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J Biol Chem, Vol. 274, Issue 30, 20970-20976, July 23, 1999


Control of Actin Filament Length and Turnover by Actin Depolymerizing Factor (ADF/Cofilin) in the Presence of Capping Proteins and ARP2/3 Complex*

Fariza RessadDagger , Dominique DidryDagger , Coumaran Egile§, Dominique PantaloniDagger , and Marie-France CarlierDagger

From the Dagger  Dynamique du Cytosquelette, Laboratoire díEnzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette, France and the § Unité de Pathogénicité Microbienne Moléculaire, Institut Pasteur, Paris, France

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The effect of Arabidopsis thaliana ADF1 and human ADF on the number of filaments in F-actin solutions has been examined using a seeded polymerization assay. ADF did not sever filaments in a catalytic fashion, but decreased the steady-state length distribution of actin filaments in correlation with its effect on actin dynamics. The increase in filament number was modest as compared with the large increase in filament turnover. ADF did not decrease the length of filaments shorter than 1 µm. ADF promoted the rapid turnover of gelsolin-capped filaments in a manner dependent on the number of pointed ends. To explain these results, we propose that, as a consequence of the cooperative binding of ADF to F-actin, two populations of energetically different filaments coexist in solution pending a flux of subunits from one to the other. The ADF-decorated filaments depolymerize rapidly from their pointed ends, while undecorated filaments polymerize. ADF also promotes rapid turnover of gelsolin-capped filaments in the presence of the pointed end capper Arp2/3 complex. It is shown that the Arp2/3 complex steadily generates new barbed ends in solutions of gelsolin-capped filaments, which represents an important aspect of its function in actin-based motility.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Actin-binding proteins of the actin depolymerizing factor (ADF)1/cofilin family play an essential and unique role in the regulation of cell motility (1-3). ADF/cofilins localize to regions of the cells where filaments are highly dynamic (4-7). ADF/cofilins are responsible for the rapid turnover of actin filaments in cortical actin assembly in yeast (8), lamellipodia or growth cone extension, or the actin comet tail of Listeria (9, 10). In vitro biochemical studies have shed light on the mechanism of ADF function in vivo. When added to solutions of pure F-actin, ADF/cofilins enhance the turnover of actin filaments up to values similar to those observed in vivo, by increasing the rate of depolymerization of actin filaments from their pointed ends (9), which is the rate-limiting step in the treadmilling cycle of F-actin at steady state. It was then proposed that ADF accelerated the treadmilling of F-actin, i.e. increased the steady-state rate of barbed end growth, which drives lamellipodium extension or Listeria propulsion. In support to the proposed model, the steady-state concentration of ATP-G-actin, which feeds barbed end growth, was found increased by ADF (11). The effect of ADF on filament turnover is mediated by its specific interaction with the ADP-bound forms of G- and F-actin, and the subsequent modification of their dynamic properties. While ADF association with G-actin is a simple, rapid, bimolecular reaction, its binding to F-actin is more complex and shows a high degree of kinetic cooperativity (12). The cooperative binding is associated with a change in structure of the filament, visualized in EM as a change in twist (13). The ability to modulate the angular disorder of the filament is another unique property of ADF/cofilin (14). The structural change of the filament imposed by ADF results in a functional sorting of the filaments, because the structure of the ADF-F-actin filament is not compatible with the binding of myosin or tropomyosin (15) or drugs like phalloidin (9). All ADFs from different organisms from amoeba to man share similar biochemical properties and control the dynamics of actin filaments by the same general mechanism, with quantitative differences that specify the functional properties of the different ADFs (9, 12, 16-18).

The change in structure and dynamics of the filament is likely to be associated with a change in the mechanical properties of the filaments, making them more easily fragmented when submitted to shearing forces. The intricate complexity of ADF effects on actin structure and dynamics and the fact that these effects are not independent of each other raised discrepancies between different groups concerning the real cellular function of ADF and the molecular mechanism by which it is fulfilled. The enhancement of filament turnover in bulk solutions of F-actin in vitro was viewed as the sole consequence of the increased number of filaments due to a severing action of ADF (19-23), or of the enhanced dynamics of individual filaments (9), or of both (11, 18). As emphasized earlier, a severing effect of ADF cannot by itself elicit depolymerization of F-actin (11). In vivo, the agonist effect of ADF on motility processes such as extension of the lamellipodium, which require rapid barbed end growth of individual filaments anchored at the plasma membrane, cannot be accounted for by fragmentation and is likely to be rather mediated by the effect of ADF on the dynamic parameters of F-actin.

