Complex, time-varying responses have been observed widely in cell signaling, but how specific dynamics are generated or regulated is largely unknown. One major obstacle has been that high-throughput screens for identifying pathway components are typically incompatible with the live-cell assays used to monitor dynamics. Here, we address this challenge by performing a drug screen for altered Erk signaling dynamics in primary mouse keratinocytes. We screened a library of 429 kinase inhibitors, monitoring Erk activity over 5 h in more than 80,000 single live cells. The screen revealed both known and uncharacterized modulators of Erk dynamics, including inhibitors of non-EGFR receptor tyrosine kinases (RTKs) that increased Erk pulse frequency and overall activity. Using drug treatment and direct optogenetic control, we demonstrate that drug-induced changes to Erk dynamics alter the conditions under which cells proliferate. Our work opens the door to highthroughput screens using live-cell biosensors and reveals that cell proliferation integrates information from Erk dynamics as well as additional permissive cues. Ras | Erk | MAP kinase | cell signaling | dynamics | drug screen

Figure S1, related to Figure 1. Data processing pipeline for microscopy data of Erk activity. (A,C,E) Schematics of each component of the data processing pipeline that takes in multichannel TIFF image stacks of H2B-RFP and KTR-BFP as inputs, and returns quantitative data about single-cell Erk dynamics. (B) Images of H2B and KTR images where nuclei have been automatically identified (red circles). Nuclei are excluded that only express one of the two components (yellow arrows). (D) Filtering and processing of Erk activity trajectories. Representative trajectory of nuclear KTR fluorescence from a single keratinocyte over time (upper panel) is h-minima transformed, inverted, and subjected to peak-finding (lower panel). Erk activity pulse timing (blue triangles), pulse prominence (red vertical bars), and pulse width (pink horizontal bars) are obtained for each trajectory. (F) For quantifying well-averaged Erk activity, cytosolic regions are computed by a combination of dilation and erosion operations to define regions outside of nuclei. The ratio of the mean cytosolic and nuclear pixel intensities is used as a quantitative metric of the overall Erk activity offset at each timepoint. (G) Quantifying Erk activity offset for wells of keratinocytes stimulated with EGF demonstrates that a pulsatile Erk response can be sensitively detected for EGF concentrations as low as 50 pg/mL.