Mammalian Brain Tubulin Research Articles

Affinity purification of tubulin from plant materials.

In the plant cytoskeleton research, mammalian brain tubulin has been widely used to study plant microtubule-interacting proteins in vitro since purification of tubulins from plant sources is generally considered to be challenging and time-consuming. A convenient method for affinity purification of tubulins was devised, which utilized the TOG domains of yeast Stu2 tubulin-binding protein as an affinity ligand (Widlund et al., 2012). We showed that this so-called TOG tubulin affinity chromatography worked efficiently with plant materials, especially actively-dividing cultured cells (Hotta et al., 2016). Plant tubulins purified with the TOG method is highly assembly-competent and thus can be used in various in vitro experiments. Here, we summarize purification strategies of native or tagged plant tubulins as well as an in vitro pull-down assay to monitor their polymerization activity.

All tubulins are not alike: Heterodimer dissociation differs among different biological sources

Tubulin, the subunit of microtubules, is a noncovalent heterodimer composed of one α- and one β-tubulin monomer. Both tubulins are encoded by multiple genes or composed of different isotypes, which are differentially expressed in different tissues and in development. Tubulin αβ dimers are found throughout the eukaryotes and, although very similar, are known to differ among organisms. We seek to investigate tubulins from different tissues and different organisms for a basic physical characteristic: heterodimer stability and monomer exchange between heterodimers. We previously showed that mammalian brain tubulin heterodimers reversibly dissociate, following the mass action law. Dissociation yields native monomers that can exchange with added tubulin to form new heterodimers. Here, we compared the dissociation of tubulins from multiple sources, including mammalian (rat) brain, cultured human cells (HeLa cells), chicken brain, chicken erythrocytes, and the protozoan Leishmania We used fluorescence-detected analytical ultracentrifugation to measure tubulin dissociation over a >1000-fold range in concentration and found that tubulin heterodimers from different biological sources differ in Kd by as much as 150-fold under the same conditions. Furthermore, when fluorescent tracer tubulins from various sources were titrated with unlabeled tubulin from a single source (rat brain tubulin), heterologous dimerization occurred, exhibiting similar affinities, in some cases binding even more strongly than with autologous tubulin. These results provide additional insight into the regulation of heterodimer formation of tubulin from different biological sources, revealing that monomer exchange appears to contribute to the sorting of α- and β-tubulin monomers that associate following tubulin folding.

Stu2, the Budding Yeast XMAP215/Dis1 Homolog, Promotes Assembly of Yeast Microtubules by Increasing Growth Rate and Decreasing Catastrophe Frequency

Stu2 is the budding yeast member of the XMAP215/Dis1 family of microtubule polymerases. It is essential in cell division, allowing proper spindle orientation and metaphase chromosome alignment, as well as spindle elongation during anaphase. Despite Stu2 having a phenotype that suggests it promotes microtubule growth, like the other members of the XMAP215/Dis1 family, previous studies with purified Stu2 indicate only that it antagonizes microtubule growth. One potential explanation for these contradictory findings is that the assays were performed with mammalian brain tubulin, which may not be the right substrate to test the activity of Stu2 given that yeast and brain tubulins are quite divergent in sequence and that the vertebrate tubulins are subject to many post-translational modifications. To test this possibility, we reconstituted the activity of Stu2 with purified budding yeast tubulin. We found that Stu2 accelerated microtubule growth in yeast tubulin by severalfold, similar to the acceleration reported for XMAP215 in porcine brain tubulin. Furthermore, Stu2 accelerated polymerization in yeast tubulin to a much greater extent than in porcine brain tubulin, and the concentration of Stu2 required to reach 50% maximum activity in yeast tubulin was nearly 2 orders of magnitude lower than that in porcine brain tubulin. We conclude that Stu2 is a microtubule polymerase, like its relatives, and that its activity is considerably higher in yeast tubulin compared with mammalian brain tubulin. The biochemical properties of Stu2 reported here account for many of the phenotypes of Stu2 observed in cells.

