Acta histochemica. Supplementband最新文献

The microtubule system of normal and microtubule-poisoned amoebae of Dictyostelium discoideum has been investigated both by indirect immunofluorescence with antibodies to microtubule proteins and electron microscopy. Nocodazole, like some other microtubule poisons, destroys most of the microtubules in both interphase and dividing cells resulting in an inhibition of nuclear and cell division. The microtubule organizing centres, however, continue to duplicate once or twice. The daughter organizing centres segregate, they seem to be connected with nuclear material, that splits partly, too, forming more or less extended nuclear clefts. This segregation, at least over short distances, takes place without intranuclear microtubules. Duplication of microtubule organizing centres is not strictly correlated with nuclear division and cytokinesis. Microtubule poisons are able to uncouple these events. Different levels of regulation should be responsible for microtubule organizing centre, nuclear, and cell division.

The microfilament system is thought to provide motor elements needed for plasma membrane dynamics. This article focuses on two protein components that may play key roles in this process: (1) Profilin, a G-actin binding protein which is considered as the source of actin subunits necessary for rapid changes in the amount of actin filaments. Our data demonstrate that profilin is synthesized even in terminally differentiated blood cells of a high dynamic potential. In addition, we show that plasma membrane-associated profilin in fibroblasts is unevenly distributed and is concentrated in areas that are highly motile. (2) The filament-forming myosin which is the classical motor protein in the microfilament system. We show that interfering with myosin filaments by microinjecting antibodies causes brush border-type microvilli on epithelial cells to loose their upright position. This result, together with our previous observations on the effects of anti-myosin injection into fibroblastic and epithelial cells (loss of stress fibers and cellular contact sites, increase in locomotory activity, delay of cytokinesis), suggests that bipolar myosin filaments are needed to maintain a specific cortical tension which is lost upon antibody binding.

The microfilament pattern in the somatic follicle cells of Drosophila ovarian follicles has been studied by staining F-actin with rhodaminyl-phalloidin. Parallel microfilament bundles were observed at the basal side of the follicle cells at all analyzed stages, but the organization of the microfilaments was found to undergo characteristic changes during development. At previtellogenic and early vitellogenic stages the microfilaments formed very long and thin bundles which were oriented perpendicular to the long axis of the follicle. Actin-containing cell protrusions formed only at one side of each cell indicating a planar circular cell polarity (best seen at stages 7 and 8). At later stages densely packed parallel microfilaments were observed at the basal end of the follicle cells. This pattern was maintained until stage 14 when the microfilament bundles became thinner and more widely spaced and finally disintegrated. During late vitellogenic stages the cell shape differed basally and apically: while apically the cells formed regular and very precise patterns the basal cell borders were convoluted. When stage 10 follicles were isolated in simple saline solutions the diameter of the oocyte decreased during 30 min culture. This contraction, which was presumably due to the activities of the basal microfilament bundles, could be inhibited by cytochalasins as well as azide or dinitrophenol. The reaction was most likely induced by the in vitro culture conditions, since there is no evidence that the contraction also takes place in loco.

1. The Triton-extraction procedure is well suited for the demonstration of the cytoskeleton by TEM. A good preservation of the fine cytoskeletal network depends on optimal drying. 2. Immunogold-labeling is a useful method for visualization of intermediate filaments (IF) and microfilaments (MF). Labelled and unlabelled filaments are discernible. 3. Various methods were tested for a direct gold-labeling of MF with phallotoxin-gold preparations: The application of phalloidin-gold complexes results in an intensive unspecific staining of cytoskeletal filaments. A modified coupling procedure for the binding of phallacidin to transferrin was necessary to avoid the agglomeration of gold particles. The direct labeling of the cytoskeleton by the application of phallacidin-transferrin-gold complexes to Triton-extracted cells demonstrated an enrichment of gold particles on the both types of filaments, but MF and IF were not discernible. The treatment with phallacidin-polylysin-gold complexes demonstrated labelled filaments, but without sufficient intensity.

The intermediate filament (IF) subunit protein vimentin is efficiently cleaved in vitro by purified human immunodeficiency virus type 1 (HIV-1) protease. Immunological data confirm that identical sites are cleaved when vimentin is polymerized into filaments or occurs as protofilaments. Preformed filaments require 10 times more protease to achieve the same extent of cleavage seen with protofilaments, suggesting that the cleavage sites are partially masked in IFs. The primary cleavage gives rise to molecule lacking most of the tail domain and which not only remains in preformed filaments, but also is capable of polymerizing into essentially normal 10 nm filaments. However, these filaments of the vimentin primary cleavage product show a propensity to form large lateral aggregates. The three secondary cleavage products of vimentin additionally lack portions of the head domain, are almost quantitatively released from preformed filaments and are not capable of forming filaments de novo. These results confirm and extend previous data obtained with desmin and provide a limit for that portion of the tail domain of type III IF subunit proteins that play a role in IF formation and stability. Microinjection of HIV-1 protease into cultured human skin fibroblasts resulted in a large increase in the percentage of cells with an altered and abnormal distribution of vimentin IFs. Most commonly, the IFs were observed to have collapsed into a clump with a juxtanuclear localization. The efficient cleavage of vimentin observed in vitro and the ability of microinjected HIV-1 protease to alter IF distribution in vivo suggest that IF proteins may serve as substrates within HIV-1 infected cells and may play a role in viral infection.

The amounts of the different forms of actin (G-actin, F-actin) can be measured biochemically following lysis of the cells by the DNase I inhibition assay. The existing methodology for studying G-actin in unstimulated platelets was found to be inappropriate for studies during ADP-induced platelet activation. However, this problem was overcome by a simple modification of the procedure in which formaldehyde was added to the buffer used to lyse the activated platelets. Using this modification the G-actin values obtained immediately after lysis did not change during storage of the lysates on ice for more than 30 minutes. The results show rapid conversion of G-actin to F-actin in association with the shape change during ADP-stimulated activation. Examination of unstimulated platelets using the modified procedure enabled identification of a pool of actin that is rapidly dissociated to G-actin in the absence of formaldehyde, the existence of which had not previously been recognised.

A panel of monoclonal antibodies specific to alpha- or beta-tubulin subunits was used to study the location of tubulin molecules in microtubules. Limited proteolysis of tubulin with trypsin and chymotrypsin followed by immunoblotting demonstrated that the antibodies discriminated between structural domains of tubulin subunits. Antibodies against N-terminal domains were tested for their ability to interfere with the formation of microtubules in vitro. Although the antibodies exhibited similar association constants when tested on immobilized tubulin, they differed in their inhibitory effect on microtubule assembly. The sedimentation assay using microtubules prepared from purified tubulin showed an almost undetectable binding of the antibodies with the strongest inhibitory power to the microtubules. Immunofluorescence staining of unfixed detergent-extracted cells revealed that antibodies to determinants on C-terminal domains labelled microtubules, but these were not decorated with antibodies against N-terminal domains. The same results were obtained after a microinjection of antibodies into living cells. The data indicate that while parts of C-terminal domains of both subunits are exposed on the exterior of microtubules, considerable regions of the N-terminal domains are not. The surface regions of N-terminal domains appear to be involved in the formation of microtubules.

The ultrastructure and protein content of goldfish Mauthner cells (M-cells) at different functional states induced by natural vestibular stimulations were studied. 2-h stimulation, usually causing a fatiguing of the fishes, was found to be accompanied by ultrastructural changes within M-cells and a decreased content of cytoskeletal proteins. After training by short stimulations resulting in a long-term adaptation of the fishes, the ultrastructure and protein content of M-cells could not be distinguished qualitatively and quantitatively from those of non-adapted fishes. When the adapted fishes were stimulated for 2 h the content of a 70-kDa protein was found to be increased. In addition, the content of a 42-kDa protein, obviously actin, was elevated in this case. Correspondingly, electron microscopic analysis demonstrated a significantly increased resistance of the cytoskeleton to fatiguing stimulation. The data obtained indicate that the neural cytoskeleton is a central target of fatiguing stimulation. We suppose that the 70-kDa protein is responsible for the adaptive properties of the cytoskeleton. This protein is assumed to be identical with one of the so-called heat-shock proteins of non-neural cells which have the same electrophoretic mobility and are also able to protect the cytoskeleton under stress conditions.

Paired helical filaments (PHF) were isolated from the cerebral cortex of patients with Alzheimer's disease (AD) by a combination of SDS treatment and density gradient centrifugation according to the method of Ihara et al. (1983). The protein component of the preparation was extracted with formic acid and Balb/c mice were used for immunization. Hybridoma supernatants were screened by immunocytochemical staining, by an ELISA assay, and by immunoblotting of SDS-PAGE, the latter both using the PHF preparation as antigen. One hybridoma which showed a strong reactivity with PHF in both the ELISA assay and immunocytochemistry was then used to produce ascites fluid in Balb/c mice. Antibodies reacted immunocytochemically with neurofibrillary tangles and neurites involved in plaque formation in AD but did not show a cross-reaction to human control brain and rat brain. The results indicate that the antibody which has been raised reacts with an antigen component of PHF.