T cell receptor ligation induces the formation of dynamically regulated signaling assemblies

Bunnell, Stephen C.; Hong, Deli; Kardon, Julia R.; Yamazaki, Tetsuo; McGlade, C. Jane; Barr, Valarie A.; Samelson, Lawrence E.

doi:10.1083/jcb.200203043

Cited by 576 publications

(828 citation statements)

References 55 publications

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“…Zap70 is rapidly localized in patches (15 s) whose intensity reaches a steady state at 3 min; Zap70 remains associated with these laterally immobile clusters for 30 min (59). In contrast, adaptor signaling proteins such as LAT, Grb2, Gads, and SLP-76 show similar initiation timing but disappear rapidly, within 1-3 min, from these structures (61). This phenomenon was also observed by Houtman et al (62), who demonstrated that early Zap70 phosphorylation showed maximal effect of 50% at 19 s following anti-CD3 T cell stimulation, occurred maximally at 30 s, and persisted throughout the 120-s experiment.…”

Section: Discussionmentioning

confidence: 56%

Recombinant Anti-CD4 Antibody 13B8.2 Blocks Membrane-Proximal Events by Excluding the Zap70 Molecule and Downstream Targets SLP-76, PLCγ1, and Vav-1 from the CD4-Segregated Brij 98 Detergent-Resistant Raft Domains

Chentouf

Ghannam

Bès

et al. 2007

The Journal of Immunology

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The biological effects of rIgG1 13B8.2, directed against the CDR3-like loop on the D1 domain of CD4, are partly due to signals that prevent NF-κB nuclear translocation, but the precise mechanisms of action, particularly at the level of membrane proximal signaling, remain obscure. We support the hypothesis that rIgG1 13B8.2 acts by interfering with the spatiotemporal distribution of signaling or receptor molecules inside membrane rafts. Upon cross-linking of Jurkat T lymphocytes, rIgG1 13B8.2 was found to induce an accumulation/retention of the CD4 molecule inside polyoxyethylene-20 ether Brij 98 detergent-resistant membranes at 37°C, together with recruitment of TCR, CD3ζ, p56 Lck, Lyn, and Syk p70 kinases, linker for activation of T cells, and Csk-binding protein/phosphoprotein associated with glycosphingolipid adaptor proteins, and protein kinase Cθ, but excluded Zap70 and its downstream targets Src homology 2-domain-containing leukocyte protein of 76 kDa, phospholipase Cγ1, and p95vav. Analysis of key upstream events such as Zap70 phosphorylation showed that modulation of Tyr292 and Tyr319 phosphorylation occurred concomitantly with 13B8.2-induced Zap70 exclusion from the membrane rafts. 13B8.2-induced differential raft partitioning was epitope, cholesterol, and actin dependent but did not require Ab hyper-cross-linking. Fluorescence confocal imaging confirmed the spatiotemporal segregation of the CD4 complex inside rafts and concomitant Zap70 exclusion, which occurred within 10–30 s following rIgG1 13B8.2 ligation, reached a plateau at 1 min, and persisted until the end of the 1-h experiment. The differential spatiotemporal partitioning between the CD4 receptor and the Zap70-signaling kinase inside membrane rafts interrupts the proximal signal cross-talk leading to subsequent NF-κB nuclear translocation and explains how baculovirus-expressed CD4-CDR3-like-specific rIgG1 13B8.2 acts to induce its biological effects.

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Section: Discussionmentioning

confidence: 56%

Recombinant Anti-CD4 Antibody 13B8.2 Blocks Membrane-Proximal Events by Excluding the Zap70 Molecule and Downstream Targets SLP-76, PLCγ1, and Vav-1 from the CD4-Segregated Brij 98 Detergent-Resistant Raft Domains

Chentouf

Ghannam

Bès

et al. 2007

The Journal of Immunology

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“…The puncta containing interacting STIM1-CFP and Orai1-YFP did not colocalize with clusters containing phosphotyrosine that mark the sites of TCR clustering and activation ( Figure 1E; Bunnell et al, 2002). Thus, after TCR engagement, STIM1-CFP and Orai1-YFP closely interact in structures that are distinct from the signaling complexes that form at the activated TCR.…”

