A Lipidomics Approach to Measure Phosphatidic Acid Species in Subcellular Membrane Fractions Obtained from Cultured Cells.

Over the last decade, lipids have emerged as possessing an ever-increasing number of key functions, especially in membrane trafficking. For instance, phosphatidic acid (PA) has been proposed to play a critical role in different steps along the secretory pathway or during phagocytosis. To further investigate in detail the precise nature of PA activities, we need to identify the organelles in which PA is synthesized and the PA subspecies involved in these biological functions. Indeed, PA, like all phospholipids, has a large variety based on its fatty acid composition. The recent development of PA sensors has helped us to follow intracellular PA dynamics but has failed to provide information on individual PA species. Here, we describe a method for the subcellular fractionation of RAW264.7 macrophages that allows us to obtain membrane fractions enriched in specific organelles based on their density. Lipids from these membrane fractions are precipitated and subsequently processed by advanced mass spectrometry-based lipidomics analysis to measure the levels of different PA species based on their fatty acyl chain composition. This approach revealed the presence of up to 50 different species of PA in cellular membranes, opening up the possibility that a single class of phospholipid could play multiple functions in any given organelle. This protocol can be adapted or modified and used for the evaluation of other intracellular membrane compartments or cell types of interest.

Mono- and Poly-unsaturated Phosphatidic Acid Regulate Distinct Steps of Regulated Exocytosis in Neuroendocrine Cells.

Specific forms of fatty acids are well known to have beneficial health effects, but their precise mechanism of action remains elusive. Phosphatidic acid (PA) produced by phospholipase D1 (PLD1) regulates the sequential stages underlying secretory granule exocytosis in neuroendocrine chromaffin cells, as revealed by pharmacological approaches and genetic mouse models. Lipidomic analysis shows that secretory granule and plasma membranes display distinct and specific composition in PA. Secretagogue-evoked stimulation triggers the selective production of several PA species at the plasma membrane near the sites of active exocytosis. Rescue experiments in cells depleted of PLD1 activity reveal that mono-unsaturated PA restores the number of exocytotic events, possibly by contributing to granule docking, whereas poly-unsaturated PA regulates fusion pore stability and expansion. Altogether, this work provides insight into the roles that subspecies of the same phospholipid may play based on their fatty acyl chain composition.

Regulation of Phospholipase D by Arf6 during FcγR-Mediated Phagocytosis.

Phagocytosis is an essential element of the immune response, assuring the elimination of pathogens, cellular debris, and apoptotic and tumoral cells. Activation of phagocytosis by the FcγR stimulates phospholipase D (PLD) activity and triggers the production of phosphatidic acid (PA) at the plasma membrane of macrophages, but the regulatory mechanisms involved are still not clearly understood. In this study, we examined the role of the small GTPase Arf6 in the activation of the PLD isoforms during FcγR-mediated phagocytosis. In RAW 264.7 macrophage cells, expressed Arf6-GFP partially colocalized with PLD1-hemagglutinin on intracellular membrane-bound vesicles and with PLD2-hemagglutinin at the plasma membrane. Both PLD isoforms were found to interact with Arf6 during FcγR-mediated phagocytosis as seen by immunoprecipitation experiments. In macrophages stimulated for phagocytosis, Arf6 was observed to be associated with nascent phagosomes. RNA interference knockdown of Arf6 reduced the amount of active Arf6 associated with phagosomes, revealed by the MT2-GFP probe that specifically binds to Arf6-GTP. Arf6 silencing concomitantly decreased PLD activity as well as the levels of PA found on phagosomes and phagocytic sites as shown with the PA probe Spo20p-GFP. Altogether, our results indicate that Arf6 is involved in the regulation of PLD activity and PA synthesis required for efficient phagocytosis.

Protein⁻Phospholipid Interaction Motifs: A Focus on Phosphatidic Acid.

Cellular membranes are composed of thousands of different lipids usually maintained within a narrow range of concentrations. In addition to their well-known structural and metabolic roles, signaling functions for many lipids have also emerged over the last two decades. The latter largely depend on the ability of particular classes of lipids to interact specifically with a great variety of proteins and to regulate their localization and activity. Among these lipids, phosphatidic acid (PA) plays a unique role in a large repertoire of cellular activities, most likely in relation to its unique biophysical properties. However, until recently, only incomplete information was available to model the interaction between PA and its protein partners. The development of new liposome-based assays as well as molecular dynamic simulation are now providing novel information. We will review the different factors that have shown to modulate the capacity of PA to interact with specific domains in target proteins.

Comparative Characterization of Phosphatidic Acid Sensors and Their Localization during Frustrated Phagocytosis.

