Michelyne Breton

Rafts Promote Assembly and Atypical Targeting of a Nonenveloped Virus, Rotavirus, in Caco-2 Cells

ABSTRACT Rotavirus follows an atypical pathway to the apical membrane of intestinal cells that bypasses the Golgi. The involvement of rafts in this process was explored here. VP4 is the most peripheral protein of the triple-layered structure of this nonenveloped virus. High proportions of VP4 associated with rafts within the cell as early as 3 h postinfection. In the meantime a significant part of VP4 was targeted to the Triton X-100-resistant microdomains of the apical membrane, suggesting that this protein possesses an autonomous signal for its targeting. At a later stage the other structural rotavirus proteins were also found in rafts within the cells together with NSP4, a nonstructural protein required for the final stage of virus assembly. Rafts purified from infected cells were shown to contain infectious particles. Finally purified VP4 and mature virus were shown to interact with cholesterol- and sphingolipid-enriched model lipid membranes that changed their phase preference from inverted hexagonal to lamellar structures. Together these results indicate that a direct interaction of VP4 with rafts promotes assembly and atypical targeting of rotavirus in intestinal cells.

Induction by lysophospholipids of CoA-dependent arachidonyl transfer between phospholipids in rat platelet homogenates

Rat platelet homogenates are able to catalyze CoA-mediated, ATP-independent transfer of arachidonic acid from platelet phospholipids to added lysophospholipids. Homogenates of platelets prelabelled with radioactive arachidonic or oleic acid were incubated in the presence of CoA and various lysophospholipids. Transfer observed with arachidonic acid-labelled platelets was dependent on the lysophospholipid added. When 1-alkenyl- or 1-acyllysophosphatidylethanolamine was used, there was a more efficient arachidonyl transfer from phosphatidylcholine than from phosphatidylinositol to the phosphatidylethanolamine fraction. Lysophosphatidylserine also accepted arachidonyl from phosphatidylcholine. Addition of lysophosphatidylcholine resulted in a decrease in the labelling of phosphatidylinositol and to a lesser extent of phosphatidylethanolamine with concomitant transfer to phosphatidylcholine. Lysophosphatidylinositol and lysophosphatic acid did not act as substrate for this transfer reaction. Free, non-radioactive arachidonic acid did not compete for the labelled arachidonic acid transfer. This pathway may play a major role in the synthesis of arachidonyl species of phosphatidylethanolamine and phosphatidylserine and for the arachidonyl transfer to the phosphatidylethanolamine plasmologen in stimulated platelets.

Oxidant-induced arachidonic acid release and impairment of fatty acid acylation in vascular smooth muscle cells

