Title: Phospholipase D and phosphatidic acid in the biogenesis and cargo loading of extracellular vesicles
Abstract:Extracellular vesicles released by viable cells (exosomes and microvesicles) have emerged as important organelles supporting cell-cell communication. Because of their potential therapeutic significanc...Extracellular vesicles released by viable cells (exosomes and microvesicles) have emerged as important organelles supporting cell-cell communication. Because of their potential therapeutic significance, important efforts are being made toward characterizing the contents of these vesicles and the mechanisms that govern their biogenesis. It has been recently demonstrated that the lipid modifying enzyme, phospholipase D (PLD)2, is involved in exosome production and acts downstream of the small GTPase, ARF6. This review aims to recapitulate our current knowledge of the role of PLD2 and its product, phosphatidic acid, in the biogenesis of exosomes and to propose hypotheses for further investigation of a possible central role of these molecules in the biology of these organelles. Extracellular vesicles released by viable cells (exosomes and microvesicles) have emerged as important organelles supporting cell-cell communication. Because of their potential therapeutic significance, important efforts are being made toward characterizing the contents of these vesicles and the mechanisms that govern their biogenesis. It has been recently demonstrated that the lipid modifying enzyme, phospholipase D (PLD)2, is involved in exosome production and acts downstream of the small GTPase, ARF6. This review aims to recapitulate our current knowledge of the role of PLD2 and its product, phosphatidic acid, in the biogenesis of exosomes and to propose hypotheses for further investigation of a possible central role of these molecules in the biology of these organelles. Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), the most abundant membrane phospholipid, to generate phosphatidic acid (PA) and choline. There are six different mammalian proteins designated as PLDs (Fig. 1). Most of our knowledge about PLD biology refers to the PLD1 and PLD2 isoenzymes. PLDs participate in the normal maintenance of cellular membranes (1.Frohman M.A. Sung T.C. Morris A.J. Mammalian phospholipase D structure and regulation.Biochim. Biophys. Acta. 1999; 1439: 175-186Crossref PubMed Scopus (277) Google Scholar) and in a variety of physiological cellular functions, such as cell migration and proliferation, vesicle trafficking, cytoskeleton remodeling, and morphogenesis (2.Exton J.H. Regulation of phospholipase D.FEBS Lett. 2002; 531: 58-61Crossref PubMed Scopus (234) Google Scholar, 3.Jenkins G.M. Frohman M.A. Phospholipase D: a lipid centric review.Cell. Mol. Life Sci. 2005; 62: 2305-2316Crossref PubMed Scopus (392) Google Scholar, 4.Lee C.S. Kim K.L. Jang J.H. Choi Y.S. Suh P.G. Ryu S.H. The roles of phospholipase D in EGFR signaling.Biochim. Biophys. 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Cloning and initial characterization of a human phospholipase D2 (hPLD2).J. Biol. Chem. 1998; 273: 12846-12852Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 8.Hiroyama M. Exton J.H. Localization and regulation of phospholipase D2 by ARF6.J. Cell. Biochem. 2005; 95: 149-164Crossref PubMed Scopus (32) Google Scholar). For example, the activation of ARF6 by ARF nucleotide-binding site opener (ARNO), which is an ARF6-specific guanine-nucleotide exchange factor (GEF), results in increased activation of PLD and induces epithelial cell migration (9.Santy L.C. Casanova J.E. Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D.J. Cell Biol. 2001; 154: 599-610Crossref PubMed Scopus (314) Google Scholar). Stimulation of chromaffin cells triggers the translocation of ARF6 from secretory granules to the plasma membrane and the concomitant activation of PLD at the plasma membrane (10.Caumont A.S. 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It has also been reported that ARF6 can colocalize with PLD1 and PLD2 in membrane ruffles (12.Honda A. Nogami M. Yokozeki T. Yamazaki M. Nakamura H. Watanabe H. Kawamoto K. Nakayama K. Morris A.J. Frohman M.A. et al.Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation.Cell. 1999; 99: 521-532Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar, 13.Powner D.J. Hodgkin M.N. Wakelam M.J.O. Antigen-stimulated activation of phospholipase D1b by Rac1, ARF6, and PKC alpha in RBL-2H3 cells.Mol. Biol. Cell. 2002; 13: 1252-1262Crossref PubMed Scopus (66) Google Scholar). In spite of the attempts of many groups to identify the PLD binding site for ARF (14.Kim J.H. Lee S.D. Han J.M. Lee T.G. Kim Y. Park J.B. Lambeth J.D. Suh P.G. Ryu S.H. Activation of phospholipase D1 by direct interaction with ADP-ribosylation factor 1 and RalA.FEBS Lett. 1998; 430: 231-235Crossref PubMed Scopus (89) Google Scholar, 15.Selvy P.E. Lavieri R.R. Lindsley C.W. Brown H.A. Phospholipase D: enzymology, functionality, and chemical modulation.Chem. Rev. 2011; 111: 6064-6119Crossref PubMed Scopus (249) Google Scholar), to date, evidence for direct PLD-ARF interaction is missing. PLD1 and PLD2 are known to differentially localize in the cell, possibly through specific posttranslational modifications. PLD1 (16.Sugars J.M. Cellek S. Manifava M. Coadwell J. Ktistakis N.T. Fatty acylation of phospholipase D1 on cysteine residues 240 and 241 determines localization on intracellular membranes.J. Biol. Chem. 1999; 274: 30023-30027Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) and PLD2 (17.Xie Z. Ho W.T. Exton J.H. Functional implications of post-translational modifications of phospholipases D1 and D2.Biochim. Biophys. Acta. 2002; 1580: 9-21Crossref PubMed Scopus (20) Google Scholar) can be palmitoylated on two cysteine residues in their PH domain, facilitating the sorting of these isoforms into different compartments. At steady-state, PLD1 preferentially localizes to the perinuclear area, potentially associated with the endoplasmic reticulum, the Golgi apparatus, and/or late endosomes (18.Colley W.C. Sung T.C. Roll R. Jenco J. Hammond S.M. Altshuller Y. Bar-Sagi D. Morris A.J. Frohman M.A. Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization.Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar, 19.Sung T.C. Zhang Y. Morris A.J. Frohman M.A. Structural analysis of human phospholipase D1.J. Biol. Chem. 1999; 274: 3659-3666Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). PLD2 is most often reported to be associated with the plasma membrane (6.Exton J.H. Phospholipase D - structure, regulation and function.Rev. Physiol. Biochem. Pharmacol. 