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O C Martin

Publications and source records attributed to O C Martin.

31 records · Page 2Linked to original sources
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↗
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↗
PubMed1991

A novel fluorescent ceramide analogue for studying membrane traffic in animal cells: accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor.

A series of ceramide analogues bearing the fluorophore boron dipyrromethene difluoride (BODIPY) were synthesized and evaluated as vital stains for the Golgi apparatus, and as tools for studying lipid traffic between the Golgi apparatus and the plasma membrane of living cells. Studies of the spectral properties of several of the BODIPY-labeled ceramides in lipid vesicles demonstrated that the fluorescence emission maxima were strongly dependent upon the molar density of the probes in the membrane. This was especially evident using N-[5-(5,7-dimethyl BODIPY)-1-pentanoyl]-D-erythro-sphingosine (C5-DMB-Cer), which exhibited a shift in its emission maximum from green (integral of 515 nm) to red (integral of 620 nm) wavelengths with increasing concentrations. When C5-DMB-Cer was used to label living cells, this property allowed us to differentiate membranes containing high concentrations of the fluorescent lipid and its metabolites (the corresponding analogues of sphingomyelin and glucosylceramide) from other regions of the cell where smaller amounts of the probe were present. Using this approach, prominent red fluorescent labeling of the Golgi apparatus, Golgi apparatus-associated tubulovesicular processes, and putative Golgi apparatus transport vesicles was seen in living human skin fibroblasts, as well as in other cell types. Based on fluorescence ratio imaging microscopy, we estimate that C5-DMB-Cer and its metabolites were present in Golgi apparatus membranes at concentrations up to 5-10 mol %. In addition, the concentration-dependent spectral properties of C5-DMB-Cer were used to monitor the transport of C5-DMB-lipids to the cell surface at 37 degrees C.

4-Chloro-7-nitrobenzofurazan↗
PubMed1989

Molecular trapping of a fluorescent ceramide analogue at the Golgi apparatus of fixed cells: interaction with endogenous lipids provides a trans-Golgi marker for both light and electron microscopy.

We have previously shown that a fluorescent derivative of ceramide, N-(epsilon-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl)-D-eryth ro-sphingosin e (C6-NBD-Cer), vitally stains the Golgi apparatus of cells (Lipsky, N. G., and R. E. Pagano. 1985. Science (Wash. DC). 228:745-747). In the present paper we demonstrate that C6-NBD-Cer also accumulates at the Golgi apparatus of fixed cells and we explore the mechanism by which this occurs. When human skin fibroblasts were fixed with glutaraldehyde and then incubated with C6-NBD-Cer at 2 degrees C, the fluorescent lipid spontaneously transferred into the cells, labeling the Golgi apparatus as well as other intracellular membranes. Subsequent incubations with defatted BSA at 24 degrees C removed excess C6-NBD-Cer from the cells such that fluorescence was then detected only at the Golgi apparatus. Similar results were obtained using other cell types. A method for visualizing the fluorescent lipid at the electron microscopic level, based on the photoconversion of a fluorescent marker to a diaminobenzidine product (Sandell, J. H., and R. H. Masland, 1988. J. Histochem. Cytochem. 36:555-559), is described and evidence is presented that C6-NBD-Cer was localized to the trans cisternae of the Golgi apparatus. While accumulation occurred in cells fixed in various ways, it was inhibited when fixation protocols that extract or modify cellular lipids were used. In addition, Filipin, which forms complexes with cellular cholesterol, labeled the Golgi apparatus of fixed cells and inhibited accumulation of C6-NBD-Cer at the Golgi apparatus. These results are discussed in terms of a simple model based on the physical properties of C6-NBD-Cer and its interactions with endogenous lipids of the Golgi apparatus. Possible implications of these findings for metabolism and transport of (fluorescent) sphingolipids in vivo are also presented.

4-Chloro-7-nitrobenzofurazan↗
PubMed1988

A series of fluorescent N-acylsphingosines: synthesis, physical properties, and studies in cultured cells.

