Sensory stimuli and cilium trafficking defects trigger the release of ciliary extracellular vesicles from multiple ciliary locations

The primary cilium is a signaling organelle that extends from many cell types to detect and relay extracellular signals. Beyond its signaling role, the cilium also produces cilia-derived extracellular vesicles (cEVs), although the mechanisms underlying their biogenesis and functions remain poorly understood. We characterized the cEV biogenesis in vivo using ciliated sensory neurons of C. elegans. In response to sensory cues, interruption of the intraflagellar transport (IFT) -a ciliary trafficking machinery carrying cargoes along the cilium-occurs together with ciliary membrane fission, resulting in the release of cEVs. Similarly, mutants disrupting IFT and ciliary receptor trafficking also enhance cEV production. To investigate how IFT influences the rate and location of cEV biogenesis, we selected a membrane marker that spans the entire length of the ciliary membrane independently of IFT. Single-molecule tracking demonstrates that the tetraspanin TSP-6 enters and diffuses within the cilia and does it independently of IFT. Lack of receptor retrieval or receptor entry in the cilium induces membrane budding from ciliary or periciliary membranes, respectively. Prior to fission, these membrane buds get enriched in TSP-6 as well as signaling receptors. Coupling receptor buildup with their export by cEVs provides a mechanism to preserve ciliary function and to modulate ciliary signaling. HIGHLIGHTS- The cone-shaped tetraspanin TSP-6 crosses the transition zone and moves by passive diffusion within the ciliary membrane, independently of IFT. - The production of ciliary EVs loaded with TSP-6 increases upon acute sensory stimulation or when IFT of ciliary membrane proteins is disrupted. - Depending on the nature of the perturbations, ciliary EVs remove excess material from the distal, proximal or periciliary segments of the cilia.