Multi-colour DNA-qPAINT reveals how Csk nano-clusters regulate T-cell receptor signalling

2019 ◽  
Author(s):  
Sabrina Simoncelli ◽  
Juliette Griffié ◽  
David J. Williamson ◽  
Jack Bibby ◽  
Cara Bray ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Single Molecule ◽  
Spatial Organization ◽  
Regulatory Element ◽  
Tyrosine Phosphatase ◽  
Super Resolution ◽  
Cell Receptor ◽  
Knock Out ◽  
Spatio Temporal

AbstractPhosphorylation of the negative regulatory element of the tyrosine kinase Lck by Csk down-modulates T-cell receptor induced signalling. Being constitutively active, Csk spatial organization is responsible for regulating this signalling interaction. Here, we used stoichiometrically accurate, multiplexed, single-molecule super-resolution microscopy (DNA-qPAINT) to image the nanoscale spatial architecture of Csk and two binding partners implicated in its membrane association – PAG and TRAF3. Combined with a newly developed co-clustering analysis framework, we provide a powerful resource for dissecting signalling pathways regulated by spatio-temporal organisation. We found that Csk forms nanoscale clusters proximal to the plasma membrane that are lost post-stimulation and re-recruited at later time points. Unexpectedly, these clusters do not directly co-localise with PAG at the membrane, but instead provide a ready pool of monomers to down-regulate signalling. By generating CRISPR/Cas9 knock-out T-cells, our data also identify that protein tyrosine phosphatase non-receptor type 22 (PTPN22) is essential for Csk nanocluster re-recruitment and for maintenance of the synaptic PAG population.

scholarly journals Clus-DoC: a combined cluster detection and colocalization analysis for single-molecule localization microscopy data

2016 ◽  
Vol 27 (22) ◽  
pp. 3627-3636 ◽  
Author(s):  
Sophie V. Pageon ◽  
Philip R. Nicovich ◽  
Mahdie Mollazade ◽  
Thibault Tabarin ◽  
Katharina Gaus
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Single Molecule ◽  
Cell Activation ◽  
Spatial Organization ◽  
Focal Adhesions ◽  
Cell Receptor ◽  
Intact Cells ◽  
Analysis Strategy ◽  
Signaling Activity

Advances in fluorescence microscopy are providing increasing evidence that the spatial organization of proteins in cell membranes may facilitate signal initiation and integration for appropriate cellular responses. Our understanding of how changes in spatial organization are linked to function has been hampered by the inability to directly measure signaling activity or protein association at the level of individual proteins in intact cells. Here we solve this measurement challenge by developing Clus-DoC, an analysis strategy that quantifies both the spatial distribution of a protein and its colocalization status. We apply this approach to the triggering of the T-cell receptor during T-cell activation, as well as to the functionality of focal adhesions in fibroblasts, thereby demonstrating an experimental and analytical workflow that can be used to quantify signaling activity and protein colocalization at the level of individual proteins.

Altered enteroendocrine cell expression in T cell receptor alpha chain knock-out mice

2000 ◽  
Vol 51 (2) ◽  
pp. 112-120 ◽  
Author(s):  
Deborah C. Rubin ◽  
Huiying Zhang ◽  
Peiqing Qian ◽  
Robinna G. Lorenz ◽  
Karen Hutton ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Enteroendocrine Cell ◽  
Knock Out ◽  
Receptor Alpha ◽  
Knock Out Mice ◽  
Cell Expression ◽  
Receptor Alpha Chain ◽  
Cell Receptor Alpha

scholarly journals Determination of Interaction Kinetics between the T Cell Receptor and Peptide-Loaded MHC Class II via Single-Molecule Diffusion Measurements

2012 ◽  
Vol 103 (2) ◽  
pp. L17-L19 ◽  
Author(s):  
Markus Axmann ◽  
Johannes B. Huppa ◽  
Mark M. Davis ◽  
Gerhard J. Schütz
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Mhc Class Ii ◽  
Single Molecule ◽  
Cell Receptor ◽  
Class Ii ◽  
Interaction Kinetics

scholarly journals Early T cell receptor signals globally modulate ligand:receptor affinities during antigen discrimination

