In Bioluminescent Optogenetics (BL‐OG) a biological, rather than a physical, light source is used to activate light‐sensing opsins, such as channelrhodopsins or pumps. This is commonly achieved by utilizing a luminopsin (LMO), a fusion protein of a light‐emitting luciferase tethered to a light‐sensing opsin. Light of the wavelength matching the activation peak of the opsin is emitted by the luciferase upon application of its small molecule luciferin, resulting in activation of the fused opsin and subsequent effects on membrane potential. Using optimized protocols for culturing, transforming, and testing primary neurons in multi electrode arrays, we systematically defined parameters under which changes in neuronal activity are specific to bioluminescent activation of opsins, rather than due to off‐target effects of either the luciferin or its solvent on neurons directly, or on opsins directly. We further tested if there is a direct effect of bioluminescence on neurons. Critical for assuring specific BL‐OG effects are testing the concentration and formulation of the luciferin against proper controls, including testing effects of vehicle on LMO expressing and of luciferin on nonLMO expressing targets.
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 (Δ
- NSF-PAR ID:
- 10360583
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Biology
- Volume:
- 5
- Issue:
- 12
- ISSN:
- 2701-0198
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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