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Synaptic-vesicle exocytosis is mediated by the vesicular Ca2+ sensor synaptotagmin-1. Synaptotagmin-1 interacts with the SNARE protein syntaxin-1A and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how these interactions contribute to triggering membrane fusion. Using PC12 cells from Rattus norvegicus and artificial supported bilayers, we show that synaptotagmin-1 interacts with the polybasic linker region of syntaxin-1A independent of Ca2+ through PIP2. This interaction allows both Ca2+-binding sites of synaptotagmin-1 to bind to phosphatidylserine in the vesicle membrane upon Ca2+ triggering. We determined the crystal structure of the C2B domain of synaptotagmin-1 bound to phosphoserine, allowing development of a high-resolution model of synaptotagmin bridging two different membranes. Our results suggest that PIP2 clusters organized by syntaxin-1 act as molecular beacons for vesicle docking, with the subsequent Ca2+ influx bringing the vesicle membrane close enough for membrane fusion. [ABSTRACT FROM AUTHOR]

Synaptotagmin-1 is the main Ca2+ sensor of neuronal exocytosis. It binds to both Ca2+ and the anionic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), but the precise cooperativity of this binding is still poorly understood. Here, we used microscale thermophoresis to quantify the cooperative binding of PIP2 and Ca2+ to synaptotagmin-1. We found that PIP2 bound to the well conserved polybasic patch of the C2B domain with an apparent dissociation constant of ~20 μm. PIP2 binding reduced the apparent dissociation constant for Ca2+ from ~250 to <5 μm. Thus, our data show that PIP2 makes synaptotagmin-1 >40-fold more sensitive to Ca2+. This interplay between Ca2+, synaptotagmin-1, and PIP2 is crucial for neurotransmitter release. [ABSTRACT FROM AUTHOR]

Neuronal exocytosis is catalysed by the SNAP receptor protein syntaxin-1A, which is clustered in the plasma membrane at sites where synaptic vesicles undergo exocytosis. However, how syntaxin-1A is sequestered is unknown. Here we show that syntaxin clustering is mediated by electrostatic interactions with the strongly anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Using super-resolution stimulated-emission depletion microscopy on the plasma membranes of PC12 cells, we found that PIP2 is the dominant inner-leaflet lipid in microdomains about 73 nanometres in size. This high accumulation of PIP2 was required for syntaxin-1A sequestering, as destruction of PIP2 by the phosphatase synaptojanin-1 reduced syntaxin-1A clustering. Furthermore, co-reconstitution of PIP2 and the carboxy-terminal part of syntaxin-1A in artificial giant unilamellar vesicles resulted in segregation of PIP2 and syntaxin-1A into distinct domains even when cholesterol was absent. Our results demonstrate that electrostatic protein-lipid interactions can result in the formation of microdomains independently of cholesterol or lipid phases. [ABSTRACT FROM AUTHOR]

Synaptotagmin-1 triggers Ca2+-sensitive, rapid neurotransmitter release by promoting interactions between SNARE proteins on synaptic vesicles and the plasma membrane. How synaptotagmin-1 promotes this interaction is unclear, and the massive increase in membrane fusion efficiency of Ca2+-bound synaptotagmin-1 has not been reproduced in vitro. However, previous experiments have been performed at relatively high salt concentrations, screening potentially important electrostatic interactions. Using functional reconstitution in liposomes, we show here that at low ionic strength SNARE-mediated membrane fusion becomes strictly dependent on both Ca2+ and synaptotagmin-1. Under these conditions, synaptotagmin-1 functions as a distance regulator that tethers the liposomes too far from the plasma membrane for SNARE nucleation in the absence of Ca2+, but while bringing the liposomes close enough for membrane fusion in the presence of Ca2+. These results may explain how the relatively weak electrostatic interactions between synaptotagmin-1 and membranes substantially accelerate fusion. [ABSTRACT FROM AUTHOR]

Summary: Dual-color fluorescence-burst analysis (DCFBA) was applied to measure the quaternary structure and high-affinity binding of the bacterial motor protein SecA to the protein-conducting channel SecYEG reconstituted into lipid vesicles. DCFBA is an equilibrium technique that enables the direct observation and quantification of protein-protein interactions at the single molecule level. SecA binds to SecYEG as a dimer with a nucleotide- and preprotein-dependent dissociation constant. One of the SecA protomers binds SecYEG in a salt-resistant manner, whereas binding of the second protomer is salt sensitive. Because protein translocation is salt sensitive, we conclude that the dimeric state of SecA is required for protein translocation. A structural model for the dimeric assembly of SecA while bound to SecYEG is proposed based on the crystal structures of the Thermotoga maritima SecA-SecYEG and the Escherichia coli SecA dimer. [ABSTRACT FROM AUTHOR]

We determined the diffusion coefficients ( D) of (macro)molecules of different sizes (from ∼0.5 to 600 kDa) in the cytoplasm of live Escherichia coli cells under normal osmotic conditions and osmotic upshift. D values decreased with increasing molecular weight of the molecules. Upon osmotic upshift, the decrease in D of NBD-glucose was much smaller than that of macromolecules. Barriers for diffusion were found in osmotically challenged cells only for GFP and larger proteins. These barriers are likely formed by the nucleoid and crowding of the cytoplasm. The cytoplasm of E. coli appears as a meshwork allowing the free passage of small molecules while restricting the diffusion of bigger ones. [ABSTRACT FROM AUTHOR]

In eukaryotes, most intracellular membrane fusion reactions are mediated by the interaction of SNARE proteins that are present in both fusing membranes. However, the minimal number of SNARE complexes needed for membrane fusion is not known. Here we show unambiguously that one SNARE complex is sufficient for membrane fusion. We performed controlled in vitro Förster resonance energy transfer (FRET) experiments and found that liposomes bearing only a single SNARE molecule are still capable of fusion with other liposomes or with purified synaptic vesicles. Furthermore, we demonstrated that multiple SNARE complexes do not act cooperatively, showing that synergy between several SNARE complexes is not needed for membrane fusion. Our findings shed new light on the mechanism of SNARE-mediated membrane fusion and call for a revision of current views of fusion events such as the fast release of neurotransmitters. [ABSTRACT FROM AUTHOR]

Export of messenger RNA from the transcription site in the nucleus and mRNA targeting to the translation site in the cytoplasm are key regulatory processes in protein synthesis. In yeast, the mRNA-binding proteins Nab2p and Nab4p/Hrp1p accompany transcripts to their translation site, where the karyopherin Kap104p mediates both their dissociation from the mRNA and their transport back into the nucleus. We found that Kap104p localized to the distal bud tip and the bud neck during cell division, resulting in a localized release of translation-competent mRNA and increased protein synthesis in the emerging daughter cell. Temporally and spatially coordinated localization of Kap104p is a new mechanism for the asymmetric distribution of protein synthesis in dividing cells. [ABSTRACT FROM AUTHOR]

The mechanism of pore formation of lytic peptides, such as melittin from bee venom, is thought to involve binding to the membrane surface, followed by insertion at threshold levels of bound peptide. We show that in membranes composed of zwitterionic lipids, i.e. phosphatidylcholine, melittin not only forms pores but also inhibits pore formation. We propose that these two modes of action are the result of two competing reactions: direct insertion into the membrane and binding parallel to the membrane surface. The direct insertion of melittin leads to pore formation, whereas the parallel conformation is inactive and prevents other melittin molecules from inserting, hence preventing pore formation. [ABSTRACT FROM AUTHOR]

Abstract: Dual-color fluorescence-burst analysis (DCBFA) enables to study leakage of fluorescently labeled (macro) molecules from liposomes that are labeled with a second, spectrally non-overlapping fluorophore. The fluorescent bursts that reside from the liposomes diffusing through the focal volume of a confocal microscope will coincide with those from the encapsulated size-marker molecules. The internal concentration of size-marker molecules can be quantitatively calculated from the fluorescence bursts at a single liposome level. DCFBA has been successfully used to study the effective pore-size of the mechanosensitive channel of large-conductance MscL and the pore-forming mechanism of the antimicrobial peptide melittin from bee venom. In addition, DCFBA can be used to quantitatively measure the binding of proteins to liposomes and to membrane proteins. In this paper, we provide an overview of the method and discuss the experimental details of DCFBA. [Copyright &y& Elsevier]

The effect of osmotic stress on the intracellular diffusion of proteins in Escherichia coli was studied, using a pulsed version of fluorescence recovery after photo-bleaching, pulsed-FRAP. This method employs sequences of laser pulses which only partly bleach the fluorophores in a cell. Because the cell size and geometry are taken into account, pulsed-FRAP enables to measure diffusion in very small cells of different shapes. We found that upon an osmotic upshock from 0.15 to 0.6 Osm, imposed by NaCl or sorbitol, the apparent intracellular diffusion ( D) of mobile green fluorescent protein (GFP) decreased from 3.2 to 0.4 μm2 s−1, whereas the membrane permeable glycerol had no effect. Exposing E. coli cells to higher osmolalities (> 0.6 Osm) led to compartmentalization of the GFP into discrete pools, from where the GFP could not escape. Although free diffusion through the cell was hindered, the mobility of GFP in these pools was still relatively high ( D∼0.4 μm2 s−1). The presence of osmoprotectants restored the effect of osmotic stress on the protein mobility and apparent compartmentalization. Also, lowering the osmolality from 0.6 Osm back to 0.15 Osm restored the mobility of GFP. The implications of these findings in terms of heterogeneities and diffusive barriers inside the cell are discussed. [ABSTRACT FROM AUTHOR]

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are membrane-associated trafficking proteins that confer identity to lipid membranes and facilitate membrane fusion. These functions are achieved through the complexing of Q-SNAREs with a specific cognate target R-SNARE, leading to the fusion of their associated membranes. These SNARE complexes then dissociate so that the Q-SNAREs and R-SNAREs can repeat this cycle. Whilst the basic function of SNAREs has been long appreciated, it is becoming increasingly clear that the cell can control the localisation and function of SNARE proteins through posttranslational modifications (PTMs), such as phosphorylation and ubiquitylation. Whilst numerous proteomic methods have shown that SNARE proteins are subject to these modifications, little is known about how these modifications regulate SNARE function. However, it is clear that these PTMs provide cells with an incredible functional plasticity; SNARE PTMs enable cells to respond to an ever-changing extracellular environment through the rerouting of membrane traffic. In this Review, we summarise key findings regarding SNARE regulation by PTMs and discuss how these modifications reprogramme membrane trafficking pathways. [ABSTRACT FROM AUTHOR]

The intracellular pathogens Listeria monocytogenes, Salmonella enterica, Shigella spp. and Staphylococcus aureus are major causes of foodborne illnesses. Following the ingestion of contaminated food or beverages, pathogens can invade epithelial cells, immune cells and other cell types. Pathogens survive and proliferate intracellularly via two main strategies. First, the pathogens can remain in membrane-bound vacuoles and tailor organellar trafficking to evade host-cell defenses and gain access to nutrients. Second, pathogens can rupture the vacuolar membrane and proliferate within the nutrient-rich cytosol of the host cell. Although this virulence strategy of vacuolar escape is well known for L. monocytogenes and Shigella spp., it has recently become clear that S. aureus and Salmonella spp. also gain access to the cytosol, and that this is important for their survival and growth. In this Review, we discuss the molecular mechanisms of how these intracellular pathogens rupture the vacuolar membrane by secreting a combination of proteins that lyse the membranes or that remodel the lipids of the vacuolar membrane, such as phospholipases. In addition, we also propose that oxidation of the vacuolar membrane also contributes to cytosolic pathogen escape. Understanding these escape mechanisms could aid in the identification of new therapeutic approaches to combat foodborne pathogens. [ABSTRACT FROM AUTHOR]

• CD36 is expressed by cancer cells and cancer-associated immune cells. • Lipid uptake by CD36 can both promote and inhibit cancer progression. • Thrombospondin-1 binding to CD36 can both promote and inhibit cancer progression. • Oxidized low-density lipoprotein binding promotes cancer progression. The membrane protein CD36 is a lipid transporter, scavenger receptor, and receptor for the antiangiogenic protein thrombospondin 1 (TSP1). CD36 is expressed by cancer cells and by many associated cells including various cancer-infiltrating immune cell types. Thereby, CD36 plays critical roles in cancer, and it has been reported to affect cancer growth, metastasis, angiogenesis, and drug resistance. However, these roles are partly contradictory, as CD36 has been both reported to promote and inhibit cancer progression. Moreover, the mechanisms are also partly contradictory, because CD36 has been shown to exert opposite cellular effects such as cell division, senescence and cell death. This review provides an overview of the diverse effects of CD36 on tumor progression, aiming to shed light on its diverse pro- and anti-cancer roles, and the implications for therapeutic targeting. [ABSTRACT FROM AUTHOR]

