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Treatment of Vicia faba root tip cells with specific inhibitors to cyclin-dependent kinases leads to abnormal spindle formation

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PLANT JOURNAL 1998年第6期16卷 697-707页

作者： Binarová, P Dolezel, J Draber, P Heberle-Bors, E Strnad, M Bögre, L Univ Vienna Vienna Bioctr Inst Microbiol & Genet A-1030 Vienna Austria De Montfort Univ Norman Borlaug Ctr Plant Sci Inst Expt Bot Prague 14220 Czech Republic Acad Sci Czech Republ Inst Microbiol CR-14220 Prague Czech Republic De Montfort Univ Norman Borlaug Ctr Plant Sci Inst Expt Bot ASCR Olomouc Czech Republic ASCR Inst Mol Genet Prague 14220 Czech Republic Palacky Univ Inst Expt Bot Lab Growth Regulators Olomouc 78371 Czech Republic

Many events during cell division are triggered by an evolutionary conserved regulator the cyclin-dependent kinase (Cdk). Here we used two novel drugs, the purine analogues bohemine and roscovitine, to study the role of Cdks in cell cycle progression and microtubule organisation in Vicia faba root tip cells. Both drugs inhibited the activity of immunopurified Vicia faba and alfalfa Cdc2-kinase. The transcript levels of an A and B type cyclin, as well as of the cdc2 genes, declined in treated root tips, white the mRNA level of a D-type cyclin gene was not affected. An observed transient arrest at the G1/S and G2/M regulatory points indicated that inhibition of the Cdc2-kinase had an effect on both transitions. In contrast to the regular bipolar spindle in untreated cell, in drug-treated metaphase cells abnormally short and dense kinetochore microtubule fibres were observed. These microtubules were randomly arranged in the vicinity of the kinetochores and connected the chromosomes. Thus, the chromosomes were not aligned on the metaphase plate but were arranged in a circle, with kinetochores pointing inwards and chromosome arms pointing outwards, gamma-Tubulin, which plays a role in microtubule nucleation, also localised to the centre of the monopolar spindle. The observed abnormalities in mitosis, after inhibition of Cdc2-kinase by specific drugs, suggest a role for this enzyme in regulating some of the steps leading to a bipolar spindle structure.

关键词： CDC2蛋白激酶/拮抗剂和抑制剂 CDC2蛋白激酶/遗传学细胞周期/药物作用细胞周期蛋白A/遗传学细胞周期蛋白B/遗传学酶抑制剂/药理学豆科/细胞学分生组织/细胞学分生组织/药物作用分生组织/超微结构微管/药物作用微管/超微结构有丝分裂纺锤体/药物作用植物根/细胞学植物根/药物作用植物根/超微结构植物药用蛋白激酶类/代谢嘌呤类/药理学转录遗传

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Tubulin binding sites on γ-tubulin:: Identification and molecular characterization

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BIOCHEMISTRY 1999年第48期38卷 15712-15720页

作者： Llanos, R Chevrier, V Ronjat, M Meurer-Grob, P Martinez, P Frank, R Bornens, M Wade, RH Wehland, J Job, D CEA Grenoble Dept Biol Mol & Struct CS INSERMU366 F-38054 Grenoble 9 France IBS LMES F-38027 Grenoble France AG Mol Recognit D-38124 Braunschweig Germany Gesell Biotechnol Forsch GmbH Div Cell Biol & Immunol D-38124 Braunschweig Germany Inst Curie CNRS UMR 144 Sect Rech F-75231 Paris 05 France

gamma-Tubulin is essential to microtubule organization in eukaryotic cells. It is believed that gamma-tubulin interacts with tubulin to accomplish its cellular functions. However, such an interaction has been difficult to demonstrate and to characterize at the molecular level. gamma-Tubulin is a poorly soluble protein, not amenable to biochemical studies in a purified form as yet. Therefore basic questions concerning the existence and properties of tubulin binding sites on gamma-tubulin have been difficult to address. Here we have performed a systematic search for tubulin binding sites on gamma-tubulin using the SPOT peptide technique. We find a specific interaction of tubulin with six distinct domains on gamma-tubulin. These domains are clustered in the central part of the gamma-tubulin primary amino acid sequence. Synthetic peptides corresponding to the tubulin binding domains of gamma-tubulin bind with nanomolar K(d)s to tubulin dimers. These peptides do not interfere measurably with microtubule assembly in vitro and associate with microtubule along the polymer length. On the tertiary structure, the gamma-tubulin peptides cluster to surface regions on both sides of the molecule. Using SPOT analysis, we also find peptides interacting with gamma-tubulin in both the alpha- and beta-tubulin subunits. The tubulin peptides cluster to surface regions on both sides of the alpha- and beta- subunits. These data establish gamma-tubulin as a tubulin ligand with unique tubulin-binding properties and suggests that gamma-tubulin and tubulin dimers associate through lateral interactions.

关键词：氨基酸序列结合部位二聚作用微管/化学微管/超微结构模型分子分子序列数据肽碎片/化学肽谱蛋白质亚型/化学蛋白质结构三级表面等离激元微管蛋白/分析微管蛋白/化学人类

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Translational regulation of ribonucleotide reductase by eukaryotic initiation factor 4E links protein synthesis to the control of DNA replication

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第50期274卷 35991-35998页

作者： Abid, MR Li, Y Anthony, C De Benedetti, A Louisiana State Univ Med Ctr Dept Biochem & Mol Biol Shreveport LA 71130 USA

Ribonucleotide reductase synthesizes dNDPs, a specific and limiting step in DNA synthesis, and can participate in neoplastic transformation when overexpressed, The small subunit (ribonucleotide reductase 2 (RNR2)) was cloned as a major product in a subtraction library from eukaryotic initiation factor 4E (eIF4E)-transformed cells (Chinese hamster ovary-4E (CHO-4E)), CHO-4E cells have 20-40-fold elevated RNR2 protein, reflecting an increased distribution of RNR2 mRNA to the heavy polysomes, CHO-4E cells display an altered cell cycle with shortened S phase, similar to cells selected for RNR2 overexpression with hydroxyurea, The function of ribonucleotide reductase as a checkpoint component of S progression was studied in yeast in which elevated eIF4E rescued S-arrested rnr2-68(ts) cells, by increasing recruitment of its mRNA to polysomes, Crosses between rnr2-68(ts) and mutant eIF4E (cdc33-1(ts)) engendered conditional synthetic lethality, with extreme sensitivity to hydroxyurea and the microtubule depolymerizing agent, benomyl. The double mutant (cdc33-1 rnr2-68) also identified a unique terminal phenotype, arrested with small bud and a randomly distributed single nucleus, which is distinct from those of both parental single mutants. This phenotype defines eIF4E and RNR2 as determinants in an important cell cycle checkpoint, in early/mid-S phase. These results also provide a link between protein and DNA synthesis and provide an explanation for cell cycle alterations induced by elevated eIF4E.

关键词：苯菌灵/药理学 CHO细胞细胞周期/生理学克隆分子仓鼠亚科 DNA复制真核细胞起始因子4E 成纤维细胞生长因子/遗传学基因表达调控/药物作用羟基脲/药理学微管/药物作用微管/超微结构鸟氨酸脱羧酶/遗传学肽起始因子类/遗传学肽起始因子类/代谢多核糖体/代谢蛋白质生物合成 RNA 信使/遗传学重组蛋白质类/代谢核苷酸还原酶类/生物合成核苷酸还原酶类/遗传学 S期酿酒酵母菌/遗传学转录遗传转染动物

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Tau phosphorylation at serine 396 and serine 404 by human recombinant tau protein kinase II inhibits tau's ability to promote microtubule assembly

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第32期275卷 24977-24983页

作者： Evans, DB Rank, KB Bhattacharya, K Thomsen, DR Gurney, ME Sharma, SK Pharmacia Corp Prot Sci Kalamazoo MI 49007 USA

In Alzheimer's disease, hyperphosphorylated tau is an integral part of the neurofibrillary tangles that form within neuronal cell bodies and fails to promote microtubule assembly, Dysregulation of the brain-specific tau protein kinase II is reported to play an important role in the pathogenesis of Alzheimer's disease (Patrick, G. N., Zukerberg, L., Nikolic, M., De La Monte, S., Dikkes, P., and Tsai, L.-H, (1999) Nature 402, 615-622), We report here that in vitro phosphorylation of human tau by human recombinant tau protein kinase II severely inhibits the ability of tau to promote microtubule assembly as monitored by tubulin polymerization. The ultrastructure of tau-mediated polymerized tubulin was visualized by electron microscopy and compared with phosphorylated tau, Consistent with the observed slower kinetics of tubulin polymerization, phosphorylated tau is compromised in its ability to generate microtubules. Moreover, me show that phosphorylation of microtubule-associated tau results in tau's dissociation from the microtubules and tubulin depolymerization, Mutational studies with human tau indicate that phosphorylation by tau protein kinase II at serine 396 and serine 404 is primarily responsible for the functional loss of tau-mediated tubulin polymerization. These in vitro results suggest a possible role for tau protein kinase II-mediated tau phosphorylation in initiating the destabilization of microtubules.

关键词：氨基酸取代细胞系昆虫动力学微管/生理学微管/超微结构诱变定点磷酰化蛋白质丝氨酸苏氨酸激酶/代谢重组蛋白质类/代谢丝氨酸底物特异性转染微管蛋白/生理学微管蛋白/超微结构 tau蛋白质类/拮抗剂和抑制剂 tau蛋白质类/化学 tau蛋白质类/代谢动物人类

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Effect of the microtubule polymerizing agent taxol on contraction, Ca²⁺ transient and L-type Ca²⁺ current in rat ventricular myocytes

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JOURNAL OF PHYSIOLOGY-LONDON 1999年第2期516卷 409-419页

作者： Howarth, FC Calaghan, SC Boyett, MR White, E Univ Leeds Sch Biomed Sci Leeds LS2 9JT W Yorkshire England

1. Microtubules form part of the cytoskeleton. Their role in adult ventricular myocytes is not well understood although microtubule proliferation has previously been linked with reduced contractile function. 2. We investigated the effect of the anti-tumour drug taxol, a known microtubule polymerizing agent, on Ca2+ handling in adult rat ventricular myocytes. 3. Treatment of cells with taxol caused proliferation of microtubules. 4. In taxol-treated cells there was a reduction in the amplitude of contraction, no significant effect on the amplitude of L-type Ca2+ current, but a significant reduction in the amplitude of the Ca2+ transient. 5. Caffeine was used to release Ca2+ from the sarcoplasmic reticulum (SR). There was a significant reduction in the ratio of electrically stimulated: caffeine-induced Ca2+ transients in taxol-treated cells. This observation is consistent with the hypothesis that taxol reduces fractional SR Ca2+ release. 6. We suggest that the negative inotropic effect of taxol may, at least in part, be the result of reduced release of Ca2+ from the SR. Microtubules may be important regulators of Ca2+ handling in the heart.

关键词：抗肿瘤药植物/药理学咖啡因/药理学钙通道/药物作用钙通道/代谢钙通道 L型钙信号/药物作用秋水仙碱/药理学抑制化学性电刺激心室/细胞学心室/药物作用心室/超微结构微管/药物作用微管/超微结构心肌收缩/药物作用心肌/细胞学心肌/代谢心肌/超微结构紫杉酚/药理学膜片钳术磷酸二酯酶抑制剂/药理学大鼠 Wistar 肌浆网/药物作用肌浆网/代谢动物男(雄)性大鼠

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OPTIC-NERVE GLIOMA IN AN 18-MONTH-OLD CHILD

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AMERICAN JOURNAL OF OPHTHALMOLOGY 1975年第4期79卷 589-595页

