Superresolution microscopy localizes endogenous Dvl2 to Wnt signaling-responsive biomolecular condensates
Antonia Schubert1,2,3, Oksana Voloshanenko1, Franziska Ragaller1
1Division of Signaling and Functional Genomics, German Cancer Research Center and Department of Cell and Molecular Biology, Heidelberg University and BioQuant, 69120 Heidelberg, Germany.
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.
Insights
Dishevelled (Dvl) proteins form dynamic Wnt signaling condensates at the centrosome. This study reveals their physiological regulation and cell cycle dependence using super-resolution microscopy of endogenous Dvl2.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Dishevelled (Dvl) proteins are crucial for Wnt signaling pathways, impacting development, homeostasis, and disease.
- Previous research on overexpressed Dvl showed cytoplasmic puncta, but physiological mechanisms remain unclear.
Purpose of the Study:
- To investigate the endogenous function and localization of Dishevelled 2 (Dvl2) in Wnt signaling.
- To characterize the dynamic formation and regulation of Dvl2-containing biomolecular condensates at the single-molecule level.
Main Methods:
- Genome engineering of endogenous Dvl2 fused to mEos3.2 fluorescent protein.
- Super-resolution microscopy techniques, including photoactivated localization microscopy (PALM) and DNA-PAINT.
- Live-cell imaging and biochemical assays to assess Wnt pathway functionality.
Main Results:
- Endogenous Dvl2 forms sparse, single supramolecular condensates per cell at physiological levels, unlike overexpressed Dvl.
- These Dvl2 condensates colocalize with Wnt pathway components at centrosomal structures (gamma-tubulin, CEP164).
- Condensate formation is Wnt-dependent and exhibits cell cycle-specific organization and patterns.
Conclusions:
- Dvl2 localization into supramolecular condensates is dynamically regulated by Wnt signaling and cell state.
- This study provides single-molecule resolution insights into physiologically relevant biomolecular condensates in Wnt pathways.
