A Fluorometric activity assay for Light-Regulated Cyclic- Nucleotide- Monophosphate actuators

Abstract

As a transformative approach in neuroscience and cell biology, optogenetics grants control over manifold cellular events with unprecedented spatiotemporal defi nition, reversibility, and noninvasiveness. Sensory photoreceptors serve as genetically encoded, light-regulated actuators and hence embody the cornerstone of optogenetics. To expand the scope of optogenetics, ever more naturally occurring photoreceptors are being characterized, and synthetic photoreceptors with customized, light-regulated function are being engineered. Perturbational control over intracellular cyclic-nucleotide-monophosphate (cNMP) levels is achieved via sensory photoreceptors that catalyze the making and breaking of these second messengers in response to light. To facilitate discovery, engineering and quantitative characterization of such lightregulated cNMP actuators, we have developed an effi cient fl uorometric assay. Both the formation and the hydrolysis of cNMPs are accompanied by proton release which can be quantifi ed with the fl uorescent pH indicator 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfl uorescein (BCECF). This assay equally applies to nucleotide cyclases, e.g., blue-light-activated bPAC, and to cNMP phosphodiesterases, e.g., red-lightactivated LAPD. Key benefi ts include potential for parallelization and automation, as well as suitability for both purifi ed enzymes and crude cell lysates. The BCECF assay hence stands to accelerate discovery and characterization of light-regulated actuators of cNMP metabolism.