Vesicle-vesicle fusion promoted by the Atg8-family autophagy protein LC3C: relevance of the N-terminal region.

Among the MAP1LC3/LC3 subfamily of Atg8 proteins, LC3B and LC3C constitute the most and least studied members, respectively, LC3B being generally considered as an autophagosomal marker and a canonical representative of the LC3 subfamily. In several recent studies, LC3C has emerged as an important modulator in various processes of cell homeostasis. Our own research data demonstrate that LC3C induces higher levels of tethering and of intervesicular lipid mixing than LC3B. LC3C contains a peculiar N-terminal region, different from the other Atg8-family protein members. Using a series of mutants, we have shown that the N-terminal region of LC3C is responsible for the enhanced vesicle tethering, membrane perturbation and vesicle-vesicle fusion activities of LC3C as compared to LC3B.Abbreviations: ATG: autophagy related; GABARAP: gamma-aminobutyric acid receptor associated protein; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PC: phosphatidyl choline; PE: phosphatidylethanolamine; PEmal: maleimide-derivatized PE; PtdIns: phosphatidylinositol.

The N-terminal region of the ATG8 autophagy protein LC3C is essential for its membrane fusion properties.

Autophagy is a catabolic process in which a double-membrane organelle, the autophagosome (AP), engulfs cellular components that will be degraded in the lysosomes. ATG8 protein family members participate at various stages of AP formation. The present study compares the capacity to induce lipid-vesicle tethering and fusion of two ATG8 family members, LC3B and LC3C, with model membranes. LC3B is the most thoroughly studied ATG8 protein. It is generally considered as an autophagosomal marker and a canonical representative of the LC3 subfamily. LC3C is less studied, but recent data have reported its implication in various processes, crucial to cellular homeostasis. The results in this paper show that LC3C induces higher levels of tethering and of intervesicular lipid mixing than LC3B. As the N-terminus of LC3C is different from that of the other family members, various mutants of the N-terminal region of both LC3B and LC3C were designed, and their activities compared. It was concluded that the N-terminal region of LC3C was responsible for the enhanced vesicle tethering, membrane perturbation and vesicle-vesicle fusion activities of LC3C as compared to LC3B. The results suggest a specialized function of LC3C in the AP expansion process.

Effects of a N-Maleimide-derivatized Phosphatidylethanolamine on the Architecture and Properties of Lipid Bilayers.

N-maleimide-derivatized phospholipids are often used to facilitate protein anchoring to membranes. In autophagy studies, this is applied to the covalent binding of Atg8, an autophagy protein, to a phosphatidylethanolamine (PE) in the nascent autophagosome. However, the question remains on how closely the N-maleimide PE derivative (PE-mal) mimicks the native PE in the bilayer. In the present paper, spectroscopic and calorimetric techniques have been applied to vesicles containing either PE or PE-mal (together with other phospholipids) to compare the properties of the native and derivatized forms of PE. According to differential scanning calorimetry, and to infrared spectroscopy, the presence of PE-mal did not perturb the fatty acyl chains in the bilayer. Fluorescence spectroscopy and microscopy showed that PE-mal did not alter the bilayer permeability either. However, fluorescence emission polarization of the Laurdan and DPH probes indicated an increased order, or decreased fluidity, in the bilayers containing PE-mal. In addition, the infrared spectral data from the phospholipid phosphate region revealed a PE-mal-induced conformational change in the polar heads, accompanied by increased hydration. Globally considered, the results suggest that PE-mal would be a reasonable substitute for PE in model membranes containing reconstituted proteins.

Lipids in Mitochondrial Macroautophagy: Phase Behavior of Bilayers Containing Cardiolipin and Ceramide.

Cardiolipin (CL) is a key lipid for damaged mitochondrial recognition by the LC3/GABARAP human autophagy proteins. The role of ceramide (Cer) in this process is unclear, but CL and Cer have been proposed to coexist in mitochondria under certain conditions. Varela et al. showed that in model membranes composed of egg sphingomyelin (eSM), dioleoyl phosphatidylethanolamine (DOPE), and CL, the addition of Cer enhanced the binding of LC3/GABARAP proteins to bilayers. Cer gave rise to lateral phase separation of Cer-rich rigid domains but protein binding took place mainly in the fluid continuous phase. In the present study, a biophysical analysis of bilayers composed of eSM, DOPE, CL, and/or Cer was attempted to understand the relevance of this lipid coexistence. Bilayers were studied by differential scanning calorimetry, confocal fluorescence microscopy, and atomic force microscopy. Upon the addition of CL and Cer, one continuous phase and two segregated ones were formed. In bilayers with egg phosphatidylcholine instead of eSM, in which the binding of LC3/GABARAP proteins hardly increased with Cer in the former study, a single segregated phase was formed. Assuming that phase separation at the nanoscale is ruled by the same principles acting at the micrometer scale, it is proposed that Cer-enriched rigid nanodomains, stabilized by eSM:Cer interactions formed within the DOPE- and CL-enriched fluid phase, result in structural defects at the rigid/fluid nanointerfaces, thus hypothetically facilitatingLC3/GABARAP protein interaction.

