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Distinct temporal-spatial roles for rho kinase and myosin light chain kinase in epithelial purse-string wound closure

2005; Elsevier BV; Volume: 128; Issue: 4 Linguagem: Inglês

10.1053/j.gastro.2005.01.004

1528-0012

John M. Russo, Peter Florian, Le Shen, W. Vallen Graham, Maria Tretiakova, Alfred H. Gitter, Randall J. Mrsny, Jerrold R. Turner,

Cell Adhesion Molecules Research

Background & Aims: Small epithelial wounds heal by purse-string contraction of an actomyosin ring that is regulated by myosin light chain (MLC) kinase (MLCK) and rho kinase (ROCK). These studies aimed to define the roles of these kinases in purse-string wound closure. Methods: Oligocellular and single-cell wounds were created in intestinal epithelial monolayers. Fluorescence imaging and electrophysiologic data were collected during wound closure. Human biopsies were studied immunohistochemically. Results: Live-cell imaging of enhanced green fluorescent protein-β-actin defined rapid actin ring assembly within 2 minutes after wounding. This progressed to a circumferential ring within 8 minutes that subsequently contracted and closed the wound. We therefore divided this process into 2 phases: ring assembly and wound contraction. Activated rho and ROCK localized to the wound edge during ring assembly. Consistent with a primary role in the assembly phase, ROCK inhibition prevented actin ring assembly and wound closure. ROCK inhibition after ring assembly was complete had no effect. Recruitment and activation of MLCK occurred after ring assembly was complete and coincided with ring contraction. MLCK inhibition slowed and then stopped contraction but did not prevent ring assembly. MLCK inhibition also delayed barrier function recovery. Studies of human colonic biopsy specimens suggest that purse-string wound closure also occurs in vivo, because MLC phosphorylation was enhanced surrounding oligocellular wounds. Conclusions: These results suggest complementary roles for these kinases in purse-string closure of experimental and in vivo oligocellular epithelial wounds; rho and ROCK are critical for actin ring assembly, while the activity of MLCK drives contraction. Background & Aims: Small epithelial wounds heal by purse-string contraction of an actomyosin ring that is regulated by myosin light chain (MLC) kinase (MLCK) and rho kinase (ROCK). These studies aimed to define the roles of these kinases in purse-string wound closure. Methods: Oligocellular and single-cell wounds were created in intestinal epithelial monolayers. Fluorescence imaging and electrophysiologic data were collected during wound closure. Human biopsies were studied immunohistochemically. Results: Live-cell imaging of enhanced green fluorescent protein-β-actin defined rapid actin ring assembly within 2 minutes after wounding. This progressed to a circumferential ring within 8 minutes that subsequently contracted and closed the wound. We therefore divided this process into 2 phases: ring assembly and wound contraction. Activated rho and ROCK localized to the wound edge during ring assembly. Consistent with a primary role in the assembly phase, ROCK inhibition prevented actin ring assembly and wound closure. ROCK inhibition after ring assembly was complete had no effect. Recruitment and activation of MLCK occurred after ring assembly was complete and coincided with ring contraction. MLCK inhibition slowed and then stopped contraction but did not prevent ring assembly. MLCK inhibition also delayed barrier function recovery. Studies of human colonic biopsy specimens suggest that purse-string wound closure also occurs in vivo, because MLC phosphorylation was enhanced surrounding oligocellular wounds. Conclusions: These results suggest complementary roles for these kinases in purse-string closure of experimental and in vivo oligocellular epithelial wounds; rho and ROCK are critical for actin ring assembly, while the activity of MLCK drives contraction. Like all epithelia, a primary function of the gastrointestinal epithelium is to provide a barrier to the noxious luminal milieu. This barrier must be maintained despite local epithelial damage that occurs regularly as a result of minor trauma, inflammation, and the physiologic shedding of apoptotic cells.1Madara J.L. Maintenance of the macromolecular barrier at cell extrusion sites in intestinal epithelium physiological rearrangement of tight junctions.J Membr Biol. 1990; 116: 177-184Crossref PubMed Scopus (206) Google Scholar, 2Abreu M.T. Palladino A.A. Arnold E.T. Kwon R.S. McRoberts J.A. Modulation of barrier function during Fas-mediated apoptosis in human intestinal epithelial cells.Gastroenterology. 2000; 119: 1524-1536Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 3Gitter A.H. Bendfeldt K. Schulzke J.D. Fromm M. Leaks in the epithelial barrier caused by spontaneous and TNF-alpha-induced single-cell apoptosis.FASEB J. 2000; 14: 1749-1753Crossref PubMed Scopus (232) Google Scholar, 4Rosenblatt J. Raff M.C. Cramer L.P. An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism.Curr Biol. 2001; 11: 1847-1857Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 5Bruewer M. Luegering A. Kucharzik T. Parkos C.A. Madara J.L. Hopkins A.M. Nusrat A. Proinflammatory cytokines disrupt epithelial barrier function by apoptosis-independent mechanisms.J Immunol. 2003; 171: 6164-6172Crossref PubMed Scopus (733) Google Scholar Thus, rapid and efficient closure of these wounds and restoration of barrier integrity are critical to normal function and prevention of disease. Epithelial wound closure occurs by 2 distinct mechanisms: lamellipodia-dependent cell migration and coordinated purse-string contraction.6Nusrat A. Delp C. Madara J.L. Intestinal epithelial restitution. Characterization of a cell culture model and mapping of cytoskeletal elements in migrating cells.J Clin Invest. 1992; 89: 1501-1511Crossref PubMed Scopus (209) Google Scholar, 7Bement W.M. Forscher P. Mooseker M.S. A novel cytoskeletal structure involved in purse string wound closure and cell polarity maintenance.J Cell Biol. 1993; 121: 565-578Crossref PubMed Scopus (274) Google Scholar Larger wounds tend to heal by extension of lamellipodia from cells surrounding the wound followed by the migration of these cells to close the wound.6Nusrat A. Delp C. Madara J.L. Intestinal epithelial restitution. Characterization of a cell culture model and mapping of cytoskeletal elements in migrating cells.J Clin Invest. 1992; 89: 1501-1511Crossref PubMed Scopus (209) Google Scholar, 8Fenteany G. Janmey P.A. Stossel T.P. Signaling pathways and cell mechanics involved in wound closure by epithelial cell sheets.Curr Biol. 2000; 10: 831-838Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar In contrast, small epithelial wounds, defined operationally as those <10 cells in diameter, close by purse-string contraction of a ring of actin filaments that form a continuous arc encircling the wound edge.4Rosenblatt J. Raff M.C. Cramer L.P. An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism.Curr Biol. 2001; 11: 1847-1857Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 7Bement W.M. Forscher P. Mooseker M.S. A novel cytoskeletal structure involved in purse string wound closure and cell polarity maintenance.J Cell Biol. 1993; 121: 565-578Crossref PubMed Scopus (274) Google Scholar, 9Florian P. Schoneberg T. Schulzke J.D. Fromm M. Gitter A.H. Single-cell epithelial defects close rapidly by an actinomyosin purse string mechanism with functional tight junctions.J Physiol. 2002; 545: 485-499Crossref PubMed Scopus (70) Google Scholar The force of this contraction is transmitted across cells via adherens junctions,10Danjo Y. Gipson I.K. Actin 'purse string' filaments are anchored by E-cadherin-mediated adherens junctions at the leading edge of the epithelial wound, providing coordinated cell movement.J Cell Sci. 1998; 111: 3323-3332Crossref PubMed Google Scholar resulting in rapid restoration of barrier function.9Florian P. Schoneberg T. Schulzke J.D. Fromm M. Gitter A.H. Single-cell epithelial defects close rapidly by an actinomyosin purse string mechanism with functional tight junctions.J Physiol. 