Genetically Encoded pH Sensor for Tracking Surface Proteins through Endocytosis

Authors

  • Anmol Grover,

    1. Department of Biological Sciences and Molecular Biosensor and Imaging Center, Carnegie Mellon University (USA)
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  • Dr. Brigitte F. Schmidt,

    1. Molecular Biosensor and Imaging Centre, Carnegie Mellon University (USA)
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  • Dr. Russell D. Salter,

    1. Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA (USA)
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  • Dr. Simon C. Watkins,

    1. Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA (USA)
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  • Dr. Alan S. Waggoner,

    1. Department of Biological Sciences and Molecular Biosensor and Imaging Center, Carnegie Mellon University (USA)
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  • Dr. Marcel P. Bruchez

    Corresponding author
    1. Department of Chemistry, Department of Biological Sciences and Molecular Biosensor and Imaging Center, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213 (USA)
    • Department of Chemistry, Department of Biological Sciences and Molecular Biosensor and Imaging Center, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213 (USA)
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  • This project was supported by grants from the National Center for Research Resources (5U54RR022241-08) and the National Institute of General Medical Sciences (8 U54 GM103529-08) from the National Institutes of Health. We would like to thank Jonathan W. Jarvik for the gift of stable ADRB2 cells, Yehuda Creeger for help with flow cytometry, and Haibing Teng for help with confocal microscopy. We would like to thank Lauren Ernst and Chris Szent-Gyorgyi for helpful discussions, and Kristen McConnell for assistance preparing the illustration. Microscopes used for the project were acquired under NIH grants 1S10RR024716 and 1S10RR026766.

Abstract

original image

Traffic cam: A tandem dye prepared from a FRET acceptor and a fluorogenic donor functions as a cell surface ratiometric pH indicator, which upon internalization serves to follow protein trafficking during endocytosis. This sensor was used to analyze agonist-dependent internalization of β2-adrenergic receptors (see scheme). It was also used as a surrogate antigen to reveal direct surface-to-endosome antigen transfer between dendritic cells (not shown).

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