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Valencia Potter, Michael Robichaux, Theodore G Wensel; Utilization of Super Resolution Nanoscopy to Localize CEP290 within the Connecting Cilium of Rod Photoreceptor Cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):356.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations to CEntrosomal Protein of 290kDa (CEP290) are linked to Leber Congenital Amaurosis (LCA) and other ciliopathies that result in retinal degeneration. In the retina, CEP290 has been proposed to be a regulator of ciliary trafficking and a structural scaffold within the connecting cilium (CC) of rod photoreceptor cells. However, its localization and function within the CC are unclear. In this study, we used super resolution nanoscopy to localize CEP290 in the rods of murine retina. Super resolution nanoscopy has the potential to resolve subcellular details such as the localization of CEP290 relative to the microtubule doublets of the axoneme. We hypothesize that CEP290 localizes just peripherally to the microtubule doublets of the CC.
For this study, we employed Structured Illuminated Microscopy (SIM) and Stochastic Optical Reconstruction Microscopy (STORM). For SIM, murine cryosections were immunostained with antibodies for CEP290 and acetylated a tubulin as a structural marker of the CC. For STORM, retinas were first stained with whole-mount immunostaining procedures using the same set of antibodies as SIM. Ultra-thin sections were prepared for imaging and reconstructed CEP290 and acetylated a tubulin clusters were identified. CC data from the SIM and STORM experiments were measured and analyzed with Image J.
We find that CEP290 does localize peripherally to the microtubule doublets of the CC. In SIM, CEP290 staining appears to extend 80nm beyond the regions of a acetylated tubulin staining. In STORM, which has higher resolution, CEP290 staining extends 20nm beyond the boundaries of a acetylated tubulin staining.
Our results are consistent with our hypothesis that CEP290 localizes peripherally to the microtubule doublets of the CC. Additionally, CEP290 localizes in close proximity to the microtubule doublets. Super resolution nanoscopy modalities SIM and STORM allow for 110nm and 20nm resolutions, respectively, and prove to be invaluable in clarifying the subcellular details of the CC. The use of SIM and STORM provides vast opportunities to precisely localize proteins throughout the CC.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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