scholarly journals RhoB regulates cell migration through altered focal adhesion dynamics

Open Biology ◽  
2012 ◽  
Vol 2 (5) ◽  
pp. 120076 ◽  
Author(s):  
Francisco M. Vega ◽  
Audrey Colomba ◽  
Nicolas Reymond ◽  
Mairian Thomas ◽  
Anne J. Ridley
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Rho Gtpase ◽  
Focal Adhesions ◽  
Β1 Integrin ◽  
Membrane Ruffling ◽  
Similar Phenotype ◽  
And Migration ◽  
Directional Cell Migration ◽  
Chemotactic Gradient

The Rho GTPase RhoB has been shown to affect cell migration, but how it does this is not clear. Here we show that cells depleted of RhoB by RNAi are rounded and have defects in Rac-mediated spreading and lamellipodium extension, although they have active membrane ruffling around the periphery. Depletion of the exchange factor GEF-H1 induces a similar phenotype. RhoB-depleted cells migrate faster, but less persistently in a chemotactic gradient, and frequently round up during migration. RhoB-depleted cells have similar numbers of focal adhesions to control cells during spreading and migration, but show more diffuse and patchy contact with the substratum. They have lower levels of surface β1 integrin, and β1 integrin activity is reduced in actin-rich protrusions. We propose that RhoB contributes to directional cell migration by regulating β1 integrin surface levels and activity, thereby stabilizing lamellipodial protrusions.

scholarly journals Targeting and transport: How microtubules control focal adhesion dynamics

2012 ◽  
Vol 198 (4) ◽  
pp. 481-489 ◽  
Author(s):  
Samantha Stehbens ◽  
Torsten Wittmann
Keyword(s):  
Extracellular Matrix ◽  
Cell Migration ◽  
Focal Adhesion ◽  
Molecular Mechanisms ◽  
Rho Gtpase ◽  
Focal Adhesions ◽  
Matrix Metalloproteases ◽  
Force Generation ◽  
Diverse Group ◽  
Directional Cell Migration

Directional cell migration requires force generation that relies on the coordinated remodeling of interactions with the extracellular matrix (ECM), which is mediated by integrin-based focal adhesions (FAs). Normal FA turnover requires dynamic microtubules, and three members of the diverse group of microtubule plus-end-tracking proteins are principally involved in mediating microtubule interactions with FAs. Microtubules also alter the assembly state of FAs by modulating Rho GTPase signaling, and recent evidence suggests that microtubule-mediated clathrin-dependent and -independent endocytosis regulates FA dynamics. In addition, FA-associated microtubules may provide a polarized microtubule track for localized secretion of matrix metalloproteases (MMPs). Thus, different aspects of the molecular mechanisms by which microtubules control FA turnover in migrating cells are beginning to emerge.

scholarly journals Local Myo9b RhoGAP activity regulates cell motility

2020 ◽  
pp. jbc.RA120.013623
Author(s):  
Sandra Angela Hemkemeyer ◽  
Veith Vollmer ◽  
Vera Schwarz ◽  
Birgit Lohmann ◽  
Ulrike Honnert ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Motility ◽  
Cell Morphology ◽  
Actin Polymerization ◽  
Rho Gtpase ◽  
Leading Edge ◽  
Cell Extension ◽  
Local Inhibition ◽  
And Migration ◽  
Directional Cell Migration

To migrate, cells assume a polarized morphology, extending forward with a leading edge with their trailing edge retracting back toward the cell body. Both cell extension and retraction critically depend on the organization and dynamics of the actin cytoskeleton, and the small, monomeric GTPases Rac and Rho are important regulators of actin. Activation of Rac induces actin polymerization and cell extension whereas activation of Rho enhances acto-myosin II contractility and cell retraction. To coordinate migration, these processes must be carefully regulated. The myosin Myo9b, a Rho GTPase activating protein (GAP), negatively regulates Rho activity and deletion of Myo9b in leukocytes impairs cell migration through increased Rho activity. However, it is not known whether cell motility is regulated by global or local inhibition of Rho activity by Myo9b. Here, we addressed this question by using Myo9b-deficient macrophage-like cells that expressed different recombinant Myo9b constructs. We found that Myo9b accumulates in lamellipodial extensions generated by Rac-induced actin polymerization as a function of its motor activity. Deletion of Myo9b in HL-60 derived macrophages altered cell morphology and impaired cell migration. Reintroduction of Myo9b or Myo9b motor and GAP mutants revealed that local GAP activity rescues cell morphology and migration. In summary, Rac activation leads to actin polymerization and recruitment of Myo9b, which locally inhibits Rho activity to enhance directional cell migration. In summary, Rac activation leads to actin polymerization and recruitment of Myo9b, which locally inhibits Rho activity to enhance directional cell migration.

