Polyphosphoinositides are present in plasma membranes isolated from fusogenic carrot cells

Fusogenic carrot cells grown in suspension culture were labeled 12 hours with myo-[2-³H]inositol. Plasma membranes were isolated from the prelabeled fusogenic carrot cells by both aqueous polymer two-phase partitioning and Renografin density gradients. With both methods, the plasma membrane-enriched fractions, as identified by marker enzymes, were enriched in [³H]inositol-labeled phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP₂). An additional [³H]inositol-labeled lipid, lysophosphatidylinositol monophosphate, which migrated between PIP and PIP₂ on thin layer plates, was found primarily in the plasma membrane-rich fraction of the fusogenic cells. This was in contrast to lysophosphatidylinositol which is found primarily in the lower phase, microsomal/mitochrondrial-rich fraction.     

Metabolic pathways for the degradation of phosphatidic acid in isolated nuclei from cerebellar cells

The aim of the present research was to analyse the pathways for phosphatidic acid metabolism in purified nuclei from cerebellar cells. Lipid phosphate phosphatase and diacylglyceride lipase activities were detected in nuclei from cerebellar cells. It was observed that DAGL activity makes up 50% of LPP activity and that PtdOH can also be metabolised to lysophosphatidic acid. With a nuclear protein content of approximately 40 μg, the production of diacylglycerol and monoacylglycerol was linear for 30 min and 5 min, respectively, whereas it increased with PtdOH concentrations of up to 250 μM. LysoPtdOH, sphingosine 1-phosphate and ceramide 1-phosphate, which are alternative substrates for LPP, significantly reduced DAG production from PA. DAG and MAG production increased in the presence of Triton X-100 (1 mM) whereas no modifications were observed in the presence of ionic detergent sodium deoxycholate. Ca²⁺ and Mg²⁺ stimulated MAG production without affecting DAG formation whereas fluoride and vanadate inhibited the generation of both products. Specific PtdOH-phospholipase A1 and PtdOH-phospholipase A2 were also detected in nuclei. Our findings constitute the first reported evidence of active PtdOH metabolism involving LPP, DAGL and PtdOH-selective PLA activities in purified nuclei prepared from cerebellar cells.     

Cooperative phosphoinositide and peptide binding by PSD-95/discs large/ZO-1 (PDZ) domain of polychaetoid, Drosophila zonulin

PDZ domains are well known protein-protein interaction modules that, as part of multidomain proteins, assemble molecular complexes. Some PDZ domains have been reported to interact with membrane lipids, in particular phosphatidylinositol phosphates, but few studies have been aimed at elucidating the prevalence or the molecular details of such interactions. We screened 46 Drosophila PDZ domains for phosphoinositide-dependent cellular localization and discovered that the second PDZ domain of polychaetoid (Pyd PDZ2) interacts with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) at the plasma membrane. Surface plasmon resonance binding experiments with recombinant protein established that Pyd PDZ2 interacts with phosphatidylinositol phosphates with apparent affinities in the micromolar range. Electrostatic interactions involving an extended positively charged surface of Pyd PDZ2 are crucial for the PtdIns(4,5)P(2)-dependent membrane interactions as shown by a combination of three-dimensional modeling, mutagenesis, binding, and localization studies. In vivo localization studies further suggested that both lipid and peptide binding contribute to membrane localization. We identified the transmembrane protein Crumbs as a Pyd PDZ2 ligand and probed the relation between peptide and PtdIns(4,5)P(2) binding. Contrary to the prevalent view on PDZ/peptide/lipid binding, we did not find competition between peptide and lipid ligands. Instead, preloading the protein with the 10-mer Crb3 peptide increased the apparent affinity of Pyd PDZ2 for PtdIns(4,5)P(2) 6-fold. Our results suggest that membrane localization of Pyd PDZ2 may be driven by a combination of peptide and PtdIns(4,5)P(2) binding, which raises the intriguing possibility that the domain may coordinate protein- and phospholipid-mediated signals.     

