Imaging Ca2+ activity in mammalian cells and zebrafish with a novel red-emitting aequorin variant

Abstract

Ca2+ monitoring with aequorin is an established bioluminescence technique, whereby the photoprotein emits blue light when it binds to Ca2+. However, aequorin’s blue emission and low quantum yield limit its application for in vivo imaging because blue-green light is greatly attenuated in animal tissues. In earlier work, aequorin was molecularly fused with green, yellow, and red fluorescent proteins, producing an emission shift through bioluminescence resonance energy transfer (BRET). We have previously shown that the chimera tandem dimer Tomato-aequorin (tdTA) emits red light in mammalian cells and across the skin and other tissues of mice [1]. In this work, we varied the configuration of the linker in tdTA to maximize energy transfer. One variant, named Redquorin, improved BRET from aequorin to tdTomato to almost a maximumvalue, and the emission above 575 nm exceeded 73% of total counts. By pairing Redquorin with appropriate synthetic coelenterazines, agonist-induced and spontaneous Ca2+ oscillations in single HEK-293 cells were imaged. In addition, we also imaged Ca2+ transients associated with twitching behavior in developing zebrafish embryos expressing Redquorin during the segmentation period. Furthermore, the emission profile of Redquorin resulted in significant luminescence crossing a blood sample, a highly absorbing tissue. This new tool will facilitate in vivo imaging of Ca2+ from deep tissues of animals.