Membrane lipids couple synaptotagmin to SNARE-mediated granule fusion in insulin-secreting cells.

Insulin secretion depends on the Ca2+-regulated fusion of granules with the plasma membrane. A recent model of Ca2+-triggered exocytosis in secretory cells proposes that lipids in the plasma membrane couple the calcium sensor Syt1 to the membrane fusion machinery (Kiessling et al., 2018). Specifically, Ca2+-mediated binding of Syt1's C2 domains to the cell membrane shifts the membrane-anchored SNARE syntaxin-1a to a more fusogenic conformation, straightening its juxtamembrane linker. To test this model in live cells and extend it to insulin secretion, we enriched INS1 cells with a panel of lipids with different acyl chain compositions. Fluorescence lifetime measurements demonstrate that cells with more disordered membranes show an increase in fusion efficiency, and vice versa. Experiments with granules purified from INS1 cells and recombinant SNARE proteins reconstituted in supported membranes confirmed that lipid acyl chain composition determines SNARE conformation and that lipid disordering correlates with increased fusion. Addition of Syt1's C2AB domains significantly decreased lipid order in target membranes and increased SNARE-mediated fusion probability. Strikingly, Syt's action on both fusion and lipid order could be partially bypassed by artificially increasing unsaturated phosphatidylserines in the target membrane. Thus, plasma membrane lipids actively participate in coupling Ca2+/synaptotagmin-sensing to the SNARE fusion machinery in cells.

A novel in vitro system of supported planar endosomal membranes (SPEMs) reveals an enhancing role for cathepsin B in the final stage of Ebola virus fusion and entry.

Ebola virus (EBOV) causes a hemorrhagic fever with fatality rates up to 90%. The EBOV entry process is complex and incompletely understood. Following attachment to host cells, EBOV is trafficked to late endosomes/lysosomes where its glycoprotein (GP) is processed to a 19-kDa form, which binds to the EBOV intracellular receptor Niemann-Pick type C1. We previously showed that the cathepsin protease inhibitor, E-64d, blocks infection by pseudovirus particles bearing 19-kDa GP, suggesting that further cathepsin action is needed to trigger fusion. This, however, has not been demonstrated directly. Since 19-kDa Ebola GP fusion occurs in late endosomes, we devised a system in which enriched late endosomes are used to prepare supported planar endosomal membranes (SPEMs), and fusion of fluorescent (pseudo)virus particles is monitored by total internal reflection fluorescence microscopy. We validated the system by demonstrating the pH dependencies of influenza virus hemagglutinin (HA)-mediated and Lassa virus (LASV) GP-mediated fusion. Using SPEMs, we showed that fusion mediated by 19-kDa Ebola GP is dependent on low pH, enhanced by Ca2+, and augmented by the addition of cathepsins. Subsequently, we found that E-64d inhibits full fusion, but not lipid mixing, mediated by 19-kDa GP, which we corroborated with the reversible cathepsin inhibitor VBY-825. Hence, we provide both gain- and loss-of-function evidence that further cathepsin action enhances the fusion activity of 19-kDa Ebola GP. In addition to providing new insights into how Ebola GP mediates fusion, the approach we developed employing SPEMs can now be broadly used for studies of virus and toxin entry through endosomes. IMPORTANCE Ebola virus is the causative agent of Ebola virus disease, which is severe and frequently lethal. EBOV gains entry into cells via late endosomes/lysosomes. The events immediately preceding fusion of the viral and endosomal membranes are incompletely understood. In this study, we report a novel in vitro system for studying virus fusion with endosomal membranes. We validated the system by demonstrating the low pH dependencies of influenza and Lassa virus fusion. Moreover, we show that further cathepsin B action enhances the fusion activity of the primed Ebola virus glycoprotein. Finally, this model endosomal membrane system should be useful in studying the mechanisms of bilayer breaching by other enveloped viruses, by non-enveloped viruses, and by acid-activated bacterial toxins.

Serinc5 Restricts HIV Membrane Fusion by Altering Lipid Order and Heterogeneity in the Viral Membrane.

