A kinetic analysis of the assembly of microtubules in vitro

Engelborghs, Yves; Maeyer, L. C. M. De; Overbergh, N.

doi:10.1016/0014-5793(77)80411-0

Cited by 59 publications

(30 citation statements)

References 11 publications

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“…The effect of taxol on endosperm is consistent with data in vitro (1, 2, 45, 52, 59, 63, 65) and expected on the basis of MT properties (20,24,25,28,54). We believe that our experiments provide evidence for two major effects of taxol: enhancement of lateral associations between MTs and a rapid shift of MTtubulin equilibrium toward polymerization.…”

Section: Discussionsupporting

confidence: 88%

“…Low temperature, as judged from IGS stain, either completely disassembles MTs or leaves a few long, scattered ones, as predicted from studies in vitro (24,25); mitotic cells treated with taxol either retain (if they are exposed to the low temperature in taxol solution) or 3 A critical evaluation of the IGS method is beyond the scope of this paper. This is a new technique (17) providing a permanent label which permits prolonged observations; due to the optical clarity of the Haemanthus spindle, the morphology of a single chromosomal fiber can be often followed.…”

Section: Cold Resistancementioning

confidence: 99%

“…We do not know at present whether mitosis in taxol-treated cells progresses at temperatures which normally arrest chromosome movements (43). The ability of taxol to prevent the disassembly of MTs in Haemanthus and to restore nearly normal spindle organization at low temperature (0 °-4°C) draws attention to the close similarity of the effects of taxol in vivo and in vitro (24,25,65).…”

Section: Cold Resistancementioning

confidence: 99%

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Action of taxol on mitosis: modification of microtubule arrangements and function of the mitotic spindle in Haemanthus endosperm.

Molè-Bajer¹,

Bajer²

1983

The Journal of Cell Biology

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We have studied the effect of taxol on mitosis in Haemanthus endosperm. ImmunoGold Stain (IGS), a new immunocytochemical method (17), was used to visualize microtubules (MTs) in the light microscope. Observations on MT arrangements were correlated with studies in vivo. Chromosome movements are affected in all stages of mitosis which progresses over at least 104 range of taxol concentrations. The three most characteristic effects on MTs are: (a) enhancement of the lateral associations between MTs, seen especially during the reorganization of the polar region of the spindle, (b) promotion of MT assembly, leading to the formation of additional MTs in the spindle and MT arrays in the cytoplasm, and (c) an increase in MT stability, demonstrated in their increased cold resistance. In this report, the emphasis is on the primary, immediate effects, occurring in the first 30 min of taxol action.Effects are detected after a few mins, are reversible, and are concentration/time dependent. The spindle and phragmoplast are remarkably modified due to the enhancement of lateral associations of MTs and the formation of abundant nonkinetochore and polar, asterlike MTs. The equatorial region of the interzone in anaphase may be entirely depleted of MTs, and the spindle may break perpendicular to the spindle axis. Mitosis is completed in these conditions, providing evidence for the motile autonomy of each half-spindle. Trailing chromosome arms in anaphase are often stretched and broken. Chromosome fragments are transported away from the polar regions, i.e., in the direction opposite to that expected (5, 6). This supplies the first direct evidence of pushing by elongating MTs in an anastral higher plant spindle. These observations draw attention to the relation between the lateral association of MT ends to assembly/disassembly and to the role of such an interaction in spindle function and organization.Taxol, a low molecular weight microcyclic alkaloid, was isolated from the bark of the western yew (Taxus brevifolia) by Wani et al. in 1971 (68). It is a potent drug which lowers the critical concentration of tubulin subunits which are required for the polymerization of MTs and shortens the lag time for microtubule (MT) assembly in vitro (59). Taxol binds specifically and reversibly to polymerized tubulin, i.e., MTs, and affects the allosteric transition of the receptor from the dimeric to polymeric state (52). Taxol-treated MTs are stabilized through either a lowered rate of dissociation (52, 61, 65) or a decreased rate of steady-state tubulin flux (66). All work on taxol, except that of Wolniak et al. (69), has been done on animal cells, and the data on mitosis in progress are still fragmentary (14,15,16). In animal culture cells, taxol treatment results in the formation of abundant and unusually arranged MTs (2,15,16,45,60,63). In such exponentially growing cultures, the cell cycle (in G2) and/or mitosis is inhibited by taxol (1,16,58,60). The short duration of mitosis and the small spindle make it difficult to study the ini...

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Section: Discussionsupporting

confidence: 88%

Section: Cold Resistancementioning

confidence: 99%

Section: Cold Resistancementioning

confidence: 99%

See 1 more Smart Citation

Action of taxol on mitosis: modification of microtubule arrangements and function of the mitotic spindle in Haemanthus endosperm.

Molè-Bajer¹,

Bajer²

1983

The Journal of Cell Biology

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“…Furthermore, it has been shown both theoretically and experimentally that the MAPs function in the in vitro assembly reaction by associating with the tubulin subunit lattice after tubulin polymerization has occurred (33), thereby serving to decrease the reverse rate constant of the assembly reaction (11,25) . The polymer is therefore stabilized, and the equilibrium shifts to favor assembly .…”

Section: Discussionmentioning

confidence: 99%

Structure and composition of the cytoskeleton of nucleated erythrocytes I. The presence of microtubule-associated protein 2 in the marginal band.

