Imaging interorganelle contacts at a glance.

Eukaryotic cells are compartmentalized into membrane-bound organelles that must coordinate their responses to stimuli. One way that organelles communicate is via membrane contact sites (MCSs), sites of close apposition between organelles used for the exchange of ions, lipids and information. In this Cell Science at a Glance article and the accompanying poster, we describe an explosion of new methods that have led to exciting progress in this area and discuss key examples of how these methods have advanced our understanding of MCSs. We discuss how diffraction-limited and super-resolution fluorescence imaging approaches have provided important insight into the biology of interorganelle communication. We also describe how the development of multiple proximity-based methods has enabled the detection of MCSs with high accuracy and precision. Finally, we assess how recent advances in electron microscopy (EM), considered the gold standard for detecting MCSs, have allowed the visualization of MCSs and associated proteins in 3D at ever greater resolution.

Imaging Giardia intestinalis cellular organisation using expansion microscopy reveals atypical centrin localisation.

Advanced imaging of microorganisms, including protists, is challenging due to their small size. Specimen expansion prior to imaging is thus beneficial to increase resolution and cellular details. Here, we present a sample preparation workflow for improved observations of the single-celled eukaryotic pathogen Giardia intestinalis (Excavata, Metamonada). The binucleated trophozoites colonize the small intestine of humans and animals and cause a diarrhoeal disease. Their remarkable morphology includes two nuclei and a pronounced microtubular cytoskeleton enabling cell motility, attachment and proliferation. By use of expansion and confocal microscopy, we resolved in a great detail subcellular structures and organelles of the parasite cell. The acquired spatial resolution enabled novel observations of centrin localization at Giardia basal bodies. Interestingly, non-luminal centrin localization between the Giardia basal bodies was observed, which is an atypical eukaryotic arrangement. Our protocol includes antibody staining and can be used for the localization of epitope-tagged proteins, as well as for differential organelle labelling by amino reactive esters. This fast and simple technique is suitable for routine use without a superresolution microscopy equipment.

Double infection of Nicotiana benthamiana with AMV and WCMV increases both virus concentrations and synergistically changes both host organelle ultrastructure and chlorophyll content.

To clarify the synergistic pathogenic mechanism of Nicotiana benthamiana double infection with alfalfa mosaic virus (AMV) and white clover mosaic virus (WCMV), AMV and WCMV co-inoculation of N. benthamiana as treatment and single inoculation of AMV or WCMV and phosphate buffer solution (pH 7.0, PBS) as control, respectively. The concentrations and the relative expression of AMV and WCMV coat proteins were determined by a double antibody sandwich enzyme-linked immune sorbent assay (DAS-ELISA) and real-time fluorescence quantitative PCR (RT-qPCR) in a double infection of N. benthamiana with AMV and WCMV. Meanwhile, virion morphology, ultrastructure morphology, and chlorophyll content were observed and determined by electron microscopy. The results showed that the diseased symptoms were more serious, and virus concentration and relative expression of AMV and WCMV coat proteins were also higher in N. benthamiana double infection with AMV and WCMV than in AMV or WCMV single infection. The main symptoms manifested as severe mottle mosaic, shrinkage, and chlorosis. The concentrations of AMV and WCMV were 182.23 pg/mL and 148.77 pg/mL of double infection with AMV and WCMV, which were 1.75-fold and 1.62-fold than AMV and WCMV single infection, respectively. The relative expression of AMV and WCMV coat proteins was 4.25-fold and 2.50-fold than the single virus infection, respectively. Electron microscopy also observed that chloroplast malformation, cell membrane deformation, contents dissolution, grana lamella disorder, fat granules increased and enlarged, starch granules enlarged, and mitochondria were seriously malformed in a double infection of N. benthamiana with AMV and WCMV. The chlorophyll content was significantly lower for double infection with AMV and WCMV than for AMV or WCMV single-infected and CK, reduced by 31.52 %, 22.83 %, and 76.09 %, respectively. This is the first report of a double infection of N. benthamiana with AMV and WCMV that increases both virus concentrations and synergistically changes both host organelle ultrastructure and chlorophyll content.

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.

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.

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.

A high-resolution phase-contrast microscopy system for label-free imaging in living cells.

