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PLoS Biology Jan 2004Previous data suggested that anastral spindles, morphologically similar to those found in oocytes, can assemble in a centrosome-independent manner in cells that contain...
Previous data suggested that anastral spindles, morphologically similar to those found in oocytes, can assemble in a centrosome-independent manner in cells that contain centrosomes. It is assumed that the microtubules that build these acentrosomal spindles originate over the chromatin. However, the actual processes of centrosome-independent microtubule nucleation, polymerisation, and sorting have not been documented in centrosome-containing cells. We have identified two experimental conditions in which centrosomes are kept close to the plasma membrane, away from the nuclear region, throughout meiosis I in Drosophila spermatocytes. Time-lapse confocal microscopy of these cells labelled with fluorescent chimeras reveals centrosome-independent microtubule nucleation, growth, and sorting into a bipolar spindle array over the nuclear region, away from the asters. The onset of noncentrosomal microtubule nucleation is significantly delayed with respect to nuclear envelope breakdown and coincides with the end of chromosome condensation. It takes place in foci that are close to the membranes that ensheath the nuclear region, not over the condensed chromosomes. Metaphase plates are formed in these spindles, and, in a fraction of them, some degree of polewards chromosome segregation takes place. In these cells that contain both membrane-bound asters and an anastral spindle, the orientation of the cytokinesis furrow correlates with the position of the asters and is independent of the orientation of the spindle. We conclude that the fenestrated nuclear envelope may significantly contribute to the normal process of spindle assembly in Drosophila spermatocytes. We also conclude that the anastral spindles that we have observed are not likely to provide a robust back-up able to ensure successful cell division. We propose that these anastral microtubule arrays could be a constitutive component of wild-type spindles, normally masked by the abundance of centrosome-derived microtubules and revealed when asters are kept away. These observations are consistent with a model in which centrosomal and noncentrosomal microtubules contribute to the assembly and are required for the robustness of the cell division spindle in cells that contain centrosomes.
Topics: Animals; Animals, Genetically Modified; Cell Division; Cell Membrane; Cell Nucleus; Centrosome; Chromatin; Cytokinesis; Demecolcine; Drosophila; Female; Fluorescent Dyes; Male; Metaphase; Microscopy, Confocal; Microtubules; Molecular Sequence Data; Spermatocytes; Spindle Apparatus; Time Factors; Transgenes
PubMed: 14758368
DOI: 10.1371/journal.pbio.0020008 -
The Journal of Cell Biology Feb 1984During the short-term culturing of mouse spermatogenic cells, flagella were generated by round spermatids previously lacking tails. Unseparated germ cells were obtained...
During the short-term culturing of mouse spermatogenic cells, flagella were generated by round spermatids previously lacking tails. Unseparated germ cells were obtained by enzymatic treatments and round spermatids (greater than 90% pure) were purified by unit gravity sedimentation. As determined by Nomarski or phase-contrast microscopy, no cells had flagella immediately after isolation; flagella were first clearly detected after 6 1/2 h of culture in Eagle's minimal essential medium containing 10% fetal bovine serum and 6 mM lactate. After 24 h, approximately 20% of round spermatids had formed flagella. Multinucleated round spermatids often formed multiple flagella, the number never exceeding the number of nuclei per symplast. Round spermatids were the only spermatogenic cells capable of tail formation. Flagella elongation was blocked by 1 microM demecolcine, an inhibitor of tubulin polymerization. Indirect immunofluorescence localized tubulin in the flagella. As seen by scanning electron microscopy, flagella developed as early as 2 h after culture and continued to elongate over the next 20 h, reaching lengths of at least 19 micron. Transmission electron microscopy demonstrated that flagella formed in culture resembled flagella from Golgi-phase round spermatids in situ; the flagella consisted of "9+2" axonemes lacking other accessory structures such as outer dense fibers and the fibrous sheath. As determined by acridine orange staining of the developing acrosomes, all spermatids that formed flagella in culture were Golgi-phase spermatids. By these criteria, the structures are indeed true flagella, corresponding in appearance to what others have described for early mammalian spermatid flagella in situ. We believe this is the first substantiated report of limited in vitro differentiation by isolated mammalian spermatids.
Topics: Animals; Cells, Cultured; Flagella; Fluorescent Antibody Technique; Kinetics; Male; Mice; Microscopy, Electron; Microscopy, Electron, Scanning; Spermatids; Spermatozoa
PubMed: 6363426
DOI: 10.1083/jcb.98.2.619 -
The EMBO Journal Dec 2002Trichostatin A (TSA) inhibits all histone deacetylases (HDACs) of both class I and II, whereas trapoxin (TPX) cannot inhibit HDAC6, a cytoplasmic member of class II...
Trichostatin A (TSA) inhibits all histone deacetylases (HDACs) of both class I and II, whereas trapoxin (TPX) cannot inhibit HDAC6, a cytoplasmic member of class II HDACs. We took advantage of this differential sensitivity of HDAC6 to TSA and TPX to identify its substrates. Using this approach, alpha-tubulin was identified as an HDAC6 substrate. HDAC6 deacetylated alpha-tubulin both in vivo and in vitro. Our investigations suggest that HDAC6 controls the stability of a dynamic pool of microtubules. Indeed, we found that highly acetylated microtubules observed after TSA treatment exhibited delayed drug-induced depolymerization and that HDAC6 overexpression prompted their induced depolymerization. Depolymerized tubulin was rapidly deacetylated in vivo, whereas tubulin acetylation occurred only after polymerization. We therefore suggest that acetylation and deacetylation are coupled to the microtubule turnover and that HDAC6 plays a key regulatory role in the stability of the dynamic microtubules.
Topics: Acetylation; Animals; Antineoplastic Agents, Phytogenic; Demecolcine; Ethanol; Histone Deacetylase 6; Histone Deacetylases; Immunoblotting; Mice; Microscopy, Fluorescence; Microtubules; NIH 3T3 Cells; Peptides; Plasmids; Precipitin Tests; Protein Binding; Time Factors; Transfection; Tubulin
PubMed: 12486003
DOI: 10.1093/emboj/cdf682 -
Infection and Immunity Jul 1997The mechanisms which enable entry into cultured human epithelial cells by Klebsiella pneumoniae were compared with those of Salmonella typhi Ty2. K. pneumoniae 3091,... (Comparative Study)
Comparative Study
The mechanisms which enable entry into cultured human epithelial cells by Klebsiella pneumoniae were compared with those of Salmonella typhi Ty2. K. pneumoniae 3091, isolated from a urine sample of a patient with a urinary tract infection, invaded human epithelial cells from the bladder and ileocecum and persisted for days in vitro. Electron microscopic studies demonstrated that K. pneumoniae was always contained in endosomes. The internalization mechanism(s) triggered by K. pneumoniae was studied by invasion assays conducted with different inhibitors that act on prokaryotic and eukaryotic cell structures and processes. Chloramphenicol inhibition of bacterial uptake revealed that bacterial de novo protein synthesis was essential for efficient invasion by K. pneumoniae and S. typhi. Interference with receptor-mediated endocytosis by g-strophanthin or monodansylcadaverine and inhibition of endosome acidification by monensin reduced the number of viable intracellular K. pneumoniae cells, but not S. typhi cells. The depolymerization of microfilaments by cytochalasin D inhibited the uptake of both bacteria. Microtubule depolymerization caused by colchicine, demecolcine, or nocodazole and the stabilization of microtubules with taxol reduced only the invasion ability of K. pneumoniae. S. typhi invasion was unaffected by microtubule depolymerization or stabilization. These data suggest that the internalization mechanism triggered by K. pneumoniae 3091 is strikingly different from the solely microfilament-dependent invasion mechanism exhibited by many of the well-studied enteric bacteria, such as enteroinvasive Escherichia coli, Salmonella, Shigella, and Yersinia strains.
Topics: Bacterial Proteins; Cadaverine; Cells, Cultured; Chloramphenicol; Colchicine; Cytochalasin D; Demecolcine; Endocytosis; Epithelial Cells; Epithelium; Humans; Ionophores; Klebsiella pneumoniae; Microtubules; Monensin; Nocodazole; Nucleic Acid Synthesis Inhibitors; Ouabain; Paclitaxel; Protein Synthesis Inhibitors; Salmonella typhi; Urinary Tract; Urinary Tract Infections
PubMed: 9199471
DOI: 10.1128/iai.65.7.2950-2958.1997 -
Molecular and Cellular Biology Mar 2007The mouse gene Recql is a member of the RecQ subfamily of DEx-H-containing DNA helicases. Five members of this family have been identified in both humans and mice, and...