Sloboda¹,

Dickersin²

1980

The Journal of Cell Biology

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The marginal band (MB) of nucleated erythrocytes is composed of a bundle of microtubules that encircles the cell immediately beneath the plasma membrane. When cells are lysed in buffer containing Triton X-100 the MB microtubules remain intact, and the nucleus remains suspended at the cell center by a filamentous network called the trans-MB material that connects the nucleus to the peripheral MB. When these lysed cells are prepared for indirect immunofluorescence by use of an antibody to chick brain microtubule-associated protein 2 (MAP 2), intense staining of the MB results; no staining is evident in the areas occupied by the nucleus or the trans-MB material. Controls demonstrate that the staining is specific, because no staining occurs with fluorescent goat antirabbit serum alone or when nonimmune serum is used as the first antibody. Furthermore, the fluorescence of the MB is not affected by pretreatment of the immune serum with purified tubulin, but staining is prevented by pretreatment of the immune serum with purified MAP 2. To determine which protein component of the MB was responsible for the positive immunofluorescence results, 125I-protein A staining was used after the protein components of the isolated cytoskeleton had been resolved by SDS-polyacrylamide gels. Controls showed that the antiserum could react on SDS gels with MAP 2 from purified chick brain microtubules. The results with the cytoskeletal proteins demonstrated that the antiserum reacted only with a high molecular weight protein having a molecular weight similar, but not identical, to that of chick brain MAP 2. Thus, it is concluded that a protein with antigenic characteristics similar to those of chick brain MAP 2 is a component of the MB. The results are discussed in terms of the possible function of MAP 2 in the MB.

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“…Microtubule protein was purified from pig brain homogenates according to the method of Shelanski et al [l I], modified as previously described [12]. Glycerol was used only in the first polynierization to increase the yield.…”

Section: Materials a N D Methodsmentioning

confidence: 99%

A Quantitatiove Desdcription of Microtubule Formadtion in the Presence of Tubulin-Colchicine

Lambeir¹,

Ensgelborghs²

1983

Eur J Biochem

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The overall polymerization of microtubule protein in the presence of tubulin-colchicine is described by competition between an intrinsically unaltered nucleation process and the process of propagation inhibited by the binding of tubulin-colchicine to the microtubule ends.The inhibition of propagation can be quantified with the binding constant previously determined [Lambeir and Engelborghs (1980) Eur. J . Biochcm. 109, 619-6241. A quantitativc description of the competition between nucleation and propagation follows from the kinetic theory of Oosawa.Comparison of several subsequent cycles of polymerization/depolymerization shows that a fraction of cold-stable complexes are formed.An equilibrium derivation is presented which shows the enhanced nucleation upon binding of inhibiting proteins, by the increase of the nucleation parameter A [Oosawa and Asakura (1 975 The kinetic and equilibrium derivations presented here are generally applicable to all capping factors, e.g. some of the actin-binding proteins.Colchicine is a very potent inhibitor of microtubule assembly [I]. Margolis and Wilson [2] showed that the tubulincolchicine complex binds to the microtubule ends. In this way binding interferes with further elongation and also with dissociation. These authors proposed an irreversible binding in order to explain the very high efficiency of colchicine inhibition. Sternlicht and Ringel [3] showed, however, that more radioactive colchicine can be found in microtubules than can be accounted for by saturation of the ends only. These authors, therefore, proposed a copolymerization model. However, true copolymerization predicts a decrease of the unpolymerized tubulin concentration below its original critical concentration (Oosawa and Asakura f41). This is not observed. Therefore incorporation has to be explained by a mechanism other than copolymerization.Lambeir and Engelborghs [ S -71 have analysed the binding of tubulin-colchicine to microtubule ends in conditions close to the steady state. These authors showed that the binding is reversible. They analysed their data, assuming that a site occupied with tubulin-colchicine shows a negligible rate of further polymerization, but neighbouring sites on the same end are not influenced. (It should be clear that a single microtubule end has probably many binding sites for tubulin. As the microtubule is built from 13 protofilaments, a maximum of 13 binding sites can be assumed. Considering the staircase-like structure of the ends, a lower value seems more likely.) Formally, therefore, the rate constant for growth to a microtubule end decreases linearly with the saturation of that end with tubulin-colchicine. In this way these authors were able to determine the binding constant. In this model incorporation of tubulin-colchicine in the microtubules is accepted to be a consequence of trapping of tubulin-colchicine during fast growth at neighbouring sites.The question we want to answer here is whether the binding constant, previously determined at steady state, is still applicabl...

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A kinetic analysis of the assembly of microtubules in vitro

Cited by 59 publications

References 11 publications

Action of taxol on mitosis: modification of microtubule arrangements and function of the mitotic spindle in Haemanthus endosperm.

Action of taxol on mitosis: modification of microtubule arrangements and function of the mitotic spindle in Haemanthus endosperm.

Structure and composition of the cytoskeleton of nucleated erythrocytes I. The presence of microtubule-associated protein 2 in the marginal band.

A Quantitatiove Desdcription of Microtubule Formadtion in the Presence of Tubulin-Colchicine

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