Hot on the trail of molecular optogenetic signaling activators
Summary by Leanne Iannucci: William Benman, Erin E. Berlew, Hao Deng, Caitlyn Parke, Ivan A. Kuznetsov, Bomyi Lim, Arndt F. Siekmann, Brian Y. Chow, & Lukasz J. Bugaj. Temperature-responsive optogenetic probes of cell signaling. Nature Chemical Biology, 2022. 18(2): p. 152-160.
Image credit: Midjourney
Understanding how cells communicate with each other during development, growth, and repair is essential to develop biologically informed therapeutics. However, many current experimental paradigms do not have sufficient resolution to elucidate the mechanisms governing these processes. Molecular optogenetics is emerging as a powerful toolset for the investigation of cell signaling. These tools allow for the precise control of molecular function in space and time by using light to regulate biological processes. The photoreceptor BcLOV4, which was recently isolated from fungus Botryis cinerea [1], has been identified as having high potential for application in the biomedical space due to its rapid, blue light-controlled movement between the cellular cytoplasm and membrane. Benman et al. [2] sought to characterize this tool’s specific applicability to two essential cellular signaling pathways: Ras and phosphatidyl inositol-3-kinase (PI3K).
First, the authors fused the BcLOV4 photoreceptor to activators of their two pathways of interest (BcLOV-SOS (Ras) and BcLOV-iSH (PI3K)) and expressed these engineered proteins in a mouse fibroblast cell line. Groups of cells were then exposed to 5 minutes of blue LED light for a set intensity ranging from low to high. In response, an intensity-dependent increase in Ras and PI3K signaling was observed, respectively. When exposing the engineered cells to light for a longer duration (1 hour), the authors were surprised to observe decay in pathway activation after 20 minutes. That is, the pathway activation levels decreased over the long-term illumination, despite all variables being held constant. Further, they ruled out the possibility of a negative feedback loop’s influence on this phenomenon by performing the experiment with a different, but analogous light-sensitive protein (iLID) and observing sustained pathway activation. Finally, after briefly illuminating the BcLOV4-containing cells and allowing the signal to decay, the researchers also observed that re-illuminating the sample could not restore the signaling to its original peak level.
Based on these unexpected findings and the knowledge that LED-based illumination can cause unintended heat generation, the authors then hypothesized that BcLOV4’s activity might be temperature-dependent. Using their custom-engineered “optoPlate”, the authors simultaneously controlled the local temperature and sample illumination of cells seeded into a 96 well plate. The authors varied temperature or light exposure, while keeping the opposite variable constant. They observed that the decay rate of the targeted signaling pathway increased with both increasing light intensity and increasing temperature.
Notably, BcLOV4-controlled signaling had its lowest rate of decay at lower (<30C) temperatures. As organisms such as Drosophila and zebrafish have preferred temperatures in this range, the authors then experimentally showed that these BcLOV4 tools activated signaling in these model systems. Further, the authors showed that the temperature-responsiveness could be leveraged to generate orthogonal blue-light optogenetic systems. By linking signaling pathways to blue-light optogenetic tools such as BcLOV4 as well as tools that are not temperature-sensitive, (e.g., iLID or Cry2), investigation of multiple signaling pathways can be multiplexed with only one required illumination wavelength and a change in temperature.
Glantz, S.T., et al., Directly light-regulated binding of RGS-LOV photoreceptors to anionic membrane phospholipids. Proceedings of the National Academy of Sciences, 2018. 115(33): p. E7720-E7727.
Benman, W., et al., Temperature-responsive optogenetic probes of cell signaling. Nature Chemical Biology, 2022. 18(2): p. 152-160.