2002; 144: 1-94Crossref PubMed Google Scholar, 18.Colley W.C. Sung T.C. Roll R. Jenco J. Hammond S.M. Altshuller Y. Bar-Sagi D. Morris A.J. Frohman M.A. Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization.Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar), but also localizes in the cytosolic cell interior (12.Honda A. Nogami M. Yokozeki T. Yamazaki M. Nakamura H. Watanabe H. Kawamoto K. Nakayama K. Morris A.J. Frohman M.A. et al.Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation.Cell. 1999; 99: 521-532Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar) and vesicular compartments (20.Divecha N. Roefs M. Halstead J.R. D'Andrea S. Fernandez-Borga M. Oomen L. Saqib K.M. Wakelam M.J.O. D'Santos C. Interaction of the Type I alpha PIPkinase with phospholipase D: a role for the local generation of phosphatidylinositol 4,5-bisphosphate in the regulation of PLD2 activity.EMBO J. 2000; 19: 5440-5449Crossref PubMed Google Scholar). PLD2 has been shown to participate in the biogenesis of extracellular vesicles (EVs)/exosomes in two different cell types (21.Ghossoub R. Lembo F. Rubio A. Gaillard C.B. Bouchet J. Vitale N. Slavik J. Machala M. Zimmermann P. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.Nat. Commun. 2014; 5: 3477Crossref PubMed Scopus (329) Google Scholar, 22.Laulagnier K. Grand D. Dujardin A. Hamdi S. Vincent-Schneider H. Lankar D. Salles J.P. Bonnerot C. Perret B. Record M. PLD2 is enriched on exosomes and its activity is correlated to the release of exosomes.FEBS Lett. 2004; 572: 11-14Crossref PubMed Scopus (163) Google Scholar). EVs are limited by a lipid bilayer, and can be released by any type of cell. EVs have the same topology as the cell, and they contain cytosolic components and various types of nucleic acids. They also carry transmembrane proteins and lipids. In the last years, it became clear that EVs are true organelles, supporting cell-to-cell communication over short or long distances (23.Tkach M. Thery C. Communication by extracellular vesicles: where we are and where we need to go.Cell. 2016; 164: 1226-1232Abstract Full Text Full Text PDF PubMed Scopus (2005) Google Scholar, 24.Maas S.L.N. Breakefield X.O. Weaver A.M. Extracellular vesicles: unique intercellular delivery vehicles.Trends Cell Biol. 2017; 27: 172-188Abstract Full Text Full Text PDF PubMed Scopus (806) Google Scholar). Moreover, the concept of "disease EVs" was validated in various pathological contexts, like cancer, neurodegeneration, and cardiovascular disease (25.Pitt J.M. Kroemer G. Zitvogel L. Extracellular vesicles: masters of intercellular communication and potential clinical interventions.J. Clin. Invest. 2016; 126: 1139-1143Crossref PubMed Scopus (299) Google Scholar). These "bad EVs" can transport pathogenic molecules (including proteins, lipids, and nucleic acids) and participate in disease progression. In cancer, for example, bad EVs promote carcinogenesis, tumor growth, and angiogenesis, suppress immune response, mold the premetastatic niche, and impair response to therapy. According to the current nomenclature, one distinguishes three types of EVs, namely exosomes, microvesicles, and apoptotic bodies. Microvesicles originate from direct budding at the cell surface and their diameter can vary between 50 and 1,000 nm. Apoptotic bodies result from cell fragmentation and can reach a 5,000 nm in diameter. Apoptosis is not covered in this review. Exosomes find their origin in endosomal membranes. They originate from intraluminal vesicles (ILVs) that accumulate inside late endosomes and multivesicular bodies (MVBs). MVB fusion with the plasma membrane allows ILV secretion in the extracellular environment, where these small vesicles are then designated as "exosomes". Their size can vary from 40 to 150–200 nm. The term exosome is abusively used in the literature. It often refers to the fraction of the secretome pelleting at 100,000 g (further referred to as "exosome-enriched EVs" in this document). Yet, this fraction can be contaminated with small vesicles that are not of exosomal origin, and complementary methods need to be used to prove the exosomal nature of EVs. Our understanding of the molecular mechanisms controlling exosome production is far from complete and some of these mechanisms appear to vary between cell types (26.van Niel G. D'Angelo G. Raposo G. Shedding light on the cell biology of extracellular vesicles.Nat. Rev. Mol. Cell Biol. 2018; 19: 213-228Crossref PubMed Scopus (3352) Google Scholar). Several molecules, including various specific Rabs, cortactin, and SNAREs, have been described to control the fusion of "secretory" MVBs with the plasma membrane. The endosomal sorting complex required for transport (ESCRT) was the first described mechanism for the formation of ILVs and MVBs (27.Henne W.M. Buchkovich N.J. Emr S.D. The ESCRT pathway.Dev. Cell. 2011; 21: 77-91Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar, 28.Hanson P.I. Cashikar A. Multivesicular body morphogenesis.Annu. Rev. Cell Dev. Biol. 2012; 28: 337-362Crossref PubMed Scopus (403) Google Scholar). It consists of four multimeric complexes (ESCRT-0, -I, -II, and -III) and associated proteins (e.g., VPS4 and ALIX). ESCRTs are assembled in an orderly manner at the endosome. ESCRT-0, -I, and -II recognize and sequester ubiquitinated membrane proteins, while the ESCRT-III complex is responsible for membrane budding and abscission to form ILVs (29.Raiborg C. Stenmark H. The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins.Nature. 2009; 458: 445-452Crossref PubMed Scopus (1001) Google Scholar, 30.Hurley J.H. Boura E. Carlson L-A. Rozycki B. Membrane budding.Cell. 2010; 143: 875-887Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Besides ESCRTs, neutral sphingomyelinase 2 (nSMase2) (31.Trajkovic K. Hsu C. Chiantia S. Rajendran L. Wenzel D. Wieland F. Schwille P. Bruegger B. Simons M. Ceramide triggers budding of exosome vesicles into multivesicular endosomes.Science. 