Journal article
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
Biochemical and biophysical research communications, Vol.459(3), pp.405-410
04/10/2015
DOI: 10.1016/j.bbrc.2015.02.117
PMID: 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.
Details
- Title: Subtitle
- 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
- Creators
- Sophie Cadra - 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, FranceAlexia Gucciardi - 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, FranceMarie-Pierre Valignat - 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, FranceOlivier Theodoly - 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, FranceAldo Vacaflores - Graduate Program in Immunology, University of Iowa, Iowa City, IA, USAJon C D Houtman - Graduate Program in Immunology, University of Iowa, Iowa City, IA, USA; Department of Microbiology, University of Iowa, Iowa City, IA, USAAnnemarie C Lellouch - 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
- Resource Type
- Journal article
- Publication Details
- Biochemical and biophysical research communications, Vol.459(3), pp.405-410
- DOI
- 10.1016/j.bbrc.2015.02.117
- PMID
- 25735979
- ISSN
- 0006-291X
- eISSN
- 1090-2104
- Grant note
- name: Association pour la Recherche contre le Cancer; name: ANR Jeune Chercheur Programme «DissecTion», award: ANR-09-JCJC-0091; name: Fondation de Recherche Medicale; name: CNRS Programme Prise de Risques; name: Provence-Alpes Côte d’Azur
- Language
- English
- Date published
- 04/10/2015
- Academic Unit
- Microbiology and Immunology; Internal Medicine
- Record Identifier
- 9984094395102771
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