RNA genome packaging and capsid assembly of bluetongue virus visualized in host cells.

Unlike those of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and ssRNA viruses, the mechanism of genome packaging of dsRNA viruses is poorly understood. Here, we combined the techniques of high-resolution cryoelectron microscopy (cryo-EM), cellular cryoelectron tomography (cryo-ET), and structure-guided mutagenesis to investigate genome packaging and capsid assembly of bluetongue virus (BTV), a member of the Reoviridae family of dsRNA viruses. A total of eleven assembly states of BTV capsid were captured, with resolutions up to 2.8 Å, with most visualized in the host cytoplasm. ATPase VP6 was found underneath the vertices of capsid shell protein VP3 as an RNA-harboring pentamer, facilitating RNA packaging. RNA packaging expands the VP3 shell, which then engages middle- and outer-layer proteins to generate infectious virions. These revealed "duality" characteristics of the BTV assembly mechanism reconcile previous contradictory co-assembly and core-filling models and provide insights into the mysterious RNA packaging and capsid assembly of Reoviridae members and beyond.

A serotonergic axon-cilium synapse drives nuclear signaling to alter chromatin accessibility.

Chemical synapses between axons and dendrites mediate neuronal intercellular communication. Here, we describe a synapse between axons and primary cilia: the axo-ciliary synapse. Using enhanced focused ion beam-scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between brainstem serotonergic axons and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, the 5-hydroxytryptamine receptor 6 (5-HTR6). Using a cilia-targeted serotonin sensor, we show that opto- and chemogenetic stimulation of serotonergic axons releases serotonin onto cilia. Ciliary 5-HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway, which modulates nuclear actin and increases histone acetylation and chromatin accessibility. Ablation of this pathway reduces chromatin accessibility in CA1 pyramidal neurons. As a signaling apparatus with proximity to the nucleus, axo-ciliary synapses short circuit neurotransmission to alter the postsynaptic neuron's epigenetic state.

In Situ Structure of Neuronal C9orf72 Poly-GA Aggregates Reveals Proteasome Recruitment.

Protein aggregation and dysfunction of the ubiquitin-proteasome system are hallmarks of many neurodegenerative diseases. Here, we address the elusive link between these phenomena by employing cryo-electron tomography to dissect the molecular architecture of protein aggregates within intact neurons at high resolution. We focus on the poly-Gly-Ala (poly-GA) aggregates resulting from aberrant translation of an expanded GGGGCC repeat in C9orf72, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. We find that poly-GA aggregates consist of densely packed twisted ribbons that recruit numerous 26S proteasome complexes, while other macromolecules are largely excluded. Proximity to poly-GA ribbons stabilizes a transient substrate-processing conformation of the 26S proteasome, suggesting stalled degradation. Thus, poly-GA aggregates may compromise neuronal proteostasis by driving the accumulation and functional impairment of a large fraction of cellular proteasomes.

The Fly Brain Atlas.

The brain's synaptic networks endow an animal with powerfully adaptive biological behavior. Maps of such synaptic circuits densely reconstructed in those model brains that can be examined and manipulated by genetic means offer the best prospect for understanding the underlying biological bases of behavior. That prospect is now technologically feasible and a scientifically enabling possibility in neurobiology, much as genomics has been in molecular biology and genetics. In Drosophila, two major advances are in electron microscopic technology, using focused ion beam-scanning electron microscopy (FIB-SEM) milling to capture and align digital images, and in computer-aided reconstruction of neuron morphologies. The last decade has witnessed enormous progress in detailed knowledge of the actual synaptic circuits formed by real neurons. Advances in various brain regions that heralded identification of the motion-sensing circuits in the optic lobe are now extending to other brain regions, with the prospect of encompassing the fly's entire nervous system, both brain and ventral nerve cord.

ATG4 family proteins drive phagophore growth independently of the LC3/GABARAP lipidation system.

The sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step of PINK1/Parkin mitophagy. The ATG4 family of proteases are thought to regulate autophagosome formation exclusively by processing the ubiquitin-like ATG8 family (LC3/GABARAPs). We discover that human ATG4s promote autophagosome formation independently of their protease activity and of ATG8 family processing. ATG4 proximity networks reveal a role for ATG4s and their proximity partners, including the immune-disease protein LRBA, in ATG9A vesicle trafficking to mitochondria. Artificial intelligence-directed 3D electron microscopy of phagophores shows that ATG4s promote phagophore-ER contacts during the lipid-transfer phase of autophagosome formation. We also show that ATG8 removal during autophagosome maturation does not depend on ATG4 activity. Instead, ATG4s can disassemble ATG8-protein conjugates, revealing a role for ATG4s as deubiquitinating-like enzymes. These findings establish non-canonical roles of the ATG4 family beyond the ATG8 lipidation axis and provide an AI-driven framework for rapid 3D electron microscopy.

Lipid droplet dynamics are essential for the development of the malaria parasite Plasmodium falciparum.

Lipid droplets (LDs) are organelles that are central to lipid and energy homeostasis across all eukaryotes. In the malaria-causing parasite Plasmodium falciparum the roles of LDs in lipid acquisition from its host cells and their metabolism are poorly understood, despite the high demand for lipids in parasite membrane synthesis. We systematically characterised LD size, composition and dynamics across the disease-causing blood infection. Applying split fluorescence emission analysis and three-dimensional (3D) focused ion beam-scanning electron microscopy (FIB-SEM), we observed a decrease in LD size in late schizont stages. LD contraction likely signifies a switch from lipid accumulation to lipid utilisation in preparation for parasite egress from host red blood cells. We demonstrate connections between LDs and several parasite organelles, pointing to potential functional interactions. Chemical inhibition of triacylglyerol (TAG) synthesis or breakdown revealed essential LD functions for schizogony and in counteracting lipid toxicity. The dynamics of lipid synthesis, storage and utilisation in P. falciparum LDs might provide a target for new anti-malarial intervention strategies.

The multi-scale architecture of mammalian sperm flagella and implications for ciliary motility.

Motile cilia are molecular machines used by a myriad of eukaryotic cells to swim through fluid environments. However, available molecular structures represent only a handful of cell types, limiting our understanding of how cilia are modified to support motility in diverse media. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image sperm flagella from three mammalian species. We resolve in-cell structures of centrioles, axonemal doublets, central pair apparatus, and endpiece singlets, revealing novel protofilament-bridging microtubule inner proteins throughout the flagellum. We present native structures of the flagellar base, which is crucial for shaping the flagellar beat. We show that outer dense fibers are directly coupled to microtubule doublets in the principal piece but not in the midpiece. Thus, mammalian sperm flagella are ornamented across scales, from protofilament-bracing structures reinforcing microtubules at the nano-scale to accessory structures that impose micron-scale asymmetries on the entire assembly. Our structures provide vital foundations for linking molecular structure to ciliary motility and evolution.

Volume electron microscopy analysis of synapses in primary regions of the human cerebral cortex.

Functional and structural studies investigating macroscopic connectivity in the human cerebral cortex suggest that high-order associative regions exhibit greater connectivity compared to primary ones. However, the synaptic organization of these brain regions remains unexplored. In the present work, we conducted volume electron microscopy to investigate the synaptic organization of the human brain obtained at autopsy. Specifically, we examined layer III of Brodmann areas 17, 3b, and 4, as representative areas of primary visual, somatosensorial, and motor cortex. Additionally, we conducted comparative analyses with our previous datasets of layer III from temporopolar and anterior cingulate associative cortical regions (Brodmann areas 24, 38, and 21). 9,690 synaptic junctions were 3D reconstructed, showing that certain synaptic characteristics are specific to particular regions. The number of synapses per volume, the proportion of the postsynaptic targets, and the synaptic size may distinguish one region from another, regardless of whether they are associative or primary cortex. By contrast, other synaptic characteristics were common to all analyzed regions, such as the proportion of excitatory and inhibitory synapses, their shapes, their spatial distribution, and a higher proportion of synapses located on dendritic spines. The present results provide further insights into the synaptic organization of the human cerebral cortex.

In situ structure of intestinal apical surface reveals nanobristles on microvilli.

