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  • 1
    Electronic Resource
    Electronic Resource
    [s.l.] : Nature Publishing Group
    Nature 357 (1992), S. 442-442 
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] ONCE again beads have been used to trick Mother Nature into revealing how actin moves things around on the cell surface1'2. On page 515 of this issue2, Forscher and his collaborators illustrate how polycationic beads, which bind to the dorsal surface of nerve cell growth cones, can induce actin ...
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  • 2
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 0886-1544
    Keywords: cytoskeleton ; actin ; alpha-actin ; vinuclin ; microtubules ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Flurescently labeled heavy mermoyosin, alpha-actinin, and vinculin were used to localize actin, and vinculin, respectively, in permeabilized and living cells during the process of stress fiber reassembly, which occurred when cells were removed from ATP-depleting medium (20 mM sodium azide and 10 mM 2-deoxyglucose). In 80% of the cells recovering from ATP depletion, small, scattered plaques containing actin, alpha-actinin, and vinculin were replaced by long, thin, periodic fibers within 5 minutes of removal of the inhibitors. These nascent stress fibers grew broader as recovery progressed, until they attained the thickness of stress fibers in control cells. In the other 20% of the cells, the scattered plaques aggregated within 5 minutes of reversal, and almost all the actin, alpha-actinin, and vinculin in the cell became localized in one perinuclear aggregate, with a diameter of approximaterly 15-25 μm. As recovery progressed, all aggregates resembled rings, with diameters that increased at about 0.5 μm/minute and grew to as large as 70 μm in some giant cells. As the size of the rings increased, fibers radiated outward from them and sometimes spanned the diamater of te rings. The shape of the cells did not change during this time. By 1 hour after reversal, the rings were no longer present and all cells had networks of stress fibers. Indirect immunofluorescence techniques used to localize tubulin and vimentin indicated that microtubules and intermediate filaments were not constituents of the rings, and the rings were not closely apposed to the substrate, judging from reflection contrast optics. The rapid rearrangement of attachment plaques into a perinuclear aggregate that spreads radially in the cytoplasm occurs at the same speed as fibroblast and chromosomal movement, but is unlike other types of intracytoplasmic motility.
    Additional Material: 24 Ill.
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  • 4
    ISSN: 0886-1544
    Keywords: sarcoplasmic reticulum ; mitochondira ; mitotic spindle ; cytoskeleton ; cytokinesis ; fluorescent membrane dyes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The dynamic changes of the endoplasmic reticulum (ER) in interphase and mitotic cells was detected by the vital fluorescent dye 3,3′-dihexyloxacarbocyanine iodide. Two types of arrays characterize the continuous ER system in the non-muscle PtK2 cell: (1) a lacy network of irregular polygons and (2) long strands of ER that are found aligned along stress fibers. In cross-striated myotubes there was a periodic localization of fluorescence over each I-band corresponding to the positions of the terminal cisternae of the sarcoplasmic reticulum (SR). In contrast to the arrangement in muscle cells, the aligment of the long strands of ER along stress fibers showed no strict periodicity that could be correlated with the sarcomeric units of the stress fibers. The ER and SR arrays seen in living cells were also detected in fixed cells stained with antibodies directed against proteins of the endoplasmic reticulum and sarcoplasmic reticulum, respectively. Observations of vitally stained PtK2 cells at 1 to 2 minute intervals using low light level video cameras and image processing techniques enabled us to see the polygonal ER units form and undergo changes in their shapes. During cell division, the ER, rhodamine 123-stained mitochondria, and phagocytosed fluorescent beads were excluded from the mitotic spindle while soluble proteins were not. No obvious concentration or alignment of membranes could be found associated with the contractile proteins in the cleavage furrow. After completion of cell division there was a redeployment of the ER network in each daughter cell.
    Additional Material: 16 Ill.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 14 (1989), S. 271-287 
    ISSN: 0886-1544
    Keywords: actin-membrane interaction ; adhesion plaque ; vinculin ; integrin ; fibroblasts ; epithelial cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: 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 B sulfonyl chloride. The fluorescently labeled protein was then chromatographed on Sephadex G-25 and DEAE-cellulose in order to remove free dye and denatured protein. The fluorescent talin was able to bind purified vinculin and was localized in adhesion plaques, membrane ruffles, microspikes, and polygonal networks in acetone-permeabilized nonmuscle cells. In cells that were double-stained with fluorescent talin and an affinity-purified anti-talin an-tibody, a one-to-one correspondence of adhesion plaque staining was seen. Living epithelial cells (PtK2) were microinjected during interphase with fluorescent talin. Computer-enhanced video microscopy was used to document adhesion plaque dynamics such as (1) changes in plaque shape, (2) alterations in plaque positions, and (3) the appearance, growth, and dissolution of plaques. In cells that were followed during mitosis, the adhesion plaques disappeared during cell rounding and then subsequently reappeared upon spreading of the two daughter cells. Treatment of microinjected cells with DMSO in order to disassemble stress fibers resulted in an altered localization of the fluorescent talin. Upon recovery of the cell from the drug, the talin was visualized in its characteristic submembraneous position. These results are the first to document the role and distribution of talin in dynamic processes occurring in living microinjected nonmuscle cells.
