1Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202; and 2Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York 13210
Submitted 17 January 2003 ; accepted in final form 17 October 2003
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ABSTRACT |
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tissue transfection; plasmids; cytoskeleton; talin; immunofluorescence
The cytoskeletal proteins vinculin, talin, and -actinin have been implicated in the physical coupling of actin filaments to
-integrins (5, 6, 9, 28, 36, 38). Talin and
-actinin bind to the cytoplasmic domain of
-integrins as well as to actin filaments and thereby have the potential to provide direct mechanical coupling between integrin proteins and the actin cytoskeleton (6, 10, 36). Vinculin also binds to actin filaments, talin, and
-actinin, but there is no evidence that it can bind directly to
-integrins (10). Vinculin has been proposed to strengthen connections between integrins and actin filaments to support force transfer from the cytoskeleton to the extracellular matrix (7, 15, 34). There is evidence that the binding of vinculin to actin and talin can be actively regulated by phosphoinositides and inhibited by acidic phospholipids (20, 53), suggesting that the structural links between actin filaments and integrins may be regulated in some cell types.
We previously hypothesized (2326, 31) that force development in smooth muscle in response to contractile stimulation involves cytoskeletal reorganization and the remodeling of structural linkages between actin filaments and integrin proteins at membrane-associated dense plaque sites. Contractile activation stimulates actin polymerization in tracheal smooth muscle and other smooth muscle tissues, and the polymerization of actin is required for tension generation in tracheal muscle (2, 8, 30, 31). In the present study, we evaluated the effects of stimulation with acetylcholine (ACh) on the localization of the cytoskeletal "linker" proteins vinculin and talin in freshly dissociated smooth muscle cells obtained from canine tracheal smooth muscle. Both proteins were found to redistribute to the membrane in response to stimulation with ACh, suggesting that contractile stimulation promotes the recruitment of these proteins to the smooth muscle cell membrane.
The proteins focal adhesion kinase (FAK) and paxillin, a substrate for FAK, localize to the focal adhesion sites of cultured cells and have been implicated in regulating focal adhesion assembly and stress fiber formation during cell adhesion (1, 5, 32, 37, 48). Both paxillin and FAK bind to peptides mimicking -integrin cytoplasmic domains (35). In tracheal smooth muscle they undergo tyrosine phosphorylation in response to contractile stimulation, and this phosphorylation increases concurrently with force development (33, 40, 52). The depletion of paxillin from tracheal smooth muscle by antisense oligonucleotides inhibits force development and disrupts actin polymerization but does not affect myosin light chain (MLC) phosphorylation or myosin ATPase activity (43). Furthermore, the expression of nonphosphorylatable paxillin mutants in tracheal muscle that suppress ACh-induced paxillin tyrosine phosphorylation also inhibits tension development and actin polymerization, without affecting MLC phosphorylation (42). These findings suggest that paxillin plays an important role in regulating tension development in smooth muscle but that it is not involved in regulating contractile protein activation or cross-bridge cycling. Evidence from the present study indicates that FAK and paxillin also redistribute to the membrane in response to contractile stimulation. We therefore postulated that paxillin might be involved in regulating changes in the organization of the actin cytoskeleton or the linkage of actin filaments to integrin proteins.
In the present study, we evaluated the role of paxillin in regulating the localization of cytoskeletal proteins that may mediate the formation of linkages between actin filaments and integrins. Antisense oligonucleotides (ODNs) were used to deplete tissues of paxillin, and the effects of paxillin depletion on the relocalization of FAK, talin, and vinculin in response to contractile stimulation were determined. Paxillin depletion by antisense depressed contractile force and inhibited the recruitment of vinculin, but not of FAK or talin, to the membrane in response to stimulation with ACh, suggesting that vinculin translocation to the membrane depends on paxillin. We further evaluated the role of paxillin in recruiting cytoskeletal proteins to the membrane by transfecting smooth muscle tissues with plasmids encoding mutant paxillin protein in which the focal adhesion targeting sequence was deleted (paxillin LIM3 dl) (4). Expression of the mutant paxillin LIM3 dl also inhibited the contraction of tracheal tissues and prevented the redistribution of paxillin and vinculin to the membrane in freshly isolated smooth muscle cells during stimulation.
Our results demonstrate that the contractile stimulation of smooth muscle causes the recruitment of vinculin, talin, FAK, and paxillin to the smooth muscle cell membrane. When the agonist-stimulated recruitment of paxillin and vinculin to the membrane is prevented, force development is inhibited, suggesting that this redistribution is required for tension development in smooth muscle. Furthermore, we find that the targeting of vinculin to the smooth muscle membrane in response to contractile stimulation depends on the relocalization of paxillin to the membrane.
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METHODS |
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ODNs dissolved in Tris-EDTA buffer or plasmids encoding wild-type or mutant paxillin protein in which the focal adhesion targeting sequence was deleted (dl LIM3 444494; Ref. 4) were introduced into muscle strips according to methods previously described (4143). The strips were then transferred to organ baths containing PSS at 37°C and attached to Grass transducers for the measurement of isometric force. Tissue strips were then removed from the organ baths, and cells were dissociated from them for the analysis of intracellular protein localization as described in Dissociation of airway smooth muscle cells for evaluation of protein localization. In other experiments, after completion of force measurements, muscle strips were frozen with liquid N2-cooled tongs and pulverized under liquid N2 with a mortar and pestle for biochemical analysis.
Introduction of ODNs or plasmids into tracheal smooth muscle. Antisense and sense ODNs to paxillin were designed as previously described (43): paxillin antisense, 5'-GCCATTTAGGGCCTCACT-3'; paxillin sense, 5'-AGTGAGGCCCTAAATGGC-3'. Phosphorothiolated ODNs were obtained from Life Technologies (Rockville, MD) or Integrated DNA Technologies (Coralville, IA).
cDNA constructs encoding chicken wild-type paxillin or a paxillin mutant (paxillin LIM3 dl) were described previously (3, 4). The paxillin LIM3 dl cDNA encodes a mutant paxillin protein in which the LIM3 domain (444494) is deleted (4). Mutant and wild-type paxillin cDNA were subcloned into the mammalian expression vector pcDNA3 (Invitrogen, San Diego, CA). Escherichia coli (Bluescript) transformed with wild-type and mutant paxillin cDNA was grown in Luria-Bertani broth overnight. The bacterial cells were harvested by centrifugation at 4,000 rpm for 15 min at 4°C, and the plasmids were purified by standard methodology.
Paxillin antisense or sense ODNs or plasmids were introduced into tracheal smooth muscle strips by the method of reversible permeabilization, which we described previously (41, 43). After the optimal muscle length was determined, muscle strips were placed on metal hooks under tension to maintain them at optimal length. Strips were then 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, with 10 µM ODNs (antisense or sense ODNs) or 10 µg/ml plasmids (LIM3 dl mutant or wild type); solution 3 (at 4°C for 30 min) containing (in mM) 0.1 EGTA, 5 Na2ATP, 120 KCl, 10 MgCl2, and 20 TES; solution 4 (at 22°C for 60 min) containing (in mM) 110 NaCl, 3.4 KCl, 0.8 MgSO4, 25.8 NaHCO3, 1.2 KH2PO4, and 5.6 dextrose. Solutions 13 were aerated with 100% O2 to maintain a pH of 7.1, and solution 4 was aerated with 95% O2-5% CO2 to maintain a pH of 7.4. After 30 min in solution 4, CaCl2 was added gradually to reach a final concentration of 2.4 mM. The strips were then transferred to DMEM containing 5 mM Na2ATP, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 µM ODNs (antisense or sense) or 10 µg/ml plasmids (paxillin LIM3 dl mutant or wild-type paxillin) and incubated for 2 days at 37°C and 5% CO2. The media were changed every other day. The inclusion of ODNs or plasmids in the incubation medium was done to compensate for the degradation of DNA molecules by nucleases in cells (17, 50). ODNs and DNA molecules can be taken up by endocytosis, thus helping to maintain necessary levels in cells (39, 50). In preliminary experiments we found that the addition of plasmids or ODNs to the DMEM incubation medium enhanced their effectiveness, as evidenced by better expression of recombinant paxillin or enhanced downregulation of target proteins.
The efficiency of tissue transfection was evaluated by immunostaining cells dissociated from plasmid-treated tracheal tissues with antibody to chicken paxillin, which is specific for the recombinant protein, and determining the percentage of positively stained cells. Consistent with observations in previous studies (42), we found that 90% of cells dissociated from the transfected tissues expressed the recombinant proteins, indicating that the plasmids were incorporated into most of the cells throughout the tissue. (see Fig. 8).
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Dissociation of airway smooth muscle cells for evaluation of protein localization. After completion of the force measurements, smooth muscle cells were enzymatically dissociated from tracheal muscle strips for the analysis of cellular protein distribution by confocal microscopy. Tracheal muscle strips were minced and transferred to 5 ml of dissociation solution (composition in mM: 130 NaCl, 5 KCl, 1.0 CaCl2, 1.0 MgCl2, 10 HEPES, 0.25 EDTA, 10 D-glucose, and 10 taurine, pH 7) with collagenase (type I, 400 U/ml), papain (type IV, 30 U/ml), bovine serum albumin (1 mg/ml), and dithiothreitol (DTT; 1 mM). All enzymes were obtained from Sigma (St. Louis, MO). The strips were then placed in a 37°C shaking water bath at 80 oscillations/min for 40 min. The strips were then washed three times with a HEPES-buffered saline solution (composition in mM: 130 NaCl, 5 KCl, 1.0 CaCl2, 1.0 MgCl2, 20 HEPES, and 10 D-glucose, pH 7.4) and triturated with a pipette to liberate individual smooth muscle cells from the tissue. The solution containing the dissociated cells was poured over glass coverslips, and the cells were allowed to adhere to the coverslips for 2 h at room temperature. Cells on coverslips were incubated in HEPES-buffered saline solution containing 20 mM 2,3-butanedione monoxime (BDM) to prevent shortening. In other experiments, cells were incubated in HEPES-buffered saline solution without BDM to verify that BDM treatment did not alter the cellular distribution of proteins of interest. Cells were stimulated with ACh (105 M) for 5 min at 37°C or left unstimulated and used as controls. Stimulated and unstimulated cells were fixed for 10 min in 4% paraformaldehyde (vol/vol) in phosphate-buffered saline (composition in mM: 137 NaCl, 4.3 Na2HPO4, 1.4 KH2PO4, and 2.7 KCl, pH 7.4).
