Phosphoinositide analysis by liquid chromatography-mass spectrometry.

The phosphoinositides are a highly dynamic group of molecules implicated in many cellular control processes; however, the analysis of many of these structures has proven very difficult and time-consuming, with limited sensitivity and/or discrimination. Recent developments in LCMS now provide the prospect of routine structural and quantitative analysis of all the known phosphoinositides (and possibly some as yet unidentified structures) at high sensitivity in any biological sample. The procedures described here give very high extraction recovery from a variety of biological matrices and enable chromatographic resolution of most phosphoinositides as their native structures. When coupled with the accurate mass and fragmentation capabilities of an MS, full structural and isomeric identification can be achieved.

Rhabdomere biogenesis in Drosophila photoreceptors is acutely sensitive to phosphatidic acid levels.

Phosphatidic acid (PA) is postulated to have both structural and signaling functions during membrane dynamics in animal cells. In this study, we show that before a critical time period during rhabdomere biogenesis in Drosophila melanogaster photoreceptors, elevated levels of PA disrupt membrane transport to the apical domain. Lipidomic analysis shows that this effect is associated with an increase in the abundance of a single, relatively minor molecular species of PA. These transport defects are dependent on the activation state of Arf1. Transport defects via PA generated by phospholipase D require the activity of type I phosphatidylinositol (PI) 4 phosphate 5 kinase, are phenocopied by knockdown of PI 4 kinase, and are associated with normal endoplasmic reticulum to Golgi transport. We propose that PA levels are critical for apical membrane transport events required for rhabdomere biogenesis.

Lipidomic analysis of phospholipids and related structures by liquid chromatography-mass spectrometry.

Lipids are highly dynamic molecules with many different roles ranging from structural to signaling. Alterations in particular lipid levels change cellular behavior. In humans, inappropriate changes may manifest as diseases such as Alzheimer's, atherosclerosis, diabetes, obesity, and even cancer. This has led to a great interest in monitoring lipid changes both during normal cellular processes and in disease situations. With the advent of rapid mass spectrometry, it has become possible to analyze many lipids within a single sample with unsurpassed sensitivity and detail. Coupling this with liquid chromatography potentially enables the complete profiling of all the phospholipids, both major and minor, from a single sample within a single analysis run.

An intracellular motif of GLUT4 regulates fusion of GLUT4-containing vesicles.

BACKGROUND: Insulin stimulates glucose uptake by adipocytes through increasing translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane. Fusion of GLUT4-containing vesicles at the cell surface is thought to involve phospholipase D activity, generating the signalling lipid phosphatidic acid, although the mechanism of action is not yet clear. RESULTS: Here we report the identification of a putative phosphatidic acid-binding motif in a GLUT4 intracellular loop. Mutation of this motif causes a decrease in the insulin-induced exposure of GLUT4 at the cell surface of 3T3-L1 adipocytes via an effect on vesicle fusion. CONCLUSION: The potential phosphatidic acid-binding motif identified in this study is unique to GLUT4 among the sugar transporters, therefore this motif may provide a unique mechanism for regulating insulin-induced translocation by phospholipase D signalling.

Lipidomic analysis of signaling pathways.

