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J M Mullins

Publications and source records attributed to J M Mullins.

36 records · Page 2Linked to original sources
PubMed1990

Nuclear and mitotically enhanced epitope.

Salt-extracted proteins of taxol-stabilized microtubules from Chinese hamster ovary cells arrested at mitosis were used to immunize mice for hybridoma production. From a group of related monoclonal antibodies (MAbs), one, C9, recognized an epitope on antigens localized by immunofluorescence microscopy to interphase centrosomes and nuclei. The availability of the nuclear antigen was cell cycle-dependent; however, permeabilization of cells before fixation revealed that the antigen was present throughout the cell cycle. The nuclear antigen was exposed during prophase and was released from the nucleus upon nuclear envelope breakdown filling the cytoplasm of the mitotic cell. Antigenic material re-accumulated at daughter nuclei and was concealed during G1 phase. Detergent extraction of the cytoplasmic antigen from mitotic cells enabled localization of antigens to centrosomes, kinetochores, and the furrowing region/midbody. Immunoblot analysis of cells of a variety of species of origin identified an approximate 250 kD polypeptide as corresponding to the nuclear antigen, whereas polypeptides of 107/117 kD as well as approximately 250 kD accounted for the mitotic cytoplasmic antigens. No polypeptides could be associated with antigens at centrosomes, kinetochores, or midbodies. This MAb joins the antibody preparations previously reported that describe nuclear antigens, or epitopes on antigens, enhanced at mitosis.

Animals↗
PubMed1989

Internalization of IgE receptors on rat basophilic leukemic cells by phorbol ester. Comparison with endocytosis induced by receptor aggregation.

Phorbol myristate acetate (PMA) can induce a rapid and significant decrease in the expression of IgE receptors on RBL-2H3 cells. Fluorescence microscopy confirmed that the down-regulation is due to internalization of receptors. The endocytotic response to PMA shares several characteristics with endocytosis induced by immunochemical aggregation of surface-bound monomeric IgE: the rates of internalization both have a t1/2 of about 5 min, a maximum of 35% of the surface-bound IgE can be endocytosed by the action of PMA (50% by receptor aggregation), endocytosis is sustained for at least up to 60 min, neither stimulus requires extracellular Ca2+ and endocytosis induced by either stimulus is an active process, i.e., is dependent on temperature and cellular energy. Biochemical studies revealed some differences between the endocytotic responses to the two stimuli. After prolonged treatment of cells with dexamethasone, only endocytosis induced by PMA is inhibited. Cells depleted of protein kinase C by prolonged exposure to PMA can sustain a significant endocytotic response to aggregation of IgE receptors, but become completely desensitized to PMA. These data suggest that different biochemical pathways mediate the signals from the two stimuli and that protein kinase C is directly involved in endocytosis induced by PMA but does not have a major role in endocytosis induced by receptor aggregation.

Animals↗
PubMed1989

Induction of random microtubule polymerization in cold and drug-treated PtK1 cells following hyperosmotic shock treatment.

The effects of hypertonic sucrose on spindle and interphase microtubule (MT) arrays of PtK1 cells were investigated by incubating cells in complete culture medium at 4 degrees or 37 degrees C, with or without hypertonic sucrose, nocodazole or vinblastine (VLB). Results from anti-tubulin immunofluorescence showed that sucrose-induced alterations of spindle morphology seen at 37 degrees C did not occur at cold temperatures, but cold-induced MT loss was diminished. Application of warm hypertonic sucrose following depolymerization of MTs by nocodazole or cold resulted in the formation of a "feltwork" of randomly oriented, short MTs throughout the cytoplasm. These results, and those obtained substituting VLB for nocodazole, suggest that the effects of sucrose depend on the cytoplasmic concentration of soluble tubulin and support the hypothesis that osmotic factors are involved in effects of hypertonic sucrose on MT organization.

Animals↗
PubMed1987

Cytochalasin B-induced redistribution of cytokeratin filaments in PtK1 cells.

Indirect immunofluorescence demonstrated a dramatic reorganization of cytokeratin filaments produced by cytochalasin B (CB) treatment of PtK1 cells. Much of the normal cytokeratin network became arranged into a latticework consisting of bundles of cytokeratin filaments that radiated from, and interconnected, distinct foci. Electron microscopy showed foci to be dense granular regions through which bundles of cytokeratin filaments looped. Composition of the foci included actin, myosin, and alpha-actinin, as shown by labeling with rhodamine phalloidin or specific antisera. Simultaneous treatment with CB and colchicine was not required for lattice formation, but did produce more extensive development than did CB alone. In cells treated only with CB, the microtubule network remained intact, even in regions of extensive lattice formation. These results contrast sharply with those of Knapp et al (J. Cell Biol. 97:1788 [1983b]), who found lattice formation dependent upon simultaneous CB and colchicine treatment. Time-course and dose-response studies of CB treatment showed lattice formation to follow disruption of stress fibers and the concentration of actin into distinct patches that marked the location of lattice foci. Overall results suggest a structural association between microfilaments and cytokeratin filaments that produces the lattice pattern upon CB-induced disruption of stress fibers. Lattice formation was not limited to a specific cell-cycle stage, since G1, G2, and M cells displayed the lattice. Treatment of cells with dihydro-CB and experiments with enucleated cells showed that lattice formation was dependent upon neither the inhibition of sugar transport nor the nuclear extrusion effects of CB.

