scholarly journals Discrete LAT condensates encode antigen information from single pMHC:TCR binding events

2021 ◽  
Author(s):  
Darren B. McAffee ◽  
Mark K. O'Dair ◽  
Jenny J. Lin ◽  
Shalini T. Low-Nam ◽  
Kiera B. Wilhelm ◽  
...  
Keyword(s):  
Phase Transition ◽  
T Cells ◽  
Single Molecule ◽  
Dwell Time ◽  
Imaging Techniques ◽  
Two Dimensional ◽  
Prominent Feature ◽  
Antigen Specificity ◽  
Molecular Binding ◽  
Binding Event

LAT assembly into a two-dimensional protein condensate is a prominent feature of antigen discrimination by T cells. Here, we use single-molecule imaging techniques to resolve the spatial position and temporal duration of each pMHC:TCR molecular binding event while simultaneously monitoring LAT condensation at the membrane. An individual binding event is sufficient to trigger a LAT condensate, which is self-limiting, and neither its size nor lifetime is correlated with the duration of the originating pMHC:TCR binding event. Only the probability of the LAT condensate forming is related to the pMHC:TCR binding dwell time. LAT condenses abruptly, but after an extended delay from the originating binding event. A LAT mutation that facilitates phosphorylation at the PLC-γ1 recruitment site shortens the delay time to LAT condensation and alters T cell antigen specificity. These results identify a role for the LAT protein condensation phase transition in setting antigen discrimination thresholds in T cells.

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 Probe the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility

2021 ◽  
Author(s):  
Zhongwen Chen ◽  
Dongmyung Oh ◽  
Kabir H. Biswas ◽  
Ronen Zaidel-Bar ◽  
Jay T. Groves
Keyword(s):  
Signal Transduction ◽  
Single Molecule ◽  
Cellular Response ◽  
Dwell Time ◽  
Signaling System ◽  
Molecular Binding ◽  
Downstream Signaling ◽  
Functional Consequences ◽  
Signaling Modules ◽  
Signaling Efficiency

AbstractClustering of ligand:receptor complexes on the cell membrane is widely presumed to have functional consequences for subsequent signal transduction. However, it is experimentally challenging to selectively manipulate receptor clustering without altering other biochemical aspects of the cellular system. Here, we develop a microfabrication strategy to produce substrates displaying mobile and immobile ligands that are separated by roughly one micron and thus experience an identical cytoplasmic signaling state, enabling precision comparison of downstream signaling reactions. Applying this approach to characterize the ephrinA1:EphA2 signaling system reveals that EphA2 clustering enhances receptor phosphorylation. Single molecule imaging clearly resolves increased molecular binding dwell time at EphA2 clusters for both Grb2:SOS and NCK:NWASP signaling modules. This type of intracellular comparison enables a substantially higher degree of quantitative analysis than is possible when comparisons must be made between different cells and essentially eliminates the effects of cellular response to ligand manipulation.

scholarly journals Direct single molecule measurement of TCR triggering by agonist pMHC in living primary T cells

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Geoff P O'Donoghue ◽  
Rafal M Pielak ◽  
Alexander A Smoligovets ◽  
Jenny J Lin ◽  
Jay T Groves
Keyword(s):  
T Cells ◽  
Single Molecule ◽  
Lipid Membranes ◽  
Antigenic Peptides ◽  
Tcr Signaling ◽  
Membrane Recruitment ◽  
Molecular Binding ◽  
Entire Cell ◽  
Antigen Presenting

T cells discriminate between self and foreign antigenic peptides, displayed on antigen presenting cell surfaces, via the TCR. While the molecular interactions between TCR and its ligands are well characterized in vitro, quantitative measurements of these interactions in living cells are required to accurately resolve the physical mechanisms of TCR signaling. We report direct single molecule measurements of TCR triggering by agonist pMHC in hybrid junctions between live primary T cells and supported lipid membranes. Every pMHC:TCR complex over the entire cell is tracked while simultaneously monitoring the local membrane recruitment of ZAP70, as a readout of TCR triggering. Mean dwell times for pMHC:TCR molecular binding of 5 and 54 s were measured for two different pMHC:TCR systems. Single molecule measurements of the pMHC:TCR:ZAP70 complex indicate that TCR triggering is stoichiometric with agonist pMHC in a 1:1 ratio. Thus any signal amplification must occur downstream of TCR triggering.

Proteome Analysis of CD4+ T Cells Reveals Differentially Expressed Proteins in Infertile Polycystic Ovary Syndrome Patients

2020 ◽  
Vol 20 ◽  
Author(s):  
Fatemeh Nasri ◽  
Maryam Zare ◽  
Mehrnoosh Doroudchi ◽  
Behrouz Gharesi-Fard
Keyword(s):  
Mass Spectrometry ◽  
T Cells ◽  
T Lymphocytes ◽  
Gel Electrophoresis ◽  
Cd4 T Cells ◽  
Polycystic Ovary ◽  
Proteome Analysis ◽  
Two Dimensional ◽  
Ovary Syndrome ◽  
Two Dimensional Gel Electrophoresis

Background: Polycystic ovary syndrome (PCOS) is the most frequent endocrine disorder affecting 6–7% of premenopausal women. Recent studies revealed that the immune system especially CD4+ T helper cells are important in the context PCOS. Proteome analysis of CD4+ T lymphocytes can provide valuable information regarding the biology of these cells in the context of PCOS. Objective: To investigate immune dysregulation in CD4+ T lymphocytes at the protein level in the context of PCOS using two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS). Methods: In the present study, we applied two-dimensional gel electrophoresis / mass spectrometry to identify proteins differentially expressed by peripheral blood CD4+ T cells in ten PCOS women compared with ten healthy women. Western blot technique was used to confirm the identified proteins. Results: Despite the overall proteome similarities, there were significant differences in the expression of seven spots between two groups (P <0.05). Three proteins, namely phosphatidylethanolamine-binding protein 1, proteasome activator complex subunit 1 and triosephosphate isomerase 1 were successfully identified by Mass technique and confirmed by western blot. All characterized proteins were over-expressed in CD4+ T cells from patients compared to CD4+ T cells from controls (P <0.05). In-silico analysis suggested that the over-expressed proteins interact with other proteins involved in cellular metabolism especially glycolysis and ferroptosis pathway. Conclusion: These findings suggest that metabolic adjustments in CD4+ T lymphocytes, which is in favor of increased glycolysis and Th2 differentiation are important in the context of PCOS.

Temperature Effect on the Two-Dimensional Phase Transition Kinetics of Adenine Adsorbed at the Hg Electrode/Aqueous Solution Interface

2003 ◽  
Vol 68 (8) ◽  
pp. 1407-1419 ◽  
Author(s):  
Claudio Fontanesi ◽  
Roberto Andreoli ◽  
Luca Benedetti ◽  
Roberto Giovanardi ◽  
Paolo Ferrarini
Keyword(s):  
Phase Transition ◽  
Aqueous Solution ◽  
Phase Change ◽  
Temperature Effect ◽  
Transition Rate ◽  
Effect Of Temperature ◽  
Two Dimensional ◽  
Solution Interface ◽  
Phase Transition Kinetics ◽  
Kinetics Of

The kinetics of the liquid-like → solid-like 2D phase transition of adenine adsorbed at the Hg/aqueous solution interface is studied. Attention is focused on the effect of temperature on the rate of phase change; an increase in temperature is found to cause a decrease of transition rate.

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