scholarly journals A real-time, click chemistry imaging approach reveals stimulus-specific subcellular locations of phospholipase D activity

2019 ◽  
Vol 116 (31) ◽  
pp. 15453-15462 ◽  
Author(s):  
Dongjun Liang ◽  
Kane Wu ◽  
Reika Tei ◽  
Timothy W. Bumpus ◽  
Johnny Ye ◽  
...  
Keyword(s):  
Real Time ◽  
Phospholipase D ◽  
Second Messengers ◽  
Primary Alcohols ◽  
Fluorescent Reporter ◽  
Lipid Trafficking ◽  
Tyrosine Kinase Signaling ◽  
Inverse Electron Demand ◽  
Transphosphatidylation Reaction ◽  
Phospholipid Transport

The fidelity of signal transduction requires spatiotemporal control of the production of signaling agents. Phosphatidic acid (PA) is a pleiotropic lipid second messenger whose modes of action differ based on upstream stimulus, biosynthetic source, and site of production. How cells regulate the local production of PA to effect diverse signaling outcomes remains elusive. Unlike other second messengers, sites of PA biosynthesis cannot be accurately visualized with subcellular precision. Here, we describe a rapid, chemoenzymatic approach for imaging physiological PA production by phospholipase D (PLD) enzymes. Our method capitalizes on the remarkable discovery that bulky, hydrophilic trans-cyclooctene–containing primary alcohols can supplant water as the nucleophile in the PLD active site in a transphosphatidylation reaction of PLD’s lipid substrate, phosphatidylcholine. The resultant trans-cyclooctene–containing lipids are tagged with a fluorogenic tetrazine reagent via a no-rinse, inverse electron-demand Diels–Alder (IEDDA) reaction, enabling their immediate visualization by confocal microscopy in real time. Strikingly, the fluorescent reporter lipids initially produced at the plasma membrane (PM) induced by phorbol ester stimulation of PLD were rapidly internalized via apparent nonvesicular pathways rather than endocytosis, suggesting applications of this activity-based imaging toolset for probing mechanisms of intracellular phospholipid transport. By instead focusing on the initial 10 s of the IEDDA reaction, we precisely pinpointed the subcellular locations of endogenous PLD activity as elicited by physiological agonists of G protein-coupled receptor and receptor tyrosine kinase signaling. These tools hold promise to shed light on both lipid trafficking pathways and physiological and pathological effects of localized PLD signaling.

scholarly journals Structures of an engineered phospholipase D with specificity for secondary alcohol transphosphatidylation: Insights into plasticity of substrate binding and activation

2021 ◽  
Author(s):  
Ariela Samantha ◽  
Jasmina Damnjanović ◽  
Yugo Iwasaki ◽  
Hideo Nakano ◽  
Alice Vrielink
Keyword(s):  
Phospholipase D ◽  
Catalytic Mechanism ◽  
Inositol Phosphate ◽  
Substrate Binding ◽  
Secondary Alcohol ◽  
Molecular Size ◽  
Underlying Mechanism ◽  
Head Group ◽  
Wild Type ◽  
Primary Alcohols

Phospholipase D (PLD) is an enzyme useful for the enzymatic modification of phospholipids.  In the presence of primary alcohols, the enzyme catalyses transphosphatidylation of the head group of phospholipid substrates to synthesize a modified phospholipid product.  However, the enzyme is specific for primary alcohols and thus the limitation of the molecular size of the acceptor compounds has restricted the type of phospholipid species that can be synthesised.  An engineered variant of PLD from Streptomycesantibioticus termed TNYR SaPLD was developed capable of synthesizing 1-phosphatidylinositol with positional specificity of up to 98%. To gain a better understanding of the substrate binding features of the TNYR SaPLD, crystal structures have been determined for the free enzyme and its complexes with phosphate, phosphatidic acid and 1-inositol phosphate.  Comparisons of these structures with the wild-type SaPLD show a larger binding site able to accommodate a bulkier secondary alcohol substrate as well as changes to the position of a flexible surface loop proposed to be involved in substrate recognition.  The complex of the active TNYR SaPLD with 1-inositol phosphate reveals a covalent intermediate adduct with the ligand bound to H442 rather than to H168, the proposed nucleophile in the wild type enzyme.  This structural feature suggests that the enzyme exhibits plasticity of the catalytic mechanism different from what has been reported to date for PLDs.  These structural studies provide insights into the underlying mechanism that governs the recognition of myo-inositol by TNYR SaPLD, and an important foundation for further studies of the catalytic mechanism.

