Probing the mechanism of incorporation of fluorescently labeled actin into stress fibers.
AuthorsAmato PA, Taylor DL
JournalJ Cell Biol
PubMed ID3949874
'The mechanism of actin incorporation into and association with stress fibers of 3T3 and WI38 fibroblasts was examined by fluorescent analog cytochemistry, fluorescence recovery after photobleaching (FRAP), image analysis, and immunoelectron microscopy. Microinjected, fluorescein-labeled actin (AF-actin) became associated with stress fibers as early as 5 min post-injection. There was no ... More
Oriented reconstitution of red cell membrane proteins and assessment of their transmembrane disposition by immunoquenching of fluorescence.
AuthorsDarmon A, Bar-Noy S, Ginsburg H, Cabantchik ZI
JournalBiochim Biophys Acta
PubMed ID3893545
'The two major membrane glycoproteins of human red cells, glycophorin and band 3, the anion exchange protein, were isolated from cells exofacially labeled with fluorescein and reconstituted into vesicles with defined transmembrane disposition. Uniform orientation of polypeptides was accomplished by two procedures: Vesicles with single protein units were obtained by ... More
Absence of Na+,K(+)-ATPase regulation of endosomal acidification in K562 erythroleukemia cells. Analysis via inhibition of transferrin recycling by low temperatures.
AuthorsSipe DM, Jesurum A, Murphy RF
JournalJ Biol Chem
PubMed ID1847374
'Transferrin (Tf) acidification has been shown to be limited to pH 6 in murine Balb/c 3T3 fibroblasts, human A549 epidermoid carcinoma cells, and Chinese hamster ovary cells and is followed by alkalinization during recycling. In contrast, Tf acidification in the human erythroleukemic cell line K562 proceeds to below pH 5.5, ... More
Talin dynamics in living microinjected nonmuscle cells.
AuthorsHock RS, Sanger JM, Sanger JW
JournalCell Motil Cytoskeleton
PubMed ID2515003
'To investigate the role of talin in the anchoring of actin-containing stress fibers to the cell membrane of nonmuscle cells, a fluorescent analog of the adhesion plaque protein talin was developed, characterized, and microinjected into living cells. Purified chicken gizzard talin was covalently labeled with the fluorescent dye lissamine rhodamine ... More
A simple fluorescent method for simultaneous determination of aortic permeability to horseradish peroxidase and bovine serum albumin.
AuthorsHollis TM, Katora ME, Montini J
JournalJ Histochem Cytochem
PubMed ID6798104
'Differences in regional aortic net uptake of bovine serum albumin (BSA) and horseradish peroxidase (HP) have been examined by means of conjugation of these molecules to the fluorescent protein tracers fluorescein isothiocyanate (FITC) and lissamine rhodamine B (RB200). Using male Wistar rats, uptake of FITC-BSA under steady state conditions in ... More
Phosphorylation of the vasodilator-stimulated phosphoprotein regulates its interaction with actin.
AuthorsHarbeck B, Hüttelmaier S, Schluter K, Jockusch BM, Illenberger S
JournalJ Biol Chem
PubMed ID10882740
'The vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cyclic nucleotide-dependent kinases in platelets and other cardiovascular cells. It promotes actin nucleation and binds to actin filaments in vitro and associates with stress fibers in cells. The VASP-actin interaction is salt-sensitive, arguing for electrostatic interactions. Hence, phosphorylation may significantly alter ... More
Association of microtubules and intermediate filaments in chicken gizzard cells as detected by double immunofluorescence.
AuthorsGeiger B, Singer SJ
JournalProc Natl Acad Sci U S A
PubMed ID7001467
'By double indirect immunofluorescence, using guinea pig and rabbit antibodies to tubulin and to desmin, we have simultaneously labeled microtubules and intermediate filaments in cultured chicken embryo gizzard cells. At the resolution of the light microscope there was extensive but not complete superposition of the labeling patterns for the two ... More
Translocation of NLS-BSA conjugates into nuclei of permeabilized mammalian cells can be supported by protoplast extract. An experimental system for studying plant cytosolic factors involved in nuclear import.
AuthorsBroder YC, Stanhill A, Zakai N, Friedler A, Gilon C, Loyter A
JournalFEBS Lett
PubMed ID9276462
'An heterologous experimental system, which allows the study of the yet unknown cytosolic factors involved in nuclear import of nuclear localization signal (NLS)-containing proteins in plants, has been established. The ability of plant cell extract to substitute mammalian cytosol and to promote translocation of NLS-containing proteins into nuclei of permeabilized ... More
Two-color labeling of temporally defined protein populations in mammalian cells.
AuthorsBeatty KE, Tirrell DA,
JournalBioorg Med Chem Lett
PubMed ID18774715
'The proteome undergoes complex changes in response to disease, drug treatment, and normal cellular signaling processes. Characterization of such changes requires methods for time-resolved protein identification and imaging. Here, we describe the application of two reactive methionine (Met) analogues, azidohomoalanine (Aha) and homopropargylglycine (Hpg), to label two protein populations in ... More
Formation of membrane domains during the activation of protein kinase C.
AuthorsYang L, Glaser M
JournalBiochemistry
PubMed ID8909294
'The lateral membrane organization of phosphatidylserine, diacylglycerol, substrate, and Ca(2+)-dependent protein kinase C in large unilamellar vesicles was investigated by using fluorescence digital imaging microscopy. The formation of phosphatidylserine domains could be induced by either Ca2+, the MARCKS peptide, or protein kinase C. However, only Ca2+ could induce diacylglycerol to ... More
Identical distribution of fluorescently labeled brain and muscle actins in living cardiac fibroblasts and myocytes.
