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Fig. 6. Delay in CAR formation is dependent upon Cdc16, Mad2 and Zfs1. A and B: synchronisation by transient arrest in S phase using 12 mM hydroxyurea and release at 25°C (A) or 35.5°C (B). (C-E): (C-E) synchronisation by cdc25-22 temperature arrest and release. (A) Synchronised myo2-gc cdc16-116 cells were released in the presence (open symbols) or absence (filled symbols) of 100 µg/ml TBZ. The microtubule-depolymerisation-induced delay in the appearance of Myo2 rings (green circles) was also seen using this synchronisation technique (compare open green circles in A with the closed ones), as expected, no binucleate cells (blue diamonds) were observed in TBZ. (B) In the absence of functional Cdc16 there was no delay in the appearance of single septa [compare filled (DMSO) and open (TBZ) black squares] or multiple septa [filled (DMSO) and open (TBZ) red squares]. Synchronised zfs1{Delta} myo2-gc cdc25-22 (D) or mad2{Delta} myo2-gc cdc25-22 (E) cells were released into DMSO (filled symbols), 100 µg/ml TBZ (open symbols) or 25 µg/ml MBC (yellow filled symbols). In contrast to control myo2-gc cdc25-22 cells (C), no delay was seen.