Alp7 Potentiates Microtubule Tip Tracking by the Processive Plus End Polymerase Alp14

TOG-family polymerases track microtubule plus ends in vivo and modulate microtubule dynamics. Several different model mechanisms for the molecular mechanism of action of these proteins have been proposed (Kerssemakers et al. (2006) Nature 442:709; Al-Bassam et al (2006) JCB 172:1009; Brouhard et al. (2008) Cell 132:79). To test candidate models in more detail, we have expressed and purified Alp14 and Alp7 using a baculovirus system and reconstituted their activities in vitro using dynamically unstable microtubules built from purified single isoform (a1b) S.pombe tubulin. Alp14 deletion mutants show an in vivo defect in microtubule assembly (Sato et al. (2004) MCB 15:1609), consistent with an effect on microtubule dynamics. We find that in vitro, Alp14 accelerates the shrinkage of GMPCPP microtubules 2x and the growth of GTP-microtubules up to 10x. Changes in growth rate within a growth phase correspond to changes in the amount of Alp14 at the tip. The acceleration of growth by Alp14 causes a decrease in catastrophe frequency consistent with extension of the GTP-cap. Remarkably, although Alp14 binds to mammalian brain tubulin, it does not accelerate the growth of mammalian brain microtubules. Instead, Alp14 is competitively inhibited by mammalian brain tubulin. Tip tracking by Alp14 is tightly linked to the catalysis of microtubule growth: Alp14 loses its tip tracking ability upon the addition of 10% mammalian brain tubulin. The addition of the TACC-protein Alp7 restores the tip-tracking ability of Alp14, but not its ability to enhance the microtubule growth rate. This result is consistent with reports that Alp7 is a localization factor of Alp14 in vivo (Sato et al. (2004) MCB 15:1609). On dynamic S.pombe microtubules, Alp7 enhances the processivity of Alp14, causing sustained fast growth and correspondingly reduced catastrophe.

Differences in Paclitaxel and Docetaxel Interactions with Tubulin Detected by Mutagenesis of Yeast Tubulin

Differences in Paclitaxel and Docetaxel Interactions with Tubulin Detected by Mutagenesis of Yeast Tubulin

Antimitotic antifungal compound benomyl inhibits brain microtubule polymerization and dynamics and cancer cell proliferation at mitosis, by binding to a novel site in tubulin.

The antifungal agent benomyl [methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate] is used throughout the world against a wide range of agricultural fungal diseases. In this paper, we investigated the interaction of benomyl with mammalian brain tubulin and microtubules. Using the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid, benomyl was found to bind to brain tubulin with a dissociation constant of 11.9 +/- 1.2 microM. Further, benomyl bound to at a novel site, distinct from the well-characterized colchicine and vinblastine binding sites. Benomyl altered the far-UV circular dichroism spectrum of tubulin and reduced the accessibility of its cysteine residues to modification by 5,5'-dithiobis-2-nitrobenzoic acid, indicating that benomyl binding to tubulin induces a conformational change in the tubulin. Benomyl inhibited the polymerization of brain tubulin into microtubules, with 50% inhibition occurring at a concentration of 70-75 microM. Furthermore, it strongly suppressed the dynamic instability behavior of individual brain microtubules in vitro as determined by video microscopy. It reduced the growing and shortening rates of the microtubules but did not alter the catastrophe or rescue frequencies. The unexpected potency of benomyl against mammalian microtubule polymerization and dynamics prompted us to investigate the effects of benomyl on HeLa cell proliferation and mitosis. Benomyl inhibited proliferation of the cells with an IC(50) of 5 microM, and it blocked mitotic spindle function by perturbing microtubule and chromosome organization. The greater than expected actions of benomyl on mammalian microtubules and mitosis together with its relatively low toxicity suggest that it might be useful as an adjuvant in cancer chemotherapy.

Understanding tubulin-Taxol interactions: mutations that impart Taxol binding to yeast tubulin.