2002; 545: 485-499Crossref PubMed Scopus (70) Google Scholar This mechanism of wound closure is highly conserved developmentally and evolutionarily and has been demonstrated in embryonic chick wing buds11Martin P. Lewis J. Actin cables and epidermal movement in embryonic wound healing.Nature. 1992; 360: 179-183Crossref PubMed Scopus (374) Google Scholar and closure of plasma membrane wounds within single Xenopus oocytes.12Bement W.M. Mandato C.A. Kirsch M.N. Wound-induced assembly and closure of an actomyosin purse string in Xenopus oocytes.Curr Biol. 1999; 9: 579-587Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar A primary regulator of actomyosin contraction is phosphorylation of myosin light chain (MLC). Phosphorylation is typically accomplished by myosin light chain kinase (MLCK), which phosphorylates MLC at serine 19, thereby activating actomyosin contraction. Although recent studies have identified additional kinases that can phosphorylate MLC,13Chew T.L. Masaracchia R.A. Goeckeler Z.M. Wysolmerski R.B. Phosphorylation of non-muscle myosin II regulatory light chain by p21-activated kinase (gamma-PAK).J Muscle Res Cell Motil. 1998; 19: 839-854Crossref PubMed Scopus (172) Google Scholar, 14Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase).J Biol Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1711) Google Scholar, 15Jin Y. Blue E.K. Dixon S. Hou L. Wysolmerski R.B. Gallagher P.J. Identification of a new form of death-associated protein kinase that promotes cell survival.J Biol Chem. 2001; 276: 39667-39678Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 16Komatsu S. Ikebe M. ZIP kinase is responsible for the phosphorylation of myosin II and necessary for cell motility in mammalian fibroblasts.J Cell Biol. 2004; 165: 243-254Crossref PubMed Scopus (77) Google Scholar, 17Yamashiro S. Totsukawa G. Yamakita Y. Sasaki Y. Madaule P. Ishizaki T. Narumiya S. Matsumura F. Citron kinase, a Rho-dependent kinase, induces di-phosphorylation of regulatory light chain of myosin II.Mol Biol Cell. 2003; 14: 1745-1756Crossref PubMed Scopus (166) Google Scholar MLCK is the primary kinase responsible for MLC phosphorylation in intestinal epithelial cells.18Turner J.R. Angle J.M. Black E.D. Joyal J.L. Sacks D.B. Madara J.L. Protein kinase C-dependent regulation of transepithelial resistance the roles of myosin light chain and myosin light chain kinase.Am J Physiol. 1999; 277: C554-C562Crossref PubMed Google Scholar, 19Clayburgh D.R. Rosen S. Witkowski E.D. Wang F. Blair S. Dudek S. Garcia J.G.N. Alverdy J.C. Turner J.R. A differentiation-dependent splice variant of myosin light chain kinase, MLCK1, regulates epithelial tight junction permeability.J Biol Chem. 2004; 279: 55506-55513Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar Rho kinase (ROCK) can also regulate MLC phosphorylation, either by direct MLC phosphorylation14Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase).J Biol Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1711) Google Scholar or by inhibition of MLC phosphatase leading to increased MLC phosphorylation.20Kimura K. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase).Science. 1996; 273: 245-248Crossref PubMed Scopus (2476) Google Scholar, 21Ito M. Nakano T. Erdodi F. Hartshorne D.J. Myosin phosphatase structure, regulation and function.Mol Cell Biochem. 2004; 259: 197-209Crossref PubMed Scopus (386) Google Scholar Thus, although it is reasonable to hypothesize that MLC phosphorylation drives actomyosin contraction in epithelial purse-string wound closure, available data from disparate systems provide conflicting descriptions of the roles of rho, ROCK, MLCK, and MLC during this process.4Rosenblatt J. Raff M.C. Cramer L.P. An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism.Curr Biol. 2001; 11: 1847-1857Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 9Florian P. Schoneberg T. Schulzke J.D. Fromm M. Gitter A.H. Single-cell epithelial defects close rapidly by an actinomyosin purse string mechanism with functional tight junctions.J Physiol. 