Functional Nanoscale Organization of Signaling Molecules Downstream of the T Cell Antigen Receptor (original) (raw)
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Mechanisms of localized activation of the T cell antigen receptor inside clusters
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2015
The T cell antigen receptor (TCR) has been shown to cluster both before and upon engagement with cognate antigens. However, the effect of TCR clustering on its activation remains poorly understood. Here, we used two-color photo-activated localization microscopy (PALM) to visualize individual molecules of TCR and ZAP-70, as a marker of TCR activation and phosphorylation, at the plasma membrane of uniformly activated T cells. Imaging and second-order statistics revealed that ZAP-70 recruitment and TCR activation localized inside TCR clusters. Live cell PALM imaging showed that the extent of localized TCR activation decreased, yet remained significant, with cell spreading. Using dynamic modeling and Monte-Carlo simulations we evaluated possible mechanisms of localized TCR activation. Our simulations indicate that localized TCR activation is the result of long-range cooperative interactions between activated TCRs, or localized activation by Lck and Fyn. Our results demonstrate the role of molecular clustering in cell signaling and activation, and are relevant to studying a wide range of multi-molecular complexes. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.
Development of nanoscale structure in LAT-based signaling complexes
Journal of Cell Science, 2016
The adapter molecule Linker for Activation of T cells (LAT) plays a critical role in forming signaling complexes induced by stimulation of the T cell receptor (TCR). These multi-molecular complexes are dynamic structures that activate highly regulated signaling pathways. Previously, we demonstrated nanoscale structure in LAT-based complexes where the adapter SLP-76 localizes to the periphery of LAT clusters. In this study, we show that initially LAT and SLP-76 are randomly dispersed throughout the clusters that form upon TCR engagement. The segregation of LAT and SLP-76 develops near the end of the spreading process. The local concentration of LAT also increases at the same time. Both changes require TCR activation and an intact actin cytoskeleton. These results demonstrate that the nanoscale organization of LAT-based signaling complexes is dynamic and indicates that different kinds of LAT-based complexes appear at different times during T cell activation.
T cell receptor ligation induces the formation of dynamically regulated signaling assemblies
The Journal of Cell Biology, 2002
cell antigen receptor (TCR) ligation initiates tyrosine kinase activation, signaling complex assembly, and immune synapse formation. Here, we studied the kinetics and mechanics of signaling complex formation in live Jurkat leukemic T cells using signaling proteins fluorescently tagged with variants of enhanced GFP (EGFP). Within seconds of contacting coverslips coated with stimulatory antibodies, T cells developed small, dynamically regulated clusters which were enriched in the TCR, phosphotyrosine, ZAP-70, LAT, Grb2, Gads, and SLP-76, excluded the lipid raft marker enhanced yellow fluorescent protein-GPI, and T were competent to induce calcium elevations. LAT, Grb2, and Gads were transiently associated with the TCR. Although ZAP-70-containing clusters persisted for more than 20 min, photobleaching studies revealed that ZAP-70 continuously dissociated from and returned to these complexes. Strikingly, SLP-76 translocated to a perinuclear structure after clustering with the TCR. Our results emphasize the dynamically changing composition of signaling complexes and indicate that these complexes can form within seconds of TCR engagement, in the absence of either lipid raft aggregation or the formation of a central TCR-rich cluster.
Hierarchical nanostructure and synergy of multimolecular signalling complexes
Nature communications, 2016
Signalling complexes are dynamic, multimolecular structures and sites for intracellular signal transduction. Although they play a crucial role in cellular activation, current research techniques fail to resolve their structure in intact cells. Here we present a multicolour, photoactivated localization microscopy approach for imaging multiple types of single molecules in fixed and live cells and statistical tools to determine the nanoscale organization, topology and synergy of molecular interactions in signalling complexes downstream of the T-cell antigen receptor. We observe that signalling complexes nucleated at the key adapter LAT show a hierarchical topology. The critical enzymes PLCγ1 and VAV1 localize to the centre of LAT-based complexes, and the adapter SLP-76 and actin molecules localize to the periphery. Conditional second-order statistics reveal a hierarchical network of synergic interactions between these molecules. Our results extend our understanding of the nanostructure...