We have successfully used mutagenesis to engineer Taxol (paclitaxel) binding activity in Saccharomyces cerevisiae tubulin. Taxol, a successful antitumor agent, acts by promoting tubulin assembly and stabilizing microtubules. Several structurally diverse antimitotic compounds, including the epothilones, compete with Taxol for binding to mammalian microtubules, suggesting that Taxol and these compounds share an overlapping binding site. However, Taxol has no effect on tubulin or microtubules from S. cerevisiae, whereas epothilone does. After considering data on Taxol binding to mammalian tubulin and recent modeling studies, we have hypothesized that differences in five key amino acids are responsible for the lack of Taxol binding to yeast tubulin. After changing these amino acids to those found in mammalian brain tubulin, we observed Taxol-related activity in yeast tubulin comparable to that in mammalian tubulin. Importantly, this experimental system can be used to reveal tubulin interactions with Taxol, the epothilones, and other Taxol-like compounds.

Epothilone and paclitaxel: unexpected differences in promoting the assembly and stabilization of yeast microtubules.

Paclitaxel (Taxol) and the epothilones are antimitotic agents that promote the assembly of mammalian tubulin and stabilization of microtubules. The epothilones competitively inhibit the binding of paclitaxel to mammalian brain tubulin, suggesting that the two types of compounds share a common binding site in tubulin, despite the lack of structural similarities. It is known that paclitaxel does not stabilize microtubules formed in vitro from Saccharomyces cerevisiae tubulin; thus, it would be expected that the epothilones would not affect yeast microtubules. However, we found that epothilone A and B do stimulate the formation of microtubules from purified yeast tubulin. In addition, epothilone B severely dampens the dynamics of yeast microtubules in vitro in a manner similar to the effect of paclitaxel on mammalian microtubules. We used current models describing paclitaxel and epothilone binding to mammalian beta-tubulin to explain why paclitaxel apparently fails to bind to yeast tubulin. We propose that three amino acid substitutions in the N-terminal region and at position 227 in yeast beta-tubulin weaken the interaction of the 3'-benzamido group of paclitaxel with the protein. These results also indicate that mutagenesis of yeast tubulin could help define the sites of interaction with paclitaxel and the epothilones.

Isolation of tubulin polyglutamylase from Crithidia; binding to microtubules and tubulin, and glutamylation of mammalian brain alpha- and beta-tubulins.

Trypanosomatids have a striking cage-like arrangement of submembraneous microtubules. We previously showed that alpha- and beta- tubulins of these stable microtubules are extensively modified by polyglutamylation. Cytoskeletal microtubular preparations obtained by Triton extraction of Leishmania tarentolae and Crithidia fasciculata retain an enzymatic activity that incorporates radioactive glutamic acid in a Mg2+-ATP-dependent manner into alpha- and beta-tubulins. The tubulin polyglutamylase is extracted by 0.25 M salt. The Crithidia enzyme can be purified by ATP-affinity chromatography, glycerol-gradient centrifugation and ion-exchange chromatography. After extraction from the microtubular cytoskeleton the glutamylase forms a complex with alphabeta tubulin, but behaves after removal of tubulin as a globular protein with a molecular mass of 38x10(3). In highly enriched fractions a corresponding band is the major polypeptide visible in SDS-PAGE. The enzyme from Crithidia recognises mammalian brain tubulin, where it incorporates glutamic acid preferentially into the more acidic variants of both alpha- and beta-tubulins. Synthetic peptides with an oligoglutamyl side chain, corresponding to the carboxy-terminal end of brain alpha- and beta-tubulins, are accepted by the enzyme, albeit at low efficiency. The polyglutamylase elongates the side chain by up to 3 and 5 residues, respectively. Other properties of the tubulin polyglutamylase are also discussed.

