scholarly journals Critical Roles of Phosphorylation and Actin Binding Motifs, but Not the Central Proline-rich Region, for Ena/Vasodilator-stimulated Phosphoprotein (VASP) Function during Cell Migration

2002 ◽  
Vol 13 (7) ◽  
pp. 2533-2546 ◽  
Author(s):  
Joseph J. Loureiro ◽  
Douglas A. Rubinson ◽  
James E. Bear ◽  
Gretchen A. Baltus ◽  
Adam V. Kwiatkowski ◽  
...  
Keyword(s):  
Cell Motility ◽  
Cell Movement ◽  
Protein Family ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Binding Motif ◽  
Conserved Sequence ◽  
Cellular Processes ◽  
Sequence Elements

The Ena/vasodilator-stimulated phosphoprotein (VASP) protein family is implicated in the regulation of a number of actin-based cellular processes, including lamellipodial protrusion necessary for whole cell translocation. A growing body of evidence derived largely from in vitro biochemical experiments using purified proteins, cell-free extracts, and pathogen motility has begun to suggest various mechanistic roles for Ena/VASP proteins in the control of actin dynamics. Using complementation of phenotypes in Ena/VASP-deficient cells and overexpression in normal fibroblasts, we have assayed the function of a panel of mutants in one member of this family, Mena, by mutating highly conserved sequence elements found in this protein family. Surprisingly, deletion of sites required for binding of the actin monomer-binding protein profilin, a known ligand of Ena/VASP proteins, has no effect on the ability of Mena to regulate random cell motility. Our analysis revealed two features essential for Ena/VASP function in cell movement, cyclic nucleotide-dependent kinase phosphorylation sites and an F-actin binding motif. Interestingly, expression of the C-terminal EVH2 domain alone is sufficient to complement loss of Ena/VASP function in random cell motility.

scholarly journals Actin-depolymerizing Factor and Cofilin-1 Play Overlapping Roles in Promoting Rapid F-Actin Depolymerization in Mammalian Nonmuscle Cells

2005 ◽  
Vol 16 (2) ◽  
pp. 649-664 ◽  
Author(s):  
Pirta Hotulainen ◽  
Eija Paunola ◽  
Maria K. Vartiainen ◽  
Pekka Lappalainen
Keyword(s):  
Cell Motility ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Depolymerizing Factor ◽  
Cytoskeletal Dynamics ◽  
Nonmuscle Cells ◽  
In Cells

Actin-depolymerizing factor (ADF)/cofilins are small actin-binding proteins found in all eukaryotes. In vitro, ADF/cofilins promote actin dynamics by depolymerizing and severing actin filaments. However, whether ADF/cofilins contribute to actin dynamics in cells by disassembling “old” actin filaments or by promoting actin filament assembly through their severing activity is a matter of controversy. Analysis of mammalian ADF/cofilins is further complicated by the presence of multiple isoforms, which may contribute to actin dynamics by different mechanisms. We show that two isoforms, ADF and cofilin-1, are expressed in mouse NIH 3T3, B16F1, and Neuro 2A cells. Depleting cofilin-1 and/or ADF by siRNA leads to an accumulation of F-actin and to an increase in cell size. Cofilin-1 and ADF seem to play overlapping roles in cells, because the knockdown phenotype of either protein could be rescued by overexpression of the other one. Cofilin-1 and ADF knockdown cells also had defects in cell motility and cytokinesis, and these defects were most pronounced when both ADF and cofilin-1 were depleted. Fluorescence recovery after photobleaching analysis and studies with an actin monomer-sequestering drug, latrunculin-A, demonstrated that these phenotypes arose from diminished actin filament depolymerization rates. These data suggest that mammalian ADF and cofilin-1 promote cytoskeletal dynamics by depolymerizing actin filaments and that this activity is critical for several processes such as cytokinesis and cell motility.

scholarly journals Villin Severing Activity Enhances Actin-based Motility In Vivo

2007 ◽  
Vol 18 (3) ◽  
pp. 827-838 ◽  
Author(s):  
Céline Revenu ◽  
Matthieu Courtois ◽  
Alphée Michelot ◽  
Cécile Sykes ◽  
Daniel Louvard ◽  
...  
Keyword(s):  
Shigella Flexneri ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Loss Of Function ◽  
Mesenchymal Transition ◽  
Capping Protein ◽  
Function Strategy ◽  
In Cells

Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition.

scholarly journals STIP1/HOP Regulates the Actin Cytoskeleton through Interactions with Actin and Changes in Actin-Binding Proteins Cofilin and Profilin

2020 ◽  
Vol 21 (9) ◽  
pp. 3152 ◽  
Author(s):  
Samantha Joy Beckley ◽  
Morgan Campbell Hunter ◽  
Sarah Naulikha Kituyi ◽  
Ianthe Wingate ◽  
Abantika Chakraborty ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Binding Proteins ◽  
Major Effect ◽  
Vital Role ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Nuclear Accumulation ◽  
Actin Binding Proteins ◽  
Hsp90 Chaperone

Cell migration plays a vital role in both health and disease. It is driven by reorganization of the actin cytoskeleton, which is regulated by actin-binding proteins cofilin and profilin. Stress-inducible phosphoprotein 1 (STIP1) is a well-described co-chaperone of the Hsp90 chaperone system, and our findings identify a potential regulatory role of STIP1 in actin dynamics. We show that STIP1 can be isolated in complex with actin and Hsp90 from HEK293T cells and directly interacts with actin in vitro via the C-terminal TPR2AB-DP2 domain of STIP1, potentially due to a region spanning two putative actin-binding motifs. We found that STIP1 could stimulate the in vitro ATPase activity of actin, suggesting a potential role in the modulation of F-actin formation. Interestingly, while STIP1 depletion in HEK293T cells had no major effect on total actin levels, it led to increased nuclear accumulation of actin, disorganization of F-actin structures, and an increase and decrease in cofilin and profilin levels, respectively. This study suggests that STIP1 regulates the cytoskeleton by interacting with actin, or via regulating the ratio of proteins known to affect actin dynamics.

Structure of the 34 kDa F-actin-bundling protein ABP34 fromDictyostelium discoideum

2015 ◽  
Vol 71 (9) ◽  
pp. 1835-1849 ◽  
Author(s):  
Min-Kyu Kim ◽  
Ji-Hye Kim ◽  
Ji-Sun Kim ◽  
Sa-Ouk Kang
Keyword(s):  
Domain Structure ◽  
Hydrophobic Interactions ◽  
Convex Surface ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Helical Structures ◽  
Binding Model ◽  
Conserved Sequence ◽  
Ef Hand

The crystal structure of the 34 kDa F-actin-bundling protein ABP34 fromDictyostelium discoideumwas solved by Ca2+/S-SAD phasing and refined at 1.89 Å resolution. ABP34 is a calcium-regulated actin-binding protein that cross-links actin filaments into bundles. Itsin vitroF-actin-binding and F-actin-bundling activities were confirmed by a co-sedimentation assay and transmission electron microscopy. The co-localization of ABP34 with actin in cells was also verified. ABP34 adopts a two-domain structure with an EF-hand-containing N-domain and an actin-binding C-domain, but has no reported overall structural homologues. The EF-hand is occupied by a calcium ion with a pentagonal bipyramidal coordination as in the canonical EF-hand. The C-domain structure resembles a three-helical bundle and superposes well onto the rod-shaped helical structures of some cytoskeletal proteins. Residues 216–244 in the C-domain form part of the strongest actin-binding sites (193–254) and exhibit a conserved sequence with the actin-binding region of α-actinin and ABP120. Furthermore, the second helical region of the C-domain is kinked by a proline break, offering a convex surface towards the solvent area which is implicated in actin binding. The F-actin-binding model suggests that ABP34 binds to the side of the actin filament and residues 216–244 fit into a pocket between actin subdomains −1 and −2 through hydrophobic interactions. These studies provide insights into the calcium coordination in the EF-hand and F-actin-binding site in the C-domain of ABP34, which are associated through interdomain interactions.

scholarly journals βcap73-ARF6 Interactions Modulate Cell Shape and Motility after Injury In Vitro

2003 ◽  
Vol 14 (10) ◽  
pp. 4155-4161 ◽  
Author(s):  
Kathleen N. Riley ◽  
Angel E. Maldonado ◽  
Patrice Tellier ◽  
Crislyn D'Souza-Schorey ◽  
Ira M. Herman
Keyword(s):  
Western Blot ◽  
Molecular Mechanisms ◽  
Active Form ◽  
Leading Edge ◽  
Dominant Negative ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Capping Protein ◽  
Actin Capping Protein

