Architecture of CTPS filament networks revealed by cryo-electron tomography.

The cytoophidium is a novel type of membraneless organelle, first observed in the ovaries of Drosophila using fluorescence microscopy. In vitro, purified Drosophila melanogaster CTPS (dmCTPS) can form metabolic filaments under the presence of either substrates or products, and their structures that have been analyzed using cryo-electron microscopy (cryo-EM). These dmCTPS filaments are considered the fundamental units of cytoophidia. However, due to the resolution gap between light and electron microscopy, the precise assembly pattern of cytoophidia remains unclear. In this study, we find that dmCTPS filaments can spontaneously assemble in vitro, forming network structures that reach micron-scale dimensions. Using cryo-electron tomography (cryo-ET), we reconstruct the network structures formed by dmCTPS filaments under substrate or product binding conditions and elucidate their assembly process. The dmCTPS filaments initially form structural bundles, which then further assemble into larger networks. By identifying, tracking, and statistically analyzing the filaments, we observed distinct characteristics of the structural bundles formed under different conditions. This study provides the first systematic analysis of dmCTPS filament networks, offering new insights into the relationship between cytoophidia and metabolic filaments.

Genomic transfer via membrane vesicle: a strategy of giant phage phiKZ for early infection.

During infection, the giant phiKZ phage forms a specialized structure at the center of the host cell called the phage nucleus. This structure is crucial for safeguarding viral DNA against bacterial nucleases and for segregating the transcriptional activities of late genes. Here, we describe a morphological entity, the early phage infection (EPI) vesicle, which appears to be responsible for earlier gene segregation at the beginning of the infection process. Using cryo-electron microscopy, electron tomography (ET), and fluorescence microscopy with membrane-specific dyes, we demonstrated that the EPI vesicle is enclosed in a lipid bilayer originating, apparently, from the inner membrane of the bacterial cell. Our investigations further disclose that the phiKZ EPI vesicle contains both viral DNA and viral RNA polymerase (vRNAP). We have observed that the EPI vesicle migrates from the cell pole to the center of the bacterial cell together with ChmA, the primary protein of the phage nucleus. The phage DNA is transported into the phage nucleus after phage maturation, but the EPI vesicle remains outside. We hypothesized that the EPI vesicle acts as a membrane transport agent, efficiently delivering phage DNA to the phage nucleus while protecting it from the nucleases of the bacterium. IMPORTANCE: Our study shed light on the processes of phage phiKZ early infection stage, expanding our understanding of possible strategies for the development of phage infection. We show that phiKZ virion content during injection is packed inside special membrane structures called early phage infection (EPI) membrane vesicles originating from the bacterial inner cell membrane. We demonstrated the EPI vesicle fulfilled the role of the safety transport unit for the phage genome to the phage nucleus, where the phage DNA would be replicated and protected from bacterial immune systems.

Intracellular Ebola virus nucleocapsid assembly revealed by in situ cryo-electron tomography.

Filoviruses, including the Ebola and Marburg viruses, cause hemorrhagic fevers with up to 90% lethality. The viral nucleocapsid is assembled by polymerization of the nucleoprotein (NP) along the viral genome, together with the viral proteins VP24 and VP35. We employed cryo-electron tomography of cells transfected with viral proteins and infected with model Ebola virus to illuminate assembly intermediates, as well as a 9 Å map of the complete intracellular assembly. This structure reveals a previously unresolved third and outer layer of NP complexed with VP35. The intrinsically disordered region, together with the C-terminal domain of this outer layer of NP, provides the constant width between intracellular nucleocapsid bundles and likely functions as a flexible tether to the viral matrix protein in the virion. A comparison of intracellular nucleocapsids with prior in-virion nucleocapsid structures reveals that the nucleocapsid further condenses vertically in the virion. The interfaces responsible for nucleocapsid assembly are highly conserved and offer targets for broadly effective antivirals.