Since their invention about two decades ago, super-resolution microscopes have become a method of choice in cell biology. Owing to a spatial resolution below 50 nm, smaller than the size of most organelles, and an order of magnitude better than the diffraction limit of conventional light microscopes, super-resolution microscopy is a powerful technique for resolving intracellular trafficking. In this review we discuss discoveries in endocytosis and phagocytosis that have been made possible by super-resolution microscopy – from uptake at the plasma membrane, endocytic coat formation, and cytoskeletal rearrangements to endosomal maturation. The detailed visualization of the diverse molecular assemblies that mediate endocytic uptake will provide a better understanding of how cells ingest extracellular material. Super-resolution microscopy is widely available to most cell biologists and has become the method of choice for studying endocytic and phagocytic trafficking. Current live imaging techniques allow 3D super-resolution microscopy with a temporal resolution of seconds to visualize uptake and trafficking processes at the whole-cell level. Quantification strategies combined with super-resolution microscopy have enabled a quantitative understanding of receptor clustering, the appearance of signaling lipids, and the dynamics of F-actin assembly on endosomes and phagosomes. Super-resolution microscopy is often combined with techniques such as electron microscopy and TIRF to enable more accurate localization of endocytic proteins in relation to stage of uptake, membrane curvature, and protein–protein interactions. Although generally of lower spatial resolution compared with other super-resolution microscopy techniques, structured illumination microscopy has at present yielded most new insights in endocytic trafficking because it combines improved spatial resolution with a reasonably high temporal resolution and can be readily used for live cell imaging applications. [ABSTRACT FROM AUTHOR]

The Golgi membrane protein GOLM1/GP73/GOLPH2 has been found to impact cytokine production in both infectious disease and cancer. In viral infections, GOLM1 levels are increased, and this lowers the production of type I interferons and other inflammatory cytokines. However, elevated GOLM1 expression levels due to mutations are linked to a higher production of interleukin (IL)‐6 during Candida infections, potentially explaining an increased susceptibility to candidemia in individuals carrying these mutations. In cancer, the protease Furin produces a soluble form of GOLM1 that has oncogenic properties by promoting the production of the chemokine CCL2 and suppressing the production of inflammatory cytokines such as IL‐12 and interferon gamma. This review will focus on the role of GOLM1 in cytokine production, highlighting how it can both promote and inhibit cytokine production. It is crucial to understand this in order to effectively target GOLM1 for therapeutic purposes in diseases associated with abnormal cytokine production, including cancer and infectious disease. [ABSTRACT FROM AUTHOR]

In this article the author discusses the molecular events inherent in exocytosis of synaptic vesicles. They believe that reconstitution of a complex cellular event in vitro, such as protein-mediated membrane fusion, is a significant step toward a complete mechanistic knowledge underlying membrane merger. They examine the study of Kyoung and colleagues which offers a unique tool for studying membrane fusion through the researchers' introduction of triple-fluorescence single-liposome assay.

3D printing is gaining traction in research and development as a way to quickly, cheaply, and easily manufacture polydimethylsiloxane (PDMS) molds. The most commonly used method is resin printing, which is relatively expensive and requires specialized printers. This study shows that polylactic acid (PLA) filament printing is a cheaper, more readily available alternative to resin printing, that does not inhibit the curing of PDMS. As a proof of concept, a PLA mold for PDMS-based wells was designed, and 3D printed. We introduce an effective method to smooth the printed PLA mold, based on chloroform vapor treatment. After this chemical post-processing step, the smoothened mold was used to cast a ring of PDMS prepolymer. The PDMS ring was attached to a glass coverslip after oxygen plasma treatment. The PDMS–glass well showed no leakage and was well suited to its intended use. When used for cell culturing, monocyte-derived dendritic cells (moDCs) showed no morphological anomalies, as tested by confocal microscopy, nor did they show an increase in cytokines, as tested using ELISA. This underlines the versatility and strength of PLA filament printing and exemplifies how it can be valuable to a researcher's toolset. [ABSTRACT FROM AUTHOR]

A correction to the article "Nuclear Transport Factor Directs Localization of Protein Synthesis During Mitosis," published in a previous issue of "Nature Cell Biology," is presented.

Chromogranin A (CgA) is a 439 amino acid protein secreted by neuroendocrine cells. Proteolytic processing of CgA results in the production of different bioactive peptides. These peptides have been associated with inflammatory bowel disease, diabetes, and cancer. One of the chromogranin A-derived peptides is ∼52 amino acid long Pancreastatin (PST: human (h)CgA 250–301 , murine (m)CgA 263–314). PST is a glycogenolytic peptide that inhibits glucose-induced insulin secretion from pancreatic islet β-cells. In addition to this metabolic role, evidence is emerging that PST also has inflammatory properties. This review will discuss the immunomodulatory properties of PST and its possible mechanisms of action and regulation. Moreover, this review will discuss the potential translation to humans and how PST may be an interesting therapeutic target for treating inflammatory diseases. [Display omitted] • Pancreastatin (PST) is a glycogenolytic peptide that inhibits glucose-induced insulin secretion from pancreatic islet β-cells. • PST increases pro-inflammatory cytokines in murine macrophages (in vitro and in vivo). • The receptor for PST on immune cells and PST's mechanism of action are still unknown. • PST's function is altered by naturally occurring single nucleotide polymorphisms. • The discovery of PST inhibitors (PSTNΔ3 and PSTCΔ8) may be useful to increase knowledge of the PST mechanism of action. [ABSTRACT FROM AUTHOR]

5-methoxy tryptophan (5-MTP) is an anti-fibrotic metabolite made by fibroblasts and epithelial cells, present in a micromolar concentrations in human blood, and is associated with the progression of fibrotic kidney disease, but the mechanism is unclear. Here, we show by microscopy and functional assays that 5-MTP influences mitochondria in human peripheral blood monocyte-derived macrophages. As a result, the mitochondrial membranes are more rigid, more branched, and are protected against oxidation. The macrophages also change their metabolism by reducing mitochondrial import of acyl-carnitines, intermediates of fatty acid metabolism, driving glucose import. Moreover, 5-MTP increases the endocytosis of collagen by macrophages, and experiments with inhibition of glucose uptake showed that this is a direct result of their altered metabolism. However, 5-MTP does not affect the macrophages following pathogenic stimulation, due to 5-MTP degradation by induced expression of indole-amine oxygenase-1 (IDO-1). Thus, 5-MTP is a fibrosis-protective metabolite that, in absence of pathogenic stimulation, promotes collagen uptake by anti-inflammatory macrophages by altering the physicochemical properties of their mitochondrial membranes. Schematic overview of the effects of 5-MTP on human monocyte derived macrophages. 5-MTP promotes mitochondrial fusion, protecting against oxidative stress, and increases glucose uptake which drive collagen uptake. However, IDO is able to negate any anti-inflammatory effects by breaking down 5-MTP. [Display omitted] • 5-MTP increases collagen uptake in macrophages. • 5-MTP protects against mitochondrial oxidative stress. • Macrophages increase their sugar metabolism in the presence of 5-MTP. • Macrophages are able to breakdown 5-MTP via indoleamine oxygenase (IDO). [ABSTRACT FROM AUTHOR]

