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Fig. 4. Don3 and Cdc42 activities are required for assembly of the cytokinetic contractile actomyosin ring. (A-O) don3-as cells expressing Cdc15-GFP (A-E) were stained with 10 µg/ml Calcofluor White (F-J). Merged images of the septation zone are shown as magnifications (K-O), GFP (green) and Calcofluor White (red). Different stages of actomyosin ring assembly and septum formation are shown. (A,F,K) Cdc15-GFP assembles at the neck of the mother bud. (B,G,L) Cdc15-GFP accumulates in the actomyosin ring during primary septum formation. (C,H,M) After completion of the first septum Cdc15-GFP disappeared. (D,I,N) A second actomyosin ring is visible before secondary septum intitiation. (E,J,O) During secondary septum formation Cdc15-GFP colocalises with the inward growing septum. (P-T) don3-as cells expressing Cdc15-GFP were grown in the presence of 1 µM NA-PP1 and released from inhibition after 8 hours. (P,S) In the presence of the inhibitor, Cdc15-GFP was detectable only in the primary septa of the newly formed buds (arrows). (Q,T) Upon inhibitor release, Cdc15-GFP accumulation in all cells at the site where the secondary septum was formed (arrow heads in the magnification). (R,U) Cdc15-GFP expressed in Pcrg::cdc42 cells. Under repressing conditions Cdc15-GFP behaves similar as shown in panel M. After shifting the cells to arabinose-containing medium cells start to build the secondary septum. In cells whose the second septum is stained with Calcofluor-White (U), Cdc15-GFP can be observed there, too (R) (arrowheads in the magnification).
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