The T Cell Receptor Triggering Apparatus Is Composed of Monovalent or Monomeric Proteins

Abstract

Understanding the component stoichiometry of the T cell antigen receptor (TCR) triggering apparatus is essential for building realistic models of signal initiation. Recent studies suggesting that the TCR and other signaling-associated proteins are preclustered on resting T cells relied on measurements of the behavior of membrane proteins at interfaces with functionalized glass surfaces. Using fluorescence recovery after photobleaching, we show that, compared with the apical surface, the mobility of TCRs is significantly reduced at Jurkat T cell/glass interfaces, in a signaling-sensitive manner. Using two biophysical approaches that mitigate these effects, bioluminescence resonance energy transfer and two-color coincidence detection microscopy, we show that, within the uncertainty of the methods, the membrane components of the TCR triggering apparatus, i.e. the TCR complex, MHC molecules, CD4/Lck and CD45, are exclusively monovalent or monomeric in human T cell lines, implying that TCR triggering depends only on the kinetics of TCR/pMHC interactions. These analyses also showed that constraining proteins to two dimensions at the cell surface greatly enhances random interactions versus those between the membrane and the cytoplasm. Simulations of TCR-pMHC complex formation based on these findings suggest how unclustered TCR triggering-associated proteins might nevertheless be capable of generating complex signaling outputs via the differential recruitment of cytosolic effectors to the cell membrane.

Imaging the cell surface and its organization down to the level of single molecules

“…CD4/Lck, CD45 and MHC class II. These proteins have all previously been claimed to form dimers, but gave strong signals for monomers in the TCCD analysis (figure 5a) [46]. The simple organization of the triggering apparatus of the T cell suggested by these studies is shown in figure 5b.…”

“…Although BRET requires heterologous protein expression, the luminescence detection mode allows molecular interactions to be studied at nearnative expression levels so that the observations are relevant to the behaviour of the native proteins, which can also be studied directly using the TCCD method. The TCCD measurements discussed above showed with 95% confidence that all analysed proteins are monomeric [46]. When combined with the orthogonal BRET method for which, in the case of the analysis of the TCR, the upper limit on the fraction of oligomers was estimated to be 10 per cent, these two techniques allow us to be confident that these assignments represent the state of the proteins at the cell surface.…”

“…When combined with the orthogonal BRET method for which, in the case of the analysis of the TCR, the upper limit on the fraction of oligomers was estimated to be 10 per cent, these two techniques allow us to be confident that these assignments represent the state of the proteins at the cell surface. An additional, unexpected outcome of the BRET experiments is the extent to which these membrane-bound molecules form non-specific associations within the membrane, owing to an increase in their effective concentrations versus cytosolic proteins, whose encounters are likely to be much less frequent [46]. This is likely to have important implications for the architecture of receptor signalling pathways.…”

“…The 'ground state' measurements of the TCR triggering apparatus using TCCD and DySCo have been complemented with bioluminescence resonance energy transfer (BRET)-based experiments [46,48]. Although BRET requires heterologous protein expression, the luminescence detection mode allows molecular interactions to be studied at nearnative expression levels so that the observations are relevant to the behaviour of the native proteins, which can also be studied directly using the TCCD method.…”

“…A crucial difference between the TCCD and PALM experiments is that these were done at different locations, that is, at the apical surface of the T cell and the basal cell/glass interface, respectively (figure 4b). Using fluorescence recovery after photo-bleaching (FRAP) measurements, it has since been possible to show that contact with a functionalized glass surface in fact alters the behaviour of the TCR, indicating that it is important to make measurements at the apical surface of resting cells [46]. FRAP measurements at the apical surface showed that the TCR is largely if not completely monovalent or monomeric, whereas approximately 40 per cent of the receptors at the basal membrane contacting the glass surface become immobile in a signalling-dependent manner, explaining the discordant findings [46].…”