High spatial resolution confocal microscopy of young MDCK cells stained with the lipophilic probe 1,1'-dihexadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (DiIC(16)) revealed a reticulated fluorescence pattern on the apical membrane. DiIC(16) was delivered as crystals to live cells to minimize possible solvent perturbations of the membrane lipids. The ratio of the integrated fluorescence ... More
Activated K-Ras and H-Ras display different interactions with saturable nonraft sites at the surface of live cells.
AuthorsNiv H, Gutman O, Kloog Y, Henis YI
JournalJ Cell Biol
PubMed ID12021258
'Ras-membrane interactions play important roles in signaling and oncogenesis. H-Ras and K-Ras have nonidentical membrane anchoring moieties that can direct them to different membrane compartments. Ras-lipid raft interactions were reported, but recent studies suggest that activated K-Ras and H-Ras are not raft resident. However, specific interactions of activated Ras proteins ... More
A calsequestrin-like protein in the endoplasmic reticulum of the sea urchin: localization and dynamics in the egg and first cell cycle embryo.
AuthorsHenson JH, Begg DA, Beaulieu SM, Fishkind DJ, Bonder EM, Terasaki M, Lebeche D, Kaminer B
JournalJ Cell Biol
PubMed ID2663877
'Using an antiserum produced against a purified calsequestrin-like (CSL) protein from a microsomal fraction of sea urchin eggs, we performed light and electron microscopic immunocytochemical localizations on sea urchin eggs and embryos in the first cell cycle. The sea urchin CSL protein has been found to bind Ca++ similarly to ... More
Involvement of raft-like plasma membrane domains of Entamoeba histolytica in pinocytosis and adhesion.
AuthorsLaughlin RC, McGugan GC, Powell RR, Welter BH, Temesvari LA
JournalInfect Immun
PubMed ID15322032
'Lipid rafts are highly ordered, cholesterol-rich, and detergent-resistant microdomains found in the plasma membrane of many eukaryotic cells. These domains play important roles in endocytosis, secretion, and adhesion in a variety of cell types. The parasitic protozoan Entamoeba histolytica, the causative agent of amoebic dysentery, was determined to have raft-like ... More
High-avidity CTL exploit two complementary mechanisms to provide better protection against viral infection than low-avidity CTL.
AuthorsDerby M, Alexander-Miller M, Tse R, Berzofsky J
JournalJ Immunol
PubMed ID11160212
'Previously, we observed that high-avidity CTL are much more effective in vivo than low-avidity CTL in elimination of infected cells, but the mechanisms behind their superior activity remained unclear. In this study, we identify two complementary mechanisms: 1) high-avidity CTL lyse infected cells earlier in the course of a viral ... More
The orientation of first cleavage in the sea urchin embryo, Lytechinus variegatus, does not specify the axes of bilateral symmetry.
AuthorsSummers RG, Piston DW, Harris KM, Morrill JB
JournalDev Biol
PubMed ID8608864
'One blastomere of the two-cell stage sea urchin embryo (Lytechinus variegatus) was labeled with an intracellular fluorescent lineage tracing stain to determine, from the lineage of that blastomere, the orientation of the first cleavage furrow with regard to the axes of bilateral symmetry in the gastrula and pluteus larva. Two ... More
Intracellular dye heterogeneity determined by fluorescence lifetimes.
AuthorsPackard BS, Karukstis KK, Klein MP
JournalBiochim Biophys Acta
PubMed ID6546351
'The cellular localization of a fluorescent probe molecule depends on both the chemical structure of the dye and the cellular environment. To study the number and types of environments in an epithelial cell line, we have measured in Madin-Darby canine kidney (MDCK) cells the fluorescence lifetimes of three structurally distinct ... More
The movement of fluorescent endocytic tracers in Plasmodium falciparum infected erythrocytes.
AuthorsHaldar K, Uyetake L
JournalMol Biochem Parasitol
PubMed ID1371847
'The fluorescent lipophilic probe 1,1''-dihexadecyl-3-3''-3-3''- tetramethylindocarbocyanine (diIC16) inserted in the red cell surface, functioned as a non-exchangeable lipid marker which was not metabolised or toxic in plasmodial cultures. Invasion by Plasmodium falciparum resulted in the internalisation of the lipid, suggesting the uptake of red cell membrane components during parasite entry. ... More
Carbocyanine dyes with long alkyl side-chains: broad spectrum inhibitors of mitochondrial electron transport chain activity.
AuthorsAnderson WM, Trgovcich-Zacok D
JournalBiochem Pharmacol
PubMed ID7763312
'Certain indocarbocyanine, thiacarbocyanine, and oxacarbocyanine dyes possessing short alkyl side-chains (one to five carbons) are potent inhibitors of mammalian mitochondrial NADH-ubiquinone reductase (EC 1.6.99.3) activity (Anderson et al., Biochem Pharmacol 41: 677-684, 1991; Anderson et al., Biochem Pharmacol 45: 691-696, 1993; Anderson et al., Biochem Pharmacol 45: 2115-2122, 1993), and ... More
Fatty acid-albumin complexes and the determination of the transport of long chain free fatty acids across membranes.
AuthorsCupp D, Kampf JP, Kleinfeld AM
JournalBiochemistry
PubMed ID15078093
'Understanding the mechanism that governs the transport of long chain free fatty acids (FFA) across lipid bilayers is critical for understanding transport across cell membranes. Conflicting results have been reported for lipid vesicles; most investigators report that flip-flop occurs within the resolution time of the method (<5 ms) and that ... More
Structural aspects of the association of FcepsilonRI with detergent-resistant membranes.
AuthorsField KA, Holowka D, Baird B
JournalJ Biol Chem
PubMed ID9880557
'We recently showed that aggregation of the high affinity IgE receptor on mast cells, FcepsilonRI, causes this immunoreceptor to associate rapidly with specialized regions of the plasma membrane, where it is phosphorylated by the tyrosine kinase Lyn. In this study, we further characterize the detergent sensitivity of this association on ... More
Membrane interactions of a constitutively active GFP-Ki-Ras 4B and their role in signaling. Evidence from lateral mobility studies.
AuthorsNiv H, Gutman O, Henis YI, Kloog Y
JournalJ Biol Chem
PubMed ID9880539
'Membrane anchorage of Ras proteins in the inner leaflet of the plasma membrane is an important factor in their signaling and oncogenic potential. Despite these important roles, the precise mode of Ras-membrane interactions is not yet understood. It is especially important to characterize these interactions at the surface of intact ... 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
Movement of a fluorescent lipid label from a labeled erythrocyte membrane to an unlabeled erythrocyte membrane following electric-field-induced fusion.
AuthorsSowers AE
JournalBiophys J
PubMed ID3986281
'A short burst of electric field pulses was used to induce nearly simultaneous fusion among 50% or more of a population composed of unlabeled erythrocytes and erythrocytes labeled with the fluorescent lipid analogue DiI (1,1''-dihexadecyl-3,3,3'''',3''-tetra-methylindo carbocyanine perchlorate). Fusion products that ended in an hourglass shape were selected for analysis. The ... More
Caspase dependence of target cell damage induced by cytotoxic lymphocytes.
AuthorsSarin A, Haddad EK, Henkart PA
JournalJ Immunol
PubMed ID9743340
'Since the CTL secreted granule protease granzyme B can activate multiple target caspases, it has been proposed that this pathway is responsible for CTL-induced cytolysis of Fas-negative targets. However, target lysis via the granule exocytosis pathway is completely resistant to caspase inhibitors. To test the possibility that granzymes trigger a ... More
Electrofusion of dissimilar membrane fusion partners depends on additive contributions from each of the two different membranes.
AuthorsSowers AE
JournalBiochim Biophys Acta
PubMed ID2804115
'Rabbit erythrocyte ghost (REG) membranes and human erythrocyte ghosts (HEG) were aligned into contact by dielectrophoresis and fused with an electric pulse in REG + REG, HEG + HEG, and REG + HEG combinations. REG + HEG fusion yields were approximately midway between fusion yields for REG + REG and ... More
Determination of the sidedness of carbocyanine dye labeling of membranes.
AuthorsWolf DE
JournalBiochemistry
PubMed ID3994976
'We have found that 2,4,6-trinitrobenzenesulfonate (TNBS) is an effective quencher of the fluorescence of the 1,1''-dialkyl-3,3,3'',3''-tetramethylindocarbocyanines (CNdiI''s). This quenching appears to occur by complex formation rather than a collisional mechanism. By use of this quenching, we have studied the transbilayer asymmetry of CNdiI labeling for large unilamellar membranes of egg ... More
Use of lectins and polyethylene glycol for fusion of glycolipid-containing liposomes with eukaryotic cells.
AuthorsSzoka F, Magnusson KE, Wojcieszyn J, Hou Y, Derzko Z, Jacobson K
JournalProc Natl Acad Sci U S A
PubMed ID6940182
'Efficient fusion of phospholipid vesicles with monolayer cultures of eukaryotic cells was accomplished by attaching glycolipid-containing vesicles to the cell surface by using a lectin displaying binding for both the cell surface and the glycolipid, followed by treatment with polyethylene glycol. Fusion was inferred from the transfer of fluorescent lipid ... More
Lipid domains and lipid/protein interactions in biological membranes.
AuthorsTocanne JF, Cézanne L, Lopez A, Piknova B, Schram V, Tournier JF, Welby M
JournalChem Phys Lipids
PubMed ID8001179
'In the fluid mosaic model of membranes, lipids are organized in the form of a bilayer supporting peripheral and integral proteins. This model considers the lipid bilayer as a two-dimensional fluid in which lipids and proteins are free to diffuse. As a direct consequence, both types of molecules would be ... More
Fluorescence studies on the mechanism of liposome-cell interactions in vitro.