Griseofulvin: A novel interaction with bovine brain tubulin

Griseofulvin is an anti-fungal drug whose mechanism of action is directed against microtubules. Although it inhibits the assembly of mammalian brain tubulin, its binding to tubulin has not been directly measured successfully. We have examined the interaction of griseofulvin with tubulin fluorometrically by measuring the quenching of tubulin tryptophan fluorescence by griseofulvin. From Scatchard analysis, we found that griseofulvin bound to tubulin at one class of binding site with an affinity constant of 1.26 ± 0.19 × 10 4M −1, and the binding was largely reversible. Griseofulvin caused a major change in the conformation of tubulin in that it increased the sulfhydryl titer of tubulin approximately 2-fold. The drug affected both the α and β subunits of tubulin equally. Interestingly, griseofulvin did not increase the sulfhydryl titer of the tubulin-colchicine complex although the binding site of griseofulvin was distinctly different from that of colchicine. The change of conformation of tubulin upon interaction with griseofulvin did not affect the exposure of hydrophobic areas on tubulin as shown by binding of bis-5,5′-[8( N-phenyl)aminonapthalene-1-sulfonic acid (BisANS). Even in combination with colchicine, griseofulvin had very little effect on BisANS binding to tubulin. Thus, griseofulvin appears to interact with tubulin in a manner that is very different from that of many other tubulin ligands.

Distribution of acetylated tubulin in cultured cells and tissues from the Atlantic cod ( Gadus morhua). Role of acetylation in cold adaptation and drug stability

The Atlantic cod (Gadus morhua) is a poikilothermic animal living at temperatures between 2-15°C. Isolated cod brain tubulin is, in contrast to mammalian brain tubulin, posttranslationally modified by acetylation to a high extent. To investigate the role of acetylation in cold adaptation, microtubules were isolated by a taxol-dependent procedure from different organs of the cod, and cells from different tissues were cultured. All cells from skin and brain were able to grow between 4°C and room temperature. Microtubules in the cultured cells were sometimes severed near the periphery of the cells. Microtubules in brain cells were in general more stable to vinblastine and colchicine, when compared to skin cells. Acetylated microtubules were found only in brain cells, in peripheral nerves on scales and in nerves of the intestinal tract and in microtubules isolated from neuronal tissue. Our results show that acetylated microtubules are found both in the central and peripheral nervous system, but that there is no correlation between acetylation and cold-adaptation.

Chapter 50 In Vitro Polymerization of Tubulin from Echinoderm Sperm Flagellar Microtubules

Publisher Summary The purpose of this chapter is to outline two basic methods for the purification and in vitro polymerization of tubulin from echinoderm sperm flagellar microtubules and to summarize the uses and advantages of this system. Methods were developed for the purification and assembly of tubulin and microtubule-associated proteins (MAPS), by depolymerization at 0°C and repolymerization at 37°C, in Mg 2+ /GTP-containing buffers. Relative to the basal body (minus) and the distal (plus) ends of cilia and flagella, microtubules were defined kinetically as having plus and minus ends; the plus end corresponds to the fast-growing end with a higher on rate for tubulin assembly and it is distal relative to the cell center, whereas the minus end is a slow-growing end generally associated with the centrosomes/spindle poles in both interphase and mitotic cells. Flagellar doublet microtubules could be solubilized by sonication in Mg 2+ /GTP-containing buffers at 0°C and repolymerized by elevating the temperature to 37°C; the conditions and kinetic parameters of flagellar tubulin assembly in vitro were essentially identical to those for mammalian brain tubulin. A method developed by combining the fractionation procedure used to selectively solubilize tubulin from the B-subfiber and the need to protect the GTP binding site during solubilization to answer the question of how tubulin isoforms might influence tubulin assembly and microtubule structure. The methods described in the chapter are solubilization of doublet tubules by sonication, thermal fractionation of doublet tubules into crude, soluble B-Tubulin, and cycled polymerization and depolymerization of soluble doublet tubulin. Native doublet microtubules are complex structures consisting of an A-tubule with 13 protofilaments and an incomplete B-tubule with 10 protofilaments that are attached to the A-tubule in a specific pattern. Soluble doublet tubulin polymerizes in vitro to give singlet microtubules with variable numbers of protofilaments and these microtubules are cold labile.

Use of N,N'-polymethylenebis(iodoacetamide) derivatives as probes for the detection of conformational differences in tubulin isotypes.

