Live-cell imaging of membrane proteins by a coiled-coil labeling method—Principles and applications

In situ investigations in living cell membranes are important to elucidate the dynamic behaviors of membrane proteins in complex biomembrane environments. Protein-specific labeling is a key technique for the detection of a target protein by fluorescence imaging. The use of post-translational labeling methods using a genetically encodable tag and synthetic probes targeting the tag offer a smaller label size, labeling with synthetic fluorophores, and precise control of the labeling ratio in multicolor labeling compared with conventional genetic fusions with fluorescent proteins. This review focuses on tag-probe labeling studies for live-cell analysis of membrane proteins based on heterodimeric peptide pairs that form coiled-coil structures. The robust and simple peptide-peptide interaction enables not only labeling of membrane proteins by noncovalent interactions, but also covalent crosslinking and acyl transfer reactions guided by coiled-coil assembly. A number of studies have demonstrated that membrane protein behaviors in live cells, such as internalization of receptors and the oligomeric states of various membrane proteins (G-protein-coupled receptors, epidermal growth factor receptors, influenza A M2 channel, and glycopholin A), can be precisely analyzed using coiled-coil labeling, indicating the potential of this labeling method in membrane protein research.