The molecular architecture of the nuclear basket.

The nuclear pore complex (NPC) is the sole mediator of nucleocytoplasmic transport. Despite great advances in understanding its conserved core architecture, the peripheral regions can exhibit considerable variation within and between species. One such structure is the cage-like nuclear basket. Despite its crucial roles in mRNA surveillance and chromatin organization, an architectural understanding has remained elusive. Using in-cell cryo-electron tomography and subtomogram analysis, we explored the NPC's structural variations and the nuclear basket across fungi (yeast; S. cerevisiae), mammals (mouse; M. musculus), and protozoa (T. gondii). Using integrative structural modeling, we computed a model of the basket in yeast and mammals that revealed how a hub of nucleoporins (Nups) in the nuclear ring binds to basket-forming Mlp/Tpr proteins: the coiled-coil domains of Mlp/Tpr form the struts of the basket, while their unstructured termini constitute the basket distal densities, which potentially serve as a docking site for mRNA preprocessing before nucleocytoplasmic transport.

The beginning of iron corrosion - high-resolution visualization with 3D electron tomography.

Understanding the genesis and early-phase reactions of iron corrosion is essential for the early detection, mitigation and prevention of metal degradation. In this work, high-resolution 3D tomography of metallic iron oxidation was acquired using high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM). In particular, dendritic capillaries (<0.5 nm) were observed during the initial oxidation of fresh nanoscale zero-valent iron due to the differential oxygen diffusion and iron atoms migration. This observation led to the proposal of a nanoscale "pothole" model for early-phase corrosion, wherein hollowing out of the metal nanoparticle and formation of nanovoids beneath the iron/oxide interface through Kirkendall effect. Coalescence of the nanocapillaries results in the ultimate collapse of metal structure and/or functional failure. Using nanoscale zero-valent iron as a research model, this work provides unprecedented insights into the nano- and atomic-scale mechanisms of iron oxidation, paving the way for advanced detection and prevention strategies for iron corrosion.

Large attachment organelle mediates interaction between Nanobdellota archaeon YN1 and its host.

DPANN archaea are an enigmatic superphylum that are difficult to isolate and culture in the laboratory due to their specific culture conditions and apparent ectosymbiotic lifestyle. Here, we successfully isolated and cultivated a coculture system of a novel Nanobdellota archaeon YN1 and its host Sulfurisphaera ohwakuensis YN1HA. We characterized the coculture system by complementary methods, including metagenomics and metabolic pathway analysis, fluorescence microscopy, and high-resolution electron cryo-tomography (cryoET). We show that YN1 is deficient in essential metabolic processes and requires host resources to proliferate. CryoET imaging revealed an enormous attachment organelle present in the YN1 envelope that forms a direct interaction with the host cytoplasm, bridging the two cells. Together, our results unravel the molecular and structural basis of ectosymbiotic relationship between YN1 and YN1HA. This research broadens our understanding of DPANN biology and the versatile nature of their ectosymbiotic relationships.

An image processing pipeline for electron cryo-tomography in RELION-5.

Electron tomography of frozen, hydrated samples allows structure determination of macromolecular complexes that are embedded in complex environments. Provided that the target complexes may be localised in noisy, three-dimensional tomographic reconstructions, averaging images of multiple instances of these molecules can lead to structures with sufficient resolution for de novo atomic modelling. Although many research groups have contributed image processing tools for these tasks, a lack of standardisation and interoperability represents a barrier for newcomers to the field. Here, we present an image processing pipeline for electron tomography data in RELION-5, with functionality ranging from the import of unprocessed movies to the automated building of atomic models in the final maps. Our explicit definition of metadata items that describe the steps of our pipeline has been designed for interoperability with other software tools and provides a framework for further standardisation.

Observation of Vacuoles in Arabidopsis Developing Pollen Grains with Whole-Cell Electron Tomography.

Arabidopsis thaliana developing pollen grains serve as an excellent system for studying vacuole dynamics. Here, we present a methodological approach that utilizes the serial tomography package in Etomo software from IMOD to generate whole-cell tomograms on A. thaliana developing pollens for visualizing vacuoles on the whole-cell scale. In order to understand the vacuole dynamics along with the pollen maturation, we also introduce a sampling method aimed at harvesting the pollen grains at various stages, marked by the vegetative nucleus or generative cell. The cryo-fixation/freeze-substitution technique can then be applied to preserve the fine structures of the pollen grains and facilitate detailed ultrastructure examination. Through this method, large-volume whole-cell electron tomograms regarding vacuolar morphologies and ultrastructural changes during pollen development and maturation have been obtained. Overall, the method presented here provides valuable insights into the dynamic nature of vacuoles in Arabidopsis developing pollen.

Whole-Cell Electron Tomography Analysis of Vacuole Biogenesis.

Vacuoles in plant cells are the most prominent organelles that harbor distinctive features, including lytic function, storage of proteins and sugars, balance of cell volume, and defense responses. Despite their dominant size and functional versatility, the nature and biogenesis of vacuoles in plants per se remain elusive and several models have been proposed. Recently, we used the whole-cell 3D electron tomography (ET) technique to study vacuole formation and distribution at nanometer resolution and demonstrated that small vacuoles are derived from multivesicular body maturation and fusion. Good sample preparation is a critical step to get high-quality electron tomography images. In this chapter, we provide detailed sample preparation methods for high-resolution ET in Arabidopsis thaliana root cells, including high-pressure freezing, subsequent freeze-substitution fixation, embedding, and serial sectioning.

High-Pressure Freezing and Low-Temperature Processing of Seeds for Electron Microscopy and Electron Tomography.

High-pressure freezing/freeze substitution has been used to preserve biological samples for ultrastructure study instead of chemical fixation. For most plant samples, the water content is too high and cannot be properly preserved during cryofixation. Additionally, the cell wall is a barrier that prevents the substitution of water with the resin. In this chapter, we will discuss modified high-pressure freezing and subsequent processing protocols based on our routinely used methodology for examining Arabidopsis seeds in transmission electron microscopy and electron tomography.

Endocytosis in malaria parasites: An ultrastructural perspective of membrane interplay in a unique infection model.

Malaria remains a major global threat, representing a severe public health problem worldwide. Annually, it is responsible for a high rate of morbidity and mortality in many tropical developing countries where the disease is endemic. The causative agent of malaria, Plasmodium spp., exhibits a complex life cycle, alternating between an invertebrate vector, which transmits the disease, and the vertebrate host. The disease pathology observed in the vertebrate host is attributed to the asexual development of Plasmodium spp. inside the erythrocyte. Once inside the red blood cell, malaria parasites cause extensive changes in the host cell, increasing membrane rigidity and altering its normal discoid shape. Additionally, during their intraerythrocytic development, malaria parasites incorporate and degrade up to 70 % of host cell hemoglobin. This mechanism is essential for parasite development and represents an important drug target. Blocking the steps related to hemoglobin endocytosis or degradation impairs parasite development and can lead to its death. The ultrastructural analysis of hemoglobin endocytosis on Plasmodium spp. has been broadly explored along the years. However, it is only recently that the proteins involved in this process have started to emerge. Here, we will review the most important features related to hemoglobin endocytosis and catabolism on malaria parasites. A special focus will be given to the recent analysis obtained through 3D visualization approaches and to the molecules involved in these mechanisms.

Quantitative Analysis of Rhodobacter sphaeroides Storage Organelles via Cryo-Electron Tomography and Light Microscopy.