To understand the role that ARF6 plays in regulating isoactin dynamics and cell motility, we transfected endothelial cells (EC) with HA-tagged ARF6: the wild-type form (WT), a constitutively-active form unable to hydrolyze GTP (Q67L), and two dominant-negative forms, which are either unable to release GDP (T27N) or fail to bind nucleotide (N122I). Motility was assessed by digital imaging microscopy before Western blot analysis, coimmunoprecipitation, or colocalization studies using ARF6, β-actin, or β-actin-binding protein-specific antibodies. EC expressing ARF6-Q67L spread and close in vitro wounds at twice the control rates. EC expressing dominant-negative ARF6 fail to develop a leading edge, are unable to ruffle their membranes (N122I), and possess arborized processes. Colocalization studies reveal that the Q67L and WT ARF6-HA are enriched at the leading edge with β-actin; but T27N and N122I ARF6-HA are localized on endosomes together with the β-actin capping protein, βcap73. Coimmunoprecipitation and Western blot analyses reveal the direct association of ARF6-HA with βcap73, defining a role for ARF6 in signaling cytoskeletal remodeling during motility. Knowledge of the role that ARF6 plays in orchestrating membrane and β-actin dynamics will help to reveal molecular mechanisms regulating actin-based motility during development and disease.

Small-Molecule Screen Identifies ROS as Key Regulators of Actin Dynamics in Neutrophils

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4641-4641
Author(s):  
Hidenori Hattori ◽  
Kulandayan K Subramanian ◽  
Hongbo R. Luo
Keyword(s):  
Small Molecule ◽  
Actin Polymerization ◽  
Neutrophil Migration ◽  
Physiological Role ◽  
Leading Edge ◽  
Actin Dynamics ◽  
Functional Screening ◽  
Cellular Processes

Abstract Precise spatial and temporal control of actin polymerization and depolymerization is essential for mediating various cellular processes such as migration, phagocytosis, vesicle trafficking and adhesion. In this study, we used a small-molecule functional screening approach to identify novel regulators of actin dynamics during neutrophil migration. Here we show that NADPH-oxidase dependent Reactive Oxygen Species act as negative regulators of actin polymerization. Neutrophils with pharmacologically inhibited oxidase or isolated from Chronic Granulomatous Disease (CGD) patient and mice displayed enhanced F-actin polymerization, multiple pseudopods formation and impaired chemotaxis. ROS localized to pseudopodia and inhibited actin polymerization by driving actin glutathionylation at the leading edge of migrating cells. Consistent with these in vitro results, adoptively transferred CGD murine neutrophils also showed impaired in vivo recruitment to sites of inflammation. Together, these results present a novel physiological role for ROS in regulation of action polymerization and shed new light on the pathogenesis of CGD.

scholarly journals Myocardin-Related Transcription Factor A Activation by Competition with WH2 Domain Proteins for Actin Binding

2016 ◽  
Vol 36 (10) ◽  
pp. 1526-1539 ◽  
Author(s):  
Julia Weissbach ◽  
Franziska Schikora ◽  
Anja Weber ◽  
Michael Kessels ◽  
Guido Posern
Keyword(s):  
Serum Response Factor ◽  
De Novo ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Serum Stimulation ◽  
Competitive Mechanism ◽  
Related Transcription Factor ◽  
Simultaneous Inhibition ◽  
Serum Response

The myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF)-mediated gene expression. Activation of MRTF-A occurs in response to alterations in actin dynamics and critically requires the dissociation of repressive G-actin–MRTF-A complexes. However, the mechanism leading to the release of MRTF-A remains unclear. Here we show that WH2 domains compete directly with MRTF-A for actin binding. Actin nucleation-promoting factors, such as N-WASP and WAVE2, as well as isolated WH2 domains, including those of Spire2 and Cobl, activate MRTF-A independently of changes in actin dynamics. Simultaneous inhibition of Arp2-Arp3 or mutation of the CA region only partially reduces MRTF-A activation by N-WASP and WAVE2. Recombinant WH2 domains and the RPEL domain of MRTF-A bind mutually exclusively to cellular and purified G-actinin vitro. The competition by different WH2 domains correlates with MRTF-SRF activation. Following serum stimulation, nonpolymerizable actin dissociates from MRTF-A, andde novoformation of the G-actin–RPEL complex is impaired by a transferable factor. Our work demonstrates that WH2 domains activate MRTF-A and contribute to target gene regulation by a competitive mechanism, independently of their role in actin filament formation.

scholarly journals Inhibition of platelet function by targeting the platelet cytoskeleton via the LIMK-signaling pathway

