Roles of Rho-associated Kinase and Myosin Light Chain Kinase in Morphological and Migratory Defects of Focal Adhesion Kinase-null Cells
2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês
10.1074/jbc.m204429200
ISSN1083-351X
AutoresBor-Huah Chen, Jason T. C. Tzen, Anne R. Bresnick, Hong‐Chen Chen,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoFibroblasts derived from focal adhesion kinase (FAK)-null mouse embryos have a reduced migration rate and an increase in the number and size of peripherally localized adhesions (Ilic, D., Furuta, Y., Kanazawa, S., Takeda, N., Sobue, K., Nakatsuji, N., Nomura, S., Fujimoto, J., Okada, M., and Yamamoto, T. (1995)Nature 377, 539–544). In this study, we have found that Y27632, a specific inhibitor for Rho-associated kinase (Rho-kinase), dramatically reversed the round cell morphology of FAK−/−cells to a spread fibroblast-like shape in 30 min and significantly enhanced their motility. The effects of Y27632 on the FAK−/− cell morphology and motility were concomitant with reorganization of the actin cytoskeleton and redistribution of focal adhesions. Conversely, the expression of the constitutively active Rho-kinase in FAK+/+ cells led to round cell shape and inhibition of cell motility. Furthermore, coincident with the formation of cortical actin filaments, myosin light chain (MLC), Ser-19-phosphorylated MLC, and MLC kinase mainly accumulated at the FAK−/− cell periphery. We found that the disruption of actin filaments by cytochalasin D prevented the peripheral accumulation of MLC kinase and that inhibition of myosin-mediated contractility by 2,3-butanedione monoxime induced FAK−/−cells to spread. Taken together, our results suggest that Rho-kinase may mediate the formation of cortical actomyosin filaments at the FAK−/− cell periphery, which further recruits MLC kinase to the cell periphery and generates a non-polar contractile force surrounding the cell, leading to cell rounding and decreased motility. Fibroblasts derived from focal adhesion kinase (FAK)-null mouse embryos have a reduced migration rate and an increase in the number and size of peripherally localized adhesions (Ilic, D., Furuta, Y., Kanazawa, S., Takeda, N., Sobue, K., Nakatsuji, N., Nomura, S., Fujimoto, J., Okada, M., and Yamamoto, T. (1995)Nature 377, 539–544). In this study, we have found that Y27632, a specific inhibitor for Rho-associated kinase (Rho-kinase), dramatically reversed the round cell morphology of FAK−/−cells to a spread fibroblast-like shape in 30 min and significantly enhanced their motility. The effects of Y27632 on the FAK−/− cell morphology and motility were concomitant with reorganization of the actin cytoskeleton and redistribution of focal adhesions. Conversely, the expression of the constitutively active Rho-kinase in FAK+/+ cells led to round cell shape and inhibition of cell motility. Furthermore, coincident with the formation of cortical actin filaments, myosin light chain (MLC), Ser-19-phosphorylated MLC, and MLC kinase mainly accumulated at the FAK−/− cell periphery. We found that the disruption of actin filaments by cytochalasin D prevented the peripheral accumulation of MLC kinase and that inhibition of myosin-mediated contractility by 2,3-butanedione monoxime induced FAK−/−cells to spread. Taken together, our results suggest that Rho-kinase may mediate the formation of cortical actomyosin filaments at the FAK−/− cell periphery, which further recruits MLC kinase to the cell periphery and generates a non-polar contractile force surrounding the cell, leading to cell rounding and decreased motility. Rho-associated kinase focal adhesion kinase FAK-null cells myosin light chain MLC kinase myelin basic protein 2,3-butanedione monoxime differential interference contrast tetramethylrhodamine isothiocyanate fluorescein isothiocyanate green fluorescent protein. Rho GTPases including Rho, Rac, and Cdc42 are key modulators of the actin cytoskeleton (1Takai Y. Sasaki T. Tanaka K. Nakanishi H. Trends Biochem. Sci. 1995; 20: 227-231Abstract Full Text PDF PubMed Scopus (367) Google Scholar, 2Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar, 3Kaibuchi K. Kuroda S. Amano M. Annu. Rev. Biochem. 