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1.
Life Sci ; 273: 119300, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33662433

RESUMO

AIMS: Plasma hyperlipidemia is a protective factor in amyotrophic lateral sclerosis (ALS) while cholesterol-lowering drugs aggravate the pathology. We hypothesize that this phenomenon can be linked with membrane lipid alterations in the neuromuscular junctions (NMJs) occurring before motor neuron loss. METHODS: Neurotransmitter release in parallel with lipid membrane properties in diaphragm NMJs of SOD1G93A (mSOD) mice at nine weeks of age (pre-onset stage) were assessed. KEY FINDINGS: Despite on slight changes in spontaneous and evoked quantum release of acetylcholine, extracellular levels of choline at resting conditions, an indicator of non-quantum release, were significantly increased in mSOD mice. The use of lipid-sensitive fluorescent probes points to lipid raft disruption in the NMJs of mSOD mice. However, content of cholesterol, a key raft component was unchanged implying another pathway responsible for the loss of raft integrity. In the mSOD mice we found marked increase in levels of raft-destabilizing lipid ceramide. This was accompanied by enhanced ability to uptake of exogenous ceramide in NMJs. Acute and chronic administration of 25-hydroxycholesterol, whose levels increase due to hypercholesterolemia, recovered early alterations in membrane properties. Furthermore, chronic treatment with 25-hydroxycholesterol prevented increase in ceramide and extracellular choline levels as well as suppressed lipid peroxidation of NMJ membranes and fragmentation of end plates. SIGNIFICANCE: Thus, lipid raft disruption likely due to ceramide accumulation could be early event in ALS which may trigger neuromuscular abnormalities. Cholesterol derivative 25-hydroxycholesterol may serve as a molecule restoring the membrane and functional properties of NMJs at the early stage.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Modelos Animais de Doenças , Hidroxicolesteróis/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Superóxido Dismutase-1/fisiologia , Acetilcolina/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Ceramidas/metabolismo , Colesterol/metabolismo , Feminino , Masculino , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Junção Neuromuscular , Transmissão Sináptica
2.
Methods Mol Biol ; 2187: 37-46, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770500

RESUMO

The discovery of dynamic platforms in cell membranes, called lipid rafts or detergent resistant membrane domains, opened a new chapter on studies of membrane cell biology. Indeed, the analysis of lipid rafts enabled innovative ways to understand cellular and molecular mechanisms regulating normal and pathological processes. Lipid rafts have been studied in most cell types, where they work by providing transient and fluid architectural scaffolding platforms regulating a spectrum of important signaling pathways, including receptor activities, protein-protein interactions, posttranslational modifications of proteins and lipids and the function of ion channels. In this chapter, we will explain how to isolate these membrane domains from neural tissue samples and perform further analysis of proteins and lipids.


Assuntos
Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/patologia , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/patologia , Animais , Membrana Celular/metabolismo , Membrana Celular/patologia , Canais Iônicos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Mapas de Interação de Proteínas/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Transdução de Sinais/fisiologia
3.
Methods Mol Biol ; 2187: 47-86, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770501

RESUMO

Biomimetic lipid bilayer systems are a useful tool for modeling specific properties of cellular membranes in order to answer key questions about their structure and functions. This approach has prompted scientists from all over the world to create more and more sophisticated model systems in order to decipher the complex lateral and transverse organization of cellular plasma membranes. Among a variety of existing biomembrane domains, lipid rafts are defined as small, dynamic, and ordered assemblies of lipids and proteins, enriched in cholesterol and sphingolipids. Lipid rafts appear to be involved in the development of Alzheimer's disease (AD) by affecting the aggregation of the amyloid-ß (Aß) peptide at neuronal membranes thereby forming toxic oligomeric species. In this review, we summarize the laboratory methods which allow to study the interaction of Aß with lipid rafts. We describe step by step protocols to form giant (GUVs) and large unilamellar vesicles (LUVs) containing raft-mimicking domains surrounded by membrane nonraft regions. Using fluorescence microscopy GUV imaging protocols, one can design experiments to visualize micron-scale raft-like domains, to determine the micron-scale demixing temperature of a given lipid mixture, construct phase diagram, and photogenerate domains in order to assess the dynamics of raft formation and raft size distribution. LUV fluorescence spectroscopy protocols with proper data analysis can be used to measure molecular packing of raft/nonraft regions of the membrane, to report on nanoscale raft formation and determine nanoscale demixing temperature. Because handling of the Aß requires dedicated laboratory experience, we present illustrated protocols for Aß-stock aliquoting, Aß aqueous solubilization, oligomer preparation, determination of the Aß concentration before and after filtration. Thioflavin binding, dynamic light scattering, and transmission electron microscopy protocols are described as complementary methods to detect Aß aggregation kinetics, aggregate sizes, and morphologies of observed aggregates.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Bicamadas Lipídicas/metabolismo , Doença de Alzheimer/metabolismo , Animais , Biomimética/métodos , Membrana Celular/metabolismo , Humanos , Laboratórios , Microdomínios da Membrana/metabolismo , Lipossomas Unilamelares/metabolismo
4.
Methods Mol Biol ; 2187: 87-98, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770502

