Transfer and amplification of a mutant beta-tubulin gene results in colcemid dependence: use of the transformant to demonstrate regulation of beta-tubulin subunit levels by protein degradation.

Total genomic DNA from a temperature-sensitive, colcemid-resistant Chinese hamster ovary (CHO) cell mutant expressing an electrophoretic variant beta-tubulin was used to transform wild-type CHO cells to colcemid-resistant cells at 37 degrees C. Southern blot analysis of the transformant demonstrated the three- to fivefold amplification of one of many beta-tubulin sequences compared with that of the wild type or mutant, thereby identifying a functional tubulin gene in CHO cells. This amplification of one tubulin-coding sequence resulted in a threefold increase in two beta-tubulin mRNA species, suggesting that both species may be encoded by a single gene. Pulse-chase experiments showed that in the transformant, total beta-tubulin was synthesized and degraded faster than in the revertant or wild-type cells, so that the steady-state levels of beta-tubulin and alpha-tubulin were unchanged in the transformant compared with those of wild-type, mutant, or revertant cells. Increased ratios of mutant to wild-type beta-tubulin made the transformant dependent on microtubule-depolymerizing drugs for growth at 37 but not 34 degrees C and supersensitive to the microtubule-stabilizing drug taxol at 34 degrees C.     

Characterization and Intracellular Distribution of Microtubule-Associated Protein 2 in Differentiating Human Neuroblastoma Cells

The use of a panel of monoclonal antibodies (mAbs) directed against different determinants of microtubule-associated protein 2 (MAP2) enabled us to identify two distinct high-molecular-mass MAP2 species (270 and 250 kDa) and a substantial amount of MAP2c (70 kDa) in human neuroblastoma cells. The 250-kDa MAP2 species appears to be confined to the human neuroblastoma cells and was not observed in microtubules (MTs) from bovine and rat brain, mouse neuroblastoma, or MTs from human cerebellum. A new overlay method was developed, which demonstrates binding of tubulin to human neuroblastoma high-molecular-mass MAP2 by exposing nitrocellulose-bound MT proteins under polymerization conditions to tubulin. Bound tubulin was detected with a mAb directed against beta-tubulin. The binding of tubulin to MAP2 could be abolished by a peptide homologous to positions 426-445 of the C-terminal region of beta-tubulin. Immunological cross-reactivity with several mAbs directed against bovine brain MAP2, taxol-promoted coassembly into MTs, and immunocytochemical visualization within cells were further criteria utilized to characterize these proteins as true MAPs. Indirect immunofluorescence with anti-MAP2 and anti-beta-tubulin mAbs demonstrated that there is a change in the spatial organization of MTs during induced cell differentiation, as indicated by the appearance of MT bundles and the redistribution of MAP2.     

Both carboxy-terminal tails of alpha- and beta-tubulin are essential, but either one will suffice

Microtubules (MTs) are organized into distinct systems essential for cell shape, movement, intracellular transport, and division. Electron crystallographic analyses provide little information about how MTs produce diverse structures and functions, perhaps because they failed to visualize the last 10 residues of the alpha- and the last 18 of the beta-tubulin C-terminal tails (CTTs), which likely play a role in MT diversity. CTTs define conserved, nonallelic isotypes in mammals, are major sites of posttranslational modifications (PTMs), are binding sites for microtubule-associated proteins (MAPs), and determine MT motor processivity. Using mutagenesis and homologous gene replacement in Tetrahymena thermophila, we analyzed mutations, deletions, tail switches, and tail duplications of alpha- and beta-tubulin CTTs. We demonstrate that a tail is required for the essential function of both alpha- and beta-tubulin. However, the two tails are interchangeable, and cells grow normally with either an alpha or a beta tail on both tubulins. In addition, an alpha gene containing a duplicated alpha C terminus rescues a lethal mutant lacking all known posttranslational modification sites on the beta C terminus but cannot rescue deletion of the beta tail. Thus, tubulin tails have a second essential function that is not associated with posttranslational modification.     

