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Kwangsic Joo, Jongshin Kim, Seok Hyun Lee, Joon Kim; ARL13b and CCDC41 is a key molecules for docking and tethering of a primary ciliary vesicle in human retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2961.
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© ARVO (1962-2015); The Authors (2016-present)
Dysfunction of primary cilium, also known as a ciliopathy, causes diverse hereditary retinal degenerations such as X-linked retinitis pigmentosa and Leber’s congenital amaurosis. Recent reports show that human ciliopathy-related genes, such as CEP164 and CCDC41, are related to the centriole-vesicular docking step. However, the process of centriole-vesicular docking was not clearly defined. Here, we show the correlation and interaction between key molecules of ciliogenesis, ARL13b, CCDC41, CEP164 and CP110 in the centriole-vesicular docking step.
We used human RPE1 cellline stably expressing EGFP-tagged smoothened (Smo-GFP) was established as previously reported. Cells were transfected with 5-10nM siRNAsand human ARL13b and CCDC41 cDNAs cloned into plasmid vectors using Lipofectamine. For indirect immunofluorescence, cells were fixed in paraformaldehyde 8 minutes at room temperature and then methanol for 2 minutes at -20 'C. Primary antibodies and secondary antibodies (Alexa 488-, 594- or 647-conjugated) were applied for 1 h at room temperature. Immunoprecipitation were performed with Anti-FLAG M2 affinity gel.
ARL13b colocalizes with single Smo-GFP dot representing a primary ciliary vesicle and does not overlap with centriole marker γ-Tubulin and distal appendage marker CEP164. ARL13b depletion inhibits ciliogenesis and accumulates Smo-GFP vesicles, illustrating that ARL13b play a key role in formation of the primary ciliary vesicle. Moreover, immunofluorescence staining and co-immunoprecipitation reveals an overlap and interaction between exogenously expressed EGFP-tagged ARL13b and FLAG-tagged CCDC41, whereas mutant forms of FLAG-CCDC41 cDNAs do not overlap and interact with EGFP-ARL13b cDNAs.
The recruitment of ARL13b to the primary ciliary vesicle is indispensable for the selective formation of the primary ciliary vesicle in human RPE1 cells. And, CCDC41 is a key tether for maintaining ARL13b into the primary ciliary vesicle.
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