RESUMO

Extracellular vesicles (EVs) are secreted by eukaryotic cells and serve as carriers for a variety of cell signaling factors, including RNAs, proteins, and lipids. We described a unique population of EVs, the membrane of which is highly enriched with the sphingolipid ceramide. We suggested that ceramide in the EV membrane is organized in ceramide-rich platforms (CRPs), a type of lipid raft that mediates interaction of ceramide with ceramide-associated proteins (CAPs). Here, we describe methods using anti-ceramide antibody to isolate ceramide-enriched EVs and detect exosomes after uptake into recipient cells. In addition, we discuss methods for EV analysis using nanoparticle tracking and mass spectrometry. The methods can be extended to the isolation of other types of EVs and "mobile rafts" transported by EVs from donor to recipient cells using antibodies against lipids specific for these EVs.


Assuntos
Ceramidas/metabolismo , Vesículas Extracelulares/metabolismo , Animais , Anticorpos/metabolismo , Linhagem Celular , Exossomos/metabolismo , Humanos , Espectrometria de Massas/métodos , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Camundongos , Nanopartículas/metabolismo , Transdução de Sinais/fisiologia , Esfingolipídeos/metabolismo
5.
Methods Mol Biol ; 2187: 99-112, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770503

RESUMO

Lipid rafts (LRs) represent cellular microdomains enriched in sphingolipids and cholesterol which may fuse to form platforms in which signaling molecules can be organized and regulated (Simons and Ikonen, Nature 387:569-572, 1997; Pike, Biochem J 378:281-292, 2004; Grassme et al., J Immunol 168: 300-307, 2002; Cheng et al., J Exp Med 190:1549-1550, 1999; Kilkus et al., J Neurosci Res 72(1) 62-75, 2003). In a proposed Model 1 (Cheng et al., J Exp Med 190:1549-1550, 1999) the LR has a well-ordered central core composed mainly of cholesterol and sphingolipids that is surrounded by a zone of decreasing lipid order. Detergents such as Triton X-100 can solubilize the core (and a significant amount of phosphoglyceride), but the LRs will be insoluble at 4 °C and be enriched in a well-characterized set of biomarkers. Model 2 proposes that the LRs are homogeneous, but there is selectivity in the lipids (and proteins) extracted by the 1% Triton X-100. Model 3 proposes LRs with distinct lipid compositions are highly structured and can be destroyed by binding molecules such as beta-methylcyclodextrin or filipin. These may be Caveolin in some cell types but not in brain. Since it is unlikely that two LR preparations will be exactly the same this review will concentrate on LRs defined as "small (50 nm) membranous particles which are insoluble in 1% Triton X-100 at 4 °C and have a low buoyant density (Simons and Ikonen, Nature 387:569-572, 1997; Pike, Biochem J 378:281-292, 2004; Grassme et al., J Immunol 168: 300-307, 2002; Cheng et al., J Exp Med 190:1549-1550, 1999; Kilkus et al., J Neurosci Res 72(1):62-75, 2003; Testai et al., J Neurochem 89:636-644, 2004). We will present a generic method for isolating LRs for both lipidomic, proteomic, and cellular signaling analysis [1-6].


