Lipid Composition Of Vesicles Research Articles

Analysis of lipid composition of cell-bound membrane vesicles (CBMVs) derived from endothelial cells

Cell-bound membrane vesicles (CBMVs), a novel type of membrane vesicles, has been identified through a series of characterization tools. However, the lipid composition of CBMVs has not yet been characterized. This study focuses on the differences between the lipid composition of CBMVs and cell membranes. In order to determine the lipid composition of CBMVs and cell membranes of Human umbilical vein endothelial cells (HUVECs) and find out differential metabolites, this study was carried out by isolating CBMVs lipids and characterizing them using high-performance liquid chromatography tandem secondary mass spectrometry (LC-MS/MS). The results showed the presence of 213 up-regulated and 726 down-regulated lipids in CBMVs compared to cell membranes which produced CBMVs. There are lipids expressed in CBMVs and not in cell membranes: DGDG 18:0_8:0, DGDG O-8:0_16:1, DGDG O-26:7_26:7, DGDG O-16:3_26:7, TG 15:4_21:5_22:5;4O, PC 49:11, PG 19:5_38:10, PI 60:14, PI 44:9, PI 25:2, PI 43:5, PI 50:10, PS 55:10. DGDG (digalactosyl diglyceride), MGDG (monogalactosyl diglyceride) belongs to galactosyl diglyceride, promotes fat catabolism, which also has antioxidant and anti-inflammatory effects, and unsaturated diacylglycerols are a class of antioxidant compounds, which enables CBMVs to have a therapeutic potential.

Efficiency of transcription and translation of cell-free protein synthesis systems in cell-sized lipid vesicles with changing lipid composition determined by fluorescence measurements.

To develop artificial cell models that mimic living cells, cell-sized lipid vesicles encapsulating cell-free protein synthesis (CFPS) systems are useful for protein expressions or artificial gene circuits for vesicle-vesicle communications. Therefore, investigating the transcriptional and translational properties of CFPS systems in lipid vesicles is important for maximizing the synthesis and functions of proteins. Although transcription and translation using CFPS systems inside lipid vesicles are more important than that outside lipid vesicles, the former processes are not investigated by changing the lipid composition of lipid vesicles. Herein, we investigated changes in transcription and translation using CFPS systems inside giant lipid vesicles (approximately 5-20μm in diameter) caused by changing the lipid composition of lipid vesicles containing neutral, positively, and negatively charged lipids. After incubating for 30min, 1h, 2h, and 4h, the transcriptional and translational activities in these lipid vesicles were determined by detecting the fluorescence intensities of the fluorogenic RNA aptamer on the 3'-untranslated region of mRNA (transcription) and the fluorescent protein sfCherry (translation), respectively. The results revealed that transcriptional and translational activities in a lipid vesicle containing positively charged lipids were high when the protein was synthesized using the CFPS system inside the lipid vesicle. Thus, the present study provides an experimental basis for constructing complex artificial cell models using bottom-up approaches.

Protein-lipid interactions and protein anchoring modulate the modes of association of the globular domain of the Prion protein and Doppel protein to model membrane patches.

The Prion protein is the molecular hallmark of the incurable prion diseases affecting mammals, including humans. The protein-only hypothesis states that the misfolding, accumulation, and deposition of the Prion protein play a critical role in toxicity. The cellular Prion protein (PrPC) anchors to the extracellular leaflet of the plasma membrane and prefers cholesterol- and sphingomyelin-rich membrane domains. Conformational Prion protein conversion into the pathological isoform happens on the cell surface. In vitro and in vivo experiments indicate that Prion protein misfolding, aggregation, and toxicity are sensitive to the lipid composition of plasma membranes and vesicles. A picture of the underlying biophysical driving forces that explain the effect of Prion protein - lipid interactions in physiological conditions is needed to develop a structural model of Prion protein conformational conversion. To this end, we use molecular dynamics simulations that mimic the interactions between the globular domain of PrPC anchored to model membrane patches. In addition, we also simulate the Doppel protein anchored to such membrane patches. The Doppel protein is the closest in the phylogenetic tree to PrPC, localizes in an extracellular milieu similar to that of PrPC, and exhibits a similar topology to PrPC even if the amino acid sequence is only 25% identical. Our simulations show that specific protein-lipid interactions and conformational constraints imposed by GPI anchoring together favor specific binding sites in globular PrPC but not in Doppel. Interestingly, the binding sites we found in PrPC correspond to prion protein loops, which are critical in aggregation and prion disease transmission barrier (β2-α2 loop) and in initial spontaneous misfolding (α2-α3 loop). We also found that the membrane re-arranges locally to accommodate protein residues inserted in the membrane surface as a response to protein binding.

