Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity.

Vesicle-inducing protein in plastids 1 (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-remodeling functions. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket on one end of the ring. Inside the ring's lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Using cryo-correlative light and electron microscopy (cryo-CLEM), we observe oligomeric VIPP1 coats encapsulating membrane tubules within the Chlamydomonas chloroplast. Our work provides a structural foundation for understanding how VIPP1 directs thylakoid biogenesis and maintenance.

Cellular Electron Cryotomography: Toward Structural Biology In Situ.

Electron cryotomography (ECT) provides three-dimensional views of macromolecular complexes inside cells in a native frozen-hydrated state. Over the last two decades, ECT has revealed the ultrastructure of cells in unprecedented detail. It has also allowed us to visualize the structures of macromolecular machines in their native context inside intact cells. In many cases, such machines cannot be purified intact for in vitro study. In other cases, the function of a structure is lost outside the cell, so that the mechanism can be understood only by observation in situ. In this review, we describe the technique and its history and provide examples of its power when applied to cell biology. We also discuss the integration of ECT with other techniques, including lower-resolution fluorescence imaging and higher-resolution atomic structure determination, to cover the full scale of cellular processes.

Enhanced SARS-CoV-2 entry via UPR-dependent AMPK-related kinase NUAK2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remodels the endoplasmic reticulum (ER) to form replication organelles, leading to ER stress and unfolded protein response (UPR). However, the role of specific UPR pathways in infection remains unclear. Here, we found that SARS-CoV-2 infection causes marginal activation of signaling sensor IRE1α leading to its phosphorylation, clustering in the form of dense ER-membrane rearrangements with embedded membrane openings, and XBP1 splicing. By investigating the factors regulated by IRE1α-XBP1 during SARS-CoV-2 infection, we identified stress-activated kinase NUAK2 as a novel host-dependency factor for SARS-CoV-2, HCoV-229E, and MERS-CoV entry. Reducing NUAK2 abundance or kinase activity impaired SARS-CoV-2 particle binding and internalization by decreasing cell surface levels of viral receptors and viral trafficking likely by modulating the actin cytoskeleton. IRE1α-dependent NUAK2 levels were elevated in SARS-CoV-2-infected and bystander non-infected cells, promoting viral spread by maintaining ACE2 cell surface levels and facilitating virion binding to bystander cells.

An aldehyde-crosslinking mitochondrial probe for STED imaging in fixed cells.

Fluorescence labeling of chemically fixed specimens, especially immunolabeling, plays a vital role in super-resolution imaging as it offers a convenient way to visualize cellular structures like mitochondria or the distribution of biomolecules with high detail. Despite the development of various distinct probes that enable super-resolved stimulated emission depletion (STED) imaging of mitochondria in live cells, most of these membrane-potential-dependent fluorophores cannot be retained well in mitochondria after chemical fixation. This lack of suitable mitochondrial probes has limited STED imaging of mitochondria to live cell samples. In this study, we introduce a mitochondria-specific probe, PK Mito Orange FX (PKMO FX), which features a fixation-driven cross-linking motif and accumulates in the mitochondrial inner membrane. It exhibits high fluorescence retention after chemical fixation and efficient depletion at 775 nm, enabling nanoscopic imaging both before and after aldehyde fixation. We demonstrate the compatibility of this probe with conventional immunolabeling and other strategies commonly used for fluorescence labeling of fixed samples. Moreover, we show that PKMO FX facilitates correlative super-resolution light and electron microscopy, enabling the correlation of multicolor fluorescence images and transmission EM images via the characteristic mitochondrial pattern. Our probe further expands the mitochondrial toolkit for multimodal microscopy at nanometer resolutions.

Precision in situ cryo-correlative light and electron microscopy of optogenetically-positioned organelles.

Unambiguous targeting of cellular structures for in situ cryo-electron microscopy in the heterogeneous, dense, and compacted environment of the cytoplasm remains challenging. Here we have developed a cryogenic correlative light and electron microscopy (cryo-CLEM) workflow which combines thin cells grown on a mechanically defined substratum to rapidly analyse organelles and macromolecular complexes by cryo-electron tomography (cryo-ET). We coupled these advancements with optogenetics to redistribute perinuclear-localised organelles to the cell periphery, allowing visualisation of organelles otherwise positioned in cellular regions too thick for cryo-ET. This reliable and robust workflow allows for fast in situ analyses without the requirement for cryo-focused ion beam milling. Using this protocol, cells can be frozen, imaged by cryo-fluorescence microscopy and be ready for batch cryo-ET within a day.

Live-cell imaging and CLEM reveal the existence of ACTN4-dependent ruffle-edge lamellipodia acting as a novel mode of cell migration.

α-Actinin-4 (ACTN4) expression levels are correlated with the invasive and metastatic potential of cancer cells; however, the underlying mechanism remains unclear. Here, we identified ACTN4-localized ruffle-edge lamellipodia using live-cell imaging and correlative light and electron microscopy (CLEM). BSC-1 cells expressing EGFP-ACTN4 showed that ACTN4 was most abundant in the leading edges of lamellipodia, although it was also present in stress fibers and focal adhesions. ACTN4 localization in lamellipodia was markedly diminished by phosphoinositide 3-kinase inhibition, whereas its localization in stress fibers and focal adhesions remained. Furthermore, overexpression of ACTN4, but not ACTN1, promoted lamellipodial formation. Live-cell analysis demonstrated that ACTN4-enriched lamellipodia are highly dynamic and associated with cell migration. CLEM revealed that ACTN4-enriched lamellipodia exhibit a characteristic morphology of multilayered ruffle-edges that differs from canonical flat lamellipodia. Similar ruffle-edge lamellipodia were observed in A549 and MDA-MB-231 invasive cancer cells. ACTN4 knockdown suppressed the formation of ruffle-edge lamellipodia and cell migration during wound healing in A549 monolayer cultures. Additionally, membrane-type 1 matrix metalloproteinase was observed in the membrane ruffles, suggesting that ruffle-edge lamellipodia have the ability to degrade the extracellular matrix and may contribute to active cell migration/invasion in certain cancer cell types.

Correlative light-electron microscopy methods to characterize the ultrastructural features of the replicative and dormant liver stages of Plasmodium parasites.

BACKGROUND: The infection of the liver by Plasmodium parasites is an obligatory step leading to malaria disease. Following hepatocyte invasion, parasites differentiate into replicative liver stage schizonts and, in the case of Plasmodium species causing relapsing malaria, into hypnozoites that can lie dormant for extended periods of time before activating. The liver stages of Plasmodium remain elusive because of technical challenges, including low infection rate. This has been hindering experimentations with well-established technologies, such as electron microscopy. A deeper understanding of hypnozoite biology could prove essential in the development of radical cure therapeutics against malaria. RESULTS: The liver stages of the rodent parasite Plasmodium berghei, causing non-relapsing malaria, and the simian parasite Plasmodium cynomolgi, causing relapsing malaria, were characterized in human Huh7 cells or primary non-human primate hepatocytes using Correlative Light-Electron Microscopy (CLEM). Specifically, CLEM approaches that rely on GFP-expressing parasites (GFP-CLEM) or on an immunofluorescence assay (IFA-CLEM) were used for imaging liver stages. The results from P. berghei showed that host and parasite organelles can be identified and imaged at high resolution using both CLEM approaches. While IFA-CLEM was associated with more pronounced extraction of cellular content, samples' features were generally well preserved. Using IFA-CLEM, a collection of micrographs was acquired for P. cynomolgi liver stage schizonts and hypnozoites, demonstrating the potential of this approach for characterizing the liver stages of Plasmodium species causing relapsing malaria. CONCLUSIONS: A CLEM approach that does not rely on parasites expressing genetically encoded tags was developed, therefore suitable for imaging the liver stages of Plasmodium species that lack established protocols to perform genetic engineering. This study also provides a dataset that characterizes the ultrastructural features of liver stage schizonts and hypnozoites from the simian parasite species P. cynomolgi.

3D-CLEM Reveals that a Major Portion of Mitotic Chromosomes Is Not Chromatin.

Recent studies have revealed the importance of Ki-67 and the chromosome periphery in chromosome structure and segregation, but little is known about this elusive chromosome compartment. Here we used correlative light and serial block-face scanning electron microscopy, which we term 3D-CLEM, to model the entire mitotic chromosome complement at ultra-structural resolution. Prophase chromosomes exhibit a highly irregular surface appearance with a volume smaller than metaphase chromosomes. This may be because of the absence of the periphery, which associates with chromosomes only after nucleolar disassembly later in prophase. Indeed, the nucleolar volume almost entirely accounts for the extra volume found in metaphase chromosomes. Analysis of wild-type and Ki-67-depleted chromosomes reveals that the periphery comprises 30%-47% of the entire chromosome volume and more than 33% of the protein mass of isolated mitotic chromosomes determined by quantitative proteomics. Thus, chromatin makes up a surprisingly small percentage of the total mass of metaphase chromosomes.

Indirect Correlative Light and Electron Microscopy (iCLEM): A Novel Pipeline for Multiscale Quantification of Structure From Molecules to Organs.

Correlative light and electron microscopy (CLEM) methods are powerful methods that combine molecular organization (from light microscopy) with ultrastructure (from electron microscopy). However, CLEM methods pose high cost/difficulty barriers to entry and have very low experimental throughput. Therefore, we have developed an indirect correlative light and electron microscopy (iCLEM) pipeline to sidestep the rate-limiting steps of CLEM (i.e., preparing and imaging the same samples on multiple microscopes) and correlate multiscale structural data gleaned from separate samples imaged using different modalities by exploiting biological structures identifiable by both light and electron microscopy as intrinsic fiducials. We demonstrate here an application of iCLEM, where we utilized gap junctions and mechanical junctions between muscle cells in the heart as intrinsic fiducials to correlate ultrastructural measurements from transmission electron microscopy (TEM), and focused ion beam scanning electron microscopy (FIB-SEM) with molecular organization from confocal microscopy and single molecule localization microscopy (SMLM). We further demonstrate how iCLEM can be integrated with computational modeling to discover structure-function relationships. Thus, we present iCLEM as a novel approach that complements existing CLEM methods and provides a generalizable framework that can be applied to any set of imaging modalities, provided suitable intrinsic fiducials can be identified.

Where in the cell is my protein?

The application of cryo-correlative light and cryo-electron microscopy (cryo-CLEM) gives us a way to locate structures of interest in the electron microscope. In brief, the structures of interest are fluorescently tagged, and images from the cryo-fluorescent microscope (cryo-FM) maps are superimposed on those from the cryo-electron microscope (cryo-EM). By enhancing cryo-FM to include single-molecule localization microscopy (SMLM), we can achieve much better localization. The introduction of cryo-SMLM increased the yield of photons from fluorophores, which can benefit localization efforts. Dahlberg and Moerner (2021, Annual Review of Physical Chemistry, 72, 253-278) have a recent broad and elegant review of super-resolution cryo-CLEM. This paper focuses on cryo(F)PALM/STORM for the cryo-electron tomography community. I explore the current challenges to increase the accuracy of localization by SMLM and the mapping of those positions onto cryo-EM images and maps. There is much to consider: we need to know if the excitation of fluorophores damages the structures we seek to visualize. We need to determine if higher numerical aperture (NA) objectives, which add complexity to image analysis but increase resolution and the efficiency of photon collection, are better than lower NA objectives, which pose fewer problems. We need to figure out the best way to determine the axial position of fluorophores. We need to have better ways of aligning maps determined by FM with those determined by EM. We need to improve the instrumentation to be easier to use, more accurate, and ice-contamination free. The bottom line is that we have more work to do.

Correlating cryo-super resolution radial fluctuations and dual-axis cryo-scanning transmission electron tomography to bridge the light-electron resolution gap.

