Correspondence to: Sally H. Zigmond, Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018. Tel:(215) 898-4559 Fax:(215) 898-8780 E-mail:szigmond{at}sas.upenn.edu.
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Abstract |
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We find that profilin contributes in several ways to Cdc42-induced nucleation of actin filaments in high speed supernatant of lysed neutrophils. Depletion of profilin inhibited Cdc42-induced nucleation; re-addition of profilin restored much of the activity. Mutant profilins with a decreased affinity for either actin or poly-L-proline were less effective at restoring activity. Whereas Cdc42 must activate Wiskott-Aldrich Syndrome protein (WASP) to stimulate nucleation by the Arp2/3 complex, VCA (verpolin homology, cofilin, and acidic domain contained in the COOH-terminal fragment of N-WASP) constitutively activates the Arp2/3 complex. Nucleation by VCA was not inhibited by profilin depletion. With purified N-WASP and Arp2/3 complex, Cdc42-induced nucleation did not require profilin but was enhanced by profilin, wild-type profilin being more effective than mutant profilin with reduced affinity for poly-L-proline.
Nucleation by the Arp2/3 complex is a function of the free G-actin concentration. Thus, when profilin addition decreased the free G-actin concentration, it inhibited Cdc42- and VCA-induced nucleation. However, when profilin was added with G-actin in a ratio that maintained the initial free G-actin concentration, it increased the rate of both Cdc42- and VCA-induced nucleation. This enhancement, also seen with purified proteins, was greatest when the free G-actin concentration was low. These data suggest that under conditions present in intact cells, profilin enhances nucleation by activated Arp2/3 complex.
Key Words: actin polymerization, nucleation, Cdc42, leukocytes, profilin
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Introduction |
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Neutrophils provide the body's first line of defense against bacterial infection. They are produced in the bone marrow and are carried passively in the circulation throughout the body. At sites of injury or infection, they are activated to move and directed to the site of infection by chemoattractants. This activation is reversible: upon removal of chemoattractant, the cells become round and immobile. Activation involves actin polymerization: 10 s after addition of chemoattractant, the F-actin level doubles (0.5 µM during most of the F-actin rise. However, when peak F-actin levels are reached, the free G-actin is calculated to decline to
0.15 µM (see Materials and Methods for details).
The chemoattractant-induced F-actin appears to be turning over rapidly, since upon removal of the chemoattractant (or addition of cytochalasin), the concentration of F-actin returns to basal levels, with a half-time between 3 and 10 s (
However, treadmilling of monomers within a filament probably accounts for a small part of the F-actin dynamics. The increase in F-actin in neutrophils correlates with a rapid increase in filament number (
Cdc42, a member of the Rho family of small GTPases, induces actin polymerization in a high speed supernatant of cell extracts (
Cdc42 stimulates actin nucleation through binding to Wiskott-Aldrich Syndrome protein (WASP)1 or its ubiquitous relative N-WASP (
WASP and N-WASP are also activated by other factors including PIP2, phosphorylation, and clustering ( (
In this paper, we focus on the role of profilin in Cdc42Arp2/3-induced actin nucleation. We show that the presence of profilin is critical for Cdc42-induced nucleation in cell supernatant. We confirm previous studies showing that profilin is not essential for Cdc42-induced nucleation with pure proteins and that addition of profilin can inhibit nucleation (
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Materials and Methods |
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Reagents
Rabbit pAb, raised against the glutathione S-transferase (GST)human profilin I or VASP peptide (NH2-CEAFVQELRKRGSP-COOH), were used for immunodepletion of profilin or VASP from supernatants. Recombinant VASP was a gift from Dr. M.-F. Carlier (Centre National de la Recherche Scientifique, Gif-sur-Yvette, France). This VASP was able to restore Listeria motility to VASP-depleted supernatants (
Supernatant of Neutrophil Lysates
High speed supernatant of lysed rabbit peritoneal exudate neutrophils (supernatant) was obtained as described by
Arp2/3 complex was depleted from supernatant using beads coupled to CA (the COOH-terminal fragment of N-WASP containing cofilin and the acidic tail). Supernatant was incubated with GSACA beads or control GST beads (1:3, vol/vol) for 1 h on a rocker at 4°C. The beads were removed by centrifugation and fresh beads were added to the supernatant; again at a ratio of 1:3. This was repeated one more time (final of three times).
