scholarly journals Fractionation of Golgi, Endoplasmic Reticulum, and Plasma Membrane from Cultured Cells in a Preformed Continuous Iodixanol Gradient

2002 ◽  
Vol 2 ◽  
pp. 1435-1439 ◽  
Author(s):  
John Graham
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cultured Cells ◽  
Density Range ◽  
Cultured Cell ◽  
Golgi Membranes ◽  
Cell Homogenate ◽  
Membrane Compartments

A continuous iodixanol gradient within the range 0-30% (w/v) iodixanol can resolve the major membrane compartments of the endoplasmic reticulum, Golgi membranes, and plasma membrane from a postnuclear supernatant prepared from a cultured cell homogenate. The precise density range of the gradient and the centrifugation conditions (100,000-200,000gfor 2-16 h) vary with the type of cell and the requirements of the separation. The strategy is widely used to study the processing of proteins within cells.

Characterization of endoplasmic reticulum by co-localization of BiP and dicarbocyanine dyes

1992 ◽  
Vol 101 (2) ◽  
pp. 315-322 ◽  
Author(s):  
M. Terasaki ◽  
T.S. Reese
Keyword(s):  
Endoplasmic Reticulum ◽  
Cultured Cells ◽  
Protein A ◽  
Epithelial Cell Line ◽  
Cultured Fibroblasts ◽  
Whole Cells ◽  
Membrane Compartments ◽  
Immunoglobulin Binding Protein ◽  
Immunoglobulin Binding ◽  
Peripheral Regions

The original concept of endoplasmic reticulum derived from the observation of a reticular network in cultured fibroblasts by electron microscopy of whole cells. It was previously reported that the fluorescent dye, DiOC6(3), stains a similar network as well as mitochondria and other organelles in living cells. Here, we investigate the significance of the structures labeled by DiO6(3) in CV-1 cells, a monkey epithelial cell line. First, we show that the network stained in living CV-1 cells is preserved by glutaraldehyde fixation and then we co-label it with an antibody against BiP (immunoglobulin binding protein), a protein commonly accepted to be present in the endoplasmic reticulum. Anti-BiP labeled the same network as that labeled by DiOC6(3), so this network now is identified as being part of the endoplasmic reticulum. DiOC6(3) labels many other membrane compartments in addition to the endoplasmic reticulum. This, along with its lipophilic properties, suggests that DiOC6(3) stains all intracellular membranes. However, the extensive reticular network in the thin peripheral regions of cultured cells is easily distinguished from these other membranes. Thus, staining by DiOC6(3) is a useful method for localizing the endoplasmic reticulum, particularly in thin peripheral regions of cultured cells.

Incorporation in vivo of [32P]orthophosphate and [Me-3H]choline into rough microsomal, Golgi, and plasma membranes of rat liver

1977 ◽  
Vol 55 (8) ◽  
pp. 876-885 ◽  
Author(s):  
Patricia L. Chang ◽  
John R. Riordan ◽  
Mario A. Moscarello ◽  
Jennifer M. Sturgess
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Marker Enzyme ◽  
Golgi Membranes ◽  
Endomembrane Flow

To study membrane biogenesis and to test the validity of the endomembrane flow hypothesis, incorporation of 32P and [Me-3H]choline in vivo into membranes of the rat liver was followed. Rough microsomal, Golgi-rich, and plasma membrane fractions were monitored with marker enzyme assays and shown with morphometric analysis to contain 82% rough microsomes, at least 70% Golgi complexes, and 88% plasma membranes, respectively. Membrane subfractions from the rough microsomal and Golgi-rich fractions were prepared by sonic disruption.At 5 to 30 min after 32P injection, the specific radioactivity of phosphatidylcholine was higher in the rough microsomal membranes than in the Golgi membranes. From 1 to 3 h, the specific activity of phosphatidylcholine in Golgi membranes became higher and reached the maximum at about 3 h. Although the plasma membrane had the lowest specific radioactivity throughout 0.25–3 h, it increased rapidly thereafter to attain the highest specific activity at 5 h. Both rough microsomal and plasma membranes reached their maxima at 5 h.The specific radioactivity of [32P]phosphatidylethanolamine in the three membrane fractions was similar to that of [32P]phosphatidylcholine except from 5 to 30 min, when the specific radioactivity of phosphatidylethanolamine in the Golgi membranes was similar to the rough microsomal membranes.At 15 min to 5 h after [Me-3H]choline injection, more than 90% of the radioactivity in all the membranes was acid-precipitable. The specific radioactivities of the acid-precipitated membranes, expressed as dpm per milligram protein, reached the maximum at 3 h. After [Me-3H]choline injection, the specific radioactivity of phosphatidylcholine separated from the lipid extract of the acid-precipitated membranes (dpm per micromole phosphorus) did not differ significantly in the three membrane fractions. The results indicated rapid incorporation of choline into membrane phosphatidylcholine by the rough endoplasmic reticulum, Golgi, and plasma membranes simultaneously.The data with both 32P and [Me-3H]choline precursors did not support the endomembrane flow hypothesis. The Golgi complexes apparently synthesized phosphatidylethanolamine and incorporated choline into phosphatidylcholine as well as the endoplasmic reticulum. The results are discussed with relevance to current hypotheses on the biogenesis and transfer of membrane phospholipids.