The host restriction factor, Serinc5, incorporates into budding HIV particles and inhibits their infection by an incompletely understood mechanism. We have previously reported that Serinc5 but not its paralogue, Serinc2, blocks HIV cell entry by membrane fusion, specifically by inhibiting fusion pore formation and dilation. A body of work suggests that Serinc5 may alter the conformation and clustering of the HIV fusion protein, Env. To contribute an additional perspective to the developing model of Serinc5 restriction, we assessed Serinc2 and Serinc5's effects on HIV pseudoviral membranes. By measuring pseudoviral membrane thickness via cryo-electron microscopy and order via the fluorescent dye, FLIPPER-TR, Serinc5 was found to increase membrane heterogeneity, skewing the distribution toward a larger fraction of the viral membrane in an ordered phase. We also directly observed for the first time the coexistence of membrane domains within individual viral membrane envelopes. Using a total internal reflection fluorescence-based single particle fusion assay, we found that treatment of HIV pseudoviral particles with phosphatidylethanolamine (PE) rescued HIV pseudovirus fusion from restriction by Serinc5, which was accompanied by decreased membrane heterogeneity and order. This effect was specific for PE and did not depend on acyl chain length or saturation. Together, these data suggest that Serinc5 alters multiple interrelated properties of the viral membrane─lipid chain order, rigidity, line tension, and lateral pressure─which decrease the accessibility of fusion intermediates and disfavor completion of fusion. These biophysical insights into Serinc5 restriction of HIV infectivity could contribute to the development of novel antivirals that exploit the same weaknesses.

Drunken lipid membranes, not drunken SNARE proteins, promote fusion in a model of neurotransmitter release.

Alcohol affects many neuronal proteins that are upstream or down-stream of synaptic vesicle fusion and neurotransmitter release. Less well studied is alcohol's effect on the fusion machinery including SNARE proteins and lipid membranes. Using a SNARE-driven fusion assay we show that fusion probability is significantly increased at 0.4% v/v (68 mM) ethanol; but not with methanol up to 10%. Ethanol appears to act directly on membrane lipids since experiments focused on protein properties [circular dichroism spectrometry, site-directed fluorescence interference contrast (sdFLIC) microscopy, and vesicle docking results] showed no significant changes up to 5% ethanol, but a protein-free fusion assay also showed increased lipid membrane fusion rates with 0.4% ethanol. These data show that the effects of high physiological doses of ethanol on SNARE-driven fusion are mediated through ethanol's interaction with the lipid bilayer of membranes and not SNARE proteins, and that methanol affects lipid membranes and SNARE proteins only at high doses.

SARS-CoV-2 requires acidic pH to infect cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry starts with membrane attachment and ends with spike (S) protein-catalyzed membrane fusion depending on two cleavage steps, namely, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time three-dimensional single-virion tracking, we show that fusion and genome penetration require virion exposure to an acidic milieu of pH 6.2 to 6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2-overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2-expressing cells in the acidic milieu of the nasal cavity.

Complexin-1 and synaptotagmin-1 compete for binding sites on membranes containing PtdInsP2.

Complexin-1 is an essential protein for neuronal exocytosis that acts to depress spontaneous fusion events while enhancing evoked neurotransmitter release. In addition to binding soluble N-ethylmaleimide-sensitive factor attachment protein receptors, it is well established that complexin associates with membranes in a manner that depends upon membrane curvature. In the present work, we examine the membrane binding of complexin using electron paramagnetic resonance spectroscopy, fluorescence anisotropy, and total internal reflection fluorescence microscopy. The apparent membrane affinity of complexin is found to strongly depend upon the concentration of protein used in the binding assay, and this is a result of a limited number of binding sites for complexin on the membrane interface. Although both the N- and C-terminal regions of complexin associate with the membrane interface, membrane affinity is driven by its C-terminus. Complexin prefers to bind liquid-disordered membrane phases and shows an enhanced affinity toward membranes containing phosphatidylinositol 4-5-bisphosphate (PI(4,5)P2). In the presence of PI(4,5)P2, complexin is displaced from the membrane surface by proteins that bind to or sequester PI(4,5)P2. In particular, the neuronal calcium sensor synaptotagmin-1 displaces complexin from the membrane but only when PI(4,5)P2 is present. Complexin and synaptotagmin compete on the membrane interface in the presence of PI(4,5)P2, and this interaction may play a role in calcium-triggered exocytosis by displacing complexin from its fusion-inhibiting state.

SARS-CoV-2 requires acidic pH to infect cells.