This chapter outlines methods that can be applied to determine the levels of lipids in cells and tissues. In particular, the methods focus upon the extraction and analysis of those lipids critical for monitoring signal transduction pathways. The methods address the analysis of the phosphoinositides, the lipid agonists lysophosphatidic acid and sphingosine 1-phosphate, and the neutral lipid messengers diacylglycerol and ceramide. Additionally, because of the increasing need to determine the dynamics of signaling, the analysis of phospholipids synthesis using stable isotope methods is described. The use of these methods as described or adaptation to permit both approaches should allow investigators to determine changes in signaling lipids and to better understand such processes in most cell types. The increasing appreciation of the central roles played by lipid signaling pathways has dispelled the misconception that lipids are inert structural components that are involved solely in keeping a cell intact. Advances in our understanding of cell-signaling pathways have identified particular lipids that act to regulate the functions of a number of proteins either by controlling enzyme activity directly, or by localizing proteins to particular intracellular compartments where they perform a specialized role. These lipid-binding domains (e.g., PH, PX, FYVE) have been found in many proteins, and considerable detail is recorded of the structural basis of lipid protein interaction. Additional lipid-binding domains exist, which remain less well characterized (e.g., those that bind phosphatidic acid [PA] or ceramide); however, the important regulatory roles that these lipids play and the pathways involving these messengers are increasingly appreciated. While the downstream targets are thus being defined, the actual changes in lipid concentration in a stimulated cell or membrane are less characterized. The primary reason for this lack has been a deficiency in methodology. Much of the reported studies of lipid messengers in stimulated cells have depended upon monitoring changes in radio-labeled cells. Many well-documented problems are associated with this type of methodology, including lack of isotopic equilibrium, distinct pools with different turnover rates, and inadequate separation of radio-labeled metabolites; however, much important information has been generated. The second approach has been to make use of the lipid-binding properties of the target protein domains and to generate a tagged fusion protein, generally GFP, which permits identification of a region rich in a signaling lipid (Guillou et al., 2007). This has proved useful in monitoring PI-3-kinase activation in stimulated cells; however, considerable caveats must be raised, not least the problems associated with lipid specificity and the fact that many of these domains have associated protein-binding regions that can compromise the findings. A further problem associated with these two methodologies is that they tend to group lipids together and take no account of the multiple acyl chain structures that occur in all lipids. These concerns point to the need to determine actual changes in lipid compositions. Until relatively recently, such an analysis was unachievable; however, advances in both chromatographic separation and mass spectrometry (MS) have permitted the development of lipidomic analysis. This chapter outlines a number of methods that allow determination of changes in signaling lipids. Adaptation of the methods here for the analysis of other molecules should be relatively straightforward in the future. Much of the lipidomic research in the United Kingdom is focused upon signaling lipidomics, with particular foci upon phosphoinositide-related signaling in Birmingham and Cambridge (Wakelam) and London (Larijani), upon eicosanoids in Cardiff (O'Donnell), and steroids in London (Griffiths). Meanwhile, the use of stable isotopes has been particularly developed in Southampton (Postle).

Stable adhesion and migration of human neutrophils requires phospholipase D-mediated activation of the integrin CD11b/CD18.

The pathways regulating integrin-mediated adhesion during neutrophil migration are incompletely defined. Using a flow-based model in which human neutrophils rolling on P-selectin were activated to migrate by the chemoattractant peptide fMLP, we investigated the role of phospholipase D (PLD). fMLP-stimulated PLD generation of phosphatidate (PtdOH); while inhibition of PtdOH production with butan-1-ol had no effect on the initial immobilisation of rolling neutrophils (supported by activation of constitutively surface-expressed beta(2)-integrin CD11b/CD18) it impaired longer-term stability of adhesion and reduced the rate of migration (supported by activation of de novo-exocytosed CD11b/CD18). PtdOH regulated these processes by controlling activation of exocytosed CD11b/CD18, and appeared to act by directly stimulating phosphatidylinositol 4-phosphate 5-kinase type I to generate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Cell-permeable PtdIns(4,5)P(2) recovered migration of neutrophils after PLD inhibition; PtdIns(4,5)P(2) appeared to act by promoting talin binding to CD18 and hence activating CD11b/CD18, as migration was inhibited when neutrophils were loaded with peptides previously shown to block the interaction between PtdIns(4,5)P(2) and talin or talin and CD18. Thus, these data indicate that PLD-synthesised PtdOH stimulates the generation of PtdIns(4,5)P(2), which in turn mediates talin binding to, and activation of, CD11b/CD18 required for neutrophil stable adhesion and migration.

Antineutrophil cytoplasm antibody-stimulated neutrophil adhesion depends on diacylglycerol kinase-catalyzed phosphatidic acid formation.

Patients with certain forms of systematic vasculitis, such as Wegener's granulomatosis, have circulating antineutrophil cytoplasmic antibodies (ANCA). These inappropriately stimulate circulating neutrophils adhere to and thereby obstruct small vessels. This, together with ANCA-induced degranulation and an oxidative burst, leads to local tissue damage. The signaling pathways that are activated by ANCA IgG are distinct from those that are involved in normal neutrophil activation. This study shows that diacylglycerol kinase is selectively activated by ANCA and that the generated phosphatidic acid is responsible for promoting neutrophil adhesion, in part through integrin activation. The data presented point to diacylglycerol kinase alpha as a novel but selective target for the development of drugs to treat this potentially fatal disorder.

PLCgamma is enriched on poly-phosphoinositide-rich vesicles to control nuclear envelope assembly.

Nuclear envelope assembly is an essential event in each cell cycle but the proteins and lipids involved in its regulation remain mostly unknown. Assembly involves membrane fusions but neither specific SNAREs nor Rab GTPases have been identified in its control. We report that a precursor membrane population (MV1) required for NE assembly has a unique lipid composition consisting prominently of poly-phosphatidylinositides. The lipid composition was determined by adapting HPLC electrospray ionisation tandem mass spectrometry to phosphoinositide analysis, revealing the capacity of this technique to document dynamic lipid transitions of functional importance in natural membrane populations. MV1 is >100-fold enriched in endogenous PLCgamma and >25-fold enriched in the PLC substrate phosphatidylinositol bisphosphate (PtdInsP2) compared to the second membrane population, derived largely from endoplasmic reticulum (ER), that contributes most of the NE. During NE formation PLCgamma becomes transiently phosphorylated at the tyrosine 783 site indicative of its activation. In addition specific inhibition of PLCgamma blocks nuclear envelope formation. In vivo, PLCgamma is concentrated on vesicles of similar size to purified MV1. These associate with nuclei during the period of NE formation and are distinct from ER membranes. The unprecedented concentration of PLCgamma and its substrate PtdInsP2 in a subset of membranes that binds to only two regions of the nucleus, and activation of PLCgamma by GTP during initial stages of NE formation provide a mechanism for temporal control of NE assembly and offer an explanation for how such a process of membrane fusion can be spatially regulated.

Analysis of intact phosphoinositides in biological samples.

It is now apparent that each of the known, naturally occurring polyphosphoinositides, the phosphatidylinositol monophosphates (PtdIns3P, PtdIns4P, PtdIns5P), phosphatidylinositol bisphosphates [PtdIns(3,4)P(2), PtdIns(3,5)P(2), PtdIns(4,5)P(2)], and phosphatidylinositol trisphosphate [PtdIns(3,4,5)P(3)], have distinct roles in regulating many cellular events, including intracellular signaling, migration, and vesicular trafficking. Traditional identification techniques require [(32)P]inorganic phosphate or [(3)H]inositol radiolabeling, acidified lipid extraction, deacylation, and ion-exchange head group separation, which are time-consuming and not suitable for samples in which radiolabeling is impractical, thus greatly restricting the study of these lipids in many physiologically relevant systems. Thus, we have developed a novel, high-efficiency, buffered citrate extraction methodology to minimize acid-induced phosphoinositide degradation, together with a high-sensitivity liquid chromatography-mass spectrometry (LC-MS) protocol using an acetonitrile-chloroform-methanol-water-ethylamine gradient with a microbore silica column that enables the identification and quantification of all phosphoinositides in a sample. The liquid chromatograph is sufficient to resolve PtdInsP(3) and PtdInsP(2) regioisomers; however, the PtdInsP regioisomers require a combination of LC and diagnostic fragmentation to MS(3). Data are presented using this approach for the analysis of phosphoinositides in human platelet and yeast samples.

lazaro encodes a lipid phosphate phosphohydrolase that regulates phosphatidylinositol turnover during Drosophila phototransduction.

An essential step in Drosophila phototransduction is the hydrolysis of phosphatidylinositol 4,5 bisphosphate PI(4,5)P2 by phospholipase Cbeta (PLCbeta) to generate a second messenger that opens the light-activated channels TRP and TRPL. Although the identity of this messenger remains unknown, recent evidence has implicated diacylglycerol kinase (DGK), encoded by rdgA, as a key enzyme that regulates its levels, mediating both amplification and response termination. In this study, we demonstrate that lazaro (laza) encodes a lipid phosphate phosphohydrolase (LPP) that functions during phototransduction. We demonstrate that the synergistic activity of laza and rdgA regulates response termination during phototransduction. Analysis of retinal phospholipids revealed a reduction in phosphatidic acid (PA) levels and an associated reduction in phosphatidylinositol (PI) levels. Together our results demonstrate the contribution of PI depletion to the rdgA phenotype and provide evidence that depletion of PI and its metabolites might be a key signal for TRP channel activation in vivo.

Diacylglycerol induces fusion of nuclear envelope membrane precursor vesicles.

Purified membrane vesicles isolated from sea urchin eggs form nuclear envelopes around sperm nuclei following GTP hydrolysis in the presence of cytosol. A low density subfraction of these vesicles (MV1), highly enriched in phosphatidylinositol (PtdIns), is required for nuclear envelope formation. Membrane fusion of MV1 with a second fraction that contributes most of the nuclear envelope can be initiated without GTP by an exogenous bacterial PtdIns-specific phospholipase C (PI-PLC) which hydrolyzes PtdIns to form diacylglycerides and inositol 1-phosphate. This PI-PLC hydrolyzes a subset of sea urchin membrane vesicle PtdIns into diglycerides enriched in long chain, polyunsaturated species as revealed by a novel liquid chromatography-mass spectrometry analysis. Large unilammelar vesicles (LUVs) enriched in PtdIns can substitute for MV1 in PI-PLC induced nuclear envelope formation. Moreover, MV1 prehydrolyzed with PI-PLC and washed to remove inositols leads to spontaneous nuclear envelope formation with MV2 without further PI-PLC treatment. LUVs enriched in diacylglycerol mimic prehydrolyzed MV1. These results indicate that production of membrane-destabilizing diglycerides in membranes enriched in PtdIns may facilitate membrane fusion in a natural membrane system and suggest that MV1, which binds only to two places on the sperm nucleus, may initiate fusion locally.

Phospholipase D2 stimulates integrin-mediated adhesion via phosphatidylinositol 4-phosphate 5-kinase Igamma b.

Cellular adhesion can be regulated by, as yet, poorly defined intracellular signalling events. Phospholipase D enzymes generate the messenger lipid phosphatidate and here we demonstrate that suppression of this reaction inhibits cellular adhesion. This effect was reversed by the addition of cell-permeable analogues of either phosphatidate or phosphatidylinositol 4,5-bisphosphate. By contrast, neither diacylglycerol nor lysophosphatidic acid were able to reverse this effect suggesting that phosphatidate itself acts directly on a target protein(s) to regulate adhesion rather than as the result of its conversion to either of these metabolite lipids. Antibodies that block beta1 and beta2 integrin-substrate interactions inhibited adhesion stimulated by both phosphatidate and phosphatidylinositol 4,5-bisphosphate indicating that these lipids regulate beta1 and beta2 integrin-mediated adhesion. In vivo, these lipids can be generated by phospholipase D2 and phosphatidylinositol 4-phosphate 5-kinase Igamma b, respectively, and over-expression of catalytically-functional forms of these enzymes dose-dependently stimulated adhesion while siRNA depletion of PLD2 levels inhibited adhesion. Furthermore the ability of over-expressed phospholipase D2 to stimulate adhesion was inhibited by a dominant-negative version of phosphatidylinositol 4-phosphate 5-kinase Igamma b. Consistent with this, phosphatidylinositol 4-phosphate 5-kinase Igamma b-mediated adhesion was dependent upon phospholipase D2's product, phosphatidate indicating that phosphatidylinositol 4-phosphate 5-kinase Igamma b is downstream of, and necessary for, phospholipase D2's regulation of adhesion. It is likely that this phospholipase D2-generated phosphatidate directly stimulates phosphatidylinositol 4-phosphate 5-kinase Igamma b to generate phosphatidylinositol 4,5-bisphosphate as this mechanism has previously been demonstrated in vitro. Thus, our data indicates that during the initial stages of adhesion, phospholipase D2-derived phosphatidate stimulates phosphatidylinositol 4-phosphate 5-kinase Igamma b to generate phosphatidylinositol 4,5-bisphosphate and that consequently this inositol phospholipid promotes adhesion through its regulation of cell-surface integrins.

Phospholipase D activity is essential for actin localization and actin-based motility in Dictyostelium.

PLD (phospholipase D) activity catalyses the generation of the lipid messenger phosphatidic acid, which has been implicated in a number of cellular processes, particularly the regulation of membrane traffic. In the present study, we report that disruption of PLD signalling causes unexpectedly profound effects on the actin-based motility of Dictyostelium. Cells in which PLD activity is inhibited by butan-1-ol show a complete loss of actin-based structures, accompanied by relocalization of F-actin into small clusters, and eventually the nucleus, without a visible fall in levels of F-actin. Addition of exogenous phosphatidic acid reverses the effects of butan-1-ol, confirming that these effects are caused by inhibition of PLD. Loss of motility correlates with complete inhibition of endocytosis and a reduction in phagocytosis. Inhibition of PLD caused a major decrease in the synthesis of PtdIns(4,5)P2, which could again be reversed by exogenously applied phosphatidic acid. Thus the essential role of PLD signalling in both motility and endocytosis appears to be mediated directly via regulation of PtdIns(4)P kinase activity. This implies that localized PLD-regulated synthesis of PtdIns(4,5)P2 is essential for Dictyostelium actin function.

Assays to study phospholipase D regulation by inositol phospholipids and ADP-ribosylation factor 6.

