Receptor and membrane recycling can occur with unaltered efficiency despite dramatic Rab5(q79l)-induced changes in endosome geometry.
AuthorsCeresa BP, Lotscher M, Schmid SL
JournalJ Biol Chem
PubMed ID11136733
'Current models for sorting in the endosomal compartment suggest that endosomal geometry plays a significant role as membrane-bound proteins accumulate in tubular regions for recycling, and lumenal markers accumulate in large vacuolar portions for delivery to lysosomes. Rab5, a small molecular weight GTPase, functions in the formation and maintenance of ... More
Phospholipid translocation and miltefosine potency require both L. donovani miltefosine transporter and the new protein LdRos3 in Leishmania parasites.
AuthorsPérez-Victoria FJ, Sánchez-Cañete MP, Castanys S, Gamarro F
JournalJ Biol Chem
PubMed ID16785229
'The antitumor drug miltefosine has been recently approved as the first oral drug active against visceral leishmaniasis. We have previously identified the L. donovani miltefosine transporter (LdMT) as a P-type ATPase involved in phospholipid translocation at the plasma membrane of Leishmania parasites. Here we show that this protein is essential ... More
Imaging of total intracellular calcium and calcium influx and efflux in individual resting and stimulated tumor mast cells using ion microscopy.
'Ion microscopy was employed to investigate intracellular total calcium concentrations and calcium influx, and efflux in resting and antigen-stimulated tumor mast cells (RBL-2H3 cells). The nucleus, a perinuclear region which included the Golgi apparatus (Golgi region), and the remaining cytoplasm were spatially resolved with the Cameca IMS-3f ion microscope in ... More
Differential targeting of glucosylceramide and galactosylceramide analogues after synthesis but not during transcytosis in Madin-Darby canine kidney cells.
Authorsvan Genderen I, van Meer G
JournalJ Cell Biol
PubMed ID7593186
'A short-chain analogue of galactosylceramide (6-NBD-amino-hexanoyl-galactosylceramide, C6-NBD-GalCer) was inserted into the apical or the basolateral surface of MDCK cells and transcytosis was monitored by depleting the opposite cell surface of the analogue with serum albumin. In MDCK I cells 32% of the analogue from the apical surface and 9% of ... More
Formation of membrane domains created during the budding of vesicular stomatitis virus. A model for selective lipid and protein sorting in biological membranes.
AuthorsLuan P, Yang L, Glaser M
JournalBiochemistry
PubMed ID7543280
'Vesicular stomatitis virus buds from domains of the plasma membrane that have a unique protein and lipid composition. Fluorescence digital imaging microscopy and resonance energy transfer were used to determine how the two viral envelope-associated proteins, the G and the M proteins, could alter the lateral distribution of lipids in ... More
Chlamydia trachomatis interrupts an exocytic pathway to acquire endogenously synthesized sphingomyelin in transit from the Golgi apparatus to the plasma membrane.
AuthorsHackstadt T, Rockey DD, Heinzen RA, Scidmore MA
JournalEMBO J
PubMed ID8605892
'Chlamydia trachomatis acquires C6-NBD-sphingomyelin endogenously synthesized from C6-NBD-ceramide and transported to the vesicle (inclusion) in which they multiply. Here we explore the mechanisms of this unusual trafficking and further characterize the association of the chlamydial inclusion with the Golgi apparatus. Endocytosed chlamydiae are trafficked to the Golgi region and begin ... More
Sorting of sphingolipids in the endocytic pathway of HT29 cells.
AuthorsKok JW, Babia T, Hoekstra D
JournalJ Cell Biol
PubMed ID2071671
'The intracellular flow and fate of two fluorescently labeled sphingolipids, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]hexanoyl glucosyl sphingosine (C6-NBD-glucosylceramide) and C6-NBD-sphingomyelin, was examined in the human colon adenocarcinoma cell line HT29. After their insertion into the plasma membrane at low temperature and subsequent warming of the cells to 37 degrees C, both sphingolipid analogues ... More
Clathrin polymerization is not required for bulk-phase endocytosis in rat fetal fibroblasts.
AuthorsCupers P, Veithen A, Kiss A, Baudhuin P, Courtoy PJ
JournalJ Cell Biol
PubMed ID7962055
'To assess the role of clathrin in the bulk endocytic flow of rat foetal fibroblasts, the rate of internalization of fluid-phase and membrane-lipid tracers were compared, under control conditions and after inhibition of endocytic clathrin-coated pit formation. After intracellular potassium depletion or upon cell transfer into 0.35 M NaCl, the ... More
Hierarchy of mechanisms involved in generating Na/K-ATPase polarity in MDCK epithelial cells.