AuthorsMcKenna N, Meigs JB, Wang YL
JournalJ Cell Biol
PubMed ID3965475
'We have investigated whether living muscle and nonmuscle cells can discriminate between microinjected muscle and nonmuscle actins. Muscle actin purified from rabbit back and leg muscles and labeled with fluorescein isothiocyanate, and nonmuscle actin purified from lamb brain and labeled with lissamine rhodamine B sulfonyl chloride, were co-injected into chick ... More
Spectra and fluorescence lifetimes of lissamine rhodamine, tetramethylrhodamine isothiocyanate, texas red, and cyanine 3.18 fluorophores: influences of some environmental factors recorded with a confocal laser scanning microscope.
AuthorsBrismar H, Trepte O, Ulfhake B
JournalJ Histochem Cytochem
PubMed ID7608524
'We report on the spectra and fluorescence lifetimes of four commonly used fluorophores: lissamine rhodamine (LRSC); tetramethyl rhodamine isothiocyanate (TRITC); Texas Red; and cyanine 3.18 (Cy-3). Fluorescence lifetime recordings revealed that these spectrally overlapping fluorophores can be individually detected by their lifetimes, indicating that at least four fluorophores can be ... More
Incorporation of fluorescently labeled actin and tropomyosin into muscle cells.
'The two major proteins in the I-bands of skeletal muscle, actin and tropomyosin, were each labeled with fluorescent dyes and microinjected into cultured cardiac myocytes and skeletal muscle myotubes. Actin was incorporated along the entire length of the I-band in both types of muscle cells. In the myotubes, the incorporation ... More
Use of resonance energy transfer to monitor membrane fusion.
AuthorsStruck DK, Hoekstra D, Pagano RE
JournalBiochemistry
PubMed ID7284312
'An assay for vesicle--vesicle fusion involving resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl), the energy donor, and rhodamine, the energy acceptor, has been developed. The two fluorophores are coupled to the free amino group of phosphatidylethanolamine to provide analogues which can be incorporated into a lipid vesicle bilayer. When both fluorescent lipids ... More
Incorporation of fluorescently labeled contractile proteins into freshly isolated living adult cardiac myocytes.
'When fluorescently labeled contractile proteins are injected into embryonic muscle cells, they become incorporated into the cells'' myofibrils. In order to determine if this exchange of proteins is unique to the embryonic stage of development, we isolated adult cardiac myocytes and microinjected them with fluorescently labeled actin, myosin light chains, ... More
Polyanionic agents as inhibitors of phagosome-lysosome fusion in cultured macrophages: evolution of an alternative interpretation.
AuthorsGoren MB, Vatter AE, Fiscus J
JournalJ Leukoc Biol
PubMed ID3468191
'Various natural and synthetic substances classified as polyanionics have been implicated in antagonizing phagosome-lysosome fusion in cultured macrophages. The phenomenon has been judged by comparing the transfer of selected markers from secondary lysosomes to phagosomes in control and in "polyanion" cells. Our earlier studies showed that use of one of ... More
Sulfhydryl site-specific cross-linking and labeling of monoclonal antibodies by a fluorescent equilibrium transfer alkylation cross-link reagent.
'The site-specific intramolecular cross-linking of sulfhydryls of monoclonal antibodies via a new class of "equilibrium transfer alkylation cross-link (ETAC) reagents" is described. Following complete or partial reduction of interchain disulfides with dithiothreitol (DTT), two murine IgG2a monoclonal antibodies, 225.28S and 5G6.4, were reacted with alpha,alpha-bis[(p-tolylsulfonyl)methyl]-m-aminoacetophenone (ETAC 1a) and a fluorescent ... More
Optimized conditions to couple two water-soluble biomolecules through alkylamine thiolation and thioetherification.
AuthorsMeunier L, Bourgerie S, Mayer R, Roche AC, Monsigny M
JournalBioconjug Chem
PubMed ID10077469
'A simple method for introducing, in buffered saline, a reactive sulfhydryl group on water-soluble molecules bearing an alkyl-amino group is described. This method is based on the use of two water-soluble reagents: 2-iminothiolane and 6,6''-dithiodinicotinic acid. The first one is open upon reaction with an amino group, and the generated ... More
Interaction of fluorescently-labeled contractile proteins with the cytoskeleton in cell models.
AuthorsSanger JW, Mittal B, Sanger JM
JournalJ Cell Biol
PubMed ID6540785
'To determine if a living cell is necessary for the incorporation of actin, alpha-actinin, and tropomyosin into the cytoskeleton, we have exposed cell models to fluorescently labeled contractile proteins. In this in vitro system, lissamine rhodamine-labeled actin bound to attachment plaques, ruffles, cleavage furrows and stress fibers, and the binding ... More
Binding and distribution of fluorescently labeled filamin in permeabilized and living cells.
AuthorsMittal B, Sanger JM, Sanger JW
JournalCell Motil Cytoskeleton
PubMed ID3690693
'This study reports the first development of a fluorescently labeled filamin. Smooth muscle filamin was labeled with fluorescent dyes in order to study its interaction with stress fibers and myofibrils, both in living cells and in permeabilized cells. The labeled filamin bound to the Z bands of isolated cross-striated myofibrils ... More
Contractile basis of ameboid movement. VII. The distribution of fluorescently labeled actin in living amebas.
