From proteins to processes: an integrated analysis of mitotic spindle function

Mitosis, the process by which cells segregate their chromosomes into two equal masses, underlies the proliferation of life. Animal cells facilitate this by generating a mitotic spindle - a symmetrical structure, composed of many thousands of protein polymers, microtubules, which dynamically grow and shrink, exerting force on the chromosomes. A clear connection exists between aging, several chronic conditions, and the regulation of microtubules, with improper chromosome segregation and abnormal spindles being hallmarks of old and diseased cells.

The Wakefield lab has recently made advances both in our understanding of the molecular mechanisms underpinning mitotic spindle robustness and in the biochemical isolation of protein complexes required for mitotic microtubule organisation (Hayward et al., Developmental Cell, 2014; Palumbo et al., Current Biology, 2015). Central to this PhD project is a new fast isolation method, termed Photo-Cleavable Protein Complex Affinity Purification. This technique allows fluorescent versions of mitotic protein complexes to be isolated intact from cells, for use in biophysical and microscopy-based assays, overcoming the traditional limitations of reductionist approaches.

In a collaboration between the Wakefield and Soeller labs, the PhD student will isolate a GFP-labelled protein complex termed TACC/TOG, important in spindle formation, from cells and explore both its physical properties and cellular role, bridging the traditional gap between cell and developmental biology and in vitro biochemistry. This project will have three strands:
(i) In vivo purified GFP-labelled TACC/TOG will be incubated with fluorescent microtubules, immobilised on coverslips, and subjected to super-resolution microscopy-based techniques, in order to determine with molecular resolution how TACC/TOG interacts biophysically with microtubules. This aspect of the project will be extended by further characterisation of the biophysical properties of TACC/TOG alone and in combination with in vivo purified -TuRC, the master regulator of microtubule nucleation.
(ii) Structure-function studies of TACC/TOG, through the expression and purification of individual subunits, will determine the regions of the proteins critical for binding microtubules and the -TuRC.
(iii) The above techniques will allow peptides to be designed in order to interfere with TACC/TOG function when injected into cells. Undertaking such perturbation experiments in developing fruit fly embryos that possess fluorescent microtubules and DNA will determine the over-arching role of TACC/TOG in the cell.

In summary, through combining super-resolution microscopy, cell biology, proteomics and a novel biochemical method, this project aims to determine the way in which proteins co-ordinate their functions within the context of a mitotic animal cell system.

Academic Supervisors:

Professor James Wakefield, Bioscience (University of Exeter)
Professor Christian Soeller, Physics (University of Exeter)
Charlie Jeynes, ISSF2 Fellow

Location: University of Exeter, Streatham Campus, Exeter

About the award:

This project is one of a number which are funded within the Carlota Palmer PhD programme. This four-year programme, run under the auspices of the Centre for Biomedical Modelling and Analysis, will commence in September 2016. The studentships will provide funding for a stipend (currently £16,165 per annum), research costs and UK/EU tuition fees for four years. Further details can be found here: http://www.exeter.ac.uk/bma/phd/

Entry requirements:

Applicants should have obtained, or be about to obtain, a First or Upper Second Class UK Honours degree, or the equivalent qualifications gained outside the UK, in an appropriate area of science or technology. Applicants with a Lower Second Class degree will be considered if they also have Master’s degree or have significant relevant non-academic experience. If English is not your first language you will need to have achieved at least 6.5 in IELTS (and no less than 6.0 in any section) by the start of the project (alternative tests may be acceptable, see http://www.exeter.ac.uk/postgraduate/apply/english/).