To sort out the involvement of the different properties of ADF in its function, the number of filament ends in F-actin solutions in the absence and presence of ADF was measured using the capping protein as a tool (11). The increase in number of ends due to ADF was too modest to account for the 30-fold increase in turnover rate. However, the presence of the capping protein could introduce a bias in those measurements, in part due to its ability to nucleate filaments, hence to affect the length distribution as well. The present work was undertaken with the goal to understand the effects of ADF on the structure and dynamic properties of the actin filament in a comprehensive fashion. The dependence of the number of filaments on ADF and F-actin concentration, in the presence and absence of capping proteins like gelsolin, or of the Arp2/3 complex which has been reported to bind to pointed ends with high affinity (24), has been examined in detail, in combination with turnover measurements. It is found that the cooperativity in ADF binding to F-actin, associated with the large change in dynamic properties of the filament, generates two populations of filaments of very different stabilities and dynamic properties, which coexist in solution. The energetic difference between the ADF-decorated and the bare filaments promotes a flux of subunits from one to the other. ADF also promotes fast turnover of gelsolin-capped filaments in the presence of Arp2/3 complex, due to the continuous generation of barbed ends by Arp2/3 at steady state. The mechanism of rapid turnover of actin filaments, somewhat different from the classical treadmilling process, is discussed in view of the biological function of ADF in living cells.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Proteins-- Actin was purified from rabbit muscle acetone powder (25) and isolated as CaATP-G-actin by Sephadex G-200 chromatography (26) in G buffer (5 mM Tris-Cl-, pH 7.8, 0.1 mM CaCl2, 0.2 mM ATP, 1 mM dithiothreitol, 0.01% NaN3). CaATP-G-actin was converted into MgATP-G-actin by addition of 1 molar equivalent and 10 µM excess MgCl2. Actin was pyrenyl-labeled as described (27).

Profilin and thymosin beta 4 were purified from bovine spleen as described (28). Recombinant ADF1 from Arabidopsis thaliana and human ADF were expressed in Escherichia coli and purified as described (9).

Arp2/3 complex was purified from bovine brain by ion exchange and affinity chromatography as described elsewhere2 and stored at -80 °C in 10 mM Tris-Cl- buffer, pH 7.5, supplemented with 0.2 M KCl, 0.2 mM ATP, 0.2 mM dithiothreitol, and 0.2 M sucrose. Capping protein beta 2 (the homolog of CapZ) was purified from bovine erythrocytes as described (29). Gelsolin purified from human plasma was a kind gift from Dr. Yukio Doi (Kyoto).

Protein concentrations were determined spectrophotometrically using extinction coefficients epsilon 0.1% of 0.617 cm-1 at 290 nm for actin, 0.89 and 0.63 cm-1 at 278 nm for A. thaliana ADF1 and human ADF, respectively (9), and 1.0 for profilin. The concentration of the Arp2/3 complex and of the capping protein were determined using the Bradford assay (Bio-Rad), with bovine serum albumin as a standard.

Actin was polymerized by addition of 0.1 M KCl and 1 mM MgCl2 to Mg-G-actin. Barbed end capped filaments were polymerized by addition of 0.1 M KCl and 2 mM MgCl2 to a solution of CaATP-G-actin in G buffer containing either gelsolin at the desired gelsolin:actin ratio.

Measurement of the Number of Filaments Using Seeded Polymerization and Dilution-induced Depolymerization Assays-- Solutions of 10% pyrenyl-labeled F-actin at different concentrations were obtained by serial dilution of a stock F-actin solution polymerized at 20 µM for 1 h, split in two samples that were supplemented, 3 h later, with either ADF at a given concentration or with an identical volume of buffer. The F-actin and F-actin + ADF solutions were then used as seeds of filament growth by diluting them 20-30-fold into either F buffer (dilution-induced depolymerization assay) or solutions of Mg-G-actin (10% pyrenyl labeled) at the desired concentrations that were supplemented with 0.1 M KCl and 1 mM MgCl2 just before adding the seeds (seeded filament growth assay). When seeds capped by gelsolin (at the indicated gelsolin:actin ratio) were used, care was taken to add CaCl2 in excess over the EGTA coming from the Mg-G-actin solution, to avoid dissociation of the capping protein from the capped barbed ends during the growth assay. Gelsolin-capped and standard filaments used as seeds were prepared at least 3 h in advance and were split into ADF-free and ADF-containing seeds 30 min before use. The amount of gelsolin-capped F-actin added as seeds was adjusted at each gelsolin:actin ratio, so as to work at a constant number of pointed ends added to the growth assay. The initial rates of filament depolymerization or elongation were measured from the change in fluorescence of pyrenyl-labeled actin, using a Spex Fluorolog2 spectrofluorimeter with excitation and emission wavelengths of 366 and 387 nm, respectively.

