Microscopy Conference 2023

Crosshair: semiautomated targeting for electron microscopy

Abstract text (incl. figure legends and references) Volume EM brings new insights on cells and tissues ultrastructure in 3D. However the techniques allowing volume EM are low throughput. Increasing the throughput can be achieved, for instance, via targeting approaches that enable replicates and multiplication of observations. Here we are presenting a newly developed workflow to target structures of interest within a resin-embedded block. The block has to be first trimmed and subsequently imaged by means of X-rays with a microCT machine. The X-ray tomogram generated is a 3D map, from which we can calculate the location of a specific structures of interest using the Fiji plugin called Crosshair (https://github.com/K-Meech/crosshair). The targeting of the structures of interest is then achieved by the use of a motorized ultramicrotome designed by the EMBL electronic and mechanical workshops that allows precise angular movements. An example of the application of this workflow to find Plasmodium falciparum-infected red blood cells in bioengineered 3D microvessels (that mimic the brain blood barrier) is also shown. Reference: Kimberly Meechan, Wei Guan, Alfons Riedinger, Vera Stankova, Azumi Yoshimura, Rosa Pipitone, Arthur Milberger, Helmuth Schaar, Inés Romero-Brey, Rachel Templin, Christopher J Peddie, Nicole L Schieber, Martin L Jones, Lucy Collinson and Yannick Schwab. Crosshair, semi-automated targeting for electron microscopy with a motorised ultramicrotome. https://doi.org/10.1101/2022.06.02.491151. Accepted to be published in eLife.

LSLB: Late breaking abstracts

Poster

A streamlined and ice contamination free cryo-electron tomography workflow to improve throughput and data quality

Abstract text (incl. figure legends and references) Cryo-electron tomography (cryo-ET) is a powerful technique that allows researchers to obtain 3D structures at nano-meter resolution in their near-native state. In recent years the technique has gained popularity and a large number of complexes have been resolved. This has improved our understanding of the molecular foundation of viral infections1, skeletal muscles2 and Parkinson&quot;s disease3. To obtain such a high resolution 3D structure, 1000-2000 copies of a protein complex need to be averaged in a process called subtomogram averaging4,5. This means that a high number of high quality tomograms are needed. Unfortunately the workflow is very error-prone and this has limited the data output6. To acquire high quality tomograms there are two main challenges: keeping the sample ice contamination-free and targeting the region of interest (ROI). At Delmic we developed two workflow solutions called CERES and METEOR to improve the entire cryo-ET sample preparation workflow. The CERES Ice Defence System is based on the tools developed by Tacke et al7 and consists of multiple innovative tools that are tailored to minimize ice contamination during sample handling (CERES Clean Station), transfer (CERES Vitri-Lock) and FIB milling (CERES Ice Shield). METEOR is an integrated fluorescence light microscope (FLM) that greatly enhances the ROI targeting inside the cryo-FIB/SEM while reducing the number of handling steps required. We show that these innovative tools can lead to improved cryo-ET data quality and throughput. Klein, S. et al. SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography. Nature Communications 2020 11:1 11, 1–10 (2020). Wang, Z. et al. The molecular basis for sarcomere organization in vertebrate skeletal muscle. Cell 184, 2135-2150.e13 (2021). Watanabe, R. et al. The In Situ Structure of Parkinson&quot;s Disease-Linked LRRK2. Cell 182, 1508-1518.e16 (2020). Chen, M. et al. A complete data processing workflow for cryo-ET and subtomogram averaging. Nature Methods 2019 16:11 16, 1161–1168 (2019). Scaramuzza, S. &amp; Castaño-Díez, D. Step-by-step guide to efficient subtomogram averaging of virus-like particles with Dynamo. PLoS Biol 19, e3001318 (2021). Lau, K., Jonker, C., Liu, J. &amp; Smeets, M. The Undesirable Effects and Impacts of Ice Contamination Experienced in the Cryo-Electron Tomography Workflow and Available Solutions. Micros Today 30, 30–35 (2022). Tacke, S. et al. A streamlined workflow for automated cryo focused ion beam milling. J Struct Biol 213, 107743 (2021).

