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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 3 (1983), S. 1-19 
    ISSN: 0886-1544
    Keywords: cytoplasmic transport ; Saltation ; microtubules ; keratocytes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We report the first direct demonstration that the cytoplasmic transport of organelles and vesicles (collectively called particles) takes place along microtubules. Living keratocytes from the corneal stroma of the frog, Rana pipiens, were observed with Allen video-enhanced constrast, differential interference constrast (AVEC-DIC) microscopy [Allen et al, 1981]. In sufficiently thin regions of these cells a network of linear elements was visible. When particles were observed in motion, they always moved along these linear elements. The linear elements remained intact and in focus on the microscope when lysed in a cell lysis solution that stabilized microtubules. Preparations were then fixed in formaldehyde, washed with phosphate-buffered saline (PBS), incubated with rabbit antitubulin, washed with PBS, stained with rhodamine-conjugated goat antirabbit, and washed with PBS. The extracted cells continued to remain in place and in focus on the microscope throughout these procedures. The same cells were then observed using epifluorescence optics and a silicon-intensified target (SIT) video camera. A network of fluorescent linear elements was seen to correspond in number, form, and position to the linear elements seen in the live AVEC-DIC image. Taken together, the AVEC-DIC and fluorescence microscopy observations prove that the linear elements along which particles move are microtubules (MTLEs). The observed particle speeds, pause times, and distances moved varied widely, even for the same particle on the same microtubule. Particles were also observed to switch from one microtubule to another as they were transported. The polarity of the microtubules did not seem to affect the particle direction, since particles were observed to move in both directions on the same MTLE. When not in motion these particles behaved as if anchored to the microtubules since they showed negligible Brownian motion. Finally, it was observed that an elongate particle could move onto two intersecting linear elements such that it was deformed into an inverted “Y” shape. This indicates that there may be more than a single site of attachment between the force generator and the particle.
    Additional Material: 9 Ill.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 2 (1982), S. i 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 6 (1986), S. 1-1 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 5 (1985), S. 81-101 
    ISSN: 0886-1544
    Keywords: fast axonal transport ; isolated axoplasm ; video microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The development of AVEC-DIC microscopy and the application of this method to the study of fast axonal transport in isolated axoplasm extruded from the giant axon of the squid Loligo pealei provides a new paradigm for analyzing the intracellular transport of membranous organelles. The size of the axon, the number of transported particles, and the absence of permeability barriers like the plasma membrane in this preparation permit many experiments that are difficult or impossible to perform using other model systems. The use and features of this preparation are described in detail and a number of properties are evaluated for the first time. The process of extrusion is characterized. Particle movement is evaluated both in the interior of extruded axoplasm and along individual fibrils that extend from the periphery of perfused axoplasm. The role of divalent cations, particularly Ca2+, and the effects of elevated Ca2+ on axoplasmic organization and transport are analyzed. A series of pharmacological agents and polypeptides that alter cytoskeletal organization are used to examine the role of microfilaments and microtubules in fast transport. Finally, the effects of depleting ATP and of adding ATP analogues are discussed. The extruded axoplasm preparation is shown to be an invaluable model system for biochemical and pharmacological analyses of the molecular mechanisms of intracellular transport.
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  • 5
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We present a high-resolution electron microscopic study of the sidearms on microtubules and vesicles that are suggested to form the crossbridges which produce the microtubule-based vesicle transport in squid axoplasm. The sidearms were found attached to the surfaces of the anterogradely transported vesicles in the presence of ATP. These sidearms were made of one to three filaments of uniform diameter. Each filament measured 5-6 nm in width and 30-35 nm in length. The filaments in some of the sidearms had splayed apart by pivoting at their base, thereby assuming a “V” shape. The spread configuration illustrated the independence of the individual filaments. The filaments in other sidearms were closely spaced and oriented parallel to each other, a pattern called the compact configuration. In axoplasmic buffer containing AMP-PNP, structures indistinguishable from the filaments of the sidearms on the vesicles were observed attached to microtubules. Pairs of filaments, thought to represent the basic functional unit, were observed attached to adjacent protofilaments of the microtubules by their distal tips. These data support a model of vesicle movement in which a pair of filaments within a sidearm forms two crossbridges and moves a vesicle by “walking” along the protofilaments of the microtubule.
