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
