Identification of Ncd tail domain-binding sites on the tubulin dimer

The Drosophila non-claret disjunctional (Ncd) kinesin-like protein is required for spindle assembly in oocytes and spindle maintenance in early embryos. Through the action of ATP-dependent microtubule (MT)-binding sites in the head and ATP-independent MT-binding sites in the tail, Ncd may bundle and, perhaps, slide MTs relative to each other. Our previous work on the MT-binding site of the Ncd tail domain demonstrated that this site, like the MT-binding sites of tau, contains basic residues flanked by proline residues and can promote MT assembly and stability. Here, we characterize the interactions of a monomeric Ncd tail protein with subtilisin-digested MTs in order to identify sites on the tubulin dimer that interact with the Ncd tail. The results provide evidence for four such binding sites per tubulin dimer and support the hypothesis that each binding site consists of a cluster of acidic residues in the C-terminal regions of alpha- and beta-tubulin.     

Tubulin isotypes and their interaction with microtubule associated proteins

Summary To assay the functional significance of the multiple but closely related - and -tubulin polypeptides (termed isotypes) that are expressed in mammalian cells, we have generated a number of sera that uniquely discriminate among these isotypes. These sera have been used to demonstrate that there is no subcellular sorting of either - or -tubulin isotypes among microtubules of diverse function, either in cells growing in culture or in tissues consisting of cell types that contain specialized kinds of microtubule. In spite of this failure to segregate between functionally distinct kinds of microtubule, the fact that isotype-specific amino acid sequences have been strictly conserved over extensive periods of evolutionary time argues persuasively for a functional role for the different tubulin gene products. One possibility is that they are required for specific interactions with microtubule associated proteins (MAPs), and that tubulin isotypes have coevolved with different cell type-specific MAPs with which they must interact. We have tested this hypothesis by examining the distribution of -tubulin isotypes in mammalian cerebellum in relationship to the known patterns of expression of a number of MAPs, and find that these patterns correlate in the case of M 2 and MAP 3, and M 6 and MAP 1 a. These data, plus emerging data based on a structural analysis of tau, MAP 1 b and MAP 2 obtained via sequence determination of cloned cDNAs, are discussed in terms of the possible functional significance of tubulin isotype/MAP interactionsin vivo.     

Tubulin subunit sorting: Analysis of the mechanisms involved in the segregation of unique tubulin isotypes within microtubule copolymers

Summary Vertebrate cells contain biochemical and genetic isotypes of tubulin which are expressed in unique combinations in different tissues and cell types. To determine if mixtures of tubulin isotypes assemblein vitro to form different classes of microtubules, we analyzed the composition of microtubule copolymers assembled from mixtures of chicken brain and erythrocyte tubulin. During microtubule elongation brain tubulin assembled onto the ends of microtubules faster than erythrocyte tubulin, resulting in copolymers with continually changing ratios of isotypes along their lengths. Unlike examples of microtubule assembly where the rate of polymerization depends on the association rate constant (k⁺) and the subunit concentration, the rate and extent of sorting in copolymers appear to depend on the dissociation rate constant (k^–), which governs the rate at which subunits are released from tubulin oligomers and microtubules and thereby made available for reassembly into copolymers. The type of microtubule seed used to initiate elongation was also found to influence the composition of copolymers, indicating that polymerization favors association of subunits of the same isotype.     

The phylogenetic distribution of tubulin:Tyrosine ligase

Summary The post-translational addition of tyrosine toa-tubulin, catalyzed by tubulin:tyrosine ligase, has been previously reported in mammals and birds. The present study demonstrated that significant ligase activity was present in representative organisms from several other major vertebrate classes (chondrichthyes through reptiles) and that both substrate and enzyme from all vertebrates investigated were compatible with mammalian ligase and tubulin in the tyrosination reaction. None of the invertebrate tissues examined showed incorporation of tyrosine, phenylalanine or dihydroxyphenylalanine intoa tubulin under conditions allowing significant incorporation of these compounds in vertebrate supernatant samples. The failure of invertebrate tubulin to incorporate tyrosine in vitro did not appear to be due to saturation of the carboxyl terminal position with tyrosine or the presence of a soluble inhibitor of ligase activity.Although tubulin amino acid composition has been highly conserved throughout evolution, a major evolutionary divergence is described based upon biochemical differences whereby invertebrate tubulin cannot be tyrosinated or posttranslationally modified with phenylalanine or dihydroxyphenylalanine under conditions suitable for the incorporation of these compounds by vertebratea tubulin.     

Why do tubulin gene families lack diversity in flagellate/ciliate protists?

Summary Considerable amino acid sequence diversity is found among tubulin isotypes encoded by tubulin gene families in animal, higher plant, and fungal systems. In contrast, relatively little diversity is found among the isotypes produced by the gene families in a number of flagellate or ciliate protists. It is possible that proper assembly of the axoneme requires a homogeneous pool of tubulin subunits and that the axoneme thus provides a stringent selection against amino acid replacement substitutions among tubulin genes in these systems.     

Direct interaction between prion protein and tubulin

Recently published data show that the prion protein in its cellular form (PrP(C)) is a component of multimolecular complexes. In this report, zero-length cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) allowed us to identify tubulin as one of the molecules interacting with PrP(C) in complexes observed in porcine brain extracts. We found that porcine brain tubulin added to these extracts can be cross-linked with PrP(C). Moreover, we observed that the 34 kDa species identified previously as full-length diglycosylated prion protein co-purifies with tubulin. Cross-linking of PrP(C) species separated by Cu(2+)-loaded immobilized metal affinity chromatography confirmed that only the full-length protein but not the N-terminally truncated form (C1) binds to tubulin. By means of EDC cross-linking and cosedimentation experiments, we also demonstrated a direct interaction of recombinant human PrP (rPrP) with tubulin. The stoichiometry of cosedimentation implies that rPrP molecules are able to bind both the alpha- and beta-isoforms of tubulin composing microtubule. Furthermore, prion protein exhibits higher affinity for microtubules than for unpolymerized tubulin.     

Taxanes with high potency inducing tubulin assembly overcome tumoural cell resistances

We have found that four taxanes with chemical modifications at positions C10 and C13 were active against all types of taxane resistant cell lines, resistant by P-gp overexpression, by mutations in the β-tubulin binding site or by overexpression of the highly dynamic βIII-tubulin isotype.We have characterized the interaction of taxanes with high activity on chemotherapy resistant tumoural cells with microtubules, and also studied their cellular effects. The biochemical property enhanced in comparison with other taxanes is their potency at inducing tubulin assembly, despite the fact that their interactions with the microtubule binding sites (pore and luminal) are similar as studied by NMR and SAXS. A differential interaction with the S7–S9 loop (M-loop) is responsible for their enhanced assembly induction properties. The chemical changes in the structure also induce changes in the thermodynamic properties of the interaction, indicating a higher hydrophilicity and also explaining their properties on P-gp and βIII overexpressing cells and on mutant cells.The effect of the compounds on the microtubular network is different from those observed with the classical (docetaxel and paclitaxel) taxanes, inducing different bundling in cells with microtubules being very short, indicating a very fast nucleation effect and reflecting their high assembly induction power.     

New insights into microtubule structure and function from the atomic model of tubulin

The structure of tubulin has recently been solved by electron crystallography of zinc-induced tubulin sheets. Because tubulin was studied in a polymerized state, the model contains information on the interactions between monomers that give rise to the αβ dimer as well as contacts between adjacent dimers that result in the structure of the protofilament. The model includes the binding site of taxol, an anti-cancer agent that acts by stabilizing microtubules. The present tubulin model gives the first structural framework for understanding microtubule polymerization and its regulation by nucleotides and anti-mitotic drugs at the molecular level. Received: 15 December 1997 / Revised version: 25 January 1998 / Accepted: 2 February 1998     

A 2-step synthesis of Combretastatin A-4 and derivatives as potent tubulin assembly inhibitors

A series of combretastatin derivatives were designed and synthesised by a two-step stereoselective synthesis by use of Wittig olefination followed by Suzuki cross-coupling. Interestingly, all new compounds (2a-2i) showed potent cell-based antiproliferative activities in nanomolar concentrations. Among the compounds, 2a, 2b and 2e were the most active across three cancer cell lines. In addition, these compounds inhibited the polymerisation of tubulin in vitro more efficiently than CA-4. They caused cell cycle arrest in G₂/M phase further confirming their ability to inhibit tubulin polymerisation.     

Prion protein inhibits microtubule assembly by inducing tubulin oligomerization

A growing body of evidence points to an association of prion protein (PrP) with microtubular cytoskeleton. Recently, direct binding of PrP to tubulin has also been found. In this work, using standard light scattering measurements, sedimentation experiments, and electron microscopy, we show for the first time the effect of a direct interaction between these proteins on tubulin polymerization. We demonstrate that full-length recombinant PrP induces a rapid increase in the turbidity of tubulin diluted below the critical concentration for microtubule assembly. This effect requires magnesium ions and is weakened by NaCl. Moreover, the PrP-induced light scattering structures of tubulin are cold-stable. In preparations of diluted tubulin incubated with PrP, electron microscopy revealed the presence of approximately 50 nm disc-shaped structures not reported so far. These unique tubulin oligomers may form large aggregates. The effect of PrP is more pronounced under the conditions promoting microtubule formation. In these tubulin samples, PrP induces formation of the above oligomers associated with short protofilaments and sheets of protofilaments into aggregates. Noticeably, this is accompanied by a significant reduction of the number and length of microtubules. Hence, we postulate that prion protein may act as an inhibitor of microtubule assembly by inducing formation of stable tubulin oligomers.     

NMR assignment of PN2-3, a tubulin interaction subdomain of the CPAP protein

We report the NMR assignment of the PN2-3 subdomain of the CPAP protein. It has been previously shown that this motif interacts with tubulin, inhibits microtubule nucleation from the centrosome and depolymerizes taxol-stabilized microtubules. Marie-Jeanne Clément and Philippe Savarin contributed equally.     

Turbidity as a probe of tubulin polymerization kinetics: a theoretical and experimental re-examination

We report here an examination of the validity of the experimental practice of using solution turbidity to study the polymerization kinetics of microtubule formation. The investigative approach proceeds via numerical solution of model rate equations to yield the time dependence of each microtubule species, followed by the calculation of the time- and wavelength-dependent turbidity generated by the calculated distribution of rod lengths. The wavelength dependence of the turbidity along the time course is analyzed to search for generalized kinetic regimes that satisfy a constant proportionality relationship between the observed turbidity and the weight concentration of polymerized tubulin. An empirical analysis, which permits valid interpretation of turbidity data for distributions of microtubules that are not long relative to the wavelength of incident light, is proposed. The basic correctness of the simulation work is shown by the analysis of the experimental time dependence of the turbidity wavelength exponent for microtubule formation in taxol-supplemented 0.1 M Pipes buffer (1 mM GTP, 1 mM EGTA, 1 mM MgSO4, pH 6.4). We believe that the general findings and principles outlined here are applicable to studies of other fibril-forming systems that use turbidity as a marker of polymerization progress.     

Colchicine-binding activity of carrot tubulin at different culture age

Tubulin contents in the extract from cultured carrot cells at different growth phases were investigated by measuring colchicine-binding activity. The addition of vinblastine and dithiothreitol to the reaction mixture appreciably improved the stability of both free and colchicine-bound tubulins. Colchicine-binding activity in the cell extract obtained from stationary phase was more labile than that from log phase though the extract showed higher affinity to colchicine. After purification, however, tubulin from the cells at different growth phases showed the same affinity and its colchicine-binding activity was much more stable than in crude extract. The colchicine-binding activity in the crude extract was corrected for the decay during measurement and apparent difference in the affinity so that the activity in the cells containing different kind and amount of interefering substances could be compared. The corrected amount of colchicine that binds to the 100,000×g extract was 46 pmol/10⁵ cells at log phase. It decreased with the progression of culture age from linear to stationary phase. Combining the data with the morphological observation, it was suggested that the log phase cells contained larger free tubulin pool than the linear or stationary phase cells.     