A novel acetylation of β-tubulin by San modulates microtubule polymerization via down-regulating tubulin incorporation

Dynamic instability is a critical property of microtubules (MTs). By regulating the rate of tubulin polymerization and depolymerization, cells organize the MT cytoskeleton to accommodate their specific functions. Among many processes, posttranslational modifications of tubulin are implicated in regulating MT functions. Here we report a novel tubulin acetylation catalyzed by acetyltransferase San at lysine 252 (K252) of β-tubulin. This acetylation, which is also detected in vivo, is added to soluble tubulin heterodimers but not tubulins in MTs. The acetylation-mimicking K252A/Q mutants were incorporated into the MT cytoskeleton in HeLa cells without causing any obvious MT defect. However, after cold-induced catastrophe, MT regrowth is accelerated in San-siRNA cells while the incorporation of acetylation-mimicking mutant tubulins is severely impeded. K252 of β-tubulin localizes at the interface of α-/β-tubulins and interacts with the phosphate group of the α-tubulin-bound GTP. We propose that the acetylation slows down tubulin incorporation into MTs by neutralizing the positive charge on K252 and allowing tubulin heterodimers to adopt a conformation that disfavors tubulin incorporation.     

Yeast Num1p associates with the mother cell cortex during S/G2 phase and affects microtubular functions

The NUM1 gene is involved in the control of nuclear migration in Saccharomyces cerevisiae. The content of NUM1 mRNA fluctuates during the cell cycle, reaching a maximum at S/G2 phase, and the translation product Num1p associates with the cortex of mother cells mainly during S, G2, and mitosis, as seen by indirect immunofluorescence. The nuclear spindle in NUM1-deficient large-budded cells often fails to align along the mother/bud axis, while abnormally elongated astral microtubules emanate from both spindle pole bodies. A num1 null mutation confers temperature sensitivity to the cold-sensitive alpha-tubulin mutant tub1- 1, and shows synthetic lethality with the beta-tubulin mutant alleles tub2-402, tub2-403, tub2-404, and tub2-405. Deletion mapping has defined three functionally important Num1p regions: a potential EF hand Ca2+ binding site, a cluster of potential phosphorylation sites and a pleckstrin homology domain. The latter domain appears to be involved in targeting Num1p to the mother cell cortex. Our data suggest that the periodically expressed NUM1 gene product controls nuclear migration by affecting astral microtubule functions.     

Paclitaxel resistance in cells with reduced beta-tubulin

We previously described the isolation of colcemid resistant Chinese hamster ovary cell lines containing alpha- and beta-tubulin mutations that increase microtubule assembly and stability. By analyzing colcemid sensitive revertants from one of the beta-tubulin mutants, we now find that loss or inactivation of the mutant allele represents the most common mechanism of reversion. Consistent with this loss, the revertants have 35% less tubulin at steady state, no evidence for the presence of a mutant polypeptide, and a normal extent of tubulin polymerization. In addition to the loss of colcemid resistance, the revertant cells exhibit increased resistance to paclitaxel relative to wild-type cells. This paclitaxel resistance can be suppressed by transfecting the revertant cells with a cDNA for wild-type beta-tubulin, indicating that the reduction in tubulin in the revertant cells is responsible for the resistance phenotype. We propose that reducing tubulin levels may represent a novel mechanism of paclitaxel resistance.     

Tubulin composition and microtubule nucleation of a griseofulvin-resistant Chinese hamster ovary cell mutant with abnormal spindles

A griseofulvin-resistant Chinese hamster ovary (CHO) mutant (Grs-2) which has an altered beta-tubulin subunit as well as wild-type beta-tubulin is temperature-sensitive (ts) for growth at 40.5 degrees C. This growth defect appears to result from the formation of abnormal mitotic spindles at the non-permissive temperature (Abraham, I et al., J cell biol 97 (1983) 1055) [19]. Light microscopy of spindles isolated from mutant cells cultured at the permissive temperature showed a typical bipolar morphology, whereas spindles isolated at the non-permissive temperature were multipolar. In order to study the role of tubulin in spindle formation, we analyzed the tubulin composition of the multipolar spindles. Two-dimensional gels and immunoblotting analysis of one-dimensional electrophoretic gels stained with monoclonal anti-Chinese hamster brain beta-tubulin antibody revealed that both mutant and wild-type beta-tubulins were present in similar proportions in both bipolar spindles at 37 degrees C and multipolar spindles at 40.5 degrees C. The ratio between wild-type and mutant tubulin in spindles was also found to be the same as in the cytoplasmic microtubule network in interphase cells, providing evidence that the mutant beta-tubulin appeared to be incorporated in a similar manner into both interphase and mitotic microtubule structures. In vitro microtubule polymerization onto centrosomes prepared from mutant Grs-2 demonstrated that 80% of the sites for microtubule nucleation were without centrioles, suggesting fragmentation of pericentriolar material away from centrioles. This may be one of the causes of multipolar spindle formation in the mutant cells. These results, therefore, suggest that abnormal formation of spindles in mutant cells is due not to the presence of the mutant tubulin per se, but to the abnormal behavior of this mutant tubulin in the cellular environment during mitosis or abnormal interaction with other components in the spindle at 40.5 degrees C.     

