Temperature-Responsive Optogenetic Probes of Ras and PI3K Signaling

Optogenetic signaling probes have served as powerful tools to dissect the role of spatiotemporal 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 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 basal signal perturbation and dynamic range of induction. However, we were surprised to discover that BcLOV4 responds not only to light, but also to temperature, such that membrane localization and pathway stimulation spontaneously decay —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. Our model reveals that BcLOV4 permits stable activation at lower temperatures (< 30C), but activation decays at higher temperatures. Our model also provides a predictive model for the use of BcLOV-based tools. In addition to use in mammalian cells, we show that BcLOV4-based tools function robustly and stably in Drosophila and zebrafish, which are not amenable to stoichiometric expression tuning, and which are cultured at permissive temperatures. Ongoing protein engineering efforts will permit tuning of the temperature-dependence of BcLOV4 for its application across an even wider range of experimental systems and conditions.