Membrane-enclosed Compartments Research Articles

The efficient magneto-mechanical actuation of cancer cells using a very low concentration of non-interacting ferrimagnetic hexaferrite nanoplatelets

Magneto-mechanical actuation (MMA) using the low-frequency alternating magnetic fields (AMFs) of magnetic nanoparticles internalized into cancer cells can be used to irreparably damage these cells. However, nanoparticles in cells usually agglomerate, thus greatly augmenting the delivered force compared to single nanoparticles. Here, we demonstrate that MMA also decreases the cell viability, with the MMA mediated by individual, non-interacting nanoparticles. The effect was demonstrated with ferrimagnetic (i.e., permanently magnetic) barium-hexaferrite nanoplatelets (NPLs, ∼50 nm wide and 3 nm thick) with a unique, perpendicular orientation of the magnetization. Two cancer-cell lines (MDA-MB-231 and HeLa) are exposed to the NPLs in-vitro under different cell-culture conditions and actuated with a uniaxial AMF. TEM analyses show that only a small number of NPLs internalize in the cells, always situated in membrane-enclosed compartments of the endosomal-lysosomal system. Most compartments contain 1–2 NPLs and only seldom are the NPLs found in small groups, but never in close contact or mutually oriented. Even at low concentrations, the single NPLs reduce the cell viability when actuated with AMFs, which is further increased when the cells are in starvation conditions. These results pave the way for more efficient in-vivo MMA at very low particle concentrations.

Coacervate Droplets for Synthetic Cells.

The design and construction of synthetic cells - human-made microcompartments that mimic features of living cells - have experienced a real boom in the past decade. While many efforts have been geared toward assembling membrane-bounded compartments, coacervate droplets produced by liquid-liquid phase separation have emerged as an alternative membrane-free compartmentalization paradigm. Here, the dual role of coacervate droplets in synthetic cell research is discussed: encapsulated within membrane-enclosed compartments, coacervates act as surrogates of membraneless organelles ubiquitously found in living cells; alternatively, they can be viewed as crowded cytosol-like chassis for constructing integrated synthetic cells. After introducing key concepts of coacervation and illustrating the chemical diversity of coacervate systems, their physicochemical properties and resulting bioinspired functions are emphasized. Moving from suspensions of free floating coacervates, the two nascent roles of these droplets in synthetic cell research are highlighted: organelle-like modules and cytosol-like templates. Building the discussion on recent studies from the literature, the potential of coacervate droplets to assemble integrated synthetic cells capable of multiple life-inspired functions is showcased. Future challenges that are still to be tackled in the field are finally discussed.

O-197 Vertical transmission of maternal mitochondrial DNA via extracellular vesicles (EVs) modulates embryo bioenergetics during the periconceptional period

Abstract Study question Does maternal endometrial mitochondrial (mt) DNA cargo of EVs modulate embryo bioenergetics during embryo implantation? Summary answer We demonstrate the vertical transmission of maternal mtDNA within endometrial-derived EVs and their uptake by the trophoblast which reduces mitochondrial respiration and ATP production. What is known already The release and uptake of membrane-enclosed compartments with specific cargos, commonly known as EVs, represents a novel cell-to-cell communication mechanism in physiological and pathogenic conditions. EVs are generally classified into three populations based on their biogenetic pathways, composition, and physical characteristics: apoptotic bodies (ABs), microvesicles (MVs), and exosomes (EXOs). Among other contents, EVs contain single- and double-stranded DNA, with their relative abundance varying depending on the cell and vesicle type. The vertical transmission of EV-associated DNA has been proposed as a novel genetic material transfer mechanism that may impact genome evolution and tumorigenesis. Study design, size, duration Prospective observational multicenter analysis in which EVs were obtained from endometrial fluid from healthy donors aged 18–35 years (n = 10) during the receptive phase of their natural cycle and under hormonal replacement therapy in pre-receptive (P + 2), receptive (P + 5), post-receptive (P + 8) stages (n = 13). Participants/materials, setting, methods Endometrial EVs isolated using ultracentrifugation and classified according to `parameters obtained from electron microscopy, Western blotting, and size distribution analysis. DNA copy number identified using high throughput sequencing. EV-associated DNA tagged with 5-ethynyl-2’-deoxyuridine was followed by confocal imaging after co-incubation of EVs with murine embryos (n = 200). ATP levels assessed using the FLASC luciferase reporter system, and the Seahorse XFE96 extracellular flux analyzer used to measure embryo oxygen consumption rate (OCR) (n = 400). Main results and the role of chance The human endometrium secretes all three EV types - ABs, MVs, and EXOs - into the human endometrial fluid. Deep sequencing revealed that EVs encapsulated nuclear and mtDNA. When analyzing endometrial biopsies, we observed the reduced mtDNA content of endometrial cells and the activation of mitochondrial clearance mechanisms, which coincided with the time of embryo implantation together with specific enrichment in endometrial MVs secreted during the periconceptional period. EVs were internalized and DNA was transferred to the cytoplasm and nuclei of trophectoderm of murine embryos. We analyzed ATP concentrations in murine embryos and found a significant reduction in ATP levels following the coculture of embryos with a combination of all EVs types compared to control embryos cultured without endometrial EVs (p < 0.001). Finally, we demonstrated a reduction in the OCR in embryos treated with endometrial EVs obtained during the receptive phase compared with the pre-receptive phase. In conclusion, maternal EVs modulate the bioenergetics of the preimplantation embryo by increasing the embryo's metabolic rate and oxygen consumption during the periconceptional period. Limitations, reasons for caution These results were obtained using a combination of a human endometrial model and a murine embryo model. Wider implications of the findings Our results suggest that the vertical transmission of maternal mtDNA encapsulated within EVs to the trophectoderm might energetically assist the preimplantation embryo through the implantation process. Trial registration number N/A