2017 ◽  
Vol 114 (46) ◽  
pp. 12190-12195 ◽  
Author(s):  
Rafal M. Pielak ◽  
Geoff P. O’Donoghue ◽  
Jenny J. Lin ◽  
Katherine N. Alfieri ◽  
Nicole C. Fay ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Single Molecule ◽  
Cell Activation ◽  
Cell Receptor ◽  
Physical Constraints ◽  
Molecular Binding ◽  
Antigen Presenting

Antigen discrimination by T cells occurs at the junction between a T cell and an antigen-presenting cell. Juxtacrine binding between numerous adhesion, signaling, and costimulatory molecules defines both the topographical and lateral geometry of this cell–cell interface, within which T cell receptor (TCR) and peptide major histocompatibility complex (pMHC) interact. These physical constraints on receptor and ligand movement have significant potential to modulate their molecular binding properties. Here, we monitor individual ligand:receptor binding and unbinding events in space and time by single-molecule imaging in live primary T cells for a range of different pMHC ligands and surface densities. Direct observations of pMHC:TCR and CD80:CD28 binding events reveal that the in situ affinity of both pMHC and CD80 ligands for their respective receptors is modulated by the steady-state number of agonist pMHC:TCR interactions experienced by the cell. By resolving every single pMHC:TCR interaction it is evident that this cooperativity is accomplished by increasing the kinetic on-rate without altering the off-rate and has a component that is not spatially localized. Furthermore, positive cooperativity is observed under conditions where the T cell activation probability is low. This TCR-mediated feedback is a global effect on the intercellular junction. It is triggered by the first few individual pMHC:TCR binding events and effectively increases the efficiency of TCR scanning for antigen before the T cell is committed to activation.

scholarly journals In vitro reconstitution of T cell receptor-mediated segregation of the CD45 phosphatase

2017 ◽  
Vol 114 (44) ◽  
pp. E9338-E9345 ◽  
Author(s):  
Catherine B. Carbone ◽  
Nadja Kern ◽  
Ricardo A. Fernandes ◽  
Enfu Hui ◽  
Xiaolei Su ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Signaling ◽  
T Cell Receptor ◽  
Spatial Organization ◽  
Cell Receptor ◽  
Binding Energies ◽  
Size Difference ◽  
T Cell Signaling ◽  
Receptor Ligand ◽  
Membrane Interfaces

T cell signaling initiates upon the binding of peptide-loaded MHC (pMHC) on an antigen-presenting cell to the T cell receptor (TCR) on a T cell. TCR phosphorylation in response to pMHC binding is accompanied by segregation of the transmembrane phosphatase CD45 away from TCR–pMHC complexes. The kinetic segregation hypothesis proposes that CD45 exclusion shifts the local kinase–phosphatase balance to favor TCR phosphorylation. Spatial partitioning may arise from the size difference between the large CD45 extracellular domain and the smaller TCR–pMHC complex, although parsing potential contributions of extracellular protein size, actin activity, and lipid domains is difficult in living cells. Here, we reconstitute segregation of CD45 from bound receptor–ligand pairs using purified proteins on model membranes. Using a model receptor–ligand pair (FRB–FKBP), we first test physical and computational predictions for protein organization at membrane interfaces. We then show that the TCR–pMHC interaction causes partial exclusion of CD45. Comparing two developmentally regulated isoforms of CD45, the larger RABC variant is excluded more rapidly and efficiently (∼50%) than the smaller R0 isoform (∼20%), suggesting that CD45 isotypes could regulate signaling thresholds in different T cell subtypes. Similar to the sensitivity of T cell signaling, TCR–pMHC interactions with Kds of ≤15 µM were needed to exclude CD45. We further show that the coreceptor PD-1 with its ligand PD-L1, immunotherapy targets that inhibit T cell signaling, also exclude CD45. These results demonstrate that the binding energies of physiological receptor–ligand pairs on the T cell are sufficient to create spatial organization at membrane–membrane interfaces.

scholarly journals CD8+ T-cell clones deficient in the expression of the CD45 protein tyrosine phosphatase have impaired responses to T-cell receptor stimuli.

