Role of mto2 in temporal and spatial regulation of cytoplasmic microtubule nucleation in Schizosaccharomyces pombe

Abstract

The microtubule [MT] cytoskeleton of S. pombe is a highly dynamic network of filaments that facilitates intracellular transport, determines cell polarity and plays an essential role in chromosome separation during mitosis. In fission yeast, MTs are nucleated in a temporally and spatially regulated manner from sites called Microtubule Organising Centres [MTOCs], through the activity of both the g-tubulin complex [g-TuC] and the Mto1/2 complex. The Mto1/2 complex determines the localisation of the g-TuC at MTOCs, which change throughout the cell cycle. As cells enter mitosis the cytoplasmic array of MT bundles depolymerise. They are replaced by the intranuclear mitotic spindle and cytoplasmic spindle pole bodyderived astral MTs that in turn give way to the formation of the post-anaphase array. Although much is known about the properties of each type of MT array, the mechanism by which the timing of MT nucleation at different MTOCs is regulated over the cell cycle remains unclear. In the Mto1/2 complex, Mto1 is thought to provide the primary interaction with the g-TuC, and Mto2 functions by reinforcing this interaction. Due to the lack of structural information for the Mto1/2 complex, the molecular mechanism of Mto1/2- mediated assembly of the g-TuC at MTOCs is unknown. The aim of my study is to investigate the possibility that the Mto1/2 complex is able to promote g-TuC assembly by forming a direct template. In addition, I will attempt to determine the molecular role of Mto2 within the Mto1/2 complex and examine ways in which regulation of Mto2 may influence the function the Mto1/2 complex at specific MTOCs. As part of the investigation into the mechanism of Mto2 function, an in vitro analysis of recombinant protein demonstrated that in the absence of Mto1, purified Mto2 is able to self-interact as a tetramer. I have confirmed this interaction in vivo and have also shown that Mto2 forms a dimer as cells enter mitosis. However, in the context of an Mto1/2 complex the significance of the change in Mto2 oligomeric state remains unknown. Hydrodynamic analysis of a truncated form of the Mto1/2 complex suggests that it may form a heterotetramer, a hypothesis which is consistent with the equimolar levels of Mto2 and Mto1 protein within the cell. This information provides some structural insight as to how the Mto1/2 complex may interact with the g-TuC at MTOCs. Further analysis of the Mto1/2 complex revealed that in vivo, the Mto1-Mto2 interaction is disrupted during mitosis. This was found to correlate with the hyperphosphorylation of Mto2, which occurs as cells enter mitosis. Subsequently, an in vitro kinase assay demonstrated that phosphorylation of the Mto1/2 complex reduces the stability of the complex. Mass spectrometry techniques and sequence conservation were used to identify several phosphorylated residues within Mto2 and the ability of these mutants to bind to Mto1 was analysed in vivo and in vitro. In summary, in this study I have uncovered a mechanism which allows fission yeast cells to regulate the nucleation of cytoplasmic MT nucleation in a cell-cycle dependent manner, through a phosphorylation-dependent remodelling of the Mto1/2 complex.