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R E Pagano

Publications and source records attributed to R E Pagano.

At least 37 records · Page 2Linked to original sources
PubMed1997

Changes in the spectral properties of a plasma membrane lipid analog during the first seconds of endocytosis in living cells.

N-[5-(5, 7-dimethyl Bodipy)-1-pentanoyl]-D-erythro-sphingosylphosphorylcholine (C5-DMB-SM), a fluorescent analog of sphingomyelin, has been used in a study of the formation of very early endosomes in human skin fibroblasts. This lipid exhibits a shift in its fluorescence emission maximum from green (approximately 515 nm) to red (approximately 620 nm) wavelengths with increasing concentrations in membranes. When cells were incubated with 5 microM C5-DMB-SM at 4 degrees C and washed, only plasma membrane fluorescence (yellow-green) was observed. When these cells were briefly (< or = 1 min) warmed to 37 degrees C to allow internalization to occur, and then incubated with defatted bovine serum albumin (back-exchanged) at 11 degrees C to remove fluorescent lipids from the plasma membrane, C5-DMB-SM was distributed in a punctate pattern throughout the cytoplasm. Interestingly, within the same cell some endosomes exhibited green fluorescence, whereas others emitted red-orange fluorescence. Furthermore, the red-orange endosomes were usually seen at the periphery of the cell, while the green endosomes were more uniformly distributed throughout the cytoplasm. This mixed population of endosomes was seen after internalization times as short as 7 s and was also seen over a wide range of C5-DMB-SM concentrations (1-25 microM). Control experiments established that the variously colored endosomes were not induced by changes in pH, membrane potential, vesicle size, or temperature. Quantitative fluorescence microscopy demonstrated that the apparent concentration of the lipid analog in the red-orange endosomes was severalfold higher than its initial concentration at the plasma membrane, suggesting selective internalization (sorting) of the lipid into a subset of early endosomes. Colocalization studies using C5-DMB-SM and either anti-transferrin receptor antibodies or fluorescently labeled low-density lipoprotein further demonstrated that this subpopulation of endosomes resulted from receptor-mediated endocytosis. We conclude that the spectral properties of C5-DMB-SM can be used to distinguish unique populations of early endosomes from one another and to record dynamic changes in their number and distribution within living cells.

Cell Membrane↗
PubMed1997

Visualization of peptides derived from liposome-encapsulated proteins in the trans-Golgi area of macrophages.

Exogenous proteins are generally not presented through the major histocompatibility complex (MHC) class I pathway, yet several recent studies show that particle-associated antigens induce a CD8+ T-cell response. Therefore, a pathway must exist in vivo for the presentation of exogenous antigens on class I molecules. In the present study, we investigated the intracellular fate of liposome-encapsulated Texas Red (TR)-conjugated protein in cultured bone marrow-derived macrophages (BMs). After phagocytosis of liposomes, the fluorescent liposomal protein, initially associated with the liposomal lipids in phagosomes, later entered the cytoplasm, and the processed protein was subsequently visualized in the trans-Golgi as a fluorescent peptide. Experiments performed with BMs from transporter associated with antigen processing (TAP1) knock-out mice demonstrated that the translocation of peptides into the trans-Golgi area was dependent upon TAP1 protein. We conclude that delivery of liposomal proteins or peptides to the cytoplasm of phagocytes and subsequent transport of peptides to the Golgi via the classical MHC class I pathway involving TAP proteins might explain the known propensity of liposomal antigens to induce cytotoxic T-lymphocytes (CTLs).

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Use of a fluorescent analog of CDP-DAG in human skin fibroblasts: characterization of metabolism, distribution, and application to studies of phosphatidylinositol turnover.

