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.

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 Mapping the stochastic sequence of individual ligand-receptor binding events to cellular activation: T cells act on the rare events

2019 ◽  
Vol 12 (564) ◽  
pp. eaat8715 ◽  
Author(s):  
Jenny J. Y. Lin ◽  
Shalini T. Low-Nam ◽  
Katherine N. Alfieri ◽  
Darren B. McAffee ◽  
Nicole C. Fay ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Single Molecule ◽  
Cellular Response ◽  
Dwell Time ◽  
Cell Activation ◽  
Cell Receptor ◽  
Cellular Activation ◽  
Spatially Correlated ◽  
Order Of Magnitude

T cell receptor (TCR) binding to agonist peptide major histocompatibility complex (pMHC) triggers signaling events that initiate T cell responses. This system is remarkably sensitive, requiring only a few binding events to successfully activate a cellular response. On average, activating pMHC ligands exhibit mean dwell times of at least a few seconds when bound to the TCR. However, a T cell accumulates pMHC-TCR interactions as a stochastic series of discrete, single-molecule binding events whose individual dwell times are broadly distributed. With activation occurring in response to only a handful of such binding events, individual cells are unlikely to experience the average binding time. Here, we mapped the ensemble of pMHC-TCR binding events in space and time while simultaneously monitoring cellular activation. Our findings revealed that T cell activation hinges on rare, long–dwell time binding events that are an order of magnitude longer than the average agonist pMHC-TCR dwell time. Furthermore, we observed that short pMHC-TCR binding events that were spatially correlated and temporally sequential led to cellular activation. These observations indicate that T cell antigen discrimination likely occurs by sensing the tail end of the pMHC-TCR binding dwell time distribution rather than its average properties.

PKC-θ selectively controls the adhesion-stimulating molecule Rap1

Blood ◽  
2008 ◽  
Vol 112 (12) ◽  
pp. 4617-4627 ◽  
Author(s):  
Thomas Letschka ◽  
Veronika Kollmann ◽  
Christa Pfeifhofer-Obermair ◽  
Christina Lutz-Nicoladoni ◽  
Gerald J. Obermair ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
T Cell ◽  
T Lymphocytes ◽  
T Cell Activation ◽  
Cell Activation ◽  
Spatial Organization ◽  
Specific Interaction ◽  
Cell Receptor ◽  
Small Gtpase ◽  
Intercellular Interaction

Abstract The antigen-specific interaction of a T cell with an antigen-presenting cell (APC) results in the formation of an immunologic synapse (IS) between the membranes of the 2 cells. β2 integrins on the T cell, namely, leukocyte function-associated antigen 1 (LFA-1) and its counter ligand, namely, immunoglobulin-like cell adhesion molecule 1 (ICAM-1) on the APC, critically stabilize this intercellular interaction. The small GTPase Rap1 controls T-cell adhesion through modulating the affinity and/or spatial organization of LFA-1; however, the upstream regulatory components triggered by the T-cell receptor (TCR) have not been resolved. In the present study, we identified a previously unknown function of a protein kinase C-θ (PKC-θ)/RapGEF2 complex in LFA-1 avidity regulation in T lymphocytes. After T-cell activation, the direct phosphorylation of RapGEF2 at Ser960 by PKC-θ regulates Rap1 activation as well as LFA-1 adhesiveness to ICAM-1. In OT-II TCR-transgenic CD4+ T cells, clustering of LFA-1 after antigen activation was impaired in the absence of PKC-θ. These data define that, among other pathways acting on LFA-1 regulation, PKC-θ and its effector RapGEF2 are critical factors in TCR signaling to Rap1. Taken together, PKC-θ sets the threshold for T-cell activation by positively regulating both the cytokine responses and the adhesive capacities of T lymphocytes.

scholarly journals CD8 Co-Receptor Enhances T-Cell Activation without Any Effect on Initial Attachment

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 429
Author(s):  
Philippe Robert ◽  
Laurent Limozin ◽  
P. Anton van der Merwe ◽  
Pierre Bongrand
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Single Molecule ◽  
High Speed ◽  
Cell Activation ◽  
Cell Receptor ◽  
Flow Chamber ◽  
Jurkat Cells ◽  
Single Molecule Level ◽  
Major Histocompatibility Antigens

