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Am J Physiol Heart Circ Physiol 285: H1528-H1536, 2003. First published June 12, 2003; doi:10.1152/ajpheart.00188.2003
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Downregulation of profilin with antisense oligodeoxynucleotides inhibits force development during stimulation of smooth muscle

Dale D. Tang and Jian Tan

Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202

Submitted 28 February 2003 ; accepted in final form 6 June 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The actin-regulatory protein profilin has been shown to regulate the actin cytoskeleton and the motility of nonmuscle cells. To test the hypothesis that profilin plays a role in regulating smooth muscle contraction, profilin antisense or sense oligodeoxynucleotides were introduced into the canine carotid smooth muscle by a method of reversible permeabilization, and these strips were incubated for 2 days for protein downregulation. The treatment of smooth muscle strips with profilin antisense oligodeoxynucleotides inhibited the expression of profilin; it did not influence the expression of actin, myosin heavy chain, and metavinculin/vinculin. Profilin sense did not affect the expression of these proteins in smooth muscle tissues. Force generation in response to stimulation with norepinephrine or KCl was significantly lower in profilin antisense-treated muscle strips than in profilin sense-treated strips or in muscle strips not treated with oligodeoxynucleotides. The depletion of profilin did not attenuate increases in phosphorylation of the 20-kDa regulatory light chain of myosin (MLC20) in response to stimulation with norepinephrine or KCl. The increase in F-actin/G-actin ratio during contractile stimulation was significantly inhibited in profilin-deficient smooth muscle strips. These results suggest that profilin is a necessary molecule of signaling cascades that regulate carotid smooth muscle contraction, but that it does not modulate MLC20 phosphorylation during contractile stimulation. Profilin may play a role in the regulation of actin polymerization or organization in response to contractile stimulation of smooth muscle.

cytoskeleton; actin-binding proteins; actin polymerization; myosin light chain phosphorylation; contraction

VASOCONSTRICTORS have been shown to play an important role in the pathogenesis of essential hypertension (35, 45, 49). However, the mechanisms by which vasoconstrictors regulate force development in vascular smooth muscle are not completely understood. Although smooth muscle contraction has long been thought to depend solely on the regulation of the 20-kDa regulatory light chain of myosin (MLC20) that in turn modulates actomyosin cross-bridge cycling, recent studies reveal that a pool of actin monomers is stimulated to polymerize into actin filaments in vascular and airway smooth muscle tissues in response to contractile stimulation (3, 12, 30, 34) and in cultured smooth muscle cells (1, 25). The inhibition of actin polymerization by specific inhibitors such as latrunculin depresses force development in smooth muscle activated by contractile stimuli (12, 34, 61), even though increases in MLC20 phosphorylation in response to contractile stimulation are not depressed (34). These studies suggest that the polymerization of actin filaments per se is an essential cellular event during smooth muscle contraction. However, the mechanisms that regulate actin filament polymerization in smooth muscle are largely unknown.

Profilin is an actin-regulatory protein that regulates actin dynamics, the organization of actin cytoskeleton, and the movement of a number of cultured cells, including fibroblasts, epithelial cells, leukocytes, and platelets (6, 9, 20, 22, 32, 33, 57). In vitro biochemical studies have shown that profilin binds to unpolymerized globular actin (G-actin) and influences actin dynamics by sequestering actin monomers (8, 9), enhancing actin filament assembly at barbed ends (20, 32) and promoting the nucleation of actin polymerization mediated by Cdc42, neuronal Wiskott-Aldrich syndrome protein (N-WASP) and the actin-related protein (Arp2/3) complex (7, 23, 60).

In this study, we have evaluated the role of profilin in regulating smooth muscle contraction by depleting carotid smooth muscle tissues of profilin protein with antisense oligodeoxynucleotides. We then assessed the effect of profilin depletion on active force, phosphorylation of MLC20, and the F-actin/G-actin ratio, an index of actin filament polymerization, in response to contractile stimulation. We found that the downregulation of profilin with antisense inhibited force generation, but it did not affect MLC20 phosphorylation, suggesting that profilin is essential for smooth muscle contraction and that it does not regulate contractile protein activation. However, the depletion of profilin inhibited increases in the F-actin/G-actin ratio in response to agonist stimulation, indicating that profilin is necessary for actin dynamics during contractile stimulation of smooth muscle.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Preparation of smooth muscle tissues. Mongrel dogs (20–25 kg) were anesthetized with pentobarbital sodium (30 mg/kg iv) and quickly exsanguinated. All procedures were performed according to the guideline of Institutional Animal Care and Use Committee, Indiana University School of Medicine. A 6- to 8-cm segment of carotid was immediately removed and immersed in physiological saline solution (PSS) at 22°C (composition in mM: 110 NaCl, 3.4 KCl, 2.4 CaCl2, 0.8 MgSO4, 25.8 NaHCO3, 1.2 KH2PO4, and 5.6 glucose). The solution was aerated with 95% O2-5% CO2 to maintain a pH of 7.4. Circumferential strips of smooth muscle (3 mm wide, 8–10 mm long, and 0.2 mm thick) were dissected from the carotid after removal of the endothelium, intimal, and adventitial layers. The use of an appropriately sized strip was critical for maintaining muscle contractility during the incubation period and for the successful introduction of oligonucleotides throughout the muscle strip.

Measurement of force development in smooth muscle. Each muscle strip was placed in PSS at 37°C in a 25-ml organ bath and attached to a Grass force transducer that had been connected to a Gould recorder. Smooth muscle strips were stretched until the passive tension reached 2 g, and these strips were then equilibrated at 37°C for 30 min. The muscle strips were stimulated with 40 mM KCl for three to four times to optimize contractile response to agonist stimulation. These muscle strips were then stimulated with norepenephrine (NE) or KCl for a different time period and were frozen using liquid N2-cooled tongs for biochemical analysis.

Oligodeoxynucleotides (ODNs) dissolved in Tris-EDTA buffer were introduced into the muscle strips according to experimental procedures described previously (53, 54). Muscle strips were then incubated for 2 days with ODNs in Dulbecco's modified Eagle's medium (DMEM). The strips were returned to PSS at 37°C in 25-ml organ baths and attached to Grass force transducers for the measurement of isometric force.

