Click chemistry and optogenetic approaches to visualize and manipulate phosphatidic acid signaling

Abstract

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–Gq 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. 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–Gq 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. Lipids have many important functions in cells, including as major components of membranes, compounds for energy storage, and messenger molecules for signal transduction (1Eyster K.M. The membrane and lipids as integral participants in signal transduction: Lipid signal transduction for the non-lipid biochemist.Adv. Physiol. Educ. 2007; 31: 5-16Crossref PubMed Scopus (139) Google Scholar). Lipids are typically classified by their head group and backbone, with each class containing multiple species with variable composition of their acyl tails. Signaling lipids often have pleiotropic effects, which can arise both from this acyl chain diversity and also from what we term locational diversity, that is, the diversity of subcellular localizations that most lipids can assume (2Harayama T. Riezman H. Understanding the diversity of membrane lipid composition.Nat. Rev. Mol. Cell Biol. 2018; 19: 281-296Crossref PubMed Scopus (595) Google Scholar). In cell signaling pathways, protein localizations are carefully regulated by various factors, and proper lipid localization — both to and within the correct membrane — is crucial for enabling the intended lipid–protein interactions to occur. Both the hydrophilic head group and lipid acyl chains can be important in determining protein-binding affinities (3Corradi V. Sejdiu B.I. Mesa-Galloso H. Abdizadeh H. Noskov S. Yu. Marrink S.J. Tieleman D.P. Emerging diversity in lipid–protein interactions.Chem. Rev. 2019; 119: 5775-5848Crossref PubMed Scopus (171) Google Scholar) and biophysical properties of lipids, such as lateral and flip-flop movements in the bilayer (4Coreta-Gomes F.M. Vaz W.L.C. Moreno M.J. Effect of acyl chain length on the rate of phospholipid flip-flop and intermembrane transfer.J. Membr. Biol. 2018; 251: 431-442Crossref PubMed Scopus (2) Google Scholar, 5Schuhmacher M. Grasskamp A.T. Barahtjan P. Wagner N. Lombardot B. Schuhmacher J.S. Sala P. Lohmann A. Henry I. 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Frohman M.A. A common lipid links Mfn-mediated mitochondrial fusion and SNARE-regulated exocytosis.Nat. Cell Biol. 2006; 8: 1255-1262Crossref PubMed Scopus (325) Google Scholar). Further metabolism of PA is catalyzed via three other pathways: (1) dephosphorylation to DAG by PA phosphatase/lipin, (2) hydrolysis to LPA by phospholipase A, and (3) conversion to CDP-DAG by CDP-DAG synthase. These enzymes, with their isoform-specific localizations and acyl chain preferences (10Zegarlinska J. Piaścik M. Sikorski A.F. Czogalla A. Phosphatidic acid – a simple phospholipid with multiple faces.Acta Biochim. Pol. 2018; 65: 163-171Crossref PubMed Scopus (36) Google Scholar, 15Massart J. Zierath J.R. Role of diacylglycerol kinases in glucose and energy homeostasis.Trends Endocrinol. Metab. 2019; 30: 603-617Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar), diversify the pools of PA in cells and thus contribute to its pleiotropy. Several recent findings have reinvigorated the field, helping us to better understand PA production and metabolism, notably the first high-resolution structures of mammalian PLDs (16Bowling F.Z. Salazar C.M. Bell J.A. Frohman M.A. structure of into by and Chem. Biol. 2020; PubMed Scopus Google Scholar, C.M. P. T. M. PLD structures enable and of Chem. Biol. 2020; PubMed Scopus Google Scholar) and Wang H. Bell J.A. structure of a phosphatidic acid 2020; PubMed Scopus Google Scholar). many to these enzymes affect pools of PA in PA pools are for various cellular As is the for other pleiotropic lipids, tools for and pools of PA be crucial for these However, a major is the of the PA pathways, as to are often by to in other as a in a in other to from such tools these (1) cells to the and (2) be to cellular endogenous tools have the to in this to chemical tools can have in certain such as and and can have better by functions of enzymes from roles In the of PA metabolism, PLD J. Phospholipase D and the phosphatidic acid in of PLD the of phospholipid and Lipid Signaling in Scholar) and N. P. M. T. Vitale N. for phosphatidic in Cell Scholar, Wang M. P. S. production of the signaling lipid phosphatidic acid by phospholipase the of signaling in Cell. Biol. PubMed Scopus Google Scholar, N. E. T. D. S. Vitale N. of phosphatidic acid and their localization Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) are tools to understand of PA pools and to visualize the cellular of PA. However, these tools have to several important are pools of PA, as to pools by other cells PLD pools of PA affect cellular these we have the of and protein and to a suite of chemical tools for studying PA (Fig. The first set of methods of the pools of PA are These for Imaging PLD Activity with Clickable Alcohols via and click to and the of PLD within cells Baskin J.M. A for imaging cellular phosphatidic acid Chem. PubMed Scopus Google Scholar, Baskin J.M. Clickable enable imaging of phospholipase D Sci. PubMed Scopus Google Scholar, D. R. J. Baskin J.M. A click imaging reveals subcellular of phospholipase D Natl. Acad. Sci. U. S. A. 2019; PubMed Scopus Google Scholar, D. Baskin J.M. Imaging phospholipase D with alcohols via in Scholar). The set of and small tools spatiotemporal manipulation of PA signaling by R. Baskin J.M. of phosphatidic acid signaling with phospholipase Cell Biol. 2020; PubMed Scopus Google Scholar, R. J. A. D. Baskin J.M. of phosphatidic acid Sci. 2021; 7: PubMed Scopus Google Scholar). The first of these PLD to PA subcellular and the photoswitchable PA shape and bioactivity is controlled by a light-mediated the we highlight applications of these their ability to biological functions of PA the of a chemical to lipid signaling pathways. The functions of the two PLD for PA signaling, and are regulated in and F.Z. Frohman M.A. and of phospholipase Biol. 2021; PubMed Scopus Google Scholar). In to to subcellular in to can also its and phosphorylation F.Z. Frohman M.A. and of phospholipase Biol. 2021; PubMed Scopus Google Scholar). the localizations of the PLD enzymes the are PA. A to the of PLDs of these enzymes are PA signaling such imaging we the of PLD PLDs PA via hydrolysis of as a E. S. P. M.A. is intracellular messenger to 2019; Full Text Full Text PDF PubMed Scopus Google Scholar). with PLDs use these of as a to a transphosphatidylation of the as the lipid head is to the pools of lipids S. Transphosphatidylation by phospholipase Biol. Chem. Full Text PDF PubMed Google Scholar). The transphosphatidylation are typically by a (Fig. this lipid reporter of PLD is in on organelle endogenous PLD We to a transphosphatidylation which of the within a both the localization and of PLD and In alcohols with are as PLD The lipid with these are subsequently tagged with enabling by We by alcohols click and and that both are by PLDs Baskin J.M. A for imaging cellular phosphatidic acid Chem. PubMed Scopus Google Scholar, Baskin J.M. Clickable enable imaging of phospholipase D Sci. PubMed Scopus Google Scholar). The lipids can be to molecules the which are in cells, (Fig. and The signal in each cell can be by both of which enable of PLD this we a in PLD the cells in the Baskin J.M. Clickable enable imaging of phospholipase D Sci. PubMed Scopus Google Scholar), which have transphosphatidylation a complex of PLD the within and the of that enable of the click in cells, we also that the lipid can be and tagged in for by which a precise of PLD Baskin J.M. A for imaging cellular phosphatidic acid Chem. PubMed Scopus Google Scholar, Baskin J.M. Clickable enable imaging of phospholipase D Sci. PubMed Scopus Google Scholar). the lipids can be tagged with a which by to lipid species by and their acyl chain Baskin J.M. Clickable enable imaging of phospholipase