scholarly journals Revisiting T7 RNA polymerase transcription in vitro with the Broccoli RNA aptamer as a simplified real-time fluorescent reporter

2020 ◽  
pp. jbc.RA120.014553
Author(s):  
Zachary J Kartje ◽  
Helen I Janis ◽  
Shaoni Mukhopadhyay ◽  
Keith T Gagnon
Keyword(s):  
Rna Polymerase ◽  
Real Time ◽  
High Throughput Screening ◽  
In Vitro Transcription ◽  
T7 Rna Polymerase ◽  
Rna Aptamer ◽  
Reaction Conditions ◽  
Fluorescent Reporter ◽  
Transcription In Vitro

Methods for rapid and high-throughput screening of transcription in vitro to examine reaction conditions, enzyme mutants, promoter variants, and small molecule modulators can be extremely valuable tools. However, these techniques may be difficult to establish or inaccessible to many researchers. To develop a straightforward and cost-effective platform for assessing transcription in vitro, we used the “Broccoli” RNA aptamer as a direct, real-time fluorescent transcript readout. To demonstrate the utility of our approach, we screened the effect of common reaction conditions and components on bacteriophage T7 RNA polymerase (RNAP) activity using a common quantitative PCR instrument for fluorescence detection. Several essential conditions for in vitro transcription by T7 RNAP were confirmed with this assay, including the importance of enzyme and substrate concentrations, co-variation of magnesium and nucleoside triphosphates, and the effects of several typical additives. When we used this method to assess all possible point mutants of a canonical T7 RNAP promoter, our results coincided well with previous reports. This approach should translate well to a broad variety of bacteriophage in vitro transcription systems and provides a platform for developing fluorescence-based readouts of more complex transcription systems in vitro.

scholarly journals The temporal relationship between phospholipase activation, diradylglycerol formation and superoxide production in the human neutrophil

1990 ◽  
Vol 271 (1) ◽  
pp. 209-213 ◽  
Author(s):  
N T Thompson ◽  
J E Tateson ◽  
R W Randall ◽  
G D Spacey ◽  
R W Bonser ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Human Neutrophil ◽  
Second Messengers ◽  
Superoxide Production ◽  
Human Neutrophils ◽  
Inositol 1,4,5 Trisphosphate ◽  
Phosphatidate Phosphohydrolase ◽  
Time Courses ◽  
Rapid Activation ◽  
Stimulation Of

Fluctuations in the amounts of choline, inositol 1,4,5-trisphosphate (IP3) and diradylglycerol have been used to monitor phospholipase activation in the human neutrophil. Stimulation of human neutrophils by formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) resulted in a rapid activation of both phosphatidylinositol 4,5-bisphosphate breakdown by phospholipase C and phosphatidylcholine breakdown by phospholipase D. Diradylglycerol accumulation occurred more slowly than that of either choline or IP3 and was inhibited by 30 mM-butanol, suggesting that the bulk was derived from the phospholipase D pathway via phosphatidate phosphohydrolase. Consistent with this is the observation that choline and diradylglycerol are produced in similar amounts. 1,2-Diacylglycerol (DAG) and 1-O-alkyl-2-acyl-sn-glycerol species accumulated with different time courses, indicating that one or more steps in the phospholipase D pathway was selective for the diacyl species. Superoxide production by fMet-Leu-Phe-stimulated neutrophils paralleled DAG accumulation over the first 5 min, but thereafter this production stopped, despite the fact that DAG remained elevated. We conclude that DAG derived from the phospholipase D pathway is only one of the second messengers important in controlling this functional response.

scholarly journals A Multiplexed Fluorescent Assay for Independent Second-Messenger Systems

2013 ◽  
Vol 18 (7) ◽  
pp. 797-806 ◽  
Author(s):  
Paul H. Tewson ◽  
Anne Marie Quinn ◽  
Thomas E. Hughes
Keyword(s):  
Real Time ◽  
Second Messengers ◽  
Basic Research ◽  
Second Messenger ◽  
Cell Types ◽  
Live Cell ◽  
Multiple Components ◽  
Second Messenger Systems ◽  
G Protein Coupled ◽  
Cell Signaling Pathways

