INTRODUCTION

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Profilin and actin depolymerizing factor (ADF¹/cofilin) are essential actin-binding proteins that play a key role in the control of actin dynamics and actin-based motility processes. These two proteins fulfill their functions by interacting with actin in different ways.

Profilin binds ATP-G-actin specifically. Although profilin does not bind F-actin, the profilin-ATP-G-actin complex contributes to filament assembly, at the barbed end exclusively (1-4). When filaments are assembled at steady state in the presence of profilin, ATP-G-actin and profilin-ATP-G-actin can both be considered as polymerizable actin monomers undergoing monomer-polymer exchange at the barbed ends, therefore profilin lowers the steady-state concentration of ATP-G-actin (3). When all barbed ends are blocked by capping proteins, profilin only sequesters G-actin.

ADF/cofilin, on the other hand, specifically recognizes the ADP-bound form of both G- and F-actin. Hence, ADF too participates in filament assembly, but in a manner different from profilin; ADF-bound F-actin dissociates much faster than F-actin from the pointed ends of actin filaments, which results in a large enhancement of filament treadmilling (5). Actin-based motility of Listeria monocytogenes in the cytoplasm of infected cells, or the forward movement of the leading edge, which are supported by the treadmilling of actin filaments, are therefore enhanced by ADF (6). The effect of ADF on filament turnover is accompanied by the partial depolymerization of F-actin. The mechanistic Scheme I (see Fig. 1), which was proposed (5) to account for the effects of ADF on actin dynamics, implies the following. First, the steady-state pool of unassembled actin consists of ADP-G-actin and ATP-G-actin both in the free and ADF-bound states. Since ADF has a 100-fold higher affinity for ADP-G-actin than for ATP-G-actin under physiological ionic conditions (5, 7), a large proportion of ADP-G-actin is thought to be in complex with ADF, while ATP-G-actin is thought to be essentially free. Next, ADF is in rapid equilibrium with G-actin (7), so that nucleotide exchange, which is strongly inhibited on ADF-ADP-G-actin complex, occurs on free ADP-G-actin so as to regenerate ATP-G-actin which polymerizes better than ADP-G-actin. Finally, the establishment of a faster treadmilling by ADF implies that the steady-state concentrations of ADP-G-actin and ATP-G-actin increase until the fluxes of nucleotide exchange and polymerization onto barbed ends become equal to the large dissociation flux of ADF-ADP-actin from the pointed ends.

To assess the validity of Scheme I (shown in Fig. 1) and to model the effects of ADF on actin dynamics, it is necessary to elaborate methods allowing to measure the concentrations of the different G-actin species at steady state in the presence of ADF. In particular, the steady-state concentration of ATP-G-actin, [G_T], is an important parameter, since it directly controls the treadmilling flux, i.e. the rate of subunit addition to barbed ends at steady state, which equals k₊^B·[B]·([G_T]  C_C^B), where k₊^B is the rate constant for association of ATP-G-actin to barbed ends, [B] is the concentration of non-capped barbed ends, and C_C^B is the critical concentration for actin assembly at the barbed ends.

Once the steady-state parameters for actin assembly in the presence of ADF alone are known, the complexity of the system can be increased by adding profilin in addition to ADF, and using the same methods, to determine how the dynamics of actin filaments are affected by the presence of the two proteins, and discover which new regulatory properties arise from the cumulated effects of the two proteins.

