The Molecular Basis of Chromosome Periphery Function, Structure and Composition

Background: The purpose of mitosis is to divide our DNA equally between two new daughter cells, an event that is essential for life. The DNA content of just one cell extends over two metres in length, if unravelled. This presents a challenge for cells that need to divide. To make mitosis as efficient as possible our DNA is condensed into 46 compact structures known as chromosomes (with a famous 'X' shape). However, even assembled into chromosomes DNA separation during mitosis can go wrong, with some cells receiving an abnormal amount of DNA. This is known as aneuploidy, and can lead to cancer and birth defects. It is critical that we fully understand each and all of the mechanisms that lead to aneuploidy, so that we can prevent or treat it.
My research focuses on the mitotic chromosome periphery (MCP) a sheath that covers the entire outer surface of mitotic chromosomes, like a thick winter glove (the MCP) on a hand (the chromosome). The MCP was discovered over 100 years ago, however until recently no one has been able to remove it and therefore test if or how the MCP might be an important component of chromosomes. I recently discovered the first ever way to remove the MCP through a protein called Ki67 (a key component of the sheath). I went on to show that the MCP is important for mitotic chromosomes to function correctly and, most recently, that it may also have a role in aneuploidy.
Despite these advances we still do not know; what the sheath is made of, what its full functions are, what it looks like (its structure) and finally its contribution to diseases. The role of Ki67 in each of these 'unknowns' is also unclear, but is important as Ki67 has been implicated in the progression of some cancers and is currently being tested as a drug target.

Aims: The goal of this proposal is to build on my unpublished data and investigate how the MCP (and Ki67) contributes to aneuploidy. This is important, because a better understanding of this sheath may reveal new preventative measure or treatments for cancer.

Aim 1 - Function) Use microscopy experiments, including 3D correlative light electron microscopy (3DCLEM), an advanced imaging tool that I recently developed, to determine how and why the MCP can cause aneuploidy.
Aim 2 - Structure) Investigate what the MCP looks like on a nanometre scale (a human hair is ~50,000nm in diameter, the MCP is ~100nm thick) and determine how its structure is important for its function. This will be performed using CryoCLEM, one of the most powerful imaging tools in the world and that few people can perform.
Aim 3 - Composition) Investigate what the MCP is made of and explore if other components also have important functions, like Ki67, that might be used as a drug target during future studies.

Host Institute and Collaborators: My lab will be based at the University of Nottingham Biodiscovery Institute III. This brand new £25million building contains state of the art equipment and hundreds of disease experts, including; medical doctors, cancer researchers and drug discovery specialists. This mix of expertise is designing to maximise research impact, making the progression from basic research (using cells in a dish) to treatments (for patients), as efficient as possible. I am fortunate to have an existing network of internationally renowned collaborators. Since my arrival at UoN this network has expanded to include cancer biologists such as Anna Grabowska and Alan McIntyre and technical experts such as Kenton Arkill and Robert Brandt (industrial collaboration with ThermoFisher).

Since I discovered how to remove the MCP this field has been revitalised, with several labs also confirming that the MCP is a critical component of mitotic chromosomes. My contribution to this revival of interest has been significant and using my skills, comprehensive knowledge of the MCP and important collaborations, I am in a unique position to drive this research and become a world leader in this field.

The molecular triggers of aneuploidy are poorly understood, but with an aging population and continued increases in incidences of cancer, these are 21st century challenges that need to be confronted. My research will begin to address this by defining the molecular basis by which the mitotic chromosome periphery (MCP), the least understood chromosome compartment, contributes to aberrant cell division and aneuploidy. This will be achieved through three main objectives and implementation of multiple state-of-the-art tools and technologies.

Aim 1: Determine the impact of mitotic chromosome periphery (MCP) load on aneuploidy.
Rationale: Recent evidence indicates a link between the MCP and the maintenance of ploidy, however this is yet to be formally tested. I hypothesise that MCP abundance and its impact on chromosome positioning is a critical part of the mechanism.
How: Depletion and over expression experiments (of Ki67) followed by a microscopy based phenotypic analysis to score for aberrancies consistent with aneuploidy. Cells will also be analysed using 3D-CLEM to tease out the mechanistic link between MCP load, chromosome positioning and mitotic errors.

Aim 2: Resolve the higher-order structure of the chromosome periphery.
Rationale: Details of MCP ultra-structure remain poorly defined but a recent report identified a critical link between its structure and function, with Ki67 configured as a 'brush'. However, this needs to be further tested.
How: CryoCLEM of chromosomes lacking or overexpressing Ki67

Aim 3: Define the 'peripherome'.
Rationale: The full list of proteins at the surface of chromosomes remains incomplete, but many more are predicted to exist. I hypothesise that more cPerPs remain unidentified and that some of these will have critical functions, like Ki67 and nucleolin.
How: Using BioID (proximity proteomics) to reveal the MCP interactome in addition to Ki67 dependent and Ki67 independent MCP components.