Abstract
A genetically encoded sensor for parallel measurements of phosphatidylinositol 3-kinase activity and hydrogen peroxide (H(2)O(2)) levels (termed PIP-SHOW) was developed. Upon elevation of local phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) concentration, the sensor translocates from the cytosol to the plasma membrane, while a ratiometric excitation change rapidly and simultaneously reports changes in the concentration of H(2)O(2). The dynamics of PIP(3) and H(2)O(2) generation were monitored in platelet-derived growth factor-stimulated fibroblasts and in T-lymphocytes after formation of an immunological synapse. We suggest that PIP-SHOW can serve as a prototype for many fluorescent sensors with combined readouts.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Agammaglobulinaemia Tyrosine Kinase
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Animals
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Bacterial Proteins / chemistry*
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Fluorescent Dyes / chemistry*
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HeLa Cells
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Humans
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Hydrogen Peroxide / metabolism*
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Luminescent Proteins / chemistry*
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Mice
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Microscopy, Fluorescence
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NIH 3T3 Cells
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Oxidation-Reduction
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Phosphatidylinositol 3-Kinases / metabolism
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Phosphatidylinositol Phosphates / metabolism*
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Platelet-Derived Growth Factor / pharmacology
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Platelet-Derived Growth Factor / physiology
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Protein Structure, Tertiary
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Protein-Tyrosine Kinases / chemistry*
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Recombinant Fusion Proteins / chemistry*
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Signal Transduction
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Spectrometry, Fluorescence
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T-Lymphocytes, Helper-Inducer / enzymology
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T-Lymphocytes, Helper-Inducer / metabolism
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Time-Lapse Imaging
Substances
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Bacterial Proteins
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Fluorescent Dyes
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Luminescent Proteins
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Phosphatidylinositol Phosphates
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Platelet-Derived Growth Factor
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Recombinant Fusion Proteins
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phosphatidylinositol 3,4,5-triphosphate
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yellow fluorescent protein, Bacteria
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Hydrogen Peroxide
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Phosphatidylinositol 3-Kinases
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Protein-Tyrosine Kinases
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Agammaglobulinaemia Tyrosine Kinase