Progression of herpesvirus infection remodels mitochondrial organization and metabolism.

Viruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized to the perinuclear area, a significant increase in the number and clustering of endoplasmic reticulum-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.

Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography.

Plus-strand RNA viruses are proficient at remodeling host cell membranes for optimal viral genome replication and the production of infectious progeny. These ultrastructural alterations result in the formation of viral membranous organelles and may be observed by different imaging techniques, providing nanometric resolution. Guided by confocal and electron microscopy, this study describes the generation of wide-field volumes using cryogenic soft-X-ray tomography (cryo-SXT) on SARS-CoV-2-infected human lung adenocarcinoma cells. Confocal microscopy showed accumulation of double-stranded RNA (dsRNA) and nucleocapsid (N) protein in compact perinuclear structures, preferentially found around centrosomes at late stages of the infection. Transmission electron microscopy (TEM) showed accumulation of membranous structures in the vicinity of the infected cell nucleus, forming a viral replication organelle containing characteristic double-membrane vesicles and virus-like particles within larger vesicular structures. Cryo-SXT revealed viral replication organelles very similar to those observed by TEM but indicated that the vesicular organelle observed in TEM sections is indeed a vesiculo-tubular network that is enlarged and elongated at late stages of the infection. Overall, our data provide additional insight into the molecular architecture of the SARS-CoV-2 replication organelle.

Chaperonin CCT controls extracellular vesicle production and cell metabolism through kinesin dynamics.

Cell proteostasis includes gene transcription, protein translation, folding of de novo proteins, post-translational modifications, secretion, degradation and recycling. By profiling the proteome of extracellular vesicles (EVs) from T cells, we have found the chaperonin complex CCT, involved in the correct folding of particular proteins. By limiting CCT cell-content by siRNA, cells undergo altered lipid composition and metabolic rewiring towards a lipid-dependent metabolism, with increased activity of peroxisomes and mitochondria. This is due to dysregulation of the dynamics of interorganelle contacts between lipid droplets, mitochondria, peroxisomes and the endolysosomal system. This process accelerates the biogenesis of multivesicular bodies leading to higher EV production through the dynamic regulation of microtubule-based kinesin motors. These findings connect proteostasis with lipid metabolism through an unexpected role of CCT.

Shedding Light on Osteosarcoma Cell Differentiation: Impact on Biomineralization and Mitochondria Morphology.

Osteosarcoma (OS) is the most common primary malignant bone tumor and its etiology has recently been associated with osteogenic differentiation dysfunctions. OS cells keep a capacity for uncontrolled proliferation showing a phenotype similar to undifferentiated osteoprogenitors with abnormal biomineralization. Within this context, both conventional and X-ray synchrotron-based techniques have been exploited to deeply characterize the genesis and evolution of mineral depositions in a human OS cell line (SaOS-2) exposed to an osteogenic cocktail for 4 and 10 days. A partial restoration of the physiological biomineralization, culminating with the formation of hydroxyapatite, was observed at 10 days after treatment together with a mitochondria-driven mechanism for calcium transportation within the cell. Interestingly, during differentiation, mitochondria showed a change in morphology from elongated to rounded, indicating a metabolic reprogramming of OS cells possibly linked to an increase in glycolysis contribution to energy metabolism. These findings add a dowel to the genesis of OS giving new insights on the development of therapeutic strategies able to restore the physiological mineralization in OS cells.

Collective magnetotaxis of microbial holobionts is optimized by the three-dimensional organization and magnetic properties of ectosymbionts.

Over the last few decades, symbiosis and the concept of holobiont-a host entity with a population of symbionts-have gained a central role in our understanding of life functioning and diversification. Regardless of the type of partner interactions, understanding how the biophysical properties of each individual symbiont and their assembly may generate collective behaviors at the holobiont scale remains a fundamental challenge. This is particularly intriguing in the case of the newly discovered magnetotactic holobionts (MHB) whose motility relies on a collective magnetotaxis (i.e., a magnetic field-assisted motility guided by a chemoaerotaxis system). This complex behavior raises many questions regarding how magnetic properties of symbionts determine holobiont magnetism and motility. Here, a suite of light-, electron- and X-ray-based microscopy techniques [including X-ray magnetic circular dichroism (XMCD)] reveals that symbionts optimize the motility, the ultrastructure, and the magnetic properties of MHBs from the microscale to the nanoscale. In the case of these magnetic symbionts, the magnetic moment transferred to the host cell is in excess (102 to 103 times stronger than free-living magnetotactic bacteria), well above the threshold for the host cell to gain a magnetotactic advantage. The surface organization of symbionts is explicitly presented herein, depicting bacterial membrane structures that ensure longitudinal alignment of cells. Magnetic dipole and nanocrystalline orientations of magnetosomes were also shown to be consistently oriented in the longitudinal direction, maximizing the magnetic moment of each symbiont. With an excessive magnetic moment given to the host cell, the benefit provided by magnetosome biomineralization beyond magnetotaxis can be questioned.