Recent advances in studies on magnetosome-associated proteins composing the bacterial geomagnetic sensor organelle.

Magnetotactic bacteria (MTB) generate a membrane-enclosed subcellular compartment called magnetosome, which contains a biomineralized magnetite or greigite crystal, an inner membrane-derived lipid bilayer membrane, and a set of specifically targeted associated proteins. Magnetosomes are formed by a group of magnetosome-associated proteins encoded in a genomic region called magnetosome island. Magnetosomes are then arranged in a linear chain-like positioning, and the resulting magnetic dipole of the chain functions as a geomagnetic sensor for magneto-aerotaxis motility. Recent metagenomic analyses of environmental specimens shed light on the sizable phylogenetical diversity of uncultured MTB at the phylum level. These findings have led to a better understanding of the diversity and conservation of magnetosome-associated proteins. This review provided an overview of magnetosomes and magnetosome-associated proteins and introduced recent topics about this fascinating magnetic bacterial organelle. This article is protected by copyright. All rights reserved.

Phase separation can regulate membrane curvature generation by bar-proteins.

Protein phase separation enables confining protein activities in cells without requiring a membrane-enclosed compartment. Phase separation of membrane-associated proteins has been found to play central roles in cellular processes such as signaling, autophagy, and endocytosis. How phase separation regulates functionalities of membrane binding proteins is therefore a key to unravel mechanistic details of these pathways. Recently we discovered that endophilin, a member of the membrane curvature generating Bin/Amphiphysin/Rvs (BAR) protein family, undergoes liquid-liquid phase separation in association with the adapter protein lamellipodin (LPD) via multivalent interactions. Our findings suggest that endophilin-LPD phase separation plays an important role in the non-canonical endocytic pathway known as Fast Endophilin Mediated Endocytosis (FEME). We further observe that membrane curvature generation properties of endophilin are remarkably altered upon multivalent interactions with the C-terminal domain of LPD. In model membrane systems, while endophilin alone could generate tubules from negatively charged liposomes, introduction of LPD caused apparent shortening of tubule length. Investigating the membrane nanostructures with electron microscopy revealed that membrane adhesion was promoted by endo-LDP interactions on the membrane surface. Earlier, using optical microscopy, we found that LPD-mediated multivalent interactions assemble endophilin into clusters on the membrane surface. Since, the organization of endophilin on the membrane is closely related to its curvature generation functions, modulating it by a second protein could be a cellular mechanism to regulate the membrane curvature generation.

Bottom-Up Assembly of Bioinspired, Fully Synthetic Extracellular Vesicles.

