Genetically encoded probes for phosphatidic acid

Provided for non-commercial research and educational use only. Not for reproduction, distribution or commercial use. This chapter was originally published in the book Methods In Cell Biology. The copy attached is provided by Elsevier for the author’s benefit and for the benefit of the author’s institution, for noncommercial research, and educational use. This includes without limitation use in instruction at your institution, distribution to specific colleagues, and providing a copy to your institution’s administrator. All other uses, reproduction and distribution, including without limitation commercial reprints, selling or licensing copies or access, or posting on open internet sites, your personal or institution’s website or repository, are prohibited. For exceptions, permission may be sought for such use through Elsevier's permission site at: http://www.elsevier.com/locate/permissionusematerial From Nicolas Vitale, Genetically Encoded Probes for Phosphatidic Acid. In: Paolo and Wenk, editors: Methods In Cell Biology, Vol 108, USA: Academic Press; 2012, p. 445-459. ISBN:978-0-12-386487-1 © Copyright 2012 Elsevier Inc. Academic Press. CHAPTER 20 Genetically Encoded Probes for Phosphatidic Acid Nawal Kassas, Petra Tryoen-Tóth, Matthias Corrotte, Tamou Thahouly, Marie-France Bader, Nancy J. Grant and Nicolas Vitale Institut des Neurosciences Cellulaires et Int egratives, CNRS UPR3212, Strasbourg, France Abstract I. Phosphatidic Acid: A Rapid Overview A. Rationale II. Choice of the PA-Probes III. Specific Binding of PA to Probes A. Expression of Recombinant PA Probes B. In vitro Lipid Binding Assay IV. Imaging PA in Cells A. Choice of Cell Types B. PC12 Cell Culture Conditions and Transfection C. RAW 264.7 Macrophage Culture Conditions and Transfection D. Imaging PA in PC12 Cells E. Imaging PA in RAW 264.7 Macrophage and Phagocytosis Assay F. Image Analysis and Results in PC12 Cells G. Results in RAW 264.7 Macrophages V. Summary and Conclusion Acknowledgments References Abstract 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 METHODS IN CELL BIOLOGY, VOL 108 Copyright 2012, Elsevier Inc. All rights reserved. 445 0091-679X/10 $35.00 DOI 10.1016/B978-0-12-386487-1.00020-1 446 Nawal Kassas et al. 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. I. Phosphatidic Acid: A Rapid Overview Phosphatidic acid (PA) is the simplest diacyl-glycerophospholipid and consists of an alcohol and a glycerol moiety, which has two fatty acids and a phosphate group esterified at positions 1, 2, and 3, respectively. In general, a saturated fatty acid is bond to carbon 1, a saturated fatty acid to carbon 2, and a phosphate group to carbon 3. PA is found only in limited amounts in biological membranes, yet is crucial for cell survival. It is only the recent development of liquid-chromatography coupled to mass spectrometry techniques that have allowed a more precise idea of the relative levels of the different PA species (Oliveira et al., 2010) and provided some idea of the PA composition in different subcellular compartments (Shulga et al., 2010). The physiological importance of PA is related to its central role in glycerophospholipid synthesis, in addition to diverse functions in membrane dynamics and lipid signaling (Athenstaedt and Daum, 1999). In mammalian two different pathways are known for the de novo synthesis of PA, the glycerol 3-phosphate pathway or the dihydroxyacetone phosphate pathway (Roth, 2008). The signaling PA is produced through three major pathways: the hydrolysis of phosphatidylcholine by phospholipase D (PLD), the acylation of lysophosphatidic acid by lysoPA-acyltransferase (LPAAT), and the phosphorylation of diacylglycerol (DAG) by DAG kinase (Roth, 2008). PA is degraded by conversion into DAG by lipid phosphate phosphohydrolases (LPPs) or into lyso-PA by phospholipase A (PLA) (Roth, 2008). These potent enzymes maintain at extremely low levels PA concentrations in the cell. PA, like other lipid signals can serve as membrane docking sites, which serve to recruit specific proteins. PA can activate proteins either by directly stimulating their enzymatic activity or by indirectly targeting them to the site where they are required to function. Alternatively, PA can inactivate proteins by depleting them from the place where they are active. In this way, the endoplasmic reticulum (ER) sequesters the yeast transcriptional repressor Opi1p (Loewen et al., 2004). When PA levels decrease, Opi1p is no longer retained in the ER and translocates to the nucleus where it represses target gene expression (Loewen et al., 2004). PA can also directly inhibit 20. Genetically Encoded Probes for Phosphatidic Acid 447 enzymes such as the protein phosphatase-1 (Jones and Hannun, 