Cryo soft X-ray tomography to explore Escherichia coli nucleoid remodeling by Hfq master regulator.

The bacterial chromosomic DNA is packed within a membrane-less structure, the nucleoid, due to the association of DNA with proteins called Nucleoid Associated Proteins (NAPs). Among these NAPs, Hfq is one of the most intriguing as it plays both direct and indirect roles on DNA structure. Indeed, Hfq is best known to mediate post-transcriptional regulation by using small noncoding RNA (sRNA). Although Hfq presence in the nucleoid has been demonstrated for years, its precise role is still unclear. Recently, it has been shown in vitro that Hfq forms amyloid-like structures through its C-terminal region, hence belonging to the bridging family of NAPs. Here, using cryo soft X-ray tomography imaging of native unlabeled cells and using a semi-automatic analysis and segmentation procedure, we show that Hfq significantly remodels the Escherichia coli nucleoid. More specifically, Hfq influences nucleoid density especially during the stationary growth phase when it is more abundant. Our results indicate that Hfq could regulate nucleoid compaction directly via its interaction with DNA, but also at the post-transcriptional level via its interaction with RNAs. Taken together, our findings reveal a new role for this protein in nucleoid remodeling in vivo, that may serve in response to stress conditions and in adapting to changing environments.

Hydroxyapatite nanoparticles-cell interaction: New approaches to disclose the fate of membrane-bound and internalised nanoparticles.

Hydroxyapatite nanoparticles are popular tools in bone regeneration, but they have also been used for gene delivery and as anticancer drugs. Understanding their mechanism of action, particularly for the latter application, is crucial to predict their toxicity. To this end, we aimed to elucidate the importance of nanoparticle membrane interactions in the cytotoxicity of MG-63 cells using two different types of nanoparticles. In addition, conventional techniques for studying nanoparticle internalisation were evaluated and compared with newer and less exploited approaches. Hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles were used as suspensions or compacted as specular discs. Comparison between cells seeded on the discs and those supplemented with the nanoparticles allowed direct interaction of the cell membrane with the material to be ruled out as the main mechanism of toxicity. In addition, standard techniques such as flow cytometry were inconclusive when used to assess nanoparticles toxicity. Interestingly, the use of intracellular calcium fluorescent probes revealed the presence of a high number of calcium-rich vesicles after nanoparticle supplementation in cell culture. These structures could not be detected by transmission electron microscopy due to their liquid content. However, by using cryo-soft X-ray imaging, which was used to visualise the cellular ultrastructure without further treatment other than vitrification and to quantify the linear absorption coefficient of each organelle, it was possible to identify them as multivesicular bodies, potentially acting as calcium stores. In the study, an advanced state of degradation of the hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles within MG-63 cells was observed. Overall, we demonstrate that the combination of fluorescent calcium probes together with cryo-SXT is an excellent approach to investigate intracellular calcium, especially when found in its soluble form.

Evaluation of the Role of Bacterial Amyloid on Nucleoid Structure Using Cryo-Soft X-Ray Tomography.

Bacterial chromosomal DNA is packed within a non-membranous structure, the nucleoid, thanks to nucleoid associated proteins (NAPs). The role of bacterial amyloid has recently emerged among these NAPs, particularly with the nucleoid-associated protein Hfq that plays a direct role in DNA compaction. In this chapter, we present a 3D imaging technique, cryo-soft X-ray tomography (cryo-SXT) to obtain a detailed 3D visualization of subcellular bacterial structures, especially the nucleoid. Cryo-SXT imaging of native unlabeled cells enables observation of the nucleoid in 3D with a high resolution, allowing to evidence in vivo the role of amyloids on DNA compaction. The precise experimental methods to obtain 3D tomograms will be presented.

Lethal Interactions of Atomically Precise Gold Nanoclusters and Pseudomonas aeruginosa and Staphylococcus aureus Bacterial Cells.

