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Thermal Steadiness along with Destruction Kinetics regarding Patulin throughout Highly Acid Circumstances: Impact involving Cysteine.
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.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.The study of the structure and dynamics of membrane domains in vivo is a challenging task. #link# 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.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 565-72, 2005; Jacobi J et al, Cell Signal 2952-61, 2017; Rotolo J et al, J Clin Invest 1221786-1790, 2012; Truman JP et al, PLoS One 5e12310, 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 2952-61, 2017; Truman JP et al, PLoS One 5e12310, 2010; van Hell AJ et al, Cell Signal 3486-91, 2017; Hajj C, Haimovitz-Friedman A, Handb Exp Pharmacol 216115-130, 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.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.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.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.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. link2 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.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 phosphatidylcholineceramide 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.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 387569-572, 1997; Pike, Biochem J 378281-292, 2004; Grassme et al., J Immunol 168 300-307, 2002; Cheng et al., J Exp Med 1901549-1550, 1999; Kilkus et al., J Neurosci Res 72(1) 62-75, 2003). In a proposed Model 1 (Cheng et al., J Exp Med 1901549-1550, 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. link3 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 learn more 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 387569-572, 1997; Pike, Biochem J 378281-292, 2004; Grassme et al., J Immunol 168 300-307, 2002; Cheng et al., J Exp Med 1901549-1550, 1999; Kilkus et al., J Neurosci Res 72(1)62-75, 2003; Testai et al., J Neurochem 89636-644, 2004). We will present a generic method for isolating LRs for both lipidomic, proteomic, and cellular signaling analysis [1-6].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.
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