PURPOSE: To explore the roles of ZO-1 in corneal fibroblasts and myofibroblasts in a model of wounding. METHODS: Antibodies were used to identify ZO-1 in cultured rabbit corneal fibroblasts by immunocytochemistry, Western blot analysis, and immunoprecipitation. For colocalization studies, antibodies to beta-catenin, cadherins, connexins, integrins, alpha-actinin, and cortactin were used. G- and F-actin were identified by DNase and rhodamine phalloidin, respectively. To study ZO-1 localization during cell migration, confluent corneal fibroblasts were subjected to scrape-wounding and evaluated by immunocytochemistry. RESULTS: As predicted from previous studies, ZO-1 colocalized with cadherins and connexin 43 in intercellular junctions. The study revealed a new finding: ZO-1 was also detected at the leading edge of lamellipodia, especially in motile wounded fibroblasts and in freshly plated fibroblasts, before the formation of cell-cell contacts. In fibroblast lysates, ZO-1 largely partitioned to the detergent-soluble fraction compared with myofibroblast lysates, indicating that much of the fibroblast ZO-1 is not associated with insoluble structural components. Lamellipodial ZO-1 colocalized with G-actin, alpha-actinin, and cortactin, which are proteins involved with actin remodeling and cell migration. Integrins alpha5beta1 and alphavbeta3 also localized to the leading edge of migrating fibroblasts, and the association of ZO-1 with integrin was confirmed by immunoprecipitation. Finally, alkaline phosphatase treatment of fibroblast lysate decreased the molecular mass of ZO-1 in lysates of cells grown in serum, demonstrating that, in activated fibroblasts, ZO-1 is phosphorylated. CONCLUSIONS: ZO-1's appearance at the leading edge of migrating fibroblasts makes it a candidate for a role in the initiation and organization of integrin-dependent fibroblast adhesion complexes formed during migration and adhesion. Further, phosphorylation of ZO-1 may regulate its cellular localization.
PURPOSE: To explore the roles of ZO-1 in corneal fibroblasts and myofibroblasts in a model of wounding. METHODS: Antibodies were used to identify ZO-1 in cultured rabbit corneal fibroblasts by immunocytochemistry, Western blot analysis, and immunoprecipitation. For colocalization studies, antibodies to beta-catenin, cadherins, connexins, integrins, alpha-actinin, and cortactin were used. G- and F-actin were identified by DNase and rhodamine phalloidin, respectively. To study ZO-1 localization during cell migration, confluent corneal fibroblasts were subjected to scrape-wounding and evaluated by immunocytochemistry. RESULTS: As predicted from previous studies, ZO-1 colocalized with cadherins and connexin 43 in intercellular junctions. The study revealed a new finding: ZO-1 was also detected at the leading edge of lamellipodia, especially in motile wounded fibroblasts and in freshly plated fibroblasts, before the formation of cell-cell contacts. In fibroblast lysates, ZO-1 largely partitioned to the detergent-soluble fraction compared with myofibroblast lysates, indicating that much of the fibroblast ZO-1 is not associated with insoluble structural components. Lamellipodial ZO-1 colocalized with G-actin, alpha-actinin, and cortactin, which are proteins involved with actin remodeling and cell migration. Integrins alpha5beta1 and alphavbeta3 also localized to the leading edge of migrating fibroblasts, and the association of ZO-1 with integrin was confirmed by immunoprecipitation. Finally, alkaline phosphatase treatment of fibroblast lysate decreased the molecular mass of ZO-1 in lysates of cells grown in serum, demonstrating that, in activated fibroblasts, ZO-1 is phosphorylated. CONCLUSIONS: ZO-1's appearance at the leading edge of migrating fibroblasts makes it a candidate for a role in the initiation and organization of integrin-dependent fibroblast adhesion complexes formed during migration and adhesion. Further, phosphorylation of ZO-1 may regulate its cellular localization.
Authors: Edward G Tall; Audrey M Bernstein; Noelynn Oliver; Julia L Gray; Sandra K Masur Journal: Invest Ophthalmol Vis Sci Date: 2010-04-14 Impact factor: 4.799
Authors: Gautam S Ghatnekar; Michael P O'Quinn; L Jane Jourdan; Abhijit A Gurjarpadhye; Robert L Draughn; Robert G Gourdie Journal: Regen Med Date: 2009-03 Impact factor: 3.806
Authors: Vivien J Coulson-Thomas; Yvette M Coulson-Thomas; Tarsis F Gesteira; Claudia A Andrade de Paula; Celia R W Carneiro; Valdemar Ortiz; Leny Toma; Winston W-Y Kao; Helena B Nader Journal: Exp Cell Res Date: 2013-02-08 Impact factor: 3.905
Authors: Ariadna Mendoza-Naranjo; Peter Cormie; Antonio E Serrano; Rebecca Hu; Shay O'Neill; Chiuhui Mary Wang; Christopher Thrasivoulou; Kieran T Power; Alexis White; Thomas Serena; Anthony R J Phillips; David L Becker Journal: PLoS One Date: 2012-05-15 Impact factor: 3.240