Rapid, high-yield purification of cell surface membrane using colloidal magnetite coated with polyvinylamine: sedimentation versus magnetic isolation

A new technique for the magnetic isolation of external plasma membrane from Dictyostelium discoideum is described and compared to a previously published procedure employing sedimentation of silica-coated plasma membrane. The magnetic isolation technique involves coating intact cells with a polyvinylamine-magnetite colloid and overcoating with polyacrylate to form a dense pellicle. The magnetite pellicle totally coated the cells and was not internalized. Coated cells were lysed and membrane fragments retrieved from the cell homogenate using a diverging field electromagnet. The membrane obtained in such a manner was analyzed for marker enzyme activity and cell surface label. The plasma membrane was obtained in high yield (42%) with an average purification of 8-fold. The polyvinylamine-magnetite pellicle shielded the external plasma membrane face to proteolysis by papain and pronase. It also acted as a barrier to alpha-methylmannoside in concanavalin A-carbohydrate competition studies.     

Periodic fluctuations in oxygen consumption comparing HeLa (cancer) and CHO (non-cancer) cells and response to external NAD(P)+/NAD(P)H

Oxygen consumption in the presence of cyanide was utilized as a measure of plasma membrane electron transport in Chinese hamster ovary (CHO) and human cervical carcinoma (HeLa) cell lines. Both intact cells and isolated plasma membranes carry cyanide-insensitive NADH(P)H oxidases at their external membrane surfaces (designated ECTO-NOX proteins). Regular oscillatory patterns of oxygen consumption with period lengths characteristic of those observed for rates of NADH oxidation by ECTO-NOX proteins were observed to provide evidence for transfer of protons and electrons to reduce oxygen to water. The oscillations plus the resistance to inhibition by cyanide identify the bulk of the oxygen consumption as due to ECTO-NOX proteins. With intact CHO cells, oxygen consumption was enhanced by but not dependent upon external NAD(P)H addition. With intact HeLa cells, oxygen consumption was inhibited by both NADH and NAD⁺ as was growth. The results suggest that plasma membrane electron transport from internal donors to oxygen as an external acceptor is mediated through ECTO-NOX proteins and that electron transport to molecular oxygen may be differentially affected by external pyridine nucleotides depending on cell type.     

Immunocytological localization of an epitope-tagged plasma membrane proton pump (H(+)-ATPase) in phloem companion cells.

In higher plants, the plasma membrane proton pump (H(+)-ATPase) is encoded by a surprisingly large multigene family whose members are expressed in different tissues. Using an 18-amino acid epitope tag derived from the animal oncogene c-Myc, we have performed immunocytolocalization measurements of the protein expressed by one member of this family, AHA3 (Arabidopsis H(+)-ATPase isoform 3). Immunofluorescence studies with tissue sections of transgenic plants have revealed that c-Myc-tagged AHA3 is restricted to the plasma membrane of phloem companion cells, whereas other AHA isoproteins are more widely distributed in the plasma membrane of other cell types. Electron microscopy with immunogold-labeled tissue sections suggests that there is a high concentration of proton pumps in the plasma membrane of companion cells but a much lower concentration in the plasma membrane of sieve elements. Due to plasmodesmata connecting the plasma membrane of these two adjacent cell types, it is likely that the proton motive force generated by the proton pump in companion cells can serve to power the uptake of sugar by proton-coupled symporters in either the companion cell or sieve element cell. The abundance of the proton pump in the plasma membrane of companion cells supports an apoplastic model for phloem loading in which the metabolic energy that drives sugar uptake is consumed by AHA3 at the companion cell plasma membrane. These experiments with a genetically altered integral plasma membrane protein demonstrate the utility of using a short c-Myc sequence as an epitope tag in Arabidopsis. Furthermore, our results demonstrate that, using genes encoding individual members of a gene family, it is possible to label plasma membrane proteins immunologically in specific, differentiated cell types of higher plants.     

One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A

We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membrane proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment of plasma membrane marker 5′-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5′-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1 was enriched about threefold relative to that of the original membranes. In similar experiments, this method produced 13-fold enrichment of 5′-nucleotidase activity with 45% recovery of the activity from a total cell lysate of PC-3 cells and 7.1-fold enrichment of 5′-nucleotidase activity with 33% recovery of the activity from a total cell lysate of HeLa cells. These results suggest that this one-step purification method can be used to isolate total plasma membrane proteins from tissue or cells for the identification of membrane biomarkers.     

Applications of second harmonic generation (SHG)/sum-frequency generation (SFG) imaging for biophysical characterization of the plasma membrane

The plasma membrane is a lipid bilayer of < 10 nm width that separates intra- and extra-cellular environments and serves as the site of cell-cell communication, as well as communication between cells and the extracellular environment. As such, biophysical phenomena at and around the plasma membrane play key roles in determining cellular physiology and pathophysiology. Thus, the selective visualization and characterization of the plasma membrane are crucial aspects of research in wide areas of biology and medicine. However, the specific characterization of the plasma membrane has been a challenge using conventional imaging techniques, which are unable to effectively distinguish between signals arising from the plasma membrane and those from intracellular lipid structures. In this regard, interface-specific second harmonic generation (SHG) and sum-frequency generation (SFG) imaging demonstrate great potential. When combined with exogenous SHG/SFG active dyes, SHG/SFG can specifically highlight the plasma membrane as the most prominent interface associated with cells. Furthermore, SHG/SFG imaging can be readily extended to multimodal multiphoton microscopy with simultaneous occurrence of other multiphoton phenomena, including multiphoton excitation and coherent Raman scattering, which shed light on the biophysical properties of the plasma membrane from different perspectives. Here, we review traditional and current applications, as well as the prospects of long-known but unexplored SHG/SFG imaging techniques in biophysics, with special focus on their use in the biophysical characterization of the plasma membrane.     

Glucose recruits K(ATP) channels via non-insulin-containing dense-core granules

beta cells rely on adenosine triphosphate-sensitive potassium (K(ATP)) channels to initiate and end glucose-stimulated insulin secretion through changes in membrane potential. These channels may also act as a constituent of the exocytotic machinery to mediate insulin release independent of their electrical function. However, the molecular mechanisms whereby the beta cell plasma membrane maintains an appropriate number of K(ATP) channels are not known. We now show that glucose increases K(ATP) current amplitude by increasing the number of K(ATP) channels in the beta cell plasma membrane. The effect was blocked by inhibition of protein kinase A (PKA) as well as by depletion of extracellular or intracellular Ca(2+). Furthermore, glucose promoted recruitment of the potassium inward rectifier 6.2 to the plasma membrane, and intracellular K(ATP) channels localized in chromogranin-positive/insulin-negative dense-core granules. Our data suggest that glucose can recruit K(ATP) channels to the beta cell plasma membrane via non-insulin-containing dense-core granules in a Ca(2+)- and PKA-dependent manner.     

Interaction with Gbetagamma is required for membrane targeting and palmitoylation of Galpha(s) and Galpha(q)

Peripheral membrane proteins utilize a variety of mechanisms to attach tightly, and often reversibly, to cellular membranes. The covalent lipid modifications, myristoylation and palmitoylation, are critical for plasma membrane localization of heterotrimeric G protein alpha subunits. For alpha(s) and alpha(q), two subunits that are palmitoylated but not myristoylated, we examined the importance of interacting with the G protein betagamma dimer for their proper plasma membrane localization and palmitoylation. Conserved alpha subunit N-terminal amino acids predicted to mediate binding to betagamma were mutated to create a series of betagamma binding region mutants expressed in HEK293 cells. These alpha(s) and alpha(q) mutants were found in soluble rather than particulate fractions, and they no longer localized to plasma membranes as demonstrated by immunofluorescence microscopy. The mutations also inhibited incorporation of radiolabeled palmitate into the proteins and abrogated their signaling ability. Additional alpha(q) mutants, which contain these mutations but are modified by both myristate and palmitate, retained their localization to plasma membranes and ability to undergo palmitoylation. These findings identify binding to betagamma as a critical membrane attachment signal for alpha(s) and alpha(q) and as a prerequisite for their palmitoylation, while myristoylation can restore membrane localization and palmitoylation of betagamma binding-deficient alpha(q) subunits.     

Plasma membrane specializations in the cells of the kidney distal segment of the lamprey, Lampetra japonica (von Martens)

The unique and highly specialized structural features of the plasma membrane in the cells of the kidney distal segment of the lamprey, Lampetra japonica, were studied by electron microscopy. The cells of the distal segment are largely filled by a continuous network of cytoplasmic tubules which are derived from the basolateral plasma membrane. Thin sections and freeze-fracture replicas of the membrane of the cytoplasmic tubules show spirally wound parallel rows of particles. The rows are approximately 17 nm apart and are wound at a pitch of approximately 45 degrees with respect to the major axis of the tubules. Another type of membrane specialization was found in the freeze-cleaved surface of the basolateral plasma membrane. It consists of large square aggregations of membrane particles containing 100-400 cuboidal subunits. The distribution of these particles in this cell, as well as in other systems in which they have been noted, suggests a polarization of membrane activity.     

Salt effects on membranes of the hypodermis and mesophyll cells of Avicennia germinans (Avicenniaceae): a freeze-fracture study

Freeze-fracture electron microscopy was used to investigate intramembranous particle (IMP) densities and particle distributions in the plasma membrane and tonoplast of the cells of secreting and nonsecreting leaves of Avicennia germinans (L.) Steam. Intramembranous particle densities of the protoplasmic (P) and exoplasmic (E) face of the plasma membrane and tonoplast were significantly higher in hypodermal cells of secreting leaves than of nonsecreting leaves. In contrast, no significant differences in the frequency of intramembranous particles were found in any membrane faces of secreting or nonsecreting mesophyll cells. However, particle densities were higher in the plasma membrane and tonoplast of the mesophyll cells, compared to the hypodermal cells, with the exception of the P-face of hypodermal plasma membranes of secreting tissue, which had the highest particle density measured. Particle distributions were dispersed and no discernible patterns such as paracrystalline arrays or other multi-IMP structures were observed. Results support the hypothesis that secretion is coupled to changes in membrane ultrastructure, and the possibility that salt secretion is an active process driven by integral membrane proteins such as the H⁺/ATPase. Additionally, the hypodermal cells of the leaf may function as storage reservoirs for salt as well as water, suggesting a regulatory role in salt secretion.     

Docosahexaenoic acid (DHA) alters the phospholipid molecular species composition of membranous vesicles exfoliated from the surface of a murine leukemia cell line

Previously, we presented evidence that the vesicles routinely exfoliated from the surface of T27A tumor cells arise from vesicle-forming regions of the plasma membrane and possess a set of lateral microdomains distinct from those of the plasma membrane as a whole. We also showed that docosahexaenoic acid (DHA, or 22:6n-3), a fatty acyl chain known to alter microdomain structure in model membranes, also alters the structure and composition of exfoliated vesicles, implying a DHA-induced change in microdomain structure on the cell surface. In this report we show that enrichment of the cells with DHA reverses some of the characteristic differences in composition between the parent plasma membrane and shed microdomain vesicles, but does not alter their phospholipid class composition. In untreated cells, DHA-containing species were found to be a much greater proportion of the total phosphatidylethanolamine (PE) pool than the total phosphatidylcholine (PC) pool in both the plasma membrane and the shed vesicles. After DHA treatment, the proportion of DHA-containing species in the PE and PC pools of the plasma membrane were elevated, and unlike in untreated cells, their proportions were equal in the two pools. In the vesicles shed from DHA-loaded cells, the proportion of DHA-containing species of PE was the same as in the plasma membrane. However, the proportion of DHA-containing species of PC in the vesicles (0.089) was much lower than that found in the plasma membrane (0.194), and was relatively devoid of species with 16-carbon acyl components. These data suggested that DHA-containing species of PC, particularly those having a 16-carbon chain in the sn-1 position, were preferentially retained in the plasma membrane. The data can be interpreted as indicating that DHA induces a restructuring of lateral microdomains on the surface of living cells similar to that predicted by its behavior in model membranes.     

Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells

To study the intracellular transport of newly synthesized sphingolipids in epithelial cells we have used a fluorescent ceramide analog, N-6[7-nitro-2,1,3-benzoxadiazol-4-yl] aminocaproyl sphingosine (C6-NBD-ceramide; Lipsky, N. G., and R. E. Pagano, 1983, Proc. Natl. Acad. Sci. USA, 80:2608-2612) as a probe. This ceramide was readily taken up by filter-grown Madin-Darby canine kidney (MDCK) cells from liposomes at 0 degrees C. After penetration into the cell, the fluorescent probe accumulated in the Golgi area at temperatures between 0 and 20 degrees C. Chemical analysis showed that C6-NBD-ceramide was being converted into C6-NBD-sphingomyelin and C6-NBD-glucosyl-ceramide. An analysis of the fluorescence pattern after 1 h at 20 degrees C by means of a confocal scanning laser fluorescence microscope revealed that the fluorescent marker most likely concentrated in the Golgi complex itself. Little fluorescence was observed at the plasma membrane. Raising the temperature to 37 degrees C for 1 h resulted in intense plasma membrane staining and a loss of fluorescence from the Golgi complex. Addition of BSA to the apical medium cleared the fluorescence from the apical but not from the basolateral plasma membrane domain. The basolateral fluorescence could be depleted only by adding BSA to the basal side of a monolayer of MDCK cells grown on polycarbonate filters. We conclude that the fluorescent sphingomyelin and glucosylceramide were delivered from the Golgi complex to the plasma membrane where they accumulated in the external leaflet of the membrane bilayer. The results also demonstrated that the fatty acyl labeled lipids were unable to pass the tight junctions in either direction. Quantitation of the amount of NBD-lipids delivered to the apical and the basolateral plasma membranes during incubation for 1 h at 37 degrees C showed that the C6-NBD-glucosylceramide was two- to fourfold enriched on the apical as compared to the basolateral side, while C6-NBD-sphingomyelin was about equally distributed. Since the surface area of the apical plasma membrane is much smaller than that of the basolateral membrane, both lipids achieved a higher concentration on the apical surface. Altogether, our results suggest that the NBD-lipids are sorted in MDCK cells in a way similar to their natural counterparts.     

Compactin (ML-236B) reduces the content of filipin-cholesterol complexes in the plasma membrane of chronic lymphocytic leukemia cells

The polyene antibiotic filipin was used to visualize the presence and distribution of cholesterol in the plasma membrane of glutaraldehyde-fixed human chronic lymphocytic leukemia (CLL) cells. Both compactin (ML-236B), a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and 25-hydroxycholesterol reduced the content of filipin-cholesterol complexes in the plasma membrane of CLL cells grown in media supplemented with either 15% delipidized horse serum or 15% normal (whole) horse serum. The reduction due to compactin was reversed by the concomitant addition of mevalonolactone. The ability of compactin to reduce the relative cholesterol content (as judged by filipin labeling) in CLL cells grown in lipoprotein-containing (normal) serum suggest that either CLL cells are different from other cells in that they predominantly utilize endogenously synthesized cholesterol for incorporation into the plasma membrane, or that a separate pool of endogenously synthesized cholesterol provides cholesterol for the plasma membrane.     

The role of biomembranes in chromium (III)-induced toxicity in vitro

The role of biomembranes in the chronic toxicity of environmentally occurring chromium acetate hydroxide was investigated by using primary human fibroblasts. Transport of chromium acetate hydroxide across the plasma membrane of the cell, and the effects of chromium (III) ions on the plasma membrane as well as other intracellular membranes, were determined during six weeks of continuous exposure by using atomic absorption spectrometry, observation of cell morphology, membrane integrity assays (for lactate dehydrogenase leakage and lysosomal membrane disruption), and mitochondrial assays (for mitochondrial dehydrogenase activity and mitochondrial transmembrane potential analysis). The type of cell death induced by long-term exposure was determined in terms of phosphatidylserine externalisation, caspase-3 activation, and chromatin fragmentation. Chromium acetate hydroxide, at a concentration of 100 micromol/l, accumulated in exposed cells, inflicting plasma membrane damage and suppressing mitochondrial function. Antioxidant co-enzyme Q, at a concentration of 10 micromol/l, partially prevented plasma membrane damage and mitochondrial dysfunction. Exposure to chromium acetate hydroxide produced apoptosis, necrosis and an intermediate type of cell death in primary human fibroblasts. These results show that the plasma membrane and mitochondrial membrane are important targets for chronic chromium acetate hydroxide toxicity, and that this in vitro system holds promise for studying the toxicity resulting from long-term exposure to metal ions.     

Plasma membrane structure in spermatogenic cells of the swordtail (teleostei,Xiphophorus helleri)

The plasma membrane of spermatogenic cells of the teleost Xiphophorus helleri was examined ultrastructurally and cytochemically in order to characterize the temporal development of the membrane specializations characteristic of the mature spermatozoon. Mature sperm display a mosaic distribution of Concanavalin A and Ricinus comrnunis I binding sites; the anterior region of the head displays an intense binding that is not seen in other surface regions. This asymmetric binding is evident in early spermatids and the area of lectin binding appears associated with the plasma membrane overlying the nucleus. Transmission electron microscopy reveals that the plasma membrane over the anterior region of the head is characterized by an ordered glycocalyx and a tight adherence to the underlying nucleus. Additional membrane differentiations were revealed both in the midpiece region where a “submitochondrial net” is attached to the plasma membrane and at the base of the axoneme where the plasma membrane possesses a “collar-like” arrangement of circumferential rings. The possible functions of these differentiations, as well as their correlation to differentiations seen in sperm of other animal groups, are discussed.     

Death by apoptosis in salivary glands of females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

The salivary glands of females of the tick Rhipicephalus sanguineus at three feeding stages: unfed, engorged, and at day three post-engorgement, were subjected to cytochemical methods of enzymatic analysis and cell viability. Comparing glands at these stages, was observed distinct staining patterns in cells of different types of acini, specially in degenerating types III, II, I, which were affected in this sequence by cell death. This study also revealed changes in: nuclei, staining intensity for acid phosphatase and ATPase activities, and permeability of the plasma membrane. Acid phosphatase activity was inversely proportional to that of ATPase, while ATPase activity was always proportional to membrane integrity. The glands of unfed females exhibited high metabolic activity and cells with intact nucleus and plasma membrane, suggesting that the presence of acid phosphatase detected in these individuals may participate in the normal physiology of some acini, as they were not undergoing degeneration. In acini I and II of engorged females, we observed cells with intact membranes, as well as changes characterized by nuclear changes, decrease in ATPase activity, and stronger acid phosphatase activity. At day three post-engorgement, degeneration progressed to more advanced stages, loss of membrane integrity was observed in most cells (of some type I acini, most type II acini, and all type III acini), as well as prominent nuclear changes, decrease in ATPase activity, and intense acid phosphatase activity, resulting in apoptotic bodies. During the death of cells nuclear changes preceded cytoplasmic ones in the following sequence: nuclear changes, loss of ATPase activity, loss of integrity of the plasma membrane, increase in acid phosphatase activity, and formation of apoptotic bodies. The presence of acid phosphatase with a secondary role (late) during cell death, degrading final cell remnants, characterized this process in the glands of R. sanguineus females as atypical or non-classic apoptosis.     

Rapid stiffening of integrin receptor-actin linkages in endothelial cells stimulated with thrombin: a magnetic bead microrheology study

By using magnetic bead microrheology we study the effect of inflammatory agents and toxins on the viscoelastic moduli of endothelial cell plasma membranes in real time. Viscoelastic response curves were acquired by applying short force pulses of ~500 pN to fibronectin-coated magnetic beads attached to the surface membrane of endothelial cells. Upon addition of thrombin, a rapid stiffening of the membrane was observed within 5 s, followed by recovery of the initial deformability within 2 min. By using specific inhibitors, two known pathways by which thrombin induces actin reorganization in endothelial cells, namely activation of Ca2+-calmodulin-dependent myosin light chain kinase and stimulation of Rho/Rho-kinase, were excluded as possible causes of the stiffening effect. Interestingly, the cytotoxic necrotizing factor of Escherichia coli, a toxin which, in addition to Rho, activates the GTPases Rac and CDC42Hs, also induced a dramatic stiffening effect, suggesting that the stiffening may be mediated through a Rac- or Cdc42Hs-dependent pathway. This work demonstrates that magnetic bead microrheometry is not only a powerful tool to determine the absolute viscoelastic moduli of the composite cell plasma membrane, but also a valuable tool to study in real time the effect of drugs or toxins on the viscoelastic parameters of the plasma membrane.     

Identification of a calmodulin-regulated soybean Ca(2+)-ATPase (SCA1) that is located in the plasma membrane

Ca(2)+-ATPases are key regulators of Ca(2+) ion efflux in all eukaryotes. Animal cells have two distinct families of Ca(2+) pumps, with calmodulin-stimulated pumps (type IIB pumps) found exclusively at the plasma membrane. In plants, no equivalent type IIB pump located at the plasma membrane has been identified at the molecular level, although related isoforms have been identified in non-plasma membrane locations. Here, we identify a plant cDNA, designated SCA1 (for soybean Ca(2+)-ATPase 1), that encodes Ca(2+)-ATPase and is located at the plasma membrane. The plasma membrane localization was determined by sucrose gradient and aqueous two-phase membrane fractionations and was confirmed by the localization of SCA1p tagged with a green fluorescent protein. The Ca(2+)-ATPase activity of the SCA1p was increased approximately sixfold by calmodulin (K(1/2) approximately 10 nM). Two calmodulin binding sequences were identified in the N-terminal domain. An N-terminal truncation mutant that deletes sequence through the two calmodulin binding sites was able to complement a yeast mutant (K616) that was deficient in two endogenous Ca(2+) pumps. Our results indicate that SCA1p is structurally distinct from the plasma membrane-localized Ca(2+) pump in animal cells, belonging instead to a novel family of plant type IIB pumps found in multiple subcellular locations. In plant cells from soybean, expression of this plasma membrane pump was highly and rapidly induced by salt (NaCl) stress and a fungal elicitor but not by osmotic stress.     

Spatial and temporal regulation of protein kinase D (PKD)

Protein kinase D (PKD; also known as PKCmicro) is a serine/threonine kinase activated by diacylglycerol signalling pathways in a variety of cells. PKD has been described previously as Golgi-localized, but herein we show that it is present within the cytosol of quiescent B cells and mast cells and moves rapidly to the plasma membrane after antigen receptor triggering. The membrane redistribution of PKD requires the diacylglycerol-binding domain of the enzyme, but is independent of its catalytic activity and does not require the integrity of the pleckstrin homology domain. Antigen receptor signalling initiates in glycosphingolipid-enriched microdomains, but membrane-associated PKD does not co-localize with these specialized structures. Membrane targeting of PKD is transient, the enzyme returns to the cytosol within 10 min of antigen receptor engagement. Strikingly, the membrane-recycled PKD remains active in the cytosol for several hours. The present work thus characterizes a sustained antigen receptor-induced signal transduction pathway and establishes PKD as a serine kinase that temporally and spatially disseminates antigen receptor signals away from the plasma membrane into the cytosol.     

The role of the pleckstrin homology domain in membrane targeting and activation of phospholipase Cbeta(1)

Current studies involve an investigation of the role of the pleckstrin homology (PH) domain in membrane targeting and activation of phospholipase Cbeta(1) (PLCbeta(1)). Here we report studies on the membrane localization of the isolated PH domain from the amino terminus of PLCbeta(1) (PLCbeta(1)-PH) using fluorescence microscopy of a green fluorescent protein fusion protein. Whereas PLCbeta(1)-PH does not localize to the plasma membrane in serum-starved cells, it undergoes a rapid but transient migration to the plasma membrane upon stimulation of cells with serum or lysophosphatidic acid (LPA). Regulation of the plasma membrane localization of PLCbeta(1)-PH by phosphoinositides was also investigated. PLCbeta(1)-PH was found to bind phosphatidylinositol 3-phosphate most strongly, whereas other phosphoinositides were bound with lower affinity. The plasma membrane localization of PLCbeta(1)-PH induced by serum and LPA was blocked by wortmannin pretreatment and by LY294002. In parallel, activation of PLCbeta by LPA was inhibited by wortmannin, by LY294002, or by the overexpression of PLCbeta(1)-PH. Microinjection of betagamma subunits of G proteins in serum-starved cells induced the translocation of PLCbeta(1)-PH to the plasma membrane. These results demonstrate that a cooperative mechanism involving phosphatidylinositol 3-phosphate and the Gbetagamma subunit regulates the plasma membrane localization and activation of PLCbeta(1)-PH.     

Phosphatidylserine exposure during early primary necrosis (oncosis) in JB6 cells as evidenced by immunogold labeling technique

Apoptotic cell death is characterized by the early exposure of phosphatidylserine (PS) at the outer surface of the plasma membrane. The aim of the present study was to examine whether PS exposure also occurs during oncosis (early primary necrosis) and to localize PS at the subcellular level, applying a pre-embedding immunogold labeling technique with biotin conjugated annexin V. The issue was addressed by using caspase-8 deficient, Bcl-2 overexpressing JB6 cells, which die by oncosis when stimulated with synthetic dsRNA. We observed by fluorescence microscopy that oncotic cells with preserved plasma membrane integrity showed PS exposure (annexin+/propidium iodide-). The data was confirmed on the ultrastructural level and PS was localized in oncosis at the outer leaflet of the continuous plasma membrane with preserved trilamellar structure. In postoncotic necrotic cells the immunogold labels were found on the plasma membrane and on the intracellular membranes of the cells, which underwent plasma membrane disruption. In conclusion, this study reveals that PS externalization occurs not only in apoptosis but also in oncosis at least in our cell model system.