The mouse gene Recql is a member of the RecQ subfamily of DEx-H-containing DNA helicases. Five members of this family have been identified in both humans and mice, and mutations in three of these, BLM, WRN, and RECQL4, are associated with human diseases and a cellular phenotype that includes genomic instability. To date, no human disease has been associated with mutations in RECQL and no cellular phenotype has been associated with its deficiency. To gain insight into the physiological function of RECQL, we disrupted Recql in mice. RECQL-deficient mice did not exhibit any apparent phenotypic differences compared to wild-type mice. Cytogenetic analyses of embryonic fibroblasts from the RECQL-deficient mice revealed aneuploidy, spontaneous chromosomal breakage, and frequent translocation events. In addition, the RECQL-deficient cells were hypersensitive to ionizing radiation, exhibited an increased load of DNA damage, and displayed elevated spontaneous sister chromatid exchanges. These results provide evidence that RECQL has a unique cellular role in the DNA repair processes required for genomic integrity. Genetic background, functional redundancy, and perhaps other factors may protect the unstressed mouse from the types of abnormalities that might be expected from the severe chromosomal aberrations detected at the cellular level.
Topics: Alleles; Animals; Cells, Cultured; Chromosomal Instability; DNA Damage; Demecolcine; Electroporation; Embryonic Stem Cells; Fibroblasts; Fluorescent Dyes; In Situ Hybridization, Fluorescence; Indoles; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Knockout; RNA, Messenger; Radiation, Ionizing; RecQ Helicases; Sister Chromatid Exchange; Tissue Distribution
PubMed: 17158923
DOI: 10.1128/MCB.01620-06 -
Molecular Biology of the Cell Apr 1997It is well established that microtubules interact with intracellular membranes of eukaryotic cells. There is also evidence that tubulin, the major subunit of...
It is well established that microtubules interact with intracellular membranes of eukaryotic cells. There is also evidence that tubulin, the major subunit of microtubules, associates directly with membranes. In many cases, this association between tubulin and membranes involves hydrophobic interactions. However, neither primary sequence nor known posttranslational modifications of tubulin can account for such an interaction. The goal of this study was to determine the molecular nature of hydrophobic interactions between tubulin and membranes. Specifically, I sought to identify a posttranslational modification of tubulin that is found in membrane proteins but not in cytoplasmic proteins. One such modification is the covalent attachment of the long chain fatty acid palmitate. The possibility that tubulin is a substrate for palmitoylation was investigated. First, I found that tubulin was palmitoylated in resting platelets and that the level of palmitoylation of tubulin decreased upon activation of platelets with thrombin. Second, to obtain quantities of palmitoylated tubulin required for protein structure analysis, a cell-free system for palmitoylation of tubulin was developed and characterized. The substrates for palmitoylation were nonpolymerized tubulin and tubulin in microtubules assembled with the slowly hydrolyzable GTP analogue guanylyl-(alpha, beta)-methylene-diphosphonate. However, tubulin in Taxol-assembled microtubules was not a substrate for palmitoylation. Likewise, palmitoylation of tubulin in the cell-free system was specifically inhibited by the antimicrotubule drugs Colcemid, podophyllotoxin, nocodazole, and vinblastine. These experiments identify a previously unknown posttranslational modification of tubulin that can account for at least one type of hydrophobic interaction with intracellular membranes.
Topics: Animals; Blood Platelets; Brain Chemistry; Cell Membrane; Cell-Free System; Demecolcine; Guanosine Triphosphate; Humans; Male; Microtubules; Nocodazole; Paclitaxel; Palmitoyl Coenzyme A; Podophyllotoxin; Protein Processing, Post-Translational; Rats; Rats, Sprague-Dawley; Substrate Specificity; Swine; Thrombin; Tubulin; Vinblastine
PubMed: 9247643
DOI: 10.1091/mbc.8.4.621 -
The Journal of Clinical Investigation Oct 1993We recently demonstrated that pathologically relevant inflammatory microcrystals, namely triclinic monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD)...
