RESUMEN
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.
RESUMEN
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.
Asunto(s)
Fusión de Membrana , Proteínas Asociadas a Microtúbulos , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Autofagia , LípidosRESUMEN
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. Moreover, Cer is increasingly being recognized as an autophagy activator, although its mechanism of action is unclear. In the present review, the proposed Cer roles in autophagy are summarized, together with some biophysical properties of Cer in membranes. Possible pathways for Cer activation of autophagy are discussed, including specific protein binding of the lipid, and Cer-dependent perturbation of bilayer properties. Cer generation of lateral inhomogeneities (domain formation) is given special attention. Recent biophysical results, including fluorescence and atomic force microscopy data, show Cer-promoted enhanced binding of LC3/GABARAP to lipid bilayers. These observations could be interpreted in terms of the putative formation of Cer-rich nanodomains.
Asunto(s)
Ceramidas , Esfingolípidos , Ceramidas/metabolismo , Esfingolípidos/metabolismo , Membrana Dobles de Lípidos/química , Autofagia , MitofagiaRESUMEN
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.
Asunto(s)
Autofagia , Proteínas Asociadas a Microtúbulos , Humanos , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Autofagosomas/metabolismo , Lípidos , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 12 Relacionada con la Autofagia , Proteína 5 Relacionada con la Autofagia/genéticaRESUMEN
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.
Asunto(s)
Cardiolipinas , Proteínas Asociadas a Microtúbulos , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia , Ceramidas , Proteínas Asociadas a Microtúbulos/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Asociadas a Microtúbulos , Fosfolípidos , Autofagia , Cardiolipinas/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfolípidos/metabolismo , Unión Proteica , Saccharomyces cerevisiae/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Asociadas a Microtúbulos , Mitofagia , Neuroblastoma , Humanos , Autofagia/fisiología , Carbonil Cianuro m-Clorofenil Hidrazona , Cardiolipinas/metabolismo , Ácido gamma-Aminobutírico , Proteínas Asociadas a Microtúbulos/metabolismo , Rotenona/farmacología , Liposomas UnilamelaresRESUMEN
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.
Asunto(s)
Autofagosomas/genética , Autofagia/genética , Membrana Dobles de Lípidos/química , Fagocitosis/genética , Autofagosomas/química , Biofisica , Comunicación Celular/genética , Lisosomas/química , Lisosomas/genética , Fusión de Membrana/genéticaRESUMEN
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. We have applied quantitative biophysical techniques to the study of CL interaction with various Atg8 human orthologs, namely LC3B, GABARAPL2 and GABARAP. We have found that LC3B interacts preferentially with CL over other di-anionic lipids, that CL-LC3B binding occurs with positive cooperativity, and that the CL-LC3B interaction relies only partially on electrostatic forces. CL-induced increased membrane fluidity appears also as an important factor helping LC3B to bind CL. The LC3B C terminus remains exposed to the hydrophilic environment after protein binding to CL-enriched membranes. In intact U87MG human glioblastoma cells rotenone-induced autophagy leads to LC3B translocation to mitochondria and subsequent delivery of mitochondria to lysosomes. We have also observed that GABARAP, but not GABARAPL2, interacts with CL in vitro. However neither GABARAP nor GABARAPL2 were translocated to mitochondria in rotenone-treated U87MG cells. Thus the various human Atg8 orthologs might play specific roles in different autophagic processes.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Autofagia , Cardiolipinas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitofagia , Secuencia de Aminoácidos , Proteínas Reguladoras de la Apoptosis , Autofagia/efectos de los fármacos , Familia de las Proteínas 8 Relacionadas con la Autofagia/química , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Dronabinol/farmacología , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Asociadas a Microtúbulos/química , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Mitofagia/efectos de los fármacos , Presión , Unión Proteica/efectos de los fármacos , Rotenona/farmacología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
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 Δ9-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. By using a wide array of experimental approaches we show that THC (but not nutrient deprivation) increases the dihydroceramide:ceramide ratio in the endoplasmic reticulum of glioma cells, and this alteration is directed to autophagosomes and autolysosomes to promote lysosomal membrane permeabilization, cathepsin release and the subsequent activation of apoptotic cell death. These findings pave the way to clarify the regulatory mechanisms that determine the selective activation of autophagy-mediated cancer cell death.