Phospholipase D (PLD) is an enzyme implicated in the regulation of both exocytic and endocytic vesicle trafficking as well as many other processes. Consistent with this, the small GTPase Arf6 and regulated changes in inositol phospholipids levels are two factors that regulate both PLD and vesicle trafficking. Here we describe three methodologies through which the activation of PLD by Arf6 and inositol phospholipids may be investigated. The first method described is an in vitro protocol that allows the analysis of purified proteins or cell lysates. Furthermore, this protocol can be used to analyze the effects of different inositol phospholipids by changing the composition of the substrate vesicle. The major advantage of this protocol lies in the ability to analyze the effects of direct interactions on PLD activation by using pure proteins and lipids. The other two methods are in vivo protocols for the analysis of PLD activation in response to extracellular stimuli. Modification of cellular composition using overexpression/deletion or knockout of specific genes can be utilized with these protocols to characterize PLD activation pathways. The first of these methods uses the detection of radiolabeled PLD products and can be used for most cell types whereas the second of these two protocols is used to measure PLD products when radiolabeling of cells is not possible, such as freshly isolated cells that will not survive long enough to attain radiochemical equilibrium.

Breast cancer cell-derived EMMPRIN stimulates fibroblast MMP2 release through a phospholipase A(2) and 5-lipoxygenase catalyzed pathway.

Metalloproteinases (MMP) produced by both cancer and normal stromal fibroblast cells play a critical role in the metastatic spread of tumours, however little is known of the regulation of their release. In this report we demonstrate that breast cancer cells in culture release apparently full length soluble EMMPRIN that promotes the release of pro-MMP2 from fibroblasts. The generation of MMP2 is mediated by activation of phospholipase A(2) and 5-lipoxygenase. These results suggest that the production of soluble EMMPRIN, phospholipase A(2) and 5-lipoxygenase activities are sites for potential therapeutic intervention.

TAPAS-1, a novel microdomain within the unique N-terminal region of the PDE4A1 cAMP-specific phosphodiesterase that allows rapid, Ca2+-triggered membrane association with selectivity for interaction with phosphatidic acid.

Here we identify an 11-residue helical module in the unique N-terminal region of the cyclic AMP-specific phosphodiesterase PDE4A1 that determines association with phospholipid bilayers and shows a profound selectivity for interaction with phosphatidic acid (PA). This module contains a core bilayer insertion unit that is formed by two tryptophan residues, Trp(19) and Trp(20), whose orientation is optimized for bilayer insertion by the Leu(16):Val(17) pairing. Ca(2+), at submicromolar levels, interacts with Asp(21) in this module and serves to gate bilayer insertion, which is completed within 10 ms. Selectivity for interaction with PA is suggested to be achieved primarily through the formation of a charge network of the form (Asp(21-):Ca(2+):PA(2-):Lys(24+)) with overall neutrality at the bilayer surface. This novel phospholipid-binding domain, which we call TAPAS-1 (tryptophan anchoring phosphatidic acid selective-binding domain 1), is here identified as being responsible for membrane association of the PDE4A1 cAMP-specific phosphodiesterase. TAPAS-1 may not only serve as a paradigm for other PA-binding domains but also aid in detecting related phospholipid-binding domains and in generating simple chimeras for conferring membrane association and intracellular targeting on defined proteins.

Generation of diacylglycerol molecular species through the cell cycle: a role for 1-stearoyl, 2-arachidonyl glycerol in the activation of nuclear protein kinase C-betaII at G2/M.

Protein kinase C (PKC) is a family of 11 isoenzymes that are differentially involved in the regulation of cell proliferation. PKC-betaII, a mitotic lamin kinase, has been shown previously to translocate to the nucleus at G(2)/M and this was coupled to the generation of nuclear diacylglycerol. However, it is not clear how isoenzyme selective translocation and nuclear targeting is achieved during cell cycle. To investigate further the role of nuclear diacylglycerol we measured PKC isoenzyme translocation and analysed diacylglycerol species at different stages of the cell cycle in U937 cells synchronized by centrifugal elutriation. Translocation of PKC-betaII to the membrane fraction, an indicator of activation, occurred at S and G(2)/M, although PKC-betaII was targeted to the nucleus only at G(2)/M. Levels of nuclear diacylglycerol, specifically tetraunsaturated species, increased during G(2)/M. By contrast, there were no obvious changes in nuclear phosphatidic acid species or mass. 1-stearoyl, 2-arachidonyl glycerol (SAG), the major polyunsaturated nuclear diacylglycerol, was able to activate classical PKC isoenzymes (PKC-alpha and beta), but was less effective for activation of novel isoenzymes (PKC-delta), in an in vitro PKC assay. We propose that PKC-betaII nuclear translocation during G(2)/M phase transition is mediated in part by generation of SAG at the nucleus.