Molecular Probes

Section 13.3 — Sphingolipids, Steroids, Lipopolysaccharides and Related Probes

Sphingolipids

Structure and Activity

Sphingolipids are essential components of the plasma membrane of eukaryotic cells, where they are typically found in the outer leaflet. Although particularly abundant in mammalian cells, sphingolipids are also present in Saccharomyces cerevisiae, other fungi and plants. Sphingolipids differ from phospholipids in being based on a lipophilic amino alcohol (sphingosine, Figure 13.3) rather than glycerol. Sphingolipids play important roles in signal transduction processes (Chapter 17). Genetic defects in enzymes in the metabolic pathways of sphingolipid synthesis and degradation, including those involved in type I Gaucher's (Ashkenazi) disease, type A Niemann–Pick disease and Krabbe's disease, and other lysosomal storage diseases, can be detected at the cellular level using our fluorescent analogs of sphingolipids. Ceramides are the biological building blocks of more complex sphingolipids. Metabolism of ceramides typically occurs in Golgi and endoplasmic reticulum membranes, and fluorescent ceramide analogs (Section 12.4) are important probes for measuring the intracellular distribution and transport of the labeled molecules in live cells. If the hydroxyl group of the ceramide is esterified to phosphocholine, the sphingolipid is a sphingomyelin (Figure 13.3). The main pathway of sphingomyelin biosynthesis in mammalian cells is based on the transfer of phosphocholine from glycerophosphocholine to ceramide, catalyzed by sphingomyelin synthase in the Golgi membrane. Synthesis is followed by exocytosis of the sphingomyelin to the plasma membrane, apparently via a vesicular pathway and flip-flop to the outer membrane. Sphingomyelinases, which are functionally analogous to phospholipase C in their chemistry, hydrolyze sphingomyelins back to ceramides. Generation of ceramides by hydrolysis of sphingomyelins appears to play a role in mediating the effects of exposure to tumor necrosis factor–α (TNF-α), γ-interferon and several other agents, all of which induce an apoptosis-like cell death. Molecular Probes' extensive assortment of reagents for following the diverse morphological and biochemical changes that occur during apoptosis are described in Section 15.5. Sensitive fluorescence-based measurements of sphingomyelinase activity using totally natural, unlabeled sphingomyelin as the substrate can be carried out using our Amplex Red Sphingomyelinase Assay Kit (A12220), described below.

In glycosylsphingolipids, the free hydroxyl group of the ceramide is glycosylated to give a sphingosyl glycoside (cerebroside, ) or a ganglioside (). These glycosphingolipids form cell-type–specific patterns at the cell surface that change with cell growth, differentiation, viral transformation and oncogenesis. Glycosphingolipids interact at the cell surface with toxins, viruses and bacteria, as well as with receptors and enzymes and are involved in cell-type–specific adhesion processes. Gangliosides modulate the trophic factor–stimulated dimerization, tyrosine phosphorylation and subsequent signal transduction events of several tyrosine kinase receptors. Ganglioside G_M1 has antineurotoxic, neuroprotective and neurorestorative effects on various central neurotransmitter systems. Gangliosides, including ganglioside G_M1, partition into lipid rafts — detergent-insoluble, sphingolipid- and cholesterol-rich membrane microdomains that form lateral assemblies in the plasma membrane. Molecular Probes offers Vybrant Lipid Raft Labeling Kits (V34403, V34404, V34405; see below), as well as Alexa Fluor dye conjugates of subunit B of cholera toxin (Section 7.7), a protein that selectively binds to ganglioside G_M1 in lipid rafts. We also offer sphingolipid arrays on nitrocellulose membranes (SphingoStrips, Section 17.4) for analyzing sphingolipid–protein interaction specificity.

BODIPY Sphingolipids

Ceramides (N-acylsphingosines), like diacylglycerols, are lipid second messengers that function in signal transduction processes. The concentration-dependent spectral properties of Molecular Probes' BODIPY FL C₅-ceramide (D3521, B22650; ), BODIPY FL C₅-sphingomyelin (D3522, ) and BODIPY FL C₁₂-sphingomyelin (D7711) make them particularly suitable for investigating sphingolipid transport and metabolism, in addition to their applications as structural markers for the Golgi complex (Section 12.4, ). BODIPY FL C₅-ceramide can be visualized by fluorescence microscopy (, , ) or by electron microscopy following diaminobenzidine (DAB) photoconversion to an electron-dense product. (Product Highlight: Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents).

