The ability of the human malaria parasite Plasmodium falciparum to remodel its host red blood cell (RBC) is central to the parasite’s ability to survive within the circulation of its host and cause disease. Following invasion, the parasite exports proteins into the RBC cytoplasm where they remodel the membrane skeleton and alter the cell’s biophysical properties. Many of the exported proteins are trafficked through parasite derived structures called Maurer’s clefts in the cytoplasm of the RBC. These clefts act as an intermediate compartment for proteins en route to the RBC membrane such as the major virulence protein, Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1). From the Maurer’s clefts, PfEMP1 is delivered to the RBC membrane through a poorly understood process, which may involve trafficking via a physical connection and/or vesicle mediated trafficking. Electron-tomography of 3D7 infected RBCs reveals a tube-like structure, called the tether, that connects the Maurer’s clefts to the RBC membrane. To date, the formation and function of these tether structures is unclear, with only one protein, the Membrane Associated Histidine Rich Protein 2 (MAHRP2) known to locate at these structures. In this study, we show that deletion of MAHRP2 leads to a loss of tethers, reduced Maurer’s cleft immobilization at the RBC membrane and an increase in Maurer’s cleft size. Lastly, we show that deletion of MAHRP2 impacts PTP2 related vesicle formation, and a reduced ability to cytoadhere to the endothelial ligands CD36.

Trophoblast cells line the surface of placental villi, facilitating the exchange of nutrients, gases, and wastes between the maternal and fetal circulations. The fusion of cytotrophoblast (CTB) cells into the surrounding multinucleated syncytiotrophoblast (STB), is accompanied by a shift in cellular ultrastructure (subcellular architecture). Mitochondria undergo a remarkable decrease in size and alteration in morphology following trophoblast differentiation, and have thus been the subject of investigations due to their crucial role in producing energy for placental development. Observing this shift in structure has relied on the use of electron microscopy, which has offered insights into underlying mitochondrial functions. Since the initial use of electron microscopy to study villous trophoblasts in the 1950s, novel techniques have emerged that have the capacity to interrogate placental ultrastructure with unprecedented resolution. This review discusses the evolution of electron microscopy techniques to study the placenta over the last 70 years. Moreover, we discuss emerging methods for resolving 3D organelle structure within the placenta, which offer more physiologically pertinent information and context for complex topologies. Further, we discuss advanced methods of cryo-electron tomography (cryo-ET) that present the placental field with an exciting opportunity to determine the complex relationship between mitochondrial architecture and protein structure in the human placenta. By specifically focusing on mitochondrial imaging, we showcase the capacity for volume electron microscopy and cryo-ET to reveal the role of organelle structure in placental development.

NanT and NanX are bacterial transporters that import the sialic acids, N-acetylneuraminate and 2,7-anhydro-n-acetylneuraminate, respectively. Here, we used complementary biophysical and computational approaches to structurally characterise Escherichia coli NanX. Size exclusion chromatography, analytical ultracentrifugation and low-resolution cryo-electron microscopy reveal that NanX exists in both monomeric and dimeric states following purification. Molecular modelling and substrate docking identify key residues likely involved in 2,7-anhydro-n-acetylneuraminate recognition. Using this information, we engineered a mutant NanX transporter that can import the NanT-specific substrate, N-acetylneuraminate, which we verified using a bacterial growth assay. These data identify amino acids involved in major facilitator superfamily mediated sialic acid transport and offer a new research perspective of its metabolism.

Abstract Mitochondria adapt their structure through fusion and fission, yet how their morphology and cristae architecture change as cells differentiate remains unclear. The human placenta is a valuable model for studying mitochondrial diversity within a single tissue. As epithelial trophoblast cells of the placenta differentiate, their accompanying mitochondria morphologically and functionally transform. We use array tomography to characterise mitochondrial volume and network complexity. Cryo-electron tomography reveals two distinct subpopulations of mitochondria within preparations enriched for progenitor cytotrophoblasts, and a singular, homogeneous population in the differentiated syncytiotrophoblast. We showcase the 3D topology of individual cristae through a standardised metric of “curvedness”. Proteomic analysis of isolated mitochondria identifies reduced levels of proteins involved in mitochondrial dynamics and cristae organisation complexes in the syncytiotrophoblast, accompanied by variations in electron transport chain subunits, ATP synthase, and supercomplex components. This study highlights the advantages of combining multimodal imaging with proteomic analyses, to elucidate the process of mitochondrial morphology and cristae architecture remodelling as cells differentiate.

