KIF13B controls ciliary protein content by promoting endocytic retrieval and suppressing release of large extracellular vesicles from cilia.

Dynamic control of ciliary membrane protein content is crucial for the organelle's homeostasis and signaling function and involves removal of ciliary components by intraflagellar transport (IFT) and BBSome-mediated export, endocytic retrieval, and/or extracellular vesicle (EV) shedding. We report that the kinesin-3 motor KIF13B regulates ciliary protein composition and EV shedding in cultured kidney epithelial cells, with effects that vary over time. In early stages of ciliation, Kif13b^-/- cells aberrantly accumulate polycystin-2 (PC2) within cilia and release large EVs enriched with CCDC198 and the centriole distal appendage protein CCDC92, which also localizes to the ciliary tip. These cells also produce fewer small EVs through the neutral sphingomyelinase 2 pathway. Upon cilia maturation, Kif13b^-/- cells accelerate large EV release of numerous ciliary proteins, including PC2, BBSome, and IFT components, which correlates with gradual depletion of CCDC92 and PC2 from the ciliary tip and shaft, respectively. Furthermore, over time, Kif13b^-/- cells show an upregulation in the release of small EVs, which differ in composition from wild-type small EVs. Specifically, mutant small EVs lack several proteins that are enriched in small EVs from BBSome-deficient cells, including palmitoyl transferase ZDHHC5, which localizes to cilia where it accumulates upon BBSome dysfunction and regulates ciliary length and PC2 levels. Our results suggest that KIF13B acts at the level of centriole distal appendages to limit ciliary protein entry and promote endocytic retrieval downstream of the BBSome, thereby suppressing EV release from cilia. Furthermore, the ciliary localization of CCDC198 and ZDHHC5 indicates they are potential novel ciliopathy candidates.