Our range of BODIPY sphingolipids also includes the long-wavelength light–excitable BODIPY TR ceramide (D7540, ), as well as BODIPY FL C₅-lactosylceramide (D13951), BODIPY FL C₅-ganglioside G_M1 (B13950, ) and three BODIPY FL cerebrosides (glycosylated ceramides; D7519, D7547, D7548). All of Molecular Probes' sphingolipids (NBD- and BODIPY(R)-Dye–Labeled Sphingolipids) are prepared from D-erythro-sphingosine and therefore have the same stereochemical conformation as natural biologically active sphingolipids.

Complexing fluorescent lipids with bovine serum albumin (BSA) facilitates cell labeling by eliminating the need for organic solvents to dissolve the lipophilic probe — the BSA-complexed probe can be directly dissolved in water. We offer four BODIPY sphingolipid–BSA complexes for the study of lipid metabolism and trafficking, including BODIPY FL C₅-ceramide, BODIPY TR C₅-ceramide, BODIPY FL C₅-ganglioside G_M1 and BODIPY FL C₅-lactosylceramide, each complexed with defatted BSA (B22650, B34400, B34401, B34402).

BODIPY FL C₅-ceramide has been used to investigate the linkage of sphingolipid metabolism to protein secretory pathways and neuronal growth. Internalization of BODIPY FL C₅-sphingomyelin (D3522) from the plasma membrane of human skin fibroblasts results in a mixed population of labeled endosomes that can be distinguished based on the concentration-dependent green (~515 nm) or red (~620 nm) emission of the probe (). BODIPY C₅-sphingomyelin has also been used to assess sphingomyelinase gene transfer and expression in hematopoietic stem and progenitor cells. Our BODIPY FL C₅-lactosylceramide, BODIPY FL C₅-ganglioside G_M1 and BODIPY FL cerebrosides should be useful tools for the study of glycosphingolipid transport and signaling pathways in cells and for diagnosis of lipid-storage disorders such as Niemann–Pick disease, Gaucher's disease, G_M1 gangliosidosis, Morquio's syndrome and type IV mucolipidosis (ML-IV). Addition of BODIPY FL C₅-lactosylceramide to the culture medium of cells from patients with sphingolipid-storage diseases (sphingolipidosis) results in fluorescent product accumulation in lysosomes, whereas this probe accumulates in the Golgi apparatus of normal cells and cells from patients with other storage diseases. BODIPY FL C₅-ganglioside G_M1 has been shown to form cholesterol-enhanced clusters in membrane complexes with amyloid β-protein in a model of Alzheimer's disease amyolid fibrils. Colocalization of fluorescent cholera toxin B conjugates (Section 7.7) and BODIPY FL C₅-ganglioside G_M1 observed by fluorescence microscopy provides a direct indication of the association of these molecules in lipid rafts (, ). Studies by Martin and Pagano have shown that the internalization routes for BODIPY FL C₅-glucocerebroside (D7548) follow both endocytic and nonendocytic pathways and are quite different from those for BODIPY FL C₅-sphingomyelin.

NBD Sphingolipids

NBD C₆-ceramide (N1154, ) and NBD C₆-sphingomyelin (N3524) analogs predate their BODIPY counterparts and have been extensively used for following sphingolipid metabolism in cells and in multicellular organisms. As with BODIPY FL C₅-ceramide, we also offer NBD C₆-ceramide complexed with defatted BSA (N22651) to facilitate cell loading without the use of organic solvents to dissolve the probe. Koval and Pagano have prepared NBD analogs of both the naturally occurring D-erythro and the nonnatural L-threo stereoisomers of sphingomyelin and have compared their intracellular transport behavior in Chinese hamster ovary (CHO) fibroblasts.

NBD C₆-ceramide lacks the useful concentration-dependent optical properties of the BODIPY FL analog and is less photostable; however, the fluorescence of NBD C₆-ceramide is apparently sensitive to the cholesterol content of the Golgi apparatus, a phenomenon that is not observed with BODIPY FL C₅-ceramide. If NBD C₆-ceramide–containing cells are starved for cholesterol, the NBD C₆-ceramide that accumulates within the Golgi apparatus appears to be severely photolabile but this NBD photobleaching can be reduced by stimulation of cholesterol synthesis. Thus, NBD C₆-ceramide may be useful in monitoring the cholesterol content of the Golgi apparatus in live cells.