作者： MIKI, H HIRANO, A MONTEFIORE HOSP & MED CTR ALBERT EINSTEIN COLL MED DEPT OPHTHALMOL BRONX NY USA MONTEFIORE HOSP & MED CTR ALBERT EINSTEIN COLL MED DEPT PATHOL BRONX NY USA

An optic nerve glioma in an 18 month old child was examined by both light and electron microscopy. The tumor revealed the characteristic features of uniform benign and fibrillary astrocytoma. Rosenthal fibers and calcium deposits were found within numerous intracellular glial processes. The above features indicated a slow growing tumor of long duration, confirming the generally supported assumption of the congenital nature of optic nerve glioma. One unsuspected feature was the presence of fenestrated blood vessels.

关键词：钙质沉着症/病理学毛细血管/超微结构细胞核/超微结构结缔组织/超微结构细胞质/超微结构神经胶质瘤/病理学显微镜检查电子微管/超微结构线粒体/超微结构视神经/病理学视神经/超微结构周围神经系统肿瘤/病理学人类婴儿

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Microtubule reduction in Alzheimer's disease and aging is independent of τ filament formation

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AMERICAN JOURNAL OF PATHOLOGY 2003年第5期162卷 1623-1627页

作者： Cash, AD Aliev, G Siedlak, SL Nunomura, A Fujioka, H Zhu, XW Raina, AK Vinters, HV Tabaton, M Johnson, AB Paula-Barbosa, M Avila, J Jones, PK Castellani, RJ Smith, MA Perry, G Case Western Reserve Univ Inst Pathol Cleveland OH 44106 USA Case Western Reserve Univ Dept Anat Cleveland OH 44106 USA Case Western Reserve Univ Dept Epidemiol & Biostat Cleveland OH 44106 USA Asahikawa Med Coll Dept Psychiat & Neurol Asahikawa Hokkaido 078 Japan Univ Calif Los Angeles Dept Pathol & Lab Med Los Angeles CA 90024 USA Univ Genoa Dept Neurosci Genoa Italy Albert Einstein Coll Med Dept Pathol Bronx NY 10467 USA Porto Med Sch Dept Anat Oporto Portugal Univ Autonoma Madrid Ctr Mol Biol Madrid Spain Michigan State Univ Ctr Clin E Lansing MI 48824 USA

Biochemical studies show that phosphorylated T, like that found in paired helical filaments (PHFs), does not promote microtubule assembly leading to the view that PHF formation leads to microtubule deficiency in Alzheimer's disease (AD). However, although this issue is one of the most important aspects to further understanding the cell biology of AD, no quantitative examination of microtubule diminution in AD and its relationship with PHFs has been performed. To examine this issue directly, we undertook a morphometric study of brain biopsy specimens from AD and control cases. Ultrastructural analysis of neurons was performed to compare the microtubule assembly state in neurons of diseased and control cases and to examine the effect of PHF accumulation. We found that both number and total length of microtubules were significantly and selectively reduced in pyramidal neurons from AD in comparison to control cases (P = 0.000004) but that this decrement in microtubule density was surprisingly unrelated to PHFs (P = 0.8). Further, we found a significant age-dependent decrease in microtubule density with aging in the control cases (P = 0.016). These findings suggest that reduction in microtubule assembly is not dependent on T abnormalities of AD and aging.

关键词：衰老/生理学阿尔茨海默病/病理学活组织检查脑/病理学脑肿瘤/病理学脑积水/病理学显微镜检查电子微管/病理学微管/超微结构神经元/病理学锥体细胞/病理学 tau蛋白质类/代谢老年人女(雌)性人类男(雄)性中年人

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The Ncd tail domain promotes microtubule assembly and stability

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 1999年第1期258卷 39-43页

作者： Karabay, A Walker, RA Virginia Polytech Inst & State Univ Dept Biol Blacksburg VA 24061 USA

Non-claret disjunctional (Ncd) is a Drosophila kinesin-like motor required for spindle assembly and maintenance in oocytes and early embryos. Ncd has an ATP-independent microtubule binding site in the N-terminal tail domain as well as an ATP-dependent microtubule binding site in the C-terminal motor domain. The Ncd tail domain shares many properties with the microtubule-associated proteins that regulate microtubule assembly, including microtubule binding and bundling activity and an abundance of basic and proline residues. Given these similarities, we examined the ability of Ncd tail domain proteins to promote MT assembly and stability. The results indicate that the Ncd tail domain can promote MT assembly and stabilize MTs against conditions that induce MT disassembly, and suggest that Ncd may influence MT dynamics within the spindle. (C) 1999 Academic Press.