Immunofluorescence staining. Stimulated and unstimulated smooth muscle cells on coverslips were washed three times in Tris-buffered saline (TBS) containing 50 mM Tris, 150 mM NaCl, and 0.1% NaN3 and permeabilized in 0.2% Triton X-100 dissolved in TBS for 2 min. Cells were washed again in TBS and placed in a blocking solution containing 2% goat serum and 1% bovine serum albumin for 1 h at room temperature. Cells were washed repeatedly and incubated with primary antibody against vinculin, talin, paxillin, or FAK for 1 h at 37°C. The primary antibodies used in this study were as follows: vinculin MAb clone Vin-115 and talin MAb clone 8D4 (Sigma), paxillin MAb clone 349 and FAK MAb clone 77 (Transduction Laboratories, Lexington, KY), chicken paxillin polyclonal antibody (3), and vinculin polyclonal antibody (43). The specificity of antibodies used for immunofluorescence analysis was documented by Western blot, except for that of the chicken paxillin antibody, which does not react well on Western blot (Fig. 1B). Cells were washed again and incubated with a secondary antibody conjugated to a fluorescent green (Alexa 488) or red (Alexa 546) fluoroprobe (Molecular Probes, Eugene, OR) for 30 min at 37°C. In some experiments cells were double-labeled by reacting primary antibodies with secondary antibodies conjugated to different fluorescent dyes. Cells were washed to remove excess antibody, and coverslips were mounted onto slides with mounting medium.
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Confocal microscopy and image analysis. The cellular localization of fluorescently labeled vinculin, paxillin, talin, and FAK was assessed in the dissociated smooth muscle cells with a Zeiss LSM 510 laser scanning confocal microscope with an Apo x63 oil-immersion objective (NA 1.4). Alexa 488-labeled (green) proteins were excited with a 488-nm argon laser light, and fluorescence emissions were collected at 500550 nm. The fluorescence of Alexa 546-labeled (red) proteins was excited with a helium/neon laser at 543 nm, and emissions were collected at 565615 nm. The optical pinhole was set to resolve optical sections of 1 µm in cell thickness. The plane of focus was set midway between the bottom and top of the cell. Fluorescence intensity measurements were standardized among all cells compared within a single experiment by maintaining the same confocal settings for each fluoroprobe.
Images of smooth muscle cells were analyzed for regional differences in fluorescence intensity of stained proteins by quantifying the pixel intensity with a series of 610 cross-sectional line scans along the entire length of each cell (Fig. 1A). The area of the nucleus was excluded from the analysis. The ratio of pixel intensities between the cell periphery and interior was determined for each line scan by taking the ratio of the average maximum pixel intensity at the cell periphery to the minimum pixel intensity in the cell interior. The ratios of pixel intensities between the cell periphery and the cell interior for all of the 610 line scans performed on a given cell were averaged to obtain a single value for the ratio for each cell. The ratio of fluorescence intensity at the cell periphery to that at the cell interior was compared in cells at rest and cells stimulated with ACh (105 M).
Measurement of intracellular Ca2+ in freshly dissociated cells. Freshly dissociated smooth muscle cells were allowed to settle on coverslips for 60 min and were washed with HEPES-buffered saline solution. Cells were then loaded with the fluorescent Ca2+-sensitive indicator fluo 3 (Molecular Probes) by incubating them in 5 µM fluo 3-AM [diluted from a stock containing 2 mM fluo 3-AM and 0.025% (wt/vol) Pluronic F-127 in dimethyl sulfoxide] for 45 min, followed by a 30-min wash in PSS to allow time for the deesterification of the indicator. Intracellular Ca2+ was then evaluated within 45 min with a Zeiss LSM 510 laser scanning confocal microscope. Cells were observed with a Zeiss plan-Apochromat x40 1.2 NA water-immersion objective. Fluo 3 fluorescence was excited by the 488-nm line of a 60-mW argon ion laser. The emitted fluorescence was detected by the confocal detector at wavelengths >515 nm.
Immunoblot analysis. Pulverized smooth muscle strips were mixed with 50 µl of extraction buffer containing 20 mM Tris-HCl at pH 7.4, 2% Triton X-100, 0.2% SDS, 2 mM EDTA, phosphatase inhibitors (in mM: 2 sodium orthovanadate, 2 molybdate, and 2 sodium pyrophosphate), and protease inhibitors (in mM: 2 benzamidine, 0.5 aprotinin, and 1 phenylmethylsulfonyl fluoride). Samples were boiled for 5 min and then centrifuged (14,000 rpm, 15 min), and the supernatant was collected. Supernatants were then boiled in sample buffer [1.5% DTT, 2% SDS, 80 mM Tris·HCl (pH 6.8), 10% glycerol, and 0.01% bromphenol blue] for 5 min, and proteins were separated by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose. Membranes were then probed with monoclonal antibodies to paxillin, vinculin, talin, or FAK, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (Ig; Amersham Life Sciences). Proteins were visualized by enhanced chemiluminescence (ECL) and quantified by scanning densitometry.