Assuntos
Detergentes/química , Exossomos/metabolismo , Lipídeos/isolamento & purificação , Microdomínios da Membrana/metabolismo , Animais , Biomarcadores/metabolismo , Encéfalo/metabolismo , Caveolinas/metabolismo , Linhagem Celular Tumoral , Colesterol/metabolismo , Filipina/metabolismo , Humanos , Camundongos , Octoxinol/química , Proteômica/métodos , Transdução de Sinais/fisiologia , Esfingolipídeos/metabolismo , beta-Ciclodextrinas/metabolismo
6.
Methods Mol Biol ; 2187: 113-129, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770504

RESUMO

This chapter will discuss methods for analyses of the rates of sphingomyelin synthesis and turnover associated with lipid rafts or plasma membrane. These methods involve the use of fluorescently (NBD-C6-ceramide or NBD-C6-Sphingomyelin)) or radioactively labeled substrates ([3H-methyl]-phosphatidylcholine, [3H-acyl]-ceramide, [14C-methyl]-sphingomyelin) to quantify in vitro the activity of the sphingomyelin synthase (SMS) (also known as phosphatidylcholine:ceramide phosphocholine transferase), acid sphingomyelinase (the endosomal/lysosomal (L-SMase) and the secretory (S-SMase) forms) and neutral sphingomyelinase-2 (nSMase-2). These methods allow to quantify changes in the activity of enzymes that affect the SM-to-ceramide ratio on the plasma membrane, and consequently, the lipid rafts biophysical properties, dynamics, and raft-associated receptor clustering and signaling events. Specific attention is paid to challenges caused by the fact that SMS and nSMase-2 are integral/membrane bound proteins and how to avoid the use of detergent that suppress their specific activities.


Assuntos
Bioensaio/métodos , Membrana Celular/metabolismo , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Esfingomielinas/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/metabolismo , Animais , Ceramidas/metabolismo , Endossomos/metabolismo , Humanos , Lisossomos/metabolismo , Fosfatidilcolinas/metabolismo , Transdução de Sinais/fisiologia , Esfingomielina Fosfodiesterase , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo
7.
Methods Mol Biol ; 2187: 131-145, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770505

RESUMO

The traditional methods to study lipid rafts and their association with membrane proteins are based mainly on the isolation of a detergent-resistant membrane by biochemical fractionation. However, the use of detergents may induce lipid segregation and/or redistribution of membrane proteins during the process of sample preparation. Here, we describe a detergent-free method to study the glycolipid and growth factor receptor interaction and their association with lipid rafts. This method combines the biochemical and immunoblotting tools with confocal microscopic imaging, which allows for evaluation and verification of the membrane protein interaction and association with the lipid rafts components in a multifaceted manner.


Assuntos
Glicolipídeos/metabolismo , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Animais , Fracionamento Celular/métodos , Células Cultivadas , Detergentes/metabolismo , Proteínas de Membrana/metabolismo , Camundongos
8.
Methods Mol Biol ; 2187: 147-186, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770506

RESUMO

Lipid rafts are heterogeneous membrane domains enriched in cholesterol, sphingolipids, and gangliosides that serve as sorting platforms to compartmentalize and modulate signaling pathways. Death receptors and downstream signaling molecules have been reported to be recruited into these raft domains during the triggering of apoptosis. Here, we provide two protocols that support the presence of Fas/CD95 in lipid rafts during apoptosis, involving lipid raft isolation and confocal microscopy techniques. A detailed protocol is provided for the isolation of lipid rafts, by taking advantage of their resistance to Triton X-100 solubilization at 4 °C, followed by subsequent sucrose gradient centrifugation and analysis of the protein composition of the different gradient fractions by Western blotting. In addition, we also provide a detailed protocol for the visualization of the coclustering of Fas/CD95 death receptor and lipid rafts, as assessed by using anti-Fas/CD95 antibodies and fluorescent dye-conjugated cholera toxin B subunit that binds to ganglioside GM1, a main component of lipid rafts, by immunofluorescence and confocal microscopy. These protocols can be extended to any protein of interest to be analyzed for its association to lipid rafts.


Assuntos
Apoptose/fisiologia , Centrifugação/métodos , Lipídeos de Membrana/química , Microdomínios da Membrana/química , Microscopia de Fluorescência/métodos , Sacarose/química , Receptor fas/metabolismo , Linhagem Celular Tumoral , Toxina da Cólera/metabolismo , Gangliosídeo G(M1)/metabolismo , Humanos , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Octoxinol/química , Ligação Proteica/fisiologia , Transdução de Sinais/fisiologia , Receptor fas/química
9.
Methods Mol Biol ; 2187: 187-206, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770507

RESUMO

Numerous G protein-coupled receptors (GPCRs) and GPCR-signaling molecules reside in lipid rafts and thus, are inherently regulated in these microdomains. However, the limitations of current methods to investigate lipid raft biology and GPCR activity in situ have hindered the complete understanding of the molecular underpinnings of GPCR trafficking and signaling, especially in the whole organism. This book chapter details an innovative in vivo approach to study the crucial role of lipid rafts on the workings of GPCRs in the mouse kidney. This protocol involves the use of a modified mini osmotic pump to deliver an agent that selectively disrupts the lipid raft in the kidney.