High-fat diets: You are what you eat….your extracellular vesicles too!

Recent works indicate that the lipid composition of extracellular vesicles (EVs) can modify their biological functions and their incorporation into recipient cells. In particular high-fat diets affect EV biogenesis, EV lipid composition, EV targeting and consequently the cross-talk between tissues. This review connects different research topics to show that a vicious circle is established during the development of high-fat diet-induced obesity, connecting the alteration of lipid metabolism, the composition of extracellular vesicles and the spread of deleterious lipids between tissues, which participates in NAFLD/NASH and diabetes development. According to the studies described in this review, it is urgent to take an interest in this question as the modulation of EV lipid composition could be an important factor to take into account during the therapeutic management of patients suffering from metabolic syndrome and related pathologies such as obesity and diabetes. Furthermore, as lipid modification of EVs is a strategy currently being tested to enable better integration into their target tissue or cell, it is important to consider the impact of these lipid modifications on the homeostasis of these targets.

Changes in lipid composition of host-derived extracellular vesicles following Salmonella infection.

This study delves into the previously unexplored territory of extracellular vesicle (EV) cargo and composition, specifically focusing on lipid composition changes in EVs following Salmonella infection. EVs play crucial roles in intercellular communication, carrying a variety of biomolecules. Investigating how these EV cargo lipids change post-infection with Salmonella is significant for understanding the molecular mechanisms underlying lipid trafficking during infection. Given the impact of lipid composition on EV function, this research uncovers distinct differences in the lipid profiles of EVs at different time points post-infection and between infected and uninfected macrophages. This study identified lipids that are differentially abundant in EVs produced by the host during infection, offering novel insights into the dynamics of lipid profiles in EVs during cellular processes and infections. This work advances our understanding of host-pathogen interactions, specifically lipid-mediated EV functions during infection.

Vitamin D Modulates Lipid Composition of Adipocyte-Derived Extracellular Vesicles Under Inflammatory Conditions.

Adipocyte-derived extracellular vesicles (AdEVs) convey lipids that can play a role in the energy homeostasis. Vitamin D (VD) has been shown to limit the metabolic inflammation as it decreases inflammatory markers expression in adipose tissue (AT). However, VD effect on adipocytes-derived EVs has never been investigated. Thus, the aim of this study is to evaluate the AdEVs lipid composition by LC-MS/MS approach in 3T3-L1 cells treated with VD or/and pro-inflammatory factor (tumor necrosis factor α[TNFα]). Among all lipid species, four are highlighted (glycerolipids, phospholipids, lysophospholipids, and sphingolipids) with a differential content between small (sEVs) and large EVs (lEVs). This study also observes that VD alone modulates EV lipid species involved in membrane fluidity and in the budding of membrane. EVs treated with VD under inflammatory conditions have different lipid profiles than the control group, which is more pronounced in lEVs. Indeed, 25 lipid species are significantly modulated in lEVs, compared with only seven lipid species in sEVs. This study concludes that VD, alone or under inflammatory conditions, is associated with specific lipidomic signature of sEVs and lEVs. These observations reinforce current knowledge on the anti-inflammatory effect of VD.

The lipid composition of extracellular vesicles: applications in diagnostics and therapeutic delivery.

Extracellular vesicles (EVs), including exosomes, with nanoscale sizes, biological origins, various functions, and unique lipid and protein compositions have been introduced as versatile tools for diagnostic and therapeutic medical applications. Numerous studies have reported the importance of the lipid composition of EVs and its influence on their mechanism of action. For example, changes in the lipidomic profile of EVs have been shown to influence the progression of various diseases, including ovarian malignancies and prostate cancer. In this review, we endeavored to examine differences in the lipid content of EV membranes derived from different cell types to characterize their capabilities as diagnostic tools and treatments for diseases like cancer and Alzheimer's disease. We additionally discuss designing functionalized vesicles, whether synthetically by hybrid methods or by changing the lipid composition of natural EVs. Lastly, we provide an overview of current and potential biomedical applications and perspectives on the future of this growing field.

Vesicles made by several methods contain the ratio of phospholipids in the stock solution.

