T H E J O U R N A L O F C E L L B I O L O G Y Supplemental material Courtheoux et al., http://www.jcb.org/cgi/content/full/jcb.200902093/dc1 S1
Figure S2. Visualization of multiple merotelic attachments. (A, top) Kymograph of a rad21-k1 ndc80-gfp cdc11-gfp cell showing two merotelic attachments at the spindle center, each of which is stretched into two dots. (right) A line scan confirms the presence of four dots (2 2 Kts). (bottom) Automated tracking analysis of SPBs (red) and Kt position. (B, top) Kymograph of a rad21-k1 ndc80-gfp cdc11-cfp cell showing three merotelic Kts at the spindle center, each of which is stretched into two dots. (right) A line scan confirms the presence of six dots (2 3 Kts). (bottom) Automated tracking analysis of the SPBs (red) and the Kt position. Green, Kts at the poles; blue, light blue, and purple, the three merotelic Kts. Figure S1. A combination of live cell microscopy and laser ablation to visualize merotelic attachment. (A, top) Live cen1-gfp (red) ndc80-cfp (green) cells during anaphase. (bottom) Representation of an amphitelic Kt attachment. (B, top) Live rad21-k1 cen1-gfp (red) ndc80-cfp (green) cells during anaphase. (bottom) Representation of a merotelic Kt attachment. Schematic representations of fission yeast cell shape are shown (dashed ovals). The boxed region indicates the enlarged region in C. (C) Enlarged images of the merotelic attachment shown in B. (D) Image series of a rad21-k1 ndc80-gfp (green) cdc11-cfp (blue) in live cells stained with Hoechst (DNA; red). Note the presence of a stretched merotelic Kt at the 405-s time point. (E) Quantification of Hoechst signal was performed by integrated intensity on the defined area minus the noise at the 405-s time point. The data shown are representative of a single experiment, which was repeated in several cells (n = 8). (F) Simultaneous laser ablation of cells within the same field is shown. Image series of a field of cells showing eight individual wt SV40-GFP-atb2 mitotic cells (red ovals) before and after laser ablation (blue dots; impact at the 20-s time point). Note that the spindles are simultaneously and reproducibly cut in all cells. S2
Figure S3. Merotelic attachment leads to cut/asymmetric division/multipolar spindles and cell death in the absence of Ase1. (A) Image series of a rad21-k1 (MTs) ndc80-gfp (Kts) cell during mitosis. Example of a cell with a merotelic (Mero) attachment (white arrows) during mitotic progression up to the completion of cytokinesis and cell abscission (left, bright field picture). One of the daughter cells (top) undergoes a successful second round of mitosis and cytokinesis, whereas the other (bottom right), although alive, is delayed in G2. (B) Image series of a rad21-k1 ase1 atb2-gfp (MTs) ndc80-gfp (Kts) cell during cell cycle progression after asymmetric spindle collapse. The two SPBs are positioned at one extremity of the cell; mitosis is followed by cytokinesis and abscission (bright field picture). The top cell possesses two sets of chromosomes as judged by the ndc80-gfp signal and undergoes a second round of cytokinesis before cell death. The bottom cell is aneuploid and dies. (C) Image series of a rad21-k1 ase1 ndc80-gfp (Kts) cdc11-cfp (SPBs) Myo2 GFP (ring) after asymmetric spindle collapse and cytokinesis. After replication of the two existing SPBs, cells with multipolar spindles are produced. The inset shows an enlarged view of the SPBs and Kts at 4 min. S3
