Microscopy Conference 2023

Prof.

Session chairs

Microscopy helps to diagnose and to understand diseases. The session will cover all applications of electron microscopy in this field, from high-resolution work by cryo-electron microscopy to routine diagnostic electron microscopy using thin section or negative staining electron microscopy.

Zooming into the spread and entry of influenza A viruses by correlative light and electron microscopy

Abstract text (incl. figure legends and references) Influenza A viruses (IAV) cause annual epidemics and occasional pandemics typically resulting from zoonotic transfers. While it has been well established that the IAV are highly pleomorphic forming filamentous virions typically found in human isolates and spherical particles after lab adaptation, the benefits of filamentous virions in spread and entry inside lungs are not fully understood. Here, we used reverse genetics to create spherical and filamentous reporter viruses with the same genetic background. We apply cryo-correlative light and electron tomography to elucidate IAV entry in endosomes and established a correlative light and scanning electron microscopy to study IAV spread in a quantitative manner using plaque assays. Our data show differences in the spread of spherical and filamentous virions in different cell lines.

LS 6: Pathology, pathogens and diagnostics

Invited talk

Dissecting the lifecycle of uropathogens by directly analyzing patient samples with cryo-ET

Abstract text (incl. figure legends and references) Urinary tract infections (UTIs) are among the most common bacterial infections in humans. Besides being generally considered to be extracellular infections and easily treated with antibiotics, they are often incompletely resolved. About 25% of patients experience a recurrent UTI and the resulting repetitive administration of antibiotics contributed to higher antibiotic resistance rates in uropathogens. Up to 90% of UTIs are caused by uropathogenic E. coli (UPEC), which can proliferate in the bladder lumen but are also capable of invading uroepithelial cells where they form intracellular bacterial communities, protecting the pathogen from host defenses and antibiotics. By using an integrative approach of mass spectrometry, (cryo-correlative) light microscopy, cryo-focused ion beam (FIB) milling and cryo-electron tomography (cryo-ET), we aim to dissect the different infection stages of UPEC in vitro and directly in patient samples. In a first step, we resolved the architecture of the most abundant protein in our urine, uromodulin, and could show how it serves as a multivalent antagonist against UPEC adhesion. However, many bacteria can overcome the uromodulin barrier and infect bladder epithelial cells to form intracellular communities. We optimized an infection protocol and transferred it to cells seeded on electron microscopy (EM) grids. By using cryo-fluorescent light microscopy, we correlate our subsequent cryo-FIB milling step to different maturation stages of intracellular bacterial communities to reveal their native in situ architecture and composition. To transfer our insights to actual patients, we are working on analyzing infected cells found in patient urine and in bladder biopsies with cryo-ET.

Plenary

Prof. Dr.

The role of vaccinia virus protein A6 for correct assembly

Abstract text (incl. figure legends and references) Introduction: Enveloped viruses acquire their membrane from the host, but for the large DNA-virus vaccinia virus (VACV), member of the poxviridae, the origin and biogenesis of its membrane remains controversial until this date. By electron microscopy (EM) newly synthesized VACV membranes appear as short, half-moon shaped units (the crescents) that grow into membrane spheres, the immature virus (IV). Upon DNA-uptake, the IV matures in to the infectious brick-shaped mature virus (MV) [1]. The gene product of A6, a 43 kDa protein, is one of five VACV proteins that seem to be required for viral membrane biogenesis and the formation of infectious virus, collectively called virus membrane associated proteins (VMAPs) [2]. Objectives: It is known, that the VMAPs play an essential role in the assembly of the VACV. In this study, the role of A6 in VACV-assembly was further investigated. Materials &amp; methods: Hela cells were infected with a recombinant VACV where the synthesis of A6 can be conditionally induced. Cells infected with or without A6 synthesis were fixed at different time points and prepared for EM-imaging. Complementary EM-imaging methods were applied: conventional thin section EM, EM-immuno-labeling and scanning transmission electron tomography. Results: When the expression of A6 is blocked, IV- and MV-formation is absent [3]. Instead aberrant virally induced structures accumulate in a time dependent manner; fragmented core accumulations, and large spherical membranes, resembling IVs, but devoid of viral core proteins. Conclusion: It seems that A6 plays a role in the interaction of core protein and crescents. Reference: [1] DOI: 10.1016/j.virol.2015.02.003 [2] DOI: 10.1128/JVI.02405-16 [3] DOI: 10.1128/JVI.00877-09

Abstract talk

Influence of iron overload on neurons in artificially induced Alzheimer&quot;s disease

Abstract text (incl. figure legends and references) Introduction: Organotypic cultures (OTC) represent a promising method to study neurodegenerative diseases with the advantage of preservation of original architecture and connections. We established Alzheimer"s disease (AD) condition ex-vivo in the hippocampus region of the porcine brain and the human brain. We designed a simple protocol to generate the fibrils from the amyloid beta (Aβ) peptide prior to our treatments. We used this as our model to study the influence of iron accumulation in AD pathology. Iron is an essential and most abundant trace element in the human body. Iron deposition in AD is correlated with free radical damage, oxidative stress, amyloid beta plaques, etc1,2. Several studies report the increase in toxicity of AD due to iron overload3. Hence, we hypothesize that the unexplained toxicity due to the accumulation of iron in the brain could be due to its effect on the structure of the Aβ fibrils. To understand the same, we plan to mimic the iron accumulation in the brain using ferric citrate as the iron (Fe+3) donor in our ex-vivo 3D model - organotypic culture induced with Aβ. Objectives: To establish an authentic ex-vivo Alzheimer"s model in the human brain to enable the closer study of AD pathology. To establish iron overload in the brain using ferric citrate as the iron donor. To study the effect of iron overload on the structure of Aβ fibrils. Materials and methods: Human brain specimen was obtained either from the glioma surgery or from the post-autopsy routine of patients. The specimen was sectioned into 160µm thickness using vibratome. The slices were immediately shifted onto a porous membrane over neurobasal media and maintained for several days. To induce artificial AD and iron overload conditions in the culture ex-vivo, the exogenous Aβ1-42 peptides and ferric citrate were added respectively to the serum-free neurobasal media. Harvested sections were processed accordingly for immunofluorescent staining and electron microscopy imaging. The embedded tissue was sectioned into 70nm thickness and used for routine electron microscopy and negative contrasting. We plan to use ATUMtome to produce serial sections for electron tomography to study the effect of iron on the fibrils" structure in detail. Results: We compared the morphology of the samples obtained from post-mortem porcine samples with OTC samples and found that the cell integrity and the neuronal connections are viable and the cells are in an active state. The slices exposed to Aβ peptides polymerized and formed aggregates and displayed a mesh-like appearance in the extracellular matrix when observed in the electron microscope. Having established this model, we now plan to induce iron accumulation in the brain to characterize the structure of Aβ fibrils in former condition using 2D electron micrographs and perform electron tomography to understand the structural changes in the fibrils in 3D. Conclusion: Organotypic culture systems have been a long-standing tool in neuroscience. However, in neuropathological conditions like AD, most of the studies opt for transgenic mice models to study the disease. We hope that this ex-vivo model system of AD from the human brain with slight modifications serves as an excellent alternative to studying the AD pathological conditions and the factors contributing to it. (The project is funded by FWF – Austrian Science fund - P 29370).

SARS-CoV2 induced membrane remodelling and assembly revisited in high-pressure frozen and freeze substituted cells

Abstract text (incl. figure legends and references) The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative of the still ongoing COVID-19 pandemic, is a highly pathogenic β-coronavirus. During the replication of this virus, viral RNA (vRNA) synthesis takes place in double-membrane vesicles (DMVs). The newly synthesized vRNA exit the DMVs, presumably through a molecular pore (1). Virion assembly is initiated by clustering of the nucleocapsid protein complexed with genomic vRNA at the cytoplasmic face of single membrane vesicle (SMV) membranes. This induces membrane curvature and budding of virions into the SMV lumen. Important structural insights into the SARS-CoV2 replication cycle have so far been obtained by EM. Mainly chemically fixed, conventionally dehydrated samples were used for room temperature TEM or frozen hydrated samples were analyzed by cryoTEM. We analyzed remodeling of cellular structures upon SARS-CoV-2 infection in high-pressure frozen (HPF), freeze substituted (FS) and Epon embedded cells, aiming for high-quality structural preservation and membrane visibility. That is a pre-requisite for scanning transmission electron microscopy (STEM) tomography to visualize the different stages of SARS-CoV2 virion assembly in 3D. Cells were cultivated on carbon-coated sapphire discs, infected with SARS-CoV-2 for 24h, and inactivated with 4 % PFA for 1 hour at 4°C. Immediately, samples were prepared by HPF, FS (5% H2O, 0.2% OsO4 and 0.1% UA in acetone for 17h) and Epon embedding, as published in (2). For TEM, 70 nm thick sections were collected on glow-discharged grids coated with a carbon reinforced formvar film and imaged with a JEOL JEM-1400 TEM at 120 kV acceleration voltage. For STEM tomography, 700-800 nm thick sections were cut, collected on freshly glow-discharged grids with parallel bars, shortly immersed into a colloidal gold-suspension, and coated with a 5 nm carbon layer. A series of 97 STEM bright-field-images (-72° to +72° with 1.5° increment) was recorded with a JEOL JEM-2100F at 200 kV acceleration voltage. Tomograms were reconstructed using the IMOD software package (3,4). Through HPF-FS and Epon embedding, membrane profiles were well preserved. The clear membrane visibility allowed characterization of the cytopathological alterations in SARS-CoV-2 infected cells, such as the compartmentation of the cytoplasm. Further, DMVs and SMVs could be well resolved, showing the power of the HPF-FS technique. Both membranes of the DMVs appeared often tightly apposed and showed minimal deformation. Although imaging of numerous DMVs, the postulated nuclear pore connecting the DMV interior with the cytoplasm discovered by cryoTEM (1) was not detected by TEM. In one case, we identified a structure in a STEM tomogram that resembled the molecular pore. However, compared to multiple pores per DMV detected by (1), only a single pore spanned the membranes of the DMV in our study. Furthermore, a detailed view into the virus budding process at SMVs (Fig1) allowed temporal resolution of the assembly process. We show that HPF-FS allows visualization of structures related to SARS-CoV2 infection comparable to advanced cryoEM techniques, opening the door for further studies. STEM tomography provided a detailed insight into SARS-CoV-2 virion assembly. Fig1: Virtual STEM section of SARS-CoV2 virion assembly (1) Wolff et al. (2020) Science (2) Read et al. (2019) J Virol. (3) Wieland et al (2021) Histochem Cell Biol. (4) Kremer et al. (1996) J Struct Biol.

LS 3: Imaging of large volumes and plastic section tomography

The emerging key features of latency in tobamoviruses

Abstract text (incl. figure legends and references) Introduction Tobacco mosaic virus (TMV), the first plant virus imaged in electron microscopy, is the type member of the genus Tobamovirus, family Virgaviridae. Typically, tobamoviruses cause systemic infections with severe symptoms on the host leading to major yield loss. For new emerging and highly virulent members such as the tomato brown rugose fruit virus infecting tomato quarantine measures, apply. The diseased phenotype can consist of mosaic patterns, leaf mottling and/or deformations in early infection stages that develop to reduced growth and necrosis in new emerging leaves. All diseased tissues are associated with major disruptions and reorganizations in their ultrastructure. The tropical plant Hoya ssp. is susceptible to both virulent as well as latent tobamoviruses. Hoya tobamovirus 2 (HoTV-2 ) has been isolated from asymptomatic hoya plants (Gaafar et al., 2020) and serves as model system to investigate the ultrastructure of latent, asymptomatic tobamovirus infections. Objectives Analysis of serological relationship of HoTV-2 with other tobamoviruses Identification of cellular features during latent (HoTV-2) compared to virulent (TMV) tobamovirus infections. Characterization of viral interferences in mixed infections comprising HoTV-2 and TMV Analyses of cellular ultrastructure in compatible and resistant tobacco species Identification of HoTV-2 virions and viral RNA respectively in associated compartments of infected cells using IEM Material and Methods HoTV-2 was isolated from infected hoya plants using a bioassay. Therefore, healthy Nicotiana ssp. were mechanically inoculated with HoTV-2 infected hoya leaves. Negative stain and ISEM were used for virus detection in plant sap homogenates (Richert-Pöggeler et al., 2018; Gaafar et al., 2020). Embedding and generation of ultrathin sections were performed as described in (Erokhina et al., 2017). Results HoTV-2 is serological cross-reacting with Youcai mosaic virus (YoMV) antiserum (DSMZ, Germany) but not with other tested tobamoviruses. Whereas phenotypically no differences of systemic HoTV-2 infections are visible when the virus is transmitted among asterid hosts from its primary host (Apocynyceae, Gentianales) to susceptible Nicotiana ssp. (Solanaceae, Solanales), the cellular ultrastructure indicates differences in viral replication. Only in Solanales virus particles were detected. The number of virions was significantly reduced in latent (HoTV-2) compared to virulent (TMV) tobamovirus infections. YoMV antiserum can be used in IEM, but lacks specificty for efficient immunogold labeling. Conclusions Ultrastructural analysis provided evidence that the asymptomatic phenotype is associated with reduced viral replication. References Erokhina, T. N., Lazareva, E. A., Richert-Pöggeler, K. R., Sheval, E. V., Solovyev, A. G., and Morozov, S. Y. (2017). Subcellular Localization and Detection of Tobacco mosaic virus ORF6 Protein by Immunoelectron Microscopy. Biochemistry (Mosc) 82, 60–66. doi: 10.1134/S0006297917010060 Gaafar, Y., Richert-Pöggeler, K. R., Hartrick, J., Lüddecke, P., Maaß, C., Schuhmann, S., et al. (2020). A new tobamovirus infecting Hoya spp. New Dis. Rep. 42, 10. doi: 10.5197/j.2044-0588.2020.042.010 Richert-Pöggeler, K. R., Franzke, K., Hipp, K., and Kleespies, R. G. (2018). Electron Microscopy Methods for Virus Diagnosis and High Resolution Analysis of Viruses. Front Microbiol 9, 3255. doi: 10.3389/fmicb.2018.03255

LS 6
Pathology, pathogens and diagnostics

Appointment

Session chairs

Description

Programme

LS 6Pathology, pathogens and diagnostics

Appointment

Session chairs

Description

Programme

LS 6
Pathology, pathogens and diagnostics