Quantitative analysis of Arp2/3 and NPFs localization during apical constriction. (A) Maximum intensity projections of 10 planes spanning a total Z-depth of 5 μm, depicting C. elegans gastrulation from a ventral view with plasma membranes fluorescently labeled (mex-5p::mScarlet-I::PH). Ea and Ep cells are pseudocolored to visualize their internalization over time. The diagram to the right that shows Ea and Ep, along with their neighboring cells, is used throughout the paper to depict cellular and subcellular protein localization. (B) Micrographs from time-lapse movies depicting localization of Arp2/3 (ARX-2::TagRFP), WAVE (GFP-C1^3xFlag::GEX-3), WASP (GFP::WSP-1A), and WASH (WSHC-5::mNG-C1^3xFlag) from a ventral view. White arrowheads point to Ea and Ep cells. White arrows point to vesicle-like structures in the cytoplasm. The diagrams underneath each micrograph highlight the observed localization in Ea, Ep, and neighboring cells. (C) Diagrams representing the four regions of interest for quantification: the Ep-P₄ contact, the other cell–cell contacts, the cytoplasm, and a line scan within the cytoplasm. (D) Violin plots depicting normalized fluorescence intensity of Arp2/3, WAVE, WASP, and WASH at Ep-P₄ contacts, other cell–cell contacts, and the cytoplasm. Insets of Arp2/3 and WASP highlight the difference between the signal at the other cell–cell contacts and the cytoplasm. Measurements were collected 6 min after the division of neighboring mesoderm precursor cells (MSx) (center dot, mean; vertical line, standard deviation (s.d.); outline, the distribution of the data; n ≥ 10 embryos). (E) Representative line scan measurements of Arp2/3, WAVE, WASP, and WASH. (F) Violin plots depicting the differences between the maximum and minimum gray values along the line scan (center dot, mean; vertical line, s.d.; outline, the distribution of the data; n ≥10 embryos). Statistical tests for experiments in D and F were chosen based on the normality and variance of the data. (D) For Arp2/3 and WASP, Welch’s ANOVA was followed by post-hoc Dunnett’s tests; for WAVE, one-way ANOVA was followed by Post-hoc Tukey’s tests; for WASH, Kruskal–Wallis test was followed by post-hoc Dunn’s test. (F) Kruskal–Wallis test was followed by post-hoc Dunn’s test. *P < 0.05, **P < 0.01, ****P < 0.0001. Scale bar: 5 µm.