Bacterial cytoplasmic organelles are diverse and serve many varied purposes. Here, we employed Rhodobacter sphaeroides to investigate the accumulation of carbon and inorganic phosphate in the storage organelles, polyhydroxybutyrate (PHB) and polyphosphate (PP), respectively. Using cryo-electron tomography (cryo-ET), these organelles were observed to increase in size and abundance when growth was arrested by chloramphenicol treatment. The accumulation of PHB and PP was quantified from three-dimensional (3D) segmentations in cryo-tomograms and the analysis of these 3D models. The quantification of PHB using both segmentation analysis and liquid chromatography and mass spectrometry (LCMS) each demonstrated an over 10- to 20-fold accumulation of PHB. The cytoplasmic location of PHB in cells was assessed with fluorescence light microscopy using a PhaP-mNeonGreen fusion-protein construct. The subcellular location and enumeration of these organelles were correlated by comparing the cryo-ET and fluorescence microscopy data. A potential link between PHB and PP localization and possible explanations for co-localization are discussed. Finally, the study of PHB and PP granules, and their accumulation, is discussed in the context of advancing fundamental knowledge about bacterial stress response, the study of renewable sources of bioplastics, and highly energetic compounds.

Polysome collapse and RNA condensation fluidize the cytoplasm.

The cell interior is packed with macromolecules of mesoscale size, and this crowded milieu significantly influences cellular physiology. Cellular stress responses almost universally lead to inhibition of translation, resulting in polysome collapse and release of mRNA. The released mRNA molecules condense with RNA-binding proteins to form ribonucleoprotein (RNP) condensates known as processing bodies and stress granules. Here, we show that polysome collapse and condensation of RNA transiently fluidize the cytoplasm, and coarse-grained molecular dynamic simulations support this as a minimal mechanism for the observed biophysical changes. Increased mesoscale diffusivity correlates with the efficient formation of quality control bodies (Q-bodies), membraneless organelles that compartmentalize misfolded peptides during stress. Synthetic, light-induced RNA condensation also fluidizes the cytoplasm. Together, our study reveals a functional role for stress-induced translation inhibition and formation of RNP condensates in modulating the physical properties of the cytoplasm to enable efficient response of cells to stress conditions.

Accurate size-based protein localization from cryo-ET tomograms.

Cryo-electron tomography (cryo-ET) combined with sub-tomogram averaging (STA) allows the determination of protein structures imaged within the native context of the cell at near-atomic resolution. Particle picking is an essential step in the cryo-ET/STA image analysis pipeline that consists in locating the position of proteins within crowded cellular tomograms so that they can be aligned and averaged in 3D to improve resolution. While extensive work in 2D particle picking has been done in the context of single-particle cryo-EM, comparatively fewer strategies have been proposed to pick particles from 3D tomograms, in part due to the challenges associated with working with noisy 3D volumes affected by the missing wedge. While strategies based on 3D template-matching and deep learning are commonly used, these methods are computationally expensive and require either an external template or manual labelling which can bias the results and limit their applicability. Here, we propose a size-based method to pick particles from tomograms that is fast, accurate, and does not require external templates or user provided labels. We compare the performance of our approach against a commonly used algorithm based on deep learning, crYOLO, and show that our method: i) has higher detection accuracy, ii) does not require user input for labeling or time-consuming training, and iii) runs efficiently on non-specialized CPU hardware. We demonstrate the effectiveness of our approach by automatically detecting particles from tomograms representing different types of samples and using these particles to determine the high-resolution structures of ribosomes imaged in vitro and in situ.

3D imaging photocatalytically degraded micro- and nanoplastics.

