Thus, even genetically identical cells display significant variations in protein and mRNA abundance, and like a consequence, may additionally present differences within their signaling responses. Be trigger of this kind of heterogeneity in protein abundance, po pulation regular measurements are certainly not sufficient for investigating all or practically nothing responses. single cell meas urement procedures capable of capturing the dynamics of digital signal transduction are desired. Right here, we use movement cytometry to measure EGF induced, single cell ERK activation responses in the HEK293 cell population. We observe bimodal response distributions in cell populations which can be typically considered to indicate switch like behavior in single cells. Surprisingly, an ERK cascade signaling model incorporating detrimental feedback and also a graded, analog single cell dose response is shown for being consistent with all the observed population responses.
Our model examination suggests that this kind of a conversion of analog responses in single cells to digital responses at the population degree is due to protein abundance vari means, which gives rise to a broad distribution of ERK pathway activation thresholds and RasGTP levels. Thus, bimodal response distributions usually do not always imply digital single cell signaling. this kind of distributions can arise from the interplay in between protein selleck SP600125 expression noise and adverse suggestions mediated, analog single cell responses. Outcomes Analyses of ERK responses to EGF in individual cells and populations We implemented a flow cytometry primarily based phosphorylation assay to find out the kinetics and dose response of ERK activation by EGF in HEK293 cells. We display that population averages obtained from FCPA success corres pond very well to common Western blot measurements of activated ppERK amounts in cell populations.
Yet, the FCPA also reveals how person cells contribute to this collective population response. The maximize in indicate values of ppERK was dose dependent following two minutes of EGF stimulation, suggesting that analog signaling has occurred in personal cells. Having said that, a fraction of cells contain ppERK amounts just like individuals from the basal state. We refer to this attribute in the distribution selleck chemical as being a shoulder. Whilst the height of this shoulder decreases with expanding EGF dose, its pos ition remains 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 behavior. Higher EGF doses grow the fraction of cells with substantial ppERK on the cost from the ERK off popula tion. Hence, within a dose dependent manner, EGF increases the probability that a cell can have ERK turned on.