scholarly journals Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum

2008 ◽  
Vol 182 (4) ◽  
pp. 685-701 ◽  
Author(s):  
Elizabeth L. Axe ◽  
Simon A. Walker ◽  
Maria Manifava ◽  
Priya Chandra ◽  
H. Llewelyn Roderick ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Dynamic Equilibrium ◽  
Membrane Vesicles ◽  
Amino Acid Starvation ◽  
Autophagosome Formation ◽  
Double Membrane ◽  
Dynamic Relationship ◽  
Golgi Membranes ◽  
Membrane Compartments ◽  
Phosphatidylinositol 3

Autophagy is the engulfment of cytosol and organelles by double-membrane vesicles termed autophagosomes. Autophagosome formation is known to require phosphatidylinositol 3-phosphate (PI(3)P) and occurs near the endoplasmic reticulum (ER), but the exact mechanisms are unknown. We show that double FYVE domain–containing protein 1, a PI(3)P-binding protein with unusual localization on ER and Golgi membranes, translocates in response to amino acid starvation to a punctate compartment partially colocalized with autophagosomal proteins. Translocation is dependent on Vps34 and beclin function. Other PI(3)P-binding probes targeted to the ER show the same starvation-induced translocation that is dependent on PI(3)P formation and recognition. Live imaging experiments show that this punctate compartment forms near Vps34-containing vesicles, is in dynamic equilibrium with the ER, and provides a membrane platform for accumulation of autophagosomal proteins, expansion of autophagosomal membranes, and emergence of fully formed autophagosomes. This PI(3)P-enriched compartment may be involved in autophagosome biogenesis. Its dynamic relationship with the ER is consistent with the idea that the ER may provide important components for autophagosome formation.

scholarly journals Movement of plasma-membrane sterols to the endoplasmic reticulum in cultured cells

1987 ◽  
Vol 248 (1) ◽  
pp. 237-242 ◽  
Author(s):  
J P Slotte ◽  
E L Bierman
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Muscle ◽  
Plasma Membrane ◽  
Smooth Muscle Cells ◽  
Human Skin ◽  
Cultured Cells ◽  
Muscle Cells ◽  
Skin Fibroblasts ◽  
Human Skin Fibroblasts ◽  
Arterial Smooth Muscle Cells

The spontaneous turnover of plasma-membrane sterols, as measured by their transfer to the endoplasmic reticulum, was measured in quiescent cultured human skin fibroblasts and monkey arterial smooth-muscle cells. The plasma-membrane sterol pool was pulse-labelled with trace amounts of either [3H]desmosterol or [3H]cholesterol. We then measured the enzymic conversion of [3H]desmosterol into [3H]cholesterol and of [3H]cholesterol into [3H]cholesteryl esters in intact cells. Depending on the probe used, markedly different transfer or conversion rates were found in these cells. In quiescent human skin fibroblasts, incubated in a serum-free medium, about 1.1% of the plasma-membrane [3H]desmosterol was converted into [3H]cholesterol/h, whereas in monkey arterial smooth-muscle cells the corresponding rate was 0.4%. Under similar experimental conditions, these cells esterified less than 0.02% (fibroblasts) and 0.12% (smooth-muscle cells) of the plasma-membrane [3H]cholesterol/h. The movement of sterols from the plasma membrane to the endoplasmic reticulum, as measured by the conversion of [3H]desmosterol into [3H]cholesterol was not blocked by colchicine, but was markedly enhanced by 3% (w/v) dimethyl sulphoxide. In all, these results indicate that plasma-membrane sterols of cultured cells are continuously transferred to the interior of the cell at a rate substantially higher than previously appreciated. This turnover of plasma-membrane sterol molecules took place even when there was no mass transfer of sterols into the cells.

