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Biomedical subjects

M Osborn

Publications and source records attributed to M Osborn.

At least 271 records · Page 15Linked to original sources
PubMed1978

Microtubule system of isolated fish melanophores as revealed by immunofluorescence microscopy.

The microtubule system of melanophores of the angelfish, Pterophyllum scalare, has been studied using antibodies prepared against purified porcine brain tubulin in indirect immunofluorescence microscopy. Melanophores were freed from the surrounding tissue components of isolated scales by mild enzymatic digestion and then allowed to settle on a glass cover slip. In both the dispersed and the aggregated states large numbers of fluorescent fibers are seen. The number and the astral arrangement of these fibers, which run from the central region to the periphery of the cell, are striking. The system of fluorescent fibers is replaced by diffuse fluorescence of moderate intensity after cold treatment, but is restored after rewarming the cells. Differences in the immunofluorescence profiles between cells with dispersed and aggregated pigment are discussed in relation to electron microscopic data available for this system.

Animals↗
PubMed1978

Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK2 cell.

PtK2 cells were grown on gold grids and treated with Triton X-100 in a microtubule stabilizing buffer. The resulting cytoskeletons were fixed with glutaraldehyde and subjected to the indirect immunofluorescence procedure using monospecific tubulin antibodies. Grids were examined first by fluorescence microscopy, and the display of fluorescent cytoplasmic microtubules was recorded. The grids were then stained with uranyl acetate and the display of fibrous structures recorded by electron microscopy. Thus the display of cytoplasmic microtubular structures in the light microscope and the electron microscope can be compared within the same cytoskeleton. The results show a direct correspondence of the fluorescent fibers in the light microscope with uninterrupted fibers of diameter approximately 550 A in the electron microscope. This is the diameter reported for a single microtubule decorated around its circumference by two layers of antibody molecules. Thus under optimal conditions immunofluorescence microscopy can visualize individual microtubules.

Animals↗
PubMed1978

The distribution of actin cables and microtubules in hybrids between malignant and non-malignant cells, and in tumours derived from them.

Cell fusion provides a genetic test for linkage between cellular phenotypic markers and malignancy. We have examined hybrid cells in which malignancy is suppressed and malignant segregants derived from them, to test whether the reappearance of malignancy is regularly associated with loss or abnormality of actin cables or microtubules. Immunofluorescence studies did not reveal any difference between the malignant and non-malignant hybrids in the number and distribution of microtubules that they contained. All the hybrids, whether tumorigenic or not, contained elaborate patterns of cytoplasmic microtubules. Furthermore, no consistent relationship was observed between the absence of actin cables and malignancy.

Actins↗
PubMed1978

Specific visualization of the distribution of the calcium dependent regulatory protein of cyclic nucleotide phosphodiesterase (modulator protein) in tissue culture cells by immunofluorescence microscopy: mitosis and intercellular bridge.

Monospecific antibodies against the homogeneous Ca++ dependent regulatory protein of cyclic nucleotide phosphodiesterase (CDR protein) from bovine brain were used in indirect immunofluorescence microscopy to visualize the cytoplasmic organization of this key regulatory protein in growing tissue culture cells. Although cells during interphase reveal only a weak general cytoplasmic fluorescence, a dramatic reorganization of CDR protein occurs with the onset of mitosis. Throughout the different mitotic stages CDR protein is strongly concentrated in the two polar parts of each half spindle. After completion of telophase (CDR protein appears at both cytoplasmic ends of the intercellular bridge which still connects the two daughter cells. Parallel use of monospecific antibodies against CDR protein and tubulin emphasizes the spatial restriction in the localization of CDR protein during mitosis and early G1 phase of the cell cycle.

Calmodulin↗
PubMed1978

The intermediate-sized filaments in rat kangaroo PtK2 cells. I. Morphology in situ.