Microvilli are actin-bundle-supported membrane protrusions essential for absorption, secretion, and sensation. Microvilli defects cause gastrointestinal disorders; however, mechanisms controlling microvilli formation and organization remain unresolved. Here, we study microvilli by vitrifying the Caenorhabditis elegans larvae and mouse intestinal tissues with high-pressure freezing, thinning them with cryo-focused ion-beam milling, followed by cryo-electron tomography and subtomogram averaging. We find that many radial nanometer bristles referred to as nanobristles project from the lateral surface of nematode and mouse microvilli. The C. elegans nanobristles are 37.5 nm long and 4.5 nm wide. Nanobristle formation requires a protocadherin family protein, CDH-8, in C. elegans. The loss of nanobristles in cdh-8 mutants slows down animal growth and ectopically increases the number of Y-shaped microvilli, the putative intermediate structures if microvilli split from tips. Our results reveal a potential role of nanobristles in separating microvilli and suggest that microvilli division may help generate nascent microvilli with uniformity.

Precise Fabrication and Manipulation of Individual Polymer Nanofibers.

Polymer nanofibers hold promise in a wide range of applications owing to their diverse properties, flexibility, and cost effectiveness. In this study, we introduce a polymer nanofiber drawing process in a scanning electron microscope and focused ion beam (SEM/FIB) instrument with in situ observation. We employed a nanometer-sharp tungsten needle and prepolymer microcapsules to enable nanofiber drawing in a vacuum environment. This method produces individual polymer nanofibers with diameters as small as ∼500 nm and lengths extending to millimeters, yielding nanofibers with an aspect ratio of 2000:1. The attachment to the tungsten manipulator ensures accurate transfer of the polymer nanofiber to diverse substrate types as well as fabrication of assembled structures. Our findings provide valuable insights into ultrafine polymer fiber drawing, paving the way for high-precision manipulation and assembly of polymer nanofibers.

Directed Self-Assembly of Oxide Nanocomposites by Ion-Beam Lithography.

Vertically aligned self-assembled nanocomposite films have provided a unique platform to study magnetoelectric effects and other forms of coupling between complex oxides. However, the distribution in the locations and sizes of the phase-separated nanostructures limits their utility. In this work, we demonstrate a process to template the locations of the self-assembled structure using ion lithography, which is effective for general insulating substrates. This process was used to produce a nanocomposite consisting of fin-shaped vertical nanostructures of ferroelectric BiFeO3 and ferrimagnetic CoFe2O4 with a feature size of 100 nm on (111)-oriented SrTiO3 substrates. Cross-sectional imaging of the three-phase perovskite-spinel-substrate epitaxial interface reveals the selective nucleation of CoFe2O4 in the trenches of the patterned substrate, and the magnetic domains of CoFe2O4 were manipulated by applying an external magnetic field.

Defect Density and Atomic Defect Recognition in the Middle Layer of a Trilayer MoS2 Stack.

Molybdenum disulfide (MoS2) is one of the most intriguing two-dimensional materials, and moreover, its single atomic defects can significantly alter the properties. These defects can be both imaged and engineered using spherical and chromatic aberration-corrected high-resolution transmission electron microscopy (CC/CS-corrected HRTEM). In a few-layer stack, several atoms are vertically aligned in one atomic column. Therefore, it is challenging to determine the positions of missing atoms and the damage cross-section, particularly in the not directly accessible middle layers. In this study, we introduce a technique for extracting subtle intensity differences in CC/CS-corrected HRTEM images. By exploiting the crystal structure of the material, our method discerns chalcogen vacancies even in the middle layer of trilayer MoS2. We found that in trilayer MoS2 the middle layer's damage cross-section is about ten times lower than that in the monolayer. Our findings could be essential for the application of few-layer MoS2 in nanodevices.

A single transcription factor is sufficient to induce and maintain secretory cell architecture.

We hypothesized that basic helix-loop-helix (bHLH) MIST1 (BHLHA15) is a "scaling factor" that universally establishes secretory morphology in cells that perform regulated secretion. Here, we show that targeted deletion of MIST1 caused dismantling of the secretory apparatus of diverse exocrine cells. Parietal cells (PCs), whose function is to pump acid into the stomach, normally lack MIST1 and do not perform regulated secretion. Forced expression of MIST1 in PCs caused them to expand their apical cytoplasm, rearrange mitochondrial/lysosome trafficking, and generate large secretory granules. Mist1 induced a cohort of genes regulated by MIST1 in multiple organs but did not affect PC function. MIST1 bound CATATG/CAGCTG E boxes in the first intron of genes that regulate autophagosome/lysosomal degradation, mitochondrial trafficking, and amino acid metabolism. Similar alterations in cell architecture and gene expression were also caused by ectopically inducing MIST1 in vivo in hepatocytes. Thus, MIST1 is a scaling factor necessary and sufficient by itself to induce and maintain secretory cell architecture. Our results indicate that, whereas mature cell types in each organ may have unique developmental origins, cells performing similar physiological functions throughout the body share similar transcription factor-mediated architectural "blueprints."

Focused ion beam-SEM 3D study of osteodentin in the teeth of the Atlantic wolfish Anarhichas lupus.

The palette of mineralized tissues in fish is wide, and this is particularly apparent in fish dentin. While the teeth of all vertebrates except fish contain a single dentinal tissue type, called orthodentin, dentin in the teeth of fish can be one of several different tissue types. The most common dentin type in fish is orthodentin. Orthodentin is characterized by several key structural features that are fundamentally different from those of bone and from those of osteodentin. Osteodentin, the second-most common dentin type in fish (based on the tiny fraction of fish species out of ∼30,000 extant fish species in which tooth structure was so far studied), is found in most Selachians (sharks and rays) as well as in several teleost species, and is structurally different from orthodentin. Here we examine the hypothesis that osteodentin is similar to anosteocytic bone tissue in terms of its micro- and nano-structure. We use Focused Ion Beam-Scanning Electron Microscopy (FIB/SEM), as well as several other high-resolution imaging techniques, to characterize the 3D architecture of the three main components of osteodentin (denteons, inter-denteonal matrix, and the transition zone between them). We show that the matrix of osteodentin, although acellular, is extremely similar to mammalian osteonal bone matrix, both in general morphology and in the three-dimensional nano-arrangement of its mineralized collagen fibrils. We also document the presence of a complex network of nano-channels, similar to such networks recently described in bone. Finally, we document the presence of strings of hyper-mineralized small 'pearls' which surround the denteonal canals, and characterize their structure.

Melanin granules morphology and distribution in human black hair investigated by focused ion beam scanning electron microscopy: Differences between Asian and Caucasian hair.

Melanin granules (melanosomes) in Asian and Caucasian black hairs were investigated by focused ion beam scanning electron microscopy (FIB-SEM). This technique facilitates a direct evaluation of the three-dimensional distribution and morphology of melanin granules without requiring their isolation from hair. Three-dimensional reconstructed images of melanin granule distribution in hair samples were obtained using serial SEM images observed by FIB-SEM. Melanin granules in black hair tended to be three-dimensionally dense in the outer periphery of the cortex. The morphometric parameters of melanin granules were calculated using the reconstructed three-dimensional images. The results confirmed that melanin granules in Caucasian black hair were much smaller those in Asian black hair. Moreover, it was indicated that the relative frequency distribution of the volume of melanin granules was significantly different between Asians and Caucasians.

Tooth acellular extrinsic fibre cementum incremental lines in humans are formed by parallel branched Sharpey's fibres and not by its mineral phase.