We studied the uptake metabolism, and distribution of a fluorescent analog of CDP-diacylglycerol [cytidine diphosphate-1, 2-oleoyl, (N-(4-nitrobenzo-2-oxa-1,3-diazole) aminocaproyl) diacylglycerol; CDP-NBD-DAG]. When cells were incubated with CDP-NBD-DAG for 60 min at 11 degrees C and washed, the fluorescent lipid was localized to the plasma membrane. However, upon warming to 37 degrees C, the fluorescent lipid redistributed into various intracellular membranes and was metabolized primarily to fluorescent analogs of DAG and phosphatidylcholine (PC), although small amounts of fluorescent phosphatidic acid and phosphatidylinositol (PI) were also formed. The incorporation of 32Pi into some of the fluorescent lipids was also determined in order to assess their turnover. Stimulation of cells with platelet-derived growth factor enhanced the synthesis of fluorescent PI relative to unstimulated cells by approximately 68%, while the synthesis of fluorescent PC was unaffected. In addition, the incorporation of 32Pi into fluorescent PI was enhanced. Stimulation of cells with interleukin-1 beta enhanced the synthesis of both fluorescent PI (approximately 88%) and PC (approximately 250%) compared to non-stimulated cells, but with less incorporation of 32Pi into fluorescent PI. Finally, incubation of CDP-NBD-DAG-treated cells with inhibitors of phosphatidic acid phosphohydrolase and DAG kinase resulted in a dramatic increase in the amount of fluorescent PI formed (approximately 64% of all the CDP-NBD-DAG metabolites). We conclude that CDP-NBD-DAG can be used for the de novo synthesis of fluorescent PI, and in combination with 32P labeling, provides a convenient method for studying PI turnover.

4-Chloro-7-nitrobenzofurazan↗
PubMed1996

Purification and characterization of UDP-glucose:ceramide glucosyltransferase from rat liver Golgi membranes.

We present a method for solubilizing and purifying UDP-Glc:ceramide glucosyltransferase (EC 2.4.1.80; glucosylceramide synthase (GCS) from a rat liver and present data on its substrate specificity. A Golgi membrane fraction was isolated, washed with N-lauroylsarcosine, and subsequently treated with 3[3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate to solubilize the enzyme. GCS activity was monitored throughout purification using UDP-Glc and a fluorescent ceramide analog as substrates. Purification of GCS was achieved via a two-step dye-agarose chromatography procedure using UDP-Glc to elute the enzyme. This resulted in an enrichment > 10,000-fold relative to the starting homogenate. The enzyme was further characterized by sedimentation on a glycerol gradient, I labeling, and SDS-polyacrylamide gel electrophoresis. which demonstrated that two polypeptides (60-70 kDa) corresponded closely with GCS activity. Purified GCS was found to require exogenous phospholipids for activity, and optimal results were obtained using dioleoyl phosphatidylcholine. Studies of the substrate specificity of the purified enzyme demonstrated that it was stereospecific and dependent on the nature and chain length of the N-acyl-spingosine or -sphinganine substrate. UDP-Glc was the preferred hexose donor, but TDP-glucose and CDP-glucose were also efficiently used. This study provides a basis for molecular characterization of this key enzyme in glycosphingolipid biosynthesis.

Animals↗
PubMed1995

Ceramide as a modulator of endocytosis.

The effects of ceramide analogs on the uptake of markers for fluid-phase (horseradish peroxidase, HRP) and receptor-mediated (low density lipoprotein, LDL) endocytosis were studied in Chinese hamster fibroblasts. N-Hexanoyl-D-erythro-sphingosine (C6-Cer) decreased the uptake of HRP in a dose-dependent manner. Internalization was inhibited > 40% with 25 microM C6-Cer, relative to controls, and was apparent within 5 min. Internalization of HRP was also inhibited by other Cer analogs and by treatment with agents that raise levels of endogenous Cer (sphingomyelinase or the glycosphingolipid synthesis inhibitor, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP)), but not by N-hexanoyl-D-erythrosphinganine (C6-dihydro-Cer) or sphingosine. Internalization of LDL was also inhibited by C6-Cer in a concentration-dependent manner, but was less pronounced than the effect on HRP internalization (10% versus 40% inhibition with 25 microM C6-Cer), suggesting that ceramide might affect fluid-phase and receptor-mediated endocytosis to different extents. C6-Cer also slowed HRP and LDL transport from endosomes to lysosomes as studied by analysis of endocytic vesicles on Percoll density gradients and induced a redistribution of endocytic organelles as determined by fluorescence microscopy of intact cells using appropriate markers. This resulted in decreased degradation of 125I-labeled LDL in the presence of C6-Cer. These results suggest that endogenous ceramide may modulate endocytosis.

Animals↗
PubMed1995

Both sphingolipids and cholesterol participate in the detergent insolubility of alkaline phosphatase, a glycosylphosphatidylinositol-anchored protein, in mammalian membranes.

SPB-1, a Chinese hamster ovary cell variant defective in serine palmitoyltransferase activity for sphingolipid synthesis, provides a useful system for studying the effects of sphingolipids and/or cholesterol deprivation on cellular functions and membrane properties. To investigate whether there was an interaction among sphingolipids, cholesterol, and glycosylphosphatidylinositol (GPI)-anchored proteins in biological membranes, we introduced human placental alkaline phosphatase (PLAP) in SPB-1 and in wild type cells by stable transfection and examined the effects of sphingolipid and/or cholesterol deprivation on the solubility of PLAP in Triton X-100. Although the PLAP solubility of the membranes isolated from the control cells in Triton X-100 was only 10%, deprivation of sphingolipid and cholesterol further enhanced the solubility, which reached 50% when both sphingolipids and cholesterol were deprived. The enhanced solubility was suppressed to the control level by metabolic complementation with exogenous sphingosine and cholesterol. The sphingolipid and cholesterol content of the isolated membranes changed independently, eliminating the possibility that sphingolipid deprivation induced a reduction in cellular cholesterol and enhanced PLAP solubility and vice versa. It was also unlikely that the enhanced solubility was due to structural changes in PLAP molecules since, regardless of sphingolipid and cholesterol deprivations, almost all PLAP had the GPI-anchor moiety and there were no differences in the apparent molecular weight of the protein in supernatant and precipitate fractions of the detergent-treated membranes. In addition, the expression level of caveolin in the isolated membranes was not significantly affected by sphingolipids and/or cholesterol depletion. These results indicated that both sphingolipids and cholesterol were involved in the PLAP insolubility and suggested that these lipids coordinately played a role in formation of Triton X-100-resistant complexes.

Acyltransferases↗
PubMed1995

A Chinese hamster ovary cell mutant defective in the non-endocytic uptake of fluorescent analogs of phosphatidylserine: isolation using a cytosol acidification protocol.

Transmembrane movement of phosphatidylserine (PS) and various PS analogs at the plasma membrane is thought to occur by an ATP-dependent, protein-mediated process. To isolate mutant CHO cells defective in this activity, we first obtained conditions which inhibited the endocytic, but not the non-endocytic pathway of lipid internalization since PS may enter cells by a combination of these two pathways. We found that acidic treatment of cells, which blocks clathrin-dependent endocytosis, enhanced the energy-dependent uptake of 1-palmitoyl-2-(6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl -sn- glycero-3-phosphoserine (C6-NBD-PS) in CHO cells from donor vesicles (liposomes) by about twofold. Control experiments demonstrated that the enhanced uptake of C6-NBD-PS at acidic pH was not due to: (a) an increase in the capacity of the plasma membrane to incorporate C6-NBD-PS from the donor vesicles; (b) a decrease in the rate of loss of C6-NBD-PS from the cells; or (c) fusion or engulfment of the donor vesicles. When cytosolic acidification (to pH 6.3) was imposed without acidification of the extracellular medium, C6-NBD-PS uptake by intact cells was increased by about 50% compared to control values determined in the absence of acidification. These results suggested that a protein and energy dependent system(s) for transbilayer movement of the fluorescent PS was stimulated by cytosolic acidification. A screening method for mutant cells defective in the non-endocytic uptake of fluorescent PS analogs with replica cell colonies at acidic pH was then devised. After selection of mutagenized CHO-K1 cells by in situ screening, we obtained a mutant cell line in which uptake of fluorescent PS analogs was reduced to about 25% of the wild type level at either pH 6.0 or 7.4. Control experiments demonstrated that the reduced uptake of fluorescent PS analogs in the mutant cells was unrelated to multidrug resistance, and that endocytosis of another plasma membrane lipid marker occurred normally in the mutant cells. These results suggested that a non-endocytic pathway responsible for uptake of fluorescent PS analogs was specifically affected in the mutant cells.

