Periodic Reporting for period 3 - ANEUPLOIDY (Molecular origins of aneuploidies in healthy and diseased human tissues)
Reporting period: 2023-04-01 to 2024-09-30
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.
A close collaboration between our four Synergy groups resulted in 9 joint publications between at least two groups, covering several exciting findings. Using the expertise of the Kops lab in single-cell DNA sequencing and organoids, we have shown that chromosomes have different segregation error frequencies that correlate with their location in the interphase nucleus, and in combination with the expertise of the Tolić lab in high-end imaging, we have found a "danger zone" for chromosomes behind spindle poles (Klaasen et al., Nature 2022). Collaboration between the Kops and Tolić labs continued with the finding that centromere chromatin is organized into a two-domain structure in mitosis, with lagging chromosomes in cancer cells having the two subdomains bound to opposite spindle poles (Sacristan et al., Cell 2024). This teamwork is currently extended to explore the chromosome instability in patient-derived organoids and organoids from a genetically engineered mouse model for chromosomal instability (CiMKi), generated by the Kops lab. Development of a mathematical model based on a novel concept of macro-karyotype by the Pavin group, in collaboration with MIT and the Tolić lab, has shown that tumor karyotypes can be explained by proliferation-driven evolution of aneuploid cells (Ban et al., Biophys J 2023). This theoretical approach has been extended to describe karyotype evolution in organoids during tumor development, in a collaboration between the Pavin and Kops groups. A teamwork between the Tolić and Pavin groups revealed that poleward flux of kinetochore fibers promotes chromosome alignment (Risteski et al., Cell Rep 2022). In this work the Tolić lab developed speckle microscopy in human spindles and showed that kinetochore fiber flux is length-dependent, and the Pavin group introduced a flux-driven centering model that provides a physical explanation for chromosome alignment. This collaboration further showed that the formation of the mitotic spindle relies on a network-to-bundles transition of microtubules driven by kinetochores and chromosomes (Matković et al., Nat Commun 2022). Continuing studies on the chirality of the mitotic spindle, the Pavin and Tolić groups developed a novel method to quantify spindle shape (Ivec et al., Biophys J 2021). In total, we published 16 original papers in top journals such as Nature, Curr Biol, J Cell Biol, eLife, and 7 reviews. The Synergy team is now exploring the mechanisms of chromosome congression and how the spindle adapts to polyploidy, including the formation of multipolar spindles.
The next steps will be to combine our experimental and theoretical approaches developed from the beginning of the project, including long-term live-cell imaging by lattice light-sheet microscopy, patient-derived organoids, and the theoretical model based on the macro-karyotype concept, to study the mechanisms of chromosome instability and karyotype evolution. The knowledge and expertise obtained by studying human cell lines will be transferred to studies of chromosome segregation in organoids (mouse and human), for which we optimized genome editing, immunofluorescence imaging, and live-cell imaging . We will study the mechanisms of chromosome congression to the spindle midplane and how defects in this process lead to chromosome mis-segregation. As a frequent congression defect observed in cancer cells, we will explore unaligned chromosomes that remain at the spindle pole and prolong mitosis, focusing on the mechanisms of how they lead to chromosome mis-segregation. Rescue of mitotic errors will be used as a validation of the discovered mechanisms. Another important source of mitotic errors are multipolar spindles. We will study the mechanisms of formation of multipolar spindles and their transformation into bipolar spindles, which promote cell survival. The daughter cells coming from multipolar divisions are expected to have substantially perturbed karyotypes and we will study how these karyotypes affect cell proliferation and how they evolve. Finally, we aim to reveal how karyotypes in unperturbed pre-tumor organoids differ between patients and how they evolve during organoid growth.