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.

Phase behaviour of C18-N-acyl sphingolipids, the prevalent species in human brain.

Lipidomic analysis of the N-acyl components of sphingolipids in different mammalian tissues had revealed that brain tissue differed from all the other samples in that SM contained mainly C18:0 and C24:1N-acyl chains, and that the most abundant Cer species was C18:0. Only in the nervous system was C18:0 found in sizable proportions. The high levels of C18:0 and C16:0, respectively in brain and non-brain SM, were important because SM is by far the most abundant sphingolipid in the plasma membrane. In view of these observations, the present paper is devoted to a comparative study of the properties of C16:0 and C18:0 sphingolipids (SM and Cer) pure and in mixtures of increasing complexities, using differential scanning calorimetry, confocal microscopy of giant unilamellar vesicles, and correlative fluorescence microscopy and atomic force microscopy of supported lipid bilayers. Membrane rigidity was measured by force spectroscopy. It was found that in mixtures containing dioleoyl phosphatidylcholine, sphingomyelin and cholesterol, i.e. representing the lipids predominant in the outer monolayer of cell membranes, lateral inhomogeneities occurred, with the formation of rigid domains within a continuous fluid phase. Inclusion of saturated Cer in the system was always found to increase the rigidity of the segregated domains. C18:0-based sphingolipids exhibit hydrocarbon chain-length asymmetry, and some singularities observed with this N-acyl chain, e.g. complex calorimetric endotherms, could be attributed to this property. Moreover, C18:0-based sphingolipids, that are typical of the excitable cells, were less miscible with the fluid phase than their C16:0 counterparts. The results could be interpreted as suggesting that the predominance of C18:0 Cer in the nervous system would contribute to the tightness of its plasma membranes, thus facilitating maintenance of the ion gradients.

Ceramide enhances binding of LC3/GABARAP autophagy proteins to cardiolipin-containing membranes.

Macroautophagy, or autophagy, is a process in which cell macromolecules, or even organelles, are engulfed in a double-membrane vesicle, the autophagosome, and directed to a lysosome. Among autophagy-related proteins, LC3/GABARAP constitute a protein family derived from yeast Atg8. They play important roles in autophagosome formation, binding future cargo organelles and promoting autophagosome growth. The involvement of specific lipids in this process is poorly understood. The present study explores the interaction of LC3/GABARAP proteins with phospholipid monolayers and bilayers based on phosphatidylcholine or on sphingomyelin. Cardiolipin is found to be essential for the protein interaction with such bilayers, as measured through gradient centrifugation experiments, while ceramide markedly increases binding. Giant unilamellar vesicles examined under confocal fluorescence microscopy reveal that ceramide segregates laterally into very rigid domains, while GABARAP binds only the more fluid regions, suggesting that the enhancing role of ceramide is exerted by the minority of ceramide molecules dispersed in the fluid phase. Although in further autophagy steps the LC3/GABARAP proteins are covalently bound to a phospholipid, this is not the case in our system, thus it is proposed that the observed ceramide effects would correspond to very early stages in the process, such as cargo recognition.

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.

LC3 subfamily in cardiolipin-mediated mitophagy: a comparison of the LC3A, LC3B and LC3C homologs.

Externalization of the phospholipid cardiolipin (CL) to the outer mitochondrial membrane has been proposed to act as a mitophagy trigger. CL would act as a signal for binding the LC3 macroautophagy/autophagy proteins. As yet, the behavior of the LC3-subfamily members has not been directly compared in a detailed way. In the present contribution, an analysis of LC3A, LC3B and LC3C interaction with CL-containing model membranes, and of their ability to translocate to mitochondria, is described. Binding of LC3A to CL was stronger than that of LC3B; both proteins showed a similar ability to colocalize with mitochondria upon induction of CL externalization in SH-SY5Y cells. Besides, the double silencing of LC3A and LC3B proteins was seen to decrease CCCP-induced mitophagy. Residues 14 and 18 located in the N-terminal region of LC3A were shown to be important for its recognition of damaged mitochondria during rotenone- or CCCP-induced mitophagy. Moreover, the in vitro results suggested a possible role of LC3A, but not of LC3B, in oxidized-CL recognition as a counterweight to excessive apoptosis activation. In the case of LC3C, even if this protein showed a stronger CL binding than LC3B or LC3A, the interaction was less specific, and colocalization of LC3C with mitochondria was not rotenone dependent. These results suggest that, at variance with LC3A, LC3C does not participate in cargo recognition during CL-mediated-mitophagy. The data support the notion that the various LC3-subfamily members might play different roles during autophagy initiation, identifying LC3A as a novel stakeholder in CL-mediated mitophagy. Abbreviations: ACTB/ß-actin: actin beta; Atg8: autophagy-related 8; CL: cardiolipin; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DMSO: dimethyl sulfoxide; DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DTT: DL-dithiothreitol; FKBP8: FKBP prolyl isomerase 8; GABARAP: GABA type A receptor associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GFP: green fluorescent protein; IMM: inner mitochondrial membrane; LUV/LUVs: large unilamellar vesicle/s; MAP1LC3A/LC3A: microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP1LC3C/LC3C: microtubule associated protein 1 light chain 3 gamma; NME4/NDPK-D/Nm23-H4: NME/NM23 nucleoside diphosphate kinase 4; O/A: oligomycin A + antimycin A; OMM: outer mitochondrial membrane; PA: phosphatidic acid; PC: phosphatidylcholine; PG: phosphatidylglycerol; PINK1: PTEN induced putative kinase 1; PtdIns4P: phosphatidylinositol-4-phosphate; Rho-PE: lissamine rhodamine phosphatidylethanolamine; SUV/SUVs: small unilamellar vesicle/s.

