scholarly journals Taxol-induced rose microtubule polymerization in vitro and its inhibition by colchicine.

1984 ◽  
Vol 99 (1) ◽  
pp. 141-147 ◽  
Author(s):  
L C Morejohn ◽  
D E Fosket
Keyword(s):  
Binding Site ◽  
Cultured Cells ◽  
Critical Concentration ◽  
Relative Sensitivity ◽  
Mammalian Brain ◽  
Molar Ratios ◽  
Microtubule Polymerization ◽  
Colchicine Binding Site ◽  
Brain Tubulin

Tubulin was isolated from cultured cells of rose (Rosa, sp.cv. Paul's scarlet) by DEAE-Sephadex A50 chromatography, and the taxol-induced polymerization of microtubules in vitro was characterized at 24 degrees C by turbidity development, sedimentation analysis, and electron microscopy. Numerous, short microtubules were formed in the presence of taxol, and maximum levels of turbidity and polymer yield were obtained at approximately 2:1 molar ratios of taxol to tubulin. The critical concentration of rose tubulin for polymerization in saturating taxol was estimated to be 0.21 mg/ml. Colchicine inhibited the taxol-induced polymerization of tubulin as shown by sedimentation assays; however, much higher concentrations of colchicine were required for the inhibition of taxol-induced rose tubulin assembly than for inhibition of taxol-induced mammalian brain tubulin assembly. On the basis of the relative sensitivity of rose tubulin assembly to taxol and its insensitivity to colchicine, we propose that the taxol-binding site(s) on plant and animal tubulins have been more conserved over evolution than the colchicine-binding site(s).

Inability to detect Chlamyodomonas microtubule assembly in vitro: possible implications to the in vivo regulation of microtubule assembly

1975 ◽  
Vol 17 (3) ◽  
pp. 669-681
Author(s):  
K.W. Farrell ◽  
R.G. Burns
Keyword(s):  
Column Chromatography ◽  
Divalent Cation ◽  
Microtubule Assembly ◽  
Mammalian Brain ◽  
Wide Range ◽  
In Vitro Assembly ◽  
Brain Extracts ◽  
Brain Tubulin

It has been demonstrated that the in vitro assembly of microtubules from Chlamydomonas preparations does not occur under a wide range of conditions, including those efficacious for mammalian brain tubulin. This incompetence of Chlamydomonas extracts to form microtubules is independent of the tubulin concentration, the presence of added nucleotides or an added seed, temperature, or the concentration of divalent cation. However, an amorphous aggregate was observed under certain conditions, who composition was mainly tubulin. The in vitro reassembly of microtubules in gerbil brain extracts is inhibited by Chlamydomonas preparations. Fractionation of the Chlamydomonas extracts by column chromatography suggests that the inhibitory component is Chlamydomonas tubulin itself. The mechanism of this inhibition is unknown, but reassembly experiments indicate that the 2 types of tubulins cannot copolymerize. We suggest that the Chlamydomonas tubulin, derived from a cytoplasmic pool, requires to be activated prior to its in vivo polymerization into microtubules.

scholarly journals The Microtubule Plus-End Proteins EB1 and Dynactin Have Differential Effects on Microtubule Polymerization

2003 ◽  
Vol 14 (4) ◽  
pp. 1405-1417 ◽  
Author(s):  
Lee A. Ligon ◽  
Spencer S. Shelly ◽  
Mariko Tokito ◽  
Erika L.F. Holzbaur
Keyword(s):  
Cultured Cells ◽  
Cell Types ◽  
Microtubule Dynamics ◽  
In Vitro Assays ◽  
Nucleation Effect ◽  
Microtubule Polymerization ◽  
The Individual ◽  
Different Cell Types ◽  
Dynactin Complex

