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.

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.

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

-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 -maleimide PE derivative (PE-mal) mimicks the native PE in the bilayer.

Ceramide regulation of autophagy: A biophysical approach.

Specific membrane lipids play unique roles in (macro)autophagy. Those include phosphatidylethanolamine, to which LC3/GABARAP autophagy proteins become covalently bound in the process, or cardiolipin, an important effector in mitochondrial autophagy (or mitophagy). Ceramide (Cer), or N-acyl sphingosine, is one of the simplest sphingolipids, known as a stress signal in the apoptotic pathway.

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.

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.

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.

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.

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.

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.

Dihydroceramide accumulation mediates cytotoxic autophagy of cancer cells via autolysosome destabilization.

Autophagy is considered primarily a cell survival process, although it can also lead to cell death. However, the factors that dictate the shift between these 2 opposite outcomes remain largely unknown. In this work, we used Δ-tetrahydrocannabinol (THC, the main active component of marijuana, a compound that triggers autophagy-mediated cancer cell death) and nutrient deprivation (an autophagic stimulus that triggers cytoprotective autophagy) to investigate the precise molecular mechanisms responsible for the activation of cytotoxic autophagy in cancer cells.

Human Atg8-cardiolipin interactions in mitophagy: Specific properties of LC3B, GABARAPL2 and GABARAP.

The phospholipid cardiolipin (CL) has been proposed to play a role in selective mitochondrial autophagy, or mitophagy. CL externalization to the outer mitochondrial membrane would act as a signal for the human Atg8 ortholog subfamily, MAP1LC3 (LC3). The latter would mediate both mitochondrial recognition and autophagosome formation, ultimately leading to removal of damaged mitochondria.

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.

Fluorescent polyene ceramide analogues as membrane probes.

Three ceramide analogues have been synthesized, with sphingosine-like chains containing five conjugated double bonds. Pentaene I has an N-palmitoyl acyl chain, while the other two pentaenes contain also a doxyl radical, respectively, at C5 (Penta5dox) and at C16 (Penta16dox) positions of the N-acyl chain. Pentaene I maximum excitation and emission wavelengths in a phospholipid bilayer are 353 and 478 nm, respectively.

In situ synthesis of fluorescent membrane lipids (ceramides) using click chemistry.

Ceramide analogues containing azide groups either in the polar head or in the hydrocarbon chains are non-fluorescent. When incorporated into phospholipid bilayers, they can react in situ with a non-fluorescent 1,8-naphthalimide using click chemistry giving rise to fluorescent ceramide derivatives emitting at ≈440 nm. When incorporated into giant unilamellar vesicles, two-photon excitation at 760 nm allows visualization of the ceramide-containing bilayers.

Recruitment of a phospholipase C/sphingomyelinase into non-lamellar lipid droplets during hydrolysis of lipid bilayers.

When giant unilamellar vesicles (GUVs) composed of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, and cholesterol are treated with PlcHR(2), a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa, the initial stages of lipid hydrolysis do not cause large changes in vesicle morphology (Ibarguren et al., 2011). However, when hydrolysis progresses confocal fluorescence microscopy reveals the formation of lipid aggregates, whose morphology is not compatible with that of bilayers.

Phospholipases C and sphingomyelinases: Lipids as substrates and modulators of enzyme activity.

This review article deals with phospholipases C (PLC), sphingomyelinases (SMases) and related lipases. Bacterial PC-preferring PLC and PI-specific PLC, bacterial SMases and PLC/SMases, eukaryotic SMases and ceramide phosphorylinositol hydrolases are discussed. The aim of the review is to offer a coherent description of lipid-protein interactions for the above enzymes, considering that (a) the enzyme activity is influenced by the physical properties of the substrate lipid, (b) the enzyme activity is modulated by non-substrate lipids, (c) enzyme end-products often change the physical properties of the lipid matrix, hence the enzyme activity.

Model systems of precursor cellular membranes: long-chain alcohols stabilize spontaneously formed oleic acid vesicles.

Oleic acid vesicles have been used as model systems to study the properties of membranes that could be the evolutionary precursors of more complex, stable, and impermeable phospholipid biomembranes. Pure fatty acid vesicles in general show high sensitivity to ionic strength and pH variation, but there is growing evidence that this lack of stability can be counterbalanced through mixtures with other amphiphilic or surfactant compounds. Here, we present a systematic experimental analysis of the oleic acid system and explore the spontaneous formation of vesicles under different conditions, as well as the effects that alcohols and alkanes may have in the process.

Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting ordered-disordered and gel-fluid domains.

The binding and early stages of activity of a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa on giant unilamellar vesicles (GUV) have been monitored using fluorescence confocal microscopy. Both the lipids and the enzyme were labeled with specific fluorescent markers. GUV consisted of a mixture of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and cholesterol in equimolar ratios, to which 5-10 mol% of the enzyme end-product ceramide and/or diacylglycerol were occasionally added.

Electroformation of giant unilamellar vesicles from native membranes and organic lipid mixtures for the study of lipid domains under physiological ionic-strength conditions.

Giant unilamellar vesicles (GUVs) constitute a cell-sized model membrane system that allows direct visualization of particular membrane-related phenomena, such as domain formation, at the level of single vesicles using fluorescence microscopy-related techniques. Currently available protocols for the preparation of GUVs work only at very low salt concentrations, thus precluding experimentation under physiological conditions. In addition, the GUVs thus obtained lack membrane compositional asymmetry.

Ceramide-enriched membrane domains in red blood cells and the mechanism of sphingomyelinase-induced hot-cold hemolysis.

Hot-cold hemolysis is the phenomenon whereby red blood cells, preincubated at 37 degrees C in the presence of certain agents, undergo rapid hemolysis when transferred to 4 degrees C. The mechanism of this phenomenon is not understood. PlcHR 2, a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa, that is the prototype of a new phosphatase superfamily, induces hot-cold hemolysis.

Cholesterol displacement by ceramide in sphingomyelin-containing liquid-ordered domains, and generation of gel regions in giant lipidic vesicles.

Fluorescence confocal microscopy and differential scanning calorimetry are used in combination to study the phase behaviour of bilayers composed of PC:PE:SM:Chol equimolecular mixtures, in the presence or absence of 10 mol% egg ceramide. In the absence of ceramide, separate liquid-ordered and liquid-disordered domains are observed in giant unilamellar vesicles. In the presence of ceramide, gel-like domains appear within the liquid-ordered regions.

Membrane organization and ionization behavior of the minor but crucial lipid ceramide-1-phosphate.

Ceramide-1-phosphate (Cer-1-P), one of the simplest of all sphingophospholipids, occurs in minor amounts in biological membranes. Yet recent evidence suggests important roles of this lipid as a novel second messenger with crucial tasks in cell survival and inflammatory responses. We present a detailed description of the physical chemistry of this hitherto little explored membrane lipid.

Giant unilamellar vesicles electroformed from native membranes and organic lipid mixtures under physiological conditions.

In recent years, giant unilamellar vesicles (GUVs) have become objects of intense scrutiny by chemists, biologists, and physicists who are interested in the many aspects of biological membranes. In particular, this "cell size" model system allows direct visualization of particular membrane-related phenomena at the level of single vesicles using fluorescence microscopy-related techniques. However, this model system lacks two relevant features with respect to biological membranes: 1), the conventional preparation of GUVs currently requires very low salt concentration, thus precluding experimentation under physiological conditions, and 2), the model system lacks membrane compositional asymmetry.