Loading of oligodeoxynucleotides and organ culture. Antisense ODNs with the following sequence were designed to selectively suppress profilin expression in canine vascular smooth muscle: 5'-TCCTGCAGCAGTGAGTCC-3'. In some protocols, the following sequence of sense oligonucleotides was used as a control: 5'-GGACTCACTGCTGCAGGA-3'. According to sequence matching results obtained from The National Center for Biotechnology Information, these sequences are not homologous to sequences of any other contractile proteins, cytoskeletal proteins, or actin-associated proteins. The antisense molecule targeted to a region of mRNA that is unique to profilin. The phosphorothioate ODNs were synthesized and purified by Invitrogen (Carlsbad, CA). A homologous region was confirmed for canine profilin mRNA by using RT-PCR to amplify a 199-bp fragment from canine carotid mRNA. The 5'-primer used for RT-PCR was 5'-GGACTCACTGCTGCAGGA-3'. The 3'-primer used for RT-PCR was 5'-TCAGTACTGGGAACGCCG-3'.

The ODNs were introduced into carotid smooth muscle strips by reversible permeabilization using methods we have previously used for the introduction of focal adhesion kinase and paxillin antisense into tracheal muscle strips (53, 54). After determination of contractile response, each muscle strip was attached to a metal mount at the appropriate length. The strips were placed in 0.5-ml tubes and incubated successively in each of the following solutions: solution 1 (at 4°C for 120 min) containing (in mM) 10 EGTA, 5 Na2ATP, 120 KCl, 2 MgCl2, and 20 N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid (TES); solution 2 (at 4°C overnight) containing (in mM) 0.1 EGTA, 5 Na2ATP, 120 KCl, 2 MgCl2, and 20 TES plus 10 µM antisense or sense ODNs; solution 3 (at 4°C for 30 min) containing (in mM) 0.1 EGTA, 5 Na2ATP, 120 KCl, 10 MgCl2, and 20 TES. The strips were then transferred to 25-ml organ baths containing solution 4 (at 22°C for 60 min, 110 mM NaCl, 3.4 mM KCl, 0.8 mM MgSO4, 25.8 mM NaHCO3, 1.2 mM KH2PO4, and 5.6 mM dextrose). Solutions 1–3 were maintained at pH 7.1 and aerated with 100% O2. Solution 4 was maintained at pH 7.4 and was aerated with 95% O2-5% CO2. After 30 min in solution 4, CaCl2 was added gradually to reach a final concentration of 2.4 mM. The strips were then incubated for 2 days in DMEM containing 5 mM Na2ATP, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 µM antisense or 10 µM sense oligonucleotides, which were kept at 37°C, 5% CO2. The media were changed every other day. Reversible permeabilization is believed to load large amounts of ODNs into smooth muscle strips (29, 53, 54). The addition of ODNs in media was to compensate for the degradation of oligonucleotides in cells (16, 53, 54, 56). ODNs can be taken up into cells by endocytosis, maintaining appropriate intracellular ODN concentration (46, 56).

Immunoblot analysis. Pulverized muscle strips were mixed with 50 µl of extraction buffer containing 10% glycerol, 2 mM EDTA, 20 mM Tris · HCl at pH 7.6, 10 mM DTT, phosphatase inhibitors (2 mM sodium orthovanadate, 2 mM molybdate, and 2 mM sodium pyrophosphate) and protease inhibitors (2 mM benzamidine, 0.5 mM aprotinin and 1 mM phenylmethylsulfonyl fluoride). Each sample was kept on ice for 1 h and then centrifuged for the collection of supernatant. Muscle extracts were boiled in sample buffer [1.5% dithiothreitol, 2% SDS, 80 mM Tris · HCl (pH 6.8), 10% glycerol and 0.01% bromophenol blue] for 5 min and then separated by SDS-PAGE. Proteins were transferred to nitrocellulose, after which the membrane was cut into several parts for immunobloting of different proteins. The nitrocellulose membrane was blocked with 2% gelatin for 1 h and probed with polyclonal antibody against profilin (Cytoskeleton; Denver, CO) followed by horseradish peroxidase (HRP)-conjugated anti-rabbit Ig (ICN Biomedicals; Irvine, CA). Nitrocellulose membranes were then stripped of bound antibodies. Actin was probed using a monoclonal antibody against actin (clone 1A4, Sigma) followed by HRP-conjugated anti-mouse IgG (Amersham Pharmacia Biotech; Piscataway, NJ). Myosin heavy chain was detected with monoclonal antibody against smooth muscle myosin heavy chain (clone: hSM-V, Sigma). Polyclonal antibody against metavinculin/vinculin was used to probe each of the proteins followed by HRP-conjugated anti-rabbit IgG (ICN). Proteins were visualized by enhanced chemiluminescence and quantitated by scanning densitometry. Densitometric values of profilin, actin, myosin heavy chain, and metavinculin/vinculin were determined for sense-treated and antisense-treated strips and normalized to those of no ODN-treated strips. The ratios of these proteins were calculated to verify that changes in protein expression were selective for profilin.

Analysis of MLC20 phosphorylation. Muscle strips were rapidly frozen at desired time points after contractile stimulation and then immersed in acetone containing 10% (wt/vol) trichloroacetic acid and 10 mM DTT (acetone-TCA-DTT) that was precooled with dry ice. Strips were thawed in acetone-TCA-DTT at room temperature for 1 h and then washed four times with acetone-DTT. Proteins were extracted for 2 h in 8 M urea, 20 mM Tris base, 22 mM glycine, and 10 mM DTT. The MLC20 was separated by glycerol-urea polyacrylamide gel electrophoresis and transferred to nitrocellulose. The membranes were blocked with 5% milk and incubated with polyclonal affinity-purified rabbit myosin light chain antibody. The primary antibody was reacted with HRP-conjugated anti-rabbit IgG (ICN). Unphosphorylated and phosphorylated bands of MLC20 were visualized by ECL and quantitated by scanning densitometry. MLC20 phosphorylation was calculated as the ratio of phosphorylated MLC20 to total MLC20.