AuthorsTaylor DL, Wang YL, Heiple JM
JournalJ Cell Biol
PubMed ID6893200
'The technique of molecular cytochemistry has been used to follow the distribution of fluorescently labeled actin in living Chaos carolinensis and Amoeba proteus during ameboid movement and various cellular processes. The distribution of 5-iodoacetamidofluorescein-labeled actin was compared with that of Lissamine rhodamine B sulfonyl chloride-labeled ovalbumin microinjected into the same ... More
Salvage of glucosylceramide by recycling after internalization along the pathway of receptor-mediated endocytosis.
AuthorsKok JW, Eskelinen S, Hoekstra K, Hoekstra D
JournalProc Natl Acad Sci U S A
PubMed ID2690077
'To examine the (intra)cellular fate of a glycolipid, normally residing at the cell surface, a fluorescent analog of glucosylceramide, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosylsp hingosine (C6-NBD-glucosylceramide), was inserted into the plasma membrane of baby hamster kidney cells at low temperature. Upon warming the cells to 37 degrees C, part of the glycolipid analog was ... More
Fibronectin receptor exhibits high lateral mobility in embryonic locomoting cells but is immobile in focal contacts and fibrillar streaks in stationary cells.
AuthorsDuband JL, Nuckolls GH, Ishihara A, Hasegawa T, Yamada KM, Thiery JP, Jacobson K
JournalJ Cell Biol
PubMed ID2971668
'The dynamic process of embryonic cell motility was investigated by analyzing the lateral mobility of the fibronectin receptor in various locomotory or stationary avian embryonic cells, using the technique of fluorescence recovery after photobleaching. The lateral mobility of fibronectin receptors, labeled by a monoclonal antibody, was defined by the diffusion ... More
Preparation and characterization of a new molecular cytochemical probe: 5-iodoacetamidofluorescein-labeled actin.
AuthorsWang YL, Taylor DL
JournalJ Histochem Cytochem
PubMed ID6107318
'The new technique of molecular cytochemitry (Taylor DL, Wang YL (1978): Proc Natl Acad Sci USA 75:857) requires the use of functional fluorescent analogs of cellular components with optimal fluorescence characteristics. An analog of actin suitable for this technique is prepared by reacting purified rabbit striated muscle actin with 5-iodoacetamidofluorescein ... More
Localization and mobility of gelsolin in cells.
AuthorsCooper JA, Loftus DJ, Frieden C, Bryan J, Elson EL
JournalJ Cell Biol
PubMed ID2834402
'To investigate the physiologic role of gelsolin in cells, we have studied the location and mobility of gelsolin in a mouse fibroblast cell line (C3H). Gelsolin was localized by immunofluorescence of fixed and permeabilized cells and by fluorescent analog cytochemistry of living cells and cells that were fixed and/or permeabilized. ... More
Multiple spectral parameter imaging.
AuthorsWaggoner A, DeBiasio R, Conrad P, Bright GR, Ernst L, Ryan K, Nederlof M, Taylor D
JournalMethods Cell Biol
PubMed ID2648118
Preparation, assay, and microinjection of fluorescently labeled cytoskeletal proteins: actin, alpha-actinin, and vinculin.
AuthorsKreis TE
JournalMethods Enzymol
PubMed ID3102901
A-CAM: a 135-kD receptor of intercellular adherens junctions. II. Antibody-mediated modulation of junction formation.
AuthorsVolk T, Geiger B
JournalJ Cell Biol
PubMed ID3095334
'Intercellular adherens junctions between cultured lens epithelial cells are highly Ca2+-dependent and are readily dissociated upon chelation of extracellular Ca2+ ions. Addition of Ca2+ to EGTA-treated cells results in the recovery of cell-cell junctions including the reorganization of adherens junction-specific cell adhesion molecule (A-CAM), vinculin, and actin (Volk, T., and ... More
Isolation of the dorsal, ventral and intracellular domains of HeLa cell plasma membranes following adhesion to a gelatin substrate.
AuthorsMason PW, Jacobson BS
JournalBiochim Biophys Acta
PubMed ID4063367
'The plasma membrane is a complex organelle responsible for many cellular functions. In addition to mediating the exchange of components with the extracellular fluid, the plasma membrane is involved in cell adhesion to matrix proteins in vivo and in vitro. In vitro, adherent cells have three distinct plasma membrane domains ... More
Kinetics of gelsolin interaction with phalloidin-stabilized F-actin. Rate constants for binding and severing.
AuthorsKinosian HJ, Selden LA, Estes JE, Gershman LC
JournalBiochemistry
PubMed ID8987989
'The kinetics of gelsolin interaction with actin filaments have been investigated using two fluorescent probes, tetramethylrhodamine isothiocyanate-labeled phalloidin bound to F-actin and N-(1-pyrenyl)iodoacetamide-labeled actin. We have also analyzed the F-actin severing by gelsolin using an assay for actin filaments which measures the polymerization rate of monomeric actin added to the ... More
Effect of cholesterol and charge on pore formation in bilayer vesicles by a pH-sensitive peptide.
AuthorsNicol F, Nir S, Szoka FC
JournalBiophys J
PubMed ID8968598
'The effect of cholesterol on the bilayer partitioning of the peptide GALA (WEAALAEALAEALAEHLAEALAEALEALAA) and its assembly into a pore in large unilamellar vesicles composed of neutral and negatively charged phospholipids has been determined. GALA undergoes a conformational change from a random coil to an amphipathic alpha-helix when the pH is ... More
A novel fluorescent pH indicator for the acidic range.
AuthorsMarchesini S, Gatt S, Agmon V, Giudici ML, Monti E
JournalBiochem Int
PubMed ID1417891
'Reaction of a commercial preparation of lissamine-rhodamine B sulfonyl chloride with primary amines gives rise to two sulfonamide derivatives, of which one behaves as a pH indicator with maximal fluorescence emission in the acidic range and a pKa of about 4.6. The structure of this derivative and the mechanism of ... More
A peripheral benzodiazepine receptor targeted agent for in vitro imaging and screening.
