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Imaging Phosphoinositide Dynamics in Living Cells

  • Anne Wuttke
  • Olof Idevall-Hagren
  • Anders TengholmEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words:

Phosphatidylinositol 4,5-bisphosphate Phosphatidylinositol 3,4,5-trisphosphate Phospholipase C PI3-kinase Pleckstrin homology domain Ca2+ Green fluorescent protein Total internal reflection fluorescence microscopy Insulin-secreting cell 

Notes

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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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Anne Wuttke
    • 1
  • Olof Idevall-Hagren
    • 1
  • Anders Tengholm
    • 1
    Email author
  1. 1.Department of Medical Cell BiologyUppsala UniversityUppsalaSweden

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