Real-time measurement of endosomal acidification by a novel genetically encoded biosensor

  • Michela Serresi
  • Ranieri Bizzarri
  • Francesco Cardarelli
  • Fabio Beltram
Original Paper

Abstract

Genetically encoded fluorescent proteins are optimal reporters when used to monitor cellular processes as they can be targeted to any subcellular region by fusion to a protein of interest. Here, we present the pH-sensitive fluorescent protein E1GFP which is ideally suited to monitor pH changes in dynamic intracellular structures in real time with high spatio temporal resolution. E1GFP is a ratiometric pH indicator by emission with a pK close to 6.0. We describe an application of this novel pH reporter in the measurement of pH changes along the endo-lysosomal pathway. By fusing E1GFP to the HIV-Tat protein which is endowed with cell-penetrating properties, we were able to monitor multi-step endocytosis from the initial cell-surface binding through to the intracellular endocytic network in real time. This represents a framework for the application of E1GFP to the in situ detection of pH changes involved in dynamic biological phenomena.

Figure

The green fluorecent protein variant, E1GFP, is a ratiometric pH-indicator by emission with a pK close to 6.0 and is therefore particularly suitable for pH detection below neutrality. Upon excitation of the neutral state of the chromophore (~400-410 nm), E1GFP emission properties are strongly dependent on the environmental pH. We describe an application of this novel pH-reporter in the measurement of pH changes along the endo-lysosomal pathway. By fusing E1GFP to the HIV-Tat protein, which is endowed with cell-penetrating properties, we were able to monitor in real-time multi-step endocytosis from the initial cell-surface binding through to the intracellular endocytic network.

Keywords

Biosensor Endocytosis GFP In vivo imaging pH measurement Tat-HIV protein 

Supplementary material

ESM 1

(MOV 453 KB).

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Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Michela Serresi
    • 1
  • Ranieri Bizzarri
    • 1
    • 2
  • Francesco Cardarelli
    • 1
    • 2
  • Fabio Beltram
    • 1
    • 2
  1. 1.NEST, Scuola Normale Superiore and Italian Institute of TechnologyPisaItaly
  2. 2.NEST, Scuola Normale Superiore and CNR-INFMPisaItaly

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