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J R McIntosh

Publications and source records attributed to J R McIntosh.

At least 109 records · Page 6Linked to original sources
PubMed1980

Evidence for microtubule subunit addition to the distal end of mitotic structures in vitro.

HeLa cells blocked in metaphase with 0.04 micrograms/ml of the microtubule poison nocodazole were shown to contain large numbers of microtubules with typical mitotic organization but no cenriole. Lysis of nocodazole-poisoned cells in a microtubule reassembly buffer containing 0.5 M PIPES, 2.5% dimethyl sulfoxide, 1 mM EDTA, 1 mM MgCl2, 1 mM GTP, 1% Triton X-165, 0.5% sodium deoxycholate, 0.2% SDS, pH 6.9, preserved metaphase aster structures 5 micrograms in diameter surrounded only by a thin, fibrous cell remnant. Inclusion of 2 mg/ml porcine brain microtubule protein in the lysis buffer produced asters up to 20 micrometers in diameter with a birefringent retardation of 5-6 nm. In these large asters the central microtubules had normal morphology, but peripheral microtubules were clearly abnormal. Our interpretation is that in high PIPES lysis buffer, exogenous brain tubulin adds to the distal ends of preexisting aster microtubules to form abnormal microtubules. This observation supports the assumptions made by Borisy and by Summers and Kirschner in their interpretation of growth experiments to determine the microtubule polarity in mitotic structures.

Benzimidazoles↗
PubMed1980

Isolation and partial characterization of a cage of filaments that surrounds the mammalian mitotic spindle.

Mitotic cells have been detergent extracted under conditions that support microtubule assembly. When HeLa cells are lysed in the presence of brain tubulin, mitotic-arrested cells nucleate large asters and true metaphase cells yield spindles that remain enclosed within a roughly spherical cage of filamentous material. Detergent-extracted mitotic Chinese hamster ovary (CHO) cells show a similar, insoluble cage but the mitotic apparatus is only occasionally stabilized. In later stages of mitosis, HeLa cages are observed in elongated and furrowed configurations. In the terminal stages of cell division, two daughter filamentous networks are connected by the intercellular bridge. When observed in the electron microscope the cages include fibers 7-11 nm in diameter. The polypeptide composition of cages isolated from mitotic HeLa cells is complex, but the major polypeptides are a group with mol wt ranging from 43,000-60,000 daltons and a high molecular weight polypeptide. CHO cells contain a subset of these proteins which includes a major 58,000-dalton and a high molecular weight polypeptide. Two different antisera directed against the vimentin-containing intermediate filaments bind to polypeptides in the electrophoretic profiles of isolated HeLa and CHO cages and stain the cages, as visualized by indirect immunofluorescence. These results suggest that the HeLa and CHO cages include intermediate filaments of the vimentin type. The polypeptide composition of HeLa cages suggests that they also contain tonofilaments. The cages apparently form as the cells enter mitosis. We propose that these filamentous cages maintain the structural continuity of the cytoplasm while the cell is in mitosis.

Animals↗
PubMed1980

Polarity of midbody and phragmoplast microtubules.

A newly discovered method (Heidemann and McIntosh, 1980, Nature [Lond.] 286:517) for displaying the molecular polarity of microtubules (MTs) has been slightly modified and applied to the midbodies of cultured mammalian cells and the phragmoplasts of Haemanthus endosperm. The method involves the decoration of preexisting MTs in lysed cells with curved ribbons of tubulin protofilaments; the direction of curvature of these C-shaped appendages as seen in cross section reflects the intrinsic polarity of the MTs. In travsverse sections of midbodies from HeLa and PtK cells, we find that essentially all the MTs in a given region of the structures have the same direction of hook curvature, and hence the same polarity. The midbody MTs that lie on one side of the spindle equator show the opposite polarity from those on the other side, indicating that the midbody is constructed from two families of antiparallel MTs. Midbody MTs are arranged with their fast-growing ends overlapping at the spindle equator, consistent with the hypothesis that the midbody is formed by the interdigitation of aster MTs. The polarities of the MTs from the phragmoplast of endosperm cells are the same as those found in the mammalian midbody. Our results eliminate one model for mitosis, but are consistent with others. The systematic and reproducible polarities observed favor the concept that MT polarity is an important factor in the formation and/or the function of these two mitotic structures.

Cell Division↗
PubMed1980

The effects of applied electric fields on Micrasterias. I. Morphogenesis and the pattern of cell wall deposition.

Applied electric fields of approximately 14 V/cm have profound effects on the morphogenesis of the unicellular green alga, Micrasterias denticulata. This field corresponds to a potential drop of 15-40 mV across lobes oriented perpendicular to the applied field. These lobes show a galvanotropism toward the cathode. Lobes growing parallel to the field are stunted to varying degrees, depending on their orientation. As shown by other investigators, most cell wall material is normally deposited at the tips of growing lobes. If, however, cell expansion is osmotically inhibited in electric fields, wall material also accumulates along the cathode-facing (CF) sides of lobes oriented perpendicular to the field. Similarly, in cells growing under the influence of an applied field, radioactively labelled glucose and the methyl groups from methionine are incorporated along the CF sides of lobes as well as the lobe tips. This is also true when the label is added immediately after cells are removed from the fields, indicating that the wall-depositing machinery itself has been temporarily altered by the field. These results demonstrate that applied electric fields can be a valuable tool in elucidating the mechanisms of growth localization in Micrasterias cells.

Cell Wall↗
PubMed1979

Three-dimensional structure of the central mitotic spindle of Diatoma vulgare.

Central mitotic spindles in Diatoma vulgare have been investigated using serial sections and electron microscopy. Spindles at both early stages (before metaphase) and later stages of mitosis (metaphase to telophase) have been analyzed. We have used computer graphics technology to facilitate the analysis and to produce stereo images of the central spindle reconstructed in three dimensions. We find that at prometaphase, when the nuclear envelope is dissassembling, the spindle is constructed from two sets of polar microtubules (MTs) that interdigitate to form a zone of overlap. As the chromosomes become organized into the metaphase configuration, the polar MTs, the spindle, and the zone of overlap all elongate, while the number of MTs in the central spindle decreases from greater than 700 to approximately 250. Most of the tubules lost are short ones that reside near the spindle poles. The previously described decrease in the length of the zone of overlap during anaphase central spindle elongation is clearly demonstrated in stereo images. In addition, we have used our three-dimensional data to determine the lengths of the spindle MTs at various times during mitotis. The distribution of lengths is bimodal during prometaphase, but the short tubules disappear and the long tubules elongate as mitosis proceeds. The distributions of MT lengths are compared to the length distributions of MTs polymerized in vitro, and a model is presented to account for our findings about both MT length changes and microtubule movements.

Anaphase↗
PubMed1979

Cross-sectional structure of the central mitotic spindle of Diatoma vulgare. Evidence for specific interactions between antiparallel microtubules.

During the transition from prometaphase to metaphase, the cross-sectional area of the central spindle of Diatoma decreases by a factor of nearly two, both at the poles and at the region of overlapping microtubules (MTs) near the spindle equator. The density of spindle MT packing stays approximately constant throughout mitosis. Optical diffraction analysis of electron micrographs shows that the packing of the MTs at the poles at all stages of mitosis is similar to that expected for a two-dimensional liquid. Analysis of the region of overlap reveals more packing regularity: during prometaphase, a square packing emerges that displays sufficient organization by late metaphase to generate five orders of diffraction; during anaphase the packing in the overlap region shifts to hexagonal; at telophase, it returns to square. From the data provided by serial section reconstructions of the central spindle, it is possible to identify the polarity of almost every spindle MT, that is, to identify one pole with which the MT is associated. Near neighbor analyses of MTs in cross sections of the overlap region show that MTs prefer antiparallel near neighbors. These near neighbors are most often found at a spacing of approximately 40 nm center-to-center, while parallel near neighbors in the zone of overlap are spaced essentially at random. These results are evidence for a specific interaction between antiparallel MTs. In some sections definite bridges between MTs can be seen. Our findings show that certain necessary conditions for a sliding filament model of anaphase spindle elongation are met.

Anaphase↗
PubMed1977

A comparison of the distribution of actin and tubulin in the mammalian mitotic spindle as seen by indirect immunofluorescence.

Rabbit antibodies against actin and tubulin were used in an indirect immunofluorescence study of the structure of the mitotic spindle of PtK1 cells after lysis under conditions that preserve anaphase chromosome movement. During early prophase there is no antiactin staining associated with the mitotic centers, but by late prophase, as the spindle is beginning to form, a small ball of actin antigenicity is found beside the nucleus; After nuclear envelope breakdown, the actiactin stains the region around each mitotic center, and becomes organized into fibers that run between the chromosomes and the poles. Colchicine blocks this organization, but does not disrupt the staining at the poles. At metaphase the antiactin reveals a halo of ill-defined radius around each spindle pole and fibers that run from the poles to the metaphase plate. Antitubulin shows astral rays, fibers running from chromosomes to poles, and some fibers that run across the metaphase plate. At anaphase, there is a shortening of the antiactin-stained fibers, leaving a zone which is essentially free of actin-staining fluorescence between the separating chromosomes. Antitubulin stains the region between chromosomes and poles, but also reveals substantial fibers running through the zone between separating chromosomes. Cells fixed during cytokinesis show actin in the region of the cleavage furrow, while antitubulin reveals the fibrous spindle remnant that runs between daughter cells. These results suggest that actin is a component of the mammalian mitotic spindle, that the distribution of actin differs from that of tubulin and that the distributions of these two fibrous proteins change in different ways during anaphase.

Actins↗
PubMed1977

On the mechanism of anaphase spindle elongation in Diatoma vulgare.

