Imaging techniques for studying virus–cell interactions: a review of current methods and challenges

Advances in electron microscopy: A qualitative view of instrumentation development for macromolecular imaging and tomography

Structural features of the salivary gland hypertrophy virus of the tsetse fly revealed by cryo-electron microscopy and tomography

In the recent years, cryo-electron microscopy (cryo-EM) single-particle analysis (SPA) has greatly benefited from a multitude of technological advances cumulating in breaking the atomic-resolution barrier for biological macromolecules. Since then, structural insights by cryo-EM have led to driving rational design of new vaccines and therapeutics due to high throughput and high-resolution screening and data collection technologies. Additionally, cryo-electron tomography (cryo-ET) has become an indispensable tool for structural cell biology. In this presentation we elaborate on the latest Thermo Fisher Scientific innovations for 300kV cryo electron microscopy for SPA and cryo-ET to advance more workflow efficiency and performance. Our ongoing mission is to further improve the connectivity between the cryo-FIB and cryo-TEM for an even more streamlined tomography workflow that is safer, faster and cleaner. Data acquisition and processing can be a significant cryo-ET bottleneck. Our integrated solution, combining Thermo Scientific Tomography 5 and Tomo Live, automates pre-acquisition tasks, enhances productivity with predictive tracking and enables rapid quality assessment. Robust and optimized motion correction helps produce crisp tomograms and image quality needed for sub-tomogram averaging. Results are displayed with new quality metrics on a web platform, making the workflow user-friendly and facilitating structural biology advancements.

Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy or tomography, fluorescence microscopy must be performed while maintaining the specimen vitrified at liquid-nitrogen temperatures and in a dry environment during imaging and transfer. Here we present instrumentation, software and an experimental workflow that improves the ease of use, throughput and performance of correlated cryo-fluorescence and cryo-electron microscopy. The new cryo-stage incorporates a specially modified high-numerical aperture objective lens and provides a stable and clean imaging environment. It is combined with a transfer shuttle for contamination-free loading of the specimen. Optimized microscope control software allows automated acquisition of the entire specimen area by cryo-fluorescence microscopy. The software also facilitates direct transfer of the fluorescence image and associated coordinates to the cryo-electron microscope for subsequent fluorescence-guided automated imaging. Here we describe these technological developments and present a detailed workflow, which we applied for automated cryo-electron microscopy and tomography of various specimens.

Inorganic storage granules have long been recognized in bacterial and eukaryotic cells but were only recently identified in archaeal cells. Here, we report the cellular organization and chemical compositions of storage granules in the Euryarchaeon, Archaeoglobus fulgidus strain VC16, a hyperthermophilic, anaerobic, and sulfate-reducing microorganism. Dense granules were apparent in A. fulgidus cells imaged by cryo electron microscopy (cryoEM) but not so by negative stain electron microscopy. Cryo electron tomography (cryoET) revealed that each cell contains one to several dense granules located near the cell membrane. Energy dispersive X-ray (EDX) spectroscopy and scanning transmission electron microscopy (STEM) show that, surprisingly, each cell contains not just one but often two types of granules with different elemental compositions. One type, named iron sulfide body (ISB), is composed mainly of the elements iron and sulfur plus copper; and the other one, called polyphosphate body (PPB), is composed of phosphorus and oxygen plus magnesium, calcium, and aluminum. PPBs are likely used for energy storage and/or metal sequestration/detoxification. ISBs could result from the reduction of sulfate to sulfide via anaerobic energy harvesting pathways and may be associated with energy and/or metal storage or detoxification. The exceptional ability of these archaeal cells to sequester different elements may have novel bioengineering applications.

New technologies are ready to revolutionize glomerular imaging and significantly improve or replace immunofluorescence and electron microscopy, which have driven research and diagnosis of glomerular diseases for over 50 years. Advanced forms of transmission and scanning electron microscopy have revealed the detailed spatial relationships of the glomerular basement membrane, podocytes, and endothelial cells. These may be overshadowed by super resolution microscopy (SRM), which combines the advantages of immunofluorescence and electron microscopy, offers high resolution identification of specific molecules, and images large, physiologically relevant volumes of the glomerulus. Rapidity, ease of use and low cost with some types of SRM make them potentially suitable for routine diagnosis. SRM visualizes structures below the classical diffraction limit of conventional light microscopy by adding a time variable to either the illumination of the specimen, or to the fluorescence signal emitted by it. Ensemble techniques vary illumination and include Structured Illumination Microscopy (SIM) and Stimulation Emission Depletion Microscopy (STED). Single molecule localization techniques vary the light emission by fluorescence labels in the specimen, and include Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM). Technologies such as expansion microscopy and genetic labeling can also create effective super resolution imaging by non-optical, specialized preparation techniques. All technologies require dark field fluorescence and some require computer image analysis and reconstruction. Replicating successful application in other areas of biology, SIM, STED, and STORM have visualized normal and nephrotic disease podocytes, and have confirmed their appearances to be similar to those seen by electron microscopy, but with added new information on cell configuration and protein localization. STORM has also localized podocyte cytoskeleton and adhesion proteins, and glomerular basement membrane proteins at a resolution never before possible. These pioneering efforts show the promise of super resolution microscopy, and lay the groundwork for future study and new diagnostic tools for glomerular diseases.

As cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) continue to advance, the ability to visualize cellular and organismal structures with unprecedented clarity is redefining the landscape of structural biology. Breakthroughs in imaging technology, sample preparation and image processing now enable the detailed elucidation of cellular architecture, macromolecular organization, and dynamic biological processes at sub-nanometer resolution. Recent methodological advances have propelled the field to new frontiers, facilitating the investigation of complex biological questions across scales-from macromolecular complexes to organism-wide structural insights. This review explores rapidly emerging trends, highlights key innovations that are pushing the boundaries of in situ structural biology, and addresses persistent challenges in expanding the applicability of cryo-EM and cryo-ET across diverse biological systems.

Ebola virus is a highly pathogenic filovirus causing severe hemorrhagic fever with high mortality rates. It assembles heterogenous, filamentous, enveloped virus particles containing a negative-sense, single-stranded RNA genome packaged within a helical nucleocapsid (NC). We have used cryo-electron microscopy and tomography to visualize Ebola virus particles, as well as Ebola virus-like particles, in three dimensions in a near-native state. The NC within the virion forms a left-handed helix with an inner nucleoprotein layer decorated with protruding arms composed of VP24 and VP35. A comparison with the closely related Marburg virus shows that the N-terminal region of nucleoprotein defines the inner diameter of the Ebola virus NC, whereas the RNA genome defines its length. Binding of the nucleoprotein to RNA can assemble a loosely coiled NC-like structure; the loose coil can be condensed by binding of the viral matrix protein VP40 to the C terminus of the nucleoprotein, and rigidified by binding of VP24 and VP35 to alternate copies of the nucleoprotein. Four proteins (NP, VP24, VP35, and VP40) are necessary and sufficient to mediate assembly of an NC with structure, symmetry, variability, and flexibility indistinguishable from that in Ebola virus particles released from infected cells. Together these data provide a structural and architectural description of Ebola virus and define the roles of viral proteins in its structure and assembly.

Recent advances in microscopy techniques enabled nanoscale discoveries in biology. In particular, electron microscopy reveals important cellular structures with nanometer resolution, yet it is hard, and sometimes impossible to resolve specific protein localizations. Super-resolution fluorescence microscopy techniques developed over the recent years allow for protein-specific localization with ~ 20 nm precision are overcoming this limitation, yet it remains challenging to place those in cells without a reference frame. Correlative light and electron microscopy (CLEM) approaches have been developed to place the fluorescence image in the context of a cellular structure. However, combining imaging methods such as super resolution microscopy and transmission electron microscopy necessitates a correlation using fiducial markers to locate the fluorescence on the structures visible in electron microscopy, with a measurable precision. Here, we investigated different fiducial markers for super-resolution CLEM (sCLEM) by evaluating their shape, intensity, stability and compatibility with photoactivatable fluorescent proteins as well as the electron density. We further carefully determined limitations of correlation accuracy. We found that spectrally-shifted FluoSpheres are well suited as fiducial markers for correlating single-molecule localization microscopy with transmission electron microscopy.