AuthorsSzoka F, Jacobson K, Derzko Z, Papahadjopoulos D
JournalBiochim Biophys Acta
PubMed ID7397162
'Sonicated unilamellar liposomes containing fluorescent lipid analogs or biotinyl phosphatidylethanolamine as a ligand for fluorescein avidin have been used to study the mechanism of interaction of phospholipid vesicles with eucaryotic cells. Microscopy revealed that after short incubations the fluorescence was associated with the cell surface in a punctate as opposed ... More
Partitioning behavior of indocarbocyanine probes between coexisting gel and fluid phases in model membranes.
AuthorsSpink CH, Yeager MD, Feigenson GW
JournalBiochim Biophys Acta
PubMed ID2317494
'Gel-fluid partition coefficients, Kp, were measured for a series of indocarbocyanine dyes in multilamellar lipid vesicles. The dyes examined had alkyl chain lengths from 12 to 22 carbons. Fluorescence quenching by a spin-labeled phosphatidylcholine-enriched fluid phase created a large difference in quantum yield for indocarbocyanine fluorescence between fluid and gel ... More
Binding of anticoagulant vitamin K-dependent protein S to platelet-derived microparticles.
AuthorsDahlbäck B, Wiedmer T, Sims PJ
JournalBiochemistry
PubMed ID1463747
'Vitamin K-dependent protein S is an anticoagulant plasma protein serving as cofactor to activated protein C in degradation of coagulation factors Va and VIIIa on membrane surfaces. In addition, it forms a noncovalent complex with complement regulatory protein C4b-binding protein (C4BP), a reaction which inhibits its anticoagulant function. Both forms ... More
Effect of microvilli on lateral diffusion measurements made by the fluorescence photobleaching recovery technique.
AuthorsWolf DE, Handyside AH, Edidin M
JournalBiophys J
PubMed ID7104441
Lipid lateral diffusion in the surface membrane of cells and in multibilayers formed from plasma membrane lipids.
AuthorsJacobson K, Hou Y, Derzko Z, Wojcieszyn J, Organisciak D
JournalBiochemistry
PubMed ID7295677
Changes in the organization of the mouse egg plasma membrane upon fertilization and first cleavage: indications from the lateral diffusion rates of fluorescent lipid analogs.
AuthorsWolf DE, Edidin M, Handyside AH
JournalDev Biol
PubMed ID7250512
Calorimetric investigation of the phase partitioning of the fluorescent carbocyanine probes in phosphatidylcholine bilayers.
AuthorsEthier MF, Wolf DE, Melchior DL
JournalBiochemistry
PubMed ID6838845
Lipid vesicle-cell interactions: analysis of a model for transfer of contents from adsorbed vesicles to cells.
AuthorsBlumenthal R, Ralston E, Dragsten P, Leserman LD, Weinstein JN
JournalMembr Biochem
PubMed ID6897439
Membrane asymmetry in epithelia: is the tight junction a barrier to diffusion in the plasma membrane?
AuthorsDragsten PR, Blumenthal R, Handler JS
JournalNature
PubMed ID7322203
Lateral diffusion of lipids in model and natural membranes.
AuthorsTocanne JF, Dupou-Cézanne L, Lopez A
JournalProg Lipid Res
PubMed ID8022844
Lipid lateral diffusion and membrane organization.
AuthorsTocanne JF, Dupou-Cézanne L, Lopez A, Tournier JF
JournalFEBS Lett
PubMed ID2680602
It is shown that investigating the lateral motion of lipids in biological membranes can provide useful information on membrane lateral organization. After labeling membranes with extrinsic or intrinsic lipophilic fluorescent probes, fluorescence recovery after photobleaching experiments strongly suggests that specialized cells like spermatozoa, eggs and epithelia exhibit surface membrane regionalization ... More
Fluorescence quenching in model membranes. 1. Characterization of quenching caused by a spin-labeled phospholipid.
AuthorsLondon E, Feigenson GW
JournalBiochemistry
PubMed ID6261807
A new method is described to evaluate contact between fluorophors and lipids in model membranes. This method utilizes a nitroxide spin-labeled phosphatidylcholine to quench th fluorescence from a variety of membrane-bound molecules by a static process. The distance dependence of the fluorescence quenching is analyzed in terms of simple models. ... More
Unconstrained lateral diffusion of concanavalin A receptors on bulbous lymphocytes.
AuthorsWu ES, Tank DW, Webb WW
JournalProc Natl Acad Sci U S A
PubMed ID6956904
The lateral diffusion coefficient, D, of concanavalin A receptors and receptor complexes on the surface of lymphocytes and RDM4 lymphomas is enhanced by several orders of magnitude to D greater than 5 X 10(-9)cm2/sec by induction of swelling of the cells to bulbous form. Treatments with concanavalin A or 7-nitrobenz-2-oxa-1,3-diazole-phallacidin ... More
Lateral diffusion in membranes.