Synaptic transmission is mediated by the release of neurotransmitters, which involves exo-endocytotic cycling of synaptic vesicles. To maintain synaptic function, synaptic vesicles are refilled with thousands of neurotransmitter molecules within seconds after endocytosis, using the energy provided by an electrochemical proton gradient. However, it is unclear how transmitter molecules carrying different net charges can be efficiently sequestered while maintaining charge neutrality and osmotic balance. We used singlevesicle imaging to monitor pH and electrical gradients and directly showed different uptake mechanisms for glutamate and g-aminobutyric acid (GABA) operating in parallel. In contrast to glutamate, GABA was exchanged for protons, with no other ions participating in the transport cycle. Thus, only a few components are needed to guarantee reliable vesicle filling with different neurotransmitters. [ABSTRACT FROM AUTHOR]

Aim: Defects in hepatic glycogen synthesis contribute to post‐prandial hyperglycaemia in type 2 diabetic patients. Chromogranin A (CgA) peptide Catestatin (CST: hCgA352‐372) improves glucose tolerance in insulin‐resistant mice. Here, we seek to determine whether CST induces hepatic glycogen synthesis. Methods: We determined liver glycogen, glucose‐6‐phosphate (G6P), uridine diphosphate glucose (UDPG) and glycogen synthase (GYS2) activities; plasma insulin, glucagon, noradrenaline and adrenaline levels in wild‐type (WT) as well as in CST knockout (CST‐KO) mice; glycogen synthesis and glycogenolysis in primary hepatocytes. We also analysed phosphorylation signals of insulin receptor (IR), insulin receptor substrate‐1 (IRS‐1), phosphatidylinositol‐dependent kinase‐1 (PDK‐1), GYS2, glycogen synthase kinase‐3β (GSK‐3β), AKT (a kinase in AKR mouse that produces Thymoma)/PKB (protein kinase B) and mammalian/mechanistic target of rapamycin (mTOR) by immunoblotting. Results: CST stimulated glycogen accumulation in fed and fasted liver and in primary hepatocytes. CST reduced plasma noradrenaline and adrenaline levels. CST also directly stimulated glycogenesis and inhibited noradrenaline and adrenaline‐induced glycogenolysis in hepatocytes. In addition, CST elevated the levels of UDPG and increased GYS2 activity. CST‐KO mice had decreased liver glycogen that was restored by treatment with CST, reinforcing the crucial role of CST in hepatic glycogenesis. CST improved insulin signals downstream of IR and IRS‐1 by enhancing phospho‐AKT signals through the stimulation of PDK‐1 and mTORC2 (mTOR Complex 2, rapamycin‐insensitive complex) activities. Conclusions: CST directly promotes the glycogenic pathway by (a) reducing glucose production, (b) increasing glycogen synthesis from UDPG, (c) reducing glycogenolysis and (d) enhancing downstream insulin signalling. [ABSTRACT FROM AUTHOR]

In autophagy, LC3‐positive autophagophores fuse and encapsulate the autophagic cargo in a double‐membrane structure. In contrast, lipidated LC3 (LC3‐II) is directly formed at the phagosomal membrane in LC3‐associated phagocytosis (LAP). In this study, we dissected the effects of autophagy inhibitors on LAP. SAR405, an inhibitor of VPS34, reduced levels of LC3‐II and inhibited LAP. In contrast, the inhibitors of endosomal acidification bafilomycin A1 and chloroquine increased levels of LC3‐II, due to reduced degradation in acidic lysosomes. However, while bafilomycin A1 inhibited LAP, chloroquine did not. Finally, EACC, which inhibits the fusion of autophagosomes with lysosomes, promoted LC3 degradation possibly by the proteasome. Targeting LAP with small molecule inhibitors is important given its emerging role in infectious and autoimmune diseases. [ABSTRACT FROM AUTHOR]

Catestatin (CST) is a bioactive cleavage product of the neuroendocrine prohormone chromogranin A (CgA). Recent findings show that CST can exert anti-inflammatory and antiadrenergic effects by suppressing the inflammatory actions of mammalian macrophages. However, recent findings also suggest that macrophages themselves are major CST producers. Here, we hypothesize that macrophages produce CST in an inflammation-dependent manner and thereby might self-regulate inflammation in an autocrine fashion. CST is associated with pathological conditions hallmarked by chronic inflammation, including autoimmune, cardiovascular, and metabolic disorders. Since intraperitoneal injection of CST in mouse models of diabetes and inflammatory bowel disease has been reported to be beneficial for mitigating disease, we posit that CST should be further investigated as a candidate target for treating certain inflammatory diseases. Catestatin (CST), a 21-amino acid peptide derived from proteolytic cleavage of the prohormone chromogranin A, has been reported to have anti-inflammatory and antiadrenergic functions in mice. CST is thought to be mainly co-released with catecholamines by neuroendocrine cells. Recent studies suggest that macrophages are also a source of CST and that the anti-inflammatory effects of CST might be attributable to CST produced by macrophages, at least in mice. We propose that CST produced by macrophages might suppress neuronal and neuroendocrine activity, in an inflammation-dependent manner. [ABSTRACT FROM AUTHOR]

Exocytosis of neurosecretory vesicles is mediated by the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins syntaxin-1, synaptobrevin and SNAP-25, with synaptotagmin functioning as the major Ca2+ sensor for triggering membrane fusion. Here we show that bovine chromaffin granules readily fuse with large unilamellar liposomes in a SNARE-dependent manner. Fusion is enhanced by Ca2+, but only when the target liposomes contain phosphatidylinositol-4,5-bisphosphate and when polyphosphate anions, such as nucleotides or pyrophosphate, are present. Ca2+-dependent enhancement is mediated by endogenous synaptotagmin-1. Polyphosphates operate by an electrostatic mechanism that reverses an inactivating cis association of synaptotagmin-1 with its own membrane without affecting trans binding. Hence, the balancing of trans- and cis-membrane interactions of synaptotagmin-1 could be a crucial element in the pathway of Ca2+-dependent exocytosis. [ABSTRACT FROM AUTHOR]

