Ccw12p localizes to areas of active cell wall synthesis and ccw12 Δ cells display bud lysis. (A) Cell lysis phenotypes. Wt (SEY6211) and ccw12 Δ mutant (MEY12B) strains, exponentially growing in YPD (a, b) or YPD supplemented with 1 M sorbitol (c, d), were stained with the vital dye methylene blue to identify dead cells (details are described in Methods): (a) wt cells display a typical ellipsoidal shape; (b) mutant cells show a pronounced round morphology and lyse as small budded cells (16% of ccw12 Δ cells vs. 3% of wt cells); (c) mutant cells round morphology is partially reverted in presence of osmotic stabilization; (d) After hypotonic shock (transfer to YPD) lysis as small budded cell is observed(41% of ccw12 Δ cells vs. 8% of wt cells) and (e-f) cell lysis occurs after completion of cytokinesis as shown by DAPI staining (panel e and f represent the same cells that have been stained with methylene blue and DAPI). (B) Localization of Ccw12p during vegetative growth. To exclude artefacts due to over-expression of CCW12-GFP, Ccw12p-GFP is expressed from plasmid pCCW12-GFP in mutant MEY12B. (a) Ccw12p-GFP is enriched at sites of emerging buds. The arrow marks the site of bud emergence. When cells proceed in the cell cycle, Ccw12p-GFP is specifically enriched in small (b) and medium-sized (c) buds. (d) After cytokinesis Ccw12p-GFP marks the septum.