Aggregation of microtubule subunit protein. Effects of divalent cations, colchicine, and vinblastine

Weisenberg, Richard C.; Timasheff, Serge N.

doi:10.1021/bi00823a012

Cited by 232 publications

(82 citation statements)

References 20 publications

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“…Currently we do not know if similar structures also occur in our assembly system that is free of accessory proteins. Earlier work has shown, however, that pure tubulin can form structures that are double rings of 26 tubulin molecules (24,25). These double rings were formed at a temperature of 20-25' in the absence of the Ca2+ chelator EGTA, and at Mg2+ concentrations similar to those we are using.…”

Section: Discussionsupporting

confidence: 71%

In vitro assembly of pure tubulin into microtubules in the absence of microtubule-associated proteins and glycerol.

Herzog¹,

Weber²

1977

Proc. Natl. Acad. Sci. U.S.A.

118

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Microtubule protein from porcine cerebrum was fractionated into pure tubulin and microtubule-associated proteins by chromatography on phosphocellulose. In agreement with previous studies, pure tubulin does not form microtubules to a significant extent at 370 in normal assembly buffers, which are characterized by a low concentration of Mg2+ ions. If, however, the Mg2+ concentration is raised to approximately 10 mM, rapid and extensive self-assembly of pure tubulin into microtubules is observed, provided the tubulin concentration is above 2.5 mg/ml. At a protein concentration of 3 mg/ml, the lag period is 1.5 min and the assembly process is virtually complete after 6 min at 370. These microtubules are like normal microtubules-sensitive to calcium ions, colchicine, and low temperature.The controlled dynamic process of assembly and disassembly of microtubules in vivo is an intriguing but still poorly understood feature of eukaryotic cells. Therefore, much attention has been focussed on the in vitro assembly of microtubules (for recent reviews see refs. 1 and 2). It has been generally assumed that in vitro assembly of tubulin into microtubules requires the presence of at least one microtubule-associated protein (3-9). The protein(s) necessary for initiation of microtubule formation has either specifically been called the r factor (3,5,6) or is assumed without further specification to be a microtubule-associated protein (4-6) or one or both of the high-molecular-weight proteins that are especially predominant in the microtubuleassociated protein (MAP) fraction (4, 7, 9). Some reports assume that in vitro assembly of tubulin has an absolute requirement for a MAP (3, 5, 6, 9), while others indicate that an extremely poor but still observable assembly could be noticed in the absence of MAPs (4,[7][8][9].Recently Lee and Timasheff reported (10) that tubulin free of higher-molecular-weight polypeptides can assemble into microtubules provided an assembly buffer containing high concentrations of glycerol (3.4 M) and Mg2+ ions (16 mM) is used. Their studies used tubulin purified by the ammonium sulfate, DEAE-Sephadex method rather than by the polymerization-depolymerization procedure. The critical tubulin concentration in this system was found to be higher (1.2 mg/ml) than in the assembly system involving MAPs (0.2 mg/ml) (11)(12)(13). This finding challenged the previous assumption that tubulin assembly can only occur when preexisting nucleation centers or protein factors required to assemble such nucleation centers are present. The finding of Lee and Timasheff (10) has been confirmed in two different buffer systems with homogeneous tubulin separated from MAPs by phosphocellulose chromatography. Erickson and Voter (14) used the high glycerol, high Mg2+ buffer previously described (10), whereas we Because such buffer conditions have been criticized as unphysiological (6), we wondered if self-assembly of homogeneous tubulin can be achieved reproducibly in the absence of glycerol. Here we show that phosphocellulose-pur...

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Section: Discussionsupporting

confidence: 71%

In vitro assembly of pure tubulin into microtubules in the absence of microtubule-associated proteins and glycerol.

Herzog¹,

Weber²

1977

Proc. Natl. Acad. Sci. U.S.A.

118

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“…We have found that the presence of GTP and/or MgCI2 stabilizes colchicine binding to vinblastine-precipitated protein.7 GTP stabilization has previously been reported for microtubule protein isolated from brain.13 When vinblastineprecipitated protein is resuspended in ST, bound to 3H-colchicine, and placed in the cold for 24 hours, 85 per cent of the original binding activity is retained in the presence of GTP and/or Mg, whereas only 50 per cent of the activity remains in the absence of either or both of these factors. Although we have been unable to detect the presence of bound guanine nucleotides on vinblastine-precipitated protein, the protein will bind some 3H-GTP in vitro.…”

mentioning

confidence: 55%

Isolation of Microtubule Protein from Cultured Mouse Neuroblastoma Cells

Olmsted¹,

Carlson²,

Klebe³

et al. 1970

Proc. Natl. Acad. Sci. U.S.A.

