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      Identification of Three Distinct Functional Sites of Insulin-mediated GLUT4 Trafficking in Adipocytes Using Quantitative Single Molecule Imaging

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          We developed a novel approach allowing intracellular GLUT4 dynamics to be analyzed directly at the single molecule level using Quantum dot to quantitatively establish the behavioral nature of GLUT4. With this approach, we defined the actual steps at which insulin signals directly converge and impact the process of dynamic GLUT4 trafficking events.


          Insulin stimulation of glucose uptake is achieved by redistribution of insulin-responsive glucose transporters, GLUT4, from intracellular storage compartment(s) to the plasma membrane in adipocytes and muscle cells. Although GLUT4 translocation has been investigated using various approaches, GLUT4 trafficking properties within the cell are largely unknown. Our novel method allows direct analysis of intracellular GLUT4 dynamics at the single molecule level by using Quantum dot technology, quantitatively establishing the behavioral nature of GLUT4. Our data demonstrate the predominant mechanism for intracellular GLUT4 sequestration in the basal state to be “static retention” in fully differentiated 3T3L1 adipocytes. We also directly defined three distinct insulin-stimulated GLUT4 trafficking processes: 1) release from the putative GLUT4 anchoring system in storage compartment(s), 2) the speed at which transport GLUT4-containing vesicles move, and 3) the tethering/docking steps at the plasma membrane. Intriguingly, insulin-induced GLUT4 liberation from its static state appeared to be abolished by either pretreatment with an inhibitor of phosphatidylinositol 3-kinase or overexpression of a dominant-interfering AS160 mutant (AS160/T642A). In addition, our novel approach revealed the possibility that, in certain insulin-resistant states, derangements in GLUT4 behavior can impair insulin-responsive GLUT4 translocation.

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          Most cited references 51

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          Measurements of trajectories of individual proteins or lipids in the plasma membrane of cells show a variety of types of motion. Brownian motion is observed, but many of the particles undergo non-Brownian motion, including directed motion, confined motion, and anomalous diffusion. The variety of motion leads to significant effects on the kinetics of reactions among membrane-bound species and requires a revision of existing views of membrane structure and dynamics.
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            Semiconductor quantum dots (QDs) are nanometer-sized fluorescent probes suitable for advanced biological imaging. We used QDs to track individual glycine receptors (GlyRs) and analyze their lateral dynamics in the neuronal membrane of living cells for periods ranging from milliseconds to minutes. We characterized multiple diffusion domains in relation to the synaptic, perisynaptic, or extrasynaptic GlyR localization. The entry of GlyRs into the synapse by diffusion was observed and further confirmed by electron microscopy imaging of QD-tagged receptors.
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                Author and article information

                Role: Monitoring Editor
                Mol Biol Cell
                Mol. Bio. Cell
                Molecular Biology of the Cell
                The American Society for Cell Biology
                1 August 2010
                : 21
                : 15
                : 2721-2731
                *Tohoku University Biomedical Engineering Research Organization, Sendai, Miyagi, 980-8575, Japan;
                Graduate School of Biomedical Engineering, Tohoku University, Sendai, Miyagi, 980-8575, Japan;
                §World Premier International Research Center, Immunology Frontier Research Center, Osaka University, Suita, Osaka, 565-0871, Japan;
                Department of Physics, Graduate School of Science, University of Tokyo, Tokyo, 113-0033, Japan; and
                Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Kawaguchi, Saitama, 332-0012, Japan
                Author notes
                Address correspondence to: Makoto Kanzaki ( kanzaki@ ).

                These authors contributed equally to this work.

                © 2010 by The American Society for Cell Biology
                Membrane Trafficking

                Molecular biology


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