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Engineered light-dependent switches provide uniquely powerful opportunities to investigate and control cell regulatory mechanisms. Existing tools offer high spatiotemporal resolution, reversibility and repeatability. Cellular optogenetics applications remain limited with diffusible targets as the response of the actuator is difficult to independently validate. Blue light levels commonly needed for actuation can be cytotoxic, precluding long-term experiments. We describe a simple approach overcoming these obstacles. Resonance energy transfer can be used to constitutively or dynamically modulate actuation sensitivity. This simultaneously offers on-line monitoring of light-dependent switching and precise quantification of activation-relaxation properties in intact living cells. Applying this approach to different LOV2-based switches reveals that flanking sequences can lead to relaxation times up to 11-fold faster than anticipated. In situ-measured parameter values guide the design of target-inhibiting actuation trains with minimal blue-light exposure, and context-based optimisation can increase sensitivity and experimental throughput a further 10-fold without loss of temporal precision.

Protein tyrosine phosphatases regulate a myriad of essential subcellular signaling events, yet they remain difficult to study in their native biophysical context. Here we develop a minimally disruptive optical approach to control protein tyrosine phosphatase 1B (PTP1B)-an important regulator of receptor tyrosine kinases and a therapeutic target for the treatment of diabetes, obesity, and cancer-and we use that approach to probe the intracellular function of this enzyme. Our conservative architecture for photocontrol, which consists of a protein-based light switch fused to an allosteric regulatory element, preserves the native structure, activity, and subcellular localization of PTP1B, affords changes in activity that match those elicited by post-translational modifications inside the cell, and permits experimental analyses of the molecular basis of optical modulation. Findings indicate, most strikingly, that small changes in the activity of PTP1B can cause large shifts in the phosphorylation states of its regulatory targets.

Engineering light-sensitive protein regulators has been a tremendous multidisciplinary challenge. Optogenetic regulators of MAPKs, central nodes of cellular regulation, have not previously been described. Here we present OptoJNKi, a light-regulated JNK inhibitor based on the AsLOV2 light-sensor domain using the ubiquitous FMN chromophore. OptoJNKi gene-transfer allows optogenetic applications, whereas protein delivery allows optopharmacology. Development of OptoJNKi suggests a design principle for other optically regulated inhibitors. From this, we generate Optop38i, which inhibits p38MAPK in intact illuminated cells. Neurons are known for interpreting temporally-encoded inputs via interplay between ion channels, membrane potential and intracellular calcium. However, the consequences of temporal variation of JNK-regulating trophic inputs, potentially resulting from synaptic activity and reversible cellular protrusions, on downstream targets are unknown. Using OptoJNKi, we reveal maximal regulation of c-Jun transactivation can occur at unexpectedly slow periodicities of inhibition depending on the inhibitor's subcellular location. This provides evidence for resonance in metazoan JNK-signalling circuits.

The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution. The ability to precisely and reversibly control nuclear translocation would, therefore, allow dissecting and engineering cellular networks. Here we develop a genetically encoded, light-inducible nuclear localization signal (LINuS) based on the LOV2 domain of Avena sativa phototropin 1. LINuS is a small, versatile tag, customizable for different proteins and cell types. LINuS-mediated nuclear import is fast and reversible, and can be tuned at different levels, for instance, by introducing mutations that alter AsLOV2 domain photo-caging properties or by selecting nuclear localization signals (NLSs) of various strengths. We demonstrate the utility of LINuS in mammalian cells by controlling gene expression and entry into mitosis with blue light.