Extracellular vesicles (EVs) are lipid membrane-enclosed compartments released by cells for intercellular communication in homeostasis and disease. Studies have shown great therapeutic potential of EVs, including but not limited to regenerative and immunomodulatory therapies. Additionally, EVs are promising next-generation drug delivery systems due to their biocompatibility, low immunogenicity, and inherent target specificity. However, clinical application of EVs is so far limited due to challenges in scaling up production, high heterogeneity, batch-to-batch variation, and limited control over composition. Although attaining a fundamental characterization of EVs' functions is a compelling goal, these limitations have hindered a full understanding. Therefore, there is rising interest in exploiting the beneficial properties of EVs while gaining better control over their production and composition. Herein, we describe a method for the bottom-up assembly of bioinspired, fully synthetic vesicles that mimic the most important biophysical and biochemical properties of natural EVs.

A new enzymatic assay to quantify inorganic pyrophosphate in plasma.

Inorganic pyrophosphate (PPi) is a crucial extracellular mineralization regulator. Low plasma PPi concentrations underlie the soft tissue calcification present in several rare hereditary mineralization disorders as well as in more common conditions like chronic kidney disease and diabetes. Even though deregulated plasma PPi homeostasis is known to be linked to multiple human diseases, there is currently no reliable assay for its quantification. We here describe a PPi assay that employs the enzyme ATP sulfurylase to convert PPi into ATP. Generated ATP is subsequently quantified by firefly luciferase-based bioluminescence. An internal ATP standard was used to correct for sample-specific interference by matrix compounds on firefly luciferase activity. The assay was validated and shows excellent precision (< 3.5%) and accuracy (93-106%) of PPi spiked into human plasma samples. We found that of several anticoagulants tested only EDTA effectively blocked conversion of ATP into PPi in plasma after blood collection. Moreover, filtration over a 300,000-Da molecular weight cut-off membrane reduced variability of plasma PPi and removed ATP present in a membrane-enclosed compartment, possibly platelets. Applied to plasma samples of wild-type and Abcc6-/- rats, an animal model with established low circulating levels of PPi, the new assay showed lower variability than the assay that was previously in routine use in our laboratory. In conclusion, we here report a new and robust assay to determine PPi concentrations in plasma, which outperforms currently available assays because of its high sensitivity, precision, and accuracy.

Plasma Chemokine C-C Motif Ligand 2 as a Potential Biomarker for Prostate Cancer.

PurposeSerum levels of the polypeptide chemokine C-C motif ligand 2 (CCL2) have previously shown potential as a prostate cancer diagnostic and prognostic biomarker. Plasma CCL2 levels may be superior to serum levels as a biomarker because of their potentially lower signal-to-noise ratio.Materials and MethodsBefore initiating a large comparative study of plasma and serum CCL2 levels, we performed a prospective, diagnostic pilot study Of 133 individuals from a clinically relevant population. CCL2 plasma levels were measured using a validated assay kit. Plasma was obtained independently of digital rectal examination.ResultsIn this pilot study, we found no relationship between CCL2 plasma values and risk of proven prostate cancer, whereas previous studies found a strong diagnostic relationship between CCL2 serum values and prostate cancer.ConclusionOur contribution to the existing literature strengthens the idea that early in the pathological process, CCL2 mainly circulates in large, membrane-enclosed compartments, whereas plasma CCL2 levels increase markedly during disease progression. We conclude that whereas plasma CCL2 levels are not useful as a diagnostic measure, a ratio of CCL2 plasma to serum levels may prove useful as a marker of disease progression, which warrants further study.

Method uncovers synaptic vesicles’ hidden proteome

Synaptic vesicles are tiny, membrane-enclosed compartments that package and release messenger compounds, such as neurotransmitters, in neurons. Researchers have identified common proteins associate...

Organelle-Specific Autophagy in Cellular Aging and Rejuvenation.

The health of a cell requires proper functioning, regulation, and quality control of its organelles, the membrane-enclosed compartments inside the cell that carry out its essential biochemical tasks. Aging commonly perturbs organelle homeostasis, causing problems to cellular health that can spur the initiation and progression of degenerative diseases and related pathologies. Here, we discuss emerging evidence indicating that age-related defects in organelle homeostasis stem in part from dysfunction of the autophagy-lysosome system, a pivotal player in cellular quality control and damage clearance. We also highlight natural examples from biology where enhanced activity of the autophagy-lysosome system might be harnessed to erase age-related organelle damage, raising potential implications for cellular rejuvenation.

