Incorporation of the High‐Molecular‐Weight Microtubule‐Associated Protein 2 (MAP2) into Microtubules at Steady State <i>in vitro</i>

Manso-Martínez, Rafael; Villasanté, Alfredo; Ávila, Jesús

doi:10.1111/j.1432-1033.1980.tb04502.x

Cited by 12 publications

(11 citation statements)

References 23 publications

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“…According to Margolis and Wilson (7) approximately 3.5% of the microtubule length will be labelled at the primary assembly end. 15/zg of protein MAP 2 were added simultaneously to the radioactive nucleotide to saturate the microtubule lattice (14). When podophyllotoxin is added together with the chase GTP, the average labelled length fraction of the resulting microtubules will increase gradually with the time of treatment if disassembly occurs exclusively or preferentially from the disassembly end (see Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Before being used, samples of protein stored in liquid nitrogen were subjected to a new cycle of polymerization/ depolymerization as previously reported (14). Microtubule protein was used for experiments at a concentration of 1.1-1.6 mg/ml.…”

Section: Preparation Of Microtubule Protein From Porcine Brainmentioning

confidence: 99%

“…The concentration of tubulin dimers was deduced considering that 75% of the total protein is tubulin (M r for the heterodimer 110 000). Protein MAP 2 was purified as previously reported (14).…”

Section: Preparation Of Microtubule Protein From Porcine Brainmentioning

confidence: 99%

“…Since all incubations terminated at a single time, the addition of drug and radioactive and chase GTP was performed in a staggered time sequence. At the end of the incubation the microtubules were pelleted through a 50% sucrose cushion and the protein content and radioactivity incorporated determined as previously described (14). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was performed as indicated by Laemmli (18).…”

Section: Treatment Of Microtubule Samples and Analysis Of The Incorpomentioning

confidence: 99%

“…These authors, however, introduce a limitation in its validity: when the molar ratio of tubulin to colchicine-tubulin complex addition falls below a critical value, the poisoning effect of the drug-tubulin complex renders the microtubule assembly ends incompetent for assembly. During experiments aimed to study the incorporation and distribution of the microtubule-associated protein 2 (MAP 2) into microtubules assembled to steady-state (14) it was observed that podophyllotoxin induces a rapid loss of subunitis from the microtubule when used at superstoichiometric concentrations relative to tubulin. Since this result could not be explained totally on the basis of the proposed models, the poisoning effect of podophyllotoxin on the microtubule assembly-disassembly process was further studied.…”

Section: Introductionmentioning

confidence: 99%

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Podophyllotoxin poisoning of microtubules at steady-state: effect of substoichiometric and superstoichiometric concentrations of drug

Manso-Martínez

1982

Mol Cell Biochem

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Depolymerization kinetics of microtubules assembled to steady-state by podophyllotoxin treatment show a dose-dependent effect of this mitotic poison on the net rate of microtubule disassembly. Pulse-chase experiments with microtubules at steady-state indicate that the depolymerization effect induced by superstoichiometric concentrations of podophyllotoxin relative to tubulin is polar and time-dependent, i.e. the rate of tubulin loss decreases along with the time of treatment in the presence of the drug. Under these conditions the rate of microtubule disassembly is much faster than one could expect from a unique effect of drug-tubulin complex on the microtubule assembly end. Podophyllotoxin-tubulin complex is not able to induce active depolymerization of microtubules, while free podophyllotoxin is. These results are consistent with the hypothesis that this drug acts on the microtubule assembly-disassembly process by two different mechanisms: 1) as a free drug, it actively promotes polar depolymerization of microtubules, and 2) as a drug-tubulin complex, it retards the addition of subunits into the microtubule ends.

