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Synaptic vesicle (SV) fusion with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. The effect of persistent high or low exocytosis activity on intermediate steps remains unknown. Using spray-mixing plunge-freezing cryo-electron tomography we observe events following synaptic stimulation at nanometer resolution in near-native samples. Our data suggest that during the stage that immediately follows stimulation, termed early fusion, PM and SV membrane curvature changes to establish a point contact. The next stage—late fusion—shows fusion pore opening and SV collapse. During early fusion, proximal tethered SVs form additional tethers with the PM and increase the inter-SV connector number. In the late-fusion stage, PM-proximal SVs lose their interconnections, allowing them to move toward the PM. Two SNAP-25 mutations, one arresting and one disinhibiting spontaneous release, cause connector loss. The disinhibiting mutation causes loss of membrane-proximal multiple-tethered SVs. Overall, tether formation and connector dissolution are triggered by stimulation and respond to spontaneous fusion rate manipulation. These morphological observations likely correspond to SV transition from one functional pool to another. Synaptic vesicle (SV) fusion with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. The effect of persistent high or low exocytosis activity on intermediate steps remains unknown. Using spray-mixing plunge-freezing cryo-electron tomography we observe events following synaptic stimulation at nanometer resolution in near-native samples. Our data suggest that during the stage that immediately follows stimulation, termed early fusion, PM and SV membrane curvature changes to establish a point contact. The next stage—late fusion—shows fusion pore opening and SV collapse. During early fusion, proximal tethered SVs form additional tethers with the PM and increase the inter-SV connector number. In the late-fusion stage, PM-proximal SVs lose their interconnections, allowing them to move toward the PM. Two SNAP-25 mutations, one arresting and one disinhibiting spontaneous release, cause connector loss. The disinhibiting mutation causes loss of membrane-proximal multiple-tethered SVs. Overall, tether formation and connector dissolution are triggered by stimulation and respond to spontaneous fusion rate manipulation. These morphological observations likely correspond to SV transition from one functional pool to another.

In contrast to many eukaryotic organisms, trypanosomes only contain a single mitochondrion per cell. Within that singular mitochondrion, the protist carries a single mitochondrial genome that consists of a complex DNA network, the kinetoplast DNA (kDNA). Segregation of the replicated kDNA is coordinated by the basal body of the cell’s single flagellum. The tripartite attachment complex (TAC) forms a physical connection between the proximal end of the basal body and the kDNA. This allows anchoring of the kDNA throughout the cell cycle and couples kDNA segregation with the separation of the basal bodies prior to cell division. Over the past years, several components of the TAC have been identified. To shed light on the structure of the cytoplasmic part of the TAC (known as the exclusion zone), we performed cryo-electron tomography on whole cells. This allowed us to acquire three-dimensional high-resolution images of the exclusion zonein situ. We observed that the exclusion zone filaments offer great mechanical flexibility for basal body movement. We measured the dimensions of the individual structural elements of the area, as well as the overall orientation and positioning of the basal bodies towards the mitochondrial kDNA pocket. Using a combination of experimental data and modelling, we generated a structural model of the exclusion zone protein p197. Our findings suggest that the majority of p197 consists of a string of spectrin-like repeats. We propose that these structural units provide the architecture of a molecular spring and that they are required in the TAC to withstand the mechanical forces generated through basal body repositioning events during kDNA segregation and motility of the organism.

SummarySince the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, investigation of the SARS-CoV-2 infection in the native cellular context is scarce, and there is a lack of comprehensive knowledge on SARS-CoV-2 replicative cycle. Understanding the genome replication, assembly and egress of SARS-CoV-2, a multistage process that involves different cellular compartments and the activity of many viral and cellular proteins, is critically important as it bears the means of medical intervention to stop infection. Here, we investigated SARS-CoV-2 replication in Vero cells under the near-native frozen-hydrated condition using a unique correlative multi-modal, multi-scale cryo-imaging approach combining soft X-ray cryo-tomography and serial cryoFIB/SEM volume imaging of the entire SARS-CoV-2 infected cell with cryo-electron tomography (cryoET) of cellular lamellae and cell periphery, as well as structure determination of viral components by subtomogram averaging. Our results reveal at the whole cell level profound cytopathic effects of SARS-CoV-2 infection, exemplified by a large amount of heterogeneous vesicles in the cytoplasm for RNA synthesis and virus assembly, formation of membrane tunnels through which viruses exit, and drastic cytoplasm invasion into nucleus. Furthermore, cryoET of cell lamellae reveals how viral RNAs are transported from double-membrane vesicles where they are synthesized to viral assembly sites; how viral spikes and RNPs assist in virus assembly and budding; and how fully assembled virus particles exit the cell, thus stablishing a model of SARS-CoV-2 genome replication, virus assembly and egress pathways.

