Simulation of Stress Fiber Remodeling and Mixed-Mode Focal Adhesion Assembly During Cell Spreading and for Cells Adhered to Elastic Substrates

2011 ◽  
Author(s):  
William Ronan ◽  
Vikram S. Deshpande ◽  
Robert M. McMeeking ◽  
J. Patrick McGarry
Keyword(s):  
Actin Cytoskeleton ◽  
Focal Adhesion ◽  
Physical Environment ◽  
Mixed Mode ◽  
Computational Models ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Stress Fiber ◽  
Cellular Processes ◽  
Elastic Substrates

Cell spreading is governed by two cooperative cellular processes: the association of binding proteins to form focal adhesions, and the active remodeling of the actin cytoskeleton as the cell spreads [1]. The interaction between these two processes is poorly understood, and previous computational models have only examined each process in isolation. Previous studies have established that cells possess the ability to sense and react to their physical environment, for example cells seeded on substrates of varying stiffness exhibit a different cytoskeletal response [2,3].

scholarly journals B-RAF Regulation of Rnd3 Participates in Actin Cytoskeletal and Focal Adhesion Organization

2008 ◽  
Vol 19 (2) ◽  
pp. 498-508 ◽  
Author(s):  
R. Matthew Klein ◽  
Laurie S. Spofford ◽  
Ethan V. Abel ◽  
Arisa Ortiz ◽  
Andrew E. Aplin
Keyword(s):  
Actin Cytoskeleton ◽  
Focal Adhesion ◽  
Constitutive Expression ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Mitogen Activated Protein Kinase ◽  
Actin Stress Fiber ◽  
Lim Kinase ◽  
Stress Fiber Formation

The actin cytoskeleton controls multiple cellular functions, including cell morphology, movement, and growth. Accumulating evidence indicates that oncogenic activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 (MEK/ERK1/2) pathway is accompanied by actin cytoskeletal reorganization. However, the signaling events contributing to actin cytoskeleton remodeling mediated by aberrant ERK1/2 activation are largely unknown. Mutant B-RAF is found in a variety of cancers, including melanoma, and it enhances activation of the MEK/ERK1/2 pathway. We show that targeted knockdown of B-RAF with small interfering RNA or pharmacological inhibition of MEK increased actin stress fiber formation and stabilized focal adhesion dynamics in human melanoma cells. These effects were due to stimulation of the Rho/Rho kinase (ROCK)/LIM kinase-2 signaling pathway, cumulating in the inactivation of the actin depolymerizing/severing protein cofilin. The expression of Rnd3, a Rho antagonist, was attenuated after B-RAF knockdown or MEK inhibition, but it was enhanced in melanocytes expressing active B-RAF. Constitutive expression of Rnd3 suppressed the actin cytoskeletal and focal adhesion effects mediated by B-RAF knockdown. Depletion of Rnd3 elevated cofilin phosphorylation and stress fiber formation and reduced cell invasion. Together, our results identify Rnd3 as a regulator of cross talk between the RAF/MEK/ERK and Rho/ROCK signaling pathways, and a key contributor to oncogene-mediated reorganization of the actin cytoskeleton and focal adhesions.

scholarly journals pH sensing by FAK-His58 regulates focal adhesion remodeling

2013 ◽  
Vol 202 (6) ◽  
pp. 849-859 ◽  
Author(s):  
Chang-Hoon Choi ◽  
Bradley A. Webb ◽  
Michael S. Chimenti ◽  
Matthew P. Jacobson ◽  
Diane L. Barber
Keyword(s):  
Focal Adhesion ◽  
Conformational Changes ◽  
Cancer Metastasis ◽  
Ph Sensor ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Initial Step ◽  
Ph Sensing ◽  
Cellular Processes ◽  
Dynamics Simulations

Intracellular pH (pHi) dynamics regulates diverse cellular processes, including remodeling of focal adhesions. We now report that focal adhesion kinase (FAK), a key regulator of focal adhesion remodeling, is a pH sensor responding to physiological changes in pH. The initial step in FAK activation is autophosphorylation of Tyr397, which increased with higher pHi. We used a genetically encoded biosensor to show increased pH at focal adhesions as they mature during cell spreading. We also show that cells with reduced pHi had attenuated FAK-pY397 as well as defective cell spreading and focal adhesions. Mutagenesis studies indicated FAK-His58 is critical for pH sensing and molecular dynamics simulations suggested a model in which His58 deprotonation drives conformational changes that may modulate accessibility of Tyr397 for autophosphorylation. Expression of FAK-H58A in fibroblasts was sufficient to restore defective autophosphorylation and cell spreading at low pHi. These data are relevant to understanding cancer metastasis, which is dependent on increased pHi and FAK activity.

