Abstract
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 Ca2+ indicator,
BRAC, which is composed of Ca2+-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 Ca2+ dynamics at the single-cell level with temporal resolution
at 1 Hz. Moreover, BRAC signals were acquired by ratiometric imaging capable of canceling out
Ca2+-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.
