Lee M , Nagashima K , Yoon J , Sun J , Wang Z , Carpenter C , Lee HK , Hwang YS , Westlake CJ , Daar IO .

NIH HHS , NCI NIH HHS

	Figure 1. CEP97 associates with Dyrk1a. (A) Exogenous CEP97 interacts with Dyrk1a. The indicated mRNAs were injected into Xenopus embryos, and co-IPs were conducted. (B) Endogenous CEP97 interacts with Dyrk1a. Endogenous Dyrk1a was precipitated from HEK293T cell lysates and immunoblotted with the indicated antibodies. (C) Schematic diagram of CEP97 deletion mutants and depiction of ability in co-IP with Dyrk1a. (D) The aa region 541–565 of CEP97 is required for Dyrk1 interaction. Using embryos expressing the indicated proteins, reciprocal co-IPs were performed. (E) Schematic diagram of Dyrk1a deletion mutants and depiction of ability in co-IP with CEP97. Representative domains are indicated: WT; N, N-terminus; KD, kinase domain; PEST, domain rich in proline, glutamate, serine, and threonine; His, histidine repeat; ST, serine/threonine-rich region; C, C-terminus. (F) His region of Dyrk1a is required for CEP97 interaction. Embryo lysates expressing the indicated proteins were used for reciprocal co-IP assay. (G) Dyrk1a kinase activity is not necessary for CEP97–Dyrk1a interaction. The indicated mRNAs were injected into embryos, and lysates were used for co-IPs. (H) Embryo lysates expressing the indicated protein were incubated with or without λ PPase at 30°C for 30 min and analyzed in 6% polyacrylamide gel. PPI, phosphatase inhibitor. (I) Dyrk1a-mediated CEP97 protein shift was reversed by λ PPase. The indicated recombinant proteins were incubated with active Dyrk1a and/or λ PPase at 30°C for 30 min. (J) CEP97 amino acid sequence comparison among different species. The region including the residues that Dyrk1a phosphorylates is conserved among different species. P, phosphorylation; conservation: fully conserved (*), strongly conserved (:), weakly conserved (.). Numbers marked on left side of Western blot images represent protein molecular weight (kilodaltons).
	Figure 2. CEP97-Dyrk1a modulates multiciliation in Xenopus MCCs. (A and D) The acetylated tubulin signal in MCCs is decreased upon CEP97 knockdown (A) and Dyrk1 knockdown (D). A cocktail of mRNAs and morpholinos was injected into one ventral blastomere in eight-cell stage embryos, cultured to stage 27, and fixed. Multicilia were visualized by antiacetylated tubulin staining (magenta), and membrane GFP (green) was used as a tracer. Scale bars, 50 µm. Con, control. (B and E) Quantification of relative acetylated tubulin intensity in A (number of images for quantification, n = 40; embryos per group from three independent experiments for each condition, n = 20) and D (number of images for quantification, n = 32; embryos per group from three independent experiments for each condition, n = 16). ****, P < 0.0001; one-way ANOVA. Error bars indicate ±SD. (C) Immunoblot of the indicated exogenous proteins expressed for the rescue experiment in A. Numbers marked on left side of Western blot images represent protein molecular weight (kilodaltons). (F) SEM and TEM of embryonic epidermis at stage 26. SEM revealed decreased number and length of multicilia in CEP97 and Dyrk1a morphant MCCs. The indicated MOs were injected into both ventral blastomeres at the eight-cell stage. Red arrows point to mispositioned basal bodies. Scale bars, 5 µm (SEM) and 1 µm (TEM). (G) Quantification of cilia length per MCC, n = 10; embryo per group, n = 5; ****, P < 0.0001, unpaired two-tailed t test. Error bars indicate ±SD.
	Figure 3. Abnormal basal body migration and docking in CEP97 and Dyrk1a morphant MCCs. (A) Defective basal body migration and docking to the apical surface of MCCs upon knockdown of CEP97 and Dyrk1a. The injected embryos were fixed at stage 25 and stained with phalloidin (green) to visualize apical F-actin. Cent-RFP (magenta) marks basal bodies. Arrow marks apical surface. Serial z-stack confocal images were projected in the y–z plane. Scale bars, 3 µm. (B) Apical actin meshwork decreases in CEP97 and Dyrk1a morphant MCCs. Embryos were fixed at stage 25. Images were generated by maximum-intensity projection of serial confocal z-stack images from surface (0 µm) to subapical (−2.5 µm). Cent-RFP (magenta) marks basal bodies. Phalloidin (green) stains apical F-actin. Antiacetylated tubulin antibody stains cilia. Scale bars, 10 µm. (C) Quantification of phalloidin intensity in an MCC in B. ****, P < 0.0001, one-way ANOVA; MCCs, n = 45; embryos per group from one representative of three independent experiments, n = 9. Data are mean ± SD. (D) Basal bodies contain distal appendages in CEP97 and Dyrk1a morphant MCCs. Embryos injected with the indicated mRNAs and MOs were harvested at stage 27. Images were generated using 3D-SIM. Images in white dotted squares are magnified to the right. Data are representative of three independent experiments. Scale bars, large images = 2 µm; small images = 0.3 µm.
	Figure 4. CEP97 forms a complex with Dyrk1a and Plk1. (A) The subcellular colocalization of CEP97 and Plk1 in migrating MCCs. Embryos injected with the indicated mRNAs were fixed at stage 19 and transversely sectioned. CEP97-mCherry, magenta; Plk1-GFP, green. DAPI was used to stain nuclei. Scale bars, 5 µm. (B) The phosphorylation of CEP97 is required for CEP97-Plk1 interaction. The embryos were injected with the indicated mRNAs, and reciprocal co-IPs were performed. (C) CEP97 mutant lacking aa 541–565 does not associate with Plk1. Reciprocal co-IPs were performed with embryo lysates as indicated. (D) Schematic diagram of Xenopus Plk1 deletion mutants. (E and F) PB domain is required for CEP97-Plk1 binding. CEP97 WT (E), but not CEP97 SA (F), associates with WT Plk1 and PB alone. The indicated mRNAs were injected into one-cell stage embryos, and reciprocal co-IPs were performed. (G) CEP97 mediates Dyrk1a-Plk1 interaction. Embryos injected with the indicated MOs or mRNA were lysed at stage 16 for co-IPs. (H) Quantification of co-IP protein bands in G. Co-IP protein band density was normalized to input. Graph was generated from three independent reciprocal co-IPs. ***, P < 0.001; one-way ANOVA. Data are mean ± SD. (I) The His region of Dyrk1a is necessary for Dyrk1a–Plk1 interaction. The embryo lysates expressing the indicated exogenous proteins were used to perform co-IPs. Numbers marked on left side of Western blot images represent protein molecular weight (kilodaltons).
	Figure 5. CEP97-Dyrk1a modulates centriole disengagement. (A) The subcellular localization of CEP97 to deuterosomes. The mRNAs of CEP97-HA and GFP-Ccdc78 (deuterosome marker) were injected into two ventral blastomeres at the eight-cell stage. Embryos were fixed at stage 19 and transverse sectioned, followed by immunofluorescence with HA probe (red), anti-GFP (cyan), and anti-centrin (magenta) antibodies. Images were generated by 3D-SIM. Scale bar, 1 µm. (B) Defective centriole disengagement upon knockdown of CEP97 and Dyrk1a. The indicated combinations of the mRNAs and MOs were injected into one blastomere of eight-cell stage embryos and fixed at stage 23. Cent-RFP marks basal bodies (red). GFP-Ccdc78 marks deuterosomes (cyan). Cilia are visualized by acetylated tubulin antibody staining. Images in white dotted square are magnified to the right. Rightmost images are 3D projections. Scale bars, large images = 20 µm; small images = 5 µm. Con, control. (C) Quantification of completed centriole disengagement in MCCs in B. Cent-RFP expressing MCCs in a field were considered for quantification. Graph is mean percentage ± SD of MCCs per image. Image numbers for quantification, n = 30; embryos per group from three independent experiments for each condition, n = 25; ***, P < 0.001; ****, P < 0.0001; one-way ANOVA.
	Figure 6. Separase is required for centriole disengagement in Xenopus MCCs. (A and B) Knockdown of Separase (Sepa; A) and expression of mutant Separase (B) caused centriole disengagement defects. The indicated mRNAs and MOs were injected into one ventral blastomere of eight-cell stage embryos, and embryos were fixed at stage 23. GFP-Ccdc78, cyan; Cent-RFP, red. Antiacetylated tubulin antibody stains cilia. Images in white dotted square are magnified to the right. Rightmost images are 3D projections. Scale bars, large images = 20 µm; small images = 5 µm. (C) Quantification of completed centriole disengagement in MCCs in A. Graph is mean percentages ± SD of MCCs per image. Images for analysis, n = 30; embryos per group from three independent experiments, n = 20; ****, P < 0.0001; one-way ANOVA. (D) Immunoblot of the indicated exogenous proteins in A. Con, control. (E) Quantification of completed centriole disengagement in MCCs in B. Graph is mean percentage ± SD of MCCs per image. Images for analysis, n = 30; embryos per group from three independent experiments, n = 22; ****, P < 0.0001; one-way ANOVA. (F) Immunoblot of the indicated exogenous proteins in B. Numbers marked on left side of Western blot images represent protein molecular weight (kilodaltons).
	Figure S1. CEP97 knockout causes ciliogenesis defects in MCCs. (A) The antisense MOs and morpholino-resistant CEP97 construct nucleotide sequence are aligned with the X. laevis CEP97 target sequence. (B) The verification of morpholino knockdown efficacy. Immunoblot shows morpholino-resistant (MO resi) CEP97 expression in morpholino coinjected embryos, but WT CEP97 expression is compromised by morpholinos. Numbers marked on left side of Western blot images represent protein molecular weight (kilodaltons). (C) CEP97 knockout phenocopies CEP97 knockdown effect on MCC cilia formation. Cas9 protein with or without CEP97 targeting gRNA was coinjected with Cent-RFP mRNA to one ventral blastomere of four-cell stage embryos, and the embryos were fixed at stage 27. Cilia was visualized by immunofluorescence using acetylated tubulin antibody. Phalloidin (green) marks apical F-actin. Scale bars, 5 µm. (D) Confirmation of CEP97 genetic disruption by DNA-sequencing data using genomic DNAs of embryos in C.
