Original Paper

Analytical and Bioanalytical Chemistry

, 393:1123

First online:

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

  • Michela SerresiAffiliated withNEST, Scuola Normale Superiore and Italian Institute of Technology Email author 
  • , Ranieri BizzarriAffiliated withNEST, Scuola Normale Superiore and Italian Institute of TechnologyNEST, Scuola Normale Superiore and CNR-INFM
  • , Francesco CardarelliAffiliated withNEST, Scuola Normale Superiore and Italian Institute of TechnologyNEST, Scuola Normale Superiore and CNR-INFM
  • , Fabio BeltramAffiliated withNEST, Scuola Normale Superiore and Italian Institute of TechnologyNEST, Scuola Normale Superiore and CNR-INFM

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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.

https://static-content.springer.com/image/art%3A10.1007%2Fs00216-008-2489-7/MediaObjects/216_2008_2489_Figa_HTML.gif
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