Distribution of glutamylated alpha and beta-tubulin in mouse tissues using a specific monoclonal antibody, GT335.

A monoclonal antibody (GT335) directed against polyglutamylated tubulin was obtained by immunization with a synthetic peptide which mimics the structure of the polyglutamylated site of alpha-tubulin. This peptide corresponds to the C-terminal sequence Glu441-Gly448 and was chemically modified by the addition of two glutamyl units at Glu445. The specificity of GT335 was assayed by direct and competitive enzyme-linked immunosorbent assay (ELISA) against tubulin and several synthetic peptides differing either by the structure of the added polyglutamyl chain or by their amino acid sequence. Further characterization was carried out by immunoblotting detection after one- or two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The epitope appears to be formed by at least two constituents: a basic motif of monoglutamylation which is retained in the polyglutamylated forms independent of their degree of glutamylation, and some elements of the polypeptide chain close to the site of glutamylation. Given the specificity of GT335 and the delineation of its epitope, our results indicate that, in addition to alpha and beta' (class III)-tubulin, other beta-tubulin isotypes are also glutamylated. This antibody has been used to analyze the cell and tissue distributions of glutamylated tubulin. In mouse brain extracts, GT335 reacts strongly with alpha-tubulin and, to a lesser extent, with beta' (class III) and beta-tubulin. The same reactivity is also observed with cultured neurons whereas astroglial cells exhibit only low levels of glutamylated tubulin. In non-nervous mouse tissues such as spleen, lung or testis, glutamylation was shown to involve only beta-tubulin, but at far lower levels than in brain.     

Tubulin-tyrosine ligase has a binding site on beta-tubulin: a two- domain structure of the enzyme

Tubulin-tyrosine ligase and alpha beta-tubulin form a tight complex which is conveniently monitored by glycerol gradient centrifugation. Using two distinct ligase monoclonal antibodies, several subunit-specific tubulin monoclonal antibodies, and chemical cross-linking, a ligase-binding site was identified on beta-tubulin. This site is retained when the carboxy-terminal domains of both tubulin subunits are removed by subtilisin treatment. The ligase-tubulin complex is also formed when ligase is added to alpha beta-tubulin carrying the monoclonal antibody YL 1/2 which binds only to the carboxyl end of tyrosinated alpha-tubulin. The beta-tubulin-binding site described here explains the extreme substrate specificity of ligase, which does not act on other cellular proteins or carboxy-terminal peptides derived from detyrosinated alpha-tubulin. Differential accessibility of this site in tubulin and in microtubules seems to explain why ligase acts preferentially on unpolymerized tubulin. Ligase exposed to V8-protease is converted to a nicked derivative. This is devoid of enzymatic activity but still forms the complex with tubulin. Gel electrophoresis documents both 30- and a 14-kD domains, each which is immunologically and biochemically distinct and seems to cover the entire molecule. The two domains interact tightly under physiological conditions. The 30-kD domain carries the binding sites for beta-tubulin and ATP. The 14-kD domain can possibly form an additional part of the catalytic site as it harbors the epitope for the monoclonal antibody ID3 which inhibits enzymatic activity but not the formation of the ligase-tubulin complex.     

Reversible polyglutamylation of alpha- and beta-tubulin and microtubule dynamics in mouse brain neurons.

The relationship between microtubule dynamics and polyglutamylation of tubulin was investigated in young differentiating mouse brain neurons. Selective posttranslational labeling with [3H]glutamate and immunoblotting with a specific monoclonal antibody (GT335) enabled us to analyze polyglutamylation of both alpha and beta subunits. Nocodazole markedly inhibited incorporation of [3H]glutamate into alpha- and beta-tubulin, whereas taxol had no effect for alpha-tubulin and a stimulating effect for beta-tubulin. These results strongly suggest that microtubule polymers are the preferred substrate for polyglutamylation. Chase experiments revealed the existence of a reversal reaction that, in the case of alpha-tubulin, was not affected by microtubule drugs, suggesting that deglutamylation of this subunit can occur on both polymers and soluble tubulin. Evidence was obtained that deglutamylation of alpha-tubulin operates following two distinct rates depending on the length of the polyglutamyl chain, the distal units (4th-6th) being removed rapidly whereas the proximal ones (1st-3rd) appearing much more resistant to deglutamylation. Partition of glutamylated alpha-tubulin isoforms was also correlated with the length of the polyglutamyl chain. Forms bearing four to six units were recovered specifically in the polymeric fraction, whereas those bearing one to three units were distributed evenly between polymeric and soluble fractions. It thus appears that the slow rate component of the deglutamylation reaction offers to neurons the possibility to maintain a basal level of glutamylated alpha-tubulin in the soluble pool independently of microtubule dynamics. Finally, some differences observed in the glutamylation of alpha- and beta-tubulin suggest that distinct enzymes are involved.     

