Using inducible degradation to characterise the roles of TRIP13 and CEP57 in mitosis
Thesis or Dissertation
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DNA and centrosomes are both semi-conservatively replicated once per cell cycle and it is essential that the DNA and centrosomes are divided equally between daughter cells in mitosis. When this fails, it can lead to aneuploidy, which is a hallmark of cancer. Whilst centrosomes are not essential for mitosis in some organisms, centrosome dysfunction also leads to incorrect cell division and is highly prevalent in some cancers. Evolutionary conserved checkpoints exist throughout the cell cycle to halt the progression of cells that have undergone damage in the previous cell cycle stage to ensure the fidelity of transmission of DNA. In mitosis, the Spindle Assembly Checkpoint (SAC) ensures that sister chromatid separation does not occur prematurely, which would lead to unequal separation of DNA. The SAC has evolved to arrest cells until all kinetochores have been correctly attached to the mitotic spindle. The centrosome is the microtubule organising centre (MTOC) and in cycling cells it facilitates the assembly of the bi-polar spindle during mitosis. In mammalian cells, inheritance of an incorrect number of centrosomes arrests cells in G1 through the action of p53. Over the last few decades, our understanding of signalling pathways, such as the SAC and centrosome cycle, has been greatly expanded by removing specific proteins from a system and examining the consequences. The function of a protein can be targeted directly or indirectly by upstream perturbation at either the DNA or RNA level. This can lead to secondary effects that may complicate the interpretation of phenotypes. Whilst small molecule inhibitors act at the protein level and are often rapid and reversible, they can also have off-target effects, and the design and validation of new inhibitors can take several years. It is also unlikely that a small molecule inhibitor can be designed for every protein, especially non-enzymatic proteins that lack a clearly targetable domain. More recently, acute degradation techniques have provided an attractive alternative for conditional protein inactivation that mitigate some of the challenges and limitations of previous techniques. The Auxin Inducible Degron (AID) system was developed in 2007 to target specific proteins for acute and rapid degradation. With the advent of CRISPR/Cas9, AID presents a specific system that, in theory, enables the targeting of any protein at the endogenous level for rapid and complete knock-down. Due to the acute nature, it provides an elegant system to study cell cycle regulators. In this thesis, I have used AID to characterise the roles of two genes that function in different pathways in mitosis: TRIP13 and CEP57. Mutations in TRIP13 and CEP57 both lead to a rare autosomal recessive disease known as Mosaic Variegated Aneuploidy (MVA) syndrome. Affected individuals suffer from developmental disorders, microcephaly and in some cases a prevalence to cancer from an early age. With the help of the adaptor protein p31comet, TRIP13 has been shown in-vitro to catalyse a conformational change in the mitotic checkpoint complex (MCC) protein Mad2. At the start of my PhD, the in-vivo role of TRIP13 in the SAC was partially characterised (unpublished data) in my laboratory. Dr Chiara Marcozzi had generated TRIP13 knock-out (KO) cell lines but had observed clonal variation in mitotic timing and changes to the levels of other SAC proteins, the phenotype of TRIP13 remained unclear. I used TRIP13 as a proof of principle to setup my AID system and to confirm the phenotype observed after removal of TRIP13. Interestingly, I find that TRIP13 is not essential to initially activate the SAC in contrast to previously published literature. My results indicate instead that TRIP13 plays a role in both maintenance and silencing of the SAC as TRIP13-AID cells no longer arrest in response to microtubule depolymerising agents. I also showed that depletion of TRIP13 rapidly leads to a change in the levels of Mad2 and p31comet, showing that the changes are not a side effect of CRISPR mediated KO and instead implicating TRIP13 in the stability of these two other SAC regulators. The role of CEP57 in mitosis has not been previously well described. CEP57 has been implicated in a number of different pathways that could have an effect on mitosis including the SAC, centriole disengagement and interacting with microtubules. Crucially all other genes identified to cause MVA have been SAC regulators. Therefore, I targeted CEP57 with AID in order to gain further insights into its function. My preliminary data confirms that CEP57 depletion leads to changes to the number of chromosomes inherited from the previous cell cycle. However, I find that mitotic timing did not change upon acute or chronic depletion of CEP57. I also confirm that in human cells CEP57 localises to the centrosome and not the kinetochore. These data indicate that CEP57 is unlikely to function in the SAC. Instead, I find that acute depletion of CEP57 leads to premature disengagement of centrioles in metaphase rather than telophase. I show that inhibition of the centriole disengagement regulator rescues this timing defect, and that depletion of CEP57 in interphase does not lead to centriole disengagement until the next mitosis. The centrosome is surrounded by a complex mass of proteins known as the pericentriolar (PCM). In G2 and mitosis, the PCM expands to enable increased microtubule nucleation. I show that loss of CEP57 leads to lower levels of pericentriolar matrix (PCM), most clearly in Cdk5rap2, at the centrosome during mitosis. My preliminary data indicates a direct link between CEP57 depletion and aneuploidy, and points toward a role for CEP57 in the expansion of the PCM during mitosis.
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Institute of Cancer Research (University Of London)