Characterization and in vitro polymerization of Tetrahymena tubulin

Tetrahymena tubulin was purified from the cell extract using DEAE-Sephadex A-50 ion-exchanger and ammonium sulfate precipitation. About 2.2% of the total protein in the 20,000 X g supernatant was recovered as DEAE-Sephadex-purified tubulin fraction. Applying the temperature-dependent polymerization-depolymerization method to this fraction in the presence of Tetrahymena outer fibers as a seed, almost pure tubulin was obtained. Tetrahymena tubulin dimer showed different behavior on SDS-polyacrylamide gels from porcine brain tubulin, and showed very low affinity for colchicine, amounting to about one-twentieth of the binding to porcine brain tubulin. The tubulin fraction failed to polymerize into microtubules by itself. Addition of a small amount of the ciliary outer fiber fragment induced polymerization as demonstrated by viscometric measurements, but the reconstituted microtubules were very unstable in the absence of glycerol. Microtubule-depolymerizing agents such as Ca2+ ions, low temperature, or colchicine all inhibited in vitro polymerization. Although Tetrahymena tubulin purified by the polymerization-depolymerization method could copolymerize with porcine brain microtubules, the DEAE-Sephadex-purified tubulin fraction suppressed the initial rate of porcine brain microtubule assembly in vitro. There seemed to be no differences between cytoplasmic tubulin and outer fiber tubulin in colchicine binding activity or SDS-gel electrophoretic behavior, or between the fine structure of both reconstituted microtubules observed by electron microscopy.     

Bundling of microtubules in vitro by a high molecular weight protein prepared from the squid axon

A high molecular weight protein has been partially purified from sheaths of squid giant axons. This protein fraction was capable of restoring the membrane excitability of the squid axon which had been destroyed by internal perfusion of microtubule poison, when perfused along with microtubule proteins (Matsumoto et al. (1979) J. Biochem. 86, 1155-1158). This protein, designated as 260 K protein, was purified by gel filtration and Con A-Sepharose affinity chromatography. The apparent molecular weight of the axonal protein was estimated to be 260,000 by electrophoresis in the presence of sodium dodecylsulfate. This protein was revealed to be a glycoprotein. When phosphocellulose-purified tubulin was incubated with 260 K protein at 36 degrees C in the presence of dimethylsulfoxide, turbidity of the solution was much increased. 260 K protein co-sedimented with microtubles assembled from purified tubulin. Light microscopic and electron microscopic observations revealed that the high turbidity was due to bundling of microtubules which was caused by 260 K protein. On the other hand, the effect of this protein on the turbidity increase was not so prominent when microtubules were assembled from microtubule proteins consisting of tubulin and microtubule-associated proteins. High shear and low shear viscometry and co-sedimentation experiments revealed that 260 K protein had little effect on actin polymerization under the same medium conditions as used in tubulin polymerization.     

The presence of an adenosine-5'-triphosphatase dependent on 6S tubulin and calcium ions in rat brain microtubules.

An ATPase activity was found in rat brain microtubules prepared by successive cycles of polymerization and depolymerization. On phosphocellulose column chromatography, the ATPase activity was recovered in the fraction eluted with 0.6 M KCl and containing the microtubule associated proteins. The ATPase activity was markedly stimulated by the addition of purified brain 6S tubulin, and the stimulation was dependent on the presence of Ca2+ ions. Approximately 50 pmol of purified 6S tubulin was required for the maximal stimulation in the presence of 8 microgram of microtubule associated proteins. The specific activity was 8 to 13 nmol of ATP hydrolyzed per min per mg of protein at 37 degrees C, and the Km value for ATP was 3 X 10(-5) M in the presence of added tubulin.     

Resolution of cyclic AMP stimulated protein kinase from polymerization-purified brain microtubules.

