Dietary antioxidant curcumin inhibits microtubule assembly through tubulin binding

Curcumin, a component of turmeric, has potent antitumor activity against several tumor types. However, its molecular target and mechanism of antiproliferative activity are not clear. Here, we identified curcumin as a novel antimicrotubule agent. We have examined the effects of curcumin on cellular microtubules and on reconstituted microtubules in vitro. Curcumin inhibited HeLa and MCF-7 cell proliferation in a concentration-dependent manner with IC(50) of 13.8 +/- 0.7 microm and 12 +/- 0.6 microm, respectively. At higher inhibitory concentrations (> 10 microm), curcumin induced significant depolymerization of interphase microtubules and mitotic spindle microtubules of HeLa and MCF-7 cells. However, at low inhibitory concentrations there were minimal effects on cellular microtubules. It disrupted microtubule assembly in vitro, reduced GTPase activity, and induced tubulin aggregation. Curcumin bound to tubulin at a single site with a dissociation constant of 2.4 +/- 0.4 microm and the binding of curcumin to tubulin induced conformational changes in tubulin. Colchicine and podophyllotoxin partly inhibited the binding of curcumin to tubulin, while vinblastine had no effect on the curcumin-tubulin interactions. The data together suggested that curcumin may inhibit cancer cells proliferation by perturbing microtubule assembly dynamics and may be used to develop efficacious curcumin analogues for cancer chemotherapy.     

The benzophenanthridine alkaloid sanguinarine perturbs microtubule assembly dynamics through tubulin binding. A possible mechanism for its antiproliferative activity

Sanguinarine has been shown to inhibit proliferation of several types of human cancer cell including multidrug-resistant cells, whereas it has minimal cytotoxicity against normal cells such as neutrophils and keratinocytes. By analyzing the antiproliferative activity of sanguinarine in relation to its effects on mitosis and microtubule assembly, we found that it inhibits cancer cell proliferation by a novel mechanism. It inhibited HeLa cell proliferation with a half-maximal inhibitory concentration of 1.6 +/- 0.1 microM. In its lower effective inhibitory concentration range, sanguinarine depolymerized microtubules of both interphase and mitotic cells and perturbed chromosome organization in mitotic HeLa cells. At concentrations of 2 microM, it induced bundling of interphase microtubules and formation of granular tubulin aggregates. A brief exposure of HeLa cells to sanguinarine caused irreversible depolymerization of the microtubules, inhibited cell proliferation, and induced cell death. However, in contrast with several other microtubule-depolymerizing agents, sanguinarine did not arrest cell cycle progression at mitosis. In vitro, low concentrations of sanguinarine inhibited microtubule assembly. At higher concentrations (> 40 microM), it altered polymer morphology. Further, it induced aggregation of tubulin in the presence of microtubule-associated proteins. The binding of sanguinarine to tubulin induces conformational changes in tubulin. Together, the results suggest that sanguinarine inhibits cell proliferation at least in part by perturbing microtubule assembly dynamics.     

Rotenone inhibition of spindle microtubule assembly in mammalian cells

Rotenone, a potent inhibitor of mitochondrial respiration is also an effective antimitotic agent. The addition of either rotenone or Colcemid to exponentially growing Chinese hamster ovary cells resulted in a dramatic increase in mitotic index after 90 min. When the cultures were washed free of the drugs, mitosis was completed and the cells progressed into G 1 at approximately the same rate. Further similarity of rotenone-arrested cells to Colcemid-induced mitotic inhibition was apparent at the ultrastructural level. Mitotic cells treated by either drug contained monopolar spindles with chromosomes grouped around centriole pairs near the cell center. Occasional microtubules were seen near the kinetochore and centrioles. These observations, along with the fact that rotenone inhibited the binding of ³H-colchicine to isolated bovine brain tubulin, suggested that rotenone inhibited mitosis by binding directly to tubulin to prevent microtubule assembly.     

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.     

Mechanism of mitotic arrest induced by dolastatin 15 involves loss of tension across kinetochore pairs

Dolastatin 15 (DL15) is a potent, tubulin-targeted, vinca-site binding, anticancer agent that induces mitotic arrest and inhibit cell proliferation in a variety of cell types. Several analogs of DL15, including LU 103793 and tasidotin, have been progressed to clinical trials for different types of cancer. DL15 has been known to interfere with cellular microtubules and purified tubulin in vitro. However, the molecular mechanism with which the peptide arrests cells in mitosis is poorly understood. This study reports a possible antimitotic mechanism of action of DL15. DL15 inhibited HeLa cell proliferation in a concentration-dependent manner with a half-maximal inhibitory concentration (IC₅₀) of 2.8 ± 0.3 nM, induced mitotic arrest, disrupted cellular microtubules near its IC₅₀ for cell proliferation, and inhibited the re-polymerization of cellular microtubules. By staining the centrosomes of DL15-treated cells with anti-γ tubulin antibodies, the study found a significant reduction in interpolar distances in mitotic HeLa cells, indicating a disruption in the normal assembly dynamics of the microtubules. The study further found that DL15 induced a loss of tension across the kinetochore pairs as indicated by a reduction in interkinetochore distance. In response to this loss of tension, the tension-sensing checkpoint protein BuBR1 accumulated at the kinetochores, promoting mitotic arrest. In vitro, DL15 promoted formation of curved and fragmented polymers of microtubule proteins and inhibited tubulin decay in a manner similar to vinca-site binding agents such as phomopsin A. Together, the data indicate that the mitotic arrest induced by DL15 involves a loss of tension across the kinetochore pairs due to disruption of normal assembly dynamics of microtubules.     

