Abstract
Purpose :
Mutations in CEP290 cause Leber Congenital Amaurosis (LCA) and ciliopathies that result in retinal degeneration. CEP290 has been proposed to regulate ciliary trafficking and function in a structural role for primary cilia; however, its localization and function in the connecting cilium (CC), the modified primary cilium of photoreceptor cells, is unclear. The goal of this study was to determine the location of CEP290 within the rod CC of murine retina, and compare the results to the distribution in primary cilia of epithelial cells, in order to provide insights into CEP290 function, and to test the hypothesis that its localization and possibly function may differ between these cell types.
Methods :
To obtain sub-diffraction resolution, we used Structured Illuminated Microscopy (SIM) and Stochastic Optical Reconstruction Microscopy (STORM). For SIM, murine cryosections were immunostained with antibodies for CEP290, and antibodies to other CC proteins were used to identify subcellular boundaries. For STORM, retinas were first immunostained as whole-mounts using the same set of antibodies for 2-color STORM. Ultra-thin sections were prepared for single-molecule localization imaging. The length of CEP290 staining along the CC was measured. For cell culture studies, RPE cells were serum starved for 36 hours to induce ciliation, fixed, and immunostained for SIM and STORM.
Results :
In SIM images and STORM reconstructions of rod cilia, we found that in rods CEP290 localizes throughout the length of the CC (length of 1231 ± 175 nm, n= 34), with a width corresponding to the cytoplasmic radial subcompartment of the CC between the microtubule doublets of the axoneme and the ciliary membrane. We observed nonuniform CEP290 clusters near the axoneme. In hRPE1 cells, CEP290 localizes to the base of the cilium (length of 269 ± 65nm, n=57), positioned between the basal body and ciliary axoneme.
Conclusions :
Our results support the hypothesis that CEP290 has a unique localization pattern in rod photoreceptor cells. Its distribution along the full length of the CC is not consistent with a function restricted to forming structures such as Y-shaped links at the base of the CC. The contrast between its spatial distribution in rod CC and RPE cells suggests that it may participate in different supramolecular complexes in those two cell types.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.