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.
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.
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.
Sophie
S
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, France.
Alexia
A
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, France.
Marie-Pierre
MP
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, France.
Olivier
O
Theodoly
Graduate Program in Immunology, University of Iowa, Iowa City, IA, USA.
Aldo
A
Vacaflores
Graduate Program in Immunology, University of Iowa, Iowa City, IA, USA; Department of Microbiology, University of Iowa, Iowa City, IA, USA.
Jon C D
JC
Houtman
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.
Annemarie C
AC
Lellouch
D000595:Amino Acid Sequence; D015374:Biosensing Techniques / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D015398:Signal Transduction; D059010:Single-Cell Analysis / Q000379:methods; D013601:T-Lymphocytes / Q000201:enzymology* / Q000276:immunology; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000378:metabolism*
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human
Biosensor; Fluorescence resonance energy transfer (FRET); Signal transduction; Syk family kinase; T-cell receptor; Zeta Associated Protein-70 kD (ZAP-70)