In humans, the growth pattern of the acellular extrinsic fibre cementum (AEFC) has been useful to estimate the age-at-death. However, the structural organization behind such a pattern remains poorly understood. In this study tooth cementum from seven individuals from a Mexican modern skeletal series were analyzed with the aim of unveiling the AEFC collagenous and mineral structure using multimodal imaging approaches. The organization of collagen fibres was first determined using: light microscopy, transmission electron microscopy (TEM), electron tomography, and plasma FIB scanning electron microscopy (PFIB-SEM) tomography. The mineral properties were then investigated using: synchrotron small-angle X-ray scattering (SAXS) for T-parameter (correlation length between mineral particles); synchrotron X-ray diffraction (XRD) for L-parameter (mineral crystalline domain size estimation), alignment parameter (crystals preferred orientation) and lattice parameters a and c; as well as synchrotron X-ray fluorescence for spatial distribution of calcium, phosphorus and zinc. Results show that Sharpey's fibres branched out fibres that cover and uncover other collagen bundles forming aligned arched structures that are joined by these same fibres but in a parallel fashion. The parallel fibres are not set as a continuum on the same plane and when they are superimposed project the AEFC incremental lines due to the collagen birefringence. The orientation of the apatite crystallites is subject to the arrangement of the collagen fibres, and the obtained parameter values along with the elemental distribution maps, revealed this mineral tissue as relatively homogeneous. Therefore, no intrinsic characteristics of the mineral phase could be associated with the alternating AEFC incremental pattern.

Honeycomb gold specimen supports enabling orthogonal focussed ion beam-milling of elongated cells for cryo-ET.

Cryo-focussed ion beam (FIB)-milling is a powerful technique that opens up thick, cellular specimens to high-resolution structural analysis by electron cryotomography (cryo-ET). FIB-milled lamellae can be produced from cells on grids, or cut from thicker, high-pressure frozen specimens. However, these approaches can put geometrical constraints on the specimen that may be unhelpful, particularly when imaging structures within the cell that have a very defined orientation. For example, plunge frozen rod-shaped bacteria orient parallel to the plane of the grid, yet the Z-ring, a filamentous structure of the tubulin-like protein FtsZ and the key organiser of bacterial division, runs around the circumference of the cell such that it is perpendicular to the imaging plane. It is therefore difficult or impractical to image many complete rings with current technologies. To circumvent this problem, we have fabricated monolithic gold specimen supports with a regular array of cylindrical wells in a honeycomb geometry, which trap bacteria in a vertical orientation. These supports, which we call "honeycomb gold discs", replace standard EM grids and when combined with FIB-milling enable the production of lamellae containing cross-sections through cells. The resulting lamellae are more stable and resistant to breakage and charging than conventional lamellae. The design of the honeycomb discs can be modified according to need and so will also enable cryo-ET and cryo-EM imaging of other specimens in otherwise difficult to obtain orientations.

Cell-induced collagen alignment in a 3D in vitro culture during extracellular matrix production.

The bone extracellular matrix consists of a highly organized collagen matrix that is mineralized with carbonated hydroxyapatite. Even though the structure and composition of bone have been studied extensively, the mechanisms underlying collagen matrix organization remain elusive. In this study, we used a 3D cell culture system in which osteogenic cells deposit and orient the collagen matrix that is subsequently mineralized. Using live fluorescence imaging combined with volume electron microscopy, we visualize the organization of the cells and collagen in the cell culture. We show that the osteogenically induced cells are organizing the collagen matrix during development. Based on the observation of tunnel-like structures surrounded by aligned collagen in the center of the culture, we propose that osteoblasts organize the deposited collagen during migration through the culture. Overall, we show that cell-matrix interactions are involved in collagen alignment during early-stage osteogenic differentiation and that the matrix is organized by the osteoblasts in the absence of osteoclast activity.

Association of liver fibrosis with extrahepatic cancer in steatotic liver disease patients with PNPLA3 I148M GG genotype.

