Auto-luminescent genetically-encoded ratiometric indicator for real-time Ca~(2+) imaging at the single cell level

摘要

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. We have applied this strategy 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. With this BRAC, we succeeded 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. Taking advantage of the bioluminescence imaging property that does not require external excitation light, BRAC might become a powerful tool applicable in conjunction with so-called optogenetic technology by which we can control cellular and protein function by light illumination.