2008; 319: 1244-1247Crossref PubMed Scopus (2286) Google Scholar) and also syndecans, syntenin (32.Baietti M.F. Zhang Z. Mortier E. Melchior A. Degeest G. Geeraerts A. Ivarsson Y. Depoortere F. Coomans C. Vermeiren E. et al.Syndecan-syntenin-ALIX regulates the biogenesis of exosomes.Nat. Cell Biol. 2012; 14: 677-685Crossref PubMed Scopus (1110) Google Scholar), heparanase (33.Roucourt B. Meeussen S. Bao J. Zimmermann P. David G. Heparanase activates the syndecan-syntenin-ALIX exosome pathway.Cell Res. 2015; 25: 412-428Crossref PubMed Scopus (213) Google Scholar), SRC (34.Imjeti N.S. Menck K. Egea-Jimenez A.L. Lecointre C. Lembo F. Bouguenina H. Badache A. Ghossoub R. David G. Roche S. et al.Syntenin mediates SRC function in exosomal cell-to-cell communication.Proc. Natl. Acad. Sci. USA. 2017; 114: 12495-12500Crossref PubMed Scopus (84) Google Scholar), and ARF6 (21.Ghossoub R. Lembo F. Rubio A. Gaillard C.B. Bouchet J. Vitale N. Slavik J. Machala M. Zimmermann P. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.Nat. Commun. 2014; 5: 3477Crossref PubMed Scopus (329) Google Scholar) were shown to participate in the budding of ILVs that will significantly contribute to the pool of exosomal vesicles (Fig. 2). In a model of human mammary carcinoma cells (MCF-7), the depletion of ARF6 or ARNO, an ARF6 GEF, leads to a decrease in several exosomal proteins, such as syntenin, ALIX, CD63, and syndecan, and affects ILV budding (21.Ghossoub R. Lembo F. Rubio A. Gaillard C.B. Bouchet J. Vitale N. Slavik J. Machala M. Zimmermann P. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.Nat. Commun. 2014; 5: 3477Crossref PubMed Scopus (329) Google Scholar). Investigation of the ARF6 effectors implicated in this process pointed to a role of PLD2 and excluded other ARF6 effectors, such as PLD1 and also phosphatidylinositol 4-phosphate 5-kinase (PIP5K)α or PIP5Kγ, enzymes producing phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] (21.Ghossoub R. Lembo F. Rubio A. Gaillard C.B. Bouchet J. Vitale N. Slavik J. Machala M. Zimmermann P. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.Nat. Commun. 2014; 5: 3477Crossref PubMed Scopus (329) Google Scholar). It is noteworthy that it has been proposed that PI(4,5)P2, the product of PIP5Ks, can activate PLD2, which leads to enhanced PA formation able to activate further PIP5Ks (12.Honda A. Nogami M. Yokozeki T. Yamazaki M. Nakamura H. Watanabe H. Kawamoto K. Nakayama K. Morris A.J. Frohman M.A. et al.Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation.Cell. 1999; 99: 521-532Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar, 20.Divecha N. Roefs M. Halstead J.R. D'Andrea S. Fernandez-Borga M. Oomen L. Saqib K.M. Wakelam M.J.O. D'Santos C. Interaction of the Type I alpha PIPkinase with phospholipase D: a role for the local generation of phosphatidylinositol 4,5-bisphosphate in the regulation of PLD2 activity.EMBO J. 2000; 19: 5440-5449Crossref PubMed Google Scholar). However, this does not seem to happen on the limiting membrane of MVBs, as the knockdown of PIP5Ks did not impact on syntenin intra-endosomal budding (21.Ghossoub R. Lembo F. Rubio A. Gaillard C.B. Bouchet J. Vitale N. Slavik J. Machala M. Zimmermann P. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.Nat. Commun. 2014; 5: 3477Crossref PubMed Scopus (329) Google Scholar). These data are consistent with the observations that ARF is able to activate PLD in the absence of PI(4,5)P2 (35.Vinggaard A.M. Jensen T. Morgan C.P. Cockcroft S. Hansen H.S. Didecanoyl phosphatidylcholine is a superior substrate for assaying mammalian phospholipase D.Biochem. 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On the contrary, ARF6-PLD2 supports the budding of syntenin cargo-laden ILVs inside the MVBs (see the pathway in Fig. 2B) (21.Ghossoub R. Lembo F. Rubio A. Gaillard C.B. Bouchet J. Vitale N. Slavik J. Machala M. Zimmermann P. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.Nat. Commun. 2014; 5: 3477Crossref PubMed Scopus (329) Google Scholar). This model is in line with the observations of Laulagnier et al. (38.Laulagnier K. Motta C. Hamdi S. Roy S. Fauvelle F. Pageaux J.F. Kobayashi T. Salles J.P. Perret B. Bonnerot C. et al.Mast cell- and dendritic cell-derived exosomes display a specific lipid composition and an unusual membrane organization.Biochem. J. 2004; 380: 161-171Crossref PubMed Scopus (0) Google Scholar) showing that stimulation of RBL-2H3 cells with ionomycin boosts the release of exosome-enriched EVs in a PLD2-dependent manner. Indeed, ionomycin is an ionophore that induces the hydrolysis of PIP2 and the release of calcium to the cytoplasm. One could speculate that PIP2 hydrolysis would impair the recycling pathway and may indirectly favor the formation of ILVs. Moreover, high levels of intra-cytosolic calcium could facilitate the MVB-plasma membrane fusion events necessary for the secretion of ILVs as exosomes (39.Hay J.C. Calcium: a fundamental regulator of intracellular membrane fusion?.EMBO Rep. 2007; 8: 236-240Crossref PubMed Scopus (123) Google Scholar). Interestingly, syntenin also directly interacts with PA. While ARNO appears to be the preferred ARF6 GEF for inward budding (away from the cytosol, with formation of ILVs inside the MVB) (21.Ghossoub R. Lembo F. Rubio A. Gaillard C.B. Bouchet J. Vitale N. Slavik J. Machala M. Zimmermann P. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.Nat. Commun. 2014; 5: 3477Crossref PubMed Scopus (329) Google Scholar), the stimulator of ARF6 that supports recycling pathways (necessitating endosomal outward budding, toward the cytosol) still needs to be identified. Clearly, syntenin-ALIX interaction and ESCRT are necessary for the exosomal pathway. As ESCRT was recently shown to also stimulate inward budding (40.McCullough J. Clippinger A.K. Talledge N. Skowyra M.L. Saunders M.G. Naismith T.V. Colf L.A. Afonine P. Arthur C. Sundquist W.I. et al.Structure and membrane remodeling activity of ESCRT-III helical polymers.Science. 2015; 350: 1548-1551Crossref PubMed Scopus (155) Google Scholar), it would be interesting to test for the impact of ALIX and ESCRT on the syntenin recycling pathway. PA (the product of PLD) represents the simplest phospholipid (Fig. 3). Despite its simple structure and relatively low abundance [1–4% of the total of phospholipids (41.Welti R. Li W.Q. Li M.Y. Sang Y.M. Biesiada H. Zhou H.E. Rajashekar C.B. 