Section: After Tcr Activation Orai1-yfp and Stim1-cfp Are Seen In Pumentioning

confidence: 92%

“…Because the puncta containing STIM1-CFP and Orai1-YFP showed positive FRET indicating energy transfer, we conclude that the two proteins were within 10 nm of each other (Szollosi et al, 1998). The puncta containing interacting STIM1-CFP and Orai1-YFP did not colocalize with clusters containing phosphotyrosine that mark the sites of TCR clustering and activation ( Figure 1E; Bunnell et al, 2002). Thus, after TCR engagement, STIM1-CFP and Orai1-YFP closely interact in structures that are distinct from the signaling complexes that form at the activated TCR.…”

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confidence: 98%

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Dynamic Movement of the Calcium Sensor STIM1 and the Calcium Channel Orai1 in Activated T-Cells: Puncta and Distal Caps

Barr

Bernot

Srikanth

et al. 2008

MBoC

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129

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The proteins STIM1 and Orai1 are the long sought components of the store-operated channels required in T-cell activation. However, little is known about the interaction of these proteins in T-cells after engagement of the T-cell receptor. We found that T-cell receptor engagement caused STIM1 and Orai1 to colocalize in puncta near the site of stimulation and accumulate in a dense structure on the opposite side of the T-cell. FRET measurements showed a close interaction between STIM1 and Orai1 both in the puncta and in the dense cap-like structure. The formation of cap-like structures did not entail rearrangement of the entire endoplasmic reticulum. Cap formation depended on TCR engagement and tyrosine phosphorylation, but not on channel activity or Ca 2؉ influx. These caps were very dynamic in T-cells activated by contact with superantigen pulsed B-cells and could move from the distal pole to an existing or a newly forming immunological synapse. One function of this cap may be to provide preassembled Ca 2؉ channel components to existing and newly forming immunological synapses. INTRODUCTIONT-cell activation in response to antigen induces and requires the elevation of intracellular Ca 2ϩ (Hogan et al., 2003;Quintana et al., 2005;Feske, 2007). T-cell receptor (TCR) engagement leads to activation of well-documented signal transduction pathways that cause a rapid release of Ca 2ϩ from the endoplasmic reticulum (ER; Samelson, 2002;Panyi et al., 2004;Gwack et al., 2007a). The depletion of Ca 2ϩ from this internal store then leads to the opening of ion channels in the plasma membrane (PM) allowing an influx of Ca 2ϩ from outside the cell. The store-operated channel responsible for Ca 2ϩ influx in T-cells is known as the Ca 2ϩ release-activated Ca 2ϩ (CRAC) channel, which has been studied by electrophysiological techniques for many years (Lewis, 2001;Prakriya and Lewis, 2003;Cahalan et al., 2007;Hewavitharana et al., 2007;Hogan and Rao, 2007;Putney, 2007a).Sustained elevation of cytosolic Ca 2ϩ is required for complete T-cell activation (Iezzi et al., 1998;Lewis, 2001;Hogan et al., 2003;Feske, 2007). High levels of intracellular Ca 2ϩ are necessary to maintain the interaction between a T-cell and antigen-presenting cell (APC) that leads to formation of the specialized contact surface known as the immunological synapse (IS). Increased Ca 2ϩ levels are also required for the activation of transcription factors. In particular, elevated Ca 2ϩ maintains prolonged nuclear accumulation of nuclear factor of activated T-cells (NFAT) by activating the phosphatase calcineurin that dephosphorylates NFAT, allowing it to translocate to the nucleus and activate genes such as IL2 (Hogan et al., 2003;Macian, 2005). Several hours of Ca 2ϩ influx are required to complete the T-cell activation program, which involves expression of a large number of activation-associated genes (Lewis, 2001;Macian et al., 2002;Hogan et al., 2003).Although sustained Ca 2ϩ influx in T-cells has been studied for decades, the proteins involved have only been ident...

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“…Results based on light and fluorescence microscopy of proteins and lipids, including refinements of the established fluorescence recovery after photobleaching methods and new single particle tracking methods, have led some investigators to conclude that lipid rafts comprise a minor fraction of the cell surface and others to postulate that Ͼ50% of the plasma membrane consists of lipid rafts (Kenworthy et al, 2000;Edidin, 2001). Although biochemical analyses imply a rather unitary and stable composition for lipid rafts (Brown and London, 1998), evidence from biophysical and fluorescence imaging studies raises the possibility that raft fractions may be made up of a collection of separate membrane domains with a common ability to resist detergent solubilization (Edidin, 2001;Anderson and Jacobson, 2002;Bunnell et al, 2002).We have provided new evidence for microdomain organization of plasma membrane components based upon highresolution electron microscopy and immunogold labeling of native membrane sheets (Wilson et al, 2000(Wilson et al, , 2001. Our model system is a rat mast cell tumor line, the RBL-2H3 cell, that is rich in surface expression of the high-affinity IgE receptor, Fc⑀RI (Ͼ200,000 receptors/cell).…”

