Molecular Probes

Section 17.4 — Probes for Lipid Metabolism and Signaling

Lipids and lipid metabolites are abundant in cells and have both a structural role and a role in cell regulation. Phospholipases, in particular, play an important part in cellular signaling processes via the generation of second messengers such as diacylglycerols, arachidonate and inositol 1,4,5-triphosphate (Ins 1,4,5-P₃, I3716; Section 17.2). In addition, phospholipase A₂ activation is a key step in inflammation processes, making these enzymes important therapeutic targets, and phospholipase A₂ plays a major role in the pathogenesis of acute respiratory distress syndrome (ARDS). Enzymatic processing of sphingolipids and phospholipids produces a wide range of physiological and pathological effects, including apoptosis, inflammatory responses, cell differentiation and proliferation.

Phospholipases are classified according to the cleavage site on the phospholipid substrate (Figure 17.23). There are at least three types of fluorescence-based phospholipase detection methods:

• Continuous methods, which permit direct fluorometric monitoring of enzymatic activity using self-quenching or excimer-forming probes
• Methods that continuously detect nonfluorescent product formation from natural phospholipids such as the fatty acid component with our ADIFAB reagent or choline with our Amplex Red Phospholipase Assay Kits
• Discontinuous methods, which require resolution of fluorescent substrates and products by TLC, HPLC or other separation techniques

Table 17.2 summarizes Molecular Probes' products for fluorescence-based phospholipase assays. Other applications for our wide range of fluorescent phospholipids are described in Chapter 13.

Phospholipases A₁ and A₂

Molecular Probes exclusively supplies a variety of unique fluorescent and fluorogenic substrates for phospholipases. Several of these probes may also be useful for detection of intracellular and secreted phospholipases and phospholipid-binding proteins by monitoring changes in their fluorescence intensity or polarization properties.

BODIPY Dye Phospholipase A Substrates

Our Patented phospholipase A substrate — bis-BODIPY FL glycerophosphocholine (bis-BODIPY FL C₁₁-PC, B7701) — has been specifically designed to allow continuous monitoring of phospholipase A action and to be spectrally compatible with argon-ion laser excitation sources. When this probe is incorporated into cell membranes, the proximity of the BODIPY FL fluorophores on adjacent phospholipid acyl chains causes fluorescence self-quenching (Figure 17.24). Separation of the fluorophores upon hydrolytic cleavage of one of the acyl chains by either phospholipase A₁ or A₂ results in increased fluorescence. Bis-BODIPY FL C₁₁-PC has been developed in collaboration with Elizabeth Simons, who has successfully employed it for flow cytometric detection of phospholipase A activity in neutrophils. Using bis-BODIPY FL C₁₁-PC and indo-1 simultaneously, researchers in the Simons lab at Boston University have demonstrated that a rise in intracellular Ca²⁺ precedes phospholipase A activation in immune complex–stimulated cells. More recently, bis-BODIPY FL C₁₁-PC has been used to detect phospholipase A₂ activation induced by tumor necrosis factor (TNF) and for imaging G-protein–coupled phospholipase A activity associated with plant cell plasma membranes.

Specificity for phospholipase A₂ versus phospholipase A₁ can be obtained using phospholipids with nonhydrolyzable, ether-linked alkyl chains in the sn-1 position. A 1-O-alkyl–substituted phospholipid containing the BODIPY FL fluorophore (D3771, ) is a useful substrate for a phospholipase A₂–specific chromatographic assay. β-BODIPY FL C₅-HPC (D3803) has been used to quantitatively delineate a discontinuous increase of Ca²⁺-dependent cytosolic phospholipase A₂ (cPLA₂) activity during zebrafish embryogenesis. The analytical method developed for this study uses a fluorescence image scanner to quantitatively detect the free BODIPY FL dye–labeled fatty acid generated by the action of cPLA₂ ().

PED6 Phospholipase A₂ Substrate

PED6 (D23739, ) is a fluorogenic substrate for phospholipase A₂ incorporating a BODIPY FL dye–labeled sn-2 acyl chain and a dinitrophenyl quencher group (Figure 17.24). Cleavage of the dye-labeled acyl chain by phospholipase A₂ eliminates the intramolecular quenching effect of the dinitrophenyl group, resulting in a corresponding fluorescence increase. Continuous kinetic assays show PED6 to be a good substrate for both secreted and cytosolic phospholipase A₂ and platelet-activating factor acetylhydrolase. PED6 has been used by Steven Farber and co-workers for in vivo analysis of intestinal lipid metabolism in zebrafish larvae as a basis for identifying and screening mutant phenotypes (). PED6 is also useful for high-throughput screening of potential phospholipase A₂ inhibitors or activators.