Polymerization-deploymerization purified microtubules from mouse brain contain, in addition to tubulin, several minor proteins, including protein kinase activity. The protein kinase copurifies with microtubules in constant proportion to tubulin through two, three, or four cycles of polymerization; it can be resolved from tubulin by gel filtration chromatography and has an apparent molecular weight of 280,000. Its activity is stimulated 7-fold by cyclic AMP, and resembles the soluble brain protein kinase described by Miyamoto $\text{et al.}$ (1). The microtubule preparation serves as an endogenous substrate for this protein kinase; both 6S and 30S tubulin are substrates for phosphorylation to the extent of about 0.10 ± 0.05 moles/mole.     

Isolation and characterization of a unique 15 kilodalton trypanosome subpellicular microtubule-associated protein.

A protein of 15 kDa (p15) was isolated from Trypanosoma brucei subpellicular microtubules by tubulin affinity chromatography. The protein bound tubulin specifically both in its native form and after SDS-PAGE in tubulin overlay experiments. p15 promoted both the in vitro polymerization of purified calf brain tubulin and the bundling of preformed mammalian microtubules. Immunolabeling identified p15 at multiple sites along microtubule polymers comprising calf brain tubulin and p15 as well as on the subpellicular microtubules of cryosectioned trypanosomes. Antibodies directed against p15 did not cross react with mammalian microtubules. It is suggested that p15 is a trypanosome-specific microtubule-associated protein (MAP) that contributes to the unique organization of the subpellicular microtubules.     

Interaction of oncodazole (R 17934), a new antitumoral drug, with rat brain tubulin.

Oncodazole (R 17934), methyl [5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl] carbamate (I), a new synthetic drug with anti-tumoral activity, inhibits the polymerization of rat brain tubulin $\text{in vitro}$. It has no depolymerizing effect on preformed microtubules $\text{in vitro}$. Binding studies by means of molecular sieving and equilibrium dialysis indicates that the drug binds to purified rat brain tubulin in a mole to mole ratio. Finally the drug competitively inhibits colchicine binding to purified rat brain tubulin. From these results the conclusion may be drawn that oncodazole is a true microtubule inhibitor.     

Purification of tau,a microtubule-associated protein that induces assembly of microtubules from purified tubulin

Tau, a microtubule-associated protein which copurifies with tubulin through successive cycles of polymerization and depolymerization, has been isolated from tubulin by phosphocellulose chromatography and purified to near homogeneity. The purified protein is seen to migrate during electrophoresis on acrylamide gels as four closely spaced bands of apparent molecular weights between 55,000 and 62,000. Specific activity for induction of microtubule formation from purified tubulin has been assayed by quantitative electron microscopy and is seen to be enhanced three- to fourfold in the purified tau when compared with the unfractionated microtubule-associated proteins. Nearly 90% of available tubulin at 1 mg/ml is found to be polymerizable into microtubules with elevated levels of tau. Moreover, the critical concentration for polymerization of the reconstituted tau + tubulin system is seen to be a function of tau concentration and may be lowered to as little as 30 μg of tubulin per ml. Under depolymerizing conditions, 50% of the tubulin at only 1 mg/ml may be driven into ring structures. A separate purification procedure for isolation of tau directly from cell extracts has been developed and data from this purification suggest that tau is present in the extract in roughly the same proportion to tubulin as is found in microtubules purified by cycles of assembly and disassembly. Tau is sufficient for both nucleation and elongation of microtubules from purified tubulin and hence the reconstituted tau + tubulin system defines a complete microtubule assembly system under standard buffer conditions. In an accompanying paper (Cleveland et al., 1977) the physical and chemical properties of tau are discussed and a model by which tau may function in microtubule assembly is presented.     

The regulatory effect of Tau protein on polymerization of MCF7 microtubules in vitro

