Experimental and computational investigation of kinetochore protein dynamics

Thesis

The ultimate goal of mitosis is to achieve faithful chromosome segregation (CS) to form two genetically identical daughter nuclei. Kinetochore (KT) is one of the most vital machinery that governs and regulates CS. How the different KT proteins regulate such a complex process with robustness and precision has been an active area of research. Here, I a present systemapproach using experimental and computational techniques to investigate the KT proteins in Drosophila melanogaster syncytial embryos.

The first aim of chapter 4 is to build a temporal dynamic map of some of the key KT proteins (associated with KMN network, Chromosomal Passenger Complex, Spindle Checkpoint and Polo) to understand loading and unloading kinetics. Although the scope of this thesis is to investigate the dynamics between Nuclear Envelope Breakdown (NEBD) and Anaphase (AO), the dynamics are plotted over an entire mitotic cycle for sake of completeness. Central to getting the dynamics is a robust image analysis method that is discussed in Chapter 3. The aim of chapter 3 was to compare various image analysis approaches to select the most appropriate one. The second aim of chapter 4 is to develop computational/mathematical approach using differential equation to identify protein inter-dependencies using their dynamics. The predicted protein interdependencies were experimentally validated using KMN network protein kinetics. An additional goal of chapter 4 is to develop a computational method using rate kinetic analysis to predict the number and timing of biological events that shape the loading and unloading dynamics of Polo that were hypothesised to be monophasic and biphasic respectively. The aim of chapter 5 and 6 was to experimentally verify and characterise the biological events governing Polo loading and unloading dynamics as hypothesised in chapter 4. The scope and aim of chapter 7, is to build an initial quantitative model using Polo dynamics to simulate the experimental findings with the lens of Error Correction (EC) and Spindle Assembly Checkpoint (SAC). The aim of chapter 8 is to build software called DAPPER and describe its functionality using Polo and BubR1 protein-protein interaction (PPI) data that is experimentally obtained. It is a part of future work to experimentally and computational investigate Polo PPI in relation to other KT and more broadly other mitotic proteins using DAPPER. Chapter 9 covers the discussion and future work.

Authors: Ladak, YN

• DOI: 10.5287/ora-8ro4gdm1r
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English