Localization of ZO-1 in the nucleolus of corneal fibroblasts.
OBJECTIVE
Within the multidomain structure of ZO-1 are motifs responsible for ZO-1's localization to intercellular junctions and its newly demonstrated localization to the leading edge of lamellipodia in corneal fibroblasts. Since ZO-1 also has two nuclear localization signals, this study was undertaken to determine whether stimuli associated with wounding would induce nuclear translocation of ZO-1


METHODS
Immunocytochemistry and immunoblot analysis were used to localize endogenous and exogenous ZO-1 in nuclear and cytoplasmic sites in corneal fibroblasts and 293T fibroblasts, with and without myc-ZO-1 transfection. Cells were serum starved by growth for 48 hours in DMEM/F12 with 0.2% FBS and subsequently were either scrape wounded or treated with 10% FBS, PDGF, or FGF-2 for 6 hours. For immunoblot analysis, after lysis, the nuclear and cytosolic fractions were separated and analyzed by SDS-PAGE. Cells on companion coverslips were fixed with 3% p-formaldehyde and permeabilized with 1% Triton before immunocytochemical detection of ZO-1 and nuclear proteins.


RESULTS
ZO-1 was rarely detected in the nucleus of serum-starved corneal fibroblasts. In contrast, it colocalized with nucleolin in the nucleoli of corneal fibroblasts after serum-starved cells were treated with 10% FBS, PDGF, or FGF-2. Immunoblot analysis confirmed the immunocytochemical results: Little ZO-1 was detected in the nuclear fraction of lysates of serum-starved cells, but ZO-1 was found in the nuclear fractions of rabbit corneal and 293T fibroblasts treated with 10% FBS, PDGF, or FGF-2. Furthermore in scrape-wounded corneal fibroblasts, ZO-1 was localized to nucleoli of both serum-starved and serum-treated cells.


CONCLUSIONS
Localization of ZO-1 to nucleoli of corneal and 293T fibroblasts under proliferative and promigratory conditions suggests a physiologically significant interaction of ZO-1 with proteins in nucleoli during the healing process.
Benezra
Miriam
M
Department of Ophthalmology, Mount Sinai School of Medicine, New York, New York 10029-6574, USA. benezram@mskcc.org
Greenberg
Roseanne S
RS
Masur
Sandra K
SK
D000818:Animals; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology; D002466:Cell Nucleolus / Q000378:metabolism*; D003315:Cornea / Q000378:metabolism*; D065306:Corneal Injuries; D003600:Cytosol; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D015151:Immunoblotting; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D011817:Rabbits; D014162:Transfection; D014945:Wound Healing / Q000502:physiology; D062826:Zonula Occludens-1 Protein; D000097796:Nucleolin
D008565:Membrane Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D062826:Zonula Occludens-1 Protein; D000097796:Nucleolin