Actomyosin contractility modulates Wnt signaling through adherens junction stability

Abstract

Actomyosin contractility can influence the canonical Wnt signaling pathway in processes like mesoderm differentiation and tissue stiffness during tumorigenesis. We identified that increased nonmuscle myosin II activation and cellular contraction inhibited Wnt target gene transcription in developing Drosophila imaginal disks. Genetic interactions studies were used to show that this effect was due to myosin-induced accumulation of cortical F-actin resulting in clustering and accumulation of E-cadherin to the adherens junctions. This results in E-cadherin titrating any available β-catenin, the Wnt pathway transcriptional coactivator, to the adherens junctions in order to maintain cell-cell adhesion under contraction. We show that decreased levels of cytoplasmic β-catenin result in insufficient nuclear translocation for full Wnt target gene transcription. Previous studies have identified some of these interactions, but we present a thorough analysis using the wing disk epithelium to show the consequences of modulating myosin phosphatase. Our work elucidates a mechanism in which the dynamic promotion of actomyosin contractility refines patterning of Wnt transcription during development and maintenance of epithelial tissue in organisms.

FIGURE 1:

Myosin phosphatase regulates NMII and Wg activity during wing development. (A, A′) Dll-lacZ expression in control (hh-Gal4 driving gfp) (A) and hh-Gal4 driving gfp and mypt-75D-RNAi (A′) third-instar wing imaginal disks. (A″) Dll expression area of the wing pouch, in the anterior (GFP negative), and posterior (GFP and MYPT-75D-RNAi positive) domains (n = 7). (B, B′) Wg protein expression in wild type (B) and GFP-marked actin flip-out clones driving mypt-75D-RNAi (B’). (C–C″′) Arm stabilization pattern in wild type (C arrows) and in flip-out clones driving flw-RNAi (C′ arrowheads). Fluorescence intensity plot of Arm and GFP along the D/V boundary of the wing pouch (C″), with average Arm intensity compared in wild type and gfp with flw-RNAi expressing tissue (C″′). (D, D′) p-MyoII stained in flw-RNAi flip-out clones. Cross-section seen in D′ is the magnified dashed line area of D. (E) Adult wings of wild type, and hh-Gal4 driving mypt-75D-RNAi, flw-RNAi, or sqh^EE (arrowheads mark loss of bristles and wing margins). Data presented as mean ± SEM; **p = 0.0029, ***p < 0.0001. Scale bars: (A–C) 50 µm, (D) 100 µm, (D′) 20 µm, (E) 300 µm.

FIGURE 2:

NMII regulates Wg activity during wing development. (A) hh>sqh^EE, gfp stained for Dll-lacZ expression. (B, B′) Total Wg in sqh^EE flip-out clones, and cross-section showing cell constriction. (C, C′) GFP flip-out clones driving sqh^EE stained for F-actin and Arm. (C′) Cross-section shows apical F-actin and Arm (C′, arrowhead vs. arrow) (dashed lines of C corresponding to section location and depth of C′). (D) Average fluorescence intensity of the apical surface of the wild-type and constricting columnar epithelial cells shown in C′. Data presented as mean ± SEM; ***p < 0.0001. Scale bars: (A) 50 µm, (B–C′) 20 µm.

FIGURE 3:

NMII activity inhibits Wg activation by reducing nuclear Arm independently of the destruction complex. (A) Salivary glands from control or dpp>flw-RNAi stained for fz3 expression to demonstrate the graded response to Wg in expression of the fz3-lacZ reporter. The white box highlights the general area within glands that we examined in B. (B) Localization of Dsh-GFP and FLAG-Axin in the proximal cells of the salivary gland, represented by the general dashed line area of A. (C–C″) Effects of flw-RNAi on ectopic Dll-lacZ in wing imaginal disks: (C) RFP marked flip-out clones expressing Fz-Arr and GFP or flw-RNAi (C′) GFP-positive axin^null MARCM clones and flw-RNAi in axin^null MARCM clones. (C″) Arm^S10 flip out clones with GFP or flw-RNAi (arrowhead identifies distinct loss of Dll expression). (D–E′) Effects of flw-RNAi on Arm distribution in GFP-marked axin^null cells, at nuclear (D) and apical sections (E). (D, E) DAPI was used to identify nuclei, and F-actin to mark the edges and junctions of the cell. (D′) Percentage of nuclear Arm in cells was measured as an intensity plot (dotted line D) in wild type (n = 16), axin^null (n = 20), and axin^null, flw-RNAi cells (n = 15). (E′) Percentage of junctional Arm in cells was measured as an intensity plot (dotted line E) in wild-type (n = 13), axin^null (n = 13), and axin^null, flw-RNAi cells (n = 13). Data presented as mean ± SEM; ***p < 0.0001. Scale bars: (A) 100 µm, (B–C″) 50 µm, (D) 5 µm.

