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  • dynein  (2)
  • epitope-tagging  (1)
  • 1
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 1 (1981), S. 499-515 
    ISSN: 0886-1544
    Keywords: dynein ; tubulin ; axonemes ; microtubules ; microtubule-associated proteins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Microtubule-associated proteins (MAPs), isolated from brain tubulin, bound to and saturated outer fibers of Chlamydomonas flagella. MAPs present on these microtubules prevented the subsequent recombination of dynein. MAPs also bound to intact axonemes and thus did not specifically bind to the dynein binding sites on the A subfiber. A molar ratio of 1 mole MAP2 per 27 moles tubulin dimers at saturation of the outer fibers with MAP2 suggested that MAPs could effectively interfere with dynein recombination only if the MAPs were near the dynein binding sites to sterically prevent binding. However, electron microscopic observations indicated that MAPs were not localized but, instead, were dispersed around the outer fibers. In addition, MAP2 present at saturating amounts on in vitro assembled brain microtubules had no significant effect on dynein binding. Dynein-decorated microtubules contained clusters of arms suggesting that there may be cooperative interaction between the arms during dynein binding. Because the A subfiber of axonemes contains sites to which dynein preferentially attaches, MAPs may prevent recombination by interfering with cooperative binding to these specific sites. Dynein presumably binds with equal affinity to any protofilament on in vitro assembled microtubules, and, therefore, the MAPs may not be capable of effectively interfering with cooperative binding of dynein to these microtubules.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 25 (1993), S. 158-170 
    ISSN: 0886-1544
    Keywords: acetylation ; epitope-tagging ; flagella ; tubulin isoforms ; microtubules ; rubisco ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Following the discovery of acetylated α-tubulin in the flagella of Chlamydomonas, many studies have documented the presence of acetylated α-tubulin in a variety of evolutionarily divergent organisms. While this posttranslational modification may define an isoform with a unique function, the primary effect of α-tubulin acetylation remains unknown. To study the function of α-tubulin acetylation, we have transformed Chlamydomoas, a organism in which almost all of the flagellar tubulin ad a subset of the cytoplasmic microtubules are acetylated, with a α1-tubulin gene whose product cannot be acetylated. Specifically, the codon for lysine 40, the lysine that is acetylated, has been replaced with the codons of nonacetylatable amino acids. To distinguish mutagenized α-tubulin from that produced by the two endogenous α-tubulin genes, mutant α-tubulin was tagged with an epitope from influenza virus hemagglutinin. Utilizing the constitutive Chlamydomonas rubisco small subunit S2 promoter, we have obtained in selected clones high levels of nonacetylatable α-tubulin expression approximating 50-70% of the total flagellar α-tubulin. Immunofluorescence and immunoblot analysis of transformed cells indicated that nonacetylatable α-tubulin could assemble, along with endogenous α-tubulin, into both cytoplasmic and flagellar microtubules. However, no gross phenotypic effects were observed, suggesting that the effect of α-tubulin acetylation is subtle. © 1993 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 4 (1984), S. 431-441 
    ISSN: 0886-1544
    Keywords: dynein ; chromatophores ; permeabilization ; melanosomes ; motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Teleost chromatophores are filled with individual pigment granules that rapidly aggregate to the cell center or become dispersed throughout the cytoplasm in response to environmental stimuli. Microtubules appear to be required for pigment aggregation (movement toward the cell center), and recent findings have suggested that a dynein-like ATPase may participate in force production. Based on previous studies, however, it has been argued that pigment aggregation does not require energy directly, a view that supports the involvement of an elastic component in granule movement. To examine this point further, we have reinvestigated the energy requirements for pigment aggregation using both intact cells and detergent-permeabilized cell models of Fundulus melanophores. Poisons of oxidative phosphorylation, namely, 2,4 dinitrophenol and NaCN, reversibly inhibit melanosome aggregation in response to adrenaline. Inhibition of movement results directly from depletion of intracellular ATP, since pigment translocation can be reactivated in permeabilized cells by the addition of exogenous ATP to the lysis buffer. Non-hydrolyzable analogues, including β,γ-imidoadenosine-5′-triphosphate (AMPPNP), β,γ-methylene adenosine-5′-triphosphate (AMPPCP), and ATPγS, will not substitute for ATP in reactivation of movement. Similarly, other nucleotides such as ADP, AMP, GTP, CTP, and ITP, have limited ability to support melanosome aggregation in metabolically poisoned cells subjected to detergent lysis. ATP itself has no effect on intact cells. These results indicate that melanosome aggregation is ATP-dependent and energy-driven, and are consistent with a role for a force-transducing ATPase in particle movement.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
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