Analysis of the molecular mechanisms of mitotic spindle assembly and checkpoint signalling using the HaloTag system

Thesis

Timely alignment and accurate chromosome segregation in mitosis are vital for genomic stability and are required to prevent the emergence of aneuploid cells, a hallmark of many cancers. Central to these processes are kinetochores, large protein complexes which connect mitotic chromosomes to spindle-associated microtubules and act as hubs for spindle assembly checkpoint (SAC) signalling. The SAC ensures that correctly bioriented chromosomes are discriminated from unaligned chromosomes in a process involving the mitotic kinases Aurora B and MPS1. These interact with and phosphorylate the kinetochore microtubule binding protein NDC80 and SAC scaffold protein KNL1, respectively. This thesis uses the synthetic HaloTag sequence integrated into the endogenous genomic loci encoding NDC80, KNL1 and the Aurora B activator and targeting protein INCENP to enable accurate visualisation and rapid PROTAC-mediated degradation in living cells. Using this system, the roles of these proteins in SAC activity, kinetochore function, and genomic stability were investigated. Integration of the mStayGold fluorescent marker into the MPS1 and BUB1 checkpoint proteins in these HaloTag backgrounds enabled the live readout of SAC activity. In metaphase, microtubule pulling forces separate Aurora B at the inner centromere from its targets at the outer kinetochore. Using super-resolution STED microscopy, I show that NDC80 at kinetochores and Aurora B and CPC Survivin/Borealin at inner centromeres are collapsed together in prometaphase and become spatially segregated in metaphase. In contrast, no discernible separation was detected between the distal and proximal ends of the kinetochore, marked by CENP-A and NDC80 respectively. Structure-guided analysis revealed that Aurora B localisation to inner centromeres is crucial for SAC signalling in prometaphase. Targeted degradation of NDC80 prevents microtubules from attaching to chromosomes, resulting in constitutive SAC signalling and delayed mitotic progression. By contrast, either degradation of the SAC scaffold KNL1 or the Aurora B activator INCENP reduced SAC activity, with KNL1 loss shortening mitosis. Proteomic analysis of INCENP-dependent Aurora B phosphorylation sites in mitosis identified the kinetochore subunit DSN1 and the inner centromere kinesin MCAK. The data presented in this thesis support a spatial separation model for Aurora B in SAC signalling and demonstrate an important role for NDC80 in silencing of the SAC.

Authors: Gold, DBH

• DOI: 10.5287/ora-zbd5jwa1n
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: INCENP; mitosis; spindle assembly checkpoint; live cell microscopy; MPS1; PROTAC; STED Microscopy; aurora B; NDC80; HaloTag; KNL1; chromosome passenger complex
• Subjects: Biochemistry