    Additional Material: 13 Ill.
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  • 6
    ISSN: 0886-1544
    Keywords: cardiac muscle ; actin dynamics ; α-actinin ; vinculin ; microinjection ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: 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, α-actinin, and vinculin. Each of these proteins was incorporated into the adult cardiomyocytes and was colocalized with the cells'native proteins, despite the fact that the labeled proteins were prepared from noncardiac tissues. Within 10 min of injection, α-actinin was incorporated into Z-bands surrounding the site of injection. Similarly, 30 sec after injection, actin was incorporated into the entire I-bands at the site of injection. Following a 3-h incubation, increased actin fluorescence was noted at the intercalated disc. Vinculin exchange was seen in the intercalated discs, as well as in the Z-bands throug hout the cells. Myosin light chains required 4-6 h after injection to become incorporated into the A-bands of the adult muscle. Nonspecific proteins, such as fluorescent BSA, showed no association with the myofibrils or the former intercalated discs. When adult cells were maintained in culture for 10 days, they retain the ability to incorporate these contractile proteins into their myofibrils. T-tubules and the sarcoplasmic reticulum could be detected in periodic arrays in the freshly isolated cells using the membrane dye WW781 and DiOC3[3], respectively. In conclusion, the myofibrils in adult, as in embryonic, muscle cells are dynamic structures, permitting isoform transitions without dismantling of the myofibrils.
    Additional Material: 9 Ill.
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  • 7
    ISSN: 0886-1544
    Keywords: endoplasmic reticulum ; carbocyanine dyes ; mitosis ; cell division ; membranous organelles ; confocal microscopy ; microtubules ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The distribution and dynamics of the membranous organelles in two cell types were investigated during cell division. Live cells (either PtK2 or LLC-PK1) labeled with the vital dye 3,3′-dihexyloxacarbocyanine iodide [DiOC6(3)] were observed via serial optical sectioning with the laser-scanning confocal microscope. Z-series of labeled, dividing cells were collected every 1-2 minutes throughout mitosis, beginning at prophase and extending to the spreading of the daughter cells. Membrane distribution began to change from the onset of prophase in both cell types. When the mitotic spindle formed in prometaphase, fine tubular membranes, similar to those extending out to the edges of interphase cells aligned along the kinetochore spindle fibers. The lacy polygonal network typical of interphase cells persisted beneath the spindle, and a membrane network was also associated with the dorsal layer of the cell. As PtK2 cells reached metaphse, their spindles were nearly devoid of membrane staining, whereas the spindles of LLC-PK1 cells contained many tubular and small vesicular membranous structures. X-Z series of the LLC-PK1 metaphase spindle revealed a small cone of membranes that was separated from the rest of the cytoplasm by kinetochore MTs. In both cell types, as chromosome separation proceeded, the interzone remained nearly devoid of membranes until the onset of anaphase B. At this time the elongating interzonal microtubules were closely associated with the polygonal network of endoplasmic reticulum. Cytokinesis caused a compression, and then an exclusion of organelles from the midbody. Immunofluorescence staining with anti-tubulin antibodies suggested that spindle membranes were associated with microtubules throughout mitosis. In addition, taxol induced a dense and extensive collection of small vesicles to collect at the spindle poles of both cell types. Nocodazole treatment induced a distinct loss of organization of the membranous components of the spindles. Together these results suggest that microtubules organize the membrane distribution in mitotic cells, and that this organization may vary in different cell types depending on the quantity of microtubules within the spindle. © 1993 Wiley-Liss, Inc.
    Additional Material: 14 Ill.
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  • 8
    ISSN: 0886-1544
    Keywords: cleavage furrows ; cytokinesis ; actin ; phalloidin ; myosin ; filamin ; talin ; attachment plaques ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: PtK2 cells of exceptionally large size were microinjected with fluorescently labeled probes for actin, myosin, filamin, and talin in order to follow the assembly of the contractile proteins into the cleavage furrows. Whereas in cells of normal size, there is usually a diffuse pattern of localization of proteins in the cleavage furrow, in these large, flat cells the labeled proteins localized in fibers in the cleavage furrow. Often, the fibers were striated in a pattern comparable to that measured in the stress fibers of the same cell type. The presence of talin in discrete plaques along fibers in the cleavage furrows of the large cells suggests a further similarity between cleavage furrow and stress fiber structure. The presence of filamin in the cleavage furrows also suggests the possibility of an overlapping mechanism in addition to that of a talin mediated mechanism for the attachment of actin filaments to the cell surfaces in the cleavage furrow. A model is presented that emphasizes the interrelationships between stress fibers, myofibrils, and cleavage furrows. © 1994 Wiley-Liss, Inc.
