Fluorescence-based selection of gene-corrected hematopoietic stem and progenitor cells from acid sphingomyelinase-deficient mice: implications for Niemann-Pick disease gene therapy and the development of improved stem cell gene transfer procedures.
AuthorsErlich S, Miranda SR, Visser JW, Dagan A, Gatt S, Schuchman EH
JournalBlood
PubMed ID9864149
'The general utility of a novel, fluorescence-based procedure for assessing gene transfer and expression has been demonstrated using hematopoietic stem and progenitor cells. Lineage-depleted hematopoietic cells were isolated from the bone marrow or fetal livers of acid sphingomyelinase-deficient mice, and retrovirally transduced with amphotropic or ecotropic vectors encoding a normal ... More
Changes in the spectral properties of a plasma membrane lipid analog during the first seconds of endocytosis in living cells.
AuthorsChen CS, Martin OC, Pagano RE
JournalBiophys J
PubMed ID8994591
'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 ... More
Structural and functional regulation of tight junctions by RhoA and Rac1 small GTPases.
AuthorsJou TS, Schneeberger EE, Nelson WJ
JournalJ Cell Biol
PubMed ID9660866
'Tight junctions (TJ) govern ion and solute diffusion through the paracellular space (gate function), and restrict mixing of membrane proteins and lipids between membrane domains (fence function) of polarized epithelial cells. We examined roles of the RhoA and Rac1 GTPases in regulating TJ structure and function in MDCK cells using ... More
Measurement of spontaneous transfer and transbilayer movement of BODIPY-labeled lipids in lipid vesicles.
AuthorsBai J, Pagano RE
JournalBiochemistry
PubMed ID9220970
'An assay was developed to study the spontaneous transfer and transbilayer movement (flip-flop) of lipid analogs labeled with the fluorescent fatty acid, 5-(5,7-dimethyl BODIPY)-1-pentanoic acid (C5-DMB-) in large unilamellar lipid vesicles comprised of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC). The assay is based on the concentration-dependent changes in fluorescence intensity that occur when ... More
The recycling endosome of Madin-Darby canine kidney cells is a mildly acidic compartment rich in raft components.
AuthorsGagescu R, Demaurex N, Parton RG, Hunziker W, Huber LA, Gruenberg J
JournalMol Biol Cell
PubMed ID10930469
'We present a biochemical and morphological characterization of recycling endosomes containing the transferrin receptor in the epithelial Madin-Darby canine kidney cell line. We find that recycling endosomes are enriched in molecules known to regulate transferrin recycling but lack proteins involved in early endosome membrane dynamics, indicating that recycling endosomes are ... More
Modified low density lipoprotein delivers substrate for ceramide formation and stimulates the sphingomyelin-ceramide pathway in human macrophages.
AuthorsKinscherf R, Claus R, Deigner HP, Nauen O, Gehrke C, Hermetter A, Russwurm S, Daniel V, Hack V, Metz J
JournalFEBS Lett
PubMed ID9094423
'Exposure of human blood monocytes derived macrophages to modified (oxidized or acetylated) LDL induced a approximately 40% elevation (60 pmol/10(6) cells) of the endogenous level of the sphingolipid ceramide. A rise of both neutral and acidic SMase activity was found after treatment with oxidized LDL (250 and 80%), while addition ... More
Characterization of a regulated form of phospholipase D in the yeast Saccharomyces cerevisiae.
AuthorsElla KM, Dolan JW, Meier KE
JournalBiochem J
PubMed ID7741711
'Phospholipase D (PLD), which is present in bacterial, plant and animal cells, can serve as an important element of signal-transduction pathways. This study examined the potential role of this enzyme in the regulation of Saccharomyces cerevisiae. An assay in vitro using a fluorescent 1-acyl-2-alkyl glycerophosphocholine as substrate was used to ... More
ISC1-encoded inositol phosphosphingolipid phospholipase C is involved in Na+/Li+ halotolerance of Saccharomyces cerevisiae.
AuthorsBetz C, Zajonc D, Moll M, Schweizer E
JournalEur J Biochem
PubMed ID12180980
'In Saccharomyces cerevisiae, toxic concentrations of Na+ orLi+ ions induce the expression of the cation-extrusion ATPase gene, ENA1. Several well-studied signal transduction pathways are known correlating high salinity to the transcriptional activation of ENA1. Nevertheless, information on the actual sensing mechanism initiating these pathways is limited. Here, we report that ... More
Sphingomyelinase activity associated with human plasma low density lipoprotein.