Profilin was depleted with PLP beads. PLP (1030 kD; SigmaAldrich) was coupled to CNBractivated Sepharose 4B (Amersham Pharmacia Biotech) according to manufacturer's instructions. After washing with buffer (20 mM Tris-HCl, pH 7.4, 150 mM KCl. and 0.2 mM ATP), PLP beads or control Separose beads were incubated with supernatants (1:3, vol/vol) on a rocker at 4°C for 30 min.
VASP was depleted by immunoprecipitation. Protein A beads were incubated with anti-VASP or 0.1% BSA at 4°C overnight amd then pelleted by centrifugation. After washing with IP buffer (135 mM KCl, 10 mM NaCl, 2 mM MgCl2, 2 mM EGTA, 10 mM Hepes, pH 7.1.), the beads were incubated with supernatants at 4°C for 4 h. The extent of depletion was determined by Western blots with a standard curve derived from serial dilutions of control supernatant run on the same gel and quantified with a PhosphorImager using the ImageQuant program (Molecular Dynamics).
In each case, the concentration of the supernatant was adjusted with IP buffer or concentrated via Centricon 10 (Amicon) to 3 or 4 mg/ml protein for assays of F-actin and nucleation.
Preparation of Profilin Mutants
The site-directed mutation replacing Arg-74 with glutamic acid (R74E mutant) and His-133 with serine (H133S mutant) was performed using QuikChangeTM sitedirected mutagenesis kit (Stratagene). The mutagenic oligonucleotides (mutated bases are underlined) 5'-GTTCGGTGATCGAGGACTCACTGCTGCAGGATGG-3' and 5'-GTTATGAAATGGCCTCCTCCCTTCGGCGTTCCC-3' and their reverse complements were used. The GSTprofilin cDNA in pGEX2T (
For expression of the GST fusion proteins, E. coli strain BL21 (DE3) was freshly transformed with the DNA constructs. Expression of the proteins was induced with 0.2 mM IPTG at 30°C for 3 h. Cells were then lysed by sonication in 50 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, 1 mM DTT, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. After centrifugation at 10,000 g for 10 min at 4°C, the GSTprofilin was purified by binding to glutathione-Sepharose beads (Amersham Pharmacia Biotech). Profilin was cleaved from the beads with thrombin. After removal of thrombin by benzamidine beads, profilin was dialyzed against 10 mM Tris-HCl, pH 7.6, and 0.1 mM DTT.
The properties of the profilin mutants were characterized by their ability to bind proteins in neutrophil supernatant. Equal amounts (100 µg) of GSTH133S profilin and GSTR74E profilin on 50 µl glutathione-Sepharose beads (Amersham Pharmacia Biotech) were incubated with 400 µl supernatant (7 µg/µl) for 2 h at 4°C. After washing with IP buffer, proteins bound to the beads were separated on SDS-PAGE, blotted onto Immobilon-P Transfer Membrane (Millipore), and probed with various antibodies. The affinities of wild-type and H133S profilin for G-actin were both determined to be 0.1 µM by their ability to sequester actin from the pointed end and decrease the concentration of gelsolin-capped F-actin. The H133S mutant was able to support filament elongation as it was comparable to wild-type profilin in stimulating the rate of Listeria movement in platelet extracts (
Purification of Recombinant Proteins
Recombinant Cdc42 was expressed in a baculovirus insect cell expression system (S as described by
S-activated Cdc42. The Cdc42 used in these experiments, based on protein concentration (Bio-Rad Laboratories), had three to tenfold lower activity than that used in previous studies reported from this lab (
S. Partitioning in Triton X-115 suggested that this Cdc42 was less fully prenylated than that used previously. Experiments comparing nucleation activity and the time course of polymerization in supernatant using two different preparations (i.e., old versus new) gave similar results given the three to tenfold difference in apparent concentration.
Recombinant N-WASP was expressed in a baculovirus insect cell expression system and purified using FPLC on HiTrap-Heparin column according to the published methods of
F-actin Determination
F-actin was quantified from TRITC-phalloidin staining of pelleted material as described originally by
Assays of Nucleation Sites in Supernatant
Pyrenyl-actin assays of nucleation were performed as described previously by 90%, the initial rate of polymerization is primarily due to barbed-end elongation and thus the initial rate was considered proportional to the number of free barbed ends.
Time Course of Polymerization with Purified Proteins
The time course of polymerization induced by N-WASP or VCA in the presence of Arp2/3 complex was followed by using G-actin that contained 10% or less pyrenylactin. Lower enrichments of labeled G-actin (3% or 1%) were used in experiments in which a high concentration of profilin was present. The mixture was placed, without dilution, into a cuvette and the pyrenylactin fluorescence was monitored continuously. In this assay, nucleation activity is detected from the decrease in the lag time before polymerization begins, or the decrease in the time to achieve half-maximal polymerization. Unlike the assay of nucleation sites described above, the rate of polymerization at any given time is the product of the number of barbed ends present and the concentration of G-actin above the critical concentration. With time, the concentration of G-actin decreases to the critical concentration and the fluorescence reaches a plateau.
Calculation of Free G-actin
In supernatants at a concentration of 3 mg/ml protein, there is 4 µM profilin, 12 µM G-actin, and 17.5 µM thymosin ß4 (
0.52 µM. The concentration of "active" (able to bind G-actin) latrunculin A and vitamin D binding protein (VDBP), was determined from their ability to decrease the initial rate of polymerization of pyrenylactin from spectrinactin seeds (using a Kd for latrunculin A of 0.1 µM, and for VDBP of 1 nM). Using these values, we calculate the concentration of free G-actin in supernatant before or after addition of each protein.
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Results |
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Cdc42 Acts through the Arp2/3 Complex to Induce New Actin Filaments in Neutrophil Supernatant
Cdc42, charged with GTPS (Cdc42) and added to supernatant of lysed neutrophils, increases the number of actin filaments (
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Profilin Depletion Inhibited Cdc42induced Actin Nucleation
To investigate the role of profilin in Cdc42induced actin nucleation, we treated supernatant with beads coupled to PLP. The PLP bead treatment depleted >90% of the profilin, >90% of the VASP, and 510% of the actin in the supernatant. There was no detectable depletion of WASP, WIP, WAVE, Arp3, or IQGAP (Fig 2). PLP treatment severely inhibited the ability of Cdc42 to induce actin nucleation (Fig 3 a). The number of nucleation sites induced by Cdc42 in PLP-treated supernatant was reduced to 10 ± 12% (n = 7) of mock-treated supernatant.
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If the ability of Cdc42 to increase F-actin levels depends on its ability to create new filaments, profilin depletion should also inhibit Cdc42induced actin polymerization. Indeed, PLP treatment decreased the Cdc42-induced increase in F-actin to 11.2 ± 4.8% (n = 12) of control level (Fig 3 b). Polymerization was not merely slowed since even after 16 min of Cdc42 induction, the F-actin level increased only slightly (Fig 3 c). Supplementing the supernatant with 100 µg/ml brain lipid and/or 0.1 µM F-actin did not increase the response to Cdc42.
To determine if the inhibition was due to profilin and/or VASP depletion, we investigated whether re-addition of these proteins would restore activity. Addition of 12 µM recombinant profilin restored 60% of Cdc42-induced nucleation and
50% actin polymerization (Fig 3c and Fig d). Purified spleen profilin and recombinant profilin were similar in their ability to restore activity. Higher concentrations of profilin inhibited both nucleation and polymerization (see below), even though control supernatant contains about 4 µM profilin. Inclusion of 0.5 µM G-actin with 1.5 µM profilin restored Cdc42-induced nucleation to >80% of the control level (data not shown). Higher concentrations of profilinactin increased nucleation above control levels (see below). Thus, complete restoration of activity can be achieved by replacing both the depleted profilin and the depleted actin.
Addition of VASP (up to 1 µM) had no effect on nucleation or polymerization, with or without profilin. The conclusion that VASP is not required for Cdc42-induced polymerization was supported by experiments in which immunodepletion of >90% VASP, with no detectable depletion of profilin, had no effect on actin polymerization (data not shown).
To Restore Activity, Profilin Must Be Able to Bind Actin and Polyproline
Profilin can simultaneously bind actin and PLP. To determine whether binding to one or both of these sites was required to restore nucleation, we used site-directed mutagenesis to create two profilin mutants. The R74E mutant has a decreased affinity for actin (
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The H133S profilin had a reduced ability to restore Cdc42-induced nucleation or polymerization to PLP-treated supernatant (Fig 3 d). At concentrations >2 µM, H133S profilin, like wild-type profilin, inhibited nucleation. No restoration (or inhibition) of nucleation was seen with up to 32 µM R74E profilin. Thus, both the PLP- and actin-binding sites of profilin are needed to enhance Cdc42-induced nucleation. Profilin might function at two independent sites, one binding to the proline-rich domain, the other to actin, however, this seems not to be the case since simultaneous addition of H133S and R74E to PLP-depleted supernatant did not restore activity (data not shown). Thus, the same molecule of profilin must simultaneously bind a proline-rich domain and actin.
Profilin Was Not Required for Nucleation in Supernatants Induced by VCA
Cdc42 stimulates nucleation by binding to a member of the WASP family which then activates nucleation by the Arp2/3 complex (
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Profilin Modulation of Nucleation Induced by Cdc42, Lipids, N-WASP, and the Arp2/3 Complex
To characterize how profilin enhanced the ability of Cdc42 to activate WASP, we examined its effect on nucleation induced by purified proteins. Cdc42 in combination with phosphotidylinositol lipids stimulate N-WASP to activate the Arp2/3 complex in vitro (
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With the pure proteins, the time course of actin polymerization could be directly monitored in the cuvette containing G-actin spiked with pyrenylactin. The combination of Cdc42 and lipid added to N-WASP, Arp2/3 complex, and G-actin increased nucleation as detected by a decrease in the lag time before polymerization, as described previously by
The effects of profilin on Cdc42 activation of purified N-WASP were small compared with the effects in the cell supernatant. In the supernatant, a reservoir of G-actin buffered with thymosin ß4 allows addition of a low concentration of profilin to form profilinactin without significantly decreasing the free G-actin. To determine if a G-actin reservoir would enhance the effects of profilin, we examined the effect of profilin in the presence of 10 µM thymosin ß4 and 6 µM G-actin (Kd = 1 µM). Under these conditions, addition of 0.5 µM profilin will form 0.45 µM profilinactin, and only decreasing the free G-actin from 10.88 µM. Indeed, addition of profilin decreased the long lag (900 s to half maximal polymerization) by 64% (±6% range in two experiments) and increased the maximal rate of polymerization (Fig 6 c). H133S profilin was less effective than wild-type profilin, decreasing the lag by 55 ± 3% and having no effect on the maximal rate. In the presence of the thymosin-actin buffer, profilin also decreased the lag for polymerization induced by VCA, but in this case the decrease by wild-type or H133S profilin was similar (47 ± 3%) (Fig 6 d). Thus, the presence of a G-actin reservoir accounts for some of the differences between the effects of profilin in supernatant and with pure proteins.
