RESUMO
Correlative light and electron microscopy (CLEM) can provide valuable information about a biological sample by giving information on the specific localization of a molecule of interest within an ultrastructural context. In this work, we describe a simple CLEM method to obtain high-resolution images of neurotransmitter receptor distribution in synapses by electron microscopy (EM). We use hippocampal organotypic slices from a previously reported mouse model expressing a modified AMPA receptor (AMPAR) subunit that binds biotin at the surface (Getz et al., 2022). This tag can be recognized by StreptAvidin-Fluoronanogold™ conjugates (SA-FNG), which reach receptors at synapses (synaptic cleft is 50-100nm thick). By using pre-embedding labeling, we found that SA-FNG reliably bind synaptic receptors and penetrate around 10-15µm in depth in live tissue. However, the silver enhancement was only reaching the surface of the slices. We show that permeabilization with triton is highly effective at increasing the in depth-gold amplification and that the membrane integrity is well preserved. Finally, we also apply high-resolution electron tomography, thus providing important information about the 3D organization of surface AMPA receptors in synapses at the nanoscale.
Assuntos
Hipocampo , Receptores de AMPA , Sinapses , Animais , Camundongos , Hipocampo/metabolismo , Hipocampo/citologia , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Sinapses/ultraestrutura , Proteínas de Membrana/metabolismo , Ouro/química , Microscopia Eletrônica/métodos , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismoRESUMO
Infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, leads to profound remodeling of cellular membranes, promoting viral replication and virion assembly. A full understanding of this drastic remodeling and the process of virion morphogenesis remains lacking. In this study, we applied room temperature transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) tomography to visualize the SARS-CoV-2 replication factory in Vero cells, and present our results in comparison with published cryo-EM studies. We obtained cryo-EM-like clarity of the ultrastructure by employing high-pressure freezing, freeze substitution (HPF-FS) and embedding, allowing room temperature visualization of double-membrane vesicles (DMVs) in a near-native state. In addition, our data illustrate the consecutive stages of virion morphogenesis and reveal that SARS-CoV-2 ribonucleoprotein assembly and membrane curvature occur simultaneously. Finally, we show the tethering of virions to the plasma membrane in 3D, and that accumulations of virus particles lacking spike protein in large vesicles are most likely not a result of defective virion assembly at their membrane. In conclusion, this study puts forward a room-temperature EM technique providing near-native ultrastructural information about SARS-CoV-2 replication, adding to our understanding of the interaction of this pandemic virus with its host cell.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Chlorocebus aethiops , Humanos , Células Vero , Pandemias , Vírion/ultraestruturaRESUMO
A-kinase anchoring protein 79-human/150-rodent (AKAP79/150) organizes signaling proteins to control synaptic plasticity. AKAP79/150 associates with the plasma membrane and endosomes through its N-terminal domain that contains three polybasic regions and two Cys residues that are reversibly palmitoylated. Mutations abolishing palmitoylation (AKAP79/150 CS) reduce its endosomal localization and association with the postsynaptic density (PSD). Here we combined advanced light and electron microscopy (EM) to characterize the effects of AKAP79/150 palmitoylation on its postsynaptic nanoscale organization, trafficking, and mobility in hippocampal neurons. Immunogold EM revealed prominent extrasynaptic membrane AKAP150 labeling with less labeling at the PSD. The label was at greater distances from the spine membrane for AKAP150 CS than WT in the PSD but not in extra-synaptic locations. Immunogold EM of GFP-tagged AKAP79 WT showed that AKAP79 adopts a vertical, extended conformation at the PSD with its N-terminus at the membrane, in contrast to extrasynaptic locations where it adopts a compact or open configurations of its N- and C-termini with parallel orientation to the membrane. In contrast, GFP-tagged AKAP79 CS was displaced from the PSD coincident with disruption of its vertical orientation, while proximity and orientation with respect to the extra-synaptic membrane was less impacted. Single-molecule localization microscopy (SMLM) revealed a heterogeneous distribution of AKAP150 with distinct high-density, nano-scale regions (HDRs) overlapping the PSD but more prominently located in the extrasynaptic membrane for WT and the CS mutant. Thick section scanning transmission electron microscopy (STEM) tomography revealed AKAP150 immunogold clusters similar in size to HDRs seen by SMLM and more AKAP150 labeled endosomes in spines for WT than for CS, consistent with the requirement for AKAP palmitoylation in endosomal trafficking. Hidden Markov modeling of single molecule tracking data revealed a bound/immobile fraction and two mobile fractions for AKAP79 in spines, with the CS mutant having shorter dwell times and faster transition rates between states than WT, suggesting that palmitoylation stabilizes individual AKAP molecules in various spine subpopulations. These data demonstrate that palmitoylation fine tunes the nanoscale localization, mobility, and trafficking of AKAP79/150 in dendritic spines, which might have profound effects on its regulation of synaptic plasticity.
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Biological systems are the sum of their dynamic three-dimensional (3D) parts. Therefore, it is critical to study biological structures in 3D and at high resolution to gain insights into their physiological functions. Electron microscopy of metal replicas of unroofed cells and isolated organelles has been a key technique to visualize intracellular structures at nanometer resolution. However, many of these methods require specialized equipment and personnel to complete them. Here, we present novel accessible methods to analyze biological structures in unroofed cells and biochemically isolated organelles in 3D and at nanometer resolution, focusing on Arabidopsis clathrin-coated vesicles (CCVs). While CCVs are essential trafficking organelles, their detailed structural information is lacking due to their poor preservation when observed via classical electron microscopy protocols experiments. First, we establish a method to visualize CCVs in unroofed cells using scanning transmission electron microscopy tomography, providing sufficient resolution to define the clathrin coat arrangements. Critically, the samples are prepared directly on electron microscopy grids, removing the requirement to use extremely corrosive acids, thereby enabling the use of this method in any electron microscopy lab. Secondly, we demonstrate that this standardized sample preparation allows the direct comparison of isolated CCV samples with those visualized in cells. Finally, to facilitate the high-throughput and robust screening of metal replicated samples, we provide a deep learning analysis method to screen the "pseudo 3D" morphologies of CCVs imaged with 2D modalities. Collectively, our work establishes accessible ways to examine the 3D structure of biological samples and provide novel insights into the structure of plant CCVs.
Assuntos
Cáusticos , Vesículas Revestidas por Clatrina , Cáusticos/análise , Clatrina , Vesículas Revestidas por Clatrina/química , Vesículas Revestidas por Clatrina/ultraestrutura , Endocitose/fisiologia , Imageamento TridimensionalRESUMO
Megapinosomes are endocytic organelles found in human macrophage colony-stimulating factor (M-CSF) monocyte-derived M macrophages. They are large (several microns) and have a complex internal structure that is connected with the cytosol and consists of interconnected knots and concave bridges with sizes in the range of 100 nm. We called this structure trabecular meshwork. The luminal part of the megapinosome can be connected with luminal tubules and cisterns that form the megapinosome complex. The structures are especially well visible in scanning electron tomography when macrophages are prepared by high-pressure freezing and freeze substitution. Our research received a new impulse after studying the literature on hematopoietic cells, where very similar, most likely homologous, structures have been published in peritoneal macrophages as well as in megakaryocytes and blood platelets. In platelets, they serve as membrane storage that is used for structural changes of platelets during activation.
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Endocitose , Macrófagos , Megacariócitos , Humanos , Malha TrabecularRESUMO
Fluorescence lifetime imaging microscopy (FLIM) allows the characterization of cellular metabolism by quantifying the rate of free and unbound nicotinamide adenine dinucleotide hydrogen (NADH). This study delineates the correlative imaging of cells with FLIM and electron microscopy (EM). Human fibroblasts were cultivated in a microscopy slide bearing a coordinate system and FLIM measurement was conducted. Following chemical fixation, embedding in Epon and cutting with an ultramicrotome, tomograms of selected cells were acquired with a scanning transmission electron microscope (STEM). Correlative imaging of antimycin A-treated fibroblasts shows a decrease in fluorescence lifetime as well as swollen mitochondria with large cavities in STEM tomography. To our knowledge, this is the first correlative FLIM and EM workflow. Combining the high sensitivity of FLIM with the high spatial resolution of EM could boost the research of pathophysiological processes involving cell metabolism, such as cancer, neurodegenerative disorders, and viral infection.
Assuntos
Tomografia com Microscopia Eletrônica , Imagem Óptica , Humanos , Microscopia Eletrônica , Microscopia de Fluorescência , Fluxo de TrabalhoRESUMO
Structural changes of two patient-derived glioblastoma cell lines after Zika virus infection were investigated using scanning transmission electron tomography on high-pressure-frozen, freeze-substituted samples. In Zika-virus-infected cells, Golgi structures were barely visible under an electron microscope, and viral factories appeared. The cytosol outside of the viral factories resembled the cytosol of uninfected cells. The viral factories contained largely deranged endoplasmic reticulum (ER), filled with many so-called replication organelles consisting of a luminal vesicle surrounded by the ER membrane. Viral capsids were observed in the vicinity of the replication organelles (cell line #12537 GB) or in ER cisternae at large distance from the replication organelles (cell line #15747 GB). Near the replication organelles, we observed many about 100-nm-long filaments that may represent viral ribonucleoprotein complexes (RNPs), which consist of the RNA genome and N protein oligomers. In addition, we compared Zika-virus-infected cells with cells infected with a phlebovirus (sandfly fever Turkey virus). Zika virions are formed in the ER, whereas phlebovirus virions are assembled in the Golgi apparatus. Our findings will help to understand the replication cycle in the virus factories and the building of the replication organelles in glioblastoma cells.
Assuntos
Retículo Endoplasmático/metabolismo , Glioblastoma/metabolismo , Glioblastoma/virologia , Organelas/metabolismo , Ribonucleoproteínas/metabolismo , Zika virus/metabolismo , Tomografia com Microscopia Eletrônica , Humanos , Células Tumorais Cultivadas , Replicação ViralRESUMO
Quantitative structural characterization of nanomaterials is important to tailor their functional properties. Corrosion of AgAu-alloy nanoparticles (NPs) results in porous structures, making them interesting for applications especially in the fields of catalysis and surface-enhanced Raman spectroscopy. For the present report, structures of dealloyed NPs were reconstructed three-dimensionally using scanning transmission electron microscopy tomography. These reconstructions were evaluated quantitatively, revealing structural information such as pore size, porosity, specific surface area, and tortuosity. Results show significant differences compared to the structure of dealloyed bulk samples and can be used as input for simulations of diffusion or mass transport processes, for example, in catalytic applications.
RESUMO
The application of both fluorescence and electron microscopy results in a powerful combination of imaging modalities called "correlative light and electron microscopy" (CLEM). Whereas conventional transmission electron microscopy (TEM) tomography is only able to image sections up to a thickness of ~300nm, scanning transmission electron microscopy (STEM) tomography at 200kV allows the analysis of sections up to a thickness of 900nm in three dimensions. In the current study we have successfully integrated STEM tomography into CLEM as demonstrated for human retinal pigment epithelial 1 (RPE1) cells expressing various fluorescent fusion proteins which were high-pressure frozen and then embedded in Lowicryl HM20. Fluorescently labeled gold nanoparticles were applied onto resin sections and imaged by fluorescence and electron microscopy. STEM tomograms were recorded at regions of interest, and overlays were generated using the eC-CLEM software package. Through the nuclear staining of living cells, the use of fluorescently labeled gold fiducials for the generation of overlays, and the integration of STEM tomography we have markedly extended the application of the Kukulski protocol (Kukulski et al., 2011, 2012). Various fluorescently tagged proteins localizing to different cellular organelles could be assigned to their ultrastructural compartments. By combining STEM tomography with on-section CLEM, fluorescently tagged proteins can be localized in three-dimensional ultrastructural environments with a volume of at least 2.7×2.7×0.5µm.
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Tomografia com Microscopia Eletrônica , Nanopartículas Metálicas , Ouro , Humanos , Microscopia Eletrônica , Microscopia de FluorescênciaRESUMO
Electron tomography has been widely applied to three-dimensional (3D) morphology characterization and chemical analysis at the nanoscale. A HAADF-EDS bimodal tomographic (HEBT) reconstruction technique has been developed to extract high resolution element-specific information. However, the reconstructed elemental maps cannot be directly converted to quantitative compositional information. In this work, we propose a quantification approach for obtaining elemental weight fraction maps from the HEBT reconstruction technique using the physical parameters extracted from a Monte Carlo code, MC X-ray. A similar quantification approach is proposed for the EDS-STEM tomographic reconstruction. The performance of the two quantitative reconstruction methods, using the simultaneous iterative reconstruction technique, are evaluated and compared for a simulated dataset of a two-dimensional phantom sample. The effects of the reconstruction parameters including the number of iterations and the weight of the HAADF signal are discussed. Finally, the two approaches are applied to an experimental dataset to show the 3D structure and quantitative elemental maps of a particle of flux melted metal-organic framework glass.
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The interpretation of cell biological processes hinges on the elucidation of the underlying structures. Their three-dimensional analysis using electron tomography has extended our understanding of cellular organelles tremendously. The investigations depend on the availability of appropriate instruments for data recording. So far, such investigations have been done to a great extent on 300 keV transmission electron microscopes. Here we show the implementation of STEM tomography on a 200 kV FEG transmission electron microscope, including the tuning of the condenser for forming a beam with a small illumination aperture, dual-axis data recording, and evaluation of the maximum sample thickness and quality of the data. Our results show that the approach is accomplishable and promising, with high reliability, and reaching excellent data quality from plastic sections with a thickness of at least 900 nm.
Assuntos
Tomografia com Microscopia Eletrônica/instrumentação , Tomografia com Microscopia Eletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Animais , Rim/diagnóstico por imagem , Camundongos , Software , Inclusão do TecidoRESUMO
The megapinosome is an endocytic cell organel that we observed in human macrophages with electron microscopy. In a previous work we showed that it is formed by an endocytic event that we called megapinocytosis. The megapinosome is filled with a membrane surrounded trabecular meshwork that is topologically part of the cytosol. In this work we used scanning transmission electron tomography on high pressure frozen and freeze substituted human macrophages in order to unravel the three-dimensional structure of both the megapinosome and the adjacent structures. The megapinosome consists of the trabecular meshwork and the lacunae which are connected with and topologically equivalent to the cytosol. The surrounding lumen is topologically equivalent to the structures of the vesicular pathway. In addition, we show the connections of the trabecular meshwork with the cytosol and the connection of the megapinosomes to a complex tubular and cisternal system covering a large part of the macrophages that we named megapinosome complex. We assume that our methodological approach, based on high pressure freezing from a defined physiological state and three-dimensional imaging, renders the tubular components of the macrophages better visible than the classical two-dimensional imaging of chemically fixed cells used as a "blueprint" for textbook illustrations. The cell biological functions of the megapinosome are largely enigmatic. Probably, megapinosomes assures storage of surface membranes that can be promptly made available when a macrophage needs to change shape to move through a tissue, to uptake extracellular material or dead cells as well as to fight against microbes.
Assuntos
Macrófagos/metabolismo , Microscopia Eletrônica/métodos , Citosol/metabolismo , Endocitose/fisiologia , PressãoRESUMO
Human cytomegalovirus (HCMV) entry into susceptible cells is a fast intricate process that is not fully understood. Although, previous studies explored different aspects of this process by means of biochemical and inhibitors assays, a clear morphological characterization of its steps at the ultrastructural level is still lacking. We attempted to characterize those intermediates involved during HCMV entry by developing a methodological approach that resulted in optimal ultrastructure preservation and allowed for 3D imaging. It involves rapid freezing and cryosubstitution which ensure a clear visibility of membranous leaflets as well as retained membranous continuity. Likewise, it delivered a reproducible optimization of the growth and infection conditions that are pivotal towards maintaining biologically active enriched input virus particles. Data acquisition was achieved through STEM tomography in a 3D context. Indeed, several intermediates that characterize HCMV entry-related events were observed both extra- and intracellularly. Some of the cell-membrane associated viral particles that we referred to as "Pinocchio particles" were morphologically altered in comparison to the cell-free virions. We were also able to characterize intracellular fusion intermediates taking place between the viral envelope and the vesicular membranes. Furthermore, inhibiting actin polymerization by Latrunculin-A enabled us to spot fusion-like intermediates of the viral envelope with the host cell plasma membrane that we did not observe in the untreated infected cells. Our data also suggests that Dyngo-4a; a dynamin-2 inhibitor, does not interfere with the internalization of the HCMV into the host cells as previously deduced.
Assuntos
Citomegalovirus/fisiologia , Tomografia com Microscopia Eletrônica/métodos , Vírion/fisiologia , Internalização do Vírus , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Membrana Celular/virologia , Células Cultivadas , Citomegalovirus/ultraestrutura , Dinamina II/antagonistas & inibidores , Dinamina II/metabolismo , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Fibroblastos/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Hidrazonas/farmacologia , Imageamento Tridimensional/métodos , Naftóis/farmacologia , Reprodutibilidade dos Testes , Vírion/ultraestruturaRESUMO
Scanning transmission electron microscopic (STEM) tomography of high-pressure frozen, freeze-substituted semi-thin sections is one of multiple approaches for three-dimensional recording and visualization of electron microscopic samples. Compared to regular TEM tomography thicker sample sections can be investigated since chromatic aberration due to inelastic scattering is not a limit. The method is ideal to investigate subcellular compartments or organelles such as synapses, mitochondria, or microtubule arrangements. STEM tomography fills the gap between single-particle electron cryo-tomography, and methods that allow investigations of large volumes, such as serial block-face SEM and FIB-SEM. In this article, we discuss technical challenges of the approach and show some applications in cell biology. It is ideal to use a 300-kV electron microscope with a very small convergence angle of the primary beam ("parallel" beam). These instruments are expensive and tomography is rather time consuming, and therefore, access to such a high-end microscope might be difficult. In this article, we demonstrate examples of successful STEM tomography in biology using a more standard 200-kV microscope equipped with a field emission tip.
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Tomografia com Microscopia Eletrônica/métodos , Congelamento , Glioblastoma/patologia , Citoplasma/patologia , Humanos , PressãoRESUMO
Immunogold labeling of permeabilized whole-mount cells or thin-sectioned material is widely used for the subcellular localization of biomolecules at the high spatial resolution of electron microscopy (EM). Those approaches are well compatible with either 3-dimensional (3D) reconstruction of organelle morphology and antigen distribution or with rapid cryofixation-but not easily with both at once. We describe here a specimen preparation and labeling protocol for animal cell cultures, which represents a novel blend of specifically adapted versions of established techniques. It combines the virtues of reliably preserved organelle ultrastructure, as trapped by rapid freezing within milliseconds followed by freeze-substitution and specimen rehydration, with the advantages of robust labeling of intracellular constituents in 3D through means of pre-embedding NANOGOLD-silver immunocytochemistry. So obtained thin and semi-thick epoxy resin sections are suitable for transmission EM imaging, as well as tomographic reconstruction and modeling of labeling patterns in the 3D cellular context.
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Microscopia Eletrônica de Transmissão/métodos , Microscopia Imunoeletrônica/métodos , Tomografia/métodos , Animais , Antígenos/química , Células CACO-2 , Criopreservação/métodos , Compostos de Epóxi/química , Congelamento , Ouro/química , Células HeLa , Humanos , Imuno-Histoquímica , Nanopartículas/química , Pressão , Prata/químicaRESUMO
Vanadium pentoxide (V2O5) is proposed and investigated as a cathode material for lithium-ion (Li-ion) batteries. However, the dissolution of V2O5 during the charge/discharge remains as an issue at the V2O5-electrolyte interface. In this work, we present a heterogeneous nanostructure with carbon nanotubes supported V2O5/titanium dioxide (TiO2) multilayers as electrodes for thin-film Li-ion batteries. Atomic layer deposition of V2O5 on carbon nanotubes provides enhanced Li storage capacity and high rate performance. An additional TiO2 layer leads to increased morphological stability and in return higher electrochemical cycling performance of V2O5/carbon nanotubes. The physical and chemical properties of TiO2/V2O5/carbon nanotubes are characterized by cyclic voltammetry and charge/discharge measurements as well as electron microscopy. The detailed mechanism of the protective TiO2 layer to improve the electrochemical cycling stability of the V2O5 is unveiled.