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Biomedical subjects

J W Sanger

Publications and source records attributed to J W Sanger.

At least 73 records · Page 4Linked to original sources
PubMed1986

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 and permeabilized cells incorporated the alpha-actinin regardless of whether the alpha-actinin was isolated from nonmuscle, skeletal, or smooth muscle, or whether it was labeled with different fluorescent dyes. The living muscle cells could beat up to 5 d after injection. Rest-length sarcomeres in beating myotubes and cardiac myocytes were approximately 1.9-2.4 microns long, as measured by the separation of fluorescent bands of alpha-actinin. There were areas in nearly all beating cells, however, where narrow bands of alpha-actinin, spaced 0.3-1.5 micron apart, were arranged in linear arrays giving the appearance of minisarcomeres. In myotubes, alpha-actinin was found exclusively in these closely spaced arrays for the first 2-3 d in culture. When the myotubes became contraction-competent, at approximately day 4 to day 5 in culture, alpha-actinin was localized in Z-bands of fully formed sarcomeres, as well as in minisarcomeres. Video recordings of injected, spontaneously beating myotubes showed contracting myofibrils with 2.3 microns sarcomeres adjacent to noncontracting fibers with finely spaced periodicities of alpha-actinin. Time sequences of the same living myotube over a 24-h period revealed that the spacings between the minisarcomeres increased from 0.9-1.3 to 1.6-2.3 microns. Embryonic cardiac myocytes usually contained contractile networks of fully formed sarcomeres together with noncontractile minisarcomeres in peripheral areas of the cytoplasm. In some cells, individual myofibrils with 1.9-2.3 microns sarcomeres were connected in series with minisarcomeres. Double labeling of cardiac myocytes and myotubes with alpha-actinin and a monoclonal antibody directed against adult chicken skeletal myosin showed that all fibers that contained alpha-actinin also contained skeletal muscle myosin. This was true whether alpha-actinin was present in Z-bands of fully formed sarcomeres or present in the closely spaced beads of minisarcomeres. We propose that the closely spaced beads containing alpha-actinin are nascent Z-bands that grow apart and associate laterally with neighboring arrays containing alpha-actinin to form sarcomeres during myofibrillogenesis.

Actinin↗
PubMed1986

Observations of microfilament bundles in living cells microinjected with fluorescently labelled contractile proteins.

Fluorescently labelled contractile proteins (alpha-actinin and filamin) were used to study the dynamic nature of three types of microfilament bundles: myofibrils, stress fibres and polygonal networks. Cultured muscle and non-muscle cells that were microinjected with fluorescent alpha-actinin rapidly incorporated the labelled protein into Z-bands, stress fibre densities and the polygonal foci. Living, injected cells were then observed for varying periods of time, and changes in orientation and periodicity of the myofibrils, stress fibres and polygonal networks were recorded. Permeabilized cells were also reacted with fluorescently labelled proteins and with contractile protein antibodies in order to analyse further the changes taking place in the myofibrils and stress fibres. In both living cardiac myocytes and living skeletal muscle myotubes, contractile myofibrils were present in the same cell with non-contractile nascent myofibrils. The periodicities of small Z-bodies in the nascent non-contractile myofibrils were shorter than the Z-band spacings in the contractile myofibrils, yet both types of myofibrils contained muscle myosin. Over a period of 24 h, a nascent myofibril in a living, microinjected myotube was observed to grow from Z-body spacings of 0.9-1.3 micron to full sarcomere spacings (2.3 microns). During the same time, nascent myofibrils appeared de novo and Z-band alignment became more ordered in the fully formed myofibrils. Stress fibres were not observed to undergo the predictable type of growth seen in myofibrils, but stress fibre periodicities did change in some fibres; some shortened while others lengthened. The orientation of fibres shifted in cytoplasm of both mobile cells and stationary cells. Attachment plaques and foci also changed position and in some cases subdivided and/or disappeared. Models of stress fibres and polygonal networks are presented that suggest that the changes in the periodicities of the dense bodies in stress fibres and the distances between polygonal foci are related to the movement of the interdigitating actin and myosin filaments.

Actin Cytoskeleton↗
PubMed1985

Alpha actinin distribution and extracellular matrix products during somitogenesis and neurulation in the chick embryo.

A discrete stage in two different morphogenetic processes has been examined employing fluorescently labelled alpha-actinin as a probe to localize native alpha-actinin and antibodies to localize fibronectin and collagen type I. The stage of somitogenesis examined is the transition from the compact mesenchymal somitic mass to the epithelial somitic vesicle (ie, epithelialization of the somite). The stage of neurulation examined is the transition from the relatively flat neuroepithelium to the approximation of the neural folds. Before these morphogenetic movements begin, the neuroepithelium is sitting upon a basal lamina and interstitial collagen, and the somite is surrounded by a meshwork of interstitial collagen. During both of these processes, the cells become narrowed at their apices in the region of the tissue that is becoming concave, and alpha-actinin is localized in the apices. The localization of intracellular alpha-actinin and extracellular fibronectin, and the distribution of collagen, suggest that there is a coordinated appearance and distribution of these molecules that is temporally associated with these discrete morphogenetic events.

Actinin↗
PubMed1985

Midbody sealing after cytokinesis in embryos of the sea urchin Arabacia punctulata.

Cytokinesis consists of a contractile phase followed by sealing of the connecting midbody to form two separated cells. To determine how soon the midbody sealed after cleavage furrow contraction, the fluorescent dye Lucifer Yellow CH(457.3 M.W.) was microinjected into cells at various intervals after cleavage had begun. Mitotic PtK2 cells were recorded with video-microscopy so that daughter cells in the epithelial sheet could be identified for several hours after cell division. One daughter cell of each pair followed was microinjected to determine whether the dye diffused into the other daughter cell. For intervals up to four hours after the beginning of cytokinesis, diffusion took place between daughter cells. After this time the dye did not spread between daughter cells. In sea urchin blastomeres of the first, second and third divisions, Lucifer Yellow passed between daughter blastomeres only during the first 15 min after cytokinesis. If one cell of a two-cell, four-cell or eight-cell embryo was microinjected more than 15 min after the last cleavage, the dye remained in the injected cell and was distributed to all progeny of that cell, resulting in blastulae that were eigher one-half, one-quarter or one-eighth fluorescent, respectively. Thus, although cleavage furrow contraction takes approximately the same amount of time in sea urchin blastomeres and PtK2 cells, the time of midbody sealing differs dramatically in the two cell types. Our results also indicate the importance of knowing the mitotic history of cells when injecting dyes into interphase cells for the purpose of detecting gap junctions.

Animals↗
PubMed1984

Actin affinity chromatography in the purification of human, avian and other mammalian plasma proteins binding vitamin D and its metabolites (Gc globulins).

The human plasma protein binding vitamin D and its metabolites (Gc globulin; group-specific component) has been isolated from human plasma by column affinity chromatography on gels to which monomeric actin was covalently attached. Rabbit skeletal-muscle G-actin was covalently coupled to amino-agarose gels before the application of human plasma. At actin/protein molar ratios of 4-8:1, excellent recovery (approximately 58%) of purified binding protein was achieved. After 0.75 M-NaCl washes, the binding protein was eluted from the columns in 3 M-guanidinium chloride, dialysed and analysed. These eluates contained the binding protein as 34-100% of the total protein, reflecting a 130-fold average purification in this single step. In the presence of Ca2+, gelsolin (another plasma protein that binds actin) was apparently retained by the affinity column, but this was prevented by chelation of plasma Ca2+. The actin affinity step also was effective in the isolation of the binding protein from rat, rabbit and chicken plasma, as indicated by autoradiographs of purified fractions analysed by gel electrophoresis after incubation with 25-hydroxy[26,27-3H]cholecalciferol. Further isolation by hydroxyapatite chromatography yielded a purified binding protein which displayed characteristic binding activity toward vitamin D metabolites and G-actin, and retained its physicochemical features. This brief purification sequence is relatively simple and efficient, and should prove to be useful to investigators studying this interesting plasma protein.

Actins↗
PubMed1984

Reduction of density and anisotropic distribution of intermediate filaments occur during avian skeletal myogenesis.

Chicken skeletal muscle taken from embryos in ovo was examined by thin-section electron microscopy. Measurements of filament diameters reveal three nonoverlapping groups of filaments: thin (actin myofibrillar) filaments with mean diameters of 5.3 +/- 0.6 nm (S.D.), thick (myosin myofibrillar) filaments with mean diameters of 15 +/- 1.4 nm, and intermediate filaments with mean diameters of 9.3 +/- 0.9 nm. During muscle development these diameters do not change. By counting the number of filaments observed in the sarcoplasm at different stages, we find that the spatial density of intermediate filaments decreases during avian myogenesis in ovo, from 91 intermediate filaments/micron 2 at 6 days to 43 intermediate filaments/micron 2 at 17 days in ovo. Initially randomly arranged, some intermediate filaments become associated with Z discs, sarcoplasmic reticulum, nuclear membrane, and the sarcolemma between 6 and 10 days in ovo. These associated intermediate filaments course both parallel and transverse to myofibrils, forming lateral connections between myofibrillar Z discs and longitudinal connections from Z disc to Z disc within myofibrils. Intermediate filaments also appear to connect Z discs with the nuclear membrane. The intermediate filament associations persist through day 17 of development, after which the presence of cytoskeletal filaments is obscured by the densely packed myofibrils and membranes. Intermediate filament distribution becomes anisotropic during development. A greater proportion of intermediate filaments in the immediate perimyofibrillar area are oriented parallel to myofibrils than in other areas, so that the majority of the intermediate filaments nearest the myofibrils course parallel to them. The longitudinal intramyofibrillar intermediate filaments persist throughout development, as shown by their existence in KI-extracted adult myofibrils.

Animals↗
PubMed1984

Formation of myofibrils in spreading chick cardiac myocytes.

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 Z bands of sarcomeres, the added alpha-actinin also labeled small particles that were usually arranged periodically in linear arrays with a spacing between particles of 0.3-2.0 micron. Actin was localized between the particles of alpha-actinin by means of fluorescein-labeled heavy meromyosin. The punctate localization of alpha-actinin was prominent in pseudopods, behind ruffles, and at the periphery of spreading cells. Long rows of particles of alpha-actinin were often parallel to one another with the alpha-actinin particles in register. These linear arrays appeared to merge laterally to form strands with broader concentrations of alpha-actinin. Other linear arrays were parallel to myofibrils in the cell and some extended outward from the ends of myofibrils. We conclude that during spreading of cardiac myocytes, myofibrils form at the cell periphery behind the extending margins of the cell, and that the aggregates of alpha-actinin found in these areas are nascent Z bands in the forming myofibrils.

Actinin↗
PubMed1984

Cell surface changes during mitosis and cytokinesis of epithelial cells.

PtK2 cells were studied with scanning electron microscopy to record changes on the cell surface during mitosis and cytokinesis. During prophase, prometaphase and metaphase, the cells remain very flat with few microvilli on their surfaces. In anaphase cells, there is a marked increase in the number of microvilli, most of which are clumped over the separating chromosomes and polar regions of the mitotic spindle leaving the surface of the interzonal spindle region relatively smooth. Microvilli appear over the interzonal spindle region in telophase and the cells also increase in height. At the beginning of cleavage, the distribution of microvilli is roughly uniform over the surface but it becomes asymmetric at the completion of cleavage when the daughter cells begin to spread. At this time most microvilli are over the daughter nuclei and the surfaces that border the former cleavage furrow. The regions of the daughter cells distal to the furrow are the first to spread and their surfaces have very few microvilli. When chromosome movement is inhibited by either Nocodazole or Taxol, microvilli formation is inhibited on the arrested cells. Nevertheless cell rounding still takes place in the normal time period. It is concluded from these observations that the signal for the onset of chromosome movement in anaphase is accompanied by a signal for the formation of microvilli. It is suggested that there is also a separate signal for the cell-rounding event in mitosis and that microvilli do not play a role in this contractile process.