Aim: A "leaky" gut barrier has been implicated in the initiation and progression of a multitude of diseases, for example, inflammatory bowel disease (IBD), irritable bowel syndrome and celiac disease. Here we show how pro‐hormone Chromogranin A (CgA), produced by the enteroendocrine cells, and Catestatin (CST: hCgA352‐372), the most abundant CgA‐derived proteolytic peptide, affect the gut barrier. Methods: Colon tissues from region‐specific CST‐knockout (CST‐KO) mice, CgA‐knockout (CgA‐KO) and WT mice were analysed by immunohistochemistry, western blot, ultrastructural and flowcytometry studies. FITC‐dextran assays were used to measure intestinal barrier function. Mice were supplemented with CST or CgA fragment pancreastatin (PST: CgA250‐301). The microbial composition of cecum was determined. CgA and CST levels were measured in blood of IBD patients. Results: Plasma levels of CST were elevated in IBD patients. CST‐KO mice displayed (a) elongated tight, adherens junctions and desmosomes similar to IBD patients, (b) elevated expression of Claudin 2, and (c) gut inflammation. Plasma FITC‐dextran measurements showed increased intestinal paracellular permeability in the CST‐KO mice. This correlated with a higher ratio of Firmicutes to Bacteroidetes, a dysbiotic pattern commonly encountered in various diseases. Supplementation of CST‐KO mice with recombinant CST restored paracellular permeability and reversed inflammation, whereas CgA‐KO mice supplementation with CST and/or PST in CgA‐KO mice showed that intestinal paracellular permeability is regulated by the antagonistic roles of these two peptides: CST reduces and PST increases permeability. Conclusion: The pro‐hormone CgA regulates the intestinal paracellular permeability. CST is both necessary and sufficient to reduce permeability and primarily acts by antagonizing PST. [ABSTRACT FROM AUTHOR]

The PIKfyve inhibitor apilimod is currently undergoing clinical trials for treatment of COVID-19. However, although apilimod might prevent viral invasion by inhibiting host cell proteases, the same proteases are critical for antigen presentation leading to T cell activation and there is good evidence from both in vitro studies and the clinic that apilimod blocks antiviral immune responses. We therefore warn that the immunosuppression observed in many COVID-19 patients might be aggravated by apilimod. [ABSTRACT FROM AUTHOR]

SNARE proteins mediate membrane fusion between synaptic vesicles and the plasma membrane. A minimized peptide SNARE model system with reduced complexity was introduced combining the native SNARE transmembrane (TMD) and linker domains with artificial coiled-coil forming peptides. Specific membrane fusion initiated by coiled-coil recognition was shown by lipid and content mixing vesicle assays. [ABSTRACT FROM AUTHOR]

Immune-cell activation by inflammatory stimuli triggers the transcription and translation of large amounts of cytokines. The transport of newly synthesized cytokines to the plasma membrane by vesicular trafficking can be rate-limiting for the production of these cytokines, and immune cells upregulate their exocytic machinery concomitantly with increased cytokine expression in order to cope with the increasing demand for trafficking. Whereas it is logical that trafficking is rate-limiting for regulated secretion where an intracellular pool of molecules is waiting to be released, the reason for this is not obvious for constitutively secreted cytokines, such as interleukin-6 (IL-6), interleukin-12 (IL-12) and tumor necrosis factor-α (TNF-α). These constitutively secreted cytokines are primarily regulated at the transcriptional and/or translational level but mounting evidence presented here shows that cells might also increase or decrease the rate of post-Golgi cytokine trafficking to modulate their production. Therefore, in this Hypothesis, we ask the question: why is there a need to limit the trafficking of constitutively secreted cytokines? We propose a model where cells monitor and adjust their production rate of cytokines by sensing the intracellular level of cytokines while they are in transit to the plasma membrane. This self-regulation of cytokine production could prevent an overshooting response of acute-phase cytokines, such as IL-6, IL-12 and TNF-α, upon acute infection. [ABSTRACT FROM AUTHOR]

We recently identified a key role for SWAP70 as the tethering factor stabilizing F-actin filaments on the surface of phagosomes in human dendritic cells by interacting both with Rho-family GTPases and the lipid phosphatidylinositol (3,4)-bisphosphate. In this study, we aimed to investigate whether this role of SWAP70 was general among immune phagocytes. Our data reveal that SWAP70 is recruited to early phagosomes of macrophages and dendritic cells from both human and mouse. The putative inhibitor of SWAP70 sanguinarine blocked phagocytosis and F-actin polymerization, supporting a key role for SWAP70 in phagocytosis as demonstrated previously with knock-down. Moreover, SWAP70 was recently shown to sequester the F-actin severing protein cofilin and we investigated this relationship in phagocytosis. Our data show an increased activation of cellular cofilin upon siRNA knockdown of SWAP70. Finally, we explored whether SWAP70 would be recruited to the immune synapse between dendritic cells and T cells required for antigen presentation, as the formation of such synapses depends on F-actin. However, we observed that SWAP70 was depleted at immune synapses and specifically was recruited to phagosomes. Our data support an essential and specific role for SWAP70 in tethering and stabilizing F-actin to the phagosomal surface in a wide range of phagocytes. [ABSTRACT FROM AUTHOR]

