Fig. 6. bem1^SH3-1 mutant cells have a mild polarity defect and the polarized localization of Sec15-GFP is disrupted in bem1^SH3-1 mutant cells. (A) The percentage of yeast mother cells with oval-shaped wild-type versus round morphology was determined as described in the Materials and Methods for wild-type (Wt) and bem1^SH3-1 mutant (bem1-SH3-1) cells. For each dataset, the yeast strains were grown in parallel and DIC images were taken to measure ratios (n) of length to width. The average and s.d. were calculated from two independent datasets, and at least 200 cells were measured per strain and dataset. (B) bem1^SH3-1 mutant (bem1-SH3-1; NY2568) and wild-type (NY2557) strains containing Sec15-GFP were grown overnight at 25°C to early log phase and treated either with DMSO alone or with 200 µM latrunculin A (Lat-A). After 15 minutes of treatment, cells were rapidly fixed by methanol/acetone and mounted for microscopy. The localization was examined by direct fluorescence microscopy. Examples of cells with a small bud are marked with large arrows and those with a large bud are marked with smaller arrows. Unbudded cells are marked with arrowheads. (C) Wild-type (Wt) and bem1^SH3-1 (SH3-1) cells were categorized based upon their pattern of Sec15-GFP polarized localization – the presumptive bud, bud tip or the bud neck. The graph depicts the percentage of the population within each category. At least 200-300 cells were scored in each strain. (D) The localization of Sec15-GFP in synchronized wild-type and bem1^SH3-1 (bem1-SH3-1) cells released from G0 at 25°C in the absence or presence of Lat-A was examined at time points of 0 and 2 hours. (E) Quantification of GFP fluorescence, used to assess the percentage of cells that exhibited polarized Sec15-GFP in the presence or absence of Lat-A treatment. At least 200 cells were scored in each strain.