Multiplexed 3D Analysis of Immune States and Niches in Human Tissue.

Tissue homeostasis and the emergence of disease are controlled by changes in the proportions of resident and recruited cells, their organization into cellular neighbourhoods, and their interactions with acellular tissue components. Highly multiplexed tissue profiling (spatial omics) 1 makes it possible to study this microenvironment in situ , usually in 4-5 micron thick sections (the standard histopathology format) 2 . Microscopy-based tissue profiling is commonly performed at a resolution sufficient to determine cell types but not to detect subtle morphological features associated with cytoskeletal reorganisation, juxtracrine signalling, or membrane trafficking 3 . Here we describe a high-resolution 3D imaging approach able to characterize a wide variety of organelles and structures at sub-micron scale while simultaneously quantifying millimetre-scale spatial features. This approach combines cyclic immunofluorescence (CyCIF) imaging 4 of over 50 markers with confocal microscopy of archival human tissue thick enough (30-40 microns) to fully encompass two or more layers of intact cells. 3D imaging of entire cell volumes substantially improves the accuracy of cell phenotyping and allows cell proximity to be scored using plasma membrane apposition, not just nuclear position. In pre-invasive melanoma in situ 5 , precise phenotyping shows that adjacent melanocytic cells are plastic in state and participate in tightly localised niches of interferon signalling near sites of initial invasion into the underlying dermis. In this and metastatic melanoma, mature and precursor T cells engage in an unexpectedly diverse array of juxtracrine and membrane-membrane interactions as well as looser "neighbourhood" associations 6 whose morphologies reveal functional states. These data provide new insight into the transitions occurring during early tumour formation and immunoediting and demonstrate the potential for phenotyping of tissues at a level of detail previously restricted to cultured cells and organoids.

A biologist's guide to planning and performing quantitative bioimaging experiments.

Technological advancements in biology and microscopy have empowered a transition from bioimaging as an observational method to a quantitative one. However, as biologists are adopting quantitative bioimaging and these experiments become more complex, researchers need additional expertise to carry out this work in a rigorous and reproducible manner. This Essay provides a navigational guide for experimental biologists to aid understanding of quantitative bioimaging from sample preparation through to image acquisition, image analysis, and data interpretation. We discuss the interconnectedness of these steps, and for each, we provide general recommendations, key questions to consider, and links to high-quality open-access resources for further learning. This synthesis of information will empower biologists to plan and execute rigorous quantitative bioimaging experiments efficiently.

Cell wall synthesis and remodelling dynamics determine division site architecture and cell shape in Escherichia coli.

The bacterial division apparatus catalyses the synthesis and remodelling of septal peptidoglycan (sPG) to build the cell wall layer that fortifies the daughter cell poles. Understanding of this essential process has been limited by the lack of native three-dimensional views of developing septa. Here, we apply state-of-the-art cryogenic electron tomography (cryo-ET) and fluorescence microscopy to visualize the division site architecture and sPG biogenesis dynamics of the Gram-negative bacterium Escherichia coli. We identify a wedge-like sPG structure that fortifies the ingrowing septum. Experiments with strains defective in sPG biogenesis revealed that the septal architecture and mode of division can be modified to more closely resemble that of other Gram-negative (Caulobacter crescentus) or Gram-positive (Staphylococcus aureus) bacteria, suggesting that a conserved mechanism underlies the formation of different septal morphologies. Finally, analysis of mutants impaired in amidase activation (ΔenvC ΔnlpD) showed that cell wall remodelling affects the placement and stability of the cytokinetic ring. Taken together, our results support a model in which competition between the cell elongation and division machineries determines the shape of cell constrictions and the poles they form. They also highlight how the activity of the division system can be modulated to help generate the diverse array of shapes observed in the bacterial domain.

CAR T cell killing requires the IFNγR pathway in solid but not liquid tumours.

