Figure 6. Direct optogenetic control over Erk dynamics reveals drug-like proliferative responses.

(A) Schematic of our strategy to implement direct optogenetic control in keratinocytes, which requires overriding their endogenous dynamics and establishing light-controlled Erk activity pulses. Endogenous Erk dynamics are first eliminated by treating cells with an EGFR inhibitor, then the OptoSOS system is used to apply light-dependent optogenetic inputs directly to Ras. (B) Light pulses designed to mimic each class of drug-induced Erk dynamics: Class 1-like suppression of Erk activity, Class 2-like low-frequency pulses, or Class 3-like high-frequency pulses. (C) Representative single-cell Erk activity traces from keratinocytes stimulated as in A. Cells’ endogenous dynamics were imaged for 1 h, then the EGFR inhibitor lapatinib was added to extinguish endogenous Erk signaling, after which blue light was toggled on and off every 15 min. (D-E) Proliferation in response to optogenetic Erk stimulation mimicking Class 1, 2, and 3 compounds (in D) or comparing pulsatile to continuous light stimulation (in E). Gray bars indicate control conditions: continuous GF-free media or complete media, both without lapatinib or light stimulation. Blue bars indicate OptoSOS keratinocytes treated with the EGFR inhibitor lapatinib, and then subjected to repeated blue light pulses at the schedules indicated. Green, purple and orange shading indicate stimuli that drive Erk dynamics similar to Class 1, 2, and 3 compounds, respectively. For all conditions, S phase entry was assessed after 22 h. Biological replicates (white circles) were assessed from flow cytometry DNA content distributions of at least 20,000 cells each. Statistics are derived using a one-sided t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).