Animals↗
PubMed1984

Analysis of myofibrillar structure and assembly using fluorescently labeled contractile proteins.

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 known to prevent self-association of alpha-actinin, however, blocked the binding of fluorescently labeled alpha-actinin to Z-bands. When tropomyosin was removed from the myofibrils, alpha-actinin then added to the thin filaments as well as the Z-bands. Actin bound in a doublet pattern to the regions of the myosin filaments where there were free cross-bridges i.e., in that part of the A-band free of interdigitating native thin filaments but not in the center of the A-band which lacks cross-bridges. In the presence of 0.1-0.2 mM ATP, no actin binding occurred. When unlabeled alpha-actinin was added first to myofibrils and then labeled actin was added fluorescence occurred not in a doublet pattern but along the entire length of the myofibril. Tropomyosin did not bind to myofibrils unless the existing tropomyosin was first removed, in which case it added to the thin filaments in the l-band. Tropomyosin did bind, however, to the exogenously added tropomyosin-free actin that localizes as a doublet in the A-band. These results indicate that the alpha-actinin present in Z-bands of myofibrils is fully complexed with actin, but can bind exogenous alpha-actinin and, if actin is added subsequently, the exogenous alpha-actinin in the Z-band will bind the newly formed fluorescent actin filaments. Myofibrillar actin filaments did not increase in length when G-actin was present under polymerizing conditions, nor did they bind any added tropomyosin. These observations are discussed in terms of the structure and in vivo assembly of myofibrils.

Actinin↗
PubMed1984

Interaction of fluorescently-labeled contractile proteins with the cytoskeleton in cell models.

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 could not be blocked by prior exposure to unlabeled actin. Fluorescently labeled alpha-actinin also bound to ruffles, attachment plaques, cleavage furrows, and stress fibers. The periodicity of fluorescent alpha-actinin along stress fibers was wider in gerbil fibroma cells than it was in PtK2 cells. The fluorescent alpha-actinin binding in cell models could not be blocked by the prior addition of unlabeled alpha-actinin suggesting that alpha-actinin was binding to itself. While there was only slight binding of fluorescent tropomyosin to the cytoskeleton of interphase cells, there was stronger binding in furrow regions of models of dividing cells. The binding of fluorescently labeled tropomyosin could be blocked by prior exposure of the cell models to unlabeled tropomyosin. If unlabeled actin was permitted to polymerize in the stress fibers in cell models, fluorescently labeled tropomyosin stained the fibers. In contrast to the labeled contractile proteins, fluorescently labeled ovalbumin and BSA did not stain any elements of the cytoskeleton. Our results are discussed in terms of the structure and assembly of stress fibers and cleavage furrows.

Actinin↗
PubMed1983

Microinjection of Lucifer yellow CH into sea urchin eggs and embryos.

Eggs and embryos of Arbacia punctulata were microinjected with the fluorescent dye, Lucifer yellow CH, using a simple pressure injection system. When injected into eggs that were subsequently fertilized, the dye was distributed throughout all cells of the developing embryo. If one cell of a two-cell embryo was injected, dye did not diffuse into the uninjected blastomere. During subsequent development, all progeny of the injected cell contained dye resulting in an embryo that was half-fluorescent. Blue light irradiation of a two-cell embryo, one cell of which had been injected with Lucifer yellow, caused the injected blastomere to stop further divisions while the uninjected blastomere developed normally and was free of dye. These results indicate that the first two blastomeres of Arbacia embryos are not electrically coupled, nor up to the time of hatching, is there any coupling between cells in one half of the first cleavage plane and cells in the other half.