关键词：果蝇蛋白质类驱动蛋白/代谢显微镜检查电子微管/超微结构猪动物

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The 65-kDa carrot microtubule-associated protein forms regularly arranged filamentous cross-bridges between microtubules

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 1999年第26期96卷 14931-14936页

作者： Chan, J Jensen, CG Jensen, LCW Bush, M Lloyd, CW John Innes Ctr Plant Sci Res Dept Cell Biol Norwich NR4 7UH Norfolk England Univ Auckland Sch Med Dept Anat Auckland New Zealand

In plants, cortical microtubules (MTs) occur in characteristically parallel groups maintained up to one microtubule diameter apart by fine filamentous cross-bridges. However, none of the plant microtubule-associated proteins (MAPs) so far purified accounts for the observed separation between MTs in cells, We previously isolated from carrot cytoskeletons a MAP fraction including 120- and 65-kDa MAPs and have now separated the 65-kDa carrot MAP by sucrose density centrifugation, MAP65 does not induce tubulin polymerization but induces the formation of bundles of parallel MTs in a nucleotide-insensitive manner. The bundling effect is inhibited by porcine MAP2, but, unlike MAP2, MAP65 is heat-labile, In the electron microscope, MAP65 appears as filamentous crossbridges, maintaining an intermicrotubule spacing of 25-30 nm, Microdensitometer-computer correlation analysis reveals that the cross-bridges are regularly spaced, showing a regular axial spacing that is compatible with a symmetrical helical superlattice for 13 protofilament MTs. Because MAP65 maintains in vitro the inter-MT spacing observed in plants and is shown to decorate cortical MTs, it is proposed that th is MAP is important for the organization of the cortical array in vivo.

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FINE-STRUCTURE OF VASCULAR ENDOTHELIUM IN CULTURE

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JOURNAL OF ULTRASTRUCTURE RESEARCH 1975年第1期50卷 22-32页

作者： HAUDENSCHILD, CC COTRAN, RS GIMBRONE, MA FOLKMAN, J HARVARD UNIV CHILDRENS HOSP MED CTR MED SCH DEPT SURG BOSTON MA 02115 USA HARVARD UNIV CHILDRENS HOSP MED CTR MED SCH DEPT PATHOL BOSTON MA USA PETER BENT BRIGHAM HOSP DEPT SURG BOSTON MA USA PETER BENT BRIGHAM HOSP DEPT PATHOL BOSTON MA 02115 USA

Human umbilical cord vein endothelial cells were harvested by collagenase perfusion, cultured in Medium 199 with 20% fetal calf serum, and subcultured for 19 passages over one year. By phase microscopy, cultured endothelial cells were small and polygonal and in successful cultures, formed a monolayer of closely packed cells. By electron microscopy, cultured endothelium retained fine structural characteristics of in vivo umbilical vein endothelium. Specific endothelial granules first described by Weibel and Palade ( 30 ), were found regularly in all passages. Microtubules (250 Å), two types of filaments with diameters of 100 Å and 60–70 Å, respectively, pinocytotic vesicles, multivesicular bodies, and cellular junctions were distributed in a way that cultured endothelium could be distinguished by morphologic criteria from the most probable contaminants, smooth muscle cells, and fibroblasts.

关键词：细胞培养的 DNA/生物合成内皮/代谢内皮/超微结构显微镜检查电子显微镜检查相差微管/超微结构时间因素脐带/代谢脐带/超微结构女(雌)性人类妊娠

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