Several microtubule-binding proteins including EB1, dynactin, APC, and CLIP-170 localize to the plus-ends of growing microtubules. Although these proteins can bind to microtubules independently, evidence for interactions among them has led to the hypothesis of a plus-end complex. Here we clarify the interaction between EB1 and dynactin and show that EB1 binds directly to the N-terminus of the p150Glued subunit. One function of a plus-end complex may be to regulate microtubule dynamics. Overexpression of either EB1 or p150Glued in cultured cells bundles microtubules, suggesting that each may enhance microtubule stability. The morphology of these bundles, however, differs dramatically, indicating that EB1 and dynactin may act in different ways. Disruption of the dynactin complex augments the bundling effect of EB1, suggesting that dynactin may regulate the effect of EB1 on microtubules. In vitro assays were performed to elucidate the effects of EB1 and p150Glued on microtubule polymerization, and they show that p150Gluedhas a potent microtubule nucleation effect, whereas EB1 has a potent elongation effect. Overall microtubule dynamics may result from a balance between the individual effects of plus-end proteins. Differences in the expression and regulation of plus-end proteins in different cell types may underlie previously noted differences in microtubule dynamics.

scholarly journals THE MECHANISM OF ACTION OF COLCHICINE

1973 ◽  
Vol 58 (3) ◽  
pp. 709-719 ◽  
Author(s):  
Leslie Wilson ◽  
Isaura Meza
Keyword(s):  
Binding Site ◽  
Vinca Alkaloid ◽  
Binding Activity ◽  
Temperature Dependent ◽  
Binding Reaction ◽  
First Order ◽  
Colchicine Binding Site ◽  
Thermal Depolymerization ◽  
Brain Tubulin ◽  
Vinblastine Sulfate

The thermal depolymerization procedure of Stephens (1970. J. Mol. Biol. 47:353) has been employed for solubilization of Strongylocentrotus purpuratus sperm tail outer doublet microtubules with the use of a buffer during solubilization which is of optimal pH and ionic strength for the preservation of colchicine binding activity of chick embryo brain tubulin. Colchicine binding values were corrected for first-order decay during heat solubilization at 50°C (t½ = 5.4 min) and incubation with colchicine at 37°C in the presence of vinblastine sulfate (t½ = 485 min). The colchicine binding properties of heat-solubilized outer doublet tubulin were qualitatively identical with those of other soluble forms of tubulin. The solubilized tubulin (mol wt, 115,000) bound 0.9 ± 0.2 mol of colchicine per mol of tubulin, with a binding constant of 6.3 x 105 liters/mol at 37°C. The colchicine binding reaction was both time and temperature dependent, and the binding of colchicine was prevented in a competitive manner by podophyllotoxin (Ki = 1.3 x 10-6 M). The first-order decay of colchicine binding activity was substantially decreased by the addition of the vinca alkaloids, vinblastine sulfate or vincristine sulfate, thus demonstrating the presence of a vinca alkaloid binding site(s) on the outer doublet tubulin. Tubulin contained within the assembled microtubules did not decay. Intact outer doublet microtubules bound less than 0.001 mol of colchicine per mol of tubulin contained in the microtubules, under conditions where soluble tubulin would have bound 1 mol of colchicine per mol of tubulin (saturating concentration of colchicine, no decay of colchicine binding activity). The presence of colchicine had no effect on the rate of solubilization of outer doublet microtubules during incubation at 37°C. Therefore, the colchicine binding site on tubulin is blocked (not available to bind colchicine) when the tubulin is in the assembled outer doublet microtubules.

scholarly journals Microtubule assembly in cytoplasmic extracts of Xenopus oocytes and eggs.

1987 ◽  
Vol 105 (5) ◽  
pp. 2191-2201 ◽  
Author(s):  
D L Gard ◽  
M W Kirschner
Keyword(s):  
Xenopus Oocytes ◽  
Critical Concentration ◽  
Specific Inhibitor ◽  
Bovine Brain ◽  
High Rate ◽  
Microtubule Assembly ◽  
Microtubule Networks ◽  
Brain Tubulin

We have investigated the differences in microtubule assembly in cytoplasm from Xenopus oocytes and eggs in vitro. Extracts of activated eggs could be prepared that assembled extensive microtubule networks in vitro using Tetrahymena axonemes or mammalian centrosomes as nucleation centers. Assembly occurred predominantly from the plus-end of the microtubule with a rate constant of 2 microns.min-1.microM-1 (57 s-1.microM-1). At the in vivo tubulin concentration, this corresponds to the extraordinarily high rate of 40-50 microns.min-1. Microtubule disassembly rates in these extracts were -4.5 microns.min-1 (128 s-1) at the plus-end and -6.9 microns.min-1 (196 s-1) at the minus-end. The critical concentration for plus-end microtubule assembly was 0.4 microM. These extracts also promoted the plus-end assembly of microtubules from bovine brain tubulin, suggesting the presence of an assembly promoting factor in the egg. In contrast to activated eggs, assembly was never observed in extracts prepared from oocytes, even at tubulin concentrations as high as 20 microM. Addition of oocyte extract to egg extracts or to purified brain tubulin inhibited microtubule assembly. These results suggest that there is a plus-end-specific inhibitor of microtubule assembly in the oocyte and a plus-end-specific promoter of assembly in the eggs. These factors may serve to regulate microtubule assembly during early development in Xenopus.

scholarly journals The pyrrolopyrimidine colchicine-binding site agent PP-13 reduces the metastatic dissemination of invasive cancer cells in vitro and in vivo

2019 ◽  
Vol 160 ◽  
pp. 1-13 ◽  
Author(s):  
Pauline Gilson ◽  
Morgane Couvet ◽  
Laetitia Vanwonterghem ◽  
Maxime Henry ◽  
Julien Vollaire ◽  
...  
Keyword(s):  
Binding Site ◽  
Cancer Cells ◽  
Invasive Cancer ◽  
Metastatic Dissemination ◽  
Colchicine Binding Site

scholarly journals Tau protein function in living cells.

1986 ◽  
Vol 103 (6) ◽  
pp. 2739-2746 ◽  
Author(s):  
D G Drubin ◽  
M W Kirschner
Keyword(s):  
Tau Protein ◽  
Protein Function ◽  
Mammalian Brain ◽  
Protein Activity ◽  
Neuronal Morphogenesis ◽  
Microtubule Polymerization ◽  
A Cell ◽  
Nerve Cell Differentiation

Tau protein from mammalian brain promotes microtubule polymerization in vitro and is induced during nerve cell differentiation. However, the effects of tau or any other microtubule-associated protein on tubulin assembly within cells are presently unknown. We have tested tau protein activity in vivo by microinjection into a cell type that has no endogenous tau protein. Immunofluorescence shows that tau protein microinjected into fibroblast cells associates specifically with microtubules. The injected tau protein increases tubulin polymerization and stabilizes microtubules against depolymerization. This increased polymerization does not, however, cause major changes in cell morphology or microtubule arrangement. Thus, tau protein acts in vivo primarily to induce tubulin assembly and stabilize microtubules, activities that may be necessary, but not sufficient, for neuronal morphogenesis.

scholarly journals The periodic association of MAP2 with brain microtubules in vitro.

1979 ◽  
Vol 80 (2) ◽  
pp. 266-276 ◽  
Author(s):  
H Kim ◽  
L I Binder ◽  
J L Rosenbaum
Keyword(s):  
Critical Concentration ◽  
Microtubule Assembly ◽  
Molar Ratio ◽  
Thin Sections ◽  
Microtubule Associated Proteins ◽  
Heat Stable ◽  
Associated Proteins ◽  
Brain Microtubules ◽  
Brain Tubulin

Several high molecular weight polypeptides have been shown to quantitatively copurify with brain tubulin during cycles of in vitro assembly-disassembly. These microtubule-associated proteins (MAPs) have been shown to influence the rate and extent of microtubule assembly in vitro. We report here that a heat-stable fraction highly enriched for one of the MAPs, MAP2 (mol wt approximately 300,000 daltons), devoid of MAP1 (mol wt approximately 350,000 daltons), has been purified from calf neurotubules. This MAP2 fraction stoichiometrically promotes microtubule assembly, lowering the critical concentration for tubulin assembly to 0.05 mg/ml. Microtubules saturated with MAP2 contain MAP2 and tubulin in a molar ratio of approximately 1 mole of MAP2 to 9 moles of tubulin dimer. Electron microscopy of thin sections of the MAP2-saturated microtubules fixed in the presence of tannic acid demonstrates a striking axial periodicity of 32 +/- 8 nm.

scholarly journals The in vitro assembly of flagellar outer doublet tubulin.

