TY - JOUR
T1 - ZO-1
T2 - Lamellipodial localization in a corneal fibroblast wound model
AU - Taliana, Lavinia
AU - Benezra, Miriam
AU - Greenberg, Roseanne S.
AU - Masur, Sandra K.
AU - Bernstein, Audrey M.
PY - 2005/1
Y1 - 2005/1
N2 - 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 β-catenin, cadherins, connexins, integrins, α-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, α-actinin, and cortactin, which are proteins involved with actin remodeling and cell migration. Integrins α5β1 and αvβ3 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.
AB - 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 β-catenin, cadherins, connexins, integrins, α-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, α-actinin, and cortactin, which are proteins involved with actin remodeling and cell migration. Integrins α5β1 and αvβ3 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.
UR - http://www.scopus.com/inward/record.url?scp=11144285371&partnerID=8YFLogxK
U2 - 10.1167/iovs.04-0145
DO - 10.1167/iovs.04-0145
M3 - Article
C2 - 15623760
AN - SCOPUS:11144285371
SN - 0146-0404
VL - 46
SP - 96
EP - 103
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
IS - 1
ER -