Ciliary associated extracellular vesicles modulate WNT signalling
Primary cilia are microtubule based sensory organelles that protrude from almost every cell type. Their membrane contains highly specialized receptors important for receiving and processing extracellular signals, which enables them to regulate several signalling pathways. A recently discovered characteristic is that cilia are also able to release extracellular vesicles (EVs) from the ciliary membrane. Since EVs have been shown to exert numerous functions in physiology and pathology, these findings have the potential to dramatically alter our understanding of how the primary cilium is able to regulate various signalling pathways in development and disease. In our study we focused on the release of EVs from a ciliated kidney cell line. Using control and mutant cell lines, in which ciliary trafficking was disrupted, we observed that loss of primary cilia function leads to altered EV secretion and composition in NTA (Nanoparticle Tracking Analysis) and Western blot. Cilia mutant cells released more small EVs compared to control, and their composition was also changed between mutant and control. Protein identification via mass spectrometry identified both cilia- as well as WNT signalling-associated proteins and miRNA sequencing determined WNT-related miRNAs, which were differentially expressed in small EVs isolated from the cilia mutant cell line. Because of the presence of differentially expressed WNT related molecules in these small EVs, we tested whether this would have an effect on the WNT activity of recipient cells. We observed that small EVs secreted from cilia mutant cells differentially modulated the WNT response in recipient cells compared to control. Our results highlight a possible new small EV-dependent ciliary signalling mechanism, since deficient primary cilia lead to a change in EV secretion, resulting in an altered signal transduction. These results provide us with new insights into ciliopathy disease pathogenesis. Overall design: EV preparations were obtained in triplicate from one control (WT) and two ciliary mutant (Bbs4 and Bbs6 knockout) cell lines. Cell lines were imortalised mouse kidney medullar cells as described in Hernandez-Hernandez V,  et al. (2013): Bardet-Biedl syndrome proteins control the cilia length through regulation of actin polymerization. Hum. Mol. Genet. 22(19):3858-68