©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Actin-binding Properties of the Physarum Actin-Fragmin Complex
REGULATION BY CALCIUM, PHOSPHOLIPIDS, AND PHOSPHORYLATION (*)

(Received for publication, August 10, 1994; and in revised form, November 18, 1994)

Jan Gettemans (§) Yvette De Ville (¶) Etienne Waelkens (1)(**) Joel Vandekerckhove

From the Department of Biochemistry, Laboratory for Physiological Chemistry, Universiteit Gent, B-9000 Gent, Belgium and the Division of Biochemistry, K.U. Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The actin-binding properties of the actin-fragmin complex from Physarum polycephalum microplasmodia were investigated with respect to regulation by Ca, phospholipids, and phosphorylation of the actin subunit by the endogenous actin-fragmin kinase. Fragmin possesses two high affinity actin-binding sites and probably also a third, low affinity site. Its nucleating and F-actin severing activities are inhibited by phosphatidylinositol 4,5-bisphosphate (PIP(2)). Actin-fragmin specifically binds PIP(2) which competes with actin for the Ca-sensitive site. However, PIP(2) cannot dissociate the actin-fragmin complex nor the actin(2)-fragmin trimer. Efficient F-actin nucleating activity by actin-fragmin is only observed with unphosphorylated actin-fragmin, in the absence of PIP(2) and at high Ca (>µM) concentrations. In the presence of PIP(2), actin-fragmin only caps actin filaments when unphosphorylated. The results suggest that in the cell, hydrolysis of PIP(2), concomitant with the increase of cytosolic Ca, could promote subcortical actin polymerization.


INTRODUCTION

The organization of the microfilament system is the result of specific interactions between actin and a number of actin-binding proteins(1, 2) . One class of proteins, of which gelsolin is a representative member, is thought to play an important role in the organization of the subcortical actin network in response to extracellular signals(3) . These actin-binding proteins are found throughout the eukaryotic world and display F-actin nucleating, severing, and capping activities in vitro(4) .

Gelsolin, originally isolated from macrophages(5) , is a globular monomeric protein of M(r) ± 90,000 and built up of six structurally similar segments, each consisting of about 150 amino acids. The NH(2) terminally located domain (segment 1) displays apparent ``Ca-independent'' G-actin binding properties(6) . The second segment binds F-actin and this interaction is inhibited by phosphatidylinositol 4,5-bisphosphate (PIP(2))^1; a third Ca-dependent actin binding site is located within the second half of the molecule(7) .

The three-dimensional structure of the actin-gelsolin segment 1 complex was recently described and reveals the interface of the two proteins at atomic resolution(8) . Most interacting side chains of gelsolin participate in a long alpha-helix and interact with residues belonging to subdomains 1 and 3 of actin. Ca forms a stable bridge between chelating residues belonging to both proteins and is not removed upon subsequent treatment with EGTA, explaining why this interaction was initially interpreted as Ca-independent. This actin binding region was previously postulated on the basis of amino acid sequence homology between fragmin, gelsolin, and Acanthamoeba profilin(9) .

The gelsolin-actin complex can be dissociated by PIP(2)in vitro(10) , suggesting a role for PIP(2) as a regulator of actin assembly in the subcortical region of the plasma membrane. A similar actin regulatory mechanism was previously postulated for profilin (11) and later confirmed(12) .

Fragmin is a gelsolin-like actin-binding protein, isolated from Physarum polycephalum plasmodia(13, 14) . Its sequence is very similar to the NH(2)-terminal half of gelsolin. Like gelsolin, this 45-kDa protein nucleates polymerization of actin monomers and caps and severs actin filaments in a Ca-dependent manner(15) . In the presence of micromolar calcium concentrations, fragmin forms a stable 1:2 complex with actin (A(2)bulletF). Subsequent removal of calcium converts the 1:2 complex into a 1:1 EGTA-resistant actin-fragmin complex(9) . This dimer has no severing activity. The latter (but not the 1:2 complex) is also a specific target for the endogenous actin-fragmin kinase that phosphorylates the actin subunit at residues Thr-203 (major site) and Thr-202 (minor site)(16, 17) . These residues constitute part of the actin-actin contact site in both proposed F-actin structures(18, 19) .

To obtain better insight in this complex regulatory mechanism, we studied the effects of Ca and PIP(2) on the actin nucleating and F-actin capping and severing properties of fragmin, the actin-fragmin complex, and the phosphorylated actin-fragmin complex. The results allow us to propose a more general model for the actin-regulatory activities of gelsolin-like proteins.


MATERIALS AND METHODS

Preparation of Lipids

PIP(2), phosphatidylinositol 4-monophosphate (PIP), phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) (Sigma) were dissolved in water at a concentration of 1 mg/ml. Inositol 1,4,5-trisphosphate (IP(3)) was dissolved at a concentration of 0.1 mg/ml. Lipids that were delivered in chloroform solution were dried under a stream of nitrogen and resuspended by sonication for 15 min in a Branson S75 sonicator (Branson Sonic Power Co., Danbury, CT). Phospholipid suspensions were stored at -80 °C until further use. After thawing, phospholipids were sonicated for an additional 3 min prior to use. PS remained turbid under these conditions.

Proteins

Actin was prepared from rabbit or cow muscle according to established procedures (20) and kept in G-buffer (2 mM Tris/HCl, pH 7.6, 0.2 mM ATP, 0.5 mM beta-mercaptoethanol, 0.2 mM CaCl(2), and 0.02% NaN(3)). Pyrene-labeled actin was prepared according to (21) and stored in the dark on ice.

The actin-fragmin complex and the actin-fragmin kinase were both isolated from P. polycephalum microplasmodia as described(17) .

