Abstract
Cilia regulate several developmental and homeostatic pathways that are critical to survival. Sensory cilia of photoreceptors regulate phototransduction cascade for visual processing. Mutations in the ciliary protein RPGR (retinitis pigmentosa GTPase regulator) are a prominent cause of severe blindness disorders due to degeneration of mature photoreceptors. However, precise function of RPGR is still unclear. Here we studied the involvement of RPGR in ciliary trafficking by analyzing the composition of photoreceptor sensory cilia (PSC) in Rpgrko retina. Using tandem mass spectrometry analysis followed by immunoblotting, we detected few alterations in levels of proteins involved in proteasomal function and vesicular trafficking in Rpgrko PSC, prior to onset of degeneration. We also found alterations in the levels of high molecular weight soluble proteins in Rpgrko PSC. Our data indicate RPGR regulates entry or retention of soluble proteins in photoreceptor cilia but spares the trafficking of key structural and phototransduction-associated proteins. Given a frequent occurrence of RPGR mutations in severe photoreceptor degeneration due to ciliary disorders, our results provide insights into pathways resulting in altered mature cilia function in ciliopathies.
Highlights
RPGR localizes predominantly to the transition zone (TZ) of photoreceptor and other cilia[25,26] and interacts with TZ-associated ciliary disease proteins[26,27,28,29,30,31]
To further validate the purity of the photoreceptor sensory cilium (PSC) fractions, we carried out immunoblot analysis using antibodies against marker proteins residing in cytosol, mitochondria, as well as in the cilia
We found high representation of the category of membrane trafficking and intracellular transport among the proteins that were increased in Rpgrko PSC (Tables 3 and 4 and Supplementary Figure S2)
Summary
RPGR localizes predominantly to the TZ of photoreceptor and other cilia[25,26] and interacts with TZ-associated ciliary disease proteins[26,27,28,29,30,31]. Studies using animal models indicate that Rpgr ablation or mutation results in delayed yet severe retinal degeneration[32,33,34,35]. The precise function of RPGR and the mechanism of associated photoreceptor degeneration are poorly understood. We sought to assess the role of RPGR in ciliary trafficking by testing the effect of loss of RPGR on the composition of the photoreceptor sensory cilia in mice. Our results suggest that RPGR participates in maintaining the function of mature cilia by selectively regulating (directly or indirectly) trafficking of proteins involved in distinct yet overlapping pathways
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