The integrity of tubulin molecule is not required for the activity of tubulin carboxypeptidase

Tubulin dimer, alpha-tubulin subunit, and C-terminal peptides obtained from the alpha-tubulin subunit were compared in their capabilities to act as substrates of tubulin carboxypeptidase. The results obtained indicate that the enzyme does not require the beta-tubulin subunit to release tyrosine from alpha-tubulin. The 17-Kd C-terminal peptide of the alpha-tubulin subunit was obtained and it was detyrosinated at the same rate as tubulin dimer. A smaller C-terminal peptide of 2.8-3.7 Kd showed a lower capability to act as substrate. Similar results were obtained with pancreatic carboxypeptidase A. From the analysis of the results we consider that an optimal activity of the tubulin carboxypeptidase depends mainly on the accessibility of the C-terminal end of alpha-tubulin.     

Distribution of microtubules containing post-translationally modified alpha-tubulin during Drosophila embryogenesis

The distribution of microtubules (MTs) enriched in detyrosinated alpha-tubulin (Glu-tubulin) was studied in Drosophila embryos by immunofluorescence microscopy by using a monoclonal antibody (ID5) which was raised against a 14-residue synthetic peptide spanning the carboxyterminal sequence of Glu-tubulin (Wehland and Weber: J. Cell Sci. 88:185-203, 1987). While all MT arrays contained tyrosinated alpha-tubulin (Tyr-tubulin), MTs rich in Glu-tubulin were not found during early stages of development even by using an image intensification camera. Elevated levels of microtubular Glu-tubulin were first detected after CNS condensation in neurone processes. In addition, sperm tails, which remained remarkably stable inside the embryo until late stages of development, were decorated by ID5. This was in marked contrast to the distribution of microtubule arrays containing acetylated alpha-tubulin, which could already be detected during the cellular blastoderm stage. Additional experiments with taxol suggested that the absence of MTs rich in Glu-tubulin during early stages of development was not due to the rapid turnover rate of MTs, which would be too fast for alpha-tubulin to be detyrosinated. The possible significance of the differential detyrosination and acetylation of microtubules during development is discussed.     

Structure of the C-Terminal Tail of α-Tubulin: Increase of Heterogeneity from Newborn to Adult

Abstract: A combination of posttranslational modifications contributes to the high heterogeneity of brain tubulin in mammals. In this report, the structures of the detyrosinated carboxy-terminal peptides of α-tubulin from newborn and adult mouse brain were compared. The heterogeneity of these carboxy-terminal peptides was observed to increase from newborn to adult brain tubulin. The major part of this increased heterogeneity is due to the posttranslational excision of Glu⁴⁵⁰, which makes α-tubulin nontyrosinatable (Δ-2 tubulin). The structures of the polyglutamyl side chain of the bi- and triglutamylated peptides were analyzed in this work. In polyglutamylation of α-tubulin, the first glutamyl residue can only be amide-linked to the γ-carboxyl group of Glu⁴⁴⁵, but the additional residues may be linked either to the γ- or to the α-carboxyl groups of the preceding one. By optimized reverse-phase separations and comparison with synthetic peptides corresponding to all possible linkages for the biglutamylated (γ1α2, γ1γ2) and triglutamylated (γ1α2α3, γ1γ2γ3, γ1α2γ3, γ1γ2α3, γ1γ2α2) tubulin peptides, it was possible to conclude that the mode of linkage connecting the second and third additional glutamyl residues corresponds mostly to α-bond structures, for both newborn and adult mice.     

