Superresolution microscopy localizes endogenous Dvl2 to Wnt signaling-responsive biomolecular condensates

Abstract

During organismal development, homeostasis, and disease, Dishevelled (Dvl) proteins act as key signaling factors in beta-catenin-dependent and beta-catenin-independent Wnt pathways. While their importance for signal transmission has been genetically demonstrated in many organisms, our mechanistic understanding is still limited. Previous studies using overexpressed proteins showed Dvl localization to large, punctate-like cytoplasmic structures that are dependent on its DIX domain. To study Dvl's role in Wnt signaling, we genome engineered an endogenously expressed Dvl2 protein tagged with an mEos3.2 fluorescent protein for superresolution imaging. First, we demonstrate the functionality and specificity of the fusion protein in beta-catenin-dependent and beta-catenin-independent signaling using multiple independent assays. We performed live-cell imaging of Dvl2 to analyze the dynamic formation of the supramolecular cytoplasmic Dvl2_mEos3.2 condensates. While overexpression of Dvl2_mEos3.2 mimics the previously reported formation of abundant large "puncta," supramolecular condensate formation at physiological protein levels is only observed in a subset of cells with approximately one per cell. We show that, in these condensates, Dvl2 colocalizes with Wnt pathway components at gamma-tubulin and CEP164-positive centrosomal structures and that the localization of Dvl2 to these condensates is Wnt dependent. Single-molecule localization microscopy using photoactivated localization microscopy (PALM) of mEos3.2 in combination with DNA-PAINT demonstrates the organization and repetitive patterns of these condensates in a cell cycle-dependent manner. Our results indicate that the localization of Dvl2 in supramolecular condensates is coordinated dynamically and dependent on cell state and Wnt signaling levels. Our study highlights the formation of endogenous and physiologically regulated biomolecular condensates in the Wnt pathways at single-molecule resolution.

Keywords: CRISPR; Dishevelled; Wnt signaling; biomolecular condensates; superresolution microscopy.

Fig. 1.

Generation of endogenously tagged Dvl2_mEos3.2. (A) Schematic presentation of the knock-in design and tagging strategy. (B) Validation of the integration of the donor template into the DVL2 locus. Genomic DNA was extracted from selected single-cell clones, and PCR was performed using the primers indicated in A. Upper shows correct integration into the DVL2 locus (clones 142_1 and 142_3), whereas Lower reveals any integration in the genome (clone 225); kBp denotes kilobase pairs. (C) Dvl2_mEos3.2 protein expression in clone 142_3. The tagged protein is more abundant than nontagged Dvl2 in HEK293T^DVL2_mEos. Arrowheads indicate the endogenously mEos3.2-tagged Dvl2. Beta-actin serves as a loading control. One of three independent experiments is shown.

Fig. 2.

Characterization of the endogenous Dvl2_mEos3.2 protein. (A) Correlative live-cell confocal microscopy of HEK293T^DVL2_mEos and HEK293T pcDNA DVL2_mEos3.2 shows that endogenous condensate formation is rare and limited to a subset of cells. The indicated HEK293T cells were transfected with pcDNA or pcDNA DVL2_mEos3.2 for 48 h. The same imaging settings were applied for all conditions, to enable the comparison of signal intensities of endogenous and overexpressed Dvl2_mEos3.2 protein. Green fluorescent signal in pcDNA is transfection-induced autofluorescence. In all confocal images, mEos3.2 is shown after excitation with 488 nm wavelength. Cell membrane was visualized using CellMask Deep Red membrane dye (blue). Representative images are of three replicates with imaging of three or more points of view. (Scale bar, 10 µm.) (B and C) Confocal microscopy of Dvl2 in HEK293T^DVL2_mEos (B) and wild-type (C) cells. Maximum intensity projections are shown of HEK293T^DVL2_mEos (z = 17) and wild type (z = 16). Representative images are of three or four replicates with imaging of three or more points of view. (Scale bar, 10 µm.) White boxes mark the magnified areas. (D) Current model of the activated beta-catenin–dependent Wnt signaling cascades. (E) Colocalization of Dvl3, Axin1, APC, and LRP6 with Dvl2_mEos3.2 in the condensates. Maximum intensity projections are shown of Dvl3 (z = 25), Axin1 (z = 26), APC (z = 25), and LRP6 (z = 15). Representative images are of three to five replicates with imaging of three or more points of view. (Scale bar, 10 µm.) White boxes mark the magnified areas.

Fig. 3.

Wnt activity is maintained in endogenously tagged Dvl2_mEos3.2 cells. (A) HEK293T^DVL2_mEos and HEK293T wild-type cells were transfected with pcDNA (empty vector control), Wnt3, and DVL2 together with TCF4/Wnt-firefly luciferase and actin-Renilla reporter plasmids. Induced reporter activity was determined by normalization to Wnt3 transfected HEK293T cells. Graph represents the mean ± SEM of four independent experiments. (B and C) Dvl1 and Dvl3 have no influence on the function of Dvl2_mEos3.2.HEK293T^DVL2_mEos and HEK293T wild-type cells were transfected with the indicated siRNAs against DVL1/3, or siCtl; 24 h later, cells were transfected with Wnt3a, Wnt3a_mCherry, or pcDNA; 48 h later, total cell lysates were collected and analyzed by Western blot. Note that Dvl1 is not expressed in HEK293T^DVL2_mEos and HEK293T cells (SI Appendix, Fig. S2A). One of three independent experiments is shown. Induced reporter activity was determined by normalization to HEK293T cells (siCtl.) stimulated with Wnt3. Graph represents the mean ± SEM of four independent experiments. (D) CRISPR/Cas9-mediated knockout of all Dvl paralogs (DVL1,2,3^KO) reduces Wnt3-driven TCF4/Wnt reporter activity in HEK293T^DVL2_mEos and HEK293T cells. Induced reporter activity was determined by normalization to Wnt3 transfected cells. Graph represents the mean ± SEM of three independent experiments. (E) Recruitment of Dvl2_mEos3.2 to the plasma membrane by overexpression of Fzd7 and ROR2. Live-cell confocal microscopy was performed with the same laser settings for all conditions. White boxes mark the magnified areas seen in the dual-color images. Arrowheads indicate membrane recruitment. DNA was stained by Hoechst (blue). Representative images are of three replicates with imaging of three or more points of view. (Scale bar, 10 µm; in magnifications, 25 µm.) For pcDNA control, see Fig. 4E.

Fig. 4.

Dvl2_mEos3.2 condensate formation depends on active Wnt signaling. (A) Knockdown of DVL1/3 and additional Wnt stimulus have no impact on condensate formation. HEK293T^DVL2_mEos cells were transfected with siRNAs against DVL1/3; 24 h later, cells were transfected with the indicated siRNAs and plasmids (for transfection with Wnt5a_mScarlet, nontagged Wnt3a, and further controls, see SI Appendix, Fig. S7). Representative images are of four replicates with imaging of three or more points of view. (Scale bar, 10 µm.) (B) Expression of Evi/Wls protein and its secretion capacity in HEK293T^DVL2_mEos EVI^KO cells. Total cell lysates and Blue Sepharose enriched supernatants were used for Western blot analysis with the indicated antibodies. HSC70 serves as a loading control. One of three independent experiments is shown. (C) Canonical Wnt signaling is diminished in EVI^KO cells. Induced TCF4/Wnt-firefly luciferase reporter activity was determined by normalization to Wnt3 transfected HEK293T^DVL2_mEos cells. Graph represents the mean ± SEM of three independent experiments. (D) Canonical Wnt signaling is abolished in mFZD^KO cells. Graph represents the mean ± SEM of four independent experiments. (E) Loss of Wnt signaling due to mFZD^KO reduces condensate formation. Representative images are of three replicates with imaging of three or more points of view. (Scale bar, 10 µm; in magnifications, 25 µm.) White boxes mark the magnified areas seen in the dual-color images. Images show controls (pcDNA transfected) for Fig. 2E. (F) Knockout of EVI and mFZD significantly reduces Dvl2_mEos3.2 condensate formation. The condensate count was normalized to the number of cells per image (condensates per 100 cells). The quantification via automated image analysis detects less condensates compared to manual counting. Box plot represents the normalized condensate counts for the different cell lines, with dots representing the average normalized condensate count per plate (seven plates from three individual experiments). Statistical significance was calculated using the Wilcoxon signed rank test. (G) Stimulation with recombinant (rec.) Wnt3a induces Dvl2_mEos3.2 condensate formation in EVI^KO cells. The cells were treated either with murine rec. Wnt3a (final concentration 200 ng/mL; for activity of rec. Wnt3a, see SI Appendix, Fig. S8A) or medium (control). The automatically determined condensate count was normalized to the number of cells per image and related to the count of medium-treated HEK293T^DVL2_mEos cells of the same timepoint. Box plot is for the relative, normalized condensate counts, with dots representing the average relative, normalized condensate counts from four individual experiments.