AuthorsMays RW, Siemers KA, Fritz BA, Lowe AW, van Meer G, Nelson WJ
JournalJ Cell Biol
PubMed ID7657695
'We have studied mechanisms involved in generating a polarized distribution of Na/K-ATPase in the basal-lateral membrane of two clones of MDCK II cells. Both clones exhibit polarized distributions of marker proteins of the apical and basal-lateral membranes, including Na/K-ATPase, at steady state. Newly synthesized Na/K-ATPase, however, is delivered from the ... More
The End2 mutation in CHO cells slows the exit of transferrin receptors from the recycling compartment but bulk membrane recycling is unaffected.
AuthorsPresley JF, Mayor S, Dunn KW, Johnson LS, McGraw TE, Maxfield FR
JournalJ Cell Biol
PubMed ID8376460
'We have characterized a new CHO cell line (12-4) derived from a parental line, TRVb-1, that expresses the human transferrin receptor. This mutant belongs to the end2 complementation group of endocytosis mutants. Like other end2 mutants, the endosomes in 12-4 cells show a partial acidification defect. These cells internalize LDL ... More
Polarized sphingolipid transport from the subapical compartment changes during cell polarity development.
Authorsvan IJzendoorn SC, Hoekstra D
JournalMol Biol Cell
PubMed ID10712522
'The subapical compartment (SAC) plays an important role in the polarized transport of proteins and lipids. In hepatoma-derived HepG2 cells, fluorescent analogues of sphingomyelin and glucosylceramide are sorted in the SAC. Here, evidence is provided that shows that polarity development is regulated by a transient activation of endogenous protein kinase ... More
Cholesterol depletion induces large scale domain segregation in living cell membranes.
AuthorsHao M, Mukherjee S, Maxfield FR
JournalProc Natl Acad Sci U S A
PubMed ID11698680
'Local inhomogeneities in lipid composition play a crucial role in regulation of signal transduction and membrane traffic. Nevertheless, most evidence for microdomains in cells remains indirect, and the nature of membrane inhomogeneities has been difficult to characterize. We used lipid analogs and lipid-anchored proteins with varying fluidity preferences to examine ... More
Sphingolipid metabolism in cultured fibroblasts: microscopic and biochemical studies employing a fluorescent ceramide analogue.
AuthorsLipsky NG, Pagano RE
JournalProc Natl Acad Sci U S A
PubMed ID6573674
'A fluorescent analogue of ceramide, N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-epsilon-aminocaproyl sphingosine (C6-NBD-ceramide), was used to investigate sphingolipid metabolism in Chinese hamster fibroblasts. C6-NBD-ceramide was incorporated into small unilamellar dioleoyl phosphatidylcholine vesicles and incubated with cells in monolayer culture at 2 degrees C, resulting in rapid and preferential transfer of the labeled ceramide from vesicles ... More
Sphingolipid synthesis as a target for chemotherapy against malaria parasites.
AuthorsLauer SA, Ghori N, Haldar K
JournalProc Natl Acad Sci U S A
PubMed ID7568097
'The human malaria parasite Plasmodium falciparum contains sphingomyelin synthase in its Golgi apparatus and in a network of tubovesicular membranes in the cytoplasm of the infected erythrocyte. Palmitoyl and decanoyl analogues of 1-phenyl-2-acylamino-3-morpholino-1-propanol inhibit the enzyme activity in infected erythrocytes. An average of 35% of the activity is extremely sensitive ... More
Protein-dependent translocation of aminophospholipids and asymmetric transbilayer distribution of phospholipids in the plasma membrane of ram sperm cells.
AuthorsMüller K, Pomorski T, Müller P, Zachowski A, Herrmann A
JournalBiochemistry
PubMed ID8061006
'We have investigated the transbilayer movement of phospholipids in the plasma membrane of ram sperm cells using spin- and fluorescence-labeled lipid analogues. After incorporation into the outer leaflet, phosphatidylcholine (PC) and sphingomyelin (SM) moved slowly to the inner cytoplasmic leaflet, whereas phosphatidylserine (PS) and phosphatidylethanolamine (PE) rapidly disappeared from the ... More
Applications of ratio fluorescence microscopy in the study of cell physiology.
AuthorsDunn KW, Mayor S, Myers JN, Maxfield FR
JournalFASEB J
PubMed ID8005385
'Quantitative fluorescence microscopy is becoming an increasingly important tool in the study of cell biology. Fluorescence microscopy has long been used for qualitative characterizations of subcellular distributions of proteins, lipids, nucleic acids, and ions, but quantifying these distributions is complicated by a variety of optical, biological, and physical factors. Many ... More
Transport of sphingomyelin to the cell surface is inhibited by brefeldin A and in mitosis, where C6-NBD-sphingomyelin is translocated across the plasma membrane by a multidrug transporter activity.
