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.

Advances and current challenges in calcium signaling

“…Bioluminescent GECIs use either aequorin or aequorin fused to a fluorescent protein (FP) (Shimomura et al ., ). Such indicators, for example BRAC or Nano‐lantern, enable Ca 2+ analyses via bioluminescence resonance energy transfer (BRET; Saito et al ., , ). Fluorescent GECIs consist either of a single circular permutated FP with GCaMP and Pericam as the prototypes or of a dual‐FP fluorescence resonance energy transfer (FRET)‐pair with cameleon as the founder GECI (Miyawaki et al ., ; Nagai et al ., ; Nakai et al ., ).…”

Recent developments of genetically encoded optical sensors for cell biology

“…The first such sensor used firefly luciferase as the donor and an RFP tetramer as the acceptor (Arai et al., ). The development of new GFP mutants as well as improved luciferase enzymes and substrates prompted the generation of a number of sensors based on BRET: RLuc‐GFP, RLuc‐mOrange (De et al., ), FLuc‐mKate (Branchini et al., ), FLuc‐mCherry (Iglesias and Costoya, ) and Rluc‐EYFP (Saito et al., ). The number of BRET sensors for monitoring protein‐protein interactions is now constantly increasing (Figure A).…”

Recent progress in the development of genetically encoded Ca²⁺indicators

“…As bioluminescent indicator requires no excitation light, observation can be performed free from undesired functional crosstalk with optogenetic tools. Although the problem had been the small signal of Ca 2+ sensitive Aequorin, increased emission signal was achieved in BRAC and Nano-lantern(Ca 2+ ), being the latest version of bioluminescent-based GECIs (30,31). Both BRAC and Nano-lantern(Ca 2+ ) are the cameleon like FRETtype indicator harboring CaM-M13 moiety with Renila reniformis derived improved luciferase (RLuc8) and Venus as a donor and acceptor.…”