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1.
Methods Cell Biol ; 187: 1-41, 2024.
Article En | MEDLINE | ID: mdl-38705621

Correlative light and electron microscopy (CLEM) is an approach that combines the strength of multiple imaging techniques to obtain complementary information about a given specimen. The "toolbox" for CLEM is broad, making it sometimes difficult to choose an appropriate approach for a given biological question. In this chapter, we provide experimental details for three CLEM approaches that can help the interested reader in designing a personalized CLEM strategy for obtaining ultrastructural data by using transmission electron microscopy (TEM). First, we describe chemical fixation of cells grown on a solid support (broadest approach). Second, we apply high-pressure freezing/freeze substitution to describe cellular ultrastructure (cryo-immobilization approach). Third, we give a protocol for a ultrastructural labeling by immuno-electron microscopy (immuno-EM approach). In addition, we also describe how to overlay fluorescence and electron microscopy images, an approach that is applicable to each of the reported different CLEM strategies. Here we provide step-by step descriptions prior to discussing possible technical problems and variations of these three general schemes to suit different models or different biological questions. This chapter is written for electron microscopists that are new to CLEM and unsure how to begin. Therefore, our protocols are meant to provide basic information with further references that should help the reader get started with applying a tailored strategy for a specific CLEM experiment.


Microscopy, Electron, Transmission , Humans , Microscopy, Electron, Transmission/methods , Animals , Cryoelectron Microscopy/methods , Microscopy, Electron/methods , Microscopy, Immunoelectron/methods , Microscopy, Fluorescence/methods , Freeze Substitution/methods
2.
Methods Mol Biol ; 2794: 45-62, 2024.
Article En | MEDLINE | ID: mdl-38630219

Immunocytochemistry, a method of delineating the subcellular localization of target proteins, was developed from immunohistochemistry. In principle, proteins are labeled using an antigen-antibody reaction. In order to observe under an electron microscope, the reaction product must scatter the electron beam with sufficient contrast while it is necessary to have an amplifying label that can withstand the observation. We have some detailed tips on making electron microscope samples to achieve this objective, and we would be happy to help you.


Central Nervous System , Microscopy, Immunoelectron
3.
Microsc Microanal ; 29(5): 1694-1704, 2023 09 29.
Article En | MEDLINE | ID: mdl-37584524

Electron microscopy (EM) volume reconstruction is a powerful tool for investigating the fundamental structure of brain circuits, but the full potential of this technique is limited by the difficulty of integrating molecular information. High quality ultrastructural preservation is necessary for EM reconstruction, and intact, highly contrasted cell membranes are essential for following small neuronal processes through serial sections. Unfortunately, the antibody labeling methods used to identify most endogenous molecules result in compromised morphology, especially of membranes. Cryofixation can produce superior morphological preservation and has the additional advantage of allowing indefinite storage of valuable samples. We have developed a method based on cryofixation that allows sensitive immunolabeling of endogenous molecules, preserves excellent ultrastructure, and is compatible with high-contrast staining for serial EM reconstruction.


Brain , Cryopreservation , Microscopy, Immunoelectron , Freezing , Cryopreservation/methods , Fluid Therapy , Freeze Substitution/methods
4.
Methods Cell Biol ; 177: 55-81, 2023.
Article En | MEDLINE | ID: mdl-37451776