Mechanistic similarities in docking of the FYVE and PX domains to phosphatidylinositol 3-phosphate containing membranes

Phosphatidylinositol 3-phosphate [PtdIns(3)P], a phospholipid produced by PI 3-kinases in early endosomes and multivesicular bodies, often serves as a marker of endosomal membranes. PtdIns(3)P recruits and activates effector proteins containing the FYVE or PX domain and therefore regulates a variety of biological processes including endo- and exocytosis, membrane trafficking, protein sorting, signal transduction and cytoskeletal rearrangement. Structures and PtdIns(3)P binding modes of several FYVE and PX domains have recently been characterized, unveiling the molecular basis underlying multiple cellular functions of these proteins. Here, structural and functional aspects and current mechanisms of the multivalent membrane anchoring by the FYVE and PX domains are reviewed and compared.     

Phosphatidylinositol 3-kinase and its novel product, phosphatidylinositol 3-phosphate, are present in Saccharomyces cerevisiae

The metabolism of polyphosphoinositides has been shown to be an important factor in controlling the proliferation of Saccharomyces cerevisiae. The monophosphate form of phosphatidylinositol has been assumed to be phosphatidylinositol 4-phosphate (PI-4-P). Recent evidence from our laboratory has established that a phosphatidylinositol (PI) kinase, which phosphorylates the D-3 position of the inositol ring (PI 3-kinase), is associated with many activated protein-tyrosine kinases and may play an important role in the signaling of cell proliferation (Auger, K. R., Serunian, L. A., Soltoff, S. P., Libby, P., and Cantley, L. C. (1989) Cell 57, 167-175). To determine the evolutionary conservation of this enzymatic activity, we investigated its presence in yeast. In vitro PI kinase assays of yeast cell homogenates demonstrated that PI 3-kinase activity was present. Preliminary biochemical characterization of the activity suggested that it was quite different from the mammalian enzyme yet catalyzed the same reaction, i.e. phosphorylating the D-3 hydroxyl position of the inositol ring of phosphatidyl-myo-inositol. [3H]Inositol labeling of intact yeast cells with the subsequent extraction, deacylation, and high performance liquid chromatography analysis of the lipids demonstrated that PI-3-P was as abundant as the PI-4-P isomer. The conservation of the enzymatic activity from yeast to man suggests that it has an important functional role in the cell cycle.     

Balamuthia mandrillaris: The multiple nuclei of Balamuthia amebas; their location, activity, and site of development

Multiple nuclei were first noted in the pseudopodia of Balamuthia mandrillaris amebas feeding on mammalian cells. Phase microscope observations of live amebas in vitro reveal that while many amebas have a single nucleus, others have multiple nuclear-like structures, now confirmed as nuclei with hematoxylin and Feulgen stains. In the live cultures, two nuclei located near the tip of an extended pseudopodium were seen to fuse resulting in one larger morphologic unit. Such merging of nuclei has not been previously reported. Other nuclei were located at positions that subsequently became the site for the outgrowth of an additional pseudopod branch. A newly discovered large structure, a polyploid nucleus, was located in the mid-part of the ameba. Nucleoli of uniform size were seen to develop from the central mass of chromatin and each became surrounded by a vesicular component as they moved into the protoplasm as morphologically complete nuclei.     

