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  • Wiley-Blackwell  (10,888)
  • American Meteorological Society
  • 1985-1989  (11,976)
  • 1920-1924
  • 1988  (11,976)
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  • 1985-1989  (11,976)
  • 1920-1924
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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 337-348 
    ISSN: 0886-1544
    Keywords: isoactins ; immunogold ; myofibrils ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In vertebrate skeletal muscle, the proliferating myoblasts synthesize nonmuscle isoforms of actin, and the cells begin to express muscle-specific actin isoforms during their myogenic differentiation. To study the distributions of the actin isoforms in myogenic cells and fully differentiated skeletal muscle, we prepared a peptide antibody specific for the skeletal α isoform of actin and used this antibody along with an antibody specifically reactive with nonmuscle γ actin to stain cultured myotubes and adult skeletal myofibrils by double-indirect immunofluorescence. At this level of resolution, no differences in isoform localization were seen: Both muscle and nonmuscle actins were detected in the myotubes and in the striations of mature myofibrils. Myotubes were also double-stained using immunogold electron microscopy, and the isoform distributions were determined quantitatively by counting the two sizes of gold particles that corresponded to labeling with each antibody. A quantitative analysis of immunoreactivity revealed that, although both forms were present in all actin-containing structures, nonmuscle actin was relatively more prevalent along the edges (cortical microfilaments) of the myotubes, whereas the muscle isoform predominated in the interior regions (containing forming myofibrils). Thus, we have found evidence of a heterogeneous distribution of muscle and nonmuscle actin isoforms in differentiating myogenic cells, and we have demonstrated that a nonmuscle actin isoform is a component of the muscle contractile apparatus.
    Additional Material: 6 Ill.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 172-184 
    ISSN: 0886-1544
    Keywords: chromosome movement ; spindle elongation ; micromanipulation ; mechanical properties ; mitosis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Mechanical properties of the mitotic spindle and the effects of various operations of the mitotic apparatus on the chromosome movement and spindle elongation were investigated in fertilized eggs and blastomeres of the sand dollar, Clypeaster japonicus. On the basis of results with mechanical stretching and compression of the spindle with a pair of microneedles and the behavior of an oil drop microinjected into the spindle, it was concluded that the equatorial region of the spindle is mechanically weaker than the half-spindle region. Anaphase chromosome movement occurred in the spindle from which an aster had been removed or separated with its polar end and in the spindle in which the interzonal region had been removed. This fact indicates that chromosomes move poleward in anaphase by forces generated near the kinetochores in the half-spindle. Because of the effects of separation or removal of an aster from the spindle on the spindle elongation in anaphase and the behavior of the aster, it was concluded that the spindle elongation in anaphase is caused by pulling forces generated by asters attached to the ends of the spindle.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 153-163 
    ISSN: 0886-1544
    Keywords: intracellular particle motions ; cytoplasmic streaming ; onion (Allium) epidermal cells ; video microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The endoplasmic reticulum (ER) and associated organelle and particle movements in onion (Allium cepa) bulb scale epidermal cells were observed, recorded, and analyzed using computer-assisted video (AVEC-DIC, AVEC-POL and fluorescence) microscopy. The ER is composed of two interconnected sets of filamentous membrane tubules with diameters ranging from 0.1 to 0.5 μm. The first form a more stable, stationary network of intersecting polygonal membrane tubules lying closely appressed to the plasma membrane and continuous with a second very dynamic set of longer membrane tubules that often are located parallel to each other, shifting rapidly around the cytoplasm and forming dynamic knots or organization centers. The ER, mitochondria, and spherosomes fluoresced upon chlortetracycline treatment and are therefore presumed to sequester calcium. ER and mitochrondria also stain with the fluorescent dye, rhodamine 123. Mitochrondria and spherosomes are seen to move in the cytoplasm only along paths parallel to the axis of the ER tubules. Smaller particles (0.5 μm) tend to follow these same paths but may occasionally move independently. Particles and organelles move in close, but not in direct, association with the ER tubules. In optically favored cells, actin filaments were occasionally recorded located in parallel with the ER tubules and directly associated with moving particles. Streaming ceased promptly and reversibly upon treatment with cytochalasin B, which did not visibly disrupt the ER. Short-term treatment with colchicine did not inhibit streaming or disrupt the ER network, whereas long-term (hours) colchicine treatments caused the disappearance of the stationary, cortical polygonal networks and an aggregation of still slowly moving organelles and particles onto now visible actin filaments. This suggests that microtubule breakdown disrupts the three-dimensional distribution of the ER and rearranges actin filaments in the cell's cytoplasm. Actin filaments must be directly involved in generation of movement of the particles and organelles. A three-dimensional model, based on optical sectioning of the epidermal cells, is proposed to illustrate the distribution of the endoplasmic reticulum in onion epidermal cell cytoplasm.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 185-196 
    ISSN: 0886-1544
    Keywords: mitosis ; kinetochore ; video microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We describe preliminary results from two studies exploring the dynamics of microtubule assembly and organization within chromosomal spindle fibers. In the first study, we microinjected fluorescently labeled tubulin into mitotic PtK1 cells and measured fluorescence redistribution after photobleaching (FRAP) to determine the assembly dynamics of the microtubules within the chromosomal fibers in metaphase cells depleted of nonkinetochore microtubules by cooling to 23-24°C. FRAP measurements showed that the tubulin throughout at least 72% of the microtubules within the chromosomal fibers exchanges with the cellular tubulin pool with a half-time of 77 sec. There was no observable poleward flux of subunits. If the assembly of the kinetochore microtubules is governed by dynamic instability, our results indicate that the half-life of microtubule attachment to the kinetochore is less than several min at 23-24°C.In the second study, we used high-resolution polarization microscopy to observe microtubule dynamics during mitosis in newt lung epithelial cells. We obtained evidence from 150-nm-thick optical sections that microtubules throughout the spindle laterally associate for several sec into “rods” composed of a few microtubules. These transient lateral associations between microtubules appeared to produce the clustering of nonkinetochore and kinetochore microtubules into the chromosomal fibers. Our results indicate that the chromosomal fiber is a dynamic structure, because microtubule assembly is transient, lateral interactions between microtubules are transient, and the attachment of the kinetochores to microtubules may also be transient.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 237-245 
    ISSN: 0886-1544
    Keywords: video microscopy ; digital image processing ; fluorescence photobleaching ; microtubule dynamics ; living cell dynamics ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The ability to tag biological molecules fluorescently and to detect their distribution in living cells has promoted the study of cytoplasmic organization in general and microtubule dynamics in particular. The techniques that we have selected and developed allowed the determination of spatial and temporal changes of the microtubule network in living fibroblasts at the level of individual microtubules. We have employed two general approaches for determining pattern changes: direct video microscopy and photobleaching and subsequent observation. Direct observation of fluorescent microtubules by high-definition video microscopy provided good spatial resolution at several time points, but was limited to the less congested and thinner periphery of the cell. This approach was made possible by a relatively bright, photostable reporter, xrhodamine-tubulin, and showed that microtubules underwent rounds of assembly and disassembly from their ends. Bleaching and subsequent observation of lysed cells improved the signal to noise ratio by extracting soluble chromophore and permitted observations in congested areas, but was limited to a single time interval. This approach demonstrated that microtubule domains were replaced one by one and that turnover was most rapid at the cell periphery. Antibodies specific for nonbleached chromophore can be used to enhance the signal to noise ratio further or to extend spatial resolution by the use of immunoelectron microscopy. Direct video microscopy and photo-bleaching are two approaches to the study of dynamics that have complementary strengths and wide application to the biology of living cells.
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  • 6
    ISSN: 0886-1544
    Keywords: axoplasmic transport ; motility ; microtubules ; MAPs ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Vesikin, a protein that can associate with squid axoplasmic vesicles or optic lobe microtubuies, has been implicated as a force-generating molecule involved in microtubule-dependent vesicle transport [Gilbert and Sloboda, 1986, 1988]. Because vesikin crossreacts with an antibody to porcine brain microtubule associated protein 2 (MAP 2), studies were conducted to compare squid vesikin and brain MAPs. When taxol stabilized microtubules containing vesikin as a microtubule associated protein were incubated in the presence of ATP, vesikin dissociated from the microtubule subunit lattice. This behavior would be expected for an ATP-dependent, force generating molecule that serves as a crossbridge between vesicles and microtubules. When chick brain microtubules were treated under the same conditions, MAP 2 remained bound to the microtubules while MAP 1 dissociated in a manner similar to vesikin. One dimensional peptide mapping procedures revealed that, although digestion of vesikin and MAP 2 generated several peptides common to both proteins, vesikin and MAP 2 are clearly not identical. Furthermore, the addition of vesikin or MAPS 1 and 2 to purified tubulin stimulated microtubule assembly in a manner dependent on the concentration of added protein. These findings demonstrate that brain MAPs share characteristics common to squid vesikin and support the suggestion that brain MAPs 1 and 2 might act as a force generating complex for vesicle transport in higher organisms.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 255-262 
    ISSN: 0886-1544
    Keywords: regulation of organelle transport ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Allen Video-enhanced constrast/differential interference constrast (AVEC-DIC) microscopy was used in conjunction with video intensification immunofluorescence microscopy to demonstrate that organelles and vesicle (particles) can move in either direction along microtubular linear elements in fibroblasts [Hayden et al., 1983]. Since it is not possible to determine the number of microtubules making up a linear element with light microscopy alone, AVEC-DIC microscopy was used in conjunction with whole-mount electron microscopy to show bidirectional transport along a single microtubule [Hayden and Allen, 1984]. These studies demonstrate that the structural polarity of the microtubule does not determine the direction of particle motion, and since dynein is an asymetric molecule, a simple microtubule-dynein-particle hypothesis cannot explain bidirectional transport along a single microtubule.Very little is known about regulation of particle transport in most cell types. Human embryonic lung fibroblasts grown on glass coverslips were serum-deprived for 24 hours and re-fed with serumless medium; the particle translocations/5 minutes were then determined The cells were then re-fed with either serumless medium, serum-containing medium, or serumless medium containing some bioactive factor, and the particle translocations/5 minutes were again determined for the same cells. Medium containing 10% fetal bovine serum inhibited particle translocation by 51.8%. Of the bioactive factors tested, only vasopressin produced a significant reduction in particle translocations (38%). This suggests that protein kinase C or calcium/calmodulin kinase could be involved in regulating particle transport.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 263-270 
    ISSN: 0886-1544
    Keywords: cilia ; axoneme ; ATP-induced microtubule sliding ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The dynein arms of ciliary doublet microtubules cause adjacent axonemal doublets to slide apart with fixed polarity. This suggests that there is a unique mechanochemistry to the dynein arm with unidirectional force generation in all active arms and also that not all arms are active at once during a ciliary beat. Negative stain and thin-section images of arms in axonemes treated with β, γ methylene adenosine triphosphate (AMP-PCP) show a consistent subunit construction where the globular head of the arm interacts with subfiber B of doublet N + 1. This interpretation differs from that provided by freeze etch and STEM interpretations of in situ arm construction and has implications for the mechanochemical cycle of the arm. A computer model of the arms in relation to other axonemal structures has been constructed to test these interpretations. Attachment of the head of the arm to subfiber B is directly demonstrable in splayed axonemes in AMP-PCP. About half of the doublets in an axoneme show such attachments, while half do not. This might imply that about half the doublets in an axoneme are active at any given instant and can be identified as such. This information may be useful in probing questions of how active arms differ biochemically from inactive arms and of how microtubule translocators in general become active.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 191-204 
    ISSN: 0886-1544
    Keywords: turbidity ; ciliary doublet ; biphasic ; extrusion ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Several characteristics of the sliding disintegration of Tetrahymena ciliary axonemes were found by turbidimetric assay, the ATP-regenerating system, and quantitative determination of the ATP concentration. At ATP concentrations exceeding 40 μM, the response in terms of turbidity was biphasic and could be divided into three phases. The dependence of each phase on ATP concentration was examined. The time duration of phase 2 increased with ATP concentration. When the ATP concentration was kept constant by the ATP-regenerating system, consisting of pyruvate kinase and phosphoenol pyruvate, the time duration of phase 2 increased with the concentration of phosphoenol pyruvate. On examining the change in turbidity with decreasing ATP concentration, the transition from phase 2 to phase 3 was found to occur at an ATP concentration of 40 μM.Dark-field and electron microscopy indicated the sliding disintegration to be closely correlated with the degree of tubidity. At phase 1, one or two doublets extruded from most of the axonemes, and disintegration failed to progress during phase 2. At the transition point from phase 2 to 3, at about 40 μM, ATP, other doublets were noted to extrude from the axonemes one after the other, causing turbidity to be minimal by the end of phase 3.The ATP concentration dependence of stepwise sliding disintegration suggests that each axoneme may possess the ability to regulate doublet microtubule sliding at lower or higher concentrations of ATP. In response to local differences or gradients of ATP concentration along the axoneme, the axonemes may cause localized sliding of doublets, thus subsequently generating active bending movement.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 205-218 
    ISSN: 0886-1544
    Keywords: F-actin ; actin bundling protein ; cytoimmunofluorescence ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The Ca+2-sensitive actin-binding protein isolated from Dictyostelium discoideum, 30,000-D protein (Fechheimer and Taylor: J. Biol. Chem. 259:4514-4520, 1984;) has recently been localized in filipodia of substrate-adhered amoebae (Fechheimer: J. Cell Biol. 104:1539-1551, 1987). We have determined that this protein has a Mr of 34,000 daltons and is strictly colocalized with actin filaments in both substrate-attached Dictyostelium amoebae and cultured fibroblasts. 3T3 fibroblasts, as well as normal and virally transformed rat kidney fibroblasts (NRK) contain a 34-kilodalton (kD) protein that cross-reacts specifically with antibody to the Dictyostelium bundling protein. Mammalian 34-kD protein is colocalized with F-actin in stress fibers and the cortical cytoskeleton in substratadhered fibroblasts. In substrate-adhered vegetative Dictyostelium, F-actin and 34-kD protein are concentrated in regions of the cell cortex exhibiting filipodia and membrane ridges. Multiple filipodia formed after exposure to the chemoattractant folic acid stain intensely for 34-kD protein, implying participation in the assembly of actin bundles during filipod formation. The cortex of pseudopodia also contained high concentrations of bundling protein, but pseudopod interiors did not. In contrast to vegetative Dictyostelium, F-actin and 34-kD protein were not colocalized in cells that had progressed through the development cycle. In fruiting bodies, 34-kD protein was detected by immunofluorescence microscopy only in prespore cells, while F-actin appeared in stalk cells and spores.
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