Growing evidence continues to point toward the critical role of beta tubulin isotypes in regulating some intracellular functions. Changes that were observed in the microtubules’ intrinsic dynamics, the way they interact with some chemotherapeutic agents, or differences on translocation specifications of some molecular motors along microtubules, were associated to their structural uniqueness in terms of beta tubulin isotype distributions. These findings suggest that the effects of microtubule associated proteins (MAPs) may also vary on structurally different microtubules. Among different microtubule associated proteins, Tau proteins, which are known as neuronal MAPs, bind to beta tubulin, stabilize microtubules, and consequently promote their polymerizations.In this study, in a set of well controlled experiments, the direct effect of Tau proteins on the polymerization of two structurally different microtubules, porcine brain and breast cancer (MCF7), were tested and compared. Remarkably, we found that in contrast with the promoted effect of Tau proteins on brain microtubules’ polymerization, MCF7 expressed a demoted polymerization while interacting with Tau proteins. This finding can potentially be a novel insight into the mechanism of drug resistance in some breast cancer cells.It has been reported that microtubules show destabilizing behavior in some MCF7 cells with overexpression of Tau protein when treated with a microtubules’ stabilizing agent, Taxol. This behavior has been classified by others as drug resistance, but it may instead be potentially caused by a competition between the destabilizing effect of the Tau protein and the stabilizing effect of the drug on MCF7 microtubules. Also, we quantified the polarization coefficient of MCF7 microtubules in the presence and absence of Tau proteins by the electro-orientation method and compared the values. The two significantly different values obtained can possibly be one factor considered to explain the effect of Tau proteins on the polymerization of MCF7 microtubules.     

Enhancement of tubulin assembly as monitored by a rapid filtration assay

The early kinetics of microtubule formation from lamb brain tubulin isolated by affinity chromatography can be followed by a newly developed filter assay. The rapid collection of microtubules on glass fiber filters permits the calculation of the moles of tubulin polymerized. The filter assay gives both a rate and extent of polymerization that are identical to those obtained by turbidity or sedimentation analysis, respectively. The microtubules trapped by the filter are readily depolymerized by cold (t12= 3 min) and slowly by colchicine (t1/2= 32min). Tubulin purified by affinity chromatography requires a high protein concentration (>4 mg/ml) for polymerization. Although 5m glycerol allows polymerization to occur at tubulin concentrations below 2 mg/ml, the maximum amount of microtubule formation is observed at low tubulin concentration when microtubule-associated proteins are present. These proteins are not retained by the affinity resin; however, they can be eluted from diethylaminoethyl-Sephadex by solutions containing 0.3m KCl. Microtubule-associated proteins enhance both the rate of polymerization and the total amount of tubulin polymerized as assessed by the filter assay, suggesting that they are involved in both initiation and elongation of microtubules.     

Analysis of microtubule polymerization inhibitors in sea urchin egg extracts: Evidence for a protease

Inhibitors of microtubule polymerization have been found in extracts of unfertilized sea urchin eggs using neural tubulin polymerization assays without glycerol. The inhibitory activity is partially destroyed by boiling or by reduction and carboxymethylation and is nondialyzable. When chromatographed on DEAE-cellulose, the inhibitory activity is eluted over a broad NaCl gradient and is in association with several peaks. This partially purified inhibitor is not destroyed by incubation with RNase A. When the partially purified inhibitor is incubated with brain microtubule protein under conditions which support microtubule polymerization, both high molecular weight-microtubule associated proteins and tubulin appear to be digested when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteolytic digestion as well as inhibition of microtubule polymerization depend upon similar concentrations of partially purified inhibitor present in the polymerization reaction. It appears as though at least part of the microtubule polymerization inhibitory activity present in unfertilized sea urchin eggs is due to this protease.     

Cellular studies reveal mechanistic differences between taccalonolide A and paclitaxel

Taccalonolide A is a microtubule stabilizer that has cellular effects almost identical to paclitaxel. However, biochemical studies show that, unlike paclitaxel, taccalonolide A does not enhance purified tubulin polymerization or bind tubulin/microtubules. Mechanistic studies aimed at understanding the nature of the differences between taccalonolide A and paclitaxel were conducted. Our results show that taccalonolide A causes bundling of interphase microtubules at concentrations that cause antiproliferative effects. In contrast, the concentration of paclitaxel that initiates microtubule bundling is 31-fold higher than its IC₅₀. Taccalonolide A’s effects are further differentiated from paclitaxel in that it is unable to enhance the polymerization of tubulin in cellular extracts. This finding extends previous biochemical results with purified brain tubulin to demonstrate that taccalonolide A requires more than tubulin and a full complement of cytosolic proteins to cause microtubule stabilization. Reversibility studies were conducted and show that the cellular effects of taccalonolide A persist after drug washout. In contrast, other microtubule stabilizers, including paclitaxel and laulimalide, demonstrate a much higher degree of cellular reversibility in both short-term proliferation and long-term clonogenic assays. The propensity of taccalonolide A to alter interphase microtubules at antiproliferative concentrations as well as its high degree of cellular persistence may explain why taccalonolide A is more potent in vivo than would be expected from cellular studies. The close linkage between the microtubule bundling and antiproliferative effects of taccalonolide A is of interest given the recent hypothesis that the effects of microtubule targeting agents on interphase microtubules might play a prominent role in their clinical anticancer efficacy.     

