Gene disruption and construction of C-terminally tagged strains

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1 Supplementary information Supplementary Methods Gene disruption and construction of C-terminally tagged strains A PCR-based gene targeting method (Bähler et al., 1998; Sato et al., 2005) was used for constructing gene deletion or C-terminally tagged strains under the natural promoter. Fluorescence microscopy, time-lapse analysis and image processing For time-lapse microscopy, a 35 mm glass-bottomed culture dish (MatTek Corporation, P35G C) was coated with 200 µg/ml soybean lectin (SIGMA, L1395). The culture of logarithmically growing cells (100 µl) was deposited in the well for a couple of minutes and then removed. The dish was filled with 3 ml of YE5S medium and the cells that were attached to the bottom of the well were subjected to microscopic analysis. Images were taken using an Olympus IX70 wide-field inverted epifluorescence microscope with an Olympus PlanApo 100x, NA 1.4, oil immersion objective. DeltaVision image acquisition software (softworx 3.3.0, Applied Precision, Issaquah, WA) equipped with Coolsnap HQ (Roper Scientific, Tuscon, AZ) was used for capture of live images, which were processed with Adobe Photoshop (version 7.0). The sections of images at each time point were compressed into a 2D projection using the DeltaVision maximum intensity algorithm. Deconvolution was applied before the 2D projection. Kymograph pictures derived from 2D-projected time-lapse images were constructed using softworx Imaging conditions For the observation of Mal3-GFP, 10 sections of images were taken ( z = 0.4µm). The whole cell volume was included within 10 sections. For time-lapse imaging of Mal3-GFP (Supplementary Figure S1), 10 sections of images were taken ( z = 0.3 µm) every 5 seconds. For the simultaneous observation of Mal3-GFP and Sad1-RFP, 4 sections of 1

2 images ( z = 0.4 µm) were taken at every 10 seconds. The SPBs were included in these 4 sections at every time point. For the simultaneous observation of Sad1-RFP and cen2- GFP, 8 to 10 sections of images ( z = 0.4 µm) were taken at every 30 or 60 seconds. For the kymograph analysis, 6 sections of images ( z = 0.4 µm) including the mitotic spindle were taken at every 10 seconds. For the simultaneous observation of Sad1-RFP and cen2-gfp or Bub1-GFP, 8 to 10 sections of images ( z = 0.4 µm) were taken at every 30 or 60 seconds. Visualization of Pcp1-RFP Procedures described previously (Grallert et al., 2004) were followed except that strains were grown at 26 C in the minimal medium that contained a glutamate (0.15 mm) instead of ammonium chloride and was supplemented with appropriate amino acids and bases (leucine, histidine, adenine and uracil at 0.75 % each). Supplementary Note for Figure 3B We acquired the images of early mitotic cells (the spindle length µm) from both mal3 and wild type strains (40 live samples each). The relative fluorescence intensities of Bub1-GFP dot were measured, in which GFP intensities from the nucleoplasm were used as background signals. We then picked up cells that contained the Bub1 dot with more than 6 times brighter than the nuclear background. The result we obtained by these procedures was that 13 out of 40 mal3 cells showed >6x signals from Bub1-GFP dot, whilst none of 40 wild-type cells displayed such a intensity of Bub1 dot. In Figure3B, we divided the mitotic spindle into equal three parts, where the middle region is flanked by the polar regions. In mal3 mutant cells, 10 cells showed >6x Bub1-GFP in the polar region and 3 cells in the middle. Supplementary Reference 2

3 Bähler, J., Wu, J., Longtine, M.S., Shah, N.G., McKenzie III, A., Steever, A.B., Wach, A., Philippsen, P. and Pringle, J.R. (1998) Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast, 14, Grallert, A., Krapp, A., Bagley, S., Simanis, V. and Hagan, I.M. (2004) Recruitment of NIMA kinase shows that maturation of the S. pombe spindle-pole body occurs over consecutive cell cycles and reveals a role for NIMA in modulating SIN activity. Genes Dev., 18, Sato, M., Dhut, S. and Toda, T. (2005) New drug-resistant cassettes for gene disruption and epitope tagging in Schizosaccharomyces pombe. Yeast, 22,

4 Supplementary Table S1: Strain list used in this study Strains Genotypes Derivations 513 h - leu1ura4 This study MA145 h - leu1 ura4 mal3 + -GFP-kan r This study 393 h - leu1 ura4-ds/e his1-102 ade6-216 bub1::ura4 + Dr. J.-P. Javerzat KZ2 h + leu1 ade6-210 bub1 + -GFP-kan r containing Ch16 This study KZ85 h - leu1 ura4 sad1 + -RFP-LEU2 cen2-kan r -ura4 + -lacop This study his7 + -laci-gfp KZ154 h + leu1 his2 ura4 mal3::kan r This study KZ159 h - leu1 ura4 mad1::ura4 + mal3::kan r This study KZ160 h - leu1 ura4 mad2::leu2 mal3::kanr This study KZ161 h - leu1 ura4 ade6-216 bub1::ura4 + mal3::kan r This study KZ162 h - leu1 ura4 bub3::ura4 + mal3::kan r This study KZ163 h - leu1 ura4 mph1::ura4 + mal3::kan r This study KZ164 h - leu1 ura4 mal3::kan r mad3::ura4 + This study KZ181 h - leu1 ade6-210 bub1 + -GFP-kan r sad1 + -RFP-LEU2 This study KZ186 h - leu1 ura4 mal3::kan r sad1 + -RFP-LEU2 This study cen2(d107)-kan r -ura4 + -lacop his7 + -laci-gfp KZ188 h leu1 ura4 mal3::kan r ade6-216 bub1::ura4 + This study sad1 + -RFP-LEU2 cen2(d107)-kan r -ura4 + -lacop his7 + -laci-gfp KZ190 h - leu1 mal3::kan r bub1 + -GFP-kan r nuf2 + -CFP-kan r This study KZ191 h - leu1 mal3::kan r sad1 + -RFP-LEU2 bub1 + -GFP-kan r This study KZ199 h - leu1 mal3 + -GFP-kan r nuf2 + -CFP-kan r This study 4

5 sad1 + -RFP-LEU2 KZ201 h - leu1 ura4 mad2 + -GFP-kan r sad1 + -RFP-LEU2 This study KZ202 h - leu1 ura4 mal3::kan r mad2-gfp-kan r sad1 + -RFP-LEU2 This study MT18 h - leu1 mal3::kanr mad2::leu2 sad1 + -RFP-LEU2 This study cen2(d107)-kan r -ura4 + -lacop his7 + -laci-gfp IH2675 h - pcp1 + -RFP-kan r Dr. Hagan MT25 h + his2 mal3::kan r mad2 + -GFP-LEU2 bub1 + -RFP-kan r This study MT28 h - leu1 ura4 mal3::kan r bub1::ura4 + pcp1 + -RFP-kan r This study ade cen2(D107)-kan r -ura4 + -lacop his7 + -laci-gfp TTK2 h - leu1 ura4 his7 bub1 + -GFP-kan r cut12 + -CFP-nat This study pcp1 + -RFP-kan r 5

6 Supplementary Figure S1: Time-lapse analysis of mitotic localization of Bub1-GFP with kinetochore and SPB markers Time-lapse live analysis of Mal3-GFP, Nuf2-CFP and Sad1-RFP in a single mitotic cell. Mitotic movement of Mal3-GFP (A and green in C), Nuf2-CFP (B and red in C), and Sad1-RFP (blue in C) is shown from 0 to 300 s (25 s intervals). Mal3 signals that do not overlap with Nuf2 are marked with arrowheads, whilst Nuf2 signals that do not overlap with Mal3 are marked with arrows. The bars indicate 2 µm. Supplementary Figure S2: Chromosome segregation in double mutants between mal3 and deletions in spindle checkpoint genes Indicated strains were grown in liquid cultures at 26 o C and shifted to 36 o C. Aliqots of samples were taken at 0, 4 and 6 h, fixed with formaldehyde and stained with DAPI. The percentage of anaphase cells displaying chromosome mis-segregation was counted in each preparation. Mis-segregated chromosomes are pointed with arrows. At least 50 anaphase cells per sample were observed. The bars indicate 10 µm. Supplementary Figure S3: Kinetics of mitotic spindle elongation in wild type, mal3, mal3bub1 and mal3mad2 mutants Visualization of spindle dynamics. 4 strains (wild type in black, mal3 in light and dark blue, mal3bub1 in red and mal3mad2 in yellow and orange lines) containing cen2-gfp and Sad1-RFP were grown and time-lapse live analysis was performed. Two independent examples of spindle dynamics in mal3 and mal3mad2 mutants are shown. In order to clearly present each line, O time points, when live imaging started, are plotted at different positions. 6

7 Supplementary Movies Movie S1: Time-lapse images that show Mal3-GFP during mid-mitosis. A strain containing Mal3-GFP (green) and Sad1-RFP (red) is used. Movie S2: Time-lapse images that show Mal3-GFP (green), Nuf2-CFP (red) and Sad1-RFP (blue) in a single mitotic cell. Movie S3: Time-lapse images that show dynamics of Bub1-GFP (green) in a mitotic wild type cell. Sad1 is also tagged with RFP (red). Movie S4: Time-lapse images that show dynamics of Bub1-GFP (green) in a mitotic mal3 cell. Sad1 is also tagged with RFP (red). Movie S5: Time-lapse images that show dynamics of cen2-gfp (green) during mitosis in a wild type strain. Sad1 is also tagged with RFP (red). Movie S6: Time-lapse images that show dynamics of cen2-gfp (green) during mitosis in the mal3bub1 mutant. Sad1 is also tagged with RFP (red). 7

8 A Mal3-GFP B merged +Sad1-RFP Nuf2-C FP C Supplementary Figure S1: Asakawa et al.

9 WT bub1 mal3 mal3 bub1 mal3 bub3 mal3 mph1 mal3 mad1 mal3 mad2 mal3 mad3 Supplementary Figure S2 : Asakawa et al.

10 16 Sp indle length (µm) mal3 bub1 wt mal3 mad2 #2 #1 #2 #1 mal (min) Supplementary Figure S3: Asakawa et al.