We have previously shown that the chromogranin A (CgA)-derived peptide catestatin (CST: hCgA352–372) inhibits nicotine-induced secretion of catecholamines from the adrenal medulla and chromaffin cells. In the present study, we seek to determine whether CST regulates dense core (DC) vesicle (DCV) quanta (catecholamine and chromogranin/secretogranin proteins) during acute (0.5-h treatment) or chronic (24-h treatment) cholinergic (nicotine) or peptidergic (PACAP, pituitary adenylyl cyclase activating polypeptide) stimulation of PC12 cells. In acute experiments, we found that both nicotine (60 μM) and PACAP (0.1 μM) decreased intracellular norepinephrine (NE) content and increased 3H‐NE secretion, with both effects markedly inhibited by co-treatment with CST (2 μM). In chronic experiments, we found that nicotine and PACAP both reduced DCV and DC diameters and that this effect was likewise prevented by CST. Nicotine or CST alone increased expression of CgA protein and together elicited an additional increase in CgA protein, implying that nicotine and CST utilize separate signaling pathways to activate CgA expression. In contrast, PACAP increased expression of CgB and SgII proteins, with a further potentiation by CST. CST augmented the expression of tyrosine hydroxylase (TH) but did not increase intracellular NE levels, presumably due to its inability to cause post-translational activation of TH through serine phosphorylation. Co-treatment of CST with nicotine or PACAP increased quantal size, plausibly due to increased synthesis of CgA, CgB and SgII by CST. We conclude that CST regulates DCV quanta by acutely inhibiting catecholamine secretion and chronically increasing expression of CgA after nicotinic stimulation and CgB and SgII after PACAPergic stimulation. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) constantly sample peripheral tissues for antigens, which are subsequently ingested to derive peptides for presentation to T cells in lymph nodes. To do so, DCs have to traverse many different tissues with varying oxygen tensions. Additionally, DCs are often exposed to low oxygen tensions in tumors, where vascularization is lacking, as well as in inflammatory foci, where oxygen is rapidly consumed by inflammatory cells during the respiratory burst. DCs respond to oxygen levels to tailor immune responses to such low-oxygen environments. In the present study, we identified a mechanism of hypoxia-mediated potentiation of release of tumor necrosis factor α (TNF-α), a pro-inflammatory cytokine with important roles in both anti-cancer immunity and autoimmune disease. We show in human monocyte-derived DCs (moDCs) that this potentiation is controlled exclusively via the p38/mitogen-activated protein kinase (MAPK) pathway.We identified MAPK kinase kinase 8 (MAP3K8) as a target gene of hypoxia-induced factor (HIF), a transcription factor controlled by oxygen tension, upstream of the p38/MAPK pathway. Hypoxia increased expression of MAP3K8 concomitant with the potentiation of TNF-α secretion. This potentiation was no longer observed upon siRNA silencing of MAP3K8 or with a small molecule inhibitor of this kinase, and this also decreased p38/MAPK phosphorylation. However, expression of DC maturation markers CD83, CD86, and HLA-DR were not changed by hypoxia. Since DCs play an important role in controlling T-cell activation and differentiation, our results provide novel insight in understanding T-cell responses in inflammation, cancer, autoimmune disease and other diseases where hypoxia is involved. [ABSTRACT FROM AUTHOR]

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein family is of vital importance for organelle communication. The complexing of cognate SNARE members present in both the donor and target organellar membranes drives the membrane fusion required for intracellular transport. In the endocytic route, SNARE proteins mediate trafficking between endosomes and phagosomes with other endosomes, lysosomes, the Golgi apparatus, the plasma membrane, and the endoplasmic reticulum. The goal of this review is to provide an overview of the SNAREs involved in endosomal and phagosomal trafficking. Of the 38 SNAREs present in humans, 30 have been identified at endosomes and/or phagosomes. Many of these SNAREs are targeted by viruses and intracellular pathogens, which thereby reroute intracellular transport for gaining access to nutrients, preventing their degradation, and avoiding their detection by the immune system. A fascinating picture is emerging of a complex transport network with multiple SNAREs being involved in consecutive trafficking routes. [ABSTRACT FROM AUTHOR]

Immune cells communicate by releasing large quantities of cytokines. Although the mechanisms of cytokine secretion are increasingly understood, quantitative knowledge of the number of cytokines per vesicle is still lacking. Here, we measured with quantitative microscopy the release rate of vesicles potentially carrying interleukin‐6 (IL‐6) in human dendritic cells. By comparing this to the total secreted IL‐6, we estimate that secretory vesicles contain about 0.5–3 IL‐6 molecules, but with a large spread among cells/donors. Moreover, IL‐6 did not accumulate within most cells, indicating that synthesis and not trafficking is the bottleneck for IL‐6 production. IL‐6 accumulated in the Golgi apparatus only in ~ 10% of the cells. Understanding how immune cells produce cytokines is important for designing new immunomodulatory drugs. [ABSTRACT FROM AUTHOR]

Cross-presentation of foreign antigen in major histocompatibility complex (MHC) class I by dendritic cells (DCs) requires activation of the NADPH-oxidase NOX2 complex. We recently showed that NOX2 is recruited to phagosomes by the SNARE protein VAMP8 where NOX2-produced reactive oxygen species (ROS) cause lipid oxidation and membrane disruption, promoting antigen translocation into the cytosol for cross-presentation. In this study, we extend these findings by showing that VAMP8 is also involved in NOX2 trafficking to endosomes. Moreover, we demonstrate in both human and mouse DCs that absence of VAMP8 leads to decreased ROS production, lipid peroxidation and antigen translocation, and that this impairs cross-presentation. In contrast, knockdown of VAMP8 did not affect recruitment of MHC class I and the transporter associated with antigen processing 1 (TAP1) to phagosomes, although surface levels of MHC class I were reduced. Thus, in addition to a secretory role, VAMP8-mediates trafficking of NOX2 to endosomes and phagosomes and this promotes induction of cytolytic T cell immune responses. [ABSTRACT FROM AUTHOR]

SNARE proteins play a crucial role in intracellular trafficking by catalyzing membrane fusion, but assigning SNAREs to specific intracellular transport routes is challenging with current techniques. We developed a novel Fo? rster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM)-based technique allowing visualization of real-time local interactions of fluorescently tagged SNARE proteins in live cells. We used FRET-FLIM to delineate the trafficking steps underlying the release of the inflammatory cytokine interleukin-6 (IL-6) from human blood- derived dendritic cells. We found that activation of dendritic cells by bacterial lipopolysaccharide leads to increased FRET of fluorescently labeled syntaxin 4 with VAMP3 specifically at the plasma membrane, indicating increased SNARE complex formation, whereas FRET with other tested SNAREs was unaltered. Our results revealed that SNARE complexing is a key regulatory step for cytokine production by immune cells and prove the applicability of FRET-FLIM for visualizing SNARE complexes in live cells with subcellular spatial resolution. [ABSTRACT FROM AUTHOR]