The present work focuses on the above issues. We show that ADF causes a 3-fold increase in the steady-state concentration of ATP-G-actin, which accounts for the enhancement of treadmilling within Scheme I (see Fig. 1). We find that profilin synergizes with ADF to increase the turnover of filaments. In the presence of both ADF and profilin, the rate of treadmilling is increased 125-fold. The ADF and profilin dependence values of the rate of treadmilling are quantitatively reproduced by computer modeling of Scheme II (shown in Fig. 9), using the experimental values (4, 5, 7) of the rate parameters for interaction of ADF and profilin with actin. The in vivo consequences of the synergy observed in vitro between ADF and profilin are discussed.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Scheme I for the acceleration of treadmilling by ADF. The larger size of the ADF-ADP-actin (D-ADF) and ATP-G-actin (T) species indicates that they are the predominant monomer forms of actin at steady state. Symbols representing the rate constants are described in Table I. The formation of the T4-ATP-G-actin complex causes actin depolymerization without affecting the dynamics of F-actin at steady state.

	MATERIALS AND METHODS

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Proteins-- Actin was purified from rabbit muscle acetone powder (8) and isolated as Ca-ATP-G-actin by gel filtration (9) on Sephadex-G200 in G buffer (5 mM Tris-Cl, pH 7.6, containing 0.2 mM ATP, 0.1 mM CaCl₂, 1 mM dithiothreitol, and 0.01% NaN₃). Mg²⁺ exchange for bound Ca²⁺ on G-actin was performed as described (4). Thymosin ₄ was purified from bovine spleen as described (10). Vertebrate profilin I was also purified from bovine spleen by poly-L-proline chromatography (10). Recombinant Arabidopsis thaliana profilin 1 was expressed in Escherichia coli and purified by poly-L-proline chromatography as described (4). Recombinant ADFs (human ADF and A. thaliana ADF₁) were expressed in E. coli and purified by DEAE-cellulose and SP-trisacryl ion exchange chromatography as described (5). Protein concentration was determined spectrophotometrically using the following values of the extinction coefficients: ₂₉₀^0.1% = 0.617 cm¹ for actin; ₂₇₇ = 0.015 µM¹.cm¹ for profilin; ₂₇₇^0.1% = 0.89 cm¹ for ADF₁, ₂₇₇^0.1% = 0.64 cm¹ for human ADF. The concentration of T₄ was determined using the bicinchoninic acid assay with bovine serum albumin as a standard.

Measurement of the Concentration of Unassembled Actin at Steady State-- The concentration of unassembled actin was determined by a sedimentation assay. Actin was polymerized at pH 7.5 in the presence of 2 mM MgCl₂ and 0.1 M KCl, and supplemented with ADF, profilin, or T₄ as indicated. Samples containing no ADF were centrifuged at 400,000 × g, 20 °C, for 30 min, after a 16-h incubation. Samples containing ADF were centrifuged similarly except within 15-30 min following addition of ADF, since the steady state is reached rapidly in the presence of ADF and the consumption of ATP is high (5), precluding long incubation times. It was verified that results identical to those presented here were obtained in the presence of an ATP-regenerating system (5 mM creatine phosphate, creatine kinase). The concentration of unassembled actin in the supernatants was determined by scanning the Coomassie Blue-stained SDS gels of electrophoresed samples (Arcus II, Agfa Corp., Orangeburg, NY) and comparing to actin standards electrophoresed on the same gel. The NIH Image analysis program was used.

Measurement of the Treadmilling Rate of Actin Filaments-- The rate of filament treadmilling was estimated using two different methods (5), as follows. First, measurement of the rate of ATP hydrolysis that accompanies the association of ATP-G-actin subunits to the barbed ends at steady state yields the treadmilling rate. G-actin (15 µM) was equilibrated in G buffer containing [-³²P]ATP (Amersham Pharmacia Biotech), and polymerized. When steady state was reached, the solution was split into several samples supplemented with the desired concentrations of ADF and profilin. The steady state rate of ATP hydrolysis was measured by removing 50-µl aliquots from the solution at intervals, for periods of up to 6 h, quenching the reaction in 10 mM molybdate, 1 N HCl and processing for extraction of the -³²P-labeled phosphomolybdate complex (5). Second, the turnover of filaments was estimated by measuring the rate at which fluorescently labeled -ADP-F-actin filaments assembled from -ATP-G-actin subunits became non-fluorescent ADP-F-actin filaments following addition of ATP in the medium, via the consecutive steps of dissociation of -ADP-actin from the pointed end, exchange of ATP for bound -ADP on G-actin in the medium (the fluorescence of -ADP is 5-fold lower in the free than in the actin-bound state), and association of ATP-G-actin to barbed ends. The experiments were carried out at a concentration of 15 µM -ADP-F-actin polymerized under physiological ionic conditions in the presence of 50 µM -ATP, and supplemented with ADF and/or profilin, as indicated, 10 min before the addition of 0.5 mM ATP in the medium. The fluorescence of -ADP was monitored at 20 °C in a Spex spectrofluorimeter with excitation and emission wavelengths of 350 and 410 nm, respectively.