    Additional Material: 11 Ill.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 1-24 
    ISSN: 0886-1544
    Keywords: myofibrillogenesis ; directionality ; non-muscle myosin II ; myosin ; α-actinin ; Z-bodies ; zeugmatin ; titin ; C-protein ; premyofibril ; nascent myofibril ; mature myofibril ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: When cardiac muscle cells are isolated from embryonic chicks and grow in culture they attach to the substrate as spherical cells with disrupted myofibrils, and over several days in culture, they spread and extend lamellae. Based on antibody localizations of various cytoskeletal proteins within the spreading cardiomyocyte, three types of myofibrils have been identified: 1) fully formed mature myofibrils that are centrally positioned in the cell, 2) premyofibrils that are closest to the cell periphery, and 3) nascent myofibrils located between the premyofibrils and the mature myofibrils. Muscle-specific myosin is localized in the A-bands in the mature, contractile myofibrils, and along the nascent myofibrils in a continuous pattern, but it is absent from the premyofibrils. Antibodies to non-muscle isoforms of myosin IIB react with the premyofibrils at the cell periphery and with the nascent myofibrils, revealing short bands of myosin between closely spaced bands of α-actinin. In the areas where the nascent myofibrils border on the mature myofibrils, the bands of non-muscle myosin II reach lengths matching the lengths of the mature A-bands. With the exception of a small transition zone consisting of one myofibril, or sometimes several sarcomeres, bordering the nascent myofibrils, there is no reaction of these non-muscle myosin IIB antibodies with the mature myofibrils in spreading myocytes. C-protein is found only in the mature myofibrils, and its presence there may prevent co-polymerization of non-muscle and muscle myosins. Antibodies directed against the non-muscle myosin isoforms, IIA, do not stain the cardiomyocytes. In contrast to the cardiomyocytes, the fibroblasts in these cultures stain with antibodies to both non-muscle myosin IIA and IIB. The premyofibrils near the leading edge of the lamellae show no reaction with antibodies to either titin or zeugmatin, whereas the nascent myofibrils and mature myofibrils do. The spacings of the banded α-actinin staining range from 0.3 to 1.4 μm in the pre- and nascent myofibrils and reach full spacings (1.8-2.5 μm) in the mature myofibrils. Based on these observations, we propose a premyofibril model in which non-muscle myosin IIB, titin, and zeugmatin play key roles in myofibrillogenesis. This model proposes that pre- and nascent myofibrils are composed of minisarcomeres that increase in length, presumably by the concurrent elongation of actin filaments, the loss of the non-muscle myosin II filaments, the fusion of dense bodies or Z-bodies to form wide Z-bands, and the capture and alignment of muscle myosin II filaments to form the full spacings of mature myofibrils. © 1994 Wiley-Liss, Inc.
    Additional Material: 16 Ill.
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  • 10
    ISSN: 0886-1544
    Keywords: thymosin β4 ; actin ; stress fibers ; cleavage furrows ; cytokinesis ; cell spreading ; PtK2 cells ; microinjection ; transfection ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Thymosin β4 (Tβ4) binds to G-actin in vitro and inhibits actin polymerization. We studied the effects of incresing Tβ4 concentration within living PtK2 cells, comparing its effects on the disassembly of stress fibers and membrane-associated actin with its ability to inhibit cytokinesis and cell spreading after mitosis. We chose PtK2 cells for the study because these cells have many striking actin bundles in both stress fibers and cleavage furrows. They also have prominent concentrations of membrane-associated actin and remain flattened during mitosis. We have found that PtK2 cells contain an endogenous homologue of Tβ4 at a concentration (approximately 28 μM) sufficient to complex a third or more of the cell's unpolymerized actin. Intracellular Tβ4 concentrations were increased by three different methods: (1) microinjection of an RSV vector containing a cDNA for Tβ4; (2) transfection with the same vector; and (3) microinjection of purified Tβ4 protein. The plasmid coding for Tβ4 was microinjected into PtK2 cells together with fluorescently labeled alpha-actinin as a reporter molecule. Immediately after microinjection fluorescently labeled alpha-actinin was detected in a periodic pattern along the stress fibers just as in control cells injected solely with the reporter. However, after 13 h, cells microinjected with reporter and plasmid showed marked disassembly of the fiber bundles. PtK2 cells transfected with this RSV vector for 2-3 days showed disassembly of stress fibers as detected by rhodamine-phalloidin staining; in these cells the membrane actin was also greatly diminished or absent and the border of the cells was markedly retracted. Microinjection of pure Tβ4 protein into interphase PtK2 cells induced disassembly of the stress fibers within 10 min, while membrane actin appeared only somewhat reduced. If the PtK2 cells were mitotic, Similar microinjection of pure thymosin β4 protein at times from early prophase to metaphase resulted in an unusual pattern of delayed cytokinesis. Furrowing occurred but at a much slower rate than in controls and the amount of actin in the cleavage furrow was greatly reduced. The cells constricted to apparent completion, but after about 30 min the furrow re-gressed, forming a binucleate cell, much as after treatment with cytochalasin B or D. Postcytokinesis spreading of these Tβ4-injected cells was often inhibited. These experiments suggest that an insufficient number of actin filaments prolongs the contractile phase of cytokinesis and abolishes the final sealing process. © 1995 Wiley-Liss, Inc.
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