Principal Supervisor: Dr Andrew McAinsh - Warwick Medical School
Co-supervisor: Geraldine Hartshorne - Warwick Medical School and Nigel Burroughs - Maths
PhD project title: Origins of chromosomal abnormalities in human embryos
University of Registration: Warwick
Kinetochores are multi-protein complexes that mediate the attachment of chromosomes to the microtubule-based spindle during cell division (Santaguida and Musacchio EMBO J. 2009 28:2511-31). Their function is critical for normal disjunction of chromosomes to daughter cells. Errors in chromosome segregation that occur in human oocytes or early embryos, predispose to genetic disorders in children, underlying, for example, Trisomy 21 Down Syndrome, which is the commonest inherited cause of disability. Human eggs and embryos are known to have a very high risk of chromosomal abnormalities, with many embryos lacking the ability to survive beyond the first few days after fertilisation. The origin of this high degree of chromosomal disorder in human embryos remains uncertain although many hypotheses have been proposed.
We have been studying oocyte kinetochores at meiotic metaphase I, which is a unique cell division in which kinetochores on sister chromatids pull in the same rather than opposite directions so that a pair of sister chromatids travels to each pole. We have recently discovered an unusual age-related dissociation of the sister kinetochores in humans that differ from other species and may underlie the maternal age-related increase in chromosomal abnormalities in our species (see: Patel et al, Biology Open, 5:178-84, 2015 and also the related work in Zielinska et al; eLife, e11389, 2015). The purpose of this project is to build on this initial work and answer two key questions:
- How does the architecture and mechanical properties of the kinetochore adapt as they progress from meiosis-to-mitosis?
- What are the origin chromosome mis-segregation events during the meiotic and early mitotic divisions in the human embryo?
To address these questions human oocytes will be fixed and stained with pairs of antibodies recognising multiple components of the kinetochore structure and image stacks acquired using spinning disk confocal microscopy. We can then measure the nano-scale distances between two kinetochore components and build up a 3D model of kinetochore architecture (see: Smith et al, eLife, 2016 e161159). We can then remove pulling forces by perturbing microtubule dynamics and measure how parts of the structure adapt. i.e. does it compress, stretch or remain stiff under load. In a second series of experiments we will aim to begin analysis of embryos that are developing abnormally because of abnormal fertilisation or which failed to grow normally in the first 5 days of development. If possible, individual chromosomes will be identified and comparison will be made between kinetochores in cells with normal and abnormal numbers of chromosomes to investigate how missing or additional chromosomes might relate to alterations in kinetochore architecture. Next, we will use live cell imaging to follow the movements of chromosomes (and kinetochores) during both meiotic divisions and then the first few mitotic divisions of the human embryo. These experiments will build on our previous work in human cells (Burroughs et al, eLife, 2015 e09500) and use our existing computational biology tools (Armond et al, Bioinformatics, 2016 32:1917-9). These experiments will break new ground and allow us to probe the mechanisms that give rise to non-dysjunction during chromosome segregation in the human meiotic and subsequent (after fertilisation) mitotic divisions.
Note: This project would only use material that cannot be used for patient treatments and would otherwise be disposed of. You would need to work for part of the time in a clinical environment at University Hospitals Coventry and Warwickshire NHS Trust, and will be required to observe confidentiality and other clinical governance regulations, such as those imposed by the Human Fertilisation and Embryology Authority. You will also need to be willing to travel to/from the hospital and the Gibbet Hill site.
BBSRC Strategic Research Priority: Molecules, cells and systems
Techniques that will be undertaken during the project:
- Culture of human somatic cells and oocytes.
- Standard molecular biology.
- Quantitative immunofluorescence in human oocytes and somatic cells.
- Individual or multicolour FISH to identify chromosomes abnormalities.
- Microinjection of human oocytes
- Live cell imaging using spinning disk confocal and/or light sheet microscopy.
- Use of MATLAB software for kinetochore tracking.
Contact: Dr Andrew McAinsh, University of Warwick