Inhibition of Nucleation by Elevated Concentration of Profilin Is Due to G-actin Sequestration
In the experiments above, we have examined the effects of depleting profilin from cell supernatant. Since supernatant preparation involves a tenfold dilution of cytoplasm, the concentration of profilin in the intact cell is higher than in the supernatant. Thus, we investigated the effects of increasing the profilin concentration. Adding even 1 µM profilin to cell supernatant or purified proteins decreased the rate of nucleation (see Fig 3 d and 6 a). Since high concentrations of a profilin mutant, which does not bind G-actin (R74E), did not inhibit nucleation, it seemed likely that the inhibition was due to G-actin sequestration. Indeed, addition of latrunculin A or VDBP, both of which sequester G-actin, also inhibited nucleation (Fig 7 a). Addition of thymosin ß4 also inhibited nucleation (not shown), although, due to its lower affinity, higher concentrations were required to sequester a comparable amount free G-actin. Cdc42-induced nucleation could not be detected when the calculated free G-actin was decreased to 0.1 µM (see Materials and Methods). This concentration of G-actin appears to limit nucleation with pure proteins as well. Addition of 5 nM Arp2/3 complex and 50 nM VCA to 0.5 µM actin filaments at steady state (G-actin is at its critical concentration of 0.1 µM) resulted in little or no increase in the number of filament ends, even after a two-hour incubation (data not shown).
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Increasing the free G-actin concentration in the supernatant by addition of exogenous G-actin increased Cdc42-induced nucleation. Up to 2 µM G-actin could be added without increasing basal nucleation (nucleation in the absence of Cdc42); addition of higher concentrations caused a parallel increase in basal and agonist-induced nucleation (Fig 7 b). Combined, these data suggest that Cdc42-induced nucleation in neutrophil supernatant is a function of the free G-actin calculated to be between 0.10.8 µM (see Materials and Methods). G-actin bound to profilin or thymosin ß4 did not effectively substitute for free G-actin in this nucleation reaction.
ProfilinActin Enhanced Cdc42-induced Nucleation
With a better understanding of the G-actin requirement for nucleation, we next examined the effect of profilin added to supernatant in combination with enough G-actin to maintain a constant concentration of free G-actin. Under these conditions, the presence of profilinactin enhanced nucleation by Cdc42 without increasing basal nucleation. The effects of profilinactin were not due merely to an increased pool of G-actin, since in a parallel experiment, addition of the same amount of G-actin in combination with enough thymosin ß4 to maintain the same concentration of free G-actin did not enhance nucleation (not shown). The Cdc42-induced nucleation sites increased with increasing concentrations of profilinactin (Fig 7 c). To induce a comparable number of nucleation sites, profilinactin was required at 10 times the concentration of free G-actin. Since the concentration of profilinactin in the neutrophil cytoplasm is about 20-fold higher than that of free G-actin (20 µM versus 0.5 µM; see Materials and Methods), the contribution by profilinactin is likely to be physiologically meaningful.
Furthermore, increasing the concentration of profilinactin decreased the concentration of free G-actin needed for nucleation. To vary the concentration of free G-actin in the presence of profilinactin, 10 µM profilin was added to supernatant along with increasing concentrations of G-actin. In parallel samples, increasing concentrations of G-actin alone were added. The nucleation induced by Cdc42 was then measured. The number of nucleation sites versus the calculated free G-actin concentration is shown in Fig 7 d.
G-actin and ProfilinActin Also Stimulate Nucleation Induced by GSTVCA
To determine if stimulation by increased concentrations of profilinactin in supernatant was unique to Cdc42-induced nucleation, we examined nucleation induced by GSTVCA. Profilinactin enhanced nucleation and decreased the requirement for free G-actin, similar to GSTVCA- and Cdc42-induced nucleation (not shown). In summary, in cell supernatant, profilinactin is less efficient than free G-actin in supporting Arp2/3-mediated nucleation activated by either Cdc42 or GSTVCA, but profilinactin, in the presence of the same initial free G-actin concentration, enhances nucleation. To examine the mechanism of this enhancement we switched to a pure protein system.