Actin Cytoskeleton↗
PubMed1987

D2O induced alterations of mitosis in PtK1 cells.

Deuterium oxide (D2O) was applied to PtK1 cells to assess its effect on mammalian mitosis. Cells exposed to culture medium containing up to 50% D2O were able to enter and complete mitosis, but the duration of mitosis was increased proportionally to the concentration of D2O applied. Cells exposed to 50% D2O showed increases of more than 300% for the interval between nuclear envelope breakdown and anaphase onset, and approximately 65% for the interval between anaphase onset and initial furrowing. At a concentration of 80%, D2O acted as an inhibitor of mitosis; after 8 h exposure to this concentration, cultures showed an increase in the proportion of mulinucleate cells and an absence of mitotic figures. When applied early in anaphase, 80% D2O effectively slowed chromosome separation, prolonging anaphase for more than 60 min. Normal chromosome motion was restored when medium containing D2O was replaced with control medium. Mitotic chromosomes remained condensed throughout prolonged anaphase intervals. Immunofluoresence examination of spindles stained using a monoclonal anti-tubulin revealed no pronounced increase in microtubule polymerization after exposure of cells to 20-80% D2O.

Anaphase↗
PubMed1986

Endocytosis of aggregated immunoglobulin G by rat basophilic leukemia cells; rate, extent, and effects on the endocytosis of immunoglobulin E.

Rat basophilic leukemia (RBL) cells have distinct receptors for IgE and IgG. We assessed the endocytosis of chemically and immunochemically cross-linked mouse-IgG and its influence on the simultaneous endocytosis of IgE. We found that at 37 degrees C, aggregates of IgG and IgE were endocytosed at about the same rate with one-half of the maximal endocytosis occurring in 5 to 13 min, and the efficiency of endocytosis for both ligands ranging from 40 to 70%. We also found that endocytosis of cross-linked IgE and IgG occurred simultaneously and neither ligand significantly affected the rate or extent of endocytosis of the other. The cells accumulated the cross-linked IgG, and then released it to the extracellular environment, at a rate (less than 3%/hr) slower than the released endocytosed IgE (greater than 10%/hr). Using an assay that discriminates between unbound and receptor-bound oligomeric IgG, we found that oligomeric IgG is endocytosed with its receptor, and that the bulk of the ligand remains bound to its receptor for greater than 120 min after endocytosis. The differences in the rate of release of endocytosed IgG vs IgE suggests that the intracellular fate or pathway of these two oligomeric ligands may differ.

Animals↗
PubMed1986

Immunofluorescence analysis of sucrose-induced changes in spindle morphology.

Metaphase and anaphase PtK1 cells show spindle elongation without concomitant chromosome motion when treated with culture medium containing 0.5 M sucrose. Electron microscopy has shown sucrose-induced changes in microtubule (MT) organization, changes in trilaminar kinetochore structure, and specific kinetochore-MT associations which may account for these results. In this paper we employ double-label immunofluorescence techniques using antibodies against tubulin and the kinetochore to analyze changes in spindle microtubule and kinetochore distribution produced by sucrose treatment. Cells treated from prometaphase through anaphase with 0.5 M sucrose from 10 min to 2 h showed spindle elongation and a distinct rearrangement of spindle microtubules into bundles, with a pronounced increase in length of interpolar microtubule bundles. In sucrose-treated mitotic cells kinetochores remained as antigenically distinct structures, similar to those found in untreated interphase cells. Kinetochore determinants remained positioned within a diffuse chromatin mass, but the orientation of sister kinetochores to opposite spindle poles was lost. Instead, kinetochore pairs were found in lateral association with microtubule bundles, with several pairs of determinants associated with a single bundle in many instances. Cells released from 0.5 M sucrose treatment showed a return of the spindle to a pretreatment arrangement for both the microtubules and kinetochore determinants.

Anaphase↗
PubMed1984

Spindle-membrane associations in freeze fractured HeLa cells.

Mitotic HeLa cells, obtained by means of thymidine and nocodazole synchronization, were freeze fractured, and spindle-membrane associations were studied. Except for small vesicles most membrane structures were excluded from the spindle region. A few tubular to lamellar membrane arrays were observed that projected into the spindle region from the vicinity of the spindle poles. Concentric layers of flattened, extensively fenestrated cisternae were found in the cytoplasm surrounding the spindle. The plasma membrane overlying the midbody in early G1 cells was examined.

Freeze Fracturing↗
PubMed1982

Isolation and initial characterization of the mammalian midbody.