Effects of mitotic and tubulin mutations on microtubule architecture in actively growing protoplasts of Aspergillus nidulans

We used immunofluorescent microscopy to characterize microtubule (MT) architecture in wild-type and mutant protoplasts of Aspergillus nidulans at interphase and at mitosis. Because the visualization of MTs by immunofluorescence is technically difficult in intact hyphae of A. nidulans, we developed a method for removing the cell wall under conditions that do not perturb cell physiology, as evidenced by the fact that the resulting protoplasts undergo nuclear division at a normal rate and that cell cycle mutant phenotypes are expressed at restrictive temperature. Interphase cells exhibited an extensive network of cytoplasmic MTs. During mitosis the cytoplasmic MTs mostly disappeared and an intranuclear mitotic spindle appeared. We have previously shown that the benA 33 beta-tubulin mutation causes hyperstabilization of the mitotic spindle, and we have presented additional indirect evidence that suggested that the tubA1 and tubA4 alpha-tubulin mutations destabilize spindle MTs. In this paper, we show that the benA33 mutation increases the stability of cytoplasmic MTs as well as spindle MTs and that the tubA1 and tubA4 mutations destabilize both spindle and cytoplasmic MTs.     

Op18/stathmin mediates multiple region-specific tubulin and microtubule-regulating activities.

Oncoprotein18/stathmin (Op18) is a regulator of microtubule (MT) dynamics that binds tubulin heterodimers and destabilizes MTs by promoting catastrophes (i.e., transitions from growing to shrinking MTs). Here, we have performed a deletion analysis to mechanistically dissect Op18 with respect to (a) modulation of tubulin GTP hydrolysis and exchange, (b) tubulin binding in vitro, and (c) tubulin association and MT-regulating activities in intact cells. The data reveal distinct types of region-specific Op18 modulation of tubulin GTP metabolism, namely inhibition of nucleotide exchange and stimulation or inhibition of GTP hydrolysis. These regulatory activities are mediated via two-site cooperative binding to tubulin by multiple nonessential physically separated regions of Op18. In vitro analysis revealed that NH(2)- and COOH-terminal truncations of Op18 have opposite effects on the rates of tubulin GTP hydrolysis. Transfection of human leukemia cells with these two types of mutants result in similar decrease of MT content, which in both cases appeared independent of a simple tubulin sequestering mechanism. However, the NH(2)- and COOH-terminal-truncated Op18 mutants regulate MTs by distinct mechanisms as evidenced by morphological analysis of microinjected newt lung cells. Hence, mutant analysis shows that Op18 has the potential to regulate tubulin/MTs by more than one specific mechanism.     

Mutations of oncoprotein 18/stathmin identify tubulin-directed regulatory activities distinct from tubulin association

Oncoprotein 18/stathmin (Op18) is a recently identified phosphorylation-responsive regulator of the microtubule (MT) system. It was originally proposed that Op18 specifically regulates dynamic properties of MTs by associating with tubulin, but it has subsequently been proposed that Op18 acts simply by sequestering of tubulin heterodimers. We have dissected the mechanistic action of Op18 by generation of two distinct classes of mutants. One class has interruptions of the heptad repeats of a potential coiled-coil region of Op18, and the other involves substitution at all four phosphorylation sites with negatively charged Glu residues. Both types of mutation result in Op18 proteins with a limited decrease in tubulin complex formation. However, the MT-destabilizing activities of the coiled-coil mutants are more severely reduced in transfected leukemia cells than those of the Glu-substituted Op18 derivative, providing evidence for tubulin-directed regulatory activities distinct from tubulin complex formation. Analysis of Op18-mediated regulation of tubulin GTPase activity and taxol-promoted tubulin polymerization showed that while wild-type and Glu-substituted Op18 derivatives are active, the coiled-coil mutants are essentially inactive. This suggests that Op18-tubulin contact involves structural motifs that deliver a signal of regulatory importance to the MT system.     

