In order to study the effects of protein condensation on the Wnt-pathway I focused on the key signaling factor β-catenin in three main sections. First, I characterized the effect of β-catenin condensation on the destruction complex. Second, I determined the functional relevance of β-catenin condensation for cofactor interactions. Finally, I elucidated the effect of oncogenic levels of β-catenin on cytoplasmic and nuclear β-catenin condensates.
The destruction complex is a protein complex in the cytoplasm that is instrumental in the continuous degradation of β-catenin when the Wnt-pathway is inactive. I engineered several genetically modified cell lines that that were then characterized using several fluorescent microscopy techniques. The gathered experimental data showed that the Wnt-pathway destruction complex indeed forms protein droplets in living cells. This work also allowed me to initiate an ongoing collaboration with the lab of Prof. Madelon Maurice, an expert on destruction-complex biology. This work has led to the development of a new model for the β-catenin destruction complex that can have a high impact on the fields of WNT-signaling and protein droplet formation. Therefore, it will likely be shared in a scientific publication.
To fully understand the Wnt-pathway is crucial to understand the nature of the interactions between β-catenin and its co-factors. For several reported co-factors, the mode of interaction is unclear. A condensate model would explain how these co-factors do interact with β-catenin. I pursued a candidate-based approach where I biochemically purified several suspected β-catenin co-factors and used in-vitro droplet formation assays with β-catenin to evaluate their capacity to co-condense. I have purified over a dozen putative co-factors and have found all of these candidates to be able to form protein droplets together with β-catenin, with varying degrees of efficiency. These results indicate that condensate formation is a likely mechanism for β-catenin co-factor interaction and indicate that perturbing condensate formation of these factors may be a valid strategy to inhibit the Wnt-pathway in a disease setting. The perturbation of condensate formation is an ongoing research project that is currently active in my lab and the identification of a condensate mechanism for β-catenin co-factor interaction is part of a scientific publication currently under preparation.
Condensation into protein droplets is sensitive to protein levels, therefore it may be that the high levels of β-catenin in cancer alter the protein droplets. After engineering several colon cancer cell lines and studying the β-catenin protein droplets I found limited differences between the droplets that are formed under normal conditions and in cancer cells. In this work I developed a new system for β-catenin protein droplet visualization. This approach allowed more sensitive detection of β-catenin protein droplets and identified specific proteins that can affect the propensity of oncogenic β-catenin to form protein droplets. These results have fueled further investigation into the topic of specific condensate perturbation. The data from these studies will have a high impact on the scientific field of condensate biology and is currently under preparation for a scientific publication.
The results obtained in this project have allowed me to present at several scientific meetings and present at in-house meetings regularly. Scientific publication of the project results is pending, and one manuscript is submitted for review. The other parts of the work carried out will be part of further research publications. In addition to these outputs, I have applied for a patent based on the work carried out which may lead to a spin-off company or out-licensing. Next to these scientific dissemination activities, I have also engaged in several teaching activities concerning the work carried out. These include supervision of 4 master students in the lab, supervision of 3 master students during the writing of a literature thesis and teaching of 3 courses through plenary lectures and individual supervision.