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Phospholipase D2 modulates the secretory pathway in RBL-2H3 mast cells.PLoS One. 2015; 10: e0139888Crossref PubMed Scopus (4) Google Scholar). Whether PA might be essential for budding in endosomes and for MVB formation has not been directly investigated. One study indicates that PA is 1.8-fold enriched in exosome-enriched EVs versus lysates from PC-3 cells. Yet PA accounts for only 0.16% of the total lipids in these organelles (51.Llorente A. Skotland T. Sylvanne T. Kauhanen D. Rog T. Orlowski A. Vattulainen I. Ekroos K. Sandvig K. Molecular lipidomics of exosomes released by PC-3 prostate cancer cells.Biochim. Biophys. Acta. 2013; 1831: 1302-1309Crossref PubMed Scopus (464) Google Scholar) and other studies failed to detect PA (21.Ghossoub R. Lembo F. Rubio A. Gaillard C.B. Bouchet J. Vitale N. Slavik J. Machala M. Zimmermann P. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.Nat. Commun. 2014; 5: 3477Crossref PubMed Scopus (329) Google Scholar, 38.Laulagnier K. Motta C. Hamdi S. Roy S. Fauvelle F. Pageaux J.F. Kobayashi T. Salles J.P. Perret B. Bonnerot C. et al.Mast cell- and dendritic cell-derived exosomes display a specific lipid composition and an unusual membrane organization.Biochem. J. 2004; 380: 161-171Crossref PubMed Scopus (0) Google Scholar, 52.Brouwers J.F. Aalberts M. Jansen J.W.A. van Niel G. Wauben M.H. Stout T.A.E. Helms J.B. Stoorvogel W. Distinct lipid compositions of two types of human prostasomes.Proteomics. 2013; 13: 1660-1666Crossref PubMed Scopus (102) Google Scholar). What might explain the discrete amount of PA in exosome-enriched EVs? The team of Michel Record observed that active phosphatidate phosphatase 1 (PAP1) is present in exosome-enriched EVs derived from RBL-2H3 cells (53.Subra C. Grand D. Laulagnier K. Stella A. Lambeau G. Paillasse M. De Medina P. Monsarrat B. Perret B. Silvente-Poirot S. et al.Exosomes account for vesicle-mediated transcellular transport of activatable phospholipases and prostaglandins.J. Lipid Res. 2010; 51: 2105-2120Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). Upon the incubation of BODIPY-PA, 60% of the PA was hydrolyzed into diglycerides within 15 min. It might thus be that PA is rapidly degraded in these organelles. Alternatively, during ILV biogenesis, PA might flip-flop and be restricted to the abscission sites, the membrane domains with the highest curvature. Yet, PA might be key at several stages of exosome formation because of its biophysical properties (Fig. 4A, B) and/or due to its direct interactions with effector proteins involved in the process of ILV budding (Fig. 4C–E). Moreover, PA could be important for the transport (Fig. 4F) and plasma membrane docking (Fig. 4G) of ILV-filled MVBs. Because of its negative curvature, PA production could stimulate the initiation of ILV budding (Fig. 4A). It is noteworthy that lipid-binding data suggest that syntenin can directly interact with PA (21.Ghossoub R. Lembo F. Rubio A. Gaillard C.B. Bouchet J. Vitale N. Slavik J. Machala M. Zimmermann P. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.Nat. Commun. 2014; 5: 3477Crossref PubMed Scopus (329) Google Scholar). Irrespective of the curvature, syntenin interaction with PA could thus concentrate the syntenin/syndecan/CD63/ALIX complexes at the nascent bud, with ALIX-ESCRT further stimulating the budding process (Fig. 4C). PA could also support ILV-budding independently of ESCRT. Indeed, nSMase2 has also been proposed to interact with PA (54.Wu B.X. Clarke C.J. Matmati N. Montefusco D. Bartke N. Hannun Y.A. Identification of novel anionic phospholipid binding domains in neutral sphingomyelinase 2 with selective binding preference.J. Biol. Chem. 2011; 286: 22362-22371Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 55.Marchesini N. Luberto C. Hannun Y.A. Biochemical proRead More
Title: $Phospholipase D and phosphatidic acid in the biogenesis and cargo loading of extracellular vesicles
Abstract: Extracellular vesicles released by viable cells (exosomes and microvesicles) have emerged as important organelles supporting cell-cell communication. Because of their potential therapeutic significance, important efforts are being made toward characterizing the contents of these vesicles and the mechanisms that govern their biogenesis. It has been recently demonstrated that the lipid modifying enzyme, phospholipase D (PLD)2, is involved in exosome production and acts downstream of the small GTPase, ARF6. This review aims to recapitulate our current knowledge of the role of PLD2 and its product, phosphatidic acid, in the biogenesis of exosomes and to propose hypotheses for further investigation of a possible central role of these molecules in the biology of these organelles. Extracellular vesicles released by viable cells (exosomes and microvesicles) have emerged as important organelles supporting cell-cell communication. Because of their potential therapeutic significance, important efforts are being made toward characterizing the contents of these vesicles and the mechanisms that govern their biogenesis. It has been recently demonstrated that the lipid modifying enzyme, phospholipase D (PLD)2, is involved in exosome production and acts downstream of the small GTPase, ARF6. This review aims to recapitulate our current knowledge of the role of PLD2 and its product, phosphatidic acid, in the biogenesis of exosomes and to propose hypotheses for further investigation of a possible central role of these molecules in the biology of these organelles. Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), the most abundant membrane phospholipid, to generate phosphatidic acid (PA) and choline. There are six different mammalian proteins designated as PLDs (Fig. 1). Most of our knowledge about PLD biology refers to the PLD1 and PLD2 isoenzymes. PLDs participate in the normal maintenance of cellular membranes (1.Frohman M.A. Sung T.C. Morris A.J. Mammalian phospholipase D structure and regulation.Biochim. Biophys. Acta. 1999; 1439: 175-186Crossref PubMed Scopus (277) Google Scholar) and in a variety of physiological cellular functions, such as cell migration and proliferation, vesicle trafficking, cytoskeleton remodeling, and morphogenesis (2.Exton J.H. Regulation of phospholipase D.FEBS Lett. 2002; 531: 58-61Crossref PubMed Scopus (234) Google Scholar, 3.Jenkins G.M. Frohman M.A. Phospholipase D: a lipid centric review.Cell. Mol. Life Sci. 2005; 62: 2305-2316Crossref PubMed Scopus (392) Google Scholar, 4.Lee C.S. Kim K.L. Jang J.H. Choi Y.S. Suh P.G. Ryu S.H. The roles of phospholipase D in EGFR signaling.Biochim. Biophys. Acta. 2009; 1791: 862-868Crossref PubMed Scopus (41) Google Scholar). Most of these functions are attributed to PA generation. The catalytic domain of PLD1 and PLD2 appears to be under strict control, and a plethora of stimuli supports PLD activation. Some proteins have been reported to regulate PLD activity through protein-protein interaction and/or phosphorylation/dephosphorylation processes. Regulators of PLD activity include tyrosine and serine/threonine kinases and small GTPases, such as those of the ARF and Rho families (5.Houle M.G. Bourgoin S. Regulation of phospholipase D by phosphorylation-dependent mechanisms.Biochim. Biophys. Acta. 1999; 1439: 135-149Crossref PubMed Scopus (85) Google Scholar, 6.Exton J.H. Phospholipase D - structure, regulation and function.Rev. Physiol. Biochem. Pharmacol. 