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Biochem Biophys Res Commun. 2015 Apr 10;459(3):405-10. doi: 10.1016/j.bbrc.2015.02.117. Epub 2015 Feb 28.

ROZA-XL, an improved FRET based biosensor with an increased dynamic range for visualizing zeta associated protein 70 kD (ZAP-70) tyrosine kinase activity in live T cells.

Author information

1
Aix Marseille Université, UM 61 Laboratoire d'Adhésion et Inflammation, F-1288, Marseille, France; CNRS UMR7333, F-13288, Marseille, France; INSERM U1067, F-13288, Marseille, France.
2
Graduate Program in Immunology, University of Iowa, Iowa City, IA, USA.
3
Graduate Program in Immunology, University of Iowa, Iowa City, IA, USA; Department of Microbiology, University of Iowa, Iowa City, IA, USA.
4
Aix Marseille Université, UM 61 Laboratoire d'Adhésion et Inflammation, F-1288, Marseille, France; CNRS UMR7333, F-13288, Marseille, France; INSERM U1067, F-13288, Marseille, France. Electronic address: annemarie.lellouch@inserm.fr.

Abstract

Genetically encoded FRET based biosensors allow one to visualize the spatial and temporal evolution of specific enzyme activities in live cells. We have previously reported the creation of a FRET based biosensor specific for Zeta-Associated Protein -70 kD (ZAP-70) (Randriamampita et al., 2008), a Syk family protein tyrosine kinase. ZAP-70 is essential for early T cell receptor (TCR) signaling events, T lymphocyte development and has also been implicated in integrin mediated T lymphocyte migration. In order to facilitate the study of ZAP-70 kinase activity during dynamic phenomena such as immunological synapse formation or cell migration, we have designed and prepared a second generation of ZAP-70 specific biosensors. Here we describe a novel biosensor named ROZA-XL, that displays a 3-4 times greater dynamic range than its predecessor and possesses a robust baseline FRET value when expressed in the Jurkat human T cell line. We demonstrate that the robust behavior of this biosensor allows for rapid analysis of TCR mediated of ZAP-70 kinase activity at a single cell level, as shown in a simple end point assay in which ROZA-XL expressing cells are allowed to interact with stimulatory anti-CD3epsilon coated coverslips.

KEYWORDS:

Biosensor; Fluorescence resonance energy transfer (FRET); Signal transduction; Syk family kinase; T-cell receptor; Zeta Associated Protein-70 kD (ZAP-70)

PMID:
25735979
DOI:
10.1016/j.bbrc.2015.02.117
[Indexed for MEDLINE]

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