Binding sites of fluorescent derivatives of insulin in nuclei,rough endoplasmic reticulum,and mitochondria

Summary Intracellular insulin-binding sites were directly traced in fixed monolayer cultures of a variety of cell types with the use of two fluorescent derivatives of insulin, viz. fluorescein isothiocyanate (FITC)-labelled and tetramethyl rhodamine isothiocyanate (TMRITC)-labelled insulin. Both derivatives retained the property of stimulating DNA synthesis in fibroblasts. Insulin-binding sites were found in the nuclear envelope, nucleoplasm, nucleoli, and in mitochondria and rough endoplasmic reticulum. The identity of these structures was established by concomitant studies on the same cell by means of phase contrast optics and immunocytochemical tracing with specific antibodies to nuclei, mitochondria, or ribosomes. Binding of insulin to the nuclear and cytoplasmic structures was rapid, reversible and saturable, temperature and pH-dependent, and inhibited by an excess of native, but not other, hormones. The staining reactions were sensitive to treatment by the nonionic detergents, NP-40 and TX-100, and to trypsin and pronase, but not to DNase and RNase, suggesting that the binding sites are protein in nature.Supported by a grant from the Anti-Cancer Council of Victoria. We thank Mrs. I. Burns for technical assistance, Dr. H.A. Ward and staff for preparation of the conjugated insulins, and Prof. R.C. Nairn for advice     

Oligomeric forms of potato spindle tuber viroid (PSTV) and of its complementary RNA are present in nuclei isolated from viroid-infected potato cells

Different oligomeric forms of PSTV are detected in nuclei isolated from PSTV-infected potato cells by means of molecular hybridization, using as probes synthetic oligodeoxyribonucleotides with sequence specificity for (+)PSTV and for (?)PSTV. In addition to several species of longer-than-unit-length (?)PSTV molecules, two oligomeric forms os (+)PSTV are detected, which correspond in size to RNA strands of approximately two and three times viroid unit-length. They must be considered as the precursors os the circular and linear (+)PSTV monomers accumulating in the cell nucleus.     

At least 60 ADP-ribosylated variant histones are present in nuclei from dimethylsulfate-treated and untreated cells.

The level of histone adenosine diphospho (ADP) ribosylation was studied in isolated nuclei from mouse myeloma cells in culture. The cells were treated with dimethylsulfate (DMS), a DNA-methylating agent, and histones were analyzed using two-dimensional gel electrophoresis. Seventeen or more bands probably representing mono-to heptadeca (ADP-ribosylated) histones could be visualized for each major variant histone. DMS treatment, by increasing the number of chromatin sites undergoing repair, greatly enhanced histone ADP-ribosylation. When histones were labeled in a cell lysate rather than in isolated nuclei, mono- and oligo(ADP-ribosylated) histone forms prevailed. The presence of approximately 87 ADP-ribosylated variant histone forms in cell lysates and of approximately 170 in isolated nuclei is shown for the first time in this work. Previous studies show multiple ADP-ribosylated forms for only histone H1. The theoretical number of variegated nucleosomes is thus much higher than previously thought, provided that histone-histone contacts are not disrupted at up to a certain level of histone ADP-ribosylation.     

Phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, and the phosphoinositol sphingolipids are found in the plasma membrane and stimulate the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae.

Several plasma membrane phospholipids have been studied for their ability to modulate the activity of the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae. We show here that phosphatidylinositol phosphate (PIP), phosphatidylinositol bisphosphate (PIP2), and/or the phosphatidylinositol and PIP kinases are localized primarily in the plasma membrane. Previous in vivo studies with S. cerevisiae have shown that large, rapid, and reversible changes occur in the levels of PIP and PIP2 congruent with changes in cellular ATP levels. We demonstrate here that isolated plasma membranes exhibit the same changes in PIP and PIP2 content when they are supplied with or washed free of ATP. Using a mixed micellar assay we systematically studied the efficacy of the plasma membrane lipids in sustaining the activity of the plasma membrane H(+)-ATPase. We demonstrate for the first time that a number of plasma membrane glycerophospholipids effectively stimulate the ATPase, including PIP, PIP2, and cardiolipin. Phosphoinositol-containing sphingolipids, major components of the plasma membrane, are also shown to stimulate the ATPase at significantly lower levels than the glycerophospholipids and must also be considered as important effectors in vivo.     