D Sci. PubMed Scopus Google Scholar). Because of the of click and the of these the of in IMPACT is typically which is that the transphosphatidylation IMPACT endogenous PA production by in to and we also that other functionalized including and a can be by PLDs Baskin J.M. of phospholipid biosynthetic with a 2018; PubMed Scopus Google Scholar, C.M. Baskin J.M. A to phospholipase Chem. Biol. 2021; Scopus (3) Google Scholar). The of PLD via the pathway by Baskin J.M. of phospholipid biosynthetic with a 2018; PubMed Scopus Google Scholar, M. R. A. and imaging of in Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), it a reporter for two biosynthetic pathways. The the in of and lipid of PLD These lipids structural to a lipid of that in click and of lipid–protein we have several protein of these lipids as a first elucidating functions of and other alcohols C.M. Baskin J.M. A to phospholipase Chem. Biol. 2021; Scopus (3) Google Scholar). IMPACT is to PLD the and we imaging the subcellular localizations of PLD We that IMPACT from both and PLD the and as as and pools Baskin J.M. Clickable enable imaging of phospholipase D Sci. PubMed Scopus Google Scholar). IMPACT the membrane under certain are to F.Z. Frohman M.A. and of phospholipase Biol. 2021; PubMed Scopus Google Scholar, Phospholipase and chemical Rev. PubMed Scopus Google Scholar). We that the of membrane IMPACT arise from trafficking of lipids organelle the IMPACT The of and the for set a for the of and we this lipids the membrane trafficking to intracellular this we a of which we have IMPACT by of click We that a hydrophilic be by with a other simple alcohols and D. R. J. Baskin J.M. A click imaging reveals subcellular of phospholipase D Natl. Acad. Sci. U. S. A. 2019; PubMed Scopus Google Scholar). the lipids be tagged with a via a M. J.M. on Chem. PubMed Scopus Google Scholar, R. to cell Chem. 19: PubMed Scopus Google Scholar), enabling their by in within of of the We various compounds for high and cell and we that M. R. Wang H. B. J. for of with in 2019; PubMed Google Scholar) D. R. J. Baskin J.M. A click imaging reveals subcellular of phospholipase D Natl. Acad. Sci. U. S. A. 2019; PubMed Scopus Google Scholar). A with the by a of the click by is to on the PLD We the of is high to the reporter lipids are via transphosphatidylation their trafficking to other organelle D. R. J. Baskin J.M. A click imaging reveals subcellular of phospholipase D Natl. Acad. Sci. U. S. A. 2019; PubMed Scopus Google Scholar). reporter lipids the membrane endogenous PLDs with a a of which PLDs the membrane Vitale N. Huang P. S. A.J. Frohman M.A. Regulation of phospholipase subcellular of multiple membrane Cell Biol. PubMed Scopus Google Scholar). we the of these lipid by imaging the we their trafficking from the membrane to the and subsequently to the complex (Fig. that the trafficking of reporter lipids the click that the in the head group to the the phospholipid trafficking the reporter lipids via the first intracellular of the lipids the this the of a for lipid also the of for imaging and of intracellular phospholipid we set to it to the subcellular localizations of PLD by physiological PLD enzymes can from multiple of including and kinases Phospholipase and chemical Rev. PubMed Scopus Google Scholar, J.M. A.J. Frohman M.A. for and protein in of phospholipase Biol. Cell. PubMed Scopus Google Scholar). Both and can PLDs via of several intracellular including from the and (16Bowling F.Z. Salazar C.M. Bell J.A. Frohman M.A. structure of into by and Chem. Biol. 2020; PubMed Scopus Google Scholar, S. Role of D interaction in from J. PubMed Google Scholar, The of phospholipase D by and small Lett. PubMed Scopus Google Scholar, to of phospholipase D a complex with PubMed Scopus Google Scholar). As a result, both to PA physiological on with of and signaling, we in the of PLD two D. R. J. Baskin J.M. A click imaging reveals subcellular of phospholipase D Natl. Acad. Sci. U. S. A. 2019; PubMed Scopus Google Scholar). of the a in IMPACT