2002). In other cases, PA appears to act in concert with other lipid second messengers. For example, when protein kinase C is stimulated, PA binds to its C2-domain allowing translocation of the kinase to the plasma membrane. Subsequently, DAG binding to the C1-domain then has a synergistic effect on protein kinase C activity (Jose LopezAndreo et al., 2003). Similarly, the pleckstrin homology (PH) domain of Son of sevenless was shown to bind both phosphatidylinositol 4,5-bisphosphate (PIP2) and PA (Zhao et al., 2007). Another interesting possibility, not directly involving protein targeting, is that the shape and negative charge of PA itself promote a membrane curvature and thereby, affect membrane topology and fusion properties (Bader and Vitale, 2009; Chernomordik and Kozlov, 2008). A. Rationale The PA synthesized by the mammalian PLD isoforms has been proposed to play important roles in many important cellular functions, in particular membrane trafficking. By measuring the production of labeled phosphatidylethanol by PLD-mediated transphosphatidylation in the presence of ethanol, an increase in PLD activity has been reported in numerous processes, including neurosecretion (Caumont et al., 1998; Humeau et al., 2001) and phagocytosis (Iyer et al., 2004). More recently numerous experiments based on the use of selective knockdown of the PLD isoforms, using a small RNA interference approach have confirmed the implication of PLD in many vesicular trafficking events (Bader and Vitale, 2009; Corrotte et al., 2006; Du et al., 2004; Huang et al., 2005). Nevertheless, the precise localization and dynamics of PA in these processes remains a major challenge. Here, we describe the characterization of two genetically encoded probes for PA and their use in studying exocytosis in neurosecretory cells and phagocytosis in macrophages. II. Choice of the PA-Probes The specificity of PA-protein interactions appears to largely rely on the unique ionization properties of the phosphomonoester headgroup of PA (Kooijman and Burger, 2009). High affinity PA-protein interactions have been described for over 20 proteins in mammalian, plant and yeast cells (for review, see Stace and Ktistakis, 2006). These include the protein kinase Raf-1 and the yeast SNARE protein Spo20p that we have used as probes to visualize PA in cells. Interestingly, when one compares the PA-binding region of the subset of proteins in which the binding region has been evaluated, no consensus sequence (sequence homology) is apparent. This is in sharp contrast to other lipid binding modules such as the PH, PX, C1, and C2 domains. One general, but not surprising, feature that arises is the presence of basic amino acid residues. In most cases, hydrophobic residues also appear to be important for PA binding. To visualize PA within cells, we have used two probes based on the PAbinding domains of the yeast SNARE protein Spo20p and the protein kinase Raf-1. 448 Nawal Kassas et al. In Saccharomyces cerevisiae, the developmentally regulated Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein Spo20p mediates the fusion of vesicles with the prospore membrane that is required for spore formation. Spo20p is positively regulated through an amphipathic helix, which confers plasma membrane or prospore membrane localization (Nakanishi et al., 2004). Genetic manipulations of phospholipid pools indicate that the likely in vivo ligand of this domain is PA (Nakanishi et al., 2004). Accordingly in vitro flotation assay have shown that Spo20p binds preferentially to PA when compared to negatively charged phosphoinositol 4,5-bisphosphate (Nakanishi et al., 2004). The minimal PA binding domain consists of a stretch of 40 residues that are predicted to form an amphipathic a-helix. Hydrophobic residues on one side of the helix, as well as basic residues appear to be critical for PA binding (Nakanishi et al., 2004). The protooncogene Raf-1 kinase plays a crucial role in several normal and pathological cellular processes. A common aspect of Raf-1 activation is its translocation to the plasma membrane. In vitro analysis of Raf-1-lipid interaction reveals two distinct phospholipid binding sites within Raf-1 kinase, of which a PA interacting domain is located between residues 390 and 423 of human Raf-1 (Ghosh et al., 2003; Rizzo et al., 2000). The tetrapeptide RKTR (residues 398401 of human Raf-1) was shown to be critical in Raf-1 for interaction with PA by a combination of alanine-scanning, deletion mutagenesis and liposome assay (Ghosh et al., 2003). III. Specific Binding of PA to Probes A. Expression of Recombinant PA Probes The Spo20p PA-binding domain (PABD) is amplified by PCR from the pEGFPC1-PABD-Spo20p construct using the forward primer 5’-CGGGATCCCTCG AGCGTCTAGAATGG-3’ and reverse primer 