Plasma membrane redox and regulation of cell growth

Summary Gene expression can be activated by external oxidants which are reduced at the cell surface by plasma membrane electron transport. The signals generated in response to the plasma membrane electron transport include activation of proton release, internal calcium changes, and change in reductant/oxidant ratio in the cytosol. H₂O₂ generated in response to ligands which bind to plasma membrane receptors can also activate protein tyrosine kinases and gene expression. Inhibition of oxygen radical generation at the cell surface in response to the mitogen, phorbol myristate acetate by retinoic acid is consistent with a role for the plasma membrane electron transport as the source for H₂O₂ in Balb 3T3 cells. Agents which affect the binding of coenzyme Q to redox sites in the plasma membrane electron transport may increase formation of semiquinone radicals in the membrane which can be a source of oxygen radicals and H₂O₂. The generation of H₂O₂ by transformed cells indicates that oncogene product expression in the plasma membrane may also increase quinone-based oxygen radical generation.     

Alterations of wheat root plasma membrane lipid composition induced by copper stress result in changed physicochemical properties of plasma membrane lipid vesicles

A response when wheat is grown in excess copper is an altered lipid composition of the root plasma membrane (PM). With detailed characterisation of the root PM lipid composition of the copper-treated plants as a basis, in the present study, model systems were used to gain a wider understanding about membrane behaviour, and the impact of a changed lipid composition.PMs from root cells of plants grown in excess copper (50 μM Cu²⁺) and control (0.3 μM Cu²⁺) were isolated using the two-phase partitioning method. Membrane vesicles were prepared of total lipids extracts from the isolated PMs, and also reference vesicles of phosphatidylcholine (PC). In a series of tests, the vesicle permeability for glucose and for protons was analysed. The vesicles show that copper stress reduced the permeability for glucose of the lipid bilayer barrier. When vesicles from stressed plants were modified by addition of lipids to resemble vesicles from control plants, the permeability for glucose was very similar to that of vesicles from control plants. The permeability for protons did not change upon stress.Electron paramagnetic resonance (EPR) of the lipid vesicles spin probed with n-doxylstearic acid (nDSA) was used to explore the lipid rotational freedom at different depth of the bilayer. The EPR measurements supported the permeability data, indicating that the copper stress resulted in more tightly packed bilayers of the PMs with reduced acyl chain motion.     

Limitations of the Colloidal Silica Method in Mapping the Endothelial Plasma Membrane Proteome of the Mouse Heart

The endothelial cell (EC) membrane is an important interface, which plays a crucial role in signal transduction. Our aim was to selectively purify luminal EC membrane proteins from the coronary vasculature of the isolated perfused mouse heart and analyze its composition with mass spectrometry (MS). To specifically label coronary ECs in the intact heart, the colloidal silica method was applied, which is based on the binding of positively charged colloidal silica to the surface of EC membranes. Transmission electron microscopy revealed the specific labeling of ECs of macro and microvessels. Two different methods of tissue homogenization (Teflon pestle and ultra blade) together with density centrifugation were used for membrane protein enrichment. Enrichment and purity was controlled by Western blot analysis using the EC-specific protein caveolin 1 and various intracellular marker proteins. The ultra blade method resulted in a tenfold enrichment of caveolin 1, while there was negligible contamination as judged by Western blot. However, protein yield was low and required pooling of ten hearts for MS. When enriched endothelial membrane proteins were digested with trypsin and analyzed by LC-MS, a total of 56 proteins could be identified, of which only 12 were membrane proteins. We conclude that coronary endothelial membranes can be conveniently labeled with colloidal silica. However, due to the ionic nature of interaction of colloidal silica with the EC membrane the shear rate required for cardiac homogenization resulted in a substantial loss of specificity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Plasma membrane redox systems in tumor cells

Summary Plasma membrane redox systems in tumor cells are analyzed, their role in proton flux and tumor cell growth is described, and the modulation of their activity by antitumor drugs and growth factors is presented. As an example of the evolution of studies in the characterization of plasma membrane redox systems in tumor cells, we summarized our own results on the model system Ehrlich ascites carcinoma.     

Plasma membrane dehydrogenases in rat brain synaptic membranes. Multiplicity and subunit composition

Plasma membrane redox enzymes have been investigated in synaptic membranes from rat brain nerve terminals. UV-Vis spectra of intact synaptic plasma membranes are presented and the presence of ab-type cytochrome, detectable at 77°K and sensitive to NADH or NADPH, is shown. The molecular characterization of rat synaptic NADH-dehydrogenases was further performed on solubilized enzymes using a recently developed nondissociating polyacrylamide gel electrophoresis technique. Synaptic plasma membranes were solubilized with 1% sodium cholate or Triton X-114 and centrifuged. The supernatant retained over 60% of the NADH-dehydrogenase activity, tested with either DCIP or ferricyanide as substrates, together with NADH. Both enzyme activities were insensitive toward rotenone. This extraction procedure also solubilized about 50% of the proteins. When submitted to polyacrylamide gel electrophoresis under nondenaturing conditions and stained for NADH-dehydrogenase activity, five bands of different mobilities were detected. The multiple NADH-dehydrogenases of synaptic plasma membranes were investigated by means of band excision and the five excised bands each submitted to amino acid analysis and to 2-D electrophoresis. The subunit composition of each band was then deduced, together with the molecular weight and pI of each respective subunit. NADH-dehydrogenases have also been purified by means of FPLC on Mono-P (chromatofocusing) followed by gel filtration on Superose 12. NADH-Dehydrogenase IV and V could be purified in their active forms by this approach.Abbreviations DCIP dichlorophenol-indophenol - FPLC fast protein liquid chromatography.     

Plasma membrane Ca-ATPases in the nervous system during development and ageing

Calcium signaling is used by neurons to control a variety of functions, including cellular differentiation, synaptic maturation, neurotransmitter release, intracellular signaling and cell death. This review focuses on one of the most important Ca(2+) regulators in the cell, the plasma membrane Ca(2+)-ATPase (PMCA), which has a high affinity for Ca(2+) and is widely expressed in brain. The ontogeny of PMCA isoforms, linked to specific requirements of Ca(2+) during development of different brain areas, is addressed, as well as their function in the adult tissue. This is based on the high diversity of variants in the PMCA family in brain, which show particular kinetic differences possibly related to specific localizations and functions of the cell. Conversely, alterations in the activity of PMCAs could lead to changes in Ca(2+) homeostasis and, consequently, to neural dysfunction. The involvement of PMCA isoforms in certain neuropathologies and in brain ageing is also discussed.     

The Changes of Cytoskeletal Proteins in Plasma of Acrylamide-Induced Rats

Acrylamide (ACR) is a known industrial neurotoxic chemical that can induce neurodegeneration. Cytoskeletal protein aggregation is a pathological hallmark of neurodegenerative disorders. This study was an initial exploration on cytoskeletal proteins in plasma as potential biomarkers of ACR neurotoxicity. Low and high ACR groups received 20 mg/kg and 40 mg/kg ACR by intraperitoneal injection in adult Wistar rats and control group received physiological saline. Rats were all killed after 8 weeks to evaluate the levels of neurofilament(NF)-L, NF-M, NF-H, β-actin, α-tubulin, β-tubulin, tau, MAP₂ proteins in plasma using both SDS-PAGE and western blotting. Compared with the control, the levels of NF-L, NF-M, NF-H, β-actin, tau, MAP₂ proteins decreased and the level of α-tubulin increased in high ACR group, the levels of α-tubulin, β-tubulin and MAP₂ increased in low ACR group. The results suggested that the changes of these proteins might be relevant to the neurotoxicity of ACR. Some of the cytoskeletal proteins in plasma might be used as marker of biological effect in ACR induced neuropathy.     

Protein-protein interactions in intracellular membrane fusion

The fusion of intracellular vesicles with their target membranes is an essential feature of the compartmental structure of eukaryotic cells. This process requires proteins that dictate the targeting of a vesicle to the correct cellular location, mediate bilayer fusion and, in some systems, regulate the precise time at which fusion occurs. Recent biophysical and structural studies of these proteins have begun to provide a foundation for understanding their functions at a molecular level.     

The G protein-coupled receptor rhodopsin in the native membrane

The higher-order structure of G protein-coupled receptors (GPCRs) in membranes may involve dimerization and formation of even larger oligomeric complexes. Here, we have investigated the organization of the prototypical GPCR rhodopsin in its native membrane by electron and atomic force microscopy (AFM). Disc membranes from mice were isolated and observed by AFM at room temperature. In all experimental conditions, rhodopsin forms structural dimers organized in paracrystalline arrays. A semi-empirical molecular model for the rhodopsin paracrystal is presented validating our previously reported results. Finally, we compare our model with other currently available models describing the supramolecular structure of GPCRs in the membrane.     

Plasma membrane signaling in HIV-1 infection

Plasma membrane is a multifunctional structure that acts as the initial barrier against infection by intracellular pathogens. The productive HIV-1 infection depends upon the initial interaction of virus and host plasma membrane. Immune cells such as CD4 + T cells and macrophages contain essential cell surface receptors and molecules such as CD4, CXCR4, CCR5 and lipid raft components that facilitate HIV-1 entry. From plasma membrane HIV-1 activates signaling pathways that prepare the grounds for viral replication. Through viral proteins HIV-1 hijacks host plasma membrane receptors such as Fas, TNFRs and DR4/DR5, which results in immune evasion and apoptosis both in infected and uninfected bystander cells. These events are hallmark in HIV-1 pathogenesis that leads towards AIDS. The interplay between HIV-1 and plasma membrane signaling has much to offer in terms of viral fitness and pathogenicity, and a better understanding of this interplay may lead to development of new therapeutic approaches. This article is part of a Special Issue entitled: Viral Membrane Proteins — Channels for Cellular Networking.     

Interactions Between Ascorbyl Free Radical and Coenzyme Q at the Plasma Membrane

A role for coenzyme Q in the stabilization of extracellular ascorbate by intact cells has beenrecently recognized. The aim of this work was to study the interactions between reducedubiquinone in the plasma membrane and the ascorbyl free radical, as an approach to understandubiquinone-mediated ascorbate stabilization at the cell surface. K-562 cells stabilized ascorbateand decreased the steady-state levels of the semiascorbyl radical. The ability of cells to reduceascorbyl free radical was inhibited by the quinone analogs capsaicin and chloroquine andstimulated by supplementing cells with coenzyme Q₁₀. Purified plasma membranes also reducedascorbyl free radical in the presence of NADH. Free-radical reduction was notobserved inquinone-depleted plasma membranes, but restored after its reconstitution with coenzyme Q₁₀.Addition of reduced coenzyme Q₁₀ to depleted membranes allowed them toreduce the signalof the ascorbyl free radical without NADH incubation and the addition of an extra amount ofpurified plasma membrane quinone reductase further stimulated this activity. Reduction wasabolished by treatment with the reductase inhibitor p-hydroximercuribenzoate and by blockingsurface glycoconjugates with the lectin wheat germ agglutinin, which supports the participationof transmembrane electron flow. The activity showed saturation kinetics by NADH andcoenzyme Q, but not by the ascorbyl free radical in the range of concentrations used. Our resultssupport that reduction of ascorbyl free radicals at the cell surface involves coenzyme Qreduction by NADH and the membrane-mediated reduction of ascorbyl free radical.     

Seasonal ultrastructural alterations in the plasma membrane produced by slow freezing in cortical tissues of mulberry (Morus bombyciz Koidz. cv. Goroji)

Seasonal alterations in the ultrastructure of the plasma membrane produced by slow freezing were examined in cortical parenchyma cells of mulberry twigs (Morus bombyciz Koidz. cv. Goroji) grown in northern Japan. In freezing-sensitive summer, freezing produced distinct aparticulate domains with accompanying inverted hexagonal_II (H_II) phase transitions in the plasma membrane. In autumn and spring, during cold acclimation and deacclimation, freezing produced aparticulate domains in the plasma membrane without accompanying H_ii phase transitions. In winter, when the twigs were freezing-tolerant, freezing did not produce ultrastructural alterations in the plasma membrane. A significant relationship was recognized between the percentages of cells with aparticulate domains in the plasma membrane, regardless of the presence or absence of H_II phase transitions, and the occurrence of freezing injury throughout all seasons and at all freezing temperatures tested in each season. The aparticulate domains in the plasma membranes were shown to be produced by the close apposition of membranes due to freezing-induced dehydration and deformation of cells. Although the precise mechanisms that cause injury as a result of the formation of aparticulate domains in the plasma membrane remain unclear, our results indicate that the development of cold acclimation paralleled the process whereby cells developed the ability to reduce and finally to prevent the formation of aparticulate domains in the plasma membrane that would otherwise result from freezing-induced cellular dehydration and deformation that brings membranes into close proximity with one another.     

Deconstructing membrane traffic

Intracellular movement of proteins and lipids between organelles is usually described in terms of cargo, carriers, traffic and docking, familiar terms that imply parallels to human activities. Over the past century, scientists have been criticized for constructing hypotheses that reflect too much of their current political and cultural values. In this article, concepts of membrane traffic are re-examined to see whether they reflect the cell’s view of the world or our own.     

Deconstructing membrane traffic

Intracellular movement of proteins and lipids between organelles is usually described in terms of cargo, carriers, traffic and docking, familiar terms that imply parallels to human activities. Over the past century, scientists have been criticized for constructing hypotheses that reflect too much of their current political and cultural values. In this article, concepts of membrane traffic are re-examined to see whether they reflect the cell’s view of the world or our own.     

Plasma membrane coenzyme Q10 and growth control

Summary Coenzyme Q is distributed among cellular membranes and it has a significant concentration at the plasma membrane. The plasma membrane contains a trans-membrane electron transport system, which is centered on coenzyme Q. This molecule is maintained reduced by NAD(P)H-dependent enzymes and can reduce other antioxidants such as tocopheroxyl quinone and ascorbate free radical. Its antioxidant property and its ability to maintain in the reduced state the other antioxidants offers a system to protect membrane components against oxidations and prevents oxidative-stress-dependent cellular damage. Growth factor withdrawal induces cell growth arrest and apoptosis through an oxidative-stress-induced pathway. Coenzyme Q can stimulate growth of different cell lines under serum deficiency, mainly by preventing apoptosis. The protection caused by coenzyme Q is independent of the Bcl-2 protein. Plasma membrane coenzyme Q appears to be essential in the regulation of the redox equilibrium of the cell and redox-dependent pathways.     

Deconstructing membrane traffic

Intracellular movement of proteins and lipids between organelles is usually described in terms of cargo, carriers, traffic and docking, familiar terms that imply parallels to human activities. Over the past century, scientists have been criticized for constructing hypotheses that reflect too much of their current political and cultural values. In this article, concepts of membrane traffic are re-examined to see whether they reflect the cell’s view of the world or our own.     

SNARE and regulatory proteins induce local membrane protrusions to prime docked vesicles for fast calcium‐triggered fusion

Synaptic vesicles fuse with the plasma membrane in response to Ca²⁺ influx, thereby releasing neurotransmitters into the synaptic cleft. The protein machinery that mediates this process, consisting of soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) and regulatory proteins, is well known, but the mechanisms by which these proteins prime synaptic membranes for fusion are debated. In this study, we applied large‐scale, automated cryo‐electron tomography to image an in vitro system that reconstitutes synaptic fusion. Our findings suggest that upon docking and priming of vesicles for fast Ca²⁺‐triggered fusion, SNARE proteins act in concert with regulatory proteins to induce a local protrusion in the plasma membrane, directed towards the primed vesicle. The SNAREs and regulatory proteins thereby stabilize the membrane in a high‐energy state from which the activation energy for fusion is profoundly reduced, allowing synchronous and instantaneous fusion upon release of the complexin clamp.     

Formation of the plasma membrane during regeneration ofParamecium caudatum

Summary Fragments ofParamecium caudatum cells obtained by merotomy were fixed in 1% OsO₄ within 5 seconds after cutting. The ultrastructure of the damaged area of the fragment was studied in oriented ultrathin sections and by scanning electron microscopy. The cytoplasm exposed by merotomy was covered during a few seconds with a new membrane. This was a typical trilaminar membrane continuous with the plasma membrane covering the undamaged surface of the cell. The surface over the wound was wrinkled into irregular grooves and ridges. The cytoplasm, mitochondria and trichocysts in the injured region were electron translucent. The cytoplasm under the new membrane contained an unusually high amount of small membrane vesicles, 20–90 nm in diameter. These were probably the remnants of subpellicular alveoli and the plasma membrane destroyed by microsectioning. The possibility that the exposed cytoplasm would be covered by mere shifting of the existing plasma membrane can be excluded. The complex structure of the cortex with its subpellicular alveoli and regularly distributed cilia provide a strong argument against this notion. It seems probable that the new membrane was built up from the available molecular material,e.g., phospholipids and proteins present in the cytoplasm. Fragments of the membrane and alveolar membranes in the form of small vesicles may have also been included into the new membrane.