Mammalian brain tubulin is an alpha beta heterodimer; both alpha and beta exist in 6-7 isotypic forms which differ in their amino acid sequences. By the use of isotype-specific monoclonal antibodies, we have previously shown that we can purify the alpha beta II, alpha beta III, and alpha beta IV tubulin dimers from bovine brain. We have also observed that these isotypes differ in their distribution in vivo and their polymerization and drug-binding properties in vitro. We have now explored the question of whether the isotypically purified dimers differ in their overall conformation using as probes compounds of the N,N'-polymethylenebis (iodoacetamide) series which are known to form discrete intrachain cross-links in beta-tubulin. These compounds have the structure ICH2CONH(CH2)nNHCOCH2I. One of these cross-links, designated beta s, is between cys12 and either cys201 or cys211. The other, designated beta*, is between cys239 and cys354. The beta* cross-link forms in alpha beta II and alpha beta IV but not in alpha beta III; this is not surprising in view of the fact that alpha beta III has serine at position 239 instead of cysteine. However, alpha beta III is also unable to form the beta s cross-link, although it appears to have all three cysteines which may be involved in the cross-link. This suggests that at least one of the sulfhydryls involved in the cross-link may be inaccessible in alpha beta III. Although both alpha beta II and alpha beta IV can form the beta s cross-link, the dependence on cross-linker chain length is different. alpha beta II forms beta s with derivatives in which n = 2, 4, 5, 6, and 7 but not with those in which n = 3 or 10. In contrast, alpha beta IV forms beta s with derivatives in which n = 2, 3, 4, 5, 6, 7, and 10. These results imply that the beta s sulfhydryls are slightly more accessible in alpha beta IV and are therefore less dependent on the conformation of the cross-linker to react with it. It appears, therefore, that the alpha beta II, alpha beta III, and alpha beta IV dimers each have unique conformations. This may help to explain the different assembly and drug-binding properties of these dimers.

Kinetics of colchicine binding to purified beta-tubulin isotypes from bovine brain.

Tubulin, the constituent protein of microtubules, is an alpha beta heterodimer; both alpha and beta exist in several isotypic forms whose functional significance is not precisely known. The antimitotic alkaloid colchicine binds to mammalian brain tubulin in a biphasic manner under pseudo-first-order conditions in the presence of a large excess of colchicine (Garland, D. L. (1978) Biochemistry 17, 4266-4272). We have studied the kinetics of colchicine binding to purified beta-tubulin isotypes and find that each of the purified beta-tubulin isotypes binds colchicine in a monophasic manner. The apparent on-rate constants for the binding of colchicine to alpha beta II-, alpha beta III-, and alpha beta IV-tubulin dimers are respectively 132 +/- 5, 30 +/- 2, and 236 +/- 7 M-1 s-1. When the isotypes are mixed, the kinetics become biphasic. Scatchard analysis revealed that the isotypes differ significantly in their affinity constants (Ka) for binding colchicine. The affinity constants are 0.24 x 10(6), 0.12 x 10(6), and 3.31 x 10(6) M-1, respectively, for alpha beta II-, alpha beta III-, and alpha beta IV-tubulin dimers. Our results are in agreement with the hypothesis that the beta-subunit of tubulin plays a major role in the interaction of colchicine with tubulin. Our binding data raise the possibility that the tubulin isotypes might play important regulatory roles by interacting differently with other non-tubulin proteins in vivo, which in turn, may regulate microtubule-based functions in living cells.

Binding of [ 3H]benzimidazole carbamates to mammalian brain tubulin and the mechanism of selective toxicity of the benzimidazole anthelmintics

