TCRs are randomly distributed on the plasma membrane of resting antigen-experienced T cells

Rossboth, Benedikt K.; Arnold, A.; Ta, Haisen; Platzer, René; Kellner, Florian; Huppa, Johannes B.; Brameshuber, Mario; Baumgart, Florian; Schütz, Gerhard J.

doi:10.1038/s41590-018-0162-7

Cited by 104 publications

(118 citation statements)

References 54 publications

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“…A recent report (73) suggested that observations of TCR clusters on the T-cell membrane (17,18,20,22,23,48) were due to imaging artifacts, and that all TCR machineries are randomly distributed on the surface of T cells. However, in that paper TCR-stimulated T cells were spread on ICAM-1-coated surfaces.…”

Section: Discussionmentioning

confidence: 99%

ERM-Dependent Assembly of T-Cell Receptor Signaling and Co-stimulatory Molecules on Microvilli Prior to Activation

Ghosh

Bartolo

Tubul

et al. 2019

Preprint

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T-cell surfaces are covered with microvilli, actin-rich and flexible protrusions. We use super-resolution microscopy to show that 90% T-cell receptor (TCR) complex molecules TCR and TCR, as well as the co-receptor CD4 and the co-stimulatory molecule CD2 reside on microvilli of human T cells. Furthermore, TCR proximal signaling molecules involved in the initial stages of the immune response, such as the protein tyrosine kinase Lck and the key adaptor molecule LAT, are also enriched on microvilli. Notably, phosphorylated proteins of the ERM (ezrin, radixin, moesin) family colocalize with these heterodimers as well as with actin filaments within the microvilli of resting T cells. This finding implies a role for one or more phosphorylated ERMs in linking the TCR complex to the actin cytoskeleton within microvilli. Indeed, expression of a dominant-negative ezrin fragment effectively redistributes TCR molecules over the whole T cell surface. Our results establish microvilli as key signaling hubs, in which the TCR complex and its proximal signaling molecules and adaptors are pre-assembled prior to activation in an ERMdependent manner. The preformed positioning of these actin-binding TCR assemblies on individual microvilli can facilitate the local transmission of TCR signals seconds after TCR occupancy and impacts the slower subsequent events that lead to the assembly of immunological synapses.

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Section: Discussionmentioning

confidence: 99%

ERM-Dependent Assembly of T-Cell Receptor Signaling and Co-stimulatory Molecules on Microvilli Prior to Activation

Ghosh

Bartolo

Tubul

et al. 2019

Preprint

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“…S4a). 3 Further, the magnitude of the localization errors hardly affects the obtained results ( Fig. S4b).…”

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confidence: 84%

“…S1) For the simulations shown in Fig. S3 and Fig S9., we used blinking statistics determined previously 2 (Rossboth et al, 2018). Localization errors were simulated by spreading these detections using a Gaussian 3 profile centered on the molecule position with a width of 30 nm, which corresponds to typical localization 4 errors achieved in SMLM experiments.…”

Section: Simulation Of Areas Of Enrichment or Depletion Of Biomoleculesmentioning

confidence: 99%

“…1 Both authors contributed equally 26 2 Institute of Applied Physics, TU Wien, Getreidemarkt 9, A-1060 Vienna, Austria. 27 3 Institute of Visual Computing and Human-Centered Technology, TU Wien, Favoritenstrasse 9-11, A-1040 28 Vienna, Austria 29 Introduction 1 Single Molecule Localization Microscopy (SMLM) has boosted our insights into cellular structures below 2 the diffraction limit of light microscopy (Schermelleh et al, 2019). Common to all SMLM variants is the 3 stochastic switching of single dye molecules between a bright and a dark state.…”

mentioning

confidence: 99%

“…S2). 3 Hence, this p-value allows for the correct interpretation of the significance level  as the probability of 4 falsely rejecting the null hypothesis.  can also be interpreted as the inevitable false positive rate for the 5 erroneous detection of overcounting-induced clustering for a random distribution of biomolecules.…”

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confidence: 99%

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Verifying molecular clusters by 2-color localization microscopy and significance testing