Immunoprecipitation of proteins. Endogenous and/or recombinant paxillin proteins were immunoprecipitated as previously described (42, 43). Pulverized muscle strips were mixed with extraction buffer containing 20 mM Tris·HCl at pH 7.4, 2% Triton X-100, 0.2% SDS, 2 mM EDTA, phosphatase inhibitors (in mM: 2 sodium orthovanadate, 2 molybdate, and 2 sodium pyrophosphate), and protease inhibitors (in mM: 2 benzamidine, 0.5 aprotinin, and 1 phenylmethylsulfonyl fluoride). Each sample was centrifuged for the collection of supernatant. Muscle extracts containing equal amounts of protein were precleared for 30 min with 50 µl of 10% protein A-Sepharose. The precleared extracts were centrifuged at 14,000 rpm for 2 min. The extracts were incubated overnight with monoclonal antibody against paxillin to immunoprecipitate endogenous paxillin and then incubated for 2 h with 125 ml of a 10% suspension of protein A-Sepharose beads conjugated to rabbit anti-mouse Ig. For recombinant paxillin immunoprecipitation, the extracts were incubated with anti-chicken paxillin antibody for 60 min followed by 2-h incubation with a 10% suspension of protein A-Sepharose beads. Immunocomplexes were washed four times in a buffer containing 50 mM Tris·HCl (pH 7.6), 150 mM NaCl, and 0.1% Triton X-100. All procedures of immunoprecipitation were performed at 4°C. The immunoprecipitates of endogenous or recombinant paxillin were separated by SDS-PAGE followed by transfer to nitrocellulose membranes. The nitrocellulose membranes were divided into two parts: the lower part was probed with antibody for paxillin, and the upper part was probed with antibody against metavinculin/vinculin or myosin heavy chain. Proteins were quantitated by scanning densitometry.
Measurement of MLC phosphorylation. Muscle strips were rapidly frozen 5 min after contractile stimulation and then immersed in acetone containing 10% (wt/vol) trichloroacetic acid and 10 mM DTT (acetone-TCA-DTT), which was precooled with dry ice. Strips were thawed in acetone-TCA-DTT at room temperature and then washed four times with acetone-DTT. Proteins were extracted for 60 min in 8 M urea, 20 mM Tris base, 22 mM glycine, and 10 mM DTT. MLCs were separated by glycerol-urea PAGE and transferred to nitrocellulose. The membranes were blocked with 5% bovine serum albumin and incubated with polyclonal affinity-purified rabbit MLC20 antibody. Unphosphorylated and phosphorylated bands of MLCs were detected by Western blotting and quantified by densitometry.
Statistical analysis. Comparisons between groups were performed with paired t-tests. Values of n refer to the number of cells used to obtain mean values. P < 0.05 was considered to be significant.
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RESULTS |
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The effect of stimulation with ACh on the cellular localization of each of the fluorescently stained proteins was assessed by quantifying the pixel intensity of fluorescence along multiple cross sections of each cell with line scans (Fig. 1A). The fluorescence intensity profile recorded from a single representative line scan is shown as an inset within each panel in Fig. 2. In unstimulated cells the fluorescence intensity profiles were relatively flat, indicating that cytoskeletal protein distribution was uniform throughout the cell. In contrast, in cells stimulated with ACh, fluorescence intensity was markedly higher at the periphery of each cell relative to the interior of the cell for all four proteins.
Ratios of fluorescence intensity between the cell periphery and the cell interior for vinculin, paxillin, FAK, and talin were calculated for unstimulated cells and for cells stimulated with ACh (Fig. 3). A total of 40 unstimulated and 40 stimulated smooth muscle cells obtained from 6 experiments were analyzed for each protein. Fluorescence intensity for all proteins was three to four times higher at the cell periphery after stimulation with ACh (P < 0.05). These results indicate that stimulation with a contractile agonist promotes the recruitment of cytoskeletal proteins to the smooth muscle cell membrane.
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Paxillin depletion by antisense depresses contractile force and inhibits cellular redistribution of vinculin, but not FAK or talin, that occurs with contractile stimulation. Vinculin binds to paxillin, and the localization of vinculin to focal adhesion sites requires a region within the COOH-terminal domain of vinculin that is near the binding site for paxillin (4, 54). We therefore questioned whether the localization of vinculin to the smooth muscle membrane would depend on its interaction with paxillin.
We evaluated the role of paxillin in vinculin localization to the smooth muscle cell membrane by depleting smooth muscle tissues of paxillin with antisense ODNs (43). In a previous study (43), we found that the depletion of paxillin from tracheal smooth muscle by treatment with antisense ODNs inhibited force development but did not prevent the increases in MLC phosphorylation, myosin ATPase activity, or intracellular Ca2+ that occur in response to contractile stimulation.
In the present study, smooth muscle strips were treated with paxillin antisense ODNs, sense ODNs, or no ODNs and incubated for 2 days to allow for the inhibition of protein expression. Protein extracts from muscle strips were then analyzed by Western blot to confirm that the paxillin antisense selectively inhibited the expression of paxillin protein.
