FIGURE 5:

Mad1 removal from sister centromeres after capture. Wild-type, nup53Δ, and nup60Δ cells expressing Mad1-3×mCherry, GFP-Tub1, and TetR-GFP and bearing MET3pr-CDC20 and GAL1pr-CEN3-TetOs were grown for 3 h at 25°C in YP medium plus 2 mM methionine, 2% raffinose, and 2% galactose to synchronize cells in metaphase and inactivate CEN3. The cells were then released into synthetic medium plus 2 mM methionine containing 2% glucose at 25°C to reactivate CEN3 and examined by live-cell epifluorescence microscopy, taking z-stacks every 10 s after centromere reactivation. Representative deconvolved, Gaussian-filtered MIPs are shown in A–D; scale bars, 1 μm. (A–D) Time series showing Mad1 disappearance from sister centromeres after capture. Note that the time intervals depicted vary across the strains in order to show key events. The blue dashed boxes surrounding the first frame of each time series highlight the corresponding regions of the expanded views shown in Supplemental Figure S5 and Supplemental Videos S3–S6. (E) Key for A–D. (F) Timing of Mad1's final disappearance from centromeres relative to the completion of capture. The completion of capture was defined as the first frame in which the centromeres could no longer be resolved from the spindle. Error bars represent SD. *p < 0.05.