Fluoromicroscopic detection of myc-tagged GLUT4 on the cell surface. Co-localization of the translocated GLUT4 with rearranged actin by insulin treatment in CHO cells and L6 myotubes.
We earlier developed a novel method to detect translocation of glucose transporter type 4 (GLUT 4) directly, quantitatively and simply using c-MYC epitope-tagged GLUT4 (GLUT4myc) Kanai F, Nishioka Y, Hayashi H, Kamohara S, Todaka M, Ebina Y: J Biol Chem 268: 14523-14526, 1993). We further developed the method to visualize GLUT4myc on the cell surface++ by fluorescence microscope using a highly sensitive immunochemical detection system in tissue culture cells stably expressing GLUT4myc. The translocation of GLUT4myc was observed on stimulation with insulin in 3T3-L1 adipocytes, CHO cells and L6 myotubes stably expressing GLUT4myc. Platelet-derived growth factor (PDGF), norepinephrine and bradykinin also triggered GLUT4 translocation in CHO-GLUT4myc cells stably expressing each receptor. To observe the distribution of GLUT4 and actin after insulin treatment, double staining for GLUT4myc and actin was performed. Translocated GLUT4myc on the cell surface was co-localized with rearranged actin in CHO cells and L6 myotubes. This result suggests that a correlation exists between GLUT4 translocation and actin rearrangement.
Asahi
Y
Y
Division of Molecular Genetics, University of Tokushima, Japan.
Hayashi
H
H
Wang
L
L
Ebina
Y
Y
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism*; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D001920:Bradykinin / Q000378:metabolism / Q000494:pharmacology; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051275:Glucose Transporter Type 4; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins; D009638:Norepinephrine / Q000378:metabolism / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010982:Platelet-Derived Growth Factor / Q000378:metabolism / Q000494:pharmacology; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D000077191:Wortmannin
D000199:Actins; D000730:Androstadienes; D004791:Enzyme Inhibitors; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010982:Platelet-Derived Growth Factor; D016271:Proto-Oncogene Proteins c-myc; C495692:SLC2A4 protein, human; C495693:Slc2a4 protein, mouse; C495694:Slc2a4 protein, rat; D001920:Bradykinin; D009638:Norepinephrine; D000077191:Wortmannin