scholarly journals SUBSURFACE CISTERNS AND THEIR RELATIONSHIP TO THE NEURONAL PLASMA MEMBRANE

1962 ◽  
Vol 13 (3) ◽  
pp. 405-421 ◽  
Author(s):  
Jack Rosenbluth
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Nerve Cell ◽  
Plasma Membranes ◽  
Granular Endoplasmic Reticulum ◽  
Supporting Cells ◽  
Golgi Membranes ◽  
Subsurface Cisterns ◽  
Central Nervous Systems ◽  
Neuronal Plasma Membrane

Subsurface cisterns (SSC's) are large, flattened, membrane-limited vesicles which are very closely apposed to the inner aspect of the plasma membranes of nerve cell bodies and the proximal parts of their processes. They occur in a variety of vertebrate and invertebrate neurons of both the peripheral and central nervous systems, but not in the surrounding supporting cells. SSC's are sheet-like in configuration, having a luminal depth which may be less than 100 A and a breadth which may be as much as several microns. They are separated from the plasmalemma by a light zone of ∼50 to 80 A which sometimes contains a faint intermediate line. Flattened, agranular cisterns resembling SSC's, but structurally distinct from both typical granular endoplasmic reticulum (ER) and from Golgi membranes, also occur deep in the cytoplasm of neurons. It is suggested that membranes which are closely apposed may interact, resulting in alterations in their respective properties. The patches of neuronal plasmalemma associated with subsurface cisterns may, therefore, have special properties because of this association, resulting in a non-uniform neuronal surface. The possible significance of SSC's in relation to neuronal electrophysiology and metabolism is discussed.

scholarly journals Absence of cytochrome P-450 and presence of autolysosomal membrane antigens on the isolation membranes and autophagosomal membranes in rat hepatocytes.

1990 ◽  
Vol 38 (11) ◽  
pp. 1571-1581 ◽  
Author(s):  
A Yamamoto ◽  
R Masaki ◽  
Y Fukui ◽  
Y Tashiro
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Nuclear Envelope ◽  
Protein A ◽  
Rat Hepatocytes ◽  
Immunogold Technique ◽  
Microscopic Evidence ◽  
Golgi Membranes ◽  
Membrane Antigens ◽  
Cytochrome P 450

We wished to determine if phenobarbital (PB)-inducible cytochrome P-450 [P-450(PB)] and autolysosomal membrane antigens could be localized immunocytochemically on the isolation membranes and the limiting membranes of autophagosomes in rat hepatocytes by the post-embedding protein A-gold method. P-450(PB) was maximally induced by PB treatment; then formation of autophagosomes and accumulation of autolysosomes were induced by cessation of PB treatment and by injection of leupeptin, respectively. P-450(PB) was detected neither on the isolation membranes nor on the limiting membranes of autophagosomes and autolysosomes. Autolysosomal membrane antigens, which were localized by the immunogold technique exclusively in post-Golgi compartments such as lysosomes, endosomes, and plasma membrane but were not found in pre-Golgi compartments such as endoplasmic reticulum (ER) and nuclear envelope, were detected in large amounts on the isolation membranes. These results suggest that the isolation membranes originate not from ER membranes but from post-Golgi membranes. We also present direct immunoelectron microscopic evidence that P-450(PB) is indeed degraded in the autolysosomes: when rats were treated with leupeptin, P-450(PB) was detected not only within the autophagosomes but also within the autolysosomes, whereas without leupeptin treatment, P-450(PB) was detectable only within the autophagosomes.

scholarly journals The mitochondria-associated endoplasmic-reticulum subcompartment (MAM fraction) of rat liver contains highly active sphingolipid-specific glycosyltransferases

2003 ◽  
Vol 371 (3) ◽  
pp. 1013-1019 ◽  
Author(s):  
Dominique ARDAIL ◽  
Iuliana POPA ◽  
Jacques BODENNEC ◽  
Pierre LOUISOT ◽  
Daniel SCHMITT ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Marker Enzyme ◽  
Sphingoid Base ◽  
Cross Contamination ◽  
Outer Leaflet ◽  
Highly Active ◽  
Golgi Membranes ◽  
Marked Preference ◽  
Intracellular Organelles

