Figure 6. Lethal mutations in ANdc80p, BNdc80p, and CNdc80p confer defects in Dam1 complex recruitment to the kinetochore.
(A) Representative images from NDC80-AID degron experiments with cells also containing wild-type NDC80 or mutation in region BNdc80p. The inner kinetochore was visualized with Mtw1-YFP, the Dam1 complex with Dad4-CFP, and the SPB with Spc110-mCherry. Cells were treated with 6 μM α-factor for two hours prior to the addition of 0.5 mM auxin for one additional hour. At time 0, cells were released from α-factor and incubated in YPD medium containing 0.5 mM auxin. Images were taken at 60 min after release from α-factor arrest. (B) Line scan traces of Mtw1-YFP and Dad4-CFP from the images in (A). Only cells with two SPBs in the same plane of focus were selected for analysis. Each line scan extended from one SPB to the other. Each point represents one pixel. (C) Summary of correlation analysis carried out between Mtw1-YFP and Dad4-CFP. For each line scan, a correlation of the positive or negative changes of pixel intensity along the line between the Mtw1-YFP and Dad4-CFP channels was calculated to examine co-localization along the spindle (see Materials and methods). Between 18 and 33 cells were analyzed for each mutant. Mtw1-YFP and Dad4-CFP fluorescence intensity raw data are included in Figure 6—source data 1. Refer to Supplementary file 1G for statistical analysis of data.
DOI: http://dx.doi.org/10.7554/eLife.21069.025
Figure 6—figure supplement 1. Mutations in regions ANdc80p, BNdc80p, or CNdc80p results in severe defects in kinetochore biorientation and attachment.
