Smooth Muscle Actin Determines Mechanical Force-induced p38 Activation
2005; Elsevier BV; Volume: 280; Issue: 8 Linguagem: Inglês
10.1074/jbc.m410819200
ISSN1083-351X
AutoresJiaxu Wang, Jennie Fan, Carol Laschinger, Pamela D. Arora, András Kapùs, Arun Seth, Christopher A. McCulloch,
Tópico(s)Nerve injury and regeneration
ResumoThe mitogen-activated protein kinase p38 is activated by mechanical force, but the cellular elements that mediate force-induced p38 phosphorylation are not defined. As α-smooth muscle actin (SMA) is an actin isoform associated with force generation in fibroblasts, we asked if SMA participates in the activation of p38 by force. Tensile forces (0.65 pn/μm2) generated by magnetic fields were applied to collagen-coated magnetite beads bound to Rat-2 cells. Immunoblotting showed that p38α was the predominant p38 isoform. Analysis of bead-associated proteins demonstrated that SMA enrichment of collagen receptor complexes required the α2β1 integrin. SMA was present almost entirely as filaments. Swinholide depolymerized SMA filaments and blocked force-induced p38 phosphorylation and force-induced increases of SMA. Knockdown of SMA (70% reduction) using RNA interference did not affect β-actin but inhibited force-induced p38 phosphorylation by 50%. Inhibition of Rho kinase blocked SMA filament assembly, force-induced increases of SMA, and force-induced p38 activation. Force application increased SMA content and enhanced the association of phosphorylated p38 with SMA filaments. Blockade of p38 phosphorylation by SB203586 abrogated force-induced increases of SMA. In cells transfected with SMA promoter-β-galactosidase fusion constructs, co-transfection with constitutively active p38 or MKK6 increased SMA promoter activity by 2.5–3-fold. Dominant negative p38 blocked force-induced activation of the SMA promoter. In SMA negative cells, there was no force-induced p38 phosphorylation. We conclude that force-induced p38 phosphorylation is dependent on an SMA filament-dependent pathway that uses a feed-forward amplification loop to synergize force-induced SMA expression with p38 activation. The mitogen-activated protein kinase p38 is activated by mechanical force, but the cellular elements that mediate force-induced p38 phosphorylation are not defined. As α-smooth muscle actin (SMA) is an actin isoform associated with force generation in fibroblasts, we asked if SMA participates in the activation of p38 by force. Tensile forces (0.65 pn/μm2) generated by magnetic fields were applied to collagen-coated magnetite beads bound to Rat-2 cells. Immunoblotting showed that p38α was the predominant p38 isoform. Analysis of bead-associated proteins demonstrated that SMA enrichment of collagen receptor complexes required the α2β1 integrin. SMA was present almost entirely as filaments. Swinholide depolymerized SMA filaments and blocked force-induced p38 phosphorylation and force-induced increases of SMA. Knockdown of SMA (70% reduction) using RNA interference did not affect β-actin but inhibited force-induced p38 phosphorylation by 50%. Inhibition of Rho kinase blocked SMA filament assembly, force-induced increases of SMA, and force-induced p38 activation. Force application increased SMA content and enhanced the association of phosphorylated p38 with SMA filaments. Blockade of p38 phosphorylation by SB203586 abrogated force-induced increases of SMA. In cells transfected with SMA promoter-β-galactosidase fusion constructs, co-transfection with constitutively active p38 or MKK6 increased SMA promoter activity by 2.5–3-fold. Dominant negative p38 blocked force-induced activation of the SMA promoter. In SMA negative cells, there was no force-induced p38 phosphorylation. We conclude that force-induced p38 phosphorylation is dependent on an SMA filament-dependent pathway that uses a feed-forward amplification loop to synergize force-induced SMA expression with p38 activation. The mechanisms by which mechanical forces regulate gene expression is of considerable biomedical importance, but the force-transduction circuits have not been defined. Mechanical force transmission through the extracellular matrix, integrins, and the cytoskeleton has been convincingly demonstrated using matrix protein-coated magnetic beads bound to integrins (1Wang N. Butler J.P. Ingber D.E. Science. 