scholarly journals Rab40/Cullin5 complex regulates EPLIN and actin cytoskeleton dynamics during cell migration and invasion

2021 ◽  
Author(s):  
Erik S Linklater ◽  
Emily Duncan ◽  
Ke Jun Han ◽  
Algirdas Kaupinis ◽  
Mindaugas Valius ◽  
...  
Keyword(s):  
Cell Migration ◽  
Actin Cytoskeleton ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Stress Fibers ◽  
Actin Dynamics ◽  
Migration And Invasion ◽  
Matrix Remodeling ◽  
Cell Migration And Invasion

Rab40b is a SOCS box containing protein that regulates the secretion of MMPs to facilitate extracellular matrix remodeling during cell migration. Here we show that Rab40b interacts with Cullin5 via the Rab40b SOCS domain. We demonstrate that loss of Rab40b/Cullin5 binding decreases cell motility and invasive potential, and show that defective cell migration and invasion stem from alteration to the actin cytoskeleton, leading to decreased invadopodia formation, decreased actin dynamics at the leading edge, and an increase in stress fibers. We also show that these stress fibers anchor at less dynamic, more stable focal adhesions. Mechanistically, changes in the cytoskeleton and focal adhesion dynamics are mediated in part by EPLIN, which we demonstrate to be a binding partner of Rab40b and a target for Rab40b/Cullin5 dependent localized ubiquitylation and degradation. Thus, we propose a model where the Rab40b/Cullin5 dependent ubiquitylation regulates EPLIN localization to promote cell migration and invasion by altering focal adhesion and cytoskeletal dynamics.

scholarly journals Piezo2 channel regulates RhoA and actin cytoskeleton to promote cell mechanobiological responses

2018 ◽  
Vol 115 (8) ◽  
pp. 1925-1930 ◽  
Author(s):  
Carlos Pardo-Pastor ◽  
Fanny Rubio-Moscardo ◽  
Marina Vogel-González ◽  
Selma A. Serra ◽  
Alexandros Afthinos ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Nuclear Translocation ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Force Transmission ◽  
Cellular Processes ◽  
Fyn Kinase ◽  
Downstream Effector ◽  
Metastatic Cells

Actin polymerization and assembly into stress fibers (SFs) is central to many cellular processes. However, how SFs form in response to the mechanical interaction of cells with their environment is not fully understood. Here we have identified Piezo2 mechanosensitive cationic channel as a transducer of environmental physical cues into mechanobiological responses. Piezo2 is needed by brain metastatic cells from breast cancer (MDA-MB-231-BrM2) to probe their physical environment as they anchor and pull on their surroundings or when confronted with confined migration through narrow pores. Piezo2-mediated Ca2+ influx activates RhoA to control the formation and orientation of SFs and focal adhesions (FAs). A possible mechanism for the Piezo2-mediated activation of RhoA involves the recruitment of the Fyn kinase to the cell leading edge as well as calpain activation. Knockdown of Piezo2 in BrM2 cells alters SFs, FAs, and nuclear translocation of YAP; a phenotype rescued by overexpression of dominant-positive RhoA or its downstream effector, mDia1. Consequently, hallmarks of cancer invasion and metastasis related to RhoA, actin cytoskeleton, and/or force transmission, such as migration, extracellular matrix degradation, and Serpin B2 secretion, were reduced in cells lacking Piezo2.

