ZO-1: lamellipodial localization in a corneal fibroblast wound model.
OBJECTIVE
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.
Taliana
Lavinia
L
Department of Ophthalmology, Mount Sinai School of Medicine, New York, NY 10029, USA.
Benezra
Miriam
M
Greenberg
Roseanne S
RS
Masur
Sandra K
SK
Bernstein
Audrey M
AM
D000818:Animals; D015153:Blotting, Western; D015820:Cadherins / Q000378:metabolism; D018929:Cell Culture Techniques; D018031:Connexin 43 / Q000378:metabolism; D003315:Cornea / Q000378:metabolism*; D065306:Corneal Injuries; D004195:Disease Models, Animal; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism*; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D007365:Intercellular Junctions; D008565:Membrane Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011554:Pseudopodia / Q000378:metabolism*; D011817:Rabbits; D014945:Wound Healing; D062826:Zonula Occludens-1 Protein
D015820:Cadherins; D018031:Connexin 43; D008565:Membrane Proteins; D010750:Phosphoproteins; D062826:Zonula Occludens-1 Protein