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  • 1
    Electronic Resource
    Electronic Resource
    Calyculin-A increases the level of protein phosphorylation and changes the shape of 3T3 fibroblasts (1991)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 18 (1991), S. 26-40 
    Details
    ISSN: 0886-1544
    Keywords: vimentin ; phosphatase inhibitors ; intermediate filaments ; stress fibers ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Calyculin-A, an inhibitor of type 1 and 2A phosphatases, was applied extracel-lularly to 3T3 fibroblasts. At 0.1 μM, calyculin-A caused a marked increase in protein phosphorylation in both the cytosolic and insoluble cellular fractions. This effect was independent of external Ca2+. An immunoprecipitate, formed with an antibody to myosin, contained several cytoskeletal components. Increased phos-phorylation following treatment with calyculin-A was observed in vimentin, the 20-kD myosin light chain, and an unidentified 440-kD component. An enhanced level of vimentin phosphorylation was found in intermediate filament preparations from treated cells.Calyculin-A also caused marked shape changes of 3T3 cells. Within minutes after addition of calyculin-A (0.1 μM) cells became rounded and lost attachment to the substratum. Stress fibers, intermediate filaments, and microtubules, prominent in the attached control cells, were not evident in the rounded cells. Shape changes were reversible and after removal of calyculin-A the rounded cells attached to the substratum, resumed a flattened shape, and were active mitotically. In the cells treated with calyculin-A an unusual “ball-like” structure was observed with transmission electron microscopy. This unique structure was 2-3 μM in diameter and was located close to the nucleus.The use of calyculin-A adds further support to the idea that cell shape is controlled, at least in part, by concerted actions of a kinase-phosphatase couple.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970180104
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  • 2
    Electronic Resource
    Electronic Resource
    Desmin is present in proliferating rat muscle satellite cells but not in bovine muscle satellite cells (1991)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 149 (1991), S. 525-535 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The presence of desmin was characterized in cultured rat and bovine satellite cells and its potential usefulness as a marker for identifying satellite cells in vitro was evaluated. In primary cultures, positive immunohistochemical staining for desmin and skeletal muscle myosin was observed in rat and bovine myotubes. A small number of mononucleated cells (20% of rat satellite cells and 5% of bovine satellite cells) were myosin-positive, indicative of post-mitotic differentiated myocytes. In bovine satellite cell cultures 13% of the mononucleated cells were desmin-positive, while 84% of the mononucleated cells in rat satellite cell cultures were desmin-positive. Rat satellite cell mass cultures and bovine satellite cell clonal density cultures were pulsed with 3H-thymidine, and autoradiographic data revealed that 〉94% of dividing rat cells were desmin-positive, suggesting that desmin is synthesized in proliferating rat satellite cells. However, no desmin was seen in cells that incorporated labeled thymidine in bovine satellite cell clones. Analysis of clonal density cultures revealed that only 14% of the mononucleated cells in bovine satellite cell colonies were desmin-positive, whereas 98% of the cells in rat satellite cell colonies were desmin-positive. Fibroblast colonies from both species were desmin-negative. In order to further examine the relationship between satellite cell differentiation and desmin expression, 5-bromo-2′-deoxyuridine (BrdU) was added to culture medium at the time of plating to inhibit differentiation. Fusion was inhibited in rat and bovine cultures, and cells continued to divide. Very few desmin-positive cells were found in bovine cultures, but greater than 90% of the cells in rat cultures stained positive for desmin. The presence of desmin and sarcomeric myosin was also evaluated in regenerating rat tibialis anterior five days after bupivicaine injection. In regenerating areas of the muscle many desmin-positive cells were present, and only a few cells stained positive for skeletal muscle myosin. Application of desmin staining to rat satellite cell growth assays indicated that rat satellite cells cultured in serum-containing medium were contaminated with fibroblasts at levels that ranged from approximately 5% in 24 hr cultures to 15% in mature cultures. In defined medium 4 day cultures contain approximately 95% to 98% desmin-positive satellite cells. The effects of combinations of insulin-like growth factor I (IGF-I), basic fibroblast growth factor (bFGF), and transforming growth factor beta (TGF-β) on rat satellite cell proliferation and differentiation were assessed by desmin staining, and results were found to be consistent with results obtained previously using conventional cell staining and counting techniques (Allen and Boxhorn, 1989). Our experiments indicate that the pattern of desmin expression in satellite cells differs between rat and bovine and that desmin can be a useful marker for cultured rat satellite cells.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041490323
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    Paper (German National Licenses)
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