The stressed life of a lipid in the Zika virus membrane.

Protein-lipid interactions modulate a plethora of physiopathologic processes and have been the subject of countless studies. However, these kinds of interactions in the context of viral envelopes have remained relatively unexplored, partially because the intrinsically small dimensions of the molecular systems escape to the current resolution of experimental techniques. However, coarse-grained and multiscale simulations may fill that niche, providing nearly atomistic resolution at an affordable computational price. Here we use multiscale simulations to characterize the lipid-protein interactions in the envelope of the Zika Virus, a prominent member of the Flavivirus genus. Comparisons between the viral envelope and simpler molecular systems indicate that the viral membrane is under extreme pressures and asymmetric forces. Furthermore, the dense net of protein-protein contacts established by the envelope proteins creates poorly solvated regions that destabilize the external leaflet leading to a decoupled dynamics between both membrane layers. These findings lead to the idea that the Flaviviral membrane may store a significant amount of elastic energy, playing an active role in the membrane fusion process.

Bicontinuous inverted cubic phase stabilization as an index of antimicrobial and membrane fusion peptide activity.

Some antimicrobial peptides (AMPs) and membrane fusion-catalyzing peptides (FPs) stabilize bicontinuous inverted cubic (QII) phases. Previous authors proposed a topological rationale: since AMP-induced pores, fusion intermediates, and QII phases all have negative Gaussian curvature (NGC), peptides which produce NGC in one structure also do it in another. This assumes that peptides change the curvature energy of the lipid membranes. Here I test this with a Helfrich curvature energy model. First, experimentally, I show that lipid systems often used to study peptide NGC have NGC without peptides at higher temperatures. To determine the net effect of an AMP on NGC, the equilibrium phase behavior of the host lipids must be determined. Second, the model shows that AMPs must make large changes in the curvature energy to stabilize AMP-induced pores. Peptide-induced changes in elastic constants affect pores and QII phase differently. Changes in spontaneous curvature affect them in opposite ways. The observed correlation between QII phase stabilization and AMP activity doesn't show that AMPs act by lowering pore curvature energy. A different rationale is proposed. In theory, AMPs could simultaneously stabilize QII phase and pores by drastically changing two particular elastic constants. This could be tested by measuring AMP effects on the individual constants. I propose experiments to do that. Unlike AMPs, FPs must make only small changes in the curvature energy to catalyze fusion. It they act in this way, their fusion activity should correlate with their ability to stabilize QII phases.

Reporter gene assay for membrane fusion of extracellular vesicles.

Extracellular vesicles (EVs) secreted by living cells are expected to deliver biological cargo molecules, including RNA and proteins, to the cytoplasm of recipient cells. There is an increasing need to understand the mechanism of intercellular cargo delivery by EVs. However, the lack of a feasible bioassay has hampered our understanding of the biological processes of EV uptake, membrane fusion, and cargo delivery to recipient cells. Here, we describe a reporter gene assay that can measure the membrane fusion efficiency of EVs during cargo delivery to recipient cells. When EVs containing tetracycline transactivator (tTA)-fused tetraspanins are internalized by recipient cells and fuse with cell membranes, the tTA domain is exposed to the cytoplasm and cleaved by tobacco etch virus protease to induce tetracycline responsive element (TRE)-mediated reporter gene expression in recipient cells. This assay (designated as EV-mediated tetraspanin-tTA delivery assay, ETTD assay), enabled us to assess the cytoplasmic cargo delivery efficiency of EVs in recipient cells. With the help of a vesicular stomatitis virus-derived membrane fusion protein, the ETTD assay could detect significant enhancement of cargo delivery efficiency of EVs. Furthermore, the ETTD assay could evaluate the effect of potential cargo delivery enhancers/inhibitors. Thus, the ETTD assay may contribute to a better understanding of the underlying mechanism of the cytoplasmic cargo delivery by EVs.

The hypervariable region of atlastin-1 is a site for intrinsic and extrinsic regulation.

Atlastin (ATL) GTPases catalyze homotypic membrane fusion of the peripheral endoplasmic reticulum (ER). GTP-hydrolysis-driven conformational changes and membrane tethering are prerequisites for proper membrane fusion. However, the molecular basis for regulation of these processes is poorly understood. Here we establish intrinsic and extrinsic modes of ATL1 regulation that involve the N-terminal hypervariable region (HVR) of ATLs. Crystal structures of ATL1 and ATL3 exhibit the HVR as a distinct, isoform-specific structural feature. Characterizing the functional role of ATL1's HVR uncovered its positive effect on membrane tethering and on ATL1's cellular function. The HVR is post-translationally regulated through phosphorylation-dependent modification. A kinase screen identified candidates that modify the HVR site specifically, corresponding to the modifications on ATL1 detected in cells. This work reveals how the HVR contributes to efficient and potentially regulated activity of ATLs, laying the foundation for the identification of cellular effectors of ATL-mediated membrane processes.

An active tethering mechanism controls the fate of vesicles.

Vesicle tethers are thought to underpin the efficiency of intracellular fusion by bridging vesicles to their target membranes. However, the interplay between tethering and fusion has remained enigmatic. Here, through optogenetic control of either a natural tether-the exocyst complex-or an artificial tether, we report that tethering regulates the mode of fusion. We find that vesicles mainly undergo kiss-and-run instead of full fusion in the absence of functional exocyst. Full fusion is rescued by optogenetically restoring exocyst function, in a manner likely dependent on the stoichiometry of tether engagement with the plasma membrane. In contrast, a passive artificial tether produces mostly kissing events, suggesting that kiss-and-run is the default mode of vesicle fusion. Optogenetic control of tethering further shows that fusion mode has physiological relevance since only full fusion could trigger lamellipodial expansion. These findings demonstrate that active coupling between tethering and fusion is critical for robust membrane merger.

Molecular and mesoscopic geometries in autophagosome generation. A review.

Autophagy is an essential process in cell self-repair and survival. The centre of the autophagic event is the generation of the so-called autophagosome (AP), a vesicle surrounded by a double membrane (two bilayers). The AP delivers its cargo to a lysosome, for degradation and re-use of the hydrolysis products as new building blocks. AP formation is a very complex event, requiring dozens of specific proteins, and involving numerous instances of membrane biogenesis and architecture, including membrane fusion and fission. Many stages of AP generation can be rationalised in terms of curvature, both the molecular geometry of lipids interpreted in terms of 'intrinsic curvature', and the overall mesoscopic curvature of the whole membrane, as observed with microscopy techniques. The present contribution intends to bring together the worlds of biophysics and cell biology of autophagy, in the hope that the resulting cross-pollination will generate abundant fruit.

Generation and Characterization of a CRISPR/Cas9-Mediated SNAP29 Knockout in Human Fibroblasts.

Loss-of-function mutations in the synaptosomal-associated protein 29 (SNAP29) lead to the rare autosomal recessive neurocutaneous cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK) syndrome. SNAP29 is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein. So far, it has been shown to be involved in membrane fusion, epidermal differentiation, formation of primary cilia, and autophagy. Recently, we reported the successful generation of two mouse models for the human CEDNIK syndrome. The aim of this investigation was the generation of a CRISPR/Cas9-mediated SNAP29 knockout (KO) in an immortalized human cell line to further investigate the role of SNAP29 in cellular homeostasis and signaling in humans independently of animal models. Comparison of different methods of delivery for CRISPR/Cas9 plasmids into the cell revealed that lentiviral transduction is more efficient than transfection methods. Here, we reported to the best of our knowledge the first successful generation of a CRISPR/Cas9-mediated SNAP29 KO in immortalized human MRC5Vi fibroblasts (c.169_196delinsTTCGT) via lentiviral transduction.

Exocytosis by vesicle crumpling maintains apical membrane homeostasis during exocrine secretion.

Exocrine secretion commonly employs micron-scale vesicles that fuse to a limited apical surface, presenting an extreme challenge for maintaining membrane homeostasis. Using Drosophila melanogaster larval salivary glands, we show that the membranes of fused vesicles undergo actomyosin-mediated folding and retention, which prevents them from incorporating into the apical surface. In addition, the diffusion of proteins and lipids between the fused vesicle and the apical surface is limited. Actomyosin contraction and membrane crumpling are essential for recruiting clathrin-mediated endocytosis to clear the retained vesicular membrane. Finally, we also observe membrane crumpling in secretory vesicles of the mouse exocrine pancreas. We conclude that membrane sequestration by crumpling followed by targeted endocytosis of the vesicular membrane, represents a general mechanism of exocytosis that maintains membrane homeostasis in exocrine tissues that employ large secretory vesicles.

Sec17/Sec18 can support membrane fusion without help from completion of SNARE zippering.

