Hibbard JVK , Vazquez N , Satija R , Wallingford JB .

R01 HD085901 NICHD NIH HHS , R01 HL117164 NHLBI NIH HHS

             cytosolic ciliogenesis

	FIGURE 1: Functionally related proteins show distinct turnover kinetics in the basal body pool. (A) Schematic representation of FRAP experimental setup. IFT43-GFP accumulates around Centrin2-BFP, a marker for basal bodies. (B) Representative images from FRAP experiments of Ift144-GFP, Ift20-GFP, and Centrin2-BFP. (C) Schematic of IFT-A and IFT-B complexes. Proteins investigated in this report are bolded. (D) FRAP recovery curves of IFT-A proteins (averaged from Ift144-GFP, GFP-Ift122, and Ift121-GFP), IFT-B1 proteins (averaged from Ift81-GFP, Ift52-mNG, and Ift46-mNG), IFT-B2 protein Ift20-GFP, structural basal body components (Cep164-GFP, Centrin2-BFP, GFP-Ofd1), and basal body regulators (GFP-Ttbk2, GFP-Ccdc66). For clarity of presentation, error bars are removed. (E) FRAP recovery curves of IFT-B proteins. Shaded area corresponds to SD. (F) FRAP recovery curves of IFT-A proteins. Shaded area corresponds to SD. (G) Schematic representation of a basal body, showing the localization of different proteins. (H) Mobile fraction quantification of basal body turnover kinetics for investigated proteins. Several statistical differences are noted in the tree; for full discussion of differences, see Supplemental Table S2.
	FIGURE 2: Ift52 is required for the recruitment and normal turnover of Ift46 at basal bodies. (A) Imaging of Ift46-mNG (left panel) and Ift46ΔC-mNG (right panel). (B) FRAP of Ift46-mNG in WT embryos compared with ift52 KD. (C) The mobile fraction value of Ift46-mNG is significantly lower on KD of Ift52. (D) FRAP recovery of GFP-Ift122, an IFT-A protein, is not affected by ift52 KD. (E) Mobile fraction quantification of GFP-Ift122 in WT and ift52 KD embryos.
	FIGURE 3: Induced cilia regeneration has no effect on IFT protein dynamics at the basal body. (A) Time course tracking cilia regrowth, predeciliation, postdeciliation and every hour up to the fourth hour timepoint. (B) Quantification of ciliary length prior to and during ciliary regeneration. (C) FRAP recovery curves of Ift121-GFP and Ift81-GFP at different timepoints pre-, post-, and during ciliary regeneration. (D) Mobile fraction values of Ift121-GFP and Ift81-GFP pre-, post-, and during ciliary regeneration.
	FIGURE 4: Cold-shock + nocodazole (CS + Noc) treatment eliminates cytosolic MTs and disrupts the dynamics of Ccdc66 at basal bodies. (A) Schematic representation of MCC depicting the two proposed mechanisms for IFT recruitment to basal bodies: active transport (top panel) and diffusion-to-capture (bottom panel). (B) Transverse sections of Xenopus MCCs stained for a-tubulin to verify efficacy of treatment. Actin (stained by Alexa Fluor 555 Phalloidin) and DAPI show the apical domain and nuclei, respectively. DMSO (mock)-treated cells are shown in the top panels and CS+Noc-treated cells in the bottom panels. (C) Cytosolic a-tubulin staining quantification for DMSO vs. CS+Noc-treated embryos. (D) Ciliary a-tubulin staining quantification for DMSO vs. CS+Noc-treated embryos. (E) Mobile fraction comparison of basal body turnover kinetics of Ccdc66 between DMSO and CS+Noc treatment.
	FIGURE 5: Cytosolic MTs are dispensable for IFT recruitment to basal bodies. (A) FRAP recovery curves of IFT-A proteins with and without CS+Noc treatment. (B) FRAP recovery curves of IFT-B proteins with and without CS+Noc treatment. (C) Mobile fraction comparison of basal body turnover kinetics of IFT-A proteins between no treatment and CS+Noc treatment. (D) Mobile fraction comparison of basal body turnover kinetics of IFT-B proteins between no treatment and CS+Noc treatment. (E) FRAP recovery curves of GFP-Ift122 with 5 and 1 µm2 bleaching regions. (F) Schematic representation of diffusion-to-capture, with modeling parameters depicted. The table displays the equation used and parameters obtained. (G) Experimental and model FRAP recovery curves of GFP-Ift122, with the curve-fitting equation line overlaid.
	Supplemental Figure 1. Half-times of basal body proteins are generally short. IFT and other basal body proteins generally display half-times less than 25 seconds.
	Supplemental Figure 2. Morpholino-based KD of ift52 is rescued by addition of Ift52. Basal body intensity of Ift46-mNG was measured in WT conditions and upon ift52 KD and ift52 KD + rescue.

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