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Query: UMLS:C0009443 (cold)
 92,137 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Electrophoresis of microtubule preparations purified from calf brain by repeated cycles of assembly and disassembly shows that they contain many proteins in addition to alpha- and beta-tubulin. These additional proteins constitute about 17% of the total material present after five cycles of assembly and disassembly. Both one-dimensional and two-dimensional (P.H. O'Farrell (1975), J. Biol. Chem. 250, 4007) electrophoretic techniques have been used to characterize them. They can be divided into two groups: one that contains proteins which remain in constant quantitative ratio to tubulin during the purification cycles, and one composed of proteins which are removed during purification, although inefficiently. Gel-filtration chromatography of cold-depolymerized microtubule preparations yields a polydisperse fraction of high molecular weight containing most of the non-tubulin proteins. This fraction contains flexible filaments about 100 A in diameter similar to those reported by R.A.B. Keats and R.H. Hall ((1975), Nature (London) 247, 418). It is suggested that these fibers are neurofilaments, and that they may be the major source of the group of inefficiently removed proteins.
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PMID:Separation and characterization of microtubule proteins from calf brain. 92 53

tub2-401 is a cold-sensitive allele of TUB2, the sole gene encoding beta-tubulin in the yeast, Saccharomyces cerevisiae. At 18 degrees C, tub2-401 cells are able to assemble spindle microtubules but lack astral microtubules. Under these conditions, movement of the spindle to the bud neck is blocked. However, spindle elongation and chromosome separation are unimpeded and occur entirely within the mother cell. Subsequent cytokinesis produces one cell with two nuclei and one cell without a nucleus. The anucleate daughter can not bud. The binucleate daughter proceeds through another cell cycle to produce a cell with four nuclei and another anucleate cell. With additional time in the cold, the number of nuclei in the nucleated cells continues to increase and the percentage of anucleate cells in the population rises. The results indicate that astral microtubules are needed to position the spindle in the bud neck but are not required for spindle elongation at anaphase B. In addition, cell cycle progression does not depend on the location or orientation of the spindle.
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PMID:Astral microtubules are not required for anaphase B in Saccharomyces cerevisiae. 140 May 81

A previous fluorescence light-microscopic study showed that the fission yeast cold-sensitive beta-tubulin mutant nda3-311 was arrested with rod-like condensed chromosomes in a mitotic state at the restrictive temperature. Upon transfer to the permissive temperature, a spindle was formed and the nucleus was divided. In the present study, we employed freeze-substitution electron microscopy to examine the ultrastructure of arrested and released nda3-311 cells. In arrested cells, a single, displaced nucleus was seen with a single spindle pole body. Therefore, spindle pole body duplication seemed to require functional beta-tubulin. The nuclear membrane was highly deformed with a leaf-like profile in cross-section, possibly due to an interaction with the rod-like, condensed chromosomes. Upon transfer to the permissive temperature, the spindle pole duplicated and the daughter spindle pole bodies rapidly migrated to the opposite ends of the nucleus, accompanied by the formation of the mitotic spindle. Elongation of the nuclear envelope occurred with concomitant spindle extension, as in a wild-type mitosis. The deformed nuclear membrane became smooth and described a convex curve. The numerous vacuoles that are seen in the arrested cells decreased in number and increased in size. Septation was completed, leaving the two divided nuclei in one half of the cell. Hexagonally arranged microtubules, apparently forming the mitotic spindle, were observed in a cross-section of a cell after return to the permissive conditions.
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PMID:The transition of cells of the fission yeast beta-tubulin mutant nda3-311 as seen by freeze-substitution electron microscopy. Requirement of functional tubulin for spindle pole body duplication. 221 69

The availability of isotype-specific antisera for beta-tubulin, coupled with genetic and biochemical analysis, has allowed the determination of beta-tubulin isotype expression and distribution in Chinese hamster ovary (CHO) cells. Using genetic manipulations involving selection for colcemid resistance followed by reversion and reselection for drug resistance, we have succeeded in isolating cell lines that exhibit three major and one minor beta-tubulin spots by two-dimensional gel electrophoresis. In concert with isotype-specific antibodies, analysis of these mutants demonstrates that CHO cells express two copies of isotype I, at least one copy of isotype IV, and very small amounts of isotype V. All three isotypes assemble into both cytoplasmic and spindle microtubules and are similar in their responses to cold, colcemid, and calcium-induced depolymerization. They have comparable turnover rates and are equally sensitive to depression of synthesis upon colchicine treatment. These results suggest that beta-tubulin isotypes are used interchangeably to assemble microtubule structures in CHO cells. However, of 18 colcemid-resistant mutants with a demonstrable alteration in beta-tubulin, all were found to have the alteration in isotype I, thus leaving open the possibility that subtle differences in isotype properties may exist.
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PMID:Expression and function of beta-tubulin isotypes in Chinese hamster ovary cells. 264 75