Recent advances in volume electron microscopy (vEM) allow unprecedented visualization of the electron-dense structures of cells, tissues and model organisms at nanometric resolution in three dimensions (3D). Light-based microscopy has been widely used for specific localization of proteins; however, it is restricted by the diffraction limit of light, and lacks the ability to identify underlying structures. Here, we describe a protocol for ultrastructural detection, in three dimensions, of a protein (Connexin 43) expressed in the intercalated disc region of adult murine heart. Our protocol does not rest on the expression of genetically encoded proteins and it overcomes hurdles related to pre-embedding and immunolabeling, such as the penetration of the label and the preservation of the tissue. The pre-embedding volumetric immuno-electron microscopy (pre-embedding vIEM) protocol presented here combines several practical strategies to balance sample fixation with antigen and ultrastructural preservation, and penetration of labeling with blocking of non-specific antigen binding sites. The small 1.4 nm gold along with surrounded silver used as a detection marker buried in the sample also serves as a functional conductive resin that significantly reduces the charging of samples. Our protocol also presents strategies for facilitating the successful cutting of the samples during serial block-face scanning electron microscopy (SBF-SEM) imaging. Our results suggest that the small gold-based pre-embedding vIEM is an ideal labeling method for molecular localization throughout the depth of the sample at subcellular compartments and membrane microdomains.


Proteins , Volume Electron Microscopy , Mice , Animals , Microscopy, Immunoelectron , Intercellular Junctions , Gold , Microscopy, Electron, Scanning
5.
J Biol Chem ; 299(9): 105091, 2023 09.
Article En | MEDLINE | ID: mdl-37516240

α-Synuclein and family members ß- and γ-synuclein are presynaptic proteins that sense and generate membrane curvature, properties important for synaptic vesicle (SV) cycling. αßγ-synuclein triple knockout neurons exhibit SV endocytosis deficits. Here, we investigated if α-synuclein affects clathrin assembly in vitro. Visualizing clathrin assembly on membranes using a lipid monolayer system revealed that α-synuclein increases clathrin lattices size and curvature. On cell membranes, we observe that α-synuclein is colocalized with clathrin and its adapter AP180 in a concentric ring pattern. Clathrin puncta that contain both α-synuclein and AP180 were significantly larger than clathrin puncta containing either protein alone. We determined that this effect occurs in part through colocalization of α-synuclein with the phospholipid PI(4,5)P2 in the membrane. Immuno-electron microscopy (EM) of synaptosomes uncovered that α-synuclein relocalizes from SVs to the presynaptic membrane upon stimulation, positioning α-synuclein to function on presynaptic membranes during or after stimulation. Additionally, we show that deletion of synucleins impacts brain-derived clathrin-coated vesicle size. Thus, α-synuclein affects the size and curvature of clathrin structures on membranes and functions as an endocytic accessory protein.


Clathrin , Monomeric Clathrin Assembly Proteins , alpha-Synuclein , alpha-Synuclein/genetics , alpha-Synuclein/metabolism , Cell Membrane/metabolism , Clathrin/chemistry , Clathrin/metabolism , Endocytosis , Microscopy, Immunoelectron , Monomeric Clathrin Assembly Proteins/metabolism , Neurons/metabolism , Presynaptic Terminals/metabolism , Synaptosomes/metabolism , Protein Transport , In Vitro Techniques , Phosphatidylinositol 4,5-Diphosphate/metabolism , Brain/cytology , Clathrin-Coated Vesicles/metabolism
6.
Int. j. morphol ; 41(3): 789-797, jun. 2023. ilus
Article En | LILACS | ID: biblio-1514318

SUMMARY: Diacylglycerol kinase (DGK) exerts balancing the intracellular level between two-second messengers, diacylglycerol and phosphatidic acid, by its phosphorylation activity. DGK ζ is often localized in cell nuclei, suggesting its involvement in the regulation of intranuclear activities, including mitosis and apoptosis. The present immunohistochemical study of rat kidneys first revealed no detection levels of DGK ζ -immunoreactivity in nuclei of most proximal tubule epithelia in contrast to its distinct occurrence in cell nuclei of collecting and distal tubules with the former more dominant. This finding suggests that DGK ζ is a key factor regulating vulnerability to acute kidney injury in various renal tubules: its low expression represents the high vulnerability of proximal tubule cells, and its distinct expression does the resistance of collecting and distal tubule cells. In addition, this isozyme was more or less localized in nuclei of cells forming glomeruli as well as in endothelial nuclei of peritubular capillaries and other intrarenal blood vessels, and epithelial nuclei of glomerular capsules (Bowman's capsules) and renal calyces, including intrarenal interstitial cells.