The binding of tritiated benzimidazole carbamates ([ 3H]BZCs) to mammalian brain tubulin was examined to investigate the kinetics of the BZC-tubulin interaction and to establish the mechanism of the selective toxicity of the BZC based anthelmintics. [ 3H]BZC binding to tubulin was markedly greater at 4° than at 37° for all ligands. The association constant ( K a ) and maximum amount of [ 3H]BZC bound ( B max) were temperature dependent for [ 3H]mebendazole ([ 3H]MBZ), [ 3H]oxibendazole ([ 3H]-OBZ) and [ 3H]oxfendazole ([ 3H]OFZ). The K a and B max values obtained for [ 3H]MBZ, [ 3H]OBZ and [ 3H]OFZ, and the comparatively weak binding of [ 3H]carbendazim, reflected the known in vitro potency of these compounds as microtubule inhibitors. Dissociation of the [ 3H]MBZ-tubulin complex was also temperature dependent, the first order dissociation rate constant being reduced by two orders of magnitude at 4° compared with that observed for 37°. These results indicate that the binding of BZCs to mammalian brain tubulin is temperature dependent and suggest that temperature induced conformational changes in the tubulin dimer influence the ability of the BZCs to form a stable BZC-tubulin complex. The temperature dependence of BZC binding and the affinity of the BZCs for mammalian tubulin are therefore unlike the BZC-tubulin interaction observed for parasitic nematodes, where optimum BZC binding occurs at 37° and results in the formation of a pseudo-irreversible complex.

Isolation of mammalian brain tubulin by amino-activated gel chromatography

In the present study, we report the isolation of the acidic structural protein tubulin using a number of amino-activated gels. Crude 100 000 g supernatant derived from sheep brain was applied to gels activated with either aminohexyl, aminoethyl, argininyl, diethylaminoethyl, lysinyl and polylysinyl residues and eluted with three distinct sequential buffer changes (pH 6.5): (i) 0.025–0.4 M morpholinoethanesulphonic acid; (ii) 0.076 and 0.379 M ammonium sulphate in 0.025 M morpholinoethanesulphonic acid; and (iii) 0.8 M sodium chloride in 0.025 M morpholinoethanesulphonic acid. Tubulin was recovered from all columns in an enriched form. However, the elution profile and purity, as judged by [ 3H] colchicine binding and electrophoresis, varied with the ligand. Hydrophobic gels, such as diethylaminoethyl and aminohexyl, required elution with high-ionic-strength buffers (0.8 M sodium chloride) and significant inhibition of [ 3H] colchicine activity resulted. This problem was avoided with the hydrophilic ligands such as arginine, polylysine and aminoethyl. Manipulation of elution conditions enabled complete elution of tubulin from arginine-activated gels in 2.5% ammonium sulphate without detectable losses of [ 3H] colchicine binding activity and with purity comparable to that achieved using diethylaminoethyl Sephacel.

Rhizoxin resistant mutants with an altered beta-tubulin gene in Aspergillus nidulans.

Rhizoxin and ansamitocin P-3 (a maytansinoid compound), potent inhibitors of mammalian brain tubulin assembly, inhibit growth of a variety of fungi including Aspergillus nidulans. Mutants of A. nidulans, benA10 which is a benomyl resistant beta-tubulin gene mutant and tubA1 which is a benomyl supersensitive alpha-tubulin gene mutant, were both sensitive to rhizoxin and ansamitocin P-3 to the same extent as wild-type strains. We isolated 18 rhizoxin resistant mutants of A. nidulans. All of these mutants were cross-resistant to ansamitocin P-3, but not to benzimidazole antimitotic drugs. These mutants mapped to two loci, rhiA and rhiB, and all of those with high resistance mapped to rhiA. The fact that the protein extracts of rhiA mutants lost rhizoxin binding affinity and that rhiA was closely linked to benA, the major beta-tubulin gene in A. nidulans, indicated that rhiA must be a structural gene for beta-tubulin and that rhiA mutants are a new class of beta-tubulin gene mutants. All of this suggested that, in A. nidulans, these antimitotic drugs bind to beta-tubulin, and that rhizoxin and ansamitocin P-3 share the same binding site but the site does not overlap with the benzimidazole binding site. Protein extracts from a rhiB mutant retained rhizoxin binding affinity, therefore this rhizoxin resistance mechanism should not be a tubulin mediated process.