Growing pains: addressing the pitfalls of plant extracellular vesicle research.

Extracellular vesicles (EVs) are small, membrane-enclosed compartments that mediate the intercellular transport of proteins and small RNAs. In plants, EVs are thought to play a prominent role in immune responses and are being championed as the long-sought-after mechanism for host-induced gene silencing. However, parallel research on mammalian EVs is raising concerns about potential pitfalls faced by all EV researchers that will need to be addressed in order to convincingly establish that EVs are the primary mediators of small RNA transfer between organisms. Here we discuss these pitfalls in the context of plant EV research, with a focus on experimental approaches required to distinguish bona fide EV cargo from merely co-purifying contaminants.

Metabolic Cross-talk Between Human Bronchial Epithelial Cells and Internalized Staphylococcus aureus as a Driver for Infection*

Staphylococcus aureus is infamous for causing recurrent infections of the human respiratory tract. This is a consequence of its ability to adapt to different niches, including the intracellular milieu of lung epithelial cells. To understand the dynamic interplay between epithelial cells and the intracellular pathogen, we dissected their interactions over 4 days by mass spectrometry. Additionally, we investigated the dynamics of infection through live cell imaging, immunofluorescence and electron microscopy. The results highlight a major role of often overlooked temporal changes in the bacterial and host metabolism, triggered by fierce competition over limited resources. Remarkably, replicating bacteria reside predominantly within membrane-enclosed compartments and induce apoptosis of the host within ∼24 h post infection. Surviving infected host cells carry a subpopulation of non-replicating bacteria in the cytoplasm that persists. Altogether, we conclude that, besides the production of virulence factors by bacteria, it is the way in which intracellular resources are used, and how host and intracellular bacteria subsequently adapt to each other that determines the ultimate outcome of the infectious process.

From Inflammasome to Exosome-Does Extracellular Vesicle Secretion Constitute an Inflammasome-Dependent Immune Response?

Inflammasomes are intracellular protein complexes of pattern recognition receptors and caspase-1, with essential functions in regulating inflammatory responses of macrophages and dendritic cells. The primary role of inflammasomes is to catalyze processing and secretion of pro-inflammatory cytokines IL-1β and IL-18. Recently, intracellular non-canonical inflammasome activation by caspases-4/5, which are also regulators of pyroptosis via processing gasdermin D, has been elucidated. Caspase-1, the effector protease of inflammasome complex, is also known to modulate secretion of large number of other proteins. Thereby, besides its known role in processing pro-inflammatory cytokines, the inflammasome turns into a universal regulator of protein secretion, which allows the danger-exposed cells to release various proteins in order to alert and guide neighboring cells. Majority of these proteins are not secreted through the conventional ER-Golgi secretory pathway. Instead, they are segregated in membrane-enclosed compartment and secreted in nanosized extracellular vesicles, which protect their cargo and guide it for delivery. Growing evidence indicates that inflammasome activity correlates with enhanced secretion of extracellular vesicles and modulation of their protein cargo. This inflammasome-driven unconventional, vesicle-mediated secretion of multitude of immunoregulatory proteins may constitute a novel paradigm in inflammatory responses. In this mini review we discuss the current knowledge and highlight unsolved questions about metabolic processes, signals, and mechanisms linking inflammasome activity with regulated extracellular vesicle secretion of proteins. Further investigations on this relationship may in the future help understanding the significance of extracellular vesicle secretion in inflammatory diseases such as atherosclerosis, gouty arthritis, asthma, Alzheimer's and many others.

Lipidomics Suggests a New Role for Ceramide Synthase in Phagocytosis.

Phagocytosis is an evolutionarily conserved biological process where pathogens or cellular debris are cleared by engulfing them in a membrane-enclosed cellular compartment called the phagosome. The formation, maturation, and subsequent degradation of a phagosome is an important immune response essential for protection against many pathogens. Yet, the global lipid profile of phagosomes remains unknown, especially as a function of their maturation in immune cells. Here, we show using mass spectrometry based quantitative lipidomics that the ceramide class of lipids, especially very long chain ceramides, are enriched on maturing phagosomes with a concomitant decrease in the biosynthetic precursors of ceramides. We thus posit a new function for the enzyme ceramide synthase during phagocytosis in mammalian macrophages. Biochemical assays, cellular lipid feeding experiments, and pharmacological blockade of ceramide synthase together show that this enzyme indeed controls the flux of ceramides on maturing phagosomes. We also find similar results in the primitive eukaryote Dictyostelium discoideum, suggesting that ceramide enrichment may be evolutionarily conserved and likely an indispensible step in phagosome maturation.

Thylakoid membranes contain a non-selective channel permeable to small organic molecules

The thylakoid lumen is a membrane-enclosed aqueous compartment. Growing evidence indicates that the thylakoid lumen is not only a sink for protons and inorganic ions translocated during photosynthetic reactions but also a place for metabolic activities, e.g. proteolysis of photodamaged proteins, to sustain efficient photosynthesis. However, the mechanism whereby organic molecules move across the thylakoid membranes to sustain these lumenal activities is not well understood. In a recent study of Cyanophora paradoxa chloroplasts (muroplasts), we fortuitously detected a conspicuous diffusion channel activity in the thylakoid membranes. Here, using proteoliposomes reconstituted with the thylakoid membranes from muroplasts and from two other phylogenetically distinct organisms, cyanobacterium Synechocystis sp. PCC 6803 and spinach, we demonstrated the existence of nonselective channels large enough for enabling permeation of small organic compounds (e.g. carbohydrates and amino acids with Mr < 1500) in the thylakoid membranes. Moreover, we purified, identified, and characterized a muroplast channel named here CpTPOR. Osmotic swelling experiments revealed that CpTPOR forms a nonselective pore with an estimated radius of ∼1.3 nm. A lipid bilayer experiment showed variable-conductance channel activity with a typical single-channel conductance of 1.8 nS in 1 m KCl with infrequent closing transitions. The CpTPOR amino acid sequence was moderately similar to that of a voltage-dependent anion-selective channel of the mitochondrial outer membrane, although CpTPOR exhibited no obvious selectivity for anions and no voltage-dependent gating. We propose that transmembrane diffusion pathways are ubiquitous in the thylakoid membranes, presumably enabling rapid transfer of various metabolites between the lumen and stroma.

Extracellular Vesicles in Human Reproduction in Health and Disease.

Extensive evidence suggests that the release of membrane-enclosed compartments, more commonly known as extracellular vesicles (EVs), is a potent newly identified mechanism of cell-to-cell communication both in normal physiology and in pathological conditions. This review presents evidence about the formation and release of different EVs, their definitive markers and cargo content in reproductive physiological processes, and their capacity to convey information between cells through the transfer of functional protein and genetic information to alter phenotype and function of recipient cells associated with reproductive biology. In the male reproductive tract, epididymosomes and prostasomes participate in regulating sperm motility activation, capacitation, and acrosome reaction. In the female reproductive tract, follicular fluid, oviduct/tube, and uterine cavity EVs are considered as vehicles to carry information during oocyte maturation, fertilization, and embryo-maternal crosstalk. EVs via their cargo might be also involved in the triggering, maintenance, and progression of reproductive- and obstetric-related pathologies such as endometriosis, polycystic ovarian syndrome, preeclampsia, gestational diabetes, and erectile dysfunction. In this review, we provide current knowledge on the present and future use of EVs not only as biomarkers, but also as therapeutic targeting agents, mainly as vectors for drug or compound delivery into target cells and tissues.

Characterization of the first planctomycetal outer membrane protein identifies a channel in the outer membrane of the anammox bacterium Kuenenia stuttgartiensis

Planctomycetes are a bacterial phylum known for their complex intracellular compartmentalization. While most Planctomycetes have two compartments, the anaerobic ammonium oxidizing (anammox) bacteria contain three membrane-enclosed compartments. In contrast to a long-standing consensus, recent insights suggested the outermost Planctomycete membrane to be similar to a Gram-negative outer membrane (OM). One characteristic component that differentiates OMs from cytoplasmic membranes (CMs) is the presence of outer membrane proteins (OMPs) featuring a β-barrel structure that facilitates passage of molecules through the OM. Although proteomic and genomic evidence suggested the presence of OMPs in several Planctomycetes, no experimental verification existed of the pore-forming function and localization of these proteins in the outermost membrane of these exceptional microorganisms. Here, we show via lipid bilayer assays that at least two typical OMP-like channel-forming proteins are present in membrane preparations of the anammox bacterium Kuenenia stuttgartiensis. One of these channel-forming proteins, the highly abundant putative OMP Kustd1878, was purified to homogeneity. Analysis of the channel characteristics via lipid bilayer assays showed that Kustd1878 forms a moderately cation-selective channel with a high current noise and an average single-channel conductance of about 170–190pS in 1M KCl. Antibodies were raised against the purified protein and immunogold localization indicated Kustd1878 to be present in the outermost membrane. Therefore, this work clearly demonstrates the presence of OMPs in anammox Planctomycetes and thus firmly adds to the emerging view that Planctomycetes have a Gram-negative cell envelope.

