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Miriam Benezra, Roseanne S. Greenberg, Sandra K. Masur; Localization of ZO-1 in the Nucleolus of Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2007;48(5):2043-2049. doi: 10.1167/iovs.06-0754.
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purpose. 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.
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