Identification of cell cycle-regulated Drosophila microtubule-associated proteins using quantitative mass spectrometry
The microtubule network is the central framework in multiple cellular processes. The microtubule array undergoes dramatic changes as cells progress through the cell cycle. In mitosis the interphase microtubule array is reorganised into the dynamic mitotic spindle which mediates chromosome segregation. This reorganisation is coordinated by microtubule associated proteins (MAPs). However, little is known about the cell cycle regulation of MAPs and how it plays a role in mitotic spindle formation. In this thesis, I describe the development of a method to determine the profiles and relative quantities of MAPs purified from mitotic and interphase Drosophila culture cells. This method utilises mass spectrometry combined with stable isotope labelling by amino acids in cell culture (SILAC) for protein quantification. This study identified MAPs whose association with microtubules increased during mitosis and revealed a new mitotic MAP, which I have named NuMAP. NuMAP localises to the nucleus in interphase and to microtubules only in mitosis, covering the entire spindle. Truncation analysis identified two protein domains sufficient but not essential for nuclear localisation and one C-terminal domain vital for microtubule localisation. Interestingly, creation of an interphase cytoplasmic pool indicated that the interphase form of NuMAP has low affinity for microtubules, suggesting a cell cycle-related post-translational modification. A deletion mutant of the NuMAP gene was generated by P element excision and will be valuable to define the role of NuMAP in fly development.