The organization of intermediate filaments (IF) and microtubules (MT) and the solubility of intermediate filament proteins and tubulin in astrocytes which develop from cerebral hemispheres of neonatal rats in culture were examined using immunocytochemical and immunochemical approaches. Results of immunocytochemical studies demonstrated that in flat astrocytes which develop after 3 weeks of culturing in serum-supplemented medium, the IF containing vimentin and glial fibrillary acidic protein (GFAP) are concentrated around the nucleus and dispersed in an irregular fashion throughout the cytoplasm. Astrocytes which develop in serum-free hormonally-defined medium irrespective of whether they are bipolar, multipolar or flattened, have IF organized as a fibrous network of filaments distributed from the nuclear regions to the cell periphery. Under both culture conditions, vimentin and GFAP are resistant to extraction with low salt buffer containing nonionic detergent, indicating that the different cytoplasmic distribution of IF is unrelated to the solubility properties of vimentin and GFAP. Double immunolabelling experiments with polyclonal antibody to GFAP and monoclonal antibody to each alpha-tubulin or beta-tubulin reveal an extensive codistribution and parallel organization of IF and MT in all morphological types of astrocytes studied. Stabilization of MT with taxol, or depolymerization of MT with colchicine, cause dramatic changes in the distribution of IF and inhibit the extension of astrocyte processes in response to dibutyryl cyclic AMP (dBcAMP). In early stages of treatment with dBcAMP, renewal of culture medium without dBcAMP produces a rapid and permanent retraction of astrocyte processes, whereas in later stages the processes only retract partially and are then restored and maintained for several days in the absence of dBcAMP. The retraction of processes is accompanied by changes of immunocytochemical staining of IF with antibody to GFAP, which appears more intense and diffuse. However, electrophoretic and immunoblot analyses of detergent-extracted proteins from parallel cultures demonstrate that neither the amount nor the solubility of GFAP and vimentin are changed. Detergent extraction in MT stabilizing conditions shows that a substantial proportion of tubulin in astrocytes cultured in serum-containing and serum-free media is assembled into MT, most of which depolymerize on treatment with low temperature and Ca2+. Following long exposure to dBcAMP the proportion of cold/Ca2+-stable MT increases. The results suggest that the IF of astrocytes in culture are dependent on MT with respect to their cytoplasmic distribution.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The organization and solubility properties of intermediate filaments and microtubules of cortical astrocytes in culture. 287 33

Calmodulin-dependent kinase activity was investigated in cold-stable microtubule fractions. Calmodulin-dependent kinase activity was enriched approximately 20-fold over cytosol in cold-stable microtubule preparations. Calmodulin-dependent kinase activity in cold-stable microtubule preparations phosphorylated microtubule-associated protein-2, alpha- and beta-tubulin, an 80,000-dalton doublet, and several minor phosphoproteins. The endogenous calmodulin-dependent kinase in cold-stable microtubule fractions was identical to a previously purified calmodulin-dependent kinase from rat brain by several criteria including (1) subunit molecular weights, (2) subunit isoelectric points, (3) calmodulin-binding properties, (4) subunit autophosphorylation, (5) calmodulin-binding subunit composition on high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (6) isolation of kinase on calmodulin affinity resin, (7) kinetic parameters, (8) phosphoamino acid phosphorylation sites on beta-tubulin, and (9) phosphopeptide mapping. Endogenous cold-stable calmodulin-dependent kinase activity was isolated from the microtubule fraction by calmodulin affinity resin column chromatography and specifically eluted with EGTA. This kinase fraction contained the calmodulin-binding, autophosphorylating rho and sigma subunits of the previously purified kinase. The rho and sigma subunits of this kinase represented the major calmodulin-binding proteins in the cold-stable microtubule fractions as assessed by denaturing and non-denaturing procedures. These results indicate that calmodulin-dependent kinase is a major calmodulin-binding enzyme system in cold-stable microtubule fractions and may play an important role in mediating some of the effects of calcium on microtubule and cytoskeletal dynamics.
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PMID:Identification of endogenous calmodulin-dependent kinase and calmodulin-binding proteins in cold-stable microtubule preparations from rat brain. 298 55

Of 173 mutants of Saccharomyces cerevisiae resistant to the antimitotic drug benomyl (BenR), six also conferred cold-sensitivity for growth and three others conferred temperature-sensitivity for growth in the absence of benomyl. All of the benR mutations tested, including the nine conditional-lethal mutations, were shown to be in the same gene. This gene, TUB2, has previously been molecularly cloned and identified as the yeast structural gene encoding beta-tubulin. Four of the conditional-lethal alleles of TUB2 were mapped to particular restriction fragments within the gene. One of these mutations was cloned and sequenced, revealing a single amino acid change, from arginine to histidine at amino acid position 241, which is responsible for both the BenR and the cold-sensitive lethal phenotypes. The terminal arrest morphology of conditional-lethal alleles of TUB2 at their restrictive temperature showed a characteristic cell-division-cycle defect, suggesting a requirement for tubulin function primarily in mitosis during the vegetative growth cycle. The TUB2 gene was genetically mapped to the distal left arm of chromosome VI, very near the actin gene, ACT1; no CDC (cell-division-cycle) loci have been mapped previously to this location. TUB2 is thus the first cell-division-cycle gene known to encode a cytoskeletal protein that has been identified in S. cerevisiae.
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PMID:Isolation and characterization of mutations in the beta-tubulin gene of Saccharomyces cerevisiae. 299 23

