PUBLICATION
Imaging Ca(2+) activity in mammalian cells and zebrafish with a novel red-emitting aequorin variant
- Authors
- Bakayan, A., Domingo, B., Miyawaki, A., Llopis, J.
- ID
- ZDB-PUB-141031-7
- Date
- 2015
- Source
- Pflugers Archiv : European journal of physiology 467(9): 2031-42 (Journal)
- Registered Authors
- Domingo Moreno, Beatriz, Llopis, Juan Francisco, Miyawaki, Atsushi
- Keywords
- none
- MeSH Terms
-
- Aequorin*
- Animals
- Bioluminescence Resonance Energy Transfer Techniques/methods*
- Calcium/analysis*
- Calcium/metabolism
- HEK293 Cells
- Humans
- Luminescent Agents*
- Mice
- Zebrafish
- PubMed
- 25355614 Full text @ Pflügers Archiv. / Eur. J. Physiol.
Citation
Bakayan, A., Domingo, B., Miyawaki, A., Llopis, J. (2015) Imaging Ca(2+) activity in mammalian cells and zebrafish with a novel red-emitting aequorin variant. Pflugers Archiv : European journal of physiology. 467(9):2031-42.
Abstract
Ca(2+) monitoring with aequorin is an established bioluminescence technique, whereby the photoprotein emits blue light when it binds to Ca(2+). However, aequorin's blue emission and low quantum yield limit its application for in vivo imaging because blue-green light is greatly attenuated in animal tissues. In earlier work, aequorin was molecularly fused with green, yellow, and red fluorescent proteins, producing an emission shift through bioluminescence resonance energy transfer (BRET). We have previously shown that the chimera tandem dimer Tomato-aequorin (tdTA) emits red light in mammalian cells and across the skin and other tissues of mice [1]. In this work, we varied the configuration of the linker in tdTA to maximize energy transfer. One variant, named Redquorin, improved BRET from aequorin to tdTomato to almost a maximum value, and the emission above 575 nm exceeded 73 % of total counts. By pairing Redquorin with appropriate synthetic coelenterazines, agonist-induced and spontaneous Ca(2+) oscillations in single HEK-293 cells were imaged. In addition, we also imaged Ca(2+) transients associated with twitching behavior in developing zebrafish embryos expressing Redquorin during the segmentation period. Furthermore, the emission profile of Redquorin resulted in significant luminescence crossing a blood sample, a highly absorbing tissue. This new tool will facilitate in vivo imaging of Ca(2+) from deep tissues of animals.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping