Mitotic spindle assembly: May the force be with you

Abstract

The research described in this thesis is focused on multiple pathways required for assembly of a bipolar mitotic spindle. Proper assembly of a bipolar mitotic spindle is essential for the generation of stable kinetochore-microtubule attachments and correct segregation of the sister chromatids. Defects in pathways controlling mitotic spindle assembly can give rise to chromosome segregation defects and as a consequence, chromosomal instability, which is a hallmark of cancer cells. Microtubule-targeting drugs like the vinca alkaloids and taxanes, have been shown to be effective anti-cancer drugs. They are thought to induce specific cytotoxic effect on cancer cells due to the induction of a mitotic delay. However, in addition to targeting mitotically active cells, microtubule poisons also cause neurotoxicity due to perturbation in microtubule dynamics in general and also drug resistance is commonly observed in patients treated with microtubule poisons. Due to these observations, novel targets to perturb mitotic progression are currently considered in the treatment of cancer, including kinases and motor proteins involved in mitotic spindle assembly.One of the promising targets for anti-cancer therapy is the mitotic kinesin Eg5. Inhibition of Eg5 results in a mitotic arrest due to the formation of monopolar spindles. In chapter 2, we describe the generation of human cancer cells that can grow in the complete absence of Eg5. By studying mitotic spindle assembly in these Eg5-independent cells (EICs), we uncovered a novel pathway for prophase centrosome separation. This pathway depends on the nuclear envelope associated pool of dynein and enables cells to assemble bipolar spindles in the absence of Eg5 activity. In chapter 3, we performed a genome-wide siRNA screen in EICs to uncover essential components for Eg5-independent bipolar spindle formation that might promote resistance to treatment with Eg5 inhibitors. In this screen, we found that the mitotic kinase Aurora A and two mitotic kinesins MCAK and Kif18b are essential for bipolarization of the mitotic spindle in EICs and in cells with reduced Eg5 activity. Inchapter 4, we show that in human cells, three mitotic motors act together to produce the right force balance for correct assembly of a bipolar mitotic spindle and chromosome segregation. Eg5 and Kif15 are in human cells the main outward force-generating kinesins, and their activity is antagonized by dynein. In chapter 5, we review the function of Kif15 in different organisms and discuss if Kif15 might be a useful target for anti-cancer therapy. In chapter 6 and 7, we show that human HAP1 cells can survive without a functional spindle assembly checkpoint (SAC). Due to the haploid nature of these cells, we used SAC-deficient HAP1 cells for a genetic screen to identify novel factors involved in chromosome congression. We found that loss of the condensin II complex, the RZZ complex and Bub1 are synthetic lethal with SAC-deficiency. Finally, in chapter 8, we summarize the results described in this thesis and propose future research to investigate several remaining questions.