Pheromone response pathway.(A) The wild-type pheromone signaling pathway. The inactive pathway is shown on the left, with the intact heterotrimeric G protein complex (Gpa1p, Ste4p, and Ste18p and the inactive MAPK cascade (Ste11p, Ste7p, and Fus3p, and the Ste5p scaffold). This leaves the transcription factor (Ste12p) unphosphorylated, leading to low transcription from P_FUS1. On the right is the active pathway, in which pheromone binding (blue circle) to the transmembrane receptor drives dissociation of the heterotrimeric G protein. Free Ste4p associates with the Ste20p kinase and the Ste5p scaffold to initiate the phosphorylation cascade resulting in Fus3p activation. Active Fus3p phosphorylates Ste12p, which in turn activates transcription from P_FUS1. (B) A schematic of the bistable pheromone response with positive feedback. A dominant active protein (the black square) is induced by pheromone treatment and, once expressed, maintains the activity of the MAPK cascade even after pheromone removal. Addition of Shokat’s Inhibitor (SI, red octagon) blocks the pheromone response downstream of the positive feedback, breaking the feedback loop and resetting the cell to its uninduced state. Note that either the inactive (top) or active (bottom) state can persist in untreated cells, depending on their history.

Effects of positive feedback on the pheromone response.(A) Diagram of the transient induction experiment used to test for bistability. (B) A strain with no positive feedback was treated with the timecourse in (A) and samples were measured by flow cytometry. The histogram shows the population distribution of YFP fluorescence. (C) As (B) in a strain with P_FUS1-STE11Δ N positive feedback. (D) As (B) in a strain with P_FUS1-STE4 positive feedback. (E) Time-lapse videomicroscopy of cells with no positive feedback and with P_FUS1-STE11Δ N. Cells were immobilized in a flow chamber and exposed to pheromone for 2 hours, then grown 6 hours in the absence of pheromone. Grey-scale DIC images are overlayed with images of YFP fluorescence, which is rendered in green. (F) Diagram of the transient inhibition experiment used to distinguish constitutive activity from bistability. (G) A strain with no positive feedback was treated with the timecourse in (F) and samples were measured by flow cytometry. The histogram shows the population distribution of YFP fluorescence, which should be directly comparable to fluorescence levels in (B)-(D). (H) As (G) in a strain with P_FUS1-STE11ΔN positive feedback. (I) As (G) in a strain with P_FUS1-STE4 positive feedback.

Positive feedback with low basal expression causes bistability.(A) P_FUS1 alleles were fused to stable yEVenus and median fluorescence was measured by flow cytometry in uninduced samples and in those treated with saturating (6 μM) pheromone for 2 hours. The “none” sample had no fluorescent reporter and shows the level of background cellular fluorescence. (B) Wildtype cells as well as P_FUS1J1-STE11ΔN integrants, with the indicated number of copies (measured by Southern blotting), were transiently induced with pheromone. Median YFP fluorescence was measured by flow cytometry. (C) As (B), but positive feedback was provided by P_FUS1J5-STE5-CPR_RAS2.

Bimodality from STE4-mediated positive feedback.(A)-(F) Wild-type cells as well as various P_FUS1-STE4 integrants, whose copy number had been mea- sured by Southern blotting, were transiently induced with pheromone. The strength of positive feedback, corresponding to induced expression levels relative to a single wild-type P_FUS1 promoter, is given along with the promoter allele and copy number in parentheses. A uniform threshold between the active and inactive states is shown as a vertical cyan line on each histogram. (G) The threshold in (A)-(F) was used to find the fraction of active cells 7 hours after removing pheromone from each strain. Median induced expression was computed by multiplying the induced fluorescence for a promoter measured in 3(A) by the number of copies in a cell. (H)-(J) Population histograms of fluorescence from a stochastic simulation of pheromone induction and recovery. The simulation was repeated with different strengths of positive feedback (none, weak, or strong).

Partial induction and inhibition of the bistable pheromone response.(A) Population distributions from stochastic simulations of pheromone induction and subsequent pathway inhibition. Simulations were performed with different levels of inhibition (shown on the x axis) and the relative frequency of different fluorescence intensities (on the y axis) are shown by color density. (B) Cells with 3×P_FUS1J2-STE4 were induced with pheromone for 1.5 hours, then split into cultures with different levels of SI and grown for 14 hours. The population distribution of fluorescence in each sample was measured by flow cytometry and used to construct a density plot as in (A). Four individual histograms are shown on the right at the SI concentrations indicated by the corresponding colored arrows. (C) Cells with 3×P_FUS1J2-STE4 and controls without feedback were transiently induced with low levels of pheromone. The population distribution of YFP fluorescence was measured by flow cytometry at the end of pheromone induction and 5 hours after removing pheromone.

Galactose induction of the active stable state.(A) Cells with gal1 Δ gal10Δ P_ACT1-GAL3 P_GAL1-STE4, without positive feedback, were grown in YEP ra3nose, induced with 160 μM galactose, and transferred to YEP dextrose to inactivate P_GAL1. Population distributions of fluorescence were measured by flow cytometry before galactose addition, at the end of galactose treatment, and 8.5 hours after removing galactose. (B) As (A), in cells with positive feedback from 3×P_FUS1J2-STE4.