Arnold

Schneider

Huesson

et al. 2019

Preprint

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While single-molecule localization microscopy (SMLM) offers the invaluable prospect to visualize cellular structures below the diffraction limit of light microscopy, its potential could not be fully capitalized due to its inherent susceptibility to blinking artifacts. Particularly, overcounting of single molecule localizations has impeded a reliable and sensitive detection of biomolecular nanoclusters. Here we introduce a 2-Color Localization microscopy And Significance Testing Approach (2-CLASTA), providing a parameter-free statistical framework for the analysis of SMLM data via significance testing methods. 2-CLASTA yields p-values for the null hypothesis of random biomolecular distributions, independent of the blinking behavior of the chosen fluorescent labels. We validated the method both by computer simulations as well as experimentally, using protein concatemers as a mimicry of biomolecular clustering. As the new approach it is not affected by overcounting artifacts, it is able to detect biomolecular clustering of various shapes at high sensitivity down to a level of dimers.

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T‐Cell Antigen Recognition – Lessons from the Past and Projections into the Future

Platzer

Huppa

2020

Encyclopedia of Life Sciences

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T‐cells are central to adaptive immunity and arguably to this date the most intensely studied cells in life sciences. Paying tribute to their developmental plasticity and the complexities associated with many of their physiological functions, numerous aspects of their physiology are still far from being understood to an extent that would be sufficient to rationally design therapies effectively targeting allergies, autoimmunity and cancer. T‐cell antigen recognition is no exception: this field took up speed with the rise of monoclonal antibodies and the first successful cloning of the T‐cell antigen receptor (TCR) genes roughly 40 years ago. In the meantime, hundreds of TCRs have been crystallised in complex with their nominal peptide/MHC (pMHC) binding partners and many TCR‐pMHC interaction kinetics have been measured. Furthermore most, if not all signalling molecules acting downstream of the TCR have been identified. Despite these accomplishments, we are still searching for convincing explanations as to how T‐cells mange to reliably detect the presence of even a single antigen on the surface of antigen‐presenting cells (APCs). Elaborating underlying mechanisms will invariably require a more advanced understanding of the molecular, subcellular and cellular context in which T‐cell antigen recognition operates. What renders this endeavour both challenging and exciting is the rather weak strength and promiscuous nature of TCR‐pMHC binding and the fact that antigenic pMHCs are typically vastly outnumbered on APC surfaces by structurally similar, yet nonstimulatory pMHCs. While research of the last 20 years has provided some clarity, it has also caused at times controversies, which need to be resolved to unleash the full potential that T‐cells offer for clinical progress. Key Concepts T‐cells are indispensable for orchestrating and executing cellular and humoral adaptive immune responses; in recurrent communication with other cells of the immune system, T‐cells continuously patrol our body in search for antigenic peptide fragments derived from pathogens or cancer‐derived neoantigens. T‐cells are exquisitely sensitive for their nominal antigen as they can detect the presence of even a single stimulatory pMHC amongst thousands of structurally similar yet nonstimulatory pMHCs on the very crowded surface of an APC. Despite considerable progress in the field over the last 40 years, the molecular, biophysical and (sub‐) cellular principles underlying the detection efficiency associated with T‐cell antigen recognition and are far from being resolved. Given the complexities inherent to processes associated with T‐cell antigen recognition, which involve (1) the short‐lived nature of key protein–protein and protein–lipid interactions and (2) mechanical forces acting within the narrow confines of the immunological synapse, we consider integrative approaches combining classical biochemistry, structural biology and genetics with biophysics, advanced live‐cell imaging and systems biology most likely to provide much‐needed answers to most fundamental questions. Easy access to both experimental/analysis modalities and primary data of published work as well as improved literacy in the areas of biophysics and systems biology will help accelerate progress in the field of T‐cell antigen recognition with immediate and far‐reaching clinical implications benefiting allergy, autoimmune and cancer patients.

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TCRs are randomly distributed on the plasma membrane of resting antigen-experienced T cells

Cited by 104 publications

References 54 publications

ERM-Dependent Assembly of T-Cell Receptor Signaling and Co-stimulatory Molecules on Microvilli Prior to Activation

ERM-Dependent Assembly of T-Cell Receptor Signaling and Co-stimulatory Molecules on Microvilli Prior to Activation

Verifying molecular clusters by 2-color localization microscopy and significance testing

T‐Cell Antigen Recognition – Lessons from the Past and Projections into the Future

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