The recognition of foreign antigens by T lymphocytes is essential to most adaptive immune responses. It is driven by specific T-cell antigen receptors (TCRs) binding to antigenic peptidemajor histocompatibility complex (pMHC) molecules on other cells1. If productive, these interactions promote the formation of an immunological synapse2,3. Here we show that synaptic TCR-pMHC binding dynamics differ significantly from TCR-pMHC binding in solution. We used single-molecule microscopy and fluorescence resonance energy transfer (FRET) between fluorescently tagged TCRs and their cognate pMHC ligands to measure the kinetics of TCRpMHC binding in situ. When compared with solution measurements, the dissociation of this complex was increased significantly (4-12-fold). Disruption of actin polymers reversed this effect, indicating that cytoskeletal dynamics destabilize this interaction directly or indirectly. Nevertheless, TCR affinity for pMHC was significantly elevated as the result of a large (about 100-fold) increase in the association rate, a likely consequence of complementary molecular orientation and clustering. In helper T cells, the CD4 molecule has been proposed to bind cooperatively with the TCR to the same pMHC complex. However, CD4 blockade had no effect on the synaptic TCR affinity, nor did it destabilize TCR-pMHC complexes, indicating that the TCR binds pMHC independently of CD4. Supplementary Information is linked to the online version of the paper at www.nature.com/nature. (refs 4-6). Nevertheless, T cells are highly specific and sensitive for antigen, able to detect even a single antigenic pMHC complex among structurally similar yet non-stimulatory pMHCs7,8. However, although this is useful for comparative purposes, these measurements do not account for the many constraints and special features of the synaptic environment and might not reflect what occurs in situ. In particular, the restricted intercellular volume should greatly accelerate the association rate and enhance serial engagement9-11. Favourable molecular alignment of TCR and MHC, as well as any molecular pre-clustering, could also drastically affect binding12-14. Although the volume effect can be approximated, the influence of the others is essentially unknown. Author ContributionsWe have therefore devised a method to measure synaptic TCR binding to pMHC directly, using single-molecule microscopy and FRET between a donor fluorophore on the TCR and an acceptor fluorophore on the peptide bound to a MHC molecule.We used T-cell blasts from two different TCR transgenic T-cell lines, 2B4 and 5c.c7, which are specific for the same moth cytochrome c peptide (MCC 88-103) bound to the class II MHC molecule IE k . To permit the use of highly sensitive and rapid total internal reflection (TIRF) microscopy, we used a modified planar lipid bilayer system as a surrogate antigenpresenting cell surface, presenting IE k complexes, ICAM-1 adhesion molecules and B7-1 co-stimulatory polypeptides (see Methods).Structural analysis of an Fab fragment derived f...

Bax is a key regulator of apoptosis that mediates the release of cytochrome c to the cytosol via oligomerization in the outer mitochondrial membrane before pore formation. However, the molecular mechanism of Bax assembly and regulation by other Bcl-2 members remains obscure. Here, by analysing the stoichiometry of Bax oligomers at the single-molecule level, we find that Bax binds to the membrane in a monomeric state and then self-assembles in <1 min. Strikingly, active Bax does not exist in a unique oligomeric state, but as several different species based on dimer units. Moreover, we show that cBid activates Bax without affecting its assembly, while Bcl-xL induces the dissociation of Bax oligomers. On the basis of our experimental data and theoretical modelling, we propose a new mechanism for the molecular pathway of Bax assembly to form the apoptotic pore.

Antigen recognition is a key event during T cell activation. Here, we introduce nanopatterned antigen arrays that mimic the antigen presenting cell surface during T cell activation. The assessment of activation related events revealed the requirement of a minimal density of 90–140 stimulating major histocompatibility complex class II proteins (pMHC) molecules per μm2. We demonstrate that these substrates induce T cell responses in a pMHC dose-dependent manner and that the number of presented pMHCs dominates over local pMHC density.