FIGURE 4:

Alp7 and Alp14, but not Dis1, delocalize from the mitotic kinetochore in ndc80-NH12. (A–C) Wild-type or ndc80-NH12 mutant cells that contain Mis6-2mRFP (kinetochore, red) and Dis1-GFP (A, green), Alp7-3GFP (B, green), or Alp14-GFP (C, green) were incubated at 36°C for 1 h and fixed with methanol. Top, images of individual mitotic cells (outlined with discontinuous lines); bottom three rows, enlarged images that cover the kinetochore regions (squares shown on top). Bottom graphs, quantification of GFP signals at kinetochores vs. those of Mis6-2mRFP. The box-and-whisker plot indicates 5th–95th percentiles, 25th and 75th percentiles, and median. Filled circles indicate outliers. The number of samples (n) is indicated for each strain. Statistical significance was determined by the Student's t test. Note that in wild-type mitotic cells, Alp7 and Alp14 foci localize to both kinetochores (internal dots) and SPBs (external dots marked by arrowheads) as previously reported (Sato et al., 2004). In sharp contrast, in ndc80-NH12 mutants, these two proteins do not colocalize with kinetochores, whereas their localization to SPBs (arrowheads) is retained. Scale bars, 5 μm.