FIGURE 4:

NMII activation increases retention of adherens junction proteins. (A, B) GFP-marked actin flip-out clones driving sqh^EE stained for (A) DE-cad and Arm (arrowheads identify apical increases) and (B) expression of DE-cad or arm. (C–D″) FRAP analysis of DE-cad::GFP and Arm::GFP in wing imaginal disks with RFP-marked flw-RNAi expressing flip-out clones. (C, D) DE-cad::GFP and Arm::GFP wing imaginal disk with squares indicating bleached regions of the wing disk. Green squares represents wild-type cell interfaces, blue for wt:flw-RNAi cell interface, and red for flw-RNAi cell interfaces. (C′) AJ DE-cad::GFP (n = 6) recovery curves of FRAP analyses show cells adjacent to or expressing flw-RNAi have significantly slower recovery rates than wild type (p = 0.0029), (C″) and greater immobile protein fractions, *p < 0.05. (D′) flw-RNAi had no effect on AJ Arm::GFP (n = 6) recovery curves from FRAP analyses (p = 0.4794), (D″) or immobile protein fractions. Data presented as mean and mean curve ± SEM. Scale bars: (A) 20 µm, (B) 50 µm, (C, D) 10 µm.

FIGURE 5:

NMII activation inhibits Wg signaling through DE-cad. (A, B) GFP-marked actin flip-out clones expressing DE-cad or DE-cad∆β, stained for (A) DE-cad, or (B) F-actin and Arm. (C) RFP-marked actin flip-out clones expressing DE-cad or DE-cad∆β, stained for Dll expression. (D) Dll-lacZ expression in RFP-marked clones of the indicated genotypes. (E) Eclosion percentage of hh>mypt-75D-RNAi and hh>mypt-75D-RNAi heterozygous for shg Drosophila. Data presented as mean ± SEM; **p = 0.0022; n ≥145. Scale bars: (A, C, D) 50 µm, (B) 20 µm.

FIGURE 6:

NMII inhibits Wg signaling by increased F-actin. (A–A″) GFP-marked actin flip-out clones expressing dia∆DAD stained to detect (A) F-actin and Dll-lacZ, (A′) F-actin and Arm, and (A″) F-actin and DE-cad. dia∆DAD induces increased apical AJ Arm (A′, arrow) and DE-cad (A″, open arrowhead), and cell contractions (A″, open arrowhead). (B) GFP-marked actin flip-out clones expressing dia-RNAi stained for F-actin and Arm. Apical sections visualized from dashed box area shown in cross-section, with clone area marked by vertical lines. (C) GFP-marked actin flip-out clones of indicated genotypes stained to detect Dll-lacZ. Dashed lines correspond to location visualized in cross-section images. Scale bars: (A, A′, C) 50 µm, (A″) 20 µm, (B) 10 µm.

FIGURE 7:

NMII activation recruits β-cat to cell membranes inhibiting Wnt signaling. (A, B) Wnt-responsive luciferase TOPFLASH assay measuring TCF/LEF reporter activity in (A) MCF7 and (B) RKO cells following Wnt3A transfection and siPP1β and siMYPT3 transfection. Letters above each column represent significant difference from corresponding columns, (p < 0.01). (C) Western blot analysis of total cellular levels of E-cad, β-cat, MYPT3, Wnt3A and PP1β. β-Tub was used as a loading control. (D) TOPFLASH assay of RKO cells transfected with combinations of E-cad, Wnt3A and siPP1β with siMYPT3. (E) Western blot analysis of total cellular levels of E-cad, β-cat, and PP1β of RKO cells from conditions in D. GAPDH was used as a loading control. (F) Western blot analysis of β-cat in cytoplasmic (Cyto), membranous (Mem), chromatin-bound (CB), and whole cell extract (WCE) fractions in MCF7 cells. GAPDH, Na⁺/K⁺ ATPase, and Histone 3 were used as loading controls for corresponding fractions. Complete fraction, see Supplemental Figure S5. (G) Quantification of relative levels of β-cat from each fraction taken from F, normalized to Control + siScramble conditions (n = 4 biological replicates). (H) MCF7 cells stained for β-cat and DAPI. (I) Average fluorescence intensity of nuclear β-cat of MCF7 cells in Control + siScramble (n = 13), Wnt3A + siScramble (n = 13), and Wnt3A + siPP1β/siMYPT3 (n = 13) transfected cells. Values normalized to the maximum fluorescence intensity equal to 100 for each cell quantified. (J) Western blot analysis of total PP1β for cells analyzed in H and I. GAPDH was used as a loading control. All data are presented as mean ± SEM. ns = not significant, *p < 0.05, ***p < 0.0001. Scale bars: 20 µm.

FIGURE 8:

NMII activation inhibits Wnt signaling in a dynamic manner across developing tissue by Arm titration to AJs. (A, A′) GFP-marked shg^null MARCM clones expressing (A) DE-cad∆β::α-cat∆VH1 (n = 18) and (A′) DE-cad∆β::α-cat∆VH1 with mypt-75D-RNAi (n = 8), stained for Dll and F-actin (arrowheads show F-actin accumulation). (B) Model for regulation of Wnt signaling by activation of NMII resulting in AJ accumulation and stabilization in response to contractile forces; see text for details. Scale bars: (A, A′) 50 µm.