Coupling optogenetic stimulation with NanoLuc-based luminescence (BRET) Ca++ sensing
| Autor: | Samuel W. Centanni, Jie Yang, Derrick Cumberbatch, Danny G. Winder, Shu-qun Shi, Carl Hirschie Johnson, Donna J. Webb |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Melanopsin Luminescence Science General Physics and Astronomy Channelrhodopsin Optogenetics 7. Clean energy Article General Biochemistry Genetics and Molecular Biology 03 medical and health sciences Nuclear magnetic resonance Fluorescence Resonance Energy Transfer Animals Humans Bioluminescence Luciferase Luciferases Multidisciplinary Chemistry General Chemistry Batrachoidiformes Fluorescence Coupling (electronics) HEK293 Cells 030104 developmental biology Microscopy Fluorescence Luminescent Measurements Biophysics Calcium sense organs HeLa Cells |
| Zdroj: | Nature Communications, Vol 7, Iss 1, Pp 1-10 (2016) Nature Communications |
| ISSN: | 2041-1723 |
| Popis: | Optogenetic techniques allow intracellular manipulation of Ca++ by illumination of light-absorbing probe molecules such as channelrhodopsins and melanopsins. The consequences of optogenetic stimulation would optimally be recorded by non-invasive optical methods. However, most current optical methods for monitoring Ca++ levels are based on fluorescence excitation that can cause unwanted stimulation of the optogenetic probe and other undesirable effects such as tissue autofluorescence. Luminescence is an alternate optical technology that avoids the problems associated with fluorescence. Using a new bright luciferase, we here develop a genetically encoded Ca++ sensor that is ratiometric by virtue of bioluminescence resonance energy transfer (BRET). This sensor has a large dynamic range and partners optimally with optogenetic probes. Ca++ fluxes that are elicited by brief pulses of light to cultured cells expressing melanopsin and to neurons-expressing channelrhodopsin are quantified and imaged with the BRET Ca++ sensor in darkness, thereby avoiding undesirable consequences of fluorescence irradiation. The coupling of optogenetics with fluorescent Ca2+ sensors is confounded by sensitivity of optogenetic probes to light used to excite the sensors. Here the authors develop a Ca2+ sensor based on bioluminescence resonance energy transfer (BRET) that monitors Ca2+ fluxes in darkness without excitation. |
| Databáze: | OpenAIRE |
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