Figure 6.

A mutation in STU2 reduces the addition of Ndc80 subunits in anaphase. (a and b) Calibrated imaging was used to calculate the copy number for the protein indicated in each MAP mutant during G1 and anaphase, similar to Fig. 2, and then was plotted as in Fig. 2 d. The stu2-11 mutant has a lower anaphase/G1 ratio than WT for Nuf2, whereas Dam1 is unaffected. The bik1Δ and bim1Δ mutants had a lower copy number for Ask1 in G1, affecting the anaphase/G1 ratio. 50–130 clusters were considered for the copy number calculation (n = 3). (c and d) Snapshots and quantification of FRAP from a single cell shows that Nuf2 does not recover during anaphase in the stu2-11 background (n > 8). (e and f) Photoconversion of Ndc80-tdEos in the stu2-11 background shows that green fluorescence does not increase in anaphase from a single cell (n > 8). Bars, 5 µm. Dotted white lines mark cell peripheries. All experiments were performed at room temperature. A two-tailed t test was used to test for statistical significance, and the error bars represent SEM. Kt, kinetochore.