We show that DNA topoisomerase II (topo II) is continuously required for mitotic chromosome changes in Schizosaccharomyces pombe. We constructed cold-sensitive (cs) or temperature-sensitive (ts) strains mutated in the genes coding for topo II (top2) and beta-tubulin (nda3). The ATP-dependent activity of the top2cs gene product is cs in vitro. The cloned top2cs gene sequence predicts an amino acid substitution. A cs top2-cs nda3 double mutant at 20 degrees C shows long, entangled chromosomes, which condense and separate upon the shift to permissive temperatures. If spindle formation is prevented at permissive temperatures, the chromosomes condense but do not separate. Thus topo II is required for final chromosome condensation; moreover, pulse-shift experiments show that topo II is required for chromatid disjuction. Experiments with ts top2-cs nda3 cells show that topo II is also required for chromosome separation in anaphase: inactivation of topo II and activation of beta-tubulin allow normal spindle formation but result in "streaked" chromosomes.
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PMID:DNA topoisomerase II is required for condensation and separation of mitotic chromosomes in S. pombe. 304 Feb 64

Microtubules in yeast are functional components of the mitotic and meiotic spindles and are essential for nuclear movement during cell division and mating. We have isolated 70 conditional-lethal mutations in the TUB1 alpha-tubulin gene of the yeast Saccharomyces cerevisiae using a plasmid replacement technique. Of the 70 mutations isolated, 67 resulted in cold-sensitivity, one resulted in temperature-sensitivity, and two resulted in both. Fine-structure mapping revealed that the mutations were located throughout the TUB1 gene. We characterized the phenotypes caused by 38 of the mutations after shifts of mutants to the nonpermissive temperature. Populations of temperature-shifted mutant cells contained an excess of large-budded cells with undivided nuclei, consistent with the previously determined role of microtubules in yeast mitosis. Several of the mutants arrested growth with a sufficiently uniform morphology to indicate that TUB1 has at least one specific role in the progression of the yeast cell cycle. A number of the mutants had gross defects in microtubule assembly at the restrictive temperature, some with no microtubules and some with excess microtubules. Other mutants contained disorganized microtubules and nuclei. There were no obvious correlations between these phenotypes and the map positions of the mutations. Greater than 90% of the mutants examined were hypersensitive to the antimicrotubule drug benomyl. Mutations that suppressed the cold-sensitive phenotypes of two of the TUB1 alleles occurred in TUB2, the single structural gene specifying beta-tubulin.
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PMID:Isolation and characterization of conditional-lethal mutations in the TUB1 alpha-tubulin gene of the yeast Saccharomyces cerevisiae. 306 84

The effect of a single 750-mg/kg oral dose of tri-o-cresyl phosphate (TOCP) on the endogenous phosphorylation of brain microtubule preparations and spinal cord neurofilaments was assessed in hens after the development of delayed neurotoxicity. Protein phosphorylation with [gamma-32P]ATP was analyzed by one-dimensional and two-dimensional gel electrophoresis, autoradiography, and microdensitometry. TOCP treatment enhanced the Ca2+- and calmodulin-dependent phosphorylation of tubulin in crude chicken brain cytosol (160% for alpha-tubulin and 140% for beta-tubulin) and cold-stable microtubules (165% and 155% for alpha- and beta-tubulin, respectively). Microtubule-associated protein 2 (MAP-2) phosphorylation was also increased in brain fractions studied--i.e., brain cytosol (145%), cold-stable microtubules (133%), and cold-labile microtubules (328%). There was significant increase in phosphorylation of a 70-kDa protein in the brain cytosol and in the cold-stable microtubule fractions. TOCP also stimulated the phosphorylation of spinal cord proteins of 70 kDa (119%) and 160 kDa (129%) in a Mg2+-dependent manner. Addition of Ca2+ and calmodulin further enhanced the phosphorylation of these 70-kDa (563%) and 160-kDa (221%) proteins as well as of 52-, 59-, and 210-kDa proteins by as much as 126%, 160%, and 196%, respectively. Two-dimensional electrophoresis was carried out to identify these proteins. They were confirmed as alpha- and beta-tubulin (52 and 59 kDa) in brain and spinal cord preparations and the neurofilament triplet proteins (70, 160, and 210 kDa) in the spinal cord preparation. The 70-kDa protein in brain was not neurofilament in origin. Peptide mapping using Staphylococcus aureus V8 protease showed the brain and spinal cord cytoskeletal proteins have identical phosphopeptide patterns in control and TOCP-treated hens, indicating that it was unlikely that the phosphorylation sites were altered by TOCP treatment.
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PMID:Calcium and calmodulin-enhanced in vitro phosphorylation of hen brain cold-stable microtubules and spinal cord neurofilament triplet proteins after a single oral dose of tri-o-cresyl phosphate. 309 May 52


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