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Imaging Phosphoinositide Dynamics in Living Cells

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Inositol Phosphates and Lipids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 645))

Abstract

To improve our understanding of the important roles played by inositol lipid derivatives in signalling and other cellular processes, it is crucial to measure phosphoinositide concentration changes in individual cells with high spatial and temporal resolution. A number of protein domains that interact with inositol lipids in a specific manner have been identified. Tagged with the green fluorescent protein or its colour variants, these protein modules can be used as probes to visualize various phosphoinositide species in different sub-cellular compartments. Here, we present protocols for fluorescence imaging of phosphoinositide dynamics in single living cells. Total internal reflection fluorescence microscopy is particularly powerful for time-lapse recordings of phosphoinositides in the plasma membrane. We demonstrate how this technique can be used to record phospholipase C- and PI3-kinase-induced changes in inositol lipids in insulin-secreting cells. These procedures should be applicable to studies of the spatio-temporal regulation of phosphoinositide metabolism in many types of cells.

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Acknowledgments

We thank Professors Tobias Meyer, Stanford University, for the GFP-PHAkt and PLCδ1PH-GFP plasmids and Roger Tsien, University of California in San Diego, for tdimer2. The authors’ work is supported by grants from Åke Wiberg’s Foundation, the European Foundation for the Study of Diabetes/MSD, the family Ernfors Foundation, Harald and Greta Jeanssons Foundations, Novo Nordisk Foundation, the Swedish Diabetes Association and the Swedish Research Council.

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Authors and Affiliations

  1. Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden

    Anne Wuttke, Olof Idevall-Hagren & Anders Tengholm

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  1. Anne Wuttke
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  2. Olof Idevall-Hagren
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  3. Anders Tengholm
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Correspondence to Anders Tengholm .

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  1. Dept. Molecular Medicine & Surgery, Rolf Luft Research Ctr. for Diabetes, Karolinska University Hospital Solna, Stockholm, Sweden

    Christopher J. Barker

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Wuttke, A., Idevall-Hagren, O., Tengholm, A. (2010). Imaging Phosphoinositide Dynamics in Living Cells. In: Barker, C. (eds) Inositol Phosphates and Lipids. Methods in Molecular Biology, vol 645. Humana Press. https://doi.org/10.1007/978-1-60327-175-2_14

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  • DOI: https://doi.org/10.1007/978-1-60327-175-2_14

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60327-174-5

  • Online ISBN: 978-1-60327-175-2

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