Aurora A, MCAK, and Kif18b promote Eg5-independent spindle formation

Roy G H P Van Heesbeen,David A Egan,Albert J R Heck,René H Medema,Cor Lieftink,Vincentius A Halim,Jonne A Raaijmakers,Daphne Lelieveld,Marvin E Tanenbaum

doi:10.1007/s00412-016-0607-4

Abstract

Inhibition of the microtubule (MT) motor protein Eg5 results in a mitotic arrest due to the formation of monopolar spindles, making Eg5 an attractive target for anti-cancer therapies. However, Eg5-independent pathways for bipolar spindle formation exist, which might promote resistance to treatment with Eg5 inhibitors. To identify essential components for Eg5-independent bipolar spindle formation, we performed a genome-wide siRNA screen in Eg5-independent cells (EICs). We find that the kinase Aurora A and two kinesins, MCAK and Kif18b, are essential for bipolar spindle assembly in EICs and in cells with reduced Eg5 activity. Aurora A promotes bipolar spindle assembly by phosphorylating Kif15, hereby promoting Kif15 localization to the spindle. In turn, MCAK and Kif18b promote bipolar spindle assembly by destabilizing the astral MTs. One attractive way to interpret our data is that, in the absence of MCAK and Kif18b, excessive astral MTs generate inward pushing forces on centrosomes at the cortex that inhibit centrosome separation. Together, these data suggest a novel function for astral MTs in force generation on spindle poles and how proteins involved in regulating microtubule length can contribute to bipolar spindle assembly.

Highlights

The bipolar spindle is a microtubule (MT)-based structure required for successful chromosome segregation during mitosis
We show that the microtubule motors MCAK and Kif18b are required for bipolar spindle assembly in Eg5-independent cells (EICs) and normal cells with reduced Eg5activity
In contrast to Eg5, which directly drives bipolar spindle assembly by sliding antiparallel MTs apart (Kashina et al 1996; Kapitein et al 2005; Tanenbaum et al 2009), we show evidence that the contribution of MCAK and Kif18b to bipolar spindle assembly is likely mediated by their function in regulating the length and number of astral MTs during mitosis

Summary

Introduction

The bipolar spindle is a microtubule (MT)-based structure required for successful chromosome segregation during mitosis. An essential and highly conserved protein for bipolar spindle assembly is kinesin-5 (Eg5 in humans). Kinesin-12 (Kif15/Hklp in humans) was identified to cooperate with Eg5 in bipolar spindle assembly (Tanenbaum et al 2009; Vanneste et al 2009). To identify genes that are required for Eg5-independent bipolar spindle assembly, we performed a genome-wide small interfering RNA (siRNA) screen in HeLa and HeLa-derived Eg5-independent cells (EICs, (Raaijmakers et al 2012). We identified the mitotic kinase Aurora A and two kinesins that regulate MT dynamics, MCAK (Kif2C/kinesin-13) and Kif18b (kinesin-8), to be essential for bipolar spindle assembly in EICs. Our data reveals two novel mechanisms that are required for Eg5-independent bipolar spindle assembly and uncovers three potential targets for combination therapy with Eg5 inhibitors

Results

20 EICs specific arrest

Discussion

Experimental procedures