Regulation of Tension-induced Mechanotranscriptional Signals by the Microtubule Network in Fibroblasts
2003; Elsevier BV; Volume: 278; Issue: 52 Linguagem: Inglês
10.1074/jbc.m309027200
ISSN1083-351X
AutoresMario D’Addario, Pamela D. Arora, Richard P. Ellen, Christopher A. McCulloch,
Tópico(s)Skin and Cellular Biology Research
ResumoMechanical loading of connective tissues induces the expression of extracellular matrix and cytoskeletal genes that are involved in matrix remodeling. These processes depend in part on force transmission through β1 integrins and actin filaments, but the role of microtubules in regulating mechanotranscriptional responses is not well defined. We assessed the involvement of microtubules in the mechanotranscriptional regulation of filamin A, an actin-cross-linking protein that protects cells against force-induced apoptosis by stabilizing cell membranes. Collagen-coated magnetite beads and magnetic fields were used to apply tensile forces to cultured fibroblasts at focal adhesions. Force enhanced recruitment of α-tubulin and the plus end microtubule-binding protein cytoplasmic linker protein-170 (CLIP-170) at focal adhesions. Immunoprecipitation studies demonstrated no direct binding of tubulin to actin or filamin A, but CLIP-170 interacted with tubulin, filamin A, and β-actin. The association of CLIP-170 with β-actin was enhanced by force. Force activated the p38 mitogen-activated protein kinase, increased filamin A expression, and induced the relocation of p38 and filamin A to focal adhesions. Disruption of microtubules with nocodazole, independent of force application, enhanced filamin A expression and Sp1-mediated filamin A promoter activity, while stabilization of microtubules with Taxol inhibited force induction of both filamin A mRNA and protein. We conclude that in response to tensile forces applied through β1 integrins and actin the microtubule network modulates mechanotranscriptional coupling of filamin A. Mechanical loading of connective tissues induces the expression of extracellular matrix and cytoskeletal genes that are involved in matrix remodeling. These processes depend in part on force transmission through β1 integrins and actin filaments, but the role of microtubules in regulating mechanotranscriptional responses is not well defined. We assessed the involvement of microtubules in the mechanotranscriptional regulation of filamin A, an actin-cross-linking protein that protects cells against force-induced apoptosis by stabilizing cell membranes. Collagen-coated magnetite beads and magnetic fields were used to apply tensile forces to cultured fibroblasts at focal adhesions. Force enhanced recruitment of α-tubulin and the plus end microtubule-binding protein cytoplasmic linker protein-170 (CLIP-170) at focal adhesions. Immunoprecipitation studies demonstrated no direct binding of tubulin to actin or filamin A, but CLIP-170 interacted with tubulin, filamin A, and β-actin. The association of CLIP-170 with β-actin was enhanced by force. Force activated the p38 mitogen-activated protein kinase, increased filamin A expression, and induced the relocation of p38 and filamin A to focal adhesions. Disruption of microtubules with nocodazole, independent of force application, enhanced filamin A expression and Sp1-mediated filamin A promoter activity, while stabilization of microtubules with Taxol inhibited force induction of both filamin A mRNA and protein. We conclude that in response to tensile forces applied through β1 integrins and actin the microtubule network modulates mechanotranscriptional coupling of filamin A. During application of mechanical loads to connective tissues, the extracellular matrix distributes forces to cellular adhesive structures including integrins; these forces are in turn directed to structural components including the cytoskeleton (for reviews, see Refs. 1Critchley D.R. Curr. Opin. Cell Biol. 2000; 12: 133-139Crossref PubMed Scopus (492) Google Scholar, 2Chien S. Li S. Shyy J.Y. Hypertension. 