Phosphatidic acid (PA) is the simplest phospholipid naturally existing in living organisms, but it constitutes only a minor fraction of total cell lipids. PA has attracted considerable attention because it is a phospholipid precursor, a lipid second messenger, and a modulator of membrane shape, and it has thus been proposed to play key cellular functions. The dynamics of PA in cells and in subcellular compartments, however, remains an open question. The recent generation of fluorescent probes for PA, by fusing GFP to PA-binding domains, has provided direct evidence for PA dynamics in different intracellular compartments. Here, three PA sensors were characterized in vitro, and their preferences for different PA species in particular lipidic environments were compared. In addition, the localization of PA in macrophages during frustrated phagocytosis was examined using these PA sensors and was combined with a lipidomic analysis of PA in intracellular compartments. The results indicate that the PA sensors display some preferences for specific PA species, depending on the lipid environment, and the localization study in macrophages revealed the complexity of intracellular PA dynamics.

Phosphatidic acid in neuronal development: a node for membrane and cytoskeleton rearrangements.

Phosphatidic acid (PA) is the simplest phospholipid naturally existing in all-living organisms. It constitutes only a minor fraction of the total cell lipids but has attracted considerable attention being both a lipid second messenger and a modulator of membrane shape. The pleiotropic functions of PA are the direct consequence of its very simple chemical structure consisting of only two acyl chains linked by ester bonds to two adjacent hydroxyl groups of glycerol, whose remaining hydroxyl group is esterified with a phosphomonoester group. Hence the small phosphate head group of PA gives it the shape of a cone providing flexibility and negative curvatures in the context of a lipid bilayer. In addition, the negatively charged phosphomonoester headgroup of PA is unique because it can potentially carry one or two negative charges playing a role in the recruitment of positively charged molecules to biomembranes. In consequence, PA has been proposed to play various key cellular functions. In the brain, a fine balance between cell growth, migration and differentiation, and cell death is required to sculpt the nervous system during development. In this review, we will summarize the various functions that have been proposed for PA in neuronal development.

Lipids in Regulated Exocytosis: What are They Doing?

The regulated secretory pathway in neuroendocrine cells ends with the release of hormones and neurotransmitters following a rise in cytosolic calcium. This process known as regulated exocytosis involves the assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, the synaptic vesicle VAMP (synaptobrevin), and the plasma membrane proteins syntaxin and SNAP-25. Although there is much evidence suggesting that SNARE proteins play a key role in the fusion machinery, other cellular elements regulating the kinetics, the extent of fusion, and the preparation of vesicle for release have received less attention. Among those factors, lipids have also been proposed to play important functions both at the level of secretory vesicle recruitment and late membrane fusion steps. Here, we will review the latest evidence supporting the concept of the fusogenic activity of lipids, and also discuss how this may be achieved. These possibilities include the recruitment and sequestration of the components of the exocytotic machinery, regulation of protein function, and direct effects on membrane topology.

Genetically encoded probes for phosphatidic acid.

In addition to forming bilayers to separate cellular compartments, lipids participate in vesicular trafficking and signal transduction. Among others, phosphatidic acid (PA) is emerging as an important signaling molecule. The spatiotemporal distribution of cellular PA appears to be tightly regulated by localized synthesis and a rapid metabolism. Although PA has been long proposed as a pleiotropic bioactive lipid, when and where PA is produced in the living cells have only recently been explored using biosensors that specifically bind to PA. The probes that we have generated are composed of the PA-binding domains of either Spo20p or Raf1 directly fused to GFP. In this chapter, we will describe the expression and purification of GST-fusion proteins of these probes, and the use of phospholipid strips to validate the specificity of their interaction with PA. We will then illustrate the use of GFP-tagged probes to visualize the synthesis of PA in the neurosecretory PC12 cells and RAW 267.4 macrophages. Interestingly, the two probes show a differential distribution in these cell types, indicating that they may have different affinities for PA or recognize different pools of PA. In conclusion, the development of a broader choice of probes may be required to adequately follow the complex dynamics of PA in different cell types, in order to determine the cellular distribution of PA and its role in various cellular processes.

The synaptic ribbon is a site of phosphatidic acid generation in ribbon synapses.

Ribbon synapses continuously transmit graded membrane potential changes into changes of synaptic vesicle exocytosis and rely on intense synaptic membrane trafficking. The synaptic ribbon is considered central to this process. In the present study we asked whether tonically active ribbon synapses are associated with the generation of certain lipids, specifically the highly active signaling phospholipid phosphatidic acid (PA). Using PA-sensor proteins, we demonstrate that PA is enriched at mouse retinal ribbon synapses in close vicinity to the synaptic ribbon in situ. As shown by heterologous expression, RIBEYE, a main component of synaptic ribbons, is responsible for PA binding at synaptic ribbons. Furthermore, RIBEYE is directly involved in the synthesis of PA. Using various independent substrate binding and enzyme assays, we demonstrate that the B domain of RIBEYE possesses lysophosphatidic acid (LPA) acyltransferase (LPAAT) activity, which leads to the generation of PA from LPA. Since an LPAAT-deficient RIBEYE mutant does not recruit PA-binding proteins to artificial synaptic ribbons, whereas wild-type RIBEYE supports PA binding, we conclude that the LPAAT activity of the RIBEYE(B) domain is a physiologically relevant source of PA generation at the synaptic ribbon. We propose that PA generated at synaptic ribbons likely facilitates synaptic vesicle trafficking.