Near-Infrared Bioluminescence Assays for Protein-Protein Interactions and Cellular Membrane Fusion in Deep Tissues...

Bioluminescence analysis using luciferase is an essential tool for studying biological processes in different cells. Split luciferase reconstitution is a technique that enables the analysis of biological events through the monitoring of protein-protein interactions. However, effective detection of cellular events in vivo remains challenging due to the limitation of light penetration into deep tissues and optical sensitivity. To address this, we developed a novel split luciferase reconstitution method using a near-infrared-emitting luciferase, Akaluc, and applied it to monitor two important biological events: G protein-coupled receptor (GPCR)/β-arrestin interactions and myogenic cell fusion in vivo. The developed split Akaluc reconstitution system demonstrated high sensitivity in detecting GPCR/β-arrestin interactions as well as myogenic cell fusion in vitro, enabling real-time insights into their temporal dynamics. Moreover, in vivo bioluminescence imaging successfully monitored GPCR/β-arrestin interactions in the mouse lung and the progression of myogenesis during mouse leg muscle regeneration. The split Akaluc reconstitution method will be a versatile tool for both in vitro and in vivo analyses of protein-protein interactions and cell fusion events. This system holds significant potential for advancing drug development, especially in the screening of GPCR-targeted therapeutic and myogenesis-promoting compounds in animal models.