Ultrasmall metal nanoclusters (NCs) are employed in an array of diagnostic and therapeutic applications due to their tunable photoluminescence, high biocompatibility, polyvalent effect, ease of modification, and photothermal stability. However, gold nanoclusters' (AuNCs') intrinsically antimicrobial properties remain poorly explored and are not well understood. Here, we share an insight into the antimicrobial action of atomically precise AuNCs based on their ability to passively translocate across the bacterial membrane. Functionalized by a hydrophilic modified-bidentate sulfobetaine zwitterionic molecule (AuNC-ZwBuEt) or a more hydrophobic monodentate-thiolate, mercaptohexanoic acid (AuNC-MHA) molecule, 2 nm AuNCs were lethal to both Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus bacteria. The bactericidal efficiency was found to be bacterial strain-, time-, and concentration-dependent. The direct visualizations of the translocation of AuNCs and AuNC-cell and subcellular interactions were investigated using cryo-soft X-ray nano-tomography, transmission electron microscopy (TEM), and scanning TEM energy-dispersive spectroscopy analyses. AuNC-MHA were identified in the bacterial cytoplasm within 30 min, without evidence of the loss of membrane integrity. It is proposed that the bactericidal effect of AuNCs is attributed to their size, which allows for efficient energy-independent translocation across the cell membrane. The internalization of both AuNCs caused massive internal damage to the cells, including collapsed subcellular structures and altered cell morphology, leading to the eventual loss of cellular integrity.

Two-Step Resist Deposition of E-Beam Patterned Thick Py Nanostructures for X-ray Microscopy.

Patterned elements of permalloy (Py) with a thickness as large as 300 nm have been defined by electron beam lithography on X-ray-transparent 50 nm thick membranes in order to characterize their magnetic structure via Magnetic Transmission X-ray Microscopy (MTXM). To avoid the situation where the fragility of the membranes causes them to break during the lithography process, it has been found that the spin coating of the resist must be applied in two steps. The MTXM results show that our samples have a central domain wall, as well as other types of domain walls, if the nanostructures are wide enough.

Monitoring reversion of hepatitis C virus-induced cellular alterations by direct-acting antivirals using cryo soft X-ray tomography and infrared microscopy.

Hepatitis C virus (HCV) is an enveloped RNA virus. One of the hallmarks of HCV infection is a rearrangement of the host cell membranes, known as the `membranous web'. Full-field cryo soft X-ray tomography (cryo-SXT) in the water-window energy range (284-543 eV) was performed on the MISTRAL beamline to investigate, in whole unstained cells, the morphology of the membranous rearrangements induced in HCV replicon-harbouring cells in conditions close to the living physiological state. All morphological alterations could be reverted by a combination of sofosbuvir/daclatasvir, which are clinically approved antivirals (direct-acting antivirals; DAAs) for HCV infection. Correlatively combining cryo-SXT and 2D synchrotron-based infrared microscopy provides critical information on the chemical nature of specific infection-related structures, which allows specific patterns of the infection process or the DAA-mediated healing process to be distinguished.

Imaging of Virus-Infected Cells with Soft X-ray Tomography.

Viruses are obligate parasites that depend on a host cell for replication and survival. Consequently, to fully understand the viral processes involved in infection and replication, it is fundamental to study them in the cellular context. Often, viral infections induce significant changes in the subcellular organization of the host cell due to the formation of viral factories, alteration of cell cytoskeleton and/or budding of newly formed particles. Accurate 3D mapping of organelle reorganization in infected cells can thus provide valuable information for both basic virus research and antiviral drug development. Among the available techniques for 3D cell imaging, cryo-soft X-ray tomography stands out for its large depth of view (allowing for 10 µm thick biological samples to be imaged without further thinning), its resolution (about 50 nm for tomographies, sufficient to detect viral particles), the minimal requirements for sample manipulation (can be used on frozen, unfixed and unstained whole cells) and the potential to be combined with other techniques (i.e., correlative fluorescence microscopy). In this review we describe the fundamentals of cryo-soft X-ray tomography, its sample requirements, its advantages and its limitations. To highlight the potential of this technique, examples of virus research performed at BL09-MISTRAL beamline in ALBA synchrotron are also presented.

Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells.

Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than conventional fluorescence microscopy. This super-resolution (SR) technique enables visualization of molecular processes in whole cells and has the potential to be used in conjunction with electron microscopy and X-ray tomography to correlate structural and functional information. A SIM microscope for cryogenically preserved samples (cryoSIM) has recently been commissioned at the correlative cryo-imaging beamline B24 at the UK synchrotron. It was designed specifically for 3D imaging of biological samples at cryogenic temperatures in a manner compatible with subsequent imaging of the same samples by X-ray microscopy methods such as cryo-soft X-ray tomography. This video article provides detailed methods and protocols for successful imaging using the cryoSIM. In addition to instructions on the operation of the cryoSIM microscope, recommendations have been included regarding the choice of samples, fluorophores, and parameter settings. The protocol is demonstrated in U2OS cell samples whose mitochondria and tubulin have been fluorescently labelled.