Fragmin was obtained from the purified actin-fragmin complex by dissociation with 6 M urea. The dissociated complex was loaded onto a 1-ml DEAE-cellulose column equilibrated in 50 mM Tris/HCl, pH 8.2, 1 mM dithiothreitol, and 0.02% NaN(3) and the subunits were eluted with a linear NaCl gradient (0-500 mM, 20 ml). The first protein peak corresponded to fragmin and the second to actin (as identified by SDS-polyacrylamide gel electrophoresis). Fractions containing fragmin were pooled, concentrated in a Speed Vac Concentrator (Savant Instruments, Farmingdale, NY), and dialyzed against a solution containing 60% glycerol, 50 mM Tris/HCl, pH 7.5, 1 mM dithiothreitol, and 0.02% NaN(3).

Phosphorylation of the Actin-Fragmin Complex

The actin-fragmin complex (5 or 10 µM) was terminally phosphorylated with purified actin-fragmin kinase (17) in G-buffer supplemented with 10 mM MgCl(2) and 1 mM ATP.

Actin Polymerization Assays (Fluorescence Spectrospcopy)

All fluorescence measurements were performed at room temperature using an SFM25 fluorimeter (Kontron Instruments, Zürich). The excitation wavelength was set at 365 nm and the emission wavelength at 388 nm with a sample volume of 700 µl. Experiments were performed as described(22, 23) .

Nucleation of Actin Polymerization

Actin (6 µM final concentration of which 10% was pyrene-labeled) was preincubated for 10 min with fragmin, actin-fragmin, or the phosphorylated actin-fragmin complex (140 nM). The polymerization was initiated by the addition of F-buffer (G-buffer with 0.1 M KCl and 2 mM MgCl(2)) from a 10 times concentrated stock solution and the increase in fluorescence was recorded in function of time. When PIP(2) was used, the nucleating protein was first preincubated with different concentrations of PIP(2) for 5 min before mixing with pyrenyl-actin for another 10 min. Polymerization was again initiated by addition of F-buffer (see above).

Capping of Actin Filaments

Preassembled unlabeled actin filaments (900 nM final concentration) were used as ``nuclei.'' These were mixed with 3 µM actin of which 25% was pyrene-labeled. Actin-fragmin or phosphorylated actin-fragmin (14 nM) was added and the increase in fluorescence was measured over time. All components were mixed first and then added to G-actin in a total volume of 700 µl. Phospholipids were preincubated with the capping proteins for 5 min at room temperature. Experiments performed at low Ca always included 2 mM EGTA.

Severing of Actin Filaments by Fragmin

Actin (8 µM, 25% pyrene-labeled) was polymerized in F-buffer for 15 min. Subsequently the filaments were precapped with the actin-fragmin complex (40 nM final concentration) in order to measure only its severing activity. The mixture was stored overnight on ice. This solution was then diluted in G-buffer to a final concentration of 400 nM, which is below the critical monomer concentration of the pointed ends. Addition of a severing protein (25 nM fragmin) will create new pointed ends and the fluorescence will drastically decrease. The effect of phospholipids on the severing activity of fragmin was investigated by preincubating fragmin with the indicated phospholipid concentrations for 5 min. Subsequently the mixture was added to the pyrene-labeled F-actin solution and the decrease in fluorescence was measured over time.

Stoichiometry of Actin-Fragmin Complex Formation

Pyrenyl G-actin (100% labeled, 150 nM final concentration) was mixed for 10 min with increasing concentrations of actin-fragmin, phosphorylated actin-fragmin, or fragmin alone (between 0 and 2-fold molar excess over actin) under nonpolymerizing conditions. The relative increase in fluorescence was measured for each mixture. These experiments were carried out in the presence of either 0.2 mM Ca or 2 mM EGTA.

The inhibition of actin(2)-fragmin trimer formation by PIP(2) was measured at a concentration of 150 nM pyrenyl-actin in G-buffer to which the same concentration of actin-fragmin complex was added. The latter was preincubated in G-buffer with increasing amounts of PIP(2) (from 1 to 170 times molar excess over AbulletF). The fluorescence of pyrenyl G-actin in the same concentration was set at 0%.

The dissociation of the pyrenyl A(2)bulletF trimer was measured at a concentration of 150 nM in G-buffer, adding increasing concentrations of PIP(2) (ranging from 1 to 100 times molar excess) for 10 min at room temperature. The variations in fluorescence were expressed as above.

Immunization and Affinity Purification of Polyclonal Antifragmin Antibodies

Fragmin (150 µg), obtained from the AbulletF complex by reversed-phase high performance liquid chromatography, was emulsified in Freund's complete adjuvant and injected subcutaneously. Twenty-four days after the initial immunization, the rabbit was again injected with 150 µg of the protein in Freund's incomplete adjuvant. The booster injection was repeated on day 45. After 66 days, the rabbit was bled and the serum was prepared. The antifragmin immunoglobulins were purified by affinity chromatography following incubation of 0.5 ml of antiserum with actin-fragmin coupled to CNBr-activated Sepharose (150 µl suspension) (Pharmacia Biotech Inc.) in 10 mM Tris/HCl, pH 7.5, 1 mM dithiothreitol, 0.02% NaN(3) (TDA buffer). The column was washed extensively with TDA buffer supplemented with 600 mM NaCl and antibodies were eluted with 1 ml of 0.2 M ethanolamine, pH 10. The eluted fraction was collected in 100 µl of 1 M Tris/HCl, pH 6.8, and dialyzed immediately against TDA buffer containing 60% glycerol and stored at -20 °C. Antifragmin IgGs were subsequently coupled to CNBr-activated Sepharose. Coupling reactions were performed according to the manufacturer's instructions.

Miscellaneous

All protein concentrations were determined by the method of Bradford (24) using bovine serum albumin as a standard.

