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M. Garlipp, F. Gonzalez-Fernandez; Cone and RPE IRBP Endocytosiss. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3074.
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Interphotoreceptor retinoid-binding protein (IRBP) is a 134kDa glycolipolprotein, and the major soluble protein component of the interphotoreceptor matrix (IPM). IRBP is synthesized by the rods and cones, and has a half-life of 11 hrs in the Xenopus IPM (reviewed in Gonzalez-Fernandez, 2003, Vision Res. 43:3021-6.). Here we use organ culture to follow the uptake of IRBP by the cells bordering the IPM.
Detached/reattached washed retina-eyecups were incubated in the dark for 1.5 hrs in presence of 8.3 Î¼M bovine IRBP labeled with alexa-647. The bovine IRBP, which was purified by ConA affinity, ion-exchange and size exclusion chromatography, was labeled with alexa-647 at a molar ratio of ~1:1. The label was shown not to affect the proteinâ€TMs ligand-binding parameters for all-trans retinol in titrations monitoring fluorescence enhancement. Uptake was studied using a Zeiss LSM 510 MetaConfocal Microscope at 633nm excitation for Alexa 647 and background auto-fluorescence control excitation of 561nm. Experimental and control fluorescence were measured at 688nm. Immunogold electron microscopy studies employed a rabbit anti-Xenopus IRBP serum to localize IRBP in non-detached retinas. All experiments utilized young adult albino Xenopus.
Dramatic cone bIRBP-alexa (647) uptake appeared as numerous vesicles crowding within the cone inner segments. Occasional large single IRBP positive vacuoles (~6 Î¼m) were also noted. Numerous scattered endocytic vesicles could also be identified in the apical RPE cytoplasm. These were clearly distinct from the RPE autofluorescence. Immunogold-EM studies were able to identify different classes of endocytic vesicles in the RPE and cones including larger possible photoreceptors multivesciular bodies.
The Xenopus eyecup offers a useful system to follow the trafficking of IPM components. The avid uptake of IRBP by the cones was particularly unexpected and points to uncharacterized role of IRBP in cone physiology. On going studies will define the function and mechanism of these interesting uptake pathways.
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