Visualization of organelles and their interactions with other features in the native cell remains a challenge in modern biology. We have introduced cryo-scanning transmission electron tomography (CSTET), which can access 3D volumes on the scale of 1 micron with a resolution of nanometers, making it ideal for this task. Here we introduce two relevant advances: (a) we demonstrate the utility of multi-color super-resolution radial fluctuation light microscopy under cryogenic conditions (cryo-SRRF), and (b) we extend the use of deconvolution processing for dual-axis CSTET data. We show that cryo-SRRF nanoscopy is able to reach resolutions in the range of 100 nm, using commonly available fluorophores and a conventional widefield microscope for cryo-correlative light-electron microscopy. Such resolution aids in precisely identifying regions of interest before tomographic acquisition and enhances precision in localizing features of interest within the 3D reconstruction. Dual-axis CSTET tilt series data and application of entropy regularized deconvolution during post-processing results in close-to-isotropic resolution in the reconstruction without averaging. The integration of cryo-SRRF with deconvolved dual-axis CSTET provides a versatile workflow for studying unique objects in a cell.

Birnaviridae Virus Factories Show Features of Liquid-Liquid Phase Separation and Are Distinct from Paracrystalline Arrays of Virions Observed by Electron Microscopy.

To gain more information about the nature of Birnaviridae virus factories (VFs), we used a recombinant infectious bursal disease virus (IBDV) expressing split-GFP11 tagged to the polymerase (VP1) that we have previously shown is a marker for VFs in infected cells expressing GFP1-10. We found that VFs colocalized with 5-ethynyl uridine in the presence of actinomycin, demonstrating they contained newly synthesized viral RNA, and VFs were visible in infected cells that were fixed and permeabilized with digitonin, demonstrating that they were not membrane bound. Fluorescence recovery after photobleaching (FRAP) a region of interest within the VFs occurred rapidly, recovering from approximately 25% to 87% the original intensity over 146 s, and VFs were dissolved by 1,6-hexanediol treatment, demonstrating they showed properties consistent with liquid-liquid phase separation. There was a lower colocalization of the VF GFP signal with the capsid protein VP2 (Manders' coefficient [MC] 0.6), compared to VP3 (MC, 0.9), which prompted us to investigate the VF ultrastructure by transmission electron microscopy (TEM). In infected cells, paracrystalline arrays (PAs) of virions were observed in the cytoplasm, as well as discrete electron dense regions. Using correlative light and electron microscopy (CLEM), we observed that the electron dense regions correlated with the GFP signal of the VFs, which were distinct from the PAs. In summary, Birnaviridae VFs contain newly synthesized viral RNA, are not bound by a membrane, show properties consistent with liquid-liquid phase separation, and are distinct from the PAs observed by TEM. IMPORTANCE Members of the Birnaviridae infect birds, fish and insects, and are responsible for diseases of significant economic importance to the poultry industry and aquaculture. Despite their importance, how they replicate in cells remains poorly understood. Here, we show that the Birnaviridae virus factories are not membrane bound, demonstrate properties consistent with liquid-liquid phase separation, and are distinct from the paracrystalline arrays of virions observed by transmission electron microscopy, enhancing our fundamental knowledge of virus replication that could be used to develop strategies to control disease, or optimize their therapeutic application.

Advanced imaging techniques: microscopy.

For decades, bacteria were thought of as "bags" of enzymes, lacking organelles and significant subcellular structures. This stood in sharp contrast with eukaryotes, where intracellular compartmentalization and the role of large-scale order had been known for a long time. However, the emerging field of Bacterial Cell Biology has established that bacteria are in fact highly organized, with most macromolecular components having specific subcellular locations that can change depending on the cell's physiological state (Barry & Gitai, 2011; Lenz & Søgaard-Andersen, 2011; Thanbichler & Shapiro, 2008). For example, we now know that many processes in bacteria are orchestrated by cytoskeletal proteins, which polymerize into surprisingly diverse superstructures, such as rings, sheets, and tread-milling rods (Pilhofer & Jensen, 2013). These superstructures connect individual proteins, macromolecular assemblies, and even two neighboring cells, to affect essential higher-order processes including cell division, DNA segregation, and motility. Understanding these processes requires resolving the in vivo dynamics and ultrastructure at different functional stages of the cell, at macromolecular resolution and in 3-dimensions (3D). Fluorescence light microscopy (fLM) of tagged proteins is highly valuable for investigating protein localization and dynamics, and the resolution power of transmission electron microscopy (TEM) is required to elucidate the structure of macromolecular complexes in vivo and in vitro. This chapter summarizes the most recent advances in LM and TEM approaches that have revolutionized our knowledge and understanding of the microbial world.

Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in Caulobacter.

Superresolution fluorescence microscopy and cryogenic electron tomography (CET) are powerful imaging methods for exploring the subcellular organization of biomolecules. Superresolution fluorescence microscopy based on covalent labeling highlights specific proteins and has sufficient sensitivity to observe single fluorescent molecules, but the reconstructions lack detailed cellular context. CET has molecular-scale resolution but lacks specific and nonperturbative intracellular labeling techniques. Here, we describe an imaging scheme that correlates cryogenic single-molecule fluorescence localizations with CET reconstructions. Our approach achieves single-molecule localizations with an average lateral precision of 9 nm, and a relative registration error between the set of localizations and CET reconstruction of ∼30 nm. We illustrate the workflow by annotating the positions of three proteins in the bacterium Caulobacter crescentus: McpA, PopZ, and SpmX. McpA, which forms a part of the chemoreceptor array, acts as a validation structure by being visible under both imaging modalities. In contrast, PopZ and SpmX cannot be directly identified in CET. While not directly discernable, PopZ fills a region at the cell poles that is devoid of electron-dense ribosomes. We annotate the position of PopZ with single-molecule localizations and confirm its position within the ribosome excluded region. We further use the locations of PopZ to provide context for localizations of SpmX, a low-copy integral membrane protein sequestered by PopZ as part of a signaling pathway that leads to an asymmetric cell division. Our correlative approach reveals that SpmX localizes along one side of the cell pole and its extent closely matches that of the PopZ region.

Correlated cryogenic fluorescence microscopy and electron cryo-tomography shows that exogenous TRIM5α can form hexagonal lattices or autophagy aggregates in vivo.

Members of the tripartite motif (TRIM) protein family have been shown to assemble into structures in both the nucleus and cytoplasm. One TRIM protein family member, TRIM5α, has been shown to form cytoplasmic bodies involved in restricting retroviruses such as HIV-1. Here we applied cryogenic correlated light and electron microscopy, combined with electron cryo-tomography, to intact mammalian cells expressing YFP-rhTRIM5α and found the presence of hexagonal nets whose arm lengths were similar to those of the hexagonal nets formed by purified TRIM5α in vitro. We also observed YFP-rhTRIM5α within a diversity of structures with characteristics expected for organelles involved in different stages of macroautophagy, including disorganized protein aggregations (sequestosomes), sequestosomes flanked by flat double-membraned vesicles (sequestosome:phagophore complexes), sequestosomes within double-membraned vesicles (autophagosomes), and sequestosomes within multivesicular autophagic vacuoles (amphisomes or autolysosomes). Vaults were also seen in these structures, consistent with their role in autophagy. Our data 1) support recent reports that TRIM5α can form both well-organized signaling complexes and nonsignaling aggregates, 2) offer images of the macroautophagy pathway in a near-native state, and 3) reveal that vaults arrive early in macroautophagy.

Twenty years of traffic: A 2020 vision of cellular electron microscopy.

The last 20 years have seen incredible advances in electron microscopy (EM). Cryoelectron microscopy can now resolve protein structures to a previously unimaginable resolution but the advances in cellular EM are just as significant. I will take this opportunity to briefly summarize some of the new developments in cellular EM.

Identification and demonstration of roGFP2 as an environmental sensor for cryogenic correlative light and electron microscopy.

