Bem1 and GEF localization are not required for symmetry breaking. (A) Cells with indicated genotypes were plated on YPD media and grown at 23°C for 3 d. Cells were grown overnight in liquid culture and then diluted to an OD of 1. This and a series of 10-fold dilution were spotted left to right. (B) Polarization of GFP-Cdc42 wild type, axl2Δ rax1Δ, and rsr1Δ cells after release from G1 arrest into media containing DMSO or LatA. The percentage of cells with polarized GFP-Cdc42 was determined at different time points (given in minutes) after release. The plots show means from averaging three experiments, and error bars correspond to SEMs. More than 80 cells were counted per time point per experiment. (C) Maximum projections of representative polarized cells from A. (D) Representative kymographs of polarizing GFP-Cdc42 in axl2Δ rax1Δ and rsr1Δ cells in LatA. Note the unstable polar cap in axl2Δ rax1Δ relative to that in rsr1Δ. See also Fig. S1 and Videos 1 and 2. (E) Quantification of cap duration in LatA of polarized GFP-Cdc42 in axl2Δ rax1Δ and rsr1Δ cells from videos such as the ones shown in D and E. The maximum intensity of the polar caps was measured and plotted over time and was fitted to Gaussian curves. The duration of the cap is reported as the full width half maximum of the Gaussian fit. Boxes, SEM; whiskers, SD; horizontal lines, median values. P < 10−6. For each dataset, n ≥ 20 cells taken from three experiments. (F and G) Time-lapse imaging of Cdc24-GFP in mid–log phase axl2Δ rax1Δ bem1Δ (F) and rsr1Δ bem1Δ (G) cells. Notice the lack of Cdc24 polarization in G despite successful cell polarization and budding compared with strong Cdc24 polarization in F. Arrows point to incipient bud sites. White outlines indicate the perimeter of the budded cell. See also Videos 3 and 4. (H) Kymographs of polarizing cells from F and G. Shown at the bottom are intensity profiles taken from the time point immediately before bud emergence, indicated in the kymograph by arrows. (I) Quantification of polarization of Cdc24-GFP in axl2Δ rax1Δ bem1Δ and rsr1Δ bem1Δ cells before bud emergence. Intensity at the emergent bud site is normalized to cortical intensity of the rear half of the cell. Three data points are shown for each cell, corresponding to time points 2, 4, and 6 min before bud emergence. n = 11 cells for axl2Δ rax1Δ bem1Δ and 17 cells for rsr1Δ bem1Δ. Boxes, SEM; whiskers, SD; horizontal lines, median values. P < 10−8. (J) Maximum projections of representative cells with polarized Cdc24-GFP in axl2Δ rax1Δ and rsr1Δ cells in LatA or DMSO. (K) Quantification of polarization of Cdc24-GFP in axl2Δ rax1Δ and rsr1Δ cells in LatA 50 min after pheromone arrest release from maximum projections of z-stack images. Cells with polarized GFP-Cdc24 were identified, and cortex masks were generated from a separate fluorescent channel (mCherry-Cdc42; see Materials and methods). Mean intensity of the cortex within the mask was determined in 10° increments centered on the polar cap and normalized to the rear half as in I. Normalized profiles were averaged over n > 18 cells. Error bars correspond to SEMs. For comparison of values at 0°, P < 0.001. Bars, 5 µm.
