Intervesicular Lipid Mixing Research Articles

Effect of ATG12–ATG5-ATG16L1 autophagy E3-like complex on the ability of LC3/GABARAP proteins to induce vesicle tethering and fusion

In macroautophagy, the autophagosome (AP) engulfs portions of cytoplasm to allow their lysosomal degradation. AP formation in humans requires the concerted action of the ATG12 and LC3/GABARAP conjugation systems. The ATG12–ATG5-ATG16L1 or E3-like complex (E3 for short) acts as a ubiquitin-like E3 enzyme, promoting LC3/GABARAP proteins anchoring to the AP membrane. Their role in the AP expansion process is still unclear, in part because there are no studies comparing six LC3/GABARAP family member roles under the same conditions, and also because the full human E3 was only recently available. In the present study, the lipidation of six members of the LC3/GABARAP family has been reconstituted in the presence and absence of E3, and the mechanisms by which E3 and LC3/GABARAP proteins participate in vesicle tethering and fusion have been investigated. In the absence of E3, GABARAP and GABARAPL1 showed the highest activities. Differences found within LC3/GABARAP proteins suggest the existence of a lipidation threshold, lower for the GABARAP subfamily, as a requisite for tethering and inter-vesicular lipid mixing. E3 increases and speeds up lipidation and LC3/GABARAP-promoted tethering. However, E3 hampers LC3/GABARAP capacity to induce inter-vesicular lipid mixing or subsequent fusion, presumably through the formation of a rigid scaffold on the vesicle surface. Our results suggest a model of AP expansion in which the growing regions would be areas where the LC3/GABARAP proteins involved should be susceptible to lipidation in the absence of E3, or else a regulatory mechanism would allow vesicle incorporation and phagophore growth when E3 is present.

Autophagy protein LC3C binding to phospholipid and interaction with lipid membranes

Autophagy is a process in which parts of the eukaryotic cell are selectively degraded in the lysosome. The materials to be catabolized are first surrounded by a double-membrane structure, the autophagosome. Autophagosome generation is a complex event, in which many proteins are involved. Among the latter, yeast Atg8 or its mammalian orthologues are essential in autophagosome membrane elongation, shaping and closure. A subfamily of the human Atg8 orthologues is formed by the proteins LC3A, LC3B, and LC3C. Previous studies suggest that, at variance with the other two, LC3C does not participate in cardiolipin-mediated mitophagy. The present study was devoted to exploring the binding of LC3C to lipid vesicles, bilayers and monolayers, and the ensuing protein-dependent perturbing effects, in the absence of the mitochondrial lipid cardiolipin. All Atg8 orthologues are covalently bound to a phospholipid prior to their involvement in autophagosome elongation. In our case, a mutant in the C-terminal amino acid, LC3C G126C, together with the use of a maleimide-derivatized phosphatidyl ethanolamine, ensured LC3C lipidation, up to 100% under certain conditions. Ultracentrifugation, surface pressure measurements, spectroscopic and cryo-electron microscopic techniques revealed that lipidated LC3C induced vesicle aggregation (5-fold faster in sonicated than in large unilamellar vesicles) and inter-vesicular lipid mixing (up to 82%), including inner-monolayer lipid mixing (up to 32%), consistent with in vitro partial vesicle fusion. LC3C was also able to cause the release of 80–90% vesicular aqueous contents. The data support the idea that LC3C would be able to help in autophagosome elongation/fusion in autophagy phenomena.

