Disruption of CEP290 microtubule/membrane-binding domains causes retinal degeneration
Theodore G. Drivas, … , Erika L.F. Holzbaur, Jean Bennett
Theodore G. Drivas, … , Erika L.F. Holzbaur, Jean Bennett
Published September 24, 2013
Citation Information: J Clin Invest. 2013;123(10):4525-4539. https://doi.org/10.1172/JCI69448.
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Research Article Cell biology

Disruption of CEP290 microtubule/membrane-binding domains causes retinal degeneration

Abstract

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.

Authors

Theodore G. Drivas, Erika L.F. Holzbaur, Jean Bennett

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Figure 2

CEP290 aa 1–362 mediate peripheral membrane association.

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CEP290 aa 1–362 mediate peripheral membrane association. (A) Equal amoun...
(A) Equal amounts of each of 5 fractions, beginning from the top (row 1) and ending at the bottom (row 8) of the sucrose gradient of a membrane flotation assay performed on hTERT-RPE1 cells overexpressing CEP290 aa 1–580. Sucrose percentages and the percentage of CEP290 aa 1–590 found in each fraction are indicated. Blots were probed for GFP to detect CEP290 aa 1–580 and for the indicated controls. (B) ARL13B-positive membranes immunoprecipitated from postnuclear supernatants of hTERT-RPE1 cells expressing the indicated GFP-fused CEP290 truncations or GFP alone were probed with an anti-GFP antibody. 15% of the input fraction, 15% of the unbound fraction (UB), and the entire immunoprecipitated fraction (IP) were loaded. (C) The peripheral (Peri.) and integral (Int.) membrane protein fractions of hTERT-RPE1 cells expressing the indicated GFP-fused CEP290 truncations were isolated and probed for GFP and for the indicated controls. (D) Fluorescence microscopy images showing the localization pattern of GFP-fused full-length and truncated CEP290 constructs in hTERT-RPE1 cells stained for LAMP2 and with DAPI (blue). Scale bars: 5 μm. Insets show ×3 magnified views of areas (in boxes) illustrating lack of colocalization between CEP290 truncations and LAMP2.