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A novel ZAP-70 dependent FRET based biosensor reveals kinase activity at both the immunological synapse and the antisynapse.

Randriamampita C, Mouchacca P, Malissen B, Marguet D, Trautmann A, Lellouch AC - PLoS ONE (2008)

Bottom Line: LAT phosphorylation results in the recruitment of a signalosome including PLCgamma1, 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.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".

View Article: PubMed Central - PubMed

Affiliation: Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France.

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 PLCgamma1, 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".

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Synaptic and antisynaptic activation of ROZA.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.
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pone-0001521-g004: Synaptic and antisynaptic activation of ROZA.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.

Mentions: In order to validate the utility of ROZA for examining the evolution and subcellular distribution of ZAP-70 dependent phosphorylation during a cell-cell activation event, we followed the formation of conjugates between Jurkat clones stably expressing ROZA and Raji B cells loaded with superantigen under 40X magnification. Rapidly after conjugate formation, a synaptic clustering of the probe was observed. Such a recruitment could be due to the fact that the probe is anchored to the membrane through a palmitoylated and myristylated N-terminal sequence derived from the Src kinase Lck, that presumably targets ROZA into lipid rafts [20]. Accumulation of ROZA at the immunological synapse was generally quite marked (Fig. 4). However ZAP-70 dependent phosphorylation as revealed by the ROZA FRET change was detected before the accumulation of the probe at the synapse (data not shown). The ROZA signal was triggered and reached its maximal value within a minute of contact formation, consistent with literature reports of global LAT phosphorylation kinetics [21]. Unexpectedly, ROZA accumulation and FRET evolution was also frequently observed at the cell pole opposite to the synapse, or the “antisynapse”. In some cases, ROZA FRET and accumulation was observed first at the synapse whereas in other cases it was observed first at the antisynapse. Fig. 4 illustrates a case where both poles appeared simultaneously. Antisynaptic localisation such as we observed with ROZA has previously been observed with other synaptic molecules such as CD3, or PIP3 [22], [23]. However little information is currently available about either the dynamics or the mechanisms underlying this phenomenon.


A novel ZAP-70 dependent FRET based biosensor reveals kinase activity at both the immunological synapse and the antisynapse.

Randriamampita C, Mouchacca P, Malissen B, Marguet D, Trautmann A, Lellouch AC - PLoS ONE (2008)

Synaptic and antisynaptic activation of ROZA.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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211399&req=5

pone-0001521-g004: Synaptic and antisynaptic activation of ROZA.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.
Mentions: In order to validate the utility of ROZA for examining the evolution and subcellular distribution of ZAP-70 dependent phosphorylation during a cell-cell activation event, we followed the formation of conjugates between Jurkat clones stably expressing ROZA and Raji B cells loaded with superantigen under 40X magnification. Rapidly after conjugate formation, a synaptic clustering of the probe was observed. Such a recruitment could be due to the fact that the probe is anchored to the membrane through a palmitoylated and myristylated N-terminal sequence derived from the Src kinase Lck, that presumably targets ROZA into lipid rafts [20]. Accumulation of ROZA at the immunological synapse was generally quite marked (Fig. 4). However ZAP-70 dependent phosphorylation as revealed by the ROZA FRET change was detected before the accumulation of the probe at the synapse (data not shown). The ROZA signal was triggered and reached its maximal value within a minute of contact formation, consistent with literature reports of global LAT phosphorylation kinetics [21]. Unexpectedly, ROZA accumulation and FRET evolution was also frequently observed at the cell pole opposite to the synapse, or the “antisynapse”. In some cases, ROZA FRET and accumulation was observed first at the synapse whereas in other cases it was observed first at the antisynapse. Fig. 4 illustrates a case where both poles appeared simultaneously. Antisynaptic localisation such as we observed with ROZA has previously been observed with other synaptic molecules such as CD3, or PIP3 [22], [23]. However little information is currently available about either the dynamics or the mechanisms underlying this phenomenon.

Bottom Line: LAT phosphorylation results in the recruitment of a signalosome including PLCgamma1, 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.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".

View Article: PubMed Central - PubMed

Affiliation: Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France.

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 PLCgamma1, 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".

Show MeSH
Related in: MedlinePlus