Near-InfraredBioluminescence Assays for Protein–ProteinInteractions and Cellular Membrane Fusion in Deep Tissues Using SplitAkaluc Reconstitution

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 <i>in vivo</i> 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 <i>in vivo</i>. The developed split Akaluc reconstitution system demonstrated high sensitivity in detecting GPCR/β-arrestin interactions as well as myogenic cell fusion <i>in vitro</i>, enabling real-time insights into their temporal dynamics. Moreover, <i>in vivo</i> 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 <i>in vitro</i> and <i>in vivo</i> 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.