Analysis of F-actin/G-actin ratio. The concentration of F-actin and G-actin in smooth muscle tissues was measured using an assay kit from Cytoskeleton. Briefly, each of the carotid smooth muscle strips was homogenized in 200 µl F-actin stabilization buffer (50 mM PIPES, pH 6.9, 50 mM NaCl, 5 mM MgCl2, 5 mM EGTA, 5% glycerol, 0.1% Triton X-100, 0.1% Nonidet P-40, 0.1% Tween 20, 0.1% {beta}-mercapto-ethanol, 0.001% antifoam, 1 mM ATP, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 10 µg/ml benzamidine, and 500 µg/ml tosyl arginine methyl ester). Supernatants of the protein extracts were collected after centrifugation at 100,000 g for 60 min at 30°C. The pellets were resuspended in ice-cold distilled water plus 1 µM cytochalasin D and then incubated on ice for1hto dissociate F-actin. The resuspended pellets were gently mixed every 15 min. Supernatant (G-actin) and pellet (F-actin) fractions were subjected to analysis of immunoblot using actin antibody. The ratio of F-actin to G-actin was determined by scanning densitometry.

Statistical analysis. All statistical analysis was performed using SigmaStat software. Comparison among multiple groups was performed by one-way analysis of variance or Kruskal-Wallis one-way analysis of variance. Differences between pairs of groups were analyzed by Student-Newman-Keuls test or Dunn's method. Values of n refer to the number of experiments used to obtain each value. P < 0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
NE stimulation increases MLC20 phosphorylation and the F-actin/G-actin ratio in carotid smooth muscle. We evaluated the effect of NE stimulation on contractile protein activation and actin polymerization in vascular smooth muscle. The phosphorylation level of the MLC20 and the ratio of F-actin over G-actin were measured during contraction (0.5–15 min, 10–5 M NE) and relaxation (addition of 10–5 M phentolamine) of canine carotid smooth muscle strips. Unstimulated muscle strips were frozen in the presence of 10–5 M phentolamine to evaluate the resting level of MLC20 phosphorylation and the F-actin/G-actin ratio.

The stimulation of vascular muscle strips with NE resulted in increases in MLC20 phosphorylation. MLC20 phosphorylation in smooth muscle tissues reached a high level 2–5 min after stimulation with NE and subsequently decreased to a slightly lower level (Fig. 1B, n = 6). The increases in F-actin/G-actin ratio in carotid muscle strips were evident as early as 1 min after NE stimulation and sustained for the 15-min period (Fig. 1C, n = 6). Both MLC20 phosphorylation and the F-actin/G-actin ratio were decreased to basal levels during the relaxation of smooth muscle (Fig. 1, B and C).



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  		Fig. 1. Myosin light chain at 20 kDa (MLC20) phosphorylation and the F-actin/G-actin ratio during contraction-relaxation cycle of canine carotid smooth muscle strips. A: norepinephrine (NE, 10–5 M) increases force development; the addition of phentolamine (10–5 M) leads to the relaxation of smooth muscle. Force is expressed as percentage of maximal response to 10–5 M NE (n = 16). B: MLC20 phosphorylation was evaluated by immunoblot analysis of urea glycerol gel (see MATERIALS AND METHODS)(n = 6). C: F-actin/G-actin ratio was analyzed by a method of tissue fractionation (see MATERIALS AND METHODS) (n = 6). All values are means ± SE.

 

The actin polymerization inhibitor latrunculin A inhibits force development and the F-actin/G-actin ratio in response to NE stimulation, without affecting MLC20 phosphorylation. To determine whether actin polymerization is involved in the regulation of smooth muscle contraction, carotid smooth muscle strips were treated with 1 µM latrunculin A (dissolved in 0.1% DMSO) for 45 min or treated with 0.1% DMSO as control. Contractile force and the ratio of F-actin/G-actin in response to contractile stimulation (10–5 M NE, 5 min) were determined. MLC20 phosphorylation in response to NE stimulation was also measured in latrunculin A-treated strips or untreated strips to determine whether the treatment of muscle strips with latrunculin A affects contractile protein activation.

The treatment of carotid smooth muscle strips with latrunculin A depressed NE-stimulated contraction; force in muscle strips treated with latrunculin A was 18% of force in strips not treated with latrunculin A (n = 8, P < 0.05) (Fig. 2A). The treatment with latrunculin A did not affect MLC20 phosphorylation in response to NE stimulation; there was no significant difference in MLC20 phosphorylation in latrunculin A-treated tissues and untreated strips (P > 0.05, Fig. 2B). However, the treatment of strips with latrunculin A inhibited increases in NE-elicited actin polymerization; the F-actin/G-actin ratio in latrunculin A-treated tissues was lower than that in untreated strips (n = 4, P < 0.05) (Fig. 2C).



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  		Fig. 2. Latrunculin A (LAT-A) depresses active force and the F-actin/G-actin ratio but not MLC20 phosphorylation in response to NE stimulation. A: canine carotid smooth muscle strips were incubated with 1 µM LAT-A or DMSO (No LAT-A) for 45 min and then stimulated with 10–5 M NE for 5 min. The strips were then frozen for analysis of MLC20 phosphorylation and the ratio of F-actin/G-actin. Preincubation of muscle strips with LAT-A inhibited NE-stimulated force development (n = 8). B: treatment of strips with LAT-A did not inhibit NE-stimulated MLC20 phosphorylation (n = 4). C: LAT-A inhibited the increases in F-actin/G-actin ratio in response to NE stimulation (n = 4). All values are means ± SE. *Levels in LAT-A-treated muscle strips are significantly different from those in muscle strips not treated with LAT-A (P < 0.05).

