Meadows, J.C.; (2007) Regulation of mitotic progression by factors that influence spindle position and stability in fission yeast. Doctoral thesis , University of London.

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Abstract

It has previously been proposed that in fission yeast mitotic spindle position is monitored by a checkpoint that controls the timing of anaphase onset. This checkpoint is activated by treatment of cells with Latrunculin A (Lat A), an inhibitor of actin polymerisation. It was previously thought that Lat A imposes an anaphase delay by preventing interaction of astral microtubules with the cortical actin cytoskeleton and that this interaction was required for correct spindle orientation. By contrast I show that cells lacking Mto1, a centrosomin-like protein, have mis-orientated spindles but are not delayed in the timing of sister chromatid separation. Secondly, I show that astral microtubules are only nucleated after sister chromatids separate and that mitotic spindle orientation is instead determined by the position of the spindle pole body during interphase. Spindle pole position is controlled by interphase microtubules, which also position the nucleus in the centre of the cell. Since the position of the nucleus determines the site of septation, I propose that interphase microtubules ensure perpendicular alignment of the spindle and the axis of cell division. Thirdly, in contrast to previous findings, I find that Lat A causes an anaphase delay and mitotic spindle collapse in a proportion of cells. These results suggest that Lat A delays the onset of anaphase by disrupting mitotic spindle stability rather than by causing spindle mis-orientation. Importantly, this effect is abolished in a Lat A-insensitive actin mutant, implicating a role for actin in mitotic spindle stability. Lastly, I demonstrate that the effect of Lat A is exacerbated in cells lacking Ase1, which binds and stabilises the spindle midzone. I have uncovered two factors, Mal3, and a novel kinesin, Klp9, which are required for viability in the absence of Ase1. I present a preliminary characterisation of the role of Klp9 in the mitotic cell cycle.

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