Nanoscale Colocalization of NK Cell Activating and Inhibitory Receptors Controls Signal Integration.

Natural killer (NK) cell responses depend on the balance of signals from inhibitory and activating receptors. However, how the integration of antagonistic signals occurs upon NK cell-target cell interaction is not fully understood. Here we provide evidence that NK cell inhibition via the inhibitory receptor Ly49A is dependent on its relative colocalization at the nanometer scale with the activating receptor NKG2D upon immune synapse (IS) formation. NKG2D and Ly49A signal integration and colocalization were studied using NKG2D-GFP and Ly49A-RFP-expressing primary NK cells, forming ISs with NIH3T3 target cells, with or without the expression of single-chain trimer (SCT) H2-Dd and an extended form of SCT H2-Dd-CD4 MHC-I molecules. Nanoscale colocalization was assessed by Förster resonance energy transfer between NKG2D-GFP and Ly49A-RFP and measured for each synapse. In the presence of their respective cognate ligands, NKG2D and Ly49A colocalize at the nanometer scale, leading to NK cell inhibition. However, increasing the size of the Ly49A ligand reduced the nanoscale colocalization with NKG2D, consequently impairing Ly49A-mediated inhibition. Thus, our data shows that NK cell signal integration is critically dependent on the dimensions of NK cell ligand-receptor pairs by affecting their relative nanometer-scale colocalization at the IS. Our results together suggest that the balance of NK cell signals and NK cell responses is determined by the relative nanoscale colocalization of activating and inhibitory receptors in the immune synapse.

Author Correction: Quantitative mapping and minimization of super-resolution optical imaging artifacts.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: Nuclear pores as versatile reference standards for quantitative superresolution microscopy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nuclear pores as versatile reference standards for quantitative superresolution microscopy.

Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions. Here, we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag, HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use (1) as three-dimensional resolution standards for calibration and quality control, (2) to quantify absolute labeling efficiencies and (3) as precise reference standards for molecular counting. These cell lines will enable the broader community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.

Investigating Hepatitis C Virus Infection Using Super-Resolution Microscopy.

Super-resolution microscopy (SRM) can provide a window on the nanoscale events of virus replication. Here we describe a protocol for imaging hepatitis C virus-infected cells using localization SRM. We provide details on sample preparation, immunostaining, data collection, and super-resolution image reconstruction. We have made all efforts to generalize the protocol to make it accessible to all budding super-resolution microscopists.

Septins Recognize and Entrap Dividing Bacterial Cells for Delivery to Lysosomes.

The cytoskeleton occupies a central role in cellular immunity by promoting bacterial sensing and antibacterial functions. Septins are cytoskeletal proteins implicated in various cellular processes, including cell division. Septins also assemble into cage-like structures that entrap cytosolic Shigella, yet how septins recognize bacteria is poorly understood. Here, we discover that septins are recruited to regions of micron-scale membrane curvature upon invasion and division by a variety of bacterial species. Cardiolipin, a curvature-specific phospholipid, promotes septin recruitment to highly curved membranes of Shigella, and bacterial mutants lacking cardiolipin exhibit less septin cage entrapment. Chemically inhibiting cell separation to prolong membrane curvature or reducing Shigella cell growth respectively increases and decreases septin cage formation. Once formed, septin cages inhibit Shigella cell division upon recruitment of autophagic and lysosomal machinery. Thus, recognition of dividing bacterial cells by the septin cytoskeleton is a powerful mechanism to restrict the proliferation of intracellular bacterial pathogens.

Quantitative mapping and minimization of super-resolution optical imaging artifacts.

Super-resolution microscopy depends on steps that can contribute to the formation of image artifacts, leading to misinterpretation of biological information. We present NanoJ-SQUIRREL, an ImageJ-based analytical approach that provides quantitative assessment of super-resolution image quality. By comparing diffraction-limited images and super-resolution equivalents of the same acquisition volume, this approach generates a quantitative map of super-resolution defects and can guide researchers in optimizing imaging parameters.

Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations.

Despite significant progress, high-speed live-cell super-resolution studies remain limited to specialized optical setups, generally requiring intense phototoxic illumination. Here, we describe a new analytical approach, super-resolution radial fluctuations (SRRF), provided as a fast graphics processing unit-enabled ImageJ plugin. In the most challenging data sets for super-resolution, such as those obtained in low-illumination live-cell imaging with GFP, we show that SRRF is generally capable of achieving resolutions better than 150 nm. Meanwhile, for data sets similar to those obtained in PALM or STORM imaging, SRRF achieves resolutions approaching those of standard single-molecule localization analysis. The broad applicability of SRRF and its performance at low signal-to-noise ratios allows super-resolution using modern widefield, confocal or TIRF microscopes with illumination orders of magnitude lower than methods such as PALM, STORM or STED. We demonstrate this by super-resolution live-cell imaging over timescales ranging from minutes to hours.

VirusMapper: open-source nanoscale mapping of viral architecture through super-resolution microscopy.

The nanoscale molecular assembly of mammalian viruses during their infectious life cycle remains poorly understood. Their small dimensions, generally bellow the 300nm diffraction limit of light microscopes, has limited most imaging studies to electron microscopy. The recent development of super-resolution (SR) light microscopy now allows the visualisation of viral structures at resolutions of tens of nanometers. In addition, these techniques provide the added benefit of molecular specific labelling and the capacity to investigate viral structural dynamics using live-cell microscopy. However, there is a lack of robust analytical tools that allow for precise mapping of viral structure within the setting of infection. Here we present an open-source analytical framework that combines super-resolution imaging and naïve single-particle analysis to generate unbiased molecular models. This tool, VirusMapper, is a high-throughput, user-friendly, ImageJ-based software package allowing for automatic statistical mapping of conserved multi-molecular structures, such as viral substructures or intact viruses. We demonstrate the usability of VirusMapper by applying it to SIM and STED images of vaccinia virus in isolation and when engaged with host cells. VirusMapper allows for the generation of accurate, high-content, molecular specific virion models and detection of nanoscale changes in viral architecture.

Bacterial autolysins trim cell surface peptidoglycan to prevent detection by the Drosophila innate immune system.

Bacteria have to avoid recognition by the host immune system in order to establish a successful infection. Peptidoglycan, the principal constituent of virtually all bacterial surfaces, is a specific molecular signature recognized by dedicated host receptors, present in animals and plants, which trigger an immune response. Here we report that autolysins from Gram-positive pathogenic bacteria, enzymes capable of hydrolyzing peptidoglycan, have a major role in concealing this inflammatory molecule from Drosophila peptidoglycan recognition proteins (PGRPs). We show that autolysins trim the outermost peptidoglycan fragments and that in their absence bacterial virulence is impaired, as PGRPs can directly recognize leftover peptidoglycan extending beyond the external layers of bacterial proteins and polysaccharides. The activity of autolysins is not restricted to the producer cells but can also alter the surface of neighboring bacteria, facilitating the survival of the entire population in the infected host. DOI: http://dx.doi.org/10.7554/eLife.02277.001.