Fig. 1

ARL13B associates with FIP5 and regulates the latter’s enrichment at the ciliary base in a temporal-spatial manner. a Indicated plasmids were transfected in HEK-293T cells for 48 h and the interaction between exogenous FIP5 and ARL13B proteins was detected by immunoprecipitation. b Direct interaction between FIP5 and ARL13B was detected by GST pull-down assay. GST-fused WT ∆19, N domain, C domain and Proline-rich region of ARL13B and His-FIP5 proteins were used. c In hTERT-RPE-1 cells, endogenous ARL13B strongly associates with FIP5 during the early stage of ciliogenesis but not in non-ciliated cells or the cells with mature cilia. d FIP5-positive vesicles specifically translocate to the ciliary base during ciliogenesis. Endogenous FIP5 (upper), acetylated tubulin (Ac-Tub) and FBF1 were immunostained by corresponding antibodies and FIP5-mcherry (lower) was shown by direct fluorescence. Ac-Tub was used as a ciliary marker and FBF1 was used as the transition fiber marker. Scar bar: 10 μm. e ARL13B-EGFP RCTE stable cells were serum starved for 2 h. Structure illumination microscopic study shows that FIP5-positive vesicles locate immediately adjacent to ARL13B signal at the ciliary base in a newly synthesized cilium. Endogenous FIP5 was immunostained by antibody and ARL13B-EGFP was shown by direct fluorescence. Association of FIP5-positive vesicles and ARL13B were indicated by white triangles. Scar bars: 500 nm. f Depletion of ARL13B abolished the recruitment of FIP5-positive vesicles at the ciliary base in hTERT-RPE-1 cells. Quantification of FIP5 total intensity at mother centriole (non-ciliated cells) or cilia base (30 μm² area were measured). Scar bar: 10 μm. Center values represent mean. Error bars represent s.d. N values: cilia number accessed from at least six fields. Statistical significance was determined using unpaired Student’s t test. ** p < 0.01