4-Chloro-7-nitrobenzofurazan↗
PubMed1994

Internalization and sorting of a fluorescent analogue of glucosylceramide to the Golgi apparatus of human skin fibroblasts: utilization of endocytic and nonendocytic transport mechanisms.

We examined the uptake and intracellular transport of the fluorescent glucosylceramide analogue N-[5-(5,7-dimethyl BODIPYTM)-1-pentanoyl]-glucosyl sphingosine (C5-DMB-GlcCer) in human skin fibroblasts, and we compared its behavior to that of the corresponding fluorescent analogues of sphingomyelin, galactosylceramide, and lactosylceramide. All four fluorescent analogues were readily transferred from defatted BSA to the plasma membrane during incubation at 4 degrees C. When cells treated with C5-DMB-GlcCer were washed, warmed to 37 degrees C, and subsequently incubated with defatted BSA to remove fluorescent lipid at the cell surface, strong fluorescence was observed at the Golgi apparatus, as well as weaker labeling at the nuclear envelope and other intracellular membranes. Similar results were obtained with C5-DMB-galactosylceramide, except that labeling of the Golgi apparatus was weaker than with C5-DMB-GlcCer. Internalization of C5-DMB-GlcCer was not inhibited by various treatments, including ATP depletion or warming to 19 degrees C, and biochemical analysis demonstrated that the lipid was not metabolized during its internalization. However, accumulation of C5-DMB-GlcCer at the Golgi apparatus was reduced when cells were treated with a nonfluorescent analogue of glucosylceramide, suggesting that accumulation of C5-DMB-GlcCer at the Golgi apparatus was a saturable process. In contrast, cells treated with C5-DMB-analogues of sphingomyelin or lactosylceramide internalized the fluorescent lipid into a punctate pattern of fluorescence during warming at 37 degrees C, and this process was temperature and energy dependent. These results with C5-DMB-sphingomyelin and C5-DMB-lactosylceramide were analogous to those obtained with another fluorescent analogue of sphingomyelin in which labeling of endocytic vesicles and plasma membrane lipid recycling were documented (Koval, M., and R. E. Pagano. 1990. J. Cell Biol. 111:429-442). Incubation of perforated cells with C5-DMB-sphingomyelin resulted in prominent labeling of the nuclear envelope and other intracellular membranes, similar to the pattern observed with C5-DMB-GlcCer in intact cells. These observations are consistent with the transbilayer movement of fluorescent analogues of glucosylceramide and galactosylceramide at the plasma membrane and early endosomes of human skin fibroblasts, and suggest that both endocytic and nonendocytic pathways are used in the internalization of these lipids from the plasma membrane.

Adenosine Triphosphate↗
PubMed1994

Effects of the glucosphingolipid synthesis inhibitor, PDMP, on lysosomes in cultured cells.

The glucosphingolipid synthesis inhibitor, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) has a wide range of effects on cell physiology and morphology. Here, we studied the effects of high concentrations of PDMP on cells in culture and found that fluorescent analogs of PDMP targeted to the lysosomes of Chinese hamster ovary (CHO) cells. Overnight incubation of the cells in the presence of drug induced enlargement ("vacuolization") of the lysosomes. PDMP was toxic at high concentrations (> 30 microM); this finding was used to select CHO cells that exhibited increased resistance to PDMP (PDMPR cells). The PDMPR cells were approximately 2-fold more resistant to PDMP than the parental cells (CHO-P). PDMPR cells were resistant to a number of other drugs that are also lipophilic and possess a titratable amino group. The multidrug resistance exhibited by the PDMPR cells was distinct from that observed in cells (MDR cells) that overproduce the plasma membrane drug pump, P-glycoprotein. In addition, MDR cells were extremely sensitive to PDMP.

Amines↗
PubMed1993

Cholesterol deprivation affects the fluorescence properties of a ceramide analog at the Golgi apparatus of living cells.

