Abstract
Purpose: :
Interphotoreceptor retinoid-binding protein (IRBP), the major soluble protein component of the interphotoreceptor matrix (IPM), has access to Müller cells, photoreceptors, and RPE. The mechanism by which IRBP targets delivery and release of retinoids between these cells during the visual cycle is poorly understood. The mechanism may involve specific molecules that IRBP binds with to efficiently target retinoid removal and/or its delivery. Here, we sought to determine the spatial localization of IRBP-binding partners in the Xenopus retina.
Methods: :
Xenopus are ideal for these studies because of the large size of their photoreceptors and ability to detach their retinas in both light and dark. Full-length Xenopus IRBP (xIRBP) free of recombinant fusion tags was labeled with Alexa-Fluor(647) at a ~1:1 stoichiometry. Labeling did not interfere with retinol binding as judged by fluorescence spectroscopy. Detached retinas were washed under Xenopus Ringer’s saline to remove endogenous IRBP. Isolated retinas were then incubated with IRBP-Alexa(647) in the presence, or absence of excess unlabeled xIRBP. Unbound labeled IRBP was removed by saline washes. The fixed tissue was examined by laser scanning-confocal microscopy in cross-section, and as flat whole mounts.
Results: :
Retinas detached in light showed striking labeling of xIRBP-Alexa(647) to the cone outer segment region, and a lesser labeling of the rod outer segments. The staining was reduced in the presence of excess unlabeled IRBP. Cone outer segment staining was markedly reduced in retinas detached and incubated in the dark. In dark, increased staining was observed at the level of the outer limiting membrane.
Conclusions: :
In light, xIRBP-Alexa(647) shows significant binding to the cone outer segment, or possibly its matrix sheath. The interaction is reduced in dark. The findings suggest a specific interaction of IRBP with outer segments that may be physiologically relevant.
Keywords: photoreceptors • retinal pigment epithelium • retinoids/retinoid binding proteins