In this project, we measured the lipid composition of five-component vesicles produced via different methods. When making vesicles, it is typically assumed that the final lipid composition of the vesicles is identical to that of the stock solution. However, it is known that cholesterol incorporates poorly into vesicles made by emulsion phase transfer, and there is some evidence that PE lipids may not fully incorporate into vesicles made by electroformation. Here, we made a stock solution of a five-component mixture of DPPC, DOPC, DLPE, DOPG, and cholesterol. We produced vesicles from this stock solution using some of the most common methods: electroformation, gentle hydration, extrusion, and emulsion phase transfer. We then extracted the lipids from these vesicles using the Bligh and Dyer technique and used mass spectrometry to quantify the amount of each lipid type in the sample. We find that DOPC incorporates less readily into vesicles produced via emulsion phase transfer than other lipids do. For most of the methods we tested, the lipid composition of the vesicles is close to that of the stock solution, implying that results can be compared for vesicles made by these methods.

Membrane permeabilization can be crucially biased by a fusogenic lipid composition - leaky fusion caused by antimicrobial peptides in model membranes.

Induced membrane permeabilization or leakage is often taken as an indication for activity of membrane-active molecules, such as antimicrobial peptides (AMPs). The exact leakage mechanism is often unknown, but important, because certain mechanisms might actually contribute to microbial killing, while others are unselective, or potentially irrelevant in an in vivo situation. Using an antimicrobial example peptide (cR3W3), we illustrate one of the potentially misleading leakage mechanisms: leaky fusion, where leakage is coupled to membrane fusion. Like many others, we examine peptide-induced leakage in model vesicles consisting of binary mixtures of anionic and zwitterionic phospholipids. In fact, phosphatidylglycerol and phosphatidylethanolamine (PG/PE) are supposed to reflect bacterial membranes, but exhibit a high propensity for vesicle aggregation and fusion. We describe the implications of this vesicle fusion and aggregation for the reliability of model studies. The ambiguous role of the relatively fusogenic PE-lipids becomes clear as leakage decreases significantly when aggregation and fusion are prevented by sterical shielding. Furthermore, the mechanism of leakage changes if PE is exchanged for phosphatidylcholine (PC). We thus point out that the lipid composition of model membranes can be biased towards leaky fusion. This can lead to discrepancies between model studies and activity in true microbes, because leaky fusion is likely prevented by bacterial peptidoglycan layers. In conclusion, choosing the model membrane might implicate the type of effect (here leakage mechanism) that is observed. In the worst case, as with leaky fusion of PG/PE vesicles, this is not directly relevant for the intended antimicrobial application.

The Lipid Composition of Serum-Derived Small Extracellular Vesicles in Participants of a Lung Cancer Screening Study.

Simple SummaryMolecular components of extracellular vesicles present in serum are potential biomarkers of lung cancer, however, none of them have been validated in the context of an actual early detection of lung cancer. Here, we compared the lipid profiles of vesicles obtained from participants in a lung cancer screening study, including patients with screening-detected cancer and individuals with benign pulmonary nodules or without pathological changes. A few lipids whose levels were different between compared groups were detected, including ceramide Cer(42:1) upregulated in vesicles from cancer patients. Furthermore, a high heterogeneity of lipid profiles of extracellular vesicles was observed, which impaired the performance of classification models based on specific compounds.Molecular components of exosomes and other classes of small extracellular vesicles (sEV) present in human biofluids are potential biomarkers with possible applicability in the early detection of lung cancer. Here, we compared the lipid profiles of serum-derived sEV from three groups of lung cancer screening participants: individuals without pulmonary alterations, individuals with benign lung nodules, and patients with screening-detected lung cancer (81 individuals in each group). Extracellular vesicles and particles were purified from serum by size-exclusion chromatography, and a fraction enriched in sEV and depleted of low-density lipoproteins (LDLs) was selected (similar sized vesicles was observed in all groups: 70–100 nm). The targeted mass-spectrometry-based approach enabled the detection of 352 lipids, including 201 compounds used in quantitative analyses. A few compounds, exemplified by Cer(42:1), i.e., a ceramide whose increased plasma/serum level was reported in different pathological conditions, were upregulated in vesicles from cancer patients. On the other hand, the contribution of phosphatidylcholines with poly-unsaturated acyl chains was reduced in vesicles from lung cancer patients. Cancer-related features detected in serum-derived sEV were different than those of the corresponding whole serum. A high heterogeneity of lipid profiles of sEV was observed, which markedly impaired the performance of classification models based on specific compounds (the three-state classifiers showed an average AUC = 0.65 and 0.58 in the training and test subsets, respectively).

The Emerging World of Membrane Vesicles: Functional Relevance, Theranostic Avenues and Tools for Investigating Membrane Function.

Lipids are essential components of cell membranes and govern various membrane functions. Lipid organization within membrane plane dictates recruitment of specific proteins and lipids into distinct nanoclusters that initiate cellular signaling while modulating protein and lipid functions. In addition, one of the most versatile function of lipids is the formation of diverse lipid membrane vesicles for regulating various cellular processes including intracellular trafficking of molecular cargo. In this review, we focus on the various kinds of membrane vesicles in eukaryotes and bacteria, their biogenesis, and their multifaceted functional roles in cellular communication, host-pathogen interactions and biotechnological applications. We elaborate on how their distinct lipid composition of membrane vesicles compared to parent cells enables early and non-invasive diagnosis of cancer and tuberculosis, while inspiring vaccine development and drug delivery platforms. Finally, we discuss the use of membrane vesicles as excellent tools for investigating membrane lateral organization and protein sorting, which is otherwise challenging but extremely crucial for normal cellular functioning. We present current limitations in this field and how the same could be addressed to propel a fundamental and technology-oriented future for extracellular membrane vesicles.

Unique glycan and lipid composition of helminth-derived extracellular vesicles may reveal novel roles in host-parasite interactions

Although the study of helminth-derived extracellular vesicles (EVs) is in its infancy, proteomic studies of EVs from representatives of nematodes, cestodes and trematodes have identified homologs of mammalian EV proteins including components of the endosomal sorting complexes required for transport and heat-shock proteins, suggesting conservation of pathways of EV biogenesis and cargo loading between helminths and their hosts. However, parasitic helminth biology is unique and this is likely reflected in helminth EV composition and biological activity. This opinion article highlights two exceptional studies that identified EVs released by Heligmosomoides polygyrus and Fasciola hepatica which display differential lipid and glycan composition, respectively, when compared with EVs derived from mammalian cells. Furthermore, we discuss the potential implications of helminth EV lipid and glycan composition upon helminth infection and host pathology. Future studies, focusing on the unique composition and functional properties of helminth EVs, may prove crucial to the understanding of host-parasite communication.

Nanodomains Persist to much Higher Temperatures than Large Scale Phase Separation in Giant Plasma Membrane Vesicles and Can Respond Differently to Alterations of Plasma Membrane Lipid Composition

The formation and properties of liquid ordered (Lo) lipid domains (rafts) in the plasma membrane is still poorly understood. Giant plasma membrane vesicles (GPMV) derived from mammalian cells can undergo large scale phase separations into co-existing Lo and disordered lipid (Ld) domains with properties similar to domains in artificial lipid vesicles containing simple lipid mixtures. However, large scale phase separation in GPMV detected by light microscopy only occurs at low temperature. We compared large domain formation detected by light microscopy to nanodomain formation in GPMV using a FRET assay. We found that nanodomains can persist to physiological temperatures, much higher temperatures than large scale phase separation. Altering sterol or phospholipid structure in the plasma membrane outer leaflet of intact mammalian cells by cyclodextrin-catalyzed lipid exchange altered both GPMV lipid composition and the ability of GPMV to form ordered domains. Phospholipids and sterols that either stabilize or destabilize ordered domain formation in artificial lipid vesicles had a similar effect on ordered domain stability in GPMV. Nanodomain and large scale domain formation were generally stabilized or destabilized by lipid substitutions to a similar (but not identical) extent. These results show it is possible to alter the lipid composition and properties of membrane vesicles derived from plasma membrane. Other experiments found that after transfection and expression of soluble GFP in mammalian cells, GPMVs contained GFP in their lumen. Because membrane vesicles can be used to deliver biomolecules to cells, combining an analogous expression step with lipid exchange might allow preparation of plasma membrane vesicles with improved abilities to deliver cytosolic molecules to other cells.