Oxidative damage, which plays a major role in the early stages of atherosclerosis, is associated with arachidonic acid (AA) release in vascular smooth muscle cells (VSMC) as in other cell types. In this study, H2O2was used to investigate mechanisms of AA release from VSMC on oxidative stress. Cell treatment with H2O2inhibited AA incorporation in an inverse relationship to prolonged H2O2-induced AA release. Identical kinetics of inhibition of AA incorporation and AA release were observed after cell treatment with A[Formula: see text], a process not involving phospholipase A2(PLA2) activation as recently described (A. Cane, M. Breton, G. Béréziat, and O. Colard. Biochem. Pharmacol. 53: 327-337, 1997). AA release was not specific, since oleic acid also increased in the extracellular medium of cells treated with H2O2or A[Formula: see text] as measured by gas chromatography-mass spectrometry. In contrast, AA and oleic acid cell content decreased after cell treatment. Oleoyl and arachidonoyl acyl-CoA synthases and acyltransferases, assayed using a cell-free system, were not significantly modified. In contrast, a good correlation was observed between decreases in AA acylation and cell ATP content. The decrease in ATP content is only partially accounted for by mitochondrial damage as assayed by rhodamine 123 assay. We conclude that oxidant-induced arachidonate release results from impairment of fatty acid esterification and that ATP availability is probably responsible for free AA accumulation on oxidative stress by preventing its reesterification and/or transmembrane transport.Oxidative damage, which plays a major role in the early stages of atherosclerosis, is associated with arachidonic acid (AA) release in vascular smooth muscle cells (VSMC) as in other cell types. In this study, H2O2 was used to investigate mechanisms of AA release from VSMC on oxidative stress. Cell treatment with H2O2 inhibited AA incorporation in an inverse relationship to prolonged H2O2-induced AA release. Identical kinetics of inhibition of AA incorporation and AA release were observed after cell treatment with AlF4-, a process not involving phospholipase A2 (PLA2) activation as recently described (A. Cane, M. Breton, G. Béréziat, and O. Colard. Biochem. Pharmacol. 53: 327-337, 1997). AA release was not specific, since oleic acid also increased in the extracellular medium of cells treated with H2O2 or AlF4- as measured by gas chromatography-mass spectrometry. In contrast, AA and oleic acid cell content decreased after cell treatment. Oleoyl and arachidonoyl acyl-CoA synthases and acyltransferases, assayed using a cell-free system, were not significantly modified. In contrast, a good correlation was observed between decreases in AA acylation and cell ATP content. The decrease in ATP content is only partially accounted for by mitochondrial damage as assayed by rhodamine 123 assay. We conclude that oxidant-induced arachidonate release results from impairment of fatty acid esterification and that ATP availability is probably responsible for free AA accumulation on oxidative stress by preventing its reesterification and/or transmembrane transport.

Biosynthesis of paf-acether XVII. Regulation by the CoA-independent transacylase in human neutrophils

Treatment of intact human polymorphonuclear neutrophils (PMN) with low concentrations of phorbol myristate acetate (PMA, 1–10 ng/ml) induced paf‐acether (pat) and lyso paf formation, arachidonate release, and simultaneous inhibition of CoA‐independent lyso paf : transacylase as assayed in a cell‐free system. Inhibition of [3H]lyso paf reacylation was also observed when it was exogenously added to the PMA‐treated intact PMN. When higher concentrations of PMA (40–100 ng/ml) were used, paf biosynthesis was severely impaired and the level of the CoA‐independent transacylase activity returned to basal level. Since lyso paf appears to be the substrate for PMA‐activated paf formation (remodeling pathway), we showed that [14C]acetate was incorporated into the paf molecule. By contrast, labeling with [3H]choline was not appropriate in this model. The presented results are against the involvement of a de novo route in paf synthesis initiated by PMA and open a new possibility of an important role for the CoA‐independent transacylase in controling the level of lyso paf availability for paf formation.

Role for Actin in the Polarized Release of Rotavirus

ABSTRACT Rotaviruses are characterized by polarized release from the apical side of infected enterocytes, and the rotavirus VP4 spike protein specifically binds to the actin network at the apical pole of differentiated enterocytic cells. To determine the functional consequences of this VP4-actin interaction, fluorescence recovery after photobleaching experiments were carried out to measure the diffusional mobility of VP4 associated with the microfilaments. Results show that VP4 binds to barbed ends of microfilaments by using actin treadmilling. Actin treadmilling inhibition results in the loss of rotavirus apical preferential release, suggesting a major role for actin in polarized rotavirus release.

Hydrolysis of endogenous phospholipids by rat platelet phospholipase A2: ether or acyl bond and polar head group selectivity

Substrate specificity of platelet phospholipase A2 was investigated following Ca2+-dependent hydrolysis by endogenous enzyme of linoleate- or arachidonate-labelled platelet phospholipids. Alkylacyl, alkenylacyl and diacyl classes of ethanolamine and choline glycerophospholipids (GPE and GPC) were separated after their diacylglycerol derivation, and molecular species of diacyl-GPE were analyzed by HPLC. Hydrolysis of platelet ethanolamine and choline glycerophospholipids was dependent on Ca2+ and was maximal at neutral pH. In the presence of 0.2 mM Ca2+ the hydrolysis rate for [14C]arachidonate-labelled phospholipids was in the order diacyl-GPE greater than alkylacyl-GPE = diacyl-GPC = alkenylacyl-GPE greater than alkylacyl-GPC. In addition to being the best substrate at high Ca2+ concentration, diacyl-GPE could be degraded with Ca2+ concentrations in the micromolar range, concentrations which are unable to induce any degradation of diacyl-GPC. As a function of Ca2+ concentration, the hydrolysis rate of [14C]linoleate- and [14C]arachidonate-labelled diacyl-GPE or diacyl-GPC was identical. The five main molecular species of diacyl-GPE labelled with arachidonate or with linoleate were hydrolyzed at the same rate in the presence of 50 microM Ca2+. This study shows that platelet phospholipase A2 is specific for endogenous diacyl-GPE and is independent of fatty chain composition. These results are discussed in relation to the Ca2+ concentration observed in stimulated platelets and in relation to the lysophospholipid-induced specific transfer of arachidonate. They suggest that diacyl-GPE hydrolysis by phospholipase A2 could play a key role in stimulated platelets.

Heterogeneity of Raft-Type Membrane Microdomains Associated with VP4, the Rotavirus Spike Protein, in Caco-2 and MA 104 Cells

ABSTRACT Previous studies have shown that rotavirus virions, a major cause of infantile diarrhea, assemble within small intestinal enterocytes and are released at the apical pole without significant cell lysis. In contrast, for the poorly differentiated kidney epithelial MA 104 cells, which have been used extensively to study rotavirus assembly, it has been shown that rotavirus is released by cell lysis. The subsequent discovery that rotavirus particles associate with raft-type membrane microdomains (RTM) in Caco-2 cells provided a simple explanation for rotavirus polarized targeting. However, the results presented here, together with those recently published by another group, demonstrate that rotavirus also associates with RTM in MA 104 cells, thus indicating that a simple interaction of rotavirus with rafts is not sufficient to explain its apical targeting in intestinal cells. In the present study, we explore the possibility that RTM may have distinct physicochemical properties that may account for the differences observed in the rotavirus cell cycle between MA 104 and Caco-2 cells. We show here that VP4 association with rafts is sensitive to cholesterol extraction by methyl-β-cyclodextrin treatment in MA 104 cells and insensitive in Caco-2 cells. Using the VP4 spike protein as bait, VP4-enriched raft subsets were immunopurified. They contained 10 to 15% of the lipids present in total raft membranes. We found that the nature and proportion of phospholipids and glycosphingolipids were different between the two cell lines. We propose that this raft heterogeneity may support the cell type dependency of virus assembly and release.

Heterogeneity of arachidonate and paf-acether precursor pools in mast cells.

In mammalian cells, arachidonate release and paf-acether formation are frequently associated. The alkyl-acyl-GPC has been proposed as an important source for released arachidonic acid and arachidonate-containing alkylacyl-GPC species as unique precursor for paf-acether. However, the specificity of precursor pools either concerning arachidonic acid or paf-acether is still a matter of controversy. We studied the relationship between the precursor pools for both autacoids in antigenically-stimulated cultured mast cells. We took advantage of the particular arachidonate turnover rate in each phospholipid to investigate the role of alkyl-arachidonyl-GPC in the supply of arachidonic acid by using newly and previously [14C]arachidonate-labeled cells. The specific activity of the released arachidonate was reduced 2-fold following overnight cell incubation, whereas labeling in alkyl-arachidonoyl-GPC was only slightly modified and never corresponded to that of released arachidonate when newly or previously labeled cells were triggered with the antigen. These results are not in favor of a major role for alkyl-arachidonoyl-GPC in supplying arachidonate. In contrast, by using previously labeled cells, we demonstrated that all arachidonate-containing phospholipids were involved in the release of arachidonic acid. The pattern of alkyl chains in alkyl-arachidonoyl-GPC, as well as in total alkylacyl-GPC, is unique since it consists mainly of 18:1 (more than 55%), whereas the 16:0 represents only about 30% of total alkyl chains. Therefore, we analyzed paf-acether molecular composition in order to compare it to the alkyl composition of the precursor pools. The content in 18:1 species of paf-acether, as measured by bioassay (aggregation of rabbit platelets), was always lower than that of 16:0 species and then did not correspond to the alkyl composition of the precursor. These data suggest that the enzymes involved in paf synthesis might be specific for 16:0 alkyl chains of precursor pool.

Linoleate incorporation into rat liver membranes phospholipids: effect on plasma membrane ATPase activities and physical properties.

Plasma membrane phospholipids were modified by incubation in the presence of linoleyl-CoA with or without added lysolecithin (LPC) for various length of time. In the absence of LPC, a maximum of 10 nmoles linoleyl-phosphatidylcholine (PC) were synthesized and the ATPase specific activities were not affected whereas in the presence of LPC, when linoleyl-PC synthesis rose from 10 to 80 nmoles, the ATPase activities were decreased. The decrease was similar in the Na,K- or in the Mg-dependent-ATPase and reached maximally 30-40%. LPC by itself did not modify the ATPases. A concomitant decrease in DPH polarization was observed when linoleate was incorporated into phospholipids. We concluded that the decreased ATPase specific activities may be due to an increased fluidity of membranes produced by linoleyl- PC synthesis. We compare this modulation of ATPases by the membrane fluidity with the specific effect of linoleyl- PC species on adenylate cyclase.

Phospholipase A2-dependent and -independent pathways of arachidonate release from vascular smooth muscle cells☆

[Arg8]vasopressin (AVP), through its V1 receptor coupled to GTP-binding proteins, and aluminum fluoride (AlF4-), which directly activates GTP-binding proteins, induced the release of [3H]arachidonate from prelabeled A7r5 vascular smooth muscle-like cells. Using fura-2-loaded cells, we observed that the release induced by AVP occurred concurrently with calcium (Ca2+) mobilization from internal stores and entry of external Ca2+, whereas AlF4(-)-dependent arachidonate release was much slower and was not accompanied by intracellular Ca2+ mobilization. Arachidonate transfer from phosphatidylcholine to phosphatidylethanolamine was an early event for both agonists, but phosphatidylinositol hydrolysis was an early event for AVP-stimulated cells and a late event for cells triggered with AlF4-. In addition, phospholipase inhibitors had no effect on arachidonate release induced by AlF4-. We investigated the enzymatic pathways involved in the releases of arachidonate, which occur in such different ways. Phospholipase A2 activities were assayed in a cell-free system with various substrates, which made it possible to differentiate between cytosolic, secretory and Ca2(+)-independent phospholipases A2. The specific activities were in the order alkenyl-AA-GPE > acyl-AA-GPE > acyl-AA-GPC in the presence of Ca2+. No significant activity was observed in the presence of Ca2+ chelators and when dipalmitoyl-glycerophosphocholine was used as a substrate. Phospholipase A2 activities did not change in homogenates from stimulated cells related to control cells. However, phospholipase A2 activity increased in membrane fractions from AVP-stimulated cells. Imunodetected phosphorylated and unphosphorylated forms of cytosolic phospholipase A2 (cPLA2) also clearly increased in the membrane fractions of AVP-stimulated cells, and only the unphosphorylated form of cPLA2 was present in AlF4(-)-triggered cells. We conclude that phospholipase C and translocation of cPLA2 can account for arachidonate release with AVP stimulation, whereas neither phospholipase C nor any phospholipase A2 activity appears to be implicated in AlF4(-)-dependent arachidonate release.