Periodic Reporting for period 2 - ANEUPLOIDY (Molecular origins of aneuploidies in healthy and diseased human tissues)
Período documentado: 2021-10-01 hasta 2023-03-31
Chromosome segregation errors cause aneuploidy, a state of karyotype imbalance that accelerates tumor formation and impairs embryonic development. Even though mitotic errors have been studied extensively in cell cultures, the mechanisms generating various errors, their propagation and effects on genome integrity are not well understood. Moreover, very little is known about mitotic errors in complex tissues. The main goal of this project is to uncover the molecular origins of mitotic errors and their contribution to karyotype aberrations in healthy and diseased tissues. To achieve our goal, we have assembled an interdisciplinary team of experts in molecular and cell biology, cell biophysics, chromosomal instability in cancer, and theoretical physics. Our team is introducing novel approaches to study aneuploidy (superresolution microscopy, optogenetics, laser ablation, single cell karyotype sequencing) and applying them to state-of-the-art tissue cultures (mammalian organoids and tumoroids). In close collaboration, we are establishing assays to detect and quantify error types in cells and using them on various healthy and cancer tissues. We are exploring the molecular origins of errors, their propagation and impact on genome integrity, as well as the mechanisms that ensure high chromosome segregation fidelity in healthy tissues. Interwoven in these collaborations is the development of a theoretical model to describe the origin of errors and to quantitatively link chromosome segregation fidelity in single cells and tissues. Model and experiment are continuously inspiring each other to achieve deep understanding of how mitotic errors arise, how they propagate and how they impact on cell populations.
To uncover the origins of errors in chromosome segregation and how they propagate in tissues, we combined expertise in 3D cultures and imaging of them, in biophysical and molecular analysis of chromosome segregation, both experimental and theoretical, together with expertise in genetics and aneuploidy. We have imaged chromosome segregation in organoids, which are tiny organs grown from single cells in a gel, and quantified segregation error types and dynamics. We have further studied the molecular origins of errors by changing protein localization using laser light, and by imaging dividing cells by super-resolution microscopy after specific protein perturbations. To explore the consequences of segregation errors on karyotype evolution over multiple generations, we developed a theoretical model based on a novel approach, the solutions of which provide insight into the interplay between chromosome segregation errors, cell proliferation and cell death and their effects on the development of cancer karyotypes. An advantage of the theoretical model in comparison with experiments is that the model can provide a whole pedigree tree over many generations, which is not experimentally accessible. We developed a novel mouse model with unprecedented levels of chromosome missegregation in the adult animal, which allowed us to uncover that initial detrimental effects of random missegregation are outbalanced by clonal selection dictated by the chromosomal location and nature of certain genes. The synergy of our complementary types of expertise allowed us to study both causes and consequences of chromosome segregation errors.
The next steps will be to combine our experimental approaches to study the mechanisms of chromosome interaction with the mitotic spindle and how malfunctioning of these mechanisms results in different chromosome segregation errors, as well as how they lead to altered cell karyotypes. The knowledge and expertise gained by studying isolated cells will be transferred to studies of chromosome segregation in a 3D setting in mouse and human organoids. Our experiments will be combined with theoretical work to understand the link between the molecular origins of errors and their impact on aneuploidy in cell populations and tissues. Our theoretical model will provide an interpretation of development pathways of various tumor karyotypes, including those in mouse models of aneuploidy and in human cancers. The work of our Synergy team will continuously feed back on each other towards achieving the main goal of the project.