Wild-type or CUP-CLB cells were induced to sporulate. After 2 hr 15 min, cyclins were induced by addition of CuSO4 (50 μM). Cells were either arrested during prophase I or released from an NDT80 block 4 hr 30 min after induction of sporulation. (A) Bipolar spindle formation determined in wild-type (A22678), CUP-CLB1 (A27421), CUP-CLB3 (A22702), CUP-CLB4 (A27423) and CUP-CLB5 (A27425) during prophase I (n = 100 per time point). Images on left show spindle formation in CUP-CLB cells 4 hr after induction of sporulation; in this and all subsequent Figures microtubules are shown in green and DNA in blue. The dotted line depicts the cell membrane. (B) Microtubule–kinetochore engagement monitored during prophase I, starting at 1 hr after CuSO4 addition in wild-type (A30700), CUP-CLB1 (A30702), CUP-CLB3 (A30704), CUP-CLB4 (A30707) and CUP-CLB5 (A30708) by live cell microscopy. SPBs (marked by arrow) and heterozygous CENV-GFP dots are shown (arrowheads mark separated CENV dots). In this and all subsequent figures SPBs are in red, GFP dots are in green. (C) Top panel: representative images of wild-type (A30700) and CUP-CLB3 (A30704). Bottom panel: separation of heterozygous CENV-GFP dots in prophase I-arrested cells quantified in wild-type (A22678), CUP-CLB1 (A27421), CUP-CLB3 (A22702), CUP-CLB4 (A27423) and CUP-CLB5 (A27425) by live cell microscopy (over the duration of 8 hr, n > 100) as described in the ‘Materials and methods’. The fraction of nuclei that display sister kinetochores as separate or together for each CUP-CLB strain was compared to wild-type using a chi-square test (df 1): CUP-CLB1, χ2 = 40.77, p<0.0001; CUP-CLB3, χ2 = 34.84, p<0.0001; CUP-CLB4, χ2 = 0.1163, p=0.7330; CUP-CLB5, χ2 = 1.418, p=0.2337. (D) Segregation of sister chromatids (equational division) using heterozygous CENV-GFP dots quantified in binucleates from wild-type (A22678), CUP-CLB1 (A27421), CUP-CLB3 (A22702), CUP-CLB4 (A27423) and CUP-CLB5 (A27425) (n = 100). The fraction of binucleates that display a reductional or equational division for each CUP-CLB strain was compared to wild-type using a chi-square test (df 1): CUP-CLB1, χ2 = 45.13, p<0.0001; CUP-CLB3, χ2 = 48.22, p<0.0001; CUP-CLB4, χ2 = 1.020, p=0.3124; CUP-CLB5, χ2 = 0, p=1. (E) Wild-type (A31019) and CUP-CLB3 (A31021) cells monitored for segregation of heterozygous CENV-GFP dots with respect to Pds1 (Securin, red) degradation by live cell microscopy (n > 17). Time of Pds1 degradation set to t = 0, percent cells were plotted as a Kaplan–Meier curve. Note that for A31021, the analysis of cells that segregate sister chromatids in the first nuclear division is shown. Pds1 accumulation during prophase II is not observed using the Pds1-tdTomato construct, likely due to delayed maturation of the fluorophore (Katis et al., 2010).