Distinct mechanisms regulate Lck spatial organization in activated T cells

Phosphorylation of the T cell receptor (TCR) by the kinase Lck is the first detectable signaling event upon antigen engagement. The distribution of Lck within the plasma membrane, its conformational state, kinase activity, and protein–protein interactions all contribute to determine how efficiently Lck phosphorylates the engaged TCR. Here, we used cross-correlation raster image correlation spectroscopy and photoactivated localization microscopy to identify two mechanisms of Lck clustering: an intrinsic mechanism of Lck clustering induced by locking Lck in its open conformation and an extrinsic mechanism of clustering controlled by the phosphorylation of tyrosine 192, which regulates the affinity of Lck SH2 domain. Both mechanisms of clustering were differently affected by the absence of the kinase Zap70 or the adaptor Lat. We further observed that the adaptor TSAd bound to and promoted the diffusion of Lck when it is phosphorylated on tyrosine 192. Our data suggest that while Lck open conformation drives aggregation and clustering, the spatial organization of Lck is further controlled by signaling events downstream of TCR phosphorylation

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

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

Capturing resting T cells: the perils of PLL.

To the editor — Full understanding of lymphocyte activation will require thorough characterization of the ‘resting’ state and how it changes. Surfaces coated with the cationic homopolymer poly-L-lysine (PLL) are widely used for total internal reflection fluorescence (TIRF) imaging of the organization of surface proteins on resting lymphocytes^1,2,3,4,5 because PLL is assumed to be inert. Here we found that PLL initiated T cell signaling and profoundly altered the activity of membrane proteins such as the T cell antigen receptor (TCR). Therefore, the emerging idea that receptors and signaling proteins cluster by default^1,2,3,4,5, which has been based mostly on studies of lymphocytes interacting with PLL-coated surfaces, needs reconsideration.Supported by a Royal Society University Research Fellowship (UF120277 to S.F.L.) and Research Professorship (RP150066 to D.K.); the EPSRC (EP/L027631/1 to A.P.,); the Wellcome Trust (098274/Z/12/Z to S.J.D., and WT101609MA to R.A.F.); PA Cephalosporin Fund (C.E.); the Wolfson Imaging Centre Oxford (funded by the Wolfson Foundation and Wellcome Trust; 104924/14/Z/14); the Micron Advanced BioImaging Unit (Wellcome Trust Strategic Award 091911); the Medical Research Council (MC_UU_12010/Unit Programmes G0902418 and MC_UU_12025); an MRC/BBSRC/EPSRC award (MR/K01577X/1); and a Marie Skłodowska-Curie Intra-European grant (707348 to I.U.)

Immobilization of nonactivated unfixed platelets for real-time single-cell analysis

Existing methods for measuring the response of individual platelets to stimulation are limited. They either measure each platelet at one discrete time-point (flow cytometry) or rely on adhesive ligands to immobilize platelets that concomitantly generate activation signals (microscopy). Such methods of immobilization make it impossible to assess resting platelets, the changes that occur as platelets transition from resting to active states, or the signals generated by soluble agonists, such as ADP and thrombin, or by mechanical stimulus, independently from those generated by the adhesive ligand. Here we describe a microscopy method that allows the immobilization of platelets to a glass cover slip without triggering platelet activation. This method makes use of specific antibodies that bind platelet PECAM-1 without activating it. Platelets can therefore be immobilized to PECAM-1 antibody coated biochips without causing activation and perfused with agonists or inhibitors. Using this method, platelets can be stimulated by an array of soluble agonists at any concentration or combination, in the presence or absence of inhibitors or shear forces. This chapter describes in detail this PECAM-1 mediated immobilized platelet method and its use for measuring changes in Ca2+ signaling in individual platelets under a number of different conditions. While we focus on the measurement of Ca2+ dynamics in this chapter, it is important to consider that the basic method we describe will easily lend its self to other measures of platelet activation (integrin activation, shape change, actin dynamics, degranulation), and may, therefore, be used to measure almost any facet of platelet activation