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 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.

Characterization and engineering of photoactivated adenylyl cyclases

“…Enzyme-linked immunosorbent assays (ELISAs) are capable of precisely determining cNMP quantities even within heterogenous mixtures like cell lysates. Similarly, we developed a fluorescence-based readout that exploits that both the making and the breaking of cNMPs entail acidification of the solution (Schumacher et al, 2016). Cyclase and PDE turnover can hence be detected via the pH-sensitive fluorophore 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) (Rink et al, 1982), and this assay applies to either purified protein or crude cell lysates, thus allowing enhanced throughput.…”

Optogenetics and photopharmacology - effective tools for managing cell activity using light

“…Эти уникальные свойства, а также успехи молекулярной биологии и современных технологий обеспечили появление трёх основных направлений, в которых главным инструментом является свет, -оптогенетика, оптофар макология и оптосенсорика. Оказалось, что с помощью света можно исследовать функции клеток и клеточных ансамблей [1][2][3][4][5][6], регулировать функции дезоксирибонуклеиновой кислоты (ДНК) [7][8], модулировать проводимость и кинетику ионных каналов [9][10][11], измерять концентрации ионов [12-14] и других клеточных компонентов [15][16][17][18][19], контролировать поведение организмов [10, 20-21], а также искать новые пути лечения некоторых заболеваний [22][23].…”