SARS-CoV-2 cell entry starts with membrane attachment and ends with spike-protein (S) catalyzed membrane fusion depending on two cleavage steps, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time 3D single virion tracking, we show fusion and genome penetration requires virion exposure to an acidic milieu of pH 6.2-6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2 overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2 expressing cells in the acidic milieu of the nasal cavity. Significance Statement: Infection by SARS-CoV-2 depends upon the S large spike protein decorating the virions and is responsible for receptor engagement and subsequent fusion of viral and cellular membranes allowing release of virion contents into the cell. Using new single particle imaging tools, to visualize and track the successive steps from virion attachment to fusion, combined with chemical and genetic perturbations of the cells, we provide the first direct evidence for the cellular uptake routes of productive infection in multiple cell types and their dependence on proteolysis of S by cell surface or endosomal proteases. We show that fusion and content release always require the acidic environment from endosomes, preceded by liberation of the S1 fragment which depends on ACE2 receptor engagement. One sentence summary: Detailed molecular snapshots of the productive infectious entry pathway of SARS-CoV-2 into cells.

Flagellin outer domain dimerization modulates motility in pathogenic and soil bacteria from viscous environments.

Flagellar filaments function as the propellers of the bacterial flagellum and their supercoiling is key to motility. The outer domains on the surface of the filament are non-critical for motility in many bacteria and their structures and functions are not conserved. Here, we show the atomic cryo-electron microscopy structures for flagellar filaments from enterohemorrhagic Escherichia coli O157:H7, enteropathogenic E. coli O127:H6, Achromobacter, and Sinorhizobium meliloti, where the outer domains dimerize or tetramerize to form either a sheath or a screw-like surface. These dimers are formed by 180° rotations of half of the outer domains. The outer domain sheath (ODS) plays a role in bacterial motility by stabilizing an intermediate waveform and prolonging the tumbling of E. coli cells. Bacteria with these ODS and screw-like flagellar filaments are commonly found in soil and human intestinal environments of relatively high viscosity suggesting a role for the dimerization in these environments.

Endosomes supporting fusion mediated by vesicular stomatitis virus glycoprotein have distinctive motion and acidification.

Most enveloped viruses infect cells by binding receptors at the cell surface and undergo trafficking through the endocytic pathway to a compartment with the requisite conditions to trigger fusion with a host endosomal membrane. Broad categories of compartments in the endocytic pathway include early and late endosomes, which can be further categorized into subpopulations with differing rates of maturation and motility characteristics. Endocytic compartments have varying protein and lipid components, luminal ionic conditions and pH that provide uniquely hospitable environments for specific viruses to fuse. In order to characterize compartments that permit fusion, we studied the trafficking and fusion of viral particles pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) on their surface and equipped with a novel pH sensor and a fluorescent content marker to measure pH, motion and fusion at the single particle level in live cells. We found that the VSV-G particles fuse predominantly from more acidic and more motile endosomes, and that a significant fraction of particles is trafficked to more static and less acidic endosomes that do not support their fusion. Moreover, the fusion-supporting endosomes undergo directed motion.

Conserved arginine residues in synaptotagmin 1 regulate fusion pore expansion through membrane contact.

Synaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca2+ sensor for synchronous neurotransmitter release. In this work, an arginine containing region in the second C2 domain of synaptotagmin 1 (C2B) is shown to control the expansion of the fusion pore and thereby the concentration of neurotransmitter released. This arginine apex, which is opposite the Ca2+ binding sites, interacts with membranes or membrane reconstituted SNAREs; however, only the membrane interactions occur under the conditions in which fusion takes place. Other regions of C2B influence the fusion probability and kinetics but do not control the expansion of the fusion pore. These data indicate that the C2B domain has at least two distinct molecular roles in the fusion event, and the data are consistent with a model where the arginine apex of C2B positions the domain at the curved membrane surface of the expanding fusion pore.

ATP and large signaling metabolites flux through caspase-activated Pannexin 1 channels.

Pannexin 1 (Panx1) is a membrane channel implicated in numerous physiological and pathophysiological processes via its ability to support release of ATP and other cellular metabolites for local intercellular signaling. However, to date, there has been no direct demonstration of large molecule permeation via the Panx1 channel itself, and thus the permselectivity of Panx1 for different molecules remains unknown. To address this, we expressed, purified, and reconstituted Panx1 into proteoliposomes and demonstrated that channel activation by caspase cleavage yields a dye-permeable pore that favors flux of anionic, large-molecule permeants (up to ~1 kDa). Large cationic molecules can also permeate the channel, albeit at a much lower rate. We further show that Panx1 channels provide a molecular pathway for flux of ATP and other anionic (glutamate) and cationic signaling metabolites (spermidine). These results verify large molecule permeation directly through caspase-activated Panx1 channels that can support their many physiological roles.

Ebola virus glycoprotein interacts with cholesterol to enhance membrane fusion and cell entry.

Cholesterol serves critical roles in enveloped virus fusion by modulating membrane properties. The glycoprotein (GP) of Ebola virus (EBOV) promotes fusion in the endosome, a process that requires the endosomal cholesterol transporter NPC1. However, the role of cholesterol in EBOV fusion is unclear. Here we show that cholesterol in GP-containing membranes enhances fusion and the membrane-proximal external region and transmembrane (MPER/TM) domain of GP interacts with cholesterol via several glycine residues in the GP2 TM domain, notably G660. Compared to wild-type (WT) counterparts, a G660L mutation caused a more open angle between MPER and TM domains in an MPER/TM construct, higher probability of stalling at hemifusion for GP2 proteoliposomes and lower cell entry of virus-like particles (VLPs). VLPs with depleted cholesterol show reduced cell entry, and VLPs produced under cholesterol-lowering statin conditions show less frequent entry than respective controls. We propose that cholesterol-TM interactions affect structural features of GP2, thereby facilitating fusion and cell entry.

Distinct insulin granule subpopulations implicated in the secretory pathology of diabetes types 1 and 2.

Insulin secretion from ß-cells is reduced at the onset of type-1 and during type-2 diabetes. Although inflammation and metabolic dysfunction of ß-cells elicit secretory defects associated with type-1 or type-2 diabetes, accompanying changes to insulin granules have not been established. To address this, we performed detailed functional analyses of insulin granules purified from cells subjected to model treatments that mimic type-1 and type-2 diabetic conditions and discovered striking shifts in calcium affinities and fusion characteristics. We show that this behavior is correlated with two subpopulations of insulin granules whose relative abundance is differentially shifted depending on diabetic model condition. The two types of granules have different release characteristics, distinct lipid and protein compositions, and package different secretory contents alongside insulin. This complexity of ß-cell secretory physiology establishes a direct link between granule subpopulation and type of diabetes and leads to a revised model of secretory changes in the diabetogenic process.

Diabetes is a disease that occurs when sugar levels in the blood can no longer be controlled by a hormone called insulin. People with type 1 diabetes lose the ability to produce insulin after their immune system attacks the ß-cells in their pancreas that make this hormone. People with type 2 diabetes develop the disease when ß-cells become exhausted from increased insulin demand and stop producing insulin. ß-cells store insulin in small compartments called granules. When blood sugar levels rise, these granules fuse with the cell membrane allowing ß-cells to release large quantities of insulin at once. This fusion is disrupted early in type 1 diabetes, but later in type 2: the underlying causes of these disruptions are unclear. In the laboratory, signals that trigger inflammation and molecules called fatty acids can mimic type 1 or type 2 diabetes respectively when applied to insulin-producing cells. Kreutzberger, Kiessling et al. wanted to know whether pro-inflammatory molecules and fatty acids affect insulin granules differently at the molecular level. To do this, insulin-producing cells were grown in the lab and treated with either fatty acids or pro-inflammatory molecules. The insulin granules of these cells were then isolated. Next, the composition of the granules and how they fused to lab-made membranes that mimic the cell membrane was examined. The experiments revealed that healthy ß-cells have two types of granules, each with a different version of a protein called synaptotagmin. Cells treated with molecules mimicking type 1 diabetes lost granules with synaptotagmin-7, while granules with synaptotagmin-9 were lost in cells treated with fatty acids to imitate type 2 diabetes. Each type of granule responded differently to calcium levels in the cell and secreted different molecules, indicating that each elicits a different diabetic response in the body. These findings suggest that understanding how insulin granules are formed and regulated may help find treatments for type 1 and 2 diabetes, possibly leading to therapies that reverse the loss of different types of granules. Additionally, the molecules of these granules may also be used as markers to determine the stage of diabetes. More broadly, these results show how understanding how molecule release changes with disease in different cell types may help diagnose or stage a disease.

HIV-cell membrane fusion intermediates are restricted by Serincs as revealed by cryo-electron and TIRF microscopy.