1999; 68: 459-486Crossref PubMed Scopus (891) Google Scholar). They are critical for the cell shape changes and adhesion dynamics that drive cell migration (4Nobes C.D. Hall A. J. Cell Biol. 1999; 144: 1235-1244Crossref PubMed Scopus (1213) Google Scholar, 5Clark E.A. King W.G. Brugge J.S. Symons M. Hynes R.O. J. Cell Biol. 1998; 142: 573-586Crossref PubMed Scopus (535) Google Scholar, 6Wojciak-Stothard B. Entwistle A. Garg R. Ridley A.J. J. Cell. Physiol. 1998; 176: 150-165Crossref PubMed Scopus (355) Google Scholar, 7Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3747) Google Scholar). Among the Rho GTPase family, Rho induces the formation of focal adhesions and stress fibers (7Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3747) Google Scholar, 8Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3843) Google Scholar). Interestingly, although a basal level of Rho activity is needed for fibroblast migration, too much Rho activity impedes migration (4Nobes C.D. Hall A. J. Cell Biol. 1999; 144: 1235-1244Crossref PubMed Scopus (1213) Google Scholar, 9Takaishi K. Sasaki T. Kato M. Yamochi W. Kuroda S. Nakamura T. Takeichi M. Takai Y. Oncogene. 1994; 9: 273-279PubMed Google Scholar, 10Ridley A.J. Comoglio P.M. Hall A. Mol. Cell. Biol. 1995; 15: 1110-1122Crossref PubMed Google Scholar). It has been shown that the concerted action of two of the immediate Rho targets, Rho-associated kinase (Rho-kinase)1/ROCK and the formin homology protein mDia1, mediate the effect of Rho on matrix adhesion and the actin cytoskeleton (11Watanabe N. Kato T. Fujita A. Ishizaki T. Narumiya S. Nat. Cell Biol. 1999; 1: 136-143Crossref PubMed Scopus (727) Google Scholar). In particular, Rho-kinase was shown to stimulate myosin-driven contractility in smooth muscle and nonmuscle cells by phosphorylating, thereby inactivating myosin light chain (MLC) phosphatase (12Kimura K. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 273: 245-248Crossref PubMed Scopus (2444) Google Scholar, 13Kawano Y. Fukata Y. Oshiro N. Amano M. Nakanura T. Ito M. Matsumura F. Inagaki M. Kaibuchi K. J. Cell Biol. 1999; 147: 1023-1038Crossref PubMed Scopus (477) Google Scholar), and possibly by direct phosphorylation of MLC (14Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1685) Google Scholar, 15Kureishi Y. Kobayashi S. Amano M. Kimura K. Kanaide H. Nakano T. Kaibuchi K. Ito M. J. Biol. Chem. 1997; 272: 12257-12260Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, 16Totsukawa G. Yamakita Y. Yamashiro S. Hartshorne D.J. Sasaki Y. Matsumura F. J. Cell Biol. 2000; 150: 797-806Crossref PubMed Scopus (542) Google Scholar). In addition to Rho-kinase, MLC kinase (MLCK) is another kinase that phosphorylates the MLC in both smooth muscle and nonmuscle cells (17Kamm K.E. Stull J.T. Annu. Rev. Pharmacol. Toxicol. 1985; 25: 593-620Crossref PubMed Google Scholar, 18Kamm K.E. Stull J.T. Annu. Rev. Physiol. 1989; 51: 299-313Crossref PubMed Scopus (280) Google Scholar). The phosphorylation of MLC on Ser-19 and to a lesser extent on Thr-18 by MLCK promotes the assembly of myosin II into filaments and activates its ATPase activity, which stabilizes the actin-myosin interaction and promotes cell contractility (19Sellers J.R. Pato M.D. Adelstein R.S. J. Biol. Chem. 1981; 256: 13137-13142Abstract Full Text PDF PubMed Google Scholar, 20Sellers J.R. Spudich J.A. Sheetz M.P. J. Cell Biol. 1985; 101: 1897-1902Crossref PubMed Scopus (82) Google Scholar, 21Sellers J.R. Curr. Opin. Cell Biol. 1991; 3: 98-104Crossref PubMed Scopus (173) Google Scholar, 22Bresnick A.R. Curr. Opin. Cell Biol. 1999; 11: 26-33Crossref PubMed Scopus (315) Google Scholar). Recently, Rho-kinase and MLCK were suggested to