Measurement of the Concentration of Filament Ends in F-actin Solutions Using CapZ-- The property of CapZ to cap the barbed ends with a 10¹⁰ M¹ affinity (11) was used to count the number of filaments in an F-actin solution, using immunodetection of CapZ in the pellets of sedimented F-actin. Mg-ATP-actin (25 µM) was polymerized to steady state at pH 8.0 in the presence of 1 mM MgCl₂ and 0.1 M KCl. The solution was split in two samples, one of which was supplemented with 0.5 mM CaCl₂ and 50 nM gelsolin. The drop in viscosity due to the severing activity of gelsolin was checked to occur. When steady state was reached (30-60 min following the onset of polymerization), CapZ (25 to 50 nM) was added to the two F-actin samples. Gentle mixing was provided by either overturning the tubes or slowly pipetting the samples using the truncated tip of a 1000-µl Eppendorf pipette. The two F-actin solutions were then split into several samples containing or not Arabidopsis or human ADF (10 µM). 15 min later, the samples (400 µl) were centrifuged at 400,000 × g for 40 min at 20 °C in a Beckman TL 100 ultracentrifuge. The supernatants were removed, the walls of the tubes were thoroughly washed with G-buffer and the pellets of F-actin were resuspended in 80-120 µl of SDS-containing denaturing buffer, boiled and submitted to SDS-polyacrylamide gel electrophoresis on 10% acrylamide gels (12), followed by immunoblotting using the 5B12 anti-CapZ mouse monoclonal antibody (11) raised against the  subunit of muscle CapZ (this antibody recognizes both 1 and 2 isoforms of CapZ). A horseradish peroxidase-derivatized anti-mouse IgG raised in sheep was used as a second antibody. The amount of CapZ bound to F-actin in each sample was derived from the ECL (Amersham Pharmacia Biotech) chemiluminescence detection patterns of CapZ, with quantitative comparison with standards of pure CapZ in the appropriate concentration range.

Simulation of the Turnover of Actin Filaments in the Presence of ADF and Profilin Scheme II (Fig. 9)-- The concentrations of the different G-actin species coexisting with actin filaments at steady state in the presence of ADF and profilin can be determined by computer modeling of the establishment of steady state through the following set of kinetic steps, which describe Scheme I. ADF and profilin are considered in rapid equilibrium binding with ATP-G-actin and ADP-G-actin. The concentration of barbed ends, F_b, is constant and is not affected by ADF or profilin. On the other hand, pointed ends can be unliganded (F_p) or ADF-bound (F_p-ADF), with different dissociation rate constants and F_p + F_p-ADF = F_b = constant. The concentrations of total actin (polymerized and non-polymerized) and of free ADF were initially set. The total concentration of ADF at steady state was derived as the sum of the concentrations of free and ADF-bound species. The concentration of polymerized actin was derived as the difference between total actin and the sum of all forms of G-actin at steady state. The molar fraction of ADF-bound F-actin was equal to the fraction of ADF-bound pointed ends, [F_p-ADF]/([F_p] + [F_p-ADF]).The following set of rapid equilibria and kinetic steps were used in the modeling, where T, D, and P represent ATP-G-actin, ADP-G-actin, and profilin, respectively. 2. Kinetics of interaction of ADF with F-actin, in particular pointed ends.  3. Association and dissociation processes at filament ends.

