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      Sites of Glucose Transporter-4 Vesicle Fusion with the Plasma Membrane Correlate Spatially with Microtubules

      1 , 2 , 1 , 2 , 1 , 2 , *

      PLoS ONE

      Public Library of Science

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          Abstract

          In adipocytes, vesicles containing glucose transporter-4 (GLUT4) redistribute from intracellular stores to the cell periphery in response to insulin stimulation. Vesicles then fuse with the plasma membrane, facilitating glucose transport into the cell. To gain insight into the details of microtubule involvement, we examined the spatial organization and dynamics of microtubules in relation to GLUT4 vesicle trafficking in living 3T3-L1 adipocytes using total internal reflection fluorescence (TIRF) microscopy. Insulin stimulated an increase in microtubule density and curvature within the TIRF-illuminated region of the cell. The high degree of curvature and abrupt displacements of microtubules indicate that substantial forces act on microtubules. The time course of the microtubule density increase precedes that of the increase in intensity of fluorescently-tagged GLUT4 in this same region of the cell. In addition, portions of the microtubules are highly curved and are pulled closer to the cell cortex, as confirmed by Parallax microscopy. Microtubule disruption delayed and modestly reduced GLUT4 accumulation at the plasma membrane. Quantitative analysis revealed that fusions of GLUT4-containing vesicles with the plasma membrane, detected using insulin-regulated aminopeptidase with a pH-sensitive GFP tag (pHluorin), preferentially occur near microtubules. Interestingly, long-distance vesicle movement along microtubules visible at the cell surface prior to fusion does not appear to account for this proximity. We conclude that microtubules may be important in providing spatial information for GLUT4 vesicle fusion.

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          Most cited references 57

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          Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.

          Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive. The first true monomer was mRFP1, derived from the Discosoma sp. fluorescent protein "DsRed" by directed evolution first to increase the speed of maturation, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions. Although mRFP1 has already proven widely useful, several properties could bear improvement and more colors would be welcome. We report the next generation of monomers. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. Three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.
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            Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.

            In neural systems, information is often carried by ensembles of cells rather than by individual units. Optical indicators provide a powerful means to reveal such distributed activity, particularly when protein-based and encodable in DNA: encodable probes can be introduced into cells, tissues, or transgenic organisms by genetic manipulation, selectively expressed in anatomically or functionally defined groups of cells, and, ideally, recorded in situ, without a requirement for exogenous cofactors. Here we describe sensors for secretion and neurotransmission that fulfil these criteria. We have developed pH-sensitive mutants of green fluorescent protein ('pHluorins') by structure-directed combinatorial mutagenesis, with the aim of exploiting the acidic pH inside secretory vesicles to monitor vesicle exocytosis and recycling. When linked to a vesicle membrane protein, pHluorins were sorted to secretory and synaptic vesicles and reported transmission at individual synaptic boutons, as well as secretion and fusion pore 'flicker' of single secretory granules.
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              Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images.

              For the investigation of the molecular mechanisms involved in neurite outgrowth and differentiation, accurate and reproducible segmentation and quantification of neuronal processes are a prerequisite. To facilitate this task, we developed a semiautomatic neurite tracing technique. This article describes the design and validation of the technique. The technique was compared to fully manual delineation. Four observers repeatedly traced selected neurites in 20 fluorescence microscopy images of cells in culture, using both methods. Accuracy and reproducibility were determined by comparing the tracings to high-resolution reference tracings, using two error measures. Labor intensiveness was measured in numbers of mouse clicks required. The significance of the results was determined by a Student t-test and by analysis of variance. Both methods slightly underestimated the true neurite length, but the differences were not unanimously significant. The average deviation from the true neurite centerline was a factor 2.6 smaller with the developed technique compared to fully manual tracing. Intraobserver variability in the respective measures was reduced by a factor 6.0 and 23.2. Interobserver variability was reduced by a factor 2.4 and 8.8, respectively, and labor intensiveness by a factor 3.3. Providing similar accuracy in measuring neurite length, significantly improved accuracy in neurite centerline extraction, and significantly improved reproducibility and reduced labor intensiveness, the developed technique may replace fully manual tracing methods. Copyright 2004 Wiley-Liss, Inc.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                20 August 2012
                : 7
                : 8
                Affiliations
                [1 ]Pennsylvania Muscle Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
                [2 ]Department of Physiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
                NHLBI, NIH, United States of America
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: JMDM EMO. Performed the experiments: JMDM. Analyzed the data: JMDM YEG EMO. Wrote the paper: JMDM YEG EMO.

                Article
                PONE-D-12-11235
                10.1371/journal.pone.0043662
                3423385
                22916292

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 12
                Funding
                JMDM was supported by a training grant from the National Institutes of Health (GM07229) and a pre-doctoral fellowship from the American Heart Association. This work was also supported through a program project grant from the NIH (P01 GM087253) to YEG and EMO. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Anatomy and Physiology
                Cell Physiology
                Biochemistry
                Cytochemistry
                Cell Membrane
                Molecular Cell Biology
                Cellular Structures
                Cytoskeleton
                Signal Transduction
                Signaling Cascades
                Insulin Signaling Cascade
                Membranes and Sorting

                Uncategorized

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