 

Profilin antisense oligodeoxynucleotides inhibit the expression of profilin protein. We assessed the effect of treatment with profilin antisense oligodeoxynucleotides on the expression of profilin in canine carotid smooth muscle strips. Profilin antisense or sense oligodeoxynucleotides were introduced into smooth muscle strips by reversible permeabilization (29, 53, 54). These muscle strips were incubated for 2 days to allow for protein depletion. Protein extracted from smooth muscle tissues that had been treated with antisense or sense ODNs or had not been treated with ODNs was analyzed by Western blot to compare the expression of profilin with that of actin, myosin heavy chain (MHC), and the cytoskeletal proteins vinculin and metavinculin (a smooth muscle-specific isoform of vinculin) (4).

The expression of profilin protein was lower in muscle tissues treated with antisense ODNs than in tissues treated with sense ODNs or no ODNs (Fig. 3A). However, the expression of actin, MHC, and metavinculin/vinculin was not affected by antisense treatment. Protein expression in sense-treated and antisense-treated strips was normalized to that in strips not treated with ODNs. The expression of profilin relative to that of actin, MHC, and metavinculin/vinculin was significantly lower in antisense-treated strips than in strips not treated with ODNs or in sense-treated strips (Fig. 3B, n = 5, P < 0.01). This indicates that the decrease in profilin expression was a specific effect of the antisense treatment and that it did not result from general deterioration of the tissue during the incubation period or from nonselective effects of the antisense on protein synthesis.



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  		Fig. 3. Profilin antisense oligodeoxynucleotides (ODNs) selectively inhibit the expression of profilin in smooth muscle. A: blots of protein extracts from muscle strips that had each been incubated for 2 days without ODNs (No ODNs) or with profilin sense ODNs or profilin antisense sense ODNs were detected with antibodies against profilin, actin, myosin heavy chain (MHC), and metavinculin/vinculin. The amount of profilin was lower in antisense-treated muscle strips than in strips not treated with ODNs or sense-treated muscle strips. Similar amounts of actin, MHC, and metavinculin/vinculin were detected in all three strips. Molecular mass markers are indicated on left. B: ratios of protein expression obtained from muscle strips treated with profilin sense (open bars) or paxillin antisense (black bars). Ratios in sense-treated and antisense-treated strips are normalized to ratios obtained in muscle strips not treated with ODNs. PRF, profilin; ACT, actin; VIN, metavinculin/vinculin. Values represent means ± SE (n = 5). *Significantly lower ratios in antisense-treated strips relative to no-ODN-treated and sense-treated muscle strips (P < 0.05).

 

Profilin depletion depresses force development but not MLC20 phosphorylation in response to stimulation with NE. To determine the role of profilin in the regulation of smooth muscle contraction, we evaluated isometric force development in response to NE in muscle strips treated with profilin antisense or sense ODNs or not treated with ODNs. Force development in response to 10–5 M NE was compared before and after the 2-day incubation period. Without ODN treatment, contractile force in response to stimulation with NE for 5 min was similar to preincubation force (n = 10, P > 0.05). Force in profilin sense-treated tissues in response to NE stimulation was 96% of preincubation force (n = 10, P > 0.05). Contractile response in antisense-treated tissues was significantly reduced to 22% of preincubation force (n = 10, P < 0.01) (Fig. 4, A and B). There are no differences in tension among the three groups of strips before NE stimulation.



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  		Fig. 4. Depletion of profilin with antisense ODNs depresses contractile force but not MLC20 phosphorylation in response to stimulation with NE. Contractile responses of canine carotid smooth muscle strips were evaluated, after which profilin sense or profilin antisense was introduced into these muscle strips by reversible permeabilization, and these strips were incubated for 2 days to allow for protein depletion. Contractile responses of these muscle strips were then determined. A: representative tracings of 3 muscle strips from 1 experiment show that profilin antisense inhibited NE-induced contraction in smooth muscle strips after 2-day incubation. Contractile responses of no-ODN-treated or sense-treated muscle strips were similar to preincubation force after 2 days of incubation. B: mean active force in response to 10–5 M NE was quantitated as percentage of NE-induced force in each strip before incubation. Values are means ± SE. *Significantly lower response compared with muscles treated with sense ODNs or not treated with ODNs (n = 10, P < 0.05). C: MLC20 phosphorylation in response to NE (10–5 M, 5 min) was measured in canine carotid smooth muscle strips that had been incubated without ODNs (open bar) or with profilin sense ODNs (gray bar) or profilin antisense ODNs (black bar). Differences between antisense-treated, sense-treated, and no-ODNs-treated strips were not statistically significant (P > 0.05). Values shown are means ± SE (n = 4).

 

MLC20 phosphorylation activated by calcium/calmodulin-dependent myosin light chain kinase is a major biochemical event during smooth muscle contraction (26, 31, 53). To determine whether profilin depletion affects MLC20 phosphorylation, a subset of carotid smooth muscle strips that had been incubated with profilin antisense or sense ODNs or without ODNs was stimulated with 10–5 M NE for 5 min and then frozen for the analysis of MLC20 phosphorylation. Although active force was significantly depressed, NE stimulation resulted in a significant increase in MLC20 phosphorylation in tissues treated with profilin antisense ODNs. The average increases in MLC20 phosphorylation in strips not treated with ODNs or sense-treated and antisense-treated tissues were not significantly different 5 min after NE stimulation (Fig. 4C).

Profilin depletion inhibits contractile force without attenuating increases in MLC20 phosphorylation stimulated by KCl. We also assessed the effect of profilin depletion on force development and MLC20 phosphorylation in carotid smooth muscle strips stimulated by KCl. Force and MLC20 phosphorylation were measured 5 min after stimulation (29, 39, 40, 53, 54). Contractile force in response to KCl stimulation was lower in antisense-treated muscle strips than in muscle strips not treated with ODNs or strips treated with sense ODNs (Fig. 5A). Increases in MLC20 phosphorylation in muscle tissues not treated with ODNs were similar to those in muscle strips treated with sense or antisense ODNs (Fig. 5B, P > 0.05).



