Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) culture and sample preparation for correlative light electron microscopy.

Correlative Light Electron Microscopy (CLEM) is a powerful technique to investigate the ultrastructure of specific cells and organelles at sub-cellular resolution. Transmission Electron Microscopy (TEM) is particularly useful to the field of virology, given the small size of the virion, which is below the limit of detection by light microscopy. Furthermore, viral infection results in the rearrangement of host organelles to form spatially defined compartments that facilitate the replication of viruses. With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there has been great interest to study the viral replication complex using CLEM. In this chapter we provide an exemplary workflow describing the safe preparation and processing of cells grown on coverslips and infected with SARS-CoV-2.

Maturation and Conformational Switching of a De Novo Designed Phase-Separating Polypeptide.

Cellular compartments formed by biomolecular condensation are widespread features of cell biology. These organelle-like assemblies compartmentalize macromolecules dynamically within the crowded intracellular environment. However, the intermolecular interactions that produce condensed droplets may also create arrested states and potentially pathological assemblies such as fibers, aggregates, and gels through droplet maturation. Protein liquid-liquid phase separation is a metastable process, so maturation may be an intrinsic property of phase-separating proteins, where nucleation of different phases or states arises in supersaturated condensates. Here, we describe the formation of both phase-separated droplets and proteinaceous fibers driven by a de novo designed polypeptide. We characterize the formation of supramolecular fibers in vitro and in bacterial cells. We show that client proteins can be targeted to the fibers in cells using a droplet-forming construct. Finally, we explore the interplay between phase separation and fiber formation of the de novo polypeptide, showing that the droplets mature with a post-translational switch to largely ß conformations, analogous to models of pathological phase separation.

Roles for CEP170 in cilia function and dynein-2 assembly.

Primary cilia are essential eukaryotic organelles required for signalling and secretion. Dynein-2 is a microtubule-motor protein complex and is required for ciliogenesis via its role in facilitating retrograde intraflagellar transport (IFT) from the cilia tip to the cell body. Dynein-2 must be assembled and loaded onto IFT trains for entry into cilia for this process to occur, but how dynein-2 is assembled and how it is recycled back into a cilium remain poorly understood. Here, we identify centrosomal protein of 170 kDa (CEP170) as a dynein-2-interacting protein in mammalian cells. We show that loss of CEP170 perturbs intraflagellar transport and hedgehog signalling, and alters the stability of dynein-2 holoenzyme complex. Together, our data indicate a role for CEP170 in supporting cilia function and dynein-2 assembly.

Self-Assembly of Nanofilaments in Cyanobacteria for Protein Co-localization.

Cyanobacteria offer great potential as alternative biotechnological hosts due to their photoautotrophic capacities. However, in comparison to established heterotrophic hosts, several key aspects, such as product titers, are still lagging behind. Nanobiotechnology is an emerging field with great potential to improve existing hosts, but so far, it has barely been explored in microbial photosynthetic systems. Here, we report the establishment of large proteinaceous nanofilaments in the unicellular model cyanobacterium Synechocystis sp. PCC 6803 and the fast-growing cyanobacterial strain Synechococcus elongatus UTEX 2973. Transmission electron microscopy and electron tomography demonstrated that expression of pduA*, encoding a modified bacterial microcompartment shell protein, led to the generation of bundles of longitudinally aligned nanofilaments in S. elongatus UTEX 2973 and shorter filamentous structures in Synechocystis sp. PCC 6803. Comparative proteomics showed that PduA* was at least 50 times more abundant than the second most abundant protein in the cell and that nanofilament assembly had only a minor impact on cellular metabolism. Finally, as a proof-of-concept for co-localization with the filaments, we targeted a fluorescent reporter protein, mCitrine, to PduA* by fusion with an encapsulation peptide that natively interacts with PduA. The establishment of nanofilaments in cyanobacterial cells is an important step toward cellular organization of heterologous pathways and the establishment of cyanobacteria as next-generation hosts.

The free fatty acid-binding pocket is a conserved hallmark in pathogenic ß-coronavirus spike proteins from SARS-CoV to Omicron.

As coronavirus disease 2019 (COVID-19) persists, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) emerge, accumulating spike (S) glycoprotein mutations. S receptor binding domain (RBD) comprises a free fatty acid (FFA)-binding pocket. FFA binding stabilizes a locked S conformation, interfering with virus infectivity. We provide evidence that the pocket is conserved in pathogenic ß-coronaviruses (ß-CoVs) infecting humans. SARS-CoV, MERS-CoV, SARS-CoV-2, and VOCs bind the essential FFA linoleic acid (LA), while binding is abolished by one mutation in common cold-causing HCoV-HKU1. In the SARS-CoV S structure, LA stabilizes the locked conformation, while the open, infectious conformation is devoid of LA. Electron tomography of SARS-CoV-2-infected cells reveals that LA treatment inhibits viral replication, resulting in fewer deformed virions. Our results establish FFA binding as a hallmark of pathogenic ß-CoV infection and replication, setting the stage for FFA-based antiviral strategies to overcome COVID-19.

ESCPE-1 mediates retrograde endosomal sorting of the SARS-CoV-2 host factor Neuropilin-1.

Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed "cargoes") from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans-Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify retrograde cargo proteins of the endosomal SNX-BAR sorting complex promoting exit 1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterized host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalized with the NRP1-interacting peptide of the SARS-CoV-2 spike (S) protein. CRISPR-Cas9 deletion of ESCPE-1 subunits reduces SARS-CoV-2 infection levels in cell culture. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.

Fluorescent platinum nanoclusters as correlative light electron microscopy probes.

Correlative Imaging (CI) visualizes a single sample/region of interest with two or more imaging modalities. The technique seeks to elucidate information that may not be discernible by using either of the constituent techniques in isolation. Correlative Light Electron Microscopy (CLEM) can be employed to streamline workflows, i.e., using fluorescent signals in the light microscope (LM) to inform the user of regions which should be imaged with electron microscopy (EM). The efficacy of correlative techniques requires high spatial resolution of signals from both imaging modalities. Ideally, a single point should originate from both the fluorescence and electron density. However, many of the ubiquitously used probes have a significant distance between their fluorescence and electron dense portions. Furthermore, electron dense metal nanoparticles used for EM visualization readily quench any proximal adjacent fluorophores. Therefore, accurate registration of both signals becomes difficult. Here we describe fluorescent nanoclusters in the context of a CLEM probe as they are composed of several atoms of a noble metal, in this case platinum, providing electron density. In addition, their structure confers them with fluorescence via a mechanism analogous to quantum dots. The electron dense core gives rise to the fluorescence which enables highly accurate signal registration between epifluorescence and electron imaging modalities. We provide a protocol for the synthesis of the nanoclusters with some additional techniques for their characterization and finally show how they can be used in a CLEM set up.

Refining a correlative light electron microscopy workflow using luminescent metal complexes.

The potential for increasing the application of Correlative Light Electron Microscopy (CLEM) technologies in life science research is hindered by the lack of suitable molecular probes that are emissive, photostable, and scatter electrons well. Most brightly fluorescent organic molecules are intrinsically poor electron-scatterers, while multi-metallic compounds scatter electrons well but are usually non-luminescent. Thus, the goal of CLEM to image the same object of interest on the continuous scale from hundreds of microns to nanometers remains a major challenge partially due to requirements for a single probe to be suitable for light (LM) and electron microscopy (EM). Some of the main CLEM probes, based on gold nanoparticles appended with fluorophores and quantum dots (QD) have presented significant drawbacks. Here we present an Iridium-based luminescent metal complex (Ir complex 1) as a probe and describe how we have developed a CLEM workflow based on such metal complexes.

De Novo Designed Peptide and Protein Hairpins Self-Assemble into Sheets and Nanoparticles.

The design and assembly of peptide-based materials has advanced considerably, leading to a variety of fibrous, sheet, and nanoparticle structures. A remaining challenge is to account for and control different possible supramolecular outcomes accessible to the same or similar peptide building blocks. Here a de novo peptide system is presented that forms nanoparticles or sheets depending on the strategic placement of a "disulfide pin" between two elements of secondary structure that drive self-assembly. Specifically, homodimerizing and homotrimerizing de novo coiled-coil α-helices are joined with a flexible linker to generate a series of linear peptides. The helices are pinned back-to-back, constraining them as hairpins by a disulfide bond placed either proximal or distal to the linker. Computational modeling indicates, and advanced microscopy shows, that the proximally pinned hairpins self-assemble into nanoparticles, whereas the distally pinned constructs form sheets. These peptides can be made synthetically or recombinantly to allow both chemical modifications and the introduction of whole protein cargoes as required.

Small-residue packing motifs modulate the structure and function of a minimal de novo membrane protein.

Alpha-helical integral membrane proteins contain conserved sequence motifs that are known to be important in helix packing. These motifs are a promising starting point for the construction of artificial proteins, but their potential has not yet been fully explored. Here, we study the impact of introducing a common natural helix packing motif to the transmembrane domain of a genetically-encoded and structurally dynamic de novo membrane protein. The resulting construct is an artificial four-helix bundle with lipophilic regions that are defined only by the amino acids L, G, S, A and W. This minimal proto-protein could be recombinantly expressed by diverse prokaryotic and eukaryotic hosts and was found to co-sediment with cellular membranes. The protein could be extracted and purified in surfactant micelles and was monodisperse and stable in vitro, with sufficient structural definition to support the rapid binding of a heme cofactor. The reduction in conformational diversity imposed by this design also enhances the nascent peroxidase activity of the protein-heme complex. Unexpectedly, strains of Escherichia coli expressing this artificial protein specifically accumulated zinc protoporphyrin IX, a rare cofactor that is not used by natural metalloenzymes. Our results demonstrate that simple sequence motifs can rigidify elementary membrane proteins, and that orthogonal artificial membrane proteins can influence the cofactor repertoire of a living cell. These findings have implications for rational protein design and synthetic biology.

Prior exercise in humans redistributes intramuscular GLUT4 and enhances insulin-stimulated sarcolemmal and endosomal GLUT4 translocation.

OBJECTIVE: Exercise is a cornerstone in the management of skeletal muscle insulin-resistance. A well-established benefit of a single bout of exercise is increased insulin sensitivity for hours post-exercise in the previously exercised musculature. Although rodent studies suggest that the insulin-sensitization phenomenon involves enhanced insulin-stimulated GLUT4 cell surface translocation and might involve intramuscular redistribution of GLUT4, the conservation to humans is unknown. METHODS: Healthy young males underwent an insulin-sensitizing one-legged kicking exercise bout for 1 h followed by fatigue bouts to exhaustion. Muscle biopsies were obtained 4 h post-exercise before and after a 2-hour hyperinsulinemic-euglycemic clamp. RESULTS: A detailed microscopy-based analysis of GLUT4 distribution within seven different myocellular compartments revealed that prior exercise increased GLUT4 localization in insulin-responsive storage vesicles and T-tubuli. Furthermore, insulin-stimulated GLUT4 localization was augmented at the sarcolemma and in the endosomal compartments. CONCLUSIONS: An intracellular redistribution of GLUT4 post-exercise is proposed as a molecular mechanism contributing to the insulin-sensitizing effect of prior exercise in human skeletal muscle.

Cellular uptake and targeting of low dispersity, dual emissive, segmented block copolymer nanofibers.

Polymer-based nanoparticles show substantial promise in the treatment and diagnosis of cancer and other diseases. Herein we report an exploration of the cellular uptake of tailored, low dispersity segmented 1D nanoparticles which were prepared from an amphiphilic block copolymer, poly(dihexylfluorene)-b-poly(ethyleneglycol) (PDHF13-b-PEG227), with a crystallizable PDHF core-forming block and a 'stealth' PEG corona-forming block with different end-group functionalities. Segmented C-B-A-B-C pentablock 1D nanofibers with varied spatially-defined coronal chemistries and a selected length (95 nm) were prepared using the living crystallization-driven self-assembly (CDSA) seeded-growth method. As the blue fluorescence of PDHF is often subject to environment-related quenching, a far-red BODIPY (BD) fluorophore was attached to the PEG end-group of the coronal B segments to provide additional tracking capability. Folic acid (FA) was also incorporated as a targeting group in the terminal C segments. These dual-emissive pentablock nanofibers exhibited uptake into >97% of folate receptor positive HeLa cells by flow cytometry. In the absence of FA, no significant uptake was detected and nanofibers with either FA or BD coronal groups showed no significant toxicity. Correlative light and electron microscopy (CLEM) studies revealed receptor-mediated endocytosis as an uptake pathway, with subsequent localization to the perinuclear region. A significant proportion of the nanofibers also appeared to interact with the cell membrane in an end-on fashion, which was coupled with fluorescence quenching of the PDHF core. These results provide new insights into the cellular uptake of polymer-based nanofibers and suggest their potential use in targeted therapies and diagnostics.

