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) Scale representation of human CEP290. Known human CEP290 mutations are noted by red tick marks. The indicated domains and mutations are adapted from ref. 30. (B) Fluorescence microscopy images showing the localization pattern of GFP fusions of full-length (FL) and truncated CEP290 constructs in cycling hTERT-RPE1 cells. Cells were stained with an antibody against ARL13B and with DAPI. Scale bars: 5 μm. Insets show ×3 magnified views of areas (in boxes) of colocalization between CEP290 truncations and ARL13B. Pearson correlation coefficient for colocalization between ARL13B and CEP290 aa 1–580 is 0.53 and for CEP290 aa 1–362 is 0.65.