SummaryLarge trimeric Spikes decorate SARS-CoV-2 and bind host cells via receptor binding domains (RBDs). We report a conformation in which the trimer is ‘locked’ into a compact well-ordered form. This differs from previous structures where the RBD can flip up to recognise the receptor. In the ‘locked’ form regions associated with fusion transitions are stabilised and the RBD harbours curved lipids. The acyl chains bind a hydrophobic pocket in one RBD whilst the polar headgroups attach to an adjacent RBD of the trimer. By functional analogy with enteroviral pocket factors loss of the lipid would destabilise the ‘locked’ form facilitating receptor attachment, conversion to the postfusion state and virus infection. The nature of lipids available at the site of infection might affect the antigenicity/pathogenicity of released virus. These results reveal a potentially druggable pocket and suggest that the natural prefusion state occludes neutralising RBD epitopes, achieving conformational shielding from antibodies.HighlightsSARS-CoV-2 Spike can adopt a ‘locked’ conformation with all receptor binding domains (RBDs) down, likely to represent the prefusion resting stateThis ‘locked’ conformation is compact and stable, braced by lipid bound within a potentially druggable pocketKey neutralization epitopes are shielded in the ‘locked’ formLoss of lipid may trigger a cascade of events that lead to cell entry analogous to the role of lipids in enterovirus cell entry

The COVID-19 pandemic has had unprecedented health and economic impact, but currently there are no approved therapies. We have isolated an antibody, EY6A, from a late-stage COVID-19 patient and show it neutralises SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds tightly (KD of 2 nM) the receptor binding domain (RBD) of the viral Spike glycoprotein and a 2.6Å crystal structure of an RBD/EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues of this epitope are key to stabilising the pre-fusion Spike. Cryo-EM analyses of the pre-fusion Spike incubated with EY6A Fab reveal a complex of the intact trimer with three Fabs bound and two further multimeric forms comprising destabilized Spike attached to Fab. EY6A binds what is probably a major neutralising epitope, making it a candidate therapeutic for COVID-19.

The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.

SummaryThere are as yet no licenced therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric Spike whose receptor binding domain (RBD) recognizes angiotensin-converting enzyme 2 (ACE2), initiating conformational changes that drive membrane fusion. We find that monoclonal antibody CR3022 binds the RBD tightly, neutralising SARS-CoV-2 and report the crystal structure at 2.4 Å of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilising, CR3022 epitope is inaccessible in the prefusion Spike, suggesting that CR3022 binding would facilitate conversion to the fusion-incompetent post-fusion state. Cryo-EM analysis confirms that incubation of Spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope may be useful therapeutically, possibly in synergy with an antibody blocking receptor attachment.HighlightsCR3022 neutralises SARS-CoV-2Neutralisation is by destroying the prefusion SPIKE conformationThis antibody may have therapeutic potential alone or with one blocking receptor attachment

Summary There are as yet no licensed therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric spike whose receptor binding domain (RBD) recognizes angiotensin-converting enzyme 2, initiating conformational changes that drive membrane fusion. We find that the monoclonal antibody CR3022 binds the RBD tightly, neutralizing SARS-CoV-2, and report the crystal structure at 2.4 Å of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilizing CR3022 epitope is inaccessible in the prefusion spike, suggesting that CR3022 binding facilitates conversion to the fusion-incompetent post-fusion state. Cryogenic electron microscopy (cryo-EM) analysis confirms that incubation of spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope could be useful therapeutically, possibly in synergy with an antibody that blocks receptor attachment., Graphical Abstract, Highlights • CR3022 binds the RBD of SARS-CoV-2 and shows strong neutralization • Neutralization is by destroying the prefusion spike conformation • CR3022 binds a highly conserved epitope that is inaccessible in prefusion spike protein • CR3022 could have therapeutic potential alone or in synergy with a receptor blocker, Huo et al. find that the antibody CR3022 binds tightly to the receptor binding domain of the SARS-CoV-2 spike at a site different to that used by the receptor. CR3022 effectively neutralizes the virus, and cryo-EM reveals that it disrupts the spike. Such antibodies could have potential as COVID-19 therapeutics.

Large trimeric Spikes decorate SARS-CoV-2 and bind host cells via receptor binding domains (RBDs). We report a conformation in which the trimer is ‘locked’ into a compact well-ordered form. This differs from previous structures where the RBD can flip up to recognise the receptor. In the ‘locked’ form regions associated with fusion transitions are stabilised and the RBD harbours curved lipids. The acyl chains bind a hydrophobic pocket in one RBD whilst the polar headgroups attach to an adjacent RBD of the trimer. By functional analogy with enteroviral pocket factors loss of the lipid would destabilise the ‘locked’ form facilitating receptor attachment, conversion to the postfusion state and virus infection. The nature of lipids available at the site of infection might affect the antigenicity/pathogenicity of released virus. These results reveal a potentially druggable pocket and suggest that the natural prefusion state occludes neutralising RBD epitopes, achieving conformational shielding from antibodies.

Background Streptococcus pneumoniae is a potent human pathogen. Its pore-forming exotoxin pneumolysin is instrumental for breaching the host's epithelial barrier and for the incapacitation of the immune system. Methods and results Using a combination of life imaging and cryo-electron microscopy we show that pneumolysin, released by cultured bacteria, is capable of permeabilizing the plasmalemma of host cells. However, such permeabilization does not lead to cell lysis since pneumolysin is actively removed by the host cells. The process of pore elimination starts with the formation of pore-bearing plasmalemmal nanotubes and proceeds by the shedding of pores that are embedded in the membrane of released microvesicles. Pneumolysin prepores are likewise removed. The protein composition of the toxin-induced microvesicles, assessed by mass spectrometry, is suggestive of a Ca 2 + -triggered mechanism encompassing the proteins of the annexin family and members of the endosomal sorting complex required for transport (ESCRT) complex. Conclusions S. pneumoniae releases sufficient amounts of pneumolysin to perforate the plasmalemma of host cells, however, the immediate cell lysis, which is frequently reported as a result of treatment with purified and artificially concentrated toxin, appears to be an unlikely event in vivo since the toxin pores are efficiently eliminated by microvesicle shedding. Therefore the dysregulation of cellular homeostasis occurring as a result of transient pore formation/elimination should be held responsible for the damaging toxin action. General significance We have achieved a comprehensive view of a general plasma membrane repair mechanism after injury by a major bacterial toxin.

SNARE-complexes drive the fusion of membrane-bound vesicles with target membranes or with each other (homotypic fusion). The SNARE-proteins are subdivided into Qa, Qb, Qc and R-SNAREs depending on their position in the four-helical SNARE-bundle. Here, we review the SNAP-25 protein sub-family, which includes both the Qb and Qc SNARE-domains within a single protein. In vertebrates, this sub-family consists of SNAP-25, SNAP-23, SNAP-29 and SNAP-47, named for their apparent molecular weights. SNAP-25 and SNAP-23 are specialized for driving regulated exocytosis. SNAP-25 performs this function in the nervous system, and in neuroendocrine cells, where fast Ca2+-dependent triggering is required in order to synchronize release with an electrical signal, whereas SNAP-23 drives regulated exocytosis in most other cases that have been studied, e.g. platelet exocytosis or glucose transporter trafficking. SNAP-25 is regulated by alternative splicing, phosphorylation and by G-protein binding, and it regulates Ca2+-channels, neuronal survival and postsynaptic spine development. SNAP-23 is primarily regulated by phosphorylation within the linker connecting Qb to Qc. Cross-rescue experiments show that SNAP-25 and SNAP-23 can (at least partly) substitute for each other, whereas SNAP-29 and SNAP-47 cannot. SNAP-29 is present on intracellular membranes and performs functions in autophagosome-to-lysosome fusion, among others. An overlapping function for SNAP-47 was described; in addition, SNAP-47 mediates postsynaptic AMPA-receptor insertion. Overall, the presence of two SNARE-domains confers members of this family the ability to associate to different Qa and R-SNAREs and drive diverse membrane fusion reactions; one member of the family, SNAP-25, has been devoted entirely to Ca2+-triggered fusion and has taken on a number of additional, regulatory roles.

