Fig 1.
Pheromone concentration-dependent Ste50p shmoo tip localization ste50Δ strain, with or without Ste50-GFP on a plasmid, was treated with α-factor and imaged using epifluorescence and DIC microscopy.
(A) Response of STE50 as indicated after 4h treatment with 2μM α-factor. (B) Ste50p patch localization at the shmoo tip at the indicated concentrations of α-factor for the indicated time. (C) Number of cells with localized Ste50p polarity patches at the shmoo tip at the indicated pheromone concentrations and time (n≥100 cells, N = 3). (D) Quantified polarity patches of Ste50p at the shmoo tip with respect to the cytoplasmic amount at indicated pheromone concentrations and time (see text); N = 2: 2μM 1h (mean = 0.01, SD±0.0064; n = 35), 2μM 2h (mean = 0.035, SD±0.0224; n = 75), 4μM 1h (mean = 0.028, SD±0.0163; n = 78), 4μM 2h (mean = 0.015, SD±0.0091; n = 8); ****, p<0.0001; ns = not significant; p>0.05; one-way Anova with Tukey’s multiple comparisons. Bar represents 5μm.
Fig 2.
Cortical Ste50p patches are incipient sites for polarization.
(A) Ste50-GFP plasmid bearing yeast cells were treated with α-factor and imaged using epifluorescence and DIC still microscopy, showing cortical Ste50p foci (arrow), detectable after ~3h pheromone treatment. (B-D) Single-cell analysis by time-lapse microscopy showing nucleation of the Ste50p on the cell cortex (white arrowheads, S1–S3 Movies) before polarization for a 2nd and 1st shmoo respectively (red arrowheads indicates start of polarization), and mobile patch that stabilizes before 1st shmoo appearance (C, white arrowheads), (E) ste50p mutant that fails to form patch, R296G, has a delayed polarization (red arrowhead indicates start of polarization), time indicated, cell circumference marked in DIC image to show the start of polarization, bar 5μm (S6 Movie). (F) Percentage polarized cells at indicated times for the WT (n>200, N = 3) and the mutant (n>100, N = 3).
Fig 3.
Ste50p localizes to the shmoo tip until shmoo maturation.
Ste50p-GFP-expressing cells were treated with 2μM α-factor and imaged using time-lapse microscopy. (A-C) Single cells with indicated intervals in minutes showing Ste50p patches at the shmoo tip (white arrows); the white asterisk (*) indicates signal peak; the red arrow indicates receding of Ste50p from the shmoo (S7–S9 Movies). (Ai-Ci) Quantified GFP fluorescence at the shmoo tip (~0.2–0.3μm2 area at the tip; see S2 File) showing a peak Ste50p around 130–200 min after pheromone treatment that starts receding around 200 min (blue broken lines). (Aii-Cii) Correlation between the disappearance of Ste50p from the shmoo and the termination of polarized shmoo growth (orange broken lines indicate start of shmoo growth inhibition; see text); the major cell axis (μm) versus time (min) have been plotted (4 point rolling average); both shmoo Ste50p and growth normalized to the maximum values for each cell; linear regression fit to the linear subset of data (length when shmoo extension started until when it stopped) showing the rate of shmoo growth, slope = 0.016, 0.021 and 0.023, respectively. (Di-Fi) Cytoplasmic Ste50p retracted from the 1st shmoo and relocalized to the 2nd shmoo (pronounced around 270–320 min; white arrow indicates 1st shmoo) (Dii-Fii). Scale bar represents 5μm.
Fig 4.
Increased Ste50p expression synchronizes with shmoo polarization.
Yeast cells expressing Ste50p-GFP were treated with 2μM α-factor and followed by time-lapse microscopy for at least 8hrs. (A) Shmoo-forming cells show an increase in Ste50p expression during polarization (arrow as indicated) (S10–S12 Movies), in contrast to cells that do not form shmoo (S13–S15 Movies) (B); time as indicated, bar 5μm. (C) Quantified cellular GFP fold changes in single cells across time, showing an unimodal expression peak at ~150–270 min for single shmoo forming cells (orange, C, arrow indicates start of polarization); n = 10; N = 3, and no significant changes in no shmoo forming cells (blue, C); n-10; N = 3.; shadings are standard deviations. (D) Correlation between Ste50p expression and shmoo growth of cell in A; Pearson r = 0.9800, p<0.001. (E) Bimodal and linear increase of Ste50p expression in the cases of multiple shmoo (see text, arrows indicating the onset of 1st shmoo and 2nd shmoo).
Fig 5.
Phenotypes of yeast cells after pheromone treatment.
Part I. Yeast cells were exposed to 2μM α-factor and phenotypic changes were examined by time-lapse microscopy for eight hours. (A-G) Representative DIC and fluorescence images of each phenotypic category found, as indicated, polarization time indicated by arrow, M = mother and D = daughter, sl. = slight, cell circumference in white to show directional growth or polarization (see text), time as indicated, bar 5μm, (S16–S22 Movies). (H-N) Quantified fold induction of the Ste50p fluorescence over time for the corresponding cells in (A-G). Arrow indicates beginning of detectable polarized growth. Part II. Polarization correlates with high levels of Ste50p at G1. Yeast cells were treated with 2μM α-factor and followed by time-lapse microscopy for at least 8hrs. (A) Percentage of different phenotypes observed (n = 211 cells, N = 3). (B) Fluorescence quantified at G1 immediately after M/D separation for the indicated phenotypic groups, showing significant difference in Ste50p level among the groups; higher level correlates with polarization in the M/D shmoo group (see text), N = 2: M/D shmoo (mean = 6995, SD±5824; n = 108), M/D no shmoo (mean = 1440, SD±1142; n = 42), slight shmoo (mean = 2109, SD±1202; n = 29), replicating (mean = 6791, SD±4070; n = 17); one-way Anova followed by Tukey’s multiple comparisons. Lower panel: DIC images of the corresponding phenotypes, bar 5μm. (C) Quantified fold changes in fluorescence in mother and daughter from time-lapse movies; N = 2, n = 14; shadings are error bars for standard deviations and solid lines are means as indicated. (D) Ste50p expression at G1 for contrasting phenotypic M/D as indicated; N = 2, n = 15 in each case; unpaired Student’s t-test, ns, p = 0.7685; shmoo (mean intensity = 2759, SD±1262); no shmoo (mean intensity = 2592, SD±1763). Lower panel: DIC images of the phenotypes. (E) Time-lapse frames as indicated showing mobile Ste50p-GFP foci on the cell perimeter and their stabilization, arrows pointing at patches (S23 Movie). (F) Formation of Ste50p foci in CCA cells after prolonged pheromone treatment, but unable to polarize, time indicated (cells 1–4). Bar represents 5μm. (G) Quantified GFP for CCA cells at 0hr (initial) and 8hrs (final) of pheromone exposure, forming foci but unable to polarize; n = 9, in comparison to M or D + shmoo in (D), one-way Anova, followed by Tukey’s multiple comparison test, mean intensities, initial = 388, SD±155; final = 586, SD±164; M or D + shmoo = 2759, SD±1262. Y-axis in log scale to expand the values. (H) Representative heat map of Ste50p fluorescence versus percentage of shmoo forming cells.