(A)CHS2-GFP (YASD819) and CHS2-GFP GAL-SPA2 (YMF1660) strains were arrested in G1 phase at 24°C in YPRaff and then shifted to YPGal at 24°C to induce expression of Spa2. Subsequently, cells were released from G1 block to allow progression through the cell cycle. Samples were taken at the indicated times to determine the proportion of binucleate cells (i) and the percentage of cells with Chs2 rings at the cleavage site (ii) and (iii). (B) CHS2-GFP SPC42-EQFP (YAD380) strain together with CHS2-GFP GAL-SPA2 (YMF1660) were grown as in Fig 8A. 75 minutes after the release from G1 block cells were shifted to SC medium before being placed on the time-lapse slide to examine the localisation of Chs2 every 2 minutes as cells completed cell division at 24°C (see Methods for details). A z-stack of images was gathered. A two-dimensional projection of the three-dimensional data is shown. Scale bar: 2 ?m. The grey and black circles denote the timing of the actomyosin ring contraction. (C) chs3? control (YMF505) and chs3? GAL-SPA2 (YMF1534) cells were grown in YPRaff medium at 24°C and synchronised in G1 with alpha factor. Subsequently, cells were released in YPGal for 135 minutes from G1 block in the presence of calcofluor to visualise primary septum deposition. Samples were taken at the indicated times to determine the proportion of binucleate cells (i) and 100 cells with primary septum for each sample were examined. Examples of live cells grown with calcofluor are shown in (ii). Scale bar: 2 ?m. The relative signal intensity of primary septum was measured for 100 cells and compared with control cells, whose signal intensity was set to 100% (iii).

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