Visualization of cellular phosphoinositide pools with GFP-fused protein-domains

Tamas Balla, P. Várnai

Research output: Contribution to journalArticle

51 Citations (Scopus)

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 singlecell 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.

Original languageEnglish
JournalCurrent Protocols in Cell Biology
Issue numberSUPPL. 42
DOIs
Publication statusPublished - 2009

Fingerprint

Phosphatidylinositols
Membranes
Personnel Selection
Protein Domains
Eukaryotic Cells
Phosphoric Monoester Hydrolases
Membrane Proteins
Proteins
Phosphotransferases
Lipids
Enzymes

Keywords

  • Fluorescence microscopy
  • FRET
  • Green fluorescent proteins
  • Live-cell imaging
  • Phosphoinositide
  • Pleckstrin homology domain

ASJC Scopus subject areas

  • Cell Biology

Cite this

Visualization of cellular phosphoinositide pools with GFP-fused protein-domains. / Balla, Tamas; Várnai, P.

In: Current Protocols in Cell Biology, No. SUPPL. 42, 2009.

Research output: Contribution to journalArticle

@article{893abe4c20664bbfa4b176226aec7c33,
title = "Visualization of cellular phosphoinositide pools with GFP-fused protein-domains",
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 singlecell 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.",
keywords = "Fluorescence microscopy, FRET, Green fluorescent proteins, Live-cell imaging, Phosphoinositide, Pleckstrin homology domain",
author = "Tamas Balla and P. V{\'a}rnai",
year = "2009",
doi = "10.1002/0471143030.cb2404s42",
language = "English",
journal = "Current Protocols in Cell Biology",
issn = "1934-2500",
publisher = "John Wiley and Sons Inc.",
number = "SUPPL. 42",

}

TY - JOUR

T1 - Visualization of cellular phosphoinositide pools with GFP-fused protein-domains

AU - Balla, Tamas

AU - Várnai, P.

PY - 2009

Y1 - 2009

N2 - 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 singlecell 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.

AB - 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 singlecell 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.

KW - Fluorescence microscopy

KW - FRET

KW - Green fluorescent proteins

KW - Live-cell imaging

KW - Phosphoinositide

KW - Pleckstrin homology domain

UR - http://www.scopus.com/inward/record.url?scp=63649110520&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=63649110520&partnerID=8YFLogxK

U2 - 10.1002/0471143030.cb2404s42

DO - 10.1002/0471143030.cb2404s42

M3 - Article

C2 - 19283730

AN - SCOPUS:63649110520

JO - Current Protocols in Cell Biology

JF - Current Protocols in Cell Biology

SN - 1934-2500

IS - SUPPL. 42

ER -