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Section: Resultsmentioning

confidence: 99%

Section: Preparation Of Microtubule Protein From Porcine Brainmentioning

confidence: 99%

Section: Preparation Of Microtubule Protein From Porcine Brainmentioning

confidence: 99%

Section: Treatment Of Microtubule Samples and Analysis Of The Incorpomentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Podophyllotoxin poisoning of microtubules at steady-state: effect of substoichiometric and superstoichiometric concentrations of drug

Manso-Martínez

1982

Mol Cell Biochem

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In vivo and in vitro effects of the mitochondrial uncoupler FCCP on microtubules.

Maro¹,

Marty²,

Bornens³

1982

The EMBO Journal

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FCCP (carbonylcyanide‐p‐trifluoromethoxyphenylhydrazone), a potent uncoupler of oxidative phosphorylation, induces the complete disruption of cellular microtubules. A further analysis of this effect on BHK21 cells has shown that a decrease in the number of microtubules can be observed 15 min after adding FCCP and there is complete disruption after 60 min. Regrowth of microtubules was initiated 30 min after removal of FCCP, in marked contrast with the rapid reversion observed when microtubules are disrupted by nocodazole. A similar delay was required for the recovery of mitochondrial function as assessed by rhodamine 123 labelling. The effect of FCCP on microtubules was partially inhibited by preincubation of the cells with NaN3, suggesting that FCCP acts on microtubules through mitochondria. FCCP did not depolymerize microtubules of cells permeabilized with Triton X‐100. In vitro polymerisation of microtubule protein was only slightly diminished by concentrations of FCCP which provoke complete disassembly in vivo. SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) analysis of the microtubules polymerized in vitro in the presence of FCCP showed a reduced amount of high mol. wt. proteins, mainly MAP 2, associated with them. In an attempt to reproduce the mitochondrial effects of FCCP in vitro, we checked the effects of alkaline pH and calcium on microtubule protein polymerization in the presence of FCCP. FCCP did not influence the calcium inhibitory effect but did significantly increase the inhibitory effect of alkaline pH. We conclude that FCCP could depolymerise microtubules in vivo through a dual operation: increasing the intracellular pH by the disruption of the mitochondrial H+ gradient and decreasing the stability of microtubules by impairing the binding of microtubule‐associated proteins.

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Factors implicated in determining the structure of zinc tubulin‐sheets: Lateral tubulin–tubulin interaction is promoted by the presence of zinc

Torre

Villasanté

Corral

et al. 1981

J. Supramol. Struct. Cell. Biochem.

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Addition of increasing amounts of zinc to a cold microtubule protein solution results in the disappearance of 30 S oligomer found in the absence of that cation and in the appearance of new tubulin oligomers, 90 S and 23 S. When a microtubule protein solution is warmed in the presence of zinc, tubulin-sheets are assembled. We have tested the influence of microtubule associated proteins and the zinc:tubulin ratio on the polymerization process. Depletion of microtubule associated proteins results in wider and longer tubulin-sheets than those polymerized in the presence of microtubule associated proteins. However by increasing zinc concentration wider but shorter tubulin-sheets were found. These results suggest that microtubule associated proteins and zinc could promote nucleation of tubulin-sheets, but zinc also promotes lateral tubulin-tubulin interaction. This interpretation was confirmed when microtubule protein was assembled at a low zinc:tubulin ratio. In such conditions composite structures of microtubules and zinc tubulin-sheets are formed. These composite structures are consequence of a lateral attachment of a zinc tubulin-sheet on a microtubule protofilament.

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Incorporation of the High‐Molecular‐Weight Microtubule‐Associated Protein 2 (MAP2) into Microtubules at Steady State in vitro

Cited by 12 publications

References 23 publications

Podophyllotoxin poisoning of microtubules at steady-state: effect of substoichiometric and superstoichiometric concentrations of drug

Podophyllotoxin poisoning of microtubules at steady-state: effect of substoichiometric and superstoichiometric concentrations of drug

In vivo and in vitro effects of the mitochondrial uncoupler FCCP on microtubules.

Factors implicated in determining the structure of zinc tubulin‐sheets: Lateral tubulin–tubulin interaction is promoted by the presence of zinc

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