Three-dimensional (3D) imaging of the subcellular organisation and morphology of cells and tissues is essential for understanding biological function. Although staining is the most widely used approach for visualising biological samples under a microscope, the intracellular refractive index (RI) has been proposed as a potential biophysical marker that could supplement or even surpass the sensitivity of current histological methods. Hence, the development of new, highly sensitive, label-free techniques that can detect changes in the intracellular RI is extremely desirable for biomedical imaging. The recent development of plasmonic metamaterials, designed to mimic a traditional microscope slide, have made it possible to translate subtle changes in refractive index directly into color. This approach enables label-free visualization of tissue microstructure using standard histological slide preparation methods. Here we demonstrate ultramicrotome-assisted optical plasmon-enhanced (PE) array tomography and correlate this with electron array tomography of the same sample embedded in resin. The approach enables axially super-resolved label-free imaging of whole cells in the range of 30-200 nm and shows great potential for multimodal three-dimensional colorimetric histology at the (sub-) organelle level.

Pore-forming proteins comprise a highly diverse group of proteins exemplified by the membrane attack complex/perforin (MACPF), cholesterol-dependent cytolysin (CDC), and gasdermin superfamilies, which all form gigantic pores (>150 angstroms). A recently found family of pore-forming toxins, called CDC-like proteins (CDCLs), are wide-spread in gut microbes and are a prevalent means of antibacterial antagonism. However, the structural aspects of how CDCLs assemble a pore remain a mystery. Here, we report the crystal structure of a proteolytically activated CDCL and cryo–electron microscopy structures of a prepore-like intermediate and a transmembrane pore providing detailed snapshots across the entire pore-forming pathway. These studies reveal a sophisticated array of regulatory features to ensure productive pore formation, and, thus, CDCLs straddle the MACPF, CDC, and gasdermin lineages of the giant pore superfamilies.

Hyperspectral imaging is gaining attention in the field of disease diagnosis due to its ability to enhance tissue contrast, surpassing the capabilities of conventional brightfield imaging techniques. Typically, histological sections lack sufficient intrinsic contrast in the visible spectrum, necessitating the use of dyes or stains for adequate visualization. However, a recent breakthrough involves the application of plasmonic meta-materials as substrates for histological sections, replacing staining or labelling on traditional microscope glass slides. These nanofabricated microscope slides, shortened to nanoMslides, operate by selectively transmitting colors based on refractive index variations within the sample when illuminated with white light. This study investigates the feasibility of integration of nanoMslides for hyperspectral imaging. By employing a tunable light source, specific plasmon resonances within the slides can be selectively excited. This precise control over plasmonic interactions results in significantly heightened sensitivity and specificity, showcasing the potential for advanced applications in disease diagnosis and biomedical research.

The Type IX Secretion System exports proteins across the outer membrane (OM) of bacteria in the Bacteroidota phylum, however, the mechanistic details remain unknown. In Porphyromonas gingivalis the core components of the multi-protein complex are the Sov translocon, Attachment Complexes (PorQ, U, V, Z), PorLM molecular motors and PorKN rings. Here, we present a ~ 3.5 Å cryo-EM structure of the periplasmic rings comprising 32-33 subunits each of PorK and PorN. Additionally, we show the presence of a critical disulfide bond between PorK and the OM protein PorG that is essential for protein secretion and demonstrate that the Attachment Complexes bind to, and are localized above, the PorKN rings. Overall, each ring resembles a cogwheel with PorN forming cog-like projections that we propose engage with the PorLM motor to drive the rotation of the PorKN cogwheel together with PorG and associated Attachment Complexes, thus providing the energy to complete protein secretion and the coordinated cell surface attachment of the secreted cargo.

The Type IX Secretion System exports proteins across the outer membrane (OM) of bacteria in the Bacteroidota phylum, however, the mechanistic details remain unknown. Here, we present a ~3.5A cryo-EM structure of the periplasmic rings comprising 32-33 subunits each of PorK and PorN. Additionally, we show the presence of a critical disulfide bond between PorK and the PorG OM protein that is essential for protein secretion and demonstrate that the Attachment Complexes bind to and are localized above the PorKN rings. Overall, each ring resembles a cogwheel with PorN forming cog-like projections on the periplasmic side and the flat surface of PorK orienting towards the OM. Given these results, we propose that the PorLM motor drives the rotation of the PorKN cogwheel together with PorG and associated Attachment Complexes, potentially providing the energy to complete protein secretion and the coordinated cell surface attachment of the secreted cargo. ### Competing Interest Statement The authors have declared no competing interest.

Cryo-electron microscopy (cryoEM) grid preparation is one of the bottlenecks in using cryoEM for macromolecular structure determination. Despite significant advancements in the development of blot-free instruments, their high cost limits their widespread usage. Understanding the effects of the different parameters of the blotting instruments, particularly the Vitrobot, can lead to producing high-quality grids while saving both time and resources. In this study, we focus specifically on low magnification cryoEM images as a rapid way to evaluate ice thickness and screen the effects of different grid preparation parameters. By employing a machine learning-based approach and semi-automated image analysis, we analysed large datasets of low-magnification atlas images to quantify ice thickness and distribution across grids prepared under varied conditions. Our results show that detectable changes in ice quality often require substantial adjustments in parameters, and even then, considerable grid-to-grid variability can persist. Notably, we observed that the presence of detergent improved consistency in ice thickness. While our approach does not assess protein distribution or particle behaviour at high magnification, it offers a scalable and efficient tool for early-stage grid screening and protocol optimization.

