RGS4 controls Gαi3-mediated regulation of Bcl-2 phosphorylation on TGN38-containing intracellular membranes

We propose the use of membrane splitting by freeze-fracture for differential phospholipid analysis of protoplasmic and exoplasmic membrane leaflets (halves). Unfixed cells or tissues are quick-frozen, freeze-fractured, and platinum-carbon (Pt/C) shadowed. The Pt/C replicas are then treated with 2.5% sodium dodecyl sulfate (SDS) to solubilize unfractured membranes and to release cytoplasm or contents. While the detergent dissolves unfractured membranes, it would not extract lipids from split membranes, as their apolar domains are stabilized by their Pt/C replicas. After washing, the Pt/C replicas, along with attached protoplasmic and exoplasmic membrane halves, are processed for immunocytochemical labeling of phospholipids with antibody, followed by electron microscopic observation. Here, we present the application of the SDS-digested freeze-fracture replica labeling (SDS-FRL) technique to the transmembrane distribution of a major membrane phospholipid, phosphatidylcholine (PC), in various cell and intracellular membranes. Immunogold labeling revealed that PC is exclusively localized on the exoplasmic membrane halves of the plasma membranes, and the intracellular membranes of various organelles, e.g. nuclei, mitochondria, endoplasmic reticulum, secretory granules, and disc membranes of photoreceptor cells. One exception to this general scheme was the plasma membrane forming the myelin sheath of neurons and the Ca(2+)-treated erythrocyte membranes. In these cell membranes, roughly equal amounts of immunogold particles for PC were seen on each outer and inner membrane half, implying a symmetrical transmembrane distribution of PC. Initial screening suggests that the SDS-FRL technique allows in situ analysis of the transmembrane distribution of membrane lipids, and at the same time opens up the possibility of labeling membranes such as intracellular membranes not normally accessible to cytochemical labels without the distortion potentially associated with membrane isolation procedures.

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The inhibition of platelet cyclo-oxygenase by aspirin is associated with the acetylation of a 72kDa polypeptide in the intracellular membranes

Biochemical Journal ◽

10.1042/bj2230105 ◽

1984 ◽

Vol 223 (1) ◽

pp. 105-111 ◽

Cited By ~ 11

Author(s):

N Hack ◽

F Carey ◽

N Crawford

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Membrane System ◽

Particulate Material ◽

Intracellular Membrane ◽

Membrane Structures ◽

Major Site ◽

Intracellular Membranes ◽

Oxygenase Activity ◽

Radioactive Labelling

Previous studies by Roth & Majerus [J. Clin. Invest. (1975) 56, 624-632] showed that exposure of platelets to [acetyl-14C]aspirin resulted in the radioactive labelling of three polypeptides, two of which were in the cytosol and not saturable, whilst the third was located in particulate material, and was saturated at 30 microM-aspirin. By using high voltage free flow electrophoresis to separate a platelet mixed membrane fraction into highly purified surface and intracellular membrane subfractions, we have confirmed that the major polypeptide acetylated after exposing whole platelets to [acetyl-14C]aspirin is almost exclusively associated with intracellular membrane structures. We have shown previously that these intracellular membranes are the major site for prostanoid biosynthesis [Carey, Menashi & Crawford (1982) Biochem. J. 204, 847-851] and in the present study the extent of the radioactive labelling correlated well with inhibition of the cyclo-oxygenase activity in these intracellular membranes. In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the [14C]acetylated component, which appears to be a dimer, migrates with a mobility corresponding to 72kDa. Although cyclo-oxygenase is inhibited, there is no discernible radioactive labelling when the platelets are exposed to aromatic-ring-labelled [14C]aspirin. We suggest that the site or sites for aspirin acetylation and cyclo-oxygenase activity are structurally associated in the platelet's intracellular membranes referred to by electron microscopists as the dense tubular membrane system.