scholarly journals Calpain-mediated Proteolysis of Paxillin Negatively Regulates Focal Adhesion Dynamics and Cell Migration

2011 ◽  
Vol 286 (12) ◽  
pp. 9998-10006 ◽  
Author(s):  
Christa L. Cortesio ◽  
Lindsy R. Boateng ◽  
Timothy M. Piazza ◽  
David A. Bennin ◽  
Anna Huttenlocher
Keyword(s):  
Cell Migration ◽  
Cell Motility ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Adaptor Protein ◽  
Time Lapse ◽  
Negative Regulator ◽  
Proteolytic Fragment ◽  
Proteolytic Site ◽  
And Migration

The dynamic turnover of integrin-mediated adhesions is important for cell migration. Paxillin is an adaptor protein that localizes to focal adhesions and has been implicated in cell motility. We previously reported that calpain-mediated proteolysis of talin1 and focal adhesion kinase mediates adhesion disassembly in motile cells. To determine whether calpain-mediated paxillin proteolysis regulates focal adhesion dynamics and cell motility, we mapped the preferred calpain proteolytic site in paxillin. The cleavage site is between the paxillin LD1 and LD2 motifs and generates a C-terminal fragment that is similar in size to the alternative product paxillin delta. The calpain-generated proteolytic fragment, like paxillin delta, functions as a paxillin antagonist and impairs focal adhesion disassembly and migration. We generated mutant paxillin with a point mutation (S95G) that renders it partially resistant to calpain proteolysis. Paxillin-deficient cells that express paxillin S95G display increased turnover of zyxin-containing adhesions using time-lapse microscopy and also show increased migration. Moreover, cancer-associated somatic mutations in paxillin are common in the N-terminal region between the LD1 and LD2 motifs and confer partial calpain resistance. Taken together, these findings suggest a novel role for calpain-mediated proteolysis of paxillin as a negative regulator of focal adhesion dynamics and migration that may function to limit cancer cell invasion.

scholarly journals The phosphorylation state of MRLC is polyamine dependent in intestinal epithelial cells

2011 ◽  
Vol 300 (1) ◽  
pp. C164-C175 ◽  
Author(s):  
Mitulkumar N. Bavaria ◽  
Ramesh M. Ray ◽  
Leonard R. Johnson
Keyword(s):  
Cell Migration ◽  
Light Chain ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Focal Adhesions ◽  
Fiber Formation ◽  
Actin Cortex ◽  
And Migration

Cell migration is important to the integrity of the gastrointestinal tract for the normal movement of cells from crypt to villi and the healing of wounds. Polyamines are essential to cell migration, mucosal restitution, and, hence, healing. Polyamine depletion by α-difluoromethyl ornithine (DFMO) inhibited migration by decreasing lamellipodia and stress fiber formation and preventing the activation of Rho-GTPases. Polyamine depletion increased the association of the thick F-actin cortex with phosphorylated myosin regulatory light chain (pMRLC). In this study, we determined why MRLC is constitutively phosphorylated as part of the actin cortex. Inhibition of myosin light chain kinase (MLCK) decreased RhoA and Rac1 activities and significantly inhibited migration. Polyamine depletion increased phosphorylation of MRLC (Thr18/Ser19) and stabilized the actin cortex and focal adhesions. The Rho-kinase inhibitor Y27632 increased spreading and migration by decreasing the phosphorylation of MRLC, remodeling focal adhesions, and by activating Rho-GTPases. Thus phosphorylation of MRLC appears to be the rate-limiting step during the migration of IEC-6 cells. In addition, increased localization of RhoA with the actin cortex in polyamine-depleted cells appears to activate Rho-kinase. In the absence of polyamines, activated Rho-kinase phosphorylates myosin phosphatase targeting subunit 1 (MYPT1) at serine-668 leading to its inactivation and preventing the recruitment of phosphatase (protein phosphastase, PP1cδ) to the actomyosin cortex. In this condition, MRLC is constitutively phosphorylated and cycling does not occur. Thus activated myosin binds F-actin stress fibers and prevents focal adhesion turnover, Rho-GTPase activation, and the remodeling of the cytoskeleton required for migration.

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.

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 Characterization of the Roles of Vimentin in Regulating the Proliferation and Migration of HSCs during Hepatic Fibrogenesis

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1184 ◽  
Author(s):  
Pei-Wen Wang ◽  
Tung-Ho Wu ◽  
Tung-Yi Lin ◽  
Mu-Hong Chen ◽  
Chau-Ting Yeh ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Messenger Rna ◽  
Rho Gtpase ◽  
Focal Adhesions ◽  
Cytoskeletal Proteins ◽  
Nuclear Antigen ◽  
Dependent Manner ◽  
Hepatic Fibrogenesis ◽  
And Migration ◽  
Proliferation And Migration

The activation of hepatic stellate cells (HSCs) manifested as proliferation and migration is the pivotal event involved in liver fibrogenesis. The vimentin network, an intermediate filament (IF) system, is one of the critical cascades by which the cell morphology, growth, and motility are modulated. However, the vimentin-mediated cytoskeletal cross talk, as well as the signaling transduction, which further coordinates the cellular responses during hepatic fibrogenesis, is poorly understood. In the current study, both messenger RNA (mRNA) and the vimentin protein were significantly increased in a time-dependent manner in the dimethylnitrosamine (DMN)-exposed liver. In particular, vimentin was highly expressed in the activated HSCs. Again, the overexpressed vimentin was observed in the plasma samples derived from patients with hepatic fibrosis/cirrhosis, suggesting that vimentin may be a key factor in regulating the progression of liver fibrosis. Meanwhile, vimentin knockdown suppressed the migratory propensity, provoked morphological changes, and disturbed the focal adhesions in the HSCs due to the breakdown of associated cytoskeletal proteins. Western blotting showed that vimentin deletion inhibited proliferating cell nuclear antigen (PCNA) and arrested the Rho GTPase family, thereby impairing the HSCs’ growth as well as motility. The phosphorylated extracellular-signal regulated kinase (ERK) and AKT signals were also notably reduced in response to the silence of vimentin. Inhibitors of selected signaling pathways suppressed the migration and differentiation of activated HSCs by regulating specific serine phosphorylated sites on vimentin. Taken together, these findings revealed a novel mechanism of vimentin through which various signaling pathways controlled the proliferation, differentiation, and movement of the HSCs via the ERK/AKT and Rho cascades.

scholarly journals Cardiac fibroblasts require focal adhesion kinase for normal proliferation and migration

2009 ◽  
Vol 296 (3) ◽  
pp. H627-H638 ◽  
Author(s):  
Ana Maria Manso ◽  
Seok-Min Kang ◽  
Sergey V. Plotnikov ◽  
Ingo Thievessen ◽  
Jaewon Oh ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Adult Mouse ◽  
Cardiac Fibroblasts ◽  
Focal Adhesions ◽  
Protein Levels ◽  
Tyrosine Kinase 2 ◽  
A Cell ◽  
And Migration ◽  
Proliferation And Migration

Migration and proliferation of cardiac fibroblasts (CFs) play an important role in the myocardial remodeling process. While many factors have been identified that regulate CF growth and migration, less is known about the signaling mechanisms involved in these processes. Here, we utilized Cre-LoxP technology to obtain focal adhesion kinase (FAK)-deficient adult mouse CFs and studied how FAK functioned in modulating cell adhesion, proliferation, and migration of these cells. Treatment of FAKflox/flox CFs with Ad/Cre virus caused over 70% reduction of FAK protein levels within a cell population. FAK-deficient CFs showed no changes in focal adhesions, cell morphology, or protein expression levels of vinculin, talin, or paxillin; proline-rich tyrosine kinase 2 (Pyk2) expression and activity were increased. Knockdown of FAK protein in CFs increased PDGF-BB-induced proliferation, while it reduced PDGF-BB-induced migration. Adhesion to fibronectin was not altered. To distinguish between the function of FAK and Pyk2, FAK function was inhibited via adenoviral-mediated overexpression of the natural FAK inhibitor FAK-related nonkinase (FRNK). Ad/FRNK had no effect on Pyk2 expression, inhibited the PDGF-BB-induced migration, but did not change the PDGF-BB-induced proliferation. FAK deficiency had only modest effects on increasing PDGF-BB activation of p38 and JNK MAPKs, with no alteration in the ERK response vs. control cells. These results demonstrate that FAK is required for the PDGF-BB-induced migratory response of adult mouse CFs and suggest that FAK could play an essential role in the wound-healing response that occurs in numerous cardiac pathologies.