1997; 31: 162-169Crossref Google Scholar, 3Shyy J.Y. Chien S. Curr. Opin. Cell Biol. 1997; 9: 707-713Crossref PubMed Scopus (294) Google Scholar). Forces applied through integrins are transduced into intracellular signals that mediate the redistribution of cytoskeletal proteins and the expression of several cytoskeletal genes including α-skeletal and α-smooth muscle actins, filamin A, talin, and vinculin (4Watson P.A. FASEB J. 1991; 5: 2013-2019Crossref PubMed Scopus (284) Google Scholar, 5Tidball J.G. Spencer M.J. Wehling M. Lavergne E. J. Biol. Chem. 1999; 274: 33155-33160Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 6Wang J. Lukse E. Seth A. McCulloch C.A.G. Tissue Cell. 2001; 33: 86-96Crossref PubMed Scopus (14) Google Scholar, 7D'Addario M. Arora P. Fan J. Ganss B. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2001; 276: 31969-31977Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 8Wang J. Su M. Fan J. Seth A. McCulloch C.A.G. J. Biol. Chem. 2002; 277: 22889-22895Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 9D'Addario M. Arora P. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2002; 277: 47541-47550Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Although the mechanotranscriptional regulation of these genes has not been analyzed in depth, force-induced activation of mitogen-activated protein kinases can mediate increased binding of transcription factors to regulatory sites on the promoters of some of these genes (7D'Addario M. Arora P. Fan J. Ganss B. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2001; 276: 31969-31977Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 9D'Addario M. Arora P. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2002; 277: 47541-47550Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), thereby regulating cytoskeletal gene expression.Filamin A is an example of a cytoskeletal gene that is induced by mechanical forces (9D'Addario M. Arora P. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2002; 277: 47541-47550Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Filamins are actin-binding proteins that organize actin filaments into orthogonal networks and enhance the rigidity of the actin cytoskeleton (10Stossel T.P. Condeelis J. Cooley L. Hartwig J.H. Noegel A. Schleicher M. Shapiro S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 138-145Crossref PubMed Scopus (811) Google Scholar). Tensile force application through β1 integrins induces enhanced expression of filamin A (7D'Addario M. Arora P. Fan J. Ganss B. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2001; 276: 31969-31977Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 9D'Addario M. Arora P. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2002; 277: 47541-47550Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar); the subsequent increase of filamin A protein facilitates cell survival by mechanical stabilization of cortical actin and by prevention of cell depolarization due to excessive membrane distortion caused by high amplitude tensile forces (11Kainulainen T. Pender A. D'Addario M. Feng Y. Lekic P. McCulloch C.A.G. J. Biol. Chem. 2002; 277: 21998-22009Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). As filamin A binds integrins and other proteins enriched at cell adhesion sites (10Stossel T.P. Condeelis J. Cooley L. Hartwig J.H. Noegel A. Schleicher M. Shapiro S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 138-145Crossref PubMed Scopus (811) Google Scholar), filamin A thereby provides a good model for determining mechanotranscriptional responses transduced through β1 integrins.Cellular responses to tensile forces applied through integrins require an intact actin cytoskeleton (7D'Addario M. Arora P. Fan J. Ganss B. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2001; 276: 31969-31977Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 8Wang J. Su M. Fan J. Seth A. McCulloch C.A.G. J. Biol. Chem. 2002; 277: 22889-22895Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), but the role of the microtubule network in mediating mechanical signaling is currently not defined. Microtubules can modulate contraction and can also affect cell attachment to the extracellular matrix through regulation of turnover at adhesion sites (12Bershadsky A. Chausovsky A. Becker E. Lyubimova A. Geiger B. Curr. Biol. 1996; 6: 1279-1289Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 13Kaverina I. Rottner K. Small J.V. J. Cell Biol. 1998; 142: 181-190Crossref PubMed Scopus (254) Google Scholar, 14Kaverina I. Krylshkina O. Small J.V. J. Cell Biol. 1999; 146: 