Periodic Reporting for period 1 - Catenin-Condensation (The effect of β-catenin condensation on the Wnt-pathway)
Période du rapport: 2020-04-01 au 2022-03-31
Specialized signaling pathways allow communication between cells in the body and mis-regulation of these pathways almost invariably leads to disease. β-catenin is a key signal-transduction protein in the highly conserved Wnt-signalling pathway, regulating cell fate specification, proliferation and differentiation during development and in various adult tissues. Errors in the Wnt-pathway lead to severe developmental disorders in the embryo and cancer in the gut, breast or blood in adults. To achieve its role, β-catenin is present at the plasma membrane, in the cytoplasm and in the cell nucleus. In all these distinct sites β-catenin needs to be highly localized and organized. How this organization takes place is currently unknown. We have recently found that β-catenin can condensate into protein droplets together with other key proteins in the nucleus. In this work we aim to uncover how the condensation of β-catenin into protein droplets impacts the Wnt signalling pathway at the membrane and in the cytoplasm. Deeper knowledge of the fundamental Wnt signalling pathway can have a profound impact on the understanding of embryonic development, adult regeneration and cancer.
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.
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.
This project addressed the question if protein droplet formation plays a role in the key developmental Wnt-signaling pathway. Prior to this project nothing was known about protein droplet formation of β-catenin outside the nucleus and with different co-factors inside the nucleus. The work in this project has considerably advanced the state of the art with the following results.
1. Identification of the destruction complex in protein droplets in the cell
2. Clarification of the mechanism of interaction of a number of previously reported β-catenin co-factors with β-catenin
3. Development of a new nanobody-based imaging system for live detection of nuclear β-catenin protein droplets
4. Characterization of the liquid like properties of β-catenin protein droplets in normal and cancer cells
5. Identification of short interacting peptides that specifically perturb transcription-associated β-catenin protein droplets
These basic science advances will have a profound impact on the research field of protein droplet formation (biomolecular condensates) and will further the field of Wnt-signaling and developmental signaling. Besides impacting directly related scientific fields, the knowledge generated in this work will also impact the study of Wnt-driven cancers, because it provides mechanistic insight in previously understudied aspects. Finally, a broader impact may be the development of new therapeutic agents targeting protein droplets in cells. This work has provided the proof of principle for a peptide based therapeutical agent that directly targets protein droplets. Further research on this topic may lead to the development of other protein droplet targeting agents that can have beneficial effects in Wnt-driven disease, but also in other diseases that rely on aberrant protein droplet formation, including many different cancers, developmental disorders and neurological diseases. Taken together, the research performed under this action impacts many fields of science and may lead to new medical applications.
1. Identification of the destruction complex in protein droplets in the cell
2. Clarification of the mechanism of interaction of a number of previously reported β-catenin co-factors with β-catenin
3. Development of a new nanobody-based imaging system for live detection of nuclear β-catenin protein droplets
4. Characterization of the liquid like properties of β-catenin protein droplets in normal and cancer cells
5. Identification of short interacting peptides that specifically perturb transcription-associated β-catenin protein droplets
These basic science advances will have a profound impact on the research field of protein droplet formation (biomolecular condensates) and will further the field of Wnt-signaling and developmental signaling. Besides impacting directly related scientific fields, the knowledge generated in this work will also impact the study of Wnt-driven cancers, because it provides mechanistic insight in previously understudied aspects. Finally, a broader impact may be the development of new therapeutic agents targeting protein droplets in cells. This work has provided the proof of principle for a peptide based therapeutical agent that directly targets protein droplets. Further research on this topic may lead to the development of other protein droplet targeting agents that can have beneficial effects in Wnt-driven disease, but also in other diseases that rely on aberrant protein droplet formation, including many different cancers, developmental disorders and neurological diseases. Taken together, the research performed under this action impacts many fields of science and may lead to new medical applications.