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Studies on the bivalent-cation-activated ATPase activities of highly purified human platelet surface and intracellular membranes

Biochemical Journal ◽

10.1042/bj2330661 ◽

1986 ◽

Vol 233 (3) ◽

pp. 661-668 ◽

Cited By ~ 15

Author(s):

N Hack ◽

M Croset ◽

N Crawford

Keyword(s):

Atpase Activity ◽

Blood Platelets ◽

Present Report ◽

Surface Membrane ◽

Storage Site ◽

Intracellular Membrane ◽

Bivalent Cation ◽

Platelet Surface ◽

Intracellular Membranes ◽

Dependent Transport

Membrane-bound Ca2+-ATPases are responsible for the energy-dependent transport of Ca2+ across membrane barriers against concentration gradients. Such enzymes have been identified in sarcoplasmic reticulum of muscle tissues and in non-muscle cells in both surface membranes and endoplasmic-reticulum-like intracellular membrane complexes. In a previous study using membrane fractionation by density-gradient and free-flow electrophoresis, we reported that the intracellular membranes of human blood platelets were a major storage site for Ca2+ and involved in maintaining low cytosol [Ca2+] in the unactivated cell. In the present report we demonstrated that the intracellular membranes also exhibit a high-affinity Ca2+-ATPase which appears to be kinetically associated with the Ca2+-sequestering process. We found that both the surface membrane and the intracellular membrane exhibited a basal Mg2+-ATPase activity, but Ca2+ activation of this enzyme was confined only to the intracellular membrane. Use of Ca2+-EGTA buffers to control the extravesicle [Ca2+] allowed a direct comparison of the Ca2+-ATPase and the Ca2+-uptake process over a Ca2+ range of 0.01 microM to 1.0 mM, and it was found that both properties were maximally expressed in the range of external [Ca2+] 1-50 microM, with concentrations greater than 100 microM showing substantial inhibition. Double-reciprocal plots for the Ca2+-ATPase activity and Ca2+ uptake gave apparent Km values for Ca2+ of 0.15 and 0.13 microM respectively. However, similar plots for ATP with the enzyme revealed a discontinuity (two affinity sites, with Km 20 and 145 microM), whereas plots for the Ca2+ uptake gave a single Km value for Ca2+, 1.1 microM. Phosphorylation studies during Ca2+ uptake using [gamma-32P]ATP revealed two components of 90 and 95 kDa phosphorylated at extravesicle [Ca2+] of 3 microM. The Ca2+-ATPase activity, Ca2+ uptake and phosphorylation were all almost completely inhibited in the presence of 500 microM-Ca2+. Similar studies using mixed membranes revealed four other phosphoproteins (50, 40, 20 and 18 kDa) formed in addition to the 90 and 95 kDa components. The findings are discussed in the context of platelet Ca2+ mobilization for function and the mechanisms whereby Ca2+ homoeostasis is controlled in the unactivated cell.

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VARIATIONS IN THE STRUCTURE OF MITOCHONDRIA

The Journal of Cell Biology ◽

10.1083/jcb.2.4.305 ◽

1956 ◽

Vol 2 (4) ◽

pp. 305-312 ◽

Cited By ~ 54

Author(s):

E. W. Dempsey

Keyword(s):

Electron Microscopy ◽

Light Microscopy ◽

Internal Structure ◽

Outer Wall ◽

The Other ◽

Metabolic Processes ◽

Intracellular Membranes ◽

Janus Green ◽

Definition Of ◽

Shape Complexity

A characteristic internal structure, consisting of a double-layered outer wall enclosing a matrix-filled space through which pass double-layered membranous folds, would appear to comprise as satisfactory a definition of mitochondria for electron microscopy as their intravital affinity for Janus green affords for light microscopy. Relying for identification upon this characteristic internal structure, mitochondria appear to be pleomorphic structures which vary in size, shape, complexity, and density. They are labile also in that their number may increase or decrease under controlled conditions. The possibility therefore exists that these organelles are constantly being formed and destroyed, perhaps by their participation in metabolic processes. The problem of the origin of mitochondria is in an unsatisfactory state. New organelles unquestionably are formed in particular physiological states. The possibility that new bodies are produced by fission of ones already present does not seem adequate. On the other hand, the possible fabrication of new mitochondria out of intracellular membranes, although an attractive hypothesis, has not been adequately substantiated.