2018 ◽  
Author(s):  
Juliana Antonipillai ◽  
Sheena Rigby ◽  
Nicole Bassler ◽  
Karlheinz Peter ◽  
Ora Bernard
Keyword(s):  
Platelet Function ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Lim Kinase ◽  
Cellular Processes ◽  
New Strategy ◽  
Platelet Aggregates ◽  
Dynamic Structures ◽  
Shape Changes ◽  
Platelet Shape

AbstractActin is highly abundant in platelets, and platelet function is dependent on actin structures. Actin filaments are dynamic structures involved in many cellular processes including platelet shape changes and adhesion. The actin cytoskeleton is tightly regulated by actin-binding proteins, which include the members of the actin depolymerising factor (ADF)/cofilin family. LIM kinase (LIMK) and slingshot phosphatase (SSH-1L) regulate actin dynamics by controlling the binding affinity of ADF/cofilin towards actin. We hypothesised that inhibition of LIMK activity may prevent the changes in platelet shape during their activation and therefore their function by controlling the dynamics of Factin. Therefore, inhibition of LIMK activity may represent an attractive new strategy to control and inhibit platelet function; particularly the formation of stable platelet aggregates and thus stable thrombi.

scholarly journals Virus-Induced Cell Motility

1998 ◽  
Vol 72 (2) ◽  
pp. 1235-1243 ◽  
Author(s):  
Christopher M. Sanderson ◽  
Michael Way ◽  
Geoffrey L. Smith
Keyword(s):  
Cell Migration ◽  
Cell Motility ◽  
Uv Light ◽  
Cell Metabolism ◽  
Cell Movement ◽  
Time Lapse ◽  
Late Gene Expression ◽  
Infected Cells ◽  
And Function

ABSTRACT Many viruses induce profound changes in cell metabolism and function. Here we show that vaccinia virus induces two distinct forms of cell movement. Virus-induced cell migration was demonstrated by an in vitro wound healing assay in which infected cells migrated independently into the wound area while uninfected cells remained relatively static. Time-lapse microscopy showed that the maximal rate of migration occurred between 9 and 12 h postinfection. Virus-induced cell migration was inhibited by preinactivation of viral particles with trioxsalen and UV light or by the addition of cycloheximide but not by addition of cytosine arabinoside or rifampin. The expression of early viral genes is therefore necessary and sufficient to induce cell migration. Following migration, infected cells developed projections up to 160 μm in length which had growth-cone-like structures and were frequently branched. Time-lapse video microscopy showed that these projections were formed by extension and condensation of lamellipodia from the cell body. Formation of extensions was dependent on late gene expression but not the production of intracellular enveloped (IEV) particles. The requirements for virus-induced cell migration and for the formation of extensions therefore differ from each other and are distinct from the polymerization of actin tails on IEV particles. These data show that poxviruses encode genes which control different aspects of cell motility and thus represent a useful model system to study and dissect cell movement.

scholarly journals Reduction in SNAP-23 Alters Microfilament Organization in Myofibrobastic Hepatic Stellate Cells

2020 ◽  
Vol 20 (1) ◽  
pp. 25-37
Author(s):  
Haleigh B. Eubanks ◽  
Elise G. Lavoie ◽  
Jessica Goree ◽  
Jeffrey A. Kamykowski ◽  
Neriman Gokden ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Hepatic Stellate Cells ◽  
Cell Movement ◽  
Stellate Cells ◽  
Snare Proteins ◽  
Actin Dynamics ◽  
Critical Feature ◽  
Effector Cells

Hepatic stellate cells (HSC) are critical effector cells of liver fibrosis. In the injured liver, HSC differentiate into a myofibrobastic phenotype. A critical feature distinguishing myofibroblastic from quiescent HSC is cytoskeletal reorganization. Soluble NSF attachment receptor (SNARE) proteins are important in trafficking of newly synthesized proteins to the plasma membrane for release into the extracellular environment. The goals of this project were to determine the expression of specific SNARE proteins in myofibroblastic HSC and to test whether their alteration changed the HSC phenotype in vitro and progression of liver fibrosis in vivo. We found that HSC lack the t-SNARE protein, SNAP-25, but express a homologous protein, SNAP-23. Downregulation of SNAP-23 in HSC induced reduction in polymerization and disorganization of the actin cytoskeleton associated with loss of cell movement. In contrast, reduction in SNAP-23 in mice by monogenic deletion delayed but did not prevent progression of liver fibrosis to cirrhosis. Taken together, these findings suggest that SNAP-23 is an important regular of actin dynamics in myofibroblastic HSC, but that the role of SNAP-23 in the progression of liver fibrosis in vivo is unclear.

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