Ccw12p localizes to areas of active cell wall synthesis and ccw12Δ cells display bud lysis. (A) Cell lysis phenotypes. Wt (SEY6211) and ccw12Δ 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 ccw12Δ 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 ccw12Δ 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.
Ragni et al. BMC Genomics 2011 12:107 doi:10.1186/1471-2164-12-107