Fig. 3. Don3 and Cdc42 act independently to trigger secondary septum initiation. (A) Strain CB46 (don3-as; Pcrg::cdc42) was grown in glucose-containing medium, in which the Pcrg promoter is inactive (Cdc42, no activity). The cell clusters were washed and labelled with biotin. At the same time (0 h) Don3 activity was blocked by adding 1 µM NA-PP1 to the medium (Don3, no activity). Cdc42 expression was induced after 90 minutes by switching the carbon source of the NA-PP1-containing medium from glucose to arabinose (Cdc42, increasing activity). The fate of the cell clusters were monitored at the given time-points using fluorescence and DIC microscopy. Scale bars, 10 µm. (B) GFP-fusion of Cdc42 (upper panel) or Don3 (lower panel) expressed from the constitutive Etef-promotor in wild type cells (left panel), in the respective complementary deletion strain (central panel) and in don1 cells (right panel). Boxed areas are magnified, and arrowheads mark the localisation of GFP-Cdc42 at both septa in the wild-type or the primary septum in the mutant strains. Scale bars, 10 µm.