Bis-Pyrenyl Phospholipase A Substrates

Our bis-pyrenyl phospholipase A probes (B3781, B3782) both emit at ~470 nm, indicating that their adjacent pyrene fluorophores () form excited-state dimers (Figure 13.8). Phospholipase A–mediated hydrolysis separates the fluorophores, which then emit as monomers at ~380 nm. These substrates have proven to be effective phospholipase A₂ substrates in model membrane systems (Table 17.2); however, it has been reported that 1,2-bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine (B3781) is highly resistant to degradation by phospholipases in human skin fibroblasts. 1,2-Bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine has been used in a sensitive, continuous assay for lecithin:cholesterol acyltransferase (LCAT). These probes have several other reported applications, including investigations of protein kinase C (PKC) interactions with lipids, DNA binding to liposomes and lipid dynamics.

Singly Labeled Pyrenyl and NBD Phospholipase A Substrates

Phospholipase A₂ activity has also been measured using phospholipids labeled with a single pyrene (H361, H3809, H3810) or NBD (N3786, N3787) fluorophore (Table 17.2). Because only the sn-2 phospholipid acyl chain is labeled (, , ), these probes can discriminate between phospholipase A₂ and phospholipase A₁ activity. To obtain a direct fluorescence response to enzymatic cleavage, sufficient phospholipid must be loaded into membranes to cause either intermolecular self-quenching (NBD-acyl phospholipids) or excimer formation (pyreneacyl phospholipids). Pyrene-labeled acidic phospholipids — particularly the phosphoglycerol and phosphomethanol derivatives (H3809, H3810) — are preferred as substrates by pancreatic and intestinal phospholipase A₂, whereas phosphocholines (H361, ) are preferred by phospholipase A₂ from snake venom. A very sensitive continuous assay that uses a phosphomethanol derivative (H3810) to measure mammalian cytosolic phospholipase A₂ and human nonpancreatic phospholipase A₂ has been described. 1-Octacosanyl-2-(1-pyrenehexanoyl)-sn-glycero-3-phosphomethanol (C₂₈-O-PHPM, O7703; ) was developed for monitoring phospholipase A₂ in serum because the bis-pyrenyl probes are not specific for phospholipase A₂ and can also produce false indications of activity due to interactions with serum proteins. The cleavage product of C₂₈-O-PHPM hydrolysis by phospholipase A₂ — 1-pyrenehexanoic acid (P3840, Section 13.2) — cannot be directly distinguished from the substrate based on its fluorescence. However, this product can be readily resolved by liquid/liquid partition of the dye into aqueous methanol from chloroform/heptane. Pyrene-labeled phospholipids have been inserted into liposomes consisting of disulfide-linked polymerized phospholipids, which form a stable host matrix that is resistant to degradation by phospholipase A₂. Pyrene fluorescence is quenched by interactions with the lipid matrix and increases upon cleavage of the probe by phospholipase. This versatile approach can be adapted for detection of phospholipase A₂ by using β-py-C₁₀-HPC (H361).

ADIFAB and DAUDA: A Different View of Phospholipase A Activity

The ADIFAB fatty acid indicator (A3880, Figure 17.42) functions as a fluorescent sensor for the free fatty acid cleavage products of phospholipases. It does not require membrane loading and can be used to monitor hydrolysis of natural (rather than synthetic) substrates. Assaying lysophospholipase activity with ADIFAB yields sensitivity comparable to radioisotopic methods. Richieri and Kleinfeld have described a methodology for using the ADIFAB reagent to measure the activity of phospholipase A₂ on cell and lipid-vesicle membranes. Their assay is capable of detecting hydrolysis rates as low as 10^–12 mole/minute. Displacement of dansylundecanoic acid (DAUDA, D94) from the rat intestinal fatty acid–binding protein I-FABP is the basis of another phospholipase A₂ assay method (Table 17.2) that is conceptually similar to the detection mechanism of ADIFAB. There is more information about ADIFAB at the end of this section.

