Journal: Nature Communications

Article Title: Mutant APC reshapes Wnt signaling plasma membrane nanodomains by altering cholesterol levels via oncogenic β-catenin

doi: 10.1038/s41467-023-39640-w

Figure Lengend Snippet: To examine the effect of oncogenic APC on interactions between Wnt receptors and key lipids, cells co-expressing EGFP-tagged A , C Fzd7 or B , D LRP6 and mCherry-tagged A , B D4H or C , D pleckstrin homology (PH) domain of phospholipase C δ1 (PLC-δ1, PI(4,5)P 2 sensor) were used to perform FLIM FRET. E To examine the effect of oncogenic APC on PI(4,5)P 2 plasma membrane levels, cells expressing EGFP-tagged PLC-δ1-PH were used to measure membrane-associated EGFP fluorescence intensity using flow cytometry. EGFP plasma membrane fluorescence intensity was normalized to total EGFP fluorescence intensity. To examine the effect of oncogenic APC on the interactions between Dvl1 and key lipids, cell co-expressing EGFP-tagged F , G Dvl1 and mCherry-tagged F D4H or G PLC-δ1 were used to perform FLIM FRET. YAMC, IMCE, and IMCE βcat cells were pre-treated with mevastatin (5 µM, 24 h), MβCD (10 mM, 30 min), or phenylarsine oxide (PAO) (20 µM, 30 min) and washed, as indicated. Subsequently, cells were incubated with Wnt3a-conditioned media or control media without Wnt3a for 30 min, washed, fixed, and imaged. For FLIM-FRET experiments, the apparent FRET efficiency was calculated from FLIM data averaged per FOV (mean ± SD, n = 10–15 FOVs containing 3–8 cells were examined per condition, exact n value is shown in each graph). For flow cytometry IL PI(4,5)P 2 experiments, cells were imaged to calculate EGFP fluorescence intensity (mean ± SEM, from n = 3 independent biological replicates, total number of cells analyzed is provided below each bar). For all experiments, statistical significance was determined by two-way ANOVA and post Tukey’s multiple comparison test. Different letters indicate significant differences between WT APC (control) and mutant APC/treatment groups (experimental) ( P < 0.05). Source data are provided as a Source data file.

Article Snippet: To fluorescently label colonocytes for nanocluster analysis using STORM, colonocytes cells were blocked with 5% BSA-DPBS for 30 min at RT and, after aspirating solution, incubated with 125 μL of 10 μg/mL of primary rat IgG2B LRP6 (1:20, R and D Systems Cat# MAB2960, RRID:AB_2139440) or anti-LRP6-AF647 (1:20, R and D Systems, Cat# FAB1505R) or rat IgG2A Fzd7 (1:50, R and D Systems Cat# MAB1981-100, RRID:AB_2247464) or anti-Fzd7-AF647 (1:50, R and D Systems, Cat# FAM1981R) or anti-Dvl1-AF647 (1:20, Santa Cruz Biotechnology, Cat# sc-8025 AF647) antibody in 1% BSA-DPBS for 1 h at RT.

Techniques: Expressing, Clinical Proteomics, Membrane, Fluorescence, Flow Cytometry, Incubation, Control, Comparison, Mutagenesis

Journal: Nature Communications

Article Title: Mutant APC reshapes Wnt signaling plasma membrane nanodomains by altering cholesterol levels via oncogenic β-catenin