Assuntos
Rim/metabolismo , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Camundongos , Transporte Proteico/fisiologia , Transdução de Sinais/fisiologia
10.
Methods Mol Biol ; 2187: 207-213, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770508

RESUMO

Ceramide can be generated on cell surfaces by the activity of the acid sphingomyelinase. The unique biophysical properties of ceramide result in the self-formation of small ceramide-enriched membrane domains that spontaneously fuse to large ceramide-enriched membrane macrodomains. The present chapter describes how these domains can be labeled and thereby visualized in cells. Further, the chapter provides protocols how ceramide and sphingosine can be quantified on the surface of cells and organs.


Assuntos
Membrana Celular/metabolismo , Ceramidas/metabolismo , Imunoquímica/métodos , Lipídeos de Membrana/metabolismo , Animais , Células Cultivadas , Humanos , Microdomínios da Membrana/metabolismo , Transdução de Sinais/fisiologia , Esfingosina/metabolismo
11.
Methods Mol Biol ; 2187: 215-221, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770509

RESUMO

The prevailing mechanism of action of chemotherapeutic drugs has been challenged by the role of ceramide, a second messenger, shown to induce apoptosis, differentiation, growth arrest, senescence, and autophagy in different cells (Chabner BA, Roberts TG Jr, Nat Rev Cancer 5:65-72, 2005; Jacobi J et al, Cell Signal 29:52-61, 2017; Rotolo J et al, J Clin Invest 122:1786-1790, 2012; Truman JP et al, PLoS One 5:e12310, 2010). Certain chemotherapeutic drugs activate the acid sphingomyelinase (ASMase)/ceramide pathway, generating ceramide in the tumor endothelium and this microvascular dysfunction is crucial for the tumor response. Ceramide has fusigenic properties and as such, when generated within the plasma membrane, initiates the oligomerization of ceramide-and cholesterol-rich domains in the outer leaflet of the plasma membrane, leading to the formation of ceramide-rich microdomains/platforms (CRP) (Jacobi J et al, Cell Signal 29:52-61, 2017; Truman JP et al, PLoS One 5:e12310, 2010; van Hell AJ et al, Cell Signal 34:86-91, 2017; Hajj C, Haimovitz-Friedman A, Handb Exp Pharmacol 216:115-130, 2013) known as "signaling platform." This chapter will discuss the generation, detection, and quantitation of CRP and their possible modulation in endothelial cells, in vitro and in vivo in response to certain chemotherapeutic drugs.


Assuntos
Antineoplásicos/farmacologia , Ceramidas/metabolismo , Células Endoteliais/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Humanos , Microdomínios da Membrana/metabolismo , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esfingomielina Fosfodiesterase/metabolismo
12.
Methods Mol Biol ; 2187: 223-245, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770510

RESUMO

The study of the structure and dynamics of membrane domains in vivo is a challenging task. However, major advances could be achieved through the application of microscopic and spectroscopic techniques coupled with the use of model membranes, where the relations between lipid composition and the type, amount and properties of the domains present can be quantitatively studied.This chapter provides protocols to study membrane organization and visualize membrane domains by fluorescence microscopy both in artificial membrane and living cell models of Gaucher Disease (GD ). We describe a bottom-up multiprobe methodology, which enables understanding how the specific lipid interactions established by glucosylceramide, the lipid that accumulates in GD , affect the biophysical properties of model and cell membranes, focusing on its ability to influence the formation, properties and organization of lipid raft domains. In this context, we address the preparation of (1) raft-mimicking giant unilamellar vesicles labeled with a combination of fluorophores that allow for the visualization and comprehensive characterization of those membrane domains and (2) human fibroblasts exhibiting GD phenotype to assess the biophysical properties of biological membrane in living cells using fluorescence microscopy.


