The Aspergillus nidulans bimC4 mutation provides an excellent tool for identification of kinesin-14 inhibitors

Abstract

Centrosome amplification is a hallmark of many types of cancer cells, and clustering of multiple centrosomes is critical for cancer cell survival and proliferation. Human kinesin-14 HSET/KFIC1 is essential for centrosome clustering, and its inhibition leads to the specific killing of cancer cells with extra centrosomes. Since kinesin-14 motor domains are conserved evolutionarily, we conceived a strategy of obtaining kinesin-14 inhibitors using Aspergillus nidulans, based on the previous result that loss of the kinesin-14 KlpA rescues the non-viability of the bimC4 kinesin-5 mutant at 42°C. However, it was unclear whether alteration of BimC or any other non-KlpA protein would be a major factor reversing the lethality of the bimC4 mutant. Here we performed a genome-wide screen for bimC4 suppressors and obtained fifteen suppressor strains. None of the suppressor mutations maps to bimC. The vast majority of them contain mutations in the klpA gene, most of which are missense mutations affecting the C-terminal motor domain. Our study confirms that the bimC4 mutant is suitable for a cell-based screen for chemical inhibitors of kinesin-14. Since the selection is based on enhanced growth rather than diminished growth, cytotoxic compounds can be excluded.

The mitotic kinesin-14 KlpA contains a context-dependent directionality switch

“…Similar to the fission yeast kinesin-14 Pkl1 19 , KlpA is nonessential in wildtype cells but its loss becomes synthetically lethal with gamma tubulin mutations 20 . KlpA is an attractive model protein for dissecting the mechanism and function of kinesin-14s, as its loss-of-function mutations can be conveniently isolated as suppressors of the bimC4 mutation 21 . However, compared with other mitotic kinesin-14s such as Ncd from Drosophila melanogaster and Kar3 from Saccharomyces cerevisiae, KlpA is much less well studied.…”

The mitotic kinesin-14 KlpA contains a context-dependent directionality switch

“…Although KlpA is nonessential in wild-type cells24, its loss becomes synthetically lethal with gamma tubulin mutations28. KlpA is an attractive model protein for dissecting the mechanism and function of kinesin-14s, as its loss-of-function mutations can be conveniently isolated as suppressors of the bimC4 mutation29. However, compared with other mitotic kinesin-14s such as Ncd from Drosophila melanogaster , Pkl1 and Klp2 from Schizosaccharomyces pombe , and Kar3 from Saccharomyces cerevisiae , KlpA is much less well studied.…”

Fission yeast cells overproducing HSET/KIFC1 provides a useful tool for identification and evaluation of human kinesin-14 inhibitors

“…In fission yeast, as in other organisms, simultaneous inactivation of kinesin-5 and kinesin-14 rescues lethality resulting from kinesin-5 inhibition alone (Civelekoglu-Scholey et al, 2010;Mountain et al, 1999;O'Connell et al, 1993;Pidoux et al, 1996;Wang et al, 2015;Yukawa et al, 2017;Yukawa et al, 2018). Thus, effective inhibitors against fission yeast kinesin-14s (Pkl1 and Klp2) could be identified using cut7 temperature-sensitive mutants, as previously proposed in the Aspergillus nidulans system (Wang et al, 2015). These inhibitors might be also effective to suppress HSET activities, as HSET and Pkl1/Klp2 are structurally conserved and HSET functionally replaces for Pkl1 or Klp2 when introduced into fission yeast (Yukawa et al, 2018).…”