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A genetically encoded near-infrared fluorescent calcium ion indicator.

ABSTRACT: We report an intensiometric, near-infrared fluorescent, genetically encoded calcium ion (Ca²⁺) indicator (GECI) with excitation and emission maxima at 678?and 704?nm, respectively. This GECI, designated NIR-GECO1, enables imaging of Ca²⁺ transients in cultured mammalian cells and brain tissue with sensitivity comparable to that of currently available visible-wavelength GECIs. We demonstrate that NIR-GECO1 opens up new vistas for multicolor Ca²⁺ imaging in combination with other optogenetic indicators and actuators.

SUBMITTER: Qian Y

PROVIDER: S-EPMC6393164 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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A genetically encoded near-infrared fluorescent calcium ion indicator.

Qian Yong Y Piatkevich Kiryl D KD Mc Larney Benedict B Abdelfattah Ahmed S AS Mehta Sohum S Murdock Mitchell H MH Gottschalk Sven S Molina Rosana S RS Zhang Wei W Chen Yingche Y Wu Jiahui J Drobizhev Mikhail M Hughes Thomas E TE Zhang Jin J Schreiter Eric R ER Shoham Shy S Razansky Daniel D Boyden Edward S ES Campbell Robert E RE

Nature methods 20190121 2

We report an intensiometric, near-infrared fluorescent, genetically encoded calcium ion (Ca2+) indicator (GECI) with excitation and emission maxima at 678 and 704 nm, respectively. This GECI, designated NIR-GECO1, enables imaging of Ca2+ transients in cultured mammalian cells and brain tissue with sensitivity comparable to that of currently available visible-wavelength GECIs. We demonstrate that NIR-GECO1 opens up new vistas for multicolor Ca2+ imaging in combina ...[more]

PMID: 30664778

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Project description:Currently available genetically encoded calcium indicators (GECIs) utilize calmodulins (CaMs) or troponin C from metazoa such as mammals, birds, and teleosts, as calcium-binding domains. The amino acid sequences of the metazoan calcium-binding domains are highly conserved, which may limit the range of the GECI key parameters and cause undesired interactions with the intracellular environment in mammalian cells. Here we have used fungi, evolutionary distinct organisms, to derive CaM and its binding partner domains and design new GECI with improved properties. We applied iterative rounds of molecular evolution to develop FGCaMP, a novel green calcium indicator. It includes the circularly permuted version of the enhanced green fluorescent protein (EGFP) sandwiched between the fungal CaM and a fragment of CaM-dependent kinase. FGCaMP is an excitation-ratiometric indicator that has a positive and an inverted fluorescence response to calcium ions when excited at 488 and 405 nm, respectively. Compared with the GCaMP6s indicator in vitro, FGCaMP has a similar brightness at 488 nm excitation, 7-fold higher brightness at 405 nm excitation, and 1.3-fold faster calcium ion dissociation kinetics. Using site-directed mutagenesis, we generated variants of FGCaMP with improved binding affinity to calcium ions and increased the magnitude of FGCaMP fluorescence response to low calcium ion concentrations. Using FGCaMP, we have successfully visualized calcium transients in cultured mammalian cells. In contrast to the limited mobility of GCaMP6s and G-GECO1.2 indicators, FGCaMP exhibits practically 100% molecular mobility at physiological concentrations of calcium ion in mammalian cells, as determined by photobleaching experiments with fluorescence recovery. We have successfully monitored the calcium dynamics during spontaneous activity of neuronal cultures using FGCaMP and utilized whole-cell patch clamp recordings to further characterize its behavior in neurons. Finally, we used FGCaMP in vivo to perform structural and functional imaging of zebrafish using wide-field, confocal, and light-sheet microscopy.

Dataset Information

A genetically encoded near-infrared fluorescent calcium ion indicator.

Publications

A genetically encoded near-infrared fluorescent calcium ion indicator.

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