Measurement of Filament Turnover-- The turnover of actin filaments was measured as described previously (9, 11) from the decrease in fluorescence of epsilon -ADP-F-actin, at steady state in the presence of 50 µM epsilon -ATP, following a chase of ATP. The fluorescently labeled F-epsilon -ADP-actin solutions were supplemented with ADF 15 min before the ATP chase.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ADF Decreases the Steady-state Length of Actin Filaments-- The number of filaments in a solution of F-actin assembled at steady state was measured using them as seeds in the seeded polymerization assay described under "Materials and Methods." The behaviors of plant and human ADFs were compared. When seeds were assembled in the absence of ADF, the initial rate of filament elongation, which is indicative of the number of filaments in the seed solution, increased linearly with the concentration of F-actin present in the seed solution. On the other hand, when the seed solutions were pre-equilibrated in the presence of ADF, the initial rate of growth varied in a sigmoidal fashion with the concentration of F-actin in the seed solution (Fig. 1, a and b). At each concentration of F-actin in the seeds, a steady value of the initial rate of seeded polymerization was reached about 3 min following addition of ADF to the seed solution that is when the new steady state was established (data not shown). The rate measurements using F-actin + ADF seeds (Fig. 1) represent steady state measurements. The sigmoidal shape is consistent with the previous observation (9) that ADF causes the partial depolymerization of about 2 µM F-actin. Hence, when the actin concentration was lower than 2 µM in the F-actin seed solution containing 2 µM ADF, no filament ends could be measured in the growth assay. As the concentration of F-actin increased in the F-actin + ADF seed solution, the rate of filament growth eventually stopped increasing steeply and the curve became parallel to the control without ADF (Fig. 1, a and b). The increase in number of filaments reached at the plateau was itself a saturation function of the concentration of ADF present in the seed solution (Fig. 1c). The plant and human ADFs behaved similarly qualitatively (compare panels a and b in Fig. 1), but a larger number of ends was generated by plant ADF than by human ADF (panel c). Controls were run in which ADF at the same final concentration as in the sample was added to G-actin together with standard seeds at time 0. With ADF1 (Fig. 1c, open triangles) a notable increase in rate was recorded in the controls at high concentration of ADF, due to appreciable binding of ADF1 to the filaments during the mixing time (10-15 s). This was not observed with human ADF, which has been found to bind more slowly to F-actin (12). ADF does not behave as a severing factor which would catalytically continuously increase the number of ends with time, and which would produce more fragments when the concentration of the substrate F-actin, i.e. the number of available sites for severing, is increased, or when the concentration of the enzyme (here ADF) is increased. In contrast, ADF appears to change the steady-state length distribution of actin filaments in a manner dependent on the saturation of F-actin by ADF. The number of ends created by ADF stops increasing when the concentration of F-actin exceeds the ADF concentration. The maximum increase in number of filaments reached at saturation is moderate (about 6-fold for plant ADF, 4-fold for human ADF, Fig. 1c) as compared with the massive increase in turnover rate measured under similar conditions. Hence, in agreement with previous reports (9, 11, 18), the fragmentation activity of ADF cannot account for its effect on actin dynamics in bulk solutions.


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  		Fig. 1.   Increase in filament number in F-actin solutions in the presence of plant or human ADF. Panel a, solutions of F-actin at the indicated concentrations (total actin) containing 0 (), 1 (black-square), or 4 µM (black-triangle) ADF1 from A. thaliana were used as seeds to initiate filament growth, by 20-fold dilution into F buffer containing 1.6 µM MgATP-G-actin (10% pyrenyl-labeled). The initial rate of pyrene fluorescence increase was proportional to the number of filaments. Panel b, same experiment as in panel a, with 0 (), 6 (black-square), and 8 µM (black-triangle) human ADF. Panel c, ADF increases the number of filaments in a saturating fashion. The fold increase in the rate of filament growth from 1.6 µM G-actin derived from data in panels a and b is plotted versus the concentration of ADF1 (black-triangle) or human ADF () present in a 10 µM F-actin seed solution. The growth rate measured with seeds containing no ADF was taken equal to 1 by convention. The control curves: triangle , for ADF1; open circle , for human ADF, were obtained by 20-fold dilution of ADF alone at the indicated concentrations and of the control F-actin seeds (without ADF) into the 1.6 µM G-actin solution.

Solutions of F-actin at steady state in the presence of ADF contain a large pool of unassembled actin, which consists essentially in an increased amount of ATP-G-actin, and a high concentration (about 2 µM) of ADF-ADP-G-actin (9). ADF also greatly increases the nucleation of ADP-actin (12). One can imagine that small nuclei of ADF-ADP-G-actin dimers, for instance, exist at steady state in the seed solutions containing ADF. Such nuclei would actively elongate in the seeded polymerization assay. In this situation, the filament average length would not be actually affected by ADF, only nuclei would coexist with the filaments. To investigate this possibility, two experiments were performed. First, the effect of addition of the corresponding amount of ADF-ADP-G-actin together with the standard F-actin seeds was measured. No significant change was observed as compared with the sample. Second, the rate of dilution-induced depolymerization of the F-actin + ADF seeds was compared with the rate of depolymerization of standard F-actin seeds. In this dilution-induced depolymerization assay, only long enough filaments measurably contribute to the loss in pyrenyl-F-actin fluorescence, while nuclei are expected not to give a significant signal. The ADF-induced increase in the number of ends derived from this assay was exactly identical to the one derived from the seeded growth assay, demonstrating that the number of filaments was increased and the average filament length was decreased by ADF.

The above experiments do not provide insight in the mechanism by which the number of filaments is increased by ADF, which could be either enhanced spontaneous fragmentation or enhanced nucleation. They demonstrate that a steady number of filaments is maintained in the presence of ADF, in conditions under which practically all ADF is bound to F-ADP-actin and G-ADP-actin, which suggests that the lower average filament length results from the highly dynamic state of F-actin in the presence of ADF.

ADF Does Not Appreciably Decrease the Length of Filaments Severed by Gelsolin-- Populations of filaments of different average lengths were obtained by polymerizing actin (16 µM) in the presence of gelsolin at different gelsolin:actin ratios in the range 1:2000 to 1:100. Each F-actin solution was split in 2 samples, one of which was supplemented with 2.5 µM ADF. These two filament solutions were used as seeds to initiate polymerization from G-actin at different concentrations and derive the J(c) plots. Data displayed in Fig. 2 show that the J(c) plots obtained from F-actin and F-actin + ADF seeeds were superimposable at gelsolin:actin ratios above 1:300, that is when filaments contained less than 300 subunits (1 µm length) on average. At lower gelsolin:actin ratios (1:500, 1:1000, and 1:2000), the J(c) plot derived from F-actin + ADF seeds had a higher slope than the control plot derived from F-actin seeds, and it extrapolated to a value of the critical concentration (J = 0) lower than 0.5 µM, indicating that new uncapped barbed ends had been created by ADF in the seed solution. The association rate constant of G-actin to barbed ends is 10-fold higher than to pointed ends, hence the newly created filaments bring a large contribution to the observed rate, making the assay very sensitive to a small change in filament number. The rates of growth from F-actin seeds, J(c), and from F-actin + ADF seeds, J'((c), are expressed as a function of the concentrations of gelsolin-capped filaments, F, and of the concentration of new filaments induced by ADF, Phi , as follows,
J=k<SUP>P</SUP><SUB><UP>+</UP></SUB>F(c−C<SUP>P</SUP><SUB>C</SUB>) (Eq. 1)
J′=k<SUP>P</SUP><SUB><UP>+</UP></SUB>(F+&PHgr;)(c−C<SUP>P</SUP><SUB>C</SUB>)+k<SUP>B</SUP><SUB><UP>+</UP></SUB>&PHgr;(c−C<SUP>B</SUP><SUB>C</SUB>) (Eq. 2)
Leading to,
<FR><NU>J′</NU><DE>J</DE></FR>=1+<FR><NU>&PHgr;</NU><DE>F</DE></FR><FENCE>1+<FR><NU>k<SUP>B</SUP><SUB><UP>+</UP></SUB></NU><DE>k<SUP>P</SUP><SUB><UP>+</UP></SUB></DE></FR> · <FR><NU>c−C<SUP>B</SUP><SUB>C</SUB></NU><DE>c−C<SUP>P</SUP><SUB>C</SUB></DE></FR></FENCE> (Eq. 3)
The value of Phi  can be derived from the fit of the above equations to the data using the following values of the parameters: CCP = 0.5 µM, CCB = 0.08 µM, k+B = 10·k+P. This analysis of the data indicates that ADF increased the number of filaments by 14.5, 10.7, 5, and 0% at gelsolin:actin ratios of 1:2000, 1:1000, 1:500, and 1:250, respectively. This result is in agreement with earlier sedimentation velocity measurements (9) showing that at a gelsolin:actin ratio of 1:1000, filaments (3 µm long on average) were not appreciably shortened by ADF.


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  		Fig. 2.   Effect of ADF on the number of filaments in solutions of gelsolin-capped filaments. Solutions of F-actin, assembled at 16 µM in the presence of gelsolin at the indicated gelsolin:actin ratios, were supplemented with either 0 () or 2.5 µM ADF1 (open circle ) and used as seeds (20-150-fold dilution depending on gelsolin concentration) to initiate filament growth from G-actin (10% pyrenyl-labeled) at the indicated concentrations. The initial rate of growth J is plotted versus the concentration c of G-actin in the assay medium. At a gelsolin:actin ratio of 1:100, the plots were identical to those shown in panel d.