Authorsvan Helvoort A, Giudici ML, Thielemans M, van Meer G
JournalJ Cell Sci
PubMed ID9010786
'Sphingomyelin is a major lipid of the mammalian cell surface. The view that sphingomyelin, after synthesis in the Golgi lumen, reaches the outer leaflet of the plasma membrane on the inside of carrier vesicles has been challenged by inconsistencies in the results of transport studies. To investigate whether an alternative ... More
Activation of 5-[125I]iodonaphthyl-1-azide via excitation of fluorescent (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) lipid analogs in living cells. A potential tool for identification of compartment-specific proteins and proteins involved in intracellular transport and metabolism of lipids.
AuthorsRosenwald AG, Pagano RE, Raviv Y
JournalJ Biol Chem
PubMed ID2033068
'We describe a new technique for analysis of proteins located near fluorescent lipid analogs in intact living cells using the membrane-permeant, photoactivatable probe, 5-[125I]iodonaphthyl-1-azide ([125I]INA). [125I] INA can be activated directly with UV light or indirectly through excitation of adjacent fluorophores (photosensitizers) with visible light to modify nearby proteins covalently ... More
Transferrin receptor containing the SDYQRL motif of TGN38 causes a reorganization of the recycling compartment but is not targeted to the TGN.
AuthorsJohnson AO, Ghosh RN, Dunn KW, Garippa R, Park J, Mayor S, Maxfield FR, McGraw TE
JournalJ Cell Biol
PubMed ID8991088
'The SDYQRL motif of the cytoplasmic domain of TGN38 is involved in targeting TGN38 from endosomes to the TGN. To create a system for studying this pathway, we replaced the native transferrin receptor (TR) internalization motif (YTRF) with the SDYQRL TGN-targeting motif. The advantages of using TR as a reporter ... More
Taurocholate induces pericanalicular localization of C6-NBD-ceramide in isolated hepatocyte couplets.
AuthorsCrawford JM, Vinter DW, Gollan JL
JournalAm J Physiol
PubMed ID1987800
'The mechanisms and pathways involved in hepatocellular transport of lipid destined for biliary excretion remain poorly understood. Using fluorescence microscopy of rat hepatocyte couplets in primary culture, we examined the effects of taurocholate (TC) on the intracellular distribution of 6-N-[7-nitrobenz-2-oxa-1,3-diazol- 4-yl]aminocaproyl-sphingosine (C6-NBD-ceramide), a lipid that accumulates in the Golgi apparatus. ... More
Preparation of fluorescence-labeled GM1 and sphingomyelin by the reverse hydrolysis reaction of sphingolipid ceramide N-deacylase as substrates for assay of sphingolipid-degrading enzymes and for detection of sphingolipid-binding proteins.
AuthorsNakagawa T, Tani M, Kita K, Ito M
JournalJ Biochem (Tokyo)
PubMed ID10467178
'Sphingolipid ceramide N-deacylase is an enzyme capable of hydrolyzing the N-acyl linkages of ceramides of various sphingolipids. Recently, it was found that the enzyme catalyzes the reverse hydrolysis reaction in which free fatty acids are condensed to lyso-sphingolipids to produce sphingolipids. This paper describes a simple method for the synthesis ... More
Reversed-phase liquid chromatographic method for the determination of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labelled lipid analogues.
AuthorsFrijters CM, Tuijn CJ, Hoek FJ, Groen AK, Oude Elferink RP, Zegers BN
JournalJ Chromatogr B Biomed Sci Appl
PubMed ID9686866
'This paper reports the development of a dual column system for the simultaneous separation of fluorescent short-chain ceramide, 6-[(7-nitrobenz-2-oxa-1,3,-diazol-4-yl[NBD])amino]hexanoyl-sphingo sine and its metabolites, C6-NBD-sphingomyelin and C6-NBD-glucosylceramide, as well as the fluorescent derivatives of choline and serine phosphatides. The method enables the separation of these lipids in a single run on ... More
Epidermal growth factor receptor distribution during chemotactic responses.
AuthorsBailly M, Wyckoff J, Bouzahzah B, Hammerman R, Sylvestre V, Cammer M, Pestell R, Segall JE
JournalMol Biol Cell
PubMed ID11071913
'To determine the distribution of the epidermal growth factor (EGF) receptor (EGFR) on the surface of cells responding to EGF as a chemoattractant, an EGFR-green fluorescent protein chimera was expressed in the MTLn3 mammary carcinoma cell line. The chimera was functional and easily visualized on the cell surface. In contrast ... More
Lipid sorting in epithelial cells.
AuthorsSimons K, van Meer G
JournalBiochemistry
PubMed ID3064805
Continuous analysis of the mechanism of activated transbilayer lipid movement in platelets.
Dithionite reduction of fluorescent (NBD) phospholipids was used as the basis of a continuous assay of transbilayer lipid movement to the cell surface during platelet activation. This assay reveals that virtually all previously internalized phosphatidylserine passes through the external leaflet of the membrane within 90 s after activation with Ca2+ ... More
Rapid transport of phospholipids across the plasma membrane of Leishmania infantum.
AuthorsAraújo-Santos JM, Gamarro F, Castanys S, Herrmann A, Pomorski T
JournalBiochem Biophys Res Commun
PubMed ID12788096
The internalization of fluorescent phospholipid analogs of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM) in Leishmania infantum promastigotes was studied. We observed a rapid inward redistribution of NBD-PC, -PE, and -PS across the plasma membrane at 28 and 4 degrees C. This internalization was shown to be independent ... More
Stable Golgi-mitochondria complexes and formation of Golgi Ca(2+) gradients in pancreatic acinar cells.
We have determined the localization of the Golgi with respect to other organelles in living pancreatic acinar cells and the importance of this localization to the establishment of Ca(2+) gradients over the Golgi. Using confocal microscopy and the Golgi-specific fluorescent probe 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine, we found Golgi structures localizing to the outer ... More
Vacuole formation in mast cells responding to osmotic stress and to F-actin disassembly.
AuthorsKoffer A, Williams M, Johansen T
JournalCell Biol Int
PubMed ID12421579
Fluorescent probes were used to visualize the morphology of membranes and of F-actin in rat peritoneal mast cells, exposed to hyperosmotic medium and consequently reversed to isotonicity. Hypertonicity induced cell shrinkage followed by a regulatory volume increase, and cell alkalinization that was sensitive to amiloride, an inhibitor of the Na(+)/H(+) ... More
Functional reconstitution of sphingomyelin synthase in Chinese hamster ovary cell membranes.
AuthorsHanada K, Horii M, Akamatsu Y
JournalBiochim Biophys Acta
PubMed ID1657181
Sphingomyelin synthase (phosphatidylcholine:ceramide phosphocholinetransferase) activity in the membranes of Chinese hamster ovary cells was found to be detectable with a fluorescent ceramide analog, containing a short acyl chain, as a substrate. We developed a method for the functional reconstitution of sphingomyelin synthase in detergent-treated membranes. Treatment of membranes with 1.5% ... More
Characterization of rapid membrane internalization and recycling.
AuthorsHao M, Maxfield FR
JournalJ Biol Chem
PubMed ID10809763
Lipids and other membrane constituents recycle between the plasma membrane and intracellular endocytic compartments. In CHO cells, approximately half of the internalized C(6)-NBD-SM, a fluorescent lipid analogue widely used as a membrane maker, recycles via the endocytic recycling compartment with a t(12) of approximately 12 min (Mayor, S., Presley, J. ... More
Endocytic recycling compartments altered in cisplatin-resistant cancer cells.
AuthorsLiang XJ, Mukherjee S, Shen DW, Maxfield FR, Gottesman MM
JournalCancer Res
PubMed ID16489040
The clinical utility of cisplatin to treat human malignancies is often limited by the development of drug resistance. We have previously shown that cisplatin-resistant human KB adenocarcinoma cells that are cross-resistant to methotrexate and heavy metals have altered endocytic recycling. In this work, we tracked lipids in the endocytic recycling ... More
Trans-Golgi network and subapical compartment of HepG2 cells display different properties in sorting and exiting of sphingolipids.
AuthorsMaier O, Hoekstra D
JournalJ Biol Chem
PubMed ID12407103
In HepG2 cells, the subapical compartment (SAC) is involved in the biogenesis of membrane polarity. By contrast, direct apical transport originating from the trans-Golgi network (TGN), which may contribute to polarity establishment, has been poorly defined in these cells. Thus, although newly synthesized sphingolipids can be directly transported from the ... More
Polarized sphingolipid transport from the subapical compartment: evidence for distinct sphingolipid domains.
Authorsvan IJzendoorn SC, Hoekstra D
JournalMol Biol Cell
PubMed ID10512879
In polarized HepG2 cells, the sphingolipids glucosylceramide and sphingomyelin (SM), transported along the reverse transcytotic pathway, are sorted in subapical compartments (SACs), and subsequently targeted to either apical or basolateral plasma membrane domains, respectively. In the present study, evidence is provided that demonstrates that these sphingolipids constitute separate membrane domains ... More
Uptake and cellular localization of exogenous lipids by Giardia lamblia, a primitive eukaryote.
AuthorsStevens TL, Gibson GR, Adam R, Maier J, Allison-Ennis M, Das S
JournalExp Parasitol
PubMed ID9207743
Giardia lamblia trophozoites are unable to carry out de novo lipid synthesis. It is therefore likely that lipids are acquired from the small intestine of the host, in which the trophozoites are exposed to free and conjugated fatty acids, various sterols, phospholipids, bile acids, and bile-lipid mixed micelles. Here we ... More
Calcium-induced transbilayer scrambling of fluorescent phospholipid analogs in platelets and erythrocytes.
The non-random distribution of phospholipids in the plasma membrane of human platelets and erythrocytes is at least partially maintained by the ATP-dependent aminophospholipid translocase, but can be disturbed by a calcium-induced scrambling of lipids. Using fluorescent NBD-phospholipid analogs, we demonstrate that in both cells the aminophospholipid translocase has a slightly ... More
Detection and characterization of ceramide-1-phosphate phosphatase activity in rat liver plasma membrane.
AuthorsBoudker O, Futerman AH
JournalJ Biol Chem
PubMed ID8408075
A calcium-dependent ceramide (Cer) kinase was recently detected in human leukemia (HL-60) cells (Kolesnick, R.N., and Hemer, M.R. (1990) J. Biol. Chem. 265, 18803-18808) where it may function in terminating the regulatory effects of Cer, and in synaptic vesicles (Bajjalieh, S. M., Martin, T. F. J., and Floor, E. (1989) ... More
Glucosylceramide is synthesized at the cytosolic surface of various Golgi subfractions.
AuthorsJeckel D, Karrenbauer A, Burger KN, van Meer G, Wieland F
JournalJ Cell Biol
PubMed ID1532799
In our attempt to assess the topology of glucosylceramide biosynthesis, we have employed a truncated ceramide analogue that permeates cell membranes and is converted into water soluble sphingolipid analogues both in living and in fractionated cells. Truncated sphingomyelin is synthesized in the lumen of the Golgi, whereas glucosylceramide is synthesized ... More
Generation of lipid polarity in intestinal epithelial (Caco-2) cells: sphingolipid synthesis in the Golgi complex and sorting before vesicular traffic to the plasma membrane.
Authorsvan 't Hof W, van Meer G
JournalJ Cell Biol
PubMed ID2391372
Generation of intestinal epithelial lipid polarity was studied in Caco-2 cells. Confluent monolayers on filters incorporated the exchangeable lipid N-6-NBD-aminocaproyl-sphingosine (C6-NBD-ceramide) from liposomes. The fluorescent ceramide was converted equally to C6-NBD-glucosylceramide and C6-NBD-sphingomyelin, analogues of lipids enriched on the apical and basolateral surface, respectively, of intestinal cells in vivo. Below ... More
Formation of membrane domains by the envelope proteins of vesicular stomatitis virus.
AuthorsLuan P, Glaser M
JournalBiochemistry
PubMed ID8161502
The properties of the two envelope-associated proteins of vesicular stomatitis virus, the glycoprotein (G) and the matrix protein (M), were investigated in order to understand the mechanism of virus budding and domain formation in membranes. Fluorescence resonance energy transfer was used to study the interaction between the G protein and ... More
Different sphingolipids show differential partitioning into sphingolipid/cholesterol-rich domains in lipid bilayers.
AuthorsWang TY, Silvius JR
JournalBiophys J
PubMed ID10969009
Two fluorescence-based approaches have been applied to examine the differential partitioning of fluorescent phospho- and sphingolipid molecules into sphingolipid-enriched domains modeling membrane "lipid rafts." Fluorescence-quenching measurements reveal that N-(diphenylhexatrienyl)propionyl- (DPH3:0-)-labeled gluco- and galactocerebroside partition into sphingolipid-enriched domains in sphingolipid/phosphatidylcholine/cholesterol bilayers with substantially higher affinity than do analogous sphingomyelin, ceramide, or ... More
Bafilomycin A1 treatment retards transferrin receptor recycling more than bulk membrane recycling.