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  		Fig. 5. Profilin depletion inhibits contractile force without affecting MLC20 phosphorylation stimulated by KCl. A: smooth muscle strips were contracted with 40 mM KCl before and after a 2-day incubation without ODNs (open bar) or with profilin sense (gray bar) or with profilin antisense (black bar). Mean active force in response to 40 mM KCl was quantitated as percentage of KCl-induced force in each strip before incubation. Values are means ± SE. *Significantly lower response compared with muscles not treated with ODNs or treated with sense ODNs (n = 8) (P < 0.05). B: MLC20 phosphorylation in muscle strips incubated without ODNs (open bar) or with profilin sense (gray bar) or with profilin antisense (black bar). MLC20 phosphorylation was measured in smooth muscle strips 5 min after stimulation with 40 mM KCl. Differences among antisense-treated, sense-treated, and no-ODNs-treated strips were not statistically significant. Values shown are means ± SE (n = 4).

 

Profilin depletion inhibits the increases in the F-actin/G-actin ratio in carotid smooth muscle strips stimulated with NE. To determine whether the depletion of profilin affects actin polymerization, smooth muscle strips incubated without ODNs or with profilin sense or antisense ODNs were stimulated with 10–5 M NE for 5 min and then homogenized for the analysis of F-actin/G-actin ratio. The F-actin/G-actin ratio in response to NE stimulation was lower in profilin-depleted smooth muscle tissues than in muscle strips that had been treated with profilin sense ODNs or not treated with ODNs (Fig. 6) (n = 4, P < 0.05). We also analyzed F-actin/G-actin ratios without NE stimulation in smooth muscle strips that had been incubated without ODNs or with profilin sense or antisense ODNs. The F-actin/G-actin ratios in the absence of agonist stimulation were similar in muscle tissues incubated without ODNs or with profilin sense or antisense ODNs (n = 4, P > 0.05).



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  		Fig. 6. Profilin downregulation inhibits increases in the F-actin/G-actin ratio stimulated by NE. Canine carotid smooth muscle strips subjected to treatment with no ODNs, profilin sense, or profilin antisense ODNs for 2 days were stimulated with 10–5 M NE for 5 min (black bars) or unstimulated (open bars). F-actin/G-actin ratios of these strips were then determined. *NE-induced F-actin/G-actin ratio in antisense-treated strips is significantly lower than the value for stimulated strips not treated with ODNs or treated with sense ODNs (n = 4, P < 0.05).

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
Role of actin filament polymerization in smooth muscle contraction. Actin monomers exist in the form of free G-actin and G-actin that is sequestered by the proteins such as profilin and thymosin-{beta}4 (2, 3, 9, 10, 34, 42). Recent studies have shown that a pool of G-actin is induced to polymerize onto F-actin during contractile stimulation in smooth muscle tissues (3, 12, 30, 34) and in cultured smooth muscle cells (1, 25, 28). In the present report, actin polymerization is increased during NE stimulation and is decreased during relaxation of vascular smooth muscle (Fig. 1). The F-actin/G-actin ratio in unstimulated muscle strips is 1.8; contractile stimulation increases the F-actin/G-actin ratio by about threefold. This result is supported by previous observation that the ratio of F-actin (estimated by FITC-phalloidin staining) to G-actin (estimated by Texas Red-labeled DNase I staining) averages 2.5 ± 0.1 in unstimulated cultured smooth muscle cells and 4.5 ± 0.5 in carbachol-stimulated cells (25). The increases in the F-actin amount may facilitate increases in the length of thin filaments rather than the density of thin filaments. The studies of X-ray diffraction on thin filaments in response to contraction stimulation seem controversial; however, thin filaments undergo elongation and dynamic changes during contraction of some muscles, including mulloscan and guinea pig taeni coli smooth muscles (13, 50).

Latrunculin A binds to actin monomers and prevents their assembly into filamentous actin (14, 34). Our results show that the treatment of smooth muscle strips with latrunculin A inhibits the polymerization of actin filaments, which is consistent with previous observations (11, 34). In this study, we did not use cytochalasin D as a pharmacological tool because the treatment of smooth muscle with cytochalasin D seems not to inhibit agonist-induced actin polymerization in smooth muscle (34). This effect may result from the accelerated nucleation of new actin filaments induced by the capping of existing filaments (34, 43, 48).

In this report, the inhibition of actin polymerization by latrunculin A depresses active force in response to contractile stimulation, indicating that the polymerization of actin filaments is required for vascular smooth muscle contraction. However, agonist-stimulated MLC20 phosphorylation is normal in latrunculin A-treated tissues. These results suggest that smooth muscle cannot contract without increases in actin polymerization in response to contractile stimulation (30, 34, 44, 55). These results also indicate that the inhibition of actin polymerization does not affect MLC20 phosphorylation stimulated by agonists (30, 34).

With regard to the functional role of actin filament polymerization in smooth muscle contraction, there are several mechanisms that might contribute. First, rapid changes in the polymerization of actin filaments, as evidenced by rapid changes in the F-actin/G-actin ratio in this study, may facilitate cytoskeletal restructuring in smooth muscle cells (1, 25, 28). The restructuring of the actin cytoskeleton may enable the smooth muscle cells of hollow organs to quickly adjust to their mechanical and physiological properties in response to changes in forces in the external environment (1, 11, 12, 15, 18, 51, 54). Second, the dynamic changes in the state of actin may strengthen the connection of actin filaments to transmembrane proteins, facilitating force transmission between the actin cytoskeleton and extracellular matrix (5, 36, 37). The disruption of the attachment of the actin cytoskeleton to the integrin complex by the capping agent cytochalasin D inhibits force generation in smooth muscle stimulated by agonist (34). Third, because contractile units consist of actin and myosin filaments, newly polymerized actin might alter the length or organization of contractile units and result in changes in smooth muscle contractility and stiffness (1, 17, 19, 27). This hypothesis is supported by evidence that dynamic changes in myosin filaments in smooth muscle tissues are induced in response to contractile and mechanical stimulation (38, 59).