Polyglycylation of tubulin is essential and affects cell motility and division in Tetrahymena thermophila

We analyzed the role of tubulin polyglycylation in Tetrahymena thermophila using in vivo mutagenesis and immunochemical analysis with modification-specific antibodies. Three and five polyglycylation sites were identified at glutamic acids near the COOH termini of alpha- and beta-tubulin, respectively. Mutants lacking all polyglycylation sites on alpha-tubulin have normal phenotype, whereas similar sites on beta-tubulin are essential. A viable mutant with three mutated sites in beta-tubulin showed reduced tubulin glycylation, slow growth and motility, and defects in cytokinesis. Cells in which all five polyglycylation sites on beta-tubulin were mutated were viable if they were cotransformed with an alpha-tubulin gene whose COOH terminus was replaced by the wild-type COOH terminus of beta-tubulin. In this double mutant, beta-tubulin lacked detectable polyglycylation, while the alpha-beta tubulin chimera was hyperglycylated compared with alpha-tubulin in wild-type cells. Thus, the essential function of polyglycylation of the COOH terminus of beta-tubulin can be transferred to alpha-tubulin, indicating it is the total amount of polyglycylation on both alpha- and beta-tubulin that is essential for survival.     

Microinjection of fluorescent tubulin into plant cells provides a representative picture of the cortical microtubule array

By microinjecting rhodamine-labelled tubulin into living plant cells, it is possible to observe microtubules (MTs) directly and to see how the cortical array reorganizes itself. The validity of the conclusions drawn from such observations depends upon the assumption that most, if not all, of the native MTs are dynamic and incorporate labelled tubulin. However, if arrays also contain MTs that are not exchanging tubulin subunits, such MTs will remain unlabelled, and the labelled MT population will be under-representative of the whole array. To address this potential problem, we microinjected pea epidermal cells with rhodamine-labelled tubulin, then fixed the cells and used fluorescein-conjugated antibodies against tubulin to detect the entire MT array. The two fluorescent patterns corresponded well, confirming that the MTs labelled with exogenous tubulin were evenly distributed throughout the entire array. Also, by comparing the MT image before and after aldehyde fixation, we observed that, although some of the MTs were lost in the procedure, the fixation was able to preserve the arrangement of MTs seen in the living cell. We conclude that fluorescence analogue cytochemistry provides a valid representation of the entire cortical MT array.     

A mutant beta-tubulin confers resistance to the action of benzimidazole-carbamate microtubule inhibitors both in vivo and in vitro

The mutant BEN210 of Physarum polycephalum is highly resistant to a number of benzimidazole carbamate agents, including methylbenzimidazole-2-yl-carbamate and parbendazole. The resistance is conferred by the benD210 mutation in a structural gene for beta-tubulin. This mutant allele encodes a beta-tubulin with novel electrophoretic mobility. We have used this strain to determine whether the mutant beta-tubulin is used in microtubules and whether this usage permits microtubule polymerisation in the presence of drugs both in vivo and in vitro. In vitro assembly studies of tubulin purified from the mutant strain have shown that microtubules are formed both in the absence of drugs and in all drug concentrations tested (up to 50 microM parbendazole). In contrast, the assembly of microtubules from wild-type tubulin in vitro is totally inhibited by 2-5 microM parbendazole. Thus the resistance of BEN210 to parbendazole observed in vivo has been reproduced in vitro using tubulin purified from the mutant strain. Electrophoretic analysis of the microtubules formed in vitro has shown that both the wild-type and the mutant beta-tubulin are incorporated into the microtubules and that the proportion of mutant to wild-type beta-tubulin appears to remain constant with increasing drug concentration. This is the first demonstration of a single mutation in a tubulin structural gene causing an altered function of the gene product in vitro.     