of the a in IMPACT on intracellular (Fig. A and These that the spatiotemporal to the subcellular of endogenous PLD We have that the and of IMPACT and The of with cell is as it of cells from a with high PLD We envision applications to this with other to understand the of of PLD signaling that we have in a cell Baskin J.M. Clickable enable imaging of phospholipase D Sci. PubMed Scopus Google Scholar). we have as a in a to new regulators of PLD signaling (Fig. Huang S. R. Baskin J.M. reveals as a of PLD Natl. Acad. Sci. U. S. A. 2021; PubMed Scopus (3) Google Scholar). a these as a of PLD signaling via on these and other have the to new PLD signaling. this can the of to a signaling pathway in mammalian the subcellular is to to the subcellular PLD signaling of we these localizations for a of including the pleiotropic and such as a and D. R. J. Baskin J.M. A click imaging reveals subcellular of phospholipase D Natl. Acad. Sci. U. S. A. 2019; PubMed Scopus Google Scholar). other have to PLDs F.Z. Frohman M.A. and of phospholipase Biol. 2021; PubMed Scopus Google Scholar), and of the localizations of PLD of these important in these pathways. this we to a of the spatiotemporal dynamics of signaling of the (Fig. D. T. Baskin J.M. imaging of phospholipase D reveals spatiotemporal dynamics of GPCR–Gq Chem. Biol. Full Text Full Text PDF PubMed Scopus (2) Google Scholar). important in and multiple via both and and the can signal from S. M. Wang B. R. production by Chem. Biol. PubMed Scopus Google Scholar), the localization and of the pathway We as a and reporter of signaling and that such signaling and the of intracellular signaling. We applications of as a to imaging for and GPCR–Gq signaling pathways. A key to tools for pools of PA are approaches for these with the of biological functions to tools for PLD and a of potent of both and J. Phospholipase D and the phosphatidic acid in of PLD the of phospholipid and Lipid Signaling in Scholar, C. Frohman M.A. for PLD in and Lipid Signaling in Scholar). tools of PA is for of be to enable for it is to enable with high spatiotemporal we two on and the other lipid First, to a to enable production of PA, we on a PLD localization be controlled on We desired for PA production in cells, we mammalian are under complex and functions are F.Z. Frohman M.A. and of phospholipase Biol. 2021; PubMed Scopus Google Scholar, Phospholipase and chemical Rev. PubMed Scopus Google Scholar). we to a bacterial PLD from which with mammalian PLDs in PA via with mammalian PLDs and including interaction and interaction and for such as (16Bowling F.Z. Salazar C.M. Bell J.A. Frohman M.A. structure of into by and Chem. Biol. 2020; PubMed Scopus Google Scholar). be in mammalian cells, be in the of mammalian endogenous PLDs by and is typically by pleiotropic effects, the of is enabling the of the of PA production from other of such of endogenous PLD signaling. We that in the — that is, on — in mammalian enable of from the to a membrane for we that and M.J. of protein interactions in 7: PubMed Scopus Google Scholar). We to with the to various to it to a desired organelle in the and by to the desired organelle membrane (Fig. We this PLD and its ability to and regulated pools of PA by a R. Baskin J.M. of phosphatidic acid signaling with phospholipase Cell Biol. 2020; PubMed Scopus Google Scholar). to mammalian both hydrolysis and transphosphatidylation I. S. E. The of phospholipase D from the pathway a intermediate and Mol. Biol. PubMed Scopus Google Scholar, I. C. S. E. The first structure of a phospholipase 8: Full Text Full Text PDF PubMed Scopus Google IMPACT is also for and We a cell to signal from endogenous PLD IMPACT with in cells we that PA with a of for to organelle R. Baskin J.M. of phosphatidic acid signaling with phospholipase Cell Biol. 2020; PubMed Scopus Google Scholar). via IMPACT with we that lipids with acyl chain as mammalian that it can as a of mammalian PLDs and pools of PA in IMPACT the of of also the of the IMPACT with we a for with (Fig. R. Baskin J.M. of phosphatidic acid signaling with phospholipase Cell Biol. 2020; PubMed Scopus Google Scholar). We a with in cells, fluorescently and the cells to to cells with IMPACT The from this both and which to the and be to of mammalian PLDs in We are this to a mammalian to with properties for use in mammalian of and of the amount and subcellular localization of PA, it manipulation of cells for its which its in certain such as cell as a to we PA analogs that can be and by light. We on photoswitchable lipids, which a azobenzene in their lipid J. D. 2021; PubMed Scopus Google Scholar). lipids have as tools to functions of lipids their head group The light-controlled of azobenzene of lipid structure its and which are often with in various biological and the have to be J.A. N. J. A. N. C. D. enable of protein and vesicle Chem. Biol. PubMed Scopus Google Scholar, M. B. H. S. C. P. D. J.A. manipulation of photoswitchable 2019; PubMed Scopus (15) Google Scholar, J. S. P.C. J.A. T. Riezman H. D. of and Chem. Biol. 2019; PubMed Scopus Google Scholar, J. M.A. A. P.C. M. J.A. C. D. of lysophosphatidic acid Chem. 2020; PubMed Scopus Google Scholar). on this we PA analogs containing azobenzene in both of their lipid (Fig. R. J. A. D. Baskin J.M. of phosphatidic acid Sci. 2021; 7: PubMed Scopus Google Scholar). These PA and into both and cells, as by of lipid We in the the and we that the PA analogs be to cellular lipid metabolism and to other species that to analogs of and in cells, to of and a of cells with each with the and conversion R. J. A. D. Baskin J.M. of phosphatidic acid Sci. 2021; 7: PubMed Scopus Google Scholar). These that the PA analogs can be into cells and by endogenous enzymes as as highlight the and complex metabolism of PA in cells (10Zegarlinska J. Piaścik M. Sikorski A.F. Czogalla A. Phosphatidic acid – a simple phospholipid with multiple faces.Acta Biochim. Pol. 2018; 65: 163-171Crossref PubMed Scopus (36) Google Scholar, 11Thakur R. Naik A. Panda A. Raghu P. Regulation of membrane turnover by phosphatidic acid: Cellular functions and disease implications.Front. Cell Dev. Biol. 2019; 7: 83Crossref PubMed Scopus (10) Google Scholar). both and tools for light-controlled production of PA within cells, we to their to PA signaling highlight we to study the of PA on the signaling pathway (Fig. signaling is a by which cells can and B. pathway in and Full Text Full Text PDF PubMed Scopus Google Scholar). it is in the it cell and by the which a key protein and its to the B. N. I. Wang J. H. H. Regulation of the pathway by Full Text Full Text PDF PubMed Scopus Google Scholar). In the of is to the of cell and A recent study that PA signaling, to the phosphorylation and of H. R. B. R. Wang Regulation of the pathway by phosphatidic Cell. 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). The subcellular localization of the PA as as a of the PA these we to organelle in light-mediated and of to PA production membrane we that this regulated by a membrane of PA (Fig. and R. Baskin J.M. of phosphatidic acid signaling with phospholipase Cell Biol. 2020; PubMed Scopus Google Scholar). with a of from we that this to the amount of PA that of signaling is to the amount of PA produced in the membrane (Fig. these findings photoswitchable PA analogs in a and cell we localization. we that of both and the of the bioactivity of the PA as as their ability to be controlled by light-mediated azobenzene (Fig. R. J. A. D. Baskin J.M. of phosphatidic acid Sci. 2021; 7: PubMed Scopus Google Scholar). and photoswitchable PA analogs are tools of pools of PA with spatiotemporal precision. the pleiotropy of lipids that as signaling molecules a major for study the cell of lipid signaling. PA is a class of molecules with simple structures complex PA to study of its which is to study PA functions with to endogenous signaling, we have the of molecular to and tools that enable and of PA production in cells with high spatiotemporal

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