Figure 7.

Bir1 localizes to kinetochores that are not under tension. A temperature-sensitive cohesin mutant (mcd1-1) carrying Bir1-Venus and Ndc10-CFP (PWY347-1A) was shifted to the restrictive temperature of 37°C as described in Materials and Methods, along with a wild-type control (MCD1; PWY5-5A). (A) At the permissive temperature (22°C), Bir1 localizes between Ndc10 kinetochores in both MCD1 (Bir1, blue and Ndc10, pink) and mcd1-1 (Bir1, green and Ndc10, yellow). Tension between sister kinetochores is lost in mcd1-1 cells at the restrictive temperature (37°C), as indicated by the increase in spindle length. Average Ndc10 separations were as follows (mean ± SD): MCD1, 22°C (0.89 μm ± 0.13 μm); mcd1-1, 22°C (0.91 ± 0.14 μm); MCD1, 37°C (0.94 ± 0.13 μm); and mcd1-1, 37°C (1.56 ± 0.19 μm). Bir1 localizes between Ndc10 kinetochores in MCD1 (Bir1, blue and Ndc10, pink), but it colocalizes with kinetochores that are not under tension in mcd1-1 (Bir1, green and Ndc10, yellow). Images are scaled equally at each temperature. Bar, 1 μm. (B) Quantification of the distribution of Bir1 and Ndc10. Fluorescence was measured across ≥70 half-spindles for each strain shown. Background was subtracted, and the average half-spindle distributions were reflected about the spindle midpoint. The SE of the mean is approximately the same size as the symbols.