Gel filtration was performed on a fast protein liquid chromatography system (Pharmacia, Uppsala, Sweden), using a Superose 12 HR 10/30 column, equilibrated in 20 mM MOPS, pH 7.4, 50 mM KCl, and 0.02% NaN(3). The flow rate was kept at 0.5 ml/min. The eluate was monitored by absorbance at 280 nm.

SDS-polyacrylamide gel electrophoresis (25) was performed on 10 or 15% polyacrylamide mini slab gels according to (26) . The gels were stained with 0.25% Coomassie Brilliant Blue in 10% acetic acid or by silver staining.

The villin 133-YNVQRLLHVKGKKNVV-147 PIP(2)-binding peptide (27) was synthesized on an Applied Biosystems automated 431A Peptide Synthesizer using the Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry procedure and following the manufacturer's instructions. We have substituted the cysteine residue at the end of the peptide in the original sequence of villin (28) for a valine residue in order to prevent artifactual dimerization due to oxidation and disulfide formation. After cleavage with trifluoroacetic acid, the peptide was desalted in water through Sephadex G-25 gel filtration (30 times 2.6 cm). The eluate was monitored by UV absorbance at 254 nm. Finally, the peptide was lyophilized and stored at -20 °C until further use.


RESULTS

Actin Nucleating Activity

Phosphorylation of the Actin-Fragmin Complex Inhibits Its Actin Nucleating Activity

Fragmin and the actin-fragmin (AbulletF) complex are both known to accelerate actin polymerization(29) . Under conditions favoring actin polymerization, both proteins decrease the lag phase of actin assembly by promoting the nucleation step and induce the formation of short filaments with capped (+) ends. Fig. 1shows the rate of actin polymerization measured by fluorescence enhancement in the presence of Ca. The effect of fragmin and unphosphorylated or phosphorylated AbulletF is represented. AbulletF and fragmin drastically reduce the lag phase of polymerization. In contrast, nucleation by phosphorylated actin-fragmin (A^PbulletF) results in a lag phase comparable to that of the control (absence of nucleating protein), indicating that phosphorylation completely inhibits the nucleating activity of the complex. When the phosphorylated complex was preincubated with G-actin for more than 20 min prior to the start of polymerization, similar kinetics were observed (data not shown), suggesting that the phosphorylated actin subunit of the complex is not exchanged for free unphosphorylated actin in solution.


Figure 1: Actin nucleating activity of actin-fragmin and its inhibition by phosphorylation. Induction of actin polymerization by either fragmin (F; 140 nM) or the actin-fragmin complex (AbulletF; 140 nM) in the presence of 0.2 mM Ca. C is the control and represents the polymerization of actin (6 µM) in F buffer but in the absence of nucleating agent (notice the lag phase). A^PbulletF indicates the polymerization curve in the presence of fully phosphorylated AbulletF (140 nM).



The Actin Nucleating Activity of Fragmin Is Fully Inhibited by PIP(2); That of the Actin-Fragmin Complex Only Partially

Fig. 2depicts the effect of different concentrations of PIP(2) on the nucleating activity of fragmin (Fig. 2A) and AbulletF (Fig. 2B), respectively. We observed a decrease in the rate of actin polymerization, proportional to the PIP(2) concentration. With approximately a 100-fold molar excess of PIP(2), full inhibition was measured for fragmin (Fig. 2A). When AbulletF was used as nucleating protein (Fig. 2B), a much less pronounced inhibition was noticed even at PIP(2) levels higher than those necessary for full inhibition of fragmin (results not shown). Since intermediary concentrations of PIP(2) (i.e. 55-fold excess over fragmin or AbulletF, compare Fig. 2, A and B) resulted in a much higher inhibition of fragmin, we conclude that PIP(2) binding perturbs the interaction of actin with both high affinity actin-binding sites of fragmin. It should be noted that PIP(2) alone (in the same concentration range) produced no effect on the rate of actin polymerization (data not shown).


Figure 2: Effect of PIP(2) on the F-actin nucleating activity of fragmin and actin-fragmin in the presence of Ca. Fragmin (A) or actin-fragmin (B) were preincubated with the indicated molar excesses of PIP(2), prior to their addition to a solution of pyrene-labeled G-actin. The polymerization was induced by shifting to F buffer conditions. The control (C) consists of a polymerization mixture without nucleating protein in the absence of PIP(2).



F-actin Capping Activity

AbulletF Phosphorylation Inhibits F-actin Capping Activity in EGTA but Not in Ca

Using the falling ball assay, Maruta and Isenberg (29) demonstrated that phosphorylation of the actin-fragmin complex inhibits the capping activity in the presence of EGTA, whereas in Ca, the phosphorylated complex did retain its full activity. These results are in contrast with those reported more recently by Furuhashi and Hatano (30) who stated that the capping activity was inhibited, regardless of the presence or absence of Ca.

In view of these conflicting data, and particularly with the intention to measure the influence of PIP(2) on the capping activity (see below), we have readdressed this issue by a more quantitative assay(22) . When an F-actin (+) end capping protein is added to a mixture of unlabeled short actin filaments and pyrene-labeled G-actin monomers, it will slow down the overall polymerization rate as compared to the control (no capping protein), since elongation of filaments can only proceed from the pointed ends. From Fig. 3it can be inferred that, as expected, F-actin capping by AbulletF results in a drastic reduction of the polymerization rate. In the presence of EGTA, the fully phosphorylated AbulletF did not display any capping activity (Fig. 3A). Conversely, when 0.2 mM Ca is present, phosphorylated AbulletF capped actin filaments in a way similar to that observed for the unmodified complex (Fig. 3B). It is worth noting that two-dimensional gel analysis of the phosphorylated complex demonstrated complete phosphorylation of the actin subunit (data not shown), implying that the remaining capping activity due to unphosphorylated AbulletF can be excluded. Thus phosphorylation of the AbulletF complex in actin imposes Ca dependence on the F-actin capping activity.


