Skip to main content

Advertisement

SpringerLink
Log in

Myosin II activity is required for functional leading-edge cells and closure of epidermal sheets in fish skin ex vivo

  • Regular Article
  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

Abstract

Re-epithelialization in skin wound healing is a process in which epidermal sheets grow and close the wound. Although the actin–myosin system is thought to have a pivotal role in re-epithelialization, its role is not clear. In fish skin, re-epithelialization occurs around 500 μm/h and is 50 times faster than in mammalian skin. We had previously reported that leading-edge cells of the epidermal outgrowth have both polarized large lamellipodia and “purse string”-like actin filament cables in the scale-skin culture system of medaka fish, Oryzias latipes (Cell Tissue Res, 2007). The actin purse-string (APS) is a supracellular contractile machinery in which adherens junctions (AJs) link intracellular myosin II-including actin cables between neighboring cells. In this study, we developed a modified “face-to-face” scale-skin culture system as an ex vivo model to study epidermal wound healing, and examined the role of the actin–myosin system in the rapid re-epithelialization using a myosin II ATPase inhibitor, blebbistatin. A low level of blebbistatin suppressed the formation of APS and induced the dissociation of keratocytes from the leading edge without attenuating the growth of the epidermal sheet or the migration rate of solitary keratocytes. AJs in the superficial layer showed no obvious changes elicited by blebbistatin. However, two epidermal sheets without APSs did not make a closure with each other, which was confirmed by inhibiting the connecting AJs between the superficial layers. These results suggest that myosin II activity is required for functional leading-edge cells and for epidermal closure.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11

Similar content being viewed by others

References

  • Anderson KI, Wang YL, Small JV (1996) Coordination of protrusion and translocation of the keratocyte involves rolling of the cell body. J Cell Biol 134:1209–1218

    Article  PubMed  CAS  Google Scholar 

  • Babbin BA, Koch S, Bachar M, Conti MA, Parkos CA, Adelstein RS, Nusrat A, Ivanov AI (2009) Non-muscle myosin IIA differentially regulates intestinal epithelial cell restitution and matrix invasion. Am J Pathol 174:436–448

    Article  PubMed  CAS  Google Scholar 

  • Bement WM, Forscher P, Mooseker MS (1993) A novel cytoskeletal structure involved in purse string wound closure and cell polarity maintenance. J Cell Biol 121:565–578

    Article  PubMed  CAS  Google Scholar 

  • Bement WM, Mandato CA, Kirsch MN (1999) Wound-induced assembly and closure of an actomyosin purse string in Xenopus oocytes. Curr Biol 9:579–587

    Article  PubMed  CAS  Google Scholar 

  • Betapudi V, Rai V, Beach JR, Egelhoff T (2010) Novel regulation and dynamics of myosin II activation during epidermal wound responses. Exp Cell Res 316:980–991

    Article  PubMed  CAS  Google Scholar 

  • Bialik S, Bresnick AR, Kimchi A (2004) DAP-kinase-mediated morphological changes are localization dependent and involve myosin-II phosphorylation. Cell Death Differ 11:631–644

    PubMed  CAS  Google Scholar 

  • Brock J, Midwinter K, Lewis J, Martin P (1996) 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 135:1097–1107

    Article  PubMed  CAS  Google Scholar 

  • Clark AG, Miller AL, Vaughan E, Yu HY, Penkert R, Bement WM (2009) Integration of Single and Multicellular Wound Responses. Curr Biol 19:1389–1395

    Article  PubMed  CAS  Google Scholar 

  • Dalton BA, Steele JG (2001) Migration mechanisms: corneal epithelial tissue and dissociated cells. Exp Eye Res 73:797–814

    Article  PubMed  CAS  Google Scholar 

  • Danjo Y, Gipson IK (1998) 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 111:3323–3332

    PubMed  CAS  Google Scholar 

  • Even-Ram S, Doyle AD, Conti MA, Matsumoto K, Adelstein RS, Yamada KM (2007) Myosin IIA regulates cell motility and actomyosin-microtubule crosstalk. Nat Cell Biol 9:299–309

