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) Schematic representation of the microtubule-binding region of human CEP290 in relation to the rd16 mouse deletion (14). Truncations of CEP290 representing the part of the microtubule-binding region deleted in the rd16 mouse and the part of the microtubule-binding region maintained in the rd16 mouse were created as shown. (B) Confocal fluorescence microscopy images showing the localization pattern of GFP-tagged maintained and deleted CEP290 truncations. Cells were stained for α-tubulin (red) and pericentrin (white) and with DAPI (blue). Scale bars: 10 μm. (C) Microtubule cosedimentation assays for in vitro transcribed and translated maintained and deleted CEP290 truncations. The supernatant and microtubule pellet fractions are shown in assays performed both with and without microtubules. (D) Percentage of each truncation cosedimenting with microtubules. Data are presented as mean ± SD, n = 2. (E) Microtubule cosedimentation assays performed on full-length WT and rd16 Cep290 from mouse brain homogenate. The supernatant and microtubule pellet fractions are shown in assays performed on samples induced to polymerize microtubules and samples treated to prevent microtubule polymerization. (F) Percentage of WT and rd16 Cep290 cosedimenting with microtubules. Data are presented as mean ± SD, n = 3. Asterisks indicate statistical significance over –MT samples. *P < 0.05.