Fig. 3: Revealing nanocluster-associated molecules that regulate cluster fusion and anisotropy response.

a Median orientation and average major length of the actin patterns are plotted for the genetically manipulated tracheal cells (“OrientationJ analysis” and “Cluster analysis”). Each circle indicates different genetic conditions: control (red), RNAi lines with mild defects (gray), RNAi lines with strong defects (blue, denoted with the target genes), and mutants found after the RNAi screen (green). See Supplementary Table 2 for details. b Confocal projection images and binarized images of control, RNAi-expressing, or mutant embryos at early stage 16. More than six independent tracheal tubes were observed for each genotype. Scale bars: 2 µm (upper) and 0.5 µm (lower). c A network of protein-protein interaction between genes, whose downregulation displayed strong phenotypes. Solid lines indicate direct interaction and dotted lines indicate indirect interaction with a single interactor. The genes are grouped by representative molecular functions. d Colocalization between the proteins of interest with actin (lifeact::mScarletx2 or lifeact::GFP) at stage 15. Pearson’s correlation coefficient was calculated based on 2-channel images shown in Supplementary Fig. 5c. Boxplots represent median plus minima and maxima with lower and upper quantiles. n indicates a biologically independent sample. The two-sided Student’s t test. P = 1.46e-5 (DAAM), P = 5.43e-4 (α-Actinin), P = 2.71e-5 (Zasp52), P = 0.0321 (Myosin II), P = 4.67e-4 (ROCK) or P = 0.4951 (RhoGEF2). ^∗P < 0.05, ^∗∗P < 0.01. e Duration of the nanocluster fusion with respect to the orientation in control, zasp52 RNAi, DAAM RNAi, and myosin II^DN expressing tracheal cells at early stage 16. The numbers of fusions (n) are written below. Boxplots represent median (bar), mean (black diamond) plus minima and maxima with lower and upper quantiles. The two-sided Kruskal–Wallis test (parentheses, P = 9.23e-4 in Control, P = 0.114 in DAAM RNAi, P = 1.41e-4 in zasp52 RNAi, or P = 1.83e-5 in myosin II^DN) followed by a pairwise comparison using the two-sided Wilcoxon rank-sum test with Bonferroni adjustment of the P value (shown with bar) was performed. ^∗∗∗P < 0.001. f Confocal projection images and binarized images of control, RNAi-expressing, or mutant embryos at late stage 16. Three independent tracheal tubes are observed for each genotype. Scale bars: 2 µm (upper) and 0.5 µm (lower). Source data are provided as a Source Data file.