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Michael A Robichaux, Theodore G Wensel; Rhodopsin localization in the rod connecting cilium with STORM nanoscopy. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3102.
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© ARVO (1962-2015); The Authors (2016-present)
Rhodopsin (Rho) is the most abundant protein in the outer segment sensory cilia of rod photoreceptor cells in the retina, and its transport through these cilia is an essential process, the disruption of which is the cause and/or outcome of numerous photoreceptor dystrophies. In rods Rho must be processed in the inner segment before trafficking to the region of the connecting cilium (CC), a narrow portion of the cilia that connects the inner and outer segments. Rho is proposed to insert into the membrane near the CC region, but its distribution within the CC has not been determined due to resolution limits of conventional immunofluorescence. Thus, our purpose was to utilize super-resolution microscopy to determine the distribution of rhodopsin within and near the CC.
We used STORM localization nanoscopy to identify the localization Rho in single mouse rod cilia with super-resolution precision. Two antibodies that target either the N- or C-terminus of Rho were used for localization in addition to structural markers of the CC. Rod cilia from thin sections of mouse retina were reconstructed in both the longitudinal and transverse planes. In addition, we pre-extracted mouse retinas with Triton X-100 to test the membrane association of Rho at the CC.
In rod cilia STORM reconstructions, Rho in the CC is localized in the membrane via labeling with both N- and C-terminal antibodies. The localization pattern at the CC non-uniform with occasional bright clusters of rhodopsins. We consistently localized Rho to the basal body as a bright, tightly clustered reconstruction. Finally, we tested Rho membrane permeabilization by performing STORM localization of Rho in pre-extracted retinas, and while the outer segment membranes are removed by this method, we observed clusters of Rho that remain associated with the cilium, presumably tightly bound to non-extractable components.
The results are consistent with a model in which Rho moves to the OS through the membrane of the CC, but does so both in a random distribution, and in clusters. The strong signal from Rho at the basal body suggests a model in which post-Golgi rhodopsin transport vesicles dock at or near the basal body just prior to entry into the CC. The retention of some Rho in the CC after detergent extraction may represent tethering to the axonemal microtubules by components of the transport machinery.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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