Previous studies have established that a fluorescent analog of ceramide, N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)] -6-aminohexanoyl-D-erythro-sphingosine (C6-NBD-Cer), is a vital stain for the Golgi apparatus and a useful tool for studying the sorting and transport of sphingolipids along the secretory pathway in animal cells. Here, we examine the effects of various culture conditions on labeling of the Golgi apparatus of human skin fibroblasts by C6-NBD-Cer and demonstrate that cholesterol deprivation affects the fluorescence properties of the probe at this organelle. Labeling of the Golgi apparatus by C6-NBD-Cer was dramatically reduced in cells grown in medium containing lipoprotein-deficient serum compared to cells grown in medium containing normal serum. Quantitative fluorescence microscopy showed that this apparent reduction in labeling resulted from accelerated photo-bleaching of the fluorescent analog. C6-NBD-Cer labeling of the Golgi apparatus was restored in cholesterol-deprived cells by stimulating endogenous cholesterol biosynthesis with mevalonic acid or by adding exogenous nonlipoprotein cholesterol or low density lipoprotein to the culture medium. In addition, when cells grown in medium containing normal serum were perforated and treated with cholesterol oxidase, an apparent reduction in labeling resulted, further implicating an intracellular pool of cholesterol in the potentiation of C6-NBD-Cer fluorescence. These results demonstrate that cytological studies using C6-NBD-Cer are affected by cholesterol deprivation and suggest that this fluorescent lipid may be used to monitor cholesterol at the Golgi apparatus of living cells.

4-Chloro-7-nitrobenzofurazan↗
PubMed1993

Inhibition of glycoprotein traffic through the secretory pathway by ceramide.

Incubation of vesicular stomatitis virus-infected cells with short-chain, cell-permeable ceramide (Cer) analogs decreased the rate of viral glycoprotein transport through the Golgi complex and reduced the number of infectious virions released from cells in a concentration-dependent manner. These effects appeared to be caused directly by Cer, rather than by one of its metabolites. Cer treatment also disrupted the Golgi apparatus within 1 h, although cells treated for up to 24 h with Cer remained viable. Our results suggest that endogenous Cer may modulate secretory protein traffic and that exogenously added Cer analogs may be useful as antiviral agents.

Animals↗
PubMed1993

WIF-B cells: an in vitro model for studies of hepatocyte polarity.

We have evaluated the utility of the hepatoma-derived hybrid cell line, WIF-B, for in vitro studies of polarized hepatocyte functions. The majority (> 70%) of cells in confluent culture formed closed spaces with adjacent cells. These bile canalicular-like spaces (BC) accumulated fluorescein, a property of bile canaliculi in vivo. By indirect immunofluorescence, six plasma membrane (PM) proteins showed polarized distributions similar to rat hepatocytes in situ. Four apical PM proteins were concentrated in the BC membrane of WIF-B cells. Microtubules radiated from the BC (apical) membrane, and actin and foci of gamma-tubulin were concentrated in this region. The tight junction-associated protein ZO-1 was present in belts marking the boundary between apical and basolateral PM domains. We explored the functional properties of this boundary in living cells using fluorescent membrane lipid analogs and soluble tracers. When cells were incubated at 4 degrees C with a fluorescent analog of sphingomyelin, only the basolateral PM was labeled. In contrast, when both PM domains were labeled by de novo synthesis of fluorescent sphingomyelin from ceramide, fluorescent lipid could only be removed from the basolateral domain. These data demonstrate the presence of a barrier to the lateral diffusion of lipids between the PM domains. However, small soluble FITC-dextrans (4,400 mol wt) were able to diffuse into BC, while larger FITC-dextrans were restricted to various degrees depending on their size and incubation temperature. At 4 degrees C, the surface labeling reagent sNHS-LC-biotin (557 mol wt) had access to the entire PM, but streptavidin (60,000 mol wt), which binds to biotinylated molecules, was restricted to only the basolateral domain. Such differential accessibility of well-characterized probes can be used to mark each membrane domain separately. These results show that WIF-B cells are a suitable model to study membrane trafficking and targeting in hepatocytes in vitro.

