A cell topography-based mechanism for ligand discrimination by the T cell receptor.

The T cell receptor (TCR) initiates the elimination of pathogens and tumors by T cells. To avoid damage to the host, the receptor must be capable of discriminating between wild-type and mutated self and nonself peptide ligands presented by host cells. Exactly how the TCR does this is unknown. In resting T cells, the TCR is largely unphosphorylated due to the dominance of phosphatases over the kinases expressed at the cell surface. However, when agonist peptides are presented to the TCR by major histocompatibility complex proteins expressed by antigen-presenting cells (APCs), very fast receptor triggering, i.e., TCR phosphorylation, occurs. Recent work suggests that this depends on the local exclusion of the phosphatases from regions of contact of the T cells with the APCs. Here, we developed and tested a quantitative treatment of receptor triggering reliant only on TCR dwell time in phosphatase-depleted cell contacts constrained in area by cell topography. Using the model and experimentally derived parameters, we found that ligand discrimination likely depends crucially on individual contacts being ∼200 nm in radius, matching the dimensions of the surface protrusions used by T cells to interrogate their targets. The model not only correctly predicted the relative signaling potencies of known agonists and nonagonists but also achieved this in the absence of kinetic proofreading. Our work provides a simple, quantitative, and predictive molecular framework for understanding why TCR triggering is so selective and fast and reveals that, for some receptors, cell topography likely influences signaling outcomes.

More from less - bottom-up reconstitution of cell biology.

The ultimate goal of bottom-up synthetic biology is recreating life in its simplest form. However, in its quest to find the minimal functional units of life, this field contributes more than its main aim by also offering a range of tools for asking, and experimentally approaching, biological questions. This Review focusses on how bottom-up reconstitution has furthered our understanding of cell biology. Studying cell biological processes in vitro has a long tradition, but only recent technological advances have enabled researchers to reconstitute increasingly complex biomolecular systems by controlling their multi-component composition and their spatiotemporal arrangements. We illustrate this progress using the example of cytoskeletal processes. Our understanding of these has been greatly enhanced by reconstitution experiments, from the first in vitro experiments 70 years ago to recent work on minimal cytoskeleton systems (including this Special Issue of Journal of Cell Science). Importantly, reconstitution approaches are not limited to the cytoskeleton field. Thus, we also discuss progress in other areas, such as the shaping of biomembranes and cellular signalling, and prompt the reader to add their subfield of cell biology to this list in the future.

Myosin-II activity generates a dynamic steady state with continuous actin turnover in a minimal actin cortex.

Dynamic reorganization of the actomyosin cytoskeleton allows fast modulation of the cell surface, which is vital for many cellular functions. Myosin-II motors generate the forces required for this remodeling by imparting contractility to actin networks. However, myosin-II activity might also have a more indirect contribution to cytoskeletal dynamics; it has been proposed that myosin activity increases actin turnover in various cellular contexts, presumably by enhancing disassembly. In vitro reconstitution of actomyosin networks has confirmed the role of myosin in actin network disassembly, but the reassembly of actin in these assays was limited by factors such as diffusional constraints and the use of stabilized actin filaments. Here, we present the reconstitution of a minimal dynamic actin cortex, where actin polymerization is catalyzed on the membrane in the presence of myosin-II activity. We demonstrate that myosin activity leads to disassembly and redistribution in this simplified cortex. Consequently, a new dynamic steady state emerges in which the actin network undergoes constant turnover. Our findings suggest a multifaceted role of myosin-II in the dynamics of the eukaryotic actin cortex. This article has an associated First Person interview with the first author of the paper.

Initiation of T cell signaling by CD45 segregation at 'close contacts'.

It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the potent signaling effects of, local CD45 and kinase segregation. TCR ligands have the potential to heighten signaling simply by holding receptors in close contacts.

Single-molecule imaging reveals that small amyloid-ß1-42 oligomers interact with the cellular prion protein (PrP(C)).

Oligomers of the amyloid-ß peptide (Aß) play a central role in the pathogenesis of Alzheimer's disease and have been suggested to induce neurotoxicity by binding to a plethora of cell-surface receptors. However, the heterogeneous mixtures of oligomers of varying sizes and conformations formed by Aß42 have obscured the nature of the oligomeric species that bind to a given receptor. Here, we have used single-molecule imaging to characterize Aß42 oligomers (oAß42) and to confirm the controversial interaction of oAß42 with the cellular prion protein (PrP(C)) on live neuronal cells. Our results show that, at nanomolar concentrations, oAß42 interacts with PrP(C) and that the species bound to PrP(C) are predominantly small oligomers (dimers and trimers). Single-molecule biophysical studies can thus aid in deciphering the mechanisms that underlie receptor-mediated oAß-induced neurotoxicity, and ultimately facilitate the discovery of novel inhibitors of these pathways.

Identification of a hypochlorite-specific transcription factor from Escherichia coli.

Hypochlorite is a powerful oxidant produced by neutrophils to kill invading microorganisms. Despite this important physiological role of HOCl in fighting bacterial infections, no hypochlorite-specific stress response has been identified yet. Here, we identified a hypochlorite-responsive transcription factor, YjiE, which is conserved in proteobacteria and eukaryotes. YjiE forms unusual dodecameric ring-like structures in vitro that undergo large DNA-induced conformational changes to form dimers and tetramers as shown by transmission electron microscopy and analytical ultracentrifugation. Such smaller oligomers are predominant in hypochlorite-stressed cells and are the active species as shown by fluorescence anisotropy and analytical ultracentrifugation. YjiE regulates a large number of genes upon hypochlorite stress. Among them are genes involved in cysteine, methionine biosynthesis, and sulfur metabolism (up-regulated) and genes involved in iron acquisition and homeostasis (down-regulated), thus supposedly replenishing oxidized metabolites and decreasing the hypochlorite-mediated amplification of intracellular reactive oxygen species. As a result, YjiE specifically confers hypochlorite resistance to E. coli cells. Thus, to our knowledge, YjiE is the first described hypochlorite-specific transcription factor.