2002; 545: 485-499Crossref PubMed Scopus (70) Google Scholar, 12Bement W.M. Mandato C.A. Kirsch M.N. Wound-induced assembly and closure of an actomyosin purse string in Xenopus oocytes.Curr Biol. 1999; 9: 579-587Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 14Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase).J Biol Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1711) Google Scholar, 22Matsumura F. Ono S. Yamakita Y. Totsukawa G. Yamashiro S. Specific localization of serine 19 phosphorylated myosin II during cell locomotion and mitosis of cultured cells.J Cell Biol. 1998; 140: 119-129Crossref PubMed Scopus (195) Google Scholar, 23Brock J. Midwinter K. Lewis J. Martin P. Healing of incisional wounds in the embryonic chick wing bud characterization of the actin purse-string and demonstration of a requirement for Rho activation.J Cell Biol. 1996; 135: 1097-1107Crossref PubMed Scopus (195) Google Scholar We sought to define the recruitment, activation, and functional roles of these critical regulators in a well-characterized system of intestinal epithelial purse-string wound closure. Using intestinal epithelial cells expressing an enhanced green fluorescent protein (EGFP)-β-actin fusion protein, we were able to characterize the reorganization of actin-containing structures during purse-string closure of oligocellular epithelial wounds. These real-time observations revealed a highly orchestrated, reproducible process that could be divided into 2 distinct phases. We then used immunostaining, highly specific pharmacologic inhibitors of MLCK and ROCK, and conductance scanning analyses to define the roles of these kinases in experimental purse-string wound closure and barrier restoration. Finally, we studied human biopsy specimens from a condition of increased epithelial cell turnover: active inflammatory bowel disease. The results suggest that ROCK and MLCK serve unique, complementary roles in oligocellular epithelial wound closure. These data therefore have direct implications for understanding the mechanisms of closure for physiologic wounds that occur in healthy individuals and pathologic wounds occurring in individuals with intestinal disease. Caco-2 BBe cells24Peterson M.D. Mooseker M.S. Characterization of the enterocyte-like brush border cytoskeleton of the C2BBe clones of the human intestinal cell line, Caco-2.J Cell Sci. 1992; 102: 581-600Crossref PubMed Google Scholar expressing an EGFP/β-actin fusion protein were grown as monolayers on rat tail collagen-coated 35-mm cell culture dishes (Corning, Inc, Corning, NY). Dishes were placed on a 37°C heated stage in pH 7.4 HEPES-buffered Hanks' balanced salt solution (without bicarbonate) during wounding and subsequent imaging. Monolayers were treated with 10 μmol/L Y-27632 (Calbiochem, San Diego, CA) or 250 μmol/L PIK, an oligopeptide that is a specific membrane-permeant inhibitor of MLCK25Zolotarevsky Y. Hecht G. Koutsouris A. Gonzalez D.E. Quan C. Tom J. Mrsny R.J. Turner J.R. A membrane-permeant peptide that inhibits MLC kinase restores barrier function in in vitro models of intestinal disease.Gastroenterology. 2002; 123: 163-172Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar before wounding. Wounds were created manually using a 0.003-gauge tungsten wire (California Fine Wire, Grover Beach, CA) as described previously.7Bement W.M. Forscher P. Mooseker M.S. A novel cytoskeletal structure involved in purse string wound closure and cell polarity maintenance.J Cell Biol. 1993; 121: 565-578Crossref PubMed Scopus (274) Google Scholar Wound closure in live cells was imaged using a 63X HCX Apo immersion objective and a DMLB epifluorescence microscope (Leica Microsystems, Wetzlar, Germany) equipped with an Endow GFP bandpass emission cube (Chroma Technology Corp, Brattleboro, VT) and Roper Coolsnap HQ camera controlled by MetaMorph 6 (Universal Imaging Corp, Downingtown, PA). Serial z-stack images, at 1-μm intervals, were obtained every 2 minutes after wounding. Fixed wounds were imaged after staining using a quad bandpass 88000 filter set (Chroma Technology Corp). Wound areas were determined using MetaMorph 6 after manually tracing the wound edge. Pixel intensities were determined with MetaMorph 6 using matched samples stained and imaged under identical