The nasal cavity, which serves as the primary entry point for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is crucial in the early stages of coronavirus disease 2019 (COVID-19) infection. Despite extensive research on the molecular pathogenesis of the virus, its ultrastructural effects on the nasal and olfactory epithelium have received relatively less attention. This chapter comprehensively analyzes the ultrastructural changes in different types of cells in the nasal cavity in SARS-CoV-2-infected individuals, highlighting their role in viral entry, replication, and tissue-level damage. We explore the ultrastructure of various key cell types, including ciliated epithelial cells, goblet cells, basal cells, olfactory sensory neurons, sustentacular cells, and the leptomeningeal layers surrounding the olfactory bulb. This chapter also delves into the ultrastructure of nasal organoids, which serve as valuable models for understanding the virus’s impact at a cellular and organoid level. Through electron microscopy, we examine the cellular changes caused by SARS-CoV-2 infection, such as the remodeling of cellular organelles, disruption of the epithelial barrier, and the impact on olfactory function. This chapter aims to deepen our understanding of the virus’s tropism in the nasal cavity and its implications for disease progression, transmission, and potential therapeutic targets by providing detailed ultrastructural evidence. Illustration of SARS-CoV-2 infection in the nasal cavity, detailing ultrastructural evidence in leptomeningeal, olfactory epithelium, and respiratory epithelium, comparing infected and normal states with cellular changes and viral infiltration. https://www.w3.org/1999/xlink" content-type="colour" xlink:href="https://s3-euw1-ap-pe-df-pch-content-public-p.s3.eu-west-1.amazonaws.com/9781003748908/550bc977-6f4b-4545-82bf-2735acee8d94/content/fig6_202_1_C.jpg"/>

Cryogenic electron tomography (cryo-ET) combined with subtomogram averaging, allows in situ visualization and structure determination of macromolecular complexes at subnanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: we built a coincident three-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing FIB scanning electron microscope. We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Supramolecular signaling assemblies are of interest for their unique signaling properties. A µm scale signaling assembly, the central supramolecular signaling cluster (cSMAC), forms at the center of the interface of T cells activated by antigen-presenting cells. We have determined that it is composed of multiple complexes of a supramolecular volume of up to 0.5 µm3 and associated with extensive membrane undulations. To determine cSMAC function, we have systematically manipulated the localization of three adaptor proteins, LAT, SLP-76, and Grb2. cSMAC localization varied between the adaptors and was diminished upon blockade of the costimulatory receptor CD28 and deficiency of the signal amplifying kinase Itk. Reconstitution of cSMAC localization restored IL-2 secretion which is a key T cell effector function as dependent on reconstitution dynamics. Our data suggest that the cSMAC enhances early signaling by facilitating signaling interactions and attenuates signaling thereafter through sequestration of a more limited set of signaling intermediates. eLife digest Cells receive dozens of signals at different times and in different places. Integrating incoming information and deciding how to respond is no easy task. Signaling molecules on the cell surface pass messages inwards using chemical messengers that interact in complicated networks within the cell. One way to unravel the complexity of these networks is to look at specific groups of signaling molecules in test tubes to see how they interact. But the interior of a living cell is a very different environment. Molecules inside cells are tightly packed and, under certain conditions, they interact with each other by the thousands. They form structures known as ‘supramolecular complexes’, which changes their behavior. One such supramolecular complex is the ‘central supramolecular activation cluster’, or cSMAC for short. It forms under the surface of immune cells called T cells when they are getting ready to fight an infection. Under the microscope, the cSMAC looks like the bullseye of a dartboard, forming a crowd of signaling molecules at the center of the interface between the T cell and another cell. Its exact role is not clear, but evidence suggests it helps to start and stop the signals that switch T cells on. The cSMAC contains two key protein adaptors called LAT and SLP-76 that help to hold the structure together. So, to find out what the cSMAC does, Clark et al. genetically modified these adaptors to gain control over when the cSMAC forms. Clark et al. examined mouse T cells using super-resolution microscopy and electron microscopy, watching as other immune cells delivered the signal to switch on. As the T cells started to activate, the composition of the cSMAC changed. In the first two minutes after the cells started activating, the cSMAC included a large number of different components. This made T cell activation more efficient, possibly because the supramolecular complex was helping the network of signals to interact. Later, the cSMAC started to lose many of these components. Separating components may have helped to stop the activation signals. Understanding how T cells activate could lead to the possibility of turning them on or off in immune-related diseases. But these findings are not just relevant to immune cells. Other cells also use supramolecular complexes to control their signaling. Investigating how these complexes change over time could help us to understand how other cell types make decisions. Introduction T cell