1993; 260: 1124-1127Crossref PubMed Scopus (2457) Google Scholar, 2D'Addario M. Arora P.D. Fan J. Ganss B. Ellen R.P. McCulloch C.A. J. Biol. Chem. 2001; 276: 31969-31977Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 3Wang J. Seth A. McCulloch C.A. Am. J. Physiol. 2000; 279: H2776-H2785Crossref PubMed Google Scholar, 4Glogauer M. Arora P. Chou D. Janmey P.A. Downey G.P. McCulloch C.A. J. Biol. Chem. 1998; 273: 1689-1698Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). With these experimental approaches, critical mechano-transduction processes have been studied including force-induced activation of the mitogen-activated protein (MAP) 1The abbreviations used are: MAP, mitogen-activated protein; SMA, smooth muscle actin; RNAi, RNA interference; TGF, transforming growth factor; PBS, phosphate-buffered saline; RSV, Rous sarcoma virus; ROS, reactive oxygen species; TRITC, tetramethylrhodamine isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; siRNA, small interfering RNA; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2 terminal kinase; CMV, cytomegalovirus. kinases (2D'Addario M. Arora P.D. Fan J. Ganss B. Ellen R.P. McCulloch C.A. J. Biol. Chem. 2001; 276: 31969-31977Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 3Wang J. Seth A. McCulloch C.A. Am. J. Physiol. 2000; 279: H2776-H2785Crossref PubMed Google Scholar, 5D'Addario M. Arora P.D. Ellen R.P. McCulloch C.A. J. 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Pathol. 2001; 159: 1009-1020Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar, 13Vaughan M.B. Howard E.W. Tomasek J.J. Exp. Cell Res. 2000; 257: 180-189Crossref PubMed Scopus (399) Google Scholar, 14Arora P.D. McCulloch C.A. J. Cell. Physiol. 1994; 159: 161-175Crossref PubMed Scopus (246) Google Scholar). Interference with SMA expression inhibits force generation and the formation of focal adhesions and stress fibers (15Hinz B. Gabbiani G. Chaponnier C. J. Cell Biol. 2002; 157: 657-663Crossref PubMed Scopus (199) Google Scholar). In myofibroblasts, induction of SMA by TGF-β is dependent on the compliance of the substrate (16Arora P.D. Narani N. McCulloch C.A. Am. J. Pathol. 1999; 154: 871-882Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar), suggesting an important role for cell-generated tension in regulating gene expression (17Tomasek J.J. Gabbiani G. Hinz B. Chaponnier C. Brown R.A. Nat. Rev. Mol. Cell. 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Cell Res. 1998; 238: 481-490Crossref PubMed Scopus (68) Google Scholar). Force-induced regulation of SMA expression is also associated with p38 activation, depending on the extent of actin assembly in the specific types of cells examined (3Wang J. Seth A. McCulloch C.A. Am. J. Physiol. 2000; 279: H2776-H2785Crossref PubMed Google Scholar, 22Wang J. Chen H. Seth A. McCulloch C.A. Am. J. Physiol. 2003; 285: H1871-H1881Crossref PubMed Scopus (16) Google Scholar). As p38 activation is a prominent cellular response to applied tensile forces (2D'Addario M. Arora P.D. Fan J. Ganss B. Ellen R.P. McCulloch C.A. J. Biol. Chem. 2001; 276: 31969-31977Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 3Wang J. Seth A. McCulloch C.A. Am. J. Physiol. 2000; 279: H2776-H2785Crossref PubMed Google Scholar, 5D'Addario M. Arora P.D. Ellen R.P. McCulloch C.A. J. Biol. Chem. 2003; 278: 53090-53097Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 6D'Addario M. Arora P.D. Ellen R.P. McCulloch C.A. J. Biol. Chem. 2002; 277: 47541-47550Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), we examined the role of SMA in mediating force-induced activation of p38, and conversely, we studied the importance of p38 in regulating force-induced SMA expression. Our data indicate that when tensile forces are applied to α2β1 integrins, phosphorylated p38 binds to SMA, and there are p38-dependent increases of SMA expression. As our results showed that cells with abundant SMA exhibit enhanced force-induced activation of p38, we suggest that SMA and p38 participate in a feed-forward amplification loop to synergize force-induced SMA expression with p38 phosphorylation. Reagents—Antibodies to α-smooth muscle actin (clone 1A4), β-actin (clone AC-15), total-actin (clone AC-40), vimentin (clone VIM-13.2), vinculin (clone hVIN-1), TRITC-phalloidin, TGF-β1, DNase I, and bovine serum albumin were from Sigma. Goat anti-mouse IgG2a, goat anti-mouse IgM, and goat anti-mouse IgG1 antibodies were purchased from Caltag Laboratories (Burlingame, CA). Antibodies to p38α, p38δ, p38, ERK1/2, and JNK and the respective phospho-specific antibodies for each of these kinases were purchased from Cell Signaling Technology (New England Biolabs, Mississauga, Ontario, Canada). Antibodies to p38β and p38γ were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal antibody to GAPDH (clone 6C5) was purchased from Chemicon International (Temecula, CA). Soluble type I bovine collagen was obtained from Celltrix (Palo Alto, CA). The Rho kinase inhibitor Y27632, rat polyclonal antibodies to α1, α2, α3 integrins, DDR1 receptor, and mouse monoclonal antibody to the α2β1 integrin (clone PIE6) were purchased from Calbiochem. The Rho activation assay kit was purchased from Cytoskeleton, Inc. (Denver, CO). siRNA Preparation—We designed target-specific siRNA duplexes based on the sequence of the rat SMA gene. A 21-nucleotide SMA siRNA sequence relative to the start codon (876–896, accession number X06801) was submitted to a BLAST search against the rat genome sequence to ensure that only the SMA gene of the rat genome was targeted. An siRNA sequence (5′-GACGUAAACGGCCACAAGUUC-3′) for green fluorescent protein was synthesized as an irrelevant control. All siRNAs were produced by Qiagen. Cell Culture and Transfection for RNAi—Rat-2 cells were incubated at 37 °C in complete Hg-DMEM containing 5% fetal bovine serum and a 1:10 dilution of an antibiotic solution (0.17% w/v penicillin V, 0.1% gentamycin sulfate, and 0.01 μg/ml amphotericin). Cells were maintained in a humidified incubator gassed with 95% O2, 5% CO2 and were passaged with trypsin. One day before transfection with siRNA, cells were trypsinized, diluted with fresh medium without antibiotics, and transferred to 6-well plates for immunoblotting and 12-well plates for immunostaining. Transient transfection of siRNAs was conducted with Oligofectamine according to the supplier's instructions (Invitrogen). The entire mixture was added to the cells in one well resulting in a final concentration of 200 nm for the siRNAs. Cells were assayed 48 h after transfection. Triplicate assays were conducted for a minimum of three independent experiments. Mechanical Force Application—A force delivery system employing collagen-coated magnetite beads and magnetic fields was used to apply exogenous tensile forces in vitro as described previously (8Wang J. Su M. Fan J. Seth A. McCulloch C.A. J. Biol. Chem. 2002; 277: 22889-22895Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Briefly, magnetite beads (400 mg; Sigma) were incubated for 10 min with 1 ml of an acidic bovine collagen solution (>95% type I collagen; 3 mg/ml) at 37 °C and neutralized to pH 7.4 with 100 μl of 1 n NaOH. Under these conditions, collagen polymerizes and forms fibrils around the beads within 30 min. The beads were sonicated to eliminate clumps and were then dispersed. Analysis of bead size was performed by electronic particle counting (Coulter Channelyzer, Coulter Electronics, Hialeah, FL). Prior to incubation with cells, beads were rinsed in PBS, washed three times, resuspended in Ca2+-free buffer, and added to attached cells in complete medium for 10 min. Cells were washed three times to remove unbound beads prior to exposure to force. A ceramic permanent magnet was used to apply perpendicular forces to beads attached to the dorsal surface of cells. For all experiments, the pole face was parallel with and 2 cm from the surface of the cell culture dish. As the surface area of the magnet was larger than the culture dishes, and as the bead covering was relatively uniform for all cells, the forces applied to cells across the width of the culture dish were relatively uniform (23Glogauer M. Ferrier J. McCulloch C.A. Am. J. Physiol. 