Assuntos
Biofísica/métodos , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/metabolismo , Microscopia de Fluorescência/métodos , Membrana Celular/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Doença de Gaucher/metabolismo , Glucosilceramidas/metabolismo , Humanos , Pele/metabolismo , Lipossomas Unilamelares/metabolismo
13.
Methods Mol Biol ; 2187: 247-269, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770511

RESUMO

The use of steady-state and time-resolved fluorescence spectroscopy to study sterol and sphingolipid-enriched lipid domains as diverse as the ones found in mammalian and fungal membranes is herein described. We first address how to prepare liposomes that mimic raft-containing membranes of mammalian cells and how to use fluorescence spectroscopy to characterize the biophysical properties of these membrane model systems. We further illustrate the application of Förster resonance energy transfer (FRET) to study nanodomain reorganization upon interaction with small bioactive molecules, phenolic acids, an important group of phytochemical compounds. This methodology overcomes the resolution limits of conventional fluorescence microscopy allowing for the identification and characterization of lipid domains at the nanoscale.We continue by showing how to use fluorescence spectroscopy in the biophysical analysis of more complex biological systems, namely the plasma membrane of Saccharomyces cerevisiae yeast cells and the necessary adaptations to the filamentous fungus Neurospora crassa , evaluating the global order of the membrane, sphingolipid-enriched domains rigidity and abundance, and ergosterol-dependent properties.


Assuntos
Biofísica/métodos , Membrana Celular/metabolismo , Mamíferos/metabolismo , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Espectrometria de Fluorescência/métodos , Animais , Ergosterol/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Neurospora crassa/metabolismo , Saccharomyces cerevisiae/metabolismo , Esfingolipídeos/metabolismo , Esteróis/metabolismo
14.
Methods Mol Biol ; 2187: 271-282, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770512

RESUMO

Fluorescence-based techniques have been an integral factor in the study of cellular and model membranes. Fluorescence studies carried out on model membranes have provided valuable structural information and have helped reveal mechanistic detail regarding the formation and properties of ordered lipid domains, commonly known as lipid rafts. This chapter focuses on four techniques, based on fluorescence spectroscopy or microscopy, which are commonly used to analyze lipid rafts. The techniques described in this chapter may be used in a variety of ways and in combination with other techniques to provide valuable information regarding lipid order and domain formation, especially in model membranes.


Assuntos
Membrana Celular/metabolismo , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Microscopia de Fluorescência/métodos , Espectrometria de Fluorescência/métodos , Modelos Teóricos
15.
Methods Mol Biol ; 2187: 327-335, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770516

RESUMO

Fluorescence microscopy is a powerful and widely used tool in molecular biology. Over the years, the discovery and development of lipid-binding fluorescent probes has established new research possibilities to investigate lipid composition and dynamics in the cell. For instance, fluorescence microscopy has allowed the investigation of lipid localization and density in specific cell compartments such as membranes or organelles. Often, the characteristics and the composition of lipid-enriched structures are determined by analyzing the distribution of a fluorescently labeled lipid probe, which intercalates in lipid-enriched platforms, or specifically binds to parts of the lipid molecule. However, in many cases antibodies targeting proteins have higher specificity and are easier to generate. Therefore, we propose to use both antibodies targeting lipid transporters and lipid binding probes to better monitor lipid membrane changes. As an example, we visualize lipid rafts using the fluorescently labeled-B-subunit of the cholera toxin in combination with antibodies targeting ceramide-binding proteins CERTs, central molecules in the metabolism of sphingolipids.


Assuntos
Proteínas de Transporte/metabolismo , Imunofluorescência/métodos , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Anticorpos/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Toxina da Cólera/metabolismo , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Esfingolipídeos/metabolismo
17.
Methods Mol Biol ; 2187: 337-348, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770517

RESUMO

The detection of protein complexes by coimmunoprecipitation or two-hybrid analysis is often limited to cytosolic and soluble proteins, while interaction between membrane proteins or proteins and lipids is hampered by solubilization artefacts or absence of appropriate antibodies to detect a complex. More recently, the proximity ligation assay (PLA) using antibodies for in situ detection of protein complexes in cells and cross-linkable lipid analogs that can be endowed with molecular tags for pull-down assyas were techniques utilized to identify and monitor interaction between proteins and lipids. We have developed a novel technique termed "cross-link/PLA" combining a cross-linkable ceramide analog with PLA and anti-ceramide antibody to visualize lipid-protein complexes in ceramide-rich platforms (CRPs), a particular type of lipid raft. This chapter will discuss experimental protocols and data analysis to use cross-link/PLA for detection and visualization of lipid-protein complexes in CRPs and other types of lipid rafts.


