Temperature-Responsive Optogenetic Probes of Cell Signaling

Optogenetics provides a powerful toolset to dissect the role of signal dynamics in cells and organisms. However, such tools can be challenging to implement because they often consist of multiple components that require stoichiometric tuning and occupy valuable fluorescence channels. We thus sought to create single-component variants of optogenetic probes that work through membrane recruitment, a common mode of optogenetic control. Specifically, we engineered control of Ras/Erk and PI3K/Akt signaling by fusing pathway activators to BcLOV4, a recently characterized fungal photoreceptor that translocates to membrane lipids upon photoactivation. Our probes allowed robust activation of both pathways and compared favorably to existing 2-component probes in terms of dynamic range of induction and minimal perturbation of cell physiology. However, we discovered an unanticipated temperature sensitivity of the BcLOV4 protein, such that membrane localization and pathway stimulation spontaneously decay within ~1 hour — despite constant illumination — at a rate proportional to both the temperature and the intensity of light. We systematically characterized this unique dependency on both light and temperature, and we developed a computational model that fully predicts BcLOV4 translocation and Ras/Erk activation dynamics across a range of physiological temperatures and light conditions. In addition to use in mammalian cells, we show that BcLOV4-based tools function robustly and stably in both Drosophila and zebrafish, which are not amenable to stoichiometric expression tuning, and which are cultured at temperatures below 30 °C where BcLOV4 can be stably stimulated. Our work provides a new set of easy-to-use optogenetic probes that work across organisms, and a quantitative framework for their optimal use. Moreover, BcLOV4 provides a natural substrate for engineering a new class of tools that respond to light and/or temperature.