1995; 269: C1093-C1104Crossref PubMed Google Scholar). Constant forces (0.65 pn/μm2 projected cell area) but of varying duration were used for all experiments. Immunofluorescence—After transfection (48 h), cells were fixed with 3.7% formaldehyde, permeabilized with 0.1% Triton X-100, blocked with 0.2% bovine serum albumin, stained for SMA, and counterstained with fluorescein isothiocyanate-conjugated goat anti-mouse IgG. For double staining of SMA and actin filaments, cells were incubated with antibody to SMA, counterstained with fluorescein isothiocyanate-conjugated goat anti-mouse antibody, stained with TRITC-phalloidin, and imaged with wide field immunofluorescence microscopy. Immunoblotting—Magnetite beads with the associated focal adhesion complexes were isolated in CSKB buffer, and bead-associated proteins were recovered by magnetic selection. Beads were sonicated and washed in PBS, and the associated proteins were eluted by boiling in SDS sample buffer (62.5 mm Tris-HCl, pH 6.8, containing 2% SDS, 10% glycerol, 50 mm dithiothreitol, 0.1% w/v bromphenol blue). For cell lysates, the cells were washed with PBS and proteins solubilized in 200 μl of SDS sample buffer without bromphenol blue. Total protein was measured using the RC-DC protein assay (Bio-Rad), and equal quantities were loaded on SDS-PAGE (10% acrylamide) and transferred to nitrocellulose. Blots were blocked for 1 h with 5% skim milk in TBS and incubated with the indicated antibody (SMA, vinculin, vimentin, β-actin, GAPDH, JNK, ERK1/2, and p38) diluted 1:1000 in 0.5% Tween/TBS for 1 h at room temperature or overnight at 4 °C. Blots were washed with 0.5% Tween/TBS, incubated with appropriate second antibodies for 1 h, washed four times in Tween/TBS, and developed by chemiluminescence (ECL; Amersham Biosciences). The blots were exposed to Kodak X-Omat film, and band density was analyzed by IP Lab Gel Scientific Image Processing (Signal Analytics Corp., Vienna, VA). Immunoprecipitation—The immunoprecipitation protocol for SMA has been described previously (6D'Addario M. Arora P.D. Ellen R.P. McCulloch C.A. J. Biol. Chem. 2002; 277: 47541-47550Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Briefly, cells were scraped into RIPA buffer containing sodium vanadate (1 mm), a protease inhibitor mixture (Sigma). The cell lysate was pre-cleared, normalized for protein content, and then incubated overnight with anti-SMA antibodies at 4 °C. ImmunoPure® Plus(G) from Pierce was added, and after incubation for1.5 h at 4 °C, the resin was washed four times, and specific immunoprecipitated proteins were eluted by boiling in SDS sample buffer and processed for immunoblotting. Northern Blotting—RNA (10 μg per sample) was denatured in formaldehyde and formamide at 55 °C for 10 min and electrophoresed in 1.2% agarose gels. The SMA and β-actin cDNA probes were made by reverse transcription-PCR from Rat-2 cells using the following primers: SMA primers, 5′-ccgcaaatgcttctaagtcac-3′ (forward) and 5′-cacgagtaacaaatcaaagct-3′(reverse). For β-actin the primers were 5′-tggcctgtacactgacgtgag-3′ (forward) and 5′-tggccaggtgtcagggagata-3′ (reverse). RNA was isolated from cells (Qiagen RNeasy total RNA kit), and cDNA was produced from total RNA (2 μg per sample) using Omniscript Reverse Transcriptase. PCR products were inserted into a PCR3.9 vector using the Original TA cloning kit and sequenced to confirm the correct products (24Magnuson V.L. Young M. Schattenberg D.G. Mancini M.A. Chen D.L. Steffensen B. Klebe R.J. J. Biol. Chem. 1991; 266: 14654-14662Abstract Full Text PDF PubMed Google Scholar). Rho Activation—Rho activation was measured in Rat-2 cells cultured overnight without serum and subjected to force for 15 min. Control cells with beads but no force were also assayed for Rho activation. Lysophosphatidic acid treatment of these cells was performed as a positive control for Rho. Briefly, the assay relies on the specific binding of GTP-Rho to Rhotekin-RBD protein coupled to glutathione S-transferase beads. The amount of GTP-Rho was quantified by immunoblotting with a Rho monoclonal antibody and by comparing the bead-bound protein (active Rho) with total Rho in the cell lysates. G-actin and Immunodepletion—We determined G-actin content utilizing the specific binding of G actin to DNase I (25Arora P.D. McCulloch C.A. J. Biol. Chem. 1996; 271: 20516-20523Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 26Heacock C.S. Bamburg J.R. Anal. Biochem. 