Assuntos
Reagentes para Ligações Cruzadas/química , Ligases/metabolismo , Lipídeos de Membrana/análise , Microdomínios da Membrana/química , Proteínas de Membrana/análise , Coloração e Rotulagem/métodos , Células Cultivadas , Ceramidas/análise , Ceramidas/metabolismo , Células HEK293 , Humanos , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo
18.
Proc Natl Acad Sci U S A ; 117(33): 19943-19952, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32759206

RESUMO

The nanoscale organization of biological membranes into structurally and compositionally distinct lateral domains is believed to be central to membrane function. The nature of this organization has remained elusive due to a lack of methods to directly probe nanoscopic membrane features. We show here that cryogenic electron microscopy (cryo-EM) can be used to directly image coexisting nanoscopic domains in synthetic and bioderived membranes without extrinsic probes. Analyzing a series of single-component liposomes composed of synthetic lipids of varying chain lengths, we demonstrate that cryo-EM can distinguish bilayer thickness differences as small as 0.5 Å, comparable to the resolution of small-angle scattering methods. Simulated images from computational models reveal that features in cryo-EM images result from a complex interplay between the atomic distribution normal to the plane of the bilayer and imaging parameters. Simulations of phase-separated bilayers were used to predict two sources of contrast between coexisting ordered and disordered phases within a single liposome, namely differences in membrane thickness and molecular density. We observe both sources of contrast in biomimetic membranes composed of saturated lipids, unsaturated lipids, and cholesterol. When extended to isolated mammalian plasma membranes, cryo-EM reveals similar nanoscale lateral heterogeneities. The methods reported here for direct, probe-free imaging of nanodomains in unperturbed membranes open new avenues for investigation of nanoscopic membrane organization.


Assuntos
Microscopia Crioeletrônica/métodos , Microdomínios da Membrana/ultraestrutura , Biomimética , Colesterol/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo
19.
J Membr Biol ; 253(5): 399-423, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32833058

RESUMO

Lipids form an integral, structural, and functional part of all life forms. They play a significant role in various cellular processes such as membrane fusion, fission, endocytosis, protein trafficking, and protein functions. Interestingly, recent studies have revealed their more impactful and critical involvement in infectious diseases, starting with the manipulation of the host membrane to facilitate pathogenic entry. Thereafter, pathogens recruit specific host lipids for the maintenance of favorable intracellular niche to augment their survival and proliferation. In this review, we showcase the lipid-mediated host pathogen interplay in context of life-threatening viral and bacterial diseases including the recent SARS-CoV-2 infection. We evaluate the emergent lipid-centric approaches adopted by these pathogens, while delineating the alterations in the composition and organization of the cell membrane within the host, as well as the pathogen. Lastly, crucial nexus points in their interaction landscape for therapeutic interventions are identified. Lipids act as critical determinants of bacterial and viral pathogenesis by altering the host cell membrane structure and functions.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/epidemiologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Pneumonia Viral/epidemiologia , Esfingolipídeos/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Humanos , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Transdução de Sinais
20.
Proc Natl Acad Sci U S A ; 117(32): 18977-18983, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719116

RESUMO

Parkinson's disease is associated with α-synuclein (α-syn), a cytosolic protein enriched in presynaptic terminals. The biological function of α-syn remains elusive; however, increasing evidence suggests that the protein is involved in the regulation of synaptic vesicle fusion, signifying the importance of α-syn-lipid interactions. We show that α-syn preferentially binds to GM1-rich, liquid-ordered lipid domains on cytoplasmic membranes by using unroofed cells, which encapsulates lipid complexity and cellular topology. Moreover, proteins (Rab3a, syntaxin-1A, and VAMP2) involved in exocytosis also localize with α-syn, supporting its proposed functional role in exocytosis. To investigate how these lipid/protein interactions influence α-syn at the residue level, α-syn was derivatized with an environmentally sensitive fluorophore (7-nitrobenz-2-oxa-1,3-diazol-4-yl [NBD]) at different N- and C-terminal sites. Measurements of NBD fluorescence lifetime distributions reveal that α-syn adopts a multitude of membrane-bound conformations, which were not recapitulated in simple micelle or vesicle models, indicating an exquisite sensitivity of the protein to the complex lipid environment. Interestingly, these data also suggest the participation of the C terminus in membrane localization, which is generally overlooked and thus emphasize the need to use cellularly derived and biologically relevant membranes for biophysical characterization. Collectively, our results demonstrate that α-syn is more conformationally dynamic at the membrane interface than previously appreciated, which may be important for both its physiological and pathological functions.


Assuntos
Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , alfa-Sinucleína/química , Gangliosídeo G(M1)/metabolismo , Humanos , Cinética , Lipídeos de Membrana/química , Microdomínios da Membrana/química , Microdomínios da Membrana/genética , Ligação Proteica , Transporte Proteico , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
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