Cell death events continuously challenge epithelial barrier function, yet are crucial to eliminate old or critically damaged cells. How such apoptotic events are spatio-temporally organized to maintain epithelial homeostasis remains unclear. We observe waves of Extracellular Signal-Regulated Kinase (ERK) and AKT serine/threonine kinase (Akt) activity pulses that originate from apoptotic cells and propagate radially to healthy surrounding cells. Such a propagation requires Epidermal Growth Factor Receptor (EGFR) and matrix metalloproteinase (MMP) signaling. At the single-cell level, ERK/Akt waves act as spatial survival signals that locally protect cells in the vicinity of the epithelial injury from apoptosis for a period of 3-4 hours. At the cell population level, ERK/Akt waves maintain epithelial homeostasis (EH) in response to mild or intense insults. Disruption of this spatial signaling system results in the inability of a model epithelial tissue to ensure barrier function in response to cellular stress.
Combining single‐cell measurements of ERK activity dynamics with perturbations provides insights into the MAPK network topology. We built circuits consisting of an optogenetic actuator to activate MAPK signaling and an ERK biosensor to measure single‐cell ERK dynamics. This allowed us to conduct RNAi screens to investigate the role of 50 MAPK proteins in ERK dynamics. We found that the MAPK network is robust against most node perturbations. We observed that the ERK‐RAF and the ERK‐RSK2‐SOS negative feedback operate simultaneously to regulate ERK dynamics. Bypassing the RSK2‐mediated feedback, either by direct optogenetic activation of RAS, or by RSK2 perturbation, sensitized ERK dynamics to further perturbations. Similarly, targeting this feedback in a human ErbB2‐dependent oncogenic signaling model increased the efficiency of a MEK inhibitor. The RSK2‐mediated feedback is thus important for the ability of the MAPK network to produce consistent ERK outputs, and its perturbation can enhance the efficiency of MAPK inhibitors.
Measurements of single-cell ERK activity dynamics provide unique insights in the MAPK network topology. We built genetic circuits consisting of optogenetic actuators activating ERK from different nodes within the MAPK network together with an ERK biosensor to measure single-cell ERK dynamics. Evaluating ERK dynamics induced by different temporal optogenetic inputs, in response to a large number of perturbations, shows that the MAPK network is robust to downregulation of most of its nodes. This robustness emerges in part because of the ERK-RSK2-SOS negative feedback. Bypassing this feedback, by direct activation of the RAS/RAF/MEK/ERK submodule, or by RSK2 perturbation, breaks MAPK network robustness. Targeting the RSK2-mediated feedback in a ErbB2-dependent oncogenic signaling model greatly sensitizes ERK to MEK inhibition, allowing efficient ERK activity shutdown within a cell population. Thus, the RSK2-mediated negative feedback is a weak node of the MAPK network whose perturbation enables potent inhibition of ERK.
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