Microplastics (MPs) and nanoplastics have been an emerging global concern, with hazardous effects on plant, animal, and human health. Their small size makes it easier for them to spread to various ecosystems and enter the food chain; they are already widely found in aqueous environments and within aquatic life, and have even been found within humans. Much research has gone into understanding micro-/nanoplastic sources and environmental fate, but less work has been done to understand their degradation. Photocatalytic degradation is a promising green technique that uses visible or ultraviolet light in combination with photocatalyst to degrade plastic particles. While complete degradation, reducing plastics to small molecules, is often the goal, partial degradation is more common. We examined microscale polyethylene (PE) (125-150µm in diameter) and nanoscale polystyrene (PS) (∼300 nm in diameter) spheres both before and after degradation using multiple imaging techniques, especially electron tomography in addition to conventional electron microscopy. Electron tomography is able to image the 3D exterior and interior of the nanoplastics, enabling us to observe within aggregates and inside degraded spheres, where we found potentially open interior structures after degradation. These structures may result from differences in degradation and aggregation behavior between the different plastic types, with our work finding that PE MPs typically cracked into sharp fragments, while PS nanoplastics often fragmented into smoother, more curved shapes. These and other differences, along with interior and 3D surface images, provide new details on how the structure and aggregation of PE MPs and PS nanoplastics changes when degraded, which could influence how the resulting worn particles are collected or treated further.

Training-free CryoET Tomogram Segmentation.

Cryogenic Electron Tomography (CryoET) is a useful imaging technology in structural biology that is hindered by its need for manual annotations, especially in particle picking. Recent works have endeavored to remedy this issue with few-shot learning or contrastive learning techniques. However, supervised training is still inevitable for them. We instead choose to leverage the power of existing 2D foundation models and present a novel, training-free framework, CryoSAM. In addition to prompt-based single-particle instance segmentation, our approach can automatically search for similar features, facilitating full tomogram semantic segmentation with only one prompt. CryoSAM is composed of two major parts: 1) a prompt-based 3D segmentation system that uses prompts to complete single-particle instance segmentation recursively with Cross-Plane Self-Prompting, and 2) a Hierarchical Feature Matching mechanism that efficiently matches relevant features with extracted tomogram features. They collaborate to enable the segmentation of all particles of one category with just one particle-specific prompt. Our experiments show that CryoSAM outperforms existing works by a significant margin and requires even fewer annotations in particle picking. Further visualizations demonstrate its ability when dealing with full tomogram segmentation for various subcellular structures. Our code is available at: https://github.com/xulabs/aitom.

The GoldX Fiducial Eraser.

Gold nanoparticles with sizes in the range of 5-15 nm are a standard method of providing fiducial markers to assist with alignment during reconstruction in cryogenic electron tomography. However, due to their high electron density and resulting contrast when compared to standard cellular or biological samples, they introduce artifacts such as streaking in the reconstructed tomograms. Here, we demonstrate a tool that automatically detects these nanoparticles and suppresses them by replacing them with a local background as a post-processing step, providing a cleaner tomogram without removing any sample relevant information or introducing new artifacts or edge effects from uniform density replacements.

Elucidating bacterial coaggregation through a physicochemical and imaging surface characterization.

Bacterial coaggregation is a highly specific type of cell-cell interaction, well-documented among oral bacteria, and involves specific characteristics of the cell surface of the coaggregating strains. However, the understanding of the mechanisms promoting coaggregation in aquatic systems remains limited. This gap is critical to address, given the broad implications of coaggregation for multispecies biofilm formation, water quality, the performance of engineered systems, and diverse biotechnological applications. Therefore, this study aims to comprehensively characterize the cell surface of the coaggregating strain Delftia acidovorans 005P, isolated from drinking water, alongside a non-coaggregating strain, D. acidovorans 009P. By analyzing two strains of the same species, we aim to identify the factors contributing to the coaggregation ability of strain 005P. To achieve this, we employed a combination of physicochemical characterization, Fourier-transform infrared spectroscopy (FTIR), and advancing imaging techniques [transmission electron microscopy and cryo-electron tomography (cryo-ET)]. The coaggregating strain (005P) exhibited higher surface hydrophobicity, negative surface charge, and cell surface and co-adhesion energies than the non-coaggregating strain (009P). The chemical characterization of bacterial surfaces through FTIR revealed subtle differences, particularly in spectral regions linked to carbohydrates and phosphodiesters/amide III of proteins (860-930 cm-1 and 1212-1240 cm-1, respectively). Cryo-ET highlighted significant differences in pili structures between the strains, such as variations in length, frequency, and arrangement. The pili in the 005P strain, identified as pili-like adhesins, serve as key mediators of coaggregation. By integrating physicochemical analyses and high-resolution imaging techniques, this study conclusively links the coaggregation ability of D. acidovorans 005P to its unique pili characteristics, emphasizing their crucial role in microbial coaggregation in aquatic environments.