The conclusion that no barbed ends were created by ADF when filaments were capped by gelsolin at a gelsolin:actin ratio of at least 1:400 was confirmed by testing the G-actin sequestering effect of profilin in the absence and presence of ADF. When F-actin was capped by gelsolin at a gelsolin:actin ratio of 1:400, profilin sequestered actin (Fig. 3, circles), causing depolymerization of F-actin, in the presence and absence of ADF. The sequestering efficiency was greater in the presence of ADF, indicating that the steady-state concentration of ATP-G-actin was increased by ADF when barbed ends are capped. The increase in the steady-state concentration of ATP-G-actin by ADF has already been established when barbed ends are free (11). Using Equation 3 in Ref. 11, it was calculated that the steady-state concentration of ATP-G-actin was increased 2-fold, from 0.5 to 1 µM, by addition of 1.75 µM ADF1 to 10 µM F-actin. Different results were obtained at a gelsolin:actin ratio of 1:4000, which leaves a few barbed ends uncapped (Fig. 3, squares). Accordingly, the sequestering efficiency of profilin was greatly reduced, both in the absence and presence of ADF, due to the profilin-induced lowering of the concentration of ATP-G-actin when barbed ends are free (28). However, deriving the fraction of free barbed ends in the presence versus absence of ADF from these data is difficult because the depolymerizing effect of ADF by itself increases the sequestering effect of profilin, even though the number of barbed ends is increased by ADF. Together with the data shown in Fig. 2, these results confirm that at high enough gelsolin:actin ratio, all gelsolin-capped filaments remain capped in the presence of ADF, and no free barbed end is maintained at steady state.


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  		Fig. 3.   ADF increases the steady-state concentration of ATP-G-actin when barbed ends are capped by gelsolin, resulting in increased sequestration of actin by profilin. Gelsolin-capped filaments (10 µM F-actin, 1% pyrenyl labeled) at gelsolin:actin ratios of 1:400 (circles) and 1:4000 (squares) were supplemented with 0 (open symbols) or 1.75 µM (closed symbols) ADF1 and profilin at the indicated concentrations. The concentration of F-actin assembled at steady state was monitored by pyrene fluorescence.

ADF Increases the Turnover of Gelsolin-capped Filaments-- The effect of ADF on filament turnover was examined at different gelsolin:actin ratios ranging from 1:2000 to 1:100, as described under "Materials and Methods." The turnover was much faster in the presence than in the absence of ADF at all gelsolin concentrations (Fig. 4a). The turnover rate increased with the number of capped filaments, under conditions where the experiments described in the previous paragraph (Fig. 2, c and d) demonstrated that ADF did not create new barbed ends (gelsolin:actin < 1:300). To be absolutely sure that this rapid turnover was not mediated by the transient formation of barbed ends, due to either filament severing or nucleating activity of ADF, the turnover of gelsolin-capped, ADF-bound filaments was measured with addition of cytochalasin D (up to 150 nM) or capping protein beta 2 (up to 150 nM) together with the ATP chase. The turnover process was unaffected at all CD and gelsolin:actin ratios. In summary, ADF increases filament turnover in a pointed end-dependent fashion under conditions where the flux of subunits from the pointed ends to the barbed ends cannot occur, all barbed ends being blocked. The dependence of the turnover rate on ADF concentration, in the presence of gelsolin (gelsolin:actin = 1:300), showed a maximum at an ADF:actin ratio of 0.5 (Fig. 4b), as previously observed when barbed ends are free (9). This result was confirmed by steady-state ATPase measurements (data not shown).


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  		Fig. 4.   ADF increases the turnover of filaments capped by gelsolin. Panel a, F-actin (17 µM) was assembled from epsilon -ATP-G-actin in the presence of gelsolin at the following gelsolin:actin ratios. a, 1:300; b, 0; c, 1:1000; d, 1:300; e, 1:100, and preincubated with (b to e) or without (a) 5 µM ADF1 for 15 min before a chase of ATP was applied to the samples at time 0. The time dependence of the decrease in fluorescence of F-actin bound epsilon -ADP represents the turnover of actin filaments. Panel b, ADF concentration dependence of gelsolin-capped filament turnover. Conditions are as in panel a, with gelsolin:actin = 1:300, and ADF (in µM) as follows: 0 (a), 1 (b), 2 (c), 5 (d), 10 (e), 16 (f), and 20 (g).

ADF Elicits the Rapid Turnover of Actin Filaments in the Presence of Gelsolin and Arp2/3 Complex-- The Arp2/3 complex has been shown to cap the pointed ends of filaments with high affinity and to induce barbed end growth branching off the sides of filaments (24). The Arp2/3 complex appears recruited to regions of rapid filament turnover in response to signaling (30-33) and is thought to be responsible for the branching pattern of filaments in the lamellipodium of fibroblasts and motile keratocytes (34). Since ADF and Arp2/3 complex are both present in these motile extensions, it is important to examine how the turnover of filaments that are capped at their pointed ends may be affected by ADF. The Arp2/3 purified from bovine brain, which was used in this study, displays the characteristic delayed acceleration in the kinetics of spontaneous polymerization of actin (Fig. 5a), which has been previously observed for the Arp2/3 complex from Acanthamoeba castellanii (24).


