Abstract
The structure and function of the primary cilium as a sensory organelle depends on a motor-protein-powered intraflagellar transport system (IFT); defective IFT results in retinal degeneration and pleiotropic disorders such as the Bardet Biedl syndrome (BBS) and defective hedgehog (HH) signaling. Protein transport to the cilium involves Rab GTPases. Rab8, together with a multi protein complex of BBS proteins, recruits cargo to the basal body for transport to the cilium. Loss of Rab23 in mice recapitulates the HH phenotype but its function in HH signaling is unknown. Here we established a novel protocol, based on fluorescence recovery after photo-bleaching (FRAP), allowing the quantitative analysis of protein transport into the cilium of MDCK cells. We compared the effect of Rab8, Rab5 and Rab23 on the ciliary transport of the HH-associated transmembrane receptor Smoothened, the microtubular tip protein EB1, and the receptor protein Kim1. Ciliary FRAP confirmed the role of Rab8 in protein entry to the cilium. Dominant negative Rab5 had no impact on the ciliary transport of Smoothened or EB1, but slowed the recovery of the apical protein Kim1 in the cilium. Depletion of Rab23 or expression of dominant-negative Rab23 decreased the ciliary steady state specifically of Smoothened but not EB1 or Kim1, suggesting a role of Rab23 in protein turnover in the cilium.