1978 ◽  
Vol 79 (2) ◽  
pp. 500-515 ◽  
Author(s):  
L I Binder ◽  
J L Rosenbaum
Keyword(s):  
Gel Electrophoresis ◽  
Critical Concentration ◽  
Mammalian Brain ◽  
Basal Bodies ◽  
Temperature Dependent ◽  
Two Dimensional Gel Electrophoresis ◽  
Microtubule Associated Proteins ◽  
In Vitro Assembly ◽  
Associated Proteins

Flagellar outer doublet microtubules were solubilized by use of sonication, and the tubulin was reassembled in vitro into single microtubules containing 14 and 15 protofilaments. The tubulin assembly was dependent on both the KCl and tubulin concentrations, exhibiting a critical concentration of 0.72 mg/ml at optimum solvent conditions. Flagellar tubulin was purified by cycles of temperature-dependent assembly-disassembly and molecular sieve chromatography, and characterized by two-dimensional gel electrophoresis. Although doublet microtubules were not formed in vitro, outer doublet tubulin assembled onto intact A- and B-subfibers of outer doublet microtubules and basal bodies of Chlamydomonas; the rate of assembly from the distal ends of these structures was greater than that from the proximal ends. Microtubule-associated proteins (MAPs) from mammalian brain stimulated outer doublet tubulin assembly, decorating the microtubules with fine filamentous projections.

The filamins: properties and functions

1985 ◽  
Vol 63 (6) ◽  
pp. 397-413 ◽  
Author(s):  
Robert R. Weihing
Keyword(s):  
Actin Filaments ◽  
Critical Concentration ◽  
Three Dimensional ◽  
Actin Binding ◽  
Relative Mass ◽  
Cross Link ◽  
Homologous Proteins ◽  
Molar Ratios ◽  
Gelation Concentration

The filamins are a group of homologous proteins defined by their high native molecular weight (500 000), their amino acid compositions, their cross-reactivity to antibodies to heterologous filamins, their localization to actin networks and bundles in situ, and their ability to cross-link actin filaments in vitro into three-dimensional networks and bundles. Native filamins contain two subunits (relative mass = 250 000). Each subunit carries at least one actin-binding site and formation of bivalent dimers is therefore believed to explain filamin's ability to cross-link actin filaments. Formation of networks in vitro (corresponding to formation of macroscopic gels) has been analyzed using the theory of Flory. As predicted, a sharp transition to gel (at the critical gelation concentration of filamin) is observed when actin is mixed with increasing concentrations of filamin and the critical gelation concentration is found to vary inversely with the length of actin filaments. However, the measured values of the critical gelation concentration are all higher (2- to 14-fold) than predicted by the theory and the prediction that the critical concentration varies directly with the actin concentration was verified with only one of two techniques used. Filamin's length (160–190 nm) and flexibility (1000-fold greater than actin filaments) may make it especially well fitted to cross-link actin filaments into three-dimensional networks when present in low molar ratios (1:200 to 1:50) relative to actin. At higher molar ratios (> 1:20) it also cross-links actin filaments into bundles. Assuming that filamin actually helps organize supramolecular structures inside cells (not yet tested directly), then its concentration relative to actin may help determine whether networks or bundles are formed. Other factors that may influence its localization and function inside cells include competition with other actin-binding proteins (such as myosin and tropomyosin) for binding sites on actin and phosphorylation, which may alter its ability to bind to actin.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

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