Quantitative fluorescence microscopy for the analysis of Wnt pathway interactions and dynamics
A variety of receptors and co-receptors bind a range of specific ligands to regulate the Wnt pathway. Accurate quantification of these interactions in living cells will help us to better understand signaling specificity in complex cellular environments where multiple interactions occur. The overall aim of our research is to provide a comprehensive, quantitative analysis of ligand-receptor and receptor-(co)receptor interactions for the Wnt pathway under physiological conditions, using standardized biochemical and biophysical methods, especially advanced fluorescence microscopy techniques. We will use bioluminescence resonance energy transfer (BRET) and fluorescence fluctuation spectroscopy (FFS) approaches including axial line-scanning fluorescence correlation spectroscopy (axial lsFCS) to quantify interactions between different Wnt ligands and Frizzled (FZD) receptors. We will also address the impact of co-receptors on the binding affinity between FZDs and Wnts. In the first funding period, we have demonstrated that LRP6-mCherry expressed at low concentrations in CRISPR/Cas9 gene-edited cells as well as stable cell lines displays two- to three-fold higher affinity towards DKK1 than LRP6-mCherry overexpressed in transiently transfected cells. These results suggest that measurements on cell lines with unphysiologically high levels of receptor expression are likely to underestimate ligand-receptor binding affinities; more accurate values may be obtained by using appropriately selected stable cell lines expressing receptors at more physiological levels. Accordingly, Wnt-FZD interaction studies will be performed on stable FZD1-10-mCherry cell lines with low FZD expression levels. To extend the BRET analysis to all ten FZDs in order to quantify their relative differences in binding affinities for Wnt-3a, we will generate stable cell lines expressing low levels of Nano-luciferase tagged FZD1-10 (Nluc-FZD1-10), as well as Nluc-FZD1-10-mCherry. In initial studies, we applied eGFP-Wnt-3a conditioned medium (CM) to live HEK293T cells transiently overexpressing Nluc-FZD4 and obtained low nanomolar binding affinities (2-3 nM). We will also perform comparative analyses using both NanoBRET and axial lsFCS. Additional fluorescently tagged Wnts will be employed or, if not available, generated and tested in order to expand our analysis further. Wnts will be prepared either as CM, purified protein or purified vesicular fractions. Biophysical and biochemical experiments will be carried out to explore potential differences in their binding properties. We will use super-resolution single-molecule localization microscopy (SMLM) imaging to visualize the dynamics of activated Wnt receptors/co-receptors in time and space, with an initial focus on receptor clustering and
signalosome formation in the cell membrane. By fusing receptors (LRP6 or FZD) with the photoconvertible fluorescent protein mEosFP, the translocation of individual receptors can be tracked over time. Number and brightness (N&B) analysis will be used to analyze oligomerization (homodimerization and heterodimerization) of FZD receptors and co-receptors. In this joint project, the Davidson lab will generate and characterize Wnt signaling properties of the required constructs/transgenic cell lines and perform NanoBRET analysis together with the lab of Gunnar Schulte, while the Nienhaus lab will perform biophysical analyses using advanced fluorescence microscopy methods.
- 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.
- Wesslowski, J., P. Kozielewicz, X. Wang, H. Cui, H. Schihada, D. Kranz, M.P. Karuna, P. Levkin, J.C. Gross, M. Boutros, G. Schulte, and G. Davidson. 2020. eGFP-tagged Wnt-3a enables functional analysis of Wnt trafficking and signaling and kinetic assessment of Wnt binding to full-length Frizzled. J. Biol. Chem. 295:8759-8774. PMID: 32381507
- 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.
- Salama, M., D. Benitez-Riquelme, S. Elabd, S. Munoz., P. Zhang, M. Glanemann, M. Mione, R. Goldin, T. Soussi, G. Davidson, C. Blattner. 2019. Fam83F induces p53 stabilisation and promotes its activity. Cell Death Differ. 26:2125–2138. PMID:30692643
- 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.
- 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.
- Dörlich, R.M., Q. Chen, P.N. Hedde, V. Schuster, M. Hippler, J. Wesslowski, G. Davidson, and G.U. Nienhaus. 2015. Dual-color dual-focus line-scanning FCS for quantitative analysis of receptor-ligand interactions
in living specimens. Sci. Rep. 5:10149. PMID: 25951521.
- Chen, Q., Y. Su, J. Wesslowski, A.I. Hagemann, M. Ramialison, J. Wittbrodt, S. Scholpp, and G. Davidson. 2014. Tyrosine phosphorylation of LRP6 by Src and Fer inhibits Wnt/β-catenin signaling. EMBO Rep. 15:1254–1267. PMID: 2539190
- 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.