organization. We show that the absence of α3β1 results in an increase in stress fiber formation in keratinocytes grown in culture and at the basal face of the basal keratinocytes of α3-null epidermis. Moreover, we see a higher concentration of actin-associated proteins such as vinculin, talin, and α-actinin at focal contact sites in the α3-deficient keratinocytes. These changes in focal contact composition were not due to a change in steady-state levels of these proteins, but rather to reorganization due to α3β1 deficiency. Apart from the loss of α3β1 there is no change in expression of the other integrins expressed by the α3-null keratinocytes. However, in functional assays, α3β1 deficiency allows an increase in fibronectin and collagen type IV receptor activities. Thus, our findings provide evidence for a role of α3β1 in regulating stress fiber formation and as a trans-dominant inhibitor of the functions of the other integrins in mouse keratinocytes. These results have potential implications for the regulation of keratinocyte adhesion and migration during wound healing.

scholarly journals Delayed stress fiber formation mediates pulmonary myofibroblast differentiation in response to TGF-β

2011 ◽  
Vol 301 (5) ◽  
pp. L656-L666 ◽  
Author(s):  
Nathan Sandbo ◽  
Andrew Lau ◽  
Jacob Kach ◽  
Caitlyn Ngam ◽  
Douglas Yau ◽  
...  
Keyword(s):  
Rho Kinase ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Nuclear Accumulation ◽  
Myofibroblast Differentiation ◽  
Latrunculin B ◽  
Stress Fiber Formation ◽  
Actin Expression ◽  
Actin Stress Fibers

Myofibroblast differentiation induced by transforming growth factor-β (TGF-β) and characterized by de novo expression of smooth muscle (SM)-specific proteins is a key process in wound healing and in the pathogenesis of fibrosis. We have previously shown that TGF-β-induced expression and activation of serum response factor (SRF) is required for this process. In this study, we examined the signaling mechanism for SRF activation by TGF-β as it relates to pulmonary myofibroblast differentiation. TGF-β stimulated a profound, but delayed (18–24 h), activation of Rho kinase and formation of actin stress fibers, which paralleled SM α-actin expression. The translational inhibitor cycloheximide blocked these processes without affecting Smad-dependent gene transcription. Inhibition of Rho kinase by Y-27632 or depolymerization of actin by latrunculin B resulted in inhibition TGF-β-induced SRF activation and SM α-actin expression, having no effect on Smad signaling. Conversely, stabilization of actin stress fibers by jasplakinolide was sufficient to drive these processes in the absence of TGF-β. TGF-β promoted a delayed nuclear accumulation of the SRF coactivator megakaryoblastic leukemia-1 (MKL1)/myocardin-related transcription factor-A, which was inhibited by latrunculin B. Furthermore, TGF-β also induced MKL1 expression, which was inhibited by latrunculin B, by SRF inhibitor CCG-1423, or by SRF knockdown. Together, these data suggest a triphasic model for myofibroblast differentiation in response to TGF-β that involves 1) initial Smad-dependent expression of intermediate signaling molecules driving Rho activation and stress fiber formation, 2) nuclear accumulation of MKL1 and activation of SRF as a result of actin polymerization, and 3) SRF-dependent expression of MKL1, driving further myofibroblast differentiation.

Endothelial cytoskeletal reorganization in response to PAR1 stimulation is mediated by membrane rafts but not caveolae

2007 ◽  
Vol 293 (1) ◽  
pp. H366-H375 ◽  
Author(s):  
MaryEllen Carlile-Klusacek ◽  
Victor Rizzo
Keyword(s):  
Endothelial Cells ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Signaling Cascade ◽  
Membrane Rafts ◽  
Actin Stress Fiber ◽  
Caveolin 1 ◽  
Stress Fiber Formation ◽  
Mlc Phosphorylation ◽  
Actin Stress Fiber Formation

The vasoactive protease thrombin is a known activator of the protease-activated receptor-1 (PAR1) via cleavage of its NH2 terminus. PAR1 activation stimulates the RhoA/Rho kinase signaling cascade, leading to myosin light chain (MLC) phosphorylation, actin stress fiber formation, and changes in endothelial monolayer integrity. Previous studies suggest that some elements of this signaling pathway are localized to caveolin-containing cholesterol-rich membrane domains. Here we show that PAR1 and key components of the PAR-associated signaling cascade localize to membrane rafts and caveolae in bovine aortic endothelial cells (BAEC). To investigate the functional significance of this localization, BAEC were pretreated with filipin (5 μg/ml, 5 min) to ablate lipid rafts before thrombin (100 nM) or PAR agonist stimulation. We found that diphosphorylation of MLC and the actin stress fiber formation normally induced by PAR activation were attenuated after lipid raft disruption. To target caveolae specifically, we used a small interferring RNA approach to knockdown caveolin-1 expression. Thrombin-induced MLC phosphorylation and stress fiber formation were not altered in caveolin-1-depleted cells, suggesting that lipid rafts, but not necessarily caveolae, modulate thrombin-activated signaling pathways leading to alteration of the actin cytoskeleton in endothelial cells.

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