1033-1043Crossref PubMed Scopus (381) Google Scholar, 15Kaverina I. Krylshkina O. Beningo K. Anderson K. Wang Y.L. Small J.V. J. Cell Sci. 2002; 115: 2283-2291PubMed Google Scholar). Locally applied forces promote growth of microtubules toward substrate attachment sites (15Kaverina I. Krylshkina O. Beningo K. Anderson K. Wang Y.L. Small J.V. J. Cell Sci. 2002; 115: 2283-2291PubMed Google Scholar) as has been shown in Aplysia in which beads coated with matrix ligands induce microtubule assembly adjacent to the beads (16Thompson C. Lin C.H. Forscher P. J. Cell Sci. 1996; 109: 2843-2854PubMed Google Scholar). Thus microtubules may be involved in modulating mechanotransduction.Recent studies have established functional links between actin filaments and microtubules in a broad array of cellular processes including vesicle and organelle movement, cytokinesis, nuclear migration, contractile ring formation, and mitotic spindle alignment (17Goode B.L. Drubin D.G. Barnes G. Curr. Opin. Cell Biol. 2000; 12: 63-71Crossref PubMed Scopus (414) Google Scholar). An important element of actin-microtubule interactions that has not been characterized in detail is the interaction of these two networks in integrin-dependent mechanotransduction. We have previously shown that mechanical forces applied through β1 integrins activate the filamin A gene (9D'Addario M. Arora P. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2002; 277: 47541-47550Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Here we describe the involvement of microtubules in regulating the activation of the filamin A gene after application of mechanical forces.EXPERIMENTAL PROCEDURESCell Culture and Reagents—Human gingival fibroblasts were derived from primary explant cultures as described previously (18Pender N. McCulloch C.A.G. J. Cell Sci. 1991; 100: 187-193Crossref PubMed Google Scholar). Cells from passages 6-10 were grown as monolayer cultures in α-modified Eagle's medium containing 10% fetal bovine serum and antibiotics. Experiments involving promoter analyses used Rat-2 fibroblasts as surrogates for gingival fibroblasts as described previously (19Hui M.Z. Tenenbaum H.C. McCulloch C.A.G. J. Cell. Physiol. 1997; 172: 323-333Crossref PubMed Scopus (22) Google Scholar).Mouse anti-filamin A antibodies were obtained from Serotec (Cedarlane Laboratories, Hornby, Ontario, Canada). Mouse monoclonal antibodies to β-actin, vinculin, and α-tubulin were from Sigma. Antibodies to pp38, p38, and Sp1 were obtained from Cell Signaling Technologies (New England Biolabs, Mississauga, Ontario, Canada). Rabbit anti-cytoplasmic linker protein-170 (CLIP-170) 1The abbreviations used are: CLIP-170cytoplasmic linker proteinRTreverse transcriptionwtwild typemutmutantPIPES1,4-piperazinediethanesulfonic acidpp38phosphorylated p38GEFguanine nucleotide exchange factor. was obtained from Dr. N. Galjart (Erasmus University, Rotterdam, Netherlands). Nocodazole (1 μm, Sigma) and the microtubule stabilization agent paclitaxel (Taxol, 0.5 μm; Sigma) were used as indicated. Monoclonal antibody to αvβ3 was obtained from Calbiochem, and anti-β1 integrin was obtained from Beckman Coulter, Inc., and its use has been described elsewhere (7D'Addario M. Arora P. Fan J. Ganss B. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2001; 276: 31969-31977Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar).Force Generation—Tensile forces were applied to integrins using a model system described previously (20Glogauer M. Arora P.D. Yao G. Sokholov I. Ferrier J. McCulloch C.A.G. J. Cell Sci. 1997; 110: 11-21Crossref PubMed Google Scholar). In brief, magnetite microparticles (Fe3O4, Sigma) were incubated with purified type I bovine collagen (Vitrogen 100, Celltrix, Palo Alto, CA; 1 mg/ml); following 30-min incubations, excess non-adherent microparticles were removed by vigorous washing, and cells were supplemented with fresh α-minimum Eagle's medium. A ceramic permanent magnet (Jobmaster, Mississauga, Ontario, Canada) was placed on top of the dish to generate a perpendicular tensile force of ∼0.48 piconewtons/μm2 cell area; this force level is comparable to that applied to cells in vivo during normal function (20Glogauer M. Arora P.D. Yao G. Sokholov I. Ferrier J. McCulloch C.A.G. J. Cell Sci. 1997; 110: 11-21Crossref PubMed Google Scholar). The incubation times were specific for each individual experiment as indicated.RNA Isolation, Reverse Transcription (RT), and PCR Analysis—RNA isolation was performed with RNeasy reagents (Qiagen, Mississauga, Ontario, Canada). All RNA preparations were treated with RQ1 DNase (Promega Corp., Madison, WI) for 30 min. The RT-PCR protocol and the oligonucleotides used in the procedure have been described in detail in previous studies (7D'Addario M. Arora P. Fan J. Ganss B. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2001; 276: 31969-31977Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 9D'Addario M. Arora P. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2002; 277: 47541-47550Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 21D'Addario M. Ahmad A. Xu J.W. Menezes J. FASEB J. 1999; 13: 2203-2213Crossref PubMed Scopus (33) Google Scholar).The semiquantitative nature of the RT-PCR protocol, the precautions taken to avoid spurious reaction products, and the controls used have been described previously (7D'Addario M. Arora P. Fan J. Ganss B. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2001; 276: 31969-31977Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 21D'Addario M. Ahmad A. Xu J.W. Menezes J. FASEB J. 1999; 13: 2203-2213Crossref PubMed Scopus (33) Google Scholar). In each experiment, a non-RT control demonstrated the lack of DNA contamination.Immunoblotting, Immunofluorescence, and Immunoprecipitation— Cells were lysed, and cellular proteins were separated by SDS-PAGE and transferred to nitrocellulose (Schleicher & Schuell) as described previously (20Glogauer M. Arora P.D. Yao G. Sokholov I. Ferrier J. McCulloch C.A.G. J. Cell Sci. 1997; 110: 11-21Crossref PubMed Google Scholar). Protein concentrations were determined using the Bradford assay. Equal amounts of protein were loaded on individual lanes, separated by SDS-PAGE, transferred to nitrocellulose, and analyzed as described previously (22Arora P.D. McCulloch C.A.G. J. Biol. Chem. 1996; 271: 20516-20523Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Chemiluminescent detection was performed according to the manufacturer's instructions (Amersham Biosciences), and radiographic films were exposed for standardized lengths of time using conventional protocols.For immunofluorescence, gingival fibroblasts were grown on 10-mm glass coverslips, incubated with collagen-coated microbeads, and subjected to magnetic force application as described above. Samples were collected at standardized time points and stained as described previously (7D'Addario M. Arora P. Fan J. Ganss B. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2001; 276: 31969-31977Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The protocol used to detect protein-protein interactions by immunoprecipitation of target proteins with β-actin, filamin A, CLIP-170, and α-tubulin has been described previously (22Arora P.D. McCulloch C.A.G. J. Biol. Chem. 1996; 271: 20516-20523Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In brief, samples were treated with radioimmune precipitation assay buffer containing sodium vanadate (1 mm) and a protease inhibitor mixture (Sigma). Isolated proteins were incubated with protein G-Sepharose beads (Zymed Laboratories Inc.) that had been preincubated with antibodies to CLIP-170 or α-tubulin overnight at 4 °C. Samples were resolved by 5-20% gradient SDS-PAGE and transferred to nitrocellulose. Blots were probed with the specific antibody indicated in each figure, and ECL was carried out according to the manufacturer's instructions (Amersham Biosciences).Genomic DNA Isolation and Filamin A Promoter Construction—To generate the 3224-bp filamin A luciferase promoter construct, we isolated intact fibroblast genomic DNA using the protocol of Goelz et al. (23Goelz S. Hamilton S. Vogelstein B. Biochem. Biophys. Res. Commun. 1985; 130: 118-126Crossref PubMed Scopus (794) Google Scholar). The construction of the amplified promoter has been described in detail elsewhere (7D'Addario M. Arora P. Fan J. Ganss B. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2001; 276: 31969-31977Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 9D'Addario M. Arora P. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2002; 277: 47541-47550Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The construct was verified through restriction enzyme cleavage and ligated into the pGL2 Basic vector between HindIII and BglII, and its correct orientation was verified through diagnostic restriction enzyme digestion and sequencing performed at the DNA Sequencing Facility, Center for Applied Genomics (Hospital for Sick Children, Toronto, Ontario, Canada).To generate the final wild type 75-bp filamin A luciferase construct (pFil75wtluc), the original 3.2-kbp filamin A promoter component was cut out, blunt-ended, and isolated from an agarose gel, and the portion containing the 75-bp Sp1 fragment was fused to the luciferase reporter construct. To synthesize the final 75-bp promoter construct containing mutations at the Sp1 binding sites (pFil75mutluc), two complementary oligonucleotides (MWG Biotech, described in Refs. 7D'Addario M. Arora P. Fan J. Ganss B. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2001; 276: 31969-31977Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar and 9D'Addario M. Arora P. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2002; 277: 47541-47550Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) were hybridized in equimolar amounts and ligated into pGL2luc. Promoter scanning was used to determine the location of potentially important transcription factor binding sites, and these locations were specifically mutated (24Prestridge D.S. J. Mol. Biol. 1995; 249: 923-932Crossref PubMed Scopus (367) Google Scholar). The hybridized oligonucleotides were ligated into the pGL2 Basic luciferase vector (Promega Corp.), and the correctly ligated vector was verified through restriction enzyme digestion.Bead and Protein Isolation—Cells in normal growth medium that had reached 80-90% confluence on 60-mm tissue culture dishes were incubated with collagen-coated or bovine serum albumin-coated magnetite beads. Beads were isolated from dishes as described previously (25Plopper G. Ingber D.E. Biochem. Biophys. Res. Commun. 1993; 193: 571-578Crossref PubMed Scopus (149) Google Scholar). Cells were gently washed three times with ice-cold phosphate-buffered saline to remove unbound beads and scraped into ice-cold cytoskeleton extraction buffer (CSKB; 0.5% Triton X-100, 50 mm NaCl, 300 mm sucrose, 3 mm MgCl2, 20 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mm phenylmethylsulfonyl fluoride, 10 mm PIPES, pH 6.8). The cell bead suspension was sonicated for 10 s, and the beads were isolated from the lysate using a magnetic separation stand. The beads were resuspended in fresh, ice-cold CKSB and homogenized with a Dounce homogenizer (20 strokes), and the magnetic isolation was repeated. The beads were washed thoroughly in CSKB, sedimented with a microcentrifuge, resuspended in Laemmli sample buffer, and placed in a boiling water bath for 10 min to allow the collagen-associated complexes to dissociate from the beads. The beads were pelleted, and the lysate was collected for immunoblot analysis.Cell Transfections—Rat-2 cells were transfected using Effectene transfection reagent (Qiagen) as described previously (7D'Addario M. Arora P. Fan J. Ganss B. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2001; 276: 31969-31977Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 26Wang J. Seth A. McCulloch C.A.G. Am. J. Physiol. 2000; 279: H2776-H2785Crossref PubMed Google Scholar). Briefly, following titration experiments to determine the optimal concentration of vector needed, cells were transfected, incubated for 18-24 h, and then subjected to specific treatments (described for each individual experiment). Following each treatment, cells were processed for luciferase activity as recommended by the manufacturer (luciferase assay system, Promega Corp.). The luciferase vectors pFil3.2luc, pFil75wtluc, and pFil75mutluc are described above. To establish transfection efficiency and to provide experimental controls, a green fluorescent protein vector (pEGFPluc) was used (Clontech).Statistical Analysis—For continuous variables, means and S.E. were computed. Unpaired Student's t tests were used for comparing means between two experimental groups. Analysis of variance was used for multiple comparisons followed by Tukey's test for posthoc comparisons. Significance was set at p < 0.05. In each assay, n = 3, and each experiment was repeated at least three times.RESULTSForce-induced Recruitment of CLIP-170, α-Tubulin, and Filamin to Focal Adhesions—As force-induced increases of filamin A rely on transmission of force through focal adhesions to actin (7D'Addario M. Arora P. Fan J. Ganss B. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2001; 276: 31969-31977Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 9D'Addario M. Arora P. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2002; 277: 47541-47550Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), we determined whether the plus ends of growing microtubules are targeted to focal adhesions in response to force applied at these sites by collagen-coated magnetite beads. We examined CLIP-170, which is implicated in the targeting of the plus ends of growing microtubules to focal adhesions (27Krylyshkina O. Anderson K.I. Kaverina I. Upmann I. Manstein D.J. Small J.V. Toomre D.K. J. Cell Biol. 2003; 161: 853-859Crossref PubMed Scopus (135) Google Scholar, 28Gundersen G.G. Curr. Biol. 2002; 12: R645-R647Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The relative enrichment of CLIP-170 and α-tubulin was quantified after force application. Immunoblotting of proteins isolated from collagen beads was evaluated by densitometry and normalized to vinculin, an actin-binding protein that is a marker for focal adhesions. The relative amounts of bead-associated CLIP-170 and tubulin were increased substantially after force (>5-fold, p < 0.01; Fig. 1Ai). These data indicated that CLIP-170 and tubulin are recruited to focal adhesions by force, and they implicate microtubules in the global cytoskeletal response to tensile forces.We have shown previously that when force is applied to bovine serum albumin- or poly-l-lysine-coated magnetite beads there was no increase of filamin A transcription or enhanced filamin A protein production. In contrast, fibronectin-coated beads exhibited increased filamin RNA and protein by 4- and 3-fold, respectively, after force application (7D'Addario M. Arora P. Fan J. Ganss B. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2001; 276: 31969-31977Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The participation of β1 integrins was confirmed by preincubating fibroblasts with a monoclonal antibody (4B4) that blocks ligand-β1 integrin interactions without inhibiting cell attachment (22Arora P.D. McCulloch C.A.G. J. Biol. Chem. 1996; 271: 20516-20523Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Preincubation of cells with monoclonal antibody 4B4 (at 1:30 dilution) for 30 min followed by application of force blocked the force-induced production of filamin A protein (-fold increase of filamin A by immunoblotting and analysis by densitometry: no force controls with irrelevant antibody, 1.1 × 106 units; force-treated cells with irrelevant antibody, 4.4 × 106 units; force-treated cells with 4B4, 1.25 × 106 units). Further, in experiments utilizing bovine serum albumin- or poly-l-lysine-covered beads in which bead attachment was equivalent, there was no induction of filamin A protein following force application (no force-treated cells with collagen beads, 1.5 × 106 units; force-treated cells with bovine serum albumin-coated beads, 1.55 × 106 units; force-treated cells with poly-l-lysine-coated beads, 1.6 × 106 units).These results showed the involvement of β1 integrins but did not assess the potential participation of other integrins such as αvβ3 and did not assess the possible increase in bead-associated β1 integrin. Accordingly we isolated bead-associated proteins from untreated (no force) and force-treated fibroblasts and analyzed this material for β1 and αvβ3 integrins (Fig. 1Aii). We detected a 2-2.5-fold increase in the level of β1 integrin associated with collagen-coated magnetite beads following force application. We found very little detectable αvβ3 integrin with or without force application in bead-associated protein. Confirmation of the ability of the monoclonal antibody to detect αvβ3 integrin in these cells was confirmed by immunoblotting whole cell lysates (Fig. 1Aii, lane c).We assessed whether the collagen beads remained on the cell surface or were potentially internalized over time. Cells were incubated with magnetite