Figure S4. Merotelic attachment leads to cut/asymmetric division and cell death in the absence of Ase1. (A) Percentage of cells with a septum showing defects in chromosome segregation: wt, n = 2,466; rad21-k1, n = 1,966; ase1, n = 2,716; and rad21-k1 ase1, n = 1,368. (B) rad21-k1 is synthetically lethal with ase1 at the semipermissive temperature. rad21-k1, ase1, and rad21-k1 ase1 double mutants were streaked on YEA plates at different temperatures (25, 28, 31, 33, and 36 C). Error bars indicate SD. S4
Figure S5. Simulation of multiple merotelic attachments and reduction in spindle force generation during spindle elongation. Simulation of mitotic progression in the presence of two merotelic Kts (green and purple; SPBs are shown in red). The Kt attachment state is indicated for one of the merotelic Kts (purple). (B) Simulation of mitotic progression in the presence of three merotelic Kts (green, purple, and blue). (C) Simulation of a reduction in the spindle force generator (midzone stall force; Table I) at the onset of anaphase A (time 0) until the critical size of the spindle is reached at 7 µm (Loïodice et al., 2005). At this critical size, and as observed in vivo, force is abruptly adjusted to 0. (D) Simulation of Kt dynamics (green) and SPB position (red) when the force produced by the interdigited MTs linearly decreases. Video 1. Fluorescent time-lapse imaging of Kt dynamics in a wt S. pombe cell. Wt cells expressing Ndc80 GFP as a marker of the six Kts (green) and Cdc11 CFP as a marker of the two SPBs (red) were analyzed by time-lapse wide field microscopy. Frames were collected every 8 s and are shown at a rate of 60 frames/min. Representative frames from this video are shown in Fig. 1 A. Video 2. Fluorescent time-lapse imaging of Kt dynamics in rad21-k1 S. pombe cells. Rad21-K1 cells expressing Ndc80 GFP as a marker of the six Kts (green) and Cdc11 CFP as a marker of the two SPBs (red) were analyzed by wide field time-lapse microscopy as in Video 1. Frames were collected every 10 s and are shown at a rate of 60 frames/min. Representative frames from this video are shown in Fig. 1 B. S5
Video 3. Fluorescent time-lapse imaging of a single merotelic Kt after laser ablation. Wt (left) and rad21-k1 (right) cells expressing SV40-GFP-atb2 as a marker of -tubulin (green) and Cdc11 CFP as a marker of the two SPBs (red). Both cells were analyzed simultaneously by time-lapse microscopy as in Video 1. At the 140-s time point, cells were subjected simultaneously to laser ablation (white dots; pulse time of 35 ms). Frames were collected every 10 s and are shown at a rate of 60 frames/min. Representative frames from this video are shown in Fig. 2. Video 4. Fluorescent time-lapse imaging of an ase1 S. pombe cell expressing -tubulin. ase1 cell expressing SV40-GFP-atb2 as a marker of -tubulin (spindle) and Ndc80-GFP as a marker of the Kts (Kts are at the poles) were analyzed as in Video 1. Frames were collected every 2 s and are shown at a rate of 60 frames/min. Representative frames from this video are shown in Fig. 4 B (left). Video 5. Fluorescent time-lapse imaging of an ase1 rad21-k1 S. pombe cell expressing -tubulin. ase1 rad21-k1 cell expressing SV40-GFP-atb2 analyzed as in Video 1. Frames were collected every 2 s and are shown at a rate of 60 frames/min. Representative frames from this video are shown in Fig. 4 B (right). Video 6. Fluorescent time-lapse imaging of Kt dynamic and cytokinesis in a wt S. pombe cell. Wt cells expressing Myo2 GPF as a marker of the actomyosin