UFM1 E3 ligase promotes recycling of 60S ribosomal subunits from the ER.

Reversible modification of target proteins by ubiquitin and ubiquitin-like proteins (UBLs) is widely used by eukaryotic cells to control protein fate and cell behaviour1. UFM1 is a UBL that predominantly modifies a single lysine residue on a single ribosomal protein, uL24 (also called RPL26), on ribosomes at the cytoplasmic surface of the endoplasmic reticulum (ER)2,3. UFM1 conjugation (UFMylation) facilitates the rescue of 60S ribosomal subunits (60S) that are released after ribosome-associated quality-control-mediated splitting of ribosomes that stall during co-translational translocation of secretory proteins into the ER3,4. Neither the molecular mechanism by which the UFMylation machinery achieves such precise target selection nor how this ribosomal modification promotes 60S rescue is known. Here we show that ribosome UFMylation in vivo occurs on free 60S and we present sequential cryo-electron microscopy snapshots of the heterotrimeric UFM1 E3 ligase (E3(UFM1)) engaging its substrate uL24. E3(UFM1) binds the L1 stalk, empty transfer RNA-binding sites and the peptidyl transferase centre through carboxy-terminal domains of UFL1, which results in uL24 modification more than 150 Å away. After catalysing UFM1 transfer, E3(UFM1) remains stably bound to its product, UFMylated 60S, forming a C-shaped clamp that extends all the way around the 60S from the transfer RNA-binding sites to the polypeptide tunnel exit. Our structural and biochemical analyses suggest a role for E3(UFM1) in post-termination release and recycling of the large ribosomal subunit from the ER membrane.

Mitochondria shed their outer membrane in response to infection-induced stress.

The outer mitochondrial membrane (OMM) is essential for cellular homeostasis. Yet little is known of the mechanisms that remodel it during natural stresses. We found that large "SPOTs" (structures positive for OMM) emerge during Toxoplasma gondii infection in mammalian cells. SPOTs mediated the depletion of the OMM proteins mitofusin 1 and 2, which restrict parasite growth. The formation of SPOTs depended on the parasite effector TgMAF1 and the host mitochondrial import receptor TOM70, which is required for optimal parasite proliferation. TOM70 enabled TgMAF1 to interact with the host OMM translocase SAM50. The ablation of SAM50 or the overexpression of an OMM-targeted protein promoted OMM remodeling independently of infection. Thus, Toxoplasma hijacks the formation of SPOTs, a cellular response to OMM stress, to promote its growth.

Cryo-EM structures of the endoplasmic reticulum membrane complex.

The transmembrane α-helices of membrane proteins are in general highly hydrophobic, and they enter the lipid bilayer through a lateral gate in the Sec61 translocon. However, some transmembrane α-helices are less hydrophobic and form membrane channels or substrate-binding pockets. Insertion of these amphipathic transmembrane α-helices into the membrane requires the specific membrane-embedded insertase called the endoplasmic reticulum membrane complex (EMC), which is a multi-subunit chaperone distinct from the GET insertase complex. Four recent cryo-electron microscopy studies on the eukaryotic EMC have revealed their remarkable architectural conservation from yeast to humans; a general consensus on the substrate transmembrane helix-binding pocket; and the evolutionary link to the prokaryotic insertases of the tail-anchored membrane proteins. These structures provide a solid framework for future mechanistic investigation.

Lunapark-dependent formation of a virus-induced ER exit site contains multi-tubular ER junctions that promote viral ER-to-cytosol escape.

Viruses rearrange host membranes to support different entry steps. Polyomavirus simian virus 40 (SV40) reorganizes the endoplasmic reticulum (ER) membrane to generate focus structures that enable virus ER-to-cytosol escape, a decisive infection step. The molecular architecture of the ER exit site that might illuminate why it is ideally suited for membrane penetration is unknown. Here 3D focused ion beam scanning electron microscopy (FIB-SEM) reconstruction reveals that the ER focus structure consists of multi-tubular ER junctions where SV40 preferentially localizes, suggesting that tubular branch points are virus ER-to-cytosol penetration sites. Functional analysis demonstrates that lunapark-an ER membrane protein that typically stabilizes three-way ER junctions-relocates to the ER foci, where it supports focus formation, leading to SV40 ER escape and infection. Our results reveal how a virus repurposes the activity of an ER membrane protein to form a virus-induced ER substructure required for membrane escape and suggest that ER tubular junctions are vulnerable sites exploited by viruses for membrane penetration.

Tumor-induced reshuffling of lipid composition on the endoplasmic reticulum membrane sustains macrophage survival and pro-tumorigenic activity.

Tumor-associated macrophages (TAMs) display pro-tumorigenic phenotypes for supporting tumor progression in response to microenvironmental cues imposed by tumor and stromal cells. However, the underlying mechanisms by which tumor cells instruct TAM behavior remain elusive. Here, we uncover that tumor-cell-derived glucosylceramide stimulated unconventional endoplasmic reticulum (ER) stress responses by inducing reshuffling of lipid composition and saturation on the ER membrane in macrophages, which induced IRE1-mediated spliced XBP1 production and STAT3 activation. The cooperation of spliced XBP1 and STAT3 reinforced the pro-tumorigenic phenotype and expression of immunosuppressive genes. Ablation of XBP1 expression with genetic manipulation or ameliorating ER stress responses by facilitating LPCAT3-mediated incorporation of unsaturated lipids to the phosphatidylcholine hampered pro-tumorigenic phenotype and survival in TAMs. Together, we uncover the unexpected roles of tumor-cell-produced lipids that simultaneously orchestrate macrophage polarization and survival in tumors via induction of ER stress responses and reveal therapeutic targets for sustaining host antitumor immunity.

Disruption of the Golgi Apparatus and Contribution of the Endoplasmic Reticulum to the SARS-CoV-2 Replication Complex.

A variety of immunolabeling procedures for both light and electron microscopy were used to examine the cellular origins of the host membranes supporting the SARS-CoV-2 replication complex. The endoplasmic reticulum has long been implicated as a source of membrane for the coronavirus replication organelle. Using dsRNA as a marker for sites of viral RNA synthesis, we provide additional evidence supporting ER as a prominent source of membrane. In addition, we observed a rapid fragmentation of the Golgi apparatus which is visible by 6 h and complete by 12 h post-infection. Golgi derived lipid appears to be incorporated into the replication organelle although protein markers are dispersed throughout the infected cell. The mechanism of Golgi disruption is undefined, but chemical disruption of the Golgi apparatus by brefeldin A is inhibitory to viral replication. A search for an individual SARS-CoV-2 protein responsible for this activity identified at least five viral proteins, M, S, E, Orf6, and nsp3, that induced Golgi fragmentation when expressed in eukaryotic cells. Each of these proteins, as well as nsp4, also caused visible changes to ER structure as shown by correlative light and electron microscopy (CLEM). Collectively, these results imply that specific disruption of the Golgi apparatus is a critical component of coronavirus replication.

Rapid tool for cell nanoarchitecture integrity assessment.

With the rapid increase and accessibility of high-resolution imaging technologies of cells, the interpretation of results relies more and more on the assumption that the three-dimensional integrity of the surrounding cellular landscape is not compromised by the experimental setup. However, the only available technology for directly probing the structural integrity of whole-cell preparations at the nanoscale is electron cryo-tomography, which is time-consuming, costly, and complex. We devised an accessible, inexpensive and reliable screening assay to quickly report on the compatibility of experimental protocols with preserving the structural integrity of whole-cell preparations at the nanoscale. Our Rapid Cell Integrity Assessment (RCIA) assay is executed at room temperature and relies solely on light microscopy imaging. Using cellular electron cryo-tomography as a benchmark, we verify that RCIA accurately unveils the adverse impact of reagents and/or protocols such as those used for virus inactivation or to arrest dynamic processes on the cellular nanoarchitecture.

