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Wolfgang Baehr, Christin Hanke-Gogokhia, Houbin Zhang, Jeanne M Frederick; ARL3-GTP and its GEF ARL13b regulate photoreceptor transition zone formation. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2056.
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© ARVO (1962-2015); The Authors (2016-present)
Arf-like Protein 3 (ARL3) is a ubiquitous small GTPase--the activity of which is regulated by its GAP (RP2 protein) and GEF (ARL13b). Mutations affecting the ARL13b G-domain lead to Joubert Syndrome in human, affecting primary cilia of kidney, brain, liver and other organs. We probed the function of ARL13b in mouse photoreceptors by generating a retina-specific (prefix ret) and tamoxifen-induced (prefix tam) Arl13b knockouts.
Arl13bflox/flox mice (floxed exon 2) were provided by Tamara Caspary (Emory). Retina-specific knockout mice were obtained by mating Arl13bflox/flox mice with Six3Cre+ transgenic mice and adult tamoxifen-induced knockout mice by mating with EtCre+ transgenic mice. Mutant mice were crossed with CETN2-EGFP transgenic mice to identify photoreceptor centrioles and connecting cilia. Photoreceptor function was measured by ERG and OptoMotry; progress of retina degeneration was evaluated by optical coherence tomography, confocal immunohistochemistry, histology and TEM.
ARL3-GDP distributes within the inner segment as a soluble protein while its GEF, ARL13b, localizes to the outer segment exclusively. The distinct localizations indicate that ARL3-GTP likely forms in the transition zone as a gradient. In P10 retArl13b-/- retina, photoreceptor connecting cilia were absent centrally as indicated by the transition zone marker, CETN2-EGFP, and the retina phenotype resembles LCA. Ultrastructure performed at P6 and P10 revealed that basal bodies docked to the photoreceptor cortex but transition zones were stunted in length and outer segments were not formed. As a result, outer segment proteins mislocalized in the inner segment and ONL. In tamArl13b-/- retina at 2 weeks post-injection, ARL13b is completely depleted resulting in mislocalization of phototransduction proteins and destabilization of the axoneme.
Based on Joubert syndrome-like phenotypes of germline Arl3 and Arl13b knockouts, ARL3-GTP is key for ciliogenesis and transition zone formation in photoreceptors, as well as in primary cilia of other tissues affected by ARL13B null alleles in human.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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