5’-GCGAATTCTTAACTAGTCT TAGTGGCGTC3’, as previously described (Zeniou-Meyer et al., 2007). The Raf1 PA binding domain is amplified by PCR from the pEGFP-C1-PABD-Raf1 construct using the forward primer 5’-CGGGATCCTCCAGGCCTTCAGGAATGAGG-3’ and reverse primer 5’-GCGAATTCGCCCTCGCACCACTGGGTC-3’. The PAbinding fragment is inserted in frame into pGEX4T1 using a procedure described previously (Vitale et al., 2000a). Constructs have been verified by automated DNA sequencing. Large-scale production of chimeric GST-PABD proteins has been previously described (Vitale et al., 1996, 2001). Briefly, expression is induced at 37  C for GST-Spo20p-PABD and at 18  C for GST-Raf1-PABD. Fusion proteins are purified on glutathione-Sepharose (Vitale et al., 1998) and purity is estimated to be >98% by Coomassie blue staining of SDS-PAGE gels. The quantity of purified protein can be determined from this dye-binding assay using bovine serum albumin as a standard (Vitale et al., 2000b). Protein aliquots are stored at 80  C. 20. Genetically Encoded Probes for Phosphatidic Acid 449 B. In vitro Lipid Binding Assay The PA-binding region of Spo20p was originally characterized in yeast by successive deletions from Spo20p (Nakanishi et al., 2004), whereas the PA binding fragment of Raf1 was mapped in vitro (Ghosh et al., 2003). We have previously shown that GFP-PABD-Spo20p in lysates of transfected PC12 cells specifically binds to PA on phospholipid strips (Zeniou-Meyer et al., 2007). The GFP-Raf1PABD also appears to be specific for PA because mutations of basic residues in the binding domain (Rizzo et al., 2000) eliminate the recruitment of the probe to phagosomes in transfected RAW 264.7 macrophages (Corrotte et al., 2006). To support the specificity of these probes for PA, lipid overlay assays are performed with recombinant GST-PABD proteins. Briefly, strips containing various phospholipids, as indicated (Molecular Probes), are initially saturated with PBS 3% BSA + 0.1% Tween 20 for 4 h at 4  C. Then GST-PABD-proteins (1 mg/mL in PBS 3% BSA + 0.1% Tween20) or as a control, GST alone are incubated for 2 h at 4  C with the phospholipid strips. Subsequently, the strips are processed for Western blot analysis with an anti-GST antibody (Molecular Probes). Images are acquired with a Chemi-smart 5000 (Vilber Lourmat). Our data reveal that GST-PABD-Spo20p and GST-PABD-Raf1 bind to PA whereas they weakly bind to other acidic lipids (Fig. 1). On the other hand GST does not bind any of the lipids tested (Fig. 1). [(Fig._1)TD$IG] Fig. 1 GST-PABD-Spo20p and GST-PABD-Raf1 specifically binds to PA in vitro. (A) Layout of phospholipid strips membranes. 1: Lysophosphatidic acid, 2: Lysophosphatidylcholine, 3: Phosphatidylinositol (PtdIns), 4: PtdIns(3)P, 5: PtdIns(4)P, 6: PtdIns(5)P, 7: Phosphatidylethanolamine, 8: Phosphatidylcholine, 9: Sphingosine 1-phosphate, 10: PtdIns(3,4)P2, 11: PtdIns(3,5)P2, 12: PtdIns(4,5)P2, 13: PtdIns(3,4,5)P3, 14: Phosphatidic acid, 15: Phosphatidylserine, 16: Blank. (B) Recombinant GST-PABD-Spo20p, GSTPABD-raf1 or GST proteins were incubated with phosphlipid strips for 2 h at 4  C. Strips were subsequently processed for Western blot analysis with an anti-GST antibody. Similar results were obtained with three distinct protein preparations. 450 Nawal Kassas et al. IV. Imaging PA in Cells A. Choice of Cell Types The catecholamine-secreting PC12 cell line derived from a rat pheochromoytoma originating from chromaffin cells of the adrenal medulla has long been considered a suitable model system for studying neurosecretion and neuronal differentiation. PC12 cells contain a large number of secretory granules for storage of small molecules, processing enzymes, neuropeptides, and peptide hormones. Secretory granule exocytosis in PC12 cells is tightly regulated by calcium and occurs in response to a secretagogue (Bader et al., 2002). Because the PC12 cells are still today easier to transfect than the primary chromaffin cells, they represent a valuable model to determine the dynamics of PA synthesis during neurosecretion using genetically encoded PA probes. The murine macrophage cell line RAW 264.7 has been used extensively for characterizing the molecular machinery governing phagocytosis. Particle activation of surface receptors triggers signaling cascades that lead to the engulfment of the particle within the phagosome and its subsequent degradation. Accumulating evidence indicates that the PLD isoforms actively participate in this process (Corrotte et al., 2006, 2010; Iyer et al., 2004), and that PA production is necessary for efficient phagocytosis (Corrotte et al., 2006). One major advantage of the RAW 264.7 cell line over primary cultures of professional phagocytes such as macrophages or neutrophiles is that the cell line can be transfected