Projects Madelon Maurice
Mechanisms of Wnt signaling in stem cells, development and cancer
Wnt proteins control embryonic development in all multicellular organisms, including humans. In adults, these proteins also sustain the vital supply of stem cells that replenish various body tissues (e.g. bone, nervous system and skin repair). The interaction of Wnt proteins with cell surface receptors on receiving cells can result in a variety of cellular responses, including cell proliferation and cell fate decisions, as well as organized cell movements and establishment of tissue polarity. Deregulated Wnt signaling due to mutations is strongly linked to cancer and degenerative disease. Together, the Wnt signaling pathway holds promise for therapeutical intervention in the fields of cancer, stem cell biology and regenerative medicine.
The overall aim of our work is to understand the molecular mechanisms by which cells interpret Wnt signals received at their cell surface and how dysregulation of these events by mutations leads to cancer. In our approach we include methods at the atomic level (fluorescence spectroscopy, peptide libraries, NMR), at the molecular level in cells (biochemistry, microscopy, gene silencing, proteomics) and at the level of complex tissues in the living organism (immunohistochemistry, microscopy). By uncovering molecular details of how protein traffic, complex formation and activity direct cellular decisions we aim to provide novel clues to modulate Wnt-mediated cellular responses.
Figure 1. The canonical Wnt pathway. (OFF) In the absence of Wnt, cytoplasmic β-catenin is phosphorylated by a protein complex consisting of the scaffolding proteins Axin and APC and the kinases GSK3β and CK1β. Subsequent recognition by the ubiquitin ligase β-TrcP leads to ubiquitin-mediated degradation of β-catenin. (ON) Binding of Wnt to Fz leads to recruitment of the cytoplasmic effector protein Dvl. Phosphorylation of the Lrp cytoplasmic tail subsequently provides a docking site for Axin. Redistribution of Axin-GSK3β complexes to the plasma membrane compromises its ability to downregulate β-catenin. In turn, β-catenin accumulates, migrates to the nucleus to bind TCF and induce transcription of target genes. AJ; adherens junctions
1) Signaling relay via the Frizzled receptor
Wnt binding to heptahelical Frizzleds (Fz) mediates critical cell-cell communication events in complex tissues, including the regulation of stem cell maintenance. The molecular signaling events downstream of Fz are remarkably divergent, involving activation of β-catenin-mediated transcription, or β-catenin-independent Rac/Rho-induced cytoskeletal rearrangement or Ca2+ responses. A fundamental, unanswered question is how Wnt-Fz complexes steer downstream signaling by coupling to cytosolic effectors such as Dishevelled (Dvl). To address this issue, we use combinatorial peptide libraries which mimic the intracellular domains of Fz5 (Pepscan, Lelystad), biochemistry, imaging of cells and tissues to determine how the interaction between Dvl and different Fz members couples to downstream signaling events.
2) Ubiquitin-mediated regulation of Wnt signaling
Little is known about how posttranslational modifications drive local protein-protein interactions at the plasma membrane during Wnt signaling initiation. We identified the deubiquitinating enzyme (DUB) and tumor suppressor CYLD as a negative regulator of proximal events in the Wnt/β-catenin pathway. Mechanistically, we uncovered a positive role of Dvl ubiquitination on Wnt-induced β-catenin-mediated transcription in CYLD-depleted cells. Moreover, our results place dysregulated Wnt signaling central to development of human cylindromatosis, a skin tumor syndrome caused by CYLD mutations. We aim to uncover the molecular basis of this novel ubiquitin-driven signaling mechanism and determine its consequences for cell fate decisions and proliferation using cell and tissue culture, biochemistry and microscopy.
Figure 2. CYLD-mutant cylindroma tumors display hyperactive Wnt signaling activity. β-Catenin staining of CYLD-mutant cylindroma tumor tissue by immunohistochemistry (IHC). Tumor cells accumulate nuclear β-catenin (arrows) whereas normal human epidermis shows mainly E-cadherin-bound β-catenin in adherens junctions (asterisk). 40x Magnification is shown. (adapted from: Tauriello et al. (2010), Mol. Cell, 37, 607-619)
3) Spatio-temporal control of Wnt receptor complex assembly and endocytic trafficking
Downstream β-catenin-dependent gene activation in the Wnt signaling cascade clearly depends on Wnt-induced receptor endocytosis, but the underlying mechanisms by which membrane trafficking drives pathway activation are poorly understood. To address this issue, we examine where key signaling steps in the Wnt cascade occur, which proteins are locally involved and how they direct modification, trafficking and formation of active signaling complexes. We take a combined biochemical and microscopy (confocal, live cell and cryo-immuno EM) approach to dissect the trafficking and compartmentalization of resting and activated Wnt receptor complexes in stem cells and their differentiated progenitors. With these studies, we aim to understand how cells organize, process and tune signaling events.
Figure 3. Left; Wnt-induced Frizzled endocytosis in HEK293T cells.Fz5: Red; Wnt3a: Blue; Rab5: green. Right; Clathrin-mediated endocytosis of the Frizzled receptor. Fz5 was labeled with 15 nm gold particles and analyzed using cryo immuno-EM.
4) How missense mutations in Wnt cascade proteins lead to cancer
Single base pair alterations are a major cause of human cancer. How individual missense mutations contribute to tumor development at the protein level remains unclear however in the majority of cases. We aim to understand this fundamental question for the Wnt pathway tumor suppressors Axin and APC. Do oncogenic point mutations alter protein stability? What are the consequences for protein function in the cell? How does the mutant protein drive Wnt pathway activity and cell growth in complex tissues? We address these questions in an interdisciplinary approach using biophysics, biochemistry and animal genetics. In this work, we collaborate with Dr. Stefan Rüdiger, Bijvoet Center for Biomolecular Research, Utrecht.