Exocrine gland-resident memory CD8 T cells use mechanosensing for tissue surveillance.

Tissue-resident CD8 T cells (T) continuously scan peptide-MHC (pMHC) complexes in their organ of residence to intercept microbial invaders. Recent data showed that T lodged in exocrine glands scan tissue in the absence of any chemoattractant or adhesion receptor signaling, thus bypassing the requirement for canonical migration-promoting factors. The signals eliciting this noncanonical motility and its relevance for organ surveillance have remained unknown.

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.

Within-Host Genomic Diversity of Candida albicans in Healthy Carriers.

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.

3D structures of individual mammalian genomes studied by single-cell Hi-C.

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.

Remarkably low affinity of CD4/peptide-major histocompatibility complex class II protein interactions.

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.

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.

Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging.

Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet.

A microfluidic device for the hydrodynamic immobilisation of living fission yeast cells for super-resolution imaging.

We describe a microfluidic device designed specifically for the reversible immobilisation of (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 >25 nm 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.

Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy.

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells.

The changing point-spread function: single-molecule-based super-resolution imaging.

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.

Quantification of the effects of antibodies on the extra- and intracellular dynamics of Salmonella enterica.

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.