Phospholipase C

Amplex Red Phosphatidylcholine-Specific Phospholipase C Assay Kit

The Amplex Red Phosphatidylcholine-Specific Phospholipase C Assay Kit (A12218) provides a sensitive method for continuously monitoring phosphatidylcholine-specific phospholipase C (PC-PLC) activity in vitro using a fluorescence microplate reader or fluorometer. In this enzyme-coupled assay, PC-PLC activity is monitored indirectly using the Amplex Red reagent, a sensitive fluorogenic probe for H₂O₂ (Section 10.5, Figure 10.59). First, PC-PLC converts the phosphatidylcholine (lecithin) substrate to form phosphocholine and diacylglycerol. After the action of alkaline phosphatase, which hydrolyzes phosphocholine to inorganic phosphate and choline, choline is oxidized by choline oxidase to betaine and H₂O₂. Finally, H₂O₂, in the presence of horseradish peroxidase, reacts with the Amplex Red reagent in a 1:1 stoichiometry to generate the highly fluorescent product, resorufin. Because resorufin has absorption and fluorescence emission maxima of approximately 571 nm and 585 nm, respectively, there is little interference from autofluorescence in most biological samples. Experiments with purified PC-PLC from Bacillus cereus (E6646, see below) indicate that the Amplex Red Phosphatidylcholine-Specific Phospholipase C Assay Kit can detect PC-PLC levels as low as 0.2 mU/mL using a reaction time of one hour (Figure 17.30). One unit of PC-PLC is defined as the amount of enzyme that will liberate 1.0 micromole of water-soluble organic phosphorus from L-α-phosphatidylcholine per minute at pH 7.3 at 37°C. The Amplex Red Phosphatidylcholine-Specific Phospholipase C Assay Kit is potentially useful for detecting PC-PLC activity in cell extracts and for screening PC-PLC inhibitors. Each kit includes:

• 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
• L-α-Phosphatidylcholine (lecithin)
• Phosphatidylcholine-specific phospholipase C from Bacillus cereus
• A detailed protocol (Amplex(R) Red Phosphatidylcholine-Specific Phospholipase C 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.

D 609: A Selective Phospholipase C Inhibitor

D 609 (tricyclodecan-9-yl xanthogenate, T6615; ) is a selective inhibitor of phosphatidylcholine-specific phospholipase C. D 609 also is reported to have antiviral and antitumor activity. D 609 has a wide variety of indirect effects on cells, including stimulation of DNA synthesis, blocking DNA fragmentation during apoptosis, prevention of cell spreading and enhancing the synthesis and secretion of interleukins. D 609 is an important tool for differentiating the activities of the various PLC enzymes on phospholipids bearing different head groups and between the activities of PLC and phospholipase D (PLD).

Bacillus cereus PI-PLC

Phosphatidylinositol-specific phospholipase C (PI-PLC, EC 3.1.4.10) from Bacillus cereus cleaves phosphatidylinositol (PI), yielding water-soluble D-myo-inositol 1,2-cyclic monophosphate and lipid-soluble diacylglycerol. This enzyme also functions to release enzymes that are linked to glycosyl phosphatidylinositol (GPI) membrane anchors. Molecular Probes offers highly purified B. cereus PI-PLC (P6466), which has been used in studies of PI synthesis and export across the plasma membrane. PI-PLC generates diacylglycerols for PKC-linked signal transduction studies and provides an efficient means of releasing most GPI-anchored proteins from cell surfaces under conditions in which the cells remain viable.

Phospholipase D

Amplex Red Phospholipase D Assay Kit

The Amplex Red Phospholipase D Assay Kit (A12219) provides a sensitive method for measuring phospholipase D (PLD) activity in vitro using a fluorescence microplate reader or fluorometer. In this enzyme-coupled assay, PLD activity is monitored indirectly using the Amplex Red reagent (Section 10.5). First, PLD cleaves the phosphatidylcholine (lecithin) substrate to yield choline and phosphatidic acid. Second, choline is oxidized by choline oxidase to betaine and H₂O₂. Finally, H₂O₂, in the presence of horseradish peroxidase, reacts with the Amplex Red reagent to generate the highly fluorescent product, resorufin (excitation/emission maxima ~571/585 nm). This kit can be used to continuously assay PLD enzymes with near-neutral pH optima, whereas PLD enzymes with acidic pH optima can be assayed in a simple two-step procedure. Experiments with purified PLD from Streptomyces chromofuscus indicate that the Amplex Red Phospholipase D Assay Kit can detect PLD levels as low as 10 mU/mL using a reaction time of one hour (Figure 17.32). One unit of PLD is defined as the amount of enzyme that will liberate 1.0 µmole of choline from L-α-phosphatidylcholine per minute at pH 8.0 at 30°C. The Amplex Red Phospholipase D Assay Kit is potentially useful for detecting PLD activity in cell extracts or for screening PLD inhibitors. Each kit includes:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• H₂O₂ for use as a positive control
• Concentrated reaction buffer
• Choline oxidase from Alcaligenes sp.
• L-α-Phosphatidylcholine (lecithin)
• A detailed protocol (Amplex(R) Red Phospholipase D 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.

