Optogenetic actuator – ERK biosensor circuits identify MAPK network nodes that shape ERK dynamics

Abstract

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.

LITOS: a versatile LED illumination tool for optogenetic stimulation

“…The western blot measurement of endogenous pERK closely follows the measurement of ERK kinase activity using the ERK-KTR biosensor. Consistent with previous observations 5 , 16 , both pERK and ERK-KTR signals displayed a steep increase in ERK activity directly after light stimulation, followed by slower adaptation, that lead to pre-stimulation ERK activity levels in appropriately 20 min. The ERK-KTR signal (peak at 6 min) was slightly delayed compared to the pERK signal (peak at 3 min) (Fig.…”

“…5 d). This is consistent with the finding that a single blue light pulse switches optoFGFR on for approximately 5–10 min 5 , thus not allowing ERK to deactivate if optoFGFR is reactivated every 2 min. In contrast, low frequency light pulses evoked successive, discrete ERK pulses that all displayed adaptation to the baseline until a new light input was applied.…”

“…This was also apparent at later time points. In response to increasing light input, optoFGFR led to a transition from transient to sustained ERK dynamics, as previously observed 5 , while optoRaf always displayed adaptive ERK dynamics. The different ERK dynamics induced by optoFGFR and optoRaf inputs could either reflect different mechanisms of actuation or a difference in the ability of the two actuators to activate ERK from different levels within the MAPK network: optoFGFR triggers the activation of a full MAPK network downstream of the receptor, whereas optoRaf only triggers the activation of an isolated MEK/ERK network.…”

“…LITOS provided the time resolution to capture a transient decrease in ERK-KTR phosphorylation (Fig. 5 b,d) that we have previously shown to result from optoFGFR-calcineurin activation 5 . Altogether, these results show that LITOS is a highly flexible optogenetic device that allows one to perform complex experiments to study the dynamics of the MAPK pathway.…”

“…When combined with spectrally compatible biosensors in a fluorescence microscope, this setup can both control the cellular input using the optogenetic actuator and record the output dynamics using the biosensor 4 . This has proven to be a very powerful approach to study signaling dynamics 5 – 7 . Unfortunately, the field-of-view of the microscope’s lens system limits the number of cells that receive the light stimulation.…”