Department of Molecular Biology, Princeton University, Princeton, NJ 08544.
Department of Molecular Biology, Princeton University, Princeton, NJ 08544.
Cell Syst. 2020 Mar 25;10(3):240-253.e6. doi: 10.1016/j.cels.2020.02.005. Epub 2020 Mar 18.
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.
细胞信号中广泛存在着复杂且时变的反应，但特定动力学如何产生或调控在很大程度上仍是未知的。其中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检测不兼容。在这里，我们通过筛选 429 种激酶抑制剂文库并在超过 80000 个单个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。我们的筛选揭示了 Erk 动力学的已知和未表征调节剂，包括非表皮生长因子受体 (EGFR) 受体酪氨酸激酶 (RTK) 的抑制剂，它们增加了 Erk 脉冲频率和整体活性。通过药物处理和直接光遗传学控制，我们证明药物诱导的 Erk 动力学变化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进行高通量筛选开辟了道路，并表明细胞增殖整合了来自 Erk 动力学以及其他许可线索的信息。
