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V.L. Bonilha, M.E. Rayborn, X. Gu, S.K. Bhattacharya, J.S. Crabb, J.W. Crabb, J.G. Hollyfield; Characterization of M6a in the Retinal Pigment Epithelium Microvilli . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3051.
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Purpose:M6 is highly expressed on the surface of many CNS neurons, on the apical surface of epithelial cells of the choroid plexus and the proximal tubules of the kidney. In the retina, M6a has been shown to be present in Muller glial endfeet and in the inner plexiform layer. Two homologues have been detected in mice, M6a and M6b. M6a peptides were detected in isolated microvilli of mouse and rat retinal pigment epithelium (RPE) subjected to proteomic analysis. Described functions for M6a include neural cell adhesion, neurite growth, and ion channel. The present study was conducted to define the distribution of this protein in the mouse RPE in vivo and in vitro. Methods: Mouse whole eyecups with the RPE exposed were fixed in 4% paraformaldehyde, reacted with the M6a specific antibody followed by a secondary Alexa antibody. To analyze association with cytoskeletal components, mouse eyecups with exposed RPE were fixed in 4% paraformaldehyde and then extracted in 0.2% Triton X100(TX). Alternatively, eyecups were first extracted in a buffer containing 0.5%TX and then fixed in 4% paraformaldehyde. Cryosections of both neonatal (postnatal day 0) and adult mouse eyes processed for immunofluorescence with the M6a specific antibody were analyzed with confocal microscopy. Mouse eyecups with exposed RPE were also fixed in a mixture of 2% paraformaldehyde + 2.5% glutaraldehyde in 0.1M cacodylate buffer, reacted with M6 antibody and a secondary antibody conjugated to gold, followed by analysis with electron microscopy. Results: M6a immunoreactivity was present associated with the apical microvilli of mouse RPE with both immunofluorescence and pre–embedding immunoelectron microscopy. Most of the immunolocalization was lost when the mouse eyecups were extracted with detergent. Expression was observed from birth in mouse eyes. Conclusions: M6a is localized to the apical microvilli of the RPE and some immunoreactivity to M6a is associated with the cytoskeleton. Further studies addressing the function of this protein in the RPE are in progress.
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