The Spindle Assembly Pathways of the Drosophila Syncytial Embryo

Abstract

The mitotic spindle is a key cellular structure responsible for aligning and separating chromosomes during mitosis. It is vital that this process is performed faithfully, as errors can result in disease causing genome instability. The mitotic spindle is primarily composed of an organised mass of dynamic microtubules that form a bipolar structure. Different cells have been shown to employ varying molecular pathways to generate these microtubules; however how these multiple pathways coexist and coordinate in a single cell type is poorly understood. Using the Drosophila syncytial embryo as a model cell type, the work presented in this thesis provides evidence of multiple spindle assembly routes employed within this system. Development of a technique to disrupt the temporal controls on microtubule generation combined with high speed confocal microscopy has allowed for the direct visualisation of a chromatin mediated microtubule generation pathway, a process shown to require Ran-GTP, D-HURP and the Augmin complex, but surprisingly not by a Drosophila homologue of the vertebrate spindle assembly factor TPX2, also identified here. Disruption of centrosome assembly has also revealed the importance of Augmin in generating microtubules for spindle assembly from multiple acentrosomal microtubule organising centres. Additionally, work presented here demonstrates that spindle assembly pathways can increase their microtubule generating capacity when the activity of another pathway has been perturbed, showing a degree of coordination. This thesis demonstrates the remarkable robustness with which the mitotic spindle can assemble while providing an insight into the mechanisms behind this flexibility.