	RESULTS

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ADF Causes an Increase in the Steady-state Concentration of ATP-G-actin-- It has been established that the addition of ADF to an F-actin solution results in binding of ADF to F-actin, followed by partial depolymerization of F-actin (5, 7). The steady-state concentration of unassembled actin varies between 0.5 and 2.5 µM (for Arabidopsis ADF₁) and between 1 and 6 µM (for human ADF) as pH is increased from 6.8 to 8.2 (7). To quantitate the concentration of ATP-G-actin, which actively participates in barbed end growth at steady state, use was made of thymosin ₄ as a G-actin sequestering protein that selectively binds ATP-G-actin (13). Addition of increasing amounts of T₄ to F-actin results in depolymerization of F-actin as the T₄-ATP-G-actin (TA) complex is formed. The increase in [TA] was quantitated by analysis of the gel patterns of unassembled actin present in the supernatants of sedimented samples of F-actin assembled in the presence of ADF and T₄ (see "Materials and Methods").

(Eq. 1)

(Eq. 2)

(Eq. 3)

View larger version (18K): [in this window] [in a new window]   Fig. 2.   ADF increases the steady-state concentration of ATP-G-actin. Panel A, sequestration of ATP-G-actin by T4 in the presence of different concentrations of ADF. Actin (total concentration 9 µM) polymerized in the presence of 0 (), 5 (), 10 (), and 15 () µM ADF1 was supplemented with T4 as indicated. The amount of unassembled actin was derived from analysis of the gel patterns of the supernatants of sedimented F-actin. The value of the slope m is indicated on each line. Panel B, the steady-state concentration of ATP-G-actin is derived from the slope of plots like those shown in panel A, using Equation 3. Assays were carried out with Arabidopsis () and human () ADF. Bars indicate the scatter of the data over three experiments.

1

1

1

1

1

Profilin Synergizes with ADF to Enhance Filament Turnover-- The rate of ATP hydrolysis and the rate of exchange of F-actin-bound -ADP for ADP following an ATP chase were used as measures of treadmilling. These rates were measured at different concentrations of ADF and in the presence of increasing amounts of profilin. Fig. 3 (A and B) shows that profilin elicited a large increase in the rate of treadmilling only when ADF was present. In the absence of ADF, the increase in treadmilling rate was 25% (4), practically undetectable in the present experiment. In the presence of either plant or human ADF, the increase in treadmilling rate displayed a saturation behavior upon increasing profilin concentration (Fig. 3C). The saturation behavior is thought to be due to the gradual increase in the contribution of profilin-actin (and decrease in the contribution of G-actin) to monomer-polymer exchanges at the barbed ends. At high concentration of profilin, these reactions are fed exclusively by profilin-actin complex at its own critical concentration (3).²

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Profilin synergizes with ADF to increase the turnover of actin filaments. Panel A, ATPase measurement of filament turnover. The dependence of the ATPase rate of F-actin (15 µM total actin) on ADF concentration was measured in the presence of 0 (), 1 µM (), 2 µM (), and 5 µM () bovine profilin. Panel B, fluorescence measurement of filament turnover. F--ADP-actin (15 µM total actin) assembled at steady state in the presence of 50 µM -ATP was supplemented with: a (and inset), no addition or 2 µM profilin; b, 5 µM ADF1; c, 5 µM ADF1 and 10 µM plant profilin; d, 5 µM ADF1 and 2 µM bovine profilin. The fluorescence of bound -ADP was recorded versus time following a chase of 0.5 mM ATP applied at time zero. Note the 25% higher fluorescence of F-actin-bound -ADP when ADF is also bound to F-actin. Panel C, different properties of profilin involved in the increase in treadmilling. The steady state ATPase rate of F-actin (13.8 µM total actin) was measured in the presence of 5 µM ADF1 and the indicated concentrations of bovine spleen profilin () or Arabidopsis profilin 3 (). Circles refer to experiments carried out with Mg-actin, triangles () to those performed with Ca-actin.

