Temperature-Responsive Optogenetic Probes of Cell Signaling

Optogenetics has provided a powerful toolset to dissect the role of signal dynamics in cells and organisms using light as a precise input. Here we describe a new class of photosensor that is sensitive to both light and temperature. The BcLOV4 photoreceptor is a blue-light sensitive protein that translocates to the membrane and can be used as single-component optogenetic switch to control signaling. We used BcLOV4 to engineer optogenetic control over Ras/Erk and PI3K/Akt signaling, and we found that the probes performed well relative to comparable state-of-the-art optogenetic tools. Surprisingly, 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 dual dependency and we developed a computational model that fully predicts BcLOV4 translocation and signal dynamics across a range of experimental temperatures and light conditions. We also developed new methods to precisely control both light and temperature stimulation of cell cultured in microwell plates. In addition to use in mammalian cells, we show that our BcLOV4-based tools function robustly and stably in both Drosophila and zebrafish without the need for expression-level tuning. 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.