Tejeda-Muñoz N et al. (2022), Canonical Wnt signaling induces focal adhesion ...

XB-ART-58908

iScience 2022 Apr 15;254:104123. doi: 10.1016/j.isci.2022.104123.

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Canonical Wnt signaling induces focal adhesion and Integrin beta-1 endocytosis.

Tejeda-Muñoz N , Morselli M , Moriyama Y , Sheladiya P , Pellegrini M , De Robertis EM .

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During canonical Wnt signaling, the Wnt receptor complex is sequestered together with glycogen synthase kinase 3 (GSK3) and Axin inside late endosomes, known as multivesicular bodies (MVBs). Here, we present experiments showing that Wnt causes the endocytosis of focal adhesion (FA) proteins and depletion of Integrin β 1 (ITGβ1) from the cell surface. FAs and integrins link the cytoskeleton to the extracellular matrix. Wnt-induced endocytosis caused ITGβ1 depletion from the plasma membrane and was accompanied by striking changes in the actin cytoskeleton. In situ protease protection assays in cultured cells showed that ITGβ1 was sequestered within membrane-bounded organelles that corresponded to Wnt-induced MVBs containing GSK3 and FA-associated proteins. An in vivo model using Xenopus embryos dorsalized by Wnt8 mRNA showed that ITGβ1 depletion decreased Wnt signaling. The finding of a crosstalk between two major signaling pathways, canonical Wnt and focal adhesions, should be relevant to human cancer and cell biology.

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Genes referenced: fas gsk3b wnt8a

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	Figure 1. Wnt3a treatment for 20 min causes a major rearrangement of the actin cytoskeleton and focal adhesions in human corneal stromal (HCSF) fibroblasts(A-A″) Image of HCSF fibroblast showing that the cells attach well to fibronectin-coated plastic, have prominent F-actin cables stained by Phalloidin (in red), and abundant focal adhesion sites immunostained with vinculin (in green).(B-B″) Wnt3a treatment causes disorganization of the actin cytoskeleton, vinculin becomes associated with intracellular vesicles sometimes surrounded by F-actin (indicated by arrowheads), and few focal adhesions are visible. Similar results were obtained in three independent experiments. Insets show higher magnifications of actin and vinculin colocalization. Nuclei were stained with DAPI. Scale bars, 10 μm.(C) Quantification of the colocalization of vinculin and the F-actin marker phalloidin by Pearson’s correlation coefficient using ImageJ. Error bars denote SEM (n ≥ 3) (∗∗p < 0.01). See also Figure S1 and Video S1. Mutation in Axin1 causes extensive membrane ruffling and macropinocytosis in HCC cells, related to Figures 1 and S1, Video S2. Mimicking Wnt with the GSK3 inhibitor LiCl triggers macropinocytic cup formation in cultured Xenopus animal cap cells, related to Figures 1 and S1, Video S3. Activated Ras-GFP triggers macropinocytosis, related to Figures 1 and S1, Video S4. Increased cell motility triggered in Xenopus animal cap cells by the GSK3 inhibitor LiCl, which mimics Wnt signaling, related to Figures 1 and S1.
	Figure 2. Wnt3a treatment caused the formation of vesicles that sequestered GSK3 and the focal adhesion protein zyxin; Immunostainings of endogenous GSK3 and zyxin(A–B′) Fluorescence microscopy images in HeLa cells showing that Wnt treatment (100 ng/mL, 20 min) caused the translocation of GSK3 from the cytosol into vesicles. Note that Wnt3a protein caused the formation of prominent vesicles visible by light microscopy (arrowheads).(C–D′) The focal adhesion protein zyxin is sequestered in vesicles, as GSK3 is, by 20 min Wnt stimulation. Images were generated using a Zeiss Imager Z.1 microscope with Apotome using high magnification.(E–F) Quantification of the immunofluorescence found in vesicles (see STAR Methods). Scale bars, 10 μm. Error bars denote SEM (n ≥ 3) (∗∗p < 0.01). See also Figure S2.
	Figure 3. Integrin beta-1 is rapidly endocytosed by WntTime course (0–30 min) of Wnt3a treatment was performed in HeLa cells at a permissive temperature for endocytosis. After that, the plasma membrane was labeled with Sulfo-NHS-SS-Biotin on ice for 30 min. Pull-down assay with streptavidin-agarose beads showed that Wnt treatment induced the endocytosis of ITGβ1 after 15 min. Lanes 1–4 are HeLa Cell lysate loading controls. Lanes 5–8 show samples after pull-down with biotin agarose beads. Cell extracts were analyzed by western blot with ITGβ1 antibody. Note that cell surface ITGβ1 is endocytosed after 15 or 30 min of Wnt3a treatment. Transferrin Receptor (TfR) was used as a specificity control that is recycled independently of the Wnt pathway. Tubulin antibodies served as a control for cytoplasmic contamination. Similar results were obtained in three independent experiments. See also Figure S3.
	Figure 4. GSK3 and Integrin β-1 are protected from proteinase K digestion inside membrane-bounded organelles in digitonin-permeabilized cells, but not in the presence of Triton X-100 which solubilizes intracellular membranes(A) Diagram of steps involved in the in situ protease protection assay.(B and C) HeLa cells plated on glass coverslips were permeabilized with digitonin, treated with proteinase K to digest cytosolic proteins, stained with ITGβ1 and GSK3 antibodies, and analyzed by fluorescence microscopy. ITGβ1 and GSK3 were protease-protected within the same vesicles after treatment with Wnt3a protein for 20 min.(D and E) HeLa cells treated as described above, except for the addition of Triton X-100, used as control, which dissolves inner membranes and leads to the digestion by exogenous protease of ITGβ1 and GSK3; DAPI labels nuclei. All of the assays were performed in triplicate. Insets show higher magnification views. Scale bars, 10 μm.(F) Quantification of the number of puncta double-stained with ITGβ1 and GSK3 antibodies in protease protection assay using ImageJ. Error bars denote SEM (n ≥ 3) (∗∗p < 0.01). See also Figure S4.
	Figure 5. Integrin β-1 colocalizes together with the multivesicular endosome marker Vps4, but not with its dominant-negative point mutant Vps4-EQ(A and B) HeLa cells transiently transfected with Vps4-GFP were analyzed using fluorescence microscopy with anti- ITGβ1 antibody. Wnt3a treatment of 20 min caused the re-distribution of ITGβ1 into puncta, known to correspond to MVBs (Taelman et al., 2010), indicated by arrowheads.(C and D) Overexpression of the dominant-negative-Vps4-GFP construct containing a single mutation (EQ) in the ATP binding site blocked the induction of ITGβ1 vesicles by Wnt protein. All assays were performed in triplicate. Scale bars, 10 μm.(E) Quantification of ITGβ1 and Vps4 double-positive puncta in transfected HeLa cells. Error bars denote SEM (n ≥ 3) (∗∗p < 0.01).
	Figure 6. ITGβ1 MO inhibits Wnt signaling in a sensitized Xenopus embryo assay in which injection of xWnt8 four times into the animal pole induces a radial dorsalized phenotype(A) Control Xenopus embryos at early tail bud.(B) Embryos injected four times in the animal region at the 4-cell stage with 0.5 pg Wnt8myc mRNA consistently induced a radial head phenotype lacking any trunk development.(C) No phenotype was observed with ITGβ1 MO alone.(D) Co-injection of xITGβ1 antisense MO consistently inhibited the dorsalizing effects of xWnt8, allowing the formation of partial axial structures.(E) Microinjection of human ITGβ1 mRNA was without phenotypic effect.(F) The effect of ITGβ1 MO was specific because it was rescued by human ITGβ1 mRNA which is not targeted by the MO sequence.(G and H) Injecting the same dose (0.5 pg) of xWnt8myc mRNA into a single ventral marginal location in 4-cell embryos induces the familiar axis duplication phenotype; it is shown here to contrast with the radial dorsalized phenotype caused by four injections into the animal pole used in this sensitized assay system. Images were taken with an Axio Zoom V.16 Stereo Zoom Zeiss at low magnification. Similar results were obtained in five independent experiments. Numbers of embryos analyzed were as follows A = 140, 100%; B = 128, 97%; C = 132, 98%; D = 125, 92%; E = 129, 100%; F = 124, 85% (5 independent experiments); G = 48, 100%; H = 92, 91% complete axes (2 independent experiments). Scale bar, 500 μm. For β-catenin reporter assay in animal caps, see Figure S5.
	Figure 7. Model of the endocytosis of the focal adhesion and ITGβ1 by WntAfter Wnt treatment, Lrp6/Fz/Wnt/GSK3 signalosomes, the master regulator of cell adhesion ITGβ1, and other focal adhesion components are endocytosed by macropinocytic cups in an actin-driven process. Macropinocytosis is required for canonical Wnt signaling. Sequestration of GSK3 in MVBs is necessary for the stabilization of β-catenin that mediates the transcriptional activity of canonical Wnt.

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