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. 2022 Sep 21;221(10):e202111076. doi: 10.1083/jcb.202111076

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© 2022 Liu et al.

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Figure 5. — Retrograde IFT train-shed BBSomes pass the TZ for ciliary retrieval likely via diffusion. (A) Immunoblots of WC samples and cilia of cells indicated on the top probed with α-ARL3, α-BBS8, α-BBS1, α-BBS4, α-BBS5, and α-BBS7. α-Tubulin and acetylated (Ac)-tubulin were used to adjust the loading of WC samples and cilia, respectively. MW, molecular weight. (B) Representative TIRF images and corresponding kymograms of arl3; bbs8; ARL3:HA:YFP-TG, arl3; bbs8; ARL3Q70L:HA:YFP-TG, and arl3; bbs8; ARL3T30N:HA:YFP-TG cells. The time and transport lengths were indicated on the right and on the bottom, respectively. The ciliary base (base) and tip (tip) were shown. (C) Immunoblots of ciliary fractions of arl3; bbs8; ARL3:HA:YFP-TG, arl3; bbs8; ARL3Q70L:HA:YFP-TG, and arl3; bbs8; ARL3T30N:HA:YFP-TG cells probed with α-HA, α-IFT57 (ciliary matrix marker), α-PLD (ciliary membrane marker) and Ac-tubulin (axoneme marker). (D) Schematic representation of how IFT trains and the BBSome cycle between basal body and cilia. The diffusion of retrograde IFT train-shed BBSomes through the TZ for ciliary retrieval was shown. (E) Representative TIRF images and corresponding kymograms of ift46-1; IFT46:YFP-TG, bbs8; BBS8:YFP-TG, and arl3; bbs8; BBS8:YFP-TG cells (Videos 7, 8, and 9, 15 fps). Blue braces indicate the diffusion pattern of certain BBS8-YFP inside the TZ region of bbs8; BBS8:YFP-TG cells (the middle panel) and BBS8-YFP accumulation right above the TZ of arl3; bbs8; BBS8:YFP-TG cells (the right panel). The time was indicated on the bottom. The ciliary base (base) and tip (tip), the transition zone (TZ) and the basal body (BB) were shown. The corresponding schematic representation of how IFT46-YFP and BBS8-YFP cycle between the basal body and cilia was also shown. Source data are available for this figure: SourceData F5.