Midbodies were isolated from synchronized cultures of Chinese hamster ovary (CHO) cells and their protein composition was studied by means of SDS PAGE. Gels of the midbodies included alpha and beta tubulins as major bands (approximately 30% of the total protein) and approximately 35 other bands, none of which constituted greater than 3.5% of the total protein. Extraction of the isolated midbodies with Sarkosyl NL-30- solubilized the midbody microtubules but left the central, dense matrix zone of the midbody intact. A protein doublet of approximately 115,000 mol wt was retained preferentially by the particulate fraction containing the matrix zones, indicating it to be a component of the matrix. The 115,000 mol wt doublet was also present in gels of isolated mitotic spindles from CHO cells. The overall protein composition of the isolated spindles was very similar to that of the isolated midbodies.

Animals↗
PubMed1982

Loss of mitotic centrosomal microtubule initiation capacity at the metaphase-anaphase transition.

The microtubule initiation capacity of the mitotic centrosome was studied in PtK1 cells by using the highly reversible microtubule inhibitor nocodazole. Cells blocked with nocodazole at any stage prior to onset of anaphase completed mitosis by reforming the spindle following release from the drug. Cells treated with nocodazole immediately upon onset of sister chromatid separation and then released did not complete mitosis, but instead progressed directly to an interphase state. This anaphase-linked transition in response to treatment was clearly evident as a change in centrosomal microtubule initiation capacity coincident with commencement of sister chromatid separation. Cells blocked in very late metaphase and then released were found to have retained the enhanced centrosomal microtubule initiation capacity characteristic of early mitosis. Cells blocked after the beginning of anaphase and then released, however, displayed dramatically reduced centrosomal microtubule initiation capacity. Mitotic cells blocked with colcemid or nocodazole and lysed into microtubule protein containing buffers also exhibited stage-specific differences in their ability to initiate microtubule in the centrosomal region. Cells blocked prior to anaphase onset and lysed into microtubule protein nucleated a number of microtubules typical of that found in a metaphase aster; anaphase cells nucleated substantially fewer microtubules.

Anaphase↗
PubMed1981

Anaphase progression and furrow establishment in nocodazole-arrested PtK1 cells.

The relationship between progression through anaphase and furrow establishment was investigated in PtK1 cells using the anti-mitotic agent Nocodazole to arrest cells at different points in anaphase. The capacity of cells to furrow was compared to the kinetochore-kinetochore separation attained at the time of arrest. For the stages of anaphase examined, furrowing capacity increased directly with kinetochore-kinetochore separation until complete furrows were formed after kinetochore-kinetochore separations of 14 micron or more were reached. Furrow establishment thus occurs during a definite interval during anaphase in PtK1 cells. Results from electron microscopy of both Nocodazole-treated and control cells suggest that a population of astral microtubules may be important for furrow establishment.

Anaphase↗
PubMed1979

Effects of griseofulvin on mitosis in PtK1 cells.

The concentration dependent effects of griseofulvin (GF) on mitosis in PtK1 cells were studied using a combination of time lapse cinematography and polarization and electron microscopy. Low concentrations of GF (4 X 10(-5) M) allowed a substantial number of cells to enter and complete an apparently normal mitosis. At higher concentrations of GF (1 X 10(-4) M and 2.5 X 10(-4) M) all cells entering mitosis were arrested. Typical c-mitotic chromosome arrays were observed at 1 X 10(-4) M GF with microtubules present but no spindle formed. At 2.5 X 10(-4) M GF chromosomes did not orient toward a common center to form a c-mitotic figure, but instead remained in a loosely clustered grouping at the center of the cell. Electron microscopy showed microtubles to be absent but revealed in irregularly shaped electron dense cloud around the centrioles. Quantitative polarization microscopy of metaphase cells perfused with GF showed rapid loss of spindle birefringence after exposure to the drug. Coinciding with loss of birefringence the spindle shrank rapidly with a pronounced shortening of pole to pole distance.

Animals↗
PubMed1977

Terminal phase of cytokinesis in D-98s cells.

The events leading to the completion of cytokinesis after the formation of the midbody and intercellular bridge in D-98S cells were studied with light and electron microscopy. Pairs of daughter cells corresponding to different stages of cytokineses, as determined previously form time lapse films, were selected from embedded monolayers for serial sectioning. Separation of daughter cells is preceded by the reduction in diameter of the intercellular bridge from 1-1.5 mum to approx. 0.2 mum. Two processes contribute to this reduction: (a) The intercellular bridge becomes gradually thinner after telophase; a progressive breakdown of midbody structures accompanies this change; and (b) the more significant contribution to reduction in bridge diameter occurs through the localized constriction of a segment of the intercellular bridge.. The microtubules within the constricted portion of the bridge are forced closer together, and some microtubules disappear as this narrowing progresses. The plasma membrane over the narrowed segments is thrown into a series of wavelike ripples. Separation of daughter cells is achieved through movements of the cells which stretch and break the diameter-reduced bridge. The midbody is discarded after separation and begins to deteriorate. Occasional pairs of daughter cells were found in which incomplete karyokineses resulted in their nuclei being connected by a strand of nuclear material traversing the bridge and midbody. Such cells do not complete cytokinesis but merge together several hours after telophase. This merging of daughter cells coincides with the nearly complete breakdown of the midbody.

Bone Marrow↗