at reasonable rates either by electroporation or lipofection. The use of the PA-biosensors in RAW 264.7 cells for studying PA production during phagocytosis will be described below. B. PC12 Cell Culture Conditions and Transfection 1. Stock cells are grown in 10 cm cell culture dishes in 10 mL Dulbecco’s modified Eagle’s medium (DMEM) containing fetal bovine serum (FBS) 5%, horse serum 10%, and antibiotics (complete DMEM medium), in a humidified incubator, 5% CO2, 37  C. The dishes should be pre-coated with poly-L-lysine. Dishes are precoated with 5 mL poly-L-lysine (100 mg/mL H2O) solution and incubated 60 min at 37  C. To eliminate excess of poly-L-lysine dishes are washed with sterile water before the addition of the cells. 2. Split cells at a subcultivation ratio of 1:3 up to 1:7, as needed for experiments. Doubling time is about 30–35 h. Cells should be split when the confluency is between 70 and 80%. Wash cells with 2 mL of pre-warmed trypsin-EDTA solution (dissociation solution) then incubate them with 2 mL of cell dissociation solution at 37  C for few minutes, before collecting the cells in complete DMEM medium. 3. After centrifugation at 120 g for 5 min, cells are suspended and counted in cell culture medium without antibiotics. For the imaging experiments, cells are plated 20. Genetically Encoded Probes for Phosphatidic Acid 451 in 500 mL of this medium on coverslips precoated with poly-L-lysine in 24-well plates at a density of about 1,00,000 cells per well. 4. The day after cell plating, the GFP-PABD probes are transfected into PC12 cells using Lipofectamine 2000 following the manufacturer’s optimized protocol (Invitrogen). Briefly, 0.8 mg of DNA is diluted in 50 mL of OPTI-MEM / well, and 1.6 mL of Lipofectamine is diluted in 50 mL of OPTI-MEM/well. The diluted DNA is then mixed with the Lipofectamine solution and incubated at room temperature for 20 min. Next the DNA-Lipofectamine transfection solution is added to wells containing 500 mL of culture medium as described above. Cells are incubated with the transfection solution for 4–5 h, then medium is replaced by complete DMEM medium and cells are cultured for 24 h. C. RAW 264.7 Macrophage Culture Conditions and Transfection 1. RAW 264.7 macrophages (American Tissue Culture Collection (ATCC)) are grown in 10 cm Petri dishes containing 12 mL RPMI plus heat-inactivated FBS 10%, HEPES 10 mM, pH7.0, Na pyruvate 1 mM, b-mercaptoethanol 0.05 mM, and supplemented with streptomycin 10-6 M, and penicillin 5x104 U/L. The cells are maintained at 37  C in a 5% CO2 humidified atmosphere, and split every 2–3 days. To dilute cells, they are first detached mechanically, then diluted 1:6, and seeded at 4 x 104 cells/cm2. 2. The day before transfection, RAW264.7 cells are plated in 10 cm dishes at a dilution of 1:3 to obtain a confluency between 60 and 80% the following day. Cells can be transfected either by electroporation, or by lipofection as described above for the PC12 cells. For electroporation of the PABD plasmids, the Electrobuffer Kit and protocol (Cell Projects) is used. Cells (8 x 106) are suspended in wash buffer and collected by centrifugation at 90 g for 5 min. Cells are then suspended in 240 mL electroporation buffer B containing ATP (2.5 mM) and glutathione (4 mM) and 10 mg of plasmid. The cell suspension is electoporated in a 4 mm cuvette at 230 Vand 950 mF in a Biorad Gene Pulser (Xcell). The average pulse time is 40 ms. Cells are immediately diluted with warm culture medium without antibiotics, and then seeded at 4 x 104 cells on the glass bottom of 35 mm Microwell dishes (MatTek Corporation). Transfection efficiency by either electroporation or lipofection is about 30%. D. Imaging PA in PC12 Cells 1. The day after PABD probe transfection cultures are washed twice for 5 min with Locke buffer (HEPES 15 mM; pH 7,5; NaCl 140 mM; KCl 4,7 mM; CaCl2 2,5 mM; KH2PO4 1,2 mM; MgSO4 1,2 mM; EDTA 0,01 mM). Then cells are incubated in either Locke buffer (resting condition) or in the presence of Locke solution containing K+ 59 mM (stimulation solution) for 5 min at 37  C. 452 Nawal Kassas et al. 2. Immediately after these treatments, cells are fixed with ice-cold 4% paraformaldehyde (PFA) diluted in phosphate buffer for 10 min at room temperature. Cells are then permeabilized by incubating in 0.1% Triton X-100/4% PFA for 10 min and washed thoroughly with PBS to eliminate all fixation residues. To saturate non-specific sites cells are incubated in PBS containing 3% BSA/10% goat serum for 1 h at room temperature. 