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Optogenetics pp 93-105 | Cite as

A Fluorometric Activity Assay for Light-Regulated Cyclic-Nucleotide-Monophosphate Actuators

  • Charlotte Helene Schumacher
  • Heinz G. Körschen
  • Christopher Nicol
  • Carlos Gasser
  • Reinhard Seifert
  • Martin Schwärzel
  • Andreas MöglichEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1408)

Abstract

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 words

BCECF bPAC Cyclic nucleotide monophosphate cNMP phosphodiesterase LAPD Microtiter plate Nucleotide cyclase Optogenetics Sensory photoreceptor 

Notes

Acknowledgements

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.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Charlotte Helene Schumacher
    • 1
  • Heinz G. Körschen
    • 2
  • Christopher Nicol
    • 3
  • Carlos Gasser
    • 1
  • Reinhard Seifert
    • 2
  • Martin Schwärzel
    • 3
  • Andreas Möglich
    • 1
    • 4
    Email author
  1. 1.Institut für Biologie, Biophysikalische ChemieHumboldt-Universität zu BerlinBerlinGermany
  2. 2.Department of Molecular Sensory SystemsResearch Center CaesarBonnGermany
  3. 3.Institut für Biologie, NeurobiologieFreie Universität BerlinBerlinGermany
  4. 4.Faculty of Biology, Chemistry and Earth Sciences, Lehrstuhl für BiochemieUniversität BayreuthBayreuthGermany

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