	Figure S2. CEP97 and Dyrk1a are required for ciliation in MCCs, and CEP97 is required in GRP. (A and C) A cocktail of the indicated mRNAs and MOs was injected into one ventral blastomere of four-cell stage embryos and fixed at stage 27. Acetylated tubulin staining (magenta) marks cilia. Mem-GFP (green) was used as a tracer. Scale bars, 50 µm. Con, control. (B) Quantification of relative acetylated tubulin intensity in A (image numbers for analysis, n = 30; embryos per group from three independent experiments for each condition, n = 16). ****, P < 0.0001; one-way ANOVA. Error bars indicate ±SD. (D) Quantification of relative acetylated tubulin intensity in C (image number for analysis, n = 25; embryos per group from three independent experiments, n = 14). ****, P < 0.0001; one-way ANOVA. Error bars indicate ±SD. (E and F) Quantification of the total percentage of MCCs in A and C. No significant difference is observed in the percentage of total MCCs in CEP97 and Dyrk1a morphants. Image numbers for analysis, n = 25; one-way ANOVA. (G) TEM of multicilia in control, CEP97, and Dyrk1a morphant MCCs. Cross-sectioned TEM images do not show any structural defects in axonemes on CEP97 and Dyrk1a knockdown. Scale bars, 100 nm. (H) CEP97 knockdown, but not Dyrk1a knockdown, decreases the length of GRP cilia. A cocktail of the indicated mRNAs and MOs was injected into two dorsal blastomeres of eight-cell stage embryos and fixed at stage 16. Acetylated tubulin staining (magenta) marks GRP cilia. Mem-GFP (green) was used as a tracer. Scale bars, 50 µm. (I) Quantification of GRP cilia length in H. Measured cilia number per group, n > 800; dissected GRPs per group from three independent experiments, n = 20. ****, P < 0.0001; one-way ANOVA. Error bars indicate ±SD. A.U., arbitrary units.
	Figure S3. CEP97 localizes to deuterosome and associates with Cdc20B and Spag5. (A and B) CEP97 localizes near the basal body area (Cent-RFP) to an extent; yet CEP97 broadly localizes to the apical region of MCCs. Dyrk1a colocalizes with Cent-RFP. The indicated mRNAs were injected, and the embryos were fixed at stage 23 for confocal imaging. Scale bars, 5 µm. (C and D) The subcellular localization of CEP97 and Plk1 in migrating MCCs. The indicated synthetic mRNAs were injected into one ventral blastomere of four-cell stage embryos. The embryos were fixed at stage 19 and transverse sectioned. Cent-RFP, magenta; CEP97-GFP and Plk1-GFP, green. DAPI stains nuclei. Arrows mark the apical surface. Scale bars, 5 µm. (E) Schematic diagram of human CEP97 WT and deletion mutant (Δ577–610, corresponding to Xenopus CEP97 Δ541–565). (F and G) The indicated DNAs were transfected into HEK293T cells, and reciprocal co-IPs were performed. WT hCEP97-HA, but not Δ577-610 mutant-HA, associates with hCdc20B-Flag (F) and hSpag5-Flag (G). Numbers marked on left side of Western blot images represent protein molecular weight (kilodaltons).
	Figure S4. CEP97 and Dyrk1a do not affect one another’s deuterosome localization. (A) Dyrk1a knockdown does not affect CEP97 localization to deuterosome. The embryos injected with the indicated mRNAs and MOs were fixed at stage 19 and sectioned, followed by immunostaining and 3D-SIM. Cep97-HA, red; GFP-CCDC78, cyan. Scale bars, 500 nm. Con, control. (B) Loss of CEP97 and Dyrk1a does not change Plk1 localization to deuterosome. Embryos were fixed at stage 19 and sectioned. Plk1-HA, red; GFP-CCDC78, cyan; centrin, magenta. Images were generated by 3D-SIM. Scale bars, 500 nm.
	Figure S5. CEP97 and Dyrk1a are required for centriole disengagement in Xenopus MCCs. (A) Although defective centriole disengagement is relieved to some extent during embryonic development, a significant number of MCCs in CEP97 (46%) and Dyrk1a (55%) morphants still show incomplete centriole disengagement at stage 27. The cocktail of the mRNAs and MOs was injected into one ventral blastomere of eight-cell stage embryos and fixed at stages 23, 25, and 27. Cent-RFP (red) marks basal bodies. Deup1-GFP (cyan) marks deuterosomes. Cilia are visualized by acetylated tubulin antibody staining. Scale bars, 20 µm. Con, control. (B–D) Quantification of MCCs that completed or failed to complete centriole disengagement in A. Cent-RFP expressing MCCs in a field were considered for quantification. Bar graphs display the mean percentage of disengaged MCCs ± SE. Number of field images for measurement per group, n = 20; embryos per group from three independent experiments, n = 20; ****, P < 0.0001; one-way ANOVA.

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