AuthorsJacobson K
JournalCell Motil
PubMed ID6661764
Lateral diffusion measurements, using the photobleaching techniques, have provided unique and quantitative data on the random translational motions of proteins and lipids of membranes. Proper interpretation of this body of data can yield new insight into the structure of biomembranes. A comparative review of the lateral diffusion of membrane components ... More
Restricted movement of lipid and aqueous dyes through pores formed by influenza hemagglutinin during cell fusion.
AuthorsZimmerberg J, Blumenthal R, Sarkar DP, Curran M, Morris SJ
JournalJ Cell Biol
PubMed ID7806567
The fusion of cells by influenza hemagglutinin (HA) is the best characterized example of protein-mediated membrane fusion. In simultaneous measurements of pairs of assays for fusion, we determined the order of detectable events during fusion. Fusion pore formation in HA-triggered cell-cell fusion was first detected by changes in cell membrane ... More
The pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation.
AuthorsChernomordik LV, Frolov VA, Leikina E, Bronk P, Zimmerberg J
JournalJ Cell Biol
PubMed ID9508770
The mechanism of bilayer unification in biological fusion is unclear. We reversibly arrested hemagglutinin (HA)-mediated cell-cell fusion right before fusion pore opening. A low-pH conformation of HA was required to form this intermediate and to ensure fusion beyond it. We present evidence indicating that outer monolayers of the fusing membranes ... More
Suitability of various membrane lipophilic probes for the detection of trogocytosis by flow cytometry.
AuthorsDaubeuf S, Bordier C, Hudrisier D, Joly E,
JournalCytometry A
PubMed ID19051238
Trogocytosis is a recently discovered phenomenon whereby lymphocytes capture fragments of the plasma membrane from antigen presenting cells (APCs). Using APCs labeled with widely used fluorescent lipophilic probes, we previously described a trogocytosis analysis protocol (TRAP) useful to understand the mechanisms and biological consequences of this process and to identify ... More
Early events in polyomavirus infection: fusion of monopinocytotic vesicles containing virions with mouse kidney cell nuclei.
AuthorsGriffith GR, Marriott SJ, Rintoul DA, Consigli RA
JournalVirus Res
PubMed ID2837012
The entry of polyomavirus enclosed in monopinocytotic vesicles into mouse kidney cell nuclei was studied and evidence for a fusion mechanism was obtained. In vivo studies using the fluorescent lipophilic dye diI-C16(3) as a plasma membrane label showed that polyomavirus-infected nuclei accumulate plasma membrane, while uninfected or polyoma capsid-infected nuclei ... More
The Thy-1 antigen exhibits rapid lateral diffusion in the plasma membrane of rodent lymphoid cells and fibroblasts.
AuthorsIshihara A, Hou Y, Jacobson K
JournalProc Natl Acad Sci U S A
PubMed ID2881297
Thy-1 is a plasma membrane protein, but its primary structure lacks the typical membrane-spanning sequence. Recent studies revealed that a glycophospholipid is covalently bound to the carboxyl terminus, suggesting that the protein is integrated into the plasma membrane by this lipid moiety. Lateral diffusion of Thy-1 was measured in mouse ... More
Thermodynamics of transfer of indocarbocyanines from gel to fluid phases of phospholipid bilayers.
AuthorsSpink CH, Clouser D, O'Neil J
JournalBiochim Biophys Acta
PubMed ID8155672
Application of the regular solution model to thermal transition data obtained by differential scanning calorimetry has allowed the determination of partition coefficients, Kp, and the thermodynamics of transfer of a series of indocarbocyanine solutes between the gel and fluid phases of phospholipid bilayers. The indocarbocyanines with alkyl chain lengths of ... More
The direction of membrane lipid flow in locomoting polymorphonuclear leukocytes.
AuthorsLee J, Gustafsson M, Magnusson KE, Jacobson K
JournalScience
PubMed ID2315695
The objective of this study was to determine the direction of membrane lipid flow in locomoting cells. The plasma membrane of human polymorphonuclear leukocytes was stained with a fluorescent lipid analog dihexadecanoyl indocarbocyanine. A line was photobleached on the cell surface perpendicular to the direction of cell motion. Low-light-level fluorescence ... More
Changes in sperm plasma membrane lipid diffusibility after hyperactivation during in vitro capacitation in the mouse.
AuthorsWolf DE, Hagopian SS, Ishijima S
JournalJ Cell Biol
PubMed ID3958052
We have used the technique of fluorescence recovery after photobleaching to measure the diffusibility of the fluorescent lipid analogue, 1,1'-dihexadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate on the morphologically distinct regions of the plasma membranes of mouse spermatozoa, and the changes in lipid diffusibility that result from in vitro hyperactivation and capacitation with bovine ... More
Structural change of the endoplasmic reticulum during fertilization: evidence for loss of membrane continuity using the green fluorescent protein.
