Advanced fluorescence microscopy
In central project Z02, we will make a wide range of imaging techniques available to other projects within the CRC 1324, and we will further develop and adapt quantitative fluorescence microscopy techniques for Wnt signaling studies. We will use conventional confocal and widefield microscopy and also super-resolution techniques including stimulated emission deplection (STED) microscopy, structured illumination microscopy (SIM) and single-molecule localization microscopy (SMLM) to observe cells, tissues and entire model organisms in all three spatial dimensions. A powerful digital scanned light sheet microscope (DSLM) for fast 3D imaging with near-isotropic resolution has been built in the first funding period and is now available for Wnt research. The Z-project is located at two sites (Institute of Applied Physics in Karlsruhe and Bioquant in Heidelberg), providing complementary expertise and a wide range of imaging infrastructure to the consortium. Quantitative microscopy will be further advanced to enable measurements over extended periods of time under minimally invasive, near-physiological conditions. Fluctuation-based methods (FCS, STICS, N&B etc.) that allow us to measure fast (sub-millisecond) dynamics of biomolecules and to quantify protein-protein interactions have proven their effectiveness in the first funding period and made important contributions to several publications. We will continue to refine and apply these methods to shed light on key processes involved in Wnt signaling, i.e., secretion, transport, ligand-receptor interactions and downstream signaling. Using fluorescence imaging, we will investigate the intricate processes along the secretory pathway facilitating the release of Wnt proteins into the extracellular space. Quantitative image analysis will reveal transport
modes of secretory vesicles such as directed motion or diffusion. By using targeted photoactivation, e.g., with EosFP, we will tag proteins and track their pathways through the secretion machinery. We will further study and quantify how Wnt proteins are transported to the receiving cells, by binding to carrier proteins, or on lipoprotein particles or exovesicles or via cytonemes. We will continue with our efforts to quantify interactions between Wnt ligands, Wnt modulators and receptors in detail by using fluctuation spectroscopy and fast imaging. The key goal is to gain a clear mechanistic understanding of signalosome formation and dynamics including internalization and downstream effects. With our experiments, we will provide quantitative data on functional processes along the Wnt signaling pathway and thereby contribute to advancing our knowledge of this complex network of biomolecular interactions.
- Ambrosi, G., O. Voloshanenko, A.F. Eckert, D. Kranz, G.U. Nienhaus, and M. Boutros. 2020. Allele-specific Endogenous Tagging and Quantitative Analysis of β-Catenin in Colon Cancer Cells. bioRxiv, DOI: 10.1101/2020.06.18.159616.
- Bufe, A., A. García del Arco, M.I. Hennecke, M. Ostermaier, A. de Jaime-Soguero, Y.C. Lin, A. Ciprianidis, U. Engel, P. Beli, H. Bastians and S.P. Acebrón. 2020. Wnt signaling recruits KIF2A to the spindle to ensure chromosome congression during mitosis. bioRxiv. https://doi.org/10.1101/2020.12.22.404020
- Sunbul*, M., J. Lackner, A. Martin, D. Englert, B. Hacene, K. Nienhaus, G.U. Nienhaus* and A. Jäschke*. 2020. Super-resolution RNA imaging using a rhodamine-binding aptamer with fast exchange kinetics. Nat. Biotechnol., 39(6):686-690. PMID: 33574610
- Eckert, A.F., P. Gao, J. Wesslowski, X. Wang, J. Rath, K. Nienhaus, G. Davidson, and G.U. Nienhaus. 2020. Measuring ligand-cell surface receptor affinities with axial line-scanning fluorescence correlation spectroscopy. Elife 9:e55286. PMID: 32441251.
- Puzik, K., V. Tonnier, I. Opper, A. Eckert, L. Zhou, M.C. Kratzer, F. le Noble, G.U. Nienhaus, and D. Gradl. 2019. Lef1 regulates caveolin expression and caveolin dependent endocytosis, a process necessary for Wnt5a/Ror2 signaling during Xenopus gastrulation. Sci. Rep. 9:15645. PMID: 30060804.
- Mattes, B., Y.L. Dang, G. Greicius, L.T. Kaufmann, B. Prunsche, J. Rosenbauer, J. Stegmaier, R. Mikut, S. Ozbek, G.U. Nienhaus, A. Schug, D.M. Virshup, and S. Scholpp. 2018. Wnt/PCP controls spreading of Wnt/beta-catenin signals by cytonemes in vertebrates. Elife 7:e36953. PMID: 30060804.
- Beretta, C.A., N. Dross, L. Guglielmi, P. Bankhead, M. Soulika, J.A. Gutierrez-Triana, A. Paolini, L. Poggi, J. Falk, S. Ryu, M. Kapsimali, U. Engel, and M. Carl. 2017. Early Commissural Diencephalic Neurons Control Habenular Axon Extension and Targeting. Curr. Biol. 27:270-278. PMID: 28065605
- Engel, U. 2017. Four-Channel Super-Resolution Imaging by 3-D Structured Illumination. Methods Mol. Biol. 1663:79-94. PMID: 28924660
- Gao, P., B. Prunsche, L. Zhou, K. Nienhaus, and G.U. Nienhaus. 2017. Background suppression in fluorescence nanoscopy with stimulated emission double depletion. Nat. Photon. 11:163-169.
- Ruthnick, D., A. Neuner, F. Dietrich, D. Kirrmaier, U. Engel, M. Knop, and E. Schiebel. 2017. Characterization of spindle pole body duplication reveals a regulatory role for nuclear pore complexes. J Cell Biol. 216:2425-2442. PMID: 28659328