The scanning of surrounding tissues by T lymphocytes to detect cognate antigens requires high speed, sensitivity and specificity. T-cell receptor (TCR) co-receptors such as CD8 increase detection performance, but the exact mechanism remains incompletely understood. Here, we used a laminar flow chamber to measure at the single molecule level the kinetics of bond formation and rupture between TCR- transfected CD8+ and CD8− Jurkat cells and surfaces coated with five peptide-exposing major histocompatibility antigens (pMHCs) of varying activating power. We also used interference reflection microscopy to image the spreading of these cells dropped on pMHC-exposing surfaces. CD8 did not influence the TCR–pMHC interaction during the first few seconds following cell surface encounter, but it promoted the subsequent spreading responses, suggesting that CD8 was involved in early activation rather than binding. Further, the rate and extent of spreading, but not the lag between contact and spreading initiation, depended on the pMHC. Elucidating T-lymphocyte detection strategy may help unravel underlying signaling networks.

scholarly journals The coreceptor CD4 is expressed in distinct nanoclusters and does not colocalize with T-cell receptor and active protein tyrosine kinase p56lck

2015 ◽  
Vol 112 (13) ◽  
pp. E1604-E1613 ◽  
Author(s):  
Kyung-Ho Roh ◽  
Björn F. Lillemeier ◽  
Feng Wang ◽  
Mark M. Davis
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Protein Tyrosine Kinase ◽  
Cell Activation ◽  
Spatial Organization ◽  
Active Form ◽  
Cell Receptor ◽  
Activation Process ◽  
Dual Color ◽  
Interfacial Surfaces

CD4 molecules on the surface of T lymphocytes greatly augment the sensitivity and activation process of these cells, but how it functions is not fully understood. Here we studied the spatial organization of CD4, and its relationship to T-cell antigen receptor (TCR) and the active form of Src kinase p56lck (Lck) using single and dual-color photoactivated localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM). In nonactivated T cells, CD4 molecules are clustered in small protein islands, as are TCR and Lck. By dual-color imaging, we find that CD4, TCR, and Lck are localized in their separate clusters with limited interactions in the interfaces between them. Upon T-cell activation, the TCR and CD4 begin clustering together, developing into microclusters, and undergo a larger scale redistribution to form supramolecluar activation clusters (SMACs). CD4 and Lck localize in the inner TCR region of the SMAC, but this redistribution of disparate cluster structures results in enhanced segregation from each other. In nonactivated cells these preclustered structures and the limited interactions between them may serve to limit spontaneous and random activation events. However, the small sizes of these island structures also ensure large interfacial surfaces for potential interactions and signal amplification when activation is initiated. In the later activation stages, the increasingly larger clusters and their segregation from each other reduce the interfacial surfaces and could have a dampening effect. These highly differentiated spatial distributions of TCR, CD4, and Lck and their changes during activation suggest that there is a more complex hierarchy than previously thought.

scholarly journals PD-1 suppresses TCR-CD8 cooperativity during T-cell antigen recognition

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kaitao Li ◽  
Zhou Yuan ◽  
Jintian Lyu ◽  
Eunseon Ahn ◽  
Simon J. Davis ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Clinical Success ◽  
Clinical Intervention ◽  
Cell Receptor ◽  
Cell Interaction ◽  
Antigen Recognition ◽  
Inhibitory Function ◽  
Src Homology

AbstractDespite the clinical success of blocking its interactions, how PD-1 inhibits T-cell activation is incompletely understood, as exemplified by its potency far exceeding what might be predicted from its affinity for PD-1 ligand-1 (PD-L1). This may be partially attributed to PD-1’s targeting the proximal signaling of the T-cell receptor (TCR) and co-stimulatory receptor CD28 via activating Src homology region 2 domain-containing phosphatases (SHPs). Here, we report PD-1 signaling regulates the initial TCR antigen recognition manifested in a smaller spreading area, fewer molecular bonds formed, and shorter bond lifetime of T cell interaction with peptide-major histocompatibility complex (pMHC) in the presence than absence of PD-L1 in a manner dependent on SHPs and Leukocyte C-terminal Src kinase. Our results identify a PD-1 inhibitory mechanism that disrupts the cooperative TCR–pMHC–CD8 trimolecular interaction, which prevents CD8 from augmenting antigen recognition, explaining PD-1’s potent inhibitory function and its value as a target for clinical intervention.

