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C. Insinna, M. Humby, H. Scoma, J. C. Besharse; Circadian Rhythms Control KIF17 Mediated IFT in Vertebrate Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5451.
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In vertebrate photoreceptors, intraflagellar transport (IFT) is required to support daily biogenesis of membranes constituting the outer segment (OS), a modified sensory cilium responsible for the initial steps of visual transduction. Among the kinesin 2 family members, kinesin II and KIF17 play essential roles in photoreceptor IFT. KIF17 was recently shown to release its cargo upon phosphorylation by calcium-calmodulin-dependent protein kinase II (CaMKII), a well studied molecular switch involved in neuronal signaling. In vertebrate photoreceptors, the activity of CaMKII depends on the intracellular calcium level that is controlled in part by the dopaminergic system. Dopamine is secreted by the inner retina during the daytime and activates the photoreceptor dopamine receptor 4 (DRD4) which in turn decreases intracellular calcium levels and therefore CaMKII activity. In this study we tested the hypothesis that CaMKII-mediated phosphorylation of KIF17 regulates photoreceptor IFT in a circadian dependent manner.
Co-immunoprecipitation and co-immunolocalization of KIF17, CaMKII and IFT proteins were performed in mouse isolated photoreceptors or retinal extracts in the presence or absence of peptide treatments aimed at inhibiting CaMKII activity. Furthermore, the physiological role of KIF17 phosphorylation was assessed by analyzing the structure of photoreceptors expressing non-phosphorylatable and phospho-mimicry mutants of KIF17 in zebrafish using electron microscopy. The effect of modulating DRD4 activity on KIF17 localization was determined in mouse and zebrafish photoreceptors. Finally, the circadian expression of KIF17 was identified over 24 hours by western blotting.
Our data show that the inhibition of CaMKII results in the dissociation of KIF17 from IFT88 and depletion from the OS. Furthermore, the non-phosphorylatable KIF17 mutant was restricted to the inner segment, while the phospho-mimicry KIF17 mutant was strongly expressed in the OS along the axoneme and increased OS disc shedding. The targeting of KIF17 to the OS was also affected by treatment with a DRD4 agonist known to reduce intracellular calcium levels and CaMKII activity. KIF17 expression exhibited a strong circadian rhythm with a peak at ZT20 and a progressive decrease until ZT8.
Our results suggest that in vertebrate photoreceptors, KIF17- mediated IFT is temporally regulated by CaMKII activity and KIF17 expression levels to facilitate an efficient renewal of the sensory OS.
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