Ultrastructural Features of Membranous Replication Organelles Induced by Positive-Stranded RNA Viruses.

All intracellular pathogens critically depend on host cell organelles and metabolites for successful infection and replication. One hallmark of positive-strand RNA viruses is to induce alterations of the (endo)membrane system in order to shield their double-stranded RNA replication intermediates from detection by the host cell's surveillance systems. This spatial seclusion also allows for accruing host and viral factors and building blocks required for efficient replication of the genome and prevents access of antiviral effectors. Even though the principle is iterated by almost all positive-strand RNA viruses infecting plants and animals, the specific structure and the organellar source of membranes differs. Here, we discuss the characteristic ultrastructural features of the virus-induced membranous replication organelles in plant and animal cells and the scientific progress gained by advanced microscopy methods.

Probing the biogenesis pathway and dynamics of thylakoid membranes.

How thylakoid membranes are generated to form a metabolically active membrane network and how thylakoid membranes orchestrate the insertion and localization of protein complexes for efficient electron flux remain elusive. Here, we develop a method to modulate thylakoid biogenesis in the rod-shaped cyanobacterium Synechococcus elongatus PCC 7942 by modulating light intensity during cell growth, and probe the spatial-temporal stepwise biogenesis process of thylakoid membranes in cells. Our results reveal that the plasma membrane and regularly arranged concentric thylakoid layers have no physical connections. The newly synthesized thylakoid membrane fragments emerge between the plasma membrane and pre-existing thylakoids. Photosystem I monomers appear in the thylakoid membranes earlier than other mature photosystem assemblies, followed by generation of Photosystem I trimers and Photosystem II complexes. Redistribution of photosynthetic complexes during thylakoid biogenesis ensures establishment of the spatial organization of the functional thylakoid network. This study provides insights into the dynamic biogenesis process and maturation of the functional photosynthetic machinery.

Ultrastructure of the Mitochondria-Associated Membranes in Human Tumor Specimens.

Conventional transmission electron microscopy is an essential tool to understand the structure-function relationships and play a vital role in biological research. Mitochondria-associated membranes are linked with cancer processes in a fundamental manner. A conventional transmission electron microscopy method for preparing specimens in clinical and research settings for the study-analysis of the mitochondria-associated membranes in human tumors is presented. The sample processing includes chemical fixation by immersion, dehydration, embedding, polymerization, sectioning, and staining.

IntEResting structures: formation and applications of organized smooth endoplasmic reticulum in plant cells.

The endoplasmic reticulum (ER) is an organelle with remarkable plasticity, capable of rapidly changing its structure to accommodate different functions based on intra- and extracellular cues. One of the ER structures observed in plants is known as "organized smooth endoplasmic reticulum" (OSER), consisting of symmetrically stacked ER membrane arrays. In plants, these structures were first described in certain specialized tissues, e.g. the sieve elements of the phloem, and more recently in transgenic plants overexpressing ER membrane resident proteins. To date, much of the investigation of OSER focused on yeast and animal cells but research into plant OSER has started to grow. In this update, we give a succinct overview of research into the OSER phenomenon in plant cells with case studies highlighting both native and synthetic occurrences of OSER. We also assess the primary driving forces that trigger the formation of OSER, collating evidence from the literature to compare two competing theories for the origin of OSER: that OSER formation is initiated by oligomerizing protein accumulation in the ER membrane or that OSER is the result of ER membrane proliferation. This has long been a source of controversy in the field and here we suggest a way to integrate arguments from both sides into a single unifying theory. Finally, we discuss the potential biotechnological uses of OSER as a tool for the nascent plant synthetic biology field with possible applications as a synthetic microdomain for metabolic engineering and as an extensive membrane surface for synthetic chemistry or protein accumulation.

Dynamic remodeling of host membranes by self-organizing bacterial effectors.

During infection, intracellular bacterial pathogens translocate a variety of effectors into host cells that modify host membrane trafficking for their benefit. We found a self-organizing system consisting of a bacterial phosphoinositide kinase and its opposing phosphatase that formed spatiotemporal patterns, including traveling waves, to remodel host cellular membranes. The Legionella effector MavQ, a phosphatidylinositol (PI) 3-kinase, was targeted to the endoplasmic reticulum (ER). MavQ and the Legionella PI 3-phosphatase SidP, even in the absence of other bacterial components, drove rapid PI 3-phosphate turnover on the ER and spontaneously formed traveling waves that spread along ER subdomains inducing vesicle and tubule budding. Thus, bacteria can exploit a self-organizing membrane-targeting mechanism to hijack host cellular structures for survival.

Molecular landscape of etioplast inner membranes in higher plants.

Etioplasts are photosynthetically inactive plastids that accumulate when light levels are too low for chloroplast maturation. The etioplast inner membrane consists of a paracrystalline tubular lattice and peripheral, disk-shaped membranes, respectively known as the prolamellar body and prothylakoids. These distinct membrane regions are connected into one continuous compartment. To date, no structures of protein complexes in or at etioplast membranes have been reported. Here, we used electron cryo-tomography to explore the molecular membrane landscape of pea and maize etioplasts. Our tomographic reconstructions show that ATP synthase monomers are enriched in the prothylakoids, and plastid ribosomes in the tubular lattice. The entire tubular lattice is covered by regular helical arrays of a membrane-associated protein, which we identified as the 37-kDa enzyme, light-dependent protochlorophyllide oxidoreductase (LPOR). LPOR is the most abundant protein in the etioplast, where it is responsible for chlorophyll biosynthesis, photoprotection and defining the membrane geometry of the prolamellar body. Based on the 9-Å-resolution volume of the subtomogram average, we propose a structural model of membrane-associated LPOR.

Preparation and biological characteristics of a bovine acellular tendon fiber material.

Acellular tendon matrix is an ideal substitute for constructing tissue engineering ligaments, but using detergents causes damage to collagen and fibrin during the process of decellularization. In this study, fresh tendons were lyophilized and separated into fresh tendon fiber (FTF) bundles, and then the cellular components in FTF were removed to prepare acellular tendon fiber (ATF) without adding chemical detergent. H&E staining and DAPI fluorescence microscopy showed no nucleus and DNA residue. Compared with FTFs, the DNA content of ATFs was significantly lower without the collagen content change before and after decellularization. The microstructure of collagen fibrils in ATFs was intact under scanning electron microscopy (SEM), and the maximum tensile load and elastic modulus between FTFs and ATFs were not statistically different. The ATF bundles were cultured with SD rat tenocytes for 72 hr and cells attachment to fiber surfaces were observed under SEM. ATF bundles were then implanted into paraspinal muscles, and histological analysis showed fibroblast-like cells within the ATFs and was similar to the control group (fresh tendon autograft) in morphology. H&E staining showed that the number of lymphocytes and plasma cells in ATF was less than that in fresh tendon autograft. ATF bundles were twisted into linear fiber materials by hand, of which the maximum breaking strength was similar to silk with same diameter. These findings demonstrated that ATFs retain their original fibril structure and mechanical properties after decellularization by trypsin and pancreatic deoxyribonuclease without detergent. Lyophilized ATFs linear fiber material provides the possibility of preparing personalized ligament and other tissue engineering scaffolds.

Multiscale Electron Microscopy for the Study of Viral Replication Organelles.