AuthorsManning HC, Smith SM, Sexton M, Haviland S, Bai M, Cederquist K, Stella N, Bornhop DJ
JournalBioconjug Chem
PubMed ID16704212
'We developed a molecular imaging agent (MIA), a conjugable form of PK11195 (conPK11195) coupled to a lissamine dye (Liss-ConPK11195), which targets the peripheral benzodiazepine receptor (PBR). To determine that our compound specifically binds to this 18 kDa protein, primarily expressed on the mitochondria, we performed classic binding studies on live ... More
Effects of phorbol ester and teleocidin on Ca2+-induced fusion of liposomes.
AuthorsNam K, Kimura S, Fujiki H, Imanishi Y
JournalBiochem Biophys Res Commun
PubMed ID2610691
'The effects of different types of lipid membrane defects on Ca2+-induced fusion of liposomes containing phosphatidylserine (PS) were investigated using fluorescent probes. Teleocidin enhanced the fusion of phospholipid vesicles in an assay system using terbium/dipicolinic acid during mixing of internal aqueous phases of vesicles upon fusion. 12-O-Tetradecanoylphorbol-13-acetate (TPA) suppressed the ... More
Quantitative fluorometric assay for detection and characterization of Fc receptors.
AuthorsSchreiber AB, Haimovich J
JournalMethods Enzymol
PubMed ID6865782
Colorimetric and fluorometric assays of glutathione transferase based on 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.
AuthorsRicci G, Caccuri AM, Lo Bello M, Pastore A, Piemonte F, Federici G
JournalAnal Biochem
PubMed ID8074309
A comparison of fluorescein isothiocyanate and lissamine rhodamine (RB 200) as labels for antibody in the fluorescent antibody technique.
AuthorsMcKay IC, Forman D, White RG
JournalImmunology
PubMed ID6788685
Fluorescent reagents for the labeling of glycoconjugates in solution and on cell surfaces.
AuthorsWilchek M, Spiegel S, Spiegel Y
JournalBiochem Biophys Res Commun
PubMed ID7370030
Fluorescently labelled molecules as probes of the structure and function of living cells.
AuthorsTaylor DL, Wang YL
JournalNature
PubMed ID6987537
Multicolor laser scanning confocal immunofluorescence microscopy: practical application and limitations.
AuthorsBrelje TC, Wessendorf MW, Sorenson RL
JournalMethods Cell Biol
PubMed ID8246789
Preparation of a fluorescent analog: acetamidofluoresceinyl-labeled Dictyostelium discoideum alpha-actinin.
AuthorsSimon JR, Taylor DL
JournalMethods Enzymol
PubMed ID3029546
Fluorescent protein-dye conjugates. II. Gamma globulin conjugated with various dyes.
AuthorsChen RF
JournalArch Biochem Biophys
PubMed ID4186001
The use of microinjection and video microscopy for the study of calmodulin and calcium in living cells.
Spectral properties and relative quantum yields of new fluorescent stains.
AuthorsBedrick AE, Painter JL, Bonitz DL
JournalImmunopharmacology
PubMed ID553081
Excitation and emission spectra were obtained for thirty-two new fluorescent stains that resist fading when exposed to light. Quantum yields were determined for the fourteen stains that showed the greatest fluorescent intensity. Optimal microscopic excitatory and barrier filters, determined by the experimental results, were used to examine tissues stained with ... More
Chemistry of sulforhodamine--amine conjugates.
AuthorsCorrie JE, Davis CT, Eccleston JF
JournalBioconjug Chem
PubMed ID11312679
Commercially-available sulforhodamine sulfonyl chlorides contain two isomeric monosulfonyl chlorides. Conjugates of these isomers with amines have different properties because the sulfonamide formed from one isomer can undergo ring-closure to a colorless sultam. This chemistry has been examined for a model conjugate with methylamine and for a bioconjugate with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP. The ... More
Fluorescence quenching of dye molecules near gold nanoparticles: radiative and nonradiative effects.
AuthorsDulkeith E, Morteani AC, Niedereichholz T, Klar TA, Feldmann J, Levi SA, van Veggel FC, Reinhoudt DN, Möller M, Gittins DI
JournalPhys Rev Lett
PubMed ID12443474
The radiative and nonradiative decay rates of lissamine dye molecules, chemically attached to differently sized gold nanoparticles, are investigated by means of time-resolved fluorescence experiments. A pronounced fluorescence quenching is observed already for the smallest nanoparticles of 1 nm radius. The quenching is caused not only by an increased nonradiative ... More
Uptake and metabolism of a fluorescent sulfatide analogue in cultured skin fibroblasts.
AuthorsMonti E, Preti A, Novati A, Aleo MF, Clemente ML, Marchesini S
JournalBiochim Biophys Acta
PubMed ID1543730
The sulfatide fluorescent analogue N-lissamine rhodaminyl-(12-aminododecanoyl) cerebroside 3-sulfate was administered in the form of albumin complex to normal human skin fibroblasts and its metabolic fate was investigated. Ceramide, galactosylceramide, glucosylceramide, sphingomyelin and free acid, all containing the fluorophore lissamine rhodamine, have been synthesized as reference standards for the identification of ... More
Histological demonstration of capillaries, interstitial space and muscle fibers in heart and skeletal muscle with fluorescent dyes.
AuthorsVetterlein F, Schmidt G
JournalActa Anat (Basel)
PubMed ID6414232
A method is described which allows a clear demonstration of capillaries and muscle fibers in the heart and skeletal muscle of experimental animals. The fluorescent dyes fluorescein isothiocyanate (FITC) and lissamine rhodamine B 200 (RB 200) were conjugated with a protein of high (gamma-globulin) and low (myoglobin) molecular weight, respectively, ... More
A new fluorescence-based, hydrophobic photolabeling technique for analyzing membrane-associated proteins.
AuthorsHess D, Isenberg G
JournalFEBS Lett
PubMed ID10094472
We introduce a new, fluorescent and photoactivatable fatty acid derivative (SANU) for hydrophobic labelling of membrane-bound proteins. The technique allows fast and highly sensitive screening of hydrophobically inserting proteins analyzed by SDS-PAGE with a detection limit below 0.1 pmol. A reliable calculation of labelling efficiencies is achieved by simultaneous densitometry ... More
Synthesis, spectral properties and enzymatic hydrolysis of fluorescent derivatives of cerebroside sulfate containing long-wavelength-emission probes.
AuthorsMarchesini S, Preti A, Aleo MF, Casella A, Dagan A, Gatt S
JournalChem Phys Lipids
PubMed ID1970953
Fluorescent derivatives of cerebroside sulfate (sulfogalactosyl ceramide, sulfatide) containing long-wavelength-emission fluorophores were synthesized. For this purpose a procedure was developed for preparing a cerebroside 3-sulfate derivative with an amino group on the terminal carbon atom of its fatty acyl residue. The latter compound has been used to prepare cerebroside 3-sulfate, ... More
Novel method for covalent fluorescent labeling of plasmid DNA that maintains structural integrity of the plasmid.
We have developed a chemical strategy for covalent coupling of fluorophores to plasmid DNA. A p-azido-tetrafluoro-benzyl-lissamine conjugate was synthesized and purified. This conjugate was used to covalently associate fluorescent molecules to plasmid DNA by photoactivation. In contrast to nick-translated plasmid DNA, plasmid-lissamine conjugates appeared on gel as supercoiled DNA. Reporter ... More
Rhodamine conjugates:specific and nonspecific binding properties in immunohistochemistry.
AuthorsBrandtzaeg P
JournalAnn N Y Acad Sci
PubMed ID52316
Anionic-exchange fractions of IgG labeled with FITC, MRITC, RB200SC, or RBITC were tested on different substrates, and the resultant fluorescence was evaluated with the Ploem optical system. Conjugations with MRITC or RB200SC were found to afford the following advantages over FITC: immunofluorescence sensitivity was elevated six to seven times on ... More
The dynamic interrelationships of actin and vinculin in cultured cells.
AuthorsSchlessinger J, Geiger B
JournalCell Motil
PubMed ID6420065
The dynamic state of cytoskeletal proteins actin and vinculin was studied in living cells using microinjection of fluorescently-labeled proteins combined with fluorescence photobleaching recovery (FPR). It is shown that both proteins maintain a dynamic equilibrium between their diffusible pools in the cytoplasms and their "organized" cytoskeletal fraction. These interrelationships could ... More
Which fluorophore is brightest? A comparison of the staining obtained using fluorescein, tetramethylrhodamine, lissamine rhodamine, Texas red, and cyanine 3.18.
AuthorsWessendorf MW, Brelje TC
JournalHistochemistry
PubMed ID1429023
There are several red-emitting fluorophores available for immunofluorescence studies, including tetramethylrhodamine, lissamine rhodamine, Texas Red, and cyanine 3.18; however, it is unclear which of these is best. The present study compared the brightness of these fluorophores to that of fluorescein. Staining was attempted using a primary antibody raised against serotonin ... More
Mobility of microinjected rhodamine actin within living chicken gizzard cells determined by fluorescence photobleaching recovery.
AuthorsKreis TE, Geiger B, Schlessinger J
JournalCell
PubMed ID6891291
Rhodamine-labeled actin microinjected into living embryonic chicken gizzard cells became associated with its characteristic cytoskeletal structures. In these domains the translational diffusion coefficients (D) of rh-actin were determined in vivo by fluorescence photobleaching recovery (FPR) measurements. Two classes of actin molecules with respect to its mobilities were detected: rh-actin with ... More
Behaviour of microtubules and actin filaments in living Drosophila embryos.
AuthorsKellogg DR, Mitchison TJ, Alberts BM
JournalDevelopment
PubMed ID3248521
We describe the preparation of novel fluorescent derivatives of rabbit muscle actin and bovine tubulin, and the use of these derivatives to study the behaviour of actin filaments and microtubules in living Drosophila embryos, in which the nuclei divide at intervals of 8 to 21 min. The fluorescently labelled proteins ... More
Thyrotropin-releasing hormone increases cytosolic free Ca2+ in clonal pituitary cells (GH3 cells): direct evidence for the mobilization of cellular calcium.
AuthorsSchlegel W, Wollheim CB
JournalJ Cell Biol
PubMed ID6429159
Changes in the cytosolic free Ca2+ concentration following cell surface receptor activation have been proposed to mediate a wide variety of cellular responses. Using the specific Ca2+ chelator quin2 as a fluorescent intracellular probe, we measured the Ca2+ levels in the cytosol of clonal rat pituitary cells, GH3 cells. We ... More
Coated endosomal vesicles: sorting and recycling compartment for transferrin in BHK cells.
AuthorsEskelinen S, Kok JW, Sormunen R, Hoekstra D
JournalEur J Cell Biol
PubMed ID1802708
We have investigated receptor-mediated endocytosis of transferrin (Tf) in baby hamster kidney (BHK) cells, using fluorescence and electron microscopy, and by carrying out colocalization experiments with clathrin antibodies and a fluorescently tagged glycolipid. Early during internalization, Tf was found in small vesicles (100-150 nm in diameter) located at the cell ... More
Corequirement of specific phosphoinositides and small GTP-binding protein Cdc42 in inducing actin assembly in Xenopus egg extracts.
AuthorsMa L, Cantley LC, Janmey PA, Kirschner MW
JournalJ Cell Biol
PubMed ID9490725
Both phosphoinositides and small GTP-binding proteins of the Rho family have been postulated to regulate actin assembly in cells. We have reconstituted actin assembly in response to these signals in Xenopus extracts and examined the relationship of these pathways. We have found that GTPgammaS stimulates actin assembly in the presence ... More
A quantitative analysis of the retrograde axonal transport of 4 different fluorescent dyes in peripheral sensory and motor neurons and lack of anterograde transport in the corticospinal system.
AuthorsOgilvy CS, Borges LF
JournalBrain Res
PubMed ID2463856
Many fluorescent dye compounds are transported by axons in retrograde and anterograde directions. In the present study the uptake and retrograde axonal transport of 4 chemically related fluorescent dyes was evaluated in the peripheral nervous system of adult mice. Anterograde transport was studied in the corticospinal tract of adult rats. ... More
The coordinate organization of vinculin and of actin filaments during the early stages of fibroblast spreading on a substratum.
AuthorsDavid-Pfeuty T
JournalEur J Cell Biol
PubMed ID3922762
Cultured normal fibroblasts adhere to their support essentially through the focal adhesion plaques which are greatly enriched with the 130 000 dalton protein, vinculin, along with the newly described 215 000 dalton protein, talin, and at which actin bundles terminate. In order to explore a role for vinculin in the ... More
Formation of myofibrils in spreading chick cardiac myocytes.
AuthorsSanger JW, Mittal B, Sanger JM
JournalCell Motil
PubMed ID6391683
Cardiac myocytes were isolated from 5-6-day-old chick embryos and allowed to spread in culture. The distribution of alpha-actinin in the cells was followed for five days in culture by exposing permeabilized cells to rhodamine-labeled alpha-actinin and also by injecting the labeled alpha-actinin into living myocytes. In addition to labeling the ... More
Detection of cellulose with improved specificity using laser-based instruments.
Specific detection of cellulose has not been possible using laser based instruments such as laser scanning confocal microscopes (LSCM) and fluorescently activated cell sorters (FACS). Common cellulose dyes are nonspecific and/or nonexcitable with common lasers. Furthermore, many lasers emit wavelengths that overlap with autofluorescence from chlorophyll and other plant molecules. ... More
Effect of complement on the lateral mobility of erythrocyte membrane proteins. Evidence for terminal complex interaction with cytoskeletal components.
AuthorsLiu ZY, Sanders ME, Hu VW
JournalJ Immunol
PubMed ID2494258
The lateral mobilities of erythrocyte membrane proteins and terminal complement complexes (TCC) were measured on C-treated erythrocyte ghosts by the technique of fluorescence redistribution after photobleaching. Results showed that the lateral diffusion coefficient of the bulk membrane proteins decreased with the assembly of TCC on the membrane at low C ... More
Selective immunocytochemical detection of fluorescent analogs with antibodies specific for the fluorophore.
AuthorsLuby-Phelps K, Amato PA, Taylor DL
JournalCell Motil
PubMed ID6428748
Fluorescent analogs of cellular components are finding increasing use in the field of cell biology. The power of this technique can be augmented by the use of antibodies specific for the fluorophore to visualize selectively the fluorescent analog at the electron microscope level. Rabbit antibodies specific for fluorescein were elicited ... More
Disruption of microfilament organization in living nonmuscle cells by microinjection of plasma vitamin D-binding protein or DNase I.
Plasma vitamin D-binding protein (DBP), which binds to monomeric actin, causes the breakdown of stress fibers when it is microinjected into nonmuscle cells. Disruption of the stress fiber network is also accompanied by shape changes in the cell that resemble those seen after cytochalasin treatment. When DBP was coinjected with ... More
Reconstitution of nuclear protein transport with semi-intact yeast cells.
AuthorsSchlenstedt G, Hurt E, Doye V, Silver PA
JournalJ Cell Biol
PubMed ID8227140
We have developed an in vitro nuclear protein import reaction from semi-intact yeast cells. The reaction uses cells that have been permeabilized by freeze-thaw after spheroplast formation. Electron microscopic analysis and antibody-binding experiments show that the nuclear envelope remains intact but the plasma membrane is perforated. In the presence of ... More
Possible translocation of actin and alpha-actinin along stress fibers.
AuthorsMcKenna NM, Wang YL
JournalExp Cell Res
PubMed ID3758212
We have employed fluorescent analogue cytochemistry and fluorescence photobleaching to study the mobility of actin and alpha-actin along stress fibers. Rhodamine-labeled actin or alpha-actinin microinjected into embryonic chick cardiac fibroblasts soon became incorporated into stress fibers. A pulse of a laser microbeam was used to photobleach small spots on the ... More
Small molecule approach to studying protein tyrosine phosphatase.
AuthorsKumar S, Liang F, Lawrence DS, Zhang ZY
JournalMethods
PubMed ID15588981
Understanding the function of protein tyrosine phosphatases (PTPs) is crucial to deciphering cellular signaling in higher organisms. Of the 100 putative PTPs in human genome, only a little is known about their precise biological functions. Thus establishing novel ways to study PTP function remains a top priority among researchers. Classical ... More
Dendroaxonal transcytosis of transferrin in cultured hippocampal and sympathetic neurons.
AuthorsHémar A, Olivo JC, Williamson E, Saffrich R, Dotti CG
JournalJ Neurosci
PubMed ID9364049
Previous studies using overexpressed polymeric immunoglobulin receptor in cultured neurons have suggested that these cells may use a dendroaxonal transcytotic pathway (Ikonen et al., 1993; de Hoop et al., 1995). By using a combination of semiquantitative light microscopy, video microscopy, and a biochemical assay, we show that this pathway is ... More
A method for immunofluorescent demonstration of three coexisting neurotransmitters in rat brain and spinal cord, using the fluorophores fluorescein, lissamine rhodamine, and 7-amino-4-methylcoumarin-3-acetic acid.
AuthorsWessendorf MW, Appel NM, Molitor TW, Elde RP
JournalJ Histochem Cytochem
PubMed ID1701460
Coexistence of neurotransmitters within single nerve fibers or terminals can be convincingly demonstrated by the use of multicolor immunofluorescence. The present study examined whether three-color immunocytochemical localization of coexisting neurotransmitters can be performed using the blue fluorophore AMCA. Spectrofluorometric examination of secondary antibodies conjugated with AMCA, fluorescein, and lissamine rhodamine ... More
Analysis of cell division using fluorescently labeled actin and myosin in living PtK2 cells.
AuthorsSanger JM, Mittal B, Dome JS, Sanger JW
JournalCell Motil Cytoskeleton
PubMed ID2692841
Actin and the light chains of myosin were labeled with fluorescent dyes and injected into interphase PtK2 cells in order to study the changes in distribution of actin and myosin that occurred when the injected cells subsequently entered mitosis and divided. The first changes occurred when stress fibers in prophase ... More
A rapid and versatile method to label receptor ligands using "click" chemistry: Validation with the muscarinic M1 antagonist pirenzepine.
Tagged biologically active molecules represent powerful pharmacological tools to study and characterize ligand-receptor interactions. However, the labeling of such molecules is not trivial, especially when poorly soluble tags have to be incorporated. The classical method of coupling usually necessitates a tedious final purification step to remove the excess of reagents ... More
Molecular heterogeneity of adherens junctions.
AuthorsGeiger B, Volk T, Volberg T
JournalJ Cell Biol
PubMed ID3930512
We describe here the subcellular distributions of three junctional proteins in different adherens-type contacts. The proteins examined include vinculin, talin, and a recently described 135-kD protein (Volk, T., and B. Geiger, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 10:2249-2260). Immunofluorescent localization of the three proteins indicated that while vinculin was ... More
Fluorescent recovery after photobleaching (FRAP) of a fluorescent transferrin internalized in the late transferrin endocytic compartment of living A431 cells: theory.
AuthorsWahl P, Azizi F
JournalBiochim Biophys Acta
PubMed ID9247168
In previous works, other authors characterized a compartment (LCT) of A431 carcinoma cells in which markers of transferrin endocytose had accumulated during a long chase period. This compartment, was essentially formed by large stationary vacuoles. A few small vesicles budded from these vacuoles, rapidly saltated along microtubules and eventually fused ... More
Comparative behavior of membrane protein-antibody complexes on motile fibroblasts: implications for a mechanism of capping.
AuthorsHolifield BF, Ishihara A, Jacobson K
JournalJ Cell Biol
PubMed ID2277071
A characteristic feature of fibroblast locomotory activity is the rearward transport across the leading lamella of various materials used to mark the cell surface. The two processes most frequently invoked as explanations for this transport phenomenon, called capping, are (a) retrograde membrane flow arising from directed membrane insertion and (b) ... 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
Ionic properties of membrane association by vitamin K-dependent proteins: the case for univalency.
AuthorsMcDonald JF, Evans TC, Emeagwali DB, Hariharan M, Allewell NM, Pusey ML, Shah AM, Nelsestuen GL
JournalBiochemistry
PubMed ID9398287
Ionic properties of membrane interaction by prothrombin, protein Z, and other vitamin K-dependent proteins were studied to determine the relevance of a monovalent membrane contact mechanism between one phospholipid headgroup and a calcium-lined pore in the protein [McDonald, J. F., Shah, A. M., Schwalbe, R. A., Kisiel, W., Dahlback, B., ... More
Associations of elements of the Golgi apparatus with microtubules.
AuthorsRogalski AA, Singer SJ
JournalJ Cell Biol
PubMed ID6381504
The intracellular spatial relationships between elements of the Golgi apparatus (GA) and microtubules in interphase cells have been explored by double immunofluorescence microscopy. By using cultured cells infected with the temperature-sensitive Orsay-45 mutant of vesicular stomatitis virus and a temperature shift-down protocol, we visualized functional elements of the GA by ... More
Dynamics of beta 1 integrin-mediated adhesive contacts in motile fibroblasts.
AuthorsRegen CM, Horwitz AF
JournalJ Cell Biol
PubMed ID1280274
Motile chick skeletal fibroblasts adhere to a laminin substrate by means of clustered beta 1 integrins. These integrin "macroaggregates" are similar to classic focal contacts but do not appear dark under interference-reflection microscopy. They contain alpha 5 integrin and are associated with extracellular fibronectin. To study their behavior during cell ... More
Evaluation of biotin-dye conjugates for use in an HPLC assay to assess relative binding of biotin derivatives with avidin and streptavidin.