doi: 10.1038/s41467-023-39640-w

Figure Lengend Snippet: For in vitro FLIM-FRET experiments, cells co-expressing EGFP- and mCherry-tagged A Fzd7 or B LRP6 or C EGFP-tagged LRP6 and mCherry-tagged Fzd7 were used to perform homo- and hetero-clustering FLIM-FRET analyses, respectively. To examine the effect of oncogenic APC on the interactions between Dvl1 and Wnt receptors, cells co-expressing EGFP-tagged D Fzd7 or E LRP6 and mCherry-tagged Dvl1 were used to perform FLIM-FRET. To examine the effect of oncogenic APC on plasma membrane Wnt receptor localization, cells co-expressing EGFP-tagged F Fzd7 or G LRP6 and tH-RFP were used to perform FLIM-FRET analyses. For FLIM-FRET experiments, YAMC, IMCE, and IMCE βcat cells were pre-treated with mevastatin (5 µM, 24 h), MβCD (10 mM, 30 min), or phenylarsine oxide (PAO) (20 µM, 30 min) and washed, as indicated. Subsequently, cells were incubated with Wnt3a-conditioned media or control media without Wnt3a for 30 min, washed, fixed, and imaged. The apparent FRET efficiency was calculated from FLIM data averaged per FOV (mean ± SD, from n = 10–15 FOVs containing 3–5 cells each were examined per condition, exact n value is shown in each graph). For all experiments, statistical significance was determined by two-way ANOVA and post Tukey’s multiple comparison test. Different letters indicate significant differences between WT APC (control) and mutant APC/treatment groups (experimental) ( P < 0.05). Source data are provided as a Source data file.

Article Snippet: To fluorescently label colonocytes for nanocluster analysis using STORM, colonocytes cells were blocked with 5% BSA-DPBS for 30 min at RT and, after aspirating solution, incubated with 125 μL of 10 μg/mL of primary rat IgG2B LRP6 (1:20, R and D Systems Cat# MAB2960, RRID:AB_2139440) or anti-LRP6-AF647 (1:20, R and D Systems, Cat# FAB1505R) or rat IgG2A Fzd7 (1:50, R and D Systems Cat# MAB1981-100, RRID:AB_2247464) or anti-Fzd7-AF647 (1:50, R and D Systems, Cat# FAM1981R) or anti-Dvl1-AF647 (1:20, Santa Cruz Biotechnology, Cat# sc-8025 AF647) antibody in 1% BSA-DPBS for 1 h at RT.

Techniques: In Vitro, Expressing, Clinical Proteomics, Membrane, Incubation, Control, Comparison, Mutagenesis

Journal: Nature Communications

Article Title: Mutant APC reshapes Wnt signaling plasma membrane nanodomains by altering cholesterol levels via oncogenic β-catenin

doi: 10.1038/s41467-023-39640-w

Figure Lengend Snippet: For in vivo STORM imaging experiments, isolated single colonocytes from PDOs were fixed and labeled with primary monoclonal rat Fzd7 or mouse LRP6 antibody fluorescently labeled with Alexa Fluor 647. A Model of the formation of Wnt proteolipid condensates in ordered plasma membrane nanodomains examined via STORM imaging. B Representative bright field and Voronoi images of isolated single colonocytes from CRC-PDOs labeled with primary anti-LRP6-AF647. Quantitative analysis of Fzd7 and LRP6 C , E cluster area, D , F cluster area relative frequency, G , H total number of receptor molecules inside clusters, I , J receptor molecule absolute density, K , L percentage of receptor molecules forming part of clustered regions, M , N total number of receptor clusters, and O , P cellular receptor cluster density in isolated single colonocytes from PDOs, respectively. Cluster area was calculated from STORM data averaged per region of interest (ROI) and the respective relative frequency was calculated from individual cluster distribution data (mean ± SD, from n = 50–2274 ROIs, exact n value is shown below each bar). Data associated with the number of single receptor molecules, receptor clusters, and their density was calculated from raw fluorescence intensity images converted to text (.txt) x–y coordinate files using Clus-Doc (mean ± SD, from n = 22–66 ROIs, exact n value is shown below each bar). Different letters indicate significant differences between WT APC (control) and mutant APC groups (experimental) ( P < 0.05). Source data are provided as a Source data file.

Article Snippet: To fluorescently label colonocytes for nanocluster analysis using STORM, colonocytes cells were blocked with 5% BSA-DPBS for 30 min at RT and, after aspirating solution, incubated with 125 μL of 10 μg/mL of primary rat IgG2B LRP6 (1:20, R and D Systems Cat# MAB2960, RRID:AB_2139440) or anti-LRP6-AF647 (1:20, R and D Systems, Cat# FAB1505R) or rat IgG2A Fzd7 (1:50, R and D Systems Cat# MAB1981-100, RRID:AB_2247464) or anti-Fzd7-AF647 (1:50, R and D Systems, Cat# FAM1981R) or anti-Dvl1-AF647 (1:20, Santa Cruz Biotechnology, Cat# sc-8025 AF647) antibody in 1% BSA-DPBS for 1 h at RT.