In dendritic cells, the NADPH oxidase 2 complex (NOX2) is recruited to the phagosomal membrane during antigen uptake. NOX2 produces reactive oxygen species (ROS) in the lumen of the phagosome that kill ingested pathogens, delay antigen breakdown and alter the peptide repertoire for presentation to T cells. How the integral membrane component of NOX2, cytochrome b558 (which comprises CYBB and CYBA), traffics to phagosomes is incompletely understood. In this study, we show in dendritic cells derived from human blood-isolated monocytes that cytochrome b558 is initially recruited to the phagosome from the plasma membrane during phagosome formation. Cytochrome b558 also traffics from a lysosomal pool to phagosomes and this is required to replenish oxidatively damaged NOX2. We identified syntaxin-7, SNAP23 and VAMP8 as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediating this process. Our data describe a key mechanism of how dendritic cells sustain ROS production after antigen uptake that is required to initiate T cell responses. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor component of total plasma membrane lipids, but it has a substantial role in the regulation of many cellular functions, including exo- and endocytosis. Recently, it was shown that PI(4,5)P2 and syntaxin 1, a SNARE protein that catalyzes regulated exocytosis, form domains in the plasma membrane that constitute recognition sites for vesicle docking. Also, calcium was shown to promote syntaxin 1 clustering in the plasma membrane, but the molecular mechanism was unknown. Here, using a combination of superresolution stimulated emission depletion microscopy, FRET, and atomic force microscopy, we show that Ca2+ acts as a charge bridge that specifically and reversibly connects multiple syntaxin 1/PI(4,5)P2 complexes into larger mesoscale domains. This transient reorganization of the plasma membrane by physiological Ca2+ concentrations is likely to be important for Ca2+- regulated secretion. [ABSTRACT FROM AUTHOR]

Congenital disorders of glycosylation (CDGs) form a genetically and clinically heterogeneous group of diseases with aberrant protein glycosylation as a hallmark. A subgroup of CDGs can be attributed to disturbed Golgi homeostasis. However, identification of pathogenic variants is seriously complicated by the large number of proteins involved. As part of a strategy to identify human homologs of yeast proteins that are known to be involved in Golgi homeostasis, we identified uncharacterized transmembrane protein 199 (TMEM199, previously called C17orf32) as a human homolog of yeast V-ATPase assembly factor Vph2p (also known as Vma12p). Subsequently, we analyzed raw exome-sequencing data from families affected by genetically unsolved CDGs and identified four individuals with different mutations in TMEM199. The adolescent individuals presented with a mild phenotype of hepatic steatosis, elevated aminotransferases and alkaline phosphatase, and hypercholesterolemia, as well as low serum ceruloplasmin. Affected individuals showed abnormal N- and mucin-type O-glycosylation, and mass spectrometry indicated reduced incorporation of galactose and sialic acid, as seen in other Golgi homeostasis defects. Metabolic labeling of sialic acids in fibroblasts confirmed deficient Golgi glycosylation, which was restored by lentiviral transduction with wild-type TMEM199 . V5-tagged TMEM199 localized with ERGIC and COPI markers in HeLa cells, and electron microscopy of a liver biopsy showed dilated organelles suggestive of the endoplasmic reticulum and Golgi apparatus. In conclusion, we have identified TMEM199 as a protein involved in Golgi homeostasis and show that TMEM199 deficiency results in a hepatic phenotype with abnormal glycosylation. [ABSTRACT FROM AUTHOR]

Immune responses are initiated by the interactions between antigen-presenting cells (APCs), such as dendritic cells (DCs), with responder cells, such as T cells, via a tight cellular contact interface called the immunological synapse. The immunological synapse is a highly organized subcellular structure that provides a platform for the presentation of antigen in major histocompatibility class I and II complexes (MHC class I and II) on the surface of the APC to receptors on the surface of the responder cells. In T cells, these contacts lead to highly polarized membrane trafficking that results in the local release of lytic granules and in the delivery and recycling of T cell receptors at the immunological synapse. Localized trafficking also occurs at the APC side of the immunological synapse, especially in DCs where antigen loaded in MHC class I and II is presented and cytokines are released specifically at the synapse. Whereas the molecular mechanisms underlying polarized membrane trafficking at the T cell side of the immunological synapse are increasingly well understood, these are still very unclear at the APC side. In this review, we discuss the organization of the APC side of the immunological synapse. We focus on the directional trafficking and release of membrane vesicles carrying MHC molecules and cytokines at the immunological synapses of DCs. We hypothesize that the specific delivery of MHC and the release of cytokines at the immunological synapse mechanistically resemble that of lytic granule release from T cells. [ABSTRACT FROM AUTHOR]

Summary: PI(3,4,5)P3 is a low-abundance lipid thought to play a role in the regulation of synaptic activity; however, the mechanism remains obscure. We have constructed novel split Venus-based probes and used superresolution imaging to localize PI(3,4,5)P3 at Drosophila larval neuromuscular synapses. We find the lipid in membrane domains at neurotransmitter release sites, where it concentrates with Syntaxin1A, a protein essential for vesicle fusion. Reducing PI(3,4,5)P3 availability disperses Syntaxin1A clusters and increasing PI(3,4,5)P3 levels rescues this defect. In artificial giant unilamellar vesicles, PI(3,4,5)P3 also induces Syntaxin1A domain formation and this clustering, in vitro and in vivo, is dependent on positively charged residues in the Syntaxin1A-juxtamembrane domain. Functionally, reduced PI(3,4,5)P3 causes temperature-sensitive paralysis and reduced neurotransmitter release, a phenotype also seen in animals expressing a Syntaxin1A with a mutated juxtamembrane domain. Thus, our data indicate that PI(3,4,5)P3, based on electrostatic interactions, clusters Syntaxin1A at release sites to regulate neurotransmitter release. [Copyright &y& Elsevier]

Abstract: Microscale thermophoresis (MST) allows for quantitative analysis of protein interactions in free solution and with low sample consumption. The technique is based on thermophoresis, the directed motion of molecules in temperature gradients. Thermophoresis is highly sensitive to all types of binding-induced changes of molecular properties, be it in size, charge, hydration shell or conformation. In an all-optical approach, an infrared laser is used for local heating, and molecule mobility in the temperature gradient is analyzed via fluorescence. In standard MST one binding partner is fluorescently labeled. However, MST can also be performed label-free by exploiting intrinsic protein UV-fluorescence. Despite the high molecular weight ratio, the interaction of small molecules and peptides with proteins is readily accessible by MST. Furthermore, MST assays are highly adaptable to fit to the diverse requirements of different biomolecules, such as membrane proteins to be stabilized in solution. The type of buffer and additives can be chosen freely. Measuring is even possible in complex bioliquids like cell lysate allowing close to in vivo conditions without sample purification. Binding modes that are quantifiable via MST include dimerization, cooperativity and competition. Thus, its flexibility in assay design qualifies MST for analysis of biomolecular interactions in complex experimental settings, which we herein demonstrate by addressing typically challenging types of binding events from various fields of life science. [Copyright &y& Elsevier]