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Localization of β-glucan synthases on the membranes of cultured Lolium multiflorum (ryegrass) endosperm cells

Biochemical Journal ◽

10.1042/bj2090627 ◽

1983 ◽

Vol 209 (3) ◽

pp. 627-633 ◽

Cited By ~ 10

Author(s):

R J Henry ◽

A Schibeci ◽

B A Stone

Keyword(s):

Density Gradient ◽

Plasma Membranes ◽

Lolium Multiflorum ◽

Sucrose Density Gradient ◽

Italian Ryegrass ◽

Beta Glucan ◽

Glucan Synthase ◽

Intracellular Membranes

The distribution of beta-glucan synthases between plasma membranes and intracellular membranes of suspension-cultured Italian-ryegrass (Lolium multiflorum Lam.) endosperm cells was examined. Highly purified plasma membranes prepared from protoplasts were only slightly enriched in beta-glucan synthases assayed at 10 microM- and 1 mM-UDP-glucose. Most beta-glucan synthase was associated with intracellular membranes. These membranes were fractionated on a linear sucrose density gradient and were resolved into different membrane fractions containing beta-glucan synthases. Beta-Glucan synthases assayed at 10 microM-UDP-glucose were found in a fraction banding at a density of 1.11 g . cm-3, but most of the beta-glucan synthase assayed at 1 mM-DDP-glucose was at a density of 1.04 g . cm-3.

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Interaction of an Endosomolytic Polyamidoamine ISA23 with Vesicles Mimicking Intracellular Membranes: A SANS/EPR Study

Macromolecular Bioscience ◽

10.1002/mabi.201000040 ◽

2010 ◽

Vol 10 (8) ◽

pp. 963-973 ◽

Cited By ~ 6

Author(s):

Peter C. Griffiths ◽

Renuka Nilmini ◽

Emma Carter ◽

Patrick Dodds ◽

Damien M. Murphy ◽

...

Keyword(s):

Intracellular Membranes

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Furrow-associated microtubule arrays are required for the cohesion of zebrafish blastomeres following cytokinesis

Journal of Cell Science ◽

10.1242/jcs.111.24.3695 ◽

1998 ◽

Vol 111 (24) ◽

pp. 3695-3703 ◽

Cited By ~ 6

Author(s):

S. Jesuthasan

Keyword(s):

Confocal Microscopy ◽

Zebrafish Embryo ◽

Beta Catenin ◽

Free Medium ◽

Alpha Tubulin ◽

Intracellular Membranes

During the first few cleavages of the zebrafish embryo, daughter blastomeres are loosely associated immediately after furrow ingression, but then gradually cohere. Cohesion appears to be cadherin-dependent, as cadherin and beta-catenin are found at the membrane between cohering blastomeres, and blastomeres fail to cohere in calcium-free medium. Cadherin and beta-catenin are not initially found on the blastomere surface, but are deposited specifically at the furrow surface. An array of parallel microtubules is present on either side of the furrow tip during ingression, as seen by confocal microscopy of alpha-tubulin labelled embryos. Transient incubation of embryos in 1 microg/ml nocodazole at the start of furrowing, which causes a loss of the furrow array, inhibits the localization of beta-catenin to the furrow surface but does not prevent furrow ingression. During ingression, intracellular membranes are transported to the furrow, as shown by labelling with DiD or DiOC6(3). Concentration of these membranes near the furrow surface is microtubule-dependent. These findings suggest that microtubules are required for cohesion of blastomeres because they mediate trafficking of intracellular membranes to the furrow surface, where they are exocytosed and allow cohesion via cadherins.

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