Genetic engineering of fission yeast

S. pombe strains used in this study are listed in Table 1. PCR-based gene targeting was performed as described [90] (link). All tagged genes are under the control of endogenous promoters and integrated at their native chromosomal loci except those under the control of 3nmt1 and 41nmt1 promoters.
To construct the pom1-pom2K strain for the domain complementation experiments, the Pom1 kinase domain (amino acids 699–995) was replaced by KS-ura4 using PCR-based gene targeting to obtain strain pom1-ΔK::ura4⁺[90] (link). DNA (pom2K) encoding the Pom2 kinase domain (amino acids 518–814) was amplified and cloned into the TOPO vector. PCR fidelity was confirmed by sequencing. Then the ura4⁺ in pom1-ΔK::ura4⁺ was replaced by the pom2K fragment with 66 bp-long homologous sequences to the flanking regions of pom1 kinase domain. Positive clones were selected on 5-FOA plates and confirmed by PCR. The fusion gene was further confirmed by sequencing.
Cells were re-streaked from −80°C stock, grown 2–3 days on plates, and then inoculated into 5–15 ml YE5S liquid culture as described [91] (link). Cultures were kept in exponential phase for 36–48 h before microscopy except where specified. Strains with 3nmt1 or 41nmt1 promoter were grown in YE5S medium for at least 24 h, washed 3× with EMM5S, and then induced in EMM5S medium for 12–48 h before microscopy except where noted. Stock solutions of thiabendazole (TBZ; Sigma-Aldrich) and methyl benzimidazole-2-yl carbamate (MBC; Sigma-Aldrich) were made in DMSO at concentrations of 50 mg/ml and 5 mg/ml, respectively. The final working concentrations were 50 µg/ml for TBZ and 25 µg/ml for MBC. Cells were stained with Calcofluor and Hoechst 33342 (bisBenzimide) as described [92] (link). Stock solution of Hoechst 33342 (Sigma-Aldrich) was made in ddH₂O at 1 mg/ml and kept in the dark at 4°C.