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Journal of Virology Feb 2003The murine polyomavirus (Py) enters mouse fibroblasts and kidney epithelial cells via an endocytic pathway that is caveola-independent (as well as clathrin-independent)....
The murine polyomavirus (Py) enters mouse fibroblasts and kidney epithelial cells via an endocytic pathway that is caveola-independent (as well as clathrin-independent). In contrast, uptake of simian virus 40 into the same cells is dependent on caveola. Following the initial uptake of Py, both microtubules and microfilaments play roles in trafficking of the virus to the nucleus. Colcemid, which disrupts microtubules, inhibits the ability of Py to reach the nucleus and replicate. Paclitaxel, which stabilizes microtubules and prevents microtubule turnover, has no effect, indicating that intact but not dynamic microtubules are required for Py infectivity. Compounds that disrupt actin filaments enhance Py uptake while stabilization of actin filaments impedes Py infection. Virus particles are seen in association with actin in cells treated with microfilament-disrupting or filament-stabilizing agents at levels comparable to those in untreated cells, suggesting that a dynamic state of the microfilament system is important for Py infectivity.
Topics: 3T3 Cells; Actins; Animals; Caveolae; Cells, Cultured; Cytoskeleton; Demecolcine; Epithelial Cells; Kidney; Mice; Microtubules; Nystatin; Paclitaxel; Polyomavirus; Simian virus 40
PubMed: 12552000
DOI: 10.1128/jvi.77.4.2615-2622.2003 -
American Journal of Human Genetics Aug 2000Skin fibroblast cells from two unrelated male infants with a chromosome-instability disorder were analyzed for their response to colcemid-induced mitotic-spindle...
Skin fibroblast cells from two unrelated male infants with a chromosome-instability disorder were analyzed for their response to colcemid-induced mitotic-spindle checkpoint. The infants both had severe growth and developmental retardation, microcephaly, and Dandy-Walker anomaly; developed Wilms tumor; and one died at age 5 mo, the other at age 3 years. Their metaphases had total premature chromatid separation (total PCS) and mosaic variegated aneuploidy. Mitotic-index analysis of their cells showed the absence of mitotic block after the treatment with colcemid, a mitotic-spindle inhibitor. Bromodeoxyuridine-incorporation measurement and microscopic analysis indicated that cells treated with colcemid entered G1 and S phases without sister-chromatid segregation and cytokinesis. Preparations of short-term colcemid-treated cells contained those cells with chromosomes in total PCS and all or clusters of them encapsulated by nuclear membranes. Cell-cycle studies demonstrated the accumulation of cells with a DNA content of 8C. These findings indicate that the infants' cells were insensitive to the colcemid-induced mitotic-spindle checkpoint.
Topics: Abnormalities, Multiple; Aneuploidy; Cells, Cultured; Child, Preschool; Chromatids; Chromosome Fragility; Chromosome Segregation; DNA; Demecolcine; Fibroblasts; Flow Cytometry; Growth Disorders; Humans; Infant; Male; Metaphase; Mitotic Index; Mosaicism; Nuclear Envelope; Skin; Spindle Apparatus; Syndrome; Wilms Tumor
PubMed: 10877982
DOI: 10.1086/303022 -
The Journal of Cell Biology Nov 1985Microtubule polymer levels in mouse 3T6 fibroblasts and primary cultures of rat hepatocytes can be manipulated by treatment of cells with long term, low doses of...
Microtubule polymer levels in mouse 3T6 fibroblasts and primary cultures of rat hepatocytes can be manipulated by treatment of cells with long term, low doses of colcemid. Such treatment produces a rather uniform population of cells with microtubules of reduced lengths. Using this system, we demonstrate (a) that the rate of tubulin synthesis is sensitive to small changes (10%) in microtubule polymer mass and (b) that the percent of inhibition of synthesis is proportional to the level of soluble tubulin. Experiments with hepatocytes indicate that not only synthesis but the stability of tubulin protein was also regulated to maintain a specific level of tubulin. Treatment of hepatocytes with colcemid or other microtubule-depolymerizing drugs reduced the half-life of tubulin from 50 to 2 h, whereas taxol, which stabilizes microtubules, increased the half-life. To assess the consequences of altering microtubule polymer mass, we have analyzed the effect of controlled depolymerization of microtubules in rat hepatocytes on the processing of endocytosed ligands and found it sensitive to small changes in microtubule polymer levels.
Topics: Animals; Cells, Cultured; Demecolcine; Endocytosis; Fibroblasts; Fluorescent Antibody Technique; Homeostasis; Kinetics; Liver; Macromolecular Substances; Male; Mice; Microtubules; Rats; Rats, Inbred Strains; Tubulin
PubMed: 3902854
DOI: 10.1083/jcb.101.5.1763 -
Experimental Cell Research Mar 1989Double in situ hybridization with mercurated and biotinylated chromosome specific DNA probes in combination with digital image analysis provides a new approach to...
Double in situ hybridization with mercurated and biotinylated chromosome specific DNA probes in combination with digital image analysis provides a new approach to compare the distribution of homologous and nonhomologous chromosome targets within individual interphase nuclei. Here we have used two DNA probes representing tandemly repeated sequences specific for the constitutive heterochromatin of the human chromosomes 1 and 15, respectively, and studied the relative arrangements of these chromosome targets in interphase nuclei of human lymphocytes, amniotic fluid cells, and fibroblasts, cultivated in vitro. We have developed a 2D-image analysis approach which allows the rapid evaluation of large numbers of interphase nuclei. Models to test for a random versus nonrandom distribution of chromosome segments are discussed taking into account the three-dimensional origin of the evaluated 2D-distribution. In all three human diploid cell types the measurements of target-target and target-center distances in the 2D-nuclear image revealed that the labeled segments of the two chromosomes 15 were distributed both significantly closer to each other and closer to the center of the nuclear image than the labeled chromosome 1 segments. This result can be explained by the association of nucleolus organizer regions on the short arm of chromosome 15 with nucleoli located more centrally in these nuclei and does not provide evidence for a homologous association per se. In contrast, evaluation of the interphase positioning of the two chromosome 1 segments fits the random expectation in amniotic fluid and fibroblast cells, while in experiments using lymphocytes a slight excess of larger distances between these homologous targets was occasionally observed. 2D-distances between the labeled chromosome 1 and 15 segments showed a large variability in their relative positioning. In conclusion our data do not support the idea of a strict and permanent association of these homologous and nonhomologous targets in the cell types studied so far.
Topics: Cell Nucleolus; Cells, Cultured; Chromosomes, Human, 1-3; Chromosomes, Human, 13-15; DNA Probes; Demecolcine; Female; Heterochromatin; Humans; Image Processing, Computer-Assisted; Interphase; Male; Nucleic Acid Hybridization; Osmotic Pressure
PubMed: 2917599
DOI: 10.1016/0014-4827(89)90188-2 -
Proceedings of the National Academy of... Aug 1976The organization and growth of microtubules in cultured mouse macrophages and fibroblasts were examined by indirect immunofluorescence microscopy with antibodies to...
The organization and growth of microtubules in cultured mouse macrophages and fibroblasts were examined by indirect immunofluorescence microscopy with antibodies to microtubule protein. In macrophages, microtubules converged at a samll region at the cytocenter. During depolymerization, and repolymerization, this region acted as a microtubule organizing center. Microtubule growth was energy-dependent, but unaffected by dibutyryl-adenosine 3':5'-cyclic monophosphate, cholera toxin, or dibutyryl-guanosine 3':5'-cyclic monophosphate. Fibroblasts, which did not show such a simple microtubule organization as macrophages, contained mainly one or two, but occasionally as many as four, organizing centers during repolymerization. These microtubule organizing centers often appeared as fluorescent rings with a dark center.
Topics: Animals; Colchicine; Cold Temperature; Demecolcine; Energy Metabolism; Female; Fibroblasts; Fluorescent Antibody Technique; Macrophages; Male; Mice; Microtubules; Nucleotides, Cyclic
PubMed: 785472
DOI: 10.1073/pnas.73.8.2798 -
The Journal of Cell Biology Nov 1989Cellular repair of DNA damage due to lethal gamma irradiation was studied to reveal differences between strains and cell cycle stages that are otherwise difficult to...
Cellular repair of DNA damage due to lethal gamma irradiation was studied to reveal differences between strains and cell cycle stages that are otherwise difficult to detect. Cycling and metaphase-blocked cultures of normal fibroblasts and carcinoma cells were compared for repair of gamma sites (gamma radiation-induced nicks, breaks, and alkalilabile sites in DNA) at supralethal exposures ranging from 7 to 150 krad 137Cs radiation and at postirradiation incubations of 20-180 min. Fibroblasts from normal human skin or lung repaired gamma sites efficiently when cycling but did not repair them when blocked at mitosis. Bladder (253J) or lung (A549) carcinoma cells, unlike normal fibroblasts, repaired gamma sites efficiently even when blocked at mitosis. HeLa cells degraded their DNA soon after exposure at all doses tested, regardless of mitotic arrest. Whether the above differences in DNA repair between cell cycle stages and between strains result from differences in chromatin structure (cis effects) or from differences in the nuclear enzymatic environment (trans effects) could be resolved by placing an inert, extrachromosomal DNA molecule in the cell nucleus. Specifically, cis effects should be confined to the host chromosomes and would not be detected in the inert probe whereas trans effects should be detected in host chromosomes and inert probe DNA alike. Indeed, we found a suitable DNA molecule in the adenovirus deletion mutant dl312, which does not proliferate in the absence of E1A complementation. Gamma sites in 32P-labeled adenovirus dl312 DNA were repaired efficiently in all hosts, regardless of mitotic arrest. Failure of mitosis-arrested fibroblasts to repair gamma sites was therefore due to a cis effect of chromatin organization rather than to a trans effect such as repair enzyme insufficiency. In sharp contrast, chromosomes of mitotic carcinoma cells remained accessible to repair enzymes and nucleases alike. By means of these new tools, we should get a better understanding of higher-order chromatin management in normal and cancer cells.
Topics: Adenoviruses, Human; Cell Cycle; Cell Line; DNA Damage; DNA Repair; DNA, Neoplasm; DNA, Viral; Demecolcine; Gamma Rays; HeLa Cells; Humans; Mitosis; Tumor Cells, Cultured
PubMed: 2808517
DOI: 10.1083/jcb.109.5.1993 -
Blood Mar 1996Cells undergoing apoptosis (programmed cell death) display profound morphologic and biochemical changes in the nucleus, cytoplasm, and plasma membrane. We have shown a... (Comparative Study)
Comparative Study
Cells undergoing apoptosis (programmed cell death) display profound morphologic and biochemical changes in the nucleus, cytoplasm, and plasma membrane. We have shown a direct temporal relationship between the onset of apoptosis in Jurkat T-cell lymphoblast cultures and a greater than two-fold increase in the signal intensity of the methylene resonance (at 1.3 ppm) as observed by proton nuclear magnetic resonance spectroscopy (1H NMR). The increase in the methylene resonance intensity was seen when apoptosis was induced by serum deprivation, glucocorticoid, and doxorubicin treatment but not in necrotic (nonapoptotic) cell death. We have found similar changes in a variety of other cell lines undergoing apoptosis including the Hut 78 T-cell leukemia, JY natural killer T-cell leukemia, Daudi B-cell lymphoma, HeLa, and 3T3 fibroblast cell lines. Furthermore, this spectral change was diminished in Bcl-2 overexpressing HL-60 cell cultures treated with doxorubicin, which were relatively resistant to apoptosis, as compared to apoptotic HL-60 cultures. 1H NMR spectroscopy therefore may be useful in detecting apoptotic cell death in vivo.
Topics: 3T3 Cells; Animals; Apoptosis; Burkitt Lymphoma; Cell Line; Culture Media, Serum-Free; DNA Damage; Demecolcine; Dexamethasone; Doxorubicin; HL-60 Cells; Humans; Leukemia; Lymphocyte Subsets; Magnetic Resonance Spectroscopy; Mice; Neoplasm Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Protons; Tumor Cells, Cultured
PubMed: 8634443
DOI: No ID Found -
The Journal of Biological Chemistry Feb 1993Arrest of the human colonic cell line HT29 at the G2/M phase of the cell cycle resulted in changes in keratin assembly that were coupled with a significant increase in...
Arrest of the human colonic cell line HT29 at the G2/M phase of the cell cycle resulted in changes in keratin assembly that were coupled with a significant increase in the O-linked glycosylation and serine phosphorylation of keratin polypeptides 8 and 18 (K8/18). With mitotic arrest, enhanced keratin phosphorylation occurred preferentially on K8, whereas K18 showed a higher glycosylation level than K8. Removal of the arresting agent allowed cells to proceed through the cell cycle with a concomitant decrease in K8/18 glycosylation. In contrast, keratins isolated from S phase-enriched cells, obtained after synchronization with aphidicolin, did not show enhanced glycosylation. Tryptic peptide analysis of keratins in G2/M-arrested cells showed changes in the glycopeptide pattern of K8 and in the phosphopeptide patterns of K8 and K18. Labeling of K8/18 immunoprecipitates, isolated from G2/M-arrested cells, with [3H]galactose followed by beta-elimination showed that K8/18 glycosylation consisted of single N-acetylglucosamine residues. Threonine was identified as the site of glycosylation after comparing acid hydrolysis products of beta-eliminated and non-beta-eliminated K8 and K18. Specific cleavage at tryptophan residues indicated that K18 glycosylation and phosphorylation were restricted to the head and proximal rod domains, whereas K8 did not show the same restriction. Our results show a unique association of the single O-linked N-acetylglucosamine type of modification of keratins with mitotic arrest in HT29 cells. There was no reciprocal relationship between K8/18 glycosylation and phosphorylation, and each keratin showed a preferential G2/M cell cycle-associated increase in either serine phosphorylation or threonine glycosylation.
Topics: Aphidicolin; Cell Cycle; Cell Line; Demecolcine; Glycosylation; Humans; Isoelectric Focusing; Keratins; Mitosis; Peptide Mapping; Phosphorylation
PubMed: 7680039
DOI: No ID Found -
Cytoskeleton (Hoboken, N.J.) Mar 2011Class VI β-tubulin (β6) is the most divergent tubulin produced in mammals and is found only in platelets and mature megakaryocytes. To determine how this unique...
Class VI β-tubulin (β6) is the most divergent tubulin produced in mammals and is found only in platelets and mature megakaryocytes. To determine how this unique tubulin isotype affects microtubule assembly and organization, we expressed the cDNA in tissue culture cells under the control of a tetracycline regulated promoter. The β6 coassembled with other endogenous β-tubulin isotypes into a normal microtubule array; but once the cells entered mitosis it caused extensive fragmentation of the microtubules, disrupted the formation of the spindle apparatus, and allowed entry into G1 phase without cytokinesis to produce large multinucleated cells. The microtubule fragments persisted into subsequent cell cycles and accumulated around the membrane in a marginal band-like appearance. The persistence of the fragments could be traced to a pronounced suppression of microtubule dynamic instability. Impairment of centrosomal nucleation also contributed to the loss of a normal microtubule cytoskeleton. Incorporation of β6 allowed microtubules to resist the effects of colcemid and maytansine, but not vinblastine or paclitaxel; however, cellular resistance to colcemid or maytansine did not occur because expression of β6 prevented cell division. The results indicate that many of the morphological features of megakaryocyte differentiation can be recapitulated in non-hematopoietic cells by β6 expression and they provide a mechanistic basis for understanding these changes.
Topics: Acetylation; Animals; CHO Cells; Cell Lineage; Cricetinae; Cricetulus; Demecolcine; Humans; Maytansine; Megakaryocytes; Microtubules; Mitosis; Paclitaxel; Spindle Apparatus; Tubulin; Tubulin Modulators; Vinblastine
PubMed: 21309084
DOI: 10.1002/cm.20503 -
European Journal of Biochemistry Aug 1984Colcemid binds tubulin rapidly and reversibly in contrast to colchicine which binds tubulin relatively slowly and essentially irreversibly. At 37 degrees C the...
Colcemid binds tubulin rapidly and reversibly in contrast to colchicine which binds tubulin relatively slowly and essentially irreversibly. At 37 degrees C the association rate constant for colcemid binding is 1.88 X 10(6) M-1 h-1, about 10 times higher than that for colchicine; this is reflected in the activation energies for binding which are 51.4 kJ/mol for colcemid and 84.8 kJ/mol for colchicine. Scatchard analysis indicates two binding sites on tubulin having different affinities for colcemid. The high-affinity site (Ka = 0.7 X 10(5) M-1 at 37 degrees C) is sensitive to temperature and binds both colchicine and colcemid and hence they are mutually competitive inhibitors. The low-affinity site (Kb = 1.2 X 10(4) M-1) is rather insensitive to temperature and binds only colcemid. Like colchicine, 0.6 mol of colcemid are bound/mol of tubulin dimer (at the high-affinity site) and the reaction is entropy driven (163 J K-1 mol-1). Similar to colchicine, colcemid binding to tubulin is stimulated by certain anions (viz. sulfate and tartrate) but by a different mechanism. Colcemid binding affinity at the lower-affinity site of tubulin is increased in the presence of ammonium sulfate. Interestingly, the lower-affinity site on tubulin for colcemid, even when converted to higher affinity in presence of ammonium sulfate, is not recognized by colchicine. We conclude that tubulin possesses two binding sites, one of which specifically recognized the groups present on the B-ring of colchicine molecule and is effected by the ammonium sulfate, whereas the higher-affinity site, which could accommodate both colchicine and colcemid, possibly recognized the A and C ring of colchicine.
Topics: Ammonium Sulfate; Animals; Anions; Binding Sites; Colchicine; Demecolcine; Goats; Kinetics; Protein Binding; Sulfates; Tubulin
PubMed: 6468380
DOI: 10.1111/j.1432-1033.1984.tb08325.x