Control of membrane fusion by phospholipid head groups II. The role of phosphatidylethanolamine in mixtures with phosphatidate and phosphatidylinositol

Membrane fusion induced by Ca²⁺ and Mg²⁺ in large unilamellar vesicles composed of mixtures of phosphatidylethanolamine with phosphatidate and phosphatidylinositol was studied by means of a fluorescence assay for the intermixing of internal aqueous contents of the vesicles. The threshold concentrations of Ca²⁺ or Mg²⁺ required for fusion increased only moderately when up to 80 mol% phosphatidylethanolamine was included with phosphatidate at pH 7.4, but no fusion could be detected in vesicles containing 70 mol% phosphatidylcholine even at high concentrations of Ca²⁺ or Mg²⁺. Phosphatidate-phosphatidylethanolamine (1 : 4) vesicles could be induced to fuse by 0.1 mM Ca²⁺ in the presence of a Mg²⁺ concentration which alone was insufficient for fusion. When equimolar amounts of phosphatidylethanolamine was included with phosphatidylinositol, the vesicles were susceptible to fusion by Ca²⁺, although pure phosphatidylinositol vesicles themselves merely aggregate and do not fuse (Sundler, R. and Papahadjopoulos, D. (1981) Biochim. Biophys. Acta 649, 743–750, accompanying paper). The role of phosphatidylethanolamine acyl chains, and hence the possible involvement of the bilayer-hexagonal (H_II) transition in membrane fusion, was examined by the temperature dependence of Ca²⁺-induced fusion in phosphatidylinositol-dimyristoylphosphatidylethanolamine (1 : 1) vesicles. Fusion was strictly dependent on the gel-liquid crystalline transition of the mixture and not on the phase behavior of the phosphatidylethanolamines. Comparable fusion rates were obtained for both egg yolk phosphatidylethanolamine and dimyristoylphosphatidylethanolamine at 50°C. As the dimyristoylphosphatidylethanolamine does not convert to a non-bilayer phase in this temperature range, we conclude that the bilayer-hexagonal transition is not necessary for membrane fusion. We propose that the dehydration characteristics of the phospholipids and their metal ion complexes are the critical factors determining fusion suceptibility of phospholipid membranes.     

Phosphatidylinositol hydrolysis and phosphatidylinositol 4′,5′-diphosphate hydrolysis are separable responses during secretagogue action in the rat pancreas

Rat pancreatic fragments and acinar preparations were incubated in vitro to characterize further the changes in phosphoinositide metabolism that occur during secretagogue action. Two distinct responses were discernible. The first response, most notably involving a decrease in phosphatidylinositol content, was (a) observed at lower carbachol concentrations in dose-response studies, (b) inhibited by incubation in Ca²⁺-free media containing 1 mM EGTA, (c) associated with increases in inositol monophosphate production, and (d) provoked by all tissue secretagogues (carbachol, cholecystokinin, secretin, insulin, dibutyryl cAMP and the ionophore A23187), regardless of whether their mechanism of action primarily involved Ca²⁺ mobilization or cAMP generation. This decrease in phosphatidylinositol content was at least partly due to phospholipase C (and/or D) activation, as evidenced by the increase in inositol monophosphate. The second response, most notably involving markedly increased incorporation of ³²PO₄ into phosphatidic acid and phosphatidylinositol, was (a) observed at higher carbachol concentrations, (b) not influenced by incubation in Ca²⁺-free media containing 1 mM EGTA, and (c) associated with increases in inositol triphosphate production. This ³²PO₄ turnover response was probably largely the result of phospholipase C-mediated hydrolysis of phosphatidylinositol 4′,5′-diphosphate, which, as shown previously, also occurs at higher carbachol concentrations and is insensitive to comparable EGTA-induced Ca²⁺ deficiency. This phosphatidylinositol 4′,5′-diphosphate hydrolysis response was only observed in the action of agents (carbachol and cholecystokinin) which mobilize Ca²⁺ via activation of cell surface receptors. The present results indicate that phosphatidylinositol and phosphatidylinositol 4′,5′-diphosphate hydrolysis are truly separable responses to secretagogues acting in the rat pancreas. Furthermore, phosphatidylinositol 4′,5′-diphosphate, rather than phosphatidylinositol hydrolysis is more likely to be associated with receptor activation and Ca²⁺ mobilization.     

