Fig 2: NEK9-mediated selective autophagy of MYH9 is required for primary cilia formation.a Schematic representation of the C-terminal regions of Homo sapiens NEK9 and deletion mutants. CC, coiled-coil. b Immunoprecipitation using MEFs stably expressing wild-type or deletion mutation NEK9 constructs after serum starvation (4 h). Data are representative of three independent experiments. c The C-terminal region of Homo sapiens NEK9. The putative MYH9-binding region is colored in magenta (top). Multiple sequence alignment of NEK9 from terrestrial vertebrates (bottom). See also Fig. 1c. d Immunoblotting of wild-type or Nek9-KO MEFs stably expressing indicated constructs. e, Quantification of the intensity of the MYH9 bands in (d). Data represent the mean ± SEM of three independent experiments. f Immunofluorescence microscopy of Nek9-KO MEFs stably expressing indicated constructs after serum starvation (24 h). g Frequency of ciliated cells in f. Data represent the mean ± SEM of five independent experiments (300 cells were counted in each experiment). h Quantification of cilia length in (f). Data were collected from 100 ciliated cells for each cell-type. i Immunofluorescence microscopy of Nek9W967A MEFs in which MYH9 was depleted by two independent shRNAs (#1 and #2). See Supplementary Fig. 6e, f for the knockdown efficiency of MYH9 in these cells. j Frequency of ciliated cells in (i), as in (g). Data represent the mean ± SEM of five independent experiments (300 cells were counted in each experiment). k Quantification of cilia length in (i), as in (h). Data were collected from 100 ciliated cells for each cell-type; p values correspond to a Tukey’s multiple comparisons test; *p < 0.0001. Scale bars, 10 µm and 3 µm (insets). Solid bars indicate the medians, boxes the interquartile range (25th–75th percentile), and whiskers the 10th–90th percentile in (h, k).

Fig 3: NEK9 is degraded by selective autophagy.a Immunofluorescence microscopy of MEFs expressing GFP-NEK9 and mRuby3-GABARAP under nutrient-rich conditions and amino acid and serum starvation (2 h) conditions with or without 100 nM bafilomycin A1 (baf A1). b Quantification of the number of NEK9 puncta in (a); p values correspond to a Tukey’s multiple comparisons test. c Immunofluorescence microscopy of wild-type and Fip200-KO MEFs expressing GFP-NEK9 (top), and wild-type MEFs expressing GFP-NEK9 W967A (LIR-mutant) (bottom) after starvation (2 h). d, e Quantification of the number of NEK9 puncta in (c); p values correspond to two-tailed Mann–Whitney tests. f Wild-type and Fip200-KO MEFs were incubated under starvation conditions with or without 100 nM bafilomycin A1 for the indicated time. Whole-cell lysates were subjected to immunoblotting. g Quantification of the intensity of the NEK9 bands in (f). Data represent the mean ± SEM values of three independent experiments. h Immunoblotting of indicated organs of three-month-old Atg5+/+ (WT) and Atg5-/-;NSE-Atg5 (KO) mice. Data are representative of three biologically independent replicates. For (b, d, and e), data were collected from 100 cells for each condition. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 10th to 90th percentile. Scale bars, 10 µm and 3 µm (insets).

Fig 4: Selective autophagy of NEK9 is required for primary cilia formation in mouse kidneys.a Immunoblotting of the indicated organs of five-month-old wild-type (WT) and Nek9W967A/W967A mice (KI). Data are representative of three biologically independent replicates. b Immunohistochemistry of the cortical region of kidneys from five-month-old wild-type and Nek9W967A/W967A mice using LTL-FITC (the lumen of proximal-tubular cells) and anti-ARL13B antibody (primary cilia). Scale bars, 40 µm and 5 µm (insets). c Frequency of ciliated cells in LTL-FITC positive cells in (b). Data represent the mean ± SEM of three mice (300 cells were counted in each experiment). d Quantification of cilia length in LTL-FITC positive cells in (b). Data were collected from 100 ciliated cells for each genotype. Solid bars indicate the medians, boxes the interquartile range (25th–75th percentile), and whiskers the 10th–90th percentile. e Hematoxylin and eosin staining of the cortical region of kidneys from five-month-old wild-type and Nek9W967A/W967A mice. Scale bars, 100 µm and 10 µm (insets). f Measurement of the surface area of tubular cells in (e). Examples of measured areas are shown with broken lines in (e). Data represent the mean ± SEM of five mice (300 cells were counted in each experiment); p values correspond to two-tailed Mann–Whitney tests in (c, d, and f).

Fig 5: Autophagic degradation of NEK9–MYH9 and OFD1 is required for primary cilia formation.a FRAP analysis of GFP-actin in wild-type MEFs stably expressing FLAG or FLAG-OFD1 after serum starvation (24 h). Images were recorded at 5-s intervals following photobleaching of the indicated area, and fluorescence recovery at different time points was quantified. Data represent the mean ± SEM of 10 cells. b Immunofluorescence microscopy of wild-type or Nek9W967A MEFs after serum starvation (24 h). OFD1 or MYH9 was depleted by shRNA-mediated knockdown (shMYH9 #1 was used). Similar results were obtained using shMYH9 #2 and two independent shOFD1 (not shown). See Supplementary Fig. 8b–d for the knockdown efficiency. c Percentage of cells with centriolar satellites OFD1 in (b). Data represent the mean ± SEM of three independent experiments (100 cells were counted in each experiment). d Frequency of ciliated cells in (b). Data represent the mean ± SEM of five independent experiments (300 cells were counted in each experiment). e Quantification of cilia length in (b). Data were collected from 100 ciliated cells for each cell-type. Solid bars indicate the medians, boxes the interquartile range (25th–75th percentile), and whiskers the 10th–90th percentile. f Immunofluorescence microscopy of wild-type or Fip200-KO MEFs after serum starvation (24 h). OFD1 and/or MYH9 were depleted by shRNA-mediated knockdown. See Supplementary Fig. 8e–g for the knockdown efficiency. g Frequency of ciliated cells in (f), as in (d). h Quantification of cilia length in (f), as in (e); p values correspond to a Tukey’s multiple comparisons test; *p < 0.0001. Scale bars, 10 µm and 3 µm (insets). i Model of how autophagy drives primary cilia formation.