play distinct roles in spatial regulation of MLC phosphorylation. Rho-kinase appears to be important for MLC phosphorylation in the center of cells, and MLCK is responsible for phosphorylating MLC at the cell periphery (16Totsukawa G. Yamakita Y. Yamashiro S. Hartshorne D.J. Sasaki Y. Matsumura F. J. Cell Biol. 2000; 150: 797-806Crossref PubMed Scopus (542) Google Scholar). Focal adhesion kinase (FAK), a 125-kDa cytoplasmic tyrosine kinase localized in focal contacts, has been known to play an important role in integrin-mediated cell migration (23Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1591) Google Scholar). Fibroblasts derived from FAK-null mouse embryos are more rounded and poorly spread than their wild-type counterparts (23Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1591) Google Scholar). They show an overabundance of focal adhesions, enriched cortical actin filaments at the cell periphery, and a decreased migration rate (23Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1591) Google Scholar, 24Ren X.D. Kiosses W.B. Sieg D.J. Otey C.A. Schlaepfer D.D. Schwartz M.A. J. Cell Sci. 2000; 113: 3673-3678Crossref PubMed Google Scholar). It has been suggested that the increase in peripheral adhesions results from an inhibition of turnover in FAK−/− cells (23Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1591) Google Scholar), which may result from constitutive activation of Rho (24Ren X.D. Kiosses W.B. Sieg D.J. Otey C.A. Schlaepfer D.D. Schwartz M.A. J. Cell Sci. 2000; 113: 3673-3678Crossref PubMed Google Scholar). Because of the known involvement of Rho-kinase and MLCK in cell contractility, a major factor controlling cell migration, we hypothesize that abnormal regulation of Rho-kinase and MLCK may underlie the migratory defect of FAK−/−cells. Fetal bovine serum, non-essential amino acids, sodium pyruvate, and 2-mercaptoethanol were purchased from Invitrogen. Y27632, a specific inhibitor of Rho-kinase, was purchased fromCalbiochem. The monoclonal anti-MLCK, monoclonal anti-MLC, monoclonal anti-β-tubulin, bovine MLC, myelin basic protein (MBP), cytochalasin D, and 2,3-butanedione monoxime (BDM) were purchased from Sigma-Aldrich. The polyclonal anti-Rho-kinase and monoclonal anti-paxillin were purchased from Transduction Laboratories (Lexington, KY). The plasmid pEGFP-N1-MLCK and polyclonal anti-MLCK were described previously (25Poperechnaya A. Varlamova O. Lin P.-J. Stull J.T. Bresnick A.R. J. Cell Biol. 2000; 151: 697-707Crossref PubMed Scopus (89) Google Scholar). The plasmid pEF-Bos-myc-CA Rho-kinase was kindly provided by Dr. K. Kaibuchi (26Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (943) Google Scholar). Monoclonal anti-Ser-19-phosphorylated MLC was kindly provided by Dr. M. Ikebe (27Komatsu S. Yano T. Shibata M. Tuft R.A. Ikebe M. J. Biol. Chem. 2000; 275: 34512-34520Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). FAK+/+ and FAK−/− cells were kindly provided by Dr. D. Ilic (University of California, San Francisco, CA) and described previously (23Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1591) Google Scholar). These cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 100 μm non-essential amino acids, 1 mm sodium pyruvate, and 55 μm 2-mercaptoethanol and cultured at 37 °C in a humidified atmosphere of 5% CO2 and 95% air atmosphere. Transient transfections were performed using LipofectAMINE Plus (Invitrogen) according to the manufacturer's instructions. FAK−/− cells were grown on glass coverslips with 0.17 mm in thickness and 42 mm in diameter. The monolayer of cells was wounded by manual scratching with a pipette tip and then treated with or without 10 μm Y27632. Cells on the microscope stage were maintained at 37 °C with a humid CO2 atmosphere in a microcultivation system (model POC-R, Zeiss) with temperature and CO2 control devices (Tempcontrol 37-2 digital and CTI controller 3700 digital, Zeiss). Cells were monitored