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Plasma-membrane location of phosphatidylinositol hydrolysis in rabbit neutrophils stimulated with formylmethionyl-leucylphenylalanine.

ABSTRACT: Rabbit peritoneal neutrophils, disrupted by sonication, were separated into three subcellular fractions by sucrose-step-gradient centrifugation and these were analysed with respect to biochemical markers. They comprised a high-speed supernatant containing the cytosol, a light particulate fraction enriched in Golgi and plasma membranes and a heavy particulate fraction enriched in granules and nuclei. The light particulate fraction was further separated into its components, which were identified as Golgi membranes (galactosyltransferase activity) and plasma membranes ((radioactivity derived from labelling intact cells with [125I]di-iodosulphanilic acid diazonium salt and [3H]formylmethionyl-leucylphenylalanine ([3H]fMet-Leu-Phe) binding)). In cells prelabelled with [3H]glycerol, the hydrolysis of phosphatidylinositol due to cell stimulation with fMet-Leu-Phe (10 nM) was shown to occur in the light particulate fraction. The [32P]Pi-labelling of phosphatidate, which is an early consequence of phosphatidylinositol hydrolysis, also occurred in this fraction. Analytical sucrose-gradient centrifugation of the light particulate fraction showed that the stimulated increment in [32P]phosphatidate (and thus by implication the initial phosphatidylinositol breakdown) was localized in the plasma membrane.

SUBMITTER: Bennett JP

PROVIDER: S-EPMC1154034 | biostudies-other | 1982 Dec

REPOSITORIES: biostudies-other

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The dependence on Ca2+ of phosphatidylinositol breakdown and enzyme secretion in rabbit neutrophils stimulated by formylmethionyl-leucylphenylalanine or ionomycin.

Project description:1. We have measured the breakdown of [3H]phosphatidylinositol in rabbit neutrophils prelabelled with [3H]glycerol by a pulse-chase procedure. With a view to defining a possible causal relationship between phosphatidylinositol breakdown and enzyme secretion in these cells, we have compared the characteristics of both these processes induced by either the receptor-directed agonist formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) or the Ca2+-ionophore ionomycin. 2. The dependence on fMet-Leu-Phe concentration of phosphatidylinositol breakdown and secretion is identical (half-maximal at 0.3 nM). This is 30-fold less than that required for half-maximal occupation of receptors. 3. Both secretion and breakdown of phosphatidylinositol due to fMet-Leu-Phe are modulated by extracellular Ca2+. The sensitivity to Ca2+ of both processes is enhanced by pretreatment to deplete cell Ca2+. The concentration of Ca2+ required to cause half-maximal effects of both processes in Ca2+-depleted cells on stimulation with 1nM-fMet-Leu-Phe is 100 microM. Ionomycin-stimulated secretion and breakdown of phosphatidylinositol are completely dependent on extracellular Ca2+ over similar concentration ranges. 4. Both secretion and phosphatidylinositol breakdown due to fMet-Leu-Phe approach completion by 10s. With ionomycin these processes are slower, terminating by 2 min. 5. In the presence of [32P]Pi, labelling of [32P]phosphatidic acid reaches a maximum 15 min after stimulation with either fMet-Leu-Phe or ionomycin. This precedes the labelling of [32P]phosphatidylinositol and shows the expected precursor-product relationship. 6. We conclude from these results that in rabbit neutrophils a rise in cytosol [Ca2+] is both sufficient and necessary to cause secretion and phosphatidylinositol breakdown. In cells depleted of Ca2+, the occupation of receptors by fMet-Leu-Phe is without effect on these two processes.

| S-EPMC1163570 | biostudies-other

Co-operation of phosphatidylinositol transfer protein with phosphoinositide 3-kinase gamma in the formylmethionyl-leucylphenylalanine-dependent production of phosphatidylinositol 3,4,5-trisphosphate in human neutrophils.

Project description:Phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol 3,4,5-trisphosphate (PIP3) play an essential role in the regulation of neutrophil functions by the chemoattractant formylmethionyl-leucylphenylalanine (FMLP). Here we show that permeabilization of human neutrophils leads to loss of cytosolic components, including PI3Kgamma, and causes the loss of FMLP-dependent production of PIP3. FMLP-sensitive synthesis of PIP3 could be restored by reconstitution of permeabilized neutrophils with recombinant PI3Kgamma. Admixture of recombinant phosphatidylinositol transfer protein (PITP) to the reconstitution cocktail produced a further increase of PIP3 synthesis, whereas pertussis toxin suppressed the FMLP-dependent production of PIP3. We conclude that FMLP-sensitive PIP3 formation in human neutrophils involves the FMLP receptor, heterotrimeric G-proteins of the Gi type, PI3Kgamma and PITP.

| S-EPMC1218559 | biostudies-other

Comparison of diglyceride production from choline-containing phosphoglycerides in human neutrophils stimulated with N-formylmethionyl-leucylphenylalanine, ionophore A23187 or phorbol 12-myristate 13-acetate.

Project description:The turnover of choline-containing phosphoglycerides (PC) in response to agonist stimulation is well documented in human neutrophils. We have now compared the enzymic pathways of N-formylmethionyl-leucylphenylalanine (fMLP)-, A23187- and phorbol-12-myristate 13-acetate (PMA)-induced diglyceride (DG) and phosphatidic acid (PA) generation in these cells. In order to distinguish between phospholipase C- and D-mediated PC breakdown, human neutrophils were radiolabelled with 1-O-[3H]alkyl-2-acyl-glycero-3-phosphocholine and stimulated in the presence of ethanol or propranolol. The addition of 0.5% ethanol to the incubation mixture resulted in the production of phosphatidylethanol, indicative of phospholipase D activation, in response to all three stimuli. Concomitant with phosphatidylethanol formation was a partial block of PA production. The production of DG was also partially blocked by addition of ethanol. Propranolol (200 microM) was also used to assess the contributions of phospholipases C and D toward DG generation. Inhibition of PA phosphohydrolase by propranolol resulted in the complete abolition of DG generation when neutrophils were stimulated with fMLP. In contrast, propranolol only partially inhibited DG generation in response to A23187 and PMA. These results suggested that DG production in response to fMLP stimulation is mediated via the activation of phospholipase D, whereas A23187- or PMA-induced DG generation may involve more than one pathway. However, examination of the water-soluble choline metabolites produced indicated that phospholipase D was responsible for the production of PA and DG in response to all three stimuli.

| S-EPMC1132959 | biostudies-other

Phosphatidylinositol(4,5)bisphosphate: diverse functions at the plasma membrane.

Project description:Phosphatidylinositol(4,5) bisphosphate (PI(4,5)P2) has become a major focus in biochemistry, cell biology and physiology owing to its diverse functions at the plasma membrane. As a result, the functions of PI(4,5)P2 can be explored in two separate and distinct roles - as a substrate for phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K) and as a primary messenger, each having unique properties. Thus PI(4,5)P2 makes contributions in both signal transduction and cellular processes including actin cytoskeleton dynamics, membrane dynamics and ion channel regulation. Signalling through plasma membrane G-protein coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and immune receptors all use PI(4,5)P2 as a substrate to make second messengers. Activation of PI3K generates PI(3,4,5)P3 (phosphatidylinositol(3,4,5)trisphosphate), a lipid that recruits a plethora of proteins with pleckstrin homology (PH) domains to the plasma membrane to regulate multiple aspects of cellular function. In contrast, PLC activation results in the hydrolysis of PI(4,5)P2 to generate the second messengers, diacylglycerol (DAG), an activator of protein kinase C and inositol(1,4,5)trisphosphate (IP3/I(1,4,5)P3) which facilitates an increase in intracellular Ca2+. Decreases in PI(4,5)P2 by PLC also impact on functions that are dependent on the intact lipid and therefore endocytosis, actin dynamics and ion channel regulation are subject to control. Spatial organisation of PI(4,5)P2 in nanodomains at the membrane allows for these multiple processes to occur concurrently.

| S-EPMC7517351 | biostudies-literature

Fatty acid stimulation of membrane phosphatidylinositol hydrolysis by brain phosphatidylinositol phosphodiesterase.

Project description:The hydrolysis of membrane-bound phosphatidylinositol in rat liver microsomal fraction by the soluble phosphatidylinositol phosphodiesterase from rat brain was markedly stimulated by oleic acid or arachidonic acid. The stimulation did not require added calcium, although it was abolished by EDTA. Lysophosphatidylcholine also totally suppressed the stimulation. A possible role for the fatty acid content of a membrane in controlling phosphatidylinositol turnover is suggested.

| S-EPMC1186539 | biostudies-other

Plasma membrane phosphatidylinositol (4,5)-bisphosphate promotes Weibel-Palade body exocytosis.

Project description:Weibel-Palade bodies (WPB) are specialized secretory organelles of endothelial cells that control vascular hemostasis by regulated, Ca2+-dependent exocytosis of the coagulation-promoting von-Willebrand factor. Some proteins of the WPB docking and fusion machinery have been identified but a role of membrane lipids in regulated WPB exocytosis has so far remained elusive. We show here that the plasma membrane phospholipid composition affects Ca2+-dependent WPB exocytosis and von-Willebrand factor release. Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] becomes enriched at WPB-plasma membrane contact sites at the time of fusion, most likely downstream of phospholipase D1-mediated production of phosphatidic acid (PA) that activates phosphatidylinositol 4-phosphate (PI4P) 5-kinase γ. Depletion of plasma membrane PI(4,5)P2 or down-regulation of PI4P 5-kinase γ interferes with histamine-evoked and Ca2+-dependent WPB exocytosis and a mutant PI4P 5-kinase γ incapable of binding PA affects WPB exocytosis in a dominant-negative manner. This indicates that a unique PI(4,5)P2-rich environment in the plasma membrane governs WPB fusion possibly by providing interaction sites for WPB-associated docking factors.

| S-EPMC7442956 | biostudies-literature

Role of phosphatidylinositol 4,5-bisphosphate in regulating EHD2 plasma membrane localization.

Project description:The four mammalian C-terminal Eps15 homology domain-containing proteins (EHD1-EHD4) play pivotal roles in endocytic membrane trafficking. While EHD1, EHD3 and EHD4 associate with intracellular tubular/vesicular membranes, EHD2 localizes to the inner leaflet of the plasma membrane. Currently, little is known about the regulation of EHD2. Thus, we sought to define the factors responsible for EHD2's association with the plasma membrane. The subcellular localization of endogenous EHD2 was examined in HeLa cells using confocal microscopy. Although EHD partner proteins typically mediate EHD membrane recruitment, EHD2 was targeted to the plasma membrane independent of two well-characterized binding proteins, syndapin2 and EHBP1. Additionally, the EH domain of EHD2, which facilitates canonical EHD protein interactions, was not required to direct overexpressed EHD2 to the cell surface. On the other hand, several lines of evidence indicate that the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) plays a crucial role in regulating EHD2 subcellular localization. Pharmacologic perturbation of PIP2 metabolism altered PIP2 plasma membrane distribution (as assessed by confocal microscopy), and caused EHD2 to redistribute away from the plasma membrane. Furthermore, overexpressed EHD2 localized to PIP2-enriched vacuoles generated by active Arf6. Finally, we show that although cytochalasin D caused actin microfilaments to collapse, EHD2 was nevertheless maintained at the plasma membrane. Intriguingly, cytochalasin D induced relocalization of both PIP2 and EHD2 to actin aggregates, supporting a role of PIP2 in controlling EHD2 subcellular localization. Altogether, these studies emphasize the significance of membrane lipid composition for EHD2 subcellular distribution and offer new insights into the regulation of this important endocytic protein.

| S-EPMC3769341 | biostudies-literature

α-Synuclein plasma membrane localization correlates with cellular phosphatidylinositol polyphosphate levels.

Project description:The Parkinson's disease protein α-synuclein (αSyn) promotes membrane fusion and fission by interacting with various negatively charged phospholipids. Despite postulated roles in endocytosis and exocytosis, plasma membrane (PM) interactions of αSyn are poorly understood. Here, we show that phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3), two highly acidic components of inner PM leaflets, mediate PM localization of endogenous pools of αSyn in A2780, HeLa, SK-MEL-2, and differentiated and undifferentiated neuronal SH-SY5Y cells. We demonstrate that αSyn binds to reconstituted PIP2 membranes in a helical conformation in vitro and that PIP2 synthesizing kinases and hydrolyzing phosphatases reversibly redistribute αSyn in cells. We further delineate that αSyn-PM targeting follows phosphoinositide-3 kinase (PI3K)-dependent changes of cellular PIP2 and PIP3 levels, which collectively suggests that phosphatidylinositol polyphosphates contribute to αSyn's function(s) at the plasma membrane.

| S-EPMC7929559 | biostudies-literature

Proteomic analysis of plasma membrane and secretory vesicles from human neutrophils.

Project description:BackgroundPolymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometry-based proteomics methods.ResultsTo identify the proteins light membrane fractions enriched for plasma membrane vesicles and secretory vesicles, we employed a proteomic approach, first using MALDI-TOF (peptide mass fingerprinting) and then by HPLC-MS/MS using a 3D ion trap mass spectrometer to analyze the two vesicle populations from resting PMN. We identified several proteins that are functionally important but had not previously been recovered in PMN secretory vesicles. Two such proteins, 5-lipoxygenase-activating protein (FLAP) and dysferlin were further validated by immunoblot analysis.ConclusionOur data demonstrate the broad array of proteins present in secretory vesicles that provides the PMN with the capacity for remarkable and rapid reorganization of its plasma membrane after exposure to proinflammatory agents or stimuli.

| S-EPMC2075486 | biostudies-literature

Phosphatidylinositol (4,5) bisphosphate regulates HIV-1 Gag targeting to the plasma membrane.

Project description:A critical early event in the HIV type 1 (HIV-1) particle assembly pathway is the targeting of the Gag protein to the site of virus assembly. In many cell types, assembly takes place predominantly at the plasma membrane. Cellular factors that regulate Gag targeting remain undefined. The phosphoinositide phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2] controls the plasma membrane localization of a number of cellular proteins. To explore the possibility that this lipid may be involved in Gag targeting and virus particle production, we overexpressed phosphoinositide 5-phosphatase IV, an enzyme that depletes cellular PI(4,5)P2, or overexpressed a constitutively active form of Arf6 (Arf6/Q67L), which induces the formation of PI(4,5)P2-enriched endosomal structures. Both approaches severely reduced virus production. Upon 5-phosphatase IV overexpression, Gag was no longer localized on the plasma membrane but instead was retargeted to late endosomes. Strikingly, in cells expressing Arf6/Q67L, Gag was redirected to the PI(4,5)P2-enriched vesicles and HIV-1 virions budded into these vesicles. These results demonstrate that PI(4,5)P2 plays a key role in Gag targeting to the plasma membrane and thus serves as a cellular determinant of HIV-1 particle production.

| S-EPMC522033 | biostudies-literature

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