Genetically encoded reporters have greatly increased our understanding of biology. While fluorescent reporters have been widely used, photostability and phototoxicity have hindered their use in long‐term experiments. Bioluminescence overcomes some of these challenges but requires the addition of an exogenous luciferin limiting its use. Using a modular approach, Autonomous Molecular BioluminEscent Reporter (AMBER), an indicator of membrane potential is engineered. Unlike other bioluminescent systems, AMBER is a voltage‐gated luciferase coupling the functionalities of the Ciona voltage‐sensing domain (VSD) and bacterial luciferase, luxAB. When co‐expressed with the luciferin‐producing genes, AMBER reversibly switches the bioluminescent intensity as a function of membrane potential. Using biophysical and biochemical methods, it is shown that AMBER switches its enzymatic activity from an OFF to an ON state as a function of the membrane potential. Upon depolarization, AMBER switches from a low to a high enzymatic activity state, showing a several‐fold increase in the bioluminescence output (ΔL/L). AMBER in the pharyngeal muscles and mechanosensory touch neurons of Caenorhabditis elegans is expressed. Using the compressed sensing approach, the electropharingeogram of the C. elegans pharynx is reconstructed, validating the sensor in vivo. Thus, AMBER represents the first fully genetically encoded bioluminescent reporter without requiring exogenous luciferin addition.

1.AbstractGenetically encoded reporters have greatly increased our understanding of biology, especially in neuroscience. While fluorescent reporters have been widely used, photostability and phototoxicity have hindered their use in long-term experiments. Bioluminescence overcomes some of these challenges but requires the addition of an exogenous luciferin limiting its use. Using a modular approach we have engineered Autonomous Molecular BioluminEscent Reporter (AMBER), an indicator of membrane potential. Unlike other luciferase-luciferin bioluminescent systems, AMBER encodes the genes to express both the luciferase and luciferin. AMBER is a voltage-gated luciferase coupling the functionalities of the Ciona voltage-sensing domain (VSD) and bacterial luciferase, luxAB. When AMBER is co-expressed with the luciferin producing genes it reversibly switches the bioluminescent intensity as a function of membrane potential. Using biophysical and biochemical methods we show that AMBER modulates its enzymatic activity as a function of the membrane potential. AMBER shows several-fold increase in the luminescent (ΔL/L) signal upon switching from the off to on state when the cell is depolarized. In vivo expression of AMBER in C. elegans allowed detecting pharyngeal pumping action and mechanosensory neural activity from multiple worms simultaneously. AMBER reports neural activity of multiple animals at the same time and can be used in social behavior assays to elucidate the role of membrane potential underlying behavior.2.Significance StatementThere have been many exciting advances in the development of genetically encoded voltage indicators to monitor intracelluar voltage changes. Most sensors employ fluorescence, which requires external light, potentially causing photobleaching or overheating. Consequently, there has been interest in developing luminescence reporters. However, they require addition of an exogenous substrate to produce light intracellularly. Here, we engineered a genetically encoded bioluminescent voltage indicator, AMBER, which unlike other bioluminescent activity indicators, does not require addition of an exogenous substrate. AMBER allows a large differential signal, a high signal-to-noise ratio, and causes minimal metabolic demand on cells. We used AMBER to record voltage activity in freely-moving C. elegans, demonstrating that AMBER is a important new tool for monitoring neuronal activity during social behavior.

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