La diacilglicerol quinasa (DGK) ejerce el equilibrio del nivel intracelular entre dos segundos mensajeros, diacilglicerol y ácido fosfatídico, por su actividad de fosforilación. La DGK ζ a menudo se localiza en los núcleos celulares, lo que sugiere su participación en la regulación de las actividades intranucleares, incluidas la mitosis y la apoptosis. El presente estudio inmunohistoquímico en riñones de rata no reveló niveles de detección de inmunorreactividad de DGK ζ en los núcleos de la mayoría de los epitelios de los túbulos proximales, en contraste a la detección en los núcleos celulares de los túbulos colectores y distales, siendo el primero más dominante. Este hallazgo sugiere que DGK ζ es un factor clave que regula la vulnerabilidad a la lesión renal aguda en varios túbulos renales: su baja expresión representa la alta vulnerabilidad de las células del túbulo proximal, y su expresión distinta hace a la resistencia de las células del túbulo colector y distal. Además, esta isoenzima estaba más o menos localizada en los núcleos de las células que forman los glomérulos, así como en los núcleos endoteliales de los capilares peritubulares y otros vasos sanguíneos intrarrenales, y en los núcleos epiteliales de las cápsulas glomerulares (cápsulas de Bowman) y los cálices renales, incluidas las células intersticiales intrarrenales.


Animals , Rats , Diacylglycerol Kinase/metabolism , Kidney Tubules/metabolism , Immunohistochemistry , Microscopy, Immunoelectron , Rats, Sprague-Dawley , Diacylglycerol Kinase/ultrastructure , Kidney Tubules/ultrastructure
7.
Methods Mol Biol ; 2655: 201-210, 2023.
Article En | MEDLINE | ID: mdl-37212998

"Immunoelectron microscopy" defines a group of techniques developed for visualizing where components of cells or tissues are localized, by means of a transmission electron microscope (TEM) at a subcellular resolution. The method is based on antigen recognition by primary antibodies and subsequent visualization of recognized structures by means of electron-opaque gold granules, which are easily visible in TEM images. The potentially high resolution of this method relies on the very small size of the colloidal gold label, which consists of granules ranging from 1 to 60 nm in diameter, mostly used in the 5-15 nm sizes.


Gold Colloid , Microscopy , Immunohistochemistry , Microscopy, Immunoelectron
8.
Int J Mol Sci ; 24(8)2023 Apr 19.
Article En | MEDLINE | ID: mdl-37108688

White spot syndrome virus (WSSV) is a very large dsDNA virus. The accepted shape of the WSSV virion has been as ellipsoidal, with a tail-like extension. However, due to the scarcity of reliable references, the pathogenesis and morphogenesis of WSSV are not well understood. Here, we used transmission electron microscopy (TEM) and cryogenic electron microscopy (Cryo-EM) to address some knowledge gaps. We concluded that mature WSSV virions with a stout oval-like shape do not have tail-like extensions. Furthermore, there were two distinct ends in WSSV nucleocapsids: a portal cap and a closed base. A C14 symmetric structure of the WSSV nucleocapsid was also proposed, according to our Cryo-EM map. Immunoelectron microscopy (IEM) revealed that VP664 proteins, the main components of the 14 assembly units, form a ring-like architecture. Moreover, WSSV nucleocapsids were also observed to undergo unique helical dissociation. Based on these new results, we propose a novel morphogenetic pathway of WSSV.