Role of profilin in force generation and contractile protein activation in smooth muscle. Studies from nonmuscle cells have shown that profilin may mediate the regulation of cell migration (6, 9, 33). Our results demonstrate that profilin plays an essential role in the cellular mechanisms that mediate active force generation in smooth muscle. The treatment of smooth muscle tissues with profilin antisense oligodeoxynucleotides selectively downregulates the expression of profilin protein. The downregulation of profilin protein dramatically inhibits force generation in smooth muscle in response to contractile stimulation.

Phosphorylation of the MLC20 by Ca2+/calmodulin-regulated myosin light chain kinase initiates cross-bridge cycling and smooth muscle contraction (31, 47, 52, 58). Our results showed that phosphorylation of the MLC20 in the canine carotid smooth muscle is higher in the early stage (2–5 min) of NE stimulation and subsequently reduced to a lower level. The results are consistent with previous studies on other smooth muscle tissues (21, 39, 40, 52).

We considered the possibility that, in carotid smooth muscle, the depression of agonist-induced force generation in profilin-deficient tissues could be due to the inhibition of contractile protein activation. However, MLC20 phosphorylation in response to agonist stimulation was similar in tissues treated with no ODNs or profilin sense or antisense ODNs, whereas active force in the profilin-depleted strips was dramatically depressed. Therefore, the inhibition of active force in the profiln-deficient tissues does not result from the inhibition of MLC20 phosphorylation during contractile stimulation.

Role of profilin in actin filament polymerization in smooth muscle. From in vitro biochemical analysis, profilin has been speculated to mediate actin polymerization (20, 32, 60). Our present results demonstrate that in vascular smooth muscle, actin polymerization is impaired in the absence of profilin. Because profilin is necessary for active force development in vascular smooth muscle tissues (Figs. 4 and 5), it is probably that the inhibition of actin polymerization by antisense may contribute to the decrease in force generation in profilin-depleted tissues stimulated by contractile agonists.

There are several possibilities that profilin might regulate the polymerization of actin filaments. In response to external and internal signals, profilin might bind to the proline-rich domain of N-WASP, in combination with Cdc42 and lipid, and induce a change in the conformation of N-WASP that activates the nucleation of actin filaments by the Arp2/3 complex (24, 41, 60, 62). In addition, profilin is known to interact with actin monomers (2, 20, 57); the binding of profilin-actin complex to N-WASP may deliver actin monomers to the Arp2/3 complex nucleation site or the barbed end of a growing filament (23, 32, 60). Finally, profilin might influence actin polymerization by sequestrating actin monomers; the profilin-actin complex may prevent the association of free G-actin with the verprolin domain of N-WASP (9, 32, 60). The suppression of the binding of free actin monomers to N-WASP attenuates the nucleation of actin polymerization.

In conclusion, in canine carotid smooth muscle, contractile stimulation induces the polymerization of actin filaments, and the inhibition of actin polymerization by latrunculin A depresses active force generation. The depletion of profilin by antisense oligodeoxynucleotides dramatically depresses active force generation in response to contractile stimulation without significantly inhibiting MLC20 phosphorylation. However, the downregulation of profilin inhibits actin filament polymerization elicited by contractile stimulation. We conclude that profilin plays an essential role in the cellular processes that regulate smooth muscle contraction, but that it does not regulate the activation of contractile proteins in response to stimulation with contractile stimuli. Profilin may be important in regulating the assembly and organization of the actin cytoskeleton during contractile stimulation.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This work is supported by an American Heart Association Scientist Development Grant (to D. D. Tang).


