Imaging of High- or Low-Abundance Proteins on the Rod Outer Segment Disks

Our previous proteomic and biochemical studies suggest that the respiratory chain complexes I to V are expressed in the rod Outer Segment (OS), an organelle devoid of mitochondria, specialized for visual transduction. The expression of the cited proteins was demonstrated with several imaging techniques, even though co-localization only proved significant on retinal sections. Here we performed Immunofluorescent Confocal (CLSM) analysis on purified disks and, Transmission Electron Microscopy (TEM) immunogold analyses on bovine retinal sections. Data on co-localization of Rhodopsin and F1 Fo -ATP synthase was not significant on the isolated disks even in the presence of good labelling of the single proteins but was significant on bovine retinal sections. Indeed, the gold spheres never laid on the same discrete sites of the disk, and labelling displayed low efficiency, likely due to the hydrophobicity of the densely stacked disks, as compared to the hydrophilic antibody milieu. Data are consistent with the notion that tagging proteins with distinct fluorophores by direct labeling for co-localization is not completely unambiguous, especially when the stoichiometry of the two proteins is greatly different. This is the case for the disk, where Rhodopsin, utilized as a reference, represents more than 80% of its protein complement. The limitations of co-localization analysis have been largely investigated, and the present data confirm that co-localization on the disks surface is poor, especially on isolated disks, likely due to an exclusion process on small vesicles.