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B. Park, X. Shen, M. Samaraweera, M. Tibudan, B.Y. J. T. Yue; Golgi Alteration and Cell Loss from Overexpression of Wild Type and Mutant Optineurin in Ocular Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):207.
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To study the localization of optineurin and the consequences of overexpressing wild type and two mutants in ocular cells, including human trabecular meshwork (TM) and retinal pigmented epithelial (RPE) cells. Optineurin is a recently identified glaucoma gene. Mutations such as E50K in the optineurin gene were reported in patients with normal pressure glaucoma (NPG).
Secretion assay was performed. Localization of endogenous optineurin was examined by immunofluorescence staining and subcellular fractionation. Wild type and mutant (E50K and L157A) optineurin–GFP expressing plasmids were transfected into human TM and RPE cells and the integrity of the Golgi apparatus in the transfectants was evaluated. The number of transfected cells was counted at 24–hr intervals and changes with time were monitored. To investigate the co–distribution of optineurin with other proteins, optineurin transfectants were either co–expressed with consititutive Rab8 plasmid or were immunostained for myosin VI and transferrin receptor. Treatment for 30 min with brefeldin A, nocodazole or cytochalasin D was also carried out.
The endogenous optineurin was not secreted. It was localized in the cytosol in a diffused pattern without a distinct association with the Golgi in both human TM and RPE cells. When upregulated by transfection, optineurin formed foci around the Golgi apparatus, co–distributing with constitutive active Rab8, myosin VI, and a population of transferrin receptor. The foci were dispersed into cytosol upon brefeldin A or nocodazole treatment. Overexpression of E50K mutant resulted in a much greater number and size of the foci than those of the wild type. By contrast, only a few foci were found when cells overexpressed L157A, a non–causative mutant form for NPG. Golgi fragmentation and cell loss, observed in all optineurin overexpressing cells, appeared to be the most pronounced in the E50K transfectants.
These data implicate a possible involvement of optineurin in the Golgi integrity and vesicle trafficking in ocular cells.
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