Changes in phosphatidylinositol and phosphatidic acid in stimulated human neutrophils: Relationship to calcium mobilization,aggregation and superoxide radical generation

Human neutrophils aggregate and release mediators of inflammation, such as active oxygen species and lysosomal enzymes, when exposed to the chemoattractant, fMet-Leu-Phe, or the tumor promotor, phorbol myristate acetate. In order to ‘stage’ events which may lead to such neutrophil responses, we determined the temporal relationship between stimulus-induced changes in the endogenous phospholipids phosphatidylinositol (PI) and phosphatidic acid, the mobilization of calcium, and the onset of aggregation and generation of superoxide anion during the initial 2 min of cell activation. Within 5 s after addition of fMet-Leu-Phe (10^?7 M) neutrophils accumulated phosphatidic acid and the levels of PI decreased, as determined by two-dimensional thin-layer chromatography and phosphorus determinations. By 5 s, phosphatidic acid levels rose approximately 3.5-fold and at 15 s the loss of PI exceeded the quantity of phosphatidic acid generated. In response to phorbol myristate acetate (1 μg/ml), however, changes in PI or phosphatidic acid were not observed until after 60 s. Accumulation of phosphatidic acid in fMet-Leu-Phe-stimulated cells was not inhibited by chelation of extracellular calcium. Neutrophils exposed to either fMet-Leu-Phe or phorbol myristate acetate also showed rapid decrements in fluorescence of cell-associated chlorotetracycline (used as an indirect probe of mobilization of intracellular membrane-associated calcium) and took up ⁴⁵Ca²⁺ from the extracellular medium (under 60 s). The results indicate that changes in calcium mobilization, together with the alterations in phospholipid metabolism (under 5 s) anteceded aggregation and the generation of O^?₂ (10–15 s) induced by fMet-Leu-Phe. In contrast, when neutrophils were exposed to phorbol myristate acetate, changes in PI and phosphatidic acid (over 60 s) were observed after the mobilization of calcium (under 5 s) and the onset of O^?₂ generation and aggregation (30–35 s).     

Novel roles of phosphoinositides in signaling,lipid transport,and disease

Phosphoinositides (PPIns) are lipid signaling molecules that act as master regulators of cellular signaling. Recent studies have revealed novel roles of PPIns in myriad cellular processes and multiple human diseases mediated by misregulation of PPIn signaling. This review will present a timely summary of recent discoveries in PPIn biology, specifically their role in regulating unexpected signaling pathways, modification of signaling outcomes downstream of integral membrane proteins, and novel roles in lipid transport. This has revealed new roles of PPIns in regulating membrane trafficking, immunity, cell polarity, and response to extracellular signals. A specific focus will be on novel opportunities to target PPIn metabolism for treatment of human diseases, including cancer, pathogen infection, developmental disorders, and immune disorders.     

The Yersinia HPI is present in Serratia liquefaciens isolated from meat

AIMS: The aim of the study was to screen the Enterobacteriaceae flora of meat for the presence of bacteria harbouring the Yersinia high-pathogenicity island (HPI). METHODS AND RESULTS: Bacteria from 29 meat and 29 liver samples were isolated on violet-red bile glucose agar. A total of 197 isolates were screened for the presence of the irp2 gene, encoded within the HPI, by PCR. One isolate that was positive for irp2 gene was also positive for the fyuA, irp1, ybtP/ybtQ, ybtX/ybtS and int/asn tRNA genes by PCR. The presence of fyuA, irp1 and irp2 genes was confirmed by Southern hybridization. CONCLUSIONS: The isolate was identified as Serratia liquefaciens by sequencing of the 16S rRNA gene and by ribotyping. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a Serratia harbouring the Yersinia HPI. Serratia is a frequently occurring Enterobacteriaceae genus in chill-stored meat.     