Although most glycosphingolipids (GSLs) are thought to be located in the outer leaflet of the plasma membrane, recent evidence indicates that GSLs and their precursor, ceramide, are also associated with intracellular organelles and, particularly, mitochondria. GSL biosynthesis starts with the formation of ceramide in the endoplasmic reticulum (ER), which is transported by controversial mechanisms to the Golgi apparatus, where stepwise addition of monosaccharides on to ceramides takes place. We now report the presence of GSL-biosynthetic enzymes in a subcompartment of the ER previously characterized and termed ‘mitochondria-associated membrane’ (MAM). MAM is a membrane bridge between the ER and mitochondria that is involved in the biosynthesis and trafficking of phospholipids between the two organelles. Using exogenous acceptors coated on silica gel, we demonstrate the presence of ceramide glucosyltransferase (Cer-Glc-T), glucosylceramide galactosyltransferase and sialyltransferase (SAT) activities in the MAM. Estimation of the marker-enzyme activities showed that glycosyltransferase activities could not be ascribed to cross-contamination of MAM by Golgi membranes. Cer-Glc-T was found to have a marked preference for ceramide bearing phytosphingosine as sphingoid base. SAT activities in MAM led to the synthesis of GM3 ganglioside and small amounts of GD3. GM1 was also synthesized along with GM3 upon incubation of the fraction with exogenous unlabelled GM3, underlying the presence of other sphingolipid-specific glycosyltransferases in MAM. On the basis of our results, we propose MAM as a privileged compartment in providing GSLs for mitochondria.

scholarly journals The rough endoplasmic reticulum and the Golgi apparatus visualized using specific antibodies in normal and tumoral prolactin cells in culture.

1983 ◽  
Vol 96 (5) ◽  
pp. 1197-1207 ◽  
Author(s):  
C Tougard ◽  
D Louvard ◽  
R Picart ◽  
A Tixier-Vidal
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Rough Endoplasmic Reticulum ◽  
Primary Cultures ◽  
Gh3 Cells ◽  
Membrane Flow ◽  
Golgi Membranes ◽  
Membrane Components ◽  
Golgi Zone

Antibodies directed against membrane components of dog pancreas rough endoplasmic reticulum (A-RER) and rat liver Golgi apparatus (A-Golgi) (Louvard, D., H. Reggio, and G. Warren, 1982, J. Cell Biol. 92:92-107) have been applied to cultured rat prolactin (PRL) cells, either normal cells in primary cultures, or clonal GH3 cells. In normal PRL cells, the A-RER stained the membranes of the perinuclear cisternae as well as those of many parallel RER cisternae. The A-Golgi stained part of the Golgi membranes. In the stacks it stained the medial saccules and, with a decreasing intensity, the saccules of the trans side, as well as, in some cells, a linear cisterna in the center of the Golgi zone. It also stained the membrane of many small vesicles as well as that of lysosomelike structures in all cells. In contrast, it never stained the secretory granule membrane, except at the level of very few segregating granules on the trans face of the Golgi zone. In GH3 cells the A-RER stained the membrane of the perinuclear cisternae, as well as that of short discontinuous flat cisternae. The A-Golgi stained the same components of the Golgi zone as in normal PRL cells. In some cells of both types the A-Golgi also stained discontinuous patches on the plasma membrane and small vesicles fusing with the plasma membrane. Immunostaining of Golgi membranes revealed modifications of membrane flow in relation to either acute stimulation of PRL release by thyroliberin or inhibition of basal secretion by monensin.

scholarly journals The Endoplasmic Reticulum Membrane Is Permeable to Small Molecules

2004 ◽  
Vol 15 (2) ◽  
pp. 447-455 ◽  
Author(s):  
Sylvie Le Gall ◽  
Andrea Neuhof ◽  
Tom Rapoport
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Low Temperature ◽  
Small Molecules ◽  
Endoplasmic Reticulum Membrane ◽  
Membrane Permeation ◽  
Permeabilized Cells ◽  
Golgi Membranes ◽  
Pass Through ◽  
Er Membrane

The lumen of the endoplasmic reticulum (ER) differs from the cytosol in its content of ions and other small molecules, but it is unclear whether the ER membrane is as impermeable as other membranes in the cell. Here, we have tested the permeability of the ER membrane to small, nonphysiological molecules. We report that isolated ER vesicles allow different chemical modification reagents to pass from the outside into the lumen with little hindrance. In permeabilized cells, the ER membrane allows the passage of a small, charged modification reagent that is unable to cross the plasma membrane or the lysosomal and trans-Golgi membranes. A larger polar reagent of ∼5 kDa is unable to pass through the ER membrane. Permeation of the small molecules is passive because it occurs at low temperature in the absence of energy. These data indicate that the ER membrane is significantly more leaky than other cellular membranes, a property that may be required for protein folding and other functions of the ER.

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