Fig. 4. Cyclin B-dependent accumulation of cdc2-YFP into the nucleus after mitotic exit. (A) Cells from strains AD205 (a-b), AD207 (c, f), AD210 (d) and AD245 (e) were grown overnight at 25°C in the absence of thiamine. Cultures were incubated at 36°C for 4 hours to block the cells at the G₂/M transition (time 0). In d-f, the cdc13 gene expression was switched off by addition of 15 µM of thiamine after either 3 hours (d) or 4 hours (e-f) at 36°C. Cells were observed by LFM for 2 hours 30 minutes at RT. Cdc2-YFP nuclear fluorescence was similar during the first 30 minutes of release (from G₂/M to metaphase) in all strains (a). From anaphase B to post-cytokinesis, cdc2-YFP nuclear fluorescence (b-f, arrowheads) was dependent on the cyclin B present in the strain (cdc13p, cig1p and cig2p (b), cdc13p (c), cig2p (d), cig1p (e) or none of them (f)). Bar, 5 µm. (B) In the same experiments, DNA content was measured by FACS on fixed cells: asynchronous cultures at 25°C (AS), after 4 hours at 36°C (0) and at different time points during the release at 25°C as indicated. AD207 (a and d), AD210 (b) and AD245 (c). (C) Cdc2- and cdc13-YFP fluorescence were not observed in the nucleus of cdc10-V50 ts cells incubated for 4 hours at 36°C (AD192 and AD212). Note the cdc2-YFP aggregation in the cytoplasm of cells incubated at 36°C. This aggregation at high temperatures has also been reported recently for the GFP alone (Fukuda et al., 2000). Bar, 5 µm.