3. To delineate the plasma membrane in fixed cells, we label the SNARE protein SNAP25 with a monoclonal anti-SNAP25 antibody (Covance), diluted 1:200 in 3% BSA-PBS. A 50-mL drop of diluted antibody is placed on parafilm and coverslips with transfected PC12 cells are placed upside down on the droplets and incubated for 1 h at room temperature. Following incubation, coverslips are returned to wells and washed three times for 5 min in PBS. Incubation with secondary antibody, Alexa 555 conjugated goat anti-mouse IgG (Invitrogen) diluted to 1:1500 is carried out as described for the primary antibody. After three washes in PBS, cells are rinsed with ultrapure water, and coverslips are mounted in Mowiol-Evanol. E. Imaging PA in RAW 264.7 Macrophage and Phagocytosis Assay 1. For phagocytosis assays, the medium of RAW264.7 cells, cultured 18–24 h on glass bottom Petri dishes is changed to RPMI-BSA (medium without phenol red (Sigma R7509) supplemented with BSA 1% (Fraction V, Sigma A8806), HEPES 10 mM, pH7.0, Na pyruvate 1 mM, and b-mercaptoethanol 0.05 mM). 2. Phagocytosis is stimulated using 3-mm latex microspheres (PS05N, Bangs Laboratories) opsonized with human IgG (Invitrogen). To opsonize particles, 25 mL of microspheres (stock 10% w/v) are washed in PBS, suspended in 250 mL PBS containing 0.2 mg/mL IgG and incubated with rotation 2 h at room temperature. Particles are then washed in PBS and suspended in 250 mL RPMI-BSA to obtain a 1% solution of particles. 3. Before initiating phagocytosis, transfected cells can be identified by epifluorescence using the binoculars. Fresh RPMI-BSA medium (1 mL) pre-heated to 37  C is then placed in the Petri dishes. To initiate phagocytosis, 5 mL of the IgG-coated particle suspension is added to the dish directly over the cells. When the particles reach the cells (about 5 min), switch to the confocal mode and images can be acquired over the next 20 min. To avoid photobleaching and phototoxicity, illumination of the cells should be minimized. 1. Fluorescent Measurements/Image Acquisition To examine the cellular distribution of the PA probes, a scanning confocal microscope (Zeiss LSM 510) is used. Samples are examined with an oil immersion 63x objective (Plan Apochromat n.a. = 1.4) using an argon laser at an excitation wavelength of 488 nm for GFP and a helium/neon laser at 543 nm to excite Alexa 555 20. Genetically Encoded Probes for Phosphatidic Acid 453 fluorophore Digital images (of median confocal sections of cells) are acquired with pinhole of 1.0. F. Image Analysis and Results in PC12 Cells Multiple confocal image acquisitions (8 bits) are carried out for each experimental condition and digital images are analyzed. To determine the recruitment of the probes at the plasma membrane, the co-localization of GFP-PABD-Spo20p or GFP-PABD-Raf1 (green) with SNAP25 (red) can be visualized by generating co-localization masks and the proportion of co-localization can be quantified using the Zeiss CLSM 3.2 instrument software. Briefly, for each cell, a scatter plot of double-labelled pixels is generated and threshold levels are adjusted for each fluorophore to eliminate background-staining levels. The weighted co-localization percentages, which we use, take into account the number of pixels, the average fluorescence intensity and their frequency. For each condition, 20–30 cells were analyzed and data are expressed as the mean  SD. Similar observations were obtained from at least three different cell culture. The two GFP-tagged PA probes do not show the same cellular distribution in PC12 cells; however, their cellular localization is not altered by cell fixation (data not shown). In resting cells, the GFP-PABD-Spo20p probe is concentrated in the cell nucleus (Fig. 2a) and only about 4  1% of the PA probe co-localized with SNAP25. Upon stimulation of exocytosis, a fraction of the Spo20p fluorescent probe is efficiently recruited to the plasma membrane (Fig. 2a). The recruitment kinetics of the PA sensor reaches a maximum level after 5 min of stimulation, showing 30  4% co-localization with the SNARE protein SNAP25 (B egl e et al., 2009; ZeniouMeyer et al., 2007). The GFP-PABD-Raf1 probe on the other hand, shows a clear cytosolic distribution with faint nuclear localization. In stimulated cells only a modest co-localization of GFP-PABD-Raf1 with SNAP25 is observed (Fig. 2b). All together these results suggest that the PABD of Spo20p appears to be a more sensitive reporter for PA than that of Raf1 in the neurosecreting PC12 cells. G. Results in RAW 264.7 Macrophages As we observed in fixed PC12 cells, the PA biosensors PABD-Raf1 and PABDSpo20 do not show the same distribution in live macrophages. Prior to a phagocytic stimulation, the PABD-Raf1 probe is principally localized in the cytoplasm (Fig. 3a), whereas the PABD-Spo20p is mainly in the nucleus and not in the cytoplasm (Fig. 3d). However, the PABD-Spo20p probe is also observed at the plasma membrane in about 20% of the macrophages under resting conditions (data not shown). As PLD activity