under differential interference contrast (DIC) optics on an inverted Zeiss microscope (Axiovert 100) using Zeiss 40X LD-Apochromat objective. Time-lapse sequential micrographs were captured per minute using a cooled CCD camera (CoolSNAP fx, Roper Scientific, Inc) and analyzed by Meta Imaging SeriesTM software (version 4.5) from Universal Imaging Corporation (West Chester, PA). Cells were plated on 13-mm glass coverslips for 24 h, washed three times with phosphate-buffered saline, fixed for 10 min in phosphate-buffered saline containing 4% paraformaldehyde, and permeabilized in phosphate-buffered saline containing 0.2% Triton X-100 for 10 min. Coverslips were stained with primary antibodies for 60 min and followed by goat anti-mouse TRITC or fluorescein isothiocyanate (FITC) conjugated secondary antibodies (Jackson ImmunoReseach laboratories) at 4 μg/ml for 60 min. All antibodies used in immunofluorescence staining in this report are monoclonal including anti-paxillin (1:100), anti-MLCK (1:50), anti-MLC (1:100), anti-Ser-19-phosphorylated MLC (1:100), and anti-β-tubulin (1:100). TRITC-conjugated phalloidin (Sigma-Aldrich) at 2 μm was used to stain actin filaments. Coverslips were mounted in anti-fading solution and viewed using a Zeiss LSM-510 laser-scanning confocal microscope image system with a Zeiss 100X Plan-Apochromat objective (NA 1.4 oil). Cells were lysed with 1% Nonidet P-40 lysis buffer (1% Nonidet P-40, 20 mm Tris-HCl, pH 8.0, 137 mmNaCl, 10% glycerol, and 1 mmNa3VO4) containing protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 0.2 trypsin inhibitory units/ml aprotinin, and 20 μg/ml leupeptin). The lysates were centrifuged for 10 min at 4 °C to remove debris, and the protein concentrations were determined using the Bio-Rad protein assay. The aliquots of lysates were subjected to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose (Schleicher & Schuell). Immunoblotting was performed with polyclonal anti-MLCK (1:1000), polyclonal anti-Rho-kinase (1:1000), or monoclonal anti-Myc (1:1000) using the Amersham Biosciences enhanced chemiluminescence system for detection. For MLCK activity assays, cell lysates were incubated with 2.5 μl of monoclonal anti-MLCK for 1.5 h at 4 °C. Immunocomplexes were collected on protein A-Sepharose beads coupled with rabbit anti-mouse IgG. The beads were washed three times with 1% Nonidet P-40 lysis buffer and one time with 20 mm Tris, pH 7.4. Kinase reactions were carried out in 40 μl of kinase buffer (20 mm Tris, pH 7.4, 10 mm MgCl2, 10 mm CaCl2, 2 mm dithiothreitol, and 0.1 μm calmodulin) containing 10 μCi of [γ-32P]ATP and 10 μg of bovine MLC or MBP for 20 min at 25 °C. For Rho-kinase activity assays, cell lysates were incubated with 5 μl of polyclonal anti-Rho-kinase for 1 h at 4 °C. The immunocomplexes were washed and subjected to kinase reaction in 40 μl of kinase buffer (50 mm Tris, pH 7.4, 10 mm MgCl2, 3 mmNaCl2, 1 mm dithiothreitol, and 1 mm EDTA) in the presence of 10 μCi of [γ-32P]ATP and 10 μg of MBP. Reactions were terminated by the addition of SDS sample buffer, and proteins were resolved by SDS-polyacrylamide gel electrophoresis. Statistical analyses were performed with a Student's t test. Differences were considered to be statistically significant at p < 0.05. The constitutive activation of Rho has been found to correlate with the inhibition of focal adhesion turnover in FAK−/−cells (24Ren X.D. Kiosses W.B. Sieg D.J. Otey C.A. Schlaepfer D.D. Schwartz M.A. J. Cell Sci. 2000; 113: 3673-3678Crossref PubMed Google Scholar). Accordingly, we found that the activity of Rho-kinase, an immediate downstream target of Rho, in FAK−/− cells was ∼30% higher than that in FAK+/+ cells (Fig.1). To examine whether Rho-kinase mediates the effect of constitutively active Rho on the morphology of FAK−/− cells, a specific inhibitor for Rho-kinase, Y27632, was employed (28Uehata M. Ishizaki T. Satoh H. Ono T. Kawahara T. Morishita T. Tamakawa H. Yamagami K. Inui J. Maekawa M. Narumiya S. Nature. 