Asunto(s)
Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Ceramidas/farmacología , Lisosomas/metabolismo , Neoplasias/patología , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dronabinol/farmacología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Humanos , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestructura , Lisosomas/efectos de los fármacos , Lisosomas/ultraestructura , Modelos Biológicos , Permeabilidad , Fagosomas/efectos de los fármacos , Fagosomas/metabolismo , Fagosomas/ultraestructura , Esfingolípidos/biosíntesisRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Membrana Dobles de Lípidos/química , Proteínas Asociadas a Microtúbulos/química , Fagosomas/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Proteínas Reguladoras de la Apoptosis , Autofagia , Familia de las Proteínas 8 Relacionadas con la Autofagia , Humanos , Fusión de Membrana , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Datos de Secuencia Molecular , Fagosomas/ultraestructuraRESUMEN
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. Pentaene I does not segregate from the other lipids in the way natural ceramide does, but rather mixes with them in a selective way according to the lipid phases involved. Fluorescence confocal microscopy studies show that when lipid domains in different physical states coexist, Pentaene I emission is higher in gel than in fluid domains, and in liquid-ordered than in liquid-disordered areas. Electron paramagnetic resonance of the pentaene doxyl probes confirms that these molecules are sensitive to the physical state of the bilayer. Calorimetric and fluorescence quenching experiments suggest that the lipids under study orient themselves in lipid bilayers with their polar moieties located at the lipid-water interface. The doxyl radical in the N-acyl chain quenches the fluorescence of the pentaene group when in close proximity. Because of this property, Penta16dox can detect gel-fluid transitions in phospholipids. The availability of probes for lipids in the gel phase is important in view of novel evidence for the existence of gel microdomains in cell membranes.
Asunto(s)
Ceramidas/química , Fluorescencia , Colorantes Fluorescentes/química , Polienos/química , Ceramidas/síntesis química , Colorantes Fluorescentes/síntesis química , Estructura Molecular , Polienos/síntesis químicaRESUMEN
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. Smaller vesicles or droplets can also be seen inside the GUV. Our studies indicate that these aggregates or droplets are enriched in the non-lamellar lipid ceramide, an end-product of PlcHR(2) reaction. Moreover, the aggregates/droplets appear enriched in the hydrolytic enzyme PlcHR(2). At a final stage GUVs containing the enzyme-enriched droplets disintegrate and vanish from the microscope field. The observed non-lamellar enzyme-rich structures may be related to intermediates in the process of aggregation and fusion although the experimental design prevents vesicle free diffusion in the aqueous medium, thus actual aggregation or fusion cannot be observed.
Asunto(s)
Membrana Dobles de Lípidos/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/enzimología , Fosfolipasas de Tipo C/metabolismo , Liposomas Unilamelares/metabolismo , Ceramidas/química , Ceramidas/metabolismo , Diglicéridos/química , Diglicéridos/metabolismo , Humanos , Hidrólisis , Membrana Dobles de Lípidos/química , Pseudomonas aeruginosa/metabolismo , Liposomas Unilamelares/químicaRESUMEN
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. This approach allows a certain degree of understanding of phenomena such as: latency periods (lag times), enzyme interfacial activation, effects of intrinsic lipid curvature and of overall bilayer curvature on enzyme activity, and enzyme-promoted vesicle aggregation and fusion.
Asunto(s)
Metabolismo de los Lípidos , Esfingomielina Fosfodiesterasa/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Activación Enzimática , HumanosRESUMEN
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. Our results support the hypothesis that alcohols (in particular 10- to 14-C-atom alcohols) contribute to the stability of oleic acid vesicles under a wider range of experimental conditions. Moreover, studies of mixed oleic-acid-alkane and oleic-acid-alcohol systems using infrared spectroscopy and Langmuir trough measurements indicate that precisely those alcohols that increased vesicle stability also decreased the mobility of oleic acid polar headgroups, as well as the area/molecule of lipid.
Asunto(s)
Alcoholes/química , Membrana Celular/química , Membranas Artificiales , Ácido Oléico/química , Agua/químicaRESUMEN
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. This kind of method may be of general applicability in the study of model and cell membranes.