An Antitubulin Agent BCFMT Inhibits Proliferation of Cancer Cells and Induces Cell Death by Inhibiting Microtubule Dynamics

Using cell based screening assay, we identified a novel anti-tubulin agent (Z)-5-((5-(4-bromo-3-chlorophenyl)furan-2-yl)methylene)-2-thioxothiazolidin-4-one (BCFMT) that inhibited proliferation of human cervical carcinoma (HeLa) (IC(50), 7.2±1.8 μM), human breast adenocarcinoma (MCF-7) (IC(50), 10.0±0.5 μM), highly metastatic breast adenocarcinoma (MDA-MB-231) (IC(50), 6.0±1 μM), cisplatin-resistant human ovarian carcinoma (A2780-cis) (IC(50), 5.8±0.3 μM) and multi-drug resistant mouse mammary tumor (EMT6/AR1) (IC(50), 6.5±1μM) cells. Using several complimentary strategies, BCFMT was found to inhibit cancer cell proliferation at G2/M phase of the cell cycle apparently by targeting microtubules. In addition, BCFMT strongly suppressed the dynamics of individual microtubules in live MCF-7 cells. At its half maximal proliferation inhibitory concentration (10 μM), BCFMT reduced the rates of growing and shortening phases of microtubules in MCF-7 cells by 37 and 40%, respectively. Further, it increased the time microtubules spent in the pause (neither growing nor shortening detectably) state by 135% and reduced the dynamicity (dimer exchange per unit time) of microtubules by 70%. In vitro, BCFMT bound to tubulin with a dissociation constant of 8.3±1.8 μM, inhibited tubulin assembly and suppressed GTPase activity of microtubules. BCFMT competitively inhibited the binding of BODIPY FL-vinblastine to tubulin with an inhibitory concentration (K(i)) of 5.2±1.5 μM suggesting that it binds to tubulin at the vinblastine site. In cultured cells, BCFMT-treatment depolymerized interphase microtubules, perturbed the spindle organization and accumulated checkpoint proteins (BubR1 and Mad2) at the kinetochores. BCFMT-treated MCF-7 cells showed enhanced nuclear accumulation of p53 and its downstream p21, which consequently activated apoptosis in these cells. The results suggested that BCFMT inhibits proliferation of several types of cancer cells including drug resistance cells by suppressing microtubule dynamics and indicated that the compound may have chemotherapeutic potential.     

Human chromosomes and centrioles as nucleating sites for the in vitro assembly of microtubules from bovine brain tubulin

Treatment of HeLa cells with Colcemid at concentrations of 0.06-0.10 mug/ml leads to irreversible arrest in mitosis. Colcemid-arrested cells contained few microtubules, and many kinetochores and centrioles were free of microtubule association. When these cells were exposed to microtubule reassembly buffer containing Triton X-100 and bovine brain tubulin at 37 degrees C, numerous microtubules were reassembled at all kinetochores of metaphase chromosomes and in association with centriole pairs. When bovine brain tubulin was eliminated from the reassembly system, microtubules failed to assemble at these sites. Similarly, when EGTA was eliminated from the reassembly system, microtubules failed to polymerize. These results are consistent with other investigations of in vitro microtubule assembly and indicate that HeLa chromosomes and centrioles can serve as nucleating sites for the assembly of microtubules from brain tubulin. Both chromosomes and centrioles became displaced from their C-metaphase configurations during tubulin reassembly, indicating that their movements were a direct result of microtubule formation. Although both kinetochore- and centriole- associated microtubules were assembled and movement occurred, we did not observe direct extension of microtubules from kinetochores to centrioles. This system should prove useful for experimental studies of spindle microtubule formation and chromosome movement in mammalian cells.     

Dynein Light Chain 1 (LC8) Association Enhances Microtubule Stability and Promotes Microtubule Bundling

Dynein light chain 1 (LC8), a highly conserved protein, is known to bind to a variety of different polypeptides. It functions as a dimer, which is inactivated through phosphorylation at the Ser-88 residue. A loss of LC8 function causes apoptosis in Drosophila embryos, and its overexpression induces malignant transformation of breast cancer cells. Here we show that LC8 binds to tubulin, promotes microtubule assembly, and induces the bundling of reconstituted microtubules in vitro. Furthermore, LC8 decorates microtubules both in Drosophila embryos and in HeLa cells, increases the microtubule stability, and promotes microtubule bundling in these cells. Microtubule stability influences a number of different cellular functions including mitosis and cell differentiation. The LC8 overexpression reduces the susceptibility of microtubules to cold and nocodazole-induced depolymerization in tissue-cultured cells and increases microtubule acetylation, suggesting that LC8 stabilizes microtubules. We also show that LC8 knockdown or transfection with inhibitory peptides destabilizes microtubules and inhibits bipolar spindle assembly in HeLa cells. In addition, LC8 knockdown leads to the mitotic block in HeLa cells. Furthermore, molecular docking analysis using the crystal structures of tubulin and LC8 dimer indicated that the latter may bind at α-β tubulin junction in a protofilament at sites distinct from the kinesin and dynein binding sites. Together, we provide the first evidence of a novel microtubule-associated protein-like function of LC8 that could explain its reported roles in cellular metastasis and differentiation.     

Antimitotic sulfonamides inhibit microtubule assembly dynamics and cