©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
T Is Not a Simple G-actin Sequestering Protein and Interacts with F-actin at High Concentration (*)

(Received for publication, October 18, 1995; and in revised form, January 31, 1996)

Marie-France Carlier (1)(§) Dominique Didry (1) Inge Erk (2) Jean Lepault (2) Marleen L. Van Troys (3) Joël Vandekerckhove (3) Irina Perelroizen (1) Helen Yin (4) Yukio Doi (5) Dominique Pantaloni (1)

From the  (1)Laboratoire d'Enzymologie and (2)Centre de Génétique Moléculaire, CNRS, 91198 Gif-sur-Yvette, France, the (3)Laboratory of Physiological Chemistry, University of Gent, Belgium, the (4)University of Texas Southwestern Medical Center, Dallas, Texas 75235, and the (5)Kyoto Women's University, Kyoto 605, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Thymosin beta(4) is acknowledged as a major G-actin binding protein maintaining a pool of unassembled actin in motile vertebrate cells. We have examined the function of Tbeta(4) in actin assembly in the high range of concentrations (up to 300 µM) at which Tbeta(4) is found in highly motile blood cells. Tbeta(4) behaves as a simple G-actin sequestering protein only in a range of low concentrations (<20 µM). As the concentration of Tbeta(4) increases, its ability to depolymerize F-actin decreases, due to its interaction with F-actin. The Tbeta(4)-actin can be incorporated, in low molar ratios, into F-actin, and can be cross-linked in F-actin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. As a result of the copolymerization of actin and Tbeta(4)-actin complex, the critical concentration is the sum of free G-actin and Tbeta(4)-G-actin concentrations at steady state, and the partial critical concentration of G-actin is decreased by Tbeta(4)-G-actin complex. The incorporation of Tbeta(4)-actin in F-actin is associated to a structural change of the filaments and eventually leads to their twisting around each other. In conclusion, Tbeta(4) is not a simple passive actin-sequestering agent, and at high concentrations the ability of Tbeta(4)-actin to copolymerize with actin reduces the sequestering activity of G-actin-binding proteins. These results question the evaluation of the unassembled actin in motile cells. They account for observations made on living fibroblasts overexpressing beta-thymosins.


INTRODUCTION

It is generally thought that to elicit a motile response to extracellular signals, living cells regulate their content in F-actin, by spatially controlled changes in the steady state of filament assembly(1) . Capping proteins and G-actin binding proteins are major players in this control. In the presence of ATP, capping proteins block the dynamics at the barbed ends of actin filaments and establish the high critical concentration of the pointed ends; when barbed ends are uncapped, the effective critical concentration is close to the critical concentration of the barbed end. The changes in critical concentration, however, represent a very small amount of actin in mass (less than 1 µM), hence by themselves they cannot elicit any massive assembly of actin. These changes, however, are largely amplified by G-actin binding proteins which maintain a pool of unassembled (sequestered) actin, used for site-directed actin assembly. The concentration of actin in complex with sequestering proteins is indeed determined by the concentration of free G-actin, i.e. the critical concentration. The concentration of actin in complex with sequestering proteins is high when barbed ends are capped, and decreases locally to yield F-actin upon creation and maintenance of available barbed ends, which is thought to occur upon stimulation. A major G-actin binding protein is thymosin beta(4) (Tbeta(4)) discovered in 1991 (2) in platelets and later found to be ubiquitous in vertebrate cells (see for review, (3) and (4) ). The function of Tbeta(4) as a simple passive sequestering protein was demonstrated in vitro(5, 6, 7) as well as in vivo(8, 9, 10, 11) . The equilibrium dissociation constant for the G-actin-Tbeta(4) 1:1 complex lies in the 0.7-2.5 µM range, from measurements of its sequestering activity (5, 6, 7) as well as from direct binding studies(12, 13) . However, in the above experiments, the concentrations of Tbeta(4) used were lower than the physiological concentrations, especially those found in motile blood cells (200-500 µM in platelets and neutrophils(5, 8) ). In the present work, the function of Tbeta(4) is explored in greater detail in the higher concentration range found in motile living cells. The role of Tbeta(4) appears more complex than previously thought, because actin filaments fail to totally depolymerize in the presence of high concentrations (100-200 µM) of Tbeta(4), due to incorporation of very low amounts of Tbeta(4)-actin in the filaments. The consequences of this property of Tbeta(4) on the structure of filaments and on the regulation of actin assembly in living cells is examined.


MATERIALS AND METHODS

Proteins

Actin was purified from rabbit skeletal muscle (14) and isolated as calcium ATP-G-actin by Sephadex G-200 chromatography (15) in G buffer (5 mM Tris-Cl, pH 7.8, 0.1 mM CaCl(2), 0.2 mM ATP, 0.2 mM dithiothreitol, 0.01% NaN(3)). Actin was pyrenyl-labeled as described(16) . Thymosin beta(4) was purified from bovine spleen as follows. All operations were done at 4 °C. 400 g of frozen spleen (cut into 2 times 2 times 2-cm cubes and frozen on dry ice at the slaughterhouse, then stored at -80 °C) were homogenized with 4 volumes of cold 0.625 N HCl0(4) in a Waring blender for 2 min, then centrifuged at 20,000 times g for 15 min. The supernatant was brought to pH 4 by dropwise addition of 5 N KOH, and filtered to remove KClO(4). The solution was loaded onto a 6 times 12-cm column of Lichroprep RP18 (40-63 µm, Merck). Following a 2000-ml H(2)O wash, elution was performed with 400 ml of 33% 1-propanol in water. The eluted material was concentrated to 50 ml by rotary evaporation, brought to pH 7.8 with KOH, filtered over 0.22-µm nitrocellulose filters, and submitted to anion exchange chromatography on Q-Sepharose (2.5 times 25 cm) equilibrated in 40 mM ammonium acetate, pH 7.8. Elution was performed using a gradient from 40 mM ammonium acetate, pH 7.8, to 0.2 M ammonium acetate, 0.2 M acetic acid, pH 5.0 (400 ml times 2). Elution was monitored by absorbance at 214 nm following 10-fold dilution of an aliquot of the fractions in H(2)O. The peak of Tbeta(4) was identified by analytical HPLC (^1)on RP18 Select B (Merck, 4 times 125 mm) using a gradient from 5 to 50% acetonitrile. Fractions containing Tbeta(4) were pooled, concentrated, and final purification was achieved by preparative HPLC on RP18 Select B as described(7) . About 40 mg of pure Tbeta(4) were obtained. The purity of Tbeta(4) was checked by mass spectrometry. The concentration of Tbeta(4) was determined by the bicinchoninic acid assay, using bovine serum albumin as a standard.

[^14C]Thymosin beta(4) was chemically synthesized on a model 431A peptide synthesizer (Applied Biosystems Inc., Foster City, CA) with an additional Gly-Cys at the extreme C terminus. Previous studies have indicated that the nature of the C terminus of Tbeta(4) is not important for actin binding(17) . The C-terminal cysteine residue was then labeled with iodo-[1-^14C]acetamide (Amersham) using a 1:1.5 molar ratio of thymosin beta(4) to label. The resulting [^14C]thymosin beta(4) had a specific activity of 20,000 cpm/nmol.

Tbeta(4) was oxidized into Met^6-sulfoxide-Tbeta(4) by incubation for 1 h at room temperature in the presence of 2 M H(2)O(2) (6% v/v), immediately followed by lyophilization. Profilin was purified from bovine spleen as described (18) . Gelsolin was purified from pig plasma as described(19) . Recombinant CapG was expressed and purified as described(20) .

Steady State Measurements of F-actin

The amount of actin assembled at steady state in the presence of different amounts of Tbeta(4) or profilin was monitored by pyrene fluorescence. Actin (1% pyrenyl labeled) was polymerized at the indicated concentration in G buffer supplemented with 2 mM MgCl(2) and 0.1 M KCl. Samples of 300 µl containing Tbeta(4), profilin, gelsolin, or CapG at the indicated concentrations were incubated for 16 h at room temperature in the dark. Pyrenyl fluorescence was measured in a Spex Fluorolog 2 instrument. Excitation and emission wavelengths were 366 and 387 nm, respectively.

Barbed ends were capped by either gelsolin or CapG. Gelsolin was added at a gelsolin:actin ratio of 1:200 to 1:500. When CapG, which is a weaker capping protein, was used, it was added at a constant concentration of 120 nM in all samples, independently of actin concentration. Preliminary assays were run with each batch of CapG used in this work, to verify that the critical concentration of the pointed end was established as soon as at least 80 nM CapG was present in solution. The amount of F-actin and unassembled actin present at steady state was converted into molar concentrations by comparison of the fluorescence readings with a critical concentration curve carried out in parallel using the same actin solution, in the same concentration range as in the samples.

Initial Rate of Filament Growth or Depolymerization

The rate of filament growth off preformed F-actin seeds (either capped or uncapped) was measured as described(21) . A small aliquot (<5% of total volume) of a solution of pyrenyl-labeled F-actin (10-20 µM), preassembled at steady state at least 2 h prior to the assay, was added to a solution containing pyrenyl-labeled G-actin and Tbeta(4). The G-actin and the F-actin (seeds) solutions were identically pyrenyl-labeled. The increase or decrease in fluorescence indicating elongation or depolymerization of the seeds was measured. The rates of fluorescence increase were converted into micromolar actin assembled times s using a calibration critical concentration curve obtained with the same F-actin solutions, as described above. The initial rates of filament growth J were plotted versus the concentration of free G-actin, C. The concentration, C, of free G-actin in the presence of Tbeta(4) was calculated as a function of the total concentrations of G-actin (C(o)), and of Tbeta(4) (T(o)), and of the equilibrium dissociation complex K(T) for the Tbeta(4)-actin complex, as follows:

Measurements of the Equilibrium Dissociation Constant K(T) for the Tbeta(4)-Actin Complex

The two following methods were used to determine the value of K(T), at a low concentration of actin (leq3 µM). In the first method, samples of F-actin (3 µM) were assembled at steady state in the presence of 10 nM gelsolin or 120 nM CapG and increasing amounts of Tbeta(4). The concentration of Tbeta(4)-actin (TA) complex at steady state increased linearly with the total concentration of Tbeta(4) (T(o)) according to the following equation:

where A(c) represents the critical concentration at the pointed end. The value of K(T) was derived from the slope - A(c)/(A(c) + K(T)) of the linear decrease in the fluorescence of pyrenyl-F-actin versus T(o).

In the second method, the rate of filament growth at a given concentration C(o) of G-actin was measured in the presence of different concentrations of Tbeta(4). Since only free G-actin can appreciably participate in assembly, filament growth was inhibited due to the formation of the Tbeta(4)-actin complex(6) . Gelsolin-capped filaments were used as seeds, and the value of C(o) was chosen low enough (C(o) leq 3 µM) for the free G-actin concentration dependence of the rate of growth at the pointed ends of actin filaments to vary linearly in the range (O to C(o))(22) . Under these conditions, the fraction of Tbeta(4)-bound actin, alpha, was directly proportional to the percent of inhibition of filament growth:

where V(0), V(T(o)), and V() were the elongation rates measured in the absence or in the presence of a concentration T(o) of Tbeta(4), or at infinite concentration of Tbeta(4), respectively. (Note that V() theoretically equals the rate of depolymerization of filaments upon dilution, which was experimentally verified.) Data were analyzed within the following equation which describes the hyperbolic binding of Tbeta(4) to G-actin:

The value of K(T) was derived from the slope of 1/(1-alpha) versus [T(o)]/alpha.

Chemical Cross-linking of Tbeta4 to G-actin and F-actin at Steady State

A solution of G-actin (15 µM) was dialyzed overnight (to remove Tris and to polymerize actin) against 5 mM phosphate, pH 7.5, containing 0.2 mM CaCl(2), 2 mM MgCl(2), 0.1 M KCl, 0.2 mM dithiothreitol, 0.2 mM ATP, using Spectrapor membranes. The dialyzed F-actin sample was incubated with 200 µM^14C-Tbeta(4) (20,000 cpm/nmol) for 16 h, then supplemented with 4 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Pierce) and 4 mM sulfo-NHS at room temperature for 1 h. The reaction was stopped by addition of 20 mM Tris. The cross-linked sample was centrifuged at 400,000 times g for 30 min in the ultracentrifuge (Beckman, TL 100). The supernatant, containing covalent and non-covalent G-actin-Tbeta(4) complexes, and the pellet, containing F-actin and cross-linked F-actin-Tbeta(4) complexes, were processed separately to identify the cross-linked Tbeta(4)-actin polypeptides by SDS-polyacrylamide gel electrophoresis (23) and autoradiography. The cross-linked peptides were further characterized by cyanogen bromide cleavage for 24 h at room temperature in 70% formic acid using a 100 times molar excess of CNBr over methionine. The peptide profiles were analyzed on Tricine-SDS-polyacrylamide gels(24) .

Incorporation of Tbeta(4) into F-actin

The binding of Tbeta(4) to F-actin was measured using a sedimentation assay. Samples of F-actin (20 µM, 0.25 ml), containing different amounts of Tbeta(4) in the range 0-250 µM, were sedimented at 400,000 times g for 30 min, following 16 h incubation at room temperature. Pellets of F-actin were resuspended in a 2.5-fold smaller volume of G-buffer and assayed for Tbeta(4). Two methods were used to quantitate the amount of F-actin-bound Tbeta(4). In the first method, ^14C-Tbeta(4) was used and the amount of bound Tbeta(4) was derived from radioactivity measurements. To estimate the amount of Tbeta(4) present in the interstitial volume of the pellets and not specifically bound to F-actin, control assays were run in parallel containing different amounts of F-actin in the range 5-20 µM, and 5 µM^14C-Tbeta(4)-ox prepared by H(2)O(2) oxidation of Met^6 in the ^14C-Tbeta(4) material used in the experiments. Since Tbeta(4)-ox is known not to bind significantly to actin at this concentration(12) , it is present in the pellet as a marker of the interstitial volume. The percent of total oxidized Tbeta(4) present as a contaminant in each pellet was measured at each concentration of F-actin, providing an estimation of the contamination per unit volume of the pellet. The amounts of F-actin and unassembled actin in the samples run at different concentrations of Tbeta(4) are known (Fig. 2A). Therefore the amount of ^14C-Tbeta(4) actually bound to F-actin could be obtained after subtraction of the appropriate percent of the total ^14C-Tbeta(4) as a contaminant.


Figure 2: Tbeta(4) causes incomplete F-actin depolymerization at high concentration. A, series of samples containing F-actin at 3 µM () (data from Fig. 1), 5 µM (box), 10 µM (circle), 20 µM (down triangle), 120 nM CapG and Tbeta(4) at the indicated concentrations were incubated overnight. Data were analyzed and presented as in Fig. 1. The dotted line represents the variation of [A] + [TA] expected within . Note that the origin of the ordinate scale is set at 0.5 µM. Inset: samples of 15 µM F-actin containing 120 nM CapG and O (a, a[prime]), 40 (b, b`), 100 (c, c`), and 250 (d, d`) µM Tbeta(4) were incubated for 16 h and centrifuged at 400,000 times g for 30 min at 20 °C. The amount of actin in the electrophoresed supernatants (a-d) and pellets resuspended in the initial volume (a`-d`) is shown. 50 µl of each sample were loaded on the gel. B, comparison of the effect of Tbeta(4) on F-actin with capped and uncapped barbed ends. Actin (12.5 µM, 1% pyrenyl labeled) was assembled in the absence () or presence (circle) of 120 nM CapG and Tbeta(4) at the indicated concentrations. The concentration of F-actin at steady state was derived from pyrene fluorescence readings following overnight incubation. Each sample containing CapG was then supplemented with 0.2 mM EGTA and fluorescence was read again 3 h later (bullet). C, time course of repolymerization upon uncapping of barbed ends, at different concentrations of Tbeta(4). F-actin (12.5 µM, 1% pyrenyl labeled) was incubated overnight in the presence of 120 nM CapG and the following concentrations of Tbeta(4), in µM, top to bottom curves a-e: 44, 124, 195, 221, 248. At time 0, 0.2 mM EGTA was added to each sample, and the time course of repolymerization due to the lowering of critical concentration of G-actin was monitored by pyrene fluorescence. The emission shutter was periodically opened for 5 s and closed for 30 s.




Figure 1: Tbeta(4) is a simple G-actin sequestering protein and fully depolymerizes actin at low concentration. F-actin (3 µM, 1% pyrenyl labeled) containing 120 nM CapG was incubated overnight in the presence of the indicated amounts of Tbeta(4). The amount of unassembled actin formed at steady state, [A] + [TA], was derived from pyrene fluorescence measurements (see ``Materials and Methods'') and varied linearly with T according to , until all F-actin (2.45 µM) was depolymerized. The slope of the line was consistent with a value of 2 ± 0.2 µM for K, using a value of 0.5 µM for the critical concentration at the pointed end.



In the second method, unlabeled bovine spleen Tbeta(4) was used. Tbeta(4) present in the pellets of F-actin was assayed by HPLC of the perchloric extract of the resuspended pellets, and comparison of the peak areas of the absorbance elution diagram at 220 nm with a calibration curve using standards in the range 0-2 nmol of Tbeta(4). The proportion of contaminant Tbeta(4) trapped in the pellet was estimated as in the first method.

Electron Microscopy

The structure of actin filaments at steady state in the presence of Tbeta(4) at different concentrations was examined in the electron microscope. Samples of 3-10 µM F-actin, 14-50 nM gelsolin, and Tbeta(4) in the range 0-250 µM were prepared 16 h in advance to make sure that steady state was established. Ten microliters of each sample were deposited on a carbon coated, air-glow discharged grid. Following 10-20 s adsorption, the excess solution was blotted and the sample was negatively stained with several drops of a 2% uranyl acetate solution. Unidirectional shadowing of freeze-dried specimens was performed as described(25) . Samples were prepared and adsorbed on the grid as for negative staining, rapidly rinsed with distilled water and immediately (within 2 s) frozen by immersion into liquid nitrogen. The grid was transferred into a Cryofract (Reichert-Jung) and maintained at -85 °C for at least 2 h. The specimens were then shadowed with a 2-nm thick layer of carbon-platinum, evaporated at an angle of 45°, and coated with a 3-4-nm carbon film evaporated at 90°. Specimens were observed in a CM12 Philips electron microscope operated at 80 kV. Micrographs were recorded at a nominal magnification of 35,000.


RESULTS

Tbeta(4)Binds G-actin Selectively at Low Concentration

In living cells, the physiological ionic conditions are such that F-actin is assembled at steady state, i.e. filaments coexist with G-actin at the critical concentration. Hence in vitro measurements of F-actin at steady state lead to the closest description of the in vivo situation. The actin-sequestering activity of Tbeta(4) was monitored by the linear decrease in the concentration of F-actin at steady state versus total concentration of Tbeta(4) as described by (see ``Materials and Methods''):

When barbed ends are capped, A(c) is the critical concentration at the pointed ends of filaments, which is higher than at the barbed ends, hence Tbeta(4) sequesters G-actin more efficiently. This situation is the most favorable, for economy of material, to measure the affinity of Tbeta(4) for G-actin. Fig. 1shows that upon addition of increasing amounts of Tbeta(4) to a solution of 3 µM F-actin capped by gelsolin, the concentration of unassembled actin (A + TA) increased linearly with total beta-thymosin until all actin was depolymerized. A value of 2 ± 0.2 µM was derived for (T) from the slope of the plot, in good agreement with previous data (5, 6, 7) obtained in the same range of actin and Tbeta(4) concentrations.

Tbeta(4)Does Not Behave as a Simple G-actin Sequestering Protein at High Concentration

The same experiment as above was repeated in a range of higher concentrations of F-actin. The data, displayed in Fig. 2A, show that above a total concentration [T(o)] 20 µM, Tbeta(4) sequestered actin less efficiently than expected, and a deviation from the linear dependence of [TA] versus [T(o)] () was observed. Filaments remained stable in solution at concentrations of Tbeta(4) at which total depolymerization should have occurred. At concentrations of F-actin higher than 10 µM, an invariant curve [A] + [TA] = f([T(o)]) was obtained (E and C in Fig. 2A). The fact that the dependence of [A] + [TA] on [T(o)] becomes independent of the amounts of F-actin means that the measured amount of unassembled actin ([A] + [TA]) at each [T(o)] represents the concentration of monomers, A and TA, at equilibrium with filaments, that is the critical concentration.

It was found appropriate to verify that the ATP had not been extensively hydrolyzed during the 16-h incubation period, due to the steady-state ATPase of F-actin, and that even the most concentrated F-actin samples could be truly considered as being at steady state in the presence of ATP. G-actin (20 µM) was equilibrated in G-buffer in which the 200 µM ATP were traced by [-P]ATP, and polymerized by addition of 2 mM MgCl(2) and 0.1 M KCl as described under ``Materials and Methods.'' The solution was then split into 3 samples containing 0, 50, and 200 µM Tbeta(4). After 16 h incubation, the amounts of hydrolyzed ATP were 29, 28.8, and 31.8 µM, respectively, in the three samples. Hence free nucleotide in the medium then consisted of about 10 µM ADP and 190 µM ATP. These numbers testify that the measurements made after 16 h truly reflect a situation in which F-actin is at steady-state in ATP, hence free G-actin is ATP-G-actin at the critical concentration, and Tbeta(4) binds ATP-G-actin as described by .

Thus far the conclusion that Tbeta(4) fails to totally depolymerize F-actin at high concentration relies on fluorescence measurements of pyrenyl actin. To verify that the measurements truly reflect equilibrium values of F-actin, G-actin, and Tbeta(4)-G-actin, the following controls were carried out. 1) Identical fluorescence readings were obtained 8 h later, indicating that measurements reflected a stable situation; 2) identical results were obtained with actin solutions containing different fractions of pyrene-labeled actin; 3) sedimentation of the samples of F-actin containing different concentrations of Tbeta(4) and SDS-gel electrophoresis of the pellet and supernatant (Fig. 2A, inset) established the validity of the interpretation of fluorescence measurements in terms of F-actin and unassembled actin; 4) sedimentation velocity of Tbeta(4) at different concentrations up to 250 µM showed that Tbeta(4) was a monomeric 5-kDa protein in the whole range of concentrations investigated. Finally quantitatively identical results showing incomplete depolymerization of F-actin were obtained with chemically synthesized Tbeta(4), which eliminates the possibility that a minor contaminant present in the preparation of Tbeta(4) from spleen would be responsible for the incomplete depolymerization of F-actin at high Tbeta(4).

The fact that Tbeta(4) fails to totally depolymerize F-actin in a range of high concentrations is a first indication that it can bind to F-actin as well as to G-actin, albeit with a lower affinity. Because other actin-binding proteins like ADF/cofilin (26, 27) have been shown to bind preferentially either F- or G-actin depending on pH, the depolymerization of F-actin (20 µM) by increasing amounts of Tbeta(4) was measured at pH 6.5 and 7.8 in parallel. Practically identical curves [TA] = f([T(o)]) showing incomplete depolymerization were obtained at the two pH values (data not shown). On the other hand, the failure of Tbeta(4) to totally depolymerize F-actin at high concentration was only observed at physiological ionic strength (0.1 M KCl). In a polymerization buffer containing only 2 mM MgCl(2), addition of increasing amounts of Tbeta(4) to 20 µM F-actin led to eventual complete depolymerization, and a linear curve [TA] = f([T(o)]) was obtained (like in Fig. 1), consistent with a value of K(T) of 0.8 µM, in agreement with previous determinations at low ionic strength(12) .

The incomplete depolymerization of F-actin at high concentration of Tbeta(4) was also observed when barbed ends were uncapped. Fig. 2B shows the amount of F-actin observed at steady-state upon addition of increasing amounts of Tbeta(4) to 12 µM F-actin, with barbed ends either capped by CapG, or uncapped, in two parallel series of samples. When 0.2 mM EGTA was added to the samples containing barbed end-capped F-actin, the dissociation of CapG (28) led to the partial repolymerization of actin filaments due to the shift in critical concentration caused by uncapping. The amount of F-actin reached at the end of this relaxation process was the same as the one measured in the uncapped samples, confirming that measurements shown in Fig. 2A reflect an equilibrium situation.

The data show that the extent of repolymerization upon uncapping of barbed ends, which is the difference between the two curves, first increases with the total concentration of Tbeta(4), and reaches a finite limit of 3 µM at high concentration of Tbeta(4). The time courses of actin desequestration/repolymerization upon uncapping by EGTA, displayed in Fig. 2C, show that the repolymerization process gets slower at higher concentrations of Tbeta(4). This result is very surprising for the following reason. Upon uncapping of barbed ends due to EGTA-induced dissociation of CapG, the initial rate of repolymerization is expected to be the following:

where kistherateconstantforadditionofG-actintotheuncappedendsatconcentration [F], C^PistheconcentrationoffreeG-actinattimeofuncapping, whichisequaltothecriticalconcentrationofthepointedends, and C^Bisthecriticalconcentrationforactinassemblyatthebarbedends, whichisreacheduponcompletionoftheuncapping-linkedrepolymerizationprocess. Accordingto, theinitialrateofrepolymerizationshouldbethesameatallconcentrationsofTbeta(4). OnlytheextentofrepolymerizationshouldvaryinproportionwithTbeta(4), reflectingthedifferenceintheamountofTbeta(4)-actincomplex (TA), whenbarbedendsarecappedoruncapped, asfollows.

The Critical Concentration for Actin Assembly Decreases in the Presence of Tbeta4: Evidence for the Interaction of Tbeta4 with F-actin

The data shown in Fig. 2suggest that the Tbeta(4)-G-actin complex could be a weakly polymerizing actin species able to copolymerize with actin. If the Tbeta(4)-actin complex can undergo the monomer-polymer exchange reactions which maintain filament stability at steady-state, the global critical concentration for actin assembly is the sum of the partial critical concentrations of G-actin and Tbeta(4)-G-actin, respectively, and the contribution of Tbeta(4)-G-actin to monomer-polymer exchange is expected to decrease the critical concentration of G-actin. The following simple experiment was designed to challenge this possibility. The sequestering efficiency of profilin was used as a probe for the concentration of free G-actin at steady-state in the presence or absence of Tbeta(4). When barbed ends are capped, profilin is known indeed to be a simple G-actin sequestering protein(4, 18) . The amount of profilin-actin complex formed at steady-state, [PA], therefore is controlled by the concentration of free G-actin, i.e. the critical concentration, as described by : [PA] = [P(o)]bullet[A(c)]/([A(c)] + K). Fig. 3A shows that upon addition of increasing amounts of profilin to a solution of gelsolin-capped F-actin, the concentration of F actin at steady-state decreased linearly with a slope (negative) of 0.56, consistent with a value of 0.4 µM for the equilibrium dissociation K of the profilin-actin complex. (^2)When the same experiment was done in the presence of 50 µM Tbeta, the slope of the linear decrease in F-actin, [A] was 2.3-fold lower, consistent with a 3.8-fold lower value of [A]. Hence the partial critical concentration for actin assembly at the pointed ends is decreased by Tbeta. Evidence for the Tbeta-dependent decrease in the partial critical concentration of G-actin can also simply be derived from the analysis of the amounts of unassembled actin at different concentrations of Tbeta ([T]) shown in Fig. 2A (circle, down triangle), as follows. At each value of [T], the measured amount of unassembled actin [A] can be written:


Figure 3: Tbeta(4) causes the lowering of the critical concentration. a, F-actin (1% pyrenyl labeled) was assembled at 10 µM in the presence of 120 nM CapG and either in the absence (bullet) or presence (circle) of 50 µM Tbeta(4). Profilin was added to the samples at the indicated concentration. Samples were incubated overnight before fluorescence was measured. The linear decrease in fluorescence represents the decrease in F-actin due to the formation of profilin-actin complex. The less steep slope observed in the presence of 50 µM Tbeta(4) implies a 3.8-fold lower value of A(c) in the presence of 50 µM Tbeta(4). B, the concentration of free G-actin at steady state, or partial critical concentration of G-actin, [A], was derived from the measurements of unassembled actin, [A] + [TA], shown in Fig. 2a (circle, down triangle) using .



which leads to a quadratic equation in [A], the solution which is:

The change in [A] versus [T(o)] can therefore be derived from the measurements of [A(u)] at different values of [T(o)]. Fig. 3B shows that [A] calculated according to decreases cooperatively upon increasing [T(o)]. It is interesting to observe that 50 µM Tbeta(4), a value of [A] of 0.15 µM was derived from the data shown in Fig. 2A, that is a 3.3-fold decrease in critical concentration, in good agreement with the 3.8-fold decrease found in the experiment shown (Fig. 3A) carried out also at 50 µM Tbeta(4), and in which profilin was used to derive the value of [A]. Hence two independent methods agree quantitatively to demonstrate that the partial critical concentration of G-actin decreases as larger amounts of TA complex are formed at steady-state. This result accounts for: 1) the limited extent of repolymerization upon uncapping and 2) the slowing down in the repolymerization process upon uncapping observed in Fig. 2C. Indeed, because c(C)^P decreases from 0.5 µM to less than 0.1 µM (Fig. 3B) while C(C)^B cannot decrease by more than 0.1 µM, the value of Delta[TA] is lower than expected () and the value of V () decreases upon increasing Delta([TA])Tbeta

Incorporation of Tbeta(4)in Actin Filaments

The binding of Tbeta(4) to F-actin was quantitated using both the sedimentation assay and chemical cross-linking described under ``Materials and Methods.'' The sedimentation assays (both using chemically synthesized ^14C-Tbeta(4) and unlabeled spleen Tbeta(4)) showed evidence for a very weak, substoichiometric binding of Tbeta(4) to F-actin. The binding also appeared cooperative, as can be seen on Fig. 4. Less than 0.01 Tbeta(4) per F-actin subunit was bound at 100 µM Tbeta(4), while about 0.04 Tbeta(4) per F-actin was measured at 250 µM Tbeta(4). Although these figures are very low and indicate that the binding constant lies in the 5-10 mM range, they were significantly above the level corresponding to simple trapping of Tbeta(4) in the pellets. Typically, at 100 µM Tbeta(4), the amount of Tbeta(4) measured in the pellets was twice as high as the amount trapped in the interstitial volume.


Figure 4: Evidence for low affinity binding of Tbeta(4) to F-actin. Samples of F-actin (20 µM) capped by gelsolin (0.05 µM) containing the indicated amounts of Tbeta(4) were sedimented at 400,000 times g and the amount of bound Tbeta(4) per F-actin subunit was determined by HPLC after correction for Tbeta(4) trapped in the pellet (see ``Materials and Methods''). Inset, EDC cross-linking of Tbeta(4) to F-actin and G-actin. Samples of F-actin (15 µM) capped by gelsolin and containing either 200 µM (+) or 0(-) ^14C-Tbeta(4) were cross-linked for 1 h by EDC-sulfo-NHS as described under ``Materials and Methods.'' The samples were centrifuged and the pellets were resuspended in a volume of buffer 2.5-fold smaller than the original volume. 25 µl of supernatants (sup.) and 15 µl of the resuspended pellets (pel.) were submitted to SDS-polyacrylamide gel electrophoresis (top panel) followed by autoradiography of the gel (bottom panel).



Chemical cross-linking of Tbeta(4) to F-actin displayed in Fig. 4, inset, confirmed that at high concentration Tbeta(4) bound to F-actin. Approximately 5-10% of F-actin could be covalently cross-linked to Tbeta(4) at 200 µM^14C-Tbeta(4), leading to a 47-kDa ^14C-labeled polypeptide migrating at the same position as the covalent Tbeta(4)-G-actin complex. The mass amount of Tbeta(4)-actin cross-linked polypeptide was only about 3-4-fold lower than the mass amount of the Tbeta(4)-G-actin cross-linked polypeptide found in the supernatant of the sedimented cross-linked mixture, which rules out the possibility of a significant contamination of covalent Tbeta(4)-actin by covalent Tbeta(4)-G-actin trapped in the pellet. On the other hand, an artifact might arise if the covalently cross-linked Tbeta(4)-G-actin complex aggregated and sedimented together with F-actin. To test this possibility, G-actin (15 µM) was supplemented with 200 µMTbeta(4), followed by 1 mM MgCl(2) and 0.1 M KCl, and the mixture was immediately submitted to cross-linking. No polymerization of G-actin could occur during cross-linking due to the high amount of Tbeta(4). The sample was centrifuged at 400,000 times g. Although no pellet could be seen by the eye, any putative sedimented material was carefully resuspended. No covalent actin-Tbeta(4) adduct was observed in gel electrophoresis of the resuspended material. Therefore the cross-linking experiments also demonstrate weak binding of Tbeta(4) to F-actin. The Tricine-SDS gel patterns of the cyanogen bromide digests of the covalent ^14C-Tbeta(4)-G-actin and ^14C-Tbeta(4)-F-actin complexes were identical, which provided an indication that the contact points between Tbeta(4) and either G- or F-actin were identical.

Morphology of Actin Filaments Assembled in the Presence of Increasing Amounts of Tbeta(4)

Images of negatively stained specimens of F-actin at steady state in the presence of increasing amounts of Tbeta(4) in the range 0-250 µM are displayed in Fig. 5. In the absence of Tbeta(4), filaments showed a distinct periodicity of the two-start long pitch helix (Fig. 5a). This periodic feature was already less apparent in filaments assembled in the presence of 22 µM Tbeta(4) (Fig. 5b). Unraveling of the two-start long pitch helix was more frequent in the presence than in the absence of Tbeta(4). The mean number of unraveled areas per half-micron length of filament was 0.7 (S.D. = 0.6, n = 20 measurements) in the absence of Tbeta(4), and 1.5 (S.D. = 1.0, n = 30) in the presence of 22 µM Tbeta(4). The length of the unraveled areas also depends on Tbeta(4). The average unraveled length was 150 Å (S.D. = 40 Å, n = 18) in the absence of Tbeta(4), and 200 Å (S.D. = 80 Å, n = 18) in the presence of 22 µM Tbeta(4). In the presence of 100 µM Tbeta(4) (Fig. 5c) a small proportion of filaments twisted around each other in a rope-like or torsade fashion. The number of filaments in each torsade is not clearly defined and the individual filament cannot be recognized in the twisted polymer. In the presence of 250 µM Tbeta(4) (Fig. 5d) the proportion of intertwining and twisting of filaments increased, and few isolated filaments could be seen. The intertwining of filaments in torsades was concentration-dependent, fewer torsades being observed at a lower concentration of F-actin in the presence of 250 µM Tbeta(4). The destabilizing effect of Tbeta(4) on the structure of the individual filament can best be seen in freeze-dried and shadowed specimens (Fig. 5, e and f). In the absence of Tbeta(4), actin filaments present clear transverse striations arising from the short pitch helix. The contrast of the transverse striations is reduced in the presence of 250 µM Tbeta(4), and longitudinal depressions (arrows), which arise from the long pitch (2-start) helix, are longer and more frequent than in the absence of Tbeta(4). These observations suggest that incorporation of very few Tbeta(4) molecules in the filament creates defects in the helical arrangement of subunits causing the local destabilization of the lateral actin-actin bonds in the filament. The resulting ``opening'' of the two strands of the long pitch helix allows lateral sidewise pairing of other filaments, which leads to the observed stiffer wider ``ropes.'' As the number of defects is increased, the twisting of the torsades is increased. The Tbeta(4)-induced structural changes of actin filaments were only observed at physiological ionic strength, which correlates with the thermodynamic data. In a polymerization buffer containing only 1-2 mM MgCl(2), only single actin filaments were observed in the presence of high concentrations of Tbeta(4), under conditions where 2-3 µM F-actin remained at steady state in the presence of large amounts of Tbeta(4)-G-actin complex. Therefore the images observed in Fig. 5do not result from an artifact of background created by the presence of large amounts of Tbeta(4) and Tbeta(4)-G-actin in the solution.


Figure 5: Destabilization of the filament structure by Tbeta(4) and intertwining of actin filaments. Panels a-d, negatively stained samples of F-actin at steady state in the presence of O (a), 22 µM (b), 100 µM (c), and 250 µM (d) thymosin beta(4). Actin was polymerized in the presence of 0.1 M KCl and 1 mM MgCl(2) added to G buffer, and gelsolin in a 1:300 ratio to actin. Total actin concentration was 2.5 µM in a, 7 µM in b, 9 µM in c and d. Arrows indicate the separations between the two strands of the long pitch helix. Panels e and f, freeze dried and shadowed samples of F-actin standard (e) and F-actin in the presence of 250 µM Tbeta(4) (f). The circled arrow in the upper left corner indicates the direction of shadowing. Black arrows point to the separations between the two strands of the filament.



Control of the Tbeta(4)-Actin Complex on Filament Dynamics

The fact that an invariant curve [TA] = f([T(o)]) was obtained (Fig. 2a) suggests that the increase in concentration of Tbeta(4)-actin complex, rather than of free Tbeta(4), is linked to the lowering in critical concentration of G-actin. This point was further addressed in kinetic experiments aimed at understanding how either free Tbeta(4) or Tbeta(4)-actin complex affects the kinetic parameters for filament growth.

In a first assay, the initial rate of filament depolymerization upon 20-fold dilution in F-buffer was measured in the presence of increasing amounts of Tbeta(4). With both capped and uncapped barbed ends, the rate of depolymerization was unaffected by Tbeta(4) up to 200 µM.

In a second experiment, the initial rate of filament growth was measured in the presence of 3 µM G-actin and increasing concentrations of Tbeta(4). The inhibition of filament growth was complete at saturation by Tbeta(4), i.e. eventually filaments depolymerized when the concentration of free G-actin fell below the critical concentration. The data (Fig. 6A) analyzed as described under ``Materials and Methods'' were perfectly consistent with binding of Tbeta(4) to G-actin with an equilibrium dissociation constant of 1.6 µM, a value identical to the one derived from steady-state measurements of the sequestering activity of this protein. Therefore free Tbeta(4), in a large range of concentrations (0-100 µM) does not appear to influence the kinetic parameters for filament growth, but simply acts as a G-actin sequestering protein. The Tbeta(4)-actin complex, in the concentration range from 0 to 3 µM, does not appreciably affect the kinetic parameters for filament growth. On the other hand, different results were obtained when the inhibition of filament growth by Tbeta(4) was assayed at higher concentrations (6.5 µM, 10.5 µM, 14 µM) of G-actin. Using the value of 1.6 µM derived above for the equilibrium dissociation constant of the TA complex, the values of [TA] and [A] were calculated for each pair of total concentrations of G-actin (C(o)) and of Tbeta(4) (T(o)) (see e.g.). Since the incorporation of TA in filaments is extremely low, the process of filament growth was fed essentially by addition of free G-actin to filament pointed ends. Accordingly, the rate of elongation J varied linearly with the concentration of free G-actin, but the plots obtained at 10.5 µM and 14 µM total G-actin did not superimpose, in the region 0-3 µM free G-actin, with the regular J(c) plot obtained in the absence of Tbeta(4) (Fig. 6B). The linear J(c) plots obtained at about 90% saturation of G-actin by Tbeta(4), i.e. in a range of concentrations of free G-actin of 0-2 µM (i.e. in the presence of 20-100 µM Tbeta(4)), were characterized by a higher slope than the control J(c) curve carried out in the absence of Tbeta(4), a lower critical concentration (defined as the concentration of free G-actin at which the rate of filament growth is zero), and the same value of the ordinate intercept. At the somewhat lower value of C(o) of 6.5 µM, data clearly showed a gradual shift from the coincidence with the standard J(c) at concentrations of TA leq 3 µM, toward coincidence with the plots obtained at A(o) = 10.5 µM and 14 µM and high Tbeta(4) concentrations, as the saturation of G-actin by Tbeta(4) increased. In other words, at high concentrations Tbeta(4) is less efficient to inhibit filament growth than expected from the extent of inhibition at low concentration of Tbeta(4), a result essentially in agreement with previous observations(6) . These data demonstrate that in the presence of large concentrations of TA complex, the partial critical concentration of actin is lower, and this decrease in critical concentration is mediated by an increase in the rate constant k for association of G-actin to filament ends, while the dissociation rate constant k seems practically unchanged. This kinetic piece of data agrees with and further expands upon the data of incomplete depolymerization of actin filaments in the presence of high concentrations of TA reported in Fig. 2, A and B, the slow rate of repolymerization upon uncapping shown in Fig. 2C, and the steady state measurements of the decrease in critical concentration shown in Fig. 3.


Figure 6: Effect of Tbeta(4) and TA complex on the kinetic parameters for pointed end filament growth. The initial rate of elongation onto pointed ends was assayed as described under ``Materials and Methods.'' A, inhibition of filament growth by Tbeta(4) at low actin concentration (3.2 µM). The inset shows the analysis of the data using (see ``Materials and Methods''). B, effect of high concentrations of TA on the rate of filament growth. The rate of filament growth was measured at different concentrations of free G-actin either in the absence of Tbeta(4) (bullet), or in the presence of Tbeta(4) at three different total concentrations of G-actin (box, 6.5 µM; circle, 10.5 µM; up triangle, 14 µM). Note that at the two higher concentrations of G-actin, the rates are measured in a range of Tbeta(4) concentrations of 20-100 µM, in which 90-99% of G-actin consists of TA complex. That is [TA] = constant = 9 ± 1 and 13 ± 1.5 µM, respectively. The concentrations of free G-actin were calculated using . The curve obtained at 6.5 µM total G-actin (box) exhibits a change in regime between 3 and 5.5 µM TA during which the kinetic parameters change from those of the control curve to those operating at high TA.




DISCUSSION

The present results demonstrate that the function of Tbeta(4) in the regulation of actin assembly is more complex than previously thought. We confirm that in a range of low concentrations (<20 µM), typical of the physiological concentration of beta thymosins in many cells, Tbeta(4) acts as a simple G-actin binding protein. The 1:1 thymosin-actin complex, which may accumulate at steady state up to 4 µM when barbed ends are capped, does not participate in actin assembly and does not affect the kinetic parameters of filament growth. On the other hand, in a range of higher concentrations (20-250 µM), Tbeta(4) appears to also interact with F-actin with a very low affinity (K(D) 5-10 mM). The weak incorporation of Tbeta(4) into filaments creates defects in the structure of the polymer. These defects can be described in terms of local points of destabilization of actin-actin contacts perpendicular to the filament axis, which results in local separation of the two strands of the long-pitch helix, thus enhancing the incidental ``lateral slipping'' feature noticed by U. Aebi on standard filaments(29) . Therefore our results indicate that Tbeta(4) binding to G-actin inhibits polymerization by interfering with the formation of lateral actin-actin bonds along the short pitch helix, rather than with the formation of longitudinal bonds. At high filament concentration, the destabilized, partially unravelled filaments tend to self-associate and twist around each other to yield thick rope-like structures. The effect of Tbeta(4) on the F-actin structure may be compared to the effect of intercalating drugs on DNA structure.

The main consequence of the incorporation of Tbeta(4) in F-actin is the decrease in the partial critical concentration of G-actin. The Tbeta(4)-G-actin complex cannot be considered as a good polymerizing actin monomer since filaments containing on average 5% or less Tbeta(4)-actin subunits exhibit a destabilized structure. These unstable filaments therefore are maintained at steady state by exchanging subunits at their ends with a pool of monomers at a high critical concentration. The total critical concentration of monomeric actin is the sum of free G-actin and Tbeta(4)-G-actin at steady state. The partial critical concentration of Tbeta(4)-actin monomer is 2 orders of magnitude higher than the partial critical concentration of G-actin itself under these conditions, while the copolymer consists of less than 5% Tbeta(4)-actin subunits. These figures illustrate the fact that although Tbeta(4)-actin is a weak polymerizing species, its contribution to filament assembly, via a high partial critical concentration, helps to diminish the partial critical concentration of G-actin. Therefore, in addition to its G-actin sequestering function, Tbeta(4) also possesses the power to control the steady state of actin assembly, like capping proteins and profilin do(18) , but in contrast to profilin, it can do it at both ends. The decrease in partial critical concentration of G-actin occurs smoothly over a range of high concentrations of Tbeta(4) (only a 3-4-fold decrease was observed at 50 µM Tbeta(4)).

The following consequences can be derived from our results: the property of Tbeta(4) to decrease the concentration of G-actin at steady state causes a self-limitation of the G-actin sequestering function of Tbeta(4), but also promotes a decrease in the amount of G-actin sequestered by other G-actin binding proteins like ADF/cofilin and, when barbed ends are capped, of profilin (as illustrated in Fig. 3A). The present in vitro results and their analysis provide an explanation of in vivo observations of actin dynamics in cells overexpressing Tbeta (a variant of Tbeta(4)), which show evidence for a paradoxical decrease in the amount of unassembled actin in overexpressing cells as compared to control cells (see accompanying article, (33) ). Examination of the measured cellular amounts of Tbeta and other G-actin binding proteins leads to the conclusion that a putative 2-fold decrease in critical concentration linked to the 3-fold overexpression of Tbeta is enough to account for the observed lower amount of unassembled actin. Under these conditions, the moderate increase in Tbeta-actin complex in overexpressing cells cannot fully compensate the actual decrease in the pool of actin sequestered by other G-actin binding proteins which is linked to the decrease in steady state concentration of G-actin. It should be emphasized here that, within our model, the change in the amount of unassembled actin linked to overexpression of beta-thymosin can lead to very different situations depending on the concentration of other G-actin binding proteins in cells. Let us assume that a living cell contains Tbeta(4) and other ``bona fide'' G-actin binding proteins that do not affect the critical concentration. For simplicity, all these non-Tbeta(4) G-actin binding proteins will be collectively considered as a single species called ``GBP.'' The pool of unassembled actin consists of Tbeta(4)-actin and GBP-actin complexes. The concentration of unpolymerized actin in cells at different total concentrations of Tbeta(4) and GBP is described in a three-dimensional plot shown in Fig. 7. Iso-GBP lines outline the effect of overexpression of Tbeta(4) at different concentrations of GBP. At low concentration of GBP, the increase in Tbeta(4)-actin concentration predominates over the decrease in GBP-actin concentration, and a net increase in unassembled actin is linked to overexpression of Tbeta(4). In the intermediate range of GBP concentration, the two effects roughly compensate each other, and very little change in unassembled actin is observed upon overexpression of Tbeta(4). At high concentration of GBP, the decrease in GBP-actin complex predominates over the increase in Tbeta(4)-actin, resulting in a net decrease in unassembled actin upon overexpression of Tbeta(4). Our in vitro results therefore allow understanding of the discrepancies reported by different groups concerning the effects of Tbeta(4) overexpression on the level of actin assembly, in terms of differences in concentrations of GBPs in different cell types. It will be interesting to challenge this interpretation by detailed measurements of the amounts of GBPs in different cell types.


Figure 7: Overexpression of Tbeta(4) lead to an increase or a decrease in the concentration of unassembled actin, depending on the cellular amount of G-actin binding proteins (GBP) other than Tbeta(4). The concentration of unassembled actin was calculated as the sum of [TA] and [GBP-A] complexes. It was assumed that the values of [A] were 0.5 µM in the absence of Tbeta(4) and varied with Tbeta(4) as described in the legend to Fig. 3. The values of the equilibrium dissociation constants for TA and GBP-A complexes were assumed to be 1.6 µM and 0.5 µM, respectively. The concentration of unassembled actin was calculated as follows.

Bold lines represent the change in concentration of unpolymerized actin upon increasing Tbeta(4), at the following GBP concentrations: 0 µM, 5, 15, 25, and 40 µM (front to back).



The present in vitro data also provide an explanation for the unexplained slower rate of propulsion of Listeria in Xenopus egg extracts supplemented with F-actin together with high amounts of Tbeta(4) ( Fig. 6in (30) ). According to the proposed model for actin-based Listeria movement, the rate of actin assembly is controlled by the difference in critical concentrations between the bulk cytoplasm (where capping proteins acts to establish the high critical concentration of pointed ends) and the bacterium surface (where anchored uncapped barbed ends, characterized by a low critical concentration, are actively growing). If the critical concentration in the bulk cytoplasm is decreased by large amounts of Tbeta(4), then the rate of assembly at the bacterium surface is expected to decrease (such as observed here in Fig. 2C).

Our work raises questions concerning the actual amount of actin sequestered by Tbeta(4) in resting platelets and neutrophils and the physiological significance of the ``in vitro physiological ionic conditions.'' Clearly according to the present in vitro data, very little actin (10 µM) would be sequestered by Tbeta(4) in resting platelets or neutrophils, while in vivo data clearly indicate that at least 100 µM actin would be unpolymerized in these cells, profilin (estimated at 50 µM in platelets) and Tbeta(4) (estimated at 400 µM in platelets) being the major actin sequestering agents. Therefore some cytoplasm component, or macromolecular crowding, has to be thought of, which would limit the effects of Tbeta(4) at the high concentration that we observed in vitro. This component could either stabilize F-actin (as tropomyosin would do) and consequently prevent the incorporation of Tbeta(4)-actin in filaments, or it could screen the effects of ionic strength, thereby favoring the sequestering activity of Tbeta(4) over its interaction with F-actin. Nonetheless, the intrinsic properties of Tbeta(4) illustrated here have to be considered to some extent in the in vivo situation. It may be worth noting that complete agreement has not been reached among different groups concerning the actual value of the Tbeta(4) content of motile blood cells(5, 11) . In addition, the dilution of cytoplasm that takes place in the preparation of cellular extracts without fixation causes depolymerization of a part of the F-actin pool(31, 32) , which may lead to a somewhat overestimated concentration of unassembled actin.

