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Deciphering the origins of chromosome mis-segregation and aneuploidy in humans


Warwick Medical School

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Prof A McAinsh , Prof N Burroughs No more applications being accepted Competition Funded PhD Project (European/UK Students Only)

About the Project

The accurate segregation of chromosomes into daughter cells is a prerequisite for all eukaryotic life. In humans, this process maintains a diploid karyotype of 46 chromosome, with errors associated with a myriad of pathologies, such as tumour evolution, developmental disorders (i.e. Down’s) and recurrent miscarriage (ref 1). The chromosome movements involved in successful segregation are governed by kinetochores, large multi-protein machines that assemble on each sister chromatid (replicated chromosome) and form dynamic attachments to the microtubule-based spindle. Understanding the cell biology of chromosome segregation will be an essential step in tackling these clinical challenges.

Over the last 10 years the Burroughs and McAinsh groups have made significant contributions to our understanding of kinetochore behaviour (and thus chromosome dynamics) by pioneering the integration of image analysis and sophisticated analysis algorithms with live cell imaging (for example see, ref 2,3). We have recently expanded this work to follow kinetochore dynamics throughout mitosis using high resolution lattice light sheet microscopy and endogenous protein labelling (see figure below). This is providing new insight into the molecular origin of chromosome segregation errors.

In this project you will build on these live cell assays by using genome editing to integrate additional fluorescent markers for the telomeres (the ends of chromosomes), the periphery of chromosomes and the nuclear envelope that encloses them during interphase. With these new tools we will be able to address a number of key questions concerning how the physical organisation and behaviour of individual chromosomes drive chromosome mis-segregation:

1) What fraction and types of mis-segregating chromosomes become micronuclei, rather than normal reincorporation into the nucleus? This is of great importance, as it has been shown that micronuclei can lead to “chromothripsis”, a mutational process associated with fragmentation and rearrangements in single chromosomes. This is a major driver in the microevolution of cancer.

2) Are chromosome entanglements a contributing factor in mis-segregation, either through packing in the metaphase plate or chromatid separation at anaphase? With the telomeres (ends), centromeres and periphery of chromosomes visible we will determine how chromosome compaction and mobility influence chromosome segregation. We will modify the ability of chromosomes to physically connect and test how this impacts chromosome segregation.

3) What is the rate of karyotype evolution at the single cell level? Much work has studied this by counting chromosome number in a population using fluorescence in situ hybridisation experiments (FISH). Using our kinetochore and telomere marked chromosomes we will be able to “live count” chromosomes, and follow changes in the karyotype over time, relating this to changes at population level using FISH.

In this project you will learn how to conduct live cell imaging using state-of-the art lattice light sheet microscopy, engineer cells using CRISPR/Cas9 (McAinsh lab) and contribute to the development of image processing and analysis pipelines (Burroughs group). The balance of computational and experimental can vary depending on the students interests and abilities. Overall, you will be trained, and develop the skills, to take a multi-disciplinary approach to addressing important cell biological questions.
Techniques that will be undertaken during the project:
- CRISPR/Cas9 genome editing
- Molecular biology (PCR, molecular cloning, inc. Gibson assembly)
- Biochemical methods (preparation of protein samples, immunoblotting)
- Tissue culture techniques, cell sorting, indirect Immunofluorescence and fluorescence in situ hybridisation experiments
- Live-cell imaging (spinning disk confocal and lattice light sheet microscopy)
- Constructing analysis pipelines (using existing software: Fiji, KiT2)
- Algorithm development: coding in MATLAB, e.g. tracking, quantification

Funding Notes

Studentship includes: fees, a tax-free stipend of at least £15,009 p.a (to rise in line with UKRI recommendation); a travel allowance in year 1; a travel / conference budget; a generous consumables budget and use of a MacBook Pro for the duration of the programme. In order to apply you must ensure that you are eligible.

References

Santaguida, S., and Amon, A. (2015) Short- and long-term effects of chromosome mis-segregation and aneuploidy Nat Rev Mol Cell Biol. 8:473-85.

Burroughs, N.J., Harry, E.F. and McAinsh, A.D. (2015) Super-resolution kinetochore tracking reveals the mechanisms of human sister kinetochore directional switching. eLife 2015;10.7554/eLife.09500.

Smith C.A., McAinsh, A.D. and Burroughs, N.J. (2016) Human Kinetochores are swivel joints that mediate microtubule attachments. eLife.16159.


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