(Eq. 4)

1

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Profilin lowers the steady-state concentration of ATP-G-actin in the presence as well as in the absence of ADF. The amount of ATP-G-actin sequestered by T4 was measured as described under Fig. 2, in the absence of ADF and profilin (), or in the presence of 2 µM bovine profilin (), of 10 µM ADF1 (), or of ADF1 and profilin together (). The profilin-elicited decrease in slope, both in presence or absence of ADF, provides evidence for the lowering of ATP-G-actin concentration.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Role of the rate of nucleotide exchange in the concentration of unassembled actin in the presence of ADF. Actin (total concentration 13.5 µM) was polymerized in the presence of 5 µM ADF1 and the indicated concentrations of bovine profilin (, bottom inset) or plant profilin (, top inset). The gel patterns of unassembled actin in the supernatants (inset) were scanned and compared with standards to derive the concentrations. Each data point on the graph corresponds to the bands shown on the gels.

View larger version (40K): [in this window] [in a new window]   Fig. 6.   Schematic representation of the different forms of monomeric actin at steady state in the presence of ADF and profilin. The diagram is drawn using data obtained with Arabidopsis ADF1 and bovine profilin. The concentration of ADP-G-actin was assumed to be much smaller than that of ADF-ADP-G-actin. In the presence of ADF alone, the concentration of ADF-ADP-G-actin was calculated as the difference between total unassembled actin (1.8 µM) and the measured concentration of ATP-G-actin (0.27 µM). In the presence of both ADF and profilin, the measured amount of total unassembled actin was 0.9 µM (Fig. 5), the concentration of free ATP-G-actin was 0.11 µM, and the concentration of profilin-ATP-G-actin was such that ([ATP-G-actin] + [profilin-ATP-G-actin]  CCB) was 5-fold higher than in the absence of profilin, leading to [profilin-ATP-G-actin] = 0.7 µM. The concentration of ADF-ADP-G-actin was then calculated as the difference between total unassembled actin and total ATP-bound actin, giving 0 µM. Hence, the amount of ADF-ADP-actin is greatly depressed by profilin.

Concentration of Filament Ends in F-actin Solutions in the Presence or Absence of ADF-- Proteins of the ADF/cofilin family were early thought to cause rapid depolymerization of actin filaments via a pH-dependent severing effect (14-17), suggested by the rapid drop in fluorescence of pyrenyl- or NBD-labeled F-actin following addition of ADF. This interpretation was later reappraised (5, 7) by kinetic experiments, which showed that upon addition to F-actin, ADF first bound cooperatively to F-actin with a major quenching of fluorescence. Then, partial depolymerization to a new steady state was reached rapidly, via the ADF-enhanced depolymerization from the pointed ends. It was shown that binding of ADF to F-actin increases the twist of the filament (18). ADF also lowers the viscosity of F-actin solutions. The structural alterations parallel the decrease in thermodynamic stability of F-actin linked to ADF binding. Spontaneous fragmentation of filaments, which occurs to a low extent in standard F-actin solutions, as a result of brownian agitation, therefore is likely to be enhanced when ADF is bound to F-actin. To get an estimate as accurate as possible of the rate constants involved in the treadmilling of F-actin in the presence of ADF, it is necessary to quantitate the exact number of filaments in the absence and presence of ADF. Carrying out such measurement has thus far been an elusive problem, due to the many drawbacks linked to different methods.