Newly assembled microtubules are concentrated in the proximal and distal regions of growing axons

We have investigated the sites of microtubule (MT) assembly in neurons during axon growth by taking advantage of the relationship between the proportion of tyrosinated alpha-tubulin (tyr-tubulin) in MTs and their age. Specifically, young (newly assembled) MTs contain more tyr-tubulin than older (more long-lived) MTs. To quantify the relative proportion of tyr-tubulin in MTs, cultured rat sympathetic neurons were permeabilized under conditions that stabilize existing MTs and remove unassembled tubulin. The MTs were then double-stained with antibodies to tyr-tubulin (as a measure of the amount of tyr-tubulin in MTs) and to beta-tubulin (as a measure of total MT mass), using immunofluorescence procedures. Cells were imaged with a cooled charge-coupled device camera and the relative proportion of tyr-tubulin in the MTs was quantified by computing the ratio of the tyr-tubulin fluorescence to the beta-tubulin fluorescence using a novel application of digital image processing and analysis techniques. The amount of tyr-tubulin in the MTs was highest in the cell body and at the growth cone; peak ratios in these two regions were approximately 10-fold higher than for the axon shaft. Moving out from the cell body into the axon, the tyr-tubulin content declined over an average distance of 40 microns to reach a constant low value within the axon shaft and then rose again more distally, over an average distance of 110 microns, to reach a peak at the growth cone (average axon length = 358 microns). These observations indicate that newly assembled MTs are concentrated in the proximal and distal regions of growing axons and therefore that the cell body and growth cone are the most active sites of MT assembly dynamics in neurons that are actively extending axons.     

NEM tubulin inhibits microtubule minus end assembly by a reversible capping mechanism

Although microtubule (MT) dynamic instability is thought to depend on the guanine nucleotide (GTP vs GDP) bound to the beta-tubulin of the terminal subunit(s), the MT minus end exhibits dynamic instability even though the terminal beta-tubulin is always crowned by GTP-alpha-tubulin. As an approach toward understanding how dynamic instability occurs at the minus end, we investigated the effects of N-ethylmaleimide-modified tubulin (NTb) on elongation and rapid shortening of individual MTs. NTb preferentially inhibits minus end assembly when combined with unmodified tubulin (PCTb), but the mechanism of inhibition is unknown. Here, video-enhanced differential interference contrast microscopy was used to observe the effects of NTb on MTs assembled from PCTb onto axoneme fragments. MTs were exposed to mixtures of PCTb (25 microM) and NTb (labeled on approximately 1 Cys per monomer) in which the NTb/PCTb ratio varied from 0.025 to 1. The NTb/PCTb mixture had a slight inhibitory effect on the plus end elongation rate, but significantly inhibited or completely arrested minus end elongation. For the majority of mixtures that were assayed (0.1-1 NTb/PCTb ratio), minus end MT length remained constant until the NTb/PCTb mixture was replaced. Replacement with PCTb allowed elongation to proceed, whereas replacement with buffer or NTb caused minus ends to shorten. Taken together, the results indicate that NTb associates with both plus and minus ends and that NTb acts to reversibly cap minus ends only when PCTb is also present. Low-resolution mapping of labeled Cys residues, along with previous experiments with other Cys-reactive compounds, suggests that modification of beta-tubulin Cys(239) may be associated with the capping action of NTb.     

LIM kinase 1 coordinates microtubule stability and actin polymerization in human endothelial cells