AuthorsPresley JF, Mayor S, McGraw TE, Dunn KW, Maxfield FR
JournalJ Biol Chem
PubMed ID9153255
Treatment of Chinese hamster ovary cells with the vacuolar proton pump inhibitor bafilomycin A1 causes a 2-fold retardation in the rate of recycling of transfected human transferrin receptors back to the cell surface as measured using biochemical assays (Johnson, L. S. , Dunn, K. W., Pytowski, B., and McGraw, T. ... More
Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process.
AuthorsMayor S, Presley JF, Maxfield FR
JournalJ Cell Biol
PubMed ID8509447
A central question in the endocytic process concerns the mechanism for sorting of recycling components (such as transferrin or low density lipoprotein receptors) from lysosomally directed components; membrane-associated molecules including receptors are generally directed towards the recycling pathway while the luminal content of sorting endosomes, consisting of the acid-released ligands, ... More
PDMP blocks brefeldin A-induced retrograde membrane transport from golgi to ER: evidence for involvement of calcium homeostasis and dissociation from sphingolipid metabolism.
AuthorsKok JW, Babia T, Filipeanu CM, Nelemans A, Egea G, Hoekstra D
JournalJ Cell Biol
PubMed ID9660860
In this study, we show that an inhibitor of sphingolipid biosynthesis, D,L-threo-1-phenyl-2- decanoylamino-3-morpholino-1-propanol (PDMP), inhibits brefeldin A (BFA)-induced retrograde membrane transport from Golgi to endoplasmic reticulum (ER). If BFA treatment was combined with or preceded by PDMP administration to cells, disappearance of discrete Golgi structures did not occur. However, when ... More
Sorting of newly synthesized galactosphingolipids to the two surface domains of epithelial cells.
Authorsvan der Bijl P, Lopes-Cardozo M, van Meer G
JournalJ Cell Biol
PubMed ID8603914
The high concentration of glycosphingolipids on the apical surface of epithelial cells may be generated by selective transport from their site of synthesis to the cell surface. Previously, we showed that canine kidney MDCK and human intestinal Caco-2 cells converted a ceramide carrying the short fluorescent fatty acid C6-NBD to ... More
The accumulation and metabolism of a fluorescent ceramide derivative in Plasmodium falciparum-infected erythrocytes.
AuthorsHaldar K, Uyetake L, Ghori N, Elmendorf HG, Li WL
JournalMol Biochem Parasitol
PubMed ID1775154
We have examined the accumulation and metabolism of N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]aminocaproyl sphingosine (C6-NBD-cer) in Plasmodium falciparum FCR-3/A2-infected erythrocytes. C6-NBD-cer transferred to live infected erythrocytes at 2 degrees C to label the infected red cell surface and intracellular parasite membranes. Subsequent incubation for 30 min at 2 degrees C, resulted in a depletion ... More
A Chinese hamster ovary cell mutant defective in the non-endocytic uptake of fluorescent analogs of phosphatidylserine: isolation using a cytosol acidification protocol.
AuthorsHanada K, Pagano RE
JournalJ Cell Biol
PubMed ID7876305
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 ... More
Aggregation reroutes molecules from a recycling to a vesicle-mediated secretion pathway during reticulocyte maturation.
AuthorsVidal M, Mangeat P, Hoekstra D
JournalJ Cell Sci
PubMed ID9296387
Endocytosis of the Tf/TfR complex is essentially the only pathway active in maturing reticulocytes, while exosomes, formed by invagination of the endosomal membrane, provide a mechanism to eliminate seemingly obsolescent proteins, including the TfR, when their function is completed. In this study, we examined molecular trafficking in the recycling and ... More
Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells.
Authorsvan Meer G, Stelzer EH, Wijnaendts-van-Resandt RW, Simons K
JournalJ Cell Biol
PubMed ID3667693
To study the intracellular transport of newly synthesized sphingolipids in epithelial cells we have used a fluorescent ceramide analog, N-6[7-nitro-2,1,3-benzoxadiazol-4-yl] aminocaproyl sphingosine (C6-NBD-ceramide; Lipsky, N. G., and R. E. Pagano, 1983, Proc. Natl. Acad. Sci. USA, 80:2608-2612) as a probe. This ceramide was readily taken up by filter-grown Madin-Darby canine ... More
Polyamine regulation of plasma membrane phospholipid flip-flop during apoptosis.
AuthorsBratton DL, Fadok VA, Richter DA, Kailey JM, Frasch SC, Nakamura T, Henson PM
JournalJ Biol Chem
PubMed ID10497162
During apoptosis, phosphatidylserine (PS) is moved from the plasma membrane inner leaflet to the outer leaflet where it triggers recognition and phagocytosis of the apoptotic cell. Although the mechanisms of PS appearance during apoptosis are not well understood, it is thought that declining activity of the aminophospholipid translocase and calcium-mediated, ... More
Lysosomal involvement in cellular turnover of plasma membrane sphingomyelin.