Non-invasive marker-independent high content analysis of a microphysiological human pancreas-on-a-chip model

The increasing prevalence of diabetes, its heterogeneity, and the limited number of treatment options drive the need for physiologically relevant assay platforms with human genetic background that have the potential to improve mechanistic understanding and e\\xpedite diabetes-related research and treatment. In this study, we developed an endocrine pancreas-on-a-chip model based on a tailored microfluidic platform, which enables self-guided trapping of single human pseudo-islets. Continuous, low-shear perfusion provides a physiologically relevant microenvironment especially important for modeling and monitoring of the endocrine function as well as sufficient supply with nutrients and oxygen. Human pseudo-islets, generated from the conditionally immortalized EndoC-βH3 cell line, were successfully injected by hydrostatic pressure-driven flow without altered viability. To track insulin secretion kinetics in response to glucose stimulation in a time-resolved manner, dynamic sampling of the supernatant as well as non-invasive real-time monitoring using Raman microspectroscopy was established on-chip. Dynamic sampling indicated a biphasic glucose-stimulated insulin response. Raman microspectroscopy allowed to trace glucose responsiveness in situ and to visualize different molecular structures such as lipids, mitochondria and nuclei. In-depth spectral analyses demonstrated a glucose stimulation-dependent, increased mitochondrial activity, and a switch in lipid composition of insulin secreting vesicles, supporting the high performance of our pancreas-on-a-chip model.

Mass spectrometry of extracellular vesicles.

The review briefly summaries main features of extracellular vesicles, a joint terminology for exosomes, microvesicles, and apoptotic vesicles. These vesicles are in the center of interest in biology and medical sciences, and form a very active field of research. Mass spectrometry (MS), with its specificity and sensitivity, has the potential to identify and characterize molecular composition of these vesicles; but as yet there are only a limited, but fast-growing, number of publications that use MS workflows in this field. MS is the major tool to assess protein composition of extracellular vesicles: qualitative and quantitative proteomics approaches are both reviewed. Beside proteins, lipid and metabolite composition of vesicles might also be best assessed by MS techniques; however there are few applications as yet in this respect. The role of alternative analytical approaches, like gel-based proteomics and antibody-based immunoassays, are also mentioned. The objective of the review is to give an overview of this fast-growing field to help orient MS-based research on extracellular vesicles.

Fractional Binding: A Molecular Analog-To-Digital Converter in Ca++ Regulated Vesicle Differentiation

A critical feature of neurotransmission is that only a subset of vesicles react at a given Ca++ pulse. To achieve such a fractional response the synapse utilises a number of complex spatio-temporal and biochemical mechanisms that are still under debate but collectively appear to create distinct vesicle pools. We currently ignore whether differentiation of seemingly identical vesicles exists also for membrane trafficking vesicles.We recently established that vesicles within an ensemble display large compositional inhomogeneities(1). Annexins, as well as other Ca2+-dependent membrane binding proteins, are known to bind specific phospholipid components in cellular membranes(2). Here we investigated how the unique capability of Ca2+-regulated membrane-binding proteins to bind certain lipids enables them to discriminate between single vesicles based on their lipid compositional heterogeneity. We employed human Annexin A5 (AnxA5), which is widely used as a model system for the membrane binding properties of the Annexin superfamily(2), and our previously described single vesicle assay(3, 4). We demonstrate that heterogeneities in the lipid composition of individual vesicles within a population, in conjunction with Ca2+ concentration, regulate the fraction of vesicles recruiting Annexins with digital precision. This is a recurrent phenomenon observed in multiple vesicle samples and for different Ca2+ sensing proteins. Thus, we anticipate that the cell utilize this feature to translate analog Ca2+ influxes into a selective digital targeting of vesicle sub-populations within the ensemble for specific functions.1. J. Am. Chem. Soc. 133: 10685-10687.2. Nat Rev Mol Cell Biol. 6: 449-461.3. The EMBO Journal. 28: 3303-3314.4. Nat. Chem. Biol. 5: 835-841.

Molecular Dynamics Interactions between Silicon Electrodes and Phospholipids

In experiments involving the application of an external electric field to a biological system, the field is assumed to be uniform and isotropic far from the electrode interface, but adsorption of ions and molecules directly at the interface can complicate the structure of the charged double-layer. Reaction-kinetic models of lipid vesicle adhesion1 have utilized continuum parameters such as the rate of lipid monolayer formation and corresponding charge displacement. Experiments have suggested2 that vesicle lipid composition strongly affects the rates of lipid monolayer formation, but no atomically detailed studies have been conducted. To fill this gap we have run molecular dynamics simulations on POPS, POPC, and octane at the charged silicon interface to investigate the conformation, orientation, kinetics, and energetics of charged and uncharged molecules, respectively. This allows for direct comparisons between rates cited in the literature to those obtained in simulations, and provides researchers with additional per-molecule energetics which will further characterize lipid adhesion mechanisms near a charged interface.1 Ružic, I, Pecar-Ilic, J, DeNardis, NI (2010). Mathematical model for kinetics of organic particle adhesion at an electrified interface. Journal of Electroanalytical Chemistry 642:120-1262 Hernandez, VA, Scholz, F (2008). The lipid composition determines the kinetics of adhesion and spreading of liposomes on mercury electrodes. Bioelectrochemistry 74:149-156

