title: Organized migration of epithelial cells requires control of adhesion and protrusion through Rho kinase effectors.
abstract: Migration of epithelial cell sheets, a process involving F-actin restructuring through Rho family GTPases, is both physiologically and pathophysiologically important. Our objective was to clarify the mechanisms whereby the downstream RhoA effector Rho-associated coil-coil-forming kinase (ROCK) influences coordinated epithelial cell motility. Although cells exposed to a pharmacological ROCK inhibitor (Y-27632) exhibited increased spreading in wound closure assays, they failed to migrate in a cohesive manner. Two main phenomena were implicated: the formation of aberrant protrusions at the migrating front and the basal accumulation of F-actin aggregates. Aggregates reflected increased membrane affiliation and detergent insolubility of the actin-binding protein ezrin and enhanced coassociation of ezrin with the membrane protein CD44. While F-actin aggregation following ROCK inhibition was recapitulated by inhibiting myosin light chain (MLC) phosphorylation with the MLC kinase inhibitor ML-7, the latter did not influence protrusiveness and, in fact, significantly decreased cell migration. Our results suggest that excessive protrusiveness downstream of ROCK inhibition reflects an influence of ROCK on F-actin stability via LIM kinase 1 (LIMK-1), which phosphorylates and inactivates cofilin. Y-27632 reduced the levels of both active LIMK-1 and inactive cofilin (phospho forms), and expression of a dominant negative LIMK-1 mutant stimulated leading edge protrusiveness. Furthermore, Y-27632-induced protrusions were partially reversed by overexpression of LIMK-1 to restore cofilin phosphorylation. In summary, our results provide new evidence suggesting that adhesive and protrusive events involved in organized epithelial motility downstream of ROCK are separately coordinated through the phosphorylation of (respectively) MLC and cofilin.
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322, USA. ann.hopkins@ucd.ie
authors.forename: Ann M
authors.initials: AM
authors.lastname: Hopkins
authors.forename: A'drian A
authors.initials: AA
authors.lastname: Pineda
authors.forename: L Matthew
authors.initials: LM
authors.lastname: Winfree
authors.forename: G Thomas
authors.initials: GT
authors.lastname: Brown
authors.forename: Mike G
authors.initials: MG
authors.lastname: Laukoetter
authors.forename: Asma
authors.initials: A
authors.lastname: Nusrat
mesh_terms: D000199:Actins / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D000818:Animals; D001381:Azepines / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D051338:Cofilin 1 / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D054461:Lim Kinases; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors; D009281:Naphthalenes / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011554:Pseudopodia / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D054460:rho-Associated Kinases; D000099149:Ezrin
chemical_list: D000199:Actins; D000577:Amides; D001381:Azepines; D051338:Cofilin 1; D003598:Cytoskeletal Proteins; D018960:Hyaluronan Receptors; D047908:Intracellular Signaling Peptides and Proteins; D054461:Lim Kinases; D018994:Myosin Light Chains; D009219:Myosin-Light-Chain Kinase; D009281:Naphthalenes; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011725:Pyridines; D054460:rho-Associated Kinases; D000099149:Ezrin; C497483:CD44 protein, human; C070571:ML 7; C108830:Y 27632; C515892:LIMK1 protein, human; C515894:Limk1 protein, rat