The Experts below are selected from a list of 222 Experts worldwide ranked by ideXlab platform
Marie-france Carlier - One of the best experts on this subject based on the ideXlab platform.
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Multifunctionality of the β-thymosin/WH2 module: G-Actin sequestration, Actin filament Growth, nucleation, and severinG
Annals of the New York Academy of Sciences, 2010Co-Authors: Clotilde Husson, François-xavier Cantrelle, Pierre Roblin, Dominique Didry, Javier Perez, Eric Guittet, Carine Van Heijenoort, Louis Renault, Marie-france CarlierAbstract:The beta-thymosin/WH2 Actin-bindinG module shows an amazinG adaptation to multifunctionality. The beta-thymosins are Genuine G-Actin sequesterers of moderate affinity for G-Actin, allowinG an efficient reGulation of the G-Actin/F-Actin ratio in cells by amplifyinG chanGes in the critical concentration for filament assembly. In contrast, the first beta-thymosin domain of the protein Ciboulot makes with G-Actin a complex that supports filament Growth, such as profilin-Actin. We illustrate how the use of enGineered chimeric proteins, Actin-bindinG and polymerization assays, crystalloGraphic, NMR, and SAXS structural approaches complement each other to decipher the molecular basis for the functional versatility of these intrinsically disordered domains when they form various 1:1 complexes with G-Actin. Multifunctionality is expanded in tandem repeats of WH2 domains present in WASP family proteins and proteins involved in axis patterninG like Cordon-Bleu and Spire. The tandem repeats Generate new functions such as filament nucleation and severinG, as well as barbed end bindinG, which add up to the G-Actin sequesterinG activity. Novel reGulation pathways in Actin assembly emerGe from these additional activities.
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multifunctionality of the β thymosin wh2 module G Actin sequestration Actin filament Growth nucleation and severinG
Annals of the New York Academy of Sciences, 2010Co-Authors: Clotilde Husson, François-xavier Cantrelle, Pierre Roblin, Dominique Didry, Javier Perez, Eric Guittet, Carine Van Heijenoort, Louis Renault, Marie-france CarlierAbstract:The beta-thymosin/WH2 Actin-bindinG module shows an amazinG adaptation to multifunctionality. The beta-thymosins are Genuine G-Actin sequesterers of moderate affinity for G-Actin, allowinG an efficient reGulation of the G-Actin/F-Actin ratio in cells by amplifyinG chanGes in the critical concentration for filament assembly. In contrast, the first beta-thymosin domain of the protein Ciboulot makes with G-Actin a complex that supports filament Growth, such as profilin-Actin. We illustrate how the use of enGineered chimeric proteins, Actin-bindinG and polymerization assays, crystalloGraphic, NMR, and SAXS structural approaches complement each other to decipher the molecular basis for the functional versatility of these intrinsically disordered domains when they form various 1:1 complexes with G-Actin. Multifunctionality is expanded in tandem repeats of WH2 domains present in WASP family proteins and proteins involved in axis patterninG like Cordon-Bleu and Spire. The tandem repeats Generate new functions such as filament nucleation and severinG, as well as barbed end bindinG, which add up to the G-Actin sequesterinG activity. Novel reGulation pathways in Actin assembly emerGe from these additional activities.
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Multifunctionality of the beta-thymosin/WH2 module: G-Actin sequestration, Actin filament Growth, nucleation, and severinG.
Annals of the New York Academy of Sciences, 2010Co-Authors: Clotilde Husson, François-xavier Cantrelle, Pierre Roblin, Dominique Didry, Javier Perez, Eric Guittet, Carine Van Heijenoort, Louis Renault, Marie-france CarlierAbstract:The beta-thymosin/WH2 Actin-bindinG module shows an amazinG adaptation to multifunctionality. The beta-thymosins are Genuine G-Actin sequesterers of moderate affinity for G-Actin, allowinG an efficient reGulation of the G-Actin/F-Actin ratio in cells by amplifyinG chanGes in the critical concentration for filament assembly. In contrast, the first beta-thymosin domain of the protein Ciboulot makes with G-Actin a complex that supports filament Growth, such as profilin-Actin. We illustrate how the use of enGineered chimeric proteins, Actin-bindinG and polymerization assays, crystalloGraphic, NMR, and SAXS structural approaches complement each other to decipher the molecular basis for the functional versatility of these intrinsically disordered domains when they form various 1:1 complexes with G-Actin. Multifunctionality is expanded in tandem repeats of WH2 domains present in WASP family proteins and proteins involved in axis patterninG like Cordon-Bleu and Spire. The tandem repeats Generate new functions such as filament nucleation and severinG, as well as barbed end bindinG, which add up to the G-Actin sequesterinG activity. Novel reGulation pathways in Actin assembly emerGe from these additional activities.
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BindinG of Divalent Cation and Nucleotide to G-Actin in the Presence of Profilin
The Journal of biological chemistry, 1995Co-Authors: Irina Perelroizen, Marie-france Carlier, Dominique PantaloniAbstract:Abstract The effect of profilin, a G-Actin bindinG protein, on the mechanism of exchanGe of the tiGhtly bound metal ion and nucleotide on G-Actin, has been investiGated. 1) In low ionic strenGth buffer, profilin increases the rates of Ca and MG dissociation from G-Actin 250- and 50-fold, respectively. On the profilin-Actin complex as well as on G-Actin alone, nucleotide exchanGe is dependent on the concentration of divalent metal ion and is kinetically limited, at low concentration of metal ion, by the dissociation of the metal ion. 2) Under physioloGical ionic conditions, nucleotide exchanGe on G-Actin is 1 order of maGnitude faster than at low ionic strenGth. The rate of MGATP dissociation is increased by profilin from 0.05 s to 2 s, the rate of MGADP dissociation is increased from 0.2 s to 24 s. The dependences of the exchanGe rates on profilin concentration are consistent with a hiGh affinity (5 × 106 to 107M) of profilin for ATP-G-Actin, and a 20-fold lower affinity for ADP-GActin. Profilin bindinG to Actin lowers the affinity of metal-nucleotide by about 1 order of maGnitude. These results restrain the possible roles of profilin in Actin assembly in vivo.
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interaction of profilin with G Actin and poly l proline
Biochemistry, 1994Co-Authors: Irina Perelroizen, Dominique Didry, Jeanbaptiste Marchand, Laurent Blanchoin, Marie-france CarlierAbstract:The interaction of bovine spleen profilin with ATP- and ADP-G-Actin and poly(L-proline) has been studied by spectrofluorimetry, analytical ultracentrifuGation, and rapid kinetics in low ionic strenGth buffer. Profilin bindinG to G-Actin is accompanied by a larGe quenchinG of tryptophan fluorescence, allowinG the measurement of an equilibrium dissociation constant of 0.1-0.2 microM for the 1:1 profilin-Actin complex, in which metal ion and nucleotide are bound. Fluorescence quenchinG monitored the bimolecular reaction between G-Actin and profilin, from which association and dissociation rate constants of 45 microM-1 s-1 and 10 s-1 at 20 deGrees C could be derived. The tryptophan(s) which are quenched in the profilin-Actin complex are no lonGer accessible to solvent, which points to W356 in Actin as a likely candidate, consistent with the 3D structure of the crystalline profilin-Actin complex [Schutt, C. E., Myslik, J. C., Rozycki, M. D., Goonesekere, N. C. W., & LindberG, U. (1993) Nature 365, 810-816]. Upon bindinG poly(L-proline), the fluorescence of both tyrosines and tryptophans of profilin is enhanced 2.2-fold. A minimum of 10 prolines [three turns of poly(L-proline) helix II] is necessary to obtain bindinG (KD = 50 microM), the optimum size beinG larGer than 10. BindinG of poly(L-proline) is extremely fast, with k+ > 200 microM-1 s-1 at 10 deGrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
Richard Treisman - One of the best experts on this subject based on the ideXlab platform.
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RPEL-family rhoGAPs link Rac/Cdc42 GTP loadinG to G-Actin availability.
Nature cell biology, 2019Co-Authors: Jessica Diring, Stephane Mouilleron, Neil Q. Mcdonald, Richard TreismanAbstract:RPEL proteins, which contain the G-Actin-bindinG RPEL motif, coordinate cytoskeletal processes with Actin dynamics. We show that the ArhGAP12- and ArhGAP32-family GTPase-activatinG proteins (GAPs) are RPEL proteins. We determine the structure of the ArhGAP12/G-Actin complex, and show that G-Actin contacts the RPEL motif and GAP domain sequences. G-Actin inhibits ArhGAP12 GAP activity, and this requires the G-Actin contacts identified in the structure. In B16 melanoma cells, ArhGAP12 suppresses basal Rac and Cdc42 activity, F-Actin assembly, invadopodia formation and experimental metastasis. In this settinG, ArhGAP12 mutants defective for G-Actin bindinG exhibit more effective downreGulation of Rac GTP loadinG followinG HGF stimulation and enhanced inhibition of Rac-dependent processes, includinG invadopodia formation. Potentiation or disruption of the G-Actin/ArhGAP12 interaction, by treatment with the Actin-bindinG druGs latrunculin B or cytochalasin D, has correspondinG effects on Rac GTP loadinG. The interaction of G-Actin with RPEL-family rhoGAPs thus provides a neGative feedback loop that couples Rac activity to Actin dynamics.
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rpel family rhoGaps link rac cdc42 Gtp loadinG to G Actin availability
Nature Cell Biology, 2019Co-Authors: Jessica Diring, Stephane Mouilleron, Neil Q. Mcdonald, Richard TreismanAbstract:RPEL proteins, which contain the G-Actin-bindinG RPEL motif, coordinate cytoskeletal processes with Actin dynamics. We show that the ArhGAP12- and ArhGAP32-family GTPase-activatinG proteins (GAPs) are RPEL proteins. We determine the structure of the ArhGAP12/G-Actin complex, and show that G-Actin contacts the RPEL motif and GAP domain sequences. G-Actin inhibits ArhGAP12 GAP activity, and this requires the G-Actin contacts identified in the structure. In B16 melanoma cells, ArhGAP12 suppresses basal Rac and Cdc42 activity, F-Actin assembly, invadopodia formation and experimental metastasis. In this settinG, ArhGAP12 mutants defective for G-Actin bindinG exhibit more effective downreGulation of Rac GTP loadinG followinG HGF stimulation and enhanced inhibition of Rac-dependent processes, includinG invadopodia formation. Potentiation or disruption of the G-Actin/ArhGAP12 interaction, by treatment with the Actin-bindinG druGs latrunculin B or cytochalasin D, has correspondinG effects on Rac GTP loadinG. The interaction of G-Actin with RPEL-family rhoGAPs thus provides a neGative feedback loop that couples Rac activity to Actin dynamics.
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Structure of a Pentavalent G-ActinMRTF-A Complex Reveals How G-Actin Controls Nucleocytoplasmic ShuttlinG of a
2016Co-Authors: Stephane Mouilleron, Neil Q. Mcdonald, Carola A. Langer, Sebastian Guettler, Richard TreismanAbstract:Subcellular localization of the Actin-bindinG transcriptional coactivator MRTF-A is controlled by its interaction with monomeric Actin (G-Actin). SiGnal-induced decreases in G-Actin concentration reduce MRTF-A nuclear export, leadinG to its nuclear accumulation, whereas artificial increases in G-Actin concentration in restinG cells block MRTF-A nuclear import, retaininG it in the cytoplasm. This reGulation is dependent on three Actin-bindinG RPEL motifs in the reGulatory domain of MRTF-A. We describe the
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G-Actin reGulates the shuttlinG and PP1 bindinG of the RPEL protein Phactr1 to control actomyosin assembly
Journal of Cell Science, 2012Co-Authors: Maria Wiezlak, Stephane Mouilleron, Neil Q. Mcdonald, Jessica Diring, Jasmine V. Abella, Michael Way, Richard TreismanAbstract:Summary The Phactr family of PP1-bindinG proteins is implicated in human diseases includinG Parkinson’s, cancer and myocardial infarction. Each Phactr protein contains four G-Actin bindinG RPEL motifs, includinG an N-terminal motif, abuttinG a basic element, and a C-terminal triple RPEL repeat, which overlaps a conserved C-terminus required for interaction with PP1. RPEL motifs are also found in the reGulatory domains of the MRTF transcriptional coactivators, where they control MRTF subcellular localisation and activity by sensinG siGnal-induced chanGes in G-Actin concentration. However, whether G-Actin bindinG controls Phactr protein function – and its relation to siGnallinG – has not been investiGated. Here, we show that Rho-Actin siGnallinG induced by serum stimulation promotes the nuclear accumulation of Phactr1, but not other Phactr family members. Actin bindinG by the three Phactr1 C-terminal RPEL motifs is required for Phactr1 cytoplasmic localisation in restinG cells. Phactr1 nuclear accumulation is importin α-β dependent. G-Actin and importin α-β bind competitively to nuclear import siGnals associated with the N- and C-terminal RPEL motifs. All four motifs are required for the inhibition of serum-induced Phactr1 nuclear accumulation when G-Actin is elevated. G-Actin and PP1 bind competitively to the Phactr1 C-terminal reGion, and Phactr1 C-terminal RPEL mutants that cannot bind G-Actin induce aberrant actomyosin structures dependent on their nuclear accumulation and on PP1 bindinG. In CHL-1 melanoma cells, Phactr1 exhibits Actin-reGulated subcellular localisation and is required for stress fibre assembly, motility and invasiveness. These data support a role for Phactr1 in actomyosin assembly and suGGest that Phactr1 G-Actin sensinG allows its coordination with F-Actin availability.
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Structure of a pentavalent G-Actin*MRTF-A complex reveals how G-Actin controls nucleocytoplasmic shuttlinG of a transcriptional coactivator.
Science signaling, 2011Co-Authors: Stephane Mouilleron, Neil Q. Mcdonald, Carola A. Langer, Sebastian Guettler, Richard TreismanAbstract:Subcellular localization of the Actin-bindinG transcriptional coactivator MRTF-A is controlled by its interaction with monomeric Actin (G-Actin). SiGnal-induced decreases in G-Actin concentration reduce MRTF-A nuclear export, leadinG to its nuclear accumulation, whereas artificial increases in G-Actin concentration in restinG cells block MRTF-A nuclear import, retaininG it in the cytoplasm. This reGulation is dependent on three Actin-bindinG RPEL motifs in the reGulatory domain of MRTF-A. We describe the structures of pentavalent and trivalent G-Actin•RPEL domain complexes. In the pentavalent complex, each RPEL motif and the two interveninG spacer sequences bound an Actin monomer, forminG a compact assembly. In contrast, the trivalent complex lacked the C-terminal spacer- and RPEL-Actins, both of which bound only weakly in the pentavalent complex. Cytoplasmic localization of MRTF-A in unstimulated fibroblasts also required bindinG of G-Actin to the spacer sequences. The bipartite MRTF-A nuclear localization sequence was buried in the pentameric assembly, explaininG how increases in G-Actin concentration prevent nuclear import of MRTF-A. Analyses of the pentavalent and trivalent complexes show how Actin loads onto the RPEL domain and reveal a molecular mechanism by which Actin can control the activity of one of its bindinG partners.
Neil Q. Mcdonald - One of the best experts on this subject based on the ideXlab platform.
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RPEL-family rhoGAPs link Rac/Cdc42 GTP loadinG to G-Actin availability.
Nature cell biology, 2019Co-Authors: Jessica Diring, Stephane Mouilleron, Neil Q. Mcdonald, Richard TreismanAbstract:RPEL proteins, which contain the G-Actin-bindinG RPEL motif, coordinate cytoskeletal processes with Actin dynamics. We show that the ArhGAP12- and ArhGAP32-family GTPase-activatinG proteins (GAPs) are RPEL proteins. We determine the structure of the ArhGAP12/G-Actin complex, and show that G-Actin contacts the RPEL motif and GAP domain sequences. G-Actin inhibits ArhGAP12 GAP activity, and this requires the G-Actin contacts identified in the structure. In B16 melanoma cells, ArhGAP12 suppresses basal Rac and Cdc42 activity, F-Actin assembly, invadopodia formation and experimental metastasis. In this settinG, ArhGAP12 mutants defective for G-Actin bindinG exhibit more effective downreGulation of Rac GTP loadinG followinG HGF stimulation and enhanced inhibition of Rac-dependent processes, includinG invadopodia formation. Potentiation or disruption of the G-Actin/ArhGAP12 interaction, by treatment with the Actin-bindinG druGs latrunculin B or cytochalasin D, has correspondinG effects on Rac GTP loadinG. The interaction of G-Actin with RPEL-family rhoGAPs thus provides a neGative feedback loop that couples Rac activity to Actin dynamics.
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rpel family rhoGaps link rac cdc42 Gtp loadinG to G Actin availability
Nature Cell Biology, 2019Co-Authors: Jessica Diring, Stephane Mouilleron, Neil Q. Mcdonald, Richard TreismanAbstract:RPEL proteins, which contain the G-Actin-bindinG RPEL motif, coordinate cytoskeletal processes with Actin dynamics. We show that the ArhGAP12- and ArhGAP32-family GTPase-activatinG proteins (GAPs) are RPEL proteins. We determine the structure of the ArhGAP12/G-Actin complex, and show that G-Actin contacts the RPEL motif and GAP domain sequences. G-Actin inhibits ArhGAP12 GAP activity, and this requires the G-Actin contacts identified in the structure. In B16 melanoma cells, ArhGAP12 suppresses basal Rac and Cdc42 activity, F-Actin assembly, invadopodia formation and experimental metastasis. In this settinG, ArhGAP12 mutants defective for G-Actin bindinG exhibit more effective downreGulation of Rac GTP loadinG followinG HGF stimulation and enhanced inhibition of Rac-dependent processes, includinG invadopodia formation. Potentiation or disruption of the G-Actin/ArhGAP12 interaction, by treatment with the Actin-bindinG druGs latrunculin B or cytochalasin D, has correspondinG effects on Rac GTP loadinG. The interaction of G-Actin with RPEL-family rhoGAPs thus provides a neGative feedback loop that couples Rac activity to Actin dynamics.
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Structure of a Pentavalent G-ActinMRTF-A Complex Reveals How G-Actin Controls Nucleocytoplasmic ShuttlinG of a
2016Co-Authors: Stephane Mouilleron, Neil Q. Mcdonald, Carola A. Langer, Sebastian Guettler, Richard TreismanAbstract:Subcellular localization of the Actin-bindinG transcriptional coactivator MRTF-A is controlled by its interaction with monomeric Actin (G-Actin). SiGnal-induced decreases in G-Actin concentration reduce MRTF-A nuclear export, leadinG to its nuclear accumulation, whereas artificial increases in G-Actin concentration in restinG cells block MRTF-A nuclear import, retaininG it in the cytoplasm. This reGulation is dependent on three Actin-bindinG RPEL motifs in the reGulatory domain of MRTF-A. We describe the
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Molecular Analysis of a G-Actin Sensor
Acta Crystallographica Section A Foundations and Advances, 2014Co-Authors: Stephane Mouilleron, Neil Q. McdonaldAbstract:Actin dynamics control many aspects of cell shape and cell motility throuGh reGulatory interactions with a larGe variety of Actin-bindinG proteins. SiGnallinG to these Actin reGulators frequently involves a Rho GTPase-stimulated pathway that leads to a dramatic fluctuation in the levels of monomeric Actin (G-Actin) followinG polymerisation to F-Actin. Recent studies have identified a molecular G-Actin sensor called the RPEL domain that links RPEL-containinG proteins and their subcellular localisation to Actin dynamics. The RPEL domain contains a tandem array of typically three RPEL motifs, each of which is competent to bind a G-Actin molecule [1]. The domain is present in two otherwise unrelated protein families; the MRTF family of serum response factor (SRF) transcriptional co-activator proteins and the Phactr family of Actin and PP1 phosphatase-bindinG proteins. We have beGun to investiGate how the RPEL domain operates in both of these protein contexts and how it modulates subcellular localisation, transcriptional reGulation and actomyosin contractility. To define the molecular basis for the sensor we have reconstituted pentameric and trimeric G-Actin complexes with the RPEL domain from both MRTF-A and Phactr and used crystalloGraphy to reveal discrete supramolecular assemblies with repetitive arranGements of the G-Actin subunits around the "crankshaft"-shaped RPEL domain [2,3]. These arranGements are quite different from F-Actin intermolecular contacts and are quite unexpected. Our crystal structures reveal cooperative loadinG of G-Actin onto the RPEL domain that we show by several cell-based reporter assays to be of functional importance. These structures explain how G-Actin interaction alters the subcellular localisation of both MRTF-A and Phactr by inhibitinG nuclear import throuGh competinG with importin alpha-beta bindinG [2,3].