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Estimation of the concentration of filament ends in the presence and absence of ADF using CapZ. CapZ (25 nM) was added to F-actin (25 µM tottal actin) preassembled either in the absence of gelsolin (a-c) or in the presence of 50 nM gelsolin (d and e). 10 µM ADF1 (b and e) or human ADF (c) were added. The concentration of CapZ in the resuspended pellets of sedimented samples was estimated by immunoblot and comparison with standards (closed circles, numbered 1-4) containing the indicated amounts of CapZ. Middle panel, ECL pattern; top panel, densitometric scanning of the above; bottom panel, calibration curve and interpolation of the data (arrows). These results are from experiment 2 in Table II.

View larger version (27K): [in this window] [in a new window]   Fig. 8.   ADF causes a rapid redistribution in length of the filaments. The experiment was conducted as detailed under Fig. 7. F-actin preincubated for 20 min with 56 nM CapZ was supplemented () or not () with 10 µM ADF1 (arrow). The amount of F-actin-bound CapZ was determined after sedimenting the samples at the times indicated. Inset: immunoblot of the data shown in the main frame. a-c, CapZ standards (0.25, 0.5, and 1.0 pmol, respectively); d-f, F-actin-bound CapZ at times 15, 60, and 100 min following addition of ADF. d-f, controls without ADF; d*-f*, ADF-containing samples.

Modeling of the Dynamics of F-actin in the Presence of ADF and Profilin-- The steady-state concentrations of ATP- and ADP-G-actin and of their complexes with ADF and profilin were obtained by computer-simulation as described under "Materials and Methods," using the values of equilibrium and rate parameters listed in Table I, and a steady-state Scheme II (shown in Fig. 9), which is an extension of Scheme I incorporating the effect of profilin. The resulting dependence values of the calculated concentrations of the different G-actin species on total ADF and profilin concentrations are displayed in Fig. 10. The calculated curves show the same trend as, and in most cases a satisfactory quantitative agreement with the experimental curves, which argues in favor of the proposed Scheme II. The main features of the calculated curves that are supportive of Scheme II are (i) the increase, followed by a decrease, in ATP-G-actin concentration, as ADF concentration is increased; (ii) the accumulation of a large amount of ADF-ADP-G-actin; (iii) the severalfold increase in the ATP-bound G-actin when profilin is added to F-actin in the presence of ADF, and corresponding decrease in ADF-ADP-G-actin.

View larger version (24K): [in this window] [in a new window]   Fig. 9.   Scheme II for the combined effects of ADF and profilin on F-actin dynamics at steady state. This scheme is an extension of Scheme I incorporating the role of profilin (P) acting in synergy with ADF to increase the efficiency of treadmilling.

	DISCUSSION

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The present results allow one to understand quantitatively how the dynamics of actin assembly at steady state is controlled by ADF and profilin to elicit the rapid barbed end growth of actin filaments. It is demonstrated that the addition of ADF to F-actin solutions causes an increase in the steady-state concentration of ATP-G-actin. This increase is quantitatively consistent with the measured increase in treadmilling rate, which equals k₊^B·([ATP-G-actin]  C_C^B). The experiments confirm the validity of the model that we proposed for ADF function (5). It should be emphasized that if the large increase in turnover of actin filaments was generated by a severing activity of ADF (ADF then would have to increase the number of filaments by more than one order of magnitude), the steady-state concentration of ATP-G-actin would remain unchanged (21), because the increase in number of depolymerizing pointed ends would equal the increase in number of polymerizing barbed ends. In other words, fragmentation increases the turnover of the bulk solution of F-actin, not of the individual filaments, hence fragmentation cannot account for the effect of ADF on actin-based motile processes. In contrast, Scheme II relies on an end-specific effect of ADF on actin kinetics and leads to an increase in steady-state barbed end growth of individual filaments, which is the relevant process in actin-based motility. Modeling the ADF concentration dependence of the steady state according to this scheme generates an increase in the concentration of ATP-G-actin, followed by a decrease in quantitative agreement with experimental observations. Further measurements of the concentration of filaments using CapZ show that, while spontaneous fragmentation of actin filaments is enhanced about 3-fold by ADF, most likely as a result from the more fragile structure of ADF-F-actin, the moderate 27% increase in filament number cannot explain the large increase in turnover.

