File:Insulin-Regulates-Glut4-Confinement-in-Plasma-Membrane-Clusters-in-Adipose-Cells-pone.0057559.s011.ogv

English: Example of “fusion-with-retention” - GLUT4 vesicle fusion resulting in selective retention of GLUT4 molecules and cluster formation at the site of the fusion. Cells were co-transfected with HA-GLUT4-EOS and IRAP-pHluorin and imaged 24 h after transfection. To monitor post-fusion dispersal of individual GLUT4 molecules, cells were first illuminated with 15–20 consecutive pulses of 405 nm laser at a wide-field illumination mode to achieve near complete photoconversion of HA-GLUT4-EOS into red-fluorescent state in PM and intracellular structures. Then cells were illuminated with a 561 nm laser in TIRF mode to selectively bleach HA-GLUT4-EOS molecules present at the PM. Red channel corresponds to activated HA-GLUT4-EOS molecules; green channel corresponds to Irap-pHluorin fluorescence. Irap-pHluorin fluorescence spike corresponds to luminal pH equilibration upon fusion pore opening. Scale bar, 2 µm.