Vybrant Lipid Raft Labeling Kits

The Vybrant Lipid Raft Labeling Kits (V34403, V34404, V34405) are designed to provide convenient, reliable and extremely bright fluorescent labeling of lipid rafts in live cells. Lipid rafts are detergent-insoluble, sphingolipid- and cholesterol-rich membrane microdomains that form lateral assemblies in the plasma membrane. Lipid rafts also sequester glycophosphatidylinositol (GPI)-linked proteins and other signaling proteins and receptors, which may be regulated by their selective interactions with these membrane microdomains. Recent research has demonstrated that lipid rafts play a role in a variety of cellular processes — including the compartmentalization of cell-signaling events, the regulation of apoptosis and the intracellular trafficking of certain membrane proteins and lipids — as well as in the infectious cycles of several viruses and bacterial pathogens. Examining the formation and regulation of lipid rafts is a critical step in understanding these aspects of eukaryotic cell function.

The Vybrant Lipid Raft Labeling Kits (V34403, V34404, V34405) provide the key reagents for fluorescently labeling lipid rafts in vivo with our bright and extremely photostable Alexa Fluor dyes (, ). Live cells are first labeled with the green-fluorescent Alexa Fluor 488, orange-fluorescent Alexa Fluor 555 or red-fluorescent Alexa Fluor 594 conjugate of cholera toxin subunit B (CT-B). This CT-B conjugate binds to the pentasaccharide chain of plasma membrane ganglioside G_M1, which selectively partitions into lipid rafts. All of Molecular Probes' CT-B conjugates are prepared from recombinant CT-B and are completely free of the toxic subunit A, thus eliminating any concern for toxicity or ADP-ribosylating activity. An antibody that specifically recognizes CT-B is then used to crosslink the CT-B–labeled lipid rafts into distinct patches on the plasma membrane, which are easily visualized by fluorescence microscopy. Each Vybrant Lipid Raft Labeling Kit contains sufficient reagents to label 50 live-cell samples in a 2 mL assay, including:

• Recombinant cholera toxin subunit B (CT-B) labeled with the Alexa Fluor 488 (in Kit V34403), Alexa Fluor 555 (in Kit V34404) or Alexa Fluor 594 (in Kit V34405) dye
• Anti–cholera toxin subunit B antibody (anti–CT-B)
• Concentrated phosphate-buffered saline (PBS)
• A detailed labeling protocol (Vybrant(R) Lipid Raft Labeling Kits)

Because they are compatible with various multilabeling schemes, the Vybrant Lipid Raft Labeling Kits can also serve as important tools for identifying physiologically significant membrane proteins that associate with lipid rafts. Cells can be labeled with other live-cell probes during the lipid raft labeling protocol or immediately following the antibody crosslinking step, depending on the specific labeling requirements of the other probes. Alternatively, once the lipid rafts have been labeled and crosslinked, the cells can be fixed for long-term storage or fixed and permeabilized for subsequent labeling with antibodies or other probes that are impermeant to live cells.

Amplex Red Sphingomyelinase Assay Kit

The Amplex Red Sphingomyelinase Assay Kit (A12220) is designed for measuring sphingomyelinase activity in solution using a fluorescence microplate reader or fluorometer (Figure 13.35). This assay should be useful for screening sphingomyelinase activators or inhibitors or for detecting sphingomyelinase activity in cell and tissue extracts. The assay, which uses natural sphingomyelin as the principal substrate, employs an enzyme-coupled detection scheme in which phosphocholine liberated by the action of sphingomyelinase is cleaved by alkaline phosphatase to generate choline. Choline is, in turn, oxidized by choline oxidase, generating H₂O₂, which drives the conversion of the Amplex Red reagent (A12222, A22177; Section 10.5) to red-fluorescent resorufin (). This sensitive assay technique has been employed to detect activation of acid sphingomyelinase associated with ultraviolet radiation–induced apoptosis and to characterize an insecticidal sphingomyelinase C produced by Bacillus cereus. The Amplex Red Sphingomyelinase Assay Kit contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• H₂O₂ for use as a positive control
• Concentrated reaction buffer
• Choline oxidase from Alcaligenes sp.
• Alkaline phosphatase from calf intestine
• Sphingomyelin
• Triton X-100
• Sphingomyelinase from Staphylococcus sp.
• A detailed protocol (Amplex(R) Red Sphingomyelinase Assay Kit)

Each kit provides sufficient reagents for approximately 500 assays using a fluorescence microplate reader and a reaction volume of 200 µL per assay.

Steroids

Most steroids are neutral lipids and, as such, localize primarily within the cell's membranes, in lipid vacuoles and bound to certain lipoproteins. Our fluorescent analogs of these biomolecules, most of which are derived from our long-wavelength BODIPY dyes, NBD dyes or pyrene dyes, are highly lipophilic probes. One application of these probes is to detect enzymatic activity — either in vitro or in vivo — through hydrolysis of the fatty acid esters to fluorescent fatty acids. Although the substrates and products typically have similar fluorescence properties, they are readily extracted by an organic solvent and separated by chromatography. Molecular Probes has also developed excellent fluorometric assays for cholesterol, cholesteryl esters and enzymes that metabolize natural cholesterol derivatives; the reagents for these are available in our Amplex Red Cholesterol Assay Kits (A12216, see below).

Cholesteryl Esters

Cholesteryl esters consist of a fatty acid esterified to the 3β-hydroxyl group of cholesterol (). These very nonpolar species are the predominant lipid components of atherosclerotic plaque and low- and high-density lipoprotein (LDL and HDL) cores. We offer cholesteryl esters of three of our BODIPY fatty acids — BODIPY FL C₁₂ (C3927MP), BODIPY 542/563 C₁₁ (C12680) and BODIPY 576/589 C₁₁ (C12681) — all of which have long-wavelength visible emission and of diphenylhexatrienylpropionic acid (C7794), which has bright blue fluorescence only when it is in a lipid environment. The BODIPY FL cholesteryl ester (C3927MP) can be used as a tracer of cholesterol transport and receptor-mediated endocytosis of lipoproteins by fluorescence microscopy () and as a general nonexchangeable membrane marker. Addition of methyl β-cyclodextrin to the BODIPY FL cholesteryl ester is reported to facilitate its uptake by cells and tissues. Researchers have extensively used our BODIPY FL cholesteryl ester to measure cholesteryl ester–transfer protein (CETP) activity using fluorescence microplate readers. The longer-wavelength BODIPY 542/563 and BODIPY 576/589 cholesteryl esters likely have similar applications.

Side Chain–Modified Cholesterol Analogs

Molecular Probes offers NBD- and pyrene-labeled cholesterol analogs in which the fluorophore replaces the terminal segment of cholesterol's flexible alkyl tail. Refer to Table 13.2 for a comparison of the spectral properties of these two fluorophores. The environment-sensitive NBD fluorophore of the NBD cholesterol analog (N1148) localizes in the membrane's interior, unlike the anomalous positioning of NBD-labeled phospholipid acyl chains (Figure 13.1). As with other NBD lipid analogs, this probe is useful for investigating lipid transport processes and lipid–protein interactions. NBD cholesterol is selectively taken up by high-density lipoproteins via the scavenger receptor B1. A lipid droplet–specific protein binds unesterified NBD cholesterol with extremely high affinity (K_d = 2 nM). PMC oleate (P226) has been used to label LDL to detect receptor binding and internalization and in acetylated LDL for following receptor-mediated endocytosis by the scavenger pathway and also for selective photosensitization of cultured cells containing LDL receptors.

Amplex Red Cholesterol Assay Kit

The Amplex Red Cholesterol Assay Kit (A12216) provides an exceptionally sensitive assay for both cholesterol and cholesteryl esters in complex mixtures that is suitable for use with either fluorescence microplate readers or fluorometers. The assay provided in this kit can detect as little as 5 ng/mL (5 × 10^-4 mg/dL) cholesterol (Figure 13.38) and can accurately measure the cholesterol or cholesteryl ester content in the equivalent of 0.01 µL of human serum. The assay uses an enzyme-coupled reaction scheme in which cholesteryl esters are hydrolyzed by cholesterol esterase into cholesterol, which is then oxidized by cholesterol oxidase to yield H₂O₂ and the corresponding ketone steroidal product (Figure 10.65). The H₂O₂ is then detected using the Amplex Red reagent in combination with horseradish peroxidase (HRP). The Amplex Red cholesterol assay is continuous and requires no separation or wash steps. These characteristics make the assay particularly well suited for the rapid and direct analysis of cholesterol in blood and food samples using automated instruments. By performing two separate measurements in the presence and absence of cholesterol esterase, this assay is also potentially useful for determining the fraction of cholesterol that is in the form of cholesteryl esters within a sample. In addition, by adding an excess of cholesterol to the reaction, this assay can be used to sensitively detect the activity of cholesterol oxidase. The Amplex Red Cholesterol Assay Kit contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• H₂O₂ for use as a positive control
• Concentrated reaction buffer
• Cholesterol oxidase from Streptomyces
• Cholesterol esterase from Pseudomonas
• Cholesterol for preparation of a standard curve
• A detailed protocol (Amplex(R) Red Cholesterol Assay Kit)

Each kit provides sufficient reagents for approximately 500 assays using a fluorescence microplate reader and a reaction volume of 100 µL per assay.