The ADF-induced increase in the steady-state concentration of ATP-G-actin implies that, due to the law of mass action, the equilibria of ATP-G-actin with sequestering proteins are shifted toward complex formation, leading to an increased sequestration. Therefore, in vivo, the reported actin depolymerizing activity of ADF is due in part to its intrinsic property to partially depolymerize actin into ADF-ADP-G-actin, but also results from the increase in all complexes of ATP-G-actin with sequestering proteins like thymosin ₄.

At high concentration of ADF (above half a molar equivalent to total actin), the steady-state concentration of ATP-G-actin and the rate of treadmilling both reach a maximum and begin to decrease. As outlined in a previous paper (7), this decrease is explained by the fact that, as the ADF-ADP-G-actin complex is stabilized by ADF binding, its association to filament ends becomes predominant over its dissociation into free ADF and ADP-G-actin. The pathway leading to ATP-G-actin association to barbed ends via nucleotide exchange then is depressed and the cycle rate is slowed down as a gradual shift takes place from the steady-state cycle situation with associated ATP hydrolysis toward the equilibrium situation in which monomer-polymer exchange reactions are fed by ADF-ADP-actin (see Fig. 10C). The implication of this result may be important in vivo, to understand the effects of overexpression of ADF or of GFP-tagged ADF. Depending on the degree of overexpression and the relative original concentrations of actin and ADF, an increase or a decrease in actin dynamics might be expected to occur.

View larger version (32K): [in this window] [in a new window]   Fig. 10.   Computer-modeling of the dependence of different species of monomeric actin at steady state on ADF and profilin. The steady-state concentrations of ATP-G-actin (), ADP-G-actin (), ADF-ADP-G-actin (), profilin-ATP-G-actin (), profilin-ADP-G-actin (), and ADF-ATP-G-actin () were calculated as described under "Materials and Methods," using the equilibrium and rate parameters given in Table I. The calculations were done either varying the concentration of profilin in the absence (A) or in the presence of 3 µM ADF (B), or varying the concentration of ADF in the absence and in the presence (D) of 2 µM profilin. (actin concentration = 10 µM). The inset in panel C represents an expanded view of the dotted box in the main frame.

The experimental demonstration of the synergy between ADF and profilin in the enhancement of filament turnover is a main point of this work. Profilin alone has a weak effect on filament turnover (4); however, the property of profilin-actin to participate in barbed end assembly is sufficient to explain the synergy between ADF and profilin. These two proteins act each at one end of the actin filament, in a complementary fashion. ADF increases the flux of depolymerizing subunits from the pointed ends, while profilin increases the flux of assembly onto the barbed ends at steady state. Profilin optimizes the directionality of treadmilling in two ways as follows. First, profilin drives the ADP-bound state of actin toward the ATP-bound state, thus preventing the reverse pathway (association of ADF-ADP-actin with filament ends). Second, profilin replaces ATP-G-actin, which can associate with the two ends of the filament, by profilin-ATP-G-actin, which associates only with the barbed ends. Even in the presence of profilin, the rate-limiting step in the treadmilling cycle is still the dissociation from the pointed ends. The ADF-induced increase in the rate constant for actin dissociation from the pointed ends therefore is certainly at least 5-fold higher than the 25-fold increase found earlier (5). The measured rate of depolymerization from the pointed end was probably tempered by the reverse reaction of ADF-ADP-actin association to the pointed ends, which had not then been appreciated properly. Taking into account the slight increase (27 ± 10%) in the number of filaments observed in the presence of ADF, the conclusion that emerges from our work is that ADF increases the rate constant of depolymerization from the pointed ends at least 50-fold.