The biological effects of electromagnetic field (EMF) irradiation in the terahertz (THz) range remain ambiguous, despite numerous studies that have been conducted. In this paper, the metabolic response of Escherichia coli K 12 to EMF irradiation was examined using a 1.0 W m-2 incident synchrotron source (SS) in the range of 0.5-18.0 THz for over 90 min of continuous exposure at 25 degrees C. This continuous SS THz exposure induced periodic decreases in the cell growth after 10, 20, and 40 min of exposure compared to a time-matched control; however, the number of viable cells thereafter grew. The physiological status of treated cells immediately after exposure was assessed by using the direct plate counting technique and electron microscopy. Analysis of scanning electron microscopy (SEM) and high-resolution cryogenic transmission electron (cryo-TEM) micrographs showed that approximately 20% of the SS THz-exposed E. coli cells exhibited a deformed outer membrane, membrane perturbations, and leakage of cytosol. The proteome changes in E. coli cells after 18 h postexposure were associated with cellular response to plasma membrane regulation including phospholipid biosynthetic process and osmotic stress. The results of this study highlighted that E. coli cells can promptly activate the fundamental mechanisms in response to prolonged exposure to THz radiation that are evolutionarily developed to withstand other environmental stressors.

The immune response against Legionella longbeachae, a causative agent of the often-fatal Legionnaires’ pneumonia, is poorly understood. Here we investigated the specific roles of tissue-resident alveolar macrophages (AM) and infiltrating phagocytes during infection with this pathogen. AM were the predominant cell type that internalized bacteria one day after infection. Three and five days after infection, AM numbers were greatly reduced while there was an influx of neutrophils and later monocyte-derived cells (MC) into lung tissue. AM carried greater numbers of viable L.longbeachae than neutrophils and MC, which correlated with a higher capacity of L.longbeachae to translocate bacterial effector proteins required for bacterial replication into the AM cytosol. Cell ablation experiments demonstrated that AM promoted infection whereas neutrophils and MC were required for efficient bacterial clearance. IL-18 was important for IFN-γ production by IL-18R+ NK cells and T cells which, in turn, stimulated ROS-mediated bactericidal activity in neutrophils resulting in restriction of L.longbeachae infection. Ciliated bronchiolar epithelial cells also expressed IL-18R but did not play a role in IL-18-mediated L.longbeachae clearance. Our results have identified opposing innate functions of tissue-resident and infiltrating immune cells during L.longbeachae infection that may be manipulated to improve protective responses.

Background:Lymphedema is common after lymphatic damage in cancer treatment, with negative impacts on function and quality of life. Evidence suggests that blood vessel microvasculature is sensitive to irradiation and trauma; however, despite knowledge regarding dedicated mural blood supply to arteries and veins (vasa vasorum), equivalent blood vessels supplying lymphatics have not been characterized. We studied collecting lymphatics for dedicated mural blood vessels in our series of 500 lymphaticovenous anastomosis procedures for lymphedema, and equivalent controls. Methods:Microscopic images of lymphatics from lymphedema and control patients were analyzed for lymphatic wall vascular density. Collecting lymphatics from 20 patients with lymphedema and 10 control patients were sampled for more detailed analysis (podoplanin immunostaining, light/confocal microscopy, microcomputed tomography, and transmission electron microscopy) to assess lymphatic wall ultrastructure and blood supply. Results:Analysis revealed elaborate, dense blood microvessel networks associating with lymphatic walls in lymphedema patients and smaller equivalent vessels in controls. These vasa vasora or "arteria lymphatica" were supplied by regular axial blood vessels, parallel to lymphatic microperforators linking dermal and collecting lymphatics. Lymphatic walls were thicker in lymphedema patients than controls, with immunohistochemistry, computed tomography, transmission electron microscopy, and confocal microscopy characterizing abnormal blood vessels (altered appearance, thickened walls, elastin loss, narrow lumina, and fewer red blood cells) on these lymphatic walls. Conclusions:Dedicated blood vessels on lymphatics are significantly altered in lymphedema. A better understanding of the role of these vessels may reveal mechanistic clues into lymphedema pathophysiology and technical aspects of lymphedema microsurgery, and suggest potential novel therapeutic targets.

