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CRACKING THE CODE BEHIND MITOTIC FIDELITY: the roles of tubulin post-translational modifications and a chromosome separation checkpoint

Periodic Reporting for period 5 - CODECHECK (CRACKING THE CODE BEHIND MITOTIC FIDELITY: the roles of tubulin post-translational modifications and a chromosome separation checkpoint)

Reporting period: 2023-01-01 to 2023-06-30

During the human lifetime it is estimated that 10 000 trillion cell divisions take place by a cellular process known as ‘mitosis’ to ensure tissue homeostasis, the renewal of epithelia and an efficient immune response against pathogens. Due to the stochastic nature of chromosome/kinetochore interactions with mitotic spindle microtubules, mitosis is prone to errors that can lead to aneuploidy, a condition that results from whole chromosome mis-segregation and is the main cause of prenatal human death. Aneuploidy is also the most common abnormality in human cancers. Thus, understanding the cellular mechanisms that normally ensure mitotic fidelity is not only important for our comprehension of life, but also represents major social and economic challenges with strong implications to human health and well-being in modern societies. In this project we tested two original concepts with strong implications for chromosome segregation fidelity. The first concept was based on the “tubulin code” hypothesis, which predicts that molecular motors “read” specific modifications on spindle microtubules. In this project we found that peripheral chromosome alignment in human cells is driven by the coordinated activities of kinetochore motors that are regulated by a navigation system based on tubulin tyrosination/detyrosination of specific spindle microtubule populations. In addition, we found that tubulin tyrosination/detyrosination works as a mitotic error code to ensure proper chromosome segregation by regulating the microtubule depolymerizing enzyme MCAK. This regulation turned out to be instrumental for how cancer cells respond to the microtubule stabilizing drug taxol. The second concept was centered on the recently uncovered chromosome separation checkpoint that delays the completion of mitosis in response to incompletely separated chromosomes. In this regard, we validated the role of a chromosome separation checkpoint in human cells to spatially and temporally regulate nuclear envelope formation at the exit from mitosis. In particular, we uncovered an Aurora B-mediated surveillance mechanism that ensures proper error correction during anaphase, thereby preventing micronuclei formation. Surprisingly, we uncovered a new route for micronuclei formation in cancer cells based on the missegregation of misaligned chromosomes that satisfy the spindle-assembly checkpoint. Lastly, this project established Indian muntjac cells as a model system for mitosis, uncovering a key role for the Augmin complex in kinetochore fiber maturation and demonstrating that chromosome (mis)segregation in mammals is biased by kinetochore size. Overall, this work established a paradigm shift in our understanding of how spatial information is conveyed to faithfully segregate chromosomes during mitosis.
Objective 1- Comprehensive analysis of the tubulin code during mitosis

Work Performed and Main Results:

- Microtubule turnover analysis after experimental perturbation of tubulin (de)tyrosination; experimental perturbations of Aurora B activity and midzone localization. The impact on error correction and micronuclei formation was determined

- Systematic purification and isolation of mitotic MAPs and motors that bind selectively to tyrosinated vs. detyrosinated microtubules

- Discovered that error correction during mitosis is inhibited by microtubule detyrosination

- Uncovered that MCAK inhibition by microtubule detyrosination is critical for cancer cell response to taxol

- In vitro assays with CENP-E motor and CLASP2 were performed

- Investigated the impact of microtubule detyrosination on centrosome separation at the onset of mitosis

Objective 2- Molecular and functional dissection of a chromosome separation checkpoint

Work Performed and Main Results:

- Implemented acute endogenous protein depletion during anaphase using trim-away

- Established the cross-talk between Cdk1 and Aurora B in the regulation of the anaphase-telophase transition

- Established phosphorylation mutants for potential Aurora B substrates involved at the anaphase-telophase transition

- Validated a role for a chromosome separation checkpoint in the prevention of micronuclei formation from anaphase errors in human cells

- Uncovered a role for misaligned chromosomes that satisfy the spindle assembly checkpoint in micronuclei formation in cancer cells

Objective 3- Implementation of Indian muntjac cells as a model system for mitosis.

Work Performed and Main Results:

- Established Indian muntjac cells as a model system for molecular manipulation and the study of mitosis

- Discovered that kinetochore size is an important determinant of chromosome segregation fidelity

- Identified Augmin as a key player in kinetochore fiber maturation in mammals
Here we tested whether tubulin post-translational modifications (PTMs) generate spatial cues that guide mitotic motors on chromosomes along specific spindle microtubules. We showed that tubulin (de)tyrosination works as a navigation system that guides molecular motors on chromosomes towards specific cell locations during mitosis. Additionally, we found that tubulin tyrosination/detyrosination plays a role in mitotic spindle assembly and chromosome segregation fidelity. Related to this, we have identified mitotic MAPs and motors that show selective preference for tyrosinated or detyrosinated microtubules.
We proposed a “chromosome separation checkpoint” that operates after spindle assembly checkpoint satisfaction to delay local nuclear envelope reformation in response to incompletely separated chromosomes, including lagging chromosomes. The central player in this checkpoint is a constitutive midzone-based Aurora B phosphorylation gradient that monitors the position of chromosomes along the spindle axis during anaphase. n this project we have determined how the spatial control mediated by a midzone Aurora B gradient feeds information back to the molecular clock Cyclin B that temporally controls the anaphase-telophase transition. We also investigated the importance of this mechanism for mitotic fidelity and uncovered a new route for micronuclei formation from misaligned chromosomes that satisfy the spindle assembly checkpoint.
We have established a unique mammalian model system based on hTERT-immortalized fibroblasts from a female of the Indian muntjac, the mammal with the lowest documented chromosome number (n = 3). In particular, each kinetochore of the X chromosomes is a “super-resolved” structure that binds more than 60 microtubules, making this model ideally suited to investigate how erroneous kinetochore-microtubule attachments are corrected. Importantly, we have obtained genome sequence information of the Indian muntjac genome. With this information at hand, we have been able to combine high-resolution live-cell microscopy and micromanipulation techniques with powerful molecular tools such as RNAi or for cell biology studies of mitosis in mammals. This uncovered a role for kinetochore size in chromosome segregation fidelity and unveiled how errors are corrected at the highest possible resolution. We also generated a phenotypic gallery representing the loss-of-function of more than 60 mitotic genes in the Indian muntjac and made this resource openly available to the scientific community. From this work, Augmin was found to play a key role in kinetochore fiber maturation.
Indian muntjac fibroblast in anaphase imaged by CH-STED