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  		Fig. 5.   ADF increases the turnover of filaments capped by gelsolin at their barbed ends and by Arp2/3 complex at their pointed ends. Panel a, Arp2/3 complex purified from bovine brain nucleates F-actin assembly. MgATP-G-actin (2.5 µM, 10% pyrenyl labeled) was polymerized by addition of 0.1 M KCl and 1 mM MgCl2, in the absence (curve a) or presence (curve b) of 9.5 nM Arp2/3 complex. Panel b, F-epsilon ADP-actin (16 µM) was polymerized in the presence of gelsolin at a 1:300 gelsolin:actin ratio. The solution was split into different samples containing or not 5 µM ADF and/or 100 nM Arp2/3 complex. A chase of ATP was applied 1 h later to the samples, and the decrease in F-actin bound epsilon -ADP was monitored. Curve a, gelsolin-capped F-actin + Arp2/3, no ADF; curve b, gelsolin-capped F-actin + Arp2/3 + ADF; curve c, gelsolin-capped F-actin + ADF, no Arp2/3. Panel c, gelsolin-capped unlabeled F-actin (4 µM actin, gelsolin:actin = 1:300) was incubated for 1 h with or without 100 nM Arp2/3, as indicated. At time 0, 24 µM Tbeta 4 (from a 1.68 mM stock solution) and 6 µM ADF1 (from a 223 µM stock solution) were added simultaneously to the solution (150 µl) placed in a 1-cm light path cuvette and the absorbance at 310 nm was recorded. Panel d, gelsolin-capped F-actin seeds (16 µM actin, 1:300 gelsolin:actin ratio) were supplemented with: , 2.5 µM ADF1; triangle , 100 nM Arp2/3; open circle , 100 nM Arp2/3 and 2.5 µM ADF1, and diluted 40-fold into 10% pyrenyl-labeled G-actin at the indicated concentration in F buffer. diamond , , 0.15 µM cytochalasin D or capping protein was added to the growth assay carried out at 0.8 µM G-actin with the seeds containing Arp2/3 (diamond ) or Arp2/3 and ADF (). The initial rate of growth was measured.

The effect of ADF on the turnover of actin filaments capped at their barbed ends by gelsolin (at a gelsolin:actin ratio of 1:300) and at their pointed ends by Arp2/3 was examined next. As demonstrated above, these short filaments are not fragmented by ADF. ADF induced the rapid turnover of the barbed end-capped filaments to almost the same extent (about 2-fold difference) in the presence or absence of Arp2/3 (Fig. 5b). In an independent experiment (Fig. 5c), it was verified that gelsolin-capped F-actin (4 µM), incubated with 0.1 µM Arp2/3, depolymerizes rapidly (in 5 min) upon addition of 24 µM thymosin beta 4 and 6 µM ADF. The initial rate of depolymerization was 2.2-fold lower than in the absence of Arp2/3. In conclusion, Arp2/3-capped filaments can be induced to depolymerize rapidly from their pointed ends upon addition of ADF.

