Figure 1. A system to induce spindle misposition.
(A) Zone model of exit from mitosis. Yeast cells are partitioned into two zones: a MEN inhibitory zone in the mother cell compartment (red) and a MEN activating zone in the bud cell compartment (green). If the spindle becomes misaligned in the inhibitory zone, MEN inhibitors such as Kin4 prevent Tem1 enrichment on SPBs thereby inhibiting exit from mitosis. It is only once one SPB escapes the MEN inhibitory zone and moves into the bud cell compartment that Tem1 can become enriched at the daughter-bound SPB and the cell can exit mitosis. Note that in this model, Tem1 is shown not to localize to SPBs in cells with mispositioned spindles. This is based on the observation that Tem1-13MYC does not localize to SPBs in cells with mispositioned spindles (D’Aquino et al., 2005). (B) cMT - budneck model of exit from mitosis. If the spindle becomes misaligned in the mother compartment, cytoplasmic microtubules activate a checkpoint response through their interactions with factors at the bud neck. Once the spindle has realigned, the cytoplasmic microtubules are no longer in contact with the bud neck, the checkpoint signal is eliminated and cells exit from mitosis.(C–D) Wild type (A33138), kar9Δ (A33729) and dyn1Δ (A32922) cells harboring GFP-tagged α-tubulin were grown to mid-log in YEPD and arrested in G1 with 10 μg/mL of the α-factor pheromone at 25°C. The cultures were released into the cell cycle in YEPD and then loaded onto a Y04C CellASIC flow cell. Cells were imaged on the flow cell in synthetic complete pH 6.0 medium. (C) Quantification of the percent of anaphase cells which misposition their anaphase spindle. Anaphase was defined as any spindle measuring >2 μm. Aligned spindles were defined as those that entered anaphase with one spindle pole in the bud cell compartment. Mispositioned spindles were defined as those that entered anaphase with both spindle poles the mother cell compartment. (D) Time-lapse analysis of anaphase length. n =100 cells for each strain (E–I) osTIR1 (A35699) and osTIR1 DYN-AID kar9Δ (A35707) cells expressing GFP-tagged α-tubulin were grown in YEPD medium at 25°C and arrested in the G1 phase of the cell cycle with 10 μg/mL α-factor pheromone. Cells were released into the cell cycle in YEPD pH 6.0 medium and then monitored by live cell microscopy. Depletion of dyn1-AID was induced on a Cellasic flow cell with 100 μM auxin in SC pH 6.0 medium at 25°C. (E) Time-lapse analysis of anaphase length. Open squares indicate cells arrested in anaphase for more than 200 min. (F) Analysis of ploidy. Cells that were arrested and contained a misaligned spindle or cells that exited mitosis that contained an aligned spindle were categorized as 'euploid'. Cells that inappropriately exited mitosis and broke down the spindle in the mother cell compartment were categorized as 'multinucleate'. n=100 cells. (G–I) Montage of representative time-lapse images. The numbers at the top of the GFP images are time in minutes.