Scientific Reports, 2015
Two related models for T cell signalling initiation suggest either that T cell receptor (TCR) engagement leads to its recruitment to ordered membrane domains, often referred to as lipid rafts, where signalling molecules are enriched or that ordered TCR-containing membrane nanodomains coalesce upon TCR engagement. That ordered domains form upon TCR engagement, as they do upon lipid raft marker patching, has not been considered. The target of this study was to differentiate between those three options. Plasma membrane order was followed in live T cells at 37 °C using laurdan to report on lipid packing. Patching of the TCR that elicits a signalling response resulted in aggregation, not formation, of ordered plasma membrane domains in both Jurkat and primary T cells. The TCR colocalised with actin filaments at the plasma membrane in unstimulated Jurkat T cells, consistent with it being localised to ordered membrane domains. The colocalisation was most prominent in cells in G1 phase when the cells are ready to commit to proliferation. At other cell cycle phases the TCR was mainly found at perinuclear membranes. Our study suggests that the TCR resides in ordered plasma membrane domains that are linked to actin filaments and aggregate upon TCR engagement. Ordered membrane nanodomains, often referred to as lipid rafts, are implicated in immune cell signalling. They are considered to form by the self-aggregation of cholesterol and sphingolipids 1 and are believed to exist as liquid ordered (lo) domains, in contrast to the rest of the membrane that exists liquid disordered (ld) domains. However, both are liquid phases and diffusion can take place inside, around as well as into and out of the domains so membrane components continuously shift between domains and their surroundings. T cell signalling is initiated by Src family tyrosine kinases, Lck and Fyn, by phosphorylation of immunoreceptor tyrosine based activation motifs (ITAMs) in the CD3 subunits of the T cell receptor (TCR). Downstream signalling involves the activation of Ras and calcium pathways. All these pathways can be activated by crosslinking different lipid raft components, the ganglioside GM1 or the GPI-anchored protein CD59, suggesting a link between the aggregation of ordered membrane nanodomains and early T cell signalling 2-4. Cold stress and moderate cholesterol depletion can also lead to lipid raft aggregation and T cell activation 5,6. Formation of an immunological synapse (IS) occurs after the initial signalling events 7 and is accomplished by the transport of microclusters containing the TCR and signalling proteins along both actin filaments and microtubules to form a central supramolecular activation cluster (cSMAC) 8-10. The IS in both fixed and live T cells has been shown to contain ordered membrane domains 11,12. In addition the
Biophysical Journal, 2015
Two related models for T cell signalling initiation suggest either that T cell receptor (TCR) engagement leads to its recruitment to ordered membrane domains, often referred to as lipid rafts, where signalling molecules are enriched or that ordered TCR-containing membrane nanodomains coalesce upon TCR engagement. That ordered domains form upon TCR engagement, as they do upon lipid raft marker patching, has not been considered. The target of this study was to differentiate between those three options. Plasma membrane order was followed in live T cells at 37 °C using laurdan to report on lipid packing. Patching of the TCR that elicits a signalling response resulted in aggregation, not formation, of ordered plasma membrane domains in both Jurkat and primary T cells. The TCR colocalised with actin filaments at the plasma membrane in unstimulated Jurkat T cells, consistent with it being localised to ordered membrane domains. The colocalisation was most prominent in cells in G1 phase when the cells are ready to commit to proliferation. At other cell cycle phases the TCR was mainly found at perinuclear membranes. Our study suggests that the TCR resides in ordered plasma membrane domains that are linked to actin filaments and aggregate upon TCR engagement. Ordered membrane nanodomains, often referred to as lipid rafts, are implicated in immune cell signalling. They are considered to form by the self-aggregation of cholesterol and sphingolipids 1 and are believed to exist as liquid ordered (lo) domains, in contrast to the rest of the membrane that exists liquid disordered (ld) domains. However, both are liquid phases and diffusion can take place inside, around as well as into and out of the domains so membrane components continuously shift between domains and their surroundings. T cell signalling is initiated by Src family tyrosine kinases, Lck and Fyn, by phosphorylation of immunoreceptor tyrosine based activation motifs (ITAMs) in the CD3 subunits of the T cell receptor (TCR). Downstream signalling involves the activation of Ras and calcium pathways. All these pathways can be activated by crosslinking different lipid raft components, the ganglioside GM1 or the GPI-anchored protein CD59, suggesting a link between the aggregation of ordered membrane nanodomains and early T cell signalling 2-4. Cold stress and moderate cholesterol depletion can also lead to lipid raft aggregation and T cell activation 5,6. Formation of an immunological synapse (IS) occurs after the initial signalling events 7 and is accomplished by the transport of microclusters containing the TCR and signalling proteins along both actin filaments and microtubules to form a central supramolecular activation cluster (cSMAC) 8-10. The IS in both fixed and live T cells has been shown to contain ordered membrane domains 11,12. In addition the
Characterization of dynamic actin associations with T-cell receptor microclusters in primary T cells
T cell triggering through T-cell antigen receptors (TCRs) results in spatial assembly of the receptors on multiple length scales. This assembly is mediated by the T cell actin cytoskeleton, which reorganizes in response to TCR phosphorylation and then induces the coalescence of TCRs into microclusters, followed by their unification into a micrometer-scale structure. The exact outcomes of the association of TCRs with a dynamic and fluctuating actin network across these length scales are not well characterized, but it is clear that weak and transient interactions at the single-molecule level sum to yield significant receptor rearrangements at the plasma membrane. We used the hybrid live cell-nanopatterned supported lipid bilayer system to quantitatively probe the actin-TCR interaction in primary T cells. A specialized tracking algorithm revealed that actin slows as it passes over TCR clusters in a direction-dependent manner with respect to the resistance against TCR motion. We also observed transient actin enrichments at sites corresponding to putative TCR clusters that far exceeded pure stochastic fluctuations and described an image time-autocorrelation analysis method to quantify these accumulations.
Full control of ligand positioning reveals spatial thresholds for T cell receptor triggering
Nature nanotechnology, 2018
Elucidating the rules for receptor triggering in cell-cell and cell-matrix contacts requires precise control of ligand positioning in three dimensions. Here, we use the T cell receptor (TCR) as a model and subject T cells to different geometric arrangements of ligands, using a nanofabricated single-molecule array platform. This comprises monovalent TCR ligands anchored to lithographically patterned nanoparticle clusters surrounded by mobile adhesion molecules on a supported lipid bilayer. The TCR ligand could be co-planar with the supported lipid bilayer (2D), excluding the CD45 transmembrane tyrosine phosphatase, or elevated by 10 nm on solid nanopedestals (3D), allowing closer access of CD45 to engaged TCR. The two configurations resulted in different T cell responses, depending on the lateral spacing between the ligands. These results identify the important contributions of lateral and axial components of ligand positioning and create a more complete foundation for receptor engin...
Distinct Mechanisms regulate lck spatial Organization in activated T cells
Phosphorylation of the T cell receptor (TCR) by the kinase Lck is the first detectable signaling event upon antigen engagement. The distribution of Lck within the plasma membrane, its conformational state, kinase activity, and protein–protein interactions all contribute to determine how efficiently Lck phosphorylates the engaged TCR. Here, we used cross-correlation raster image correlation spectroscopy and photoactivated localization microscopy to identify two mechanisms of Lck clustering: an intrinsic mechanism of Lck clustering induced by locking Lck in its open conformation and an extrinsic mechanism of clustering controlled by the phosphorylation of tyrosine 192, which regulates the affinity of Lck SH2 domain. Both mechanisms of clustering were differently affected by the absence of the kinase Zap70 or the adaptor Lat. We further observed that the adaptor TSAd bound to and promoted the diffusion of Lck when it is phosphorylated on tyrosine 192. Our data suggest that while Lck open conformation drives aggregation and clustering, the spatial organization of Lck is further controlled by signaling events downstream of TCR phosphorylation.