1991 ◽  
Vol 11 (9) ◽  
pp. 4415-4422 ◽  
Author(s):  
C T Weaver ◽  
J T Pingel ◽  
J O Nelson ◽  
M L Thomas
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Protein Tyrosine Phosphatase ◽  
Transmembrane Protein ◽  
Tyrosine Phosphatase ◽  
Cell Receptor ◽  
T Cell Clones ◽  
Protein Tyrosine ◽  
Receptor Signalling ◽  
Cell Clones

CD45 is a high-molecular-weight transmembrane protein tyrosine phosphatase expressed only by nucleated cells of hematopoietic origin. To examine function, mouse CD8+ cytolytic T-cell clones were derived that had a specific defect in the expression of CD45. Northern (RNA) blot analysis indicates that the CD45 deficiency is due to either a transcriptional defect or mRNA instability. The CD45-deficient cells were greatly diminished in their ability to respond to antigen. All functional parameters of T-cell receptor signalling analyzed (cytolysis of targets, proliferation, and cytokine production) were markedly diminished. A CD45+ revertant was isolated, and the ability to respond to antigen was restored. These results support a central and immediate role for this transmembrane protein tyrosine phosphatase in T-cell receptor signalling.

scholarly journals Temporal Analysis of T-Cell Receptor-Imposed Forces Via Quantitative Single Molecule Fret Measurements

2021 ◽  
Vol 120 (3) ◽  
pp. 102a
Author(s):  
Janett Goehring ◽  
Florian Kellner ◽  
Lukas Schrangl ◽  
Rene Platzer ◽  
Hannes Stockinger ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Single Molecule ◽  
Temporal Analysis ◽  
Cell Receptor ◽  
Single Molecule Fret

scholarly journals Binding Strength and Dynamics of Invariant Natural Killer Cell T Cell Receptor/CD1d-Glycosphingolipid Interaction on Living Cells by Single Molecule Force Spectroscopy

2011 ◽  
Vol 286 (18) ◽  
pp. 15973-15979 ◽  
Author(s):  
Bianca L. Bozna ◽  
Paolo Polzella ◽  
Christian Rankl ◽  
Rong Zhu ◽  
Mariolina Salio ◽  
...  
Keyword(s):  
T Cell ◽  
Natural Killer ◽  
T Cell Receptor ◽  
Single Molecule ◽  
Killer Cell ◽  
Force Spectroscopy ◽  
Cell Receptor ◽  
Living Cells ◽  
Single Molecule Force Spectroscopy ◽  
Invariant Natural Killer

Invariant natural killer T (iNKT) cells are a population of T lymphocytes that play an important role in regulating immunity to infection and tumors by recognizing endogenous and exogenous CD1d-bound lipid molecules. Using soluble iNKT T cell receptor (TCR) molecules, we applied single molecule force spectroscopy for the investigation of the iNKT TCR affinity for human CD1d molecules loaded with glycolipids differing in the length of the phytosphingosine chain using either recombinant CD1d molecules or lipid-pulsed THP1 cells. In both settings, the dissociation of the iNKT TCR from human CD1d molecules loaded with the lipid containing the longer phytosphingosine chain required higher unbinding forces compared with the shorter phytosphingosine lipid. Our findings are discussed in the context of previous results obtained by surface plasmon resonance measurements. We present new insights into the energy landscape and the kinetic rate constants of the iNKT TCR/human CD1d-glycosphingolipid interaction and emphasize the unique potential of single molecule force spectroscopy on living cells.

Regulation of T Cell Receptor Signaling by Tyrosine Phosphatase SYP Association with CTLA-4

Science ◽  
1996 ◽  
Vol 272 (5265) ◽  
pp. 1170-1173 ◽  
Author(s):  
L. E. M. Marengere ◽  
P. Waterhouse ◽  
G. S. Duncan ◽  
H.-W. Mittrucker ◽  
G.-S. Feng ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Tyrosine Phosphatase ◽  
Cell Receptor ◽  
Receptor Signaling ◽  
T Cell Receptor Signaling ◽  
Cell Receptor Signaling
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