AuthorsTerasaki M, Jaffe LA, Hunnicutt GR, Hammer JA
JournalDev Biol
PubMed ID8903348
Green fluorescent protein (GFP) was targeted to the lumen of the endoplasmic reticulum (ER) of starfish eggs by injecting mRNA coding for a chimeric protein containing a signal sequence and the KDEL ER retention sequence. By confocal microscopy, the GFP chimeric protein was localized in intracellular cisternae (membrane sheets) and ... More
Fusion events and nonfusion contents mixing events induced in erythrocyte ghosts by an electric pulse.
AuthorsSowers AE
JournalBiophys J
PubMed ID3224147
The mechanism of membrane fusion was studied by using human erythrocyte ghosts held in close contact by alternating current-induced dielectrophoresis and inducing fusion with a single electric field pulse. Individual fusion events were followed visually using either 1,1'-dihexadecyl-3,3,3',3'-tetramethylindo carbocyanine perchlorate as a membrane-mixing label or 10-kD fluorescein isothiocyanate-dextran as a ... More
Continuous network of endoplasmic reticulum in cerebellar Purkinje neurons.
AuthorsTerasaki M, Slater NT, Fein A, Schmidek A, Reese TS
JournalProc Natl Acad Sci U S A
PubMed ID7519781
Purkinje neurons in rat cerebellar slices injected with an oil drop saturated with 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC16(3) or DiI] to label the endoplasmic reticulum were observed by confocal microscopy. DiI spread throughout the cell body and dendrites and into the axon. DiI spreading is due to diffusion in a continuous bilayer ... More
Redistribution of a major cell surface glycoprotein during cell movement.
AuthorsJacobson K, O'Dell D, Holifield B, Murphy TL, August JT
JournalJ Cell Biol
PubMed ID6386823
The distribution in living cells of an 80,000-dalton major cell surface glycoprotein of murine fibroblasts has been studied by use of monoclonal antibodies. The presence of the molecule throughout the plasma membrane and on the substrate attached surface of the cell was demonstrated by immunofluorescence. Cell growth kinetics were not ... More
Causes of nondiffusing lipid in the plasma membrane of mammalian spermatozoa.
AuthorsWolf DE, Lipscomb AC, Maynard VM
JournalBiochemistry
PubMed ID2835079
In the plasma membranes of most mammalian somatic cells, lipid is nearly completely free to diffuse laterally in the plane of the membrane. In mammalian spermatozoa and certain other highly polarized mammalian cells, a significant fraction of the plasma membrane lipid is not free to diffuse laterally. Using the technique ... More
Studies on the mechanism of polyethylene glycol-mediated cell fusion using fluorescent membrane and cytoplasmic probes.
AuthorsWojcieszyn JW, Schlegel RA, Lumley-Sapanski K, Jacobson KA
JournalJ Cell Biol
PubMed ID6826645
The mechanism by which polyethylene glycol (PEG) mediates cell fusion has been studied by examining the movements of membrane lipids and proteins, as well as cytoplasmic markers, from erythrocytes to monolayers of cultured cells to which they have been fused. Fluorescence and freeze-fracture electron microscopy and fluorescence recovery after photobleaching ... More
MHC class II-peptide complexes and APC lipid rafts accumulate at the immunological synapse.
AuthorsHiltbold EM, Poloso NJ, Roche PA
JournalJ Immunol
PubMed ID12538693
Activation of CD4(+) Th cells requires their cognate interaction with APCs bearing specific relevant MHC class II-peptide complexes. This cognate interaction culminates in the formation of an immunological synapse that contains the various proteins and lipids required for efficient T cell activation. We now show that APC lipid raft membrane ... More
PI(3)Kgamma has an important context-dependent role in neutrophil chemokinesis.
AuthorsFerguson GJ, Milne L, Kulkarni S, Sasaki T, Walker S, Andrews S, Crabbe T, Finan P, Jones G, Jackson S, Camps M, Rommel C, Wymann M, Hirsch E, Hawkins P, Stephens L
JournalNat Cell Biol
PubMed ID17173040
The directional movement of cells in a gradient of external stimulus is termed chemotaxis and is important in many aspects of development and differentiated cell function. Phophoinositide 3-kinases (PI(3)Ks) are thought to have critical roles within the gradient-sensing machinery of a variety of highly motile cells, such as mammalian phagocytes, ... 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
New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy.
AuthorsWeston SA, Parish CR
JournalJ Immunol Methods
PubMed ID2212694
16 fluorochromes were examined for their ability to label viable lymphocytes in vitro and yield fluorescence detectable by fluorescence microscopy and flow cytometry. Of these fluorochromes, four intracellular dyes were found to be suitable for in vivo migration studies. They were H33342, the well known DNA-binding dye which excites and ... More
Specificity of phosphatidylserine-containing membrane binding sites for factor VIII. Studies with model membranes supported by glass microspheres (lipospheres).
Factor VIII functions in an enzyme complex upon the activated platelet membrane where phosphatidylserine exposure correlates with expression of receptors for factor VIII. To evaluate the specificity of phosphatidylserine-containing membrane binding sites for factor VIII, we have developed a novel membrane model in which phospholipid bilayers are supported by glass ... More
Selectivity of fluorescent lipid analogues for lipid domains.
AuthorsKlausner RD, Wolf DE
JournalBiochemistry
PubMed ID7470460
We have examined the phase partition preferences of the even chain length (n = 10-22) diacyl-3'3'-indocarbo-cyanine iodides (Cn diI) incorporated in disaturated lecithin (PC) vesicles. Two parameters were used to determine this phase preference: (i) the direction of shift of the phase transition temperature (Tm) induced by the dyes and ... More
Selective accumulation of raft-associated membrane protein LAT in T cell receptor signaling assemblies.
AuthorsHarder T, Kuhn M
JournalJ Cell Biol
PubMed ID11038169
Activation of T cell antigen receptor (TCR) induces tyrosine phosphorylations that mediate the assembly of signaling protein complexes. Moreover, cholesterol-sphingolipid raft membrane domains have been implicated to play a role in TCR signal transduction. Here, we studied the assembly of TCR with signal transduction proteins and raft markers in plasma ... More
Labeled probes inserted in the macrophage membrane are transferred to the parasite surface and internalized during cell invasion by Toxoplasma gondii.
AuthorsPacheco-Soares C, De Souza W
JournalParasitol Res
PubMed ID10669130
Tachyzoites of Toxoplasma gondii attach to the macrophage surface and are internalized either by a phagocytic process, which can be inhibited by cytochalasin D, or by an active process, independent of host cell actin. Previous studies have shown that parasite attachment induces the secretion of macromolecules found in the apical ... More
Diffusion and regionalization in membranes of maturing ram spermatozoa.
AuthorsWolf DE, Voglmayr JK
JournalJ Cell Biol
PubMed ID6725394
An essential feature of the "fluid mosaic model" (Singer, S. J., and G. L. Nicolson , 1972, Science (Wash. DC)., 175:720-731) of the cell plasma membrane is the ability of membrane lipids and proteins to diffuse laterally in the plane of the membrane. Mammalian sperm are capable of overcoming free ... More
Single cell fusion events induced by influenza hemagglutinin: studies with rapid-flow, quantitative fluorescence microscopy.
AuthorsKaplan D, Zimmerberg J, Puri A, Sarkar DP, Blumenthal R
JournalExp Cell Res
PubMed ID2055263
Fusion of individual human erythrocytes to fibroblasts expressing the influenza virus hemagglutinin Cells were attached to coverslips fitted in a specially designed flow chamber mounted on a microscope stage, and fusion was triggered by rapid acidification to pH less than 5.2. Fusion between single cell pairs was monitored by a ... 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
Localised PtdIns(3,4,5)P3 or PtdIns(3,4)P2 at the phagocytic cup is required for both phagosome closure and Ca2+ signalling in HL60 neutrophils.
AuthorsDewitt S, Tian W, Hallett MB
JournalJ Cell Sci
PubMed ID16418223
Several events accompany integrin-mediated phagocytosis by myeloid cells. These include local pseudopod and phagocytic cup formation followed by Ca(2+) signalling. However, there is also a role for localised phosphatidylinositol (3,4,5) trisphosphate [PtdIns(3,4,5)P(3)] production. Here we report that in neutrophilic HL-60 cells expressing PH-Akt-GFP, binding of iC3b-coated zymosan particles (2 microm ... More
Incorporation of fluorescent gangliosides into human fibroblasts: mobility, fate, and interaction with fibronectin.
AuthorsSpiegel S, Schlessinger J, Fishman PH
JournalJ Cell Biol
PubMed ID6430916
Rhodamine- and fluorescein-labeled gangliosides were used as probes to investigate the distribution, dynamics, and fate of plasma membrane-bound gangliosides on cultured human fibroblasts. When sparse cultures of fibroblasts were incubated with the fluorescent ganglioside derivatives, their surfaces became highly fluorescent. The fluorescent gangliosides were taken up by the cells in ... More
The development of regionalized lipid diffusibility in the germ cell plasma membrane during spermatogenesis in the mouse.
AuthorsWolf DE, Scott BK, Millette CF
JournalJ Cell Biol
PubMed ID3782284
The lipids and proteins of sperm cells are highly regionalized in their lateral distribution. Fluorescence recovery after photobleaching studies of sperm membrane component lateral diffusibility have shown that the sperm plasma membrane is also highly regionalized in the extents and rates of diffusion of its surface components. These studies have ... More
Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts.