conditions. For these analyses, pixel intensities along lines perpendicular to the wound edge were plotted. The peak actin intensity, corresponding to the developing or established actin ring, was used to align multiple lines and was arbitrarily designated zero. These analyses were performed for multiple wounds. Wounds were fixed in 1% paraformaldehyde in phosphate-buffered saline at indicated times after wounding. Wound sites were labeled stereotactically to aid in identifying specific wounds after staining. After permeabilization with 0.1% Triton X-100 specific antibodies were applied. Activated rho was detected by incubation with a glutathione S-transferase (GST)-rhotekin rho binding domain fusion protein (Upstate Biotechnology, Lake Placid, NY) followed by incubation with polyclonal goat anti-GST (Amersham Biosciences, Piscataway, NJ) and then Alexa 594 donkey anti-goat immunoglobulin (Ig) G (Molecular Probes, Eugene, OR). Control experiments showed that substitution of GST/rhotekin with an irrelevant GST fusion protein did not label the wound edge but did nonspecifically label dead/damaged cells within the wound. ROCK was labeled using a mouse monoclonal anti-ROCK-I/ROK-β antibody (Becton Dickinson, Franklin Lakes, NJ) followed by Alexa-594 goat anti-mouse IgG (Molecular Probes). MLCK was detected using mouse monoclonal anti-MLCK clone K-36 (Sigma Chemical Co, St Louis, MO) followed by Alexa-594 goat anti-mouse IgG. Phosphorylated MLC was detected using affinity-purified polyclonal rabbit antisera, as described previously,26Berglund J.J. Riegler M. Zolotarevsky Y. Wenzl E. Turner J.R. Regulation of human jejunal transmucosal resistance and MLC phosphorylation by Na+-glucose cotransport.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G1487-G1493Crossref PubMed Google Scholar followed by Alexa-350 goat anti-rabbit antibody (Molecular Probes). In fixed preparations F-actin was stained using Alexa-488-phalloidin as described previously.25Zolotarevsky Y. Hecht G. Koutsouris A. Gonzalez D.E. Quan C. Tom J. Mrsny R.J. Turner J.R. A membrane-permeant peptide that inhibits MLC kinase restores barrier function in in vitro models of intestinal disease.Gastroenterology. 2002; 123: 163-172Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar Activated MLCK was detected using biotinylated PIK and Alexa-594 streptavidin as described previously25Zolotarevsky Y. Hecht G. Koutsouris A. Gonzalez D.E. Quan C. Tom J. Mrsny R.J. Turner J.R. A membrane-permeant peptide that inhibits MLC kinase restores barrier function in in vitro models of intestinal disease.Gastroenterology. 2002; 123: 163-172Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar and in Results. PIK labeling was assessed quantitatively using a fluorescent microplate reader (Synergy HT; Bio-Tek Instruments, Inc, Winooski, VT). Kinase assays were performed as described previously25Zolotarevsky Y. Hecht G. Koutsouris A. Gonzalez D.E. Quan C. Tom J. Mrsny R.J. Turner J.R. A membrane-permeant peptide that inhibits MLC kinase restores barrier function in in vitro models of intestinal disease.Gastroenterology. 2002; 123: 163-172Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar using long MLCK19Clayburgh D.R. Rosen S. Witkowski E.D. Wang F. Blair S. Dudek S. Garcia J.G.N. Alverdy J.C. Turner J.R. A differentiation-dependent splice variant of myosin light chain kinase, MLCK1, regulates epithelial tight junction permeability.J Biol Chem. 2004; 279: 55506-55513Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 27Stull J.T. Myosin minireview series.J Biol Chem. 1996; 271: 15849Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar from Caco-2 cells and recombinant intestinal epithelial MLC. PIK or vehicle was added to reaction mixtures and the reaction initiated by the addition of γ-32P-adenosine triphosphate (MP, Costa Mesa, CA) and 5 μmol/L recombinant MLC. MLC phosphorylation was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography of reaction mixtures. Monolayers were placed in a miniaturized Üssing chamber mounted on an upright light microscope. A sinusoidal electric current (100 μA/cm2, 24 Hz) was clamped