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Significance One approach to testing biological theories is to determine if they are predictive. We have developed a simple, theoretical treatment of T cell receptor (TCR) triggering that relies on just two physical principles: (i) the time TCRs spend in cell–cell contacts depleted of large tyrosine phosphatases and (ii) constraints on the size of these contacts imposed by cell topography. The theory not only distinguishes between agonistic and nonagonistic TCR ligands but predicts the relative signaling potencies of agonists with remarkable accuracy. These findings suggest that the theory captures the essential features of receptor triggering., 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.

Genomic variations in Candida albicans, a major fungal pathogen of humans, have been observed upon exposure of this yeast to different stresses and experimental infections, possibly contributing to subsequent adaptation to these stress conditions. Yet, little is known about the extent of genomic diversity that is associated with commensalism, the predominant lifestyle of C. albicans in humans. In this study, we investigated the genetic diversity of C. albicans oral isolates recovered from healthy individuals, using multilocus sequencing typing (MLST) and whole genome sequencing. While MLST revealed occasional differences between isolates collected from a single individual, genome sequencing showed that they differed by numerous single nucleotide polymorphisms, mostly resulting from short-range loss-of-heterozygosity events. These differences were shown to have occurred upon human carriage of C. albicans rather than subsequent in vitro manipulation of the isolates. Thus, C. albicans intra-sample diversity appears common in healthy individuals, higher than that observed using MLST. We propose that diversifying lineages coexist in a single human individual, and this diversity can enable rapid adaptation under stress exposure. These results are crucial for the interpretation of longitudinal studies evaluating the evolution of the C. albicans genome.

We describe a microfluidic device designed specifically for the reversible immobilisation of Schizosaccharomyces pombe (Fission Yeast) cells to facilitate live cell super-resolution microscopy. Photo-Activation Localisation Microscopy (PALM) is used to create detailed super-resolution images within living cells with a modal accuracy of >25nm in the lateral dimensions. The novel flow design captures and holds cells in a well-defined array with minimal effect on the normal growth kinetics. Cells are held over several hours and can continue to grow and divide within the device during fluorescence imaging.

T cell activation is pivotal to the initiation of the adaptive immune response which confers resistance to pathogens and plays a role in numerous diseases. Over the past decades, key protein interactions contributing to T cell activation via antigen recognition have been characterized in molecular detail, in particular the interaction of T-cell receptors (TCR) with antigenic peptide bound to the major histocompatibility complex (MHC). However, it remains unknown how this interaction is translated into intracellular signaling. Three different models of TCR triggering have been suggested which are based on aggregation, segregation or a conformational change of the TCR. Here we exploit a combination of super-resolution fluorescence microscopy and single-particle tracking to follow how the spatial distribution of proteins essential to T-cell activation like the TCR, CD45 and Lck changes at the interface of live T-cells and various surfaces. Whereas numerous studies have investigated the organization of these proteins 5-10 minutes after contact formation between the cell and the surface, our single-molecule imaging techniques enable us to study these processes immediately after contact establishment. Thus our experiments provide new insights into the molecular rearrangements that may underpin the initial events in T-cell activation.

Counting absolute numbers of proteins is of major importance in biology. This would help for example to resolve the stoechiometry of complexes, to observe aggregation phenomena or to describe the minimal signal required for a specific cellular response. The only existing tools available to biologists are bulk experiments, crystallography or photobleaching steps. Each of these methods comes with its own drawbacks: by comparing global mean values, bulk experiments do not take into account quenching or differences of brightness in a population of fluorophores; crystallography only studies the protein as a crystal and hence in a totally artificial environment; finally, the detection of photobleaching steps requires a low density of relevant complexes and a low stoechiometry (up to five monomers).Based on a TIRF (total-internal reflexion fluorescence) single-molecule microscopy approach, we set out to optimise and characterise an original method to precisely count absolute numbers of proteins in live cells.We first characterise the photo-physics (contrast, blinking, dark state, pre-activation, reversible activation, thermal or pH dependant activation, bleaching, etc.) of different photo-activable dyes. Although a blinking fluorophore allows multiple localization in PALM (photo-activable localization microscopy) or STORM (stochastic optical reconstruction microscopy) imaging, it should be only counted once when counting is at stake. We also develop a specific algorithm to avoid over- or under-counting single emitters. using a herpes simplex virus which capsid is tagged with exactly 900 photo-activable dyes, we then characterised the precision of our technique. Finally, we show the applicability of our approach to current questions in biology by investigating the oligomerisation state of various cell surface receptors in live T cells.