Spinocerebellar ataxia type 1 (SCA1), due to an unstable polyglutamine expansion within the ubiquitously expressed Ataxin-1 protein, leads to the premature degeneration of Purkinje cells (PCs), decreasing motor coordination and causing death within 10-15 years of diagnosis. Currently, there are no therapies available to slow down disease progression. As secondary cellular impairments contributing to SCA1 progression are poorly understood, here, we focused on identifying those processes by performing a PC specific proteome profiling of Sca1154Q/2Q mice at a symptomatic stage. Mass spectrometry analysis revealed prominent alterations in mitochondrial proteins. Immunohistochemical and serial block-face scanning electron microscopy analyses confirmed that PCs underwent age-dependent alterations in mitochondrial morphology. Moreover, colorimetric assays demonstrated impairment of the electron transport chain complexes (ETC) and decrease in ATPase activity. Subsequently, we examined whether the mitochondria-targeted antioxidant MitoQ could restore mitochondrial dysfunction and prevent SCA1-associated pathology in Sca1154Q/2Q mice. MitoQ treatment both presymptomatically and when symptoms were evident ameliorated mitochondrial morphology and restored the activities of the ETC complexes. Notably, MitoQ slowed down the appearance of SCA1-linked neuropathology such as lack of motor coordination as well as preventing oxidative stress-induced DNA / RNA damage and PC loss. Our work identifies a central role for mitochondria in PC degeneration in SCA1 and provides evidence for the supportive use of mitochondria-targeted therapeutics in slowing down disease progression.

1AbstractSerial block face scanning electron microscopy (SBFSEM) is an increasingly popular method for investigating the three-dimensional ultrastructure of large biological samples. Prior to imaging, samples are typically chemically fixed, stained with osmium and uranyl acetate, and subsequently embedded in resin. The purpose of staining is to provide image contrast and reduce specimen charging under the electron beam, which is detrimental to the quality of imaging. Obtaining, using, and disposing of uranyl acetate is getting increasingly cumbersome in many countries due to new regulations on the handling of radioactive substances. Therefore, we developed an alternative staining procedure that does not rely on the use of uranium or any other radioactive substance. This procedure provides excellent contrast and efficiently reduces specimen charging.

Spinocerebellar ataxia type 1 (SCA1), due to the expansion of a polyglutamine repeat within the ubiquitously expressed Ataxin-1 protein, leads to the premature degeneration of Purkinje cells (PCs), the cause of which is poorly understood. Here, we identified the unique proteomic signature of Sca1(154Q/2Q) PCs at an early stage of disease, highlighting extensive alterations in proteins associated with synaptic functioning, maintenance, and transmission. Focusing on Homer-3, a PC-enriched scaffold protein regulating neuronal activity, revealed an early decline in its expression. Impaired climbing fiber-mediated synaptic transmission diminished mTORC1 signaling, paralleling Homer-3 reduction in Sca1(154Q/2Q) PCs. Ablating mTORC1 within PCs or pharmacological inhibition of mTORC1 identified Homer-3 as its downstream target. mTORC1 knockout in Sca1(154Q/2Q) PCs exacerbated and accelerated pathology. Reinstating Homer-3 expression in Sca1(154Q/2Q) PCs attenuated cellular dysfunctions and improved motor deficits. Our work reveals that impaired mTORC1-Homer-3 activity underlies PC susceptibility in SCA1 and presents a promising therapeutic target.

The onset of action of antidepressants (ADs) usually takes several weeks, but first molecular responses to these drugs may appear already after acute administration. The Extracellular Signal-regulated Kinase/Mitogen-Activated Protein Kinase (ERK/MAPK) signaling pathway is a target of ADs and an important pathway involved in cellular plasticity. In major depressive disorder (MDD), especially the prefrontal cortex (PFC) and hippocampus (Hip) are most likely affected in depressive patients and recent work revealed a hyperactivated ERK signaling in the rat PFC after chronic stress, a precipitating factor for MDD. Strong evidences support that not only neurons but also astrocytes participate in neuronal activity and may therefore additionally be a substrate of AD action. In this study, we show by Western blot that neither fluoxetine (FLX) nor desipramine (DMI) preferentially affects the activation of one of the two ERK isoforms, ERK1 and ERK2, with respect to the other. Further immunohistochemical analysis in the PFC revealed that basal levels of phospho-activated ERK (pERK) are mostly found in neurons in contrast to very few astrocytes. Both ADs can inhibit neuronal pERK as early as 15 min after drug administration with peculiar regional and layer specificities. Contrarily, at this time point none of the two ADs shows a clear modulation of astrocytic pERK. We propose that this mechanism of action of ADs may be protective against an exacerbated cortical ERK activity that may exert detrimental effects on susceptible neuronal populations. Our findings on acute effects of AD treatment in the adult mouse PFC encourage to examine further how this treatment might influence pERK in animal models of depression to identify early targets of AD action.