    Article  PubMed  CAS  Google Scholar 

  • Farooqui R, Fenteany G (2005) Multiple rows of cells behind an epithelial wound edge extend cryptic lamellipodia to collectively drive cell-sheet movement. J Cell Sci 118:51–63

    Article  PubMed  CAS  Google Scholar 

  • Fenteany G, Janmey PA, Stossel TP (2000) Signaling pathways and cell mechanics involved in wound closure by epithelial cell sheets. Curr Biol 10:831–838

    Article  PubMed  CAS  Google Scholar 

  • Florian P, Schoneberg T, Schulzke JD, Fromm M, Gitter AH (2002) Single-cell epithelial defects close rapidly by an actinomyosin purse string mechanism with functional tight junctions. J Physiol 545:485–499

    Article  PubMed  CAS  Google Scholar 

  • Franke JD, Montague RA, Kiehart DP (2005) Nonmuscle myosin II generates forces that transmit tension and drive contraction in multiple tissues during dorsal closure. Curr Biol 15:2208–2221

    Article  PubMed  CAS  Google Scholar 

  • Gayer CP, Chaturvedi LS, Wang S, Alston B, Flanigan TL, Basson MD (2009) Delineating the signals by which repetitive deformation stimulates intestinal epithelial migration across fibronectin. Am J Physiol Gastrointest Liver Physiol 296:G876–G885

    Article  PubMed  CAS  Google Scholar 

  • Gupton SL, Waterman-Storer CM (2006) Spatiotemporal feedback between actomyosin and focal-adhesion systems optimizes rapid cell migration. Cell 125:1361–1374

    Article  PubMed  CAS  Google Scholar 

  • Hutson MS, Tokutake Y, Chang MS, Bloor JW, Venakides S, Kiehart DP, Edwards GS (2003) Forces for morphogenesis investigated with laser microsurgery and quantitative modeling. Science 300:145–149

    Article  PubMed  CAS  Google Scholar 

  • Ivanov AI, Hunt D, Utech M, Nusrat A, Parkos CA (2005) Differential roles for actin polymerization and a myosin II motor in assembly of the epithelial apical junctional complex. Mol Biol Cell 16:2636–2650

    Article  PubMed  CAS  Google Scholar 

  • Ivanov AI, Bachar M, Babbin BA, Adelstein RS, Nusrat A, Parkos CA (2007) A unique role for nonmuscle myosin heavy chain IIA in regulation of epithelial apical junctions. PLoS One 2:e658

    Article  PubMed  Google Scholar 

  • Jacinto A, Wood W, Woolner S, Hiley C, Turner L, Wilson C, Martinez-Arias A, Martin P (2002) Dynamic analysis of actin cable function during Drosophila dorsal closure. Curr Biol 12:1245–1250

    Article  PubMed  CAS  Google Scholar 

  • Kishikawa M, Suzuki A, Ohno S (2008) aPKC enables development of zonula adherens by antagonizing centripetal contraction of the circumferential actomyosin cables. J Cell Sci 121:2481–2492

    Article  PubMed  CAS  Google Scholar 

  • Komatsu S, Ikebe M (2004) ZIP kinase is responsible for the phosphorylation of myosin II and necessary for cell motility in mammalian fibroblasts. J Cell Biol 165:243–254

    Article  PubMed  CAS  Google Scholar 

  • Kuo JC, Lin JR, Staddon JM, Hosoya H, Chen RH (2003) Uncoordinated regulation of stress fibers and focal adhesions by DAP kinase. J Cell Sci 116:4777–4790

    Article  PubMed  CAS  Google Scholar 

  • Kwon YC, Baek SH, Lee H, Choe KM (2010) Nonmuscle myosin II localization is regulated by JNK during Drosophila larval wound healing. Biochem Biophys Res Commun 393:656–661

    Article  PubMed  CAS  Google Scholar 

  • Lauffenburger DA, Horwitz AF (1996) Cell migration: a physically integrated molecular process. Cell 84:359–369

