Protocol for preparing sensor molecules and analyzing heterotypic endomembrane fusion in insulin-responsive cells using live-cell imaging.

Heterotypic endomembrane fusion between static GLUT4-containing vesicles and traveling transferrin receptor-containing endosomes triggers insulin-responsive translocation of the GLUT4 glucose transporter. Here, we provide a protocol for preparing BODIPY-based fluorescent sensor molecules allowing detection of heterotypic endomembrane fusion through dequenching via streptavidin-biotin binding and ratiometrically analyzing insulin-responsive events with live-cell imaging. Although this protocol is for evaluating specific fusion processes relating GLUT4 translocation, it is also applicable to assessing other processes so long as sensor molecules can properly label target molecules. For complete details on the use and execution of this protocol, please refer to Hatakeyama et al. (2022).