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BMC Biology Jun 2020Lymphocytes circulate between peripheral lymphoid tissues via blood and lymphatic systems, and chemokine-induced migration is important in trafficking lymphocytes to...
BACKGROUND
Lymphocytes circulate between peripheral lymphoid tissues via blood and lymphatic systems, and chemokine-induced migration is important in trafficking lymphocytes to distant sites. The small GTPase Rap1 is important in mediating lymphocyte motility, and Rap1-GEFs are involved in chemokine-mediated Rap1 activation. Here, we describe the roles and mechanisms of Rap1-GEFs in lymphocyte trafficking.
RESULTS
In this study, we show that RA-GEF-1 and 2 (also known as Rapgef2 and 6) are key guanine nucleotide exchange factors (GEF) for Rap1 in lymphocyte trafficking. Mice harboring T cell-specific knockouts of Rapgef2/6 demonstrate defective homing and egress of T cells. Sphingosine-1-phosphate (S1P) as well as chemokines activates Rap1 in a RA-GEF-1/2-dependent manner, and their deficiency in T cells impairs Mst1 phosphorylation, cell polarization, and chemotaxis toward S1P gradient. On the other hand, B cell-specific knockouts of Rapgef2/6 impair chemokine-dependent retention of B cells in the bone marrow and passively facilitate egress. Phospholipase D2-dependent production of phosphatidic acid by these chemotactic factors determines spatial distribution of Rap1-GTP subsequent to membrane localization of RA-GEFs and induces the development of front membrane. On the other hand, basal de-phosphorylation of RA-GEFs is necessary for chemotactic factor-dependent increase in GEF activity for Rap1.
CONCLUSIONS
We demonstrate here that subcellular distribution and activation of RA-GEFs are key factors for a directional movement of lymphocytes and that phosphatidic acid is critical for membrane translocation of RA-GEFs with chemokine stimulation.
Topics: Animals; Cell Line; Cell Movement; Female; Guanine Nucleotide Exchange Factors; Humans; Lymphocytes; Male; Mice; Phosphatidic Acids; Phosphorylation
PubMed: 32600317
DOI: 10.1186/s12915-020-00809-0 -
International Journal of Molecular... Feb 2021Phosphatidic acid (PA) is a bioactive phospholipid capable of regulating key biological functions, including neutrophil respiratory burst, chemotaxis, or cell growth and...
Phosphatidic acid (PA) is a bioactive phospholipid capable of regulating key biological functions, including neutrophil respiratory burst, chemotaxis, or cell growth and differentiation. However, the mechanisms whereby PA exerts these actions are not completely understood. In this work, we show that PA stimulates myoblast proliferation, as determined by measuring the incorporation of [H]thymidine into DNA and by staining the cells with crystal violet. PA induced the rapid phosphorylation of Akt and ERK1/2, and pretreatment of the cells with specific small interferin RNA (siRNA) to silence the genes encoding these kinases, or with selective pharmacologic inhibitors, blocked PA-stimulated myoblast proliferation. The mitogenic effects of PA were abolished by the preincubation of the myoblasts with pertussis toxin, a Gi protein inhibitor, suggesting the implication of Gi protein-coupled receptors in this action. Although some of the effects of PA have been associated with its possible conversion to lysoPA (LPA), treatment of the myoblasts with PA for up to 60 min did not produce any significant amount of LPA in these cells. Of interest, pharmacological blockade of the LPA receptors 1 and 2, or specific siRNA to silence the genes encoding these receptors, abolished PA-stimulated myoblast proliferation. Moreover, PA was able to compete with LPA for binding to LPA receptors, suggesting that PA can act as a ligand of LPA receptors. It can be concluded that PA stimulates myoblast proliferation through interaction with LPA1 and LPA2 receptors and the subsequent activation of the PI3K/Akt and MEK/ERK1-2 pathways, independently of LPA formation.
Topics: Animals; Cell Differentiation; Cell Line; Cell Proliferation; Chemotaxis; DNA; Lysophospholipids; Mice; Myoblasts; Phosphatidic Acids; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Binding; RNA, Small Interfering; Receptors, Lysophosphatidic Acid; Signal Transduction
PubMed: 33535610
DOI: 10.3390/ijms22031452 -
Accounts of Chemical Research Nov 2022Membranes are multifunctional supramolecular assemblies that encapsulate our cells and the organelles within them. Glycerophospholipids are the most abundant component... (Review)
Review
Membranes are multifunctional supramolecular assemblies that encapsulate our cells and the organelles within them. Glycerophospholipids are the most abundant component of membranes. They make up the majority of the lipid bilayer and play both structural and functional roles. Each organelle has a different phospholipid composition critical for its function that results from dynamic interplay and regulation of numerous lipid-metabolizing enzymes and lipid transporters. Because lipid structures and localizations are not directly genetically encoded, chemistry has much to offer to the world of lipid biology in the form of precision tools for visualizing lipid localization and abundance, manipulating lipid composition, and in general decoding the functions of lipids in cells.In this Account, we provide an overview of our recent efforts in this space focused on two overarching and complementary goals: imaging and editing the phospholipidome. On the imaging front, we have harnessed the power of bioorthogonal chemistry to develop fluorescent reporters of specific lipid pathways. Substantial efforts have centered on phospholipase D (PLD) signaling, which generates the humble lipid phosphatidic acid (PA) that acts variably as a biosynthetic intermediate and signaling agent. Though PLD is a hydrolase that generates PA from abundant phosphatidylcholine (PC) lipids, we have exploited its transphosphatidylation activity with exogenous clickable alcohols followed by bioorthogonal tagging to generate fluorescent lipid reporters of PLD signaling in a set of methods termed IMPACT.IMPACT and its variants have facilitated many biological discoveries. Using the rapid and fluorogenic tetrazine ligation, it has revealed the spatiotemporal dynamics of disease-relevant G protein-coupled receptor signaling and interorganelle lipid transport. IMPACT using diazirine photo-cross-linkers has enabled identification of lipid-protein interactions relevant to alcohol-related diseases. Varying the alcohol reporter can allow for organelle-selective labeling, and varying the bioorthogonal detection reagent can afford super-resolution lipid imaging via expansion microscopy. Combination of IMPACT with genome-wide CRISPR screening has revealed genes that regulate physiological PLD signaling.PLD enzymes themselves can also act as tools for precision editing of the phospholipid content of membranes. An optogenetic PLD for conditional blue-light-stimulated synthesis of PA on defined organelle compartments led to the discovery of the role of organelle-specific pools of PA in regulating oncogenic Hippo signaling. Directed enzyme evolution of PLD, enabled by IMPACT, has yielded highly active superPLDs with broad substrate tolerance and an ability to edit membrane phospholipid content and synthesize designer phospholipids in vitro. Finally, azobenzene-containing PA analogues represent an alternative, all-chemical strategy for light-mediated control of PA signaling.Collectively, the strategies described here summarize our progress to date in tackling the challenge of assigning precise functions to defined pools of phospholipids in cells. They also point to new challenges and directions for future study, including extension of imaging and membrane editing tools to other classes of lipids. We envision that continued application of bioorthogonal chemistry, optogenetics, and directed evolution will yield new tools and discoveries to interrogate the phospholipidome and reveal new mechanisms regulating phospholipid homeostasis and roles for phospholipids in cell signaling.
Topics: Optogenetics; Phosphatidic Acids; Phosphatidylcholines; Phospholipase D; Signal Transduction
PubMed: 36278840
DOI: 10.1021/acs.accounts.2c00510 -
Phospholipase D and phosphatidic acid in the biogenesis and cargo loading of extracellular vesicles.Journal of Lipid Research Sep 2018Extracellular vesicles released by viable cells (exosomes and microvesicles) have emerged as important organelles supporting cell-cell communication. Because of their... (Review)
Review
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.
Topics: Animals; Exosomes; Extracellular Vesicles; Humans; Phosphatidic Acids; Phospholipase D; Signal Transduction
PubMed: 29853529
DOI: 10.1194/jlr.R083964 -
The Journal of Biological Chemistry Mar 2023Diacylglycerol kinase (DGK) α, which is a key enzyme in the progression of cancer and, in contrast, in T-cell activity attenuation, preferentially produces saturated...
Diacylglycerol kinase (DGK) α, which is a key enzyme in the progression of cancer and, in contrast, in T-cell activity attenuation, preferentially produces saturated fatty acid (SFA)- and/or monounsaturated fatty acid (MUFA)-containing phosphatidic acids (PAs), such as 16:0/16:0-, 16:0/18:0-, and 16:1/16:1-PA, in melanoma cells. In the present study, we searched for the target proteins of 16:0/16:0-PA in melanoma cells and identified heat shock protein (HSP) 27, which acts as a molecular chaperone and contributes to cancer progression. HSP27 more strongly interacted with PA than other phospholipids, including phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate. Moreover, HSP27 is more preferentially bound to SFA- and/or MUFA-containing PAs, including 16:0/16:0- and 16:0/18:1-PAs, than PUFA-containing PAs, including 18:0/20:4- and 18:0/22:6-PA. Furthermore, HSP27 and constitutively active DGKα expressed in COS-7 cells colocalized in a DGK activity-dependent manner. Notably, 16:0/16:0-PA, but not phosphatidylcholine or 16:0/16:0-phosphatidylserine, induced oligomer dissociation of HSP27, which enhances its chaperone activity. Intriguingly, HSP27 protein was barely detectable in Jurkat T cells, while the protein band was intensely detected in AKI melanoma cells. Taken together, these results strongly suggest that SFA- and/or MUFA-containing PAs produced by DGKα selectively target HSP27 and regulate its cancer-progressive function in melanoma cells but not in T cells.
Topics: Humans; Fatty Acids; Fatty Acids, Monounsaturated; HSP27 Heat-Shock Proteins; Phosphatidic Acids; Phosphatidylserines; Phosphatidylinositols; Phosphatidylcholines; Melanoma
PubMed: 36791913
DOI: 10.1016/j.jbc.2023.103019 -
Journal of Experimental Botany Apr 2015Phospholipase Ds (PLDs) and PLD-derived phosphatidic acids (PAs) play vital roles in plant hormonal and environmental responses and various cellular dynamics. Recent... (Review)
Review
Phospholipase Ds (PLDs) and PLD-derived phosphatidic acids (PAs) play vital roles in plant hormonal and environmental responses and various cellular dynamics. Recent studies have further expanded the functions of PLDs and PAs into plant-microbe interaction. The molecular diversities and redundant functions make PLD-PA an important signalling complex regulating lipid metabolism, cytoskeleton dynamics, vesicle trafficking, and hormonal signalling in plant defence through protein-protein and protein-lipid interactions or hormone signalling. Different PLD-PA signalling complexes and their targets have emerged as fast-growing research topics for understanding their numerous but not yet established roles in modifying pathogen perception, signal transduction, and downstream defence responses. Meanwhile, advanced lipidomics tools have allowed researchers to reveal further the mechanisms of PLD-PA signalling complexes in regulating lipid metabolism and signalling, and their impacts on jasmonic acid/oxylipins, salicylic acid, and other hormone signalling pathways that essentially mediate plant defence responses. This review attempts to summarize the progress made in spatial and temporal PLD/PA signalling as well as PLD/PA-mediated modification of plant defence. It presents an in-depth discussion on the functions and potential mechanisms of PLD-PA complexes in regulating actin filament/microtubule cytoskeleton, vesicle trafficking, and hormonal signalling, and in influencing lipid metabolism-derived metabolites as critical signalling components in plant defence responses. The discussion puts PLD-PA in a broader context in order to guide future research.
Topics: Cytoskeleton; Microtubules; Phosphatidic Acids; Phospholipase D; Plant Growth Regulators; Plant Proteins; Plants; Salicylic Acid; Signal Transduction
PubMed: 25680793
DOI: 10.1093/jxb/eru540 -
The Journal of Biological Chemistry Apr 2022The simple structure of phosphatidic acid (PA) belies its complex biological functions as both a key phospholipid biosynthetic intermediate and a potent signaling...
The simple structure of phosphatidic acid (PA) belies its complex biological functions as both a key phospholipid biosynthetic intermediate and a potent signaling molecule. In the latter role, PA controls processes including vesicle trafficking, actin dynamics, cell growth, and migration. However, experimental methods to decode the pleiotropy of PA are sorely lacking. Because PA metabolism and trafficking are rapid, approaches to accurately visualize and manipulate its levels require high spatiotemporal precision. Here, we describe recent efforts to create a suite of chemical tools that enable imaging and perturbation of PA signaling. First, we describe techniques to visualize PA production by phospholipase D (PLD) enzymes, which are major producers of PA, called Imaging Phospholipase D Activity with Clickable Alcohols via Transphosphatidylation (IMPACT). IMPACT harnesses the ability of endogenous PLD enzymes to accept bioorthogonally tagged alcohols in transphosphatidylation reactions to generate functionalized reporter lipids that are subsequently fluorescently tagged via click chemistry. Second, we describe two light-controlled approaches for precisely manipulating PA signaling. Optogenetic PLDs use light-mediated heterodimerization to recruit a bacterial PLD to desired organelle membranes, and photoswitchable PA analogs contain azobenzene photoswitches in their acyl tails, enabling molecular shape and bioactivity to be controlled by light. We highlight select applications of these tools for studying GPCR-G signaling, discovering regulators of PLD signaling, tracking intracellular lipid transport pathways, and elucidating new oncogenic signaling roles for PA. We envision that these chemical tools hold promise for revealing many new insights into lipid signaling pathways.
