Mechanisms of localized activation of the T cell antigen receptor inside clusters

Neve-Oz, Yair; Razvag, Yair; Sajman, Julia; Sherman, Eilon

doi:10.1016/j.bbamcr.2014.09.025

Cited by 29 publications

(42 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…T-tests were used and their p-values were computed by permutation (see Supplementary Materials and Methods). In good agreement with previous reports 21,32 , CD3ζ was clustered in stimulated and non-stimulated cells, and cluster parameters were significantly altered. Despite no large scale changes in the percentage of localisations found in clusters (29 ± 2% in PLL to 33 ± 1% in activated cells, P > 0.05), a significant increase in the number of clusters and a significant decrease in the size of clusters was observed from resting to activated cells (8 ± 1 clusters per region on PLL versus 20 ± 3 in activated cells; P ≤ 0.005 and 82 ± 4 nm radius versus 48 ± 2 nm after activation; P ≤ 0.0005; Fig.…”

Section: Analysis Of Protein Clustering In Primary Human T Cellssupporting

confidence: 92%

Bayesian cluster identification in single-molecule localization microscopy data

et al. 2015

View full text Add to dashboard Cite

Correspondence should be addressed to patrick. rubin-delanchy@bristol.ac.uk and dylan.owen@kcl.ac.uk Single-molecule identification-based super-resolution microscopy techniques such as photo-activated localisation microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) produce pointillist data sets of molecular coordinates. While many algorithms exist for the identification and localisation of molecules from the raw image data, methods for analysing the resulting point patterns for properties such as clustering have remained relatively under-studied. Here, we present the first model-based Bayesian approach to evaluate molecular cluster assignment proposals which, in this article, are generated by analysis based on Ripley's Kfunction. The method is also the first to take full account of the individual localisation precisions calculated for each emitter. The technique is validated using simulated and experimental data from which we characterise the clustering behaviour of CD3ζ, an important subunit of the CD3-T cell receptor complex required for T cell function, in resting and activated primary human T cells.Conventional fluorescence microscopes produce images of the distribution of fluorophores in the sample convolved with the microscope Point Spread Function (PSF). Due to diffraction, this PSF typically has a width of hundreds of nanometres meaning the resulting image has a resolution, as assessed by the Rayleigh criterion, of ~200 nm. Several strategies now exist to circumvent this resolution limit 1 . Some of these, such as Stimulated Emission Depletion (STED) microscopy, rely on narrowing the excitation spot of a confocal microscope by means of a toroidal depletion beam and the process of stimulated emission 2,3 . Despite the increased resolution, these produce conventional fluorescence images, i.e., arrays of pixels with values representing the fluorescence intensity at those locations. Quantification can be performed in the same way as for conventional microscopes.Another strategy is based on Single-Molecule Localisation Microscopy (SMLM) [4][5][6][7] . This relies on the temporal separation of the excitation of fluorophores in the sample whose PSFs would otherwise overlap at the detector.The position of each fluorophore can then be estimated from the centres of the PSFs. Many algorithms are available to extract the x-y coordinates of the molecules [8][9][10] . Each emitter can be localised to a precision between 10 and 30 nm. Common strategies for the temporal separation of molecules involve intra-molecular rearrangements to switch from dark to fluorescent states or the exploitation of non-emitting molecular radicals 11,12 . These strategies are typically pursued using photoactivatable or photoconvertible fluorescent proteins or small molecule probes coupled with a reducing buffer and immunostaining protocols 13 . We refer to all such strategies as SMLM.Unlike non-pointillist microscopy methods, SMLM imaging does not produce a conventional image. Instead, the raw data is a list of the...

show abstract

Section: Analysis Of Protein Clustering In Primary Human T Cellssupporting

confidence: 92%

Bayesian cluster identification in single-molecule localization microscopy data

et al. 2015

View full text Add to dashboard Cite

show abstract

“…TCRs (52) or through increased localized phosphorylation of TCRs by clustered Lck (26,27). Signaling efficiency in dense clusters may also be augmented by the detachment of the ITAMcontaining cytoplasmic domains of CD3ζ (53) and CD3e (54) from the membrane so that they are accessible to Lck for phosphorylation.…”

Section: Discussionmentioning

confidence: 99%

“…Similarly, Lat (23)(24)(25), Lck (26), and Zap70 (24,27) were also found to reside in nanoclusters that are extensively remodeled during T-cell activation. The link between preexisting and pMHC-induced nanoclustering and signaling activities is not clear at present and is the focus of the present study.…”

mentioning

confidence: 99%

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

Pageon

Tabarin

Yamamoto

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

209

235

View full text Add to dashboard Cite

Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR–CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR–CD3 complexes. We found that only TCR–CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR–pMHC affinity determined the density of TCR–CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR–CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination.

show abstract

“…Beyond the SPR assay, it is interesting to speculate that these tethered signaling reactions may lead to the appearance of large-scale phosphorylation patterns in cells. The appearance of spatial patterns of membrane-localized phosphorylation ( 38 ) and signaling enzymes ( 39 , 40 ) (often a proxy for phosphorylation) in T cells may be a result of tethered phosphorylation/dephosphorylation reactions. Future work in a reductionist setting can determine whether tethered reactions are sufficient to produce these patterns.…”

Section: Discussionmentioning

confidence: 99%