Gingival epithelial cell signalling and cytoskeletal responses to Porphyromonas gingivalis invasion

Microbiology ◽  
2003 ◽  
Vol 149 (9) ◽  
pp. 2417-2426 ◽  
Author(s):  
Özlem Yilmaz ◽  
Patrick A. Young ◽  
Richard J. Lamont ◽  
George E. Kenny
Keyword(s):  
Porphyromonas Gingivalis ◽  
Focal Adhesion ◽  
Cell Signalling ◽  
Focal Adhesions ◽  
Wild Type ◽  
Cortical Actin ◽  
Membrane Ruffling ◽  
Invasion Mechanism ◽  
Signalling Molecules ◽  
Gingival Epithelial Cell

Porphyromonas gingivalis, an oral pathogen, can internalize within primary gingival epithelial cells (GECs) through an invasion mechanism mediated by interactions between P. gingivalis fimbriae and integrins on the surface of the GECs. Fimbriae–integrin-based signalling events were studied by fluorescence microscopy, and the subcellular localization of integrin-associated signalling molecules paxillin and focal adhesion kinase (FAK), and the architecture of the actin and microtubule cytoskeleton were examined. GECs infected with P. gingivalis for 30 min demonstrated significant redistribution of paxillin and FAK from the cytosol to cell peripheries and assembly into focal adhesion complexes. In contrast, a fimbriae-deficient mutant of P. gingivalis did not contribute substantially to activation of paxillin or FAK. After 24 h, the majority of paxillin and FAK had returned to the cytoplasm with significant co-localization with P. gingivalis in the perinuclear region. Wild-type P. gingivalis induced nucleation of actin filaments forming microspike-like protrusions and long stable microfilaments distributed throughout the cells. Fimbriae mutants promoted a rich cortical actin meshwork accompanied by membrane ruffling dispersed along the cell membrane. Remarkable disassembly and nucleation of the actin and microtubule filamentous network was observed following 24 h infection with either wild-type or fimbriae-deficient mutants of P. gingivalis. The results show that fimbriated P. gingivalis cells induce formation of integrin-associated focal adhesions with subsequent remodelling of the actin and tubulin cytoskeleton.

scholarly journals The Abl-related Gene Tyrosine Kinase Acts through p190RhoGAP to Inhibit Actomyosin Contractility and Regulate Focal Adhesion Dynamics upon Adhesion to Fibronectin

2007 ◽  
Vol 18 (10) ◽  
pp. 3860-3872 ◽  
Author(s):  
Justin G. Peacock ◽  
Ann L. Miller ◽  
William D. Bradley ◽  
Olga C. Rodriguez ◽  
Donna J. Webb ◽  
...  
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Actin Polymerization ◽  
Focal Adhesions ◽  
Fiber Formation ◽  
Cell Periphery ◽  
Related Gene ◽  
Cell Contractility ◽  
Fibroblast Migration ◽  
Actomyosin Contractility

In migrating cells, actin polymerization promotes protrusion of the leading edge, whereas actomyosin contractility powers net cell body translocation. Although they promote F-actin–dependent protrusions of the cell periphery upon adhesion to fibronectin (FN), Abl family kinases inhibit cell migration on FN. We provide evidence here that the Abl-related gene (Arg/Abl2) kinase inhibits fibroblast migration by attenuating actomyosin contractility and regulating focal adhesion dynamics. arg−/− fibroblasts migrate at faster average speeds than wild-type (wt) cells, whereas Arg re-expression in these cells slows migration. Surprisingly, the faster migrating arg−/− fibroblasts have more prominent F-actin stress fibers and focal adhesions and exhibit increased actomyosin contractility relative to wt cells. Interestingly, Arg requires distinct functional domains to inhibit focal adhesions and actomyosin contractility. The kinase domain–containing Arg N-terminal half can act through the RhoA inhibitor p190RhoGAP to attenuate stress fiber formation and cell contractility. However, Arg requires both its kinase activity and its cytoskeleton-binding C-terminal half to fully inhibit focal adhesions. Although focal adhesions do not turn over efficiently in the trailing edge of arg−/− cells, the increased contractility of arg−/− cells tears the adhesions from the substrate, allowing for the faster migration observed in these cells. Together, our data strongly suggest that Arg inhibits cell migration by restricting actomyosin contractility and regulating its coupling to the substrate through focal adhesions.

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