Abstract
The mutations that cause Leber congenital amaurosis (LCA) lead to photoreceptor cell death at an early age, causing childhood blindness. To unravel the molecular basis of LCA, we analyzed how mutations in LCA5 affect the connectivity of the encoded protein lebercilin at the interactome level. In photoreceptors, lebercilin is uniquely localized at the cilium that bridges the inner and outer segments. Using a generally applicable affinity proteomics approach, we showed that lebercilin specifically interacted with the intraflagellar transport (IFT) machinery in HEK293T cells. This interaction disappeared when 2 human LCA-associated lebercilin mutations were introduced, implicating a specific disruption of IFT-dependent protein transport, an evolutionarily conserved basic mechanism found in all cilia. Lca5 inactivation in mice led to partial displacement of opsins and light-induced translocation of arrestin from photoreceptor outer segments. This was consistent with a defect in IFT at the connecting cilium, leading to failure of proper outer segment formation and subsequent photoreceptor degeneration. These data suggest that lebercilin functions as an integral element of selective protein transport through photoreceptor cilia and provide a molecular demonstration that disrupted IFT can lead to LCA.
Highlights
Leber congenital amaurosis (LCA; OMIM 204000) is the most severe hereditary retinal dystrophy
In order to provide a mechanistic view of the molecular perturbations in LCA, we developed a proteomics-based workflow to analyze the lebercilin interactome on a quantitative level with greatly increased sensitivity
By GST pulldown of exogenous lebercilin in bovine retina, we were able to detect endogenous intraflagellar transport (IFT) proteins (Figure 1E), which indicates that the physical association of lebercilin with IFT proteins is present in retina as well
Summary
Leber congenital amaurosis (LCA; OMIM 204000) is the most severe hereditary retinal dystrophy. Mutations in at least 15 genes lead to LCA [1,2,3] Despite this genetic heterogeneity, the clinical features of LCA are remarkably consistent. The connecting cilium (CC) is a specialized ciliary transition zone that supports selective transport of proteins and membrane vesicles, known as intraflagellar transport (IFT), between the protein-producing inner segment (IS) and the photoreceptive outer segment (OS) of the photoreceptor cell [3]. It involves 2 directional, highly conserved transport mechanisms along the ciliary microtubule tracks, essentially required in cilia of all species for the assembly
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.