Molecular and mesoscopic geometries in autophagosome generation. A review.

Autophagy is an essential process in cell self-repair and survival. The centre of the autophagic event is the generation of the so-called autophagosome (AP), a vesicle surrounded by a double membrane (two bilayers). The AP delivers its cargo to a lysosome, for degradation and re-use of the hydrolysis products as new building blocks. AP formation is a very complex event, requiring dozens of specific proteins, and involving numerous instances of membrane biogenesis and architecture, including membrane fusion and fission. Many stages of AP generation can be rationalised in terms of curvature, both the molecular geometry of lipids interpreted in terms of 'intrinsic curvature', and the overall mesoscopic curvature of the whole membrane, as observed with microscopy techniques. The present contribution intends to bring together the worlds of biophysics and cell biology of autophagy, in the hope that the resulting cross-pollination will generate abundant fruit.

Irreversible versus repairable membrane poration: differences in permeabilization elicited by Bordetella Adenylate Cyclase Toxin and its hemolysin domain in macrophages.

Rapid plasma membrane repair in response to pore-forming toxins is crucial for cell survival, but the molecular mechanisms employed by eukaryotic nucleated cells to maintain membrane integrity and the specificities of such pathways remain poorly understood. Here, we have explored the permeabilization elicited by the Bordetella pertussis adenylate cyclase toxin, a 200-kDa protein toxin with α-helical pore-forming domain that forms pores of tunable size, and evaluated the response of target macrophages to such toxin poration. We show here that the response and the fate of target macrophages depend on toxin pore width. We find that the toxin's hemolysin moiety induces a transient membrane permeabilization by forming wide enough pores allowing Ca2+ influx into the target cell cytosol. This activates a Ca2+ -dependent cellular response involving exocytosis and endocytosis steps eliminating toxin pores and restoring membrane integrity. In contrast, the full-length native toxin, at low concentrations, forms very small pores that cause insidious perturbation of cell ion homeostasis that escapes control by the macrophage membrane repair response, eventually leading to cell death. Our data reveal that permeability to Ca2+ and ATP are key elements in the membrane repair pathway for eliminating α-helical pores of bacterial origin.

Membrane Repair Mechanisms against Permeabilization by Pore-Forming Toxins.

Permeabilization of the plasma membrane represents an important threat for any cell, since it compromises its viability by disrupting cell homeostasis. Numerous pathogenic bacteria produce pore-forming toxins that break plasma membrane integrity and cause cell death by colloid-osmotic lysis. Eukaryotic cells, in turn, have developed different ways to cope with the effects of such membrane piercing. Here, we provide a short overview of the general mechanisms currently proposed for plasma membrane repair, focusing more specifically on the cellular responses to membrane permeabilization by pore-forming toxins and presenting new data on the effects and cellular responses to the permeabilization by an RTX (repeats in toxin) toxin, the adenylate cyclase toxin-hemolysin secreted by the whooping cough bacterium Bordetella pertussis, which we have studied in the laboratory.

Understanding the Mechanism of Translocation of Adenylate Cyclase Toxin across Biological Membranes.

Adenylate cyclase toxin (ACT) is one of the principal virulence factors secreted by the whooping cough causative bacterium Bordetella pertussis, and it has a critical role in colonization of the respiratory tract and establishment of the disease. ACT targets phagocytes via binding to the CD11b/CD18 integrin and delivers its N-terminal adenylate cyclase (AC) domain directly to the cell cytosol, where it catalyzes unregulated conversion of cytosolic ATP into cAMP upon activation by binding to cellular calmodulin. High cAMP levels disrupt bactericidal functions of the immune cells, ultimately leading to cell death. In spite of its relevance in the ACT biology, the mechanism by which its ≈400 amino acid-long AC domain is transported through the target plasma membrane, and is released into the target cytosol, remains enigmatic. This article is devoted to refresh our knowledge on the mechanism of AC translocation across biological membranes. Two models, the so-called "two-step model" and the recently-proposed "toroidal pore model", will be considered.

Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells.

Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of "toxin-coated bacteria" proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or "free" in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and toxin-induced Ca(2+)-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system.