During infection with positive-strand RNA viruses, viral RNA synthesis associates with modified intracellular membranes that form unique and captivating structures in the cytoplasm of the infected cell. These viral replication organelles (ROs) play a key role in the replicative cycle of important human pathogens like coronaviruses, enteroviruses, or flaviviruses. From their discovery to date, progress in our understanding of viral ROs has closely followed new developments in electron microscopy (EM). This review gives a chronological account of this progress and an introduction to the different EM techniques that enabled it. With an ample repertoire of imaging modalities, EM is nowadays a versatile technique that provides structural and functional information at a wide range of scales. Together with well-established approaches like electron tomography or labeling methods, we examine more recent developments, such as volume scanning electron microscopy (SEM) and in situ cryotomography, which are only beginning to be applied to the study of viral ROs. We also highlight the first cryotomography analyses of viral ROs, which have led to the discovery of macromolecular complexes that may serve as RO channels that control the export of newly-made viral RNA. These studies are key first steps towards elucidating the macromolecular complexity of viral ROs.

Intracellular host cell membrane remodelling induced by SARS-CoV-2 infection in vitro.

BACKGROUND INFORMATION: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection induces an alteration in the endomembrane system of the mammalian cells. In this study, we used transmission electron microscopy and electron tomography to investigate the main structural alterations in the cytoplasm of Vero cells infected with a SARS-CoV-2 isolate from São Paulo state (Brazil). RESULTS: Different membranous structures derived from the zippered endoplasmic reticulum were observed along with virus assembly through membrane budding. Also, we demonstrated the occurrence of annulate lamellae in the cytoplasm of infected cells and the presence of virus particles in the perinuclear space. CONCLUSIONS AND SIGNIFICANCE: This study contributes to a better understanding of the cell biology of SARS-CoV-2 and the mechanisms of the interaction of the virus with the host cell that promote morphological changes, recruitment of organelles and cell components, in a context of a virus-induced membrane remodelling.

Post-correlation on-lamella cryo-CLEM reveals the membrane architecture of lamellar bodies.

Lamellar bodies (LBs) are surfactant-rich organelles in alveolar cells. LBs disassemble into a lipid-protein network that reduces surface tension and facilitates gas exchange in the alveolar cavity. Current knowledge of LB architecture is predominantly based on electron microscopy studies using disruptive sample preparation methods. We established and validated a post-correlation on-lamella cryo-correlative light and electron microscopy approach for cryo-FIB milled cells to structurally characterize and validate the identity of LBs in their unperturbed state. Using deconvolution and 3D image registration, we were able to identify fluorescently labeled membrane structures analyzed by cryo-electron tomography. In situ cryo-electron tomography of A549 cells as well as primary Human Small Airway Epithelial Cells revealed that LBs are composed of membrane sheets frequently attached to the limiting membrane through "T"-junctions. We report a so far undescribed outer membrane dome protein complex (OMDP) on the limiting membrane of LBs. Our data suggest that LB biogenesis is driven by parallel membrane sheet import and by the curvature of the limiting membrane to maximize lipid storage capacity.

Better to keep in touch: investigating inter-organelle cross-talk.

The strategic importance for cellular organelles of being in contact with each other, exchanging messenger molecules, is nowadays well established. Different inter-organelle cross-talk pathways finely regulate multiple physiological cellular mechanisms, and their dysregulation has been found to underlie different pathological conditions. In the last years, a great effort has been made to study such organelle interactions, to understand their functional roles within the cell and the molecules involved in their formation and/or modulation. In this contribution, some examples of organelle cross-talk and their contributions in regulating physiological processes are presented. Moreover, the pro and cons of the available methods for a proper, reliable investigation of membrane contact sites are described.

Lipids and membrane-associated proteins in autophagy.

Autophagy is essential for the maintenance of cellular homeostasis and its dysfunction has been linked to various diseases. Autophagy is a membrane driven process and tightly regulated by membrane-associated proteins. Here, we summarized membrane lipid composition, and membrane-associated proteins relevant to autophagy from a spatiotemporal perspective. In particular, we focused on three important membrane remodeling processes in autophagy, lipid transfer for phagophore elongation, membrane scission for phagophore closure, and autophagosome-lysosome membrane fusion. We discussed the significance of the discoveries in this field and possible avenues to follow for future studies. Finally, we summarized the membrane-associated biochemical techniques and assays used to study membrane properties, with a discussion of their applications in autophagy.