Resolving intermediate states of mitotic entry
by Monica Gobran
Date of Examination:2025-09-26
Date of issue:2025-12-08
Advisor:Dr. Peter Lenart
Referee:Dr. Peter Lenart
Referee:Prof. Dr. Matthias Dobbelstein
Referee:Prof. Dr. Melina Schuh
Referee:Dr. Alexander Stein
Referee:Prof. Dr. Gregor Bucher
Referee:Dr. Johannes Soeding
Persistent Address:
http://dx.doi.org/10.53846/goediss-11684
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Abstract
English
Mitosis, the division of cells, is a fundamental biological process essential for growth, tissue repair, and the propagation of genetic material. Mitosis is tightly regulated and its dysregulation has severe consequences, underlying diseases such as cancer. A critical first step of mitosis is mitotic entry, the "point of no return", when a biochemical switch transitions the cell from an interphase to a mitotic state. This switch is mediated by mitotic kinases, which phosphorylate a large number of substrates, driving dramatic changes to cellular organization. These include the complete remodeling of the actin and microtubule cytoskeleton, the condensation of chromosomes, and the breakdown of the nuclear envelope (NEBD), mixing nuclear and cytoplasmic compartments. Despite its critical importance, many questions remain open regarding the precise mechanisms governing mitotic entry. To a large part, this is due to the fact that this dramatic transition occurs in a very short time and therefore its intermediate states are nearly impossible to access experimentally. In my thesis work, I developed novel protocols, whereby using a combination of small-molecule kinase inhibitors, I was able to drive cells into such intermediate states for the first time. By combining cellular assays and phosphoproteomics, I could resolve and characterize in detail intermediates of mitotic entry, providing critical new insights into mitotic regulation. In the first part of my thesis study, I explored the role of PLK1 in mitotic prophase. My results demonstrate that PLK1 inhibition in synchronized G2 cell populations of multiple mammalian cell lines delays mitotic entry, with significant variability observed between individual cells. To explain this variability, we used a mathematical model that recapitulates the observed phenotypic differences. Our results show that PLK1-inhibited cells are delayed in a prophase-like state characterized by low CDK1 activity, which increases gradually over hours. During this state, cells exhibit progressively condensing chromosomes, increased microtubule dynamics, and reorganization of the actin cortex, while the nuclear envelope remains intact. To further characterize this state, we employed phosphoproteomics, revealing phosphorylation of key regulators of chromatin organization and the cytoskeleton, consistent with the observed cellular phenotypes. These findings, presented in the first manuscript published in the EMBO Journal earlier this year, demonstrate that PLK1 inhibition stabilizes cells in a prophase-like state with low CDK1 activity, revealing a distinct set of early mitotic phosphorylation events. In the second part of my thesis, I investigated the role of CDK1 in mitotic progression. CDK1 is the central regulator of mitosis, and its activity is controlled by a series of feedback loops that ensure proper timing and execution of mitotic events. Our results show that when the feedback loops are disabled, CDK1 activity can be titrated to a sub-threshold level, leading to a novel state termed "low CDK mitosis". In this state, while cells apparently progress through mitosis, the nuclear envelope does not break down. Cells enter mitosis, condense their chromosomes, accumulate nuclear Cyclin B1, progress through mitosis within the same duration as in control cells, and then exit mitosis, decondense their chromosomes and completely degrade Cyclin B1. Proteomic analysis confirms that these cells return to a G1-like proteomic state after low CDK mitosis. Strikingly different from normal mitosis, the nuclear pore complex (NPC) scaffold and the nuclear lamina remain intact throughout low CDK mitosis. However, I was able to show that the nuclear-cytoplasmic transport is compromised due to partial disassembly of NPCs. I show that this partial loss of nuclear integrity releases spindle assembly factors from the nucleus and therefore the mitotic spindle assembles in the cytoplasm. However, the intact nuclear envelope prevents spindle microtubules from accessing the chromosomes, ultimately leading to mitotic exit without chromosome segregation. To further characterize this state, we performed phosphoproteomics on a pure population of cells in low CDK mitosis. Our analysis reveals that most phosphorylation events responsible for the observed mitotic changes are mediated by kinases other than CDK1. There are, however, phosphosite sequences containing the CDK1 minimal consensus motif. In these, I identified a basic amino acid-rich motif that follows CDK1 phosphorylation sites, providing new insights into CDK1 temporal regulation and substrate specificity. Together, my findings substantially advance our understanding of mitotic regulation. They highlight the critical role of PLK1 in catalyzing mitotic entry and the dynamic activation of CDK1 in ordering mitotic events. Additionally, the discovery of "low CDK mitosis" challenges the traditional view of mitosis as an all-or-none process and provides new insights into the molecular mechanisms underlying mitotic regulation in mammalian cells. This work not only contributes to our fundamental understanding of cell division but also has implications for the development of therapeutic strategies targeting mitotic kinases in cancer and other diseases.
Keywords: Mitosis; Mitotic Entry; cell division; cell cycle; CDK1; PLK1; mitotic kinases; Spatiotemporal regulation
