Unit

UNIT 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains

  1. Tamas Balla1,
  2. Péter Várnai2

Published Online: 1 MAR 2009

DOI: 10.1002/0471143030.cb2404s42

Current Protocols in Cell Biology

Current Protocols in Cell Biology

How to Cite

Balla, T. and Várnai, P. 2009. Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains. Current Protocols in Cell Biology. 42:24.4:24.4.1–24.4.27.

Author Information

  1. 1

    National Institutes of Health, Bethesda, Maryland

  2. 2

    Semmelweis University of Medical School, Budapest, Hungary

Publication History

  1. Published Online: 1 MAR 2009
  2. Published Print: MAR 2009

Abstract

This unit describes the method of following phosphoinositide dynamics in live cells. Inositol phospholipids have emerged as universal signaling molecules present in virtually every membrane of eukaryotic cells. Phosphoinositides are present in only tiny amounts as compared to structural lipids, but they are metabolically very active as they are produced and degraded by the numerous inositide kinase and phosphatase enzymes. Phosphoinositides control the membrane recruitment and activity of many membrane protein signaling complexes in specific membrane compartments, and they have been implicated in the regulation of a variety of signaling and trafficking pathways. It has been a challenge to develop methods that allow detection of phosphoinositides at the single-cell level. The only available technique in live cell applications is based on the use of the same protein domains selected by evolution to recognize cellular phosphoinositides. Some of these isolated protein modules, when fused to fluorescent proteins, can follow dynamic changes in phosphoinositides. While this technique can provide information on phosphoinositide dynamics in live cells with subcellular localization, and it has rapidly gained popularity, it also has several limitations that must be taken into account when interpreting the data. This unit summarizes the design and practical use of these constructs and also reviews important considerations for interpretation of the data obtained by this technique. Curr. Protoc. Cell Biol. 42:24.4.1-24.4.27. © 2009 by John Wiley & Sons, Inc.

Keywords:

  • phosphoinositide;
  • FRET;
  • live-cell imaging;
  • green fluorescent proteins;
  • pleckstrin homology domain;
  • fluorescence microscopy