A Novel ZAP-70 Dependent FRET Based Biosensor Reveals Kinase Activity at both the Immunological Synapse and the Antisynapse

Many hypotheses attempting to explain the speed and sensitivity with which a T-cell discriminates the antigens it encounters include a notion of relative spatial and temporal control of particular biochemical steps involved in the process. An essential step in T-cell receptor (TCR) mediated signalling is the activation of the protein tyrosine kinase ZAP-70. ZAP-70 is recruited to the TCR upon receptor engagement and, once activated, is responsible for the phosphorylation of the protein adaptor, Linker for Activation of T-cells, or LAT. LAT phosphorylation results in the recruitment of a signalosome including PLCγ1, Grb2/SOS, GADS and SLP-76. In order to examine the real time spatial and temporal evolution of ZAP-70 activity following TCR engagement in the immune synapse, we have developed ROZA, a novel FRET-based biosensor whose function is dependent upon ZAP-70 activity. This new probe not only provides a measurement of the kinetics of ZAP-70 activity, but also reveals the subcellular localization of the activity as well. Unexpectedly, ZAP-70 dependent FRET was observed not only at the T-cell -APC interface, but also at the opposite pole of the cell or “antisynapse”

A peptide model for the heparin binding site on Antithrombin III

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 1992Includes bibliographical references.by Annemarie Coffman Lellouch.Ph. D.Ph. D. Massachusetts Institute of Technology, Department of Chemistr

Synaptic and antisynaptic activation of ROZA.

<p>Sequence of events observed upon interaction of a ROZA-expressing Jurkat T-cell with superantigen-loaded Raji B cells. Left: transmitted light images; center: subcellular ROZA localization; right: ZAP-70-dependent activity in false colours, 1/R ranging from 1.25 (blue) to 1.7 (red). Time zero corresponds to the initial contact, as detected in transmitted light images. The bar corresponds to 10 microns.</p

Structure and subcellular localization of ROZA.

<p>(A) Scheme of the ROZA sequence including the N-terminal residues from p56 Lck, the linker sequence and residues 171-178 of LAT (underlined) surrounding Y175 (in bold). (B) Model showing the schematic structure of ROZA (without its membrane anchor), and illustrating that, in the absence of ZAP-70 activity, ROZA may adopt several conformations, some of them allowing FRET. Following phosphorylation of the LAT based sequence by ZAP-70, ROZA adopts a constrained conformation incompatible with FRET. (C) In Jurkat T-cells transiently transfected with ROZA, the probe is mainly located at the plasma membrane as visualized by CFP fluorescence.</p