'Isolated human plasma low density lipoprotein (LDL) was observed to possess sphingomyelinase activity. Accordingly, the formation of ceramide was catalyzed by LDL at 37 degrees C using tertiary liposomes composed of sphingomyelin (mole fraction (x) = 0.2), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (x = 0.7), 1, 2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (x = 0.1), and either the fluorescent ... More
Endocytosis is enhanced in Tangier fibroblasts: possible role of ATP-binding cassette protein A1 in endosomal vesicular transport.
AuthorsZha X, Genest J, McPherson R
JournalJ Biol Chem
PubMed ID11504722
'A human genetic disorder, Tangier disease, has been linked recently to mutations in ATP-binding cassette protein A1 (ABCA1). In addition to its function in apoprotein A-I-mediated lipid removal, ABCA1 was also shown to be a phosphatidylserine (PS) translocase that facilitates PS exofacial flipping. This PS translocation is crucial for the ... More
Use of BODIPY-labeled sphingolipids to study membrane traffic along the endocytic pathway.
AuthorsPagano RE, Chen CS
JournalAnn N Y Acad Sci
PubMed ID9668349
'In this chapter we discuss the use of BODIPY-labeled sphingolipids to study lipid transport along the endocytic pathway of cultured mammalian cells. The unique spectral properties of the BODIPY fluorophore allow the investigator to distinguish various populations of labeled endosome and lysosomes within the living cell by fluorescence microscopy, and ... More
Distribution and biophysical properties of fluorescent lipids on the surface of adult Schistosoma mansoni.
AuthorsRedman CA, Kusel JR
JournalParasitology
PubMed ID8786886
'The properties of 4 fluorescent lipid compounds in the surface membrane of adult male Schistosoma mansoni worms were examined by fluorescent microscopy and fluorescent recovery after photobleaching (FRAP). The data suggest that the probes N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s- indacene-3-pentanoyl) sphingosine (BODIPY FL ceramide) and PKH2 pass through the outer membrane and enter structures ... More
Fluorescence-based selection of gene-corrected hematopoietic stem and progenitor cells based on acid sphingomyelinase expression.
AuthorsSchuchman EH, Erlich S, Miranda SR, Dinur T, Dagan A, Gatt S
JournalMethods Enzymol
PubMed ID11070882
Synthesis of fluorescent substrates and their application to study of sphingolipid metabolism in vitro and in intact cells.
AuthorsDagan A, Agmon V, Gatt S, Dinur T
JournalMethods Enzymol
PubMed ID11070879
Applications of BODIPY-sphingolipid analogs to study lipid traffic and metabolism in cells.
AuthorsPagano RE, Watanabe R, Wheatley C, Dominguez M
JournalMethods Enzymol
PubMed ID11070900
Use of Bodipy-labeled sphingolipid and cholesterol analogs to examine membrane microdomains in cells.
AuthorsMarks DL, Bittman R, Pagano RE,
JournalHistochem Cell Biol
PubMed ID18820942
Much evidence has accumulated to show that cellular membranes such as the plasma membrane, contain multiple
Use of N-[5-(5,7-dimethyl boron dipyrromethene difluoride-sphingomyelin to study membrane traffic along the endocytic pathway.
AuthorsPagano RE, Watanabe R, Wheatley C, Chen CS
JournalChem Phys Lipids
PubMed ID11001561
We have used N-[5-(5,7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-D-erythro-sphingosylphosphorylcholine (C5-DMB-SM or 'BODIPY-SM'), a fluorescent analog of sphingomyelin, to study lipid transport along the endocytic pathway of human skin fibroblasts. The unique spectral properties of the BODIPY fluorophore allow the investigator to distinguish various populations of labeled endosomes and lysosomes within the living ... More
Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide.
AuthorsD'Angelo G, Polishchuk E, Di Tullio G, Santoro M, Di Campli A, Godi A, West G, Bielawski J, Chuang CC, van der Spoel AC, Platt FM, Hannun YA, Polishchuk R, Mattjus P, De Matteis MA,
JournalNature
PubMed ID17687330
The molecular machinery responsible for the generation of transport carriers moving from the Golgi complex to the plasma membrane relies on a tight interplay between proteins and lipids. Among the lipid-binding proteins of this machinery, we previously identified the four-phosphate adaptor protein FAPP2, the pleckstrin homology domain of which binds ... More
Cholesterol controls lipid endocytosis through Rab11.
AuthorsTakahashi M, Murate M, Fukuda M, Sato SB, Ohta A, Kobayashi T,
JournalMol Biol Cell
PubMed ID17475773
Cellular cholesterol increases when cells reach confluency in Chinese hamster ovary (CHO) cells. We examined the endocytosis of several lipid probes in subconfluent and confluent CHO cells. In subconfluent cells, fluorescent lipid probes including poly(ethylene glycol)derivatized cholesterol, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol, and fluorescent sphingomyelin analogs were internalized to pericentriolar recycling endosomes. This accumulation ... More
Fluorescent lipid probes: some properties and applications (a review).
AuthorsMaier O, Oberle V, Hoekstra D
JournalChem Phys Lipids
PubMed ID12093532
Odd as it may seem, experimental challenges in lipid research are often hampered by the simplicity of the lipid structure. Since, as in protein research, mutants or overexpression of lipids are not realistic, a considerable amount of lipid research relies on the use of tagged lipid analogues. However, given the ... More
Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy.
AuthorsXu C, Zipfel W, Shear JB, Williams RM, Webb WW
JournalProc Natl Acad Sci U S A
PubMed ID8855254
Intrinsic, three-dimensionally resolved, microscopic imaging of dynamical structures and biochemical processes in living preparations has been realized by nonlinear laser scanning fluorescence microscopy. The search for useful two-photon and three-photon excitation spectra, motivated by the emergence of nonlinear microscopy as a powerful biophysical instrument, has now discovered a virtual artist's ... More
Biochemical interactions integrating Itk with the T cell receptor-initiated signaling cascade.
Itk, a Tec family tyrosine kinase, acts downstream of Lck and phosphatidylinositol 3'-kinase to facilitate T cell receptor (TCR)-dependent calcium influxes and increases in extracellular-regulated kinase activity. Here we demonstrate interactions between Itk and crucial components of TCR-dependent signaling pathways. First, the inositide-binding pocket of the Itk pleckstrin homology domain ... More
Vectorial budding of vesicles by asymmetrical enzymatic formation of ceramide in giant liposomes.
AuthorsHolopainen JM, Angelova MI, Kinnunen PK
JournalBiophys J
PubMed ID10653795
Sphingomyelin is an abundant component of eukaryotic membranes. A specific enzyme, sphingomyelinase can convert this lipid to ceramide, a central second messenger in cellular signaling for apoptosis (programmed cell death), differentiation, and senescence. We used microinjection and either Hoffman modulation contrast or fluorescence microscopy of giant liposomes composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine ... More
A novel monoclonal antibody against the second extracellular loop of occludin disrupts epithelial cell polarity.
AuthorsTokunaga Y, Kojima T, Osanai M, Murata M, Chiba H, Tobioka H, Sawada N
JournalJ Histochem Cytochem
PubMed ID17371936
The tight junction (TJ) regulates epithelial cell polarity and paracellular permeability. In the present study, to investigate whether the second extracellular loop of occludin affects the localization of carcinoembryonic antigen (CEA) and CD26 expressed on apical membranes, and the fence function of the TJ, the human intestinal epithelial cell line ... More
Identification of an insulin-responsive, slow endocytic recycling mechanism in Chinese hamster ovary cells.
AuthorsJohnson AO, Subtil A, Petrush R, Kobylarz K, Keller SR, McGraw TE
JournalJ Biol Chem
PubMed ID9651404
In adipocytes, the insulin-regulated aminopeptidase (IRAP) is trafficked through the same insulin-regulated recycling pathway as the GLUT4 glucose transporter. We find that a chimera, containing the cytoplasmic domain of IRAP fused to transmembrane and extracellular domains of the transferrin receptor, is slowly recycled and rapidly internalized in Chinese hamster ovary ... More
Interconversion of sphingomyelin and ceramide in adult Schistosoma mansoni.
AuthorsRedman CA, Kennington S, Spathopoulou T, Kusel JR
JournalMol Biochem Parasitol
PubMed ID9497039
Fluorescent lipid analogues of the lipids ceramide and sphingomyelin, namely BODIPY FL C5-ceramide ((N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sindacene-3-pentanoyl) sphingosine) and BODIPY C5-sphingomyelin ((N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl) sphingosyl phosphocholine), respectively, were used to investigate the presence of a sphingomyelin cycle in Schistosoma mansoni adult males. The parasites were able to convert BODIPY FL C5-ceramide into a fluorescent sphingomyelin ... More
Separation of phospholipids in microfluidic chip device: application to high-throughput screening assays for lipid-modifying enzymes.
