Fig. 6. Inappropriate activation of the SIN induces cytokinesis but not EMTOC formation. (A) cdc16.116 cells were cultured in YES at 25°C then size selection was used to generate a synchronous population of late S phase/early G2 cells and the frequency of septa (A) or EMTOCs (B) was scored. The culture was split into two. One portion was constantly cultured at 25°C to monitor the cell cycle synchrony by following the septation index (A, open squares). Hydroxyurea (HU) was added to the second culture 60 minutes after elution, and it was cultured at 25°C for 260 minutes. At this time this second culture was split into two and one aliquot incubated at 36°C (A, filled circles) and the other maintained at 25°C (A, filled triangles). The grey area of the graph represents the timing of the temperature shift. Panel A shows the septation profile of each portion of the culture as indicated on the graph. (B) Failure of EMTOC formation in HU-arrested cells lacking Cdc16 function at 36°C. EMTOC formation in HU-arrested cdc16.116 cells at 36°C (filled circles) was not significantly higher than in those that retained Cdc16 function at 25°C (filled triangles), despite septum formation in over 75% of the cells following Cdc16 inactivation (filled circles in A). The frequency of binucleate cells was scored to gauge the efficacy of the S phase arrest. A minority of cells had leaked through the checkpoint arrest and re-entered the cell cycle and executed mitosis (open triangles and circles), and many of these cells contained EMTOCs (filled triangles and circles). The EMTOCs that were seen are therefore likely to be due to leak through the cell cycle arrest rather than arising from the activation of the SIN by shift to 36°C.
