Date of Award

2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Phong Tran

Abstract

Mitosis is a key event in the life of a cell, where duplicated chromosomes are separated into the daughter cells. Defects associated with chromosome segregation can lead to aneuploidy, a hallmark of cancer. Chromosome segregation is achieved by the mitotic spindle, which is composed of microtubules (MTs), motors and microtubule associated protein (MAPs). Motors such as kinesins generate forces within the spindle while MAPs perform functions such as organize the spindle pole and maintain the bipolar spindle. Both motors and MAPs contribute to spindle mechanics. Here I used the relatively simple fission yeast to address how defects in spindle mechanics affect chromosome segregation. The metaphase spindle is maintained at a constant length by an antagonistic force-balance model yet how the regulation of metaphase spindle length contribute to subsequent chromosome segregation remains unexplored. To test the force-balance model, I applied gene deletion and fast microfluidic temperature-control with live-cell imaging to monitor the effect of deleting or switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. I show that kinesin-5 cut7p and MT bundler ase1p contribute to outward pushing forces, and kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects.

Motors and MAPs cooperate to focus MTs at the spindle pole. Defects in MT focusing lead to defects in chromosome segregation, resulting in aneuploidy. The mechanism behind these observations is not well understood. Here I identified a new mechanism for aneuploidy in fission yeast. Kinesin-14 pkl1p and MAP msd1p localize to the spindle poles and focus the MT minus ends. Their absence leads to pole and MT defocusing, resulting in protrusion of MT minus ends due to cut7p-dependent pushing forces at the spindle midzone. Infrequent long MT minus end protrusions can push the already separated chromosome mass back to the cell center, where cytokinesis will `cut' the chromosome mass, creating two daughter cells with unequal chromosome content.

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