Paxillin antisense ODNs inhibited paxillin expression without affecting the expression of vinculin, MLC kinase (MLCK), or FAK (Fig. 4A). The ratios of expression of paxillin to vinculin, MLCK, and FAK were compared in antisense-treated and sense-treated tissues and expressed as a percentage of the ratios in untreated tissues. In sense-treated tissues, the ratios of paxillin to vinculin, MLCK, and FAK were 95.8 ± 5.6%, 95.3 ± 7.6%, and 99.5 ± 12.4%, respectively, and were not significantly different from those in untreated tissues (n = 4). In contrast, in the antisense-treated tissues, the ratios of expression of paxillin to vinculin, MLCK, and FAK were 24.6 ± 8.6%, 25.3 ± 7.6%, and 26.5 ± 13.2%, respectively, of the ratio in untreated tissues (n = 4). The ratios of FAK to vinculin were not significantly different in sense-treated and antisense-treated muscles from those in untreated tissues (97.6 ± 11.2% and 92.8 ± 9.8%, respectively).
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In each experiment, the effect of paxillin depletion on tension development was evaluated in tracheal muscle strips; the muscle strips were then dissociated, and cytoskeletal protein localization was evaluated in the freshly dissociated smooth muscle cells. Isometric force development in response to 105 M ACh in muscle strips before and after treatment with paxillin antisense, paxillin sense, and no ODNs is shown in Fig. 4B. In strips incubated with paxillin sense or with no ODNs there were no differences in the force developed after 5-min stimulation with ACh and the force generated before incubation (sense: 96.4 ± 8.56%, untreated: 98.5 ± 7.45%; n = 11). In contrast, contractile force was markedly reduced in tissues treated with paxillin antisense to 12.4 ± 8.2% of the force before incubation (n = 11). These results verified the effectiveness of the paxillin depletion by the antisense treatment and also demonstrated that the contractile responses of the tissues were unaffected by the procedure for loading ODNs into the tissues and by the 2-day incubation period.
After the 2-day incubation period and subsequent measurement of contractile force, smooth muscle cells were enzymatically dissociated from tissues treated with paxillin antisense ODNs, paxillin sense ODNs, or with no ODNs. Cells dissociated from smooth muscle strips treated with paxillin antisense exhibited low paxillin immunofluorescence compared with untreated strips or strips treated with paxillin sense (Fig. 5), further confirming that paxillin expression was suppressed by the treatment with paxillin antisense. There was no detectable effect of paxillin depletion by antisense on the distribution of vinculin in unstimulated smooth muscle cells.
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The effect of paxillin depletion on the localization of vinculin, talin, and FAK was compared in cells dissociated from tissue strips treated with paxillin antisense ODNs, paxillin sense ODNs, or no ODNs (Fig. 6). Paxillin depletion inhibited the redistribution of vinculin to the cell periphery in response to stimulation with ACh. In contrast, paxillin depletion did not alter the redistribution of talin or FAK in response to stimulation with ACh. In cells treated with paxillin sense and untreated cells (data not shown), vinculin, FAK, and talin all redistributed to the membrane in response to ACh stimulation.
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Figure 7 shows mean data for vinculin (A), talin (B), and FAK (C), in which the ratio of fluorescence intensity between the cell periphery and the cell interior was quantified for 20 cells from each treatment group from a total of 4 experiments. In paxillin antisense-treated cells, there was no significant difference between unstimulated and stimulated cells in the ratios of vinculin fluorescence intensity between the cell periphery and cell interior. In contrast, the distribution of talin and FAK at the membrane increased significantly relative to the cell interior in paxillin-depleted cells in response to cholinergic stimulation (P < 0.05). The increase in the ratio of talin, vinculin, and FAK at the membrane relative to the cell periphery in response to ACh was similar in untreated cells and in paxillin sense-treated cells. These results demonstrate that paxillin depletion inhibits the cellular translocation of vinculin to the membrane in response to contractile stimulation and that it does not affect the translocation of talin or FAK. Thus the presence of paxillin is required for the translocation of vinculin to the cell periphery in response to cholinergic stimulation.
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Expression of mutant paxillin with deletion of its LIM3 domain inhibits force but not MLC phosphorylation in tracheal muscle strips. The LIM3 domain of paxillin near its COOH terminus (residues 444494) is required for the localization of paxillin to focal adhesions in cultured fibroblasts during cell adhesion (4), whereas the vinculin binding site on paxillin has been localized to a contiguous stretch of 21 amino acids spanning residues 143164 near the NH2 terminus of paxillin (49). We attempted to determine whether the recruitment of paxillin to the membrane is required for tension development during contractile stimulation, whether the LIM3 domain of paxillin is required for paxillin translocation to the membrane, and whether the translocation of vinculin during cholinergic stimulation depends on the translocation of paxillin.
We expressed a chicken paxillin mutant with a deletion of the LIM3 domain (paxillin LIM3 dl; Ref. 4) in tracheal muscle strips by introducing an expression vector encoding the mutant paxillin protein into the tissues. Plasmids encoding wild-type chicken paxillin were introduced as controls. After transfection, tracheal muscle strips were incubated for 2 days to allow for expression of the recombinant proteins.
