Therefore, even genetically identical cells present considerable variations in protein and mRNA abundance, and being a outcome, may additionally present distinctions within their signaling responses. Be lead to of this kind of heterogeneity in protein abundance, po pulation average measurements are usually not enough for investigating all or nothing at all responses. single cell meas urement strategies capable of capturing the dynamics of digital signal transduction are necessary. Here, we use flow cytometry to measure EGF induced, single cell ERK activation responses in a HEK293 cell population. We observe bimodal response distributions in cell populations which are ordinarily believed to indicate switch like behavior in single cells. Surprisingly, an ERK cascade signaling model incorporating damaging feedback along with a graded, analog single cell dose response is proven to become steady with all the observed population responses.
Our model evaluation suggests that such a conversion of analog responses in single cells to digital responses on the population degree is due to protein abundance vari potential, which offers rise to a broad distribution of ERK pathway activation thresholds and RasGTP ranges. Hence, bimodal response distributions don’t automatically imply digital single cell signaling. such distributions can come up from the interplay among protein explanation “” expression noise and negative feedback mediated, analog single cell responses. Results Analyses of ERK responses to EGF in individual cells and populations We made use of a flow cytometry primarily based 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 benefits corres pond well to standard Western blot measurements of activated ppERK amounts in cell populations.
However, the FCPA also reveals how personal cells contribute to this collective population response. The improve in imply values of ppERK was dose dependent soon after two minutes of EGF stimulation, suggesting that analog signaling has occurred in personal cells. However, a fraction of cells contain ppERK ranges similar to people of the basal state. We refer to this characteristic within the distribution selleck inhibitor as being a shoulder. Although the height of this shoulder decreases with improving EGF dose, its pos ition stays unchanged, indicating a dose dependent fraction of cells failing to activate ERK. At 5 minutes immediately after EGF stimulation, the ppERK distribution is unam biguously bimodal, implying digital on off habits. Increased EGF doses enhance the fraction of cells with higher ppERK on the expense in the ERK off popula tion. Hence, within a dose dependent method, EGF increases the probability that a cell could have ERK turned on.