High-Contrast Imaging of Nanodiamonds in Cells by Energy Filtered and Correlative Light-Electron Microscopy: Toward a Quantitative Nanoparticle-Cell Analysis.

Fluorescent nanodiamonds (fNDs) represent an emerging class of nanomaterials offering great opportunities for ultrahigh resolution imaging, sensing and drug delivery applications. Their biocompatibility, exceptional chemical and consistent photostability renders them particularly attractive for correlative light-electron microscopy studies providing unique insights into nanoparticle-cell interactions. Herein, we demonstrate a stringent procedure to image and quantify fNDs with a high contrast down to the single particle level in cells. Individual fNDs were directly visualized by energy-filtered transmission electron microscopy, that is, inside newly forming, early endosomal vesicles during their cellular uptake processes as well as inside cellular organelles such as a mitochondrion. Furthermore, we demonstrate the unequivocal identification, localization, and quantification of individual fNDs in larger fND clusters inside intracellular vesicles. Our studies are of great relevance to obtain quantitative information on nanoparticle trafficking and their various interactions with cells, membranes, and organelles, which will be crucial to design-improved sensors, imaging probes, and nanotherapeutics based on quantitative data.

Modifying Self-Assembled Peptide Cages To Control Internalization into Mammalian Cells.

Nanoparticles can be used to transport a variety of biological cargoes into eukaryotic cells. Polypeptides provide a versatile material for constructing such systems. Previously, we have assembled nanoscale peptide cages (SAGEs) from de novo designed coiled-coil modules. Here, we show that the modules can be extended with short charged peptides to alter endocytosis of the assembled SAGE particles by cultured human cells in a tunable fashion. First, we find that the peptide extensions affect coiled-coil stability predictably: N-terminal polylysine and C-terminal polyglutamate tags are destabilizing; whereas, the reversed arrangements have little impact. Second, the cationic assembled particles are internalized faster and to greater extents by cells than the parent SAGEs. By contrast, anionic decorations markedly inhibit both aspects of uptake. These studies highlight how the modular SAGE system facilitates rational peptide design to fine-tune the bioactivity of nanoparticles, which should allow engineering of tailored cell-delivery vehicles.

Correlative Light and Electron Microscopy of Influenza Virus Entry and Budding.

Influenza A virus (IAV) entry is a stepwise process regulated by viral and cellular cues, facilitating cellular functions. Virus entry begins by attachment of hemagglutinin to cell surface sialic acids, followed by endocytic uptake, vesicular transport along microtubules, low-pH-mediated viral membrane fusion with the late endosomal membrane, capsid uncoating, viral ribonucleoprotein (vRNP) release, and nuclear import of vRNPs. Here we show a basic methodology to visualize incoming and egressing IAV particles by correlative light and electron microscopy (CLEM). We combine fluorescence microscopy of virus-infected human lung carcinoma A549 cells with high-pressure freezing (HPF) and in-resin fluorescence CLEM and the Tokuyasu CLEM method. This approach forms a basis to study the virus life cycle and virus-host interactions at the ultrastructural level.

Acute depletion of diacylglycerol from the cis-Golgi affects localized nuclear envelope morphology during mitosis.

Dysregulation of nuclear envelope (NE) assembly results in various cancers; for example, renal and some lung carcinomas ensue due to NE malformation. The NE is a dynamic membrane compartment and its completion during mitosis is a highly regulated process, but the detailed mechanism still remains incompletely understood. Previous studies have found that isolated diacylglycerol (DAG)-containing vesicles are essential for completing the fusion of the NE in nonsomatic cells. We investigated the impact of DAG depletion from the cis-Golgi in mammalian cells on NE reassembly. Using advanced electron microscopy, we observed an enriched DAG population of vesicles at the vicinity of the NE gaps of telophase mammalian cells. We applied a mini singlet oxygen generator-C1-domain tag that localized DAG-enriched vesicles at the perinuclear region, which suggested the existence of NE fusogenic vesicles. We quantified the impact of Golgi-DAG depletion by measuring the in situ NE rim curvature of the reforming NE. The rim curvature in these cells was significantly reduced compared with controls, which indicated a localized defect in NE morphology. Our novel results demonstrate the significance of the role of DAG from the cis-Golgi for the regulation of NE assembly.