AuthorsSutrina SL, Chen WW
JournalBiochim Biophys Acta
PubMed ID6424713
At least two isoenzymes of sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12), including lysosomal acid sphingomyelinase and nonlysosomal magnesium-dependent neutral sphingomyelinase, catalyse the degradation of sphingomyelin in cultured human skin fibroblasts. A genetically determined disorder of sphingomyelin metabolism, type A Niemann-Pick disease, is characterized by a deficiency of lysosomal acid sphingomyelinase. To ... More
Evidence against defective trans-Golgi acidification in cystic fibrosis.
AuthorsSeksek O, Biwersi J, Verkman AS
JournalJ Biol Chem
PubMed ID8663158
Defective organelle acidification has been proposed as a unifying hypothesis to explain the pleiotropic cellular abnormalities associated with cystic fibrosis. To test whether cystic fibrosis transmembrane conductance regulator (CFTR) participates in trans-Golgi pH regulation, intraluminal trans-Golgi pH was measured in stably transfected Swiss 3T3 fibroblasts (expressing CFTR or DeltaF508-CFTR) and ... More
Segregation of glucosylceramide and sphingomyelin occurs in the apical to basolateral transcytotic route in HepG2 cells.
Authorsvan IJzendoorn SC, Zegers MM, Kok JW, Hoekstra D
JournalJ Cell Biol
PubMed ID9128247
HepG2 cells are highly differentiated hepatoma cells that have retained an apical, bile canalicular (BC) plasma membrane polarity. We investigated the dynamics of two BC-associated sphingolipids, glucosylceramide (GlcCer) and sphingomyelin (SM). For this, the cells were labeled with fluorescent acyl chain-labeled 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]hexanoic acid (C6-NBD) derivatives of either GlcCer (C6-NBD-GlcCer) or ... More
Glucosylceramide synthase does not attenuate the ceramide pool accumulating during apoptosis induced by CD95 or anti-cancer regimens.
AuthorsTepper AD, Diks SH, van Blitterswijk WJ, Borst J
JournalJ Biol Chem
PubMed ID10945987
Ceramide (Cer) accumulating during the execution phase of apoptosis is generated from plasma membrane sphingomyelin (SM), which gains access to a sphingomyelinase due to phospholipid scrambling (Tepper, A. D., Ruurs, P., Wiedmer, T., Sims, P., Borst, J., and van Blitterswijk, W. J. (2000) J. Cell. Biol. 150, 155-164). To evaluate ... More
Sphingomyelin hydrolysis to ceramide during the execution phase of apoptosis results from phospholipid scrambling and alters cell-surface morphology.
Apoptosis is generally accompanied by a late phase of ceramide (Cer) production, the significance of which is unknown. This study describes a previously unrecognized link between Cer accumulation and phosphatidylserine (PS) exposure at the cell surface, a characteristic of the execution phase of apoptosis resulting from a loss of plasma ... More
Differential metabolism and trafficking of sphingolipids in differentiated versus undifferentiated HT29 cells.
AuthorsBabia T, Kok JW, Hulstaert C, de Weerd H, Hoekstra D
JournalInt J Cancer
PubMed ID8325709
Trafficking and metabolism of sphingolipids were examined in undifferentiated (G+) and differentiated (G+ reversed) HT29 human colon adenocarcinoma cell lines. Metabolic experiments employing a fluorescently labeled sphingolipid precursor, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylceramide++ + (C6-NBD-ceramide) revealed that both qualitative and quantitative differences exist in sphingolipid synthesis between the 2 cell lines. One of the ... More
The role of different P-glycoproteins in hepatobiliary secretion of fluorescently labeled short-chain phospholipids.
AuthorsFrijters CM, Tuijn CJ, Ottenhoff R, Zegers BN, Groen AK, Elferink RP
JournalJ Lipid Res
PubMed ID10552998
Class III P-glycoproteins (Pgps) mediate biliary phosphatidylcholine (PC) secretion. Recent findings that class I P-glycoproteins are able to transport several short-chain phospholipid analogues raises questions about the role of these Pgps in physiological lipid transport. We investigated the biliary secretion of C6-7-nitro-2,1, 3-benzoxadiazol-4-yl (NBD)-labeled ceramide and its metabolites in Mdr1a/b ... More
Sorting of an internalized plasma membrane lipid between recycling and degradative pathways in normal and Niemann-Pick, type A fibroblasts.