Figure 3: Effect of actin-fragmin phosphorylation on the F-actin capping activity. Panel A shows the polymerization kinetics in the presence of EGTA; panel B shows the experiments carried out in the presence of Ca. AbulletF (14 nM) indicates the actin-fragmin complex. A^PbulletF, the phosphorylated complex (14 nM). Since F-actin nuclei were added to G-actin (3 µM) to induce the polymerization, no lag phase appears. C, control, actin alone.



The F-actin Capping Activity of Actin-Fragmin and the Phosphorylated Actin-Fragmin Is Inhibited by PIP(2)

In the next experiment, we used the same assay to measure the influence of PIP(2) on the F-actin capping activity. As above, this was done in high and low Ca concentrations.

In the presence of Ca, PIP(2) inhibits the capping activity of AbulletF. A 500-fold molar excess of PIP(2) over AbulletF results in ±50% inhibition (Fig. 4A). Identical PIP(2) concentrations exert an almost total inhibition on the activity of the phosphorylated complex (Fig. 4B). Thus, both AbulletF and its phosphorylated form are inhibited by PIP(2) in their F-actin capping activities in the presence of Ca. However, the effect is much more pronounced with the phosphorylated form. As demonstrated above, a similar PIP(2)-dependent differential effect was also noticed when the nucleation activities of both fragmin and AbulletF were compared (see Fig. 2, A and B). These findings can be explained by assuming a third actin binding site in fragmin. This site is active only under conditions where excess G-actin is present, such as nucleation, but not capping (see also ``Discussion''). Furthermore, this third site is probably PIP(2)-insensitive.


Figure 4: Effect of PIP(2) and other phospholipids on the F-actin capping activity of actin-fragmin and the phosphorylated complex. F-actin capping activities were measured as in Fig. 3. A, activity of AbulletF in increasing concentrations of PIP(2). The molar excess of PIP(2) over AbulletF is shown on the right; B, the same PIP(2) concentrations as in A but now with phosphorylated AbulletF (A^PbulletF); C, protein ratios as in B but AbulletF capping activity was measured in the presence of 2 mM EGTA and the indicated PIP(2) molar excess; D, effect of various phospholipids: PIP, PI, and PC on the activity of the AbulletF complex. Concentrations of phospholipids are the same as the PIP(2) concentration resulting in 50% inhibition (500 times molar excess). E, inhibition of the activity of the phosphorylated complex (A^PbulletF) by various phospholipids including PS and PE. The effect of IP(3) (500 times molar excess over A^PbulletF) is also shown. The control (C) is as described in the legend to Fig. 3.



In the presence of EGTA (Fig. 4C), the inhibitory effect of PIP(2) on AbulletF hardly differs from that observed in Ca (Fig. 4A). These studies were not carried out for phosphorylated AbulletF, since this form does not cap F-actin in EGTA (see Fig. 3A).

We have also studied inhibition of the F-actin capping activity on AbulletF by other phospholipids (Fig. 4D). This was done using concentrations of phospholipids similar to those of PIP(2) that resulted in ±50% inhibition (see Fig. 4A). Neither PIP, PI, nor PC displayed significant inhibitory effects.

Similar effects were measured with the phosphorylated form of the AbulletF complex at high Ca concentrations (Fig. 4E). Again PIP(2) displays the highest inhibition, whereas PIP only shows a marginal effect. The other polyphosphoinositides PI, PC, PE, PS, and IP(3) were not inhibitory at all.

Inhibition of Fragmin F-actin Severing Activity by PIP(2): Comparison with Other Phospholipids

Neither AbulletF nor the phosphorylated AbulletF have intrinsic F-actin severing activity. Only fragmin can sever F-actin at high Ca concentrations(13, 14, 15) . To study the influence of PIP(2) on F-actin severing, we precapped pyrene-labeled actin filaments with the actin-fragmin complex and then decreased the actin concentration below the critical monomer concentration for the (-)-ends. Upon addition of fragmin, a large number of new(-)-ends are created, resulting in a drastic increase in the rate of actin depolymerization, since depolymerization is proportional to the number of free(-)-ends. We measured the decrease in fluorescence per min in the linear range of the curve and expressed this value in a block diagram (Fig. 5). We observed a concentration-dependent inhibition of the severing activity of fragmin, reaching maximal values when PIP(2) was present at a 300 times molar excess over fragmin.


Figure 5: Inhibition of fragmin severing activity by various phospholipids. F-actin filaments were precapped with AbulletF and then diluted below the critical concentration of the(-)-end. Severing by fragmin was measured as a decrease in fluorescence due to rapid depolymerization at the newly created pointed ends. The decrease in fluorescence per min (during the linear phase of the curve) in the presence of fragmin was set at 100% severing activity. Depolymerization in the absence of fragmin was taken as 0% severing activity. Lane 1, fragmin (25 nM); lane 2, fragmin + PIP(2) (1:300 molar ratio); lane 3, fragmin + PIP(2) (1:150 molar ratio); lane 4, fragmin + PIP(2) (1:60 molar ratio); lane 5, mixture of PIP(2) (1:150 molar ratio) and PC; lane 6, PIP(2) (1:150)/PS; lane 7, PIP(2)/IP(3) (1:150)). The following phospholipids were added in a 300-fold molar excess over fragmin: PIP, PI, PS, PC, PE, and the phosphoinositide IP(3) (lanes 8-13). Notice that inhibition of F-actin severing activity is very specific for PIP(2) and less so for PIP.