2002; 144: 1-94Crossref PubMed Google Scholar). Both ARF1 and ARF6 can activate human PLD2 (7.Lopez I. Arnold R.S. Lambeth J.D. Cloning and initial characterization of a human phospholipase D2 (hPLD2).J. Biol. Chem. 1998; 273: 12846-12852Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 8.Hiroyama M. Exton J.H. Localization and regulation of phospholipase D2 by ARF6.J. Cell. Biochem. 2005; 95: 149-164Crossref PubMed Scopus (32) Google Scholar). For example, the activation of ARF6 by ARF nucleotide-binding site opener (ARNO), which is an ARF6-specific guanine-nucleotide exchange factor (GEF), results in increased activation of PLD and induces epithelial cell migration (9.Santy L.C. Casanova J.E. Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D.J. Cell Biol. 2001; 154: 599-610Crossref PubMed Scopus (314) Google Scholar). Stimulation of chromaffin cells triggers the translocation of ARF6 from secretory granules to the plasma membrane and the concomitant activation of PLD at the plasma membrane (10.Caumont A.S. Galas M.C. Vitale N. Aunis D. Bader M.F. Regulated exocytosis in chromaffin cells. Translocation of ARF6 stimulates a plasma membrane-associated phospholipase D.J. Biol. Chem. 1998; 273: 1373-1379Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). PLD activation can be blocked by addition of the ARF inhibitor, Brefeldin A (11.Rümenapp U. Geiszt M. Wahn F. Schmidt M. Jakobs K.H. Evidence for ADP-ribosylation-factor-mediated activation of phospholipase D by m3 muscarinic acetylcholine receptor.Eur. J. Biochem. 1995; 234: 240-244Crossref PubMed Scopus (81) Google Scholar), and a synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 (10.Caumont A.S. Galas M.C. Vitale N. Aunis D. Bader M.F. Regulated exocytosis in chromaffin cells. Translocation of ARF6 stimulates a plasma membrane-associated phospholipase D.J. Biol. Chem. 1998; 273: 1373-1379Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). It has also been reported that ARF6 can colocalize with PLD1 and PLD2 in membrane ruffles (12.Honda A. Nogami M. Yokozeki T. Yamazaki M. Nakamura H. Watanabe H. Kawamoto K. Nakayama K. Morris A.J. Frohman M.A. et al.Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation.Cell. 1999; 99: 521-532Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar, 13.Powner D.J. Hodgkin M.N. Wakelam M.J.O. Antigen-stimulated activation of phospholipase D1b by Rac1, ARF6, and PKC alpha in RBL-2H3 cells.Mol. Biol. Cell. 2002; 13: 1252-1262Crossref PubMed Scopus (66) Google Scholar). In spite of the attempts of many groups to identify the PLD binding site for ARF (14.Kim J.H. Lee S.D. Han J.M. Lee T.G. Kim Y. Park J.B. Lambeth J.D. Suh P.G. Ryu S.H. Activation of phospholipase D1 by direct interaction with ADP-ribosylation factor 1 and RalA.FEBS Lett. 1998; 430: 231-235Crossref PubMed Scopus (89) Google Scholar, 15.Selvy P.E. Lavieri R.R. Lindsley C.W. Brown H.A. Phospholipase D: enzymology, functionality, and chemical modulation.Chem. Rev. 2011; 111: 6064-6119Crossref PubMed Scopus (249) Google Scholar), to date, evidence for direct PLD-ARF interaction is missing. PLD1 and PLD2 are known to differentially localize in the cell, possibly through specific posttranslational modifications. PLD1 (16.Sugars J.M. Cellek S. Manifava M. Coadwell J. Ktistakis N.T. Fatty acylation of phospholipase D1 on cysteine residues 240 and 241 determines localization on intracellular membranes.J. Biol. Chem. 1999; 274: 30023-30027Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) and PLD2 (17.Xie Z. Ho W.T. Exton J.H. Functional implications of post-translational modifications of phospholipases D1 and D2.Biochim. Biophys. Acta. 2002; 1580: 9-21Crossref PubMed Scopus (20) Google Scholar) can be palmitoylated on two cysteine residues in their PH domain, facilitating the sorting of these isoforms into different compartments. At steady-state, PLD1 preferentially localizes to the perinuclear area, potentially associated with the endoplasmic reticulum, the Golgi apparatus, and/or late endosomes (18.Colley W.C. Sung T.C. Roll R. Jenco J. Hammond S.M. Altshuller Y. Bar-Sagi D. Morris A.J. Frohman M.A. Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization.Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar, 19.Sung T.C. Zhang Y. Morris A.J. Frohman M.A. Structural analysis of human phospholipase D1.J. Biol. Chem. 1999; 274: 3659-3666Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). PLD2 is most often reported to be associated with the plasma membrane (6.Exton J.H. Phospholipase D - structure, regulation and function.Rev. Physiol. Biochem. Pharmacol. 2002; 144: 1-94Crossref PubMed Google Scholar, 18.Colley W.C. Sung T.C. Roll R. Jenco J. Hammond S.M. Altshuller Y. Bar-Sagi D. Morris A.J. Frohman M.A. Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization.Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar), but also localizes in the cytosolic cell interior (12.Honda A. Nogami M. Yokozeki T. Yamazaki M. Nakamura H. Watanabe H. Kawamoto K. Nakayama K. Morris A.J. Frohman M.A. et al.Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation.Cell. 1999; 99: 521-532Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar) and vesicular compartments (20.Divecha N. Roefs M. Halstead J.R. D'Andrea S. Fernandez-Borga M. Oomen L. Saqib K.M. Wakelam M.J.O. D'Santos C. Interaction of the Type I alpha PIPkinase with phospholipase D: a role for the local generation of phosphatidylinositol 4,5-bisphosphate in the regulation of PLD2 activity.EMBO J. 2000; 19: 5440-5449Crossref PubMed Google Scholar). PLD2 has been shown to participate in the biogenesis of extracellular vesicles (EVs)/exosomes in two different cell types (21.Ghossoub R. Lembo F. Rubio A. Gaillard C.B. Bouchet J. Vitale N. Slavik J. Machala M. Zimmermann P. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.Nat. Commun. 2014; 5: 3477Crossref PubMed Scopus (329) Google Scholar, 22.Laulagnier K. Grand D. Dujardin A. Hamdi S. Vincent-Schneider H. Lankar D. Salles J.P. Bonnerot C. Perret B. Record M. PLD2 is enriched on exosomes and its activity is correlated to the release of exosomes.FEBS Lett. 2004; 572: 11-14Crossref PubMed Scopus (163) Google Scholar). EVs are limited by a lipid bilayer, and can be released by any type of cell. EVs have the same topology as the cell, and they contain cytosolic components and various types of nucleic acids. They also carry transmembrane proteins and lipids. In the last years, it became clear that EVs are true organelles, supporting cell-to-cell communication over short or long distances (23.Tkach M. Thery C. Communication by extracellular vesicles: where we are and where we need to go.Cell. 2016; 164: 1226-1232Abstract Full Text Full Text PDF PubMed Scopus (2005) Google Scholar, 24.Maas S.L.N. Breakefield X.O. Weaver A.M. Extracellular vesicles: unique intercellular delivery vehicles.Trends Cell Biol. 2017; 27: 172-188Abstract Full Text Full Text PDF PubMed Scopus (806) Google Scholar). Moreover, the concept of "disease EVs" was validated in various pathological contexts, like cancer, neurodegeneration, and cardiovascular disease (25.Pitt J.M. Kroemer G. Zitvogel L. Extracellular vesicles: masters of intercellular communication and potential clinical interventions.J. Clin. Invest. 2016; 126: 1139-1143Crossref PubMed Scopus (299) Google Scholar). These "bad EVs" can transport pathogenic molecules (including proteins, lipids, and nucleic acids) and participate in disease progression. In cancer, for example, bad EVs promote carcinogenesis, tumor growth, and angiogenesis, suppress immune response, mold the premetastatic niche, and impair response to therapy. According to the current nomenclature, one distinguishes three types of EVs, namely exosomes, microvesicles, and apoptotic bodies. Microvesicles originate from direct budding at the cell surface and their diameter can vary between 50 and 1,000 nm. Apoptotic bodies result from cell fragmentation and can reach a 5,000 nm in diameter. Apoptosis is not covered in this review. Exosomes find their origin in endosomal membranes. They originate from intraluminal vesicles (ILVs) that accumulate inside late endosomes and multivesicular bodies (MVBs). MVB fusion with the plasma membrane allows ILV secretion in the extracellular environment, where these small vesicles are then designated as "exosomes". Their size can vary from 40 to 150–200 nm. The term exosome is abusively used in the literature. It often refers to the fraction of the secretome pelleting at 100,000 g (further referred to as "exosome-enriched EVs" in this document). Yet, this fraction can be contaminated with small vesicles that are not of exosomal origin, and complementary methods need to be used to prove the exosomal nature of EVs. Our understanding of the molecular mechanisms controlling exosome production is far from complete and some of these mechanisms appear to vary between cell types (26.van Niel G. D'Angelo G. Raposo G. Shedding light on the cell biology of extracellular vesicles.Nat. Rev. Mol. Cell Biol. 2018; 19: 213-228Crossref PubMed Scopus (3352) Google Scholar). Several molecules, including various specific Rabs, cortactin, and SNAREs, have been described to control the fusion of "secretory" MVBs with the plasma membrane. The endosomal sorting complex required for transport (ESCRT) was the first described mechanism for the formation of ILVs and MVBs (27.Henne W.M. Buchkovich N.J. Emr S.D. The ESCRT pathway.Dev. Cell. 2011; 21: 77-91Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar, 28.Hanson P.I. Cashikar A. Multivesicular body morphogenesis.Annu. Rev. Cell Dev. Biol. 2012; 28: 337-362Crossref PubMed Scopus (403) Google Scholar). It consists of four multimeric complexes (ESCRT-0, -I, -II, and -III) and associated proteins (e.g., VPS4 and ALIX). ESCRTs are assembled in an orderly manner at the endosome. ESCRT-0, -I, and -II recognize and sequester ubiquitinated membrane proteins, while the ESCRT-III complex is responsible for membrane budding and abscission to form ILVs (29.Raiborg C. Stenmark H. The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins.Nature. 2009; 458: 445-452Crossref PubMed Scopus (1001) Google Scholar, 30.Hurley J.H. Boura E. Carlson L-A. Rozycki B. Membrane budding.Cell. 2010; 143: 875-887Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Besides ESCRTs, neutral sphingomyelinase 2 (nSMase2) (31.Trajkovic K. Hsu C. Chiantia S. Rajendran L. Wenzel D. Wieland F. Schwille P. Bruegger B. Simons M. Ceramide triggers budding of exosome vesicles into multivesicular endosomes.Science. 2008; 319: 1244-1247Crossref PubMed Scopus (2286) Google Scholar) and also syndecans, syntenin (32.Baietti M.F. Zhang Z. Mortier E. Melchior A. Degeest G. Geeraerts A. Ivarsson Y. Depoortere F. Coomans C. Vermeiren E. et al.Syndecan-syntenin-ALIX regulates the biogenesis of exosomes.Nat. Cell Biol. 2012; 14: 677-685Crossref PubMed Scopus (1110) Google Scholar), heparanase (33.Roucourt B. Meeussen S. Bao J. Zimmermann P. David G. Heparanase activates the syndecan-syntenin-ALIX exosome pathway.Cell Res. 2015; 25: 412-428Crossref PubMed Scopus (213) Google Scholar), SRC (34.Imjeti N.S. Menck K. Egea-Jimenez A.L. Lecointre C. Lembo F. Bouguenina H. Badache A. Ghossoub R. David G. Roche S. et al.Syntenin mediates SRC function in exosomal cell-to-cell communication.Proc. Natl. Acad. Sci. USA. 2017; 114: 12495-12500Crossref PubMed Scopus (84) Google Scholar), and ARF6 (21.Ghossoub R. Lembo F. Rubio A. Gaillard C.B. Bouchet J. Vitale N. Slavik J. Machala M. Zimmermann P. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.Nat. Commun. 2014; 5: 3477Crossref PubMed Scopus (329) Google Scholar) were shown to participate in the budding of ILVs that will significantly contribute to the pool of exosomal vesicles (Fig. 2). In a model of human mammary carcinoma cells (MCF-7), the depletion of ARF6 or ARNO, an ARF6 GEF, leads to a decrease in several exosomal proteins, such as syntenin, ALIX, CD63, and syndecan, and affects ILV budding (21.Ghossoub R. Lembo F. Rubio A. Gaillard C.B. Bouchet J. Vitale N. Slavik J. Machala M. Zimmermann P. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.Nat. Commun. 