We present a novel technique of genetic transformation of bacterial cells mediated by high frequency electromagnetic energy (HF EME). Plasmid DNA, pGLO (5.4 kb), was successfully transformed into Escherichia coli JM109 cells after exposure to 18 GHz irradiation at a power density between 5.6 and 30 kW m(-2) for 180 s at temperatures ranging from 30 to 40 degrees C. Transformed bacteria were identified by the expression of green fluorescent protein (GFP) using confocal scanning microscopy (CLSM) and flow cytometry (FC). Approximately 90.7% of HF EME treated viable E. coli cells exhibited uptake of the pGLO plasmid. The interaction of plasmid DNA with bacteria leading to transformation was confirmed by using cryogenic transmission electron microscopy (cryo-TEM). HF EME-induced plasmid DNA transformation was shown to be unique, highly efficient, and cost-effective. HF EME-induced genetic transformation is performed under physiologically friendly conditions in contrast to existing techniques that generate higher temperatures, leading to altered cellular integrity. This technique allows safe delivery of genetic material into bacterial cells, thus providing excellent prospects for applications in microbiome therapeutics and synthetic biology.

In transmission electron microscopy (TEM), cameras are square or rectangular but beams are round so the circular lobes irradiate adjacent areas, precluding further neighboring acquisition for beam-sensitive samples. We present condenser aperture plates with square and rectangular shapes that improve the efficiency of area usage by 70% and enhance montage imaging for beam-sensitive specimens. We demonstrate the compatibility of these condenser aperture plates with high-resolution cryogenic TEM by reconstructing a 1.8-Å map of equine apo-ferritin.

Butyrophilin (BTN) molecules are emerging as key regulators of T cell immunity, however, how they trigger cell-mediated responses is poorly understood. Here, the crystal structure of a gamma-delta T cell receptor (γδTCR) in complex with BTN member 2A1 (BTN2A1) revealed that BTN2A1 engages the side of the γδTCR, leaving the apical TCR surface bioavailable. We reveal that BTN3A1 is a second γδTCR ligand, that co-engages γδTCR via binding to this accessible apical surface. BTN2A1 and BTN3A1 also directly interact with each other in cis, and structural analysis revealed formation of W-shaped heteromeric multimers. This BTN2A1–BTN3A1 interaction involved the same epitopes that BTN2A1 and BTN3A1 each use to engage γδTCR; indeed, either forced separation or locking together of BTN2A1 and BTN3A1 resulted in enhanced or abrogated γδTCR interaction, respectively. Our findings reveal a new paradigm in immune activation, whereby γδTCRs recognize dual epitopes on BTN2A1 and BTN3A1 complexes.

The immune response against Legionella longbeachae, a causative agent of the often-fatal Legionnaires' pneumonia, is poorly understood. Here, we investigated the specific roles of tissue-resident alveolar macrophages (AMs) and infiltrating phagocytes during infection with this pathogen. AMs were the predominant cell type that internalized bacteria 1 day after infection. A total of 3 and 5 days after infection, AM numbers were greatly reduced, whereas there was an influx of neutrophils and, later, monocytederived cells (MCs) into lung tissue. AMs carried greater numbers of viable L. longbeachae than neutrophils and MCs, which correlated with a higher capacity of L. longbeachae to translocate bacterial effector proteins required for bacterial replication into the AM cytosol. Cell ablation experiments demonstrated that AM promoted infection, whereas neutrophils and MC were required for efficient bacterial clearance. Interleukin (IL)-18 was important for interferon-gamma production by IL-18R+ natural killer cells and T cells, which, in turn, stimulated reactive oxygen species-mediated bactericidal activity in neutrophils, resulting in the restriction of L. longbeachae infection. Ciliated bronchiolar epithelial cells also expressed IL-18R but did not play a role in IL-18-mediated L. longbeachae clearance. Our results have identified opposing innate functions of tissue-resident and infiltrating immune cells during L. longbeachae infection that may be manipulated to improve protective responses.

Interleukin (IL-)11, an IL-6 family cytokine, has pivotal roles in autoimmune diseases, fibrotic complications, and solid cancers. Despite intense therapeutic targeting efforts, structural understanding of IL-11 signalling and mechanistic insights into current inhibitors are lacking. Here we present cryo-EM and crystal structures of the human IL-11 signalling complex, including the complex containing the complete extracellular domains of the shared IL-6 family β-receptor, gp130. We show that complex formation requires conformational reorganisation of IL-11 and that the membrane-proximal domains of gp130 are dynamic. We demonstrate that the cytokine mutant, IL-11 Mutein, competitively inhibits signalling in human cell lines. Structural shifts in IL-11 Mutein underlie inhibition by altering cytokine binding interactions at all three receptor-engaging sites and abrogating the final gp130 binding step. Our results reveal the structural basis of IL-11 signalling, define the molecular mechanisms of an inhibitor, and advance understanding of gp130-containing receptor complexes, with potential applications in therapeutic development.