Auto-luminescent genetically-encoded ratiometric indicator for real-time Ca2+ imaging at the single cell level.
BACKGROUND
Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination.


RESULTS
We applied BRET to develop an autoluminescent Ca(2+) indicator, BRAC, which is composed of Ca(2+)-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. Adjusting the relative dipole orientation of the luminescent protein's chromophores improved the dynamic range of BRET signal change in BRAC up to 60%, which is the largest dynamic range among BRET-based indicators reported so far. Using BRAC, we demonstrated successful visualization of Ca(2+) dynamics at the single-cell level with temporal resolution at 1 Hz. Moreover, BRAC signals were acquired by ratiometric imaging capable of canceling out Ca(2+)-independent signal drifts due to change in cell shape, focus shift, etc.


CONCLUSIONS
The brightness and large dynamic range of BRAC should facilitate high-sensitive Ca(2+) imaging not only in single live cells but also in small living subjects.
Saito
Kenta
K
Research Institute for Electronic Science, Hokkaido University, Kita-20, Nishi-10 Kita-ku, Sapporo, Hokkaido, Japan.
Hatsugai
Noriyuki
N
Horikawa
Kazuki
K
Kobayashi
Kentaro
K
Matsu-Ura
Toru
T
Mikoshiba
Katsuhiko
K
Nagai
Takeharu
T
D017360:Arabidopsis; D001426:Bacterial Proteins / Q000235:genetics; D002118:Calcium / Q000032:analysis*; D002147:Calmodulin / Q000235:genetics; D003584:Cytological Techniques / Q000379:methods*; D003592:Cytophotometry / Q000379:methods; D004735:Energy Transfer; D005818:Genetic Engineering; D006367:HeLa Cells; D006801:Humans; D049410:Luciferases, Renilla / Q000235:genetics; D008163:Luminescent Measurements / Q000379:methods; D008164:Luminescent Proteins / Q000235:genetics; D009219:Myosin-Light-Chain Kinase / Q000235:genetics; D010446:Peptide Fragments / Q000235:genetics; D013053:Spectrophotometry / Q000379:methods
D001426:Bacterial Proteins; D002147:Calmodulin; D008164:Luminescent Proteins; C107660:M13 protein (myosin light-chain kinase); D010446:Peptide Fragments; C054562:yellow fluorescent protein, Bacteria; D049410:Luciferases, Renilla; D009219:Myosin-Light-Chain Kinase; D002118:Calcium