There is a growing need in drug discovery and basic research to measure multiple second-messenger components of cell signaling pathways in real time and in relevant tissues and cell types. Many G-protein–coupled receptors activate the heterotrimeric protein, Gq, which in turn activates phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce two second messengers: diacylglycerol (DAG), which remains in the plasma membrane, and inositol triphosphate (IP3), which diffuses through the cytosol to release stores of intracellular calcium ions (Ca2+). Our goal was to create a series of multiplex sensors that would make it possible to simultaneously measure two different components of the Gq pathway in living cells. Here we describe new fluorescent sensors for DAG and PIP2 that produce robust changes in green or red fluorescence and can be combined with one another, or with existing Ca2+ sensors, in a live-cell assay. These assays can detect multiple components of Gq signaling, simultaneously in real time, on standard fluorescent plate readers or live-cell imaging systems.

scholarly journals Phorbol ester and bryostatin differentially regulate the hydrolysis of phosphatidylethanolamine in Ha-ras- and raf-oncogene-transformed NIH 3T3 cells

1991 ◽  
Vol 276 (2) ◽  
pp. 505-509 ◽  
Author(s):  
Z Kiss ◽  
U R Rapp ◽  
G R Pettit ◽  
W B Anderson
Keyword(s):  
Phospholipase D ◽  
Second Messengers ◽  
Transformed Cells ◽  
3T3 Cells ◽  
Intact Cells ◽  
Pkc Inhibitor ◽  
Kinase C ◽  
Nih 3T3 ◽  
Hydrolysis Of ◽  
Nih 3T3 Cells

Previously it was reported that transformation of NIH 3T3 fibroblast by the Ha-ras, v-src, v-fms, and A-raf oncogenes decreased the stimulatory effects of phorbol 12-myristate 13-acetate (PMA; ‘TPA’), an activator of protein kinase C (PKC), on the phosphorylation of an endogenous 80 kDa substrate and on 86Rb uptake [Wolfman, Wingrove, Blackshear & Macara (1987) J. Biol. Chem. 262, 16546-16552], as well as on sphingomyelin synthesis [Kiss, Rapp & Anderson (1988) FEBS Lett. 240, 221-226]. Here, we investigated how transformation affects the PMA-stimulated hydrolysis of phosphatidylethanolamine (PtdEtn), a recently characterized mechanism which may contribute to the generation of the second messengers phosphatidic acid and 1,2-diacylglycerol. The effects of PMA were compared with those of bryostatin, a non-tumour-promoter activator of PKC. Transformation of NIH 3T3 cells with Ha-ras, v-raf, or A-raf enhanced the stimulatory effect of PMA on the phospholipase D-mediated hydrolysis of PtdEtn. On the other hand, the effects of bryostatin on PtdEtn hydrolysis were only slightly increased, if at all, in cells transformed with these oncogenes. In crude membrane preparations isolated from these transformed cells, PMA, but not bryostatin, enhanced the combined stimulatory effects of ATP and the GTP analogue guanosine 5′-[gamma-thio]triphosphate on phospholipase D-mediated PtdEtn hydrolysis. The PKC inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine inhibited the stimulatory effect of PMA only in intact cells. These results indicate that transformation of cells by certain oncogenes differentially affects phospholipase D-mediated hydrolysis of PtdEtn induced by PMA and bryostatin, suggesting that the action of PMA might involve two different mechanisms.

scholarly journals H+- and Na+-elicited swift changes of the microtubule system in the biflagellated green alga Chlamydomonas

2017 ◽  
Author(s):  
Yi Liu ◽  
Mike Visetsouk ◽  
Michelle Mynlieff ◽  
Hongmin Qin ◽  
Karl F. Lechtreck ◽  
...  
Keyword(s):  
Real Time ◽  
Fresh Water ◽  
Green Alga ◽  
Dynamic Instability ◽  
Animal Cells ◽  
Reporter Protein ◽  
Fluorescent Reporter ◽  
Cellular Processes ◽  
Endogenous Level ◽  
Cytoskeletal System

AbstractThe microtubule cytoskeletal system is integral to diverse cellular processes. Although microtubules are known for dynamic instability, the system is tightly controlled in typical interphase animal cells. In contrast, diverse evidence suggests that the system is mercurial in the unicellular fresh water green alga, Chlamydomonas, but intense autofluorescence from photosynthesis pigments has hindered the investigation. By expressing a bright fluorescent reporter protein at the endogenous level, we demonstrate in real time discreet sweeping changes in algal microtubules elicited by fluctuation of intracellular H+ and Na+. These results suggest disparate sensitivity of this vital yet delicate system in diverse organisms; and illuminate how pH may drive crucial cellular processes; how plants respond to, and perhaps sense stresses; and how many species could be susceptible to accelerated changes in global environments.

scholarly journals Role of Phospholipase D in Pasteurella haemolytica Leukotoxin-Induced Increase in Phospholipase A2 Activity in Bovine Neutrophils

1999 ◽  
Vol 67 (8) ◽  
pp. 3768-3772 ◽  
Author(s):  
Zuncai Wang ◽  
Cyril R. Clarke ◽  
Kenneth D. Clinkenbeard
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Pasteurella Haemolytica ◽  
Time Dependent ◽  
Regulatory Interaction ◽  
Pneumonic Pasteurellosis ◽  
Transphosphatidylation Reaction ◽  
Bovine Neutrophils ◽  
Phospholipase A2 Activity

ABSTRACT The effects of Pasteurella haemolytica leukotoxin (LKT) on the activity of phospholipase D (PLD) and the regulatory interaction between PLD and phospholipase A2 (PLA2) were investigated in assays using isolated bovine neutrophils labeled with tritiated phospholipid substrates of the two enzymes. Exposure of [3H]lysophosphatidylcholine-labeled neutrophils to LKT caused concentration- and time-dependent production of phosphatidic acid (PA), the product of PLD. LKT-induced generation of PA was dependent on extracellular calcium. Both production of PA and metabolism of [3H]-arachidonate ([3H]AA)-labeled phospholipids by PLA2 were inhibited when ethanol was used to promote the alternative PLD-mediated transphosphatidylation reaction, resulting in the production of phosphatidylethanol rather than PA. The role of PA in regulation of PLA2 activity was then confirmed by means of an add-back experiment, whereby addition of PA in the presence of ethanol restored PLA2-mediated release of radioactivity from neutrophil membranes. Considering the involvement of chemotactic phospholipase products in the pathogenesis of pneumonic pasteurellosis, development and use of anti-inflammatory agents that inhibit LKT-induced activation of PLD and PLA2 may improve therapeutic management of the disease.

scholarly journals A Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System forClostridiumOrganisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST)

2019 ◽  
Vol 85 (14) ◽  
Author(s):  
Hannah E. Streett ◽  
Katie M. Kalis ◽  
Eleftherios T. Papoutsakis
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Real Time ◽  
Protein Localization ◽  
Anaerobic Conditions ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Content Type ◽  
Expression Activity ◽  
Microplate Reader

ABSTRACTVisualizing protein localization and characterizing gene expression activity in liveClostridiumcells is limited for lack of a real-time, highly fluorescent, oxygen-independent reporter system. Enzymatic reporter systems have been used successfully for many years withClostridiumspp.; however, these assays do not allow for real-time analysis of gene expression activity with flow cytometry or for visualizing protein localization through fusion proteins. Commonly used fluorescent reporter proteins require oxygen for chromophore maturation and cannot be used for most strictly anaerobicClostridiumorganisms. Here we show that the fluorescence-activating and absorption-shifting tag protein (FAST), when associated with the fluorogenic ligand 4-hydroxy-3-methylbenzylidene-rhodanine (HMBR; now commercially available) and other commercially available ligands, is highly fluorescent inClostridium acetobutylicumunder anaerobic conditions. Using flow cytometry and a fluorescence microplate reader, we demonstrated FAST as a reporter system by employing the promoters of theC. acetobutylicumthiolase (thl), acetoacetate decarboxylase (adc), and phosphotransbutyrylase (ptb) metabolic genes, as well as a mutant Pthland modified ribosome binding site (RBS) versions of Padcand Pptb. Flow cytometry-based sorting was efficient and fast in sorting FAST-expressing cells, and positively and negatively sorted cells could be effectively recultured. FAST was also used to tag and examine protein localization of the predicted cell division FtsZ partner protein, ZapA, to visualize the divisome localization in liveC. acetobutylicumcells. Our findings suggest that FAST can be used to further investigateClostridiumdivisomes and more broadly the localization and expression levels of other proteins inClostridiumorganisms, thus enabling cell biology studies with these organisms.IMPORTANCEFAST in association with the fluorogenic ligand HMBR is characterized as a successful, highly fluorescent reporter system inC. acetobutylicum. FAST can be used to distinguish between promoters in live cells using flow cytometry or a fluorescence microplate reader and can be used to tag and examine protein localization in live, anaerobically grown cells. Given that FAST is highly fluorescent under anaerobic conditions, it can be used in several applications of this and likely manyClostridiumorganisms and other strict anaerobes, including studies involving cell sorting, sporulation dynamics, and population characterization in pure as well as mixed cultures, such as those in various native or synthetic microbiomes and syntrophic cultures.

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