    Article  PubMed  CAS  Google Scholar 

  • Liang CC, Park AY, Guan JL (2007) In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2:329–333

    Article  PubMed  CAS  Google Scholar 

  • MacManus CF, Tipping NE, Wilson DJ (2006) A Rho-dependent actin purse-string is involved in wound repair in the early chick amnion following surgical puncture. Wound Repair Regen 14:61–65

    Article  PubMed  Google Scholar 

  • Martin P, Lewis J (1992) Actin cables and epidermal movement in embryonic wound healing. Nature 360:179–183

    Article  PubMed  CAS  Google Scholar 

  • Matsumoto R, Sugimoto M (2007) Dermal matrix proteins initiate re-epithelialization but are not sufficient for coordinated epidermal outgrowth in a new fish skin culture model. Cell Tissue Res 327:249–265

    Article  PubMed  CAS  Google Scholar 

  • Mitchison TJ, Cramer LP (1996) Actin-based cell motility and cell locomotion. Cell 84:371–379

    Article  PubMed  CAS  Google Scholar 

  • Miyake Y, Inoue N, Nishimura K, Kinoshita N, Hosoya H, Yonemura S (2006) Actomyosin tension is required for correct recruitment of adherens junction components and zonula occludens formation. Exp Cell Res 312:1637–1650

    Article  PubMed  CAS  Google Scholar 

  • Okeyo KO, Adachi T, Sunaga J, Hojo M (2009) Actomyosin contractility spatiotemporally regulates actin network dynamics in migrating cells. J Biomech 42:2540–2548

    Article  PubMed  Google Scholar 

  • Pasapera AM, Schneider IC, Rericha E, Schlaepfer DD, Waterman CM (2010) Myosin II activity regulates vinculin recruitment to focal adhesions through FAK-mediated paxillin phosphorylation. J Cell Biol 188:877–890

    Article  PubMed  CAS  Google Scholar 

  • Pelham RJ Jr, Wang Y (1997) Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc Natl Acad Sci USA 94:13661–13665

    Article  PubMed  CAS  Google Scholar 

  • Pollard TD, Borisy GG (2003) Cellular motility driven by assembly and disassembly of actin filaments. Cell 112:453–465

    Article  PubMed  CAS  Google Scholar 

  • Quilhac A, Sire JY (1999) Spreading, proliferation, and differentiation of the epidermis after wounding a cichlid fish, Hemichromis bimaculatus. Anat Rec 254:435–451

    Article  PubMed  CAS  Google Scholar 

  • Rao JN, Li J, Li L, Bass BL, Wang JY (1999) Differentiated intestinal epithelial cells exhibit increased migration through polyamines and myosin II. Am J Physiol 277:G1149–G1158

    PubMed  CAS  Google Scholar 

  • Redd MJ, Cooper L, Wood W, Stramer B, Martin P (2004) Wound healing and inflammation: embryos reveal the way to perfect repair. Philos Trans R Soc Lond B 359:777–784

    Article  CAS  Google Scholar 

  • Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, Parsons JT, Horwitz AR (2003) Cell migration: integrating signals from front to back. Science 302:1704–1709

    Article  PubMed  CAS  Google Scholar 

  • Rodriguez-Diaz A, Toyama Y, Abravanel DL, Wiemann JM, Wells AR, Tulu US, Edwards GS, Kiehart DP (2008) Actomyosin purse strings: renewable resources that make morphogenesis robust and resilient. HFSP J 2:220–237

    Article  PubMed  Google Scholar 

  • Sandquist JC, Swenson KI, Demali KA, Burridge K, Means AR (2006) Rho kinase differentially regulates phosphorylation of nonmuscle myosin II isoforms A and B during cell rounding and migration. J Biol Chem 281:35873–35883

    Article  PubMed  CAS  Google Scholar 

  • Schaub S, Bohnet S, Laurent VM, Meister JJ, Verkhovsky AB (2007) Comparative maps of motion and assembly of filamentous actin and myosin II in migrating cells. Mol Biol Cell 18:3723–3732