We have previously shown that when cultured fibroblasts are briefly incubated at 2 degrees C with a fluorescent (NBD) analogue of ceramide, N-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-epsilon-aminohexanoyl]-D-e rythro- sphingosine, fluorescent labeling of the mitochondria, endoplasmic reticulum, and nuclear envelope occurs. During further incubation at 37 degrees C, the Golgi apparatus and later the plasma membrane become intensely fluorescent. Concomitantly, the fluorescent ceramide is metabolized to fluorescent analogues of sphingomyelin and glucosylceramide [Lipsky, N. G., & Pagano, R. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2608-2612]. In the present study we synthesized fluorescent N-acylsphingosine analogues using various long-chain bases (D-erythro-sphingosine, L-erythro-sphingosine, D-threo-sphingosine, L-threo-sphingosine, D-erythro-dihydrosphingosine, L-threo-dihydrosphingosine, phytosphingosine, and 3-ketosphingosine) and fluorescent fatty acids (epsilon-NBD-aminohexanoic acid; D- or L-alpha-OH-epsilon-NBD-aminohexanoic acid; D- or L-alpha-NBD-aminohexanoic acid). Using previously described resonance energy transfer assays, we examined the rates of spontaneous transfer of these compounds between liposomes and their ability to undergo transbilayer movement. The fluorescent N-acylsphingosine analogues had half-times for spontaneous transfer of 0.3-4.0 min at 25 degrees C, and all were capable of transbilayer movement in lipid vesicles. The metabolism and intracellular distribution of analogues in cultured fibroblasts were also studied. While most of the fluorescent N-acylsphingosines were significantly metabolized to the corresponding sphingomyelin analogues, metabolism to glucosylceramide was strongly dependent on the long-chain base and the stereochemistry of the fluorescent fatty acid moiety.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Transbilayer movement of fluorescent analogs of phosphatidylserine and phosphatidylethanolamine at the plasma membrane of cultured cells. Evidence for a protein-mediated and ATP-dependent process(es).

The internalization of fluorescent analogs of phosphatidylserine and phosphatidylethanolamine following their insertion into the plasma membrane of cultured Chinese hamster fibroblasts was examined. When liposomes containing the fluorescent lipid 1,2-(palmitoyl-N-4-nitrobenzo-2-oxa-1,3-diazole-amino-caproyl) phosphatidylserine [palmitoyl-C6-NBD)-PS), were incubated with monolayer cell cultures at 2 degrees C, spontaneous transfer of the fluorescent lipid from the liposomes to the cells occurred, resulting in prominent labeling of the plasma membrane. However, if the cells were washed and warmed to 7 degrees C for 30 min, the (palmitoyl-C6-NBD)-PS also labeled numerous intracellular membranes. Evidence is presented suggesting that this internalization was not due to endocytosis, but was the result of transmembrane movement of the (palmitoyl-C6-NBD)-PS at the plasma membrane followed by translocation of lipid monomers from the plasma membrane to internal membranes. This transmembrane movement was reversibly inhibited by depletion of cellular ATP levels and was blocked by treatment with structural analogs of the lipid or by pretreatment of cells with glutaraldehyde or N-ethyl-maleimide. A fluorescent analog of phosphatidylethanolamine [palmitoyl-C6-NBD)-PE), which also exhibits transmembrane movement at the plasma membrane at 7 degrees C (Sleight, R. G., and Pagano, R. E. (1985) J. Biol. Chem. 260, 1146-1154), was further studied. Its transmembrane movement was also inhibited by depletion of cellular ATP levels, or by pretreatment of cells with N-ethylmaleimide. The transmembrane movement of the fluorescent phosphatidylserine and phosphatidylethanolamine analogs was inhibited when the unnatural D-isomers of these lipids were used, further suggesting that this process was stereospecific and therefore likely to have been protein-mediated.

4-Chloro-7-nitrobenzofurazan↗
PubMed1986

Normal- and reverse-phase HPLC separations of fluorescent (NBD) lipids.

We have developed two high-performance liquid chromatography methods for separating a number of fluorescent 4-nitrobenzo-2-oxa-1,3-diazole (NBD) analogs of glycerolipids and sphingolipids. Samples of fluorescent lipid analogs containing NBD-aminocaproyl (C6-NBD) or NBD-aminododecanoyl (C12-NBD) acyl chains were synthesized and analyzed by the following HPLC methods. An isocratic normal-phase method permitted resolution of a mixture of the 1,2-(palmitoyl, C6-NBD)-analogs of triacylglycerol, diacylglycerol, phosphatidic acid, phosphatidylethanolamine, and phosphatidylcholine in less than 10 min, while a mixture of the (C6-NBD)-labeled analogs of ceramide, glucocerebroside, and sphingomyelin was separated in approximately 15 min. This method also detected various (C6-NBD)-phosphatidylcholine and -phosphatidylethanolamine molecules which differed only in their nonfluorescent acyl (oleoyl or palmitoyl) chains, and readily separated nonfluorescent dipalmitoylphosphatidylcholine from both (C6-NBD)- and (C12-NBD)-phosphatidylcholine derivatives. An isocratic reverse-phase system permitted separation of isomers of fluorescent phosphatidylcholine, -ethanolamine, -glycerol, -inositol, -serine, and phosphatidic acid in which the NBD-fatty acid was present in either the sn-1 or sn-2 position of the glycerol backbone.

4-Chloro-7-nitrobenzofurazan↗
PubMed1985

Synthesis of fluorescent and radiolabeled analogues of phosphatidic acid.

Procedures for the synthesis of fluorescent and radiolabeled analogues of phosphatidic acid are described. The fluorophore 7-nitrobenzo-2-oxa-1,3-diazole (NBD) was coupled to 6-amino-caproic acid and 12-aminododecanoic acid by reaction of NBD-chloride with the amino acids under mild alkaline conditions at room temperature. 1,2-Dioleoyl-sn-[U-14C]glycerol 3-phosphate was prepared by acylation of sn-[U-14C]glycerol 3-phosphate with oleic acid anhydride using dimethylaminopyridine as the catalyst. This compound was converted to 1-oleoyl-sn-[U-14C]glycerol 3-phosphate by hydrolysis with phospholipase A2. The lysophosphatidic acid was reacylated with NBD-aminocaproyl imidazole or NBD-aminododecanoyl imidazole to form the fluorescent, radiolabeled analogue of phosphatidic acid. Fluorescent, non-radiolabeled analogues of phosphatidic acid were prepared by phospholipase D hydrolysis of fluorescent phosphatidylcholine.

4-Chloro-7-nitrobenzofurazan↗
PubMed1983

Intracellular translocation and metabolism of a fluorescent phosphatidic acid analogue in cultured fibroblasts.

We have investigated the metabolism and intracellular translocation of a fluorescent derivative of phosphatidic acid, 1-acyl-2-[(N-4-nitrobenzo-2-oxa-1,3-diazole)aminocaproyl] phosphatidic acid (C6-NBD-PA), and its metabolites, in Chinese hamster fibroblasts. This derivative is rapidly transferred from phospholipid vesicles to cells at 2 degrees C, and results in fluorescent labeling of the mitochondria, endoplasmic reticulum, and nuclear membrane of intact cells during its metabolism predominantly to fluorescent diglyceride (Pagano, R. E., Longmuir, K. J., Martin, O. C., and Struck, D. K. (1981) J. Cell Biol. 91, 872-877). In the present study, we show that, upon warming to 37 degrees C, the fluorescence associated with the endoplasmic reticulum was greatly reduced, while cytoplasmic lipid droplets, which were initially nonfluorescent, became intensely labeled. This altered intracellular distribution of fluorescence was accompanied by further metabolism of the fluorescent lipids to NBD-triglyceride and NBD-phosphatidylcholine. Although NBD-fatty acid was also produced, it was not re-utilized in the synthesis of other cellular lipids. Subcellular fractionation experiments demonstrated that primarily NBD-labeled triglyceride was associated with the intracellular lipid droplets, although substantial amounts of NBD-labeled phosphatidic acid, phosphatidylcholine, and diglyceride were also present in the whole cell extracts. This finding was confirmed in a separate experiment in which the fluorescent lipids associated with the intracellular lipid droplets were selectively and irreversibly photobleached in situ. Extraction and analysis of the fluorescent lipids revealed that NBD-triglyceride was preferentially photobleached. These results indicate that "sorting" of the NBD-labeled lipids into various cytoplasmic compartments accompanied their metabolism.

Animals↗
PubMed1982

Transbilayer redistribution of phosphatidylethanolamine during fusion of phospholipid vesicles. Dependence on fusion rate, lipid phase separation, and formation of nonbilayer structures.