activation is governed by spatiotemporal organization of signal transduction across scales. At the nanoscale, receptors form clusters of dozens of molecules that can coalesce into microclusters and are commonly associated with active forms of signaling intermediates (Boyle et al., 2011; Hu et al., 2016; Lillemeier et al., 2006; Schamel et al., 2005; Sherman et al., 2011; Varma et al., 2006; Yokosuka et al., 2005). This association suggests that such receptor clusters mediate efficient T cell signaling. Larger, µm scale assemblies were first described at the center and periphery of T cells activated by antigen-presenting cells (APC) for the TCR, PKCθ and LFA-1, talin, respectively, as central and peripheral supramolecular activation clusters (cSMAC and pSMAC) (Grakoui et al., 1999; Monks et al., 1998; Monks et al., 1997). µm scale of assemblies, in particular in the form of supramolecular protein complexes, provides unique biophysical and signaling properties (Banani et al., 2016; Li et al., 2012; Shin and Brangwynne, 2017). Supramolecular protein complexes play critical roles in viral sensing (Cai et al., 2014), inflammation (Franklin et al., 2014), embryonic development (Brangwynne et al., 2009), protein folding in cancer (Rodina et al., 2016), nuclear ubiquitinoylation (Marzahn et al., 2016), and chromatin compaction (Larson et al., 2017). Such complexes are readily observed by fluorescence microscopy, held together by a network of multivalent protein interactions and often have distinct phase properties (Banani et al., 2016; Li et al., 2012; Shin and Brangwynne, 2017). The cSMAC has many properties of such supramolecular protein complexes: It contains various multivalent signaling intermediates (Balagopalan et al., 2015), prominently LAT (linker of activation of T cells), components of this complex including LAT and PKCθ exchange with the remainder of the cell to a moderate extent and slowly (Roybal et al., 2015), and components of this complex can be assembled into supramolecular structures in vitro (Su et al., 2016). Therefore, understanding biophysical properties of the cSMAC and how it regulates T cell activation is of substantial importance. cSMAC function is controversial despite decades of work. Limitations in investigating the cSMAC are that its properties are largely unresolved and that its composition and/or assembly have not been systematically manipulated inside live T cells. Based on association of cSMAC formation with T cell activation conditions, the cSMAC has been proposed to enhance T cell signaling, terminate it, not be related to signaling or only upon weak stimulation or at late time points (Čemerski et al., 2008; Freiberg et al., 2002; Grakoui et al., 1999; Lee et al., 2002; Monks et al., 1997). cSMAC formation is often associated with efficient T cell activation conditions, fitting with a role in enhancing T cell signaling. Accumulation of signaling intermediates at the T cell:APC interface center is substantially reduced by blockade of the costimulatory receptor CD28 (Singleton et al., 2009; Wülfing et al., 2002), in regulatory T cells (Zanin-Zhorov et al., 2010), during thymic selection (Ebert et al., 2008), or in the absence of the signal amplifying kinase Itk (IL-2 inducible T cell kinase) (Singleton et al., 2011). To determine cSMAC properties, we have used stimulated emission depletion (STED) super-resolution microscopy and correlative light electron microscopy (CLEM). The cSMAC was composed of multiple complexes of supramolecular dimensions and associated with extensive membrane undulations. To investigate cSMAC function, we have systematically manipulated the localization of three adaptor proteins in live primary T cells: LAT (Balagopalan et al., 2015) is an integral component of the cSMAC. SLP-76 (SH2 domain-containing leucocyte protein of 76 kD) (Koretzky et al., 2006) is associated with it only during the first minute of T cell activation (Roybal et al., 2015). Grb2 (growth factor receptor-bound 2) (Jang et al., 2009) association with the cSMAC is less prevalent (Roybal et al., 2015). Interface recruitment of all three adaptors was diminished upon attenuation of T cell activation by costimulation blockade and Itk-deficiency as was IL-2 secretion, a critical T cell effector function. By fusing these adaptors with various protein domains with a strong interface localization preference we brought them back to the interface under the attenuated T cell activation conditions and restored cSMAC formation. Such restoration enhanced IL-2 secretion but only when executed to the extent and with dynamics seen under full stimulus conditions. Results μm scale LAT accumulation at the center of the T cell APC interface is associated with efficient T cell activation To investigate the function of µm scale protein accumulation at the center of the T cell:APC interface, we first cataloged protein localization events that were consistently associated with efficient T cell activation. We attenuated T cell activation through costimulation blockade (Singleton et al., 2009; Wülfing et al., 2002) and Itk deficiency. The 5C.C7 T cell receptor (TCR) recognizes the moth cytochrome C (MCC) 89–103 peptide presented by I-Ek. In the restimulation of in vitro primed 5C.C7 T cells with CH27 B cell lymphoma APCs and MCC peptide IL-2 amounts in the supernatant were reduced upon blockade of the CD28 ligands CD80 and CD86 (‘costimulation blockade’) and in T cells from Itk knock out 5C.C7 TCR transgenic mice, in particular at lower peptide concentrations (Figure 1A). As IL-2 amounts in T cell culture supernatants are determined by the difference between IL-2 generation and consumption we also determined IL-2 mRNA levels. Even at an MCC peptide concentration of 10 µM the level of IL-2 mRNA in T cells was significantly (p<0.001) reduced to less than 50% upon costimulation blockade and Itk-deficiency (Figure 1B). 