Cellular studies reveal mechanistic differences between taccalonolide A and paclitaxel

Taccalonolide A is a microtubule stabilizer that has cellular effects almost identical to paclitaxel. However, biochemical studies show that, unlike paclitaxel, taccalonolide A does not enhance purified tubulin polymerization or bind tubulin/microtubules. Mechanistic studies aimed at understanding the nature of the differences between taccalonolide A and paclitaxel were conducted. Our results show that taccalonolide A causes bundling of interphase microtubules at concentrations that cause antiproliferative effects. In contrast, the concentration of paclitaxel that initiates microtubule bundling is 31-fold higher than its IC₅₀. Taccalonolide A''s effects are further differentiated from paclitaxel in that it is unable to enhance the polymerization of tubulin in cellular extracts. This finding extends previous biochemical results with purified brain tubulin to demonstrate that taccalonolide A requires more than tubulin and a full complement of cytosolic proteins to cause microtubule stabilization. Reversibility studies were conducted and show that the cellular effects of taccalonolide A persist after drug washout. In contrast, other microtubule stabilizers, including paclitaxel and laulimalide, demonstrate a much higher degree of cellular reversibility in both short-term proliferation and long-term clonogenic assays. The propensity of taccalonolide A to alter interphase microtubules at antiproliferative concentrations as well as its high degree of cellular persistence may explain why taccalonolide A is more potent in vivo than would be expected from cellular studies. The close linkage between the microtubule bundling and antiproliferative effects of taccalonolide A is of interest given the recent hypothesis that the effects of microtubule targeting agents on interphase microtubules might play a prominent role in their clinical anticancer efficacy.Key words: taccalonolide, paclitaxel, microtubule stabilizer, microtubule targeted agent, tubulin, microtubule, laulimalide, antimitotic agent, drug persistence     

Combined enhancement of microtubule assembly and glucose metabolism in neuronal systems in vitro: decreased sensitivity to copper toxicity.

Brain cell-free extract greatly stimulates the polymerization rate of purified tubulin with a reduction of the nucleation period and without a significant alteration of the final assembly state. This effect is mimicked by neuroblastoma extract at 10-fold lower extract concentration, but not by excess muscle extract. Copper inhibits microtubule assembly in vitro but in the presence of brain extract the copper effect is suspended. Electron microscopic images showed that intact microtubules are formed and decorated by cytosolic proteins in the absence and presence of copper, while the copper alone induces the formation of S-shaped sheets and oligomeric threads. The flux of triosephosphate formation from glucose is enhanced by microtubules in brain extract, but not in muscle extract. Copper inhibits the glycolytic flux; however, the presence of microtubules not only suspends the inhibition by copper but the activation of glycolysis by microtubules is also preserved. We conclude that the organization of neuronal proteins modifies both the rates of microtubule assembly and glycolysis, and reduces their sensitivities against the inhibition caused by copper.     

Purification, characterization, and assembly properties of tubulin from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus

Tubulin was purified from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus by chromatography of an egg supernatant fraction on DEAE-Sephacel or DEAE-cellulose followed by cycles of temperature-dependent microtubule assembly and disassembly in vitro. After two assembly cycles, the microtubule protein consisted of the alpha- and beta-tubulins (greater than 98% of the protein) and trace quantities of seven proteins with molecular weights less than 55 000; no associated proteins with molecular weights greater than tubulin were observed. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on urea-polyacrylamide gradient gels, the alpha- and beta-tubulins did not precisely comigrate with their counterparts from bovine brain. Two-dimensional electrophoresis revealed that urchin egg tubulin contained two major alpha-tubulins and a single major beta species. No oligomeric structures were observed in tubulin preparations maintained at 0 degrees C. Purified egg tubulin assembled efficiently into microtubules when warmed to 37 degrees C in a glycerol-free polymerization buffer containing guanosine 5'-triphosphate. The critical concentration for assembly of once- or twice-cycled egg tubulin was 0.12-0.15 mg/mL. Morphologically normal microtubules were observed by electron microscopy, and these microtubules were depolymerized by exposure to low temperature or to podophyllotoxin. Chromatography of a twice-cycled egg tubulin preparation on phosphocellulose did not alter its protein composition and did not affect its subsequent assembly into microtubules. At concentrations above 0.5-0.6 mg/mL, a concentration-dependent "overshoot" in turbidity was observed during the assembly reaction. These results suggest that egg tubulin assembles into microtubules in the absence of the ring-shaped oligomers and microtubule-associated proteins that characterize microtubule protein from vertebrate brain.     

Perturbation of microtubule polymerization by quercetin through tubulin binding: a novel mechanism of its antiproliferative activity

The dietary flavonoid quercetin has a broad range of biological activities, including potent antitumor activity against several types of tumors. Recently, it has been shown that quercetin inhibits cancer cells proliferation by depleting cellular microtubules and perturbing cellular microtubule functions. However, the direct interactions of quercetin with tubulin and microtubules have not been examined so far. Here, we found that quercetin inhibited polymerization of microtubules and depolymerized microtubules made from purified tubulin in vitro. The binding of quercetin with tubulin was studied using quercetin fluorescence and intrinsic tryptophan fluorescence of tubulin. Quercetin bound to tubulin at a single site with a dissociation constant of 5-7 microM, and it specifically inhibited colchicine binding to tubulin but did not bind at the vinblastine site. In addition, quercetin perturbed the secondary structure of tubulin, and the binding of quercetin stimulated the intrinsic GTPase activity of soluble tubulin. Further, quercetin stabilized tubulin against decay and protected two cysteine residues of tubulin toward chemical modification by 5,5'-dithiobis-2-nitrobenzoic acid. Our data demonstrated that the binding of quercetin to tubulin induces conformational changes in tubulin and a mechanism through which quercetin could perturb microtubule polymerization dynamics has been proposed. The data suggest that quercetin inhibits cancer cells proliferation at least in part by perturbing microtubule functions through tubulin binding.     

Effects of vanadate on the assembly and disassembly of purified tubulin

Sodium-orthovanadate (100-700 microM) added to purified pig brain microtubule protein (molar ratios 13-90 moles vanadate/mole tubulin) inhibits to a considerable extent the assembly (up to 65%) and the disassembly rates (up to 60%) of microtubules, as determined by turbidimetry. Vanadate added to preformed microtubules did not appreciably alter the turbidity level of the samples, however, the disassembly rates were decreased in the same manner as when vanadate was added prior to polymerization. Microtubule protein kept on ice for 3-6 hours became more susceptible to vanadate than freshly prepared protein. The effect of vanadate was independent of the GTP concentration at which the polymerization assays were performed (0.025 to 1 mM GTP). In the presence of taxol, which increases the rate and extent of microtubule formation, vanadate had no effect on assembly rates. Disassembly was inhibited, however, much less than in the presence of vanadate alone. Electron microscopy and polyacrylamide gel electrophoresis did not reveal differences between microtubules prepared in the presence or in the absence of vanadate. This is consistent with the notion that vanadate does not interfere with the interaction between tubulin and the high-molecular weight microtubule-associated proteins. Apparently vanadate brings about an allosteric change of the microtubule protein(s) resulting in the abnormal polymerization kinetics of tubulin found in our study. The above results may be relevant for studies where the effects of vanadate on intracellular motility are interpreted as being solely due to a specific inhibition of ATPases.     

Tau protein function in living cells

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.     

Taxol-induced bundling of brain-derived microtubules

Taxol has two obvious effects in cells. It stabilizes microtubules and it induces microtubule bundling. We have duplicated the microtubule- bundling effect of taxol in vitro and report preliminary characterization of this bundling using electron microscopy, sedimentation, and electrophoretic analyses. Taxol-bundled microtubules from rat brain crude extracts were seen as massive bundles by electron microscopy. Bundled microtubules sedimented through sucrose five times faster than control microtubules. Electrophoretic analysis of control and taxol-bundled microtubules pelleted through sucrose revealed no striking differences between the two samples except for a protein doublet of approximately 100,000 daltons. Taxol-induced microtubule bundling was not produced by using pure tubulin or recycled microtubule protein; this suggested that taxol-induced microtubule bundling was mediated by a factor present in rat brain crude extracts. Taxol cross- linked rat brain crude extract microtubules were entirely labile to ATP in the millimolar range. This ATP-dependent relaxation was also demonstrated in a more purified system, using taxol-bundled microtubules pelleted through sucrose and gently resuspended. Although the bundling factor did not recycle with microtubule protein, it was apparently retained on isolated taxol-stabilized microtubules. The bundling factor was salt extracted from taxol-stabilized microtubules and its retained activity was demonstrated in an add-back experiment with assembled phosphocellulose-purified tubulin.     

Proteins from morphologically differentiated neuroblastoma cells promote tubulin polymerization

Clonal cells (N18) of the mouse neuroblastoma C-1300 can be induced to undergo a morphological differentiation characterized by the outgrowth of very long neurites (> 150 microns) that contain many microtubules. Because the marked increase in the number and length of microtubules is apparently not due to an increase in the concentration of tubulin subunits, the possible role of additional macromolecules in the regulation of tubulin polymerization during neurite formation by N18 cells was examined. Using an in vitro system where the polymerization of low concentrations (< 4 mg/ml) of purified brain tubulin requires microtubule-associated proteins (MAPs), high-speed supernates (250,000 g) from neuroblastoma and glioma cells were assayed for their ability to replace MAPs in the polymerization of brain tubulin. Only the supernates from "differentiated" N18 cells were polymerization competent. Electron microscope observations of these supernates failed to demonstrate the presence of nucleation structures (rings or disks). The active factor(s) sedimented at approximately 7S on sucrose gradient centrifugation and eluted from 4B Sepharose in the region of 170,000 mol wt proteins. Furthermore, the inactive supernates from other cells did not inhibit polymerization when tested in the presence of limiting MAPs. Thus, microtubule formation accompanying neurite outgrowth in neuroblastoma cells appears to be regulated by the presence of additional macromolecular factor(s) that may be functionally equivalent to the MAPs found with brain microtubules.     

Turnover of tubulin and the N site GTP in Chinese hamster ovary cells

Radioactively labeled tubulin from Chinese hamster ovary (CHO) cells can be isolated by co-polymerization with nonradioactive porcine brain microtubule protein. 75% of the soluble tubulin in CHO extracts co-polymerizes with the porcine protein through several cycles, without preferential loss of either CHO or porcine subunits. After phosphocellulose chromatography of the co-polymerized microtubules, the CHO tubulin is radiochemically homogeneous, as judged by SDS-polyacrylamide gel electrophoresis. CHO tubulin purified in this way has 1 mole of nucleotide per mole of protein noncovalently bound at the non-exchangeable or N site. This-layer chromatography indicates that the N site nucleotide is entirely ribo-GTP. Label and chase experiments show that the N site GTP exchanges intracellularly with a half-time of 33 hr in growing cells which have a generation time of 17 hr, while the tubulin polypeptides are degraded with a half-time of 48 hr. Intracellular hydrolysis of the gamma-phosphate of the N site nucleotide can be detected but occurs very slowly, with a half-time of 24 hr. These results suggest that the N site nucleotide may function in vivo as a stable structural co-factor of the tubulin molecule and render improbable the possibility that it has a regulatory role in microtubule assembly.