Lee C, Ma Y, Tu F, Wallingford JB.

R01 HL117164 NHLBI NIH HHS

             axoneme
             axoneme assembly
             proximal portion of axoneme
             distal portion of axoneme

	Fig. 1. A) Schematics of a representative vertebrate MCC axoneme (Lin et al., 2014), with relevant components of ciliary beating machineries indicated. ODA (outer dynein arm), IDA (inner dynein arm), CA (central apparatus), N-DRC (nexin-dynein regulatory complex), RS (radial spoke), MIP (microtubule inner protein), ODA-DC (ODA docking complex) B-E) The indicated ODAs, IDAs, radial spokes, and MIPs do not extend to the distal limit of the axoneme, as marked by membrane RFP. FH) The indicated ODA, radial spoke, and MIP are excluded from the Spef1-enriched distal domain. I) A single frame from a time-lapse movie of GFP-Ift38 and RFP-Spef1. J) A projection of 60 frames of time-lapse data reveals absence of Ift38 from the Spef1-enrched distal domain at st. 27. Scale bars = 10 m, scale bars for insets = 0.5 m.
	Fig. 2. A-C) GFP-Spef1 distal enrichment in growing axonemes at stages indicated. D-E) Eb1-GFP is enriched at the distal tip of growing axonemes at stages indicated. F) Image labeled Eb1-GFP and RFP-Spef1 G-H) The deployment of the radial spoke protein Nme5 does not reach to the Spef1-enriched domain at st. 25 but does so by st. 27. I) Plot of axoneme length versus Spef1 length, Nme5 length and the length of the gap separating them; each point represents a single axoneme. Scale bars = 10 m, scale bars for insets = 0.5 m.
	Fig. 3. A-C) Ciliary proteins fused to GFP displayed with memRFP visualizing the entire axoneme in growing axoneme. D-F) Plots of total axoneme length versus the length of the axoneme decorated by the labeled protein. Note that Cfap52 is re-plotted in the two panels to facilitate comparison. G-L) Double labeling confirms that radial spoke protiens (Nme5, Rsph1) do not display consistent differences from Cfap52, while the central apparatus proteins (Ak7, Spef2) displayed distinct kinetics, lagging behind Cfap52, Nme5 and Rsph1. Numbers of tested axonemes, cells and embryos for each protein are provided in the materials and methods. Scale bar = 5 μm, scale bar for insets = 0.5 μm.
	Fig. 4. A, B) Dnai2 and Wdr78 are clearly absent from the distal region of immature axonemes. C) ODA subunits display far slower deployment kinetics as compared to the MIP, Enkur. D) Dual labeling reveals that Enkur consistently extends distally to Dnai2 in growing axonemes, consistent with their distinct kinetics in C. E-H) IDAs display faster deployment kinetics than ODAs, and IDA sub-types display distinct kinetics. I-L) Dual labeling confirms relative rates of deployment for ODA/IDA subunits. M-P) IDAs deploy at rates slower than MIPs and radial spokes, and similar to the central apparatus. Q-T) Dual labeling confirms kinetic data on rates of IDA deployment. Note that data for Wdr78 and Dnai2 are re-plotted in multiple panels to facilitate comparison. Numbers of tested axonemes, cells and embryos for each protein are provided in the materials and methods. Scale bar = 5 μm, scale bar for insets = 0.5 μm.
	Fig. 5. A-C) Additional elements of ciliary beating machineries display unexpected kinetics, and dual labeling confirms these relative rates (D–F). Note that data for Ccdc65 and Dnai2 are re-plotted in multiple panels to facilitate comparison. G) Summary of ciliary beating machinery deployment kinetics. Graph shows the mean ratio of decorated axoneme length versus total axoneme length for each protein. H) Heatmap with adjusted p-values representing all pairwise comparisons. Significant differences are indicated in white, and no difference is indicated in dark green. Numbers of tested axonemes, cells and embryos for each protein are provided in the materials and methods. Scale bar = 5 μm, scale bar for insets = 0.5 μm.
	Fig. 4. A, B) Dnai2 and Wdr78 are clearly absent from the distal region of immature axonemes. C) ODA subunits display far slower deployment kinetics as compared to the MIP, Enkur. D) Dual labeling reveals that Enkur consistently extends distally to Dnai2 in growing axonemes, consistent with their distinct kinetics in C. E-H) IDAs display faster deployment kinetics than ODAs, and IDA sub-types display distinct kinetics. I-L) Dual labeling confirms relative rates of deployment for ODA/IDA subunits. M-P) IDAs deploy at rates slower than MIPs and radial spokes, and similar to the central apparatus. Q-T) Dual labeling confirms kinetic data on rates of IDA deployment. Note that data for Wdr78 and Dnai2 are re-plotted in multiple panels to facilitate comparison. Numbers of tested axonemes, cells and embryos for each protein are provided in the materials and methods. Scale bar = 5 μm, scale bar for insets = 0.5 μm.
	Fig. 5. A-C) Additional elements of ciliary beating machineries display unexpected kinetics, and dual labeling confirms these relative rates (D–F). Note that data for Ccdc65 and Dnai2 are re-plotted in multiple panels to facilitate comparison. G) Summary of ciliary beating machinery deployment kinetics. Graph shows the mean ratio of decorated axoneme length versus total axoneme length for each protein. H) Heatmap with adjusted p-values representing all pairwise comparisons. Significant differences are indicated in white, and no difference is indicated in dark green. Numbers of tested axonemes, cells and embryos for each protein are provided in the materials and methods. Scale bar = 5 μm, scale bar for insets = 0.5 μm.

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