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
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
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.