Microtubule (MT) destabilization promotes the formation of actin stress fibers and enhances the contractility of cells; however, the mechanism involved in the coordinated regulation of MTs and the actin cytoskeleton is poorly understood. LIM kinase 1 (LIMK1) regulates actin polymerization by phosphorylating the actin depolymerization factor, cofilin. Here we report that LIMK1 is also involved in the MT destabilization. In endothelial cells endogenous LIMK1 co-localizes with MTs and forms a complex with tubulin via the PDZ domain. MT destabilization induced by thrombin or nocodazole resulted in a decrease of LIMK1 colocalization with MTs. Overexpression of wild type LIMK1 resulted in MT destabilization, whereas the kinase-dead mutant of LIMK1 (KD) did not affect MT stability. Importantly, down-regulation of endogenous LIMK1 by small interference RNA resulted in abrogation of the thrombin-induced MTs destabilization and the inhibition of thrombin-induced actin polymerization. Expression of Rho kinase 2, which phosphorylates and activates LIMK1, dramatically decreases the interaction of LIMK1 with tubulin but increases its interaction with actin. Interestingly, expression of KD-LIMK1 or small interference RNA-LIMK1 prevents thrombin-induced microtubule destabilization and F-actin formation, suggesting that LIMK1 activity is required for thrombin-induced modulation of microtubule destabilization and actin polymerization. Our findings indicate that LIMK1 may coordinate microtubules and actin cytoskeleton.     

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.     

The human EMAP-like protein-70 (ELP70) is a microtubule destabilizer that localizes to the mitotic apparatus.

In this report, we show that the echinoderm microtubule (MT)-associated protein (EMAP) and related EMAP-like proteins (ELPs) share a similar domain organization with a highly conserved hydrophobic ELP (HELP) domain and a large tryptophan-aspartic acid (WD) repeat domain. To determine the function of mammalian ELPs, we generated antibodies against a 70-kDa human ELP and showed that ELP70 coassembled with MTs in HeLa cell extracts and colocalized with MTs in the mitotic apparatus. To determine whether ELP70 bound to MTs directly, human ELP70 was expressed and purified to homogeneity from baculovirus-infected Sf9 cells. Purified ELP70 bound to purified MTs with a stoichiometry of 0.40 +/- 0.04 mol of ELP70/mol of tubulin dimer and with an intrinsic dissociation constant of 0.44 +/- 0.13 microm. Using a nucleated assembly assay and video-enhanced differential interference contrast microscopy, we demonstrated that ELP70 reduced seeded nucleation, reduced the growth rate, and promoted MT catastrophes in a concentration-dependent manner. As a result, ELP70-containing MTs were significantly shorter than MTs assembled from tubulin alone. These data indicate that ELP70 is a novel MT destabilizer. A lateral destabilization model is presented to describe ELP70's effects on microtubules.     

Mutations affecting beta-tubulin folding and degradation

Revertants of a colcemid-resistant Chinese hamster ovary cell line with an altered (D45Y) beta-tubulin have allowed the identification of four cis-acting mutations (L187R, Y398C, a 12-amino acid in-frame deletion, and a C-terminal truncation) that act by destabilizing the mutant tubulin and preventing it from incorporating into microtubules. These unstable beta-tubulins fail to form heterodimers and are predominantly found in association with the chaperonin CCT, suggesting that they cannot undergo productive folding. In agreement with these in vivo observations, we show that the defective beta-tubulins do not stably interact with cofactors involved in the tubulin folding pathway and, hence, fail to exchange with beta-tubulin in purified alphabeta heterodimers. Treatment of cells with MG132 causes an accumulation of the aberrant tubulins, indicating that improperly folded beta-tubulin is degraded by the proteasome. Rapid degradation of the mutant tubulin does not elicit compensatory changes in wild-type tubulin synthesis or assembly. Instead, loss of beta-tubulin from the mutant allele causes a 30-40% decrease in cellular tubulin content with no obvious effect on cell growth or survival.     

Structural studies of N-terminal mutants of Connexin 32 using (1)H NMR spectroscopy

Microtubules (MTs) control cell replication, material transport and motion in eukaryotic cells, but MT role in several pathologies is still unknown. These functions are related to the MT physico-chemical properties and MT formation mode starting from tubulin molecules. This study describes a new method, based on the computer aided analysis of the electron paramagnetic resonance (EPR) spectra of selected spin probes to obtain structural and dynamical information on tubulins and MTs and the kinetics of MTs formation promoted by guanosine-5'-triphosphate (GTP). It was found that tubulin and MTs avoid radical quenching caused by ethylene glycol tetraacetic acid (EGTA). MT formation showed different kinetics as a function of tubulin concentration. At 5 mg/mL of tubulin, MTs were formed in 8 min. These results are also useful for getting information on MT-drug interactions.