High-throughput analysis of the dynamics of recycling cell surface proteins.
Recycling via the plasma membrane is a key feature that is shared by many membrane proteins. Using a combination of indirect immunofluorescence labeling and fluorescence detection using a fluorescence multiwell plate reader, we exploited the possibilities of quantitatively measuring the trafficking kinetics of transmembrane proteins. Parameters that can be studied include dynamic appearance/presence at the cell surface, recycling via the cell surface, and internalization. For the insulin-responsive glucose transporter GLUT4 (glucose transporter number 4), details are presented on how to quantitatively measure insulin-induced GLUT4 translocation toward the plasma membrane (transition state) and to analyze cell surface recycling of GLUT4 in basal and insulin-stimulated cells (steady state).
Govers
Roland
R
Faculty of Medicine, Institut national de la santé et de la recherche médicale Avenir Team, Nice, France.
James
David E
DE
Coster
Adelle C F
AC
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D002454:Cell Differentiation; D004705:Endocytosis; D019084:Fluorescent Antibody Technique, Indirect; D051275:Glucose Transporter Type 4 / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D021381:Protein Transport; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D014161:Transduction, Genetic
D051275:Glucose Transporter Type 4; D007328:Insulin; D008565:Membrane Proteins