Figure 3. Premature CLB3 expression disrupts monopolin function.
(A) Lrs4-13myc (green) localization relative to Ndc10-6HA (red) was determined in spread nuclei from wild-type (A9217), CUP-CLB3 (A26278) and CUP-CLB4 (A29643) harboring a Cdc20 depletion allele (cdc20-mn) were induced to undergo sporulation and arrested in metaphase I due to depletion of Cdc20. CuSO4 was added at 3 hr after induction of sporulation (n > 40). The fraction of spread nuclei that display colocalized, partial or mislocalized Lrs4 with respect to Ndc10 was compared to wild-type using a chi-square test (df 2): CUP-CLB4, χ2 = 1.136, p=0.5666; CUP-CLB3, χ2 = 45.84, p<0.0001. (B) Western blots for Lrs4-13myc, Clb3 and Pgk1 from wild-type (A9217) and CUP-CLB3 (A26278) cells. Cells were sporulated as described in (A).
Figure 3—figure supplement 1. Monopolin association with kinetochores is disrupted in CUP-CLB3 but not in CUP-CLB4 cells.
Wild-type (A7450), CUP-CLB3 (A28673) and CUP-CLB4 (A28674) cells carrying the cdc20-mn allele were induced to sporulate and CuSO4 (50 μM) was added 2 hr 30 min after transfer into sporulation medium. Mam1-9myc (green) localization relative to Ndc10-6HA (red) was determined in spread nuclei from metaphase I-arrested cells (n > 40). DNA is shown in blue. Using a chi-square test (df 2) the fraction of spread nuclei that display colocalized, partial or mislocalized Mam1 with respect to Ndc10 was compared to wild-type: CUP-CLB4, χ2 = 2.554, p=0.2788; CUP-CLB3, χ2 = 39.31, p<0.0001.
Figure 3—figure supplement 2. Premature Clb3 expression does not interfere with Mam1 expression.
Wild-type (A7450), CUP-CLB3 (A28673) and CUP-CLB4 (A28674) cells carrying the cdc20-mn allele were induced to sporulate and CuSO4 (50 μM) was added 2 hr 30 min after transfer into sporulation medium. Mam1 protein levels were analyzed to determine whether premature Clb3 expression interferes with Mam1 expression. Pgk1 was used as a loading control.
Figure 3—figure supplement 3. Lrs4 phosphorylation is not disrupted in CUP-CLB4 cells.
Wild-type (A26277) and CUP-CLB4 (A29643) cells carrying the cdc20-mn allele were induced to sporulate and CuSO4 (50 μM) was added 2 hr 30 min after transfer into sporulation medium. Levels of Lrs4, Clb3 and Pgk1 from cells arrested in metaphase I were examined by Western blot analysis.