Summary: LRRK2 is a kinase mutated in Parkinson’s disease, but how the protein affects synaptic function remains enigmatic. We identified LRRK2 as a critical regulator of EndophilinA. Using genetic and biochemical studies involving Lrrk loss-of-function mutants and Parkinson-related LRRK2G2019S gain-of-kinase function, we show that LRRK2 affects synaptic endocytosis by phosphorylating EndoA at S75, a residue in the BAR domain. We show that LRRK2-mediated EndoA phosphorylation has profound effects on EndoA-dependent membrane tubulation and membrane association in vitro and in vivo and on synaptic vesicle endocytosis at Drosophila neuromuscular junctions in vivo. Our work uncovers a regulatory mechanism that indicates that reduced LRRK2 kinase activity facilitates EndoA membrane association, while increased kinase activity inhibits membrane association. Consequently, both too much and too little LRRK2-dependent EndoA phosphorylation impedes synaptic endocytosis, and we propose a model in which LRRK2 kinase activity is part of an EndoA phosphorylation cycle that facilitates efficient vesicle formation at synapses. [ABSTRACT FROM AUTHOR]

In neurotransmission synaptotagmin-1 tethers synaptic vesicles to the presynaptic plasma membrane by binding to acidic membrane lipids and SNAREs and promotes rapid SNARE-mediated fusion upon Ca2+ triggering. However, recent studies suggested that upon membrane contact synaptotagmin may not only bind in trans to the target membrane but also in eis to its own membrane. Using a sensitive membrane tethering assay we have now dissected the structural requirements and concentration ranges for Ca2+-dependent and -independent c/s-binding and trans-tethering in the presence and absence of acidic phospholipids and SNAREs. Using variants of membrane-anchored synaptotagmin in which the Ca2+-binding sites in the C2 domains and a basic cluster involved in membrane binding were disrupted we show that Ca2+-depen-dent c/s-binding prevents trans-interactions if the c;'s-membrane contains 12-20% anionic phospholipids. Similarly, no trans-interactions were observable using soluble C2AB-domain fragments at comparable concentrations. At saturating concentrations, however, tethering was observed with soluble C2AB domains, probably due to crowding on the vesicle surface and competition for binding sites. We conclude that trans-interactions of synaptotagmin considered to be essential for its function are controlled by a delicate balance between eis- and trans-binding, which may play an important modulatory role in synaptic transmission. [ABSTRACT FROM AUTHOR]

We measured the lateral mobility of integral membrane proteins reconstituted in giant unilamellar vesicles (GUVs), using fluorescence correlation spectroscopy. Receptor, channel, and transporter proteins with 1 -36 transmembrane segments (lateral radii ranging from 0.5 to 4 nm) and a a-helical peptide (radius of 0.5 nm) were fluorescently labeled and incorporated into GUVs. At low protein-to-lipid ratios (i.e., 10-100 proteins per μm² of membrane surface), the diffusion coefficient D displayed a weak dependence on the hydrodynamic radius (R) of the proteins [0 scaled with ln(1/R)], consistent with the Saffman-Delbrück model. At higher protein-to lipid ratios (up to 3000 μm²), the lateral diffusion coefficient of the molecules decreased linearly with increasing the protein concentration in the membrane. The implications of our findings for protein mobility in biological membranes (protein crowding of ∼25,000 μm²) and use of diffusion measurements for protein geometry (size, oligomerization) determinations are discussed. [ABSTRACT FROM AUTHOR]

Glycosylation is an important post-translational modification for both intracellular and secreted proteins. For glycosylation to occur, cargo must be transported after synthesis through the different compartments of the Golgi apparatus where distinct monosaccharides are sequentially bound and trimmed, resulting in increasingly complex branched glycan structures. Of utmost importance for this process is the intraorganellar environment of the Golgi. Each Golgi compartment has a distinct pH, which is maintained by the vacuolar H+-ATPase (V-ATPase). Moreover, tethering factors such as Golgins and the conserved oligomeric Golgi (COG) complex, in concert with coatomer (COPI) and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion, efficiently deliver glycosylation enzymes to the right Golgi compartment. Together, these factors maintain intra-Golgi trafficking of proteins involved in glycosylation and thereby enable proper glycosylation. However, pathogenic mutations in these factors can cause defective glycosylation and lead to diseases with a wide variety of symptoms such as liver dysfunction and skin and bone disorders. Collectively, this group of disorders is known as congenital disorders of glycosylation (CDG). Recent technological advances have enabled the robust identification of novel CDGs related to membrane trafficking components. In this review, we highlight differences and similarities between membrane trafficking-related CDGs. [ABSTRACT FROM AUTHOR]

Cells are exposed to reactive oxygen species (ROS) as a by-product of mitochondrial metabolism, especially under hypoxia. ROS are also enzymatically generated at the plasma membrane during inflammation. Radicals cause cellular damage leading to cell death, as they react indiscriminately with surrounding lipids, proteins, and nucleotides. However, ROS are also important for many physiological processes, including signaling, pathogen killing and chemotaxis. The sensitivity of cells to ROS therefore likely depends on the subcellular location of ROS production, but how this affects cell viability is poorly understood. As ROS generation consumes oxygen, and hypoxia-mediated signaling upregulates expression of antioxidant transcription factor Nrf2, it is difficult to discern hypoxic from radical stress. In this study, we developed an optogenetic toolbox for organelle-specific generation of ROS using the photosensitizer protein SuperNova which produces superoxide anion upon excitation with 590 nm light. We fused SuperNova to organelle specific localization signals to induce ROS with high precision. Selective ROS production did not affect cell viability in most organelles except for the nucleus. SuperNova is a promising tool to induce locally targeted ROS production, opening up new possibilities to investigate processes and organelles that are affected by localized ROS production. [ABSTRACT FROM AUTHOR]

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 5 (Stx5) in mammals and its ortholog Sed5p in Saccharomyces cerevisiae mediate anterograde and retrograde endoplasmic reticulum (ER)-Golgi trafficking. Stx5 and Sed5p are structurally highly conserved and are both regulated by interactions with other ER-Golgi SNARE proteins, the Sec1/Munc18-like protein Scfd1/Sly1p and the membrane tethering complexes COG, p115, and GM130. Despite these similarities, yeast Sed5p and mammalian Stx5 are differently recruited to COPII-coated vesicles, and Stx5 interacts with the microtubular cytoskeleton, whereas Sed5p does not. In this review, we argue that these different Stx5 interactions contribute to structural differences in ER-Golgi transport between mammalian and yeast cells. Insight into the function of Stx5 is important given its essential role in the secretory pathway of eukaryotic cells and its involvement in infections and neurodegenerative diseases. [ABSTRACT FROM AUTHOR]