Mutations in the gene centrosomal protein 290 kDa (CEP290) cause an array of debilitating and phenotypically distinct human diseases, ranging from the devastating blinding disease Leber congenital amaurosis (LCA) to Senior-Løken syndrome, Joubert syndrome, and the lethal Meckel-Gruber syndrome. Despite its critical role in biology and disease, very little is known about CEP290’s function. Here, we have identified 4 functional domains of the protein. We found that CEP290 directly binds to cellular membranes through an N-terminal domain that includes a highly conserved amphipathic helix motif and to microtubules through a domain located within its myosin-tail homology domain. Furthermore, CEP290 activity was regulated by 2 autoinhibitory domains within its N and C termini, both of which were found to play critical roles in regulating ciliogenesis. Disruption of the microtubule-binding domain in a mouse model of LCA was sufficient to induce significant deficits in cilium formation, which led to retinal degeneration. These data implicate CEP290 as an integral structural and regulatory component of the cilium and provide insight into the pathological mechanisms of LCA and related ciliopathies. Further, these data illustrate that disruption of particular CEP290 functional domains may lead to particular disease phenotypes and suggest innovative strategies for therapeutic intervention.
Theodore G. Drivas, Erika L.F. Holzbaur, Jean Bennett
(A and B) Fluorescence microscopy images of hTERT-RPE1 cells that formed multiple cilia after transduction with lentiviral vector encoding the N terminus of CEP290. Cells were stained for either ARL13B or pericentrin and for acetylated α-tubulin and with DAPI (blue). Scale bars: 5 μm. (C) Percentage of lentivirus-transduced hTERT-RPE1 cells forming multiple cilia. At least 100 cells were counted per experiment. Data are presented as mean ± SD, n = 3.