Cytosolic Carboxypeptidase 5 Removes α- and γ-Linked Glutamates from Tubulin

Cytosolic carboxypeptidase 5 (CCP5) is a member of a subfamily of enzymes that cleave C-terminal and/or side chain amino acids from tubulin. CCP5 was proposed to selectively cleave the branch point of glutamylated tubulin, based on studies involving overexpression of CCP5 in cell lines and detection of tubulin forms with antisera. In the present study, we examined the activity of purified CCP5 toward synthetic peptides as well as soluble α- and β-tubulin and paclitaxel-stabilized microtubules using a combination of antisera and mass spectrometry to detect the products. Mouse CCP5 removes multiple glutamate residues and the branch point glutamate from the side chains of porcine brain α- and β-tubulin. In addition, CCP5 excised C-terminal glutamates from detyrosinated α-tubulin. The enzyme also removed multiple glutamate residues from side chains and C termini of paclitaxel-stabilized microtubules. CCP5 both shortens and removes side chain glutamates from synthetic peptides corresponding to the C-terminal region of β3-tubulin, whereas cytosolic carboxypeptidase 1 shortens the side chain without cleaving the peptides'' γ-linked residues. The rate of cleavage of α linkages by CCP5 is considerably slower than that of removal of a single γ-linked glutamate residue. Collectively, our data show that CCP5 functions as a dual-functional deglutamylase cleaving both α- and γ-linked glutamate from tubulin.     

Amino acid sequence requirements in the epitope recognized by the alpha-tubulin-specific rat monoclonal antibody YL 1/2

We have characterized the epitope of the rat monoclonal antibody YL 1/2 in detail using synthetic peptides and several alpha-tubulin derivatives. The epitope seems to be provided by the linear sequence spanning the carboxy-terminal residues of tyrosinated alpha-tubulin. By competitive ELISA, dipeptides covering the carboxyl end could be antigenically recognized. Three sites were deduced at the dipeptide level: a negatively charged side chain in the penultimate position followed by an aromatic residue which must carry the free carboxylate group. Experiments with longer peptides point to a further negative charge provided by a carboxylate group on the third residue from the end. Thus the tripeptide Glu-Glu-Tyr was only 5-fold less active than the octapeptide spanning the carboxy-terminal alpha-tubulin sequence. The octapeptide itself showed only a 40-fold lower activity than tyrosinated alpha-tubulin. In line with the emerging epitope requirements of YL 1/2, the Escherichia coli rec A protein, the catalytic subunit of the cyclic AMP-dependent muscle protein kinase as well as performic acid-oxidized actin were recognized by YL 1/2 in immunoblots. These results thus define the sequence requirements within a probably linear epitope and give rise to some general questions concerning experiments where monoclonal antibodies are microinjected into cells in order to assess the contribution of a known antigen to cellular physiology.     

Brain and erythrocyte microtubules from chicken contain different beta-tubulin polypeptides

beta-Tubulin subunits isolated from chicken brain tissue and erythrocytes are distinguishable as unique biochemical species by electrophoretic and peptide mapping procedures. 1) The subunits of beta-tubulin exhibit major differences in electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels that vary according to the pH and ionic strength of the gel. 2) The isoelectric points of urea-denatured beta subunits from brain tissue and erythrocytes are pH 5.1 and 5.4, respectively, whereas those of both alpha subunits are approximately pH 5.2.3) Two-dimensional peptide maps prepared with alpha-chymotrypsin or V8 protease show that alpha-tubulin peptides are indistinguishable, whereas beta-tubulin peptides are very different. Only one-third of the 15 major tyrosine-containing beta-tubulin peptides prepared with alpha-chymotrypsin are common to both beta-tubulin species. The data indicate that the beta-tubulin subunits of brain tissue and erythrocytes are biochemically distinct and may be different gene products. The presence of tubulin variants in brain tissue and erythrocytes may indicate special requirements for microtubule assembly and function in different cell types.     

Polyglutamylated alpha-tubulin can enter the tyrosination/detyrosination cycle.

We have previously identified a major modification of neuronal alpha-tubulin which consists of the posttranslational addition of a varying number of glutamyl units on the gamma-carboxyl group of glutamate residue 445. This modification, called polyglutamylation, was initially found associated with detyrosinated alpha-tubulin [Eddé, B., Rossier, J., Le Caer, J.P., Desbruyères, E., Gros, F., & Denoulet, P. (1990) Science 247, 83-85]. In this report we show that a lateral chain of glutamyl units can also be present on tyrosinated alpha-tubulin. Incubation of cultured mouse brain neurons with radioactive tyrosine, in the presence of cycloheximide, resulted in a posttranslational labeling of six alpha-tubulin isoelectric variants. Because both tyrosination and polyglutamylation occur in the C-terminal region of alpha-tubulin, the structure of this region was investigated. [3H]tyrosinated tubulin was mixed with a large excess of unlabeled mouse brain tubulin and digested with thermolysin. Five peptides, detected by their radioactivity, were purified by high-performance liquid chromatography. Amino acid sequencing and mass spectrometry showed that one of these peptides corresponds to the native C-terminal part of alpha-tubulin 440VEGEGEEEGEEY451 and that the remainders bear a varying number of glutamyl units linked to glutamate residue 445, which explains the observed heterogeneity of tyrosinated alpha-tubulin. A quantitative analysis showed that the different tyrosinated forms of alpha-tubulin represent a minor (13%) fraction of the total alpha-tubulin present in the brain and that most (80%) of these tyrosinated forms are polyglutamylated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylated and detyrosinated alpha-tubulins are co-localized in stable microtubules in rat meningeal fibroblasts