1997; 389: 990-994Crossref PubMed Scopus (2555) Google Scholar). This pharmacological reagent was reported to be specific for Rho-kinase at 10 μm (29Ishizaki T. Uehata M. Tamechika I. Keel J. Nonomura K. Maekawa M. Narumiya S. Mol. Parmacol. 2000; 57: 976-983PubMed Google Scholar). Similar to other reports in the literature (16Totsukawa G. Yamakita Y. Yamashiro S. Hartshorne D.J. Sasaki Y. Matsumura F. J. Cell Biol. 2000; 150: 797-806Crossref PubMed Scopus (542) Google Scholar, 30Maekawa M. Ishizaki T. Boku S. Watanabe N. Fujita A. Iwamatsu A. Obinata T. Ohashi K. Mizuno K. Narumiya S. Science. 1999; 285: 895-898Crossref PubMed Scopus (1296) Google Scholar), Y27632 at this concentration reduced the formation of actin stress fibers in FAK+/+cells, which became more flattened and unable to move (data not shown), supporting an essential role of Rho-kinase in stress fiber formation and cell motility. For FAK−/− cells, Y27632 promptly altered their rounded morphology to a spread fibroblast-like shape in 30 min (Fig. 2A) and significantly enhanced their motility (Fig.3). Four hours after the removal of Y27632 from the medium, the cells became less spread and finally rounded (Fig. 2B) accompanied with the recovery of Rho-kinase activity (data not shown). Importantly, the spreading of FAK−/− cells induced by Y27632 was concomitant with marked reorganization of the actin cytoskeleton from cortical actin bundles into long parallel filaments similar to those seen in polar migratory fibroblasts (Fig. 2C). In addition, paxillin, a protein localized in focal adhesions, was found from peripheral patchlike to scattered dotlike distribution upon the addition of Y27632 (Fig. 2C). These results suggest that in FAK−/− cells, the constitutive activation of Rho-kinase may be involved in the formation of cortical actin structures, abundance of peripheral adhesions, and round cell shape.Figure 2The specific Rho-kinase inhibitor Y27632 induces FAK−/−cells to spread accompanied by reorganization of actin cytoskeleton and redistribution of focal adhesions.A, FAK−/− cells were sparsely grown on glass overnight and then treated with or without 10 μmY27632. 30 min later, micrographs were taken by a cooled CCD under a differential interference contrast (DIC) microscope. Bar, 30 μm B, FAK−/− cells were treated with 10 μm Y27632 for 30 min to induce their spreading. The medium was then changed by fresh medium. After 4 h, micrographs were taken. The two micrographs in B represent the morphological change of the same cell. Bar, 10 μm.C, FAK−/− cells were treated with or without 10 μm Y27632 for 30 min and then stained for actin with fluorescein isothiocyanate (FITC)-phalloidin and for paxillin with monoclonal anti-paxillin. Bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Y27632 enhances the motility of FAK−/−cells. FAK−/− cells were grown into monolayer on glass. The monolayer of cells was wounded by manual scratching with a pipette tip and then treated with or without 10 μm Y27632. The time-lapse micrographs were taken every 1 min for 4 h to record the healing process. The representative micrographs at 0, 2, and 4 h are shown. An average migratory speed of 10 cells at the front was measured by Meta Imaging Series software, version 4.5. Values (mean ± S.E.) are from three independent experiments. Bar, 30 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It is worth noting that the Y27632-treated FAK−/− cells exhibited not only spread cell shape but also characteristics of motile cells including membrane ruffles and the formation of filopodia and lamellipodia (Figs. 2 and 3). To examine the effect of Y27632 on the motility of FAK−/− cells, wound healing assays were performed and monitored by time-lapse microscopy. We found that the motility of FAK−/− cells was indeed enhanced by Y27632 at 10 μm. Using an image analysis software, the motility of FAK−/− cells was measured at an average speed of 5 μm h−1 in the absence of Y27632 and an average speed of 20 μm h−1 in the presence of Y27632 (Fig. 3). These results suggest that constitutive activation of Rho-kinase may account for both morphological and migratory defects of FAK−/− cells. To further confirm this finding, an active truncated form of Rho-kinase with a deletion at its COOH terminus (25Poperechnaya A. Varlamova O. Lin P.