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  • Periodic Reporting for period 1 - CODECHECK (CRACKING THE CODE BEHIND MITOTIC FIDELITY: the roles of tubulin post-translational modifications and a chromosome separation checkpoint)

CODECHECK Report Summary

Project ID: 681443
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - CODECHECK (CRACKING THE CODE BEHIND MITOTIC FIDELITY: the roles of tubulin post-translational modifications and a chromosome separation checkpoint)

Reporting period: 2016-07-01 to 2017-12-31

Summary of the context and overall objectives of the project

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.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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;
We will use a systematic, multidisciplinary approach to test whether the “tubulin code” works as a navigation system that guides molecular motors on chromosomes towards specific locations. Additionally, we will investigate whether tubulin PTMs play a role in mitotic spindle assembly and error correction, while evaluating the implications for chromosome segregation fidelity.

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 (Manuscript in preparation)

- In vitro assays with CENP-E motor and CLASP2 was 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;
We propose to identify Aurora B targets on chromatin and the nuclear envelope involved in the spatiotemporal regulation of the anaphase-telophase transition. Moreover, we will investigate whether this checkpoint is required for detection and correction of errors that persist through anaphase and might lead to genomic instability.

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 (Drpic et al., under review after revision)

- Establishment of a phenotypic gallery of more than 60 mitotic genes using RNAi in Indian muntjac cells. Currently investigating error correction in this system.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Before chromosomes segregate they must congress to the cell equator in a process mediated by the microtubule minus-end-directed kinetochore motor Dynein, which moves peripheral chromosomes to the poles along astral microtubules, and the plus-end-directed kinetochore motor CENP-E/Kinesin-7 that slides polar chromosomes along spindle microtubules. Because different kinetochore motors are able to move chromosomes in opposite directions, 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. We expect until the end of the project to make progress on detailed functional characterization of these MAPs and motors.
In addition to a navigation system that guides chromosomes during mitosis to ensure faithful chromosome segregation, several checkpoints delay key cell cycle transitions until completion of a critical earlier event, providing time for error correction. The spindle assembly checkpoint (SAC) regulates the metaphase-anaphase transition by providing time for proper kinetochore-microtubule attachments. Although a robust SAC prevents massive chromosome mis-segregation, cells may progress into anaphase with few chromosomes that lag behind due to merotelic attachments (when a single kinetochore is attached to microtubules from both spindle poles), which represent a major mechanism of CIN. Recently, we have uncovered a “chromosome separation checkpoint” that operates after SAC satisfaction to delay local nuclear envelope reformation (NER) in response to incompletely separated chromosomes, including lagging chromosomes. At present, we have only an initial view of how this chromosome separation checkpoint works. 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. Until the end of this project we propose to identify molecular targets of Aurora B kinases that play a role in the anaphase-telophase transition.
We have also established a unique mammalian model system for mitosis that combines the powerful genetic tools and low chromosome number of fission yeast with the exceptional cytological features of a rat kangaroo cell. This system is 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

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