Penaeidae , White spot syndrome virus 1 , Animals , White spot syndrome virus 1/genetics , Nucleocapsid/chemistry , Nucleocapsid/metabolism , Virion/metabolism , Microscopy, Electron , Microscopy, Immunoelectron
9.
Curr Microbiol ; 80(4): 106, 2023 Feb 16.
Article En | MEDLINE | ID: mdl-36797528

Porphyromonas gingivalis is an asaccharolytic, Gram-negative, anaerobic bacterium representing a keystone pathogen in chronic periodontitis. The bacterium's energy production depends on the metabolism of amino acids, which are predominantly incorporated as dipeptides via the proton-dependent oligopeptide transporter (Pot). In this study, the localization of dipeptidyl-peptidases (DPPs) and Pot was investigated for the first time in P. gingivalis using immunoelectron microscopy with specific antibodies for the bacterial molecules and gold-conjugated secondary antibodies on ultrathin sections. High-temperature protein G and hemin-binding protein 35 were used as controls, and the cytoplasmic localization of the former and outer membrane localization of the latter were confirmed. P. gingivalis DPP4, DPP5, DPP7, and DPP11, which are considered sufficient for complete dipeptide production, were detected in the periplasmic space. In contrast, DPP3 was localized in the cytoplasmic space in accord with the absence of a signal sequence. The inner membrane localization of Pot was confirmed. Thus, spatial integration of the nutrient acquisition system exists in P. gingivalis, in which where dipeptides are produced in the periplasmic space by DPPs and readily transported across the inner membrane via Pot.


Dipeptidyl-Peptidases and Tripeptidyl-Peptidases , Porphyromonas gingivalis , Dipeptides , Microscopy, Immunoelectron , Base Composition , Phylogeny , RNA, Ribosomal, 16S , Sequence Analysis, DNA , Membrane Transport Proteins , Oligopeptides , Nutrients
10.
CEN Case Rep ; 12(1): 7-13, 2023 02.
Article En | MEDLINE | ID: mdl-35699924

We report the case of a 73-year-old-man who developed immunotactoid glomerulopathy (ITG). ITG is a rare disease characterized by proliferative glomerulonephritis and capillary wall deposits with a 10-60 nm diameter microtubular substructure. In monoclonal ITG, immunofluorescence analysis typically exhibits IgG with light chain restriction. Recent reviews recommend distinguishing monoclonal ITG from polyclonal ITG because monoclonal ITG is associated with a higher incidence of hematological disorders and better responsiveness to clone-directed therapy and renal prognosis. In our case, IgG, IgA, and IgM were negative by routine immunofluorescence; however, immunoelectron microscopy revealed positive λ chain. At 6 months after renal biopsy, the IgG λ chain was detected in the serum and urine, reflecting possible monoclonality. Therefore, it is useful to perform immunoelectron microscopy and follow-up with serum and urine protein electrophoresis and immunofixation to diagnose monoclonal ITG, even when routine immunofluorescence shows negative or nonspecific findings.


Glomerulonephritis , Humans , Aged , Microscopy, Immunoelectron , Glomerulonephritis/pathology , Kidney/pathology , Prognosis , Immunoglobulin G/analysis
11.
Methods Mol Biol ; 2565: 57-75, 2023.
Article En | MEDLINE | ID: mdl-36205887

The localization and density of any plasma membrane or intracellular protein in chromaffin cells are prerequisites for those studies designed to elucidate their contribution to cellular function within the adrenal gland and can be achieved only by immunoelectron microscopy. The most popular immunoelectron microscopic techniques involved gold particles conjugated to secondary antibodies, leading to electron-dense markers and the so-called immunogold EM method. Two main immunogold electron microscopic techniques exist: the pre-embedding immunogold, whereby the immunolabeling steps take place before samples are embedded, and the post-embedding immunogold, where the immunolabeling steps take place on embedded and sectioned samples. Pre-embedding immunogold is a very sensitive technique useful for simultaneous observation of labeled tissue at the light and electron microscopic levels. Post-embedding immunogold enables the simultaneous localization of different molecules in the cell using secondary antibodies conjugated with gold particles of different size. In this chapter, we introduce pre-embedding and post-embedding immunogold procedures used for the identification of quantitative changes in a wide range of signaling molecules in different tissues and also discuss the limitations inherent to these approaches.