scholarly journals Ca2+/Calmodulin-Dependent Protein Kinase II Is a Modulator of CARMA1-Mediated NF-κB Activation

2006 ◽  
Vol 26 (14) ◽  
pp. 5497-5508 ◽  
Author(s):  
Kazuhiro Ishiguro ◽  
Todd Green ◽  
Joseph Rapley ◽  
Heather Wachtel ◽  
Cosmas Giallourakis ◽  
...  
Keyword(s):  
T Cell ◽  
Protein Kinase ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Receptor ◽  
Tcr Signaling ◽  
Dependent Protein Kinase ◽  
Immune Synapse ◽  
Protein Kinase Ii ◽  
Dependent Protein

ABSTRACT CARMA1 is a central regulator of NF-κB activation in lymphocytes. CARMA1 and Bcl10 functionally interact and control NF-κB signaling downstream of the T-cell receptor (TCR). Computational analysis of expression neighborhoods of CARMA1-Bcl10MALT 1 for enrichment in kinases identified calmodulin-dependent protein kinase II (CaMKII) as an important component of this pathway. Here we report that Ca2+/CaMKII is redistributed to the immune synapse following T-cell activation and that CaMKII is critical for NF-κB activation induced by TCR stimulation. Furthermore, CaMKII enhances CARMA1-induced NF-κB activation. Moreover, we have shown that CaMKII phosphorylates CARMA1 on Ser109 and that the phosphorylation facilitates the interaction between CARMA1 and Bcl10. These results provide a novel function for CaMKII in TCR signaling and CARMA1-induced NF-κB activation.

scholarly journals A new HLA-linked T cell membrane molecule, related to the beta chain of the clonotypic receptor, is associated with T3.

1986 ◽  
Vol 164 (2) ◽  
pp. 458-473 ◽  
Author(s):  
Y Bushkin ◽  
D N Posnett ◽  
B Pernis ◽  
C Y Wang
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Functional Role ◽  
Cell Activation ◽  
Cell Receptor ◽  
Leukemia Cells ◽  
Alpha Beta ◽  
Beta 2 Microglobulin ◽  
T Cell Membrane ◽  
Membrane Molecule

The 38 kD molecule is noncovalently associated with beta 2 microglobulin (beta 2m)-free HLA heavy chain-like molecule, and thus forms a second heterodimer distinct from the clonotypic alpha/beta T cell receptor expressed by the same clone of leukemia cells. This second heterodimer (38 kD/HLA) is variably expressed and appears to be associated with the T3 molecule. We suggest, therefore, that it has a functional role in T cell activation.

scholarly journals T cell receptor zeta/CD3-p59fyn(T)-associated p120/130 binds to the SH2 domain of p59fyn(T).

1993 ◽  
Vol 178 (6) ◽  
pp. 2107-2113 ◽  
Author(s):  
A J da Silva ◽  
O Janssen ◽  
C E Rudd
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Intracellular Signaling ◽  
Cell Activation ◽  
Cell Receptor ◽  
Sh2 Domain ◽  
T Complex

Intracellular signaling from the T cell receptor (TCR)zeta/CD3 complex is likely to be mediated by associated protein tyrosine kinases such as p59fyn(T), ZAP-70, and the CD4:p56lck and CD8:p56lck coreceptors. The nature of the signaling cascade initiated by these kinases, their specificities, and downstream targets remain to be elucidated. The TCR-zeta/CD3:p59fyn(T) complex has previously been noted to coprecipitate a 120/130-kD doublet (p120/130). This intracellular protein of unknown identity associates directly with p59fyn(T) within the receptor complex. In this study, we have shown that this interaction with p120/130 is specifically mediated by the SH2 domain (not the fyn-SH3 domain) of p59fyn(T). Further, based on the results of in vitro kinase assays, p120/130 appears to be preferentially associated with p59fyn(T) in T cells, and not with p56lck. Antibody reprecipitation studies identified p120/130 as a previously described 130-kD substrate of pp60v-src whose function and structure is unknown. TCR-zeta/CD3 induced activation of T cells augmented the tyrosine phosphorylation of p120/130 in vivo as detected by antibody and GST:fyn-SH2 fusion proteins. p120/130 represents the first identified p59fyn(T):SH2 binding substrate in T cells, and as such is likely to play a key role in the early events of T cell activation.

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