Fig. 1: Emergence of actin nanoclusters in the apical cortex during tracheal tube expansion.

a Schematic of the tracheal tube constituted by multiple cells. The intercellular periodic circumferential actin cables appear beneath the apical (luminal) membrane. The right image shows a representative image of F-actin pattern at stage 16 visualized by phalloidin staining. Three experiments were repeated independently. Scale bar, 5 µm. b Time course of cortical actin orientation, actin cable elongation, tracheal tube expansion, and tube elongation at stage 15. The time point when the circumferential cables formed was defined as 0* min. c, d Actin pattern visualized by lifeact::GFP in the fixed embryos at stages 15 and 16, respectively. Two representative images for stage 15 are shown in (c). Lower square images indicate a magnified view of the yellow square (left) and its binarized image (right). Scale bars: 2 µm (upper) and 0.5 µm (lower). Eight and seven independent tracheal tubes were observed for stage 15 and stage 16, respectively. e, f Comparison of the cluster-fitted ellipses between stage 15 (n = 356, N = 22) and stage 16 (n = 271, N = 21) with aspect ratio (f), and angle of the major axis of clusters (e, 0° corresponds to longitudinal axis whereas 90° indicates circumferential axis of the tube). For statistical analyses, two-sided two-sample Kolmogorov–Smirnov test was performed for f (P = 3.17e-4), and two-sided Watson’s two-sample test of homogeneity was performed for e (P < 0.001). ***P < 0.001. See “Cluster analysis” for details. g Time-lapse images showing the tracheal tube of living embryos expressing lifeact::GFP in the tracheal cells. Scale bar, 2 µm. h, i Transition of dominant direction and coherency of the actin pattern every 10 min. Coherency indicates how the gradient tensor of the actin intensity distribution pattern is aligned toward the dominant direction. The coherency is used as a measure of the connectivity of the nanoclusters in this study (“OrientationJ analysis”). The mean (green line) and SD (light green region) are indicated. n = 6 (−80, −70, −60, −50, −40 min), 7 (−30 min), 9 (−20, −10, 0 min), 8 (10 min), or 5 (20 min) biologically independent samples. j, k Transition of tube diameter and length of the dorsal trunk, respectively. The mean (blue line) and range of values (light blue region) are indicated. n = 6 biologically independent samples. The dotted vertical lines in h–k indicate when the circumferential cables are formed (0* min). Source data are provided as a Source Data file.