Abstract
We introduce how to image calcium ion levels in the heart of zebrafish embryos and larvae up to 5 days post-fertilization with the photoprotein green fluorescent protein (GFP)-aequorin (GA) in the transgenic line Tg(myl7:GA). Incubation of the embryos with CTZ to obtain the functional photoprotein yields few emission counts, suggesting that, when the heart is beating, the rate of aequorin consumption is faster than that of the reconstitution with CTZ. In this chapter, we present an improved aequorin reconstitution protocol. We further describe the experimental procedure as well as the bioluminescence data analysis and processing.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Shimomura O, Johnson FH, Saiga Y (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59:223–239
Shimomura O, Johnson FH (1978) Peroxidized coelenterazine, the active group in the photoprotein aequorin. Proc Natl Acad Sci U S A 75:2611–2615
Shimomura O (2006) Bioluminescence: chemical principles and methods. World Scientific, Hackensack
Baubet V, Le Mouellic H, Campbell AK et al (2000) Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level. Proc Natl Acad Sci U S A 97:7260–7265
Bakayan A, Vaquero CF, Picazo F et al (2011) Red fluorescent protein-aequorin fusions as improved bioluminescent Ca2+ reporters in single cells and mice. PLoS One 6:e19520
Bakayan A, Domingo B, Vaquero CF et al (2017) Fluorescent protein–photoprotein fusions and their applications in calcium. Imaging 93:448–465
Brini M (2008) Calcium-sensitive photoproteins. Methods 46:160–166
Alvarez J, Montero M (2002) Measuring [Ca2+] in the endoplasmic reticulum with aequorin. Cell Calcium 32:251–260
Cheung CY, Webb SE, Love DR et al (2011) Visualization, characterization and modulation of calcium signaling during the development of slow muscle cells in intact zebrafish embryos. Int J Dev Biol 55:153–174
Vicente M, Salgado-Almario J, Soriano J et al (2019) Visualization of mitochondrial Ca2+ signals in skeletal muscle of Zebrafish embryos with bioluminescent indicators. Int J Mol Sci 20:E5409
Bakayan A, Domingo B, Miyawaki A et al (2015) Imaging Ca(2+) activity in mammalian cells and zebrafish with a novel red-emitting aequorin variant. Pflugers Arch 467:2031–2042
Vicente M, Salgado-Almario J, Collins MM et al (2021) Cardioluminescence in transgenic zebrafish embryos: a Ca2+ imaging tool to study drug effects and pathological modeling. bioRxiv:2021.05.06.442917
Kim TJ, Türkcan S, Pratx G (2017) Modular low-light microscope for imaging cellular bioluminescence and radioluminescence. Nat Protoc 12:1055–1076
Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675
Bonora M, Giorgi C, Bononi A et al (2013) Subcellular calcium measurements in mammalian cells using jellyfish photoprotein aequorin-based probes. Nat Protoc 8:2105–2118
Barrero MJ, Montero M, Alvarez J (1997) Dynamics of [Ca2+] in the endoplasmic reticulum and cytoplasm of intact HeLa cells: a COMPARATIVE STUDY*. J Biol Chem 272:27694–27699
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Vicente, M., Salgado-Almario, J., Martínez-Sielva, A., Llopis, J., Domingo, B. (2022). Optimized Aequorin Reconstitution Protocol to Visualize Calcium Ion Transients in the Heart of Transgenic Zebrafish Embryos In Vivo. In: Kim, SB. (eds) Bioluminescence. Methods in Molecular Biology, vol 2524. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2453-1_20
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2453-1_20
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2452-4
Online ISBN: 978-1-0716-2453-1
eBook Packages: Springer Protocols