Techniques: In Vivo, Imaging, Isolation, Labeling, Clinical Proteomics, Membrane, Fluorescence, Control, Mutagenesis

Journal: Nature Communications

Article Title: Mutant APC reshapes Wnt signaling plasma membrane nanodomains by altering cholesterol levels via oncogenic β-catenin

doi: 10.1038/s41467-023-39640-w

Figure Lengend Snippet: A Drosophila midgut-hindgut intestinal tissue model. ISCs/progenitor cells express humanized hFzd7 or hLRP6 under control of the UAS and esg-Gal4 TS . B Filipin III-stained midgut from Drosophila fed various cholesterol diets (red arrow, intestinal lumen). C Quantification of total cholesterol from Drosophila midgut. Cholesterol was calculated from luciferase luminescence data using the Amplex™ Red cholesterol assay and normalized to total protein (mean ± SD, from n = 3 independent biological replicates). D Filipin III fluorescence distribution of Drosophila intestinal epithelium (red arrow, ISCs). Effects of cholesterol on Wnt receptor organization. Flies co-expressing EGFP- and mCherry-tagged E LRP6 or F Fzd7 or G EGFP-LRP6 and mCherry-Fzd7 were used to perform FLIM-FRET in flies fed various cholesterol diets. FRET efficiency was calculated from FLIM data (mean ± SD, from n = 5–10 guts, ROIs analyzed provided below each bar, n value is shown in each graph). H Quantitative analysis of free cholesterol-induced βcat activation. 3T3 LL cells were pre-treated with mevastatin (5 µM, 24 h), MβCD (10 mM, 30 min), and MβCD-cholesterol (cholesterol) (10 mM, 30 min), and incubated with control or Wnt3a-conditioned media for 24 h. Luciferase luminescence was measured using a Luciferase Assay System kit. Luciferase luminescence fold change was normalized to total protein (mean ± SD, from n = 3 independent biological replicates). I Quantitative analysis of Wnt signaling activation. The percentage of TCF-LacZ+ cells is shown. Error bars represent n = 5 independent biological replicates (mean ± SD, ~100 cells analyzed per group). J Qualitative analysis of cholesterol-induced Wnt activation. TCF-LacZ+ cells activity in Drosophila posterior midguts from control ( w 1118 ; esgGal4 TS , UAS-GFP; TCF-LacZ) and hLRP6-expressing ISCs ( w 1118 ; esgGal4 TS , UAS-GFP, UAS-hLRP6; TCF-LacZ). Flies were feed a cholesterol free, standard (Std. Diet) or high cholesterol diet for 5 days. ISCs, GFP; nuclei, DAPI. Statistical significance determined by C two-way ANOVA or E – I one-way ANOVA and post hoc Tukey’s test. Different letters indicate significant differences between treatment groups ( P < 0.05). Representative images and scale bars are provided for microscopy data. Enterocytes, ECs; visceral muscle cells, muscle; intestinal stem cells, ISCs; enteroblasts, EB. Source data file provided.

Article Snippet: To fluorescently label colonocytes for nanocluster analysis using STORM, colonocytes cells were blocked with 5% BSA-DPBS for 30 min at RT and, after aspirating solution, incubated with 125 μL of 10 μg/mL of primary rat IgG2B LRP6 (1:20, R and D Systems Cat# MAB2960, RRID:AB_2139440) or anti-LRP6-AF647 (1:20, R and D Systems, Cat# FAB1505R) or rat IgG2A Fzd7 (1:50, R and D Systems Cat# MAB1981-100, RRID:AB_2247464) or anti-Fzd7-AF647 (1:50, R and D Systems, Cat# FAM1981R) or anti-Dvl1-AF647 (1:20, Santa Cruz Biotechnology, Cat# sc-8025 AF647) antibody in 1% BSA-DPBS for 1 h at RT.

Techniques: Control, Staining, Luciferase, Amplex Red Cholesterol Assay, Fluorescence, Expressing, Activation Assay, Incubation, Activity Assay, Microscopy