When other polyphosphoinositides were employed, only PIP displayed a significant inhibitory effect (Fig. 5, lane 8) while PI, PS, PC, PE, and IP(3) were inactive. Mixed polyphosphoinositides, containing PC or PS in addition to PIP(2), reduced the inhibitory effect of PIP(2) on the severing activity of fragmin (Fig. 5, lanes 5-7). A summary of the activities of fragmin, actin-fragmin, and the phosphorylated actin-fragmin complex described above is given in Table 1.



PIP(2) Binds but Does Not Dissociate the Actin-Fragmin Complex

Using gel filtration experiments, Ampe and Vandekerckhove (9) demonstrated that in the presence of Ca and actin, a stable trimer could be formed consisting of two actin molecules and fragmin. We have studied the effect of phosphoinositides on the formation and stability of the actin-fragmin complexes. Previous work has shown that PIP(2) is able to dissociate in vitro complexes formed between actin and actin-binding proteins, thus suggesting a direct link between the signaling system and the organization of actin in the cell. This was shown for profilin-actin (11) , the EGTA-resistant actin-gelsolin (31) and actin-severin complexes(23) .

When AbulletF was incubated with pyrene-labeled G-actin under nonpolymerizing conditions, an increase in fluorescence intensity was observed, due to complex formation between AbulletF and pyrenyl G-actin. At a molar ratio of 1 or higher, a plateau was seen (Fig. 6A). When this experiment was carried out in the presence of EGTA, no change in the fluorescence was measured. These results are in agreement with the existence of a second actin-binding site which is Ca-dependent, confirming previous studies(9) . The same experiment with fully phosphorylated AbulletF resulted in nearly identical plots as for AbulletF, both in high or low calcium (Fig. 6A). When fragmin was used instead of AbulletF, maximal fluorescence values were obtained at a fragmin/pyrenyl-actin molar ratio of 0.5, in accordance with the binding of two actin molecules to fragmin (Fig. 6A).


Figure 6: Stoichiometry of actin-fragmin interactions: modulation by phosphorylation, Ca, and PIP. A, pyrenyl G-actin was incubated with increasing concentrations of actin-fragmin in Ca (circle) or EGTA (box), phosphorylated actin-fragmin in Ca () or EGTA (), or with fragmin and Ca (bullet). The molar ratios of the proteins are given on the abscissa. The ordinate shows the relative fluorescence enhancement due to binding of pyrene-labeled actin. B: upper panel, the inhibitory effect of increasing amounts of PIP(2) on the formation of the trimer (A(2)bulletF) consisting of Physarum actin-fragmin (AbulletF) and pyrenyl-muscle actin (150 nM each). The first bar shows the fluorescence enhancement in the absence of PIP(2). The abscissa shows the molar ratios of PIP(2) relative to the actin-fragmin complex. Lower panel, inability of PIP(2) to dissociate the actin(2)-fragmin trimer once it is formed. The abscissa now shows the molar ratios of the A(2)bulletF trimer and PIP(2). The results are mean ± S.D. of triplicate determinations.



To study the effect of PIP(2) on A(2)bulletF trimer formation and dissociation, we preincubated the AbulletF complex with pyrenyl-actin in the presence of Ca and measured the change in fluorescence intensity after mixing the trimer with increasing concentrations of PIP(2). The phospholipid had no effect on the fluorescence intensity of the actin(2)-fragmin trimer (Fig. 6B, lower panel), suggesting it is unable to dissociate the trimer. Conversely, PIP(2) was able to inhibit the formation of a trimeric complex: when increasing amounts of PIP(2) were incubated with AbulletF, we noticed a decrease in fluorescence that leveled out to zero at a 100-fold molar excess PIP(2) (Fig. 6B, upper panel). Thus PIP(2) and the second actin molecule mutually exclude each other.

The stability of the AbulletF complex in the presence of PIP(2) was also assayed by gel filtration and immunoprecipitation. When AbulletF was incubated with PIP(2) and then passed over a Superose 12 gel filtration column, we noticed a significant increase in apparent M(r) but no dissociation into fragmin and actin (results not shown). Prolonged incubation, up to 24 h, showed no significant dissociating effect. The binding of PIP(2) to AbulletF could be inhibited when the phospholipid was first incubated with a synthetic PIP(2)-binding peptide derived from villin(27) : a 2-fold molar excess of peptide over PIP(2) resulted in a shift to the original apparent M(r) for 75% of actin-fragmin (data not shown). The remaining 25% of the actin-fragmin complex eluted from the column at the position where the PIP(2)-fragmin-actin complex elutes. In a second experiment we followed a similar approach as Janmey et al.(10) : polyclonal anti-fragmin IgGs, immobilized on Sepharose beads, were used to immunoprecipitate the actin-fragmin complex preincubated with various concentrations of PIP(2). No enrichment for actin in the supernatant was found after SDS-polyacrylamide gel electrophoresis (data not shown), consistent with the existence of a stable AbulletF complex in PIP(2).

In conclusion, these results indicate that PIP(2) binds to fragmin and to the actin-fragmin complex and inhibits further addition of actin to these proteins. However, once the complex is formed, neither the AbulletF dimer nor the A(2)bulletF trimer can be dissociated by PIP(2).


DISCUSSION

The primary structure of fragmin reveals a triple repeat of a segment of approximately 150 amino acids. Because of the high degree of sequence homology between fragmin and members of the gelsolin family (9) , we predict that these repeats fold into domain structures, each similar to the three-dimensional structure of gelsolin S1 (8) or villin 14T(32) . All three domains of fragmin can therefore be considered as potential actin-binding domains.