RESUMEN
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. Morphological examination of the GUV in the presence of enzyme reveals that, although the enzyme diffuses rapidly throughout the observation chamber, detectable enzyme binding appears to be a slow, random process, with new bound-enzyme-containing vesicles appearing for several minutes. Enzyme binding to the vesicles appears to be a cooperative process. After the initial cluster of bound enzyme is detected, further binding and catalytic activity follow rapidly. After the activity has started, the enzyme is not released by repeated washing, suggesting a "scooting" mechanism for the hydrolytic activity. The enzyme preferentially binds the more disordered domains, and, in most cases, the catalytic activity causes the disordering of the other domains. Simultaneously, peanut- or figure-eight-shaped vesicles containing two separate lipid domains become spherical. At a further stage of lipid hydrolysis, lipid aggregates are formed and vesicles disintegrate.
Asunto(s)
Esfingomielina Fosfodiesterasa/metabolismo , Fosfolipasas de Tipo C/metabolismo , Liposomas Unilamelares/química , Ceramidas/química , Colesterol/química , Diglicéridos/química , Microscopía Confocal , Microscopía Fluorescente , Pseudomonas aeruginosa/enzimología , Esfingomielina Fosfodiesterasa/química , Fosfolipasas de Tipo C/químicaRESUMEN
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. Here we show how to prepare GUVs using a new protocol based on the electroformation method either from native membranes or organic lipid mixtures at physiological ionic strength. Additionally, we describe methods to test whether membrane proteins and glycosphingolipids preserve their natural orientation after electroformation of GUVs composed of native membranes.
Asunto(s)
Membrana Celular/química , Lípidos de la Membrana/análisis , Liposomas Unilamelares/química , Membrana Celular/ultraestructura , Suministros de Energía Eléctrica , Membrana Eritrocítica/química , Membrana Eritrocítica/ultraestructura , Humanos , Lípidos/química , Lípidos de la Membrana/aislamiento & purificación , Microdominios de Membrana/química , Concentración OsmolarRESUMEN
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. We found that the sphingomyelinase, but not the phospholipase C activity, is essential for hot-cold hemolysis because the phenomenon occurs not only in human erythrocytes that contain both phosphatidylcholine (PC) and sphingomyelin (SM) but also in goat erythrocytes, which lack PC. However, in horse erythrocytes, with a large proportion of PC and almost no SM, hot-cold hemolysis induced by PlcHR 2 is not observed. Fluorescence microscopy observations confirm the formation of ceramide-enriched domains as a result of PlcHR 2 activity. After cooling down to 4 degrees C, the erythrocyte ghost membranes arising from hemolysis contain large, ceramide-rich domains. We suggest that formation of these rigid domains in the originally flexible cell makes it fragile, thus highly susceptible to hemolysis. We also interpret the slow hemolysis observed at 37 degrees C as a phenomenon of gradual release of aqueous contents, induced by the sphingomyelinase activity, as described by Ruiz-Arguello et al. [(1996) J. Biol. Chem. 271, 26616]. These hypotheses are supported by the fact that ceramidase, which is known to facilitate slow hemolysis at 37 degrees C, actually hinders hot-cold hemolysis. Differential scanning calorimetry of erytrocyte membranes treated with PlcHR 2 demonstrates the presence of ceramide-rich domains that are rigid at 4 degrees C but fluid at 37 degrees C. Ceramidase treatment causes the disapperance of the calorimetric signal assigned to ceramide-rich domains. Finally, in liposomes composed of SM, PC, and cholesterol, which exhibit slow release of aqueous contents at 37 degrees C, addition of 10 mol % ceramide and transfer to 4 degrees C cause a large increase in the rate of solute efflux.
Asunto(s)
Ceramidas/química , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Hemólisis , Esfingomielinas/química , Ceramidas/metabolismo , Ceramidas/farmacología , Humanos , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Estructura Terciaria de Proteína/efectos de los fármacos , Espectrometría de Fluorescencia , Esfingomielina Fosfodiesterasa/química , Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielinas/metabolismo , TemperaturaRESUMEN
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. The melting properties of these gel-like domains resemble those of SM:ceramide binary mixtures, suggesting Chol displacement by ceramide from SM:Chol-rich liquid-ordered regions. Thus three kinds of domains coexist within a single vesicle in the presence of ceramide: gel, liquid-ordered, and liquid-disordered. In contrast, when 10 mol% egg diacylglycerol is added instead of ceramide, homogeneous vesicles, consisting only of liquid-disordered bilayers, are observed.