Fluorescent Substrates for Phospholipase D

The products of phospholipase A₂, C and D cleavage of 1-O-alkyl-2-decanoyl-sn-glycero-3-phosphocholine labeled with the BODIPY FL fluorophore (D3771, ) can be separated and independently quantitated based on their differential migration on TLC or HPLC. Our BODIPY FL analog is preferred for this application because it is relatively photostable and the fluorescence properties of its different enzymatic products are all very similar. Researchers have taken advantage of these features to detect and quantitate phospholipase D activity in vascular smooth muscle cells, cultured mammalian cells and yeast.

EnzChek Lipase Substrate

The triacylglycerol-based EnzChek lipase substrate (E33955) offers higher throughput and better sensitivity than chromogenic (TLC or HPLC) assays, and a visible wavelength–detection alternative to pyrene-based fluorescent substrates. In the presence of lipases, the nonfluorescent EnzChek lipase substrate produces a bright, green-fluorescent product (excitation/emission maxima of ~505/515 nm) for the accurate and sensitive detection of lipase activity in solution. Furthermore, the green-fluorescent product of the EnzChek lipase substrate exhibits pH-insensitive spectra in the physiological pH range and is compatible with optics used for fluorescein detection in fluorometers.

Probes for Phosphoinositide-Mediated Cellular Functions

Phosphatidylinositol (PI or PtdIns) and its phosphorylated derivatives represent only a small fraction of eukaryotic cellular phospholipids but are functionally significant in a disproportionately large number of regulatory and signal transduction processes. The most familiar of these processes is the phospholipase C–mediated generation of the ubiquitous second messengers inositol 1,4,5-triphosphate (InsP₃) and diacylglycerol (DAG) from phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P₂; Section 17.2; Figure 17.1). Because these events are transient and spatially compartmentalized, methods for visualizing changes in the cellular localization of phosphoinositides are essential for their complete characterization. In collaboration with Echelon Biosciences, Inc. (http://www.echelon-inc.com), we have introduced an extensive collection of biologically active fluorescent phosphoinositides, anti-phosphoinositide antibodies and immobilized phosphoinositide arrays.

Fluorescent Phosphoinositides

We offer fluorescent analogs of phosphatidylinositol (PtdIns) and of its 3-, 4- and 5-phosphorylated derivatives (Table 17.3, Fluorescent Phosphoinositides and Shuttle PIP Carriers), together with Shuttle PIP carriers for delivering them into living cells. All of the fluorescent analogs are labeled with a BODIPY dye on the sn-1 acyl chain (Figure 17.33). This labeling pattern maximizes the separation of the dye from the inositol ring and ensures retention of biological activity. Each phosphoinositide analog is available with three different BODIPY dye label options (Table 17.3) for multicolor detection in combination with fluorescent Shuttle PIP carriers (see below), fluorescent Ca²⁺ indicators or GFP chimeras. compared with alternative methods based on GFP–pleckstrin homology domain chimeras, detection of intracellular phosphoinositides using these probes is not complicated by parallel detection of soluble inositol phosphates and is subject to less distributional bias. A sensitive method for analyzing these fluorescent phosphoinositides and other products of lipid-modifying enzymes has been developed using a microfluidic chip device that separates lipids based on micellar electrokinetic capillary chromatography.

Shuttle PIP Carriers

Shuttle PIP carriers are polybasic molecules that have been selected on the basis of their ability to facilitate intracellular delivery of phosphatidylinositol diphosphates and triphosphates into live cells in the form of electrostatically neutral complexes (Figure 17.34). Uptake of Shuttle PIP complexes of phosphoinositide by cells is a passive process (requiring only a few minutes of incubation) and has been demonstrated in mammalian (CHO, COS-7, MDCK, NIH 3T3) (), yeast, plant and bacterial cell types. Shuttle PIP carriers do not appear to be effective for intracellular delivery of phosphatidylinositol and its monophosphorylated derivatives. Unlabeled Shuttle PIP carriers (Shuttle PIP carrier-1 *histone H1*, S23731; Shuttle PIP carrier-2 *neomycin B sulfate*, S35900) are available, as well as the green-fluorescent Oregon Green 488 and orange-fluorescent tetramethylrhodamine conjugates of Shuttle PIP carrier-1 (S23733, S23732). Labeled Shuttle PIP carriers are designed to enable visualization of the intracellular destinations of the carriers and their phosphoinositide cargoes in contrasting fluorescent colors.

Phosphoinositide Cell-Labeling Kits

To facilitate initial evaluations, we have assembled a series of Phosphoinositide Cell-Labeling Kits (B22615, B22616, B22617), providing pre-tested combinations of fluorescent phosphoinositides and Shuttle PIP carriers. Each kit contains the following items:

• BODIPY FL dye–labeled phosphoinositide
• Corresponding unlabeled phosphoinositide
• Unlabeled and tetramethylrhodamine-labeled versions of two different Shuttle PIP carriers
• An intracellular delivery protocol (Fluorescent Phosphoinositides and Shuttle PIP Carriers)

Anti-Phosphoinositide Monoclonal Antibodies

Research has revealed the direct action of phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P₂) and phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P₃) on a diverse array of cellular functions, including actin assembly and cytoskeletal dynamics, vesicular protein trafficking, protein kinase localization and activation, cell proliferation and apoptosis. We offer mouse monoclonal IgM antibodies to PtdIns(4,5)P₂ (A21327) and PtdIns(3,4,5)P₃ (A21328) for immunocytochemical localization of these important lipid metabolites (). Both antibodies have been shown to recognize their cognate phosphoinositides in murine and human cells with only slight crossreactivity with other phosphoinositides or phospholipids (Anti-Phosphoinositide Monoclonal Antibodies).

Arrays for Detection of Phosphoinositide–Protein Interactions

Protein domains that specifically bind phosphoinositides have emerged as major determinants in localizing proteins to their site of function. These phosphoinositide-binding motifs, which include the C2 (PKC conserved region 2), PH (pleckstrin homology), FYVE (Fab1p/YOTP/Vac1p/EEA1), ENTH (epsin NH₂-terminal homology) and PX (Phox homology) domains, are found in proteins implicated in a diverse array of cellular processes, such as actin cytoskeletal organization, cell growth regulation, control of gene expression, protein transport, exocytosis and endocytosis. Through localized phosphoinositide biosynthesis within the cell, proteins containing these lipid-recognition domains can be directed to functionally appropriate sites. PIP Strips and PIP Arrays, developed by Echelon Biosciences, Inc., are designed for identification of proteins possessing phosphoinositide recognition domains and analysis of their lipid binding specificities. PIP Strips provide 100 picomole samples of 15 different phospholipids, and a blank sample, immobilized on nitrocellulose membranes (Figure 17.37). We offer PIP Strips in packages of two (P23750) or ten (P23751) strips. PIP Strips are also available in a miniaturized format (PIP MicroStrips, P23752), which have been designed to allow immersion inside a standard microcentrifuge tube. PIP Arrays provide eight different phospholipids arrayed in amounts from 100 to 1.6 picomoles (Figure 17.38), allowing assessment of the strength of a protein's binding, in addition to its lipid specificity. We offer PIP Arrays in packages of two (P23748) or five (P23749) arrays. Phosphoinositide-mediated binding of proteins to PIP Strips and PIP Arrays is typically analyzed by protein–lipid overlay assays. Proteins may be detected using standard Western blot procedures in conjunction with our high-performance alkaline phosphatase– and horseradish peroxidase–mediated signal generation systems (Section 9.4).

Phosphoinositide-Coupled Agarose Beads

Through our collaboration with Echelon Biosciences, Inc., we offer phosphoinositide–coupled agarose beads (PIP Beads). PIP Beads are designed to allow isolation of phosphoinositide-binding proteins from cell lysates or expression libraries generated by in vitro transcription/translation reactions. We offer PIP Beads coated with phosphatidylinositol (PtdIns) and each of its seven possible 3-, 4- and 5-phosphorylated derivatives (Table 17.4).

Sphingolipids

Sphingolipids include sphingomyelins, which are phospholipid analogs, as well as ceramides, glycosyl ceramides (cerebrosides), gangliosides and other derivatives (Figure 13.3). Several excellent reviews of the chemistry and biology of sphingolipids and glycosphingolipids and their role in the process of signal transduction are available.

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,