We recently demonstrated that pathologically relevant inflammatory microcrystals, namely triclinic monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals, potently stimulate a characteristic protein tyrosine phosphorylation pattern in human neutrophils that differed from that observed in response to other soluble or particulate agonists. In this study, the effects of colchicine on protein tyrosine phosphorylation induced by MSU and CPPD crystals in human blood neutrophils were investigated. Immunoblot analysis with antiphosphotyrosine antibodies demonstrated that colchicine dose-dependently inhibited the tyrosine phosphorylation of all the proteins phosphorylated in response to MSU and CPPD crystals. Other microtubule-disruptive agents such as vinblastine, nocodazole, and colcemid also inhibited crystal-induced protein tyrosine phosphorylation while lumicolchicine and trimethylcolchicinic acid were without effect. Indomethacin and phenylbutazone were similarly without effect on microcrystal-induced tyrosine phosphorylation. Colchicine, as well as the other active alkaloids, failed to inhibit the protein tyrosine phosphorylation elicited by FMLP, C5a, leukotriene B4, and unopsonized zymosan. Overall, these results demonstrate that colchicine specifically and significantly inhibits the protein tyrosine phosphorylation induced by MSU and CPPD crystals and suggest that its effects are associated, at least in part, with its interaction with microtubules. Furthermore, the use of microtubule-disrupting drugs demonstrate that the mechanisms implicated in the induction of protein tyrosine phosphorylation by microcrystals differed from those involved in response to other soluble or particulate agonists.
Topics: Blood Proteins; Calcium Pyrophosphate; Colchicine; Crystallization; Demecolcine; Humans; In Vitro Techniques; Indomethacin; Kinetics; Neutrophils; Nocodazole; Phenylbutazone; Phosphorylation; Phosphotyrosine; Tyrosine; Uric Acid; Vinblastine
PubMed: 7691884
DOI: 10.1172/JCI116759 -
American Journal of Physiology. Heart... Aug 2003Although arteriolar contraction is dependent on Ca2+-induced myosin phosphorylation, other mechanisms including Ca2+ sensitization and time-dependent phenomena such as...
Although arteriolar contraction is dependent on Ca2+-induced myosin phosphorylation, other mechanisms including Ca2+ sensitization and time-dependent phenomena such as cytoskeletal and cellular reorganization may contribute to contractile events. We hypothesized that if arteriolar smooth muscle exhibits time-dependent behavior this may be manifested in differences in relaxation after short- and long-term exposure to contractile agonists. Studies were conducted in isolated arterioles pressurized to 70 mmHg. In initial experiments (n = 10), rate of relaxation was measured after acute (5 min) or prolonged (4 h) exposure to 5 microM norepinephrine (NE). Prolonged exposure to NE resulted in significantly (P < 0.05) increased time for relaxation in physiological salt solution. Rapid relaxation of vessels exposed to NE for 4 h was observed after superfusion with 0 mM Ca2+ buffer, indicating that the alteration in relaxation was reversible and Ca2+ dependent. A similarly impaired dilation was not observed with 4-h exposure to KCl (75 mM). To determine mechanisms contributing to the effects of prolonged NE exposure, studies were performed in the presence of the microtubule depolymerizing agent demecolcine (10 microM) or a series of tyrosine phosphorylation inhibitors. Although demecolcine caused significant vasoconstriction (P < 0.05) and potentiated NE vasoconstriction, it did not prevent the effect of long-term NE exposure on relaxation. Genistein, although having no effect on acute NE-induced contraction, concentration-dependently inhibited prolonged NE constriction. Similarly, Src (PP1) and p42/44 MAP kinase (PD-98059) inhibitors prevented maintenance of long-term NE contraction. The data indicate that prolonged exposure to NE induces biochemical alterations that impair relaxation after removal of the agonist. The contractile effects are Ca2+ dependent and involve tyrosine phosphorylation but do not appear to involve the polymerization state of the microtubule network.
Topics: Animals; Antineoplastic Agents, Phytogenic; Arterioles; Calcium; Demecolcine; Enzyme Inhibitors; Flavonoids; Genistein; Male; Microtubules; Norepinephrine; Phosphorylation; Potassium Chloride; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; Signal Transduction; Tyrosine; Vasoconstrictor Agents; Vasodilation
PubMed: 12714327
DOI: 10.1152/ajpheart.00986.2002 -
European Journal of Immunology Oct 2001Precursor CD4-CD8- (DN) thymocytes rearrange their TCR-beta genes, and only those which succeed in beta-selection subsequently expand and differentiate into immature...
