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Peter W Gunning - One of the best experts on this subject based on the ideXlab platform.

  • the molecular composition of neuronal microfilaments is spatially and temporally regulated
    The Journal of Neuroscience, 1996
    Co-Authors: Ron P Weinberger, Mark A Hill, Galina Schevzov, Peter L Jeffrey, K Gordon, Peter W Gunning
    Abstract:

    The actin-based microfilament system is thought to play a critical role in neuronal development. We have determined specific changes in the composition of microfilaments accompanying neuronal morphogenesis. By using specific antibodies against the isoforms for tropomyosin (Tm) (Tm- 5 and TmBr-1/-3) and actin (beta- and gamma-actin), we found that during early morphogenesis in vivo immature growing axons contain beta- and gamma-actin and Tm-5. In particular, Tm-5 is exclusively located in the immature axonal processes relative to the neuronal cell body. In contrast, Beta-Actin and Tm-5 are absent in mature, quiescent axons. This developmental loss from axons is associated with an approximately twofold downregulation of Beta-Actin and Tm-5 levels in the brain; gamma-actin levels do not change, and this molecule is widely distributed throughout neurons during development. The loss of beta- actin and Tm-5 from axons is accompanied by a progressive appearance of TmBr-1/-3. This apparent replacement of Tm-5 with TmBr-1/-3 occurs over a 2 d time period during rat embryonic hindbrain development and is conserved in evolution between birds and mammals. The loss of Tm-5 from axons involves a redistribution of this molecule to the cell soma and dendrites. These findings suggest that specialized microfilament domains are associated with the development and maintenance of neuronal polarity. We conclude that these Tm isoforms and Beta-Actin are subject to specific patterns of segregation associated with axonal development and neuronal differentiation. This provides a potential molecular basis for the temporal and spatial specificity of microfilament function during neuronal differentiation.

  • beta and gamma actin mrnas are differentially located within myoblasts
    Journal of Cell Biology, 1993
    Co-Authors: Mark A Hill, Peter W Gunning
    Abstract:

    Actin is of fundamental importance to all eukaryotic cells. Of the six mammalian actins, beta (beta) and gamma (gamma) cytoplasmic are the isoforms found in all nonmuscle cells and differ by only four amino acids at the amino-terminal region. Both genes are regulated temporally and spatially, though no differences in protein function have been described. Using fluorescent double in situ hybridization we describe the simultaneous intracellular localization of both beta and gamma actin mRNA. This study shows that myoblasts differentially segregate the beta and gamma actin mRNAs. The distribution of gamma actin mRNA, only to perinuclear and nearby cytoplasm, suggests a distribution based on diffusion or restriction to nearby cytoplasm. The distribution of beta actin mRNA, perinuclear and at the cell periphery, implicates a peripheral localizing signal which is unique to the beta isoform. The peripheral beta actin mRNA corresponded to cellular morphologies, extending processes, and ruffling edges that reflect cell movement. Total actin and gamma actin protein steady-state distributions were identified by specific antibodies. gamma actin protein was found in both stress fibers and at the cell plasma membrane and does not correspond to its mRNA distribution. We suggest that localized protein synthesis rather than steady-state distribution functionally differentiates between the actin isoforms.

  • High level expression of transfected beta- and gamma-actin genes differentially impacts on myoblast cytoarchitecture
    The Journal of cell biology, 1992
    Co-Authors: Gafina Schevzov, Catriona Lloyd, Peter W Gunning
    Abstract:

    The impact of the human beta- and gamma-actin genes on myoblast cytoarchitecture was examined by their stable transfection into mouse C2 myoblasts. Transfectant C2 clones expressing high levels of human Beta-Actin displayed increases in cell surface area. In contrast, C2 clones with high levels of human gamma-actin expression showed decreases in cell surface area. The changes in cell morphology were accompanied by changes in actin stress-fiber organization. The Beta-Actin transfectants displayed well-defined filamentous organization of actin; whereas the gamma-actin transfectants displayed a more diffuse organization of the actin cables. The role of the Beta-Actin protein in generating the enlarged cell phenotype was examined by transfecting a mutant form of the human Beta-Actin gene. Transfectant cells were shown to incorporate the aberrant actin protein into stress-fiber-like structures. High level expression of the mutant Beta-Actin produced decreases in cell surface area and disruption of the actin microfilament network similar to that seen with transfection of the gamma-actin gene. In contrast, transfection of another mutant form of the Beta-Actin gene which encodes an unstable protein had no impact on cell morphology or cytoarchitecture. These results strongly suggest that it is the nature of the encoded protein that determines the morphological response of the cell. We conclude that the relative gene expression of beta- and gamma-actin is of relevance to the control of myoblast cytoarchitecture. In particular, we conclude that the beta- and gamma-actin genes encode functionally distinct cytoarchitectural information.

Ewald Hannappel - One of the best experts on this subject based on the ideXlab platform.

  • the β thymosins intracellular and extracellular activities of a versatile actin binding protein family
    Cytoskeleton, 2009
    Co-Authors: Hans Georg Mannherz, Ewald Hannappel
    Abstract:

    The beta-thymosins are N-terminally acetylated peptides of about 5 kDa molecular mass and composed of about 40-44 amino acid residues. The first member of the family, thymosin beta4, was initially isolated from thymosin fraction 5, prepared in five steps from calf thymus. Thymosin beta4 was supposed to be specifically produced and released by the thymic gland and to possess hormonal activities modulating the immune response. Various paracrine effects have indeed been reported for these peptides such as cardiac protection, angiogenesis, stimulation of wound healing, and hair growth. Besides these paracrine effects, it was noted that beta-thymosins occur in high concentration in the cytoplasm of many eukaryotic cells and bind to the cytoskeletal component actin. Subsequently it became apparent from in vitro experiments that they preferentially bind to monomeric (G-)actin and stabilize it in its monomeric form. Due to this ability the beta-thymosins are the main intracellular actin sequestering factor, i.e., they posses the ability to remove monomeric actin from the dynamic assembly and disassembly processes of the actin cytoskeleton that constantly occur in activated cells. In this review we will concentrate on the intracellular activity and localization of the beta-thymosins, i.e., their modulating effect on the actin cytoskeleton.

  • polymerisation of chemically cross linked actin thymosin beta 4 complex to filamentous actin alteration in helical parameters and visualisation of thymosin beta 4 binding on f actin
    Journal of Molecular Biology, 2002
    Co-Authors: Edda Ballweber, Thomas Huff, Ewald Hannappel, Harald Stephan, Markus Haener, Nicole Taschner, Daniel Stoffler, Ueli Aebi
    Abstract:

    The beta-thymosins are intracellular monomeric (G-)actin sequestering proteins forming 1:1 complexes with G-actin. Here, we analysed the interaction of thymosin beta(4) with F-actin. Thymosin beta(4) at 200 microM was chemically cross-linked to F-actin. In the presence of phalloidin, the chemically cross-linked actin:thymosin beta(4) complex was incorporated into F-actin. These mixed filaments were of normal appearance when inspected by conventional transmission electron microscopy after negative staining. We purified the chemically cross-linked actin:thymosin beta(4) complex, which polymerised only when phalloidin and the gelsolin:2-actin complex were present simultaneously. Using scanning transmission electron microscopy, the mass-per-length of control and actin:thymosin beta(4) filaments was found to be 16.0(+/-0.8) kDa/nm and 18.0(+/-0.9) kDa/nm, respectively, indicating an increase in subunit mass of 5.4 kDa. Analysis of the helical parameters revealed an increase of the crossover spacing of the two right-handed long-pitch helical strands from 36.0 to 40.5 nm. Difference map analysis of 3-D helical reconstruction of control and actin:thymosin beta(4) filaments yielded an elongated extra mass. Qualitatively, the overall size and shape of the difference mass were compatible with published data of the atomic structure of thymosin beta(4). The deduced binding sites of thymosin beta(4) to actin were in agreement with those identified previously. However, parts of the difference map might represent subtle conformational changes of both proteins occurring upon complex formation.

  • beta-Thymosins, small acidic peptides with multiple functions
    The international journal of biochemistry & cell biology, 2001
    Co-Authors: Thomas Huff, Christian S G Muller, Roland Netzker, Angela M. Otto, Ewald Hannappel
    Abstract:

    The beta-thymosins are a family of highly conserved polar 5 kDa peptides originally thought to be thymic hormones. About 10 years ago, thymosin beta(4) as well as other members of this ubiquitous peptide family were identified as the main intracellular G-actin sequestering peptides, being present in high concentrations in almost every cell. beta-Thymosins bind monomeric actin in a 1:1 complex and act as actin buffers, preventing polymerization into actin filaments but supplying a pool of actin monomers when the cell needs filaments. Changes in the expression of beta-thymosins appear to be related to the differentiation of cells. Increased expression of beta-thymosins or even the synthesis of a beta-thymosin normally not expressed might promote metastasis possibly by increasing mobility of the cells. Thymosin beta(4) is detected outside of cells in blood plasma or in wound fluid. Several biological effects are attributed to thymosin beta(4), oxidized thymosin beta(4), or to the fragment, acSDKP, possibly generated from thymosin beta(4). Among the effects are induction of metallo-proteinases, chemotaxis, angiogenesis and inhibition of inflammation as well as the inhibition of bone marrow stem cell proliferation. However, nothing is known about the molecular mechanisms mediating the effects attributed to extracellular beta-thymosins.

  • interactions of beta thymosins thymosin beta 4 sulfoxide and n terminally truncated thymosin beta 4 with actin studied by equilibrium centrifugation chemical cross linking and viscometry
    FEBS Journal, 1995
    Co-Authors: Thomas Huff, Doris Zerzawy, Ewald Hannappel
    Abstract:

    All beta-thymosins studied interact with G-actin in a bimolecular complex and inhibit the polymerization to F-actin under high salt conditions. The interactions between actin and beta-thymosins have been studied under polymerization conditions using actin labeled by a fluorescent reporter group at Cys374. Instead of labeling actin we employed equilibrium centrifugation of unlabeled G-actin, viscometry, and chemical cross-linking to investigate the interactions with several beta-thymosins, oxidized thymosin beta 4 and N-terminally truncated beta 4. The apparent dissociation constants for actin from bovine heart and beta-thymosins were 2.5, 0.1, and 2.7 microM for thymosin beta 4, [Ala1]beta 4(beta Ala4), and beta 10, respectively. Comparable apparent dissociation constants were obtained for the interaction of G-actin from rabbit skeletal muscle and thymosin beta 4 or beta Ala4. In rabbits thymosin beta Ala4 replaces beta 4 being different in amino acid residue 1 only. The apparent dissociation constant of thymosin beta 10 with actin from rabbit skeletal muscle, however, is about 10% of the value obtained with actin from bovine heart. Oxidation of thymosin beta 4 at Met6 (beta 4-sulfoxide) as well as truncation of 6 [beta 4-(7-43)] or 12 [beta 4-(13-43)] amino acid residues from the N-terminus increase apparent dissociation constants to 38-53 microM. Truncation of the first 23 amino acid residues [beta 4-(24-43)] abolishes interaction with G-actin completely. Therefore, amino acid residues between position 13 and 24 are necessary for 1-ethyl-3[3-(dimethyl-aminopropyl)-carbodiimide cross-linking of G-actin. In spite of comparable apparent dissociation constants between actin and thymosin beta 4-sulfoxide or beta 4-(7-43) or beta 4-(13-43), only beta 4-sulfoxide and not the truncated beta-thymosins inhibits actin polymerization, however, only at a 20-fold higher concentration than beta 4. Thus the first six amino acid residues are indispensable to inhibit salt-induced actin polymerization as analyzed by viscometry. While the apparent dissociation constant of the actin/thymosin beta 4 complex generated from a preformed actin/DNase-I complex is 160 microM, a fivefold excess of DNase I over the preformed actin/thymosin-beta 4 complex is necessary to observe a comparable dissociation constant.

Daniel Safer - One of the best experts on this subject based on the ideXlab platform.

  • beta thymosins from marine invertebrates primary structure and interaction with actin
    Cytoskeleton, 1997
    Co-Authors: Daniel Safer, Prokash K Chowrashi
    Abstract:

    The beta-thymosins are distributed throughout the vertebrate phyla, and all known vertebrate beta-thymosins bind actin monomers. To determine whether beta-thymosin-like peptides function as actin-binding proteins in invertebrates, we fractionated perchloric acid extracts of the gonads of both the sea urchin, Arbacia punctulata, and the scallop, Argopecten irradians, and screened the fractions for proteins which could be crosslinked to actin. In each case a peptide was isolated which crosslinks to actin from both rabbit skeletal muscle and scallop cross-striated adductor muscle; both peptides were sequenced and each was found to consist of 40 amino acid residues, compared with 41-43 residues for the vertebrate beta-thymosins. The sequences of the scallop and sea urchin beta-thymosins are 80% identical to each other, 75% identical to residues 1-40 of thymosin beta4, and 72-80% identical to residues 1-40 of other vertebrate beta-thymosins. The sea urchin peptide was found to inhibit actin polymerization and nucleotide exchange. The affinity of the sea urchin peptide for rabbit muscle actin is apparently lower than that of thymosin beta4, since about twice the concentration of sea urchin peptide is required to give inhibition of actin polymerization or nucleotide exchange equivalent to thymosin beta4.

  • beta thymosins as actin binding peptides
    BioEssays, 1994
    Co-Authors: Daniel Safer, Vivianne T Nachmias
    Abstract:

    The beta thymosins are a highly conserved family of strongly polar 5 kDa polypeptides that are widely distributed among vertebrate classes; most are now known to bind to monomeric actin and inhibit its polymerization. One beta thymosin, beta four, (T beta 4) is the predominant form in mammalian cells, present at up to 0.5 mM. Many species are known to produce at least two beta thymosin isoforms, in some cases in the same cell. Their expression can be separately regulated. When present outside the cell, the N-terminal tetrapeptide of beta four appears to affect cell cycle regulation; beta thymosins or smaller fragments derived from them may have additional regulatory functions. We suggest that many developmental changes in beta thymosin levels within cells and tissues may be related to changes in G-actin pool size.

  • Thymosin beta 4 sequesters the majority of G-actin in resting human polymorphonuclear leukocytes.
    The Journal of cell biology, 1992
    Co-Authors: Lynne Cassimeris, Daniel Safer, Vivianne T Nachmias, Sally H. Zigmond
    Abstract:

    Thymosin beta 4 (T beta 4), a 5-kD peptide which binds G-actin and inhibits its polymerization (Safer, D., M. Elzinga, and V. T. Nachmias. 1991. J. Biol. Chem. 266:4029-4032), appears to be the major G-actin sequestering protein in human PMNs. In support of a previous study by Hannappel, E., and M. Van Kampen (1987. J. Chromatography. 397:279-285), we find that T beta 4 is an abundant peptide in these cells. By reverse phase HPLC of perchloric acid supernatants, human PMNs contain approximately 169 fg/cell +/- 90 fg/cell (SD), corresponding to a cytoplasmic concentration of approximately 149 +/- 80.5 microM. On non-denaturing polyacrylamide gels, a large fraction of G-actin in supernatants prepared from resting PMNs has a mobility similar to the G-actin/T beta 4 complex. Chemoattractant stimulation of PMNs results in a decrease in this G-actin/T beta 4 complex. To determine whether chemoattractant induced actin polymerization results from an inactivation of T beta 4, the G-actin sequestering activity of supernatants prepared from resting and chemoattractant stimulated cells was measured by comparing the rates of pyrenyl-actin polymerization from filament pointed ends. Pyrenyl actin polymerization was inhibited to a greater extent in supernatants from stimulated cells and these results are qualitatively consistent with T beta 4 being released as G-actin polymerizes, with no chemoattractant-induced change in its affinity for G-actin. The kinetics of bovine spleen T beta 4 binding to muscle pyrenyl G-actin are sufficiently rapid to accommodate the rapid changes in actin polymerization and depolymerization observed in vivo in response to chemoattractant addition and removal.

  • the control of actin nucleotide exchange by thymosin beta 4 and profilin a potential regulatory mechanism for actin polymerization in cells
    Molecular Biology of the Cell, 1992
    Co-Authors: Pascal J Goldschmidtclermont, Daniel Safer, V T Nachmias, M I Furman, D Wachsstock, Thomas D Pollard
    Abstract:

    We present evidence for a new mechanism by which two major actin monomer binding proteins, thymosin beta 4 and profilin, may control the rate and the extent of actin polymerization in cells. Both proteins bind actin monomers transiently with a stoichiometry of 1:1. When bound to actin, thymosin beta 4 strongly inhibits the exchange of the nucleotide bound to actin by blocking its dissociation, while profilin catalytically promotes nucleotide exchange. Because both proteins exchange rapidly between actin molecules, low concentrations of profilin can overcome the inhibitory effects of high concentrations of thymosin beta 4 on the nucleotide exchange. These reactions may allow variations in profilin concentration (which may be regulated by membrane polyphosphoinositide metabolism) to control the ratio of ATP-actin to ADP-actin. Because ATP-actin subunits polymerize more readily than ADP-actin subunits, this ratio may play a key regulatory role in the assembly of cellular actin structures, particularly under circumstances of rapid filament turnover.

  • interaction of thymosin beta 4 with muscle and platelet actin implications for actin sequestration in resting platelets
    Biochemistry, 1992
    Co-Authors: Annemarie Weber, V T Nachmias, C R Pennise, M Pring, Daniel Safer
    Abstract:

    Quantitative measurements of the interactions of T beta 4 with muscle actin suggest that its only physiological role is monomer sequestration. T beta 4 forms a 1:1 complex with monomeric actin under physiological salt conditions. Its Kd for actin is not affected by calcium. T beta 4 binds only to actin monomers and not to filament ends or alongside the filament. T beta 4-actin complexes do not elongate actin filaments at either the barbed or the pointed end, and, unlike actobindin, T beta 4 does not specifically suppress the nucleation of polymerization. We assessed the fraction of monomeric actin that can be sequestered by T beta 4 in resting platelets. This was done on the basis of (a) its Kd of 0.4-0.7 microM for platelet actin, which had been prepared by a newly devised simpler method, and (b) the values for the concentrations of monomeric actin and of T beta 4 which we measured as 280 and 560 microM, respectively. Using the higher Kd value of 0.7 microM, the T beta 4-complexed actin is calculated to be between 70 and 240 microM, depending on the steady-state free G-actin concentration. This may vary from 0.1 to 0.5 microM, the critical concentrations for uncapped and for fully barbed-end-capped actin filaments. If the Kd in the platelet is the same as in vitro, most of the sequestered actin would be bound to T beta 4 if more than 95% of the actin filaments are capped at their barbed ends in resting platelets.

Marie-france Carlier - One of the best experts on this subject based on the ideXlab platform.

  • multifunctionality of the β thymosin wh2 module g actin sequestration actin filament growth nucleation and severing
    Annals of the New York Academy of Sciences, 2010
    Co-Authors: Clotilde Husson, Dominique Didry, Carine Van Heijenoort, Eric Guittet, François-xavier Cantrelle, Pierre Roblin, Javier Perez, Louis Renault, Marie-france Carlier
    Abstract:

    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.