Penetration enhancer-containing vesicles: Composition dependence of structural features and skin penetration ability

In this work, we focused on how composition and preparation method of vesicles might affect their morphological features and delivery performances. Penetration Enhancer-containing Vesicles, PEVs, vesicles containing a water miscible penetration enhancer (Transcutol® P; 10%, 20%, 30% v/v) and encapsulating diclofenac sodium, were formulated and compared with conventional liposomes. A cheap and unpurified commercial mixture of phospholipids, fatty acids, and triglycerides (Phospholipon® 50) was used, and the effects of this heterogeneous composition (along with the presence or absence of transcutol and the production method) on vesicle morphology, size, surface charge, drug loading, and stability were investigated. The variations in vesicle structure, bilayer thickness, and number of lamellae were assessed by TEM and Small and Wide Angle X-ray Scattering, which also proved the liquid state of the vesicular bilayer. Further, vesicles were evaluated for ex vivo (trans)dermal delivery, and their mode of action was studied performing a pre-treatment test and confocal laser scanning microscopy analyses. Results showed the formation of multi- and unilamellar vesicles that provided improved diclofenac delivery to pig skin, influenced by vesicle lipid composition and structure. Images of the qualitative CLSM analyses support the conclusion that PEVs enhance drug transport by penetrating intact the stratum corneum, thanks to a synergic effect of vesicles and penetration enhancer.

Membrane lipid composition differentially modulates the function of human plasma platelet activating factor-acetylhydrolase

Human plasma platelet activating factor-acetylhydrolase (HpPAF-AH) is a calcium-independent phospholipase that catalyzes the hydrolysis of ester bond at the sn-2 position of phospholipid substrates. The enzyme belongs to group VIIA of the phospholipase A 2 superfamily and is associated with the lipids. Circulating form of HpPAF-AH resides on the lipoprotein particles and acts on a wide variety of substrates, including oxidized phospholipids. In this study we have characterized the effect of lipid composition of the membrane vesicles on the function of purified HpPAF-AH. Lipid composition of the vesicles was varied by incorporating varying amounts of cholesterol in the matrix phospholipids, POPC and DPPC, and its effect on the membrane binding, membrane penetration and the activity of the enzyme was determined. Physicochemical properties of the phospholipid vesicles were characterized by using different fluorescent probes. For the first time our results show that (a) membrane binding of HpPAF-AH increases the activity of enzyme (interfacial activation) and (b) lipid composition of membrane vesicles, by changing the physicochemical properties, differentially modulates the binding, partial membrane penetration and the activity of the enzyme.

Role of Curvature in PEG-Mediated Fusion Between Highly Curved and Un-Curved Membranes

During neurotransmitter release, curved synaptic vesicles fuse with un-curved pre-synaptic plasma membrane, leading to the merger of two lipid bilayers and the release of neurotransmitters. Our previous efforts to model this system have employed two populations of highly curved vesicles, while others have used two populations of vesicles having ill-defined but lower curvature. Here we examine poly ethylene glycol (PEG)-triggered fusion of highly curved (SUV) with relatively un-curved (LUV) vesicles, composed of a mixture of DOPC/DOPE/sphingomeylin/DOPS/cholesterol (32/25/15/8/20), which closely models the lipid composition of synaptic vesicles. Lipid mixing (LM), contents mixing (CM) and leakage (L) time courses were fitted globally to 3- or 4-state sequential models (Weinreb, Biophys. J., 2007), from which we obtained estimates of rate constants for conversion between states as well as probabilities of LM, CM and L for each state. As expected, un-curved LUV-LUV fusion was barely detectable, while highly curved SUV-SUV fusion was reasonably efficient, saturating at ∼50% LM and ∼40% CM. Remarkably, SUV-LUV fusion was decidedly more efficient, saturating at nearly 100% LM and CM. Analysis of the fusion kinetics at different temperatures (17°-42°C) revealed complex activation thermodynamics. The rate of the first fusion intermediate formation is decidedly faster in the highly curved (stressed) SUV-SUV system than in the mixed system, with increasing temperature shifting the probability of CM toward earlier steps; whereas the probability of CM in SUV-LUV fusion shifts towards fusion pore. This suggests that mismatched curvature promotes more efficient and productive fusion events. Supported by NIGMS grants GM000678 to UNC and GM32707 to BRL.