The impacts of patatin-like phospholipase domain-containing protein 3 (PNPLA3) I148M-rs738409, methylenetetrahydrofolate reductase (MTHFR) Ala222Val-rs1801133, and aldehyde dehydrogenase 2 (ALDH2) Glu504Lys-rs671 on the outcomes of Taiwanese patients with steatotic liver disease (SLD) have remained elusive. An 8-year prospective cohort study of patients with (n = 546) and without (n = 580) SLD (controls) was undertaken in a Taiwanese tertiary care center. The 546 SLD patients comprised 306 (56.0%) men and 240 (44.0%) women with mean ages of 53.3 and 56.4 years, respectively. Compared with the controls, SLD patients had an increased frequency of the PNPLA3 I148M-rs738409 GG genotype (25.5 vs. 5.9%, p = 0.001). Among the SLD patients, 236 (43.1%) suffered cardiovascular events, 52 (9.5%) showed extrahepatic cancers, 13 (2.38%) experienced hepatic events, including hepatocellular carcinoma (n = 3, 0.5%) and liver cirrhosis (n = 8, 1.47%), and none died. The Fibrosis-4 (FIB-4) scores were associated with extrahepatic cancer (hazard ratio [HR] 1.325; 95% confidence interval [CI], 1.038-1.691) and cirrhosis development (HR 1.532; 95% CI, 1.055-2.224), and the PNPLA3 I148M-rs738409 G allele (ß = 0.158, 95% CI, 0.054-0.325) was associated with the FIB-4 score. Stratified analyses showed that the impact of the FIB-4 score on extrahepatic cancer development was evident only in SLD patients with the PNPLA3 I148M-rs738409 GG genotype (HR 1.543; 95% CI, 1.195-1.993) and not in patients with the GC or CC genotype. Moreover, the ALDH2 Glu504Lys-rs671 G allele had a dose-dependent effect on alcoholism, and the MTHFR and ALDH2 genotypes were not significantly associated with SLD patient outcomes. In conclusion, special vigilance should be exercised for emerging extrahepatic cancer in SLD patients with the PNPLA3 I148M-rs738409 GG genotype and high FIB-4 scores.

Prospective head-to-head comparison of non-invasive scores for diagnosis of fibrotic MASH in patients with type 2 diabetes.

BACKGROUND & AIMS: Non-invasive scores have been proposed to identify patients with fibrotic, metabolic dysfunction-associated steatohepatitis (MASH), who are at the highest risk of progression to complications of cirrhosis and may benefit from pharmacologic treatments. However, data in patients with type 2 diabetes (T2DM) are lacking. The aim of this multicenter prospective study was to perform a head-to-head comparison of FAST (FibroScan-aspartate aminotransferase [AST]), MAST (MRI-AST), MEFIB (magnetic resonance elastography [MRE] plus FIB-4), and FNI (fibrotic NASH index) for detecting fibrotic MASH in patients with T2DM. METHODS: A total of 330 outpatients with T2DM and biopsy-proven metabolic dysfunction-associated steatotic liver disease (MASLD) from the QUID-NASH study (NCT03634098), who underwent FibroScan, MRI-proton density fat fraction and MRE at the time of liver biopsy were studied. The main outcome was fibrotic MASH, defined as NAS ≥4 (with at least one point for each parameter) and fibrosis stage ≥2 (centrally reviewed). RESULTS: All data for score comparisons were available for 245 patients (median age 59 years, 65% male, median BMI 31 kg/m2; fibrotic MASH in 39%). FAST and MAST had similar accuracy (AUROCs 0.81 vs. 0.79, p = 0.41) but outperformed FNI (0.74; p = 0.01) and MEFIB (0.68; p <0.0001). When using original cut-offs, MAST outperformed FAST, MEFIB and FNI when comparing the percentage of correctly classified patients, in whom liver biopsy would be avoided (69% vs. 48%, 46%, 39%, respectively; p <0.001). When using cut-offs specific to our population, FAST outperformed FNI and MAST (56% vs. 40%, and 38%, respectively; p <0.001). CONCLUSION: Our findings show that FAST, MAST, MEFIB and FNI are accurate non-invasive tools to identify patients with T2DM and fibrotic MASH in secondary/tertiary diabetes clinics. Cut-offs adapted to the T2DM population should be considered. IMPACT AND IMPLICATIONS: Among patients with type 2 diabetes (T2DM), identifying those with metabolic dysfunction-associated steatohepatitis and significant fibrosis, who are the most at risk of developing clinical liver-related outcomes and who may benefit from pharmacologic treatments, is an unmet need. In this prospective multicenter study, we compared four non-invasive scores, three based on imaging (MRI or ultrasound technologies) and one on laboratory blood tests, for this purpose, using original and study-specific cut-offs. Our findings show that FAST, MAST, MEFIB and FNI are accurate non-invasive tools to identify patients with T2DM and fibrotic MASH in secondary/tertiary diabetes clinics. Cut-offs adapted to the T2DM population should be considered. TRIAL REGISTRATION NUMBER: NCT03634098.