  • structure function and evolution of the beta thymosin wh2 wasp homology2 actin binding module
    Annals of the New York Academy of Sciences, 2007
    Co-Authors: Marie-france Carlier, Maud Hertzog, Dominique Didry, Carine Van Heijenoort, Marcel Knossow, François-xavier Cantrelle, Louis Renault, Eric Guittet
    Abstract:

    beta-thymosins are acknowledged G-actin sequesterers. However, in the recent years, the conserved beta-thymosins/WH2 actin-binding module, has been identified in a large number of proteins that all interact with actin and play diverse functions in cell motility. The functional evolution of the WH2 domain has been approached by a combination of structural and biochemical methods, using thymosin beta4 (Tbeta4) and Ciboulot, a 3 beta-thymosin repeat protein from Drosophila as models. Ciboulot binds actin like Tbeta4 but promotes actin assembly like profilin. The first repeat of Ciboulot (D1) has the profilin function of the whole protein. The crystal structure of Ciboulot-actin shows that the major interaction with G-actin lies in the N-terminal amphipathic helix of D1. By point mutagenesis the sequestering activity of Tbeta4 can be changed into a profilin activity. ((1)H, (15)N)-NMR studies show that the functional switch from inhibition to promotion of actin assembly is linked to a change in the dynamics of interaction of the central and C-terminal regions of the WH2 domain with subdomains 1 and 2 of G-actin. Further systematic mutagenesis studies have been performed by engineering a series of chimeras of Ciboulot and Tbeta4. Proteins displaying either profilin function or enhanced sequestering activity compared to Tbeta4 have been characterized. The results provide insight into the structural basis for the regulation of the multiple functions of the WH2 domain.

  • control of actin dynamics by proteins made of β thymosin repeats the actobindin family
    Journal of Biological Chemistry, 2002
    Co-Authors: Maud Hertzog, Elena G Yarmola, Dominique Didry, Michael R Bubb, Marie-france Carlier
    Abstract:

    Actobindin is an actin-binding protein from amoeba, which consists of two beta-thymosin repeats and has been shown to inhibit actin polymerization by sequestering G-actin and by stabilizing actin dimers. Here we show that actobindin has the same biochemical properties as the Drosophila or Caenorhabditis elegans homologous protein that consists of three beta-thymosin repeats. These proteins define a new family of actin-binding proteins. They bind G-actin in a 1:1 complex with thermodynamic and kinetic parameters similar to beta-thymosins. Like beta-thymosins, they slow down nucleotide exchange on G-actin and make a ternary complex with G-actin and Latrunculin A. On the other hand, they behave as functional homologs of profilin because their complex with MgATP-G-actin, unlike beta-thymosin-actin, participates in filament barbed end growth, like profilin-actin complex. Therefore these proteins play an active role in actin-based motility processes. In addition, proteins of the actobindin family interact with the pointed end of actin filaments and inhibit pointed end growth, maybe via the interaction of the beta-thymosin repeats with two terminal subunits.

  • modulation of the interaction between g actin and thymosin beta 4 by the atp adp ratio possible implication in the regulation of actin dynamics
    Proceedings of the National Academy of Sciences of the United States of America, 1993
    Co-Authors: Marie-france Carlier, Catherine Jean, K J Rieger, Maryse Lenfant, Dominique Pantaloni
    Abstract:

    Abstract The interaction of G-actin with thymosin beta 4 (T beta 4), the major G-actin-sequestering protein in motile and proliferating cells, has been analyzed in vitro. T beta 4 is found to have a 50-fold higher affinity for MgATP-actin than for MgADP-actin. These results imply that in resting platelets and neutrophils, actin is sequestered by T beta 4 as MgATP-G-actin. Kinetic experiments and theoretical calculations demonstrate that this ATP/ADP dependence of T beta 4 affinity for G-actin can generate a mechanism of desequestration of G-actin by ADP, in the presence of physiological concentrations of T beta 4 (approximately 0.1 mM). The desequestration of G-actin by ADP is kinetically enhanced by profilin, which accelerates the dissociation of ATP from G-actin. Whether a local drop in the ATP/ADP ratio can allow local, transient desequestration and polymerization of actin either close to the plasma membrane, following platelet or neutrophil stimulation, or behind the Listeria bacterium in the host cell, while the surrounding cytoplasm contains sequestered ATP-G-actin, is an open issue raised by the present work.

Eric Guittet - One of the best experts on this subject based on the ideXlab platform.

  • multifunctionality of the β thymosin wh2 module g actin sequestration actin filament growth nucleation and severing
    Annals of the New York Academy of Sciences, 2010
    Co-Authors: Clotilde Husson, Dominique Didry, Carine Van Heijenoort, Eric Guittet, François-xavier Cantrelle, Pierre Roblin, Javier Perez, Louis Renault, Marie-france Carlier
    Abstract:

    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.

  • structure function and evolution of the beta thymosin wh2 wasp homology2 actin binding module
    Annals of the New York Academy of Sciences, 2007
    Co-Authors: Marie-france Carlier, Maud Hertzog, Dominique Didry, Carine Van Heijenoort, Marcel Knossow, François-xavier Cantrelle, Louis Renault, Eric Guittet
    Abstract:

    beta-thymosins are acknowledged G-actin sequesterers. However, in the recent years, the conserved beta-thymosins/WH2 actin-binding module, has been identified in a large number of proteins that all interact with actin and play diverse functions in cell motility. The functional evolution of the WH2 domain has been approached by a combination of structural and biochemical methods, using thymosin beta4 (Tbeta4) and Ciboulot, a 3 beta-thymosin repeat protein from Drosophila as models. Ciboulot binds actin like Tbeta4 but promotes actin assembly like profilin. The first repeat of Ciboulot (D1) has the profilin function of the whole protein. The crystal structure of Ciboulot-actin shows that the major interaction with G-actin lies in the N-terminal amphipathic helix of D1. By point mutagenesis the sequestering activity of Tbeta4 can be changed into a profilin activity. ((1)H, (15)N)-NMR studies show that the functional switch from inhibition to promotion of actin assembly is linked to a change in the dynamics of interaction of the central and C-terminal regions of the WH2 domain with subdomains 1 and 2 of G-actin. Further systematic mutagenesis studies have been performed by engineering a series of chimeras of Ciboulot and Tbeta4. Proteins displaying either profilin function or enhanced sequestering activity compared to Tbeta4 have been characterized. The results provide insight into the structural basis for the regulation of the multiple functions of the WH2 domain.

  • the β thymosin wh2 domain structural basis for the switch from inhibition to promotion of actin assembly
    Cell, 2004
    Co-Authors: Maud Hertzog, Dominique Didry, Carine Van Heijenoort, Martin Gaudier, Jerome Coutant, Benoit Gigant, Gerard Didelot, Thomas Preat, Marcel Knossow, Eric Guittet
    Abstract:

    The widespread beta-thymosin/WH2 actin binding domain has versatile regulatory properties in actin dynamics and motility. beta-thymosins (isolated WH2 domain) maintain monomeric actin in a "sequestered" nonpolymerizable form. In contrast, when repeated in tandem or inserted in modular proteins, the beta-thymosin/WH2 domain promotes actin assembly at filament barbed ends, like profilin. The structural basis for these opposite functions is addressed using ciboulot, a three beta-thymosin repeat protein. Only the first repeat binds actin and possesses the function of ciboulot. The region that shows the strongest interaction with actin is an amphipathic N-terminal alpha helix, present in all beta-thymosin/WH2 domains, which recognizes the ATP bound actin structure and uses the shear motion of actin linked to ATP hydrolysis to control polymerization. Crystallographic ((1)H, (15)N), NMR, and mutagenetic data reveal that the weaker interaction of the C-terminal region of beta-thymosin/WH2 domain with actin accounts for the switch in function from inhibition to promotion of actin assembly.