10 µM MCC was used for the remainder of the study. To more precisely relate the determination of IL-2 amounts in T cell culture supernatants to IL-2 mRNA generation, we determined the time course of both (Figure 1—figure supplement 1). IL-2 mRNA generation occurred during the first six hours of T cell activation, consistent with transient nuclear localization of NFkB and previous data establishing that APC contact times of less than one hour are sufficient to commit a primed T cells to proliferation (Iezzi et al., 1998). We used IL-2 mRNA generation for the remainder of the study because of its greater sensitivity to stimulus attenuation. Figure 1 with 4 supplements see all Download asset Open asset CD28 and Itk regulate IL-2 secretion and signaling organization. (A) In vitro primed 5C.C7 T cells, wild type or Itk-deficient (‘Itk ko’), were activated by CH27 APCs and the indicated concentration of MCC peptide in the absence or presence of 10 µg/ml anti-CD80 plus anti-CD86 (‘full stimulus’ or ‘costimulation blockade’). IL-2 levels in the supernatant are given relative to stimulation of wild type 5C.C7 T cells under full stimulus conditions with 10 µM MCC with SEM. 4–8 experiments were averaged per condition. Statistical significance as determined separately for each MCC peptide concentration by 1-way ANOVA is indicated. (B) Relative levels of IL-2 mRNA are given upon 5C.C7 T cell activation similar to A with only 10 µM MCC. 3–18 experiments were averaged per condition. Statistical significance as determined by 1-way ANOVA is indicated. (C) Wild type and Itk-deficient ('Itk ko') 5C.C7 T cells expressing the indicated sensors were activated by CH27 B cell APCs (10 µM MCC) in the absence or presence of 10 µg/ml anti-CD80 plus anti-CD86 ((‘full stimulus’ or ‘costimulation blockade’) and percentage occurrence of patterns of interface enrichment (Figure 1—figure supplement 2A) is given in shades of red from −40 to 420 s relative to tight cell coupling. Cluster trees are given in pink.Sensors used and source data for panel Care given in Figure 1—figure supplements 2B–4, Figure 1—source data 1. Figure 1—source data 1 Sensors used in Figure 1C,D. Publications describing the sensors used in Figure 1C,D and representative 5C.C7 T cell imaging data are given. https://cdn.elifesciences.org/articles/45789/elife-45789-fig1-data1-v2.pdf Download elife-45789-fig1-data1-v2.pdf We characterized T cell signaling organization as extensively described before (Ambler et al., 2017; Roybal et al., 2015; Singleton et al., 2009). Briefly, in vitro primed 5C.C7 T cells are retrovirally transduced to express fluorescent signaling intermediates or sensors, FACS sorted to low expression as close as possible to endogenous signaling intermediate concentrations and imaged in three dimensions over time during restimulation with APC and 10 µM MCC peptide (‘full stimulus’). In image analysis the frequency of occurrence of geometrically quantified µm scale subcellular distributions that represent underlying cell biological structures is determined (Figure 1—figure supplement 2A) (Roybal et al., 2013). Of particular interest here are accumulation at the center of the T cell APC interface (‘central’), the cSMAC, and accumulation in a µm deep ‘invagination’ at the center of the interface that likely mediates termination of early central signaling (Singleton et al., 2006). Upon costimulation blockade most sensors that displayed frequent central accumulation upon full T cell stimulation, in particular during the first two minutes of cell coupling, did less so. In Itk-deficient 5C.C7 T cells sensors with only transient early central accumulation in wild type T cells lost much of that accumulation (Figure 1C,D; Figure 1—figure supplements 2B–4, Figure 1—source data 1). Both phenotypes are indicative of reduced cSMAC formation. Efficient IL-2 secretion thus was associated with µm scale central signaling localization. LAT as a key cSMAC component displayed µm scale central localization (Figure 2A) in a biphasic pattern. At the time of tight cell coupling under full stimulus conditions 49 ± 6% of cell couples showed central LAT accumulation. After 2 min of cell coupling central LAT accumulation was only found in about 25% of cell couples (Figure 2B) and remained stable at that level. In the absence of Itk, the initial peak of central LAT accumulation was significantly (p=0.005) diminished to 26 ± 6% of cell couples with central LAT accumulation. Such reduction was more pronounced (11 ± 4%, p<0.001) upon costimulation blockade (Figure 2B; Figure 2—source data 1). Combining costimulation blockade and Itk deficiency yielded the least interface LAT accumulation (Figure 2B). Impaired activation of cytoskeletal transport processes is a likely contributor to diminished central LAT accumulation upon costimulation blockade and Itk deficiency, as enhancement of actin dynamics with active Rac and Cofilin (Roybal et al., 2016) significantly (p<0.001, Figure 2C; Figure 2—source data 1)(Roybal et al., 2016) increased central and overall LAT accumulation. Figure 2 with 2 supplements see all Download asset Open asset LAT localization and activation is regulated by costimulation and Itk. (A) An interaction of a LAT-GFP-transduced 5C.C7 T cell with a CH27 APC (10 μM MCC) is shown at the indicated time points (in minutes) relative to the time of formation of a tight cell couple. Differential interference contrast (DIC) images are shown in the top row, with top-down, maximum projections of 3-dimensional LAT-GFP fluorescence data in the bottom row. LAT-GFP fluorescence intensities are displayed in a rainbow-like false-color scale (increasing from blue to red). The scale bar corresponds to 5 µm. A corresponding video is available as Figure 2—Video 1. (B) The graphs display the percentage of cell couples with LAT accumulation in the indicated patterns (Figure 1—figure supplement 2A, ‘periphery’ is the sum of asymmetric and peripheral) relative to tight cell couple formation for wild type or Itk-deficient 5C.C7 T cells activated with CH27 APCs (10 µM MCC) in the absence or presence of 10 µg/ml anti-CD80 plus anti-CD86 (‘costimulation blockade’) as indicated. 