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E.J. Rodriguez–Boulan, V.L. Bonilha, B.–C. Shin, R.C. Mora; Raft markers in rat retinal pigment epithelium: non–polar caveolae, basolateral flotillin 2 and flotillin 1 in Golgi complex and in rab 7–enriched endosomes . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3674.
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Purpose: Membrane rafts are considered key mediators of signalling events and membrane trafficking. Here, we determined the expression and distribution in rat retinal pigmented epithelium (RPE) of caveolins and flotillins, established markers of raft microdomains. Methods: RPE in situ, primary RPE cultures (RPE–P) and the immortalized RPE cell line (RPE–J) were used for analysis by immunocytochemistry (IC), western blot and transmission electronmicroscopy (EM). Sedimentation of cold Triton X–100 cell extracts was assessed by centrifugation. Results: RPE cells express caveolins (1 and 2) as well as flotillins (1 and 2) both in vitro and in vivo and they assemble caveolae on both their apical and basolateral domains. By IC, caveolin 1 had a non–polar localization. The distribution of caveolin 2 in RPE (non–polar) was different from that found in other epithelial cells (basolateral and Golgi in kidney and only Golgi in thyroid cells). EM analysis revealed a differential surface density of the caveolae: rare in RPE in situ and frequently found in RPE–P and in RPE–J cells. In all three RPE cells, caveolae were bi–polarly localized. By IC, flotillin 2 had a basolateral distribution. Flotillin 1, in contrast, localized to Golgi complex and to a subset of rab7–containing endosomes. Flotillins were present in smaller–sized detergent resistant complexes than caveolins. Conclusions: Rat RPE cells both in vitro and in vivo assemble non–polar caveolae in striking contrast to the kidney, thyroid and intestinal epithelium where caveolae are found polarized. The data suggest a role of caveolae in bi–directional exchanges of solutes between the apical and basolateral environments separated by the RPE monolayer in the retina. The data also suggest a role for flotilin 1 in intracellular membrane trafficking through a rab 7 compartment and/or in membrane recycling between intracellular compartments (late endosomes and Golgi) and the cell surface. Supported by NIH grant EY008538.
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