Cell biologists have long sought the ability to observe intracellular structures in living cells without labels. This study presents procedures to adjust a commercially available apodized phase-contrast (APC) microscopy system for better visualizing the dynamic behaviors of various subcellular organelles in living cells. By harnessing the versatility of this technique to capture sequential images, we could observe morphological changes in cellular geometry after virus infection in real time without probes or invasive staining. The tune-up APC microscopy system is a highly efficient platform for simultaneously observing the dynamic behaviors of diverse subcellular structures with exceptional resolution.

Expression of brain-derived neurotrophic factor and formation of migrasome increases in the glioma cells induced by the adipokinetic hormone.

OBJECTIVE: It has been previously shown that brain-derived neurotrophic factor is linked with various types of cancer. Brain-derived neurotrophic factor is found to be highly expressed in multiple human cancers and associated with tumor growth, invasion, and metastasis. Adipokinetic hormones are functionally related to the vertebrate glucagon, as they have similar functionalities that manage the nutrient-dependent secretion of these two hormones. Migrasomes are new organelles that contain numerous small vesicles, which aid in transmitting signals between the migrating cells. Therefore, the aim of this study was to investigate the effects of Anax imperator adipokinetic hormone on brain-derived neurotrophic factor expression and ultrastructure of cells in the C6 glioma cell line. METHODS: The rat C6 glioma cells were treated with concentrations of 5 and 10 Anax imperator adipokinetic hormone for 24 h. The effects of the Anax imperator adipokinetic hormone on the migrasome formation and brain-derived neurotrophic factor expression were analyzed using immunocytochemistry and transmission electron microscope. RESULTS: The rat C6 glioma cells of the 5 and 10 µM Anax imperator adipokinetic hormone groups showed significantly high expressions of brain-derived neurotrophic factor and migrasomes numbers, compared with the control group. CONCLUSION: A positive correlation was found between the brain-derived neurotrophic factor expression level and the formation of migrasome, which indicates that the increased expression of brain-derived neurotrophic factor and the number of migrasomes may be involved to metastasis of the rat C6 glioma cell line induced by the Anax imperator adipokinetic hormone. Therefore, the expression of brain-derived neurotrophic factor and migrasome formation may be promising targets for preventing tumor proliferation, invasion, and metastasis in glioma.

Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles.

The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelles, the sites of replication of viral genomic RNA (vgRNA). To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain numerous vgRNA molecules along with the replication enzymes and clusters of viral double-stranded RNA (dsRNA). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of endoplasmic reticulum (ER) markers and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are encapsulated into DMVs, which have membranes derived from the host ER. These organelles merge into larger vesicle packets as infection advances. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.

Human Umbilical Vein Endothelial Cells as a Versatile Cellular Model System in Diverse Experimental Paradigms: An Ultrastructural Perspective.

Human umbilical vein endothelial cells (HUVECs) are primary cells isolated from the vein of an umbilical cord, extensively used in cardiovascular studies and medical research. These cells, retaining the characteristics of endothelial cells in vivo, serve as a valuable cellular model system for understanding vascular biology, endothelial dysfunction, pathophysiology of diseases such as atherosclerosis, and responses to different drugs or treatments. Transmission electron microscopy (TEM) has been a cornerstone in revealing the detailed architecture of multiple cellular model systems including HUVECs, allowing researchers to visualize subcellular organelles, membrane structures, and cytoskeletal elements. Among them, the endoplasmic reticulum, Golgi apparatus, mitochondria, and nucleus can be meticulously examined to recognize alterations indicative of cellular responses to various stimuli. Importantly, Weibel-Palade bodies are characteristic secretory organelles found in HUVECs, which can be easily distinguished in the TEM. These distinctive structures also dynamically react to different factors through regulated exocytosis, resulting in complete or selective release of their contents. This detailed review summarizes the ultrastructural features of HUVECs and highlights the utility of TEM as a pivotal tool for analyzing HUVECs in diverse research frameworks, contributing valuable insights into the comprehension of HUVEC behavior and enriching our knowledge into the complexity of vascular biology.

Positive-strand RNA virus genome replication organelles: structure, assembly, control.