The αβ T-cell coreceptor CD4 enhances immune responses more than 1 million-fold in some assays, and yet the affinity of CD4 for its ligand, peptide-major histocompatibility class II (pMHC II) on antigen-presenting cells, is so weak that it was previously unquantifiable. Here, we report that a soluble form of CD4 failed to bind detectably to pMHC II in surface plasmon resonance-based assays, establishing a new upper limit for the solution affinity at 2.5 mM. However, when presented multivalently on magnetic beads, soluble CD4 bound pMHC II-expressing B cells, confirming that it is active and allowing mapping of the native coreceptor binding site on pMHC II. Whereas binding was undetectable in solution, the affinity of the CD4/pMHC II interaction could be measured in 2D using CD4- and adhesion molecule-functionalized, supported lipid bilayers, yielding a 2D K d of ∼5,000 molecules/μm 2 . This value is two to three orders of magnitude higher than previously measured 2D K d values for interacting leukocyte surface proteins. Calculations indicated, however, that CD4/pMHC II binding would increase rates of T-cell receptor (TCR) complex phosphorylation by threefold via the recruitment of Lck, with only a small, 2–20% increase in the effective affinity of the TCR for pMHC II. The affinity of CD4/pMHC II therefore seems to be set at a value that increases T-cell sensitivity by enhancing phosphorylation, without compromising ligand discrimination.

The folding of genomic DNA from the beads-on-a-string-like structure of nucleosomes into higher-order assemblies is crucially linked to nuclear processes. Here we calculate 3D structures of entire mammalian genomes using data from a new chromosome conformation capture procedure that allows us to first image and then process single cells. The technique enables genome folding to be examined at a scale of less than 100 kb, and chromosome structures to be validated. The structures of individual topological-associated domains and loops vary substantially from cell to cell. By contrast, A and B compartments, lamina-associated domains and active enhancers and promoters are organized in a consistent way on a genome-wide basis in every cell, suggesting that they could drive chromosome and genome folding. By studying genes regulated by pluripotency factor and nucleosome remodelling deacetylase (NuRD), we illustrate how the determination of single-cell genome structure provides a new approach for investigating biological processes.

Based on an original molecular-based design, we present bright and photostable fluorescent organic nanoparticles (FONs) showing excellent colloidal stability in various aqueous environments. Complementary near-infrared emitting and green emitting FONs were prepared using a simple, fast and robust protocol. Both types of FONs could be simultaneously imaged at the single-particle level in solution as well as in biological environments using a monochromatic excitation and a dual-color fluorescence microscope. No evidence of acute cytotoxicity was found upon incubation of live cells with mixed solutions of FONs, and both types of nanoparticles were found internalized in the cells where their motion could be simultaneously tracked at video-rate up to minutes. These fluorescent organic nanoparticles open a novel non-toxic alternative to existing nanoparticles for imaging biological structures, compatible with live-cell experiments and specially fitted for multicolor single particle tracking.

Over the past decade, many techniques for imaging systems at a resolution greater than the diffraction limit have been developed. These methods have allowed systems previously inaccessible to fluorescence microscopy to be studied and biological problems to be solved in the condensed phase. This brief review explains the basic principles of super-resolution imaging in both two and three dimensions, summarizes recent developments, and gives examples of how these techniques have been used to study complex biological systems.

Antibodies are known to be essential in controlling Salmonella infection, but their exact role remains elusive. We recently developed an in vitro model to investigate the relative efficiency of four different human immunoglobulin G (IgG) subclasses in modulating the interaction of the bacteria with human phagocytes. Our results indicated that different IgG subclasses affect the efficacy of Salmonella uptake by human phagocytes. In this study, we aim to quantify the effects of IgG on intracellular dynamics of infection by combining distributions of bacterial numbers per phagocyte observed by fluorescence microscopy with a mathematical model that simulates the in vitro dynamics. We then use maximum likelihood to estimate the model parameters and compare them across IgG subclasses. The analysis reveals heterogeneity in the division rates of the bacteria, strongly suggesting that a subpopulation of intracellular Salmonella , while visible under the microscope, is not dividing. Clear differences in the observed distributions among the four IgG subclasses are best explained by variations in phagocytosis and intracellular dynamics. We propose and compare potential factors affecting the replication and death of bacteria within phagocytes, and we discuss these results in the light of recent findings on dormancy of Salmonella .

Among the most contentious questions in immunology is: how is the T cell receptor (TCR) triggered? using a combination of different single-molecule tracking, novel calcium signalling assays, crystallography and super-resolution imaging techniques we have demonstrated evidence to support the kinetic segregation model as the molecular mechanism behind the adaptive immune system.The phosphorylation of the T cell receptor is required for T cell activation. The kinetic segregation model predicts that this is achieved by the spatial partitioning of important components such as the TCR and Lck kinase from the CD45 phosphatase according to the difference in size of the extracellular domain of the proteins on the cell membrane. We test this prediction by imaging the formation of contacts between T cells with a model antigen presenting cells in early stage contacts at endogenous expression levels. The approach allows the interrogation of the kinetic segregation with other models based upon aggregation or conformation change of the TCR. We present the results of this study.