Thus, even genetically identical cells present considerable variations in protein and mRNA abundance, and as a end result, can also show differences within their signaling responses. Be induce of this kind of heterogeneity in protein abundance, po pulation typical measurements are usually not adequate for investigating all or absolutely nothing responses. single cell meas urement procedures capable of capturing the dynamics of digital signal transduction are necessary. Right here, we use movement cytometry to measure EGF induced, single cell ERK activation responses within a HEK293 cell population. We observe bimodal response distributions in cell populations that are commonly considered to indicate switch like habits in single cells. Surprisingly, an ERK cascade signaling model incorporating negative feedback in addition to a graded, analog single cell dose response is shown to be consistent using the observed population responses.
Our model examination suggests that this kind of a conversion of analog responses in single cells to digital responses in the population level is due to protein abundance vari skill, which provides rise to a broad distribution of ERK pathway activation thresholds and RasGTP levels. Therefore, bimodal response distributions really don’t necessarily imply digital single cell signaling. such distributions can come up in the interplay concerning protein inhibitor LY2157299 expression noise and negative feedback mediated, analog single cell responses. Results Analyses of ERK responses to EGF in individual cells and populations We applied a movement cytometry based mostly phosphorylation assay to determine the kinetics and dose response of ERK activation by EGF in HEK293 cells. We present that population averages obtained from FCPA effects corres pond well to traditional Western blot measurements of activated ppERK levels in cell populations.
However, the FCPA also reveals how person cells contribute to this collective population response. The maximize in suggest values of ppERK was dose dependent following two minutes of EGF stimulation, suggesting that analog signaling has occurred in personal cells. However, a fraction of cells have ppERK amounts similar to these from the basal state. We refer to this attribute from the distribution read what he said like a shoulder. While the height of this shoulder decreases with growing EGF dose, its pos ition remains unchanged, indicating a dose dependent fraction of cells failing to activate ERK. At 5 minutes after EGF stimulation, the ppERK distribution is unam biguously bimodal, implying digital on off conduct. Higher EGF doses raise the fraction of cells with substantial ppERK in the cost from the ERK off popula tion. Thus, inside a dose dependent manner, EGF increases the probability that a cell can have ERK turned on.