The efficiency of tissue transfection was evaluated by immunostaining cells dissociated from plasmid-treated and untreated muscle tissues with antibody to chicken paxillin, which is specific for the recombinant protein, and determining the percentage of positively stained cells. As we previously reported (42), 90% of the cells dissociated from the transfected tissues stained positively for the recombinant proteins whereas no cells dissociated from untreated tissues stained positively (Fig. 8A).
Expression of recombinant chicken paxillin proteins was also confirmed by immunoprecipitation with a paxillin antibody specific for chicken paxillin, followed by an antibody recognizing both species (Ref. 3; Fig. 8B). The mutant and wild-type chicken recombinant proteins were expressed in the tracheal muscle strips transfected with the plasmids. A faint band was also observed in muscle strips that were not treated with plasmids, which probably resulted from some cross-reactivity of the chicken paxillin antibody with the endogenous paxillin.
Paxillin immunoprecipitates were also immunoblotted for vinculin. Vinculin and metavinculin were detected in immunoprecipitates obtained from untreated fresh muscle tissues (Fig. 8B) and immunoprecipitates of both the recombinant LIM3 dl paxillin and wild-type recombinant paxillin, confirming that vinculin was associated with the mutant LIM3 dl paxillin and wild-type recombinant paxillin (4). Neither PKC- nor myosin heavy chain (MHC) are detected in paxillin immunoprecipitates, even though both proteins are present in homogenates of tracheal smooth muscle strips (42). Thus the immunoprecipitation procedure with paxillin antibody specifically concentrates paxillin and paxillin-associated proteins.
The total amount of paxillin protein expressed was compared in extracts of whole cell homogenates from fresh muscle tissues and muscle tissues incubated for 2 days with plasmids encoding wild-type paxillin, mutant LIM3 dl paxillin, or no plasmids by immunoblotting with a monoclonal paxillin antibody that reacts with both recombinant and endogenous species (Fig. 8C). The level of paxillin expression in muscle strips that had been treated with plasmids was significantly higher than that in fresh or untreated muscle strips; there were no differences in paxillin expression between fresh tissues and tissues that were incubated without plasmids (untreated). The ratios of expression of paxillin in transfected tissues to that in untransfected tissues were 2.1 ± 0.2 for LIM3 dl paxillin and 2.2 ± 0.1 for wild-type paxillin (n = 4). These results are consistent with our previous observations (42) that the expression of recombinant mutant paxillin does not inhibit the expression of endogenous paxillin.
We evaluated contractile responses to 105 M ACh in tissues treated with plasmids encoding the mutant recombinant LIM3 dl paxillin, wild-type paxillin, or no plasmids (Fig. 9A). The contractile force generated after stimulation with ACh for 5 min was markedly inhibited in tissues expressing the LIM3 dl paxillin mutant (7.4 ± 3% of preincubation force; n = 6). In muscle strips incubated with plasmids encoding the wild-type paxillin or with no plasmids, there was no significant inhibition of tension development in response to stimulation with ACh. The contractile responses were 100 ± 4% of the preincubation force for tissues not treated with plasmids and 94 ± 5% of the preincubation force for tissues expressing wild-type paxillin. There were no significant differences in force measured before incubation among the three groups.
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MLC phosphorylation was also evaluated in muscle strips incubated after treatment with plasmids encoding recombinant wild-type paxillin, LIM3 dl mutant paxillin, or no plasmids by freezing muscle strips after the measurement of contractile tension. Whereas the tension development was markedly inhibited, there were no significant differences in MLC phosphorylation in the three groups of muscles in response to stimulation with ACh (Fig. 9B).
Membrane localization of vinculin during contractile stimulation depends on paxillin localization to membrane mediated by LIM3 domain of paxillin. We evaluated whether the expression of the paxillin mutant LIM3 dl affected the redistribution of paxillin and vinculin to the membrane in response to stimulation with ACh. Tissues expressing mutant LIM3 dl paxillin protein, wild-type paxillin protein, or no recombinant protein were enzymatically dissociated. Freshly dissociated cells were double-stained for both paxillin and vinculin. Deletion of the LIM3 domain of paxillin prevented the redistribution of paxillin to the membrane in response to stimulation with ACh (Fig. 10). Vinculin redistribution to the membrane in response to ACh was also inhibited in cells expressing the paxillin LIM3 dl mutant. Redistribution of both paxillin and vinculin to the membrane in response to ACh was observed in cells expressing the wild-type recombinant paxillin, as well as in untreated cells (data not shown).
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The ratios of protein distribution for vinculin and paxillin are shown in Fig. 10B (n = 12; P < 0.05). In cells expressing wild-type recombinant paxillin, the ratio of distribution of vinculin and paxillin between the cell periphery and the cell interior was approximately four times higher than the ratio in unstimulated cells. In contrast, in cells expressing LIM3 dl paxillin, the ratio of distribution of both paxillin and vinculin remained close to 1 in both unstimulated and stimulated cells. These results indicate that the LIM3 domain of paxillin is necessary for it to localize to the membrane in smooth muscle cells in response to stimulation with ACh and that the localization of vinculin to the membrane in response to ACh stimulation depends on the membrane localization of paxillin.
Expression of the paxillin LIM3 dl mutant did not inhibit the rise in intracellular Ca2+ in freshly dissociated cells in response to stimulation with ACh. Cells were freshly dissociated from tissues that had been transfected with mutant LIM3 dl paxillin or that had been incubated for 2 days without transfection and loaded with the Ca2+ indicator fluo 3. Changes in intracellular Ca2+ in response to ACh administered to the live cells were evaluated by monitoring the tissues continuously for 1 min after stimulation with ACh by using a laser scanning confocal microscope. Twenty-six cells from tissues that had not been transfected and nineteen cells from transfected tracheal muscle strips from three different dogs were studied. An increase in Ca2+ was observed in 23 of 26 cells from the untransfected tissues and in 17 of 19 cells from tissues transfected with plasmids encoding the LIM3 dl mutant paxillin (Fig. 11).
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DISCUSSION |
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The formation of focal adhesion plaques during cell adhesion and migration has been proposed to strengthen cytoskeleton-extracellular matrix connections to support the force applied by the cytoskeleton to the extracellular matrix through integrin molecules (18). Vinculin and talin have been identified as constituents of membrane plaques in smooth muscle cells and tissues (14, 19, 38). The recruitment of cytoskeletal proteins to the adhesion sites of smooth muscle during contractile stimulation may serve an analogous function. Our observations that talin and vinculin are recruited to the smooth muscle membrane during active contraction, and our previous observations (33) that talin undergoes serine-threonine phosphorylation during the contractile activation of tracheal smooth muscle, provide evidence that the function of these proteins is actively regulated during smooth muscle contraction.
Current evidence suggests that both talin and vinculin are primary constituents of integrin-cytoskeletal connections in both smooth muscle and nonmuscle cells. Talin can bind directly to the cytoplasmic domains of -integrins and to actin filaments and can thereby form a direct link between integrin proteins and actin filaments (6, 27). Talin assembles into the initial sites of adhesion formed between integrin receptors and the extracellular matrix at the leading edge of migratory cells (11, 29). Vinculin binds to talin and to actin filaments, as well as to
-actinin and paxillin (9, 10). In cultured fibroblasts, mechanical force acts as a stimulus to recruit vinculin into integrin-extracellular matrix connections (18). The addition of vinculin to vinculin-null cells from an embryonic carcinoma cell line increases the stiffness of integrin-cytoskeleton linkages, suggesting that vinculin reinforces extracellular matrix-cytoskeleton linkages to support the transfer of mechanical stresses from the cytoskeleton across the cell surface (15).
Our data are consistent with previous studies demonstrating the presence of vinculin and talin in the adhesion junctions of smooth muscle cells and tissues (14, 19, 38). In our study, these proteins were more concentrated at membrane sites in unstimulated smooth muscle cells. However, we also demonstrate a process of active recruitment of these proteins to the membrane caused by contractile stimulation: the concentration of these proteins at the membrane increased significantly in response to contractile stimulation. The recruitment of these proteins during active contraction may strengthen existing connections or contribute to the formation of new connections between the cytoskeleton and transmembrane proteins. This may provide additional support for the transmission of force generated by the contractile apparatus to the extracellular matrix.
Role of paxillin in recruitment of cytoskeletal proteins to adhesion plaques. Paxillin functions as a scaffolding protein and has been demonstrated to play an important role in regulating cytoskeletal organization in a variety of cell types (46). The depletion of paxillin from tracheal smooth muscles by treatment with antisense ODNs dramatically inhibits active force development but does not affect MLC phosphorylation, myosin ATPase activity, or intracellular Ca2+ (43). Furthermore, the expression of nonphosphorylatable paxillin proteins in smooth muscle inhibits tension development, also without affecting MLC phosphorylation (42). These findings indicate that the role of paxillin in tension development in smooth muscle is independent of contractile protein activation or cross bridge cycling. Because paxillin has been implicated in regulating the formation of adhesion plaques in cultured cells (4), we speculated that paxillin might be important in regulating cytoskeletal linkages between actin filaments and the extracellular matrix during smooth muscle contraction. Furthermore, vinculin and FAK have been shown to bind directly to paxillin and talin binds to vinculin (21, 45, 47, 54). We evaluated the role of paxillin in the recruitment of vinculin, talin, and FAK during contractile stimulation by introducing antisense ODNs into smooth muscle tissues to selectively suppress the expression of paxillin protein. We then determined the effects of contractile stimulation on the localization of cytoskeletal proteins in cells dissociated from those strips. We found that the recruitment of vinculin to the smooth muscle membrane during contractile stimulation was inhibited by depletion of paxillin protein, whereas depletion of paxillin by antisense did not prevent the translocation of FAK or talin to the membrane in response to contractile stimulation (Fig. 7). This suggests a critical role for paxillin in regulating the organization of cytoskeletal linkages during contractile stimulation.
We used a mutant paxillin protein to evaluate whether the translocation of paxillin to the smooth muscle membrane is required for tension development. Four LIM domains have been mapped on the COOH terminus of paxillin; one of these, LIM3, is the primary determinant for the targeting of paxillin to focal adhesions in cultured fibroblasts (4). We transfected smooth muscle tissues with plasmids encoding paxillin LIM3 dl and expressed the mutant paxillin in the muscle tissues. Immunofluorescence analysis of cells freshly dissociated from these tissues demonstrated that the paxillin did not redistribute to the membrane in response to contractile stimulation; thus the LIM3 domain of paxillin is critical for the targeting of paxillin to the smooth muscle cell periphery during stimulation with ACh. The relocalization of vinculin to the membrane in response to contractile stimulation was also prevented by expression of the mutant paxillin LIM3 dl.
