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

Abstract

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”.

T cell adhesion triggers an early signaling pole distal to the immune synapse

“…S1A), revealing that active kinases are recruited at this site. This supports and extends the seminal results that we have previously obtained with the probe ROZA (Randriamampita et al, 2008). Note that, when carrying out immunofluorescence experiments, the percentage of cells that display an antisynapse is lower than that measured through live-imaging experiments, as discussed in 'Kinetics and frequency analysis' in the Materials and Methods.…”

“…By using the ZAP-70 kinase reporter ROZA, we have previously made the unexpected observation that T-cell-APC contacts trigger an increase in activity of the tyrosine kinase ZAP-70, not only at the immunological synapse but also at the pole opposite to the APC, which we called the antisynapse (Randriamampita et al, 2008). Here, we show that this very frequent and early structure has a composition similar to that of the immunological synapse and forms before the synapse.…”

“…Considering that activated ZAP-70 can be observed at the antisynapse (Randriamampita et al, 2008), we looked at the distribution of Lck, LAT and the p85 subunit of the PI3K (also known as PIK3R1), which are proteins involved in early TCR signaling. Raft distribution was also followed by using mLck, the raft-anchoring sequence of Lck comprising the first 13 amino acids of the protein fused to yellow fluorescent protein (YFP).…”

“…Cells were authenticated and tested for contamination and cultured as previously described (Randriamampita et al, 2008). Human T lymphocytes and monocytes were isolated from healthy blood donors (Etablissement Français du Sang, Paris, France; relevant ethical approvals and consent have been obtained for use of human samples) by Ficoll density gradient centrifugation and negative selection for T cells (Human T-cell isolation kit, EasySep) and CD14-positive selection (CD14 MicroBeads, Miltenyi Biotec) for monocytes.…”

Modulation of T cell signaling by the actin cytoskeleton

“…When energy is non-radically transferred from a donor fluorophore to a receptor, the energy transfer efficiency is dependent on their distance. FRET has been employed to study TCR-pMHC binding kinetics , protein-protein interactions (Zal and Gascoigne, 2004) and tyrosine phosphorylation (Randriamampita et al, 2008) in T cell activation (for details, see Wouters et al, 2001).…”