1983; 135: 22-36Crossref PubMed Scopus (49) Google Scholar). Briefly, DNase I was coupled to Aminolink (Pierce) as per the manufacturer's protocol. Cells were lysed at -10 °C in cell lysis buffer (1% Triton X-100, 10 mm Tris, 2 mm MgCl2, 0.2 mm dithiothreitol, pH 7.4) with 1 μm phalloidin to stabilize F-actin and sedimented at 14,000 × g for 1 min to remove cell debris, and the cell lysate was incubated with DNase beads for 2 min at 4 °C. Beads were rapidly washed three times, and bound protein (G-actin), supernatant (F-actin), and the total cell lysate (F plus G actin) were quantified by immunoblotting using monoclonal antibodies that recognized all actin isoforms, including β-actin and SMA. For estimation of the relative proportion of SMA in the total actin pool, we used an immunodepletion method. First, because no SMA was detected in the G-actin fraction by immunoblotting, we incubated the F-actin fraction overnight with SMA antibodies at 4 °C. SMA immunocomplexes were removed with ImmunoPure® Plus(G) (Pierce) using incubation for 1.5 h at 4 °C followed by centrifugation. The remaining actins in the supernatant were processed for total actin immunoblotting as described above. Cytoskeletal Pellets—Pellets were prepared by lysing cells in PBS containing 1% Triton X-100, 1 μm phalloidin, and inhibitors. The detergent-insoluble pellet was obtained by centrifugation (20,000 × g at 4 °C for 30 min) and solubilized by boiling in 2% SDS sample buffer. Equivalent amounts of proteins were loaded in each lane and immunoblotted for β-actin and SMA. Transfections and Reporter Gene Assays—ROS 17/2.8 cells were grown in α-DMEM with 10% fetal calf serum. Prior to transfection, cells were plated at 1 × 104 cells/well in 6-well plates and incubated in complete α-DMEM for 24 h. Cells were transiently transfected with reporter plasmids containing p547/LacZ constructs (from Dr. G. Owens) in which the whole SMA promoter is present. Cells were co-transfected with a pCMV-p38 structures. The vectors used in the co-transfection are the following, and their source is indicated in parentheses: pCMV-MKK6+ (constitutively active from Dr. J. Woodgett, University of Toronto), pCMV-p38FLAG and pCMV-38WT (constitutively active; Dr. R. J. Davis, University of Massachusetts), pCMV-p38DN (dominant negative p38 from Dr. Andras Kapas). Rous sarcoma virus (RSV) expression plasmid was normalized for equal loading using the effecting reagent according to the supplier's instructions (Qiagen). Cell extracts were prepared with a detergent lysis method. For estimates of promoter activity, β-galactosidase reporter enzyme activity was measured with an enzyme assay system (Roche Applied Science). β-Galactosidase activities were normalized to RSV luciferase activity as a transfection control. The RSV (-124 to +34) luciferase construct was provided by H. P. Elsholtz (University of Toronto) and was used as described earlier (8Wang J. Su M. Fan J. Seth A. McCulloch C.A. J. Biol. Chem. 2002; 277: 22889-22895Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). RSV luciferase assays were conducted using a luciferase assay system (Promega) according to the manufacturer's instructions. RSV luciferase activity (reflecting promoter activity) was unaffected by all treatments at all experimental times. Statistical Analyses—For quantitative data, means and standard errors were computed. When appropriate, comparisons between groups were evaluated by Student's t test or analysis of variance with statistical significance set at p < 0.05. At least three independent experiments were conducted for each condition described, and in each experiment, there were at least three replicates. SMA Incorporation into Collagen Receptor Complexes—We used Rat-2 fibroblasts and ROS 17/2.8 cells as cell models to examine the role of SMA in mechanotransduction. These cells can be induced to express SMA with appropriate stimuli (27Leavitt J. Gunning P. Kedes L. Jariwalla R. Nature. 1985; 316: 840-842Crossref PubMed Scopus (95) Google Scholar). They express abundant cell surface collagen receptors that provide attachment for collagen-coated beads and are readily transfected (2D'Addario M. Arora P.D. Fan J. Ganss B. Ellen R.P. McCulloch C.A. J. Biol. Chem. 2001; 276: 31969-31977Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 5D'Addario M. Arora P.D. Ellen R.P. McCulloch C.A. J. Biol. Chem. 2003; 278: 53090-53097Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 6D'Addario M. Arora P.D. Ellen R.P. McCulloch C.A. J. Biol. Chem. 2002; 277: 47541-47550Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). We first examined the collagen receptors that were associated with collagen beads bound to Rat-2 cells. Immunoblotting of bead-bound proteins showed that the α2 integrin, α3 integrin, and the discoidin domain receptor 1 were associated with attached