scholarly journals Different translation dynamics of β-and γ-actin regulates cell migration

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Pavan Vedula ◽  
Satoshi Kurosaka ◽  
Brittany MacTaggart ◽  
Qin Ni ◽  
Garegin Papoian ◽  
...  
Keyword(s):  
Cell Migration ◽  
Amino Acid ◽  
Single Molecule ◽  
Amino Acid Level ◽  
Focal Adhesions ◽  
Mesenchymal Cell ◽  
Cell Periphery ◽  
Pronounced Difference ◽  
Coding Sequence

β- and γ-cytoplasmic actins are ubiquitously expressed in every cell type and are nearly identical at the amino acid level but play vastly different roles in vivo. Their essential roles in embryogenesis and mesenchymal cell migration critically depend on the nucleotide sequences of their genes, rather than their amino acid sequence, however it is unclear which gene elements underlie this effect. Here we address the specific role of the coding sequence in β- and γ-cytoplasmic actins' intracellular functions, using stable polyclonal populations of immortalized mouse embryonic fibroblasts with exogenously expressed actin isoforms and their 'codon-switched' variants. When targeted to the cell periphery using the β-actin 3′UTR, β-actin and γ-actin have differential effects on cell migration. These effects directly depend on the coding sequence. Single molecule measurements of actin isoform translation, combined with fluorescence recovery after photobleaching, demonstrate a pronounced difference in β- and γ-actins' translation elongation rates in cells, leading to changes in their dynamics at the focal adhesions, impairments in actin bundle formation, and reduced cell anchoring to the substrate during migration. Our results demonstrate that coding sequence-mediated differences in actin translation play a key role in cell migration.

scholarly journals Different translation dynamics of β- and γ-actin regulates cell migration

2021 ◽  
Author(s):  
Pavan Vedula ◽  
Satoshi Kurosaka ◽  
Brittany MacTaggart ◽  
Qin Ni ◽  
Garegin A. Papoian ◽  
...  
Keyword(s):  
Cell Migration ◽  
Amino Acid ◽  
Single Molecule ◽  
Amino Acid Level ◽  
Focal Adhesions ◽  
Cell Periphery ◽  
Pronounced Difference ◽  
Translation Elongation ◽  
Coding Sequence

Abstractβ- and γ-cytoplasmic actins are ubiquitously expressed in every cell type and are nearly identical at the amino acid level but play vastly different roles in vivo. Their essential roles in embryogenesis and cell migration critically depend on the nucleotide sequences of their genes, rather than their amino acid sequence. However it is unclear which gene elements underlie this effect. Here we address the specific role of the coding sequence in β- and γ-cytoplasmic actins’ intracellular functions, using stable cell lines with exogenously expressed actin isoforms and their “codon-switched” variants. When targeted to the cell periphery using the β-actin 3′UTR, β-actin and γ-actin have differential effects on cell migration. These effects directly depend on the coding sequence. Single molecule measurements of actin isoform translation, combined with fluorescence recovery after photobleaching, demonstrate a pronounced difference in β- and γ-actins’ translation elongation rates, leading to changes in their dynamics at focal adhesions, impairments in actin bundle formation, and reduced cell anchoring to the substrate during migration. Our results demonstrate that coding sequence-mediated differences in actin translation play a key role in cell migration.

scholarly journals Smurf1 regulates tumor cell plasticity and motility through degradation of RhoA leading to localized inhibition of contractility

2006 ◽  
Vol 176 (1) ◽  
pp. 35-42 ◽  
Author(s):  
Erik Sahai ◽  
Raquel Garcia-Medina ◽  
Jacques Pouysségur ◽  
Emmanuel Vial
Keyword(s):  
Cell Migration ◽  
Tumor Cell ◽  
Rho Gtpases ◽  
Rho Kinase ◽  
Cell Movement ◽  
Leading Edge ◽  
Cell Periphery ◽  
Tumor Cell Migration ◽  
Tumor Cell Motility

Rho GTPases participate in various cellular processes, including normal and tumor cell migration. It has been reported that RhoA is targeted for degradation at the leading edge of migrating cells by the E3 ubiquitin ligase Smurf1, and that this is required for the formation of protrusions. We report that Smurf1-dependent RhoA degradation in tumor cells results in the down-regulation of Rho kinase (ROCK) activity and myosin light chain 2 (MLC2) phosphorylation at the cell periphery. The localized inhibition of contractile forces is necessary for the formation of lamellipodia and for tumor cell motility in 2D tissue culture assays. In 3D invasion assays, and in in vivo tumor cell migration, the inhibition of Smurf1 induces a mesenchymal–amoeboid–like transition that is associated with a more invasive phenotype. Our results suggest that Smurf1 is a pivotal regulator of tumor cell movement through its regulation of RhoA signaling.

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 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.

scholarly journals The dynamic interplay between ATP/ADP levels and autophagy sustain neuronal migration in vivo

2020 ◽  
Author(s):  
Bressan Cedric ◽  
Pecora Alessandra ◽  
Gagnon Dave ◽  
Snapyan Marina ◽  
Labrecque Simon ◽  
...  
Keyword(s):  
Cell Migration ◽  
Stationary Phase ◽  
Neuronal Migration ◽  
Stationary Phases ◽  
Focal Adhesions ◽  
Time Lapse ◽  
List Type ◽  
Atp Levels ◽  
Migrating Cells

AbstractCell migration is a dynamic process that entails extensive protein synthesis and recycling, structural remodeling, and a considerable bioenergetic demand. Autophagy is one of the pathways that maintain cellular homeostasis. Time-lapse imaging of autophagosomes and ATP/ADP levels in migrating cells in the rostral migratory stream of mice revealed that decrease in ATP levels force cells into the stationary phase and induce autophagy. Genetic impairment of autophagy in neuroblasts using either inducible conditional mice or CRISPR/Cas9 gene editing decreased cell migration due to the longer duration of the stationary phase. Autophagy is modulated in response to migration-promoting and inhibiting molecular cues and is required for the recycling of focal adhesions. Our results show that autophagy and energy consumption act in concert in migrating cells to dynamically regulate the pace and periodicity of the migratory and stationary phases in order to sustain neuronal migration.HighlightsADP levels dynamically change during cell migrationA decrease in ATP levels leads to cell pausing and autophagy induction via AMPKAutophagy is required to sustain neuronal migration by recycling focal adhesionsAutophagy level is dynamically modulated by migration-promoting and inhibiting cues

scholarly journals α4/α9 Integrins Coordinate Epithelial Cell Migration Through Local Suppression of MAP Kinase Signaling Pathways

2021 ◽  
Vol 9 ◽  
Author(s):  
Willow Hight-Warburton ◽  
Robert Felix ◽  
Andrew Burton ◽  
Hannah Maple ◽  
Magda S. Chegkazi ◽  
...  
Keyword(s):  
Cell Migration ◽  
Protein Complexes ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Collective Cell Migration ◽  
Keratinocyte Proliferation ◽  
Basal Keratinocytes ◽  
Keratinocyte Cell

Adhesion of basal keratinocytes to the underlying extracellular matrix (ECM) plays a key role in the control of skin homeostasis and response to injury. Integrin receptors indirectly link the ECM to the cell cytoskeleton through large protein complexes called focal adhesions (FA). FA also function as intracellular biochemical signaling platforms to enable cells to respond to changing extracellular cues. The α4β1 and α9β1 integrins are both expressed in basal keratinocytes, share some common ECM ligands, and have been shown to promote wound healing in vitro and in vivo. However, their roles in maintaining epidermal homeostasis and relative contributions to pathological processes in the skin remain unclear. We found that α4β1 and α9β1 occupied distinct regions in monolayers of a basal keratinocyte cell line (NEB-1). During collective cell migration (CCM), α4 and α9 integrins co-localized along the leading edge. Pharmacological inhibition of α4β1 and α9β1 integrins increased keratinocyte proliferation and induced a dramatic change in cytoskeletal remodeling and FA rearrangement, detrimentally affecting CCM. Further analysis revealed that α4β1/α9β1 integrins suppress extracellular signal-regulated kinase (ERK1/2) activity to control migration through the regulation of downstream kinases including Mitogen and Stress Activated Kinase 1 (MSK1). This work demonstrates the roles of α4β1 and α9β1 in regulating migration in response to damage cues.

scholarly journals The Role of the C-Terminal Lysine of S100P in S100P-Induced Cell Migration and Metastasis

Biomolecules ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1471
Author(s):  
Thamir M. Ismail ◽  
Stephane R. Gross ◽  
Tara Lancaster ◽  
Philip S. Rudland ◽  
Roger Barraclough
Keyword(s):  
Cell Migration ◽  
Focal Adhesions ◽  
Wistar Rat ◽  
Model System ◽  
Wild Type ◽  
Survival Times ◽  
Potent Inducer ◽  
Myosin Heavy Chain Isoform

S100P protein is a potent inducer of metastasis in a model system, and its presence in cancer cells of patients is strongly associated with their reduced survival times. A well-established Furth Wistar rat metastasis model system, methods for measuring cell migration, and specific inhibitors were used to study pathways of motility-driven metastasis. Cells expressing C-terminal mutant S100P proteins display markedly-reduced S100P-driven metastasis in vivo and cell migration in vitro. These cells fail to display the low focal adhesion numbers observed in cells expressing wild-type S100P, and the mutant S100P proteins exhibit reduced biochemical interaction with non-muscle myosin heavy chain isoform IIA in vitro. Extracellular inhibitors of the S100P-dependent plasminogen activation pathway reduce, but only in part, wild-type S100P-dependent cell migration; they are without effect on S100P-negative cells or cells expressing C-terminal mutant S100P proteins and have no effect on the numbers of focal adhesions. Recombinant wild-type S100P protein, added extracellularly to S100P-negative cells, stimulates cell migration, which is abolished by these inhibitors. The results identify at least two S100P-dependent pathways of migration, one cell surface and the other intracellularly-linked, and identify its C-terminal lysine as a target for inhibiting multiple migration-promoting activities of S100P protein and S100P-driven metastasis.

scholarly journals RSV attenuates epithelial cell restitution by inhibiting actin cytoskeleton-dependent cell migration

2021 ◽  
Author(s):  
Debra T Linfield ◽  
Nannan Gao ◽  
Andjela Raduka ◽  
Terri J Harford ◽  
Giovanni Piedimonte ◽  
...  
Keyword(s):  
Cell Migration ◽  
Epithelial Cell ◽  
Wound Repair ◽  
Focal Adhesions ◽  
Fiber Formation ◽  
Rsv Infection ◽  
Syncytial Virus ◽  
Tract Infections

The airway epithelium's ability to repair itself after injury, known as epithelial restitution, is an essential mechanism enabling the respiratory tract's normal functions. Respiratory Syncytial Virus (RSV) is the leading cause of lower respiratory tract infections worldwide. We sought to determine whether RSV delays the airway epithelium wound repair process both in vitro and in vivo. We found that RSV infection attenuated epithelial cell migration, a step in wound repair, promoted stress fiber formation, and mediated assembly of large focal adhesions (FA). Inhibition of Rho kinase (ROCK), a master regulator of actin function, reversed these effects. There was increased RhoA and phospho-myosin light chain (pMLC2) following RSV infection. In vivo, mice were intraperitoneally inoculated with naphthalene to induce lung injury, followed by RSV infection. RSV infection delayed re-epithelialization. There were increased concentrations of pMLC2 in day 7 naphthalene plus RSV animals which normalized by day 14. This study suggests a key mechanism by which RSV infection delays wound healing.

scholarly journals Distinct phospho-forms of cortactin differentially regulate actin polymerization and focal adhesions

2008 ◽  
Vol 295 (5) ◽  
pp. C1113-C1122 ◽  
Author(s):  
Anne E. Kruchten ◽  
Eugene W. Krueger ◽  
Yu Wang ◽  
Mark A. McNiven
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Actin Polymerization ◽  
Control Cell ◽  
Focal Adhesions ◽  
Serine Phosphorylation ◽  
Actin Binding ◽  
Actin Assembly

Cortactin is an actin-binding protein that is overexpressed in many cancers and is a substrate for both tyrosine and serine/threonine kinases. Tyrosine phosphorylation of cortactin has been observed to increase cell motility and invasion in vivo, although it has been reported to have both positive and negative effects on actin polymerization in vitro. In contrast, serine phosphorylation of cortactin has been shown to stimulate actin assembly in vitro. Currently, the effects of cortactin serine phosphorylation on cell migration are unclear, and furthermore, how the distinct phospho-forms of cortactin may differentially contribute to cell migration has not been directly compared. Therefore, we tested the effects of different tyrosine and serine phospho-mutants of cortactin on lamellipodial protrusion, actin assembly within cells, and focal adhesion dynamics. Interestingly, while expression of either tyrosine or serine phospho-mimetic cortactin mutants resulted in increased lamellipodial protrusion and cell migration, these effects appeared to be via distinct processes. Cortactin mutants mimicking serine phosphorylation appeared to predominantly affect actin polymerization, whereas mutation of cortactin tyrosine residues resulted in alterations in focal adhesion turnover. Thus these findings provide novel insights into how distinct phospho-forms of cortactin may differentially contribute to actin and focal adhesion dynamics to control cell migration.

scholarly journals Simultaneous Binding of Multiple EF-Tu Copies to Translating Ribosomes in Live Escherichia coli

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Mainak Mustafi ◽  
James C. Weisshaar
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Single Molecule ◽  
Ternary Complex ◽  
Elongation Factor ◽  
Ternary Complexes ◽  
Local Concentration ◽  
E Coli ◽  
A Site

ABSTRACT In bacteria, elongation factor Tu is a translational cofactor that forms ternary complexes with aminoacyl-tRNA (aa-tRNA) and GTP. Binding of a ternary complex to one of four flexible L7/L12 units on the ribosome tethers a charged tRNA in close proximity to the ribosomal A site. Two sequential tests for a match between the aa-tRNA anticodon and the current mRNA codon then follow. Because one elongation cycle can occur in as little as 50 ms and the vast majority of aa-tRNA copies are not cognate with the current mRNA codon, this testing must occur rapidly. We present a single-molecule localization and tracking study of fluorescently labeled EF-Tu in live Escherichia coli. Imaging at 2 ms/frame distinguishes 60% slowly diffusing EF-Tu copies (assigned as transiently bound to translating ribosome) from 40% rapidly diffusing copies (assigned as a mixture of free ternary complexes and free EF-Tu). Combining these percentages with copy number estimates, we infer that the four L7/L12 sites are essentially saturated with ternary complexes in vivo. The results corroborate an earlier inference that all four sites can simultaneously tether ternary complexes near the A site, creating a high local concentration that may greatly enhance the rate of testing of aa-tRNAs. Our data and a combinatorial argument both suggest that the initial recognition test for a codon-anticodon match occurs in less than 1 to 2 ms per aa-tRNA copy. The results refute a recent study (A. Plochowietz, I. Farrell, Z. Smilansky, B. S. Cooperman, and A. N. Kapanidis, Nucleic Acids Res 45:926–937, 2016, https://doi.org/10.1093/nar/gkw787) of tRNA diffusion in E. coli that inferred that aa-tRNAs arrive at the ribosomal A site as bare monomers, not as ternary complexes. IMPORTANCE Ribosomes catalyze translation of the mRNA codon sequence into the corresponding sequence of amino acids within the nascent polypeptide chain. Polypeptide elongation can be as fast as 50 ms per added amino acid. Each amino acid arrives at the ribosome as a ternary complex comprising an aminoacyl-tRNA (aa-tRNA), an elongation factor called EF-Tu, and GTP. There are 43 different aa-tRNAs in use, only one of which typically matches the current mRNA codon. Thus, ternary complexes must be tested very rapidly. Here we use fluorescence-based single-molecule methods that locate and track single EF-Tu copies in E. coli. Fast and slow diffusive behavior determines the fraction of EF-Tu copies that are ribosome bound. We infer simultaneous tethering of ~4 ternary complexes to the ribosome, which may facilitate rapid initial testing for codon matching on a time scale of less than 1 to 2 ms per aa-tRNA.

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