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.
Biochem Biophys Res Commun
; 459(3): 405-10, 2015 Apr 10.
Article
em En
| MEDLINE
| ID: mdl-25735979
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.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Linfócitos T
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Técnicas Biossensoriais
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Transferência Ressonante de Energia de Fluorescência
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Proteína-Tirosina Quinase ZAP-70
Tipo de estudo:
Risk_factors_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2015
Tipo de documento:
Article