Cell signaling facilitates apical constriction by basolaterally recruiting Arp2/3 via Rac and WAVE

Abstract

Apical constriction is a critical cell shape change that bends tissues. How precisely-localized actomyosin regulators drive apical constriction remains poorly understood. C. elegans gastrulation provides a valuable model to address this question. The Arp2/3 complex is essential in C. elegans gastrulation. To understand how Arp2/3 is locally regulated, we imaged embryos with endogenously-tagged Arp2/3 and its nucleation-promoting factors (NPFs). The three NPFs - WAVE, WASP, and WASH - colocalized with Arp2/3 and controlled Arp2/3 localization at distinct subcellular locations. We exploited this finding to study distinct populations of Arp2/3 and found that only WAVE depletion caused penetrant gastrulation defects. WAVE localized basolaterally with Arp2/3 at cell-cell contacts, dependent on CED-10/Rac. Establishing ectopic cell contacts recruited WAVE and Arp2/3, identifying contact as a symmetry-breaking cue for localization of these proteins. These results suggest that cell-cell signaling via Rac activates WAVE and Arp2/3 basolaterally, and that basolateral Arp2/3 is important for apical constriction.

Figure 1.. Quantitative analysis of Arp2/3 and nucleation-promoting factors (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 pseudo-colored to visualize their internalization over time. The diagram to the right 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 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, cell-cell contacts, and the cytoplasm. Insets of Arp2/3 and WASP highlight the difference between the signal at the cell-cell contacts and the cytoplasm. Measurements were collected 6 minutes 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) P-values reported in Figures D and F were calculated using either Tukey’s test or Dunnett’s test, depending on whether the variances between groups were equal. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) Scale bar: 5 μm.

Figure 2.. Three NPFs colocalize with Arp2/3 at different cellular and subcellular locations.

(A) Co-visualization of Arp2/3 with WAVE, WASP, and WASH in gastrulation stage embryos using endogenously tagged alleles. White arrowheads point to Ea and Ep cells. Scale bar: 5 μm. (B) Quantification of colocalization between Arp2/3 and NPFs, with box plots reporting Pearson correlation coefficients at Ep/P₄ contacts, cell-cell contacts, and the cytoplasm. P values were calculated with a Dunn’s test. (center line, median; box, interquartile range (IQR); whiskers, min/max range; n ≥ 10 embryos; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001)

Figure 3.. RNAi against each NPF leads to Arp2/3 reduction at different cellular and subcellular locations

(A) Micrographs from time-lapse movies depicting localization of Arp2/3 in control and WAVE, WASP, or WASH RNAi-treated embryos from a lateral view. White arrowheads point to Ea and Ep cells. The diagrams underneath each micrograph highlight the observed Arp2/3 localization in E and neighboring cells. (B) Violin plots reporting changes in Arp2/3 localization at Ep/P₄ contacts, cell-cell contacts, and the cytoplasm upon RNAi depletion of WAVE, WASP, and WASH. (center dot, mean; vertical line, s.d.; outline, the distribution of the data; n ≥ 10 embryos) (C) Violin plots reporting changes in the differences between the maximum and minimum gray values of the Arp2/3 signal along the line scan upon RNAi depletion of WAVE, WASP, and WASH. (center dot, mean; vertical line, s.d.; outline, the distribution of the data; n ≥ 10 embryos) All measurements were collected 6 minutes after the division of neighboring mesoderm precursor cells (MSx). P-values reported in Figures B and C were calculated using either an unpaired t-test or an unpaired t-test with Welch’s correction, depending on whether the variances between groups were equal. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) Scale bar: 5 μm.

Figure 4.. NPF RNAi by themselves and in combination lead to different degrees of gastrulation defects

(A) Micrographs from time-lapse DIC movies in eight different backgrounds, with time indicated on the left from the MSx cell division. E lineage cells are outlined and pseudocolored in green. Gastrulation defects (E cells dividing before being fully covered by neighboring cells) are indicated with white arrowheads. An enclosed outline and the absence of arrowheads indicate that endodermal precursors became internalized at the 2E stage, as seen in wild-type embryos. (B) The bar graph (left) and Venn diagram (right) summarize the effects of NPF RNAi, both individually and in combination, on gastrulation. The heat map represents the different penetrance levels, with darker colors indicating a higher percentage of gastrulation defects. P values were calculated using Fisher’s exact test. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001)

Figure 5.. The Rac1 GTPase CED-10 recruits WAVE and Arp2/3 at cell-cell contact and contributes to apical constriction

(A) Micrographs from time-lapse movies depicting localization of WAVE and Arp2/3 in control and CED-10 RNAi-treated embryos from a lateral view. Scale bar: 5 μm. The diagrams underneath each micrograph highlight the observed Arp2/3 localization in E and neighboring cells. (B) Violin plots reporting changes in WAVE and Arp2/3 localization at Ep/P₄ contacts, cell-cell contacts, and the cytoplasm upon RNAi depletion of CED-10. (center dot, mean; vertical line, s.d.; outline, the distribution of the data; n = 11 embryos) (C) Micrographs from time-lapse DIC movies of wild-type and CED-10 RNAi-treated embryos with time on the left from MSa/p cell division. E lineage cells are outlined and pseudocolored in green. Gastrulation defects (E cells dividing before being fully covered by neighboring cells) are indicated with white arrowheads. (D) Bar graph summarizing gastrulation defects caused by ced-10 RNAi. P-values reported in Figures B were calculated using either an unpaired t-test or an unpaired t-test with Welch’s correction, depending on whether the variances between groups were equal. P values reported in Figure D were calculated with Fisher’s exact test. ( *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001)

Figure 6.. Newly established ectopic cell-cell contact acts as a symmetry-breaking cue to recruit WAVE and Arp2/3

(A) Micrographs from time-lapse movies of chimeric embryos created by combining two embryos expressing WAVE and Arp2/3. The DIC channel of the whole chimera is shown on the left, and two fluorescence channels of a blow-up of the chimeric contact (outlined region) are shown on the right. Yellow arrows point to ectopic cell-cell contacts. (B) Violin plots reporting normalized fluorescence intensity of WAVE and Arp2/3 at ectopic/endogenous cell-cell contacts and the contact-free cell apex. (center dot, mean; vertical line, s.d.; outline, the distribution of the data; n = 15 chimeras) P values were calculated with a Tukey test. ( *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) Scale bar: 5 μm.

Figure 7.. Summary

(A) The three NPFs in C. elegans, WAVE, WASP and WASH complexes, colocalize with Arp2/3 and control Arp2/3 localization at distinct subcellular locations. (B) Cell-cell signaling makes an essential contribution to apical constriction by basolaterally recruiting Arp2/3 via Rac and WAVE. (C) Rac signaling and myosin-activating kinase are active at opposing locations in migrating and apically constricting cells.