Lipid Modulation of LC3/GABARAP-Mediated Autophagosomal Elongation

Autophagy, an important catabolic pathway involved in a broad spectrum of human diseases, implies the formation of double-membrane structures called autophagosomes (AP) which engulf material to be degraded in lytic compartments. How AP form, especially how the membrane expands and eventually closes upon itself is an area of intense research. Ubiquitin-like ATG8 has been related to both membrane expansion and membrane fusion, but the underlying molecular mechanisms are poorly understood. Here we used two minimal reconstituted systems (enzymatic and chemical conjugation) to compare the ability of human ATG8 homologues (LC3, GABARAP and GATE-16) to mediate membrane fusion. We found that both enzymatically- and chemically-lipidated forms of GATE-16 and GABARAP proteins promote extensive membrane tethering and fusion, whereas lipidated LC3 does so to a much lesser extent. Moreover, we characterize the GATE-16/GABARAP-mediated membrane fusion as a phenomenon of full membrane fusion, independently demonstrating vesicle aggregation, intervesicular lipid mixing and intervesicular mixing of aqueous content, in the absence of vesicular content leakage. Multiple fusion events give rise to large vesicles, as seen by cryo-EM observations. We also show that both vesicle diameter and selected curvature-inducing lipids (cardiolipin, diacylglycerol and lyso-phosphatidylcholine) can modulate the fusion process, smaller vesicle diameters and negative intrinsic curvature lipids (cardiolipin, diacylglycerol) facilitating fusion. These results strongly support the hypothesis of a highly bent structural fusion intermediate (“stalk”) during AP biogenesis and add to the growing body of evidence that identifies lipids as important regulators of autophagy.

Lipid Geometry and Bilayer Curvature Modulate LC3/GABARAP-Mediated Model Autophagosomal Elongation

Autophagy, an important catabolic pathway involved in a broad spectrum of human diseases, implies the formation of double-membrane-bound structures called autophagosomes (AP), which engulf material to be degraded in lytic compartments. How APs form, especially how the membrane expands and eventually closes upon itself, is an area of intense research. Ubiquitin-like ATG8 has been related to both membrane expansion and membrane fusion, but the underlying molecular mechanisms are poorly understood. Here, we used two minimal reconstituted systems (enzymatic and chemical conjugation) to compare the ability of human ATG8 homologs (LC3, GABARAP, and GATE-16) to mediate membrane fusion. We found that both enzymatically and chemically lipidated forms of GATE-16 and GABARAP proteins promote extensive membrane tethering and fusion, whereas lipidated LC3 does so to a much lesser extent. Moreover, we characterize the GATE-16/GABARAP-mediated membrane fusion as a phenomenon of full membrane fusion, independently demonstrating vesicle aggregation, intervesicular lipid mixing, and intervesicular mixing of aqueous content, in the absence of vesicular content leakage. Multiple fusion events give rise to large vesicles, as seen by cryo-electron microscopy observations. We also show that both vesicle diameter and selected curvature-inducing lipids (cardiolipin, diacylglycerol, and lyso-phosphatidylcholine) can modulate the fusion process, smaller vesicle diameters and negative intrinsic curvature lipids (cardiolipin, diacylglycerol) facilitating fusion. These results strongly support the hypothesis of a highly bent structural fusion intermediate (stalk) during AP biogenesis and add to the growing body of evidence that identifies lipids as important regulators of autophagy.

Endophilin B1/Bif-1 Stimulates BAX Activation Independently from Its Capacity to Produce Large Scale Membrane Morphological Rearrangements

Endophilin B1/BAX-interacting factor 1 (Bif-1) is a protein that cooperates with dynamin-like protein 1 (DLP1/Drp1) to maintain normal mitochondrial outer membrane (MOM) dynamics in healthy cells and also contributes to BAX-driven MOM permeabilization (MOMP), the irreversible commitment point to cell death for the majority of apoptotic stimuli. However, despite its importance, exactly how Bif-1 fulfils its proapoptotic role is unknown. Here, we demonstrate that the stimulatory effect of Bif-1 on BAX-driven MOMP and on BAX conformational activation observed in intact cells during apoptosis can be recapitulated in a simplified system consisting of purified proteins and MOM-like liposomes. In this reconstituted model system the N-BAR domain of Bif-1 reproduced the stimulatory effect of Bif-1 on functional BAX activation. This process was dependent on physical interaction between Bif-1 N-BAR and BAX as well as on the presence of the mitochondrion-specific lipid cardiolipin. Despite that Bif-1 N-BAR produced large scale morphological rearrangements in MOM-like liposomes, this phenomenon could be separated from functional BAX activation. Furthermore, DLP1 also caused global morphological changes in MOM-like liposomes, but DLP1 did not stimulate BAX-permeabilizing function in the absence or presence of Bif-1. Taken together, our findings not only provide direct evidence for a functional interplay between Bif-1, BAX, and cardiolipin during MOMP but also add significantly to the growing body of evidence indicating that components of the mitochondrial morphogenesis machinery possess proapoptotic functions that are independent from their recognized roles in normal mitochondrial dynamics.

The Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 4E10 Monoclonal Antibody Is Better Adapted to Membrane-Bound Epitope Recognition and Blocking than 2F5

The broadly neutralizing 2F5 and 4E10 monoclonal antibodies (MAbs) recognize epitopes within the membrane-proximal external region (MPER) that connects the human immunodeficiency virus type 1 (HIV-1) envelope gp41 ectodomain with the transmembrane anchor. By adopting different conformations that stably insert into the virion external membrane interface, such as helical structures, a conserved aromatic-rich sequence within the MPER is thought to participate in HIV-1-cell fusion. Recent experimental evidence suggests that the neutralizing activity of 2F5 and 4E10 might correlate with the MAbs' capacity to recognize epitopes inserted into the viral membrane, thereby impairing MPER fusogenic activity. To gain new insights into the molecular mechanism underlying viral neutralization by these antibodies, we have compared the capacities of 2F5 and 4E10 to block the membrane-disorganizing activity of MPER peptides inserted into the surface bilayer of solution-diffusing unilamellar vesicles. Both MAbs inhibited leakage of vesicular aqueous contents (membrane permeabilization) and intervesicular lipid mixing (membrane fusion) promoted by MPER-derived peptides. Thus, our data support the idea that antibody binding to a membrane-inserted epitope may interfere with the function of the MPER during gp41-induced fusion. Antibody insertion into a cholesterol-containing, uncharged virion-like membrane is mediated by specific epitope recognition, and moreover, partitioning-coupled folding into a helix reduces the efficiency of 2F5 MAb binding to its epitope in the membrane. We conclude that the capacity to interfere with the membrane activity of conserved MPER sequences is best correlated with the broad neutralization of the 4E10 MAb.

Mechanism of pH-Triggered Collapse of Phosphatidylethanolamine Liposomes Stabilized by an Ortho Ester Polyethyleneglycol Lipid

The mechanism of pH-triggered destabilization of liposomes composed of a polyethyleneglycol-orthoester-distearoylglycerol lipid (POD) and phosphatidyl ethanolamine (PE) has been studied using an ANTS/DPX leakage and a lipid-mixing assay. We developed a kinetic model that relates POD hydrolysis to liposome collapse. This minimum-surface-shielding model describes the kinetics of the pH-triggered release of POD/PE liposomes. In the model, when acid-catalyzed hydrolysis lowers the mole percentage of POD on the liposome surface to a critical level, intervesicular lipid mixing is initiated, resulting in a burst of contents release. Two phases of content leakage are observed: a lag phase and a burst phase. During the lag phase, less than 20% of liposomal contents are released and the leakage begins to accelerate when approaching to the transition point. During the burst phase, the leakage rate is dependent on interbilayer contact. The burst phase occurs when the surface density of the PEG lipid is 2.3 ± 0.6 mol%, regardless of the pH. Vesicles containing 4 mol% of a pH- insensitive PEG-lipid conjugate and 10% POD did not leak contents or collapse at any pH. These data are consistent with the stalk theory to describe the lamellar-to-inverted hexagonal phase transition and set a lower bound of ∼16 PE lipids on the external monolayer as the contact site required for lipid mixing between two bilayers.

Interaction of Phospholipases C and Sphingomyelinase with Liposomes

Phospholipases are enzymes whose substrates are, under physiological conditions, in the liquid crystalline state, in contrast to the vast majority of enzymes that work on substrates in solution. Phospholipases themselves exist most often in aqueous suspensions and must transiently dock the membranes to exert their catalytic actions. This chapter reviews a number of methods that can be used to assay and, most importantly, to evaluate the structural changes brought about by the phospholipases C, including sphingomyelinase. Apart from direct and indirect methods to assay enzyme activity, the chapter describes the applications of several fluorescence-based techniques to the study of phospholipase-induced vesicle aggregation, intervesicular lipid mixing, and intervesicular content mixing. Moreover, the methods developed in the laboratory for the preparation of vesicles with asymmetric lipid distribution and for the detection of intervesicular mixing of inner leaflet lipids are also described. This collection of methods should allow the detailed characterization of the interaction of any phospholipase C with liposomes.