Chromaffin Cells , Gold , Antibodies , Immunohistochemistry , Microscopy, Electron , Microscopy, Immunoelectron
12.
Cells ; 13(1)2023 12 28.
Article En | MEDLINE | ID: mdl-38201267

Mutations in multiple epidermal growth factor-like domain 8 (MEGF8), a multidomain transmembrane protein encoded by a gene conserved across species, cause Carpenter's syndrome, which is associated with learning disabilities, mental health issues, and left-right patterning abnormalities. MEGF8 interacts with MGRN1, a protein that functions as an E3 ubiquitin ligase and is involved in multiple physiological and pathological processes. However, the mechanism underlying the distribution of MEGF8 in the central nervous system (CNS) and its cellular and subcellular locations remain unknown. This study aimed to map MEGF8 in the mouse CNS using a new antibody. We discovered that MEGF8 was distributed in the majority of neuronal cell somata across most CNS regions. High levels of MEGF8 were expressed in the neuropils of the CNS gray matter. Immunoelectron microscopy showed that MEGF8 was present in the synapses and around the outer mitochondrial membrane. These findings show that MEGF8 is uniformly distributed throughout the mouse CNS, and its distribution indicates that it plays a substantial role in synaptic and mitochondrial functions. To the best of our knowledge, this is the first study to document MEGF8 distribution in the CNS.


Central Nervous System , Gray Matter , Animals , Mice , Microscopy, Immunoelectron , Antibodies , Cerebral Cortex , Membrane Proteins
13.
J Chem Neuroanat ; 125: 102142, 2022 11.
Article En | MEDLINE | ID: mdl-35918027

Endomorphin-2 (EM2)-immunoreactive (ir) fibers and terminals in the superficial laminae (lamina I and II) of the spinal dorsal horn (SDH) primarily come from neurons in the ipsilateral dorsal root ganglion (DRG), which are important for nociceptive information transmission and modulation. However, the morphological features of EM2-ir neurons and fibers in the DRG and terminals in the SDH under ultrastructural levels have not been completely revealed. The present study observed the distributions of EM2-ir neurons, fibers and terminals in the DRG and SDH and detected their ultrastructural features using immunoelectron microscopy. EM2-ir neurons in the DRG are primarily small or medium in size and account for 17.2% of all neurons in the DRG. EM2-ir large dense-core granule vesicles (LDCVs) are dispersed in the cytoplasm and fibers. Most of the central processes of DRG neurons were thin myelinated and unmyelinated fibers and contained a few EM2-ir LDCVs. An intensive string of EM2-ir fibers with beads and terminals were observed in the superficial laminae of the SDH, other than EM2-ir neurons. EM2-ir products were also detected sparsely in the fibers and terminals. The average diameter of terminals was 94.41 ± 18.13 nm. EM2-ir terminals formed different types of synapses, most of which were asymmetrical (91%). EM2-ir LDCVs colocalized primarily with spherical small clear vesicles in asymmetrical synapses and flat vesicles in symmetrical synapses. The average length of postsynaptic dense zones (PSDs) measured in the asymmetrical synapses was 317.00 ± 31.67 nm. These results indicate that EM2-containing structures are distributed in the cytoplasm of DRG neurons, the central processes and terminals in the SDH and provide morphological evidence for the antinociceptive effects of EM2 in the SDH.


