Abstract
Purpose :
CEP290 (centrosomal protein 290), a large multi-domain containing protein, is a key component of the transition zone in many primary and motile cilia. Mutations in Cep290 cause several ciliopathies, including Joubert Syndrome (JS) and retinal specific disorders such as Leber Congenital Amaurosis (LCA). Our goal is to determine the precise location of CEP290 and the structural effects of CEP290 defects at the nanometer scale.
Methods :
We have used a battery of electron and fluorescence microscopy techniques, including transmission, immuno-, and cryo-electron microscopy, as well as super-resolution and expansion fluorescence microscopy to examine ciliary ultrastructure in WT mice and two Cep290 mutant mouse lines with early-onset retinal degeneration: One line is virtually devoid of functional CEP290 (near-null, nn) with the N-terminus present at low levels, but localized to the base of the CC, and the other is a complete knockout (KO) of CEP290.
Results :
At post-natal day 10 in both CEP290 mutants, we observe formation of CC with Y-links, but few or no outer segment (OS) discs, instead filled by extracellular vesicles. Immunogold TEM reveals the ultrastructural location of CEP290 between the microtubules and the ciliary membrane, but not on the Y-links. Immunogold TEM and expansion microscopy suggest a spiral periodicity in CEP290 staining around the CC. In general, the ciliogenesis defects at P10 are more severe in the KO: mislocalization of specific ciliary proteins, instances of failure to form ciliary membranes (i.e., nascent ciliary structures inside the cells), and the “ciliary necklace,” periodic external protrusions seen along the CC membrane, is largely missing in the few cilia that form in the KO.
Conclusions :
These results suggest that while CEP290 is not a structural component of Y-links in the CC, it may serve a “gate keeper” function for protein trafficking and sorting. CEP290’s localization throughout the length of the CC, an observation unique to photoreceptors and observed for a subset of, but not all, ciliary transition zone proteins, may help explain the retina-specific symptoms of several CEP290 mutations. As seen in the near-null retinas in these studies, and previously in the rd16 deletion mutant, complete loss of CEP290 protein leads to more profound defects than do reduced levels of shortened forms of the protein.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.