The characteristics of liver glucose-6-phosphatase in the envelope of isolated nuclei and microsomes are identical

We have compared the characteristics of glucose-6-phosphatase (EC 3.1.3.9) in the envelope of purified nuclei and microsomes from rat liver. The latency of mannose-6-P hydrolysis, permeability to EDTA, and susceptibility of the enzyme to protease-mediated inactivation all indicated that the permeability barrier defined by the envelope in situ is significantly disrupted in isolated nuclei (i.e. in vitro). Latency of mannose-6-P hydrolysis was demonstrated to provide a quantitative measure of the degree of nuclear membrane disruption. Electron micrographs confirmed the existence of substantial regions of the envelope in vitro where the permeability barrier to EDTA was intact (i.e. an "intact component"). The kinetics of glucose-6-phosphatase catalyzed by the intact component was obtained by subtracting the contribution of enzyme in disrupted regions from the total enzymic activity of untreated nuclei. The characteristics of glucose-6-phosphatase in intact and fully disrupted membranes of nuclei were indistinguishable from microsomes with respect to (a) the kinetics of glucose-6-P hydrolysis, (b) the effects of incubations with mannose-6-P, N-ethylmaleimide, and protease from Bacillus amyloliquefaciens, (c) the extremely high latency of carbamyl phosphate:glucose phosphotransferase activity, and (d) both the patterns of response of activity and the change in latency of glucose-6-phosphatase induced by fasting, experimental diabetes, and cortisol injection. Our results show clearly that apparent differences in the glucose-6-phosphatase activity of untreated preparations of nuclei and microsomes are simply expressions of significant differences in the degree of intactness of their respective permeability barriers. Since flattened cisternae, characteristic of the rough endoplasmic reticulum in situ, are preserved in intact regions of the envelope of isolated nuclei, the present findings constitute the most direct and definitive evidence to date that the properties of glucose-6-phosphatase in the endoplasmic reticulum in situ are faithfully reproduced with intact microsomes.     

Activation of moesin, a protein that links actin cytoskeleton to the plasma membrane, occurs by phosphatidylinositol 4,5-bisphosphate (PIP2) binding sequentially to two sites and releasing an autoinhibitory linker

Many cellular processes depend on ERM (ezrin, moesin, and radixin) proteins mediating regulated linkage between plasma membrane and actin cytoskeleton. Although conformational activation of the ERM protein is mediated by the membrane PIP2, the known properties of the two described PIP2-binding sites do not explain activation. To elucidate the structural basis of possible mechanisms, we generated informative moesin mutations and tested three attributes: membrane localization of the expressed moesin, moesin binding to PIP2, and PIP2-induced release of moesin autoinhibition. The results demonstrate for the first time that the POCKET containing inositol 1,4,5-trisphosphate on crystal structure (the "POCKET" Lys-63, Lys-278 residues) mediates all three functions. Furthermore the second described PIP2-binding site (the "PATCH," Lys-253/Lys-254, Lys-262/Lys-263) is also essential for all three functions. In native autoinhibited ERM proteins, the POCKET is a cavity masked by an acidic linker, which we designate the "FLAP." Analysis of three mutant moesin constructs predicted to influence FLAP function demonstrated that the FLAP is a functional autoinhibitory region. Moreover, analysis of the cooperativity and stoichiometry demonstrate that the PATCH and POCKET do not bind PIP2 simultaneously. Based on our data and supporting published data, we propose a model of progressive activation of autoinhibited moesin by a single PIP2 molecule in the membrane. Initial transient binding of PIP2 to the PATCH initiates release of the FLAP, which enables transition of the same PIP2 molecule into the newly exposed POCKET where it binds stably and completes the conformational activation.