Ganglia, Spinal , Spinal Cord Dorsal Horn , Rats , Animals , Oligopeptides , Microscopy, Immunoelectron , Spinal Cord/physiology
14.
Methods Mol Biol ; 2470: 445-455, 2022.
Article En | MEDLINE | ID: mdl-35881365

Immuno-electron microscopy can detect and localize antigens in cells or tissues at a resolution of several nanometers. In the case of P. falciparum-infected erythrocytes, immuno-EM studies are frequently hampered by the electron-dense nature of the hemoglobin and access of antibodies to antigenic sites, particularly if the targeted protein is presented on the host cell surface or lies in proximity to the host cell cytoskeleton. Here, we describe an improved immuno-EM protocol that overcomes these problems. The improved signal to noise ratio and the enhanced access to antigenic sites now allows one to obtain information regarding target density and distribution and, hence, additional insights into the architecture and function of parasite-induced, or -affected, structures.


Malaria, Falciparum , Plasmodium falciparum , Antigen Presentation , Antigens, Protozoan , Erythrocytes/metabolism , Humans , Microscopy, Immunoelectron , Plasmodium falciparum/metabolism , Protozoan Proteins/metabolism
15.
Methods Mol Biol ; 2528: 67-89, 2022.
Article En | MEDLINE | ID: mdl-35704186

DNA-RNA hybrids can interfere with DNA replication, but the underlying intermediates and molecular mechanisms have remained elusive. Here, we describe a single molecule approach that allows to monitor DNA-RNA hybrids locus-specifically in the context of ongoing replication. Using restriction digestion, gel electrophoresis and gel elution, this workflow allows to efficiently isolate replication intermediates and to study replication dynamics across a specific genomic locus. Here, we applied this procedure to isolate a bacterial genomic locus carrying an inducible transcription-replication conflict. Moreover, we combined electron microscopy with S9.6-Gold immuno-labeling to detect DNA-RNA hybrids on the isolated replication intermediates. With some limitations, this approach may be adapted to locus-specific replication analyses in different organisms.


DNA Replication , RNA , DNA/genetics , Microscopy, Electron , Microscopy, Immunoelectron , RNA/genetics
16.
Parasitol Res ; 121(7): 2065-2078, 2022 Jul.
Article En | MEDLINE | ID: mdl-35524789

The invasive nature of Toxoplasma gondii is closely related to the properties of its cytoskeleton, which is constituted by a group of diverse structural and dynamic components that play key roles during the infection. Even if there have been numerous reports about the composition and function of the Toxoplasma cytoskeleton, the ultrastructural organization of some of these components has not yet been fully characterized. This study used a detergent extraction process and several electron microscopy contrast methods that allowed the successful isolation of the cytoskeleton of Toxoplasma tachyzoites. This process allowed for the conservation of the structures known to date and several new structures that had not been characterized at the ultrastructural level. For the first time, characterization was achieved for a group of nanofibers that allow the association between the polar apical ring and the conoid as well as the ultrastructural characterization of the apical cap of the parasite. The ultrastructure and precise location of the peripheral rings were also found, and the annular components of the basal complex were characterized. Finally, through immunoelectron microscopy, the exact spatial location of the subpellicular network inside the internal membrane system that forms the pellicle was found. The findings regarding these new structures contribute to the knowledge concerning the biology of the Toxoplasma gondii cytoskeleton. They also provide new opportunities in the search for therapeutic strategies aimed at these components with the purpose of inhibiting invasion and thus parasitism.


Toxoplasma , Cytoskeleton/ultrastructure , Microscopy, Electron , Microscopy, Immunoelectron , Microtubules , Toxoplasma/ultrastructure
17.
Autophagy ; 18(12): 3004-3022, 2022 12.
Article En | MEDLINE | ID: mdl-35387562

MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) is widely used as marker of autophagic compartments at different stages of maturation. Electron microscopy (EM) combined with immunolabeling is the only technique that can reveal the ultrastructural identity of LC3-labeled compartments. However, immuno-EM of endogenous LC3 proteins has proven difficult. Here, we test a panel of commercially available antibodies and apply different labeling conditions to present an optimized procedure for LC3 immuno-EM. Using ultrathin cryosections and protein A-colloidal gold or gold enhancement labeling, we localize endogenous LC3 in starved cells or tissues in the presence or absence of the proton pump inhibitor bafilomycin A1. We localize LC3 to early and late stage autophagic compartments that can be classified by their morphology. By on-section correlative light-electron microscopy (CLEM) we show that comparable fluorescent LC3-positive puncta can represent different autophagic intermediates. We also show that our approach is sufficiently robust to label endogenous LC3 simultaneously with other lysosomal and autophagy markers, LAMP1 or SQSTM1/p62, and can be used for quantitative approaches. Thus, we demonstrate that bafilomycin A1 treatment from 2.5 up to 24 h does not inhibit fusion between autophagosomes and lysosomes, but leads to the accumulation of LC3-positive material inside autolysosomes. Together, this is the first study presenting an extensive overview of endogenous LC3 localization at ultrastructural resolution without the need for cell permeabilization and using a commercially available antibody. This provides researchers with a tool to study canonical and non-canonical roles of LC3 in native conditions.Abbreviations: BafA1: bafilomycin A1; BSA: bovine serum albumin; BSA-c: acetylated BSA; BSA5: BSA conjugated to 5-nm gold particles; CLEM: correlative light-electron microscopy; EGFP: enhanced green fluorescent protein; EM: electron microscopy; FBS: fetal bovine serum; FSG: fish skin gelatin; GA: glutaraldehyde; IF: immunofluorescence; LAMP1: lysosomal associated membrane protein 1; LC3s: LC3 proteins; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; ON: overnight; PAG: protein A-conjugated gold particles; PAG1-3: PAG5, PAG10, PAG15, protein A conjugated to 1-3-, 5-, 10-, or 15-nm gold particles; PB: Sorensen's phosphate buffer; PBS: phosphate-buffered saline; PFA: paraformaldehyde; RT: room temperature.


Autophagy , Lysosomes , Animals , Microscopy, Immunoelectron , Lysosomes/metabolism , Microtubule-Associated Proteins/metabolism , Phosphates/metabolism
18.
Microbiol Spectr ; 10(1): e0127121, 2022 02 23.
Article En | MEDLINE | ID: mdl-35171025

The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global outbreak and prompted an enormous research effort. Still, the subcellular localization of the coronavirus in lungs of COVID-19 patients is not well understood. Here, the localization of the SARS-CoV-2 proteins is studied in postmortem lung material of COVID-19 patients and in SARS-CoV-2-infected Vero cells, processed identically. Correlative light and electron microscopy on semithick cryo-sections demonstrated induction of electron-lucent, lipid-filled compartments after SARS-CoV-2 infection in both lung and cell cultures. In lung tissue, the nonstructural protein 4 and the stable nucleocapsid N-protein were detected on these novel lipid-filled compartments. The induction of such lipid-filled compartments and the localization of the viral proteins in lung of patients with fatal COVID-19 may explain the extensive inflammatory response and provide a new hallmark for SARS-CoV-2 infection at the final, fatal stage of infection. IMPORTANCE Visualization of the subcellular localization of SARS-CoV-2 proteins in lung patient material of COVID-19 patients is important for the understanding of this new virus. We detected viral proteins in the context of the ultrastructure of infected cells and tissues and discovered that some viral proteins accumulate in novel, lipid-filled compartments. These structures are induced in Vero cells but, more importantly, also in lung of patients with COVID-19. We have characterized these lipid-filled compartments and determined that this is a novel, virus-induced structure. Immunogold labeling demonstrated that cellular markers, such as CD63 and lipid droplet marker PLIN-2, are absent. Colocalization of lipid-filled compartments with the stable N-protein and nonstructural protein 4 in lung of the last stages of COVID-19 indicates that these compartments play a key role in the devastating immune response that SARS-CoV-2 infections provoke.


