Supplemental Materials

This article contains the following supporting material:

• Supplemental Movie 1 - control. Animated time lapse images from control S2 Drosophila cells stably expressing GFP-Tubulin. From these images it is possible to calculate the time that cells spend in prometaphase, from NEBD indicated by the rapid entry of GFP-tubulin into the nuclear space to the initiation of anaphase, as chromatids start to move to the poles.
• Supplemental Movie 2 - Mad2 RNAi. Animated time lapse images from S2 Drosophila cells stably expressing GFP-Tubulin previously treated for 72 h with RNAi against Mad2. From these images it is possible to calculate the time that cells spend in prometaphase, from NEBD indicated by the rapid entry of GFP-tubulin into the nuclear space to the initiation of anaphase, as chromatids start to move to the poles. Depletion of Mad2 causes a rapid exit from mitosis soon after NEBD.
• Supplemental Figure 1 - Kinetochore accumulation of SAC proteins in the absence of Mad2. In all experiments cells were treated with RNAi against Mad2 for 72 hours. Control and RNAi treated cells were then incubated with the proteasome inhibitor MG132 (20 μM) for 2 hours to prevent mitotic exit and then with 30μM Colchicine during 45 min, fixed and stained for DNA (blue), (A) Bub1, (B) Bub3 (C) BubR1 or (D) Mad2 shown in green and the kinetochore marker Polo is shown in red. Bar is 5 μm. The results show that Mad2 is not required for the kinetochore localization of any of the checkpoint proteins that we tested.
• Supplemental Figure 2 - Timing of mitotic progression in Mad2-depleted cells. The time between NEBD and anaphase onset in control and Mad2-depleted cells was determined by time-lapse fluorescence microscopy of S2 cells stably expressing GFP-Tubulin. (A) Selected frames of both control and RNAi-treated cells show that anaphase onset occurs earlier in cells lacking Mad2 than in the control cells. Note that NEBD can be easily determined because of the rapid entry of fluorescence tubulin to the nuclear space. Bar is 5 μm. (B) Quantitative analysis of mitotic timing from at least 10 cells. The results show that control cells take on average 33±8 min from NEBD to anaphase onset displaying some variation in the timing of individual cells. However, Mad2-depleted cells complete NEBD to anaphase onset in only 11±2 min displaying little or no significant variation between individual cells.
• Supplemental Figure 3 - Following kinetochore pairs through optical stacks. Kinetochores were followed through different optical stacks (Z) to determine how they are paired. Dotted line indicates a cluster of kinetochores in which the pairing between them is undetermined.
• Supplemental Figure 4 - Depletion of BubR1 by western blot analysis. BubR1 can be depleted by over 70% by 72h, reaching a maximum depletion of 85% at 120h after the addition of the dsRNA specific for BubR1.