Membrane fusion requires R-, Qa-, Qb-, and Qc-family SNAREs that zipper into RQaQbQc coiled coils, driven by the sequestration of apolar amino acids. Zippering has been thought to provide all the force driving fusion. Sec17/αSNAP can form an oligomeric assembly with SNAREs with the Sec17 C-terminus bound to Sec18/NSF, the central region bound to SNAREs, and a crucial apolar loop near the N-terminus poised to insert into membranes. We now report that Sec17 and Sec18 can drive robust fusion without requiring zippering completion. Zippering-driven fusion is blocked by deleting the C-terminal quarter of any Q-SNARE domain or by replacing the apolar amino acids of the Qa-SNARE that face the center of the 4-SNARE coiled coils with polar residues. These blocks, singly or combined, are bypassed by Sec17 and Sec18, and SNARE-dependent fusion is restored without help from completing zippering.

A Glycoprotein Mutation That Emerged during the 2013-2016 Ebola Virus Epidemic Alters Proteolysis and Accelerates Membrane Fusion.

Genomic surveillance of viral isolates during the 2013-2016 Ebola virus epidemic in Western Africa, the largest and most devastating filovirus outbreak on record, revealed several novel mutations. The responsible strain, named Makona, carries an A-to-V substitution at position 82 (A82V) in the glycoprotein (GP), which is associated with enhanced infectivity in vitro Here, we investigated the mechanistic basis for this enhancement as well as the interplay between A82V and a T-to-I substitution at residue 544 of GP, which also modulates infectivity in cell culture. We found that both 82V and 544I destabilize GP, with the residue at position 544 impacting overall stability, while 82V specifically destabilizes proteolytically cleaved GP. Both residues also promote faster kinetics of lipid mixing of the viral and host membranes in live cells, individually and in tandem, which correlates with faster times to fusion following colocalization with the viral receptor Niemann-Pick C1 (NPC1). Furthermore, GPs bearing 82V are more sensitive to proteolysis by cathepsin L (CatL), a key host factor for viral entry. Intriguingly, CatL processed 82V variant GPs to a novel product with a molecular weight of approximately 12,000 (12K), which we hypothesize corresponds to a form of GP that is pre-triggered for fusion. We thus propose a model in which 82V promotes more efficient GP processing by CatL, leading to faster viral fusion kinetics and higher levels of infectivity.IMPORTANCE The 2013-2016 outbreak of Ebola virus disease in West Africa demonstrated the potential for previously localized outbreaks to turn into regional, or even global, health emergencies. With over 28,000 cases and 11,000 confirmed deaths, this outbreak was over 50 times as large as any previously recorded. This outbreak also afforded the largest-ever collection of Ebola virus genomic sequence data, allowing new insights into viral transmission and evolution. Viral mutants arising during the outbreak have attracted attention for their potentially altered patterns of infectivity in cell culture, with potential, if unclear, implications for increased viral spread and/or virulence. Here, we report the properties of one such mutation in the viral glycoprotein, A82V, and its interplay with a previously described polymorphism at position 544. We show that mutations at both residues promote infection and fusion activation in cells but that A82V additionally leads to increased infectivity under cathepsin-limited conditions and the generation of a novel glycoprotein cleavage product.

Regulation of a subset of release-ready vesicles by the presynaptic protein Mover.

Neurotransmitter release occurs by regulated exocytosis from synaptic vesicles (SVs). Evolutionarily conserved proteins mediate the essential aspects of this process, including the membrane fusion step and priming steps that make SVs release-competent. Unlike the proteins constituting the core fusion machinery, the SV protein Mover does not occur in all species and all synapses. Its restricted expression suggests that Mover may modulate basic aspects of transmitter release and short-term plasticity. To test this hypothesis, we analyzed synaptic transmission electrophysiologically at the mouse calyx of Held synapse in slices obtained from wild-type mice and mice lacking Mover. Spontaneous transmission was unaffected, indicating that the basic release machinery works in the absence of Mover. Evoked release and vesicular release probability were slightly reduced, and the paired pulse ratio was increased in Mover knockout mice. To explore whether Mover's role is restricted to certain subpools of SVs, we analyzed our data in terms of two models of priming. A model assuming two SV pools in parallel showed a reduced release probability of so-called "superprimed vesicles" while "normally primed" ones were unaffected. For the second model, which holds that vesicles transit sequentially from a loosely docked state to a tightly docked state before exocytosis, we found that knocking out Mover selectively decreased the release probability of tight state vesicles. These results indicate that Mover regulates a subclass of primed SVs in the mouse calyx of Held.

Mutations in membrane-fusion subunit of spike glycoprotein play crucial role in the recent outbreak of COVID-19.

Coronavirus disease-2019 (COVID-19), the ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major threat to the entire human race. It is reported that SARS-CoV-2 seems to have relatively low pathogenicity and higher transmissibility than previously outbroke SARS-CoV. To explore the reason of the increased transmissibility of SARS-CoV-2 compared with SARS-CoV, we have performed a comparative analysis on the structural proteins (spike, envelope, membrane, and nucleoprotein) of two viruses. Our analysis revealed that extensive substitutions of hydrophobic to polar and charged amino acids in spike glycoproteins of SARS-CoV2 creates an intrinsically disordered region (IDR) at the beginning of membrane-fusion subunit and intrinsically disordered residues in fusion peptide. IDR provides a potential site for proteolysis by furin and enriched disordered residues facilitate prompt fusion of the SARS-CoV2 with host membrane by recruiting molecular recognition features. Here, we have hypothesized that mutation-driven accumulation of intrinsically disordered residues in spike glycoproteins play dual role in enhancing viral transmissibility than previous SARS-coronavirus. These analyses may help in epidemic surveillance and preventive measures against COVID-19.

IKKß activation promotes amphisome formation and extracellular vesicle secretion in tumor cells.

Intracellular organelle cross-talk is a new and important research area. Under stress conditions, the coordinated action of the autophagy and endosomal systems in tumor cells is essential for maintaining cellular homeostasis and survival. The activation of the IκB kinase (IKK) complex is also involved in the regulation of stress and homeostasis in tumor cells. Here, we try to explore the effects of constitutively active IKKß subunits (CA-IKKß) on autophagy and endosomal system interactions. We confirm that CA-IKKß induces accumulation of autophagosomes and their fusion with MVBs to form amphisomes in cancer cells, and also drives the release of EVs containing autophagy components through an amphisome-dependent mechanism. We further demonstrate that CA-IKKß inhibits the expression of RAB7, thereby weakening the lysosomal-dependent degradation pathway. CA-IKKß also induces phosphorylation of SNAP23 at Ser95 instead of Ser110, which further promotes amphisome-plasma membrane fusion and sEV secretion. These results indicate that CA-IKKß drives the formation and transport of amphisomes, thereby regulating tumor cell homeostasis, which may illuminate a special survival mechanism in tumor cells under stress.

Altered mitochondrial fusion drives defensive glutathione synthesis in cells able to switch to glycolytic ATP production.

Mitochondria are highly dynamic organelles. Alterations in mitochondrial dynamics are causal or are linked to numerous neurodegenerative, neuromuscular, and metabolic diseases. It is generally thought that cells with altered mitochondrial structure are prone to mitochondrial dysfunction, increased reactive oxygen species generation and widespread oxidative damage. The objective of the current study was to investigate the relationship between mitochondrial dynamics and the master cellular antioxidant, glutathione (GSH). We reveal that mouse embryonic fibroblasts (MEFs) lacking the mitochondrial fusion machinery display elevated levels of GSH, which limits oxidative damage. Moreover, targeted metabolomics and 13C isotopic labeling experiments demonstrate that cells lacking the inner membrane fusion GTPase OPA1 undergo widespread metabolic remodeling altering the balance of citric acid cycle intermediates and ultimately favoring GSH synthesis. Interestingly, the GSH precursor and antioxidant n-acetylcysteine did not increase GSH levels in OPA1 KO cells, suggesting that cysteine is not limiting for GSH production in this context. Post-mitotic neurons were unable to increase GSH production in the absence of OPA1. Finally, the ability to use glycolysis for ATP production was a requirement for GSH accumulation following OPA1 deletion. Thus, our results demonstrate a novel role for mitochondrial fusion in the regulation of GSH synthesis, and suggest that cysteine availability is not limiting for GSH synthesis in conditions of mitochondrial fragmentation. These findings provide a possible explanation for the heightened sensitivity of certain cell types to alterations in mitochondrial dynamics.

Priming of SARS-CoV-2 S protein by several membrane-bound serine proteinases could explain enhanced viral infectivity and systemic COVID-19 infection.

The ongoing COVID-19 pandemic has already caused over a million deaths worldwide, and this death toll will be much higher before effective treatments and vaccines are available. The causative agent of the disease, the coronavirus SARS-CoV-2, shows important similarities with the previously emerged SARS-CoV-1, but also striking differences. First, SARS-CoV-2 possesses a significantly higher transmission rate and infectivity than SARS-CoV-1 and has infected in a few months over 60 million people. Moreover, COVID-19 has a systemic character, as in addition to the lungs, it also affects the heart, liver, and kidneys among other organs of the patients and causes frequent thrombotic and neurological complications. In fact, the term "viral sepsis" has been recently coined to describe the clinical observations. Here I review current structure-function information on the viral spike proteins and the membrane fusion process to provide plausible explanations for these observations. I hypothesize that several membrane-associated serine proteinases (MASPs), in synergy with or in place of TMPRSS2, contribute to activate the SARS-CoV-2 spike protein. Relative concentrations of the attachment receptor, ACE2, MASPs, their endogenous inhibitors (the Kunitz-type transmembrane inhibitors, HAI-1/SPINT1 and HAI-2/SPINT2, as well as major circulating serpins) would determine the infection rate of host cells. The exclusive or predominant expression of major MASPs in specific human organs suggests a direct role of these proteinases in e.g., heart infection and myocardial injury, liver dysfunction, kidney damage, as well as neurological complications. Thorough consideration of these factors could have a positive impact on the control of the current COVID-19 pandemic.

Measurements of Compensatory Endocytosis by Antibody Internalization and Quantification of Endocytic Vesicle Distribution in Adrenal Chromaffin Cells.

Plasma membrane proteins are amenable to endocytosis assays since they are easily labeled by reagents applied in the extracellular medium. This has been widely exploited to study constitutive endocytosis or ligand-induced receptor endocytosis. Compensatory endocytosis is the mechanism by which components of secretory vesicles are retrieved after vesicle fusion with the plasma membrane in response to cell stimulation and a rise in intracellular calcium. Luminal membrane proteins from secretory vesicles are therefore transiently exposed at the plasma membrane. Here, we described an antibody-based method to monitor compensatory endocytosis in chromaffin cells and present an image-based analysis to quantify endocytic vesicles distribution.

Cytotoxic Granule Trafficking and Fusion in Synaptotagmin7-Deficient Cytotoxic T Lymphocytes.

Granules of cytotoxic T lymphocytes (CTL) are derived from the lysosomal compartment. Synaptotagmin7 (Syt7) appears to be the calcium sensor triggering fusion of lysosomes in fibroblasts. Syt7 has been proposed to control cytotoxic granule (CG) fusion in lymphocytes and mice lacking Syt7 have reduced ability to clear infections. However, fusion of CG persists in the absence of Syt7. To clarify the role of Syt7 in CTL function, we have examined the fusion of cytotoxic granules of CD8+ T-lymphocytes from Syt7 knock-out mice. We have recorded granule fusion in living CTL, using total internal reflection microscopy. Since Syt7 is considered a high affinity calcium-sensor specialized for fusion under low calcium conditions, we have compared cytotoxic granule fusion under low and high calcium conditions in the same CTL. There was no difference in latencies or numbers of fusion events per CTL under low-calcium conditions, indicating that Syt7 is not required for cytotoxic granule fusion. A deficit of fusion in Syt7 KO CTL was seen when a high-calcium solution was introduced. Expressing wild type Syt7 in Syt7 KO lymphocytes reversed this deficit, confirming its Syt7-dependence. Mutations of Syt7 which disrupt calcium binding to its C2A domain reduced the efficacy of this rescue. We counted the cytotoxic granules present at the plasma membrane to determine if the lack of fusion events in the Syt7 KO CTL was due to a lack of granules. In low calcium there were no differences in fusion events per CTL, and granule numbers were similar. In high calcium, granule number was similar though wild type CTL exhibited significantly more fusion than Syt7 KO CTL. The modest differences in granule counts do not account for the lack of fusion in high calcium in Syt7 KO CTL. In Syt7 KO CTL expressing wild type Syt7, delivery of cytotoxic granules to the plasma membrane was comparable to that of wild type CTL. Syt7 KO CTL expressing Syt7 with deficient calcium binding in the C2A domain had significantly less fusion and fewer CG at the plasma membrane. These results indicate that Syt7 is involved in trafficking of CG to the plasma membrane.

Defective nucleotide-dependent assembly and membrane fusion in Mfn2 CMT2A variants improved by Bax.

Mitofusins are members of the dynamin-related protein family of large GTPases that harness the energy from nucleotide hydrolysis to remodel membranes. Mitofusins possess four structural domains, including a GTPase domain, two extended helical bundles (HB1 and HB2), and a transmembrane region. We have characterized four Charcot-Marie-Tooth type 2A-associated variants with amino acid substitutions in Mfn2 that are proximal to the hinge that connects HB1 and HB2. A functional defect was not apparent in cells as the mitochondrial morphology of Mfn2-null cells was restored by expression of any of these variants. However, a significant fusion deficiency was observed in vitro, which was improved by the addition of crude cytosol extract or soluble Bax. All four variants had reduced nucleotide-dependent assembly in cis, but not trans, and this was also improved by the addition of Bax. Together, our data demonstrate an important role for this region in Mfn2 GTP-dependent oligomerization and membrane fusion and is consistent with a model where cytosolic factors such as Bax are masking molecular defects associated with Mfn2 disease variants in cells.