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 8

Overexpression of either N- or C-terminal regulatory region of CEP290 ablates normal CEP290 inhibition.

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Overexpression of either N- or C-terminal regulatory region of CEP290 ab...
(A) Fluorescence microscopy fields of hTERT-RPE1 cells transduced with lentiviral empty vector (EV), or vectors encoding either the N (aa 1–580) or C terminus (aa 1966–2479) of CEP290. Cells were stained for acetylated α-tubulin (red) and pericentrin (green) to detect primary cilia and with DAPI. Arrowheads indicate primary cilia. Arrows indicate cells with multiple axonemes originating from the same focus of pericentrin. Scale bars: 10 μm. (B) Percentage of lentivirus-treated cells forming primary cilia. Data are presented as mean ± SD, n = 3. 100 cells were counted per experiment. (C) Fluorescence microscopy images of hTERT-RPE1 cells transduced with lentiviral vectors as in A. Cells were stained for acetylated α-tubulin to detect primary cilia and with DAPI. Scale bars: 5 μm. (D) Average primary cilium length for hTERT-RPE1 cells as in C. Data are presented as mean ± SD, n = 3. A total of at least 150 cilia were measured per condition. *P < 0.05.