Calcite as a Precursor of Hydroxyapatite in the Early Biomineralization of Differentiating Human Bone-Marrow Mesenchymal Stem Cells.

Biomineralization is the process by which living organisms generate organized mineral crystals. In human cells, this phenomenon culminates with the formation of hydroxyapatite, which is a naturally occurring mineral form of calcium apatite. The mechanism that explains the genesis within the cell and the propagation of the mineral in the extracellular matrix still remains largely unexplained, and its characterization is highly controversial, especially in humans. In fact, up to now, biomineralization core knowledge has been provided by investigations on the advanced phases of this process. In this study, we characterize the contents of calcium depositions in human bone mesenchymal stem cells exposed to an osteogenic cocktail for 4 and 10 days using synchrotron-based cryo-soft-X-ray tomography and cryo-XANES microscopy. The reported results suggest crystalline calcite as a precursor of hydroxyapatite depositions within the cells in the biomineralization process. In particular, both calcite and hydroxyapatite were detected within the cell during the early phase of osteogenic differentiation. This striking finding may redefine most of the biomineralization models published so far, taking into account that they have been formulated using murine samples while studies in human cell lines are still scarce.

Sample preparation strategies for efficient correlation of 3D SIM and soft X-ray tomography data at cryogenic temperatures.

3D correlative microscopy methods have revolutionized biomedical research, allowing the acquisition of multidimensional information to gain an in-depth understanding of biological systems. With the advent of relevant cryo-preservation methods, correlative imaging of cryogenically preserved samples has led to nanometer resolution imaging (2-50 nm) under harsh imaging regimes such as electron and soft X-ray tomography. These methods have now been combined with conventional and super-resolution fluorescence imaging at cryogenic temperatures to augment information content from a given sample, resulting in the immediate requirement for protocols that facilitate hassle-free, unambiguous cross-correlation between microscopes. We present here sample preparation strategies and a direct comparison of different working fiducialization regimes that facilitate 3D correlation of cryo-structured illumination microscopy and cryo-soft X-ray tomography. Our protocol has been tested at two synchrotron beamlines (B24 at Diamond Light Source in the UK and BL09 Mistral at ALBA in Spain) and has led to the development of a decision aid that facilitates experimental design with the strategic use of markers based on project requirements. This protocol takes between 1.5 h and 3.5 d to complete, depending on the cell populations used (adherent cells may require several days to grow on sample carriers).

A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography.

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 µm thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectro-tomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology.

Creation and observation of Hopfions in magnetic multilayer systems.

Among topological solitons, magnetic skyrmions are two-dimensional particle-like objects with a continuous winding of the magnetization, and magnetic Hopfions are three-dimensional objects that can be formed from a closed loop of twisted skyrmion strings. Theoretical models suggest that magnetic Hopfions can be stabilized in frustrated or chiral magnetic systems, and target skymions can be transformed into Hopfions by adapting their perpendicular magnetic anisotropy, but their experimental verification has been elusive so far. Here, we present an experimental study of magnetic Hopfions that are created in Ir/Co/Pt multilayers shaped into nanoscale disks, known to host target skyrmions. To characterize three-dimensional spin textures that distinguish Hopfions from target skyrmions magnetic images are recorded with surface-sensitive X-ray photoemission electron microscopy and bulk-sensitive soft X-ray transmission microscopy using element-specific X-ray magnetic circular dichroism effects as magnetic contrast. These results could stimulate further investigations of Hopfions and their potential application in three-dimensional spintronics devices.

Multimodal Imaging and Soft X-Ray Tomography of Fluorescent Nanodiamonds in Cancer Cells.

Multimodal imaging promises to revolutionize the understanding of biological processes across scales in space and time by combining the strengths of multiple imaging techniques. Fluorescent nanodiamonds (FNDs) are biocompatible, chemically inert, provide high contrast in light- and electron-based microscopy, and are versatile optical quantum sensors. Here it is demonstrated that FNDs also provide high absorption contrast in nanoscale 3D soft X-ray tomograms with a resolution of 28 nm in all dimensions. Confocal fluorescence, atomic force, and scanning electron microscopy images of FNDs inside and on the surface of PC3 cancer cells with sub-micrometer precision are correlated. FNDs are found inside ≈1 µm sized vesicles present in the cytoplasm, providing direct evidence of the active uptake of bare FNDs by cancer cells. Imaging artefacts are quantified and separated from changes in cell morphology caused by sample preparation. These results demonstrate the utility of FNDs in multimodal imaging, contribute to the understanding of the fate of FNDs in cells, and open up new possibilities for biological imaging and sensing across the nano- and microscale.