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) Microtubule cosedimentation assays for in vitro transcribed and translated CEP290 truncations. The supernatants (S) and microtubule pellets (P) are shown for assays performed both with (+MT) and without (–MT) microtubules. (B) Percentage of each truncation cosedimenting with microtubules. Data are presented as mean ± SD, n = 3. Symbols indicate significance as follows: *P < 0.05; †P < 0.05; ‡P < 0.05. (C) Coomassie-stained gel of microtubule cosedimentation assays performed with purified CEP290 truncation M and increasing concentrations of microtubules. The supernatants and microtubule pellets are shown. (D) Binding curve of microtubules cosedimentation assays. The fraction of truncation M present in the pellet in the absence of microtubules was subtracted from all data points. Data are presented as means ± SD, n = 3.