We have examined the distribution of acetylated alpha-tubulin using immunofluorescence microscopy in fibroblastic cells of rat brain meninges. Meningeal fibroblasts showed heterogeneous staining patterns with a monoclonal antibody against acetylated alpha-tubulin ranging from staining of primary cilia or microtubule-organising centers (MTOCs) alone to extensive microtubule networks. Staining with a broad spectrum anti-alpha-tubulin monoclonal indicated that all cells possessed cytoplasmic microtubule networks. From double-labeling experiments using an antibody against acetylated alpha-tubulin (6-11B-1) and antibodies against either tyrosinated or detyrosinated alpha-tubulin, it was found that acetylated alpha-tubulin and tyrosinated alpha-tubulin were often segregated to different microtubules. The microtubules containing acetylated but not tyrosinated alpha-tubulin were cold stable. Therefore, it appeared that in general meningeal cells possessed two subset of microtubules: One subset contained detyrosinated and acetylated alpha-tubulin and was cold stable, and the other contained tyrosinated alpha-tubulin and was cold labile. These results are consistent with the idea that acetylation and detyrosination of alpha-tubulin are involved in the specification of stable microtubules.     

Localization and Characterization of Tubulin-Like Proteins Associated with Brain Mitochondria: The Presence of a Membrane-Specific Isoform

A mitochondrial fraction, purified from pig brain, was found to contain associated polypeptides with the same electrophoretic migration and isoelectric points as the alpha- and beta-tubulin subunits present in brain microtubules. When analyzed by Western blotting these polypeptides reacted specifically with purified tubulin antibodies. The tubulin-like proteins were then visualized in mitochondrial membranes by protein A-gold complexes after the incubation of purified mitochondria with tubulin antibodies. When membrane and microtubule proteins were compared by isoelectric focussing and two-dimensional gel electrophoresis, differences were observed in the patterns of tubulin isoforms. An additional polypeptide, with the electrophoretic migration of beta-tubulin but the isoelectric point of alpha-tubulin, was found to be enriched in the mitochondrial fraction. This peptide had several Staphylococcus aureus V8 protease peptides in common with alpha-tubulin and may result from a posttranslational modification of that subunit.     

Pig sperm tail tubulin. Its extraction and characterization

Axonemal tubulin extracted from pig sperm tails has been characterized by one- and two-dimensional electrophoresis and by one-dimensional peptide mapping. The electrophoretic mobilities of its subunits after reduction and carboxymethylation were similar to those of the major subunits of pig brain tubulin. Sperm tail tubulin subunits also had roughly the same isoelectric points as pig brain tubulin subunits, except that they appeared to have a relatively larger tailing effect. The proteolytic cleavage pattern of the pig sperm tail beta-tubulin closely resembled those of both the tunicate (Ciona intestinalis) sperm beta-tubulin and pig brain beta-tubulin. The peptide pattern of pig sperm tail alpha-tubulin, however, was more similar to that of tunicate sperm tail alpha-tubulin than to that of pig brain alpha-tubulin. This supports the hypothesis put forward in a previous investigation [1] that functionally similar tubulins from taxonomically distant species can be more related than functionally dissimilar tubulins from the same species.     

Posttranslational modification of brain tubulins from the Antarctic fish Notothenia coriiceps: reduced C-terminal glutamylation correlates with efficient microtubule assembly at low temperature

We have shown previously that the tubulins of Antarctic fish assemble into microtubules efficiently at low temperatures (-2 to +2 degrees C) due to adaptations intrinsic to the tubulin subunits. To determine whether changes in posttranslational glutamylation of the fish tubulins may contribute to cold adaptation of microtubule assembly, we have characterized C-terminal peptides from alpha- and beta-tubulin chains from brains of adult specimens of the Antarctic rockcod Notothenia coriiceps by MALDI-TOF mass spectrometry and by Edman degradation amino acid sequencing. Of the four fish beta-tubulin isotypes, nonglutamylated isoforms were more abundant than glutamylated isoforms. In addition, maximal glutamyl side-chain length was shorter than that observed for mammalian brain beta tubulins. For the nine fish alpha-tubulin isotypes, nonglutamylated isoforms were also generally more abundant than glutamylated isoforms. When glutamylated, however, the maximal side-chain lengths of the fish alpha tubulins were generally longer than those of adult rat brain alpha chains. Thus, Antarctic fish adult brain tubulins are glutamylated differently than mammalian brain tubulins, resulting in a more heterogeneous population of alpha isoforms and a reduction in the number of beta isoforms. By contrast, neonatal rat brain tubulin possesses low levels of glutamylation that are similar to that of the adult fish brain tubulins. We suggest that unique residue substitutions in the primary structures of Antarctic fish tubulin isotypes and quantitative changes in isoform glutamylation act synergistically to adapt microtubule assembly to low temperatures.     

Carboxy-terminal regions on the surface of tubulin and microtubules. Epitope locations of YOL1/34, DM1A and DM1B

Tryptic and cyanogen bromide peptides of pig brain alpha- and beta-tubulin reacting with monoclonal antibodies YOL1/34, DM1A and DM1B have been isolated and identified. They all correspond to parts of the C-terminal regions of either alpha- or beta-tubulin, and those peptides reacting with a given antibody have overlapping sequences. In the case of YOL1/34, its relatively high reactivity with small peptides suggests that many of the determinants for this antibody are within the overlapping region of these peptides comprising only nine amino acids in positions alpha 414 to 422. The smallest common region of peptides reacting with the other alpha-tubulin antibody DM1A corresponds to positions alpha 426 to 450, whereby amino acids within the positions 426 and 430 appear to be particularly important for reactivity. Since the last C-terminal residues of alpha-tubulin are also accessible to antibodies and enzymes, it seems that an extensive part (35 to 40 residues) of this very acidic C-terminal domain is exposed on the surface of native tubulin dimers. In microtubules, however, the amino-terminal end of this region appears to be less accessible, as YOL1/34 reacts poorly, if at all, with intact microtubules. All of the peptides reacting with beta-tubulin monoclonal antibody DM1B were derived from the acidic C-terminal domain and they overlapped in positions beta 416 to 430. This indicates that beta-tubulin is also positioned with at least part of its acidic C-terminal domain on the surface of microtubules, since DM1B reacts with unfixed microtubules after microinjection.     

Helicity of alpha(404-451) and beta(394-445) tubulin C-terminal recombinant peptides

We have investigated the solution conformation of the functionally relevant C-terminal extremes of alpha- and beta-tubulin, employing the model recombinant peptides RL52alpha3 and RL33beta6, which correspond to the amino acid sequences 404-451(end) and 394-445(end) of the main vertebrate isotypes of alpha- and beta-tubulin, respectively, and synthetic peptides with the alpha-tubulin(430-443) and beta-tubulin(412-431) internal sequences. Alpha(404-451) and beta(394-445) are monomeric in neutral aqueous solution (as indicated by sedimentation equilibrium), and have circular dichroism (CD) spectra characteristic of nearly disordered conformation, consistent with low scores in peptide helicity prediction. Limited proteolysis of beta(394-445) with subtilisin, instead of giving extensive degradation, resulted in main cleavages at positions Thr409-Glu410 and Tyr422-Gln423-Gln424, defining the proteolysis resistant segment 410-422, which corresponds to the central part of the predicted beta-tubulin C-terminal helix. Both recombinant peptides inhibited microtubule assembly, probably due to sequestration of the microtubule stabilizing associated proteins. Trifluoroethanol (TFE)-induced markedly helical CD spectra in alpha(404-451) and beta(394-445). A substantial part of the helicity of beta(394-445) was found to be in the CD spectrum of the shorter peptide beta(412-431) with TFE. Two-dimensional 1H-NMR parameters (nonsequential nuclear Overhauser effects (NOE) and conformational C alphaH shifts) in 30% TFE permitted to conclude that about 25% of alpha(404-451) and 40% of beta(394-451) form well-defined helices encompassing residues 418-432 and 408-431, respectively, flanked by disordered N- and C-segments. The side chains of beta(394-451) residues Leu418, Val419, Ser420, Tyr422, Tyr425, and Gln426 are well defined in structure calculations from the NOE distance constraints. The apolar faces of the helix in both alpha and beta chains share a characteristic sequence of conserved residues Ala,Met(+4),Leu(+7),Tyr(+11). The helical segment of alpha(404-451) is the same as that described in the electron crystallographic model structure of alphabeta-tubulin, while in beta(394-451) it extends for nine residues more, supporting the possibility of a functional coil --> helix transition at the C-terminus of beta-tubulin. These peptides may be employed to construct model complexes with microtubule associated protein binding sites.     

Tubulin domains probed by limited proteolysis and subunit-specific antibodies

The substructure of the tubulin molecule was studied by limited proteolysis and high affinity polyclonal antibodies specific for alpha or beta-tubulin. Brief enzymatic cleavage separates the tubulin monomer into two domains of unequal size. Trypsin splits alpha-tubulin into components with Mr values of 36 X 10(3) and 14 X 10(3), chymotrypsin splits beta-tubulin into 31 X 10(3) Mr and 20 X 10(3) Mr fragments. The cleavage occurs at Arg339 (alpha) and Tyr281 (beta), as determined by sequencing several N-terminal residues of the small domains, i.e. the small domains are the C-terminal parts of the molecules, the large ones are the N-terminal parts. There is a second cleavage site of chymotrypsin within Mr 10(3) to 2 X 10(3) of the C terminus of beta-tubulin. The fragments can be separated only under denaturing conditions. They copolymerize into microtubules and incomplete microtubule walls joined by a wall junction, forming S-shapes and hooks in cross-section. The antibodies were raised against electrophoretically purified tubulin monomers. Those produced with alpha-tubulin are directed predominantly against the large domains; they are either specific for alpha-tubulin or cross-react with the large domain of beta-tubulin. Conversely, antibodies raised against beta-tubulin are directed predominantly against the small domains (beta-specific and beta-cross-reacting fractions). Thus the antibodies discriminate not only between the tubulin chains but also between the domains generated by the proteases. The complementary antigenicity correlates well with the stability of the domains. Potential sites of antigenic determinants are located within the polypeptide chains by comparing theoretical predictions with the pattern of immunoblots. Two epitopes of the alpha-cross-reacting antibodies have been located approximately. One is very close to the C terminus (within about 20 residues), the other is close to the N terminus (within about Mr 8 X 10(3) ). The epitope of the beta-cross-reacting antibody is also located within Mr 12 X 10(3) of the C terminus. The antibodies prevent microtubule assembly and cause disassembly of preformed microtubules. A variety of breakdown products are observed by electron microscopy. They include fibres of about 10 nm width, sheets with undefined substructure, thick tapered fibrous bundles and wispy filaments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tumor cells resistant to a microtubule-depolymerizing hemiasterlin analogue, HTI-286, have mutations in alpha- or beta-tubulin and increased microtubule stability

Hemiasterlins are sponge-derived tripeptides that inhibit cell growth by depolymerizing existing microtubules and inhibiting microtubule assembly. Since hemiasterlins are poor substrates for P-glycoprotein, they are attractive candidates for cancer therapy and have been undergoing clinical trials. The basis of resistance to a synthetic analogue of hemiasterlin, HTI-286 (HTI), was examined in cell populations derived from ovarian carcinoma (A2780/1A9) cells selected in HTI-286. 1A9-HTI-resistant cells (1A9-HTI(R) series) were 57-89-fold resistant to HTI. Cross-resistance (3-186-fold) was observed to other tubulin depolymerizing drugs, with collateral sensitivity (2-14-fold) to tubulin polymerizing agents. Evaluation of the percentage of polymerized and soluble tubulin in 1A9 parental and 1A9-HTI(R) cells corroborated the HTI cytotoxicity data. At 22 degrees C or 37 degrees C, in the absence of any drug, the percentage of polymerized microtubules for each of the 1A9-HTI(R) populations was greater than that in the 1A9 parental cells, consistent with more stable microtubules. Furthermore, microtubules in the 1A9-HTI(R) populations were also more resistant to depolymerization at 4 degrees C and had more acetylated and detyrosinated (Glu-tubulin) alpha-tubulin, all characteristic of more stable microtubules. The 1A9-HTI(R) cell populations exhibited either a single nucleotide change in the M40 beta-tubulin isotype, S172A, or in two cell populations where no beta-tubulin mutation was detected, mutations in the Kalpha-1 alpha-tubulin isotype, S165P and R221H in one resistant cell population and I384V in another. Unlike reports of mutations resulting in reduced drug affinity, the experimental data and location of mutations are consistent with resistance to HTI-286 mediated by microtubule-stabilizing mutations in beta- or alpha-tubulin.