Fluorescent Triacylglycerol

The fluorescent triacylglycerol (1,2-dioleoyl-3-(1-pyrenedodecanoyl)-rac-glycerol (D6562) has a pyrene fatty acid ester replacing one of the three fatty acyl residues of a natural triacylglycerol (). Pyrene has the important spectral property of forming excimers (Figure 13.8) when two fluorophores are in close proximity during the excited state. Pyrene triacylglycerols are useful for measuring cholesteryl ester transfer protein–mediated triacylglycerol transport between plasma lipoproteins. It is also an excellent substrate for lipoprotein lipase and hepatic triacylglycerol lipase.

Lipopolysaccharides

Molecular Probes offers fluorescent conjugates of lipopolysaccharides (LPS) from Escherichia coli and Salmonella minnesota (Section 16.1, Table 16.1, Product Information Sheet) with three of our Alexa Fluor dyes and with our BODIPY FL fluorophore. LPS or endotoxins are complex macromolecules present on the outer cell walls of gram-negative bacteria. Recognition of LPS by binding to the CD14 cell-surface receptor of phagocytes (Figure 16.9) is the key initiation step in the mammalian immune response to infection by gram-negative bacteria. The structural core of LPS, and the primary determinant of its biological activity, is the N-acetylglucosamine derivative, lipid A (Figure 16.10). Two plasma proteins, LPS-binding protein (LBP) and soluble CD14 (sCD14), play primary roles in transporting LPS and mediating cellular responses. If the fatty acid residues are removed from the lipid A component, the toxicity of the LPS can be reduced significantly. However, the mono- or diphosphoryl forms of lipid A are inherently toxic. In many gram-negative bacterial infections, LPS are responsible for clinically significant symptoms like fever, low blood pressure and tissue edema, which can lead to disseminated intravascular coagulation, organ failure and death. Studies also clearly indicate that LPS induce various signal transduction pathways, including those involving protein kinase C and protein myristylation, and stimulate a variety of immunochemical responses, including B lymphocyte and G-protein activation.

Our fluorescent BODIPY FL and Alexa Fluor LPS conjugates, which are labeled with succinimidyl esters of these dyes, allow researchers to follow LPS binding, transport and cell internalization processes. Lipopolysaccharide internalization activates endotoxin-dependent signal transduction in cardiomyocytes. The Alexa Fluor 488 LPS conjugates (L23351, L23356) selectively label microglia in a mixed culture containing oligodendrocyte precursors, astrocytes and microglia. A biologically active conjugate of galactose oxidase–oxidized S. minnesota LPS and our Alexa Fluor 488 hydrazide (A10436, Section 3.2; A10440, Section 14.3) has been used to elucidate molecular mechanisms of septic shock. Aggregation of the BODIPY FL derivative of S. minnesota strain R595 (L23355) in aqueous solution results in a spectral shift of the BODIPY FL dye's emission maximum to ~620 nm. Addition of LBP results in a relatively small fluorescence increase, indicating that LPS remain aggregated upon binding. Subsequent addition of sCD14 results in a large increase in fluorescence intensity and a shift in emission maximum to ~510 nm, indicating LBP-mediated transfer of monomeric LPS to sCD14. In one study, a BODIPY FL derivative of LPS from E. coli strain LCD25 (L23350) was used to measure the transfer rate of LPS from monocytes to high-density lipoprotein (HDL). Another study utilized a BODIPY FL derivative of LPS from S. minnesota to demonstrate transport to the Golgi apparatus in neutrophils, although this could have been due to probe metabolism. It has been reported that organelles other than the Golgi are labeled by some fluorescent or nonfluorescent LPS. Cationic lipids are reported to assist the translocation of fluorescent lipopolysaccharides into live cells; cell surface–bound LPS can be quenched by trypan blue (Figure 16.24). Molecular Probes' fluorescent LPS can potentially be combined with other fluorescent indicators, such as Ca²⁺-, pH- or organelle-specific stains, for monitoring intracellular localization and real-time changes in cellular response to LPS. We also offer the Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit (P20495, Section 3.2), which permits ultrasensitive analysis of unlabeled lipopolysaccharides in gel electrophoretograms (Figure 3.19, Figure 3.20, Figure 3.21).