Werner ME , Hwang P , Huisman F , Taborek P , Yu CC , Mitchell BJ .

R01 GM089970 NIGMS NIH HHS

	Figure 1. Two pools of cortical actin are observable in multiciliated cells. (A and B) Apical actin (A) forms a meshworklike network in the same plane as basal bodies, and a second pool of actin (B) localizes slightly subapically. (C and E) A late cell depicting the highly organized subapical pool of actin. The dotted line in C indicates the cross section shown in E. (D) Subapical actin in a multiciliated cell from an early embryo. (F) A diagram of subapical actin interactions with striated rootlets and basal bodies. Posterior is to the right in all panels.
	Figure 2. Cyto D and Noc treatment affects the generation of robust fluid flow but does not affect cilia structure or beat frequency. (A–C) Cyto D (A)–, mock (B)-, or Noc (C)-treated embryos stained with β-tubulin and phalloidin. Apical (A′) and subapical (A″) staining of actin with phalloidin of an embryo treated with 10 µM Cyto D. (B′ and C′) Images of maximum projection time-lapse videos acquired every 2 s over a 30-s time period of CLIP170-expressing embryos from control (B′) or Noc-treated cells (C′). (D) Quantification of flow velocity for embryos treated between stages 23 and 29 with mock, Cyto D, or Noc (n = 50, 10 flow lines from five embryos for each condition). (E) Quantification of the mean beat frequency of embryos treated between stages 23 and 29 with mock, Cyto D, or Noc (n = 75, five cilia from three individual cells on five different embryos for each condition). All error bars represent the SD of the mean.
	Figure 3. Perturbation of either actin or microtubule dynamics inhibits establishment of cilia polarity. (A–D, left) Representative images from each experimental condition. (right) Circular diagrams of mean cilia orientation (direction of arrow) as well as the variation around that mean (vector length = 1/variance) such that a short arrow represents a cell with high variance, and a long arrow represents a cell with low variance (arrow colors represent data from different embryos). (A) An untreated wild-type embryo at stage 23. (B–D) Embryos treated from stage 23–29 with 0.1% DMSO (B), 10 µM Cyto D (C), or 1 µM Noc (D). (E) Comparison of the average mean vector length (n > 60, >20 cells from at least three embryos). wt, wild type. (F) Quantification of basal body distribution by measuring the distance of individual basal bodies relative to their nearest neighbors (n > 6,000, 100–150 basal bodies from >20 cells in three embryos). (G) Angular correlation analysis for both nearest and farthest neighbor basal body pairs (n > 2,000, 100–150 basal body pairs from five cells in three embryos for each condition). All error bars represent the SD of the mean.
	Figure 4. Cytoplasmic microtubules form an organized network in multiciliated cells that becomes polarized during cilia refinement. (A) A late embryo injected with the microtubule marker EMTB-3XGFP and centrin-RFP. (B) A late embryo with EMTB-3XGFP and mCherry-CLAMP. (A and B) The boxed areas are enlarged on the right. (C and D) Triple labeling with centrin–tag BFP, RFP-CLAMP, and EMTB-3XGFP in early (C) and late (D) embryos, with circular graphs of the mean position of microtubule foci relative to the nearest basal body (n > 15, five cells from three embryos) and a diagram of microtubule basal body interactions.
	Figure 5. Loss of subapical actin results in a loss of metachronal cilia beating. (A) A cell from a polarized embryo treated with Cyto D with centrin-RFP and GFP-CLAMP and stained with phalloidin. The dotted line indicates the cross section in A’. (B) Circular diagrams showing mean cilia orientation from polarized embryos treated at stage 29 with either 0.1% DMSO (top) or 10 µM Cyto D (bottom) for 12 h before fixation. (C) Kymographs at cilia tips from videos of cilia beating from 0.1% DMSO (top; Video 2) and 10 µM Cyto D–treated embryos (bottom; Video 3). (D) A diagram of the high degree of polarized cytoskeletal organization in ciliated cells and the proposed model for the propagation of a physical signal involved in metachronal synchrony.

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