    Article  PubMed  CAS  Google Scholar 

  • Shu S, Liu X, Korn ED (2005) Blebbistatin and blebbistatin-inactivated myosin II inhibit myosin II-independent processes in Dictyostelium. Proc Natl Acad Sci USA 102:1472–1477

    Article  PubMed  CAS  Google Scholar 

  • Smutny M, Cox HL, Leerberg JM, Kovacs EM, Conti MA, Ferguson C, Hamilton NA, Parton RG, Adelstein RS, Yap AS (2010) Myosin II isoforms identify distinct functional modules that support integrity of the epithelial zonula adherens. Nat Cell Biol 12:696–702

    Article  PubMed  CAS  Google Scholar 

  • Straight AF, Cheung A, Limouze J, Chen I, Westwood NJ, Sellers JR, Mitchison TJ (2003) Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor. Science 299:1743–1747

    Article  PubMed  CAS  Google Scholar 

  • Svitkina TM, Verkhovsky AB, McQuade KM, Borisy GG (1997) Analysis of the actin-myosin II system in fish epidermal keratocytes: mechanism of cell body translocation. J Cell Biol 139:397–415

    Article  PubMed  CAS  Google Scholar 

  • Tamada M, Perez TD, Nelson WJ, Sheetz MP (2007) Two distinct modes of myosin assembly and dynamics during epithelial wound closure. J Cell Biol 176:27–33

    Article  PubMed  CAS  Google Scholar 

  • Vanable JW (1989) Integumentary potentials and wound healing. Electric fields in vertebrates repair, vol. 5. ALiss, New York, pp 171–224

  • Verkhovsky AB, Svitkina TM, Borisy GG (1999) Self-polarization and directional motility of cytoplasm. Curr Biol 9:11–20

    Article  PubMed  CAS  Google Scholar 

  • Vicente-Manzanares M, Zareno J, Whitmore L, Choi CK, Horwitz AF (2007) Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells. J Cell Biol 176:573–580

    Article  PubMed  CAS  Google Scholar 

  • Wilson CA, Tsuchida MA, Allen GM, Barnhart EL, Applegate KT, Yam PT, Ji L, Keren K, Danuser G, Theriot JA (2010) Myosin II contributes to cell-scale actin network treadmilling through network disassembly. Nature 465:373–377

    Article  PubMed  CAS  Google Scholar 

  • Yarrow JC, Perlman ZE, Westwood NJ, Mitchison TJ (2004) A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods. BMC Biotechnol 4:21

    Article  PubMed  Google Scholar 

  • Yonemura S, Itoh M, Nagafuchi A, Tsukita S (1995) Cell-to-cell adherens junction formation and actin filament organization: similarities and differences between non-polarized fibroblasts and polarized epithelial cells. J Cell Sci 108:127–142

    PubMed  CAS  Google Scholar 

  • Yonemura S, Wada Y, Watanabe T, Nagafuchi A, Shibata M (2010) alpha-Catenin as a tension transducer that induces adherens junction development. Nat Cell Biol 12:533–542

    Article  PubMed  CAS  Google Scholar 

  • Young PE, Richman AM, Ketchum AS, Kiehart DP (1993) Morphogenesis in Drosophila requires nonmuscle myosin heavy chain function. Genes Dev 7:29–41

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biomolecular Science, Faculty of Science, Toho University, Miyama 2-2-1, Funabashi, Chiba, 274-8510, Japan

    Toshiyuki Morita, Akiko Tsuchiya & Masazumi Sugimoto

Authors
  1. Toshiyuki Morita
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Akiko Tsuchiya
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Masazumi Sugimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Masazumi Sugimoto.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Morita, T., Tsuchiya, A. & Sugimoto, M. Myosin II activity is required for functional leading-edge cells and closure of epidermal sheets in fish skin ex vivo. Cell Tissue Res 345, 379–390 (2011). https://doi.org/10.1007/s00441-011-1219-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00441-011-1219-1

Keywords

Search

Navigation