Central spindles from five dividing cells (one metaphase, three anaphase, and one telophase) of Diatoma vulgare were reconstructed from serial sections. Each spindle is made up of two half-spindles that are composed almost entirely of polar microtubules. A small percentage of continuous microtubules and free microtubules were present in every stage except telophase. The half-spindles interdigitate at the midregion of the central spindle, forming a zone of overlap where the microtubules from one pole intermingle with those of the other. At metaphase the overlap zone is fairly extensive, but as elongation proceeds, the spindle poles move apart and the length of the overlap decreases because fewer microtubules are sufficiently long to reach from the pole to the zone of interdigitation. At telophase, only a few tubules are long enough to overlap at the midregion. Concurrent with the decrease in the length of the overlap zone is an increase in the staining density of the intermicrotubule matrix at the same region. These changes in morphology can most easily be explained by assuming zone mechanochemical interaction between microtubules in the overlap zone which results in a sliding apart of the two half-spindles.

Eukaryota↗
PubMed1976

Experimental autologous immune deposit nephritis in rats associated with mercuric chloride administration.

Serial administration of mercuric chloride to rats was followed by development of antibodies to tubular basement membrane and renal tubular epithelial antigen (RTE) and glomerulonephritis characterized by granular deposits of hosts IgG, C3 and RTE along the glomerular capillary walls. The glomerular fixed antibody was directed against RTE. These studies suggest that tubular injury by mercury may lead to release of RTE and autosensitization and subsequent antibody production to this antigen result in formation of and glomerular deposition of circulating immunopathogenic complexes (RTE-anti-RTE) and glomerular morphologic alterations.

Animals↗
PubMed1975

Initiation and growth of microtubules from mitotic centers in lysed mammalian cells.

Metaphase PtK1 cells, lysed into polymerization-competent microtubule protein, maintain a spindle which will gain or lose birefringence depending on the concentration of disassembled tubulin subunits used in the lysis medium. Concentrations of tubulin subunits greater than the equilibrium monomer value promote a rate and extent of birefringence increase that is proportional to the subunit concentration. Increase in spindle birefringence can be correlated with an increase in tubule number, though the relationship is not strictly linear. Increase in spindle tubule number is due to an vivo-like initiation of tubules at the mitotic centers, as well as tubulin addition onto pre-existing spindle fragments. Colcemid-treated prometaphase cells lysed into polymerization-competent tubulin develop large asters in the region of the centrioles and short tubules at kinetochores, making it unlikely that all microtubule formation in lysed cell preparations is dependent on tubulin addition to short tubule fragments. Asters can also form in colcemid-treated prometaphase cells lysed in tubulin that is incapable of spontaneous tubule initiation, suggesting that the centriolar region serves a tubule-initiator function in our lysed cell preparations. The ability of the centriole to initiate microtubule assembly is a time-dependent process-a ripening effect takes place between prophase and late prometaphase. Ripening is expressed by an increase in the number and length of tubules found associated with the centriolar region.

Cell Line↗
PubMed1975

Neuraminidase treated homologous IgG and immune deposit rental disease in inbred rats.

Immune deposit renal disease followed intravenous or intraperitoneal injections of neuraminidase treated homologous IgG or neuraminidase alone. No alterations were associated with several groups of controls. This preliminary study suggests that one mechanism by which microogranisms may be involved in the development of immune renal disease is by chemical alteration of immunoglobulin.

Animals↗
PubMed1975

Cryoglobulins. III. Further studies on the nature, incidence, clinical, diagnostic, prognostic, and immunopathologic significance of cryoproteins in renal disease.

Serial serum samples from a large number of patients with immunologic renal disease, normal healthy controls, acute infections as well as non-immunological renal disease were studied for the presence, nature and properties of cryoproteins, and these correlated with serial renal functional, morphologic, immunohistologic and clinical fingings as well as serologic observations. A high incidence of cryoproteins were found in renal disease thought to be mediated by immune complexes. Cryoprecipitates were not detected in the other patients. The presence of fibrinogen in a serum cryoprecipitate was always associated with rapidly progressive disease and poor prognosis. An association between the detection of cryoproteins and the clinical and morphological activity of disease was observed. Persistence of cryoproteinemia was associated with progression and apparent disappearance with resolution or progression to end stage renal disease. In patients with hematuria or proteinuria of questionable significance cryoprotein detection was always associated with immune complex nephritis. Renal transplantation in the presence of cryoproteinemia was associated with recurrent nephritis in the graft. Cryoproteins were found to have biologic properties attributable to antigen-antibody complexes, to contain immune complexes of antigen and antibody and to have serologic factors concentrated. The detection of serum cryoglobulins was found to be a better index of clinical and morphologic activity of immune complex renal disease than was serum complement. As in our previous studies, these proteins appear to be of diagnostic and prognostic value in renal disease and provide a method of antigen identification in these disorders.

Animals↗