Evidence for a distinct ligand binding site on tubulin discovered through inhibition by GDP of paclitaxel-induced tubulin assembly in the absence of exogenous GTP

GDP inhibits paclitaxel-induced tubulin assembly without GTP when the tubulin bears GDP in the exchangeable site (E-site). Initially, we thought inhibition was mediated through the E-site, since small amounts of GTP or Mg²⁺, which favors GTP binding to the E-site, reduced inhibition by GDP. We thought trace GTP released from the nonexchangeable site (N-site) by tubulin denaturation was required for polymer nucleation, but microtubule length was unaffected by GDP. Further, enhancing polymer nucleation reduced inhibition by GDP. Other mechanisms involving the E-site were eliminated experimentally. Upon finding that ATP weakly inhibited paclitaxel-induced assembly, we concluded that another ligand binding site was responsible for these inhibitory effects, and we found that GDP was not binding at the taxoid, colchicine, or vinca sites. There may therefore be a lower affinity site on tubulin to which GDP can bind distinct from the E- and N-sites, possibly on α-tubulin, based on molecular modeling studies.     

Dependency of microtubule-associated proteins (MAPS) for tubulin stability and assembly; use of estramustine phosphate in the study of microtubules

Summary Microtubule-associated proteins (MAPS) were separated from tubulin with several different methods. The ability of the isolated MAPs to reinduce assembly of phosphocellulose purified tubulin differed markedly between the different methods. MAPs isolated by addition of 0.35 M NaCl to taxol-stabilized microtubules stimulated tubulin assembly most effectively, while addition of 0.6M NaCl produced MAPs with a substantially lower ability to stimulate tubulin assembly. The second best preparation was achieved with phosphocellulose chromatographic separation of MAPs with 0.6 M NaCl elution.The addition of estramustine phosphate to microtubules reconstituted of MAPS prepared by 0.35 M NaCl or phosphocellulose chromatography, induced less disassembly than for microtubules assembled from unseparated proteins, and was almost without effect on microtubules reconstituted from MAPs prepared by taxol and 0.6 M NaCl. Estramustine phosphate binds to the tubulin binding part of the MAPs, and the results do therefore indicate that the MAPs are altered by the separation methods. Since the MAPs are regarded as highly stable molecules, one probable alteration could be aggregation of the MAPs, as also indicated by the results. The purified tubulin itself seemed not to be affected by the phosphocellulose purification, since the microtubule proteins were unchanged by the low buffer strenght used during the cromatography. However, the assembly competence after a prolonged incubation of the microtubule proteins at 4° C was dependent on intact bindings between the tubulin and MAPs.Abbreviations Pipes 1,4-Piperazinediethanesulfonic acid - EDTA Ethylenedinitrilo Tetraacetic Acid - MAPs Microtubule-Associated Proteins - SDS-PAGE SDS-Polyacrylamide Gel Electrophoresis     

Tubulin evolution: Ciliate-specific epitopes are conserved in the ciliary tubulin of metazoa

Summary In spite of their overall evolutionary conservation, the tubulins of ciliates display electrophoretic and structural particularities. We show here that antibodies raised againstParamecium andTetrahymena ciliary tubulins fail to recognize the cytoplasmic tubulins of all the metazoans tested. Immunoblotting of peptide maps of ciliate tubulins reveals that these antibodies react with one or very few ciliate-specific epitopes, in contrast to polyclonal antibodies against vertebrate tubulins, which are equivalent to autoantibodies and recognize several epitopes in both ciliate and vertebrate tubulins. Furthermore, we show that the anti-ciliate antibodies recognize ciliary and flagellar tubulins of metazoans ranging from sea urchin to mammals (with the exception of humans). The results support the conclusion that although duplication and specialization of tubulin genes in metazoans may have led to distinct types of tubulins, the axonemal one has remained highly conserved.     

