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Spatial organisation of microtubules within human mitotic spindles

Principal Supervisor: Dr Stephen Royle - Warwick Medical School

Co-supervisor: Dr Julia Brettschneider - Statistics

PhD project title: Spatial organisation of microtubules within human mitotic spindles

University of Registration: University of Warwick

Project outline:

Cell division is crucial for life. During development, we each start life as a single cell and become large assemblies made of ~40 trillion cells; and cell division continues in adult life to replenish dead cells.

When a cell divides, the DNA must be shared equally between the two daughter cells. This task is accomplished by the mitotic spindle, a tiny machine composed of thousands of microtubules and their associated proteins. The organisation of microtubules in the mitotic spindle is highly complex. We are trying to understand how this network is built, organised and stabilised, so that it can aid high fidelity cell division. Each chromosome is connected to the mitotic spindle by its kinetochore. This protein disc has ~20 microtubules attached to it, and these microtubules form a kinetochore fibre. These discrete subsets of microtubules are the drivers of chromosome movement during mitosis. Despite intense study, many questions remain about the organisation of kinetochore fibre microtubules, relative to other microtubules in the mitotic spindle. For example, do the microtubules that attach to the kinetochore also attach to the spindle pole? Is the number and spacing of microtubules constant along the length of the kinetochore fibre? How are these microtubules organised spatially?

You will use 3D electron microscopy (EM) methods to visualise individual microtubules within the dense network of the mitotic spindle within human cells. EM has greater resolving power than light microscopy, greater than even “super-resolution” methods. Previously, we have studied the kinetochore fibres of the mitotic spindle using 3DEM and discovered that the microtubule associated proteins form a “mesh” which appears to organise the microtubules within the fibre (Nixon et al. 2015 eLife).

The project will involve manipulating proteins that form part of this mesh to understand how the mesh influences microtubule organisation. You will use a mix of light microscopy and 3DEM to visualise individual microtubules and their associated proteins in mitotic spindles as cells go through mitosis. In order to document and explore this nano-anatomy, you will use segmentation and computer modelling to map out the mitotic spindle (see Figure 1). The resulting datasets are large and multidimensional. Together with Julia Brettschneider (Statistics) you will develop computer based image analysis pipelines in order to reproducibly analyse these data.

The project will provide strong training in modern cell biology including 3DEM, which is a much sought-after skill. In addition, you will gain and develop programming and data analysis skills which are needed in experimental science and in careers outside of the life sciences. This includes learning about the use and adaption of open source software ImageJ, R and BioC. You will also explore spatial statistical methods and image analysis tools with a view on tailoring them to your own experimental data to ensure you maximize the information from your experiments and be able to confirm those in replicates as well. The project will help you acquire the quantitative tools to tackle the questions the experiments were designed for, as well as enable you to formulate follow-up questions and plan further experiments.

Pic1

References:

  1. Nixon, F.M., Gutiérrez-Caballero, C., Hood, F.E., Booth, D.G., Prior, I.A. & Royle, S.J. (2015) The mesh is a network of microtubule connectors that stabilizes individual kinetochore fibers of the mitotic spindle eLife, 4: e07635. doi: 10.7554/eLife.07635.
  2. Royle, S.J. (2015) Super-duper resolution imaging of mitotic microtubules Nat. Rev. Mol. Cell Biol., 16: 67. doi: 10.1038/nrm3937

BBSRC Strategic Research Priority: Molecules, Cells and Systens

Techniques that will be undertaken during the project:

Cell biology: culturing cell lines, genetic manipulation using transfection, RNAi and CRISPR/Cas9.

Molecular biology: plasmid design and construction, DNA purification.

Microscopy: light microscopy and electron microscopy.

Data analysis: image analysis, segmentation, dealing with large datasets, programming using R, statistical modelling.

Contact: Dr Stephen Royle, University of Warwick