Topics: Alcohols; Click Chemistry; Optogenetics; Phosphatidic Acids; Phospholipase D; Signal Transduction
PubMed: 35276134
DOI: 10.1016/j.jbc.2022.101810 -
Advances in Biological Regulation Jan 2021Mammalian phospholipase D (PLD) generates phosphatidic acid, a dynamic lipid secondary messenger involved with a broad spectrum of cellular functions including but not... (Review)
Review
Mammalian phospholipase D (PLD) generates phosphatidic acid, a dynamic lipid secondary messenger involved with a broad spectrum of cellular functions including but not limited to metabolism, migration, and exocytosis. As a promising pharmaceutical target, the biochemical properties of PLD have been well characterized. This has led to the recent crystal structures of human PLD1 and PLD2, the development of PLD specific pharmacological inhibitors, and the identification of cellular regulators of PLD. In this review, we discuss the PLD1 and PLD2 structures, PLD inhibition by small molecules, and the regulation of PLD activity by effector proteins and lipids.
Topics: Animals; Enzyme Inhibitors; Humans; Phosphatidic Acids; Phospholipase D; Signal Transduction
PubMed: 33495125
DOI: 10.1016/j.jbior.2020.100783 -
Biomolecules Sep 2020Phosphatidic acid (PA) is a glycerophospholipid intermediate in the triglyceride synthesis pathway that has incredibly important structural functions as a component of... (Review)
Review
Phosphatidic acid (PA) is a glycerophospholipid intermediate in the triglyceride synthesis pathway that has incredibly important structural functions as a component of cell membranes and dynamic effects on intracellular and intercellular signaling pathways. Although there are many pathways to synthesize and degrade PA, a family of PA phosphohydrolases (lipin family proteins) that generate diacylglycerol constitute the primary pathway for PA incorporation into triglycerides. Previously, it was believed that the pool of PA used to synthesize triglyceride was distinct, compartmentalized, and did not widely intersect with signaling pathways. However, we now know that modulating the activity of lipin 1 has profound effects on signaling in a variety of cell types. Indeed, in most tissues except adipose tissue, lipin-mediated PA phosphohydrolase activity is far from limiting for normal rates of triglyceride synthesis, but rather impacts critical signaling cascades that control cellular homeostasis. In this review, we will discuss how lipin-mediated control of PA concentrations regulates metabolism and signaling in mammalian organisms.
Topics: Adipose Tissue; Glycerophospholipids; Humans; Metabolic Networks and Pathways; Muscle, Skeletal; Phosphatidate Phosphatase; Phosphatidic Acids; Protein Conformation; Triglycerides
PubMed: 33003344
DOI: 10.3390/biom10101386 -
Biomolecules Nov 2022Phosphatidic acid (PA) is a signaling lipid that is produced enzymatically from phosphatidylcholine (PC), lysophosphatidic acid, or diacylglycerol. Compared to PC, PA...
Phosphatidic acid (PA) is a signaling lipid that is produced enzymatically from phosphatidylcholine (PC), lysophosphatidic acid, or diacylglycerol. Compared to PC, PA lacks a choline moiety on the headgroup, making the headgroup smaller than that of PC and PA, and PA has a net negative charge. Unlike the cylindrical geometry of PC, PA, with its small headgroup relative to the two fatty acid tails, is proposed to support negatively curved membranes. Thus, PA is thought to play a role in a variety of biological processes that involve bending membranes, such as the formation of intraluminal vesicles in multivesicular bodies and membrane fusion. Using supported tubulated lipid bilayers (STuBs), the extent to which PA localizes to curved membranes was determined. STuBs were created via liposome deposition with varying concentrations of NaCl (500 mM to 1 M) on glass to form supported bilayers with connected tubules. The location of fluorescently labeled lipids relative to tubules was determined by imaging with total internal reflection or confocal fluorescence microscopy. The accumulation of various forms of PA (with acyl chains of 16:0-6:0, 16:0-12:0, 18:1-12:0) were compared to PC and the headgroup labeled phosphatidylethanolamine (PE), a lipid that has been shown to accumulate at regions of curvature. PA and PE accumulated more at tubules and led to the formation of more tubules than PC. Using large unilamellar liposomes in a dye-quenching assay, the location of the headgroup labeled PE was determined to be mostly on the outer, positively curved leaflet, whereas the tail labeled PA was located more on the inner, negatively curved leaflet. This study demonstrates that PA localizes to regions of negative curvature in liposomes and supports the formation of curved, tubulated membranes. This is one way that PA could be involved with curvature formation during a variety of cell processes.
Topics: Lipid Bilayers; Phosphatidic Acids; Lecithins; Unilamellar Liposomes; Membrane Fusion
PubMed: 36421720
DOI: 10.3390/biom12111707