AuthorsNguyen DH, Hildreth JE
JournalJ Virol
PubMed ID10708443
A number of recent studies have demonstrated the significance of detergent-insoluble, glycolipid-enriched membrane domains or lipid rafts, especially in regard to activation and signaling in T lymphocytes. These domains can be viewed as floating rafts composed of sphingolipids and cholesterol which sequester glycosylphosphatidylinositol (GPI)-linked proteins, such as Thy-1 and CD59. ... 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 cell-free system to study regulation of focal adhesions and of the connected actin cytoskeleton.
AuthorsCattelino A, Albertinazzi C, Bossi M, Critchley DR, de Curtis I
JournalMol Biol Cell
PubMed ID9950683
Assembly and modulation of focal adhesions during dynamic adhesive processes are poorly understood. We describe here the use of ventral plasma membranes from adherent fibroblasts to explore mechanisms regulating integrin distribution and function in a system that preserves the integration of these receptors into the plasma membrane. We find that ... More
Lateral diffusion of redox components in the mitochondrial inner membrane is unaffected by inner membrane folding and matrix density.
AuthorsChazotte B, Hackenbrock CR
JournalJ Biol Chem
PubMed ID2005133
We report the first lateral diffusion measurements of redox components in normal-sized, matrix-containing, intact mitoplasts (inner membrane-matrix particles). The diffusion measurements were obtained by submicron beam fluorescence recovery after photobleaching measurements of individual, intact, rat liver mitoplasts bathed in different osmolarity media to control the matrix density and the extent ... More
Characterization of electric field-induced fusion in erythrocyte ghost membranes.
AuthorsSowers AE
JournalJ Cell Biol
PubMed ID6438112
Fusion has been reported to occur in a variety of membrane systems in response to the application of certain electric currents to the medium (Zimmermann, U., 1982, Biochim. Biophys. Acta., 694:227-277). The application of a weak but continuous alternating current causes the membranes in suspension to become rearranged into the ... More
Lateral diffusion of an 80,000-dalton glycoprotein in the plasma membrane of murine fibroblasts: relationships to cell structure and function.
AuthorsJacobson K, O'Dell D, August JT
JournalJ Cell Biol
PubMed ID6386824
The lateral diffusion of an 80,000-dalton major cell surface glycoprotein of murine fibroblasts has been measured. This antigen, identified through the use of monoclonal antibodies, is an integral glycoprotein distributed through the plasma membrane as judged by immunofluorescence and immunoelectron microscopy (see preceding paper). Measurements of fluorescence recovery after photobleaching ... More
Polarity and reorganization of the endoplasmic reticulum during fertilization and ooplasmic segregation in the ascidian egg.
AuthorsSpeksnijder JE, Terasaki M, Hage WJ, Jaffe LF, Sardet C
JournalJ Cell Biol
PubMed ID8449980
During the first cell cycle of the ascidian egg, two phases of ooplasmic segregation create distinct cytoplasmic domains that are crucial for later development. We recently defined a domain enriched in ER in the vegetal region of Phallusia mammillata eggs. To explore the possible physiological and developmental function of this ... More
Endocytic sorting of lipid analogues differing solely in the chemistry of their hydrophobic tails.
AuthorsMukherjee S, Soe TT, Maxfield FR
JournalJ Cell Biol
PubMed ID10087269
To understand the mechanisms for endocytic sorting of lipids, we investigated the trafficking of three lipid-mimetic dialkylindocarbocyanine (DiI) derivatives, DiIC16(3) (1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), DiIC12(3) (1,1'- didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), and FAST DiI (1,1'-dilinoleyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate), in CHO cells by quantitative fluorescence microscopy. All three DiIs have the same head group, but differ in ... More
Pleated septate junctions in leech photoreceptors: ultrastructure, arrangement of septa, gate and fence functions.
AuthorsAschenbrenner S, Walz B
JournalCell Tissue Res
PubMed ID9662648
The leech photoreceptor forms a unicellular epithelium: every cell surrounds an extracellular "vacuole" that is connected to the remaining extracellular space via narrow clefts containing pleated septate junctions. We analyzed the complete structural layout of all septa within the junctional complex in elastic brightfield stereo electron micrographs of semithin serial ... More
A long-lived fusogenic state is induced in erythrocyte ghosts by electric pulses.
AuthorsSowers AE
JournalJ Cell Biol
PubMed ID3958050
Treatment of erythrocyte ghosts in random positions in a suspension with membrane fusion-inducing direct current electric field pulses causes the membranes to become fusogenic. Significant fusion yields are observed if the membranes are dielectrophoretically aligned into membrane-membrane contact with a weak alternating electric field as much as 5 min after ... More
A delay in membrane fusion: lag times observed by fluorescence microscopy of individual fusion events induced by an electric field pulse.
AuthorsDimitrov DS, Sowers AE
JournalBiochemistry
PubMed ID2174698
Low light level video microscopy of the fusion of DiI- (1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) labeled rabbit erythrocyte ghosts with unlabeled rabbit erythrocyte ghosts, held in stable apposition by dielectrophoresis in sodium phosphate buffers, showed reproducible time intervals (delays) between the application of a single fusogenic electric pulse and the earliest detection of ... More
Redistribution of cytoplasmic components during germinal vesicle breakdown in starfish oocytes.
AuthorsTerasaki M
JournalJ Cell Sci
PubMed ID7527047
The starfish oocyte is relatively clear optically, and its nucleus, termed the germinal vesicle, is large. These characteristics allowed studies by confocal microscopy of germinal vesicle breakdown during maturation in living oocytes. Three fluorescent probes for cytoplasmic components were used: fluorescein 70 kDa dextran, which does not cross the nuclear ... More
Spatiotemporal relationships among early events of fertilization in sea urchin eggs revealed by multiview microscopy.
AuthorsSuzuki K, Tanaka Y, Nakajima Y, Hirano K, Itoh H, Miyata H, Hayakawa T, Kinosita K
JournalBiophys J
PubMed ID7756541
Four early events of egg fertilization, changes in intracellular calcium concentration and intracellular pH, reorientation of the surface membrane, and the elevation of the fertilization envelope, were imaged in real time and in pairs in single sea urchin eggs. The paired imaging allowed the correlation of the four events spatially ... More
Evidence for the involvement of microtubules, ER, and kinesin in the cortical rotation of fertilized frog eggs.
AuthorsHouliston E, Elinson RP
JournalJ Cell Biol
PubMed ID1714912
During the first cell cycle, the vegetal cortex of the fertilized frog egg is translocated over the cytoplasm. This process of cortical rotation creates regional cytoplasmic differences important in later development, and appears to involve an array of aligned microtubules that forms transiently beneath the vegetal cortex. We have investigated ... More
Analysis of lateral redistribution of a plasma membrane glycoprotein-monoclonal antibody complex [corrected]
AuthorsIshihara A, Holifield B, Jacobson K
JournalJ Cell Biol
PubMed ID3339094
The lateral redistribution of a major murine glycoprotein, GP80, was studied on locomoting fibroblasts, using rhodamine-conjugated mAbs and ultralow light level digitized fluorescence microscopy. Confirming an earlier study (Jacobson, K., D. O'Dell, B. Holifield, T.L. Murphy, and J. T. August. 1984. J. Cell Biol. 99:1613-1623), the distribution of GP80 was ... More
Fc epsilon RI-mediated association of 6-micron beads with RBL-2H3 mast cells results in exclusion of signaling proteins from the forming phagosome and abrogation of normal downstream signaling.
AuthorsPierini L, Holowka D, Baird B
JournalJ Cell Biol
PubMed ID8830772
Cells of the mucosal mast cell line, RBL-2H3, are normally stimulated to degranulate after aggregation of high affinity receptors for IgE (Fc epsilon RI) by soluble cross-linking ligands. This cellular degranulation process requires sustained elevation of cytoplasmic Ca2+. In this study, we investigated the response of RBL-2H3 cells to 6-micron ... More
Effects of cholesterol depletion and increased lipid unsaturation on the properties of endocytic membranes.
AuthorsHao M, Mukherjee S, Sun Y, Maxfield FR
JournalJ Biol Chem
PubMed ID14734557
Lipid analogs with dialkylindocarbocyanine (DiI) head groups and short or unsaturated hydrocarbon chains (e.g. DiIC(12) and FAST DiI) enter the endocytic recycling compartment efficiently, whereas lipid analogs with long, saturated tails (e.g. DiIC(16) and DiIC(18)) are sorted out of this pathway and targeted to the late endosomes/lysosomes (Mukherjee, S., Soe, ... More
Shear stress induces a time- and position-dependent increase in endothelial cell membrane fluidity.
AuthorsButler PJ, Norwich G, Weinbaum S, Chien S
JournalAm J Physiol Cell Physiol
PubMed ID11245613
Blood flow-associated shear stress may modulate cellular processes through its action on the plasma membrane. We quantified the spatial and temporal aspects of the effects of shear stress (tau) on the lipid fluidity of 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC(16)(13)]-stained plasma membranes of bovine aortic endothelial cells in a flow chamber. A confocal ... More
Contribution of platelet microparticle formation and granule secretion to the transmembrane migration of phosphatidylserine.
AuthorsChang CP, Zhao J, Wiedmer T, Sims PJ
JournalJ Biol Chem
PubMed ID8463253
Activation of human platelets by complement proteins, C5b-9, thrombin plus collagen, or a Ca2+ ionophore results in surface exposure of phosphatidylserine (PS), accompanied by the expression of membrane catalytic activity for the tenase (VIIaIXa) and prothrombinase (VaXa) coagulation enzyme complexes. The mechanism underlying this surface exposure of PS upon platelet ... More