Apart from structural homology, fragmin shares a number of functional properties with gelsolin. For instance its ability to bind two actin molecules in the presence of Ca, thus forming an A(2)bulletF trimer. In this paper we demonstrated that A(2)bulletF is not converted into the AbulletF heterodimer upon addition of excess of fragmin (Fig. 6A). Thus the second high affinity Ca-dependent actin binding site is involved in a stable association. Similar results were previously reported for gelsolin (33) but contrast those described for severin(23) , where the actin(2)-severin complex is converted into a stable dimer upon addition of excess severin.

Fragmin also displays properties distinct from those of other members of the gelsolin family. For instance, it is only after its association with actin that the actin moiety of the complex becomes amenable for phosphorylation by the actin-fragmin kinase(17) . A similar phosphorylated gelsolin-actin complex has not been described so far (34) , nor have we been able to phosphorylate the gelsolin-actin complex with the Physarum actin-fragmin kinase(16) . This important functional difference suggests altered microfilament regulatory properties for fragmin. Therefore, we carried out F-actin nucleation, capping, and severing experiments using Ca, PIP(2), and phosphorylation as modulators. Two unexpected findings emerged from our experiments with PIP(2). First, nucleation by unphosphorylated AbulletF cannot be fully inhibited by PIP(2) (Fig. 2B). This can be explained by supposing that PIP(2) has a low affinity for the second actin binding site or by assuming the existence of a third actin binding site (see also below). The second unexpected result illustrates an important difference between the actin-gelsolin complex and AbulletF: whereas the former is dissociated by PIP(2), the latter is not.

A model incorporating these new findings is presented in Fig. 7and is based on the existence of two high affinity actin-binding domains and a third putative actin-binding site in fragmin. These domains are each differently regulated and display different affinities for actin. They are designated domains I, II, and III in Fig. 7A. This organization is reminiscent of the one described previously for severin(23) , where two lipid binding domains, two Ca binding domains, and three actin-binding sites were demonstrated.


Figure 7: Domain organization of fragmin and schematic representation of the complexes that can be formed between actin and fragmin. A, the organization and properties of the domains of fragmin. B, the different complexes that can be formed between actin and fragmin in the presence or absence of PIP(2) and Ca. Complexes 6 and 7 describe the properties of the phosphorylated forms. Severing, capping, and nucleating activities are allocated to each of these complexes. For detailed information see ``Discussion.''



In the presence of Ca, both site I and II bind actin, forming an A(2)bulletF trimer (Fig. 7B, 1). PIP(2) interferes with the formation of this complex (Fig. 7B, 2). Upon Ca depletion, only the actin on site II is released and this AbulletF heterodimer (Fig. 7B, 3) has capping activity (through the residual actin moiety) but surprisingly also possesses weak nucleating activity in EGTA (data not shown). Since nucleation requires binding of two actin molecules, two possibilities arise. Either the Ca-sensitive high affinity site II is turned into a low affinity site in the absence of Ca, or a different low affinity site exists (site III). The first possibility can be ruled out from our experiments with PIP(2). Indeed, this nucleating activity is not abolished in the presence of PIP(2) (Fig. 2B), whereas binding of actin to site II is inhibited by PIP(2) (Fig. 7B, 4). Therefore, a third actin binding site probably exists to account for the nucleating activity by AbulletF in the presence of this phospholipid (Fig. 7B, 5, hatched). Because of its low affinity, site III would be occupied only at high actin concentrations, typically found in F-actin nucleation conditions, but not in capping conditions. We further postulate that this site is only accessible when site II is free, thus at low Ca concentrations or when site II is occupied by PIP(2). Phosphorylation of actin residing on site I converts the EGTA-resistant heterodimer into a fully inactive complex with respect to nucleating and capping activity (the latter in EGTA) (Fig. 7B, 6). This phosphorylated complex displays full capping activity in Ca via site II. In the presence of PIP(2), site II is blocked and consequently no capping activity is observed (Fig. 7B, 7).

In free fragmin, site I and II are inhibited by PIP(2) (Fig. 7B, 2). Thus under nucleating conditions, we expect no activity (Fig. 2A). The higher nucleating activity of actin-fragmin as compared to fragmin (Fig. 2A) is probably due to binding of fragmin to the first actin monomer, a reaction which in the case of gelsolin, has been found to be slow(35, 36, 37) . Nor did we measure F-actin severing activity by fragmin in the presence of PIP(2) (Fig. 5), since this assumes the presence of at least two free, high affinity, actin-binding sites. Finally, there will be no capping activity because at low actin concentrations no site will be occupied.

We showed that unlike complexes of actin with gelsolin(31) , profilin (11) , severin(23) , or Cap 100(22) , the actin-fragmin complex is not dissociated by PIP(2) (Fig. 7). This property is also shared by the 45-kDa sea urchin fragmin-like protein(38) . No physiologically relevant agent that dissociates the actin-fragmin complex has been identified so far, although Ohnuma and Mabuchi (39) have mentioned a factor that dissociates the PIP(2)-resistant 45-kDa protein-actin complex.

In previous studies, the different domains and corresponding functions of gelsolin (40, 41, 42) and severin (23) were allocated. Based on the sequence homology between fragmin and gelsolin, it is reasonable to predict that the NH(2)-terminal segment of fragmin corresponds with gelsolin S1. This segment in gelsolin is involved in PIP(2) binding, since a mutant lacking the 15 COOH-terminal amino acids no longer binds PIP(2)(43) . The consensus PIP(2)-binding motif in this segment has been identified as KXXXKXKK. Fragmin, however, displays only partial homology with this region (KXXXRXLD). One of the two remaining domains (here presented as site II) contains the Ca-dependent site. Inhibition of site II by PIP(2) may be analogous to the PIP(2)-dependent F-actin binding site, involved in F-actin severing, located in the second segment of gelsolin(7) . The consensus sequence for this PIP(2)-binding motif is KXXXXKXKK(43) , but the amino acid sequence of this region in fragmin remains unknown (9) . Using deletion mutagenesis, Sun et al.(44) recently showed that the third segment of gelsolin may contain an additional polyphosphoinositide-binding site and that it does not contain a high affinity G-actin binding site. The corresponding segment in fragmin on the other hand probably binds actin monomers with low affinity, but regulation by PIP(2) is unlikely.

So far, we have little indication of how much fragmin in Physarum microplasmodia exists in the free form. Immunoprecipitation experiments with antifragmin antibodies in the presence of EGTA reveal that most fragmin is bound to actin in crude extracts. (^2)This raises the hypothesis that under these growth conditions, the actin-fragmin complex shown in Fig. 7B, 2, is predominant. Binding of A-F to PIP(2) suggests a direct physical link with the phosphoinositide signaling pathway. Based on these observations, the following models for AbulletF function in vivo could be envisaged. Although cytosolic AbulletF caps actin filaments independently of the Ca concentration, there is a difference between filaments that are capped in the presence of high or low Ca. Indeed, the former will have both site I and II on fragmin occupied with actin and therefore cannot anchor actin filaments on PIP(2)-containing membranes. However, when Ca is low, AbulletF capped filaments could attach via the unoccupied site II of fragmin. When PIP(2) is hydrolyzed, these filaments are released into the subcortical area and site II will now be occupied by actin because Ca is high. These filaments may attach again to the membrane when Ca is decreased and PIP(2) formed.

One could also consider the situation in which the AbulletF complex is associated with the membrane where it could function as an anchoring site for uncapped actin filaments and here attachment of filaments is independent of the Ca status of the cell. When PIP(2) is hydrolyzed and Ca increased, AbulletF will be released from the membrane and converted into an F-actin nucleating agent, inducing actin filaments to grow rapidly from the subcortical region. At this moment, however, we have no decisive evidence to support our hypothesis. The proposed mechanism is different from the model described previously for gelsolin activity in vivo(3, 45, 46) . There PIP(2) dissociates the gelsolin-actin complex, allowing actin to polymerize. When PIP(2) is hydrolyzed, and Ca increases, gelsolin acts as an F-actin severing protein, leading to a fast destruction of the subcortical actin network.

What is the role of AbulletF phosphorylation? At this stage it cannot be excluded that phosphorylation is a signal for intracellular translocation; alternatively, it could be the first step leading to dissociation of the complex. Its major role, however, is undoubtedly correlated with the regulation of actin binding to the AbulletF complex as is evident from the location of the phosphorylated amino acid in the actin-actin contact site(16) .

So far it is not clear if the degree of phosphorylation is altered in response to various stimuli or is correlated with stages of development of Physarum cells. Obviously, more detailed information related to these questions, as well as to the mechanism of dephosphorylation, will tell us more about the role of AbulletF phosphorylation.

In conclusion, important elements of our model will depend on the fate of the phosphorylated complex once bound to the membrane. Indeed, if PIP(2) association could function as a trigger for dephosphorylation, then this step would lead to a membrane located reactivation of AbulletF. Consecutive PIP(2) hydrolysis and Ca increase would release a fully active nucleating species.

In these models, we have not considered the interplay between AbulletF and other actin-binding proteins, that could regulate the stability of the actin filaments. Their activity will be important for the ideas which have been proposed here and future experiments will have to address these questions.


FOOTNOTES

*
This work was supported in part by grants from the National Research Foundation and the Concerted Research Action of the Flemish Community (to J. V.). 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.

§
Supported by a Concerted Research Action of the Flemish Community (GOA). To whom correspondence should be addressed: Universiteit Gent, Laboratory for Physiological Chemistry, Ledeganckstraat 35, B-9000 Gent, Belgium. Tel.: 32-9-264-52-89; Fax: 32-9-264-53-37.

Supported by a Concerted Research Action of the Flemish Community (GOA).

**
Senior research associate of the National Research Foundation (NFWO).

(^1)
The abbreviations used are: PIP(2), phosphatidylinositol 4,5-bisphosphate; AbulletF, actin-fragmin complex; A(2)bulletF, actin(2)-fragmin complex; A^PbulletF, phosphorylated actin-fragmin complex; F, fragmin; F-actin, filamentous actin; IP(3), inositol 1,4,5-trisphosphate; MOPS, 3-(N-morpholino)propanesulfonic acid; PI, phosphatidylinositol; PIP, phosphatidylinositol 4-monophosphate; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; 14T, N-terminal domain of villin.

(^2)
J. Gettemans, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. C. Ampe for critical reading of the manuscript.


