Abstract
Methods for monitoring physiological changes in cellular Ca(2+) levels have been in high demand for their utility in monitoring neuronal signaling. Recently, we introduced SCANR (Split-Tobacco Etch Virus (TEV) protease Calcium-regulated Neuron Recorder), which reports on Ca(2+) changes in cells through the binding of calmodulin and M13 to reconstitute an active TEV protease. First-generation SCANR marked all of the Ca(2+) spikes that occur throughout the lifetime of the cell, but it did not have a mechanism for controlling the time window in which recording of physiological changes in Ca(2+) occurred. Here, we explore both chemical and light-based strategies for controlling the time and place in which Ca(2+) recording occurs. We describe the adaptation of six popular chemo- and opto-genetics methods for controlling protein activity and subcellular localization to the SCANR system. We report two successful strategies, one that leverages the LOV-Jα optogenetics system for sterically controlling protein interactions and another that employs chemogenetic manipulation of subcellular protein distribution using the FKBP/FRB rapamycin binding pair.