Using two profilin species, only one of which enhances nucleotide exchange on G-actin, has been helpful to dissect the individual kinetic steps through which profilin can accelerate the treadmilling cycle. Formation of the polymerizable profilin-ATP-actin complex without increase in the rate of nucleotide exchange is sufficient to increase the rate of treadmilling 3-fold as compared with the level observed with ADF alone. A larger (5-fold) increase, however, is observed if profilin increases the rate of regeneration of ATP-actin from ADP-actin. Again, if ADF were simply a severing factor, bovine profilin could also increase the rate of treadmilling by increasing the rate of nucleotide exchange on ADP-G-actin. This effect has indeed been observed under sustained sonication (22). More specifically, we found that a 3-fold increase in steady-state ATPase rate was induced by profilin when the number of ends was increased 20-fold, corresponding to an average length of 0.3 µm, upon sustained fragmentation due to sonic vibration.⁴ Clearly, ADF does not sever filaments to that extent, yet the increase in rate due to profilin is larger. Moreover, the effect of plant profilin on the treadmilling rate cannot be explained by fragmentation, because as explained above, it requires the high ATP-G-actin concentration induced by ADF.

In conclusion, detailed measurements of rates of treadmilling as a function of filament number, and of the effect of ADF on filament number were required to quantitatively understand the mechanism of action of ADF.

CapZ has proved to be an interesting tool to measure the number of filaments in an actin solution and, in combination with gelsolin, to quantitate the spontaneous fragmentation in the absence and presence of ADF. It is remarkable that the average filament length derived from these measurements is 7.2 µm in the absence of ADF, which is the persistence length of filaments found in a previous study (23). This figure suggests that the spontaneous fragmentation-length redistribution processes that result from brownian agitation lead to the establishment of a filament length compatible with a rod-shaped polymer. The smaller average length of ADF-decorated filaments (5.7 µm) is compatible with electron microscopy data (5). Although the technique using CapZ to measure the number of filaments does not suffer from the artifacts linked to electron microscopy, it has its own biases, as follows. The experiments cannot actually discriminate whether the 27% increase in filament number induced by ADF is due to enhanced spontaneous fragmentation due to the increased fragility of ADF-F-actin, or to enhanced nucleation by CapZ in the presence of ADF. The muscle capping protein is known to weakly nucleate, at G-actin at concentrations above the critical concentration (11). In the present experiments, the G-actin concentration is equal to the critical concentration, hence nucleation by CapZ in standard F-actin solutions is very unlikely. However, the situation may be different in the presence of ADF, because ADF-ADP-G-actin nucleates very easily in the vicinity of the critical concentration (7). Hence, at steady state in the presence of ADF, the nucleation activity of ADF, enhanced by CapZ, introduces a bias in the measurements of the concentration of filament ends. The measured 27% increase in filament ends may thus be an overestimate. Second, the presence of CapZ in the assay medium interferes with the process of establishment of the distribution in length of filaments, because blockage of the barbed ends that are created by fragmentation prevents the reverse reactions of monomer-polymer exchange leading to an increase in average length. This latter bias also leads to an overestimation of the steady-state concentration of filaments.

The assembled data impose a number of constraints that are included in the simulation of actin steady state in the presence of ADF and profilin. The simulated behavior and calculated concentrations of the different actin species are in good agreement with the experimental data, in support of the proposed model.

At optimum concentrations of ADF and profilin, it takes 1 min for a 3-µm-long filament to travel a distance equal to its length. This figure lets us anticipate that the rheological properties of actin gels may be affected by actin dynamics in the presence of ADF and profilin (24). Experiments are under way to address this question.

The synergy observed in vitro between ADF and profilin may be functionally relevant in the control of actin-based motility. Both proteins are present in the lamellipodia of motile cells where monomeric actin exists in large amounts (25), are essential in pluricellular organisms, and are involved in developmental processes in which very active actin dynamics is involved (26-28).