Investigation of the Augmin Complex, the Template Dependent Microtubule Nucleator, in Drosophila Melanogaster

Abstract

In order for life to continue, proper segregation of genetic materials by the process of mitosis is essential. Mitosis is facilitated by a bipolar spindle, usually composed of a symmetrical array of microtubules (MTs). MTs consist of Tubulin polymers, and are generated in vivo with the aid of nucleators. The most predominant nucleator is the γ-Tubulin Ring Complex (γ-TuRC). During mitosis, multiple pathways contribute to proper spindle formation, including centrosome-dependent, kinetochore-dependent, and a recently discovered pathway in which new MTs are nucleated from the sides of existing MTs. This last pathway relies on the Augmin complex, a template-dependent MT nucleator which recruits γ-TuRC to the spindle. Mitotic cells lacking Augmin have weak spindles due to a lower density of MTs and in human cells, Augmin reduction also causes centrosome fragmentation. To date, very little is known about the functional properties of Augmin. I have purified individual Augmin subunits and examined their properties in vitro. I show that the Augmin subunits Dgt4 and Wac are responsible for interaction with MTs and that Dgt6, Dgt3 and Dgt5 interact with the γ-TuRC subunit Dgp71WD. I have also shown that Wac protects MTs from destabilisation. Interestingly, the Augmin complex promotes MT assembly in the absence of γ-TuRC, suggesting a novel function for Augmin. I have also successfully purified intact Augmin complex. With this, I determined some of the relationships between Augmin subunits and generated a model of how Augmin might recruit γ-TuRC to MTs. Lastly, I have determined that at least one subunit, Wac, plays a role in MT organisation beyond mitosis. This work significantly improves our understanding of the Augmin complex and provides an excellent complement of tools for further investigation.