Aquaporin-1 regulates platelet procoagulant membrane dynamics and in vivo thrombosis.

In response to collagen stimulation, platelets use a coordinated system of fluid entry to undergo membrane ballooning, procoagulant spreading, and microvesiculation. We hypothesized that water entry was mediated by the water channel aquaporin-1 (AQP1) and aimed to determine its role in the platelet procoagulant response and thrombosis. We established that human and mouse platelets express AQP1 and localize to internal tubular membrane structures. However, deletion of AQP1 had minimal effects on collagen-induced platelet granule secretion, aggregation, or membrane ballooning. Conversely, procoagulant spreading, microvesiculation, phosphatidylserine exposure, and clot formation time were significantly diminished. Furthermore, in vivo thrombus formation after FeCl3 injury to carotid arteries was also markedly suppressed in AQP1-null mice, but hemostasis after tail bleeding remained normal. The mechanism involves an AQP1-mediated rapid membrane stretching during procoagulant spreading but not ballooning, leading to calcium entry through mechanosensitive cation channels and a full procoagulant response. We conclude that AQP1 is a major regulator of the platelet procoagulant response, able to modulate coagulation after injury or pathologic stimuli without affecting other platelet functional responses or normal hemostasis. Clinically effective AQP1 inhibitors may therefore represent a novel class of antiprocoagulant antithrombotics.

Bioinspired Silicification Reveals Structural Detail in Self-Assembled Peptide Cages.

Understanding how molecules in self-assembled soft-matter nanostructures are organized is essential for improving the design of next-generation nanomaterials. Imaging these assemblies can be challenging and usually requires processing, e.g., staining or embedding, which can damage or obscure features. An alternative is to use bioinspired mineralization, mimicking how certain organisms use biomolecules to template mineral formation. Previously, we have reported the design and characterization of Self-Assembled peptide caGEs (SAGEs) formed from de novo peptide building blocks. In SAGEs, two complementary, 3-fold symmetric, peptide hubs combine to form a hexagonal lattice, which curves and closes to form SAGE nanoparticles. As hexagons alone cannot tile onto spheres, the network must also incorporate nonhexagonal shapes. While the hexagonal ultrastructure of the SAGEs has been imaged, these defects have not been observed. Here, we show that positively charged SAGEs biotemplate a thin, protective silica coating. Electron microscopy shows that these SiO2-SAGEs do not collapse, but maintain their 3D shape when dried. Atomic force microscopy reveals a network of hexagonal and irregular features on the SiO2-SAGE surface. The dimensions of these (7.2 nm ± 1.4 nm across, internal angles 119.8° ± 26.1°) are in accord with the designed SAGE network and with coarse-grained modeling of the SAGE assembly. The SiO2-SAGEs are permeable to small molecules (<2 nm), but not to larger biomolecules (>6 nm). Thus, bioinspired silicification offers a mild technique that preserves soft-matter nanoparticles for imaging, revealing structural details <10 nm in size, while also maintaining desirable properties, such as permeability to small molecules.

Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm.

We have developed a system for producing a supramolecular scaffold that permeates the entire Escherichia coli cytoplasm. This cytoscaffold is constructed from a three-component system comprising a bacterial microcompartment shell protein and two complementary de novo coiled-coil peptides. We show that other proteins can be targeted to this intracellular filamentous arrangement. Specifically, the enzymes pyruvate decarboxylase and alcohol dehydrogenase have been directed to the filaments, leading to enhanced ethanol production in these engineered bacterial cells compared to those that do not produce the scaffold. This is consistent with improved metabolic efficiency through enzyme colocation. Finally, the shell-protein scaffold can be directed to the inner membrane of the cell, demonstrating how synthetic cellular organization can be coupled with spatial optimization through in-cell protein design. The cytoscaffold has potential in the development of next-generation cell factories, wherein it could be used to organize enzyme pathways and metabolite transporters to enhance metabolic flux.