To enter a cell and establish infection, HIV must first fuse its lipid envelope with the host cell plasma membrane. Whereas the process of HIV membrane fusion can be tracked by fluorescence microscopy, the 3D configuration of proteins and lipids at intermediate steps can only be resolved with cryo-electron tomography (cryoET). However, cryoET of whole cells is technically difficult. To overcome this problem, we have adapted giant plasma membrane vesicles (or blebs) from native cell membranes expressing appropriate receptors as targets for fusion with HIV envelope glycoprotein-expressing pseudovirus particles with and without Serinc host restriction factors. The fusion behavior of these particles was probed by TIRF microscopy on bleb-derived supported membranes. Timed snapshots of fusion of the same particles with blebs were examined by cryo-ET. The combination of these methods allowed us to characterize the structures of various intermediates on the fusion pathway and showed that when Serinc3 or Serinc5 (but not Serinc2) were present, later fusion products were more prevalent, suggesting that Serinc3/5 act at multiple steps to prevent progression to full fusion. In addition, the antifungal amphotericin B reversed Serinc restriction, presumably by intercalation into the fusing membranes. Our results provide a highly detailed view of Serinc restriction of HIV-cell membrane fusion and thus extend current structural and functional information on Serinc as a lipid-binding protein.

Synaptotagmin-7 enhances calcium-sensing of chromaffin cell granules and slows discharge of granule cargos.

Synaptotagmin-7 (Syt-7) is one of two major calcium sensors for exocytosis in adrenal chromaffin cells, the other being synaptotagmin-1 (Syt-1). Despite a broad appreciation for the importance of Syt-7, questions remain as to its localization, function in mediating discharge of dense core granule cargos, and role in triggering release in response to physiological stimulation. These questions were addressed using two distinct experimental preparations-mouse chromaffin cells lacking endogenous Syt-7 (KO cells) and a reconstituted system employing cell-derived granules expressing either Syt-7 or Syt-1. First, using immunofluorescence imaging and subcellular fractionation, it is shown that Syt-7 is widely distributed in organelles, including dense core granules. Total internal reflection fluorescence (TIRF) imaging demonstrates that the kinetics and probability of granule fusion in Syt-7 KO cells stimulated by a native secretagogue, acetylcholine, are markedly lower than in WT cells. When fusion is observed, fluorescent cargo proteins are discharged more rapidly when only Syt-1 is available to facilitate release. To determine the extent to which the aforementioned results are attributable purely to Syt-7, granules expressing only Syt-7 or Syt-1 were triggered to fuse on planar supported bilayers bearing plasma membrane SNARE proteins. Here, as in cells, Syt-7 confers substantially greater calcium sensitivity to granule fusion than Syt-1 and slows the rate at which cargos are released. Overall, this study demonstrates that by virtue of its high affinity for calcium and effects on fusion pore expansion, Syt-7 plays a central role in regulating secretory output from adrenal chromaffin cells.

In vitro fusion of single synaptic and dense core vesicles reproduces key physiological properties.

Regulated exocytosis of synaptic vesicles is substantially faster than of endocrine dense core vesicles despite similar molecular machineries. The reasons for this difference are unknown and could be due to different regulatory proteins, different spatial arrangements, different vesicle sizes, or other factors. To address these questions, we take a reconstitution approach and compare regulated SNARE-mediated fusion of purified synaptic and dense core chromaffin and insulin vesicles using a single vesicle-supported membrane fusion assay. In all cases, Munc18 and complexin are required to restrict fusion in the absence of calcium. Calcium triggers fusion of all docked vesicles. Munc13 (C1C2MUN domain) is required for synaptic and enhanced insulin vesicle fusion, but not for chromaffin vesicles, correlating inversely with the presence of CAPS protein on purified vesicles. Striking disparities in calcium-triggered fusion rates are observed, increasing with curvature with time constants 0.23 s (synaptic vesicles), 3.3 s (chromaffin vesicles), and 9.1 s (insulin vesicles) and correlating with rate differences in cells.

A molecular mechanism for calcium-mediated synaptotagmin-triggered exocytosis.

The regulated exocytotic release of neurotransmitter and hormones is accomplished by a complex protein machinery whose core consists of SNARE proteins and the calcium sensor synaptotagmin-1. We propose a mechanism in which the lipid membrane is intimately involved in coupling calcium sensing to release. We found that fusion of dense core vesicles, derived from rat PC12 cells, was strongly linked to the angle between the cytoplasmic domain of the SNARE complex and the plane of the target membrane. We propose that, as this tilt angle increases, force is exerted on the SNARE transmembrane domains to drive the merger of the two bilayers. The tilt angle markedly increased following calcium-mediated binding of synaptotagmin to membranes, strongly depended on the surface electrostatics of the membrane, and was strictly coupled to the lipid order of the target membrane.

Quaternary structure of the small amino acid transporter OprG from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen that causes nosocomial infections. The P. aeruginosa outer membrane contains specific porins that enable substrate uptake, with the outer membrane protein OprG facilitating transport of small, uncharged amino acids. However, the pore size of an eight-stranded ß-barrel monomer of OprG is too narrow to accommodate even the smallest transported amino acid, glycine, raising the question of how OprG facilitates amino acid uptake. Pro-92 of OprG is critically important for amino acid transport, with a P92A substitution inhibiting transport and the NMR structure of this variant revealing that this substitution produces structural changes in the barrel rim and restricts loop motions. OprG may assemble into oligomers in the outer membrane (OM) whose subunit interfaces could form a transport channel. Here, we explored the contributions of the oligomeric state and the extracellular loops to OprG's function. Using chemical cross-linking to determine the oligomeric structures of both WT and P92A OprG in native outer membranes and atomic force microscopy, and single-molecule fluorescence of the purified proteins reconstituted into lipid bilayers, we found that both protein variants form oligomers, supporting the notion that subunit interfaces in the oligomer could provide a pathway for amino acid transport. Furthermore, performing transport assays with loop-deleted OprG variants, we found that these variants also can transport small amino acids, indicating that the loops are not solely responsible for substrate transport. We propose that OprG functions as an oligomer and that conformational changes in the barrel-loop region might be crucial for its activity.

Distinct reaction mechanisms for hyaluronan biosynthesis in different kingdoms of life.

Hyaluronan (HA) is an acidic high molecular weight cell surface polysaccharide ubiquitously expressed by vertebrates, some pathogenic bacteria and even viruses. HA modulates many essential physiological processes and is implicated in numerous pathological conditions ranging from autoimmune diseases to cancer. In various pathogens, HA functions as a non-immunogenic surface polymer that reduces host immune responses. It is a linear polymer of strictly alternating glucuronic acid and N-acetylglucosamine units synthesized by HA synthase (HAS), a membrane-embedded family-2 glycosyltransferase. The enzyme synthesizes HA and secretes the polymer through a channel formed by its own membrane-integrated domain. To reveal how HAS achieves these tasks, we determined the biologically functional units of bacterial and viral HAS in a lipid bilayer environment by co-immunoprecipitation, single molecule fluorescence photobleaching, and site-specific cross-linking analyses. Our results demonstrate that bacterial HAS functions as an obligate homo-dimer with two functional HAS copies required for catalytic activity. In contrast, the viral enzyme, closely related to vertebrate HAS, functions as a monomer. Using site-specific cross-linking, we identify the dimer interface of bacterial HAS and show that the enzyme uses a reaction mechanism distinct from viral HAS that necessitates a dimeric assembly.

Asymmetric Phosphatidylethanolamine Distribution Controls Fusion Pore Lifetime and Probability.

Little attention has been given to how the asymmetric lipid distribution of the plasma membrane might facilitate fusion pore formation during exocytosis. Phosphatidylethanolamine (PE), a cone-shaped phospholipid, is predominantly located in the inner leaflet of the plasma membrane and has been proposed to promote membrane deformation and stabilize fusion pores during exocytotic events. To explore this possibility, we modeled exocytosis using plasma membrane SNARE-containing planar-supported bilayers and purified neuroendocrine dense core vesicles (DCVs) as fusion partners, and we examined how different PE distributions between the two leaflets of the supported bilayers affected SNARE-mediated fusion. Using total internal reflection fluorescence microscopy, the fusion of single DCVs with the planar-supported bilayer was monitored by observing DCV-associated neuropeptide Y tagged with a fluorescent protein. The time-dependent line shape of the fluorescent signal enables detection of DCV docking, fusion-pore opening, and vesicle collapse into the planar membrane. Four different distributions of PE in the planar bilayer mimicking the plasma membrane were examined: exclusively in the leaflet facing the DCVs; exclusively in the opposite leaflet; equally distributed in both leaflets; and absent from both leaflets. With PE in the leaflet facing the DCVs, overall fusion was most efficient and the extended fusion pore lifetime (0.7 s) enabled notable detection of content release preceding vesicle collapse. All other PE distributions decreased fusion efficiency, altered pore lifetime, and reduced content release. With PE exclusively in the opposite leaflet, resolution of pore opening and content release was lost.