across the epithelium and the electric field generated in the mucosal bath measured with a mobile probe positioned at a constant distance of 25 μm above the apical surface. This spacing was readjusted at every measurement position as described previously.28Gitter A.H. Bertog M. Schulzke J. Fromm M. Measurement of paracellular epithelial conductivity by conductance scanning.Pflugers Arch. 1997; 434: 830-840Crossref PubMed Scopus (62) Google Scholar The apparent conductivity Ga was calculated from the scanning signal as previously described.9Florian P. Schoneberg T. Schulzke J.D. Fromm M. Gitter A.H. Single-cell epithelial defects close rapidly by an actinomyosin purse string mechanism with functional tight junctions.J Physiol. 2002; 545: 485-499Crossref PubMed Scopus (70) Google Scholar By scanning parallel to the epithelial surface, an area of at least 100 × 100 μm was chosen, where the spatial distribution of Ga was even. This value represented the conductivity of the intact epithelium (Gintact). Single-cell wounds were created using an additional glass microelectrode filled with 0.1 mol/L CaCl2. The cell touched by the microelectrode flattened, and a current pulse (1.5 μA, 0.5 seconds) was applied.9Florian P. Schoneberg T. Schulzke J.D. Fromm M. Gitter A.H. Single-cell epithelial defects close rapidly by an actinomyosin purse string mechanism with functional tight junctions.J Physiol. 2002; 545: 485-499Crossref PubMed Scopus (70) Google Scholar These single-cell lesions created a local leak, with an increased Ga in close proximity. To record the time course of the leak current, the scan of Ga along the x-axis (directly above the lesion, x = 0, and x = 10, 20, 40, 70, and 100 μm apart) was repeated in intervals of 1–1.5 minutes. With these data, the conductance associated with a single leak (gleak) was calculated by numerical integration of Ga − Gintact as described previously.9Florian P. Schoneberg T. Schulzke J.D. Fromm M. Gitter A.H. Single-cell epithelial defects close rapidly by an actinomyosin purse string mechanism with functional tight junctions.J Physiol. 2002; 545: 485-499Crossref PubMed Scopus (70) Google Scholar The leak was determined between 2 and 16 minutes. Formalin-fixed, paraffin-embedded human colonic biopsy specimens were selected from the archives of the Department of Pathology at the University of Chicago. The protocol for use of these specimens was approved by the institutional review board of the University of Chicago. Five-micrometer sections were stained by the immunoperoxidase technique using affinity-purified polyclonal rabbit anti-phosphorylated MLC as described previously.26Berglund J.J. Riegler M. Zolotarevsky Y. Wenzl E. Turner J.R. Regulation of human jejunal transmucosal resistance and MLC phosphorylation by Na+-glucose cotransport.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G1487-G1493Crossref PubMed Google Scholar, 29Turner J.R. Torres C.M. Wang H.H. Shahsafaei A. Richards W.G. Sugarbaker D. Odze R.D. Preoperative chemoradiotherapy alters the expression and prognostic significance of adhesion molecules in Barrett's-associated adenocarcinoma.Hum Pathol. 2000; 31: 347-353Crossref PubMed Scopus (13) Google Scholar Previous work has shown that experimental oligocellular wounds within epithelial monolayers heal by a mechanism that involves the assembly and contraction of multicellular actin rings or purse-strings.7Bement W.M. Forscher P. Mooseker M.S. A novel cytoskeletal structure involved in purse string wound closure and cell polarity maintenance.J Cell Biol. 1993; 121: 565-578Crossref PubMed Scopus (274) Google Scholar The process by which this occurs has not been defined. To define this process in detail, we analyzed purse-string wound closure in monolayers of Caco-2 human intestinal epithelial cells expressing EGFP-β-actin. This revealed a sequence of precisely orchestrated events that occurred with a high degree of reproducibility and was complete within 30–45 minutes (Figure 1 and Movie 1; see supplemental material online at www.gastrojournal.org). Closure occurred as a 2-phase process characterized by ring assembly followed