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G-Actin reGulates the shuttlinG and PP1 bindinG of the RPEL protein Phactr1 to control actomyosin assembly
Journal of Cell Science, 2012Co-Authors: Maria Wiezlak, Stephane Mouilleron, Neil Q. Mcdonald, Jessica Diring, Jasmine V. Abella, Michael Way, Richard TreismanAbstract:Summary The Phactr family of PP1-bindinG proteins is implicated in human diseases includinG Parkinson’s, cancer and myocardial infarction. Each Phactr protein contains four G-Actin bindinG RPEL motifs, includinG an N-terminal motif, abuttinG a basic element, and a C-terminal triple RPEL repeat, which overlaps a conserved C-terminus required for interaction with PP1. RPEL motifs are also found in the reGulatory domains of the MRTF transcriptional coactivators, where they control MRTF subcellular localisation and activity by sensinG siGnal-induced chanGes in G-Actin concentration. However, whether G-Actin bindinG controls Phactr protein function – and its relation to siGnallinG – has not been investiGated. Here, we show that Rho-Actin siGnallinG induced by serum stimulation promotes the nuclear accumulation of Phactr1, but not other Phactr family members. Actin bindinG by the three Phactr1 C-terminal RPEL motifs is required for Phactr1 cytoplasmic localisation in restinG cells. Phactr1 nuclear accumulation is importin α-β dependent. G-Actin and importin α-β bind competitively to nuclear import siGnals associated with the N- and C-terminal RPEL motifs. All four motifs are required for the inhibition of serum-induced Phactr1 nuclear accumulation when G-Actin is elevated. G-Actin and PP1 bind competitively to the Phactr1 C-terminal reGion, and Phactr1 C-terminal RPEL mutants that cannot bind G-Actin induce aberrant actomyosin structures dependent on their nuclear accumulation and on PP1 bindinG. In CHL-1 melanoma cells, Phactr1 exhibits Actin-reGulated subcellular localisation and is required for stress fibre assembly, motility and invasiveness. These data support a role for Phactr1 in actomyosin assembly and suGGest that Phactr1 G-Actin sensinG allows its coordination with F-Actin availability.
Stephane Mouilleron - One of the best experts on this subject based on the ideXlab platform.
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RPEL-family rhoGAPs link Rac/Cdc42 GTP loadinG to G-Actin availability.
Nature cell biology, 2019Co-Authors: Jessica Diring, Stephane Mouilleron, Neil Q. Mcdonald, Richard TreismanAbstract:RPEL proteins, which contain the G-Actin-bindinG RPEL motif, coordinate cytoskeletal processes with Actin dynamics. We show that the ArhGAP12- and ArhGAP32-family GTPase-activatinG proteins (GAPs) are RPEL proteins. We determine the structure of the ArhGAP12/G-Actin complex, and show that G-Actin contacts the RPEL motif and GAP domain sequences. G-Actin inhibits ArhGAP12 GAP activity, and this requires the G-Actin contacts identified in the structure. In B16 melanoma cells, ArhGAP12 suppresses basal Rac and Cdc42 activity, F-Actin assembly, invadopodia formation and experimental metastasis. In this settinG, ArhGAP12 mutants defective for G-Actin bindinG exhibit more effective downreGulation of Rac GTP loadinG followinG HGF stimulation and enhanced inhibition of Rac-dependent processes, includinG invadopodia formation. Potentiation or disruption of the G-Actin/ArhGAP12 interaction, by treatment with the Actin-bindinG druGs latrunculin B or cytochalasin D, has correspondinG effects on Rac GTP loadinG. The interaction of G-Actin with RPEL-family rhoGAPs thus provides a neGative feedback loop that couples Rac activity to Actin dynamics.
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rpel family rhoGaps link rac cdc42 Gtp loadinG to G Actin availability
Nature Cell Biology, 2019Co-Authors: Jessica Diring, Stephane Mouilleron, Neil Q. Mcdonald, Richard TreismanAbstract:RPEL proteins, which contain the G-Actin-bindinG RPEL motif, coordinate cytoskeletal processes with Actin dynamics. We show that the ArhGAP12- and ArhGAP32-family GTPase-activatinG proteins (GAPs) are RPEL proteins. We determine the structure of the ArhGAP12/G-Actin complex, and show that G-Actin contacts the RPEL motif and GAP domain sequences. G-Actin inhibits ArhGAP12 GAP activity, and this requires the G-Actin contacts identified in the structure. In B16 melanoma cells, ArhGAP12 suppresses basal Rac and Cdc42 activity, F-Actin assembly, invadopodia formation and experimental metastasis. In this settinG, ArhGAP12 mutants defective for G-Actin bindinG exhibit more effective downreGulation of Rac GTP loadinG followinG HGF stimulation and enhanced inhibition of Rac-dependent processes, includinG invadopodia formation. Potentiation or disruption of the G-Actin/ArhGAP12 interaction, by treatment with the Actin-bindinG druGs latrunculin B or cytochalasin D, has correspondinG effects on Rac GTP loadinG. The interaction of G-Actin with RPEL-family rhoGAPs thus provides a neGative feedback loop that couples Rac activity to Actin dynamics.