273

130

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Abstract. The addition of vinblastine to high-speed supernatants derived from homogenates of cultured mouse neuroblastoma cells results in the formation of a precipitate which has been characterized as microtubule protein by the following criteria: colchicine-binding activity, molecular weight, amino acid composition, and electrophoretic mobility. The method therefore permits the rapid isolation of microtubule protein from crude supernatants of neuroblastoma cells.Recent work has shown that the application of vinblastine or vincristine to various cell types results in the breakdown of microtubules into filaments and the formation of large cytoplasmic crystals which appear to be composed of microtubule protein and bound vinblastine.'-7 Since vinblastine caused this crystallization of microtubule protein in vivo, it seemed reasonable to determine if this drug would also cause the aggregation of microtubule protein in vitro.7'8 In this report, we describe studies in which the treatment of high-speed supernatants of microtubule-rich neuroblastoma cells with vinblastine causes the precipitation of colchicine-binding protein. In addition to a high specific activity of colchicine binding, the precipitate has been characterized as microtubule protein by its molecular weight, amino acid composition, and electrophoretic mobility similar to that of microtubule protein isolated from Chlamydomonas flagella.Materials and Methods. Cell culture: A murine neuroblastoma tumor line was adapted to in vitro culture in November 1967, and was maintained in either suspension or monolayer culture.9Electron microscopy: Electron microscopy of the cultured cells was carried out using the fixation, flat-embedding, and staining procedures described by Brinkley et al.1OCell fractionation: Cells from suspension cultures were harvested by low-speed centrifugation at room temperature and were washed once with ST (0.24 M sucrose, 0.01 M Tris, pH 7.0); all succeeding operations were performed at 40C. Cells, 3 X 108, were homogenized in 3.0 ml ST or ST with 20 mM MgCI2 (SMT), centrifuged at 2500 X g for 10 min, the pellet discarded, and the supernatant centrifuged at 35,000 X g for 30 min. The resulting supernatant was centrifuged at 150,000 X g for 90 min, and the 150,000 X g supernatant used for the isolation of microtubule protein.Precipitation of microtubule protein with vinblastine: Vinblastine11 was added to the 150,000 X g supernatant to a final concentration of 2 X 10-1 M. If cell homogenization was carried out in ST, MgCl2 was added to a final concentration of 2.5 mM following the addition of a vinblastine. The solution became turbid immediately but 129

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“…First, we recorded the effect of pCopN on vinblastine-induced tubulin self-assembly. Vinblastine induces the formation of helical assemblies (38) by promoting tubulin-tubulin longitudinal associations (39). The size of the oligomers is large enough to be analyzed in a high speed centrifugation assay.…”

Section: Pcopn Sequesters Tubulin In a 1:1 Complex And Delaysmentioning

confidence: 99%

Biochemical and Structural Insights into Microtubule Perturbation by CopN from Chlamydia pneumoniae

Nawrotek

Guimarães

Velours

et al. 2014

Journal of Biological Chemistry

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Background: Although parasites commonly hijack the actin cytoskeleton, the Chlamydia pneumoniae CopN protein interferes with microtubules. Results: CopN targets the ␤-tubulin longitudinal interface and inhibits microtubule assembly through a dual mechanism. Conclusion: In addition to regulating type III secretion, C. pneumoniae CopN has evolved microtubule-related specific functions. Significance: A framework for understanding the CopN role in the chlamydial infectious cycle is provided.

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Aggregation of microtubule subunit protein. Effects of divalent cations, colchicine, and vinblastine

Cited by 232 publications

References 20 publications

In vitro assembly of pure tubulin into microtubules in the absence of microtubule-associated proteins and glycerol.

In vitro assembly of pure tubulin into microtubules in the absence of microtubule-associated proteins and glycerol.

Isolation of Microtubule Protein from Cultured Mouse Neuroblastoma Cells

Biochemical and Structural Insights into Microtubule Perturbation by CopN from Chlamydia pneumoniae

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