MBC Videos and Supplemental Figures

This article contains the following supporting material:

• Supplemental Figures 1-4.pdf - Supplementary Figure 1. At high concentration Latrunculin A inhibits mitotic entry non-specifically.
  (A) A log phase culture of cdc25-22 plo1-gfp cells was incubated for 3.5 hours at 36°C and then for a further 30 minutes in the same medium either in the absence (control) or presence of 2.5 μM, 5 μM, 12.5 μM or 25 μM Latrunculin A. Cells were fixed and stained for actin containing structures. (B) + (C) Log phase cultures of cdc25-22 plo1-gfp were blocked at the restrictive temperature for 3.5 hours and and then for a further 30 minutes either in the absence (open squares) or presence of 12.5 μM (closed diamonds), 25 μM (closed circles), 50 μM (closed triangles), 100 μM (crosses) or 150μM (closed squares) Latrunculin A. The percentage of (B) binucleate cells or (C) cells with SPB-associated Plo1 was determined at the times shown after the release at permissive temperature (n=150).
  
  Supplementary Figure 2. Live imaging of Cdc13 (cyclin B) destruction.
  Synchronised cdc13-gfp cells were incubated in medium containing DAPi either in the absence (control, left panels) or presence (Lat A, right panels) of 1.25 μM Latrunculin A. Images were taken in a single focal plane for Cdc13 (green) or chromatin (blue). Time zero is the beginning of phase 2 when the spindle becomes stable in length.
  
  Supplementary Figure 3. Live imaging of sister chromatid separation in the absence of Mad2 or Bub1.
  Images from movies of (A) mad2Δ ndc80-gfp cdc11-cfp or (B) bub1Δ ndc80-gfp cdc11-cfp cells grown either in the absence (Control) or presence (Lat A) of 1.25 μM Latrunculin A. The localisation of kinetochores (green) and spindle pole bodies (red) is shown at the times indicated. Co-localisation is seen in yellow. Time zero is when the spindle becomes 2 μm in length (the beginning of phase 2).
  
  Supplementary Figure 4. Localisation of Bub1 and Mad2 relative to SPBs.
  (A) Images of mad2-gfp cdc11-cfp cells showing the localisation of Mad2 (green) and Ndc80 (red). (B) Images of bub1-gfp cdc11-cfp cells showing the localisation of Bub1 (green) and Ndc80 (red). Cells are shown in (i) G2, (ii) prometaphase (iii) metaphase (iv) anaphase and (v) telophase.
  
  
• Fig2.Video1.mov - Movement of kinetochores in mitosis. A ndc80-gfp cdc11-cfp cell was followed by live video microscopy. Images were captured every 25 seconds. Kinetochores are shown in green and spindle poles are shown in red. Movie is played at one frame per second.
  
• Fig2.Video2.mov - Movement of kinetochores in the presence latrunculin A. A ndc80-gfp cdc11-cfp cell was incubated in the presence of 1.25 μM latrunculin A and followed by live video microscopy. Images were captured every 25 seconds. Kinetochores are shown in green and spindle poles are shown in red. Movie is played at one frame per second.
  

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