Chimeric antigen receptor (CAR) therapy has had a transformative effect on the treatment of haematologic malignancies1-6, but it has shown limited efficacy against solid tumours. Solid tumours may have cell-intrinsic resistance mechanisms to CAR T cell cytotoxicity. Here, to systematically identify potential resistance pathways in an unbiased manner, we conducted a genome-wide CRISPR knockout screen in glioblastoma, a disease in which CAR T cells have had limited efficacy7,8. We found that the loss of genes in the interferon-γ receptor (IFNγR) signalling pathway (IFNGR1, JAK1 or JAK2) rendered glioblastoma and other solid tumours more resistant to killing by CAR T cells both in vitro and in vivo. However, loss of this pathway did not render leukaemia or lymphoma cell lines insensitive to CAR T cells. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell-adhesion pathways after exposure to CAR T cells. We found that loss of IFNγR1 in glioblastoma cells reduced overall CAR T cell binding duration and avidity. The critical role of IFNγR signalling in susceptibility of solid tumours to CAR T cells is surprising, given that CAR T cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumours, IFNγR signalling was required for sufficient adhesion of CAR T cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumours differ in their interactions with CAR T cells and suggests that enhancing binding interactions between T cells and tumour cells may yield improved responses in solid tumours.

VLDLR and ApoER2 are receptors for multiple alphaviruses.

Alphaviruses, like many other arthropod-borne viruses, infect vertebrate species and insect vectors separated by hundreds of millions of years of evolutionary history. Entry into evolutionarily divergent host cells can be accomplished by recognition of different cellular receptors in different species, or by binding to receptors that are highly conserved across species. Although multiple alphavirus receptors have been described1-3, most are not shared among vertebrate and invertebrate hosts. Here we identify the very low-density lipoprotein receptor (VLDLR) as a receptor for the prototypic alphavirus Semliki forest virus. We show that the E2 and E1 glycoproteins (E2-E1) of Semliki forest virus, eastern equine encephalitis virus and Sindbis virus interact with the ligand-binding domains (LBDs) of VLDLR and apolipoprotein E receptor 2 (ApoER2), two closely related receptors. Ectopic expression of either protein facilitates cellular attachment, and internalization of virus-like particles, a VLDLR LBD-Fc fusion protein or a ligand-binding antagonist block Semliki forest virus E2-E1-mediated infection of human and mouse neurons in culture. The administration of a VLDLR LBD-Fc fusion protein has protective activity against rapidly fatal Semliki forest virus infection in mouse neonates. We further show that invertebrate receptor orthologues from mosquitoes and worms can serve as functional alphavirus receptors. We propose that the ability of some alphaviruses to infect a wide range of hosts is a result of their engagement of evolutionarily conserved lipoprotein receptors and contributes to their pathogenesis.

Toxicity, uptake, and nuclear translocation of ingested micro-nanoplastics in an in vitro model of the small intestinal epithelium.

Despite mounting evidence of increasing micro- and nanoplastics (MNPs) in natural environments, food, and drinking water, little is known of the potential health hazards of MNPs ingestion. We assessed toxicity and uptake of environmentally relevant MNPs in an in vitro small intestinal epithelium (SIE). Test MNPs included 25 and 1000 nm polystyrene (PS) microspheres (PS25 and PS1K); 25, 100, and 1000 nm carboxyl modified PS spheres (PS25C, PS100C, and PS1KC), and secondary MNPs from incinerated polyethylene (PEI). MNPs were subjected to 3-phase digestion to mimic transformations in the gastrointestinal tract (GIT) and digestas applied to the SIE. Carboxylated MNPs significantly reduced viability and increased permeability to 3 kD dextran. Uptake of carboxyl PS materials was size dependent, with significantly greater uptake of PS25C. Fluorescence confocal imaging showed some PS25C agglomerates entering cells independent of endosomes (suggesting diffusion), others within actin shells (suggesting phagocytosis), and many free within the epithelial cells, including agglomerates within nuclei. Pre-treatment with the dynamin inhibitor Dyngo partially reduced PS25 translocation, suggesting a potential role for endocytosis. These findings suggest that ingestion exposures to MNPs could have serious health consequences and underscore the urgent need for additional detailed studies of the potential hazards of ingested MNPs.

Hepatitis C virus NS3-4A protease regulates the lipid environment for RNA replication by cleaving host enzyme 24-dehydrocholesterol reductase.