Comparison of the properties of tubulin from Nippostrongylus brasiliensis with mammalian brain tubulin

Tubulin was estimated to account for 16.3% and 0.25% of protein in rat brain and Nippostrongylus brasiliensis supernatants, respectively. Tubulin from N. brasiliensis and rat brain have been partially purified using polylysine agarose chromatography and high performance liquid chromatography on a gel permeation column. Western blots with α- and β-tubulin monoclonal antibodies confirmed the presence of tubulin in different fractions. The mobility of N. brasiliensis tubulin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was similar to that of rat brain tubulin. The isoelectric range for N. brasiliensis α- and β-tubulin isoforms was pH 5.4-4.8 and pH 4.8-4.7, respectively. However, for rat brain the corresponding ranges were pH 5.4-4.9 and pH 5.0-4.6, respectively. Western blots with anti-tubulin monoclonal antibodies revealed 8 isoforms of α-tubulin and 3 isoforms of β-tubulin for N. brasiliensis and 14–15 and 7–8 isoforms for rat brain α- and β-tubulins, respectively. Different peptide maps were obtained for N. brasiliensis tubulin compared with rat brain tubulin.

A monoclonal antibody against the type II isotype of beta-tubulin. Preparation of isotypically altered tubulin.

Mammalian brain tubulin consists of several isotypes of alpha and beta subunits that separate on polyacrylamide gels into three electrophoretic classes, designated alpha, beta 1, and beta 2. It has not been possible hitherto to resolve the different isotypes in a functional form. To this end, we have now isolated a monoclonal antibody, using as an immunogen a chemically synthesized peptide corresponding to the carboxyl-terminal sequence of the major tubulin isotype (type II) found in the beta 1-tubulin electrophoretic fraction. The antibody binds to beta 1 but not to alpha or beta 2. When pure tubulin from bovine brain is passed through an immunoaffinity column made from the anti-type II antibody, the tubulin that elutes in the unbound fraction is enriched greatly for the beta 2 electrophoretic variant. The tubulin that binds to the column appears to contain only alpha and beta 1, not beta 2. When these tubulin fractions are characterized by immunoblotting using the anti-type II antibody, the antibody binds only to the beta 1 band in the bound fraction, not to the beta 1 band in the unbound fraction. Using polyclonal antibodies generated against the carboxyl-termini of types I, III, and IV, we demonstrate that the beta 1 electrophoretic species is comprised of isotypes I, II, and IV, whereas the beta 2 variant is comprised exclusively of type III beta-tubulin. Further, we calculate that beta-tubulin in purified bovine brain tubulin is comprised of 3% type I, 58% type II, 25% type III, and 13% type IV tubulins.

Incorporation and turnover of labeled exogenous tubulin in the mitotic spindles of Chaetopterus oocytes and HeLa cells.

The incorporation of tubulin into mitotic spindles in situ was studied by incubating permeabilized mitotic cells in solutions containing [3H]GTP-labeled or dichlorotriazinylamino fluorescein (DTAF)-labeled tubulin. Metaphase HeLa cells or spindle-containing "minicells" from Chaetopterus oocytes were lysed in a microtubule-assembly buffer plus 0.5% Nonidet P-40, 1 mg/ml 120,000g supernatant mammalian brain tubulin, and [3H]GTP. After different periods of incubation, mitotic spindles were isolated in 2 M-glycerol-containing assembly buffer and separated from unbound counts by centrifugation through a 4 M-glycerol cushion; 3H counts per mg protein increase linearly for 8-12 min and then reach a plateau or steady state in both Chaetopterus oocytes and HeLa cells. Addition of 4 mM CaCl2 blocks or reverses incorporation. Little or no [3H]GTP is incorporated if exogenous tubulin or lysed cells are omitted from the assembly mixture. To measure the loss rate of [3H]GTP-tubulin from mitotic spindles, cells were incubated in tubulin plus [3H]GTP for 30 min, and a 20-fold excess of cold GTP (2 mM) was added. Samples were removed after incubation for different periods, and spindles were isolated as described above and counted for 3H content. [3H]GTP is lost from spindles at a rate of about 16%/min until a new steady state is reached in about 8 min. These results are consistent with an incorporation and turnover of [3H]GTP-tubulin in spindle microtubules of these lysed-cell models. The location of this newly incorporated tubulin in the spindle was investigated by incorporating fluorescent DTAF-tubulin into mitotic spindles of these lysed cell types.(ABSTRACT TRUNCATED AT 250 WORDS)