The expression of paxillin LIM3 dl also dramatically inhibited tension development in smooth muscle tissues. However, it had no effect on MLC phosphorylation and it did not prevent a rise in intracellular Ca2+ stimulated by ACh. Thus the recruitment of paxillin and vinculin to the cell periphery appears to be critical for the development of active tension in smooth muscle. These observations suggest that tension development may involve the regulation of cytoskeletal linkages between actin filaments and transmembrane molecules and that paxillin and vinculin may play a critical role in the formation of these linkages.
Molecular regulation of adhesion plaque formation. The molecular mechanisms that regulate the recruitment and assembly of focal adhesion plaques in cultured cells are controversial. Vinculin can exist in two conformations. In its inactive conformation, an intramolecular interaction between its head and tail regions masks binding sites for talin, -actinin, and F-actin. In this condition vinculin cannot form a link between actin filaments and the integrin-binding proteins talin or
-actinin. The binding of phosphatidylinositol 4,5-bisphosphate (PIP2) to vinculin results in a conformational change that exposes binding sites for
-actinin, talin, and F-actin, enabling vinculin to promote connections between actin filaments and transmembrane integrins (53). This discovery has led to the proposal that the rho-dependent synthesis of PIP2 stimulates the recruitment of vinculin to the membrane initially via an interaction between membrane phospholipids and the COOH terminus of vinculin, which inserts into the membrane (9, 20, 53). The active form of vinculin has been proposed to recruit talin and actin and thereby promote the assembly of focal adhesions.
Evidence that talin is recruited to the membrane first (11) led to the proposal that vinculin is recruited to adhesion plaques by binding to talin. The talin binding site on vinculin is localized to the NH2-terminal 258 amino acids within the head region of vinculin (21, 22). Paxillin binds selectively to the rod domain of vinculin (47). Thus an alternative possibility for the localization of vinculin to focal adhesions is that it is recruited by binding to paxillin. Wood et al. (54) identified two regions near the COOH terminus of the vinculin molecule that were required for its binding to paxillin and for focal adhesion targeting. Although these regions were not identical, they were adjacent, raising the possibility that they are the same. Subsequently, Brown et al. (4) demonstrated that paxillin can target to focal adhesions independently of its interactions with either vinculin or FAK and that the principal mechanism of targeting paxillin to focal adhesions is through its LIM3 domain.
We found that vinculin coprecipitated with endogenous, wild-type, and mutant paxillin that was immunoprecipitated from unstimulated tracheal smooth muscle strips; thus neither the LIM3 domain of paxillin nor its localization to the membrane was required for the association of paxillin with vinculin. These observations are consistent with evidence that vinculin binding motifs on paxillin are near its NH2 terminus within a contiguous stretch of 21 amino acids (44) that is distinct from the LIM domains located toward the COOH terminus of the molecule (4). Our finding that the LIM3 domain of paxillin was required for the recruitment of paxillin to the membrane during smooth muscle contraction is also consistent with the observation that this domain of paxillin is required for its recruitment to focal adhesions in cultured fibroblasts. Our results clearly showed that the redistribution of vinculin to the membrane in response to contractile stimulation in smooth muscle was inhibited in cells expressing the paxillin LIM3 dl mutant, as well as in cells depleted of paxillin by antisense. These data indicate that the translocation of vinculin to the smooth muscle cell periphery in response to contractile stimulation is dependent on the recruitment of paxillin. The paxillin binding site on vinculin is exposed when vinculin is in the inactive state (9); thus paxillin and vinculin may interact before their localization at the membrane and be recruited as a complex through the recruitment of paxillin. Paxillin itself may bind directly to -integrins (35).
In conclusion, our results demonstrate that the cytoskeletal proteins vinculin, talin, paxillin, and FAK are recruited to the smooth muscle cell periphery in response to the stimulation of freshly dissociated smooth muscle cells with ACh. We also find that the recruitment of vinculin and paxillin to the membrane during contractile stimulation is necessary for tension development in smooth muscle. These results suggest that these proteins play a critical role in regulating cytoskeleton-transmembrane linkages in response to contractile stimulation and the regulation of these linkages is essential for force transmission. We also show that the recruitment of vinculin to the cell membrane in smooth muscle depends on the recruitment of paxillin and that paxillin recruitment is mediated by the LIM3 domain of the paxillin molecule. Thus the ability of paxillin to target to the membrane and recruit vinculin is an essential step in the regulation of tension development in smooth muscle. These observations suggest a mechanism for the dynamic regulation of the cytoskeletal organization of smooth muscle cells during active contraction.
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ACKNOWLEDGMENTS |
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GRANTS
This research was supported by National Heart, Lung, and Blood Institute Grants HL-29289 and HL-74899, the American Heart Association, Midwest Affiliate, and the Canadian Lung Association.
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FOOTNOTES |
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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.
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