by wound contraction. The first characteristic event was the assembly of actin filaments at the wound edge, consistent with local actin recruitment. This typically began within 2 minutes after wounding (Figure 1A at 2 minutes, arrow). At this time, EGFP fluorescence of dead cells within the wound was also apparent. Over the next several minutes, actin accumulation at the wound edge continued, ultimately forming a ring of actin that completely encircled the wound. By 8 minutes after wounding, a stable continuous ring had formed (Figure 1A at 8 minutes, arrow). Wound edge rounding, which indicated the development of tension within the ring, marked initiation of the second phase of wound closure, contraction. This coincided with initiation of quantitatively evident wound closure, which began at 6–8 minutes (Figure 1B). At this point, the wound rapidly decreased in area, resulting in stretching of cells surrounding the wound and a subtle flattening of some cells, causing portions of the monolayer to dip out of the plane of focus (Figure 1A). Rho and ROCK play an essential role in stress fiber assembly30Ridley A.J. Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors.Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (4046) Google Scholar, 31Nobes C.D. Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia.Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3805) Google Scholar and actomyosin contraction.4Rosenblatt J. Raff M.C. Cramer L.P. An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism.Curr Biol. 2001; 11: 1847-1857Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 14Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase).J Biol Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1711) Google Scholar, 23Brock J. Midwinter K. Lewis J. Martin P. Healing of incisional wounds in the embryonic chick wing bud characterization of the actin purse-string and demonstration of a requirement for Rho activation.J Cell Biol. 1996; 135: 1097-1107Crossref PubMed Scopus (195) Google Scholar To define the roles of rho and ROCK in oligocellular epithelial purse-string wound closure, we began by defining the distributions of activated rho and ROCK during the wound closure process. We chose time points that corresponded to the initiation of the 2 phases of wound closure: ring assembly at 2 minutes after wounding and contraction at 8 minutes after wounding. To label activated rho morphologically, we adapted a well-characterized biochemical assay for detecting activated rho, binding of a GST fusion protein of the rho binding domain of rhotekin,32Ren X.D. Kiosses W.B. Schwartz M.A. Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton.EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1376) Google Scholar in a manner similar to an approach used to morphologically detect activated cdc42.33Cannon J.L. Labno C.M. Bosco G. Seth A. McGavin M.H. Siminovitch K.A. Rosen M.K. Burkhardt J.K. Wasp recruitment to the T cell:APC contact site occurs independently of Cdc42 activation.Immunity. 2001; 15: 249-259Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar As can be seen in cells away from the wound, activated rho was primarily localized to cell junctions before wounding. Within 2 minutes after wounding, when the ring assembly phase had begun, activated rho was concentrated at multiple sites along the wound edge, both in association with (Figure 2A, insets) and separate from (Figure 2A, arrows) sites of early actin accumulation. In some areas of the wound edge, actin and activated rho colocalization was apparent (Figure 2A, insets). Activated rho was also detected away from the developing actin ring (Figure 2A, top of field). An increase in cytoplasmic-activated rho was detected 8 minutes after wounding, when contraction began. However, this activated rho was primarily localized behind the actin ring (Figure 2B, inset, compare images of actin and activated rho in cells away from the wound). At 8 minutes, only small amounts of activated rho remained at the wound edge and did not colocalize with the actin ring. As seen in the live-cell imaging, damaged cells were commonly retained