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Yekaterina Gribanova, Novrouz B. Akhmedov, Debora B. Farber; Interaction of 7R and Rootletin in Mouse Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2011;52(14):920.
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© ARVO (1962-2015); The Authors (2016-present)
To identify in mouse retina potential interacting partners of 7R, a novel retinal protein characterized as a Golgi-associated protein in stably transfected HEK 293 cells.
Mouse retinal extracts were incubated with 7R antibodies and immunoprecipitated proteins were separated by SDS-PAGE and subjected to mass spectrometry. Co-localization of 7R and its interacting partners was confirmed by immunohistochemistry, electron microscopy and in situ proximity ligation assay (PLA).
Clathrin, NSF (N-ethylmaleimide sensitive fusion protein), and rootletin were identified as putative 7R interacting candidates. Based on the highest number of detected peptides and the highest sequence match score, rootletin, a coiled-coil protein found in the rootlets of photoreceptors’ cilia, was selected for further study to confirm if it was indeed a direct partner of 7R. Reciprocal co-immunoprecipitation of mouse retinal extracts with rootletin antibodies also brought down 7R and rootletin together. We then examined the distribution of 7R and rootletin in mouse retina, double -immunostaining with 7R and rootletin antibodies. Both 7R and rootletin were localized to the inner segments and partially to the cell bodies of photoreceptors. Electron microscopy showed co-localization of 7R and rootletin in the photorecptor inner segments, with 7R on the membranes of conglomerates of vesicles forming structures with rootletin fibrils. Moreover, PLA detected 7R and rootletin as interacting proteins. We also noticed that the location and intensity of the PLA signal depends on illumination. In the dark, 7R and rootletin interaction is detected mostly in the inner segments whereas after light exposure we observed a stronger fluorescent signal in the photoreceptor cell bodies, surrounding the nuclei.
Our results indicate that rootletin is an interacting partner of 7R in retinal photoreceptors. This interaction might be important for formation and anchoring of Golgi vesicles.
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