   ACKNOWLEDGMENTS
 
We thank Susan J. Gunst for useful comments and assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. D. Tang, Dept. of Cellular and Integrative Physiology, Indiana Univ. School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202 (E-mail: dtang{at}iupui.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. An SS, Laudadio RE, Lai J, Rogers RA, and Fredberg JJ. Stiffness changes in cultured airway smooth muscle cells. Am J Physiol Cell Physiol 283: C792–C801, 2002.[Abstract/Free Full Text]
  2. Babich M, Foti LR, Sykaluk LL, and Clark CR. Profilin forms tetramers that bind to G-actin. Biochem Biophys Res Commun 218: 125–131, 1996.[ISI][Medline]
  3. Barany M, Barron JT, Gu L, and Barany K. Exchange of the actin-bound nucleotide in intact arterial smooth muscle. J Biol Chem 276: 48398–48403, 2001.[Abstract/Free Full Text]
  4. Brown MC, Perrotta JA, and Turner CE. Identification of LIM3 as the principal determinant of paxillin focal adhesion localization and characterization of a novel motif on paxillin directing vinculin and focal adhesion kinase binding. J Cell Biol 135: 1109–1123, 1996.[Abstract/Free Full Text]
  5. Burridge K and Chrzanowska-Wodnicka M. Focal adhesions, contractility, and signaling. Annu Rev Cell Dev Biol 12: 463–518, 1996.[ISI][Medline]
  6. Buss F, Temm-Grove C, Henning S, and Jockusch BM. Distribution of profilin in fibroblasts correlates with the presence of highly dynamic actin filaments. Cell Motil Cytoskeleton 22: 51–61, 1992.[ISI][Medline]
  7. Carlier MF, Ducruix A, and Pantaloni D. Signalling to actin: the Cdc42-N-WASP-Arp2/3 connection. Chem Biol 6: R235–R240, 1999.[ISI][Medline]
  8. Carlier MF and Pantaloni D. Actin assembly in response to extracellular signals: role of capping proteins, thymosin beta 4 and profilin. Semin Cell Biol 5: 183–191, 1994.[Medline]
  9. Carlier MF and Pantaloni D. Control of actin dynamics in cell motility. J Mol Biol 269: 459–467, 1997.[ISI][Medline]
  10. Cassimeris L, Safer D, Nachmias VT, and Zigmond SH. Thymosin beta 4 sequesters the majority of G-actin in resting human polymorphonuclear leukocytes. J Cell Biol 119: 1261–1270, 1992.[Abstract/Free Full Text]
  11. Cipolla MJ, Gokina NI, and Osol G. Pressure-induced actin polymerization in vascular smooth muscle as a mechanism underlying myogenic behavior. FASEB J 16: 72–76, 2002.[Abstract/Free Full Text]
  12. Cipolla MJ and Osol G. Vascular smooth muscle actin cytoskeleton in cerebral artery forced dilatation. Stroke 29: 1223–1228, 1998.[Abstract/Free Full Text]
  13. Cooke P. A reversible change in the functional organization of thin filaments in smooth muscle fibers. Eur J Cell Biol 27: 55–61, 1982.[ISI][Medline]
  14. Coue M, Brenner SL, Spector I, and Korn ED. Inhibition of actin polymerization by latrunculin A. FEBS Lett 213: 316–318, 1987.[ISI][Medline]
  15. Davis MJ, Wu X, Nurkiewicz TR, Kawasaki J, Davis GE, Hill MA, and Meininger GA. Integrins and mechanotransduction of the vascular myogenic response. Am J Physiol Heart Circ Physiol 280: H1427–H1433, 2001.[Abstract/Free Full Text]
  16. Fisher TL, Terhorst T, Cao X, and Wagner RW. Intracellular disposition and metabolism of fluorescently-labeled unmodified and modified oligonucleotides microinjected into mammalian cells. Nucleic Acids Res 21: 3857–3865, 1993.[Abstract/Free Full Text]
  17. Gunst SJ, Meiss RA, Wu MF, and Rowe M. Mechanisms for the mechanical plasticity of tracheal smooth muscle. Am J Physiol Cell Physiol 268: C1267–C1276, 1995.[Abstract/Free Full Text]
  18. Gunst SJ and Tang DD. The contractile apparatus and mechanical properties of airway smooth muscle. Eur Respir J 15: 600–616, 2000.[Abstract]
  19. Gunst SJ and Wu MF. Plasticity of airway smooth muscle stiffness and extensibility: role of length-adaptive mechanisms. J Appl Physiol 90: 741–749, 2001.[Abstract/Free Full Text]
  20. Gutsche-Perelroizen I, Lepault J, Ott A, and Carlier MF. Filament assembly from profilin-actin. J Biol Chem 274: 6234–6243, 1999.[Abstract/Free Full Text]
  21. Hai CM. Length-dependent myosin phosphorylation and contraction of arterial smooth muscle. Pflügers Arch 418: 564–571, 1991.[ISI][Medline]
  22. Hartwig JH, Chambers KA, Hopcia KL, and Kwiatkowski DJ. Association of profilin with filament-free regions of human leukocyte and platelet membranes and reversible membrane binding during platelet activation. J Cell Biol 109: 1571–1579, 1989.[Abstract/Free Full Text]
  23. Higgs HN and Pollard TD. Activation by Cdc42 and PIP(2) of Wiskott-Aldrich syndrome protein (WASp) stimulates actin nucleation by Arp2/3 complex. J Cell Biol 150: 1311–1320, 2000.[Abstract/Free Full Text]
  24. Higgs HN and Pollard TD. Regulation of actin filament network formation through ARP2/3 complex: activation by a diverse array of proteins. Annu Rev Biochem 70: 649–676, 2001.[ISI][Medline]
  25. Hirshman CA and Emala CW. Actin reorganization in airway smooth muscle cells involves Gq and Gi-2 activation of Rho. Am J Physiol Lung Cell Mol Physiol 277: L653–L661, 1999.[Abstract/Free Full Text]
  26. Horowitz A, Menice CB, Laporte R, and Morgan KG. Mechanisms of smooth muscle contraction. Physiol Rev 76: 967–1003, 1996.[Abstract/Free Full Text]
  27. Hubmayr RD, Shore SA, Fredberg JJ, Planus E, Panettieri RA Jr, Moller W, Heyder J, and Wang N. Pharmacological activation changes stiffness of cultured human airway smooth muscle cells. Am J Physiol Cell Physiol 271: C1660–C1668, 1996.[Abstract/Free Full Text]
  28. Ishida T, Ishida M, Suero J, Takahashi M, and Berk BC. Agonist-stimulated cytoskeletal reorganization and signal transduction at focal adhesions in vascular smooth muscle cells require c-Src. J Clin Invest 103: 789–797, 1999.[ISI][Medline]
  29. Je HD, Gangopadhyay SS, Ashworth TD, and Morgan KG. Calponin is required for agonist-induced signal transduction–evidence from an antisense approach in ferret smooth muscle. J Physiol 537: 567–577, 2001.[Abstract/Free Full Text]
  30. Jones KA, Perkins WJ, Lorenz RR, Prakash YS, Sieck GC, and Warner DO. F-actin stabilization increases tension cost during contraction of permeabilized airway smooth muscle in dogs. J Physiol 519: 527–538, 1999.[Abstract/Free Full Text]
  31. Kamm KE and Stull JT. Regulation of smooth muscle contractile elements by second messengers. Annu Rev Physiol 51: 299–313, 1989.[ISI][Medline]
  32. Kang F, Purich DL, and Southwick FS. Profilin promotes barbed-end actin filament assembly without lowering the critical concentration. J Biol Chem 274: 36963–36972, 1999.[Abstract/Free Full Text]
  33. Mayboroda O, Schluter K, and Jockusch BM. Differential colocalization of profilin with microfilaments in PtK2 cells. Cell Motil Cytoskeleton 37: 166–177, 1997.[ISI][Medline]
  34. Mehta D and Gunst SJ. Actin polymerization stimulated by contractile activation regulates force development in canine tracheal smooth muscle. J Physiol 519: 829–840, 1999.[Abstract/Free Full Text]
  35. Miyajima E, Yamada Y, Yoshida Y, Matsukawa T, Shionoiri H, Tochikubo O, and Ishii M. Muscle sympathetic nerve activity in renovascular hypertension and primary aldosteronism. Hypertension 17: 1057–1062, 1991.[Abstract/Free Full Text]
  36. Mullins RD, Heuser JA, and Pollard TD. The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments. Proc Natl Acad Sci USA 95: 6181–6186, 1998.[Abstract/Free Full Text]
  37. Pollard TD, Blanchoin L, and Mullins RD. Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. Annu Rev Biophys Biomol Struct 29: 545–576, 2000.[ISI][Medline]
  38. Qi D, Mitchell RW, Burdyga T, Ford LE, Kuo KH, and Seow CY. Myosin light chain phosphorylation facilitates in vivo myosin filament reassembly after mechanical perturbation. Am J Physiol Cell Physiol 282: C1298–C1305, 2002.[Abstract/Free Full Text]
  39. Rembold CM and Murphy RA. Ca2+-dependent myosin phosphorylation in phorbol diester stimulated smooth muscle contraction. Am J Physiol Cell Physiol 255: C719–C723, 1988.[Abstract/Free Full Text]
  40. Rembold CM and Murphy RA. Histamine concentration and Ca2+ mobilization in arterial smooth muscle. Am J Physiol Cell Physiol 257: C122–C128, 1989.[Abstract/Free Full Text]
  41. Rohatgi R, Ma L, Miki H, Lopez M, Kirchhausen T, Takenawa T, and Kirschner MW. The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly. Cell 97: 221–231, 1999.[ISI][Medline]
  42. Safer D, Sosnick TR, and Elzinga M. Thymosin beta 4 binds actin in an extended conformation and contacts both the barbed and pointed ends. Biochemistry 36: 5806–5816, 1997.[Medline]
  43. Saito S and Karaki H. A family of novel actin-inhibiting marine toxins. Clin Exp Pharmacol Physiol 23: 743–746, 1996.[ISI][Medline]
  44. Saito SY, Hori M, Ozaki H, and Karaki H. Cytochalasin D inhibits smooth muscle contraction by directly inhibiting contractile apparatus. J Smooth Muscle Res 32: 51–60, 1996.[Medline]
  45. Schiffrin EL. Role of endothelin-1 in hypertension and vascular disease. Am J Hypertens 14: 83S–89S, 2001.[ISI][Medline]
  46. Smyth AP, Rook SL, Detmar M, and Robinson GS. Antisense oligonucleotides inhibit vascular endothelial growth factor/vascular permeability factor expression in normal human epidermal keratinocytes. J Invest Dermatol 108: 523–526, 1997.[ISI][Medline]
  47. Somlyo AP and Somlyo AV. Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and nonmuscle myosin II. J Physiol 522: 177–185, 2000.[Abstract/Free Full Text]
  48. Sugidachi A, Ogawa T, Asai F, Saito S, Ozaki H, Fusetani N, Karaki H, and Koike H. Inhibition of rat platelet aggregation by mycalolide-B, a novel inhibitor of actin polymerization with a different mechanism of action from cytochalasin-D. Thromb Haemost 79: 614–619, 1998.[ISI][Medline]
  49. Svetkey LP, Timmons PZ, Emovon O, Anderson NB, Preis L, and Chen YT. Association of hypertension with beta2- and alpha2c10-adrenergic receptor genotype. Hypertension 27: 1210–1215, 1996.[Abstract/Free Full Text]
  50. Tajima Y, Makino K, Hanyuu T, Wakabayashi K, and Amemiya Y. X-ray evidence for the elongation of thin and thick filaments during isometric contraction of a molluscan smooth muscle. J Muscle Res Cell Motil 15: 659–671, 1994.[ISI][Medline]
  51. Tang D, Mehta D, and Gunst SJ. Mechanosensitive tyrosine phosphorylation of paxillin and focal adhesion kinase in tracheal smooth muscle. Am J Physiol Cell Physiol 276: C250–C258, 1999.[Abstract/Free Full Text]
  52. Tang DC, Stull JT, Kubota Y, and Kamm KE. Regulation of the Ca2+ dependence of smooth muscle contraction. J Biol Chem 267: 11839–11845, 1992.[Abstract/Free Full Text]
  53. Tang DD and Gunst SJ. Depletion of focal adhesion kinase by antisense depresses contractile activation of smooth muscle. Am J Physiol Cell Physiol 280: C874–C883, 2001.[Abstract/Free Full Text]
  54. Tang DD, Wu MF, Opazo Saez AM, and Gunst SJ. The focal adhesion protein paxillin regulates contraction in canine tracheal smooth muscle. J Physiol 542: 501–513, 2002.[Abstract/Free Full Text]
  55. Tseng S, Kim R, Kim T, Morgan KG, and Hai CM. F-actin disruption attenuates agonist-induced [Ca2+], myosin phosphorylation, and force in smooth muscle. Am J Physiol Cell Physiol 272: C1960–C1967, 1997.[Abstract/Free Full Text]
  56. Wagner RW. Gene inhibition using antisense oligodeoxynucleotides. Nature 372: 333–335, 1994.[Medline]
  57. Wolven AK, Belmont LD, Mahoney NM, Almo SC, and Drubin DG. In vivo importance of actin nucleotide exchange catalyzed by profilin. J Cell Biol 150: 895–904, 2000.[Abstract/Free Full Text]
  58. Word RA, Tang DC, and Kamm KE. Activation properties of myosin light chain kinase during contraction/relaxation cycles of tonic and phasic smooth muscles. J Biol Chem 269: 21596–21602, 1994.[Abstract/Free Full Text]
  59. Xu JQ, Gillis JM, and Craig R. Polymerization of myosin on activation of rat anococcygeus smooth muscle. J Muscle Res Cell Motil 18: 381–393, 1997.[ISI][Medline]
  60. Yang C, Huang M, DeBiasio J, Pring M, Joyce M, Miki H, Takenawa T, and Zigmond SH. Profilin enhances Cdc42-induced nucleation of actin polymerization. J Cell Biol 150: 1001–1012, 2000.[Abstract/Free Full Text]
  61. Youn T, Kim SA, and Hai CM. Length-dependent modulation of smooth muscle activation: effects of agonist, cytochalasin, and temperature. Am J Physiol Cell Physiol 274: C1601–C1607, 1998.[Abstract/Free Full Text]
  62. Zigmond SH. Actin cytoskeleton: the Arp2/3 complex gets to the point. Curr Biol 8: R654–R657, 1998.[ISI][Medline]



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