To understand the mechanism of ADF-induced rapid turnover in the presence of gelsolin and Arp2/3, the number and nature of ends in the F-actin solution at steady state in the presence of gelsolin and Arp2/3 was examined as follows. Gelsolin-capped filaments (16 µM actin, 50 nM gelsolin) were incubated for 2 h or more with 0.1 µM Arp2/3 with or without 2.5 µM ADF1 and were used as seeds to initiate polymerization from G-actin at different concentrations and derive a J(c) plot. In principle, if Arp2/3 only caps the pointed ends of gelsolin-capped filaments, the rate of growth should be zero at all actin concentrations. Fig. 5d shows that this was not the case. The gelsolin-capped filaments preincubated with Arp2/3 did nucleate actin growth actively, in a range of concentrations extending below 0.5 µM (the pointed end critical concentration), indicating that barbed ends had been generated by addition of Arp2/3 to the solution of gelsolin-capped filaments (open triangles in Fig. 5d). Accordingly, the growth process from these seeds at 0.8 µM G-actin was greatly inhibited in the presence of either cytochalasin D or capping protein beta 2, both of which cap the barbed ends. When ADF was added to the solution of gelsolin-capped filaments containing Arp2/3, a larger number of barbed ends was found (open circles in Fig. 5d), and barbed end growth again was inhibited by cytochalasin D. Since Arp2/3 does not sever filaments (24), the generation of barbed ends by Arp2/3 in a solution of gelsolin-capped filaments is presumably due to nucleation from G-actin, which is present at 0.5 µM, the pointed end critical concentration, when Arp2/3 is added to this solution. To test this possibility, Arp2/3 (20 nM) was added to a solution of pyrenyl-labeled G-actin at 0.5 µM in polymerization buffer. Nucleation and barbed end polymerization were observed to occur much faster than in the control sample containing no Arp2/3 (data not shown). In conclusion, when Arp2/3 complex is added to gelsolin-capped filaments, barbed end nucleation occurs, thus generating non-capped barbed ends in addition to gelsolin-capped filaments. Upon addition of ADF, the number of barbed ends generated by Arp2/3 is even greater. This measurement indicates that ADF can induce pointed end depolymerization in the presence of Arp2/3, thus increasing the concentration of ATP-G-actin, thereby facilitating nucleation of barbed ends by Arp2/3. The above evidence for nucleation of barbed ends by addition of Arp2/3 to a solution of gelsolin-capped filaments allows us to understand the reactions that occur in the rapid turnover measured in the presence of gelsolin, ADF, and Arp2/3 (Fig. 5b, middle curve). The F-actin solution then contains both free (Arp2/3-generated) and gelsolin-capped barbed ends, and Arp2/3-capped pointed ends, which depolymerize rapidly upon binding ADF (Fig. 5, b and c). The possibility that binding of ADF to capped pointed ends occurs, causing pointed end dissociation of the terminal subunit is not surprising since ADF is known to bind well to DNase I-actin columns, and DNase I is a pointed end capper. The pointed end disassembly flux is balanced by assembly onto the barbed ends nucleated by Arp2/3. Overall, the present results show that actin filaments are still dynamic in the presence of ADF, barbed end capping proteins, and Arp2/3.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present work shows that two distantly related ADFs, ADF1 from A. thaliana and human ADF, control the turnover and the distribution in length of actin filaments in qualitatively similar but quantitatively different fashions. ADFs cause a moderate increase in filament number, but the time dependence, the F-actin, and the ADF concentration dependence of the increase in number are not consistent with the view that ADF works as a severing factor, and one cannot speak of a severing rate. Rather, the data indicate that as filaments are saturated by ADF, the length distribution of filaments is changed toward a shorter, limited average length. The change in length distribution accompanies the establishment of a new steady state upon addition of ADF to F-actin. Plant ADF, which is close to yeast and amoeba ADFs, caused a larger decrease in average length than human ADF. At an ADF:actin ratio of 0.5, conditions where the maximum (30-fold) increase in turnover of filaments was recorded, the number of filaments was increased 4-fold by plant ADF, and less than 2-fold by human ADF. Under physiological conditions, i.e. at an ADF:actin ratio of 0.1 to 0.2, the increase in filament number must be less significant. These figures demonstrate that the increase in filament number contributes very little in the overall increase in filament turnover elicited by ADF.

The distribution in length of all polymers is determined by their thermodynamic properties of self-assembly. These properties may be used and modulated in vivo by regulatory proteins (36). The two pathways, depolymerization-nucleation-elongation or fragmentation-reannealing, are thermodynamically equivalent in the control of the length distribution. Therefore, the following two expressions equally describe the dependence of the average length L (in µm) on the nucleation (37) or on the fragmentation equilibrium constant (38),
L=<FR><NU>1</NU><DE>m</DE></FR> · <RAD><RCD><FR><NU>C<SUB>0</SUB>−C<SUB>1</SUB></NU><DE>C<SUB>C</SUB> · &sfgr;</DE></FR></RCD></RAD> (Eq. 4)
L=<FR><NU>1</NU><DE>m</DE></FR> · <RAD><RCD><FR><NU>(C<SUB>0</SUB>−C<SUB>1</SUB>)</NU><DE>2 · K<SUB>f</SUB></DE></FR></RCD></RAD> (Eq. 5)
where m is the number of subunits per µm length (m = 360), C0 is the total actin concentration, C1 the monomer concentration, sigma  is a nucleation constant, Kf the fragmentation-reannealing equilibrium constant. Any ligand that affects the stability, i.e. the critical concentration for assembly, of the filaments also affects the value of Kf. Specifically, phalloidin, which stabilizes filaments, shifts the length distribution toward very long filaments at steady state; in contrast, capping proteins, which prevent reannealing by blocking one end, have the opposite effect and cause a decrease in average length. ADF is a less extreme case, nevertheless, in destabilizing the filament, ADF increases the fragmentation/reannealing equilibrium constant, hence decreases the average length of the filaments. The "severing" effect of ADF therefore is the normal consequence of the enhanced dynamics of the filament. The steep dependence of L on Kf shows that a ligand that tends to increase the value of Kf causes a more pronounced decrease in average length if filaments are initially long, than if they are short. In other words, when filaments are short and concentrated, fragmentation is balanced by reannealing. Accordingly, our quantitative measurements of the effect of ADF on filament number actually show that the average length is weakly reduced by ADF when filaments are shortened by the severing action of gelsolin.