Diacylglycerol effects on phosphatidylinositol-specific phospholipase C activity and vesicle fusion

Diacylglycerol increased the hydrolytic activity of phosphatidylinositol-specific phospholipase C on large unilamellar vesicles containing 5–40% phosphatidylinositol. Moreover, diacylglycerol increased the rate and extent of vesicle fusion (contents mixing) induced by the enzyme. Kinetic studies of intervesicular lipid mixing revealed that fusion was limited by the frequency of contacts involving two diacylglycerol-rich domains.

Leaky vesicle fusion induced by phosphatidylinositol-specific phospholipase C: observation of mixing of vesicular inner monolayers.

Large unilamellar vesicles containing phosphatidylinositol (PI), neutral phospholipids, and cholesterol are induced to fuse by the catalytic activity of phosphatidylinositol-specific phospholipase C (PI-PLC). PI cleavage by PI-PLC is followed by vesicle aggregation, intervesicular lipid mixing, and mixing of vesicular aqueous contents. An average of 2-3 vesicles merge into a large one in the fusion process. Vesicle fusion is accompanied by leakage of vesicular contents. A novel method has been developed to monitor mixing of lipids located in the inner monolayers of the vesicles involved in fusion. Using this method, the mixing of inner monolayer lipids and that of vesicular aqueous contents are seen to occur simultaneously, thus giving rise to the fusion pore. Kinetic studies show, for fusing vesicles, second-order dependence of lipid mixing on diacylglycerol concentration in the bilayer. Varying proportions of PI in the liposomal formulation lead to different physical effects of PI-PLC. Specifically, 30-40 mol % PI lead to vesicle fusion, while with 5-10 mol % PI only hemifusion is detected, i.e., mixing of outer monolayer lipids without mixing of aqueous contents. However, when diacylglycerol is included in the bilayers containing 5 mol % PI, PI-PLC activity leads to complete fusion.

Interactions of the HIV-1 fusion peptide with large unilamellar vesicles and monolayers. A cryo-TEM and spectroscopic study

We have examined the interaction of the human immunodeficiency virustype 1 fusion peptide (23 amino acid residues) and of a Trp-containing analog with vesicles composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine and cholesterol (molar ratio, 1:1:1). Both the native and the Trp-substituted peptides bound the vesicles to the same extent and induced intervesicular lipid mixing with comparable efficiency. Infrared reflection–absorption spectroscopy data are compatible with the adoption by the peptide of a main β-sheet structure in a cospread lipid/peptide monolayer. Cryo-transmission electron microscopy observations of peptide-treated vesicles reveal the existence of a peculiar morphology consisting of membrane tubular elongations protruding from single vesicles. Tryptophan fluorescence quenching by brominated phospholipids and by water-soluble acrylamide further indicated that the peptide penetrated into the acyl chain region closer to the interface rather than into the bilayer core. We conclude that the differential partition and shallow penetration of the fusion peptide into the outer monolayer of a surface-constrained bilayer may account for the detected morphological effects. Such single monolayer-restricted interaction and its structural consequences are compatible with specific predictions of current theories on viral fusion.

Interbilayer lipid mixing induced by the human immunodeficiency virus type-1 fusion peptide on large unilamellar vesicles: the nature of the nonlamellar intermediates

A peptide corresponding to the 23 N-terminal amino acid residues of the human immunodeficiency virus type-1 (HIV-1) gp41 has the capacity to induce intervesicular lipid mixing in large unilamellar liposomes composed of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and cholesterol (CHOL) (molar ratio, 1:1:1). Cryo-transmission electron microscopy (cryo-TEM) of diluted vesicles to which peptides has been externally added reveals a morphology that is compatible with the formation of nonlamellar lipidic aggregates during the time-course of lipid mixing. 31P-nuclear magnetic resonance and 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMADPH) steady-state anisotropy data at equilibrium indicate that the peptide is able to modulate the lipid polymorphism in pelletted membranes by: (i) promoting the thermotropic formation of inverted phases; and (ii) driving the lamellar-to-nonlamellar transition towards the formation of isotropic phases. Therefore, our combined morphological and spectroscopic data reveal the existence of a direct correlation between the ability of the externally added peptide to induce lipid-mixing in dilute liposome samples and its capacity to modulate lipid polymorphism in stacked bilayers.

Liposome fusion induced by a M(r) 18,000 protein localized to the acrosomal region of acrosome-reacted abalone spermatozoa.