Animals↗
PubMed1992

Effects of a sphingolipid synthesis inhibitor on membrane transport through the secretory pathway.

We investigated the effects of an inhibitor of sphingolipid biosynthesis, 1-phenyl-2-(decanoyl-amino)-3-morpholino-1-propanol (PDMP), on cells in culture. Two Golgi-associated enzymes were affected by incubation of cells with PDMP. The synthesis of glucosylceramide was inhibited at low concentrations of PDMP (2.5-10 microM), and in the presence of higher concentrations (greater than or equal to 25 microM), synthesis of sphingomyelin was also reduced. Transport of vesicular stomatitis virus G protein through the Golgi complex was progressively retarded by increasing concentrations of PDMP. In the presence of 75 microM PDMP, the half-times of VSV-G protein arrival at the cis, medial, and trans Golgi and the cell surface were increased 1.5-, 2.1-, 2.4-, and 2.8-fold, respectively, compared to control values. Transport of fluorescent sphingolipids, synthesized de novo at the Golgi complex from fluorescent ceramide precursors, to the cell surface was retarded by approximately 20% in the presence of 50 microM PDMP and by approximately 50% in the presence of 100 microM PDMP. Control experiments demonstrated that PDMP had minimal effects on cell morphology and physiology (including microtubule and endoplasmic reticulum structure, mitochondrial function, and endocytosis). Although incubation of cells with relatively high concentrations of PDMP was required to see the effects on protein and sphingolipid transport, use of a fluorescent analogue of PDMP demonstrated that most cell-associated PDMP was sequestered in lysosomes, while the concentration at the Golgi complex, the site of the target synthetic enzymes, was relatively low. Taken together, these results suggest that transport of proteins and sphingolipids through the secretory pathway may be coupled to sphingolipid synthesis.

Animals↗
PubMed1992

Determination of the transbilayer distribution of fluorescent lipid analogues by nonradiative fluorescence resonance energy transfer.

We measured the nonradiative fluorescence resonance energy transfer between 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) labeled lipids (amine labeled phosphatidylethanolamine or acyl chain labeled phosphatidylcholine) and rhodamine labeled lipids in large unilamellar dioleoylphosphatidylcholine vesicles. Two new rhodamine labeled lipid analogues, one a derivative of monolauroylphosphatidylethanolamine and the other of sphingosylphosphorylcholine, were found to exchange through the aqueous phase between vesicle populations but not to be capable of rapid transbilayer movement between leaflets. Energy transfer from NBD to rhodamine was measured using liposomes with symmetric or asymmetric distributions of these new rhodamine labeled lipid analogues to determine the relative contributions of energy transfer between donor and acceptor fluorophores in the same (cis) and opposite (trans) leaflets. Since the characteristic R0 values for energy transfer ranged from 47 to 73 A in all cases, significant contributions from both cis and trans energy transfer were observed. Therefore, neither of these probes acts strictly as a half-bilayer quencher of NBD lipid fluorescence. The dependence of transfer efficiency on acceptor density was fitted to a theoretical treatment of energy transfer to determine the distances of closest approach for cis and trans transfer. These parameters set limits on the positions of the fluorescent groups relative to the bilayer center, 20-31 A for NBD and 31-55 A for rhodamine, and provide a basis for future use of these analogues in measurements of transbilayer distribution and transport.

4-Chloro-7-nitrobenzofurazan↗
PubMed1992

Use of N-([1-14C]hexanoyl)-D-erythro-sphingolipids to assay sphingolipid metabolism.

An advantage of using N-([1-14C]hexanoyl)sphingolipids to assay sphingolipid metabolism is their ability to rapidly and spontaneously transfer into biological membranes without destroying membrane integrity. This property allows analysis of the activity of enzymes of sphingolipid metabolism under conditions in which the rate of product formation is not limited by availability of substrate, as is often the case with naturally occurring lipids whose rates of spontaneous transfer are extremely slow. Thus, the use of N-([1-14C]hexanoyl)sphingolipids provides an alternative means for studying sphingolipid metabolism in vitro.

Animals↗