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confidence: 99%

Markers for Detergent-resistant Lipid Rafts Occupy Distinct and Dynamic Domains in Native Membranes

Wilson

Steinberg

Liederman

et al. 2004

MBoC

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187

183

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Lipid rafts isolated by detergent extraction and sucrose gradient fractionation from mast cells are enriched for the glycosylphosphatidylinositol-linked protein Thy-1, the ganglioside GM1, palmitoylated LAT, and cross-linked IgE receptors, Fc⑀RI. This study addresses the relationship of fractionation data to the organization of raft markers in native membranes. Immunogold labeling and electron microscopy shows there is little or no colocalization of the raft markers Thy-1, GM1, and LAT with each other or with Fc⑀RI on native membrane sheets prepared from unstimulated cells. External cross-linking of Thy-1 promotes coclustering of Thy-1 with LAT, but not with GM1. Thy-1 and LAT clusters occur on membrane regions without distinctive features. In contrast, external cross-linking of Fc⑀RI and GM1 causes their redistribution to electron-dense membrane patches independently of each other and of Thy-1. The distinctive patches that accumulate cross-linked Fc⑀RI and GM1 also accumulate osmium, a stain for unsaturated lipids, and are sites for coated vesicle budding. Electron microscopy reveals a more complex and dynamic topographical organization of membrane microdomains than is predicted by biochemical analysis of detergent-resistant membranes. INTRODUCTIONOrdered regions of membrane, known as microdomains, lipid rafts, detergent-resistant membranes (DRMs), and other abbreviations are thought to be critical sites of signal propagation and membrane trafficking (Edidin, 1997(Edidin, , 2001Simons and Ikonen, 1997;Anderson, 1998; London, 1998, 2000;Jacobson and Dietrich, 1999;Langlet et al., 2000;Anderson and Jacobson, 2002). Analysis of these regions typically begins with detergent solubilization of whole cells followed by sucrose density gradient centrifugation and the recovery of detergent-resistant membranes from the light fractions of the gradient. The DRMs are enriched for caveolin, glycosylphosphatidylinositol-linked (GPI-linked) proteins, glycosphingolipids, GM1 ganglioside, and cholesterol, suggesting that these components are associated in the liquid-ordered (l o ) phase of the lipid bilayer (Schroeder et al., 1994;Ahmed et al., 1997). The interpretation of gradient centrifugation experiments remains controversial. There is evidence that detergents may force associations between components that are not colocalized in intact cells (Mayor and Maxfield, 1995), and fractionation results are known to be dramatically altered by varying the concentration of Triton X-100 (Field et al., 1999;Parolini et al., 1999), by use of alternative detergents (Montixi et al., 1998;Surviladze et al., 1998) or by omission of detergent altogether (Ilangumaran et al., 1999;Harder and Kuhn, 2000;Surviladze et al., 2001). Methods to observe membrane segregation in situ have also generated controversy. Results based on light and fluorescence microscopy of proteins and lipids, including refinements of the established fluorescence recovery after photobleaching methods and new single particle tracking methods, have led some investigators to co...

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T cell receptor ligation induces the formation of dynamically regulated signaling assemblies

Cited by 576 publications

References 55 publications

Recombinant Anti-CD4 Antibody 13B8.2 Blocks Membrane-Proximal Events by Excluding the Zap70 Molecule and Downstream Targets SLP-76, PLCγ1, and Vav-1 from the CD4-Segregated Brij 98 Detergent-Resistant Raft Domains

Recombinant Anti-CD4 Antibody 13B8.2 Blocks Membrane-Proximal Events by Excluding the Zap70 Molecule and Downstream Targets SLP-76, PLCγ1, and Vav-1 from the CD4-Segregated Brij 98 Detergent-Resistant Raft Domains

Dynamic Movement of the Calcium Sensor STIM1 and the Calcium Channel Orai1 in Activated T-Cells: Puncta and Distal Caps

Markers for Detergent-resistant Lipid Rafts Occupy Distinct and Dynamic Domains in Native Membranes

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