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

Reporting period: 2019-07-01 to 2020-12-31

During the human lifetime it is estimated that 10 000 trillion cell divisions take place to ensure tissue homeostasis, the renewal of epithelia, an efficient immune response against pathogens and sexual reproduction. At every cell division cycle, the previously replicated genome must be accurately distributed into the two daughter cells by a cellular process known as ‘mitosis’. In mitosis, the DNA condenses into chromosomes that are segregated after the establishment of stable interactions between specialized regions called kinetochores and a microtubule-based structure known as the mitotic spindle. 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, and the rate of chromosome mis-segregation (i.e. chromosomal instability or CIN) strongly correlates with tumour aggressiveness and drug resistance. 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 will test two original concepts with strong implications for chromosome segregation fidelity. The first concept is based on the “tubulin code” hypothesis, which predicts that molecular motors “read” specific modifications on spindle microtubules. How these tubulin modifications impact chromosome segregation remains unknown. The second concept is centered on the recently uncovered chromosome separation checkpoint that delays the completion of mitosis in response to incompletely separated chromosomes. Overall, this work will establish a paradigm shift in our understanding of how spatial information is conveyed to faithfully segregate chromosomes during mitosis.
The project was initial divided into 3 ground-breaking objectives . Here follows a brief report on the main results obtained for each objective for the first reporting period:

Objective 1- Comprehensive analysis of the tubulin code during mitosis;

Work Performed and 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.

- Functional characterization of mitotic MAPs

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

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

- Investigation of 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 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

- Investigating the requirement for Aurora B in the resolution of ultra-fine bridges during anaphase.

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

- Establishment of phosphorylation mutants for potential Aurora B substrates involved at the anaphase-telophase transition.

Objective 3- Implementation of Indian muntjac cells as a model system for mitosis.
We will use the unique cytological features of the Indian muntjac to investigate with unprecedented resolution the mechanisms behind the correction of erroneous kinetochore-microtubule attachments. We will further investigate whether an Aurora B-mediated chromosome separation checkpoint allows mammalian cells to deal with extra-long chromosomes to ensure mitotic fidelity

Work Performed and Results:

- Establishment of the Indian muntjac system.

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

- Establishment of a phenotypic gallery of more than 60 mitotic genes using RNAi in Indian muntjac cells. Currently investigating error correction in this system.
A critical longstanding question is how chromosomes are guided during mitosis. An exciting, but yet unexplored, possibility is that tubulin post-translational modifications (PTMs) generate spatial cues that guide mitotic motors on chromosomes along specific spindle microtubules. We have established that tubulin tyrosination/detyrosination works as a navigation system that guides molecular motors on chromosomes towards specific cell locations during mitosis. Additionally, we are currently investigating whether tubulin tyrosination/detyrosination plays a role in mitotic spindle assembly and chromosome segregation fidelity and expect this to be concluded until the end of the project. Related to this, we have identified mitotic MAPs and motors that show selective preference for tyrosinated or detyrosinated microtubules.
Recently, we have uncovered 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. In addition to this spatial control, Cyclin B1/Cdk1 work as a clock to determine the duration of anaphase. In this project we have determined how the spatial control mediated by a midzone Aurora B gradient feeds information back to the molecular clock that temporally controls the anaphase-telophase transition. We are currently investigating the importance of this mechanism for mitotic fidelity.
We have also 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). Chromosomes of the Indian muntjac are large, compound structures that resulted from DNA fusions. 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. We expect until the end of this project to elucidate the relevance of kinetochore size for chromosome segregation fidelity and how errors are corrected at the highest possible resolution. We also expect to generate a phenotypic gallery representing the loss-of-function of more than 60 mitotic genes in the Indian muntjac and make this resource openly available to the scientific community.
Indian muntjac fibroblast in anaphase imaged by CH-STED