has also been implicated in cell adhesion of RAW 264.7 macrophages (Iyer et al., 2006), it is possible that these cells are migrating cells. Following stimulation of phagocytosis, the PABD-Raf1 probe is sometimes observed in the phagocytic cup as the pseudopods extend around the particle (Fig. 3b), but is 454 Nawal Kassas et al. [(Fig._2)TD$IG] Fig. 2 Distribution of GFP-PABD-Raf1 and GFP-PABD-Spo20p in PC12 cells. Confocal immunofluorescence images showing the subcellular distribution of GFP-PABD-Raf1 and GFP-PABD-Spo20p in PC12 cells. Cells were maintained in Locke’s solution under resting conditions, or stimulated for 10 min with a depolarizing concentration of potassium. The plasma membrane-bound protein SNAP25 was visualized using anti-SNAP25 antibody. In the mask images, the black staining indicates the areas of co-localization of the PABD probes with SNAP25 as illustrated by displaying the double-labeled pixels. Bars, 5 mm. (For color version of this figure, the reader is referred to the web version of this book.) 20. Genetically Encoded Probes for Phosphatidic Acid 455 [(Fig._3)TD$IG] Fig. 3 Distribution of GFP-PABD-Raf1 and GFP-PABD-Spo20p in RAW 267.4 macrophages. Confocal images of live macrophages 12–18 h after transfection with either GFP-PABD-Raf1 (a–c) or GFP-PABD-Spo20p (d–f). Macrophages in resting conditions (a, d) and following stimulation with IgGcoated 3 mm microspheres (b, c, e, f). Stars indicate particles at different stages of phagocytosis. Bars, 5 mm. (For color version of this figure, the reader is referred to the web version of this book.) rarely found associated with internalized phagosomes (Fig. 3c), in agreement with our earlier observations of PABD-Raf1 localization in fixed RAW264.7 cells (Corrotte et al., 2006). During phagocytosis, the change in distribution of PABDSpo20p is more evident. In phagocytosing cells, there is a net increase in the probe at the plasma membrane and a corresponding decrease in the nucleus (Fig. 3e, f). As in the case of the PABD-Raf1 probe, the PABD-Spo20p probe is present in the phagosomal cup and on pseudopods during phagosome formation (Fig. 3e, f), but is less evident on internalized phagosomes (Fig. 3f). V. Summary and Conclusion Here, we have described the use of two probes for PA that can serve as tools to visualize PA synthesis at exocytotic sites in neurosecreting cells and at the phagocytic cup in macrophages in response to stimulation. In PC12 cells, the secretagogue-induced PA synthesis requires PLD1 activation (Zeniou-Meyer et al., 2007), whereas in macrophages both PLD1 and PLD2 isoforms appear to be involved in PA synthesis at the phagocytic cup (Corrotte et al., 2006). These tools have also proven valuable to validate the implication of several regulators of PLD1 in 456 Nawal Kassas et al. exocytosis. For instance, depletion of endogenous ARF6 (B egl e et al., 2009), Rac1 (Momboisse et al., 2009), or Rsk2 (Zeniou-Meyer et al., 2009) dramatically reduced the recruitment of GFP-PABD-Spo20p in stimulated PC12 cells. The specificity of these observations has also been confirmed in macrophages and neuroendocrine cells (Corrotte et al., 2006; Zeniou-Meyer et al., 2007) by using probes with point mutants that have been shown in vitro to no longer bind PA (Ghosh et al., 2003; Nakanishi et al., 2004). As mentioned in the introduction several biosynthetic or signaling pathways converge to modulate the level of PA in living cells. In order to define the source of PA visualized by these probes, a number of specific manipulations need to be performed. For instance overexpression or knockdown of PA generating enzymes (PLD, DGK, LPAAT) should increase or prevent the recruitment of the probes if these pathways are implicated in the synthesis of the PA detected by the probes. The implication of these different PA-synthesizing enzymes can also be evaluated by pharmacological inhibitors such as the recently described isoform specific PLD inhibitors (Vitale, 2010), DGK inhibitors such as R59022, and LPAAT inhibitor such as CT-32228. Alternatively, the specificity of these probes can be tested in situ by examining their recruitment in cells where PA conversion into DAG is enhanced by overexpressing LPP or PLA or reduced by using pharmacological inhibitors of these enzymes. One problem that can arise when using these probes to study PA dynamics is the observation that they can interfere with cellular functions by sequestering PA, as in the case of phagocytosis (Corrotte et al., 2006). As well, when the PABD probes are expressed at too high levels or for prolonged periods (>18 h), we have observed that they can affect cell viability both for PC12 cells and macrophages. It is therefore important to minimize the expression of the probe to a level, which permits detection of PA recruitment and minimizes deleterious effects on cellular functions and cell viability. Biological membranes contain a host of anionic lipids and are therefore generally negatively charged. The negative charge of biological membranes is an important determinant of biomembrane structure and function. It is well established that the negative charge carried by anionic lipids in biomembranes forms an important site of attraction for positively charged (carrying basic amino acids) proteins. Yet, proteins bind to individual anionic lipids with a high degree of specificity, based mostly on specific structural motives. The binding of PA to the probes described here appears to rely mainly on basic residues probably having a specific orientation towards the negative charges of PA, although the exact recognition sites remain to be determined. The transmembrane topology of phospholipids in cellular compartments remains ill defined except for PS. The presence of PS in the lumenal monolayer of the ER and Golgi complex and its cytosolic exposure at the trans-Golgi network has recently been elegantly demonstrated ultrastructurally on cryosections overlaid with PS probes (Fairn et al., 2011). This transmembrane flipping of PS is thought to contribute to the exit of cargo from the Golgi complex. Whether the PA is also present on 20. Genetically Encoded Probes for Phosphatidic Acid 457 the lumenal leaflet of subcellular compartments and the possibility that PA flipping may be involved in regulating biological functions remains to be determined. As the GFP-based PA probes described here are likely to miss a pool of PA present in the luminal/ectoplasmic leaflets of cellular membranes, an ultrastructural study using these PA-probes on cryosections is a line of investigation that may provide some answers to these questions. The difference in subcellular localization of the two PA probes merits to be mentioned. The PABD of Raf1 is mainly cytosolic, whereas the PABD of Spo20p concentrates in the nucleus. The nuclear localization of the Spo20p probe probably results from the presence of a small nuclear sequence at the amino-terminal part of the PA binding region of Spo20p (Nakanishi et al., 2004). As a probe for PA synthesis, this is however of interest because it facilitates the visualization of the recruitment of the PABD-Spo20p probe to cytoplasmic organelles and the plasma membrane. Conversely, the cytoplasmic distribution of the PABD-Raf1 probe tends to obscure observations of PA generation on organelles and the plasma membrane and this seems to be accentuated in live cells. Indeed, the recruitment of the probes to the phagosome was more easily observed in fixed macrophages (Corrotte et al., 2006) than in live macrophages (Fig. 3). For the moment, it appears that the Spo20p probe is probably be more useful as a PA biosensor than the PABD-Raf1 probe for studying PA dynamics in live cells. Another possibility to explain the differential recruitment of the two probes is that they do not recognize the same pools of PA. Variations in the length or saturation of the fatty acid chains of PA probably affect the orientation of the negatively charged polar head group of PA in the lipid bilayer. Since this head group interacts with proteins, changes in its orientation could affect the affinity of PA for binding domains of different proteins. This remains an open question that deserves to be investigated. In addition, full characterization of other potential biosensors for PA will undoubtedly prove crucial for dissecting the numerous pathways involving PA. Acknowledgments We wish to thank all the members of our laboratory that have contributed to this work. This work was supported by the Agence Nationale de la Recherche (Grant ANR-09-BLAN-0264). N.K. was supported by a grant from the CNRS-Liban. References Athenstaedt, K., and Daum, G. (1999). Phosphatidic acid, a key intermediate in lipid metabolism. Eur. J. Biochem. 266, 1–16. Bader, M. F., and Vitale, N. (2009). Phospholipase D in calcium-regulated exocytosis: lessons from chromaffin cells. Biochim. Biophys. Acta 1791, 936–941. Bader, M. F., et al. (2002). Exocytosis: the chromaffin cell as a model system. Ann. N. Y. Acad. Sci. 971, 178–183. B egl e, A., et al. (2009). ARF6 regulates the synthesis of fusogenic lipids for calcium-regulated exocytosis in neuroendocrine cells. J. Biol. Chem. 284, 4836–4845. 458 Nawal Kassas et al. Caumont, A. S., et al. (1998). Regulated exocytosis in chromaffin cells – Translocation of ARF6 stimulates a plasma membrane-associated phospholipase D. J. Biol. Chem. 273, 1373–1379. Chernomordik, L. V., and Kozlov, M. M. (2008). Mechanics of membrane fusion. Nat. Struct. Mol. Biol. 7, 675–683. Corrotte, M., et al. (2006). Dynamics and function of phospholipase D and phosphatidic acid during phagocytosis. Traffic 7, 365–377. Corrotte, M., et al. (2010). Ral isoforms are implicated in Fc gamma R-mediated phagocytosis: activation of phospholipase D by RalA. J. Immunol. 