A Fluorometric Activity Assay for Light-Regulated Cyclic-Nucleotide-Monophosphate Actuators
As a transformative approach in neuroscience and cell biology, optogenetics grants control over manifold cellular events with unprecedented spatiotemporal definition, 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 light-regulated cNMP actuators, we have developed an efficient fluorometric assay. Both the formation and the hydrolysis of cNMPs are accompanied by proton release which can be quantified with the fluorescent pH indicator 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). This assay equally applies to nucleotide cyclases, e.g., blue-light-activated bPAC, and to cNMP phosphodiesterases, e.g., red-light-activated LAPD. Key benefits include potential for parallelization and automation, as well as suitability for both purified enzymes and crude cell lysates. The BCECF assay hence stands to accelerate discovery and characterization of light-regulated actuators of cNMP metabolism.
Key wordsBCECF bPAC Cyclic nucleotide monophosphate cNMP phosphodiesterase LAPD Microtiter plate Nucleotide cyclase Optogenetics Sensory photoreceptor
Financial support by the Cluster of Excellence in Catalysis ‘Unicat’ (A.M.) of the Deutsche Forschungsgemeinschaft (DFG), through a Sofja-Kovalevskaya Award by the Alexander-von-Humboldt Foundation (A.M.), by the Caesar institute (H.G.K. and R.S.) as well as by a Heisenberg Fellowship by the DFG (M.S.) is gratefully acknowledged.
- 5.Li X, Gutierrez DV, Hanson MG, Han J, Mark MD, Chiel H, Hegemann P, Landmesser LT, Herlitze S (2005) Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin. Proc Natl Acad Sci U S A 102:17816–17821CrossRefPubMedPubMedCentralGoogle Scholar