Cryogenic correlative light and electron microscopy (cryo-CLEM) seeks to leverage orthogonal information present in two powerful imaging modalities. While recent advances in cryogenic electron microscopy (cryo-EM) allow for the visualization and identification of structures within cells at the nanometer scale, information regarding the cellular environment, such as pH, membrane potential, ionic strength, etc., which influences the observed structures remains absent. Fluorescence microscopy can potentially be used to reveal this information when specific labels, known as fluorescent biosensors, are used, but there has been minimal use of such biosensors in cryo-CLEM to date. Here we demonstrate the applicability of one such biosensor, the fluorescent protein roGFP2, for cryo-CLEM experiments. At room temperature, the ratio of roGFP2 emission brightness when excited at 425 nm or 488 nm is known to report on the local redox potential. When samples containing roGFP2 are rapidly cooled to 77 K in a manner compatible with cryo-EM, the ratio of excitation peaks remains a faithful indicator of the redox potential at the time of freezing. Using purified protein in different oxidizing/reducing environments, we generate a calibration curve which can be used to analyze in situ measurements. As a proof-of-principle demonstration, we investigate the oxidation/reduction state within vitrified Caulobacter crescentus cells. The polar organizing protein Z (PopZ) localizes to the polar regions of C. crescentus where it is known to form a distinct microdomain. By expressing an inducible roGFP2-PopZ fusion we visualize individual microdomains in the context of their redox environment.

Metallic support films reduce optical heating in cryogenic correlative light and electron tomography.

Super-resolved cryogenic correlative light and electron tomography is an emerging method that provides both the single-molecule sensitivity and specificity of fluorescence imaging, and the molecular scale resolution and detailed cellular context of tomography, all in vitrified cells preserved in their native hydrated state. Technical hurdles that limit these correlative experiments need to be overcome for the full potential of this approach to be realized. Chief among these is sample heating due to optical excitation which leads to devitrification, a phase transition from amorphous to crystalline ice. Here we show that much of this heating is due to the material properties of the support film of the electron microscopy grid, specifically the absorptivity and thermal conductivity. We demonstrate through experiment and simulation that the properties of the standard holey carbon electron microscopy grid lead to substantial heating under optical excitation. In order to avoid devitrification, optical excitation intensities must be kept orders of magnitude lower than the intensities commonly employed in room temperature super-resolution experiments. We further show that the use of metallic films, either holey gold grids, or custom made holey silver grids, alleviate much of this heating. For example, the holey silver grids permit 20× the optical intensities used on the standard holey carbon grids. Super-resolution correlative experiments conducted on holey silver grids under these increased optical excitation intensities have a corresponding increase in the rate of single-molecule fluorescence localizations. This results in an increased density of localizations and improved correlative imaging without deleterious effects from sample heating.

Quantitative 3D correlative light and electron microscopy of organelle association during autophagy.

In macroautophagy, disk-shaped double-membrane structures called phagophores elongate to form cup-shaped structures, becoming autophagosomes upon closure. These autophagosomes then fuse with lysosomes to become autolysosomes and degrade engulfed material. Autophagosome formation is reported to involve other organelles, including the endoplasmic reticulum (ER) and mitochondria. Organelles are also taken up by autophagosomes as autophagy cargos. However, few studies have performed systematic spatiotemporal analysis of inter-organelle relationships during macroautophagy. Here, we investigated the organelles in contact with phagophores, autophagosomes, and autolysosomes by using three-dimensional correlative light and electron microscopy with array tomography in cells starved 30 min. As previously reported, all phagophores associate with the ER. The surface area of phagophores in contact with the ER decreases gradually as they mature into autophagosomes and autolysosomes. However, the ER still associates with 92% of autophagosomes and 79% of autolysosomes, suggesting that most autophagosomes remain on the ER after closure and even when they fuse with lysosomes. In addition, we found that phagophores form frequently near other autophagic structures, suggesting the presence of potential hot spots for autophagosome formation. We also analyzed the contents of phagophores and autophagosomes and found that the ER is the most frequently engulfed organelle (detected in 65% of total phagophores and autophagosomes). These quantitative three-dimensional ultrastructural data provide insights into autophagosome-organelle relationships during macroautophagy.Key words: 3D-CLEM, autophagosome, electron microscopy, endoplasmic reticulum, lysosome.