The system of the intermediate-sized filaments (IF) of rat kangaroo PtK2 cells which can be specifically demonstrated by immunofluorescence microscopy using certain rabbit autoantibodies and guinea pig antibodies against bovine hoof prekeratin has been studied by electron microscopy. The characteristic ornamental, curved arrays of this system are shown after fixation in situ in both thin sections and whole-cell-preparations to represent bundles of 6 to 11 nm thick filaments extending through the whole cytoplasm, although in some cells they appear to be enriched in the perinuclear region. While many individual IF are recognized in the cytoplasm the tendency of such filaments to aggregate laterally into bundles is one of their prominent features. Among such bundle formations one form that consists of tightly packed IF cemented together in a dense osmiophilic matrix is especially conspicious. The appearance and mode of arrangement of the IF is not significantly altered in cells treated with colcemid and/or cytochalasin B. Spatial relationships of IF with microfilament-containing cables and microtubules as well as with membranous structures are also described. IF are heterogeneous in width and reveal an unstained, apparently hollow core, indicative of a tubular organization. Many IF show small, sometimes periodically arranged lateral projections which seem to be involved in IF cross-linking. Associations with polyribosomes are common. The changes in the IF system during mitosis have also been examined. The structural details of the IF as well as their possible role as cytoskeletal elements involved in the control of cell shape and cytoplasmic architecture are discussed in relation to data on various intermediate-sized filaments from other cell types. The close similarity of the IF of PtK2 cells to aggregates of prekeratin filaments is emphasized. It is suggested that PtK2 cells represent an epithelial cell line growing in a state of balanced semi-keratinization.

Animals↗
PubMed1978

The intermediate-sized filaments in rat kangaroo PtK2 cells. II. Structure and composition of isolated filaments.

When cultured cells of the rat kangaroo cell line PtK2 grown on plastic or glass surfaces are lysed and extracted with combinations of low and high salt buffers and the non-ionic detergent Triton X-100 cytoskeletal preparations are obtained that show an enrichment of 6 to 11 nm thick filaments. The arrays of these filaments have been examined by various light and electron microscopic techniques, including ultrathin sectioning, whole mount transmission electron microscopy, negative staining, and indirect immunofluorescence microscopy. In addition, 6 to 11 nm filaments isolated from these cells with similar extraction procedures and with centrifugation techniques have been examined by electron microscopy. The arrays of these isolated intermediate-sized filaments, their ultrastructure and their specific decoration by certain antibodies present in normal rabbit sera as well as by guinea pig antibodies against purified bovine prekeratin is demonstrated. When preparations enriched in these intermediate-sized filaments are examined by SDS-polyacrylamide gel electrophoresis a corresponding enrichment of three polypeptide bands with apparent molecular weights of about 45 000, 52 000 and 58 000 (the latter component sometimes appears split into two bands) is observed, besides some residual actin and a few high molecular weight bands. The morphology of the isolated filaments, their immunological reaction with antibodies decorating prekeratin-containing structures, and the sizes of their constitutive polypeptides suggest that these filaments are closely related to prekeratin-containing filaments observed in a variety of epithelial cells.

Animals↗
PubMed1977

The display of microtubules in transformed cells.

Monospecific tubulin antibodies have been used in indirect immunofluorescence microscopy on a variety of well characterized, transformed cell lines grown in tissue culture. Networks of colcemid-sensitive fibers are seen in SV40-transformed 3T3 cells, SV40-transformed rat embryo cells, HeLa cells and other transformed cell lines. In each case, greater than 90% of the cells contain visible microtubular networks, and where individual microtubules can be distinguished, they run for long distances. Documentation of these metworks is more difficult in transformed than in normal cells, because transformed cells are in general more rounded and have less well spread cytoplasm. In addition, the microtubular networks can be readily visualized in "cytoskeletons" of both normal and transformed cells, obtained by treatment of cells with nonionic detergents in a buffer which stabilizes microtubules in vitro. Addition of calcium to this buffer results in in situ fragmentation and destruction of the microtubular network. In view of these results, we conclude that transformed cells contain significant numbers of microtubules, and that in transformed cells, as in normal cells, microtubules are arranged in networks.

Animals↗
PubMed1977

Reaction of the anastral mitotic apparatus of endosperm cells of the plant Leucojum aestivum with antibodies to tubulin from porcine brain as revealed by immunofluorescence microscopy.

Structures binding an antibody against tubulin from porcine brain were localized in the giant anastral mitotic apparatus of endosperm cells of the monocotyledonous plant, Leucojum aestivum, by indirect immunofluorescence microscopy. Both continuous and chromosomal spindle fibers were strongly stained. Postive fluorescence was also noted in polar cap regions and, in prometaphase stages, to some extent at the fragmented nuclear envelope. Intermingling and branching of subfiber elements was frequently noted.

Animals↗
PubMed1977

Phalloidin-induced actin polymerization in the cytoplasm of cultured cells interferes with cell locomotion and growth.