Relationship between DNA synthesis and the increase in the level of tubulin during dedifferentiation of isolatedZinnia mesophyll cells

Summary The level of tubulin in cultured matureZinnia mesophyll cells increases to between 4 and 5 times its initial value when DNA synthesis occurs. The rapid increase in the level of tubulin requires the presence of auxin and cytokinin, as does the induction of DNA synthesis. Inhibitors of DNA synthesis suppress the rapid increase in the level of tubulin. These results imply that the rapid increase in the level of tubulin is dependent on the occurrence of DNA synthesis,i.e., on the reinitiation of the cell cycle. The presence of microtubules is not important for either the increase in the level of tubulin or for the induction of DNA synthesis, because both the increase in the level of tubulin and DNA synthesis occur even when microtubules are depolymerized by colchicine.Abbreviations APM amiprophos-methyl - araC arabinosylcytosine - BA 6-benzyladenine - BSA bovine serum albumin - DMSO dimethylsulfoxide - EGTA ethylene glycol bis(2-aminoethylether)tetraacetic acid - FITC fluorescein isothiocyanate - FUdR fluorodeoxyuridine - GA gibberellin A₃ - IAA indole-3-acetic acid - MES 2-(N-morpholino)ethanesulfonic acid - NAA 1-naphthaleneacetic acid - PBS phosphate buffered saline - PCA perchloric acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - SDS sodium dodecyl sulfate     

Direct interaction of Bcl-2 proteins with tubulin

A direct interaction between tubulin and several pro-apoptotic and anti-apoptotic members of the Bcl-2 family has been demonstrated by effects on the assembly of microtubules from pure rat brain tubulin. Bcl-2, Bid, and Bad inhibit assembly sub-stoichiometrically, whereas peptides from Bak and Bax promote tubulin polymerization at near stoichiometric concentrations. These opposite effects on microtubule assembly are mutually antagonistic. The BH3 homology domains, common to all members of the family, are involved in the interaction with tubulin but do not themselves affect polymerization. Pelleting experiments with paclitaxel-stabilized microtubules show that Bak is associated with the microtubule pellet, whereas Bid remains primarily with the unpolymerized fraction. These interactions require the presence of the anionic C-termini of alpha- and beta-tubulin as they do not occur with tubulin S in which the C-termini have been removed. While in no way ruling out other pathways, such direct associations are the simplest potential regulatory mechanism for apoptosis resulting from disturbances in microtubule or tubulin function.     

Time and cell cycle dependent formation of heterogeneous tubulin arrays induced by colchicine in Triticum aestivum root meristem

We have investigated the appearance and reorganization of tubulin-containing arrays induced by colchicine in the root meristem of wheat Triticum aestivum, using immunostaining and electron microscopy. Colchicine caused depolymerization of microtubules and formation of tubulin cortical strands composed of filamentous material only in C-mitotic cells. After prolonged exposure to the drug, both interphase and C-mitotic cells acquired needle-type bundles, arranged as different crystalloids and/or macrotubules. The unmodified tyrosinated form of alpha-tubulin was detected within microtubules in control cells, but was not found within cortical strands. It was identified, however, within needle-type bundles. The modified acetylated form of alpha-tubulin, which was absent in control cells, was detected within needle-type bundles. Thus, cortical strands were transitory arrays, transformed into needle-type bundles during prolonged exposure to colchicine. Cortical strands appeared in a cell cycle-dependent manner, whereas needle-type bundles were cell cycle stable arrays. The diverse morphological organization, intracellular distribution and stability of tubulin-containing arrays may be associated with heterogeneity of alpha-tubulin isoforms. We assume that non-microtubular arrays substitute for microtubules in conditions where normal tubulin polymerization is inhibited.