Positive-strand RNA [(+)RNA] viruses include pandemic SARS-CoV-2, tumor-inducing hepatitis C virus, debilitating chikungunya virus (CHIKV), lethal encephalitis viruses, and many other major pathogens. (+)RNA viruses replicate their RNA genomes in virus-induced replication organelles (ROs) that also evolve new viral species and variants by recombination and mutation and are crucial virus control targets. Recent cryo-electron microscopy (cryo-EM) reveals that viral RNA replication proteins form striking ringed 'crowns' at RO vesicle junctions with the cytosol. These crowns direct RO vesicle formation, viral (-)RNA and (+)RNA synthesis and capping, innate immune escape, and transfer of progeny (+)RNA genomes into translation and encapsidation. Ongoing studies are illuminating crown assembly, sequential functions, host factor interactions, etc., with significant implications for control and beneficial uses of viruses.

A Cool Look at Positive-Strand RNA Virus Replication Organelles: New Insights from Cryo-Electron Microscopy.

Positive-strand RNA viruses encompass a variety of established and emerging eukaryotic pathogens. Their genome replication is confined to specialized cytoplasmic membrane compartments known as replication organelles (ROs). These ROs derive from host membranes, transformed into distinct structures such as invaginated spherules or intricate membrane networks including single- and/or double-membrane vesicles. ROs play a vital role in orchestrating viral RNA synthesis and evading detection by innate immune sensors of the host. In recent years, groundbreaking cryo-electron microscopy studies conducted with several prototypic viruses have significantly advanced our understanding of RO structure and function. Notably, these studies unveiled the presence of crown-shaped multimeric viral protein complexes that seem to actively participate in viral RNA synthesis and regulate the release of newly synthesized RNA into the cytosol for translation and packaging. These findings have shed light on novel viral functions and fascinating macromolecular complexes that delineate promising new avenues for future research.

Automated segmentation of cell organelles in volume electron microscopy using deep learning.

Recent advances in computing power triggered the use of artificial intelligence in image analysis in life sciences. To train these algorithms, a large enough set of certified labeled data is required. The trained neural network is then capable of producing accurate instance segmentation results that will then need to be re-assembled into the original dataset: the entire process requires substantial expertise and time to achieve quantifiable results. To speed-up the process, from cell organelle detection to quantification across electron microscopy modalities, we propose a deep-learning based approach for fast automatic outline segmentation (FAMOUS), that involves organelle detection combined with image morphology, and 3D meshing to automatically segment, visualize and quantify cell organelles within volume electron microscopy datasets. From start to finish, FAMOUS provides full segmentation results within a week on previously unseen datasets. FAMOUS was showcased on a HeLa cell dataset acquired using a focused ion beam scanning electron microscope, and on yeast cells acquired by transmission electron tomography. RESEARCH HIGHLIGHTS: Introducing a rapid, multimodal machine-learning workflow for the automatic segmentation of 3D cell organelles. Successfully applied to a variety of volume electron microscopy datasets and cell lines. Outperforming manual segmentation methods in time and accuracy. Enabling high-throughput quantitative cell biology.

Modular segmentation, spatial analysis and visualization of volume electron microscopy datasets.

Volume electron microscopy is the method of choice for the in situ interrogation of cellular ultrastructure at the nanometer scale, and with the increase in large raw image datasets generated, improving computational strategies for image segmentation and spatial analysis is necessary. Here we describe a practical and annotation-efficient pipeline for organelle-specific segmentation, spatial analysis and visualization of large volume electron microscopy datasets using freely available, user-friendly software tools that can be run on a single standard workstation. The procedures are aimed at researchers in the life sciences with modest computational expertise, who use volume electron microscopy and need to generate three-dimensional (3D) segmentation labels for different types of cell organelles while minimizing manual annotation efforts, to analyze the spatial interactions between organelle instances and to visualize the 3D segmentation results. We provide detailed guidelines for choosing well-suited segmentation tools for specific cell organelles, and to bridge compatibility issues between freely available open-source tools, we distribute the critical steps as easily installable Album solutions for deep learning segmentation, spatial analysis and 3D rendering. Our detailed description can serve as a reference for similar projects requiring particular strategies for single- or multiple-organelle analysis, which can be achieved with computational resources commonly available to single-user setups.

Host Subcellular Organelles: Targets of Viral Manipulation.

Viruses have evolved sophisticated mechanisms to manipulate host cell processes and utilize intracellular organelles to facilitate their replication. These complex interactions between viruses and cellular organelles allow them to hijack the cellular machinery and impair homeostasis. Moreover, viral infection alters the cell membrane's structure and composition and induces vesicle formation to facilitate intracellular trafficking of viral components. However, the research focus has predominantly been on the immune response elicited by viruses, often overlooking the significant alterations that viruses induce in cellular organelles. Gaining a deeper understanding of these virus-induced cellular changes is crucial for elucidating the full life cycle of viruses and developing potent antiviral therapies. Exploring virus-induced cellular changes could substantially improve our understanding of viral infection mechanisms.

A tool for live-cell confocal imaging of temperature-dependent organelle dynamics.

Intracellular organelles alter their morphology in response to ambient conditions such as temperature to optimize physiological activities in cells. Observing organelle dynamics at various temperatures deepens our understanding of cellular responses to the environment. Confocal laser microscopy is a powerful tool for live-cell imaging of fluorescently labeled organelles. However, the large contact area between the specimen and the ambient air on the microscope stage makes it difficult to maintain accurate cellular temperatures. Here, we present a method for precisely controlling cellular temperatures using a custom-made adaptor that can be installed on a commercially available temperature-controlled microscope stage. Using this adaptor, we observed temperature-dependent organelle dynamics in living plant cells; morphological changes in chloroplasts and peroxisomes were temperature dependent. This newly developed adaptor can be easily placed on a temperature-controlled stage to capture intracellular responses to temperature at unprecedentedly high resolution.

Tools for assembling the cell: Towards the era of cell structural bioinformatics.

Cells consist of large components, such as organelles, that recursively factor into smaller systems, such as condensates and protein complexes, forming a dynamic multi-scale structure of the cell. Recent technological innovations have paved the way for systematic interrogation of subcellular structures, yielding unprecedented insights into their roles and interactions. In this workshop, we discuss progress, challenges, and collaboration to marshal various computational approaches toward assembling an integrated structural map of the human cell.

Genetically encoded multimeric tags for subcellular protein localization in cryo-EM.

Cryo-electron tomography (cryo-ET) allows for label-free high-resolution imaging of macromolecular assemblies in their native cellular context. However, the localization of macromolecules of interest in tomographic volumes can be challenging. Here we present a ligand-inducible labeling strategy for intracellular proteins based on fluorescent, 25-nm-sized, genetically encoded multimeric particles (GEMs). The particles exhibit recognizable structural signatures, enabling their automated detection in cryo-ET data by convolutional neural networks. The coupling of GEMs to green fluorescent protein-tagged macromolecules of interest is triggered by addition of a small-molecule ligand, allowing for time-controlled labeling to minimize disturbance to native protein function. We demonstrate the applicability of GEMs for subcellular-level localization of endogenous and overexpressed proteins across different organelles in human cells using cryo-correlative fluorescence and cryo-ET imaging. We describe means for quantifying labeling specificity and efficiency, and for systematic optimization for rare and abundant protein targets, with emphasis on assessing the potential effects of labeling on protein function.

DCX-EMAP is a core organizer for the ultrastructure of Drosophila mechanosensory organelles.

Mechanoreceptor cells develop specialized mechanosensory organelles (MOs), where force-sensitive channels and supporting structures are organized in an orderly manner to detect forces. It is intriguing how MOs are formed. Here, we address this issue by studying the MOs of fly ciliated mechanoreceptors. We show that the main structure of the MOs is a compound cytoskeleton formed of short microtubules and electron-dense materials (EDMs). In a knock-out mutant of DCX-EMAP, this cytoskeleton is nearly absent, suggesting that DCX-EMAP is required for the formation of the MOs and in turn fly mechanotransduction. Further analysis reveals that DCX-EMAP expresses in fly ciliated mechanoreceptors and localizes to the MOs. Moreover, it plays dual roles by promoting the assembly/stabilization of the microtubules and the accumulation of the EDMs in the MOs. Therefore, DCX-EMAP serves as a core ultrastructural organizer of the MOs, and this finding provides novel molecular insights as to how fly MOs are formed.