Instant Construction and Visualization of Crowded Biological Environments.

We present the first approach to integrative structural modeling of the biological mesoscale within an interactive visual environment. These complex models can comprise up to millions of molecules with defined atomic structures, locations, and interactions. Their construction has previously been attempted only within a non-visual and non-interactive environment. Our solution unites the modeling and visualization aspect, enabling interactive construction of atomic resolution mesoscale models of large portions of a cell. We present a novel set of GPU algorithms that build the basis for the rapid construction of complex biological structures. These structures consist of multiple membrane-enclosed compartments including both soluble molecules and fibrous structures. The compartments are defined using volume voxelization of triangulated meshes. For membranes, we present an extension of the Wang Tile concept that populates the bilayer with individual lipids. Soluble molecules are populated within compartments distributed according to a Halton sequence. Fibrous structures, such as RNA or actin filaments, are created by self-avoiding random walks. Resulting overlaps of molecules are resolved by a forced-based system. Our approach opens new possibilities to the world of interactive construction of cellular compartments. We demonstrate its effectiveness by showcasing scenes of different scale and complexity that comprise blood plasma, mycoplasma, and HIV.

Single-Particle Detection of Transcription following Rotavirus Entry.

ABSTRACTInfectious rotavirus particles are triple-layered, icosahedral assemblies. The outer layer proteins, VP4 (cleaved to VP8* and VP5*) and VP7, surround a transcriptionally competent, double-layer particle (DLP), which they deliver into the cytosol. During entry of rhesus rotavirus, VP8* interacts with cell surface gangliosides, allowing engulfment into a membrane vesicle by a clathrin-independent process. Escape into the cytosol and outer-layer shedding depend on interaction of a hydrophobic surface on VP5* with the membrane bilayer and on a large-scale conformational change. We report here experiments that detect the fate of released DLPs and their efficiency in initiating RNA synthesis. By replacing the outer layer with fluorescently tagged, recombinant proteins and also tagging the DLP, we distinguished particles that have lost their outer layer and entered the cytosol (uncoated) from those still within membrane vesicles. We used fluorescent in situ hybridization with probes for nascent transcripts to determine how soon after uncoating transcription began and what fraction of the uncoated particles were active in initiating RNA synthesis. We detected RNA synthesis by uncoated particles as early as 15 min after adding virus. The uncoating efficiency was 20 to 50%; of the uncoated particles, about 10 to 15% synthesized detectable RNA. In the format of our experiments, about 10% of the added particles attached to the cell surface, giving an overall ratio of added particles to RNA-synthesizing particles of between 250:1 and 500:1, in good agreement with the ratio of particles to focus-forming units determined by infectivity assays. Thus, RNA synthesis by even a single, uncoated particle can initiate infection in a cell.IMPORTANCE The pathways by which a virus enters a cell transform its packaged genome into an active one. Contemporary fluorescence microscopy can detect individual virus particles as they enter cells, allowing us to map their multistep entry pathways. Rotaviruses, like most viruses that lack membranes of their own, disrupt or perforate the intracellular, membrane-enclosed compartment into which they become engulfed following attachment to a cell surface, in order to gain access to the cell interior. The properties of rotavirus particles make it possible to determine molecular mechanisms for these entry steps. In the work described here, we have asked the following question: what fraction of the rotavirus particles that penetrate into the cell make new viral RNA? We find that of the cell-attached particles, between 20 and 50% ultimately penetrate, and of these, about 10% make RNA. RNA synthesis by even a single virus particle can initiate a productive infection.

The Multifaceted Role of SNARE Proteins in Membrane Fusion.

Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNARE-driven membrane fusion are briefly outlined.