beads coated with fluorescein isothiocyanate-labeled collagen for up to 4 h. Trypan blue quenching of the cell surface fluorescence showed that the difference in the pre- and postquench fluorescence (an estimate of the amount of cell surface fluorescein isothiocyanate-labeled collagen) at 30 min compared with 4 h of force was not significantly different (7.14 ± 1.66 fluorescence units at 30 min and 8.69 ± 2.31 fluorescence units at 4 h, p > 0.2). Therefore the collagen beads remained largely on the cell surface during force application.As tensile force is applied to cells through focal adhesions formed adjacent to the collagen beads, we assessed whether there was force-induced recruitment of filamin A protein to the bead complexes. Collagen bead-associated proteins were examined by immunoblotting and quantified by densitometry following adjustment for bead numbers. Application of tensile force caused a 3-fold increase of filamin A to the bead within 4 h (p < 0.01, Fig. 1B). Taxol treatment exerted no effect on force-induced recruitment of filamin A. Nocodazole strongly enhanced filamin A recruitment to beads independent of force application.The results described above demonstrate that force application recruits CLIP-170, α-tubulin, and filamin A to sites of force application, but they do not show whether α-tubulin or CLIP-170 is associated with actin/filamin A and whether this association is affected by force. Cell lysates were immunoprecipitated with antibodies to CLIP-170 or α-tubulin, and the immunoprecipitates were blotted for filamin A and β-actin (Fig. 1C). The specificity of the monoclonal antibody to CLIP-170 for immunoprecipitation has been described previously (29Akhmanova A. Hoogenraad C. Drabek K. Stepanova T. Verkerk T. Vermeulen W. Burgering B. De Zeeuw I. Grosveld F. Galjart N. Cell. 2001; 104: 923-935Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). CLIP-170 interacted with both filamin A and β-actin, while, as expected, α-tubulin interacted with only CLIP-170 (Fig. 1C). Force had no effect on the association of CLIP-170 with filamin A, but force increased the amount of β-actin co-precipitated by CLIP-170 by >3-fold, suggesting that interactions of actin filaments with CLIP-170 were increased during periods of applied force. As regulation of filamin A by force may be mediated by Sp1 and pp38 (9D'Addario M. Arora P. Ellen R. McCulloch C.A.G. J. Biol. Chem. 2002; 277: 47541-47550Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), we also immunoblotted immunoprecipitates of α-tubulin for Sp1, pp38, and p38 but found no detectable interactions.Effect of Microtubule Disruption—We assessed the involvement of microtubules in regulating the distribution and expression of filamin A after application of tensile forces through collagen receptors. Preliminary experiments with nocodazole (1 μm) and the microtubule stabilization agent paclitaxel (Taxol, 0.5 μm) were conducted, and cells were immunostained with antibodies to α-tubulin to verify that the microtubule network was depolymerized (nocodazole) or was stabilized (Taxol). When cells were incubated with these agents for 2-8 h (the duration of the force application experiments), microtubules were either poorly stained (after nocodazole) or were largely unaffected (Taxol, data not shown). Immunohistochemical examination of filamin A in force-treated cells showed increased overall staining intensity within 4 h of force application (Fig. 2). Pretreatment with nocodazole caused redistribution of filamin A to the cell periphery, an effect that was enhanced by force application. Taxol blocked force-induced redistribution of filamin A.Fig. 2Force application causes redistribution of filamin A. Cells were grown on glass slides, incubated with collagen-coated magnetite beads, and either untreated (A, C, and E) or subjected to a vertically directed force (B, D, and F) (0.48 piconewtons/μm2, 4 h) with a magnet. Cells were fixed with methanol, permeabilized with Triton X-100, and immunostained for filamin A. Force application increases total filamin A staining. Pretreatment with nocodazole (C and D) increases filamin staining in the cell periphery that is further enhanced by force application. There is little increase of s
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