LS 1: High-resolution cryo-EM

Biochemical and structural studies of NAPDH oxidase

Abstract text (incl. figure legends and references) Reactive oxygen species (ROS) are crucial for many cellular processes, from cell to cell signaling to redox regulation. In human cells, the NAPDH oxidase (NOX) membrane proteins are a major source of ROS and play a critical role in the innate immune system and redox regulation processes. Yet, there are large gaps in knowledge on crucial aspects of the function of the NOX proteins, especially with regards to electron transfer pathway and reaction mechanism. Structural and in-depth biochemical studies of human NOX proteins have proven difficult due to complications in heterologous expression and purification of active, stable complexes. Therefore, we have used a bacterial NOX homologue from Streptococcus pneumonia (spNOX) to gain insight into the structure and mechanism of NOX proteins. SpNOX is a small membrane of ~47 kDa and can be heterologously expressed in its active state, as confirmed by superoxide dismutase-sensitive cytochrome c reduction. The protein is stable at high concentrations and can be prepared for single particle analysis cryo-EM. Preliminary studies have allowed for the resolving of the low-resolution structure that is consistent with the structure predicted using AlphaFold2.

Prof. Dr.

SBFI-SEM and 3D reconstruction of polymer particles in a polymer matrix

Abstract text (incl. figure legends and references) The aim of the project was to perform a 3-dimensional reconstruction of isotropic and anisotropic polymer particles embedded in polymer films using volume SEM (Serial Blockface Imaging, SBFI). For this purpose, isotropic polystyrene (PS) particles with diameter of 1.4 µm were incorporated in PVA films from aqueous solution. To gain anisotropic ellipsoidal particles, the films were stretched unidirectional at different strain ratios above the glass transition point of PS. [1, 2]. Optimization of the sample preparation method for SBFI SEM included contrasting the PVA films with cesium chloride, and addition of 344 nm silica particles during film production as alignment support for the reconstruction. The particle-film samples were embedded in conductive silver epoxy resin, trimmed and cut using SBFI-SEM (Apreo VS, ThermoScientific/FEI) at 40 nm Z-axis and a total volume thickness of at least 12 µm (min. 300 slices) in low-vac mode to reduce charging artifacts. Image stacks were reconstructed using AMIRA software, utilizing the fiducial silica particles for optimized alignment of the images. Morphometric analysis of the PS particles took place after segmentation, following an optimized image data processing routine. Shrinking of the PVA films due to evaporation of residual water content despite equilibration in low vacuum led to deformation artifacts of the reconstructed PS particles. These could be corrected with an approach based on the geometric data of the silica fiducials. The morphological parameters of thus corrected 3D reconstructed PS particles of all stretching conditions correlated well with measurements of the particles isolated from the films in standard SEM. In summary, we could establish a sufficiently accurate 3D reconstruction and analysis of polymer particle/polymer film-systems using SBFI-REM. References: Ho C. C., K. A., Odell J. A., Ottewill R. H., Preparation of monodisperse ellipsoidal polystyrene particle. Colloid and Polymer Science 1993, 271, 469-479. Champion, J. A.; Katare, Y. K.; Mitragotri, S., Making polymeric micro- and nanoparticles of complex shapes. Proc Natl Acad Sci U S A 2007, 104 (29), 11901-11904.

LS 7: Advances in sample preparation

Accelerate and deepen our insights into infectious diseases through large-scale 2D and volume electron microscopy combined with artificial intelligence

Abstract text (incl. figure legends and references) The emergence of new viral variants, which can cause human diseases and pandemics, necessitates a quicker and deeper understanding of the pathogenesis and disease manifestations to develop effective treatments and preventative measures. Viral particles are in the nm range thus their observation requires the nm-resolutions of electron microscopes (EM). EM has been paramount in diagnosing Covid-19 [1] and gaining insights into its disease-causing mechanisms [2]. However, the time-consuming nature and manual operation requirement of conventional EM limit the data throughput, limiting the statistics and understanding over a timespan. Multibeam scanning electron microscope (SEM) technology such as FAST-EM (Delmic B.V., Netherlands) can overcome these bottlenecks. In this work, we apply FAST-EM whole-section imaging to human airway epithelial (HAE) cells 72 hours post-SARS-CoV-2 infection to demonstrate how more profound insights into infectious diseases can be gained faster. Using FAST-EM, we imaged a whole section of a HAE cell layer spanning an area of 270 x 270 µm2 at a 4 nm pixelsize and at 10 µs/px within only 35 minutes. The same area would take 3.5 hours to image using a single beam SEM with the same pixel size and at 2 µs/px. The EM images revealed the density and the locations of virus-like particles in the HAE cells. At 4 nm resolution hallmarks of SARS-CoV-2 such as electron-dense particles and multilamellar vesicles could be observed (Figure 1). To perform an accurate examination of the sub-cellular changes caused by different SARS-CoV-2 variants, we analyse the density of the virus-like particles as well as the features that span multiple sections (e.g. multilamellar vesicles, mitochondria, cilia) by performing array tomography and 3D data reconstruction. We employ the available auto-segmentation model empanada MitoNet [3] (Figure 2) and train new segmentation models to extract the rich information present in the FAST-EM data to facilitate downstream quantitative analyses. Our work shows the FAST-EM and artificial intelligence approach as a new and effective way of gaining insights into infectious diseases. Figure 1 Whole section FAST-EM image of HAE cell layer infected with SARS-CoV-2 enables the study of morphological changes with machine learning. (a) The morphological changes can be studied in the context of the cell layers, 270 x 45 µm², by allowing the identification of different cell types. (b dashed squares) On the cellular level, the impact of the infection is visible once zoomed in. (c, solid squares) The individual mitochondria in the cell layer are identified by employing MitoNet, allowing for quantification of morphological changes in the mitochondria. The scale bar is 20 µm (a) and 5 µm (b-c).

IMLB: Late breaking abstracts

LS 3: Imaging of large volumes and plastic section tomography

Zika virus replication in glioblastoma cells – electron microscopic tomography reveals 3D arrangement of endoplasmic reticulum, replication organelles, and viral ribonucleoproteins

Abstract text (incl. figure legends and references) The ultrastructure of two patient-derived glioblastoma cell lines infected with Zika virus (ZIKV) was investigated using scanning transmission electron microscopy (STEM) tomography of high-pressure frozen, freeze-substituted samples. Formation of viral factories containing largely deranged endoplasmic reticulum was observed. In one cell line, newly formed virions and replication organelles (ROs) consisting of luminal vesicles within the ER were found in close proximity to each other. Reconstruction of STEM tomograms showed many about 100-nm-long filaments in the vicinity of ROs, possibly representing viral ribonucleoprotein complexes (RNPs) consisting of the RNA genome and N protein oligomers of ZIKV. The second cell line showed a different morphology upon ZIKV infection, where the virions were found in ER cisternae at a large distance from replication organelles, possibly indicating a dysfunctional transport of virions or different replication kinetics. This study demonstrates the advantage of EM tomography in the investigation of structures like RNPs, which would be hard to identify in conventional two dimensional TEM. STEM tomography in particular is capable of showing the three dimensional arrangement of multiple larger structures, like the ROs, virions and RNPs encountered in viral factories. The lack of image forming lenses in STEM allows for thick specimen of up to 1 μm to be analyzed. In TEM tomgraphy, sample thickness is limited by inelastic scattering of the beam inside the sample, which leads to chromatic aberration in the image forming lenses. Since a STEM does not use image forming lenses, chromatic aberration does not occur in image acquisition. Reference: Wieland, J., Frey, S., Rupp, U. et al. Zika virus replication in glioblastoma cells: electron microscopic tomography shows 3D arrangement of endoplasmic reticulum, replication organelles, and viral ribonucleoproteins. Histochem Cell Biol 156, 527–538 (2021). https://doi.org/10.1007/s00418-021-02028-2

Ultrastructural investigation of the C-terminal region of human cytomegalovirus tegument protein UL71 and its role in secondary envelopment

Abstract text (incl. figure legends and references) Human cytomegalovirus (HCMV) is an important human pathogen which can cause serious diseases in immunocompromised patients and complications during pregnancy (Griffith et al., 2015). However, the molecular mechanisms underlying HCMV virion morphogenesis are still not well understood. In this work, an important step of HCMV morphogenesis en route to infectious virions, referred to as secondary envelopment, was investigated by electron microscopy (EM). Understanding the underlying mechanism could lead to the identification of new targets for antiviral therapy. pUL71 is a conserved herpesvirus tegument protein with 361 amino acids and plays an important role in secondary envelopment (Schauflinger et al., 2011). Several functional motifs, mainly localized in the N-terminus of pUL71, have been characterized in detail (Read et al., 2019; Fischer 2012; Metzner, 2022), but the role of the C-terminus of pUL71 is not well studied. The role of the C-terminus of pUL71 was investigated by generating and characterizing a mutant virus (UL71insHA-299) expressing a truncated version lacking the last 60 amino acids and fused to an HA epitope. A first phenotypic characterization of UL71insHA-299 after infection of human fibroblasts was performed by immunofluorescence microscopy. In contrast to wild-type virus infected cells, virus particles accumulated at the periphery of the cytoplasmic viral assembly complex at large circular structures in UL71insHA-299 infected cells. For direct analysis of secondary envelopment and these capsid accumulations and circular structures, human fibroblasts were again infected and high-pressure frozen at five days post infection, freeze substituted and embedded in epoxy resin. Thereafter the cells were ultrathin sectioned, analyzed by transmission electron microscopy (TEM) and compared with wild-type infected cells. The ultrastructural phenotype of the UL71insHA-299 mutant was very similar to that of a virus that is unable to express pUL71 (Schauflinger et al., 2011). Thus, our data show that the C-terminus of HCMV pUL71 contains functions that are necessary for sufficient virion morphogenesis and secondary envelopment. Griffiths P, Baraniak I, Reeves M. The pathogenesis of human cytomegalovirus. J Pathol. 2015 Jan;235(2):288-97. doi: 10.1002/path.4437. PMID: 25205255. Schauflinger M, Fischer D, Schreiber A, Chevillotte M, Walther P, Mertens T, von Einem J. The tegument protein UL71 of human cytomegalovirus is involved in late envelopment and affects multivesicular bodies. J Virol. 2011 Apr;85(8):3821-32. doi: 10.1128/JVI.01540-10. Epub 2011 Feb 2. PMID: 21289123; PMCID: PMC3126135. Fischer D, 2012. Dissecting Functional Motifs of the Human Cytomegalovirus Tegument Protein pUL71. Doctoral thesis, Ulm University. Read C, Schauflinger M, Nikolaenko D, Walther P, von Einem J. Regulation of Human Cytomegalovirus Secondary Envelopment by a C-Terminal Tetralysine Motif in pUL71. J Virol. 2019 Jun 14;93(13):e02244-18. doi: 10.1128/JVI.02244-18. PMID: 30996102; PMCID: PMC6580969. Annika Metzner. Role of the non-conserved C-terminus of pUL71 for human cytomegalovirus infection. 2022, Bachelor thesis, Ulm University, unpublished.

ANTEMA: a neural network-based automated nanoparticle transmission electron micrograph analysis

Abstract text (incl. figure legends and references) High-resolution transmission electron microscopy (HRTEM) is an important tool for nanoparticle analysis. This is because HRTEM images show information about the size and shape of the particles as well as their inner structure. These are all parameters that influence the physical and chemical properties of the particle and are therefore of importance for all kinds of nanoparticle applications. With high frame rates and increasing capabilities of transmission electron microscopes, large amounts data are generated. However, analysing these micrographs is often done manually which is a tedious and time-consuming process. This is especially important for in-situ TEM experiments where hundreds or thousands of frames must be analysed. As a result, only a fraction of the available data is usually quantitively analysed. To facilitate faster and more in-depth analysis of HRTEM images we have developed an automated image processing module using machine learning techniques. An important step in realizing an automated analysis of the nanoparticles in HRTEM images is to separate the particles from the noisy amorphous background. This is realized by a neural network which was trained on labelled HRTEM images by a supervised learning approach. The segmentation maps that are generated by the network are further processed to exclude particles that cross the image boundary and identify and split overlapping particles. From the resulting binary maps that include all particles in the image, shape and size-related parameters like circularity, equivalent diameter, and Feret diameter are extracted. These steps are all integrated in an automated routine which is able to analyse an HRTEM image within a few seconds. This permits the automated analysis of large quantities of data and gives more insight into nanoparticle structures compared to a manual analysis. It also avoids an operator-induced bias during manual image analysis.

Cryo-EM structure of the E. coli formate hydrogenlyase complex

Abstract text (incl. figure legends and references) The prototypical hydrogen producing enzyme, the membrane-bound formate hydrogenlyase (FHL) complex from Escherichia coli, catalyzes the conversion of formate to CO2 and H2 under anaerobic fermentative conditions. The enzyme is long known to share sequence homology with respiratory complex I, but an energy conserving character of FHL remains elusive. We aim to get a better understanding of the FHL complex and the role it plays in bacterial bioenergetics by combining high-resolution electron cryo-microscopy (cryo-EM) with complementary biophysical methods. Our cryo-EM structures reached resolutions of up to 2.6 Å and revealed important differences and similarities between FHL and complex I. We analyzed possible substrate proton pathways to the [NiFe] active site, which may play an important role in the mechanism of the enzyme. Furthermore, we identified an unpredicted metal-ion, which could play a regulatory role in binding the loosely attached formate dehydrogenase component. Since FHL is an ancestor of respiratory complex I understanding its function on a molecular level sheds light on the evolution of energy conservation from early to modern life.

Prof. Dr. rer. nat. habil.

Site-specific and advanced preparation of ultra-thin TEM samples of nanostructured materials

Abstract text (incl. figure legends and references) Advanced microanalytical investigation techniques allow to characterize the structure as well as the chemical composition of materials down to atomic scale. Especially, nanostructures with their confined length scales require a well-defined and extremely precise sample preparation to elucidate their unique characteristics [1]. Newest dual focused ion beam scanning electron microscopy (FIB-SEM) enables such indispensable site-specific sample preparation with high accuracy and precision on the nm-scale. However, beam sensitive materials and nonconductive substrates increase the challenges of an accurate preparation. In this work, we illustrate the possibilities and challenges of targeted preparation of nanostructures using the latest FIB-SEM techniques. A selection of investigated nanostructures is presented in Fig. 1. Each unique nanostructure is analyzed precisely in SEM (first column) to identify the desired TEM lamella orientation with respect to the morphology and arrangement of the nanostructure as well as its orientation towards the substrate. The first sample system are gold bipyramids (Fig. 1a) which exhibit a five-fold-twinned crystal structure with interesting optical properties [2,3,4]. The maximum extent of about ≈65nm limits significantly the position of the lamella and a high accuracy during the preparation is needed. The desired cross section must be perfectly in the center to study the present structure on the atomic scale. The final TEM studies reveal the fivefold twinned atomic structure. The lamella delimits a thin region around the center of the bipyramid, with their surfaces parallel to the central base. In this way, the diameter at the central base can be accurately determined, which shows a rounded geometry compared to the optimal structure. Fig. 1b shows AuxNi1-x nanoparticles on a α-Al2O3 substrate. In this system, the interface between the ceramic substrate and the metal is of interest due to great scientific and technological importance, with applications in electro-ceramic devices, structural composites and catalysis [5]. Therefore, the nanoparticle orientation with respect to that of the underlying substrate is identified by electron backscatter diffraction (EBSD) measurements. Afterwards, the cross section is prepared in such a way that the center of the particle is located exactly in the center of the lamella (Fig. 1b). Besides the extreme precision, the non-conductive substrate is very challenging. Additionally, advanced STEM methods are needed to image the interface between high and low atomic number materials [5]. For this purpose, lamellae with a thickness of less than 40nm and a very good surface quality are required. Besides one-dimensional structures, also single nanowires (NW) and the corresponding networks are analyzed. The electrical properties of such networks or NW in contact can be strongly improved by heat treatment to weld the corresponding contact points [6]. The presented example (Fig. 1c) shows two identified and select Ag NW after heat treatment. To investigate the welding zone between both wires, the corresponding cross section has to be precisely prepared. The achieved lamella reveals that the intersecting NW unite with each other and creating a constant internal crystalline microstructure. [1] 10.1017/S1431927613013780 [2] 10.1021/ac900276n [3] 10.1021/jacs.6b12143 [4] U. Frank et al. ACS Nano, under review [5] 10.1038/s41598-018-20377-2 [6] 10.1002/aelm.202100787

IM 3: SEM and FIB developments

Ultrastructural rearrangement of the nucleus in situ during osmotic stress

Abstract text (incl. figure legends and references) Osmotic stress causes flux of solvent into or out of cells as a result of changing environmental conditions. On second to minute time scale cells respond by adjusting their volume. How cells cope with the mechanical impact and how this process affects cellular ultrastructure remains underexplored. We combine fluorescence microscopy and electron cryo-tomography to study the structural rearrangements to the cytosolic and nuclear architecture in Dictyostelium discoideum cells during osmotic stress. These cells undergo volume changes that result in changes to the cellular protein concentration, affecting the visibility of protein complexes in the tomogram volume. We visualize specific changes to nuclear architecture, the organization of regular nuclear filaments upon nuclear shrinkage, as well as changes to the nuclear envelope membrane resulting from osmotic pressure and mechanical forces. Using subtomogram averaging, we obtain an in situ structure of the nuclear pore complex of D. discoideum and build a corresponding model from AlphaFold predicted structures of its components. D. discoideum nuclear pores have a unique structure compared to other organisms and are subject to dilation and constriction movements under different osmotic stress conditions.

Establishing pre-embedding immunogold labelling for the detection of pUL71 in herpesvirus infected cells

Abstract text (incl. figure legends and references) Morphogenesis of human cytomegalovirus (HCMV) is still only partially understood. Previous studies have shown that the HCMV tegument protein pUL71 plays an important role in viral replication and morphogenesis (1). To better understand the function of the protein, pUL71 should be localized in infected cells by pre-embedding immunogold labelling and transmission electron microscopy (TEM). In recent years, EM preparation methods have been published that combine good structural preservation by high-pressure freezing and freeze substitution with subsequent rehydration allowing immunolabeling before resin embedding (e.g., 2,3). We describe the establishment of this method in our laboratory and its adaptation for HCMV-infected fibroblasts. The major aim is to achieve the best possible ultrastructural preservation while providing sufficient permeabilization for efficient pre-embedding immunogold labeling. After high-pressure freezing of firoblasts infected with HCMV expressing an HA-tagged variant of pUL71, we tested freeze-substitution (FS) media with different concentrations of glutaraldehyde, osmium tetroxide and uranyl acetate, as well as of the detergents TritonX100 and Digitonin. We then evaluated the ultrastructural preservation of these samples by TEM. Labelling efficiency was then first tested by fluorescence microscopy experiments. Rehydration and permeabilization approaches that provide good labelling efficiency will then be adapted for pre-embedding immunogold labeling of pUL71-HA with anti-HA IgG as primary and Nanogold Fab fragments as secondary antibody. The most promising tradeoff between ultrastructural preservation, sufficient heavy metal contrast, and antibody penetration was observed with a FS solution containing 0.2% osmium tetroxide, 0.1% uranyl acetate and 5% water in acetone. Fluorescence microscopy showed specific antibody labelling of pUL71-HA after exchanging the FS medium at -20 °C for a medium containing 0.2% glutaraldehyde instead of osmium tetroxide, while it made no difference whether permeabilization was performed with TritonX or Digitonin. Immunolabeling after FS but before embedding is a promising but complex method, as the fixatives as well as the acetone can interact with the antigens and mask the epitopes. Specifically, the proteolytic effect of osmium tetroxide has a strong effect on antigenicity. However, since this effect is strongly temperature dependent, removal of osmium tetroxide at negative temperatures should have less effect on antigenicity (4,5). The identification of a suitable anti-HA antibody in fluorescence microscopy experiments by using optimized fixation methods enables us to perform immunogold labelling for detection of the antibody by TEM. 1. Schauflinger, M. et al. 2013. Analysis of human cytomegalovirus secondary envelopment by advanced electron microscopy. Cellular Microbiology. 2. Hess, M.W. et al., 2018. Combining high-pressure freezing with pre-embedding immunogold electron microscopy and tomography. Traffic. 3. Tsang, T.K. et al., 2018. High-quality ultrastructural preservation using cryofixation for 3D electron microscopy of genetically labeled tissues. eLife. 4. Flechsler, J. et al., 2020. 2D and 3D immunogold localization on (epoxy) ultrathin sections with and without osmium tetroxide. Microscopy Research and Technique. 5. Koga, D. et al., 2020. Optimizing the reaction temperature to facilitate an efficient osmium maceration procedure. Biomed. Res. 41, 161–168.

Acid sphingomyelinase deletion mutant C. elegans show formation of electron dense multilamellar bodies, accumulation of various sphingolipid species and increased resistance to Staphylococcus aureus infections

Abstract text (incl. figure legends and references) Acid sphingomyelinase (ASM; SMPD1 gene in humans) is a lysosomal enzyme that catalyses the conversion of sphingomyelin to ceramide. Harmful mutations in this gene lead to the lipid storage disease Niemann Pick Disease Types A or B, which are characterized by abnormal accumulation of sphingomyelin and cholesterol in lysosomes of various tissues. Ultrastructural analysis of patient tissues or cell lines shows the lipids arranged as multilamellar structures within the lysosome. Through its activity, ASM is contributing to lipid homeostasis. Stresses like pathogens or oxidative stress can induce the relocation of ASM from the lysosome to the plasma membrane, where it produces ceramide and thus leads to a rearrangement of ceramide-enriched domains. This process is important in many signalling cascades of the immune system. C. elegans has three ASM isoforms, ASM-1, ASM-2 and ASM-3, that are all homologous to the human ASM. Here, we used deletion mutants of the three asm genes to characterize ASM in the C. elegans sphingolipidome context and investigate their role in S. aureus infections. C. elegans is susceptible to the human pathogen S. aureus and a well-established model to study its pathogenicity. We fed single, double and triple asm mutant worms with S. aureus and quantified their survival. None of the control worms, but 40% of the asm-1 and asm-3 single or double mutants survived after 72 hours of feeding on S. aureus. asm-2 mutation did not protect the worms against S. aureus infection, and even nullified the protective effect of asm-1 and asm-3 in double or triple mutants, implying divergent roles of ASM-2 vs. ASM-1 and ASM-3 in the pathogenicity of S. aureus. Ultrastructural analysis of high-pressure frozen specimens revealed excessive lipid accumulation in asm-1 and asm-3 mutants, probably in lysosomes, which resemble the multilamellar bodies described in human Niemann-Pick patients. They were rarely observed in controls and asm-2. To confirm that this phenotype is due to a disturbed sphingolipid metabolism, we analysed the sphingolipidome of C. elegans using liquid chromatography tandem-mass spectrometry. We found that all ASMs have an impact on the levels of various sphingomyelin and ceramide species. The changes induced by asm-2 and asm-3 are very similar to each other, whereas asm-1 seems to have different roles. Our findings show that host ASM activities shape the outcome of experimental infection of C. elegans with S. aureus and that all three C. elegans ASMs have an impact on the sphingolipid homeostasis. In the next step, we want to characterize and localize the lipid species contained in the electron dense multilamellar bodies by using a combination of correlative light and electron microscopy and liquid chromatography tandem-mass spectrometry with labelled C. elegans specific iso-branched sphingolipids.

Prof.

High resolution imaging of surface dynamics

Abstract text (incl. figure legends and references) ONEM is a new hybrid microscopy technique in which an interface is probed by light in a non-invasive manner, and the structural information is read-out at high spatial resolution using electron optics. To achieve this, an ultra-thin photocathode layer is inserted in the near-field region right after the sample [2], and emitted photo-electrons are collected by the objective lens of an aberration-corrected low-energy electron microscope (LEEM) [3]. The potential applications for ONEM is in life sciences and electrochemical studies. Electrochemistry studies the processes that occur on the interface of electrodes and electrolytes due to the flow of electrons or ions. This field of study has found widespread applications in nanomanufacturing, developing batteries, hydrogen-based fuels, biosensors and etc [1]. Therefore, measuring the local changes in the formed structures over the whole area of the electrode-electrolyte interface is of great importance. Despite the significant advances in the development of optical super-resolution and electron microscopes, real-time, damage and label free imaging of dynamics in nm-resolution is yet not achieved. Here, we introduce optical near-field electron microscopy (ONEM) as a noble technique to study electrochemical processes in real time with a few nanometers of spatial resolution. ONEM will be benchmarked with other state of art techniques such as interferometric scattering microscopy (iSCAT) and liquid cell electron microscopy (LC-EM). ONEM is an ongoing project in collaboration with the Leiden University, Czech academy of sciences, and the Univerity of Vienna. References: [1] Beker, Anne France, et al. "In situ electrochemistry inside a TEM with controlled mass transport." Nanoscale 12.43 (2020): 22192-22201. [2] Marchand, Raphaël, et al. "Optical near-field electron microscopy." Physical Review Applied 16.1 (2021): 014008. [3] Geelen, D., et al. "Nonuniversal transverse electron mean free path through few-layer graphene." Physical review letters 123.8 (2019): 086802.

MSLB: Late breaking abstracts

A/Prof

Structural strategies for maximizing bacteriophage capsid capacity

Abstract text (incl. figure legends and references) Tailed bacteriophages use copies of a major capsid protein (MCP) with a common fold to build capsids capable to harbour dsDNA genomes spanning from tens to hundreds of kilobase pairs. Larger capsids are usually constructed by a higher number of MCPs, resulting in a larger triangulation number or in oblongate capsids. However, the continuous evolutionary pressure imposes phage capsids to adapt to various packaging load sizes. For instance, a relatively modest increase in the genome size cannot justify the transition to a novel capsid model, constructed from more MCPs. Instead it calls for strategies that increase the size of the capsid, while maintaining the same triangulation number. Using cryo-electron microscopy (cryoEM), we solved the architecture of the phiTE phage in several intermediate conformations. The full capsid resolved at 3.4 Å helped us to decipher the structural mechanism by which phiTE capsid is able to accommodate a genome up to 40% larger than that of phages with the same capsid geometry (T=13). The strategy combines three factors: the increase of capsomer surface without an increase in MCP molecular weight, the relative sliding between individual capsomers, and the increase of local capsomer curvature. Additionally, our cryoEM analysis offers a series of important insights into understanding the general principles of phage architecture. First, we describe two decoration proteins with distinct roles in phage stability and demonstrate that for decoration proteins belonging to the same class, a structure base classification does not mirror the classification based on amino acid alignment. Secondly, we follow the MCP conformations found in the spent empty capsid that displays an expanded geometry. Thirdly, we describe the components of the phiTE tail in the extended and contracted conformations. Finally, we show how the phiTE base plate employs a novel type of conformational change during genome ejection dictated by the reorientation of the tail fibres.

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