Many RNA viruses create specialized membranes for genome replication by manipulating host lipid metabolism and trafficking, but in most cases, we do not know the molecular mechanisms responsible or how specific lipids may impact the associated membrane and viral process. For example, hepatitis C virus (HCV) causes a specific, large-fold increase in the steady-state abundance of intracellular desmosterol, an immediate precursor of cholesterol, resulting in increased fluidity of the membrane where HCV RNA replication occurs. Here, we establish the mechanism responsible for HCV's effect on intracellular desmosterol, whereby the HCV NS3-4A protease controls activity of 24-dehydrocholesterol reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol. Our cumulative evidence for the proposed mechanism includes immunofluorescence microscopy experiments showing co-occurrence of DHCR24 and HCV NS3-4A protease; formation of an additional, faster-migrating DHCR24 species (DHCR24*) in cells harboring a HCV subgenomic replicon RNA or ectopically expressing NS3-4A; and biochemical evidence that NS3-4A cleaves DHCR24 to produce DHCR24* in vitro and in vivo We further demonstrate that NS3-4A cleaves DHCR24 between residues Cys91 and Thr92 and show that this reduces the intracellular conversion of desmosterol to cholesterol. Together, these studies demonstrate that NS3-4A directly cleaves DHCR24 and that this results in the enrichment of desmosterol in the membranes where NS3-4A and DHCR24 co-occur. Overall, this suggests a model in which HCV directly regulates the lipid environment for RNA replication through direct effects on the host lipid metabolism.

Transcriptomic and phylogenetic analysis of a bacterial cell cycle reveals strong associations between gene co-expression and evolution.

BACKGROUND: The genetic network involved in the bacterial cell cycle is poorly understood even though it underpins the remarkable ability of bacteria to proliferate. How such network evolves is even less clear. The major aims of this work were to identify and examine the genes and pathways that are differentially expressed during the Caulobacter crescentus cell cycle, and to analyze the evolutionary features of the cell cycle network. RESULTS: We used deep RNA sequencing to obtain high coverage RNA-Seq data of five C. crescentus cell cycle stages, each with three biological replicates. We found that 1,586 genes (over a third of the genome) display significant differential expression between stages. This gene list, which contains many genes previously unknown for their cell cycle regulation, includes almost half of the genes involved in primary metabolism, suggesting that these "house-keeping" genes are not constitutively transcribed during the cell cycle, as often assumed. Gene and module co-expression clustering reveal co-regulated pathways and suggest functionally coupled genes. In addition, an evolutionary analysis of the cell cycle network shows a high correlation between co-expression and co-evolution. Most co-expression modules have strong phylogenetic signals, with broadly conserved genes and clade-specific genes predominating different substructures of the cell cycle co-expression network. We also found that conserved genes tend to determine the expression profile of their module. CONCLUSION: We describe the first phylogenetic and single-nucleotide-resolution transcriptomic analysis of a bacterial cell cycle network. In addition, the study suggests how evolution has shaped this network and provides direct biological network support that selective pressure is not on individual genes but rather on the relationship between genes, which highlights the importance of integrating phylogenetic analysis into biological network studies.

Suppression of amber codons in Caulobacter crescentus by the orthogonal Escherichia coli histidyl-tRNA synthetase/tRNAHis pair.

While translational read-through of stop codons by suppressor tRNAs is common in many bacteria, archaea and eukaryotes, this phenomenon has not yet been observed in the α-proteobacterium Caulobacter crescentus. Based on a previous report that C. crescentus and Escherichia coli tRNA(His) have distinctive identity elements, we constructed E. coli tRNA(His) CUA, a UAG suppressor tRNA for C. crescentus. By examining the expression of three UAG codon- containing reporter genes (encoding a ß-lactamase, the fluorescent mCherry protein, or the C. crescentus xylonate dehydratase), we demonstrated that the E. coli histidyl-tRNA synthetase/tRNA(His) CUA pair enables in vivo UAG suppression in C. crescentus. E. coli histidyl-tRNA synthetase (HisRS) or tRNA(His) CUA alone did not achieve suppression; this indicates that the E. coli HisRS/tRNA(His) CUA pair is orthogonal in C. crescentus. These results illustrate that UAG suppression can be achieved in C. crescentus with an orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair.