摘要:

AbstractFluorescently labeled desmin was incorporated into intermediate filaments when microinjected into living tissue culture cells. The desmin, purified from chicken gizzard smooth muscle and labeled with the fluorescent dye iodoacetamido rhodamine, was capable of forming a network of 10‐nm filaments in solution. The labeled protein associated specifically with the native vimentin filaments in permeabilized, unfixed interphase and mitotic PtK2cells. The labeled desmin was microinjected into living, cultured embryonic skeletal myotubes, where it became incorporated in straight fibers aligned along the long axis of the myotubes. Upon exposure to nocodazole, microinjected myotubes exhibited wavy, fluorescent filament bundles around the muscle nuclei. In PtK2 cells, an epithelial cell line, injected desmin formed a filamentous network, which colocalized with the native vimentin intermediate filaments but not with the cytokeratin networks and microtubular arrays. Exposure of the injected cells to nocadazole or acrylamide caused the desmin network to collapse and form a perinuclear cap that was indistinguishable from vimentin caps in the same cells. During mitosis, labeled desmin filaments were excluded from the spindle area, forming a cage around it. The filaments were partitioned into two groups either during anaphase or at the completion of cytokinesis. In the former case, the perispindle desmin filaments appeared to be stretched into two parts by the elongating spindle. In the latter case, a continuous bundle of filaments extended along the length of the spindle and appeared to be pinched in two by the contracting cleavage furrow. In these cells, desmin filaments were present in the midbody where they gradually were removed as the desmin filament network became redistributed throughout the cytoplasm of the spreading daughter cell

ISSN:0886-1544    

DOI:10.1002/cm.970120302

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractThe spectrins are a family of cytoskeletal‐membrane proteins that have a wide tissue distribution. In the present study, we employed polyclonal antibodies made against mammalian and avian erythroid spectrins as well as mammalian brain spectrin to assess their presence and distributions in the mammalian heart. Western blot analyses revealed that all three antibodies were specific for a 240,000 molecular weight α‐spectrin subunit found in hamster erythrocyte ghost homogenates, whole hamster heart, and isolated hamster cardiac myofibril homogenates. Spectrin staining was absent from the Triton X‐100‐extracted supernatant fraction of myofibril preparations, suggesting that the protein is linked to the myofibril precipitate after exposure to the detergent. Frozen, unfixed, 2‐μm‐thick; sections of adult, Syrian golden hamster cardiac tissue exhibited strong immunofluorescent staining of intercalated discs and Z‐bands using all three antibodies. In addition, the mammalian erythroid spectrin antibodies showed staining of the sarcolemma, and in cross section, revealed a delicate internal network of staining that appears to surround individual myofibrils. This may be T‐tubule‐associated staining. Myofibrils isolated from cardiac myocytes using Triton X‐100 show positive Z‐band staining using all three antibodies. Double staining with Texas Red‐labeled monoclonal desmin and FITC‐labeled polyclonal spectrin antibodies revealed that both stained the myofibrillar Z‐line regions. These results demonstrate that spectrin is closely associated with the membranes, myofibrils, and intermediate fil

ISSN:0886-1544    

DOI:10.1002/cm.970120303

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractThe authors examined the molecular organization of myosin in stress fibers (microfilament bundles) of cultured mouse embryo fibroblasts. To visualize the organization of myosin filaments in these cells, fibroblast cytoskeletons were treated with gelsolin‐like protein from bovine brain (hereafter called brain gelsolin), which selectively disrupts actin filaments. As shown earlier [Verkhovsky et al., 1987], this treatment did not remove myosin from the stress fibers. The actin‐free cytoskeletons then were lightly sonicated to loosen the packing of the remaining stress fiber components and fixed with glutaraldehyde.Electron microscopy of platinum replicas of these preparations revealed dumbbell‐shaped structures of approximately 0.28 μm in length, which were identified as bipolar myosin filaments by using antibodies to fragments of myosin molecule (subfragment I and light meromyosin) and colloidal gold label. Bipolar filaments of myosin in actin‐free cytoskeletons were often organized in chains and lattices formed by end‐to‐end contacts of individual filaments at their head‐containing regions. Therefore, after extraction of actin, it was possible for the first time to display bipolar myosin filaments in the stress fibers of

ISSN:0886-1544    

DOI:10.1002/cm.970120304

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractThe spleen ofAmbystoma mexicanum(axolotl) larvae develops as a closed sac containing differentiating nucleated erythrocytes, and is typically isolated from the general circulation for about 10 days post‐hatching. Beginning 3–4 days posthatching, it can be removed intact for examination of the morphology and cytoskeletal structure of the erythropoietic cells. In the smallest (earliest) spleens, spheroidal cells predominate, while older ones contain a preponderance of cells exhibiting the flattened elliptical morphology typical of all non‐mammalian vertebrate erythrocytes. Most striking in the splenic erythroid population are cells with singly or doubly pointed morphology. Though common in the developing spleen and circulation of young larvae, pointed cells are less frequently encountered in the circulation of older larvae, indicating that they are intermediate stages in the differentiation of spheroids to flattened ellipsoids. This is supported by structural observations on cytoskeletons prepared from the splenic cells. Incomplete singly and doubly pointed marginal bands of microtubules are observed, many of which contain a pair of centrioles within or close to a pointed end, suggestive of organizing center function. The observations are consistent with a sequence of changes in cell morphology from spherical to doubly pointed to singly pointed to flattened ellipse, causally linked to stages of marginal band bioge

ISSN:0886-1544    

DOI:10.1002/cm.970120305

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractTwo posttranslational modifications of alpha‐tubulin, acetylation and detyrosination, are associated with stable microtubule (MT) populations, including those of neuronal processes. We have used a pluripotent embryonal carcinoma cell line, P19, to investigate changes in MT isotype and stability found in MT arrays during neurogenesis. This cell line has an advantage in that both commitment‐ and differentiation‐related events can be observed. Uncommitted P19 cells have minimal arrays of acetylated and detyrosinated MTs. Following neuronal induction with retinoic acid (RA), indirect immunofluorescence microscopy shows that the first MT modifications occur during commitment and before any morphological change is observed. RA‐induced cells initially polymerize a temporarily enlarged population of MTs. Included in this population is a new array of acetylated MTs arranged in a bundle of parallel MTs. This bundle is colchicine‐stable, although no MT‐associated proteins (MAPs) are detectable using a battery of anti‐MAP antibodies. Observation of MT arrays with patterns that are intermediate between the early bundles and short neurites suggests that the acetylated MT bundle subsequently extends to form a neurite. MAP 2 is first detected at about the time of neurite extension. However, at this early stage of differentiation, MAP 2 is not yet limited to dendritic processes. This report provides the first evidence that the stable MTs of mature neurons may be initiated during neuron

ISSN:0886-1544    

DOI:10.1002/cm.970120306

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970120307

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970120301

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY