Stat5 Signaling Specifies Basal versus Stress Erythropoietic Responses through Distinct Binary and Graded Dynamic Modalities
Figure 2
Measurement of binary and graded signaling responses by flow cytometry.
(A) Dose/response curves for graded and binary (switch-like) responses. The grey line shows an idealized switch response. An infinitesimally small change in stimulus such as Epo results in an increase of response from 0% to 100%. The black curve shows a graded response with nH = 1. An 81-fold increase in stimulus is required to shift the response from 10% to 90% of maximum. The green curve shows a steep, switch-like or binary dose/response curve (nH = 3). Only a 4-fold change in stimulus is required in order to generate a similar increase in response. (B) Contrasting graded and binary signaling responses: three hypothetical examples. In a graded signaling response (left panels), increasing Epo concentration results in a graded increase in p-Stat5 in individual cells, represented by increasingly darker shades of grey. Simulations of the corresponding flow-cytometric profiles show that increasing Epo concentration causes a gradual shift of the p-Stat5 fluorescence histogram to the right. A plot of the total p-Stat5 median fluorescence intensity (MFI) versus Epo concentration has Michaelian kinetics with a Hill coefficient (nH) of 1 (lower left panel, please note a log scale was used for the x-axis). In binary signaling (middle and right panels), the p-Stat5 signal in individual cells can only assume two states, either “off” (white) or “on” (black), but intermediate states (shades of grey) are unstable. Two distinct cases of binary signaling are illustrated that differ in their threshold responses. In “variable threshold” (middle panels), the threshold at which Epo causes a cell to switch from “off” to “on” varies substantially between cells of the population. Consequently, increasing Epo concentration causes a gradual increase in the number of cells that are p-Stat5+ (“on”). The simulated flow-cytometric histograms at each Epo concentration (in color) are each the sum of two histograms, corresponding to cells that are “off” (light grey histograms) and cells that are “on” (black histograms). The median fluorescence of the “on” and “off” histograms each remain unaffected by Epo concentration, but as Epo increases, the number of cells in the “on” histogram, reflected by its height, increases, with a corresponding decrease in the height of the “off” histogram. Although individual cells have binary responses, there is a graded increase in the MFI of the colored histograms representing the whole population. Therefore, a plot of total p-Stat5 MFI versus Epo concentration shows a graded response (here, the Hill coefficient is nH = 1). In the case of cells with binary responses and similar threshold (right panels), cells switch from “off” to “on” within a much narrower Epo concentration range. Consequently, the response of the whole population reflects the response of individual cells more closely. Flow-cytometric histograms representing the population tend to be in one of two principal positions, corresponding to the “on” or to the “off” states. A plot of total p-Stat5 MFI versus Epo concentration is steep, reflected by a high Hill coefficient (nH>1).