Animals↗
PubMed1983

Differences in the stress fibers between fibroblasts and epithelial cells.

In the stress fibers of two types of nonmuscle cells, epithelia (PtK2, bovine lens) and fibroblasts (Gerbil fibroma, WI-38, primary human) the spacing between sites of alpha-actinin localization differs by a factor of about 1.6 as determined by indirect immunofluorescence and ultrastructural localization with peroxidase-labeled antibody. Both methods reveal striations along the stress fibers with a center-to-center spacing in the range of 0.9 mum in epithelial cells and 1.5 mum in fibroblasts. Periodic densities spaced at comparable distances are seen in PtK2 and in gerbil fibroma cells when they are treated with tannic acid and examined in the electron microscope. In such cells, densities are found not only along stress fibers but also at cell-cell junctions, attachment plaques, and foci from which stress fibers radiate. These latter three sites all stain with alpha-actinin antibody on the light and electron microscope level. Stress fibers in the two cell types also vary in the periodicity produced by indirect immunofluorescence with tropomyosin antibodies. As is the case for alpha-actinin, the tropomyosin center-to-center banding is approximately 1.6 times as long in gerbil fibroma cells (1.7 mum) as it is in PtK2 cells (1.0 mum). These results suggest that the densities seen in the electron microscope are sites of alpha-actinin localization and that the proteins in stress fibers have an arrangement similar to that in striated muscle. We propose a sarcomeric model of stress fiber structure based on light and electron microscopic findings.

Actinin↗
PubMed1983

Light microscope observations on actin distribution during morphogenetic movements in the chick embryo.

The cellular distribution of actin during two morphogenetic processes in the chick embryo has been observed, using a high-resolution fluorescent technique, with heavy meromyosin as a probe. These cytoskeletal elements have been implicated in all cell and tissue movements in the embryo. It is now commonly accepted that microfilaments are necessary to provide the motive force for morphogenesis. Two morphogenetic movements in the early embryo have been studied at the light microscope level. During somitogenesis, the mesenchymal segmental plate becomes transformed into a meristic series of transient epithelial vesicles. Again, actin distribution is diffuse and random before the morphogenetic event. During epithelialization, actin becomes prominent in the apical regions of the epithelial cells. Cells in the somitic epithelial vesicles, the core cells, appear to be passive participants in this process, and consequently show no distinct cellular localization of actin.

Actins↗
PubMed1983

Differential response of three types of actin filament bundles to depletion of cellular ATP levels.

The effect of low levels of ATP on actin filament bundles in PtK2 cells was investigated by using 2-deoxyglucose, together with either sodium cyanide, sodium azide, or 2,4-dinitrophenol. Three actin filament systems were examined: stress fibers, cleavage rings, and dimethyl-sulfoxide (DMSO)-induced actin bundles in the nucleus. Of the three, only stress fibers disassembled when the ATP production was inhibited. The disassembly progressed slowly with the cells losing all stress fibers after about 90 min, but remaining in flat interconnected sheets. Mitotic cells that had progressed as far as metaphase when inhibitors were added, assembled cleavage rings. The process of cytokinesis took place in these cells but at a rate 5 to 10 times slower than normal, and disassembly of the cleavage ring was inhibited after the completion of cytokinesis. DMSO-induced nuclear actin bundles did not disassemble in cells depleted of ATP even when DMSO was eliminated from the medium. The peripheral aggregates of contractile proteins present in these cells became redistributed, however, and the cells flattened in the low ATP environment when DMSO was removed. Nuclear actin bundles did not form in DMSO-treated cells if the ATP inhibitors were present for as little as 5 min prior to DMSO exposure. Thus, the three types of actin filament bundles are affected in different ways by low intracellular levels of ATP. Stress fibers are most sensitive and cleavage rings, the least.