ProfilinActin Enhances Nucleation by GSTVCA-activated Arp2/3 Complex
The activation of nucleation by GSTVCA and Arp2/3 complex depends on the concentration of free G-actin, even in the presence of F-actin. Therefore, to determine if profilinactin could enhance the rate of nucleation with pure proteins, we examined the time course of polymerization of samples with profilin/G-actin ratios calculated to give the same initial free concentration of G-actin as control samples in the absence of profilin. The rationale for this strategy is as follows. At any particular concentration of barbed ends, free G-actin will elongate the filaments at a rate proportional to its concentration, independent of the presence or absence of profilin. If profilinactin is also present it will contribute to filament elongation with a rate constant 70% of that of free G-actin (
Fig 8 a shows a test of prediction b above. Profilin/G-actin ratios giving a free G-actin concentration of 0.1 µM (which on its own did not increase nucleation above background) enhanced nucleation by VCA and the Arp2/3 complex, contrary to the prediction. As in the supernatant, a much higher concentration of profilinactin than free G-actin was required to achieve a comparable number of nucleation sites. H133S profilin was as effective as wild-type profilin in this respect (not shown), consistent with there being no proline-rich region in VCA.
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Fig 8 b shows a test of prediction c above. The time course of the conversion of G-actin to F-actin was followed by using partially pyrenylated G-actin with the same initial concentration of G-actin (0.05, 0.3, or 2 µM) in the presence and absence of profilin and normalized to the same final fluorescence. In the presence of profilin, a low percentage of pyrenyl-actin was used, since profilin preferentially binds unlabeled actin, a fact that does not qualitatively alter the predictions made under the null hypothesis. Again, the outcome was contrary to the prediction: when the G-actin concentration was low, the presence of profilin accelerated nucleation. These results suggest that profilinactin either participates directly in Arp2/3-induced nucleation or enhances it in some less direct way, for example by stabilizing nascent nuclei or by increasing the concentration of a cofactor. The ability of profilinactin to stimulate Arp2/3 complex nucleation depended on the concentration of free G-actin. When the concentration of G-actin was 1 µM, the presence of profilinactin did not accelerate nucleation, presumably because any effect was masked by the more efficient G-actin.
Increasing the total G-actin pool can, if it releases an appropriate concentration of free G-actin, increase the amount of actin polymerized and the number of nucleation sites produced. To determine whether increasing the total G-actin pool might, in some unexplained way, affect the rate of nucleation, we increased the reservoir of G-actin by adding G-actin together with thymosin ß4. Since under our conditions, thymosin ß4actin does not contribute to filament elongation, we would expect its influence to be expressed via its effect of buffering the free G-actin as it is consumed. Therefore, under the null hypothesis that neither thymosin ß4 nor the total G-actin pool affect nucleation, we would predict that in the presence of thymosin ß4, compared with a control with the same initial concentration of free G-actin: (a) the concentration of free G-actin will fall more slowly and follow a higher trajectory over time; therefore (b) the concentration of barbed ends will increase more rapidly and also follow a higher trajectory; and so (c) the rate of the conversion of G-actin to F-actin will be increased.
Fig 8 c shows an experiment in which the thymosin ß4 to actin ratio was adjusted to give an initial concentration of free G-actin of 1 µM (T/A = 10 µM:6 µM), compared with a control in the absence of thymosin ß4 with an initial concentration of free G-actin of 1 µM. Again, the prediction under the null hypothesis was not born out: in the presence of thymosin ß4, the conversion of G-actin to F-actin was in fact slowed (best seen when the data are normalized to the same final fluorescence). Therefore, not only could we detect no effect of an increased total G-actin pool in enhancing the rate of nucleation, but also we conclude that either thymosin ß4 or thymosin ß4actin acts in some way to inhibit nucleation as compared with the control with the same initial concentration of free G-actin. The number of nucleation sites at plateau was, however, increased (data not shown). Thus the reservoir of G-actin did allow continued slow nucleation by the free G-actin resulting in an increase in the final level of nucleation sites.
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Discussion |
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Profilin Has Multiple Effects on Nucleation
These studies illuminate the multiple effects, both positive and negative, that profilin has on Arp2/3 complexmediated nucleation. In cell supernatant, profilin depletion profoundly inhibited nucleation induced by Cdc42, but not by VCA. Cdc42-induced nucleation was restored by addition of wild-type profilin, but not by mutant profilins unable to bind PLP or actin. PLP binding also contributed to profilin's ability to enhance Cdc42-nucleation via purified N-WASP.
Profilin binds with submicromolar affinity to the proline-rich domain of N-WASP (
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Profilin Effects on Supernatant Are Not Fully Explained by Results with Purified Proteins
Though studies with the purified proteins help define the molecular interactions, they do not fully reflect the situation in the supernatant. Addition of profilin increased Cdc42-induced nucleation in profilin-depleted supernatant more than sixfold, but with pure reagents only about twofold. Most likely, other components in the supernatant, including thymosin ß4 and cofilin, increase the importance of profilin. Other profilin-binding proteins, such as WIP, may also play a role in the supernatant.
Inhibitory Effects of Profilin on Nucleation Are Due to G-actin Sequestration
The inhibitory effects of profilin on nucleation are due in part to its sequestration of G-actin. Nucleation by Arp2/3 complex in supernatant or with pure proteins is a function of the free G-actin concentration (Fig 7;
Rather, it appears that profilinactin is less effective than free G-actin in some aspect of the nucleation reaction. G-actin binds to the verprolin domain of WASP proteins. This domain, present in all members of the family, is needed for activation of Arp2/3 by COOH-terminal fragments of WASP family proteins (
ProfilinActin Can Enhance Nucleation Independent of Polyproline Binding
Profilinactin can enhance the rate of nucleation under conditions where the free G-actin concentration was maintained, i.e., when profilin was added as profilinactin or added to a reservoir of G-actin buffered with thymosin ß4. This enhancement does not require that profilin be able to bind PLP and may result from several different actions of profilin. When F-actin, a cofactor in Arp2/3 complex-mediated nucleation, is limiting, profilinactin could enhance nucleation by its ability to elongate at barbed ends thereby increasing F-actin. Indeed, the enhancement by profilin was greater in the absence than in the presence of F-actin, suggesting that it might function by increasing F-actin or some other cofactor.
The enhancement by profilinactin was greatest, in the presence or absence of F-actin, when the free G-actin concentration was low. The presence of profilinactin allowed nucleation when the free G-actin concentration was too low to support nucleation on its own (>0.1 µM). Consistent with this result, branched filaments formed in the presence of Arp2/3 complex, VCA, and profilin/actin at a ratio expected to give a low concentration of free G-actin (0.6 µM (
10% of the verprolin domains. When G-actin is limiting, profilinactin could contribute by binding directly to the Arp2/3 complex to form a new filament and/or by barbed-end elongation to stabilize a nascent filament.
Profilin has long been known to enhance actin treadmilling. These studies show that profilin also enhances Arp2/3 complex-mediated actin nucleation. Profilin contributes both to the activation of WASP by Cdc42 and to nucleation by activated Arp2/3 complex. Profilinactin allows nucleation at decreased free G-actin concentrations. Because neutrophil cytoplasm contains about 40 µM profilin, the pool of profilinactin in a resting cell (assuming the concentration of free G-actin = 0.5 µM) is 34 µM. After chemoattractant stimulation, when the F-actin level doubles, the free G-actin level may decrease to 0.1 µM, but the profilinactin would still be present at
24 µM. This profilinactin can allow rapid nucleation of new actin filaments to continue.
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Footnotes |
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1 Abbreviations used in this paper: CA, cofilin homology and acidic tail contained in the COOH-terminal fragment of N-WASP; GST, glutathione S-transferase; PLP, poly-L-proline; VCA, verpolin homology, cofilin, and acidic domain contained in the COOH-terminal fragment of N-WASP; VDBP, vitamin D binding protein; WASP, Wiskott-Aldrich Syndrome protein.
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Acknowledgements |
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We are very grateful to Henry Higgs for helpful comments on a draft of the manuscript.
This work was supported by National Institutes of Health grant AI-19883 to S.H. Zigmond.
Submitted: 25 April 2000
Revised: 13 July 2000
Accepted: 14 July 2000
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References |
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