AuthorsLin S, Fischl AS, Bi X, Parce W
JournalAnal Biochem
PubMed ID12633607
Phospholipid molecules such as ceramide and phosphoinositides play crucial roles in signal transduction pathways. Lipid-modifying enzymes including sphingomyelinase and phosphoinositide kinases regulate the generation and degradation of these lipid-signaling molecules and are important therapeutic targets in drug discovery. We now report a sensitive and convenient method to separate these lipids ... More
Cell spreading and lamellipodial extension rate is regulated by membrane tension.
AuthorsRaucher D, Sheetz MP
JournalJ Cell Biol
PubMed ID10629223
Cell spreading and motility require the extension of the plasma membrane in association with the assembly of actin. In vitro, extension must overcome resistance from tension within the plasma membrane. We report here that the addition of either amphiphilic compounds or fluorescent lipids that expanded the plasma membrane increased the ... More
Dynamic sequestration of the recycling compartment by classical protein kinase C.
AuthorsIdkowiak-Baldys J, Becker KP, Kitatani K, Hannun YA
JournalJ Biol Chem
PubMed ID16751194
It has been previously shown that upon sustained stimulation (30-60 min) with phorbol esters, protein kinase C (PKC) alpha and betaII become sequestered in a juxtanuclear region, the pericentrion. The activation of PKC also results in sequestration of transferrin, suggesting a role for PKC in regulating endocytosis and sequestration of ... More
Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways.
AuthorsPuri V, Watanabe R, Singh RD, Dominguez M, Brown JC, Wheatley CL, Marks DL, Pagano RE
JournalJ Cell Biol
PubMed ID11481344
Sphingolipids (SLs) are plasma membrane constituents in eukaryotic cells which play important roles in a wide variety of cellular functions. However, little is known about the mechanisms of their internalization from the plasma membrane or subsequent intracellular targeting. We have begun to study these issues in human skin fibroblasts using ... 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
Macroscopic consequences of the action of phospholipase C on giant unilamellar liposomes.
Macroscopic consequences of the formation of diacylglycerol by phospholipase C (PC-PLC) in giant 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) unilamellar vesicles (GUVs, diameter 10-100 microm) were studied by phase contrast and fluorescence microscopy. PC-PLC caused a series of fast stepwise shrinkages of fluid SOPC GUVs, continuing until the vesicle disappeared beyond the optical resolution ... More
Release of phospholipids from erythrocyte membranes by taurocholate is determined by their transbilayer orientation and hydrophobic backbone.
AuthorsWüstner D, Pomorski T, Herrmann A, Müller P
JournalBiochemistry
PubMed ID9836604
Bile salts mediate a specific release of phosphatidylcholine (PC) from the canalicular membrane into the bile fluid. We utilized human red blood cells (RBC) as a model system to study the release of endogenous phospholipids as well as phospholipid analogues from plasma membranes in the presence of the bile salt ... More
Zn2+-stimulated sphingomyelinase is secreted by many cell types and is a product of the acid sphingomyelinase gene.
AuthorsSchissel SL, Schuchman EH, Williams KJ, Tabas I
JournalJ Biol Chem
PubMed ID8702487
Mammalian sphingomyelinases have been implicated in many important physiological and pathophysiological processes. Although several mammalian sphingomyelinases have been identified and studied, one of these, an acidic Zn2+-stimulated sphingomyelinase (Zn-SMase) originally found in fetal bovine serum, has received little attention since its first and only report 7 years ago. We now ... More
Internalization and sorting of a fluorescent analogue of glucosylceramide to the Golgi apparatus of human skin fibroblasts: utilization of endocytic and nonendocytic transport mechanisms.
AuthorsMartin OC, Pagano RE
JournalJ Cell Biol
PubMed ID8188745
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 ... More
Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein.
AuthorsBalda MS, Whitney JA, Flores C, González S, Cereijido M, Matter K
JournalJ Cell Biol
PubMed ID8769425
Tight junctions, the most apical of the intercellular junctions that connect individual cells in a epithelial sheet, are thought to form a seal that restricts paracellular and intramembrane diffusion. To analyze the functioning of tight junctions, we generated stable MDCK strain 2 cell lines expressing either full-length or COOH-terminally truncated ... More
WIF-B cells: an in vitro model for studies of hepatocyte polarity.
AuthorsIhrke G, Neufeld EB, Meads T, Shanks MR, Cassio D, Laurent M, Schroer TA, Pagano RE, Hubbard AL
JournalJ Cell Biol
PubMed ID7506266
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 ... More
SIRT1 regulates sphingolipid metabolism and neural differentiation of mouse embryonic stem cells through c-Myc-SMPDL3B.
Authors
JournalElife
PubMed ID34042046
Lipid accumulation controls the balance between surface connection and scission of caveolae.