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 Tension is required but not sufficient for focal adhesion maturation without a stress fiber template

2012 ◽  
Vol 196 (3) ◽  
pp. 363-374 ◽  
Author(s):  
Patrick W. Oakes ◽  
Yvonne Beckham ◽  
Jonathan Stricker ◽  
Margaret L. Gardel
Keyword(s):  
Focal Adhesion ◽  
Myosin Ii ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Matrix Remodeling ◽  
Force Transmission ◽  
Fiber Assembly ◽  
Wide Range ◽  
Structural Template

Focal adhesion composition and size are modulated in a myosin II–dependent maturation process that controls adhesion, migration, and matrix remodeling. As myosin II activity drives stress fiber assembly and enhanced tension at adhesions simultaneously, the extent to which adhesion maturation is driven by tension or altered actin architecture is unknown. We show that perturbations to formin and α-actinin 1 activity selectively inhibited stress fiber assembly at adhesions but retained a contractile lamella that generated large tension on adhesions. Despite relatively unperturbed adhesion dynamics and force transmission, impaired stress fiber assembly impeded focal adhesion compositional maturation and fibronectin remodeling. Finally, we show that compositional maturation of focal adhesions could occur even when myosin II–dependent cellular tension was reduced by 80%. We propose that stress fiber assembly at the adhesion site serves as a structural template that facilitates adhesion maturation over a wide range of tensions. This work identifies the essential role of lamellar actin architecture in adhesion maturation.

An Analysis of the Cooperative Mechano-Sensitive Feedback Between Intracellular Signaling, Focal Adhesion Development, and Stress Fiber Contractility

2011 ◽  
Vol 78 (4) ◽  
Author(s):  
Amit Pathak ◽  
Robert M. McMeeking ◽  
Anthony G. Evans ◽  
Vikram S. Deshpande
Keyword(s):  
Mechanical Loading ◽  
Focal Adhesion ◽  
Intracellular Signaling ◽  
Feedback Loop ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Mechanical Loads ◽  
Signaling Model ◽  
Wide Range

Cells communicate with their external environment via focal adhesions and generate activation signals that in turn trigger the activity of the intracellular contractile machinery. These signals can be triggered by mechanical loading that gives rise to a cooperative feedback loop among signaling, focal adhesion formation, and cytoskeletal contractility, which in turn equilibrates with the applied mechanical loads. We devise a signaling model that couples stress fiber contractility and mechano-sensitive focal adhesion models to complete this above mentioned feedback loop. The signaling model is based on a biochemical pathway where IP3 molecules are generated when focal adhesions grow. These IP3 molecules diffuse through the cytosol leading to the opening of ion channels that disgorge Ca2+ from the endoplasmic reticulum leading to the activation of the actin/myosin contractile machinery. A simple numerical example is presented where a one-dimensional cell adhered to a rigid substrate is pulled at one end, and the evolution of the stress fiber activation signal, stress fiber concentrations, and focal adhesion distributions are investigated. We demonstrate that while it is sufficient to approximate the activation signal as spatially uniform due to the rapid diffusion of the IP3 through the cytosol, the level of the activation signal is sensitive to the rate of application of the mechanical loads. This suggests that ad hoc signaling models may not be able to capture the mechanical response of cells to a wide range of mechanical loading events.

scholarly journals The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL)

2001 ◽  
Vol 154 (1) ◽  
pp. 161-176 ◽  
Author(s):  
Kip A. West ◽  
Huaye Zhang ◽  
Michael C. Brown ◽  
Sotiris N. Nikolopoulos ◽  
M.C. Riedy ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Focal Adhesion ◽  
Deletion Mutant ◽  
Morphological Changes ◽  
Cell Spreading ◽  
Small Gtpases ◽  
Random Motility ◽  
Phenotypic Changes ◽  
Rho Family ◽  
Mutant Cells

The small GTPases of the Rho family are intimately involved in integrin-mediated changes in the actin cytoskeleton that accompany cell spreading and motility. The exact means by which the Rho family members elicit these changes is unclear. Here, we demonstrate that the interaction of paxillin via its LD4 motif with the putative ARF-GAP paxillin kinase linker (PKL) (Turner et al., 1999), is critically involved in the regulation of Rac-dependent changes in the actin cytoskeleton that accompany cell spreading and motility. Overexpression of a paxillin LD4 deletion mutant (paxillinΔLD4) in CHO.K1 fibroblasts caused the generation of multiple broad lamellipodia. These morphological changes were accompanied by an increase in cell protrusiveness and random motility, which correlated with prolonged activation of Rac. In contrast, directional motility was inhibited. These alterations in morphology and motility were dependent on a paxillin–PKL interaction. In cells overexpressing paxillinΔLD4 mutants, PKL localization to focal contacts was disrupted, whereas that of focal adhesion kinase (FAK) and vinculin was not. In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif. Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLΔPBS2) induced phenotypic changes reminiscent of paxillinΔLD4 mutant cells. These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.

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