Phospho-regulation of kinetochore function during mitosis

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Accurate partition of the duplicated genome during cell division is crucial for cellular viability and organismal development. Chromosome mis-segregation is a major source of aneuploidy, and it is a hallmark of cancer, while in oocytes represents the major source of miscarriages and genetic disease. As cells enter mitosis, chromosomes undergo compaction and establish specialized connections with spindle microtubules. The connection between chromosomes and microtubules is mediated by a proteinaceous structure that associates with chromosomal DNA, called the kinetochore.  

Kinetochore composition and function needs to be tightly regulated during the cell cycle and kinetochore-specific kinases and phosphatases play a central role in this regulation. Despite advances in the identification of such kinases and phosphatases as well as their targeting mechanisms, their specific substrates and roles during mitosis are poorly understood.  

One key mitotic kinase involved in kinetochore function is polo-like kinase 1 (PLK1). While PLK1 plays critical roles during mitosis, how PLK1 achieves its different functions is not understood at the mechanistic level. Particularly, how PLK1 and counteracting phosphatases regulate kinetochore function through the different stages of mitosis is not clear. The aim of this research is to provide a mechanistic understanding of the chromosomal roles of PLK1 during mitosis by understanding how PLK1 phosphorylation of different kinetochore components impacts upon kinetochore function. 

The overarching objective of this research is to provide a mechanistic understanding of the chromosomal roles of PLK1 during mitosis by understanding how PLK1 phosphorylation of different kinetochore components impacts upon kinetochore function. My hypothesis is that different sets of substrates within the inner and outer kinetochore perform different, non-overlapping functions. Understanding why chromosome-associated PLK1 is vital at the molecular level will lead to a better understanding of mitosis. To understand why PLK1 is important during mitosis, we need a mechanistic description of the roles of PLK1 within the inner and the outer kinetochore, including its substrate proteins and the timing and impact of phosphorylation. 

The following aims will allow me to achieve the general objective:  

 AIM 1. Identification of inner- and outer-kinetochore PLK1 substrates. 

As a first step towards understanding the specific role(s) PLK1 plays in the two kinetochore locations, we will focus on identifying inner and outer kinetochore PLK1-dependent phospho-sites. To guarantee a successful outcome, we will combine in vivo and in vitro analysis of kinetochore protein phosphorylation. 

AIM 2. Characterisation of kinetochore phospho-sites underlying the chromosome-specific mitotic roles of PLK1. 

Having identified the kinetochore PLK1 substrates, the next step will be to understand the impact of kinetochore PLK1 phosphorylation events during mitosis. We will assess the effect of mutating individual phospho-sites (or combinations) on the timing of mitotic progression, spindle assembly checkpoint, and kinetochore-microtubule attachments. 

 AIM 3. Characterisation of PP2A/B56 as the PLK1-counteracting phosphatase.  

We will study the role of PP2A/B56 as the main PLK1 counteracting kinetochore phosphatase. We will identify which set of PLK1 substrates is subject to PP2A/B56 regulation and when this dephosphorylation happens during mitosis. 

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