Suppressor Analysis Uncovers That MAPs and Microtubule Dynamics Balance with the Cut7/ Kinesin-5 Motor for Mitotic Spindle Assembly in Schizosaccharomyces pombe
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The Kinesin-5 motor Cut7 in Schizosaccharomyces pombe plays essential roles in spindle pole separation, leading to the assembly of bipolar spindle. In many organisms, simultaneous inactivation of Kinesin-14s neutralizes Kinesin-5 deficiency. To uncover the molecular network that counteracts Kinesin-5, we have conducted a genetic screening for suppressors that rescue the cut7-22 temperature sensitive mutation, and identified 10 loci. Next generation sequencing analysis reveals that causative mutations are mapped in genes encoding a-, b-tubulins and the microtubule plus-end tracking protein Mal3/EB1, in addition to the components of the Pkl1/Kinesin-14 complex. Moreover, the deletion of various genes required for microtubule nucleation/polymerization also suppresses the cut7 mutant. Intriguingly, Klp2/ Kinesin-14 levels on the spindles are significantly increased in cut7 mutants, whereas these increases are negated by suppressors, which may explain the suppression by these mutations/deletions. Consistent with this notion, mild overproduction of Klp2 in these double mutant cells confers temperature sensitivity. Surprisingly, treatment with a microtubule-destabilizing drug not only suppresses cut7 temperature sensitivity but also rescues the lethality resulting from the deletion of cut7, though a single klp2 deletion per se cannot compensate for the loss of Cut7. We propose that microtubule assembly and/or dynamics antagonize Cut7 functions, and that the orchestration between these two factors is crucial for bipolar spindle assembly.
This work was supported by the Japan Society for the Promotion of Science (JSPS) [KAKENHI Scientific Research (A) (16H02503 to T.T.), a Challenging Exploratory Research grant (16K14672 to T.T.), Scientific Research (C) (16K07694 to M.Y.)], the Naito Foundation (T.T.) and the Uehara Memorial Foundation (T.T). M.Y. and T.T. designed research.M.Y. and Y.Y. performed experiments and analyzed the data.
G3:Genes, Genomes, Genetics
Genetics Society of America
Copyright © 2019 Yukawa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.