Fig. 5.

Cell cycle–dependent localization of Dvl2_mEos3.2 condensates to the centrosome. (A) Exemplary time-lapse images are of two mitotic cells that lose condensates before and regain condensates after mitosis. At 0 h, two cells marked by arrowheads in bright-field and fluorescent images harbor condensates. After 1 h to 2 h, both cells enter mitosis. No fluorescent condensates can be observed. At time point 6.5 h, the first cell has started cytokinesis (marked by an arrowhead). At 9.5 h, cell division is completed, and a new condensate starts forming (arrowheads in 9.5, 10, and 14 h). The images were taken on an A1 Nikon microscope every 15 min for 15 h under physiological conditions. Maximum intensity projections of four slices spanning a total of 3 µm are shown. (Scale bar, 10 µm.) See also Movie S1. (B) Formation of Dvl2_mEos3.2 condensates is inhibited in mitotic cells. For enrichment of mitotic cells, HEK293T^DVL2_mEos cells were synchronized by double thymidine block. Manual counting was done of mitotic cells with and without Dvl2_mEos3.2 condensates in four fields-of-view per experiment (20× magnification). Bar represents the mean of three independent experiments that are shown by dots. (C) Dvl2_mEos3.2 condensates localize to the pericentriolar matrix/centrosome in HEK293T^DVL2_mEos cells. Confocal microscopy is of HEK293T^DVL2_mEos cells with gamma-tubulin or PCM1 antibodies (magenta). Maximum intensity projections are shown of gamma-tubulin (z = 21) and PCM1 (z = 31). Representative images of three or four replicates with imaging of five or more points of view. (Scale bar, 10 µm.) White boxes mark the magnified areas. (D) Dvl2_mEos3.2 condensates localize to the mother centriole in interphase cells. HEK293T^DVL2_mEos cells were stained with the centrosomal marker CEP164 (magenta) and AAT (yellow). I–III represent magnifications as indicated by white boxes of (I) mother centriole, (II) basal body of cilia, and (III) spindle poles. Maximum intensity projections are shown (z = 30). Representative images of four replicates with imaging of six or more points of view. (Scale bar, 10 µm.) (E) SIM confirms Dvl2_mEos3.2 condensates at the centrioles (I), lack of condensate formation at cilia (II), and revealed distinct substructures (III). Multiple Z planes were acquired with 200 nm distance. Maximum intensity projections are shown (z = 7). (Scale bar, 1 µm.)

Fig. 6.

SMLM reveals repetitive shapes of Dvl2_mEos3.2 condensates at various stages of the centrosomal cycle. (A) Model of UV irradiation-induced photoconversion of mEos3.2 for CRISPR PALM including representative images of B. (B) Endogenous Dvl2_mEos3.2 unravels supramolecular condensates with distinct shapes and substructures in HEK293T^DVL2_mEos cells. Recurrent shapes include a shooting star–like lineup, C shapes, and round and donut-like shapes. Substructures within these shapes, that is, recurring patterning with smaller holes inside the larger shapes, are especially visible in the condensates labeled with “round.” Insets show widefield images of the respective condensate. Representative images of SMLM of ≥30 condensates are shown. (Scale bar, 1 µm.) (C) Model of dual-color image acquisition using CRISPR PALM in combination with DNA-PAINT including a representative image of D. (D) Repetitive shapes of Dvl2_mEos3.2 condensates in relation to the mother centriole. Dual-color, single-molecule microscopy was performed in HEK293T^DVL2_mEos cells using CRISPR PALM (mEos; green) in combination with DNA-PAINT (CEP164; magenta). Insets are schematic pictograms showing the spatial relation of the mother centriole and the respective Dvl2_mEos3.2 condensate shape. Representative images of SMLM of ≥60 condensates are shown. (Scale bar, 1 µm.)