2014; 5: 3477Crossref PubMed Scopus (329) Google Scholar). Investigation of the ARF6 effectors implicated in this process pointed to a role of PLD2 and excluded other ARF6 effectors, such as PLD1 and also phosphatidylinositol 4-phosphate 5-kinase (PIP5K)α or PIP5Kγ, enzymes producing phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] (21.Ghossoub R. Lembo F. Rubio A. Gaillard C.B. Bouchet J. Vitale N. Slavik J. Machala M. Zimmermann P. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.Nat. Commun. 2014; 5: 3477Crossref PubMed Scopus (329) Google Scholar). It is noteworthy that it has been proposed that PI(4,5)P2, the product of PIP5Ks, can activate PLD2, which leads to enhanced PA formation able to activate further PIP5Ks (12.Honda A. Nogami M. Yokozeki T. Yamazaki M. Nakamura H. Watanabe H. Kawamoto K. Nakayama K. Morris A.J. Frohman M.A. et al.Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation.Cell. 1999; 99: 521-532Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar, 20.Divecha N. Roefs M. Halstead J.R. D'Andrea S. Fernandez-Borga M. Oomen L. Saqib K.M. Wakelam M.J.O. D'Santos C. Interaction of the Type I alpha PIPkinase with phospholipase D: a role for the local generation of phosphatidylinositol 4,5-bisphosphate in the regulation of PLD2 activity.EMBO J. 2000; 19: 5440-5449Crossref PubMed Google Scholar). However, this does not seem to happen on the limiting membrane of MVBs, as the knockdown of PIP5Ks did not impact on syntenin intra-endosomal budding (21.Ghossoub R. Lembo F. Rubio A. Gaillard C.B. Bouchet J. Vitale N. Slavik J. Machala M. Zimmermann P. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.Nat. Commun. 2014; 5: 3477Crossref PubMed Scopus (329) Google Scholar). These data are consistent with the observations that ARF is able to activate PLD in the absence of PI(4,5)P2 (35.Vinggaard A.M. Jensen T. Morgan C.P. Cockcroft S. Hansen H.S. Didecanoyl phosphatidylcholine is a superior substrate for assaying mammalian phospholipase D.Biochem. J. 1996; 319: 861-864Crossref PubMed Scopus (20) Google Scholar). Basically, on endosomal membranes, ARF6-PIP5K rather supports the recycling of syntenin and associated transmembrane protein cargo, pending the coincident detection of peptide and PI(4,5)P2 by the syntenin PDZ domains (see the pathway in Fig. 2A) (36.Egea-Jimenez A.L. Gallardo R. Garcia-Pino A. Ivarsson Y. Wawrzyniak A.M. Kashyap R. Loris R. Schymkowitz J. Rousseau F. Zimmermann P. Frizzled 7 and PIP2 binding by syntenin PDZ2 domain supports Frizzled 7 trafficking and signalling.Nat. Commun. 2016; 7: 12101Crossref PubMed Scopus (24) Google Scholar, 37.Zimmermann P. Zhang Z. Degeest G. Mortier E. Leenaerts I. Coomans C. Schulz J. N'Kuli F. Courtoy P.J. David G. Syndecan recycling [corrected] is controlled by syntenin-PIP2 interaction and Arf6.Dev. Cell. 2005; 9 ([Erratum. 2005. Dev. Cell. 9: 721.]): 377-388Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). On the contrary, ARF6-PLD2 supports the budding of syntenin cargo-laden ILVs inside the MVBs (see the pathway in Fig. 2B) (21.Ghossoub R. Lembo F. Rubio A. Gaillard C.B. Bouchet J. Vitale N. Slavik J. Machala M. Zimmermann P. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.Nat. Commun. 2014; 5: 3477Crossref PubMed Scopus (329) Google Scholar). This model is in line with the observations of Laulagnier et al. (38.Laulagnier K. Motta C. Hamdi S. Roy S. Fauvelle F. Pageaux J.F. Kobayashi T. Salles J.P. Perret B. Bonnerot C. et al.Mast cell- and dendritic cell-derived exosomes display a specific lipid composition and an unusual membrane organization.Biochem. J. 2004; 380: 161-171Crossref PubMed Scopus (0) Google Scholar) showing that stimulation of RBL-2H3 cells with ionomycin boosts the release of exosome-enriched EVs in a PLD2-dependent manner. Indeed, ionomycin is an ionophore that induces the hydrolysis of PIP2 and the release of calcium to the cytoplasm. One could speculate that PIP2 hydrolysis would impair the recycling pathway and may indirectly favor the formation of ILVs. Moreover, high levels of intra-cytosolic calcium could facilitate the MVB-plasma membrane fusion events necessary for the secretion of ILVs as exosomes (39.Hay J.C. Calcium: a fundamental regulator of intracellular membrane fusion?.EMBO Rep. 2007; 8: 236-240Crossref PubMed Scopus (123) Google Scholar). Interestingly, syntenin also directly interacts with PA. While ARNO appears to be the preferred ARF6 GEF for inward budding (away from the cytosol, with formation of ILVs inside the MVB) (21.Ghossoub R. Lembo F. Rubio A. Gaillard C.B. Bouchet J. Vitale N. Slavik J. Machala M. Zimmermann P. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.Nat. Commun. 2014; 5: 3477Crossref PubMed Scopus (329) Google Scholar), the stimulator of ARF6 that supports recycling pathways (necessitating endosomal outward budding, toward the cytosol) still needs to be identified. Clearly, syntenin-ALIX interaction and ESCRT are necessary for the exosomal pathway. As ESCRT was recently shown to also stimulate inward budding (40.McCullough J. Clippinger A.K. Talledge N. Skowyra M.L. Saunders M.G. Naismith T.V. Colf L.A. Afonine P. Arthur C. Sundquist W.I. et al.Structure and membrane remodeling activity of ESCRT-III helical polymers.Science. 2015; 350: 1548-1551Crossref PubMed Scopus (155) Google Scholar), it would be interesting to test for the impact of ALIX and ESCRT on the syntenin recycling pathway. PA (the product of PLD) represents the simplest phospholipid (Fig. 3). Despite its simple structure and relatively low abundance [1–4% of the total of phospholipids (41.Welti R. Li W.Q. Li M.Y. Sang Y.M. Biesiada H. Zhou H.E. Rajashekar C.B. Williams T.D. Wang X.M. Profiling membrane lipids in plant stress responses. Role of phospholipase D alpha in freezing-induced lipid changes in Arabidopsis.J. Biol. Chem. 2002; 277: 31994-32002Abstract Full Text Full Text PDF PubMed Scopus (824) Google Scholar, 42.Voelker D.R. Organelle biogenesis and intracellular lipid transport in eukaryotes.Microbiol. Rev. 1991; 55: 543-560Crossref PubMed Google Scholar)], PA is important for membrane dynamics, i.e., fission and fusion (43.Kooijman E.E. Chupin V. de Kruijff B. Burger K.N.J. Modulation of membrane curvature by phosphatidic acid and lysophosphatidic acid.Traffic. 