ring (green), Ndc80 GFP as a marker of the six Kts (green), and Cdc11 CFP as a marker of the two SPBs (red) were analyzed by time-lapse microscopy as in Video 1. Frames were collected every 1 min and are shown at a rate of 60 frames/min. Representative frames from this video are shown in Fig. 5 A. Video 7. Fluorescent time-lapse imaging of Kt dynamic and cytokinesis in a rad21-k1 S. pombe cell. rad21-k1 cells expressing Myo2 GFP as a marker of the actomyosin ring (green), Ndc80-GFP as a marker of the six Kts (green), and Cdc11 CFP as a marker of the two SPBs (red) were analyzed as in Video 1. Frames were collected every 1 min and are shown at a rate of 60 frames/min. Representative frames from this video are shown in Fig. 5 B. Video 8. Fluorescent time-lapse imaging of Kt dynamics and cytokinesis in a rad21-k1 ase1 S. pombe cell. rad21-k1 ase1 cells expressing Myo2 GFP as a marker of the actomyosin ring (green), Ndc80-GFP as a marker of the six Kts (green), and Cdc11 CFP as a marker of the two SPBs (red) were analyzed by time-lapse microscopy as in Video 1. Frames were collected every 1 min and are shown at a rate of 60 frames/min. Representative frames from this video are shown in Fig. 5 C. Video 9. Fluorescent time-lapse imaging of Kt dynamics and cytokinesis in a rad21-k1 ase1 S. pombe cell. rad21-k1 ase1 cells expressing Myo2 GFP as a marker of the actomyosin ring (green), Ndc80-GFP as a marker of the six Kts (green), and Cdc11 CFP as a marker of the two SPBs (red) were analyzed by time-lapse microscopy as in Video 1. Frames were collected every 1 min and are shown at a rate of 60 frames/min. Representative frames from this video are shown in Fig. 5 D. S6
Table S1. S. pombe strains used in this study Strain number Genotype References TCX189 rad21-k1 cen1(d107)-kanr-ura4-laco his7-laci-gfp ndc80-cfp::kanr This study TCX177 ndc80-cfp::kanr cen1(d107)- kanr-ura4-laco his7+-laci-gfp This study ST102 ndc80-gfp::kanr cdc11-cfp::kanr ura4-d18 leu1-32 Tournier et al. (2004) TCX113 rad21-k1 ndc80-gfp::kanr cdc11-cfp::kanr This study TCX246 lys1::nmt1 atb2-gfp cdc11-cfp::kanr This study ST754 ase1::kanr Loïodice et al. (2005) TCX218 rad21-k1 ndc80-gfp::kanr cdc11-cfp::kanr ase1::kanr This study TCX220 rad21-k1 ase1::kanr ndc80-gfp::kanr cdc11-cfp::kanr SV40-gfp-atb2 This study TCX221 rad21-k1 ndc80-gfp::kanr cdc11-cfp::kanr SV40-gfp-atb2 This study ST430 rad21-k1 Tomonaga et al. (2000) TCX284 rad21-k1 ase1::kanr This study TCX276 ndc80-gfp::kanr cdc11-cfp::kanr myo2-gfp::kanr This study TCX272 rad21-k1 ndc80-gfp::kanr cdc11-cfp::kanr myo2-gfp::kanr This study TCX278 rad21-k1 ase1:: kanr ndc80-gfp::kanr cdc11-cfp::kanr myo2-gfp::kanr This study References Loïodice, I., J. Staub, T.G. Setty, N.P. Nguyen, A. Paoletti, and P.T. Tran. 2005. Ase1p organizes antiparallel microtubule arrays during interphase and mitosis in fission yeast. Mol. Biol. Cell. 16:1756 1768. doi:10.1091/mbc.e04-10-0899 Tomonaga, T., K. Nagao, Y. Kawasaki, K. Furuya, A. Murakami, J. Morishita, T. Yuasa, T. Sutani, S.E. Kearsey, F. Uhlmann, et al. 2000. Characterization of fission yeast cohesin: essential anaphase proteolysis of Rad21 phosphorylated in the S phase. Genes Dev. 14:2757 2770. doi:10.1101/gad.832000 Tournier, S., Y. Gachet, V. Buck, J.S. Hyams, and J.B. Millar. 2004. Disruption of astral microtubule contact with the cell cortex activates a Bub1, Bub3, and Mad3- dependent checkpoint in fission yeast. Mol. Biol. Cell. 15:3345 3356. doi:10.1091/mbc.e04-03-0256 S7