ARL2 GTPase is crucial for development of photoreceptor outer segments by regulating assembly of microtubule

Abstract

Purpose : ARL2, a small GTPase in the ARF protein family belonging to the RAS GTPase super family, has been suggested to regulate microtubule assembly and stability, by interacting with chaperone proteins facilitating tubulin folding during translation. ARL2 aslo interacts PDEδ and BART. Mutations of the genes encoding PDEδ and BART cause Joubert syndrome and autosomal recessive rod-cone dystrophy in human, respectively. In order to study the role of ARL2 in photoreceptor and retina, we generated a retina-specific Arl2 knockout mouse and characterized the retina phenotype.

Methods : Retina-specific knock-out mice were generated by breeding Arl2^flox/flox mice with Six3-Cre mice. Retinal function was assessed by ERGs. For immunohistochemistry (IHC), cryosections were labeled with antibodies against rhodopsin, PDE6, and GRK1, opsins, and tubulin. Western blot was performed using antibodies against rhodopsin, PDE6, cone opsins, and GRK1. Photoreceptor morphology was analyzed by EM. Microtubule and tubulin were isolated from mouse retina to assess assembly of microbubule from tubulin dimer. Assembly of tubulin dimer was analyzed by co-immunoprecipitation.

Results : Deletion of Arl2 in mouse retina (Arl2^ret-/-) caused microphthalmia. Severe retinal degeneration was observed in Arl2^ret-/- as early as P12. By P28, photoreceptors were completely degenerated and the remaining retinal cells were disorganized. ERGs were unrecordable in Arl2^ret-/- mice at P14. Microtubule was disorganized in Arl2^ret-/- photoreceptors. Tubulin failed to assemble into microtubule in Arl2^ret-/- retina, as formation of tubulin dimer was affected in the absence of ARL2. Expression of rhodopsin, cone opsins, and PDE6 were significantly downregulated, although their trafficking to outer segments appeared normal. Consequently, the length of outer segments was dramatically reduced in Arl2^ret-/-retina.

Conclusions : ARL2 plays a critical role in assembly of tubulin dimer. ARL2 regulates formation of axoneme in photoreceptors and development of photoreceptor outer segments. Additionally, ARL2 is required for proper expression of some phototransduction proteins such as rhodopsin, cone opsins, PDE6, and GRK1.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Expression of rhodopsin in Arl2^ret-/- retina (KO) was reduced, and outer segments were greatly shortened.

 

In Arl2^ret-/- retina (KO), majority of tubulin was soluble (Super) with little present in polymerized microtubule (Pellet).