2003; 4: 162-174Crossref PubMed Scopus (299) Google Scholar). The function of PA is likely in part due to its ability to induce a negative membrane curvature because of its small headgroup (forming a "cone" that might favor endosomal intraluminal budding). Moreover, PA can also directly interact with proteins (43.Kooijman E.E. Chupin V. de Kruijff B. Burger K.N.J. Modulation of membrane curvature by phosphatidic acid and lysophosphatidic acid.Traffic. 2003; 4: 162-174Crossref PubMed Scopus (299) Google Scholar, 44.Kooijman E.E. Chupin V. Fuller N.L. Kozlov M.M. de Kruijff B. Burger K.N.J. Rand P.R. Spontaneous curvature of phosphatidic acid and lysophosphatidic acid.Biochemistry. 2005; 44: 2097-2102Crossref PubMed Scopus (216) Google Scholar). Unfortunately, no consensus amino-acid sequence defines a PA-binding site. PA enables an electrostatic/hydrogen bond switch (45.Kooijman E.E. Tieleman D.P. Testerink C. Munnik T. Rijkers D.T.S. Burger K.N.J. de Kruijff B. An electrostatic/hydrogen bond switch as the basis for the specific interaction of phosphatidic acid with proteins.J. Biol. Chem. 2007; 282: 11356-11364Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar) when engaging with protein ligands and the negative charge of the PA headgroup is increased from −1 to −2 and stabilized upon formation of hydrogen bonds with lysine and arginine residues of the interacting protein (46.Kooijman E.E. Testerink C. Phosphatidic acid: an electrostatic/hydrogen-bond switch?.in: Munnik T. Lipid Signaling in Plants. Springer-Verlag, Berlin-Heidelberg2010: 203-222Crossref Scopus (16) Google Scholar). As commonly observed for protein-lipid interactions, the membrane lipid environment and the nature of the acyl chains can influence the recognition of PA by interacting proteins (47.Kassas N. Tanguy E. Thahouly T. Fouillen L. Heintz D. Chasserot-Golaz S. Bader M.F. Grant N.J. Vitale N. Comparative characterization of phosphatidic acid sensors and their localization during frustrated phagocytosis.J. Biol. Chem. 2017; 292: 4266-4279Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). PA is implicated, for instance, in regulated exocytosis in several different cell lines, such as the secretion of von Willebrand factor from endothelial cells (48.Disse J. Vitale N. Bader M.F. Gerke V. Phospholipase D1 is specifically required for regulated secretion of von Willebrand factor from endothelial cells.Blood. 2009; 113: 973-980Crossref PubMed Scopus (53) Google Scholar) and insulin from pancreatic β-cells (49.Waselle L. Gerona R.R.L. Vitale N. Martin T.F.J. Bader M.F. Regazzi R. Role of phosphoinositide signaling in the control of insulin exocytosis.Mol. Endocrinol. 2005; 19: 3097-3106Crossref PubMed Scopus (70) Google Scholar). In RBL-2H3 cells, PA controls the trafficking of glycoconjugates from the Golgi to the plasma membrane (50.Marchini-Alves C.M.M. Lorenzi V.C.B. da Silva E.Z.M. Mazucato V.M. Jamur M.C. Oliver C. Phospholipase D2 modulates the secretory pathway in RBL-2H3 mast cells.PLoS One. 2015; 10: e0139888Crossref PubMed Scopus (4) Google Scholar). Whether PA might be essential for budding in endosomes and for MVB formation has not been directly investigated. One study indicates that PA is 1.8-fold enriched in exosome-enriched EVs versus lysates from PC-3 cells. Yet PA accounts for only 0.16% of the total lipids in these organelles (51.Llorente A. Skotland T. Sylvanne T. Kauhanen D. Rog T. Orlowski A. Vattulainen I. Ekroos K. Sandvig K. Molecular lipidomics of exosomes released by PC-3 prostate cancer cells.Biochim. Biophys. Acta. 2013; 1831: 1302-1309Crossref PubMed Scopus (464) Google Scholar) and other studies failed to detect PA (21.Ghossoub R. Lembo F. Rubio A. Gaillard C.B. Bouchet J. Vitale N. Slavik J. Machala M. Zimmermann P. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.Nat. Commun. 2014; 5: 3477Crossref PubMed Scopus (329) Google Scholar, 38.Laulagnier K. Motta C. Hamdi S. Roy S. Fauvelle F. Pageaux J.F. Kobayashi T. Salles J.P. Perret B. Bonnerot C. et al.Mast cell- and dendritic cell-derived exosomes display a specific lipid composition and an unusual membrane organization.Biochem. J. 2004; 380: 161-171Crossref PubMed Scopus (0) Google Scholar, 52.Brouwers J.F. Aalberts M. Jansen J.W.A. van Niel G. Wauben M.H. Stout T.A.E. Helms J.B. Stoorvogel W. Distinct lipid compositions of two types of human prostasomes.Proteomics. 2013; 13: 1660-1666Crossref PubMed Scopus (102) Google Scholar). What might explain the discrete amount of PA in exosome-enriched EVs? The team of Michel Record observed that active phosphatidate phosphatase 1 (PAP1) is present in exosome-enriched EVs derived from RBL-2H3 cells (53.Subra C. Grand D. Laulagnier K. Stella A. Lambeau G. Paillasse M. De Medina P. Monsarrat B. Perret B. Silvente-Poirot S. et al.Exosomes account for vesicle-mediated transcellular transport of activatable phospholipases and prostaglandins.J. Lipid Res. 2010; 51: 2105-2120Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). Upon the incubation of BODIPY-PA, 60% of the PA was hydrolyzed into diglycerides within 15 min. It might thus be that PA is rapidly degraded in these organelles. Alternatively, during ILV biogenesis, PA might flip-flop and be restricted to the abscission sites, the membrane domains with the highest curvature. Yet, PA might be key at several stages of exosome formation because of its biophysical properties (Fig. 4A, B) and/or due to its direct interactions with effector proteins involved in the process of ILV budding (Fig. 4C–E). Moreover, PA could be important for the transport (Fig. 4F) and plasma membrane docking (Fig. 4G) of ILV-filled MVBs. Because of its negative curvature, PA production could stimulate the initiation of ILV budding (Fig. 4A). It is noteworthy that lipid-binding data suggest that syntenin can directly interact with PA (21.Ghossoub R. Lembo F. Rubio A. Gaillard C.B. Bouchet J. Vitale N. Slavik J. Machala M. Zimmermann P. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.Nat. Commun. 2014; 5: 3477Crossref PubMed Scopus (329) Google Scholar). Irrespective of the curvature, syntenin interaction with PA could thus concentrate the syntenin/syndecan/CD63/ALIX complexes at the nascent bud, with ALIX-ESCRT further stimulating the budding process (Fig. 4C). PA could also support ILV-budding independently of ESCRT. Indeed, nSMase2 has also been proposed to interact with PA (54.Wu B.X. Clarke C.J. Matmati N. Montefusco D. Bartke N. Hannun Y.A. Identification of novel anionic phospholipid binding domains in neutral sphingomyelinase 2 with selective binding preference.J. Biol. 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