Tβ4 Is Not a Simple G-actin Sequestering Protein and Interacts with F-actin at High Concentration

Carlier, Marie‐France; Didry, Dominique; Erk, Inge; Lepault, Jean; Troys, Marleen Van; Vandekerckhove, Joël; Perelroizen, Irina; Yin, Helen L.; Doi, Yukio; Pantaloni, Dominique

doi:10.1074/jbc.271.16.9231

Cited by 79 publications

(64 citation statements)

References 32 publications

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“…We confirm that the fraction of thymosin ␤ 4 bound to F-actin is insignificant at concentrations up to 30 M (Fig. 1C), in agreement with prior data that implied that if such binding occurred, the K d was several millimolar (10). This observation simplifies the analysis of the anisotropy data, because the anisotropy of labeled thymosin ␤ 4 will therefore only reflect binding to monomeric actin.…”

Section: Validation Of the Anisotropy Assay For Measurement Of Thesupporting

confidence: 91%

“…Other methods such as the DNase I binding assay yield the sum of A c and an indeterminate fraction of sequestered actin monomer (8). Clever uses of combinations of data have in some cases allowed investigators to subtract F-actin content from total actin and then fractionate the contributions of A c and monomer sequestration (9,10), but the results have proven controversial (11,12).…”

mentioning

confidence: 99%

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Effects of Profilin and Thymosin β4 on the Critical Concentration of Actin Demonstrated in Vitro and in Cell Extracts with a Novel Direct Assay

Yarmola

Bubb

2004

Journal of Biological Chemistry

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The free actin concentration at steady state, A c , is a variable that determines how actin regulatory proteins influence the extent of actin polymerization. We describe a novel method employing fluorescence anisotropy to directly measure A c in any sample after the addition of a trace amount of labeled thymosin ␤ 4 or thymosin ␤ 4 peptide. Using this assay, we confirm earlier theoretical work on the helical polymerization of actin and confirm the effects of actin filament-stabilizing drugs and capping proteins on A c , thereby validating the assay. We also confirm a controversial prior observation that profilin lowers the critical concentration of Mg 2؉ -actin. A general mechanism is proposed to explain this effect, and the first quantitative dose-response curve for the effect of profilin on A c facilitates its evaluation. This mechanism also predicts the effect of profilin on critical concentration in the presence of the limited amount of capping protein, which is the condition often found in cells, and the effect of profilin on critical concentration in cell extracts is demonstrated for the first time. Additionally, nonlinear effects of thymosin ␤ 4 on the steady state amount of F-actin are explained by the observed changes in A c . This assay has potential in vivo applications that complement those demonstrated in vitro.Total cellular actin is equal to the sum of the concentration of free monomer (the critical concentration, A c ), the concentration of unpolymerized actin sequestered by various actin-binding proteins, and the concentration of actin polymer or F-actin. Changes in A c induced by actin regulatory proteins influence actin polymer dynamics by an amplification mechanism that results in large changes in the amount of sequestered, unpolymerized actin. Quantitative evaluation of models of cytoskeletal function requires accurate knowledge of the value of A c . Unfortunately, A c has not yet been measured in cells and has only been estimated by very indirect methods in cell extracts (1). In vitro methods have been developed that allow for the measurement of F-actin using birefringence (2), viscosity (3), light scattering (4), centrifugation (5), binding of labeled phalloidin (6), or the fluorescence of pyrenyl-labeled actin (7). Other methods such as the DNase I binding assay yield the sum of A c and an indeterminate fraction of sequestered actin monomer (8). Clever uses of combinations of data have in some cases allowed investigators to subtract F-actin content from total actin and then fractionate the contributions of A c and monomer sequestration (9, 10), but the results have proven controversial (11,12).Based on theoretical considerations, the critical concentration is expected to be equal to the ratio of the rate constants of association and dissociation of actin subunits from actin filaments. Measurement of these rate constants using a method such as electron microscopy can be used to indirectly evaluate A c , assuming the applicability of the theory (13,14). However, the participation of actin b...

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Section: Validation Of the Anisotropy Assay For Measurement Of Thesupporting

confidence: 91%

mentioning

confidence: 99%

Effects of Profilin and Thymosin β4 on the Critical Concentration of Actin Demonstrated in Vitro and in Cell Extracts with a Novel Direct Assay

Yarmola

Bubb

2004

Journal of Biological Chemistry

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“…The data obtained from culture cells over-expressing Tb4 demonstrated its binding to F-actin and the promotion of stress fiber formation [Carlier et al, 1996;Sun et al, 1996]. These results suggested that Tb4 sequesters G-actin only at low concentration but binds to F-actin at high concentrations.…”

Section: Structure Of the Actin:thymosin B4 Complexmentioning

confidence: 88%

The β‐thymosins: Intracellular and extracellular activities of a versatile actin binding protein family

Mannherz

Hannappel

2009

Cell Motil. Cytoskeleton

109

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The b-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 b4, was initially isolated from thymosin fraction 5, prepared in five steps from calf thymus. Thymosin b4 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 b-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 b-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 b-thymosins, i.e., their modulating effect on the actin cytoskeleton. Cell Motil. Cytoskeleton 66: 839-851, 2009. '

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“…The structural basis of these functional differences between the highly similar repeats is not obvious from their primary structure. Thymosin␤4 can also be cross-linked to F-actin, albeit only at very high concentrations (Carlier et al, 1996). Hence, F-actin-binding by this module is not unprecedented (see also Ballweber et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

TetraThymosinβ Is Required for Actin Dynamics inCaenorhabditis elegansand Acts via Functionally Different Actin-binding Repeats

Troys

Ono

Dewitte

et al. 2004

MBoC

Self Cite

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Generating specific actin structures via controlled actin polymerization is a prerequisite for eukaryote development and reproduction. We here report on an essential Caenorhabditis elegans protein tetraThymosin␤ expressed in developing neurons and crucial during oocyte maturation in adults. TetraThymosin␤ has four repeats, each related to the actin monomer-sequestering protein thymosin␤4 and assists in actin filament elongation. For homologues with similar multirepeat structures, a profilin-like mechanism of ushering actin onto filament barbed ends, based on the formation of a 1:1 complex, is proposed to underlie this activity. We, however, demonstrate that tetraThymosin␤ binds multiple actin monomers via different repeats and in addition also interacts with filamentous actin. All repeats need to be functional for attaining full activity in various in vitro assays. The activities on actin are thus a direct consequence of the repeated structure. In containing both G-and F-actin interaction sites, tetraThymosin␤ may be reminiscent of nonhomologous multimodular actin regulatory proteins implicated in actin filament dynamics. A mutation that suppresses expression of tetraThymosin␤ is homozygous lethal. Mutant organisms develop into adults but display a dumpy phenotype and fail to reproduce as their oocytes lack essential actin structures. This strongly suggests that the activity of tetraThymosin␤ is of crucial importance at specific developmental stages requiring actin polymerization.

show abstract

Tβ4 Is Not a Simple G-actin Sequestering Protein and Interacts with F-actin at High Concentration

Cited by 79 publications

References 32 publications

Effects of Profilin and Thymosin β4 on the Critical Concentration of Actin Demonstrated in Vitro and in Cell Extracts with a Novel Direct Assay

Effects of Profilin and Thymosin β4 on the Critical Concentration of Actin Demonstrated in Vitro and in Cell Extracts with a Novel Direct Assay

The β‐thymosins: Intracellular and extracellular activities of a versatile actin binding protein family

TetraThymosinβ Is Required for Actin Dynamics inCaenorhabditis elegansand Acts via Functionally Different Actin-binding Repeats

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