Phalloidin, the toxic drug from the mushroom Amanita phalloides, was injected into the cytoplasm of tissue culture cells and the changes in intracellular actin distribution were followed by immunofluorescence microscopy with actin antibody. At low concentrations, phalloidin recruits the non- or less highly polymerized forms of cytoplasmic actin into stable "islands" of aggregated actin polymers and does not interfere with the preexisting thick bundles of microfilaments (stress fibers). Differential focusing shows that these islands of phalloidin-induced actin polymers occur at a level in the cytoplasm that is above the submembranous bundles of microfilaments present on the adhesive side of the cells. The pattern of cytoplasmic microtubules remains unaffected by the injection of phalloidin; however, filamin, a protein usually associated with actin in the cytoplasm, is also recruited into the islands. At higher phalloidin concentrations, contraction of the cell is observed. These results are discussed in the light of previous biochemical studies by Wieland and Faulstich and their coworkers [for a review see Wieland, T. (1977) Naturwissenschaften 64, 303-309] on the in vitro interaction of phalloidin with muscle actin, which have documented that phalloidin reacts stoichiometrically with actin, promotes actin polymerization, and stabilizes actin polymers. In addition, we show that microinjection of phalloidin interferes in a concentration-dependent manner with cell locomotion and cell growth. These results indicate that a well-balanced controlled reversible equilibrium between different polymerization states of actin may be a necessary requirement for cell locomotion and may also influence other cellular functions such as growth.

Actins↗
PubMed1977

Visualization of a system of filaments 7-10 nm thick in cultured cells of an epithelioid line (Pt K2) by immunofluorescence microscopy.

During our studies with antibodies against structural proteins of the cytoskeleton of eukaryotic cells we have observed that sera from many normal rabbits decorate a fiber system in cells of the established rat kangaroo cell line Pt K2. The display and organization of these fibers are different from those of microfilament bundles (decorated by antibody to actin) and microtubules (decorated by antibody to tubulin). This new fiber system can be further distinguished by its resistance to reorganization when cells are treated with Colcemid or cytochalasin B. The decoration of this fiber system is not detected if Pt K2 cells are fixed with formaldehyde. Such sera also appear to decorate swirls of perinuclear fibers in mouse Neuro 2a cells, and in mouse 3T3 cells treated with mitotic drugs. Comparison of the immunofluorescence pictures with electron microscopic data suggests that the sera are visualizing bundles of intermediate 7- to 10-nm filaments.

Cell Line↗
PubMed1976

The relationship between group C adenovirus tumor antigen and the adenovirus single-strand DNA-binding protein.

The group C adenoviruses code for a single-strand specific DNA-binding protein of molecular weight 72,000 daltons which is synthesized at early times after productive viral infection. Experiments were designed to determine whether this single-strand specific DNA-binding protein was expressed in adenovirus tumors and transformed cells. Two independently derived preparations of anti-sera from hamsters bearing group C adenovirus tumors were tested for antibody against the single-stranded DNA-binding proteins. One antiserum contained antibodies that reacted with these DNA-binding proteins, while the second antiserum did not contain detectable levels of antibody. Five adenovirus type 2 transformed rat cell lines were tested for the presence of the single-stranded specific DNA-binding proteins. Two of the five transformed cells expressed detectable levels of this protein. These results indicate that the group C adenovirus single-strand specific DNA-binding proteins are expressed in some, but not all, adenovirus tumors and transformed cell lines. Those transformed cell lines (type 2) containing a portion of the adenovirus genome designated by the Eco R-I-B restriction enzyme fragment express the single-strand specific DNA-binding proteins. Those cell lines missing this Eco R-I-B fragment do not contain this viral protein. Other experiments have located the structural gene of the signle-strand specific DNA-binding protein in the Eco-R-I-B DNA fragment, indicating that when this gene is present in a transformed cell, it is expressed.

Adenoviridae↗
PubMed1976

Cytoplasmic microtubules in tissue culture cells appear to grow from an organizing structure towards the plasma membrane.

A structure which appears to organize cytoplasmic microtubules in interphase mouse 3T3 cells can be visualized by immunofluorescence microscopy. Purified monospecific antibody against homogeneous tubulin from brain visualizes, in addition to cytoplasmic microtubules, a cytoplasmic polar structure as the focal point from which the microtubules seem to radiate. The structure is preserved after treatments that depolymerize cytoplasmic microtubules, i.e., exposure of cells to mitotic drugs or to low temperature. When cells recover from these treatments one end of each microtubule organizing structure acts as a nucleating center from which cytoplasmic microtubules grow toward the plasma membrane. Thus cytoplasmic microtubules assemble in vivo in an ordered unidirectional manner, and therefore the cell must be able to avoid the assembly of unwanted, unoriented, and disconnected microtubules. These results suggest that the assembly of tubulin into microtubules is regulated in vivo.

Cell Line↗