    Additional Material: 6 Ill.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 1 (1981), S. 269-272 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 4 (1984), S. 7-23 
    ISSN: 0886-1544
    Keywords: axoplasm ; elastic modulus ; viscosity ; motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A magnetic sphere viscoelastometer has been developed to peform rheological experiments in living axoplasm of Loligo pealei. The technique includes the use of a calibrated magnetic sphere viscoelastometer on surgically implanted ferro-magnetic spheres in intact squid giant axons. The axoplasm was discerned to be “living” by the biological criterion of tubulovesicular organelle motility, which was observed before and after experimentation. From these in vivo experiments, new structural characteristics of the axoplasm have been identified. First, analysis of magnetic sphere trajectories has shown the axoplasm to be a complex viscoelastic fluid. Directional experimentation showed that this material is structurally anisotropic, with a greater elastic modulus in the direction parallel to the axon long axis. Second, both magnetic sphere and in vivo capillary experiments suggested that the axoplasm is tenaciously anchored to the axolemma. Third, it was found that axoplasm could be modelled as a linear viscoelastic material in the low shear rate range of 0.0001 to 0.004 s-1. The simplest mechanical model incorporating the discovered properties of the material in this range is Burger's model.
    Additional Material: 8 Ill.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 5 (1985), S. 31-51 
    ISSN: 0886-1544
    Keywords: microtubules ; birefringence ; flow birefringence ; tubulin ; polarization microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Understanding the molecular basis of mitotic movements in living cells will require correlative experiments on intact cells, cell models, purified tubulin, and perhaps other biopolymers. Birefringence is one assay that is useful in all of these experimental situations. Heretofore, studies of birefringence changes during mitosis have lacked a quantitiative basis for interpretation in terms of microtubule number and packing density. One of the aims of this work was to establish that relationship.Purified calf brain tubulin was polymerized to equilibrium and oriented in the hydrodynamic field of a microcapillary flow birefringence apparatus. The relationship between birefringence and microtubule packing density was determined by a combination of optical, electron microscopic, and biochemical methods. The data correlate surprisingly well with those obtained by others from in vitro measurements on isolated mitotic spindles. Using the flow birefringence data, the sensitivity of polarizing microscopes for detecting microtubules was examined and found to depend on microtubule packing density, object thickness, and instrumental factors that limit both the detection and measurement of weakly birefringent objects. Because of the dependence of measurement sensitivity on object thickness, a method of measuring the thickness of microtubule bundles using the dispersion of birefringence was developed. This method is capable of measuring thickness to within two or three Airy diffraction units and does not require any assumptions regarding object symmetry.
    Additional Material: 11 Ill.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 5 (1985), S. i 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 6 (1986), S. 406-418 
    ISSN: 0886-1544
    Keywords: Intermediate filaments ; microfilaments fibroblast cell spreading ; focal center ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Spreading and fully spread chick embryo fibroblasts (CEF) were examined by double-label fluorescence microscopy using the actin-specific probe rhodamine-phalloidin and an antibody directed against CEF intermediate filaments (IF). During midspreading, a striking relationship became discernible: statistical analysis showed that approximately half of the cell population exhibited one or more phase-dense, phalloidin-binding nodules that appeared to act as foci from which IF diverged. Coincidence between actin-containing structures and IF was not limited to these centers; IF could also frequently be seen running in close parallel arrays with stress fibers.Ultrastructural analysis confirmed the presence of non-membrane-bound out-pocketings along the length of stress fibers from which 10-nm IF diverged. These structures varied in size and shape, and displayed a dense, fine fibrillar appearance. IF and microfilaments (MF) were distinguished by size and by decoration of MF with myosin subfragment-1. Other IF-MF interactions were seen in cells of all stages: IF were observed to loop through stress fibers, most frequently at the cell margins. In colchicine-treated cells, IF became redistributed into cables that often ran parallel and appeared to merge with stress fibers. Cytochalasin D-treated CEF exhibited loose aggregates of actin-containing material that appeared to be associated with IF.These results suggest the possibility of an interaction between actin-containing structures and IF, particularly during cell spreading in cultured fibroblasts.
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