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J.–Z. Chuang, M. Berryman, J. Sparrow, C.–H. Sung; Molecular and cellular characterization of CLIC4, an apical microvilli protein of retinal pigment epithelium . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3691.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: A member of chloride intracellular channel family CLIC4 was isolated from complexes containing Ezrin, an actin binding protein known to be important for the microvilli morphogenesis in RPE. The CLIC family has been implicated in chloride ion transport within various subcellular compartments. However, several lines of recent evidence indicated that CLIC4 might also be involved in other cellular processes including cytoskeletal organization and apoptosis. Here, we investigate the biochemical and cellular characterization of CLIC4 in RPE cells. Methods: The subcellular distribution of endogenous CLIC4 was examined immunocytochemically by confocal and electron microscopy (EM) and by biochemical fractionation. The interaction between Ezrin and CLIC4 was studied in the RPE–J cells. Results: CLIC4 was co–localized with Ezrin, and enriched on the RPE apical microvilli. Ultrastructural analysis indicated that CLIC4 was present on both the plasma membranes of microvilli and the membranes that surrounded the shedded outer segment discs. In addition, membrane profiles of various phagocytic compartments were also CLIC4–immunoreactive. GFP–CLIC4 transfected into rat RPE in vivo displayed enrichment on apical microvilli. Overexpression of Ezrin appeared to facilitate the membrane expression of CLIC4 in RPE–J cells. Conclusions: CLIC4 is highly enriched on the microvilli membranes of RPE in vivo. However, both native and recombinant CLIC4 are distributed to both the cytosol and the membrane fractions in RPE–J cells. The physiological function of CLIC4 and the relevance to its interaction with Ezrin remain to be investigated. (Support by NIH–EY–11307, RPB, and Steinbach Macular Degeneration Foundation)
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