COVID-19/metabolism , Lipid Metabolism/physiology , Lipids/analysis , Lung/metabolism , Nucleocapsid/analysis , SARS-CoV-2 , Adolescent , Aged , Animals , COVID-19/pathology , Child, Preschool , Chlorocebus aethiops , Disease Outbreaks , Female , Fluorescent Antibody Technique , Humans , Immunohistochemistry , Lung/cytology , Lung/pathology , Lung/ultrastructure , Male , Microscopy, Immunoelectron , Middle Aged , Nucleocapsid/metabolism , Rabbits , SARS-CoV-2/ultrastructure , Vero Cells/virology
19.
Nat Commun ; 13(1): 571, 2022 01 28.
Article En | MEDLINE | ID: mdl-35091558

Developmental osteogenesis, physiological bone remodelling and fracture healing require removal of matrix and cellular debris. Osteoclasts generated by the fusion of circulating monocytes degrade bone, whereas the identity of the cells responsible for cartilage resorption is a long-standing and controversial question. Here we show that matrix degradation and chondrocyte phagocytosis are mediated by fatty acid binding protein 5-expressing cells representing septoclasts, which have a mesenchymal origin and are not derived from haematopoietic cells. The Notch ligand Delta-like 4, provided by endothelial cells, is necessary for septoclast specification and developmental bone growth. Consistent with the termination of growth, septoclasts disappear in adult and ageing bone, but re-emerge in association with growing vessels during fracture healing. We propose that cartilage degradation is mediated by rare, specialized cells distinct from osteoclasts. Our findings have implications for fracture healing, which is frequently impaired in aging humans.


Cartilage/metabolism , Fracture Healing/physiology , Mesenchymal Stem Cells/metabolism , Osteoclasts/metabolism , Osteogenesis/physiology , Animals , Bone and Bones/cytology , Bone and Bones/metabolism , Bone and Bones/ultrastructure , Cartilage/cytology , Cells, Cultured , Chondrocytes/cytology , Chondrocytes/metabolism , Fatty Acid-Binding Proteins/genetics , Fatty Acid-Binding Proteins/metabolism , Female , Fracture Healing/genetics , Humans , Male , Mesenchymal Stem Cells/cytology , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microscopy, Immunoelectron , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Osteoclasts/cytology , Osteogenesis/genetics , RNA-Seq/methods
20.
Cell Mol Life Sci ; 79(2): 97, 2022 Jan 27.
Article En | MEDLINE | ID: mdl-35084555

Tau is a cytoskeletal protein that is expressed mainly in neurons and is involved in several cellular processes, such as microtubule stabilization, axonal maintenance, and transport. Altered tau metabolism is related to different tauopathies being the most important Alzheimer's disease where aberrant hyperphosphorylated and aggregated tau is found in the central nervous system. Here, we have analyzed that function in kidney by using tau knockout mice generated by integrating GFP-encoding cDNA into exon 1 of MAPT (here referred to as TauGFP/GFP). IVIS Lumina from PerkinElmer demonstrated GFP expression in the kidney. We then demonstrated by qPCR that the main tau isoform in the kidney is Tau4R. The GFP reporter allowed us to demonstrate that tau is found in the glomeruli of the renal cortex, and specifically in podocytes. This was further confirmed by immunohistochemistry. TauGFP/GFP mice present a podocyte cytoskeleton more dynamic as they contain higher levels of detyrosinated tubulin than wild-type mice. In addition, transmission electron microscopy studies demonstrated glomerular damage with a decrease in urinary creatinine. Our results prove that tau has an important role in kidney metabolism under normal physiological conditions.


Kidney/metabolism , Microtubules/metabolism , Podocytes/metabolism , Tauopathies/metabolism , tau Proteins/metabolism , Animals , Cytoskeleton/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Kidney/cytology , Kidney/ultrastructure , Kidney Glomerulus/metabolism , Kidney Glomerulus/ultrastructure , Mice, 129 Strain , Mice, Knockout , Mice, Transgenic , Microscopy, Confocal , Microscopy, Immunoelectron , Tauopathies/genetics , tau Proteins/genetics
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