A large increase in filament turnover was observed upon addition of ADF to gelsolin-capped short filaments, although the number of filaments remained unchanged. The turnover of the population was proportional to the steady-state number of filaments, supporting the view (11) that ADF causes a large increase in the rate of pointed ends depolymerization. The flux of subunits cannot occur, under such conditions, from the pointed to the barbed ends of the filaments, as described in the classical treadmilling process (39). This result demonstrates that ADF can shuttle subunits depolymerizing from some pointed ends to other pointed ends. To explain the energetic difference between two types of pointed ends, the cooperativity of ADF binding to the filaments was considered. The cooperative binding of ADF to F-actin (12-14) results in the coexistence of two discrete populations of filaments, the fully ADF-decorated filaments and the bare filaments. Filaments fully decorated by ADF are known to depolymerize rapidly from their pointed ends (11) and to be maintained at steady state by a high concentration of ATP-G-actin. In contrast, bare filaments depolymerize slowly from the pointed ends and are stabilized by a lower steady-state concentration of ATP-G-actin. The two populations of filaments therefore coexist in solution pending a flux of subunits from one to the other. In the absence of capping proteins, the flux mainly occurs in the traditional treadmilling fashion, from the pointed ends of ADF-bound filaments to the barbed ends of bare filaments which have a low critical concentration (0.08 µM). But when barbed ends are capped, the actin subunits depolymerizing from the ADF-bound filaments associate to the pointed ends of bare filaments, as illustrated in Fig. 6. Flux then still occurs. The filament turnover rate, in the presence as well as in the absence of gelsolin, is always limited by pointed end depolymerization, simply a higher steady-state concentration of ATP-G-actin is established when barbed ends are capped, so that the assembly flux onto pointed ends (to which actin associates with a low k+P) can balance the disassembly flux. As ADF-decorated filaments depolymerize, the released ADF rebinds cooperatively to another filament. By visiting filaments one by one, ADF elicits the turnover of the whole population, even at low ADF:actin ratios. The flux rate in fact is maximum when the concentrations of the "donor" and of the "acceptor" filaments are equal, which may account for the ADF concentration dependence of the turnover showing a maximum at an ADF:actin ratio of 0.5. This process of fiber-by-fiber renewal of the filaments shares some similarity with the dynamic instability behavior of microtubules, whose role in morphogenesis has been emphasized (40). Similarly, ADF appears to have a morphogenetic function in myofibril assembly in nematode development (41).


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  		Fig. 6.   Model for the rapid turnover of gelsolin-capped filaments in the presence of ADF. The cooperative binding of ADF to F-actin generates two populations of filaments of different stabilities, which coexist pending a flux of subunits from one to the other. The donor ADF-F-actin depolymerizes rapidly, while the acceptor undecorated F-actin polymerizes at the same rate.

ADF enhances the turnover of actin filaments in the presence of Arp2/3, even when the filament barbed ends are capped by gelsolin. Analysis of the F-actin solutions containing gelsolin and Arp2/3 shows that Arp2/3 generates new barbed ends when it is added to gelsolin-capped filaments. This piece of data is fully consistent with the nucleating activity of Arp2/3, however, it had not been foreseen initially and is not in agreement with Mullins et al. (24). In view of the present data, one of the roles of Arp2/3 complex may well be to maintain the steady occurrence of non-capped, rapidly growing barbed ends at the surface of Listeria (42), where it is recruited (31) by interacting with ActA (43). The consequence of the nucleating activity of Arp2/3 is that rapid turnover of actin filaments can be elicited upon addition of ADF to F-actin containing capping proteins and Arp2/3. In conclusion, the early view (24, 35) that in the cell Arp2/3 acts as a strong pointed end capper blocking all monomer-polymer exchange reactions at the pointed ends has to be amended. The present work accounts for the previous in vivo observations of highly dynamic actin networks in the actin tail of Listeria, in the lamellipodium, or in the actin patches in yeast, all of which are motile regions where ADF and the Arp2/3 complex are localized (11, 8, 33, 43-46).

    ACKNOWLEDGEMENT

We thank Rajaa Boujemaa for the purification of capping protein from bovine erythrocytes.

    FOOTNOTES

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

To whom correspondence should be addressed: LEBS, CNRS, Gif-sur-Yvette, France. Tel.: 33-1-69-82-34-65; Fax: 33-1-69-82-31-29; E-mail: carlier@lebs.cnrs-gif.fr.

2 F. Ressad, D. Didry, C. Egile, D. Pantaloni, and M.-F. Carlier, manuscript in preparation.

    ABBREVIATIONS

The abbreviation used is: ADF, actin depolymerizine factor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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