Actins↗
PubMed1982

Close apposition of muscle cells in the longitudinal bands of the body wall of a holothurian, Isostichopus badionotus.

Electron microscopy reveals that sarcolemmata of adjacent muscle cells form pentalaminar junctions by fusion of apposed trilaminar double leaflet membranes. These junctions appear to be candidates for low resistance pathways between muscle fibers. The muscles depolarize slowly when bathed in solutions containing elevated concentrations of KCl, and the sucrose gap method can then be used to measure the potential difference between polarized and depolarized regions. Thus the junctions which we have observed may provide the structural basis for electrical transmission through the sucrose gap.

Animals↗
PubMed1982

Effects of exogenous proteins on cytoplasmic streaming in perfused Chara cells.

Cytoplasmic streaming in characean algae is thought to be generated by interaction between subcortical actin bundles and endoplasmic myosin. Most of the existing evidence supporting this hypothesis is of a structural rather than functional nature. To obtain evidence bearing on the possible function of actin and myosin in streaming, we used perfusion techniques to introduce a number of contractile and related proteins into the cytoplasm of streaming Chara cells. Exogenous actin added at concentrations as low as 0.1 mg/ml is a potent inhibitor of streaming. Deoxyribonuclease I (DNase I), an inhibitor of amoeboid movement and fast axonal transport, does not inhibit streaming in Chara. Fluorescein-DNase I stains stress cables and microfilaments in mammalian cells but does not bind to Chara actin bundles, thus suggesting that the lack of effect on streaming is due to a surprising lack of DNase I affinity for Chara actin bundles. Heavy meromyosin (HMM) does not inhibit streaming, but fluorescein-HMM (FL-HMM), having a partially disabled EDTA ATPase, does. Quantitative fluorescence micrography provides evidence that inhibition of streaming by FL-HMM may be due to a tendency for FL-HMM to remain bound to Chara actin bundles even in the presence of MgATP. Perfusion with various control proteins, including tubulin, ovalbumin, bovine serum albumin, and irrelevant antibodies, does not inhibit streaming. These results support the hypothesis that actin and myosin function to generate cytoplasmic streaming in Chara.

Actins↗
PubMed1981

Fluorescence studies on modes of cytochalasin B and phallotoxin action on cytoplasmic streaming in Chara.

Various investigations have suggested that cytoplasmic streaming in characean algae is driven by interaction between subcortical actin bundles and endoplasmic myosin. To further test this hypothesis, we have perfused cytotoxic actin-binding drugs and fluorescent actin labels into the cytoplasm of streaming Chara cells. Confirming earlier work, we find that cytochalasin B (CB) reversibly inhibits streaming. In direct contrast to earlier investigators, who have found phalloidin to be a potent inhibitor of movement in amoeba, slime mold, and fibroblastic cells, we find that phalloidin does not inhibit streaming in Chara but does modify the inhibitory effect of CB. Use of two fluorescent actin probes, fluorescein, isothiocyanate-heavy meromyosin (FITC-HMM) and nitrobenzoxadiazole-phallacidin (NBD-Ph), has permitted visualization of the effects of CB and phalloidin on the actin bundles. FITC-HMM labeling in perfused but nonstreaming cells has revealed a previously unobserved alteration of the actin bundles by CB. Phalloidin alone does not perceptibly alter the actin bundles but does block the alteration by CB if applied as a pretreatment, NBD-Ph perfused into the cytoplasm of streaming cells stains actin bundles without inhibiting streaming. NBD-Ph staining of actin bundles is not initially observed in cells inhibited by CB but does appear simultaneously with the recovery of streaming as CB leaks from the cells. The observations reported here are consistent with the established effects of phallotoxins and CB on actin in vitro and support the hypothesis that streaming is generated by actin-myosin interactions.

Actins↗