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Structure of a Pentavalent G-ActinMRTF-A Complex Reveals How G-Actin Controls Nucleocytoplasmic ShuttlinG of a
2016Co-Authors: Stephane Mouilleron, Neil Q. Mcdonald, Carola A. Langer, Sebastian Guettler, Richard TreismanAbstract:Subcellular localization of the Actin-bindinG transcriptional coactivator MRTF-A is controlled by its interaction with monomeric Actin (G-Actin). SiGnal-induced decreases in G-Actin concentration reduce MRTF-A nuclear export, leadinG to its nuclear accumulation, whereas artificial increases in G-Actin concentration in restinG cells block MRTF-A nuclear import, retaininG it in the cytoplasm. This reGulation is dependent on three Actin-bindinG RPEL motifs in the reGulatory domain of MRTF-A. We describe the
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Molecular Analysis of a G-Actin Sensor
Acta Crystallographica Section A Foundations and Advances, 2014Co-Authors: Stephane Mouilleron, Neil Q. McdonaldAbstract:Actin dynamics control many aspects of cell shape and cell motility throuGh reGulatory interactions with a larGe variety of Actin-bindinG proteins. SiGnallinG to these Actin reGulators frequently involves a Rho GTPase-stimulated pathway that leads to a dramatic fluctuation in the levels of monomeric Actin (G-Actin) followinG polymerisation to F-Actin. Recent studies have identified a molecular G-Actin sensor called the RPEL domain that links RPEL-containinG proteins and their subcellular localisation to Actin dynamics. The RPEL domain contains a tandem array of typically three RPEL motifs, each of which is competent to bind a G-Actin molecule [1]. The domain is present in two otherwise unrelated protein families; the MRTF family of serum response factor (SRF) transcriptional co-activator proteins and the Phactr family of Actin and PP1 phosphatase-bindinG proteins. We have beGun to investiGate how the RPEL domain operates in both of these protein contexts and how it modulates subcellular localisation, transcriptional reGulation and actomyosin contractility. To define the molecular basis for the sensor we have reconstituted pentameric and trimeric G-Actin complexes with the RPEL domain from both MRTF-A and Phactr and used crystalloGraphy to reveal discrete supramolecular assemblies with repetitive arranGements of the G-Actin subunits around the "crankshaft"-shaped RPEL domain [2,3]. These arranGements are quite different from F-Actin intermolecular contacts and are quite unexpected. Our crystal structures reveal cooperative loadinG of G-Actin onto the RPEL domain that we show by several cell-based reporter assays to be of functional importance. These structures explain how G-Actin interaction alters the subcellular localisation of both MRTF-A and Phactr by inhibitinG nuclear import throuGh competinG with importin alpha-beta bindinG [2,3].
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G-Actin reGulates the shuttlinG and PP1 bindinG of the RPEL protein Phactr1 to control actomyosin assembly
Journal of Cell Science, 2012Co-Authors: Maria Wiezlak, Stephane Mouilleron, Neil Q. Mcdonald, Jessica Diring, Jasmine V. Abella, Michael Way, Richard TreismanAbstract:Summary The Phactr family of PP1-bindinG proteins is implicated in human diseases includinG Parkinson’s, cancer and myocardial infarction. Each Phactr protein contains four G-Actin bindinG RPEL motifs, includinG an N-terminal motif, abuttinG a basic element, and a C-terminal triple RPEL repeat, which overlaps a conserved C-terminus required for interaction with PP1. RPEL motifs are also found in the reGulatory domains of the MRTF transcriptional coactivators, where they control MRTF subcellular localisation and activity by sensinG siGnal-induced chanGes in G-Actin concentration. However, whether G-Actin bindinG controls Phactr protein function – and its relation to siGnallinG – has not been investiGated. Here, we show that Rho-Actin siGnallinG induced by serum stimulation promotes the nuclear accumulation of Phactr1, but not other Phactr family members. Actin bindinG by the three Phactr1 C-terminal RPEL motifs is required for Phactr1 cytoplasmic localisation in restinG cells. Phactr1 nuclear accumulation is importin α-β dependent. G-Actin and importin α-β bind competitively to nuclear import siGnals associated with the N- and C-terminal RPEL motifs. All four motifs are required for the inhibition of serum-induced Phactr1 nuclear accumulation when G-Actin is elevated. G-Actin and PP1 bind competitively to the Phactr1 C-terminal reGion, and Phactr1 C-terminal RPEL mutants that cannot bind G-Actin induce aberrant actomyosin structures dependent on their nuclear accumulation and on PP1 bindinG. In CHL-1 melanoma cells, Phactr1 exhibits Actin-reGulated subcellular localisation and is required for stress fibre assembly, motility and invasiveness. These data support a role for Phactr1 in actomyosin assembly and suGGest that Phactr1 G-Actin sensinG allows its coordination with F-Actin availability.
