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B. R. Bozard, P. S. Ganapathy, R. G. Smith, I. D. Goldman, V. Ganapathy, S. B. Smith; EM Immunolocalization of Folate Receptor alpha (FR) and Proton-Coupled Folate Transporter (PCFT) in Retinal Müller Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):6247.
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Folate, a water-soluble vitamin needed for de novo synthesis of DNA, RNA and some amino acids, is essential for cellular survival. Transport mechanisms are required for cells to obtain folate. We are interested in mechanisms by which the supportive retinal Müller glial cells (MCs) take up folate. Currently, three mechanisms are known for cellular uptake of folate: FR, RFT-1 (reduced-folate transporter-1), and PCFT. Previously, we detected FR and PCFT in MCs, by RT-PCR and immunohistochemistry. Laser scanning confocal microscopy showed FR and PCFT were both present on the plasma and nuclear membranes. FR is a GPI-linked protein that takes folate into cells via receptor-mediated endocytosis. In this study we used EM immunolocalization methods to determine whether FR and PCFT were co-localized in the endosomal cellular compartment in support of the hypothesis that the two proteins work coordinately to mediate folate uptake in MCs.
MCs were isolated from 5-7 day mouse retinas per our method (Jiang et al, 2006); FR (~40 kD) and PCFT (~50-60 kD) were detected in the cells by western blotting. For EM immunolocalization studies, cells were fixed in paraformaldehyde/glutaraldehyde, stained with uranyl acetate, dehydrated and embedded in LR White resin. Thin sections were collected on nickel grids, incubated with primary antibodies against FR and PCFT, followed by staining with 12- and 7-nm gold-labeled secondary antibodies.
Immunoblotting confirmed the presence of FR and PCFT in MCs. EM immunolocalization demonstrated FR on the plasma membrane and co-localization of FR with PCFT on the nuclear membranes of these cells as well as in vesicular bodies thought to be endosomes.
FR and PCFT co-localize in endosomes of MCs supporting the hypothesis that the two proteins are geographically well-suited to work coordinately for folate uptake. Functional studies are now underway to characterize folate uptake in these important glial cells and to determine factors that regulate their expression and activity.
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