Fig. 6: Cell fusion leads to a reduced energy state through transient, CME-dependent glucose channel internalization.

SUM-159 cells expressing VSV-G (a) or VSV-G and AP180-C (b) were induced to fused and then fixed at indicated time points. Anti-Glut1 antibodies were used to assess the subcellular localization of Glut1 by confocal microscopy. Insets depict a region in the cytoplasm at each time point. Images in a and b are representative of three independent experiments. c The glucose biosensor, iGlucoSnFR.mRuby2 (depicted in left panel, GBP glucose binding protein) fluoresces when glucose is bound. The intensity of the cytoplasmic biosensor fluorescence was monitored as cells expressing VSV-G were induced to fuse. Images are representative of nine independent experiments. d Relative cytoplasmic glucose levels were measured overtime in fusing cells. To calculate the relative changes, the fluorescence intensity in fusing cells was normalized to the intensity of control non-fusing cells. The blue area represent the SEM overtime, n = 19 cells. e VSV-G transfected SUM-159 cells were fixed before fusion, or at the indicated time after fusion was induced. Relative cytoplasmic ATP levels were measured using a luciferase-based assay (arb. units; arbitrary units). Error bars represent the SEM, n = 8, n = 16, n = 16, n = 8, and n = 8 replicates examined over two independent experiments for unwashed pre-fused, and fused cells after 5, 15, and 60 min, respectively. p values represent the differences between the pre-fused cells and each time point (5 and 15 min). **p = 0.0009, and *p = 0.0153. Statistical significance calculations were performed using a two-tailed unpaired Student’s t test. Scale bar size = 10 μm.