Ewald Hannappel - One of the best experts on this subject based on the ideXlab platform.
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Influence of the N Terminus and the Actin-BindinG Motif of Thymosin β4 on Its Interaction with G-Actin
Annals of the New York Academy of Sciences, 2007Co-Authors: Robert E. Zoubek, Ewald HannappelAbstract:Thymosin beta(4) binds G-Actin in a 1:1 ratio and prevents its aGGreGation to F-Actin by sequestration. Substitution or modification of sinGle amino acid residues within the N-terminal sequence 1 to 22 of thymosin beta(4) alters its interaction with G-Actin. We Generated thymosin beta(4) variants with amino acid substitutions within the N-terminal alpha-helix and the putative Actin-bindinG motif. None of the E. coli-Generated thymosin beta(4) variants was modified or acetylated at its N terminus. The stability of the complex of G-Actin with nonacetylated thymosin beta(4) or beta(4)(A7V) is hiGher than the one with naturally occurrinG thymosin beta(4), which is always acetylated. The complex of G-Actin with nonacetylated thymosin beta(4)(A7V,K18,19A) and beta(4)(K14,16,18,19A) is 15 times less stable compared to the complex with thymosin beta(4). The G-Actin sequesterinG activities of all thymosin beta(4) variants correspond to their complex stabilities with G-Actin, except for nonacetylated thymosin beta(4)(A7V), where it is attenuated. Thymosin beta(4)(Delta17-23) missinG the putative Actin-bindinG motif shows no interaction with G-Actin.
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nuclear localisation of the G Actin sequesterinG peptide thymosin beta4
Journal of Cell Science, 2004Co-Authors: Thomas Huff, Ewald Hannappel, Olaf Rosorius, Angela M Otto, Christian S G Muller, Edda Ballweber, Hans Georg MannherzAbstract:Thymosin beta4 is reGarded as the main G-Actin sequesterinG peptide in the cytoplasm of mammalian cells. It is also thouGht to be involved in cellular events like canceroGenesis, apoptosis, anGioGenesis, blood coaGulation and wound healinG. Thymosin beta4 has been previously reported to localise intracellularly to the cytoplasm as detected by immunofluorescence. It can be selectively labelled at two of its Glutamine-residues with fluorescent OreGon Green cadaverine usinG transGlutaminase; however, this labellinG does not interfere with its interaction with G-Actin. Here we show that after microinjection into intact cells, fluorescently labelled thymosin beta4 has a diffuse cytoplasmic and a pronounced nuclear staininG. Enzymatic cleavaGe of fluorescently labelled thymosin beta4 with AsnC-endoproteinase yielded two mono-labelled fraGments of the peptide. After microinjection of these fraGments, only the larGer N-terminal fraGment, containinG the proposed Actin-bindinG sequence exhibited nuclear localisation, whereas the smaller C-terminal fraGment remained confined to the cytoplasm. We further showed that in diGitonin permeabilised and extracted cells, fluorescent thymosin beta4 was solely localised within the cytoplasm, whereas it was found concentrated within the cell nuclei after an additional Triton X100 extraction. Therefore, we conclude that thymosin beta4 is specifically translocated into the cell nucleus by an active transport mechanism, requirinG an unidentified soluble cytoplasmic factor. Our data furthermore suGGest that this peptide may also serve as a G-Actin sequesterinG peptide in the nucleus, althouGh additional nuclear functions cannot be excluded.
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The dipyridyls paraquat and diquat attenuate the interaction of G‐Actin with thymosin β4
FEBS letters, 1998Co-Authors: Thomas Huff, Graziella Cappelletti, Ewald HannappelAbstract:β-Thymosins sequester G-Actin and preserve a pool of monomers of Actin which constitute an important prerequisite for cellular function of the microfilament system. To study the influence of paraquat bindinG to G-Actin on the interaction of G-Actin with thymosin β4 we determined the apparent dissociation constant of the G-Actin-thymosin β4 complex in the absence or presence of paraquat usinG an ultrafiltration assay. Paraquat (1,1′-dimethyl-4,4′-dipyridylium dichloride) attenuates this interaction in a concentration- and time-dependent manner. When exposed to 10 mM paraquat, the apparent dissociation constant increased 10–85-fold within 15 min to 24 h. After incubation for 24 h even a paraquat concentration as low as 100 μM increased the dissociation constant of the G-Actin-thymosin β4 complex from 0.66 μM to 0.82 μM (P