collagen beads (Fig. 1A), although in contrast, the α1 integrin was not expressed. As SMA actin filaments are associated with mature focal adhesions in human fibroblasts (19Dugina V. Fontao L. Chaponnier C. Vasiliev J. Gabbiani G. J. Cell Sci. 2001; 114: 3285-3296Crossref PubMed Google Scholar), we asked if SMA was also enriched at collagen receptor adhesion complexes in Rat-2 cells and if this enrichment required the α2β1 integrin or α3β1 integrin. Incubation of cells with α3 integrin antibody did not block the association of SMA or the focal adhesion protein vinculin with collagen-coated magnetite beads (Fig. 1B). In contrast, incubation of cells with α2β1 integrin antibody blocked the association of SMA and vinculin with collagencoated magnetite beads. By densitometry, there was a small (18%) inhibitory effect of the α2β1 integrin on the association of β-actin with collagen beads (Fig. 1C). Fluorescence microscopy showed rings of immunohistochemically stained SMA around collagen beads that co-localized with actin filaments stained by rhodamine phalloidin (Fig. 1D). Incubation with the α2β1 integrin antibody blocked the formation of SMA-stained rings around the collagen-coated beads but did not affect staining for β-actin. Thus the incorporation of SMA into the adhesion complexes that form around collagen beads, the site of force application to cells in this model system (6D'Addario M. Arora P.D. Ellen R.P. McCulloch C.A. J. Biol. Chem. 2002; 277: 47541-47550Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), is dependent on the α2β1 integrin. SMA Incorporation into Actin Filaments Is Sensitive to Swinholide—We determined whether swinholide A and latrunculin B, actin filament disrupting agents, could be used to block the association of SMA and β-actin with collagen receptor complexes. After washing away unattached beads, only cell-attached beads were used for analysis of bead-associated proteins. Pretreatment with latrunculin B (1 μm, 30 min) was not sufficient to dissociate SMA, β-actin, and vinculin from collagen beads. In contrast, swinholide A (0.1 μm; 20 min) strongly blocked the incorporation of SMA and vinculin into collagen bead complexes, while preserving the binding of β-actin (Fig. 2A). This result was not because of a lack of bead binding to cells because only proteins from bound beads were analyzed. Immunohistochemically stained SMA around collagen beads that co-localized with actin filaments stained by rhodamine phalloidin were found after latrunculin B treatment, whereas swinholide A blocked the formation of SMA-stained rings around the collagen-coated beads (Fig. 2B). Thus swinholide A but not latrunculin can be used reliably to dissociate SMA filaments from the sites of force application in Rat-2 cells. In some well differentiated fibroblast sub-types cultured in vitro, SMA comprises up to 14% of total actin content (14Arora P.D. McCulloch C.A. J. Cell. Physiol. 1994; 159: 161-175Crossref PubMed Scopus (246) Google Scholar). As the rate of assembly of SMA into actin filaments during cell spreading can be quite different from that of β-actin filaments (29Hinz B. Dugina V. Ballestrem C. Wehrle-Haller B. Chaponnier C. Mol. Biol. Cell. 2003; 14: 2508-2519Crossref PubMed Scopus (226) Google Scholar), we considered that SMA, even as a relatively low abundance actin isoform, may have an impact on the transmission of filament-dependent mechanical signals. We estimated the relative proportions of SMA and other actin isoforms in monomeric (G-actin) form by measuring the binding of G-actin to DNase I (30Hosoya H. Mabuchi I. J. Cell Biol. 1984; 99: 994-1001Crossref PubMed Scopus (39) Google Scholar) followed by actin isoform-specific immunoblotting and densitometry. In untreated Rat-2 cells, ∼20% of total actin bound to DNase I and was therefore monomeric (Fig. 2C). Treatment with swinholide A (0.1 μm; 20 min) or with latrunculin B (1 μm, 30 min) greatly increased the abundance of total actin monomers (to ∼50%) in comparison to untreated controls. In untreated samples and in samples pretreated with latrunculin B, SMA monomers were present at extremely low levels, whereas swinholide A treatment increased the amount of monomeric SMA to ∼70% (Fig. 2C). To determine the relative abundance of SMA in actin filaments, we removed G-actin wi
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