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 are typically incompatible with the live-cell assays used to monitor dynamics. Here, we address this challenge by screening a library of 429 kinase inhibitors and monitoring extracellular-regulated kinase (Erk) activity over 5 h in more than 80,000 single primary mouse keratinocytes. Our screen reveals both known and uncharacterized modulators of Erk dynamics, including inhibitors of non-epidermal growth factor receptor (EGFR) receptor tyrosine kinases (RTKs) that increase 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 high-throughput screens using live-cell biosensors and reveals that cell proliferation integrates information from Erk dynamics as well as additional permissive cues.

在细胞信号传导中已广泛观察到复杂，随时间变化的响应，但是如何产生或调节特定的动力学尚不清楚。一个主要障碍是高通量筛选通常与用于监测动力学的活细胞分析不兼容。在这里，我们通过筛选429种激酶抑制剂文库并在超过80,000个单原代小鼠角质形成细胞中监测细胞外调节激酶（Erk）活性超过5小时来解决这一挑战。我们的屏幕显示了Erk动力学的已知和未表征的调节剂，包括增加Erk脉冲频率和总体活性的非表皮生长因子受体（EGFR）受体酪氨酸激酶（RTK）抑制剂。通过药物治疗和直接光遗传学控制，我们证明了药物诱导的Erk动力学改变改变了细胞增殖的条件。我们的工作为使用活细胞生物传感器的高通量筛选打开了大门，并揭示了细胞增殖整合了来自Erk动力学信息以及其他允许的线索。