Decoding Calcium Encoding Through Bi-Directional Optogenetic Control Over Gq-Protein Signaling

Calcium is a fundamental secondary messenger responsible for relaying information from the extracellular space to the cell interior. Extracellular cues are temporally encoded through calcium signals, which often arise in the form of oscillations. These oscillations are then decoded to inform cellular decisions and regulate cellular functions. Despite its crucial role in cell signaling, the encoding and decoding of calcium oscillations is poorly understood. The current biological tools and methods used to study calcium signaling lack the temporal precision and specificity necessary to precisely manipulate, perturb, and dissect calcium signaling circuits. To address this need, we developed a new set of optogenetic techniques to regulate calcium signaling circuits and study calcium encoding and decoding. First, we created optogenetic RGS2 (opto-RGS2) for studying calcium encoding. A light-induced hetero-dimerization system was engineered to recruit the RGS2 domain to the membrane where it interacted with its cognate G protein. Using our engineered opto-RGS2 cell line, we revealed that RGS2 reduced periodicity and stochasticity of G-protein coupled calcium oscillations and acted as a feedback regulator in this signaling circuit. Our opto-RGS2 addresses the need for better tools to perturb calcium signaling circuits and will enable future studies in calcium encoding. Next, we developed an optogenetic method to dissect mechanisms of calcium decoding. The amplitude, frequency, and duty cycle of calcium oscillations are the principal components driving calcium-coupled cellular functions. However, how these components individually contribute to the overall calcium decoding is unknown. Using a mathematical model, an optogenetically-engineered cell line, and custom hardware, we optically re-created patterns of calcium oscillations that independently varied a single waveform component. By monitoring calcium-coupled transcription, we revealed that the calcium-dependent transcription factor NFAT was more sensitive to the duty cycle of calcium oscillations as opposed to the oscillation frequency. Therefore, NFAT acted primarily as a signal integrator rather than a frequency decoder as previously hypothesized. With our new optogenetic approach, we isolated the role of individual signaling components and resolved a prevailing and controversial question in calcium decoding. In summary, we developed novel optogenetic approaches and applied them to answer key questions in calcium encoding and decoding