AuthorsKoval M, Pagano RE
JournalJ Cell Biol
PubMed ID2380243
We examined the metabolism and intracellular transport of a fluorescent sphingomyelin analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl])- sphingosylphosphorylcholine (C6-NBD-SM), in both normal and Niemann-Pick, type A (NP-A) human skin fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells ... More
(Glyco)sphingolipids are sorted in sub-apical compartments in HepG2 cells: a role for non-Golgi-related intracellular sites in the polarized distribution of (glyco)sphingolipids.
Authorsvan IJzendoorn SC, Hoekstra D
JournalJ Cell Biol
PubMed ID9700158
In polarized HepG2 cells, the fluorescent sphingolipid analogues of glucosylceramide (C6-NBD-GlcCer) and sphingomyelin (C6-NBD-SM) display a preferential localization at the apical and basolateral domain, respectively, which is expressed during apical to basolateral transcytosis of the lipids (van IJzendoorn, S.C.D., M.M. P. Zegers, J.W. Kok, and D. Hoekstra. 1997. J. Cell ... More
Lipid recycling between the plasma membrane and intracellular compartments: transport and metabolism of fluorescent sphingomyelin analogues in cultured fibroblasts.
AuthorsKoval M, Pagano RE
JournalJ Cell Biol
PubMed ID2738091
We examined the metabolism and intracellular transport of the D-erythro and L-threo stereoisomers of a fluorescent analogue of sphingomyelin, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl])-sphingosylphosphorylcholine (C6-NBD-SM), in Chinese hamster ovary (CHO-K1) fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. ... More
Retarded intracellular lipid transport associated with reduced expression of Cdc42, a member of Rho-GTPases, in human aged skin fibroblasts: a possible function of Cdc42 in mediating intracellular lipid transport.
AuthorsTsukamoto K, Hirano K, Yamashita S, Sakai N, Ikegami C, Zhang Z, Matsuura F, Hiraoka H, Matsuyama A, Ishigami M, Matsuzawa Y
JournalArterioscler Thromb Vasc Biol
PubMed ID12426222
OBJECTIVE: Many cell types in atherosclerotic lesions are thought to have various biological abnormalities, such as impaired lipid homeostasis and slow cell proliferation, which may be related to senescence at cellular and individual levels. One of the common characteristics of senescent cells in vitro is the alteration of actin cytoskeletons, ... More
Inhibition and stimulation of phospholipid scrambling activity. Consequences for lipid asymmetry, echinocytosis, and microvesiculation of erythrocytes.
AuthorsKamp D, Sieberg T, Haest CW
JournalBiochemistry
PubMed ID11478914
An increase of the intracellular Ca(2+) concentration in erythrocytes is known to activate rapid nonspecific bidirectional translocation of membrane-inserted phospholipid probes and to decrease the asymmetric distribution of endogenous membrane phospholipids. These scrambling effects are now shown to be suppressed by pretreatment of cells with the essentially impermeable reagents 4,4'-diisothiocyanostilbene-2,2'-disulfonic ... More
Appearance of phosphatidylserine on apoptotic cells requires calcium-mediated nonspecific flip-flop and is enhanced by loss of the aminophospholipid translocase.
AuthorsBratton DL, Fadok VA, Richter DA, Kailey JM, Guthrie LA, Henson PM
JournalJ Biol Chem
PubMed ID9334182
Phosphatidylserine (PS), ordinarily sequestered in the plasma membrane inner leaflet, appears in the outer leaflet during apoptosis, where it triggers non-inflammatory phagocytic recognition of the apoptotic cell. The mechanism of PS appearance during apoptosis is not well understood but has been associated with loss of aminophospholipid translocase activity and nonspecific ... More
Conversion of diacylglycerol to phosphatidylcholine on the basolateral surface of epithelial (Madin-Darby canine kidney) cells. Evidence for the reverse action of a sphingomyelin synthase.
Authorsvan Helvoort A, van't Hof W, Ritsema T, Sandra A, van Meer G
JournalJ Biol Chem
PubMed ID8294425
When N-6[7-nitro-2,1,3-benzoxadiazol-4-yl]aminohexanoyl-phosphatidic acid (C6-NBD-PA) is inserted into the plasma membrane of fibroblasts, it is metabolized by the cells to C6-NBD-diacylglycerol (DG), -triacylglycerol, -phosphatidylcholine (PC), and -phosphatidylethanolamine (PE) (Pagano, R. E., Longmuir, K. J., and Martin, O. C. (1983) J. Biol. Chem. 258, 2034-2040). In Madin-Darby canine kidney (MDCK) cells incubated ... More