Aim: 

Spatially localized Ca²⁺ signaling has a crucial role in many cellular processes. Monitoring Ca²⁺ using bioluminescence does not require input of radiative energy and can be measured over long periods, with low background and good temporal resolution. In the jellyfish Aequorea victoria, binding of Ca²⁺ to the photoprotein aequorin results in intermolecular bioluminescence resonance energy transfer to GFP. A molecular fusion of aequorin with GFP resulted in intramolecular energy transfer and increased light output (1). We fused mRFP1.2 to aequorin in order to monitor Ca²⁺ changes simultaneously in several cell compartments (2). In this study, we constructed new fusions to improve signal/noise in yellow-red spectral bands.

Methods: 

The fluorescent proteins citrine, mOrange and Tomato were fused to aequorin. Transfected HeLa cells were imaged on an inverted microscope (LeicaDMIRE) with a Hamamatsu EMCCD using green, yellow and red emission filters.

Results: 

In live HeLa cells expressing the chimeras, energy transfer from aequorin to the fluorescent proteins was observed. Light output was sufficient to image cells at 2Hz with a 16x objective. Energy transfer was less efficient in red-aequorin compared to green/yellow-aequorin fusions.

Conclusion: 

Luminescent sensors with different emission peaks will improve multi-compartment monitoring of Ca²⁺ changes and facilitate study of Ca²⁺ dynamics in whole organisms.

References: (1) Baubet V et al., Proc Natl Acad Sci U S A. 97:7260-5, 2000. (2) Manjarrés IM et al., Pflugers Arch. 455:961-70, 2008