Figure 2: A decreasing gradient in tension leads to an increasing gradient in both detached kinetochores at metaphase, and mis-segregation of chromosomes in anaphase.

(A) Cartoon demonstrating a potential cellular response to low tension: a low metaphase tension (left) could lead to an increase in detached kinetochores (right). (B) Images from time series in metaphase cin8Δ mutants demonstrating dynamic attachment (see also Fig. S3A) (scale bars, 1 μm). (C) Top: Representative images of lacO-lacI-GFP spots in cells with properly attached kinetochores (left), and detached kinetochores (right) in budding yeast (scale bars, 500 nm). Bottom: Detachment rate vs tension in wild type and mutant strains. The tension and detachment rate in the Degron-Cin8 cells (dark yellow) was similar to that in cin8Δ cells (light yellow) (see also Fig. S2A, B). The detachment rate increased substantially with decreasing tension (z = −15.49, p < 0.0001, Cochran-Armitage trend test), and the detachment rate was significantly increased even when average tension was decreased by as little as 0.9 pN (Χ² = 11.91, p = 0.006). (D) Top: Representative images of lacO-lacI-GFP spots (green) and chromosomes (blue, DAPI) in anaphase cells with proper chromosome segregation (top), and representative images of lacO-lacI-GFP spots (green) and chromosomes (blue, DAPI) in anaphase cells with improper, missegregated chromosomes (bottom) (scale bars, 5 μm). Bottom: Anaphase chromosome missegregation rate vs tension in a mad2Δ strain background (triangles; z=−6.16, p<0.0001, Cochran-Armitage trend test). Note that detachment rates were similar regardless of the presence or absence of Mad2 (Fig. S3C). The anaphase missegregation rate was reduced in cells with normal Mad2 expression (circles; Χ² = 36.61, p < 0.0001). (E) Experimental protocol for deactivating Ipl1 in ipl1–321 experiments. (F) Top: Representative images of lacO-lacI-GFP spots in cells with properly attached kinetochores (left), and detached kinetochores (right) in budding yeast strains harboring the ipl-321 allele (scale bars, 500 nm). Bottom: Detachment rate vs tension in wild type and tension mutant strains in Cdc20 arrested cells at 37 °C (circles; tension measurements in Fig. S2D–E), and detachment rate vs tension in wild type and tension mutant strains in cells that harbored the ipl1–321 allele, and that were Cdc20 arrested at 37°C (crosses; tension measurements in Fig. S2D,F). The detachment rate increased substantially with decreasing tension for the Cdc20 arrested cells (Χ² = 169.70, p < 0.0001), but the inactivation of Ipl1 abrogated this dependence (Χ² = 0.27, p = 0.966). All panels: error bars=SEM.