From a structural point of view, the fact that Tbeta(4)-actin is able, although weakly, to copolymerize with actin, accounts for the difficulties encountered in the crystallization of the Tbeta(4)-actin monomer in salt-containing solutions. The observation that Tbeta(4)-actin incorporates into filaments only in high ionic strength (0.1 M KCl) assembly buffers indicates that either electrostatic bonds in the actin-Tbeta(4) interface have to be weakened, or hydrophobic bonds have to be strengthened, to allow incorporation of Tbeta(4)-actin in the filament. More detailed studies of the structure of Tbeta(4)-actin complex will challenge these expectations.


FOOTNOTES

*
This work was supported in part by the Association pour la Recherche contre le Cancer, the Ligue Nationale Française contre le Cancer, and the Association Française pour la lutte contre les Myopathies. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Laboratoire d'Enzymologie, CNRS, Gif-sur-Yvette, France. Tel.: 33-1-69-82-34-65; Fax: 33-1-69-82-31-29; carlier{at}pegase.enzy.cnrs-gif.fr.

(^1)
The abbreviations used are: HPLC, high performance liquid chromatography; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; GBP, G-actin binding protein.

(^2)
We have checked that despite the fact that profilin does not bind pyrenyl-actin, the change in fluorescence of pyrenyl-F-actin capped by gelsolin upon increasing profilin is a valid measurement of the mass amount of F-actin at steady state over periods of several hours, as long as profilin is added to preassembled F-actin. This is no longer the case when actin has been assembled in the presence of profilin. A full study and justification of this assessment is provided elsewhere (I. Perelroizen, D. Didry, H. Christensen, N. H. Chua, M.-F. Carlier, submitted for publication).


ACKNOWLEDGEMENTS

We thank Dr. Daniel Safer for helpful advice on the use of a strong anion exchange column in the purification of thymosin beta(4), and Dr. Vivianne Nachmias for a stimulating E-mail discussion on the effect of Tbeta(4) overexpression.


REFERENCES

  1. Carlier, M.-F., and Pantaloni, D. (1994) Semin. Cell Biol. 5, 183-191 [CrossRef][Medline] [Order article via Infotrieve]
  2. Safer, D., Elzinga, M., and Nachmias, V. T. (1991) J. Biol. Chem. 266, 4029-4032 [Abstract/Free Full Text]
  3. Nachmias, V. T. (1993) Curr. Opin. Cell Biol. 5, 56-62 [Medline] [Order article via Infotrieve]
  4. Sun, H.-Q., Kwiatkowska, K., and Yin, H. L. (1995) Curr. Opin. Cell Biol. 7, 102-110 [CrossRef][Medline] [Order article via Infotrieve]
  5. Weber, A., Nachmias, V. T., Pennise, C. R., Pring, M., and Safer, D. (1992) Biochemistry 31, 6179-6185 [Medline] [Order article via Infotrieve]
  6. Yu, F.-X., Lin, S.-C., Morrison-Bogorad, M., Atkinson, M. A. L., and Yin, H. L. (1993) J. Biol. Chem. 268, 502-509 [Abstract/Free Full Text]
  7. Carlier, M.-F., Jean, C., Rieger, K. J., and Lenfant, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5034-5038 [Abstract]
  8. Cassimeris, L., Safer, D., Nachmias, V. T., and Zigmond, S. H. (1992) J. Cell Biol. 119, 1261-1270 [Abstract]
  9. Sanders, M. C., Goldstein, A. L., and Wang, Y.-L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4678-4682 [Abstract]
  10. Nachmias, V. T., Cassimeris, L., Golla, R., and Safer, D. (1993) Eur. J. Cell Biol. 61, 314-320 [Medline] [Order article via Infotrieve]
  11. Yu, F.-X., Lin, S.-C., Morrison-Bogorad, M., and Yin, H. L. (1994) Cell Motil. Cytoskeleton 27, 13-25 [Medline] [Order article via Infotrieve]
  12. Jean, C., Rieger, K. J., Blanchoin, L., Carlier, M.-F., Lenfant, M., and Pantaloni, D. (1994) J. Muscle Res. Cell Motil. 15, 278-286 [Medline] [Order article via Infotrieve]
  13. Heintz, D., Reichert, A., Mihelic, M., Voelter, W., and Faulstich, H. (1993) FEBS Lett. 329, 9-12 [CrossRef][Medline] [Order article via Infotrieve]
  14. Spudich, J. A., and Watt, S. (1971) J. Biol. Chem. 246, 4866-4871 [Abstract/Free Full Text]
  15. MacLean-Fletcher, S., and Pollard, T. D. (1980) Biochem. Biophys. Res. Commun. 96, 18-27 [Medline] [Order article via Infotrieve]
  16. Kouyama, T., and Mihashi, T. (1981) Eur. J. Biochem. 114, 33-38 [Abstract]
  17. Vancompernolle, K., Goethals, M., Huet, C., Lonvard, D., and Vandekerckhove, J. (1992) EMBO J. 11, 4739-4746 [Abstract]
  18. Pantaloni, D., and Carlier, M.-F. (1993) Cell 75, 1007-1014 [Medline] [Order article via Infotrieve]
  19. Doi, Y., Kim, F., and Kido, S. (1990) Biochemistry 29, 1392-1397 [Medline] [Order article via Infotrieve]
  20. Yu, F.-X., Zhou, D., and Yin, H. L. (1991) J. Biol. Chem. 266, 19269-19275 [Abstract/Free Full Text]
  21. Carlier, M.-F., Pantaloni, D., and Korn, E. D. (1984) J. Biol. Chem. 259, 9983-9986 [Abstract/Free Full Text]
  22. Carlier, M.-F., Criquet, P., Pantaloni, D., and Korn, E. D. (1986) J. Biol. Chem. 261, 2041-2050 [Abstract/Free Full Text]
  23. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  24. Schägger, H., and Von Jagow, G. (1987) Anal. Biochem. 166, 368-379 [Medline] [Order article via Infotrieve]
  25. Lepault, J., Weiss, H., Homo, J.-C., and Lenoard, K. (1981) J. Mol. Biol. 149, 275-284 [Medline] [Order article via Infotrieve]
  26. Hayden, S. M., Miller, P. S., Brauweiler, A., and Bamburg, J. R. (1993) Biochemistry 32, 9994-10004 [Medline] [Order article via Infotrieve]
  27. Hawkins, M., Pope, B., Maciver, S. K., and Weeds, A. G. (1993) Biochemistry 32, 9985-9993 [Medline] [Order article via Infotrieve]
  28. Yu, F.-X., Johnston, P. A., Südhof, T. C., and Yin, H. L. (1990) Science 250, 1413-1415 [Medline] [Order article via Infotrieve]
  29. Aebi, U., Millonig, R., Salvo, H., and Engel, A. (1987) Ann. N. Y. Acad. Sci. 483, 100-119
  30. Marchand, J.-B., Moreau, P., Cossart, P., Carlier, M.-F., and Pantaloni, D. (1995) J. Cell Biol. 130, 331-343 [Abstract]
  31. Heptinstall, S., Glenn, J., and Spangenberg, P. (1992) Thromb. Haemostasis 68, 727-730 [Medline] [Order article via Infotrieve]
  32. Small, J. V., Herzog, M., and Anderson, K. (1995) J. Cell Biol. 129, 1275-1286 [Abstract]
  33. Sun, H. Q., Kwiatkowska, K., and Ying, H. L. (1996) J. Biol. Chem. 271, 9223-9230 [Abstract/Free Full Text]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.