-J. Stull J.T. Bresnick A.R. J. Cell Biol. 2000; 151: 697-707Crossref PubMed Scopus (89) Google Scholar, 31Ishizaki T. Naito M. Fujisawa K. Maekawa M. Watanabe N. Saito Y. Narumiya S. FEBS Lett. 1997; 404: 118-124Crossref PubMed Scopus (457) Google Scholar) was transiently expressed in FAK+/+ cells. Within 6 h after transfection, the expression of the constitutively active Rho-kinase in FAK+/+ cells led to cell rounding and inhibition of movement (Fig. 4). However, prolonged expression (>12 h) of the constitutively active Rho-kinase in FAK+/+ cells led to membrane blebbing and apoptosis (data not shown) as shown in other cell types (32Coleman M.L. Sahai E.A. Yeo M. Bosch M. Dewar A. Olson M.F. Nat. Cell Biol. 2001; 3: 339-345Crossref PubMed Scopus (985) Google Scholar, 33Sebbagh M. Renvoize C. Hamelin J. Riche N. Bertoglio J. Breard J. Nat. Cell Biol. 2001; 3: 346-352Crossref PubMed Scopus (711) Google Scholar). Although the detailed mechanism by which constitutive activation of Rho-kinase induces prominent formation of cortical actin rings at the FAK−/− cell periphery is currently unknown, these cortical actin bundles may assemble with myosin II to generate a non-polar contractile force surrounding the cell, leading to a rounded cell shape and a deficiency in cell motility. Because the phosphorylation of regulatory light chain of myosin II was known to be critical for controlling actomyosin contractility in smooth muscle and nonmuscle cells (17Kamm K.E. Stull J.T. Annu. Rev. Pharmacol. Toxicol. 1985; 25: 593-620Crossref PubMed Google Scholar), we examined the subcellular localization of MLC and its Ser-19-phosphorylated form in both FAK+/+ cells and FAK−/− cells by immunofluorescence staining (Fig.5). Using a monoclonal antibody specific for Ser-19-phosphorylated MLC (27Komatsu S. Yano T. Shibata M. Tuft R.A. Ikebe M. J. Biol. Chem. 2000; 275: 34512-34520Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), we were able to detect the active phosphorylated form of myosin II in these cells. In FAK+/+cells, MLC and phosphorylated MLC were organized into long parallel filaments with a similar distribution as actin stress fibers. In FAK−/− cells, they were mainly accumulated at the cell periphery, suggesting that active actomyosin structures are assembled at the FAK−/− cell periphery. To further examine the possibility that the actomyosin-driven contractility is responsible for round cell shape of FAK−/− cells, an inhibitor of myosin ATPase activity, 2,3-butanedione monoxime (BDM), was used to inhibit myosin-driven contractility. Indeed, BDM at 30 mm induced a morphological transition of FAK−/− cells from a round to a spread cell shape (Fig. 6A), supporting the idea that the myosin-driven contractility surrounding the FAK−/− cell is responsible for the round cell shape. However, it should be noted that although BDM-treated FAK−/− cells became spread, they did not exhibit characteristics of motile cells. Judging by time-lapse microscopy, these BDM-treated FAK−/− cells were unable to move and divide (data not shown). In addition, although BDM-treated FAK−/− cells retained their ability to form focal adhesions (indicated by paxillin localization), they had no stress fiber-like actin filaments in the center of the cells (Fig.6B).Figure 6Effect of BDM , an inhibitor of myosin contractility, on the morphology of FAK−/−cells.FAK−/− cells were treated with or without 30 mm BDM. 12 h later, the cells were visualized under a DIC microscope (A) and co-stained for actin and