REFERENCES

  1. Hartwig, J. H., and Kwiatkowski, D. J. (1991) Curr. Opin. Cell Biol. 3, 87-97 [Medline] [Order article via Infotrieve]
  2. Vandekerckhove, J., and Vancompernolle, K. (1992) Curr. Opin. Cell Biol. 4, 36-42 [Medline] [Order article via Infotrieve]
  3. Stossel, T. P. (1989) J. Biol. Chem. 264, 18261-18264 [Free Full Text]
  4. Weeds, A., and Maciver, S. (1993) Curr. Opin. Cell Biol. 5, 63-69 [Medline] [Order article via Infotrieve]
  5. Yin, H. L., and Stossel, T. P. (1979) Nature 281, 581-586
  6. Bryan, J. (1986) J. Cell Biol. 106, 1553-1562 [Abstract]
  7. Yin, H. L., Iida, K., and Janmey, P. A. (1988) J. Cell Biol. 106, 805-812 [Abstract]
  8. McLaughlin, P. J., Gooch, T. J., Mannherz, H. G., and Weeds, A. G. (1993) Nature 364, 677-683
  9. Ampe, C., and Vandekerckhove, J. (1987) EMBO J. 6, 4149-4157 [Abstract]
  10. Janmey, P. A., Iida, K., Yin, H. L., and Stossel, T. P. (1987) J. Biol. Chem. 262, 12228-12236 [Abstract/Free Full Text]
  11. Lassing, I., and Lindberg, U. (1985) Nature 314, 472-474 [Medline] [Order article via Infotrieve]
  12. Goldschmidt-Clermont, P. J., Kim, J. W., Machesky, L. M., Rhee, S. G., and Pollard, T. D. (1991) Science 251, 1231-1233 [Medline] [Order article via Infotrieve]
  13. Hasegawa, T., Takashashi, S., Hayashi, H., and Hatano, S. (1980) Biochemistry 19, 2677-2683 [Medline] [Order article via Infotrieve]
  14. Hinssen, H. (1981) Eur. J. Cell Biol. 23, 225-233 [Medline] [Order article via Infotrieve]
  15. Hinssen, H. (1981) Eur. J. Cell Biol. 23, 234-240 [Medline] [Order article via Infotrieve]
  16. Gettemans, J., De Ville, Y., Vandekerckhove, J., and Waelkens, E. (1992) EMBO J. 11, 3185-3191 [Abstract]
  17. Gettemans, J., De Ville, Y., Vandekerckhove, J., and Waelkens, E. (1993) Eur. J. Biochem. 214, 111-119 [Abstract]
  18. Holmes, K. C., Popp, D., Gebhard, W., and Kabsch, W. (1990) Nature 347, 37-44 [CrossRef][Medline] [Order article via Infotrieve]
  19. Schutt, C. E., Myslik, J. C., Rozycki, M. D., Goonesekere, N. C. W., and Lindberg, U. (1993) Nature 365, 810-816 [CrossRef][Medline] [Order article via Infotrieve]
  20. Spudich, J. A., and Watt, S. (1971) J. Biol. Chem. 246, 4866-4871 [Abstract/Free Full Text]
  21. Brenner, S. L., and Korn, E. D. (1983) J. Biol. Chem. 258, 5013-5020 [Abstract/Free Full Text]
  22. Hofmann, A., Eichinger, L., André, E., Rieger, D., and Schleicher, M. (1992) Cell Motil. Cytoskeleton 23, 133-144 [Medline] [Order article via Infotrieve]
  23. Eichinger, L., and Schleicher, M. (1992) Biochemistry 31, 4779-4787 [Medline] [Order article via Infotrieve]
  24. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  25. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  26. Matsudaira, P., and Burgess, D. R. (1978) Anal. Biochem. 87, 386-396 [Medline] [Order article via Infotrieve]
  27. Janmey, P. A., Lamb, J., Allen, P. G., and Matsudaira, P. T. (1992) J. Biol. Chem. 267, 11818-11823 [Abstract/Free Full Text]
  28. Arpin, M., Pringault, E., Finidori, J., Garcia, A., Jeltsch, J.-M., Vandekerckhove, J., and Louvard, D. (1988) J. Cell Biol. 107, 1759-1766 [Abstract]
  29. Maruta, H., and Isenberg, G. (1983) J. Biol. Chem. 258, 10151-10158 [Abstract/Free Full Text]
  30. Furuhashi, K., and Hatano, S. (1990) J. Cell Biol. 111, 1081-1087 [Abstract]
  31. Janmey, P. A., and Stossel, T. P. (1987) Nature 325, 362-364 [CrossRef][Medline] [Order article via Infotrieve]
  32. Markus, M. A., Nakayama, T., Matsudaira, P., and Wagner, G. (1994) Protein Sci. 3, 70-81 [Abstract/Free Full Text]
  33. Coué, M., and Korn, E. D. (1985) J. Biol. Chem. 260, 15033-15041 [Abstract/Free Full Text]
  34. Onoda, K., and Yin, H. L. (1993) J. Biol. Chem. 268, 4106-4112 [Abstract/Free Full Text]
  35. Selve, N., and Wegner, A. (1987) Eur. J. Biochem. 168, 111-115 [Abstract]
  36. Laham, L. E., Lamb, J. A., Allen, P. G., and Janmey, P. A. (1993) J. Biol. Chem. 268, 14202-14207 [Abstract/Free Full Text]
  37. Ditsch, A., and Wegner, A., (1994) Eur. J. Biochem. 224, 223-227 [Abstract]
  38. Ohnuma, M., and Mabuchi, I. (1993) J. Biochem. (Tokyo) 114, 718-722 [Abstract]
  39. Ohnuma, M., and Mabuchi, I. (1988) Zool. Sci. (Tokyo) 5, 691-698
  40. Kwiatkowski, D. J., Janmey, P. A., Mole, J. E., and Yin, H. L. (1985) J. Biol. Chem. 260, 15232-15238 [Abstract/Free Full Text]
  41. Bryan, J., and Hwo, S. (1986) J. Cell Biol. 102, 1439-1446 [Abstract]
  42. Chaponnier, C., Janmey, P. A., and Yin, H. L. (1986) J. Cell Biol. 103, 1473-1481 [Abstract]
  43. Yu, F. X., Sun, H. Q., Janmey, P. A., and Yin, H. L. (1992) J. Biol. Chem. 267, 14616-14621 [Abstract/Free Full Text]
  44. Sun, H. Q., Wooten, D. C., Janmey, P. A., and Yin, H. L. (1994) J. Biol. Chem. 269, 9473-9479 [Abstract/Free Full Text]
  45. Lind, S. E., Janmey, P. A., Chaponnier, C., Herbert, T-J., and Stossel, T. P. (1987) J. Cell Biol. 105, 833-842 [Abstract]
  46. Hartwig, J. H., Chambers, K. R., and Stossel, T. P. (1989) J. Cell Biol. 108, 467-479 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.