The effect of membrane fusion on the transbilayer distribution of dioleoyl- and dipalmitoylphosphatidylethanolamine (DOPE and DPPE, respectively) in phosphatidylserine (PS) vesicles was investigated. A 7-fold increase in the external pool of DOPE, as determined by labeling of the vesicle surface with 2,4,6-trinitrobenzenesulfonic acid, was observed when multilamellar vesicles (MLV) consisting of PS and DOPE were incubated with small unilamellar vesicles (SUV) of PS in the presence of Ca2+. In contrast, no significant redistribution of DPPE was seen when similar experiments were performed by using PS bilayers that contained DPPE instead of DOPE. Redistribution of neither DOPE nor DPPE could be detected during SUV-SUV fusion. By using the resonance energy transfer fusion assay for mixing of membrane lipids [Struck, D.K., Hoekstra, D., & Pagano, R. E. (1981) Biochemistry 20, 4093-4099; Hoekstra, D. (1982) Biochemistry 21, 2833-2840], it was demonstrated that fusion between SUV and MLV had actually occurred. The results also suggested that (partial) fusion of internal bilayers within the multilamellar system must have occurred. Although Mg2+-induced fusion between SUV and MLV was also observed, no redistribution of DOPE was seen in this case. It is concluded that the observed translocation of DOPE during fusion was probably mediated via inverted micellar structures, which were formed when the lipid was converted to the hexagonal (HII) phase resulting from lipid phase separation between PS and DOPE. However, induction of the hexagonal phase per se, i.e., in the absence of fusion, did not cause substantial transbilayer redistribution of DOPE, suggesting that fusion was intimately involved in this phenomenon. It is suggested that fusion represents the "driving force" for transbilayer DOPE redistribution, requiring a (partial) overlap between the kinetics of phase separation and fusion.

Calcium↗
PubMed1981

Formation of asymmetric phospholipid membranes via spontaneous transfer of fluorescent lipid analogues between vesicle populations.

A method is presented for generating artificial lipid vesicles bearing an asymmetric distribution of either of the fluorescent lipid analogues 1-acyl-2-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]caproyl]phosphatidylcholine or 1-acyl-2-[12[(7-nitro-2,1,3-benzoxadiazol-4-yl]amino]dodecanoyl]-phosphatidylcholine, in which the fluorescent lipid is located predominantly in either the outer or inner leaflet of the vesicle bilayer. The procedure is based on the observation that these lipid analogues undergo rapid spontaneous transfer (exchange) between vesicle populations [Nichols, J. W., & Pagano, R. E. (1981) Biochemistry 20, 2783-2789]. When an excess of nonfluorescent acceptor vesicles is mixed with small unilamellar vesicles containing 5 mol % fluorescent lipid, approximately 50% of the fluorescent lipid is transferred to the acceptor vesicles, whereas if fluorescent multilamellar vesicles are used, only approximately 10% of the analogues is available for transfer. These fractions of fluorescent lipid available for intervesicular transfer correspond closely to the amount of phospholipid residing in the outermost leaflet of the donor vesicles, suggesting that only fluorescent lipids present in the outer surface of the vesicles can spontaneously transfer between vesicles populations. Evidence demonstrating that the movement of the fluorescent lipid between vesicle population is the result of a net transfer process rather than lipid exchange is also presented. A novel assay based on resonance energy transfer is described for determining the size of the exchangeable fluorescent lipid pool, a measure of the degree of asymmetry of these preparations. Finally, for demonstration of the usefulness of asymmetric vesicles in distinguishing various pathways of vesicle-cell association, preliminary results are presented on their interactions with Chinese hamster fibroblasts in vitro.

Chemical Phenomena↗
PubMed1981

Metabolism and intracellular localization of a fluorescently labeled intermediate in lipid biosynthesis within cultured fibroblasts.

In this paper we report on the uptake and distribution of an exogenously supplied fluorescent phosphatidic acid analogue by Chinese hamster fibroblasts. Under appropriate in vitro incubation conditions, 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidic acid was rapidly and preferentially transferred from phospholipid vesicles to cells at 2 degrees C. However, unlike similar fluorescent derivatives of phosphatidylcholine and phosphatidylethanolamine that remain restricted to the plasma membrane under such incubation conditions (Struck, D. K., and R. E. Pagano. 1080. J. Biol. Chem. 255:5405--5410), most of the phosphatidic acid-derived fluorescence was localized at the nuclear membrane, endoplasmic reticulum, and mitochondria. This was shown by labeling cells with rhodamine-containing probes specific for mitochondria or endoplasmic reticulum, and comparing the patterns of intracellular NBD and rhodamine fluorescence. Extraction and analysis of the fluorescent lipids associated with the cells after treatment with vesicles at 2 degrees or 37 degrees C revealed that a large fraction of the fluorescent phosphatidic acid was converted to fluorescent diglyceride, phosphatidylcholine, and triglyceride. Our findings suggest that fluorescent phosphatidic acid may be useful in correlating biochemical studies of lipid metabolism in cultured cells and studies of the Intracellular localization of the metabolites by fluorescence microscopy. In addition, this compound provides a unique method for visualizing the endoplasmic reticulum in living cells.

Animals↗