185, 2942–2950. Du, G., et al. (2004). Phospholipase D2 localizes to the plasma membrane and regulates angiotensin II receptor endocytosis. Mol. Biol. Cell. 3, 1024–1030. Fairn., et al. (2011). High-resolution mapping reveals topologically distinct cellular pools of phosphatidylserine. J. Cell Biol. 194, 257–275. Ghosh, S., et al. (2003). Functional analysis of a phosphatidic acid binding domain in human Raf-1 kinase: mutations in the phosphatidate binding domain lead to tail and trunk abnormalities in developing zebrafish embryos. J. Biol. Chem. 278, 45690–45696. Huang, P., et al. (2005). Insulin-stimulated plasma membrane fusion of Glut4 glucose transporter-containing vesicles is regulated by phospholipase D1. Mol. Biol. Cell 6, 2614–2623. Humeau, Y., et al. (2001). A role for phospholipase D in neurotransmitter release. Proc. Natl. Acad. Sci. U. S. A. 93, 1941–1944. Iyer, S. S., et al. (2004). Phospholipases D1 and D2 coordinately regulate macrophage phagocytosis. J. Immunol. 173, 2615–2623. Iyer, S. S., et al. (2006). Phospholipases D1 regulates phagocyte adhesion. J. Immunol. 176, 3686–3696. Jones, J. A., and Hannun, Y. A. (2002). Tight binding inhibition of protein phosphatase-1 by phosphatidic acid. Specificity of inhibition by the phospholipid. J. Biol. Chem. 277, 15530–15538. Jose Lopez-Andreo., et al. (2003). The simultaneous production of phosphatidic acid and diacylglycerol is essential for the translocation of protein kinase Cepsilon to the plasma membrane in RBL-2H3 cells. Mol. Biol. Cell 14, 4885–4895. Kooijman, E. E., and Burger, K. N. (2009). Biophysics and function of phosphatidic acid: a molecular perspective. Biochim. Biophys. Acta 1791, 881–888. Loewen, C. J., et al. (2004). Phospholipid metabolism regulated by a transcription factor sensing phosphatidic acid. Science 304, 1644–1647. Momboisse, F., et al. (2009). betaPIX-activated Rac1 stimulates the activation of phospholipase D, which is associated with exocytosis in neuroendocrine cells. J. Cell Sci. 122, 798–806. Nakanishi, H., et al. (2004). Positive and negative regulation of a SNARE protein by control of intracellular localization. Mol. Biol. Cell 15, 1802–1815. Oliveira, T. G., et al. (2010). Phospholipase d2 ablation ameliorates Alzheimer’s disease-linked synaptic dysfunction and cognitive deficits. J. Neurosci. 30, 16419–16428. Rizzo, M. A., et al. (2000). The recruitment of Raf-1 to membranes is mediated by direct interaction with phosphatidic acid and is independent of association with Ras. J. Biol. Chem. 275, 23911–23918. Roth, M. G. (2008). Molecular mechanisms of PLD function in membrane traffic. Traffic 9, 1233–1239. Shulga, Y. V., et al. (2010). Molecular species of phosphatidylinositol-cycle intermediates in the endoplasmic reticulum and plasma membrane. Biochemistry 49, 312–317. Stace, C. L., and Ktistakis, N. T. (2006). Phosphatidic acid- and phosphatidylserine-binding proteins. Biochim. Biophys. Acta 1761, 913–926. Vitale, N., et al. (1996). ARD1, a 64-kDa bifunctional protein containing an 18-kDa GTP-binding ADPribosylation factor domain and a 46-kDa GTPase-activating domain. Proc. Natl. Acad. Sci. U. S. A. 93, 1941–1944. Vitale, N., et al. (1998). Molecular characterization of the GTPase-activating domains of ADP-ribosylation factor domain protein 1 (ARD1). J. Biol. Chem. 273, 2553–2560. Vitale, N., et al. (2000a). Specific functional interaction of human cytohesin-1 and ADP-ribosylation factor domain protein (ARD1). J. Biol. Chem 275, 21331–21339. 20. Genetically Encoded Probes for Phosphatidic Acid 459 Vitale, N., et al. (2000b). GIT proteins, A novel family of phosphatidylinositol 3,4,5-trisphosphatestimulated GTPase-activating proteins for ARF6. J. Biol. Chem 275, 13901–13906. Vitale, N., et al. (2001). Purification of ARD1 an ADP-ribosylation factor (ARF)-related protein with GTPase-activating domain. Methods Enzymol. 329, 324–334. Vitale, N. (2010). Therapeutic potentials of recently identified PLD inhibitors. Curr. Chem. Biol. 4, 244–249. Zeniou-Meyer, M., et al. (2007). Phospholipase D1 production of phosphatidic acid at the plasma membrane promotes exocytosis of large dense-core granules at a late stage. J. Biol. Chem. 282, 21746–21757. Zeniou-Meyer, M., et al. (2009). The Coffin-Lowry syndrome-associated protein RSK2 controls neuroendocrine secretion through the regulation of phospholipase D1 at the exocytotic sites. Ann. N. Y. Acad. Sci. 1152, 201–208. Zhaso, C., et al. (2007). Phospholipase D2-generated phosphatidic acid couples EGFR stimulation to Ras activation by Sos. Nat. Cell Biol. 6, 706–712.