In this investigation, studies were conducted to determine if size exclusion HPLC could be used to assess relative association rates (on-rates) and dissociation rates (off-rates) of biotin derivatives from avidin (Av) and streptavidin (SAv). For easy detection and quantification of biotin derivatives, molecules that can be detected by UV absorbance ... More
Myofibrillogenesis in living cells microinjected with fluorescently labeled alpha-actinin.
Fluorescently labeled alpha-actinin, isolated from chicken gizzards, breast muscle, or calf brains, was microinjected into cultured embryonic myotubes and cardiac myocytes where it was incorporated into the Z-bands of myofibrils. The localization in injected, living cells was confirmed by reacting permeabilized myotubes and cardiac myocytes with fluorescent alpha-actinin. Both living ... More
Behavior of a fluorescent analogue of calmodulin in living 3T3 cells.
AuthorsLuby-Phelps K, Lanni F, Taylor DL
JournalJ Cell Biol
PubMed ID4044638
We have prepared and partially characterized a lissamine-rhodamine B fluorescent analogue of calmodulin, LRB-CM. The analogue had a dye/protein ratio of approximately 1.0 and contained no free dye or contaminating labeled proteins. LRB-CM was indistinguishable from native calmodulin upon SDS PAGE and in assays of phosphodiesterase and myosin light chain ... More
Macromolecular arrangement in the aminoacyl-tRNA.elongation factor Tu.GTP ternary complex. A fluorescence energy transfer study.
AuthorsWatson BS, Hazlett TL, Eccleston JF, Davis C, Jameson DM, Johnson AE
JournalBiochemistry
PubMed ID7794902
The distance between the corner of the L-shaped transfer RNA and the GTP bound to elongation factor Tu (EF-Tu) in the aminoacyl-tRNA.EF-Tu.GTP ternary complex was measured using fluorescence energy transfer. The donor dye, fluorescein (Fl), was attached covalently to the 4-thiouridine base at position 8 of tRNAPhe, and aminoacylation yielded ... More
Recycling pathways of glucosylceramide in BHK cells: distinct involvement of early and late endosomes.
AuthorsKok JW, Hoekstra K, Eskelinen S, Hoekstra D
JournalJ Cell Sci
PubMed ID1487494
Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide, 6(-)[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosyl sphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma membrane occurs, as could be shown after treating the cells with the microtubule-disrupting agent nocodazole, which causes inhibition of the glycolipid's ... More
Experimental depression of junctional membrane permeability in mammalian cell culture. A study with tracer molecules in the 300 to 800 Dalton range.
AuthorsFlagg-Newton J, Loewenstein WR
JournalJ Membr Biol
PubMed ID41101
Cell-to-cell junctional permeability in mammalian cell cultures was probed with a series of fluorescent tracers ranging 300 to 800 in molecular weight, during treatment with metabolic inhibitors, Ca-transporting ionophore, and carbon dioxide. Treatment with the combination of cyanide and iodoacetic acid (1--2 mM each), but not with either one alone, ... More
Measurement of absolute tracer concentrations in tissue sections by using digital imaging fluorescence microscopy. Application to the study of plasma protein uptake by the arterial wall.
AuthorsWeinberg PD, Winlove CP, Parker KH
JournalJ Microsc
PubMed ID8169950
Digital imaging fluorescence microscopy (DIFM) of tissue sections was used to quantify uptake of labelled plasma proteins by the arterial wall. Several aspects of the measuring system were investigated so that absolute tracer concentrations and their local variation could be derived from digitized images. These investigations may be relevant to ... More
Permeability properties of cell-to-cell channels: kinetics of fluorescent tracer diffusion through a cell junction.
AuthorsZimmerman AL, Rose B
JournalJ Membr Biol
PubMed ID4032457
We have analyzed the intracellular and cell-to-cell diffusion kinetics of fluorescent tracers in the Chironomus salivary gland. We use this analysis to investigate whether membrane potential-induced changes in junctional permeability are accompanied by changes in cell-to-cell channel selectivity. Tracers of different size and fluorescence wavelength were coinjected into a cell, ... More
Zymogen/enzyme discrimination using peptide chloromethyl ketones.
AuthorsWilliams EB, Krishnaswamy S, Mann KG
JournalJ Biol Chem
PubMed ID2708377
Glutamylglycinylarginyl chloromethyl ketone, tyrosylglycinylarginyl chloromethyl ketone, and phenylalanylprolylarginyl chloromethyl ketone have been labeled at their amino termini using fluorescein, rhodamine-X, lissamine-rhodamine, pyrene, and the 1,5-, 2,5-, and 2,6-dimethylaminonaphthalene-1-sulfonyl moieties. These peptidyl chloromethyl ketones have also been modified by incorporation of biotin and epsilon-amino caproyl biotin. The ability of these various ... More
Stress fiber and cleavage furrow formation in living cells microinjected with fluorescently labeled alpha-actinin.
alpha-Actinins, isolated from muscle and nonmuscle sources and labeled with various fluorescent dyes, were microinjected into living PtK2 cells during interphase to observe the reformation of stress fibers following cell division. Fluorescently labeled ovalbumin and bovine serum albumin were also injected as control proteins. alpha-Actinin was incorporated into stress fibers ... 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
Analysis of myofibrillar structure and assembly using fluorescently labeled contractile proteins.
AuthorsSanger JW, Mittal B, Sanger JM
JournalJ Cell Biol
PubMed ID6699087
To study how contractile proteins become organized into sarcomeric units in striated muscle, we have exposed glycerinated myofibrils to fluorescently labeled actin, alpha-actinin, and tropomyosin. In this in vitro system, alpha-actinin bound to the Z-bands and the binding could not be saturated by prior addition of excess unlabeled alpha-actinin. Conditions ... More