Precursor CD4-CD8- (DN) thymocytes rearrange their TCR-beta genes, and only those which succeed in beta-selection subsequently expand and differentiate into immature CD4+CD8+ (DP) thymocytes. The cell subsets corresponding to the successive steps of this transition can be defined in terms of CD44 and CD25 expression. We partially synchronized the differentiation process by eliminating cycling cells with the anti-mitotic agent demecolcine. Using in vivo pulse labeling with bromodeoxyuridine, we determined the order of entry into DNA synthesis of the different DN and transitory (CD4-/lo CD8+) cell subsets. Two independent proliferation phases were identified. The first cells to enter the cell cycle were CD44-CD25lo, and CD4/CD8/TCR-/BrdU four-color staining showed that they all expressed a low density of the TCR-beta chain, an element of the pre-TCR (the TCR-alpha locus is still in germ-line configuration at this stage). Cycling of CD44+CD25+ cells was detected later, and no starting point was observed at the CD44-CD25hi stage. CD8 expression was immediately detectable in cycling cells, but they took 24 h to reach the DP stage. The study of TCR-Calpha-deficient mice showed that beta gene rearrangement occurred once proliferation had ceased at the DP stage, and that it had no influence on the DN-DP transition. These data show that precursor thymocytes undergo two independent waves of expansion, and that the second wave is restricted to cells capable of pre-TCR expression.
Topics: Animals; CD4 Antigens; CD8 Antigens; Cell Differentiation; Cell Division; Gene Rearrangement, alpha-Chain T-Cell Antigen Receptor; Genes, T-Cell Receptor delta; Hematopoiesis; Hematopoietic Stem Cells; Hyaluronan Receptors; Immunophenotyping; Kinetics; Mice; Mice, Inbred C57BL; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Interleukin-2; Regeneration; T-Lymphocytes
PubMed: 11592080
DOI: 10.1002/1521-4141(2001010)31:10<3038::aid-immu3038>3.0.co;2-3 -
Journal of Virology Jul 2006The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the viral RNA polymerase. In previous studies, we demonstrated that insertion of 19 amino... (Comparative Study)
Comparative Study
The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the viral RNA polymerase. In previous studies, we demonstrated that insertion of 19 amino acids in the hinge region of the protein had no significant effect on P protein function. In the present study, we inserted full-length enhanced green fluorescent protein (eGFP) in frame into the hinge region of P and show that the fusion protein (PeGFP) is functional in viral genome transcription and replication, albeit with reduced activity. A recombinant vesicular stomatitis virus encoding PeGFP in place of the P protein (VSV-PeGFP), which possessed reduced growth kinetics compared to the wild-type VSV, was recovered. Using the recombinant VSV-PeGFP, we show that the viral replication proteins and the de novo-synthesized RNA colocalize to sites throughout the cytoplasm, indicating that replication and transcription are not confined to any particular region of the cytoplasm. Real-time imaging of the cells infected with the eGFP-tagged virus revealed that, following synthesis, the nucleocapsids are transported toward the cell periphery via a microtubule (MT)-mediated process, and the nucleocapsids were seen to be closely associated with mitochondria. Treatment of cells with nocodazole or Colcemid, drugs known to inhibit MT polymerization, resulted in accumulation of the nucleocapsids around the nucleus and also led to inhibition of infectious-virus production. These findings are compatible with a model in which the progeny viral nucleocapsids are transported toward the cell periphery by MT and the transport may be facilitated by mitochondria.
Topics: Animals; Antineoplastic Agents; Biological Transport; Cell Line; Cricetinae; Demecolcine; Green Fluorescent Proteins; Microscopy, Confocal; Microscopy, Fluorescence, Multiphoton; Microscopy, Video; Microtubules; Mitochondria; Models, Biological; Mutant Chimeric Proteins; Nocodazole; Nucleocapsid; Phosphoproteins; Vesicular stomatitis Indiana virus; Viral Structural Proteins; Virus Replication
PubMed: 16775325
DOI: 10.1128/JVI.00211-06