47–77 cell couples from 2 to 5 independent experiments were analyzed per condition, 226 total. A statistical analysis is given in Figure 2—source data 1. (C) Itk-deficient 5C.C7 T cells were activated with CH27 APCs (10 µM MCC) in the presence of 10 µg/ml anti-CD80 plus anti-CD86 and 250 nM constitutively active Cofilin plus 1 µM constitutively active Rac1 as protein transduction reagents. LAT interface accumulation is given as in B. 30 cell couples from a single experiment were analyzed. Statistical significance is given in Figure 2—source data 1. (D) 5C.C7 T cells were activated as in B for the indicated times. Band intensities of α-LAT pY191 blots (Figure 2—figure supplement 1) as normalized to the 1 min time point under full stimulus conditions are given. 5–7 experiments were averaged per condition. Statistical significance as determined separately for each time point by 1-way ANOVA is indicated. Figure 2—source data 1 Statistical significance of differences in LAT accumulation under different T cell activation conditions is given for the indicated patterns as determined by proportion’s z-test. No entry indicates p>0.05. 0.000 indicates p<0.0005. Gray scale is used to visualize the level of significance. https://cdn.elifesciences.org/articles/45789/elife-45789-fig2-data1-v2.pdf Download elife-45789-fig2-data1-v2.pdf Attenuation of T cell activation was associated with diminished LAT phosphorylation at Y191 (Figure 2D; Figure 2—source data 1) upon costimulation blockade, in particular in combination with Itk deficiency. At 2, 5, and 10 min after tight cell coupling LAT phosphorylation was significantly (p≤0.02) reduced in Itk-deficient 5C.C7 T cells upon costimulation blockade compared to wild type 5C.C7 T cells under full stimulus conditions by 39%, 61%, and 70%, respectively. The cSMAC consists of multiple complexes of supramolecular dimensions and is associated with extensive membrane undulations To determine cSMAC properties, we stained 5C.C7 T cell:CH27 B cell APC couples for LAT and LAT phosphorylated at Y191 (‘pLAT’) and imaged them using STED super-resolution microscopy (Figure 3A). Multiple LAT/pLAT complexes formed in the cSMAC and/or beyond, on average four per cell. LAT complexes were significantly larger (p=0.04) in the cSMAC region with a supramolecular volume of 0.23 ± 0.03 µm3 than in T cells without a cSMAC (0.12 ± 0.01 µm3)(Figure 3B). pLAT complexes were similarly larger in the cSMAC region with volumes of 0.25 ± 0.03 µm3 (cSMAC) versus 0.13 ± 0.01 µm3 (non-cSMAC, p=0.007)(Figure 3B). Figure 3 with 3 supplements see all Download asset Open asset The cSMAC consists of multiple smaller complexes and is associated with extensive membrane undulations. (A) Two representative STED midplane images are given of 5C.C7 T cells activated by CH27 APCs (10 µm MCC) for 4.5 min and stained with α-LAT pY191. Staining fluorescence intensity is given in rainbow-like false-color scale (increasing from blue to red). The T cell outline is given in yellow. The scale bars correspond to 1 µm. (B) For experiments as in A LAT and LAT pY191 cluster size is given separately for cell couples with central or diffuse LAT accumulation as indicated (number of cell couples analyzed in two independent experiments in parentheses). Statistical significance as determined separately for ‘LAT’ and ‘pLAT’ by Student’s test is indicated. (C) Midplane sections are given for two EM tomograms from a CLEM experiment. 5C.C7 T cells expressing LAT-GFP (top) or LAT V3-GFP (bottom)(see Figure 4B) were activated by CH27 APCs under full stimulus (top) or costimulation blocked (bottom) conditions, respectively. Upon formation of a cell couple with central LAT-GFP or LAT V3-GFP accumulation cell were fixed and processed for EM. The T cell plasma membrane at the cellular interface is traced in blue. Videos of the entire EM tomogram reconstructions are given as Figure 3—Videos 1 and 2. (D) In cell couples processed as in C membrane undulations were determined as the ratio of the length of the plasma membrane (‘length’) to a straight-line interface diameter of the same region (‘diameter) in single images of EM sections. In cell couples with central LAT-GFP (black symbols) or LAT V3-GFP (red symbols) accumulation the interface center (‘cSMAC’) and periphery (‘pSMAC’) were measured separately. Peripheral regions were measured twice per cell, once to the left and once to the right of the central region. In control cell couples without central LAT-GFP (black symbols) or LAT V3-GFP (red symbols) accumulation (‘no the entire interface was analyzed. couples are from two independent experiments per condition. Statistical significance as determined by 1-way ANOVA is indicated To understand how LAT as a protein could the formation of multiple supramolecular complexes, we related cSMAC formation to plasma membrane with CLEM (Figure Figure supplement 1). live cell imaging of 5C.C7 T cells activated by CH27 B cell APCs we fixed upon of central LAT and processed them for electron microscopy We used two conditions, a full stimulus and activation of 5C.C7 T cells expressing a protein of LAT with the PKCθ upon costimulation blockade as an to enhanced cSMAC formation upon attenuation of T cell activation, as in in the In EM sections we measured the extent of membrane undulations in cSMAC and interface regions as the ratio of the plasma membrane length to the straight-line diameter of the region. This ratio was