paxillin (B). Bar, 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Rho-kinase and MLCK have been suggested to play distinct roles in spatial regulation of MLC phosphorylation (16Totsukawa G. Yamakita Y. Yamashiro S. Hartshorne D.J. Sasaki Y. Matsumura F. J. Cell Biol. 2000; 150: 797-806Crossref PubMed Scopus (542) Google Scholar). To investigate which kinase is responsible for phosphorylating MLC at the FAK−/− cell periphery, the subcellular localizations of Rho-kinase and MLCK were examined. Unfortunately, because of the failure of the anti-Rho-kinase antibody in immunofluorescence staining, we were unable to determine the localization of Rho-kinase in these cells. However, the localization of MLCK in FAK−/− cells was unusual, which was strongly accumulated at the cell periphery instead of a diffuse distribution as seen in FAK+/+ cells (Fig. 7A). To further confirm this phenomenon, GFP-MLCK was transiently expressed in FAK+/+ and FAK−/− cells, and the fluorescence of GFP-MLCK was visualized in living cells (Fig. 7A). Similar to endogenous MLCK in FAK−/− cells, GFP-MLCK was mainly accumulated at the cell periphery. In FAK+/+ cells, GFP-MLCK distributed both at cell periphery and the center of the cell where it assembled into long filaments. The expression and activity of MLCK was next compared between FAK+/+ and FAK−/− cells (Fig. 7B). Surprisingly, the amount of MLCK in FAK−/− cells was ∼3-fold of that in FAK+/+ cells, which was approximately correlated with the difference in MLCK activity between these two cell lines. Thus, although we cannot exclude the role of Rho-kinase in MLC phosphorylation at the FAK−/− cell periphery, the peripheral localization and high expression of MLCK in these cells render it more likely to be involved in this event. MLCK has been reported to directly interact with actin filaments (34Lin P. Luby-Phelps K. Stull J.T. J. Biol. Chem. 1997; 272: 7412-7420Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). To examine whether the formation of the cortical actin cytoskeleton is required for peripheral accumulation of MLCK, FAK−/−cells were treated with 2.5 μm cytochalasin D for 1 h to disrupt actin cytoskeleton and then co-stained for actin and MLCK (Fig. 8A). In the control experiments, GFP-MLCK was expressed in FAK−/− cells, and its peripheral localization was visualized after treatment of 30 μg/ml nocodazole, which disrupts microtubules (Fig. 8B). Our results clearly demonstrated that the disruption of cortical actin cytoskeleton but not microtubule prevented the peripheral accumulation of MLCK in FAK−/− cells, suggesting that the formation of cortical actin bundles is essential for MLCK accumulation at the cell periphery. In addition to genetic approaches with dominant negative or positive mutants of Rho-kinase, a pharmacological approach using the Rho-kinase inhibitor Y27632 has frequently been used to examine the role of Rho-kinase in cellular functions. So far, Rho-kinase has been shown to be involved in the formation of stress fibers and focal adhesions (35Leung T. Chen X.Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar, 36Amano M. Chihara K. Kimura K. Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (951) Google Scholar) and in various contractile processes including cell motility (37Fukata Y. Oshiro N. Kinoshita N. Kawano Y. Matsuoka Y. Bennett N. Matsurra Y. Kaibuchi K. J. Cell Biol. 1999; 145: 347-361Crossref PubMed Scopus (256) Google Scholar), smooth muscle contraction (12Kimura K. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 273: 245-248Crossref PubMed Scopus (2444) Google Scholar, 14Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. J. Biol. 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Unexpectedly, we found in this study that Y27632 has a positive impact on FAK−/− cells that promptly induces them to spread and facilitates their motility (Figs. 2A and3). Tog
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