Automated fiducial-based alignment of cryo-electron tomography tilt series in Dynamo.

With the advent of modern technologies for cryo-electron tomography (cryo-ET), high-quality tilt series are more rapidly acquired than processed and analyzed. Thus, a robust and fast-automated alignment for batch processing in cryo-ET is needed. While different software packages have made available several approaches for automated marker-based alignment of tilt series, manual user intervention remains necessary for many datasets, thus preventing high-throughput tomography. We have developed a MATLAB-based framework integrated into the Dynamo software package for automatic detection of fiducial markers that generates a robust alignment model with minimal input parameters. This approach allows high-throughput, unsupervised volume reconstruction. This new module extends Dynamo with a large repertory of tools for tomographic alignment and reconstruction, as well as specific visualization browsers to rapidly assess the biological relevance of the dataset. Our approach has been successfully tested on a broad range of datasets that include diverse biological samples and cryo-ET modalities.

In situ fate of Chikungunya virus replication organelles.

Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for an acute musculoskeletal disease in humans. Replication of the viral RNA genome occurs in specialized membranous replication organelles (ROs) or spherules, which contain the viral replication complex. Initially generated by RNA synthesis-associated plasma membrane deformation, alphavirus ROs are generally rapidly endocytosed to produce type I cytopathic vacuoles (CPV-I), from which nascent RNAs are extruded for cytoplasmic translation. By contrast, CHIKV ROs are poorly internalized, raising the question of their fate and functionality at the late stage of infection. Here, using in situ cryogenic-electron microscopy approaches, we investigate the outcome of CHIKV ROs and associated replication machinery in infected human cells. We evidence the late persistence of CHIKV ROs at the plasma membrane with a crowned protein complex at the spherule neck similar to the recently resolved replication complex. The unexpectedly heterogeneous and large diameter of these compartments suggests a continuous, dynamic growth of these organelles beyond the replication of a single RNA genome. Ultrastructural analysis of surrounding cytoplasmic regions supports that outgrown CHIKV ROs remain dynamically active in viral RNA synthesis and export to the cell cytosol for protein translation. Interestingly, rare ROs with a homogeneous diameter are also marginally internalized in CPV-I near honeycomb-like arrangements of unknown function, which are absent in uninfected controls, thereby suggesting a temporal regulation of this internalization. Altogether, this study sheds new light on the dynamic pattern of CHIKV ROs and associated viral replication at the interface with cell membranes in infected cells.IMPORTANCEThe Chikungunya virus (CHIKV) is a positive-stranded RNA virus that requires specialized membranous replication organelles (ROs) for its genome replication. Our knowledge of this viral cycle stage is still incomplete, notably regarding the fate and functional dynamics of CHIKV ROs in infected cells. Here, we show that CHIKV ROs are maintained at the plasma membrane beyond the first viral cycle, continuing to grow and be dynamically active both in viral RNA replication and in its export to the cell cytosol, where translation occurs in proximity to ROs. This contrasts with the homogeneous diameter of ROs during internalization in cytoplasmic vacuoles, which are often associated with honeycomb-like arrangements of unknown function, suggesting a regulated mechanism. This study sheds new light on the dynamics and fate of CHIKV ROs in human cells and, consequently, on our understanding of the Chikungunya viral cycle.