An Ask1 phosphomutant curtails the addition of Ndc80 in anaphase. (a) Normalized fluorescence intensity measurements indicate that a strain bearing the ask1-2A mutation does not compromise the assembly of the Dam1 subcomplex. For box plots (a and c), 100–150 kinetochore clusters were quantified, and for statistical analysis, a two-tailed t test was performed. The middle line represents the mean, the box represents SD, and whiskers represent SEM. (b) The ratio of fluorescence in anaphase to G1 is similar in the WT and mutant strains. Error bars indicate SEM. (c) Normalized fluorescence intensity measurements indicate that a strain bearing the ask1-2A mutation blocks the addition of Ndc80 subcomplex component Nuf2-GFP in anaphase (*, P < 0.0001). (d) The ratio of fluorescence in anaphase/G1 shows that Nuf2-GFP does not increase in intensity in the ask1-2A strain background, in contrast to the normal doubling observed in a WT strain background. Error bars indicate SEM; *, P, <0.0001. (e) Normalized fluorescence intensity measurements indicate that a strain bearing the ask1-2A mutation has addition of MIND subcomplex component Dsn1-GFP in anaphase (*, P < 0.0001). (f) The ratio of fluorescence in anaphase/G1 shows that Dsn1-GFP increases in intensity in the ask1-2A strain background. Error bars indicate SEM; *, P < 0.0001.