A M(r) 18,000 protein is secreted by abalone spermatozoa during the acrosome reaction. Immunofluorescence of acrosome-reacted sperm localizes the protein as a coating on the spent acrosomal granule hull and on the surface of the acrosomal process. The membrane of the acrosomal process fuses with the egg plasma membrane at fertilization. The M(r) 18,000 acrosomal protein aggregates negatively charged (but not neutral) large unilamellar liposomes and renders them permeable to internal probe. The M(r) 18,000 proteins from two abalone species are potent inducers of intervesicular lipid mixing in the resonance energy transfer assay, suggesting that they mediate the fusion of lipid bilayers. Predicted secondary structures of these proteins show the presence of strongly amphipathic alpha-helices that may be active in the perturbation of phospholipid bilayers. The M(r) 18,000 protein may mediate sperm-egg fusion during fertilization.

Interactions between human defensins and lipid bilayers: Evidence for formation of multimeric pores

Defensins comprise a family of broad-spectrum antimicrobial peptides that are stored in the cytoplasmic granules of mammalian neutrophils and Paneth cells of the small intestine. Neutrophil defensins are known to permeabilize cell membranes of susceptible microorganisms, but the mechanism of permeabilization is uncertain. We report here the results of an investigation of the mechanism by which HNP-2, one of 4 human neutrophil defensins, permeabilizes large unilamellar vesicles formed from the anionic lipid palmitoyloleoylphosphatidylglycerol (POPG). As observed by others, we find that HNP-2 (net charge = +3) cannot bind to vesicles formed from neutral lipids. The binding of HNP-2 to vesicles containing varying amounts of POPG and neutral (zwitterionic) palmitoyloleoylphosphatidylcholine (POPC) demonstrates that binding is initiated through electrostatic interactions. Because vesicle aggregation and fusion can confound studies of the interaction of HNP-2 with vesicles, those processes were explored systematically by varying the concentrations of vesicles and HNP-2, and the POPG:POPC ratio. Vesicles (300 microM POPG) readily aggregated at HNP-2 concentrations above 1 microM, but no mixing of vesicle contents could be detected for concentrations as high as 2 microM despite the fact that intervesicular lipid mixing could be demonstrated. This indicates that if fusion of vesicles occurs, it is hemi-fusion, in which only the outer monolayers mix at bilayer contact sites. Under conditions of limited aggregation and intervesicular lipid mixing, the fractional leakage of small solutes is a sigmoidal function of peptide concentration. For 300 microM POPG vesicles, 50% of entrapped solute is released by 0.7 microM HNP-2. We introduce a simple method for determining whether leakage from vesicles is graded or all-or-none. We show by means of this fluorescence "requenching" method that native HNP-2 induces vesicle leakage in an all-or-none manner, whereas reduced HNP-2 induces partial, or graded, leakage of vesicle contents. At HNP-2 concentrations that release 100% of small (approximately 400 Da) markers, a fluorescent dextran of 4,400 Da is partially retained in the vesicles, and a 18,900-Da dextran is mostly retained. These results suggest that HNP-2 can form pores that have a maximum diameter of approximately 25 A. A speculative multimeric model of the pore is presented based on these results and on the crystal structure of a human defensin.

Membrane interaction and conformational properties of the putative fusion peptide of PH-30, a protein active in sperm-egg fusion.

A peptide representing the putative fusion domain of PH-30, a sperm surface protein involved in sperm-egg fusion, was synthesized, and its interaction with model lipid membranes was characterized by biophysical methods. While the peptide binds to the vesicles composed of both neutral and acidic lipids, the apparent affinity is significantly higher for the latter lipid class. The intervesicular lipid mixing assay suggests that the synthetic peptide is able to induce fusion of large unilamellar vesicles. Circular dichroism and Fourier-transform infrared spectroscopy show that while in an aqueous buffer the peptide exists in an essentially unordered conformation, binding to the membranes results in a conformational transition to a beta-structure. These data indicate that the fragment identified on the alpha-subunit of PH-30 as a putative fusion peptide is indeed a good candidate for this role. However, in contrast to what has been proposed for some viral fusion peptides, the PH-30 fusion domain is highly unlikely to act as an insertional "sided" helix.