significantly (p<0.001) larger in the cSMAC ± than in the of the same T cell ± In control cell couples without cSMAC formation the ratio as measured across the entire interface was as ± as that in regions of cell couples with central LAT (Figure The data are consistent with diminished membrane undulations in 5C.C7 T cells upon costimulation blockade with a ratio of ± (Roybal et al., 2016). The cSMAC thus is a cellular region membrane undulations and multiple supramolecular complexes are associated with enhanced of signaling intermediates. To the for cSMAC in the remainder of these we used microscopy to determine central protein accumulation as a of cSMAC formation. of LAT with protein domains with pronounced interface localization preference LAT localization To determine cSMAC function, we to its formation upon costimulation blockade and in Itk-deficient T cells. To we to how cSMAC components interact within the a supramolecular complex could function through formation of protein interactions or by enhancing signaling through complex formation such that the same proteins can be assembled using and protein interaction of the large number of cSMAC components of which are (Figure 1) we the as most Impaired cSMAC formation upon costimulation blockade and Itk-deficiency at least in be by a reduction in the number of protein interaction that is the of key components such as LAT as by diminished phosphorylation (Figure We be to cSMAC formation by enhancing through the of protein interaction this and protein interaction the live cell signaling through the formation of the central region of increased protein be We increased LAT by three protein PKCθ or The PKCθ is for central interface accumulation of PKCθ et al., it central localization by (Figure supplement 1A). The domains strong central accumulation only within the first minute of cell coupling (Figure supplement as consistent with the localization of full length (Figure 1—figure supplement The interface accumulation on the first two minutes of cell coupling without a central preference (Figure supplement 1A). the of protein interaction domains to LAT is to their initial experiments to this remained as in the expression of LAT and the three LAT proteins was by FACS for the same level of proteins were at ± the endogenous level of LAT in T cells (Figure supplement with change to endogenous LAT levels in the transduced T cells. of LAT to the PKCθ yielded efficient central accumulation that was over the entire imaging time under all conditions at levels 50% of cell couples with central accumulation (Figure s after tight cell coupling such central accumulation was significantly Figure data 1) more frequent than central accumulation of LAT under full stimulus at time of LAT to the PKCθ thus central LAT accumulation the levels seen for LAT under condition. in formed upon LAT expression membrane undulations were similarly enhanced as in T cells expressing LAT (Figure cSMAC properties. Figure 4 with 4 supplements see all Download asset Open asset LAT localization can be by with protein domains with a strong interface localization (A) A of LAT-GFP is given (top) with LAT accumulation data under full stimulus conditions from Figure 2B) as a for the of the (B) top are given for the three proteins of LAT with protein localization domains as indicated. imaging data are given in the wild type or Itk-deficient 5C.C7 T cells transduced to express the LAT indicated on the top of the were activated with CH27 APCs (10 µM MCC) in the absence or presence of 10 µg/ml anti-CD80 plus anti-CD86 (‘costimulation blockade’) with different T cell activation conditions given in as indicated. the percentage of cell couples that displayed accumulation of the LAT with the indicated patterns as in Figure relative to tight cell couple formation in and red accumulation of LAT-GFP in or the central interface respectively, under the same T cell activation conditions Figure 2B) for For costimulation blocked conditions representative imaging data are given similar to Figure are available as Figure cell couples from 2 to 5 independent experiments were analyzed per condition, total. Statistical analysis is given in Figure data 1. Figure data 1 Statistical significance of differences in accumulation of as compared to LAT under different T cell activation conditions is given for the indicated patterns as determined by proportion’s z-test. No entry indicates p>0.05. 0.000 indicates p<0.0005. Gray scale is used to visualize the level of significance. Download of LAT to the domains yielded different on the T cell activation conditions. Upon a full T cell stimulus LAT in diminished central and overall accumulation compared to LAT that was across many time points (Figure Figure data that LAT not enhance assembled signaling Upon the three attenuated LAT consistently enhanced central most at most time for costimulation blockade in wild type and Itk cells (Figure Figure data 1). LAT accumulation upon attenuated T cell stimulation in was largely from LAT accumulation under full stimulus conditions Figure data 1) and central accumulation was enhanced only between 1 and 3 min after tight cell coupling. of LAT to the thus for close reconstitution of full LAT localization upon attenuated T cell of LAT to the LAT but was less (Figure Figure data 1). Upon the three attenuated T cell LAT enhanced central and overall LAT accumulation but consistently the same extent as LAT under full stimulus conditions. For in Itk-deficient 5C.C7 T cells upon costimulation blockade interface accumulation in was consistently enhanced for time point and from upon expression of LAT accumulation at the interface center only increased from a of to To that overall interface accumulation of the LAT

Background Electron cryotomography is a powerful imaging technique allowing for studying functional cellular modules in their native environment with macromolecular resolution. However, it requires access to complex and expensive instrumentation, typically a 300 kV electron cryomicroscope equipped with an energy filter. Simpler and cheaper 200 and 100 kV instruments have been successfully used for single particle cryoEM analyses, which has helped to democratize the technique and broaden access. It has not been systematically studied if 200 kV electron cryomicroscopes can deliver meaningful and interpretable data with respect to electron cryotomography applications. Methods Here, we set out to investigate if a 200 kV electron cryomicroscope without an energy filter can be utilized for in situ structural studies of mammalian cells by electron cryotomography of thin cell edges followed by extensive image analysis including segmentations, subtomogram averaging and molecular sociology studies of lipid droplets. Results We demonstrate that the resulting tomograms of thin edges of U2OS cells are of sufficient quality to annotate the contents of the cell and observe spatial inter-relationships among macromolecules. In particular, we undertook a molecular sociology analysis of lipid droplets and addressed their subcellular distribution and interactions with other organelles. Additionally, we performed subtomogram averaging of purified 70S ribosomes that resulted in ∼15 Å resolution 3D reconstruction. Finally, we examined geographical distribution and scientific output of the two most common electron cryomicroscopy platforms and deduced that 200 kV instruments are heavily underutilized with respect to electron cryotomography applications. Discussion This study demonstrates that 200 kV electron cryomicroscopes can be utilized for structural cell biology studies by electron cryotomography. Given the favorable ratio of their versatility versus costs we foresee that 200 kV electron cryomicroscopes will become workhorses of local electron cryomicroscopy facilities.

Electron cryotomography is a powerful imaging technique allowing for studying functional cellular modules in their native environment with macromolecular resolution. However, it requires access to complex and expensive instrumentation, typically a 300 kV electron cryomicroscope equipped with an energy filter. Simpler and cheaper 200 and 100 kV instruments have been successfully used for single particle cryoEM analyses, which has helped to democratize the technique and broaden access. It has not been systematically studied if 200 kV electron cryomicroscopes can deliver meaningful and interpretable data with respect to electron cryotomography applications. Here, we set out to investigate if a 200 kV electron cryomicroscope without an energy filter can be utilized for in situ structural studies of mammalian cells by electron cryotomography of thin cell edges followed by extensive image analysis including segmentations, subtomogram averaging and molecular sociology studies of lipid droplets. We demonstrate that the resulting tomograms of thin edges of U2OS cells are of sufficient quality to annotate the contents of the cell and observe spatial inter-relationships among macromolecules. In particular, we undertook a molecular sociology analysis of lipid droplets and addressed their subcellular distribution and interactions with other organelles. Additionally, we performed subtomogram averaging of purified 70S ribosomes that resulted in ~ 15 Å resolution 3D reconstruction. Finally, we examined geographical distribution and scientific output of the two most common electron cryomicroscopy platforms and deduced that 200 kV instruments are heavily underutilized with respect to electron cryotomography applications. This study demonstrates that 200 kV electron cryomicroscopes can be utilized for structural cell biology studies by electron cryotomography. Given the favorable ratio of their versatility versus costs we foresee that 200 kV electron cryomicroscopes will become workhorses of local electron cryomicroscopy facilities.

Monkeypox virus (MPXV), originally endemic in West Africa (Clade II) and Central Africa (Clade I), has recently emerged worldwide and has reinforced the need for rapid and accurate MPXV diagnostics. This review presents and critically discusses the range of virological methods for laboratory diagnosis and characterization of MPXV as well as related lessons learned and practical experience gained from the 2022 Mpox global outbreak. Real-time PCR is currently considered the diagnostic gold standard and ensures accurate and timely confirmation of suspected Mpox cases based on suspicious skin lesions, and digital PCR improves the precision of MPXV DNA quantification. Whole genome sequencing reveals the diversity within the Clade IIb outbreak and highlights the role of microevolution in the adaptation of the virus to the human host. Continuous genomic surveillance is important for better understanding of human-to-human transmission and prevention of the emergence of variola virus-like strains. Traditional virological methods such as electron microscopy and virus isolation remain essential for comprehensive virus characterization, particularly in the context of vaccine and antiviral drug development. Despite the current challenges, serological tests detecting a range of anti-MPXV antibodies are important adjunct diagnostic and research tools for confirmation of late-presenting or asymptomatic MPXV cases, contact tracing, epidemiological studies, seroepidemiological surveys, and better understanding of the role of IgG and neutralizing antibodies in the immune response to infection and vaccination. A multidisciplinary approach combining advanced molecular techniques with traditional virological methods is important for rapid and reliable diagnosis, surveillance, and control of the outbreak.