title: Auto-luminescent genetically-encoded ratiometric indicator for real-time Ca2+ imaging at the single cell level.
abstract: 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.
authors.affiliation: Research Institute for Electronic Science, Hokkaido University, Kita-20, Nishi-10 Kita-ku, Sapporo, Hokkaido, Japan.
authors.forename: Kenta
authors.initials: K
authors.lastname: Saito
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Hatsugai
authors.forename: Kazuki
authors.initials: K
authors.lastname: Horikawa
authors.forename: Kentaro
authors.initials: K
authors.lastname: Kobayashi
authors.forename: Toru
authors.initials: T
authors.lastname: Matsu-Ura
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Mikoshiba
authors.forename: Takeharu
authors.initials: T
authors.lastname: Nagai
mesh_terms: 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
chemical_list: 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