nvestigating Chromatin-Driven Spindle Assembly in the Drosophila melanogaster Syncytial Embryo

Cell division is a fundamental biological process, driven by the formation of a microtubule (MT)-based mitotic spindle that aligns the chromosomes and ensures their segregation. Research over the last twenty years has led to a list of gene products with roles in spindle formation. The challenge of post-genomic biology is to understand how these gene products work together to define biological process. We have developed methodologies, based on high-resolution fluorescence microscopy, coupled to image analysis, which allow us to quantify the generation and organisation of MTs during mitotic spindle formation in the model organism, Drosophila melanogaster (Hayward et al., Dev Cell; in revision). The goal of this project is to determine the molecular mechanisms underpinning MT organisation during the early stages of mitotic spindle formation, referred to as "nucleation". In this process, MTs are generated at specific cellular in relation to another, ensuring enough polymer is present for the cell to generate a bipolar mitotic spindle.

In the first rotation, the student will combine biochemical and cell biological techniques, affinity-purifying an antibody raised against D-HURP, which appears to generate MTs from the vicinity of the chromatin during the early stages of mitosis. They will undertake high temporal and spatial cell imaging techniques, to obtain time-lapse movies of MT organisation, both in control Drosophila embryos and those injected with the anti-D-HURP antibodies. In the second rotation, they will be introduced to computational image processing and quantitative analysis techniques required to systematically analyse their data. By the end, they will have analysed the control and anti-D-HURP movies, extracting key parameters such as MT dynamics ranging from MT nucleation, bundling to directionality, in a statistically significant and objective framework.

The main body of the PhD will see the student disrupting the other proteins known to function during spindle formation. For those for which antibodies are not available, the student will produce constructs to allow the bacterial expression and purification of pure protein, in order to generate polyclonal antibodies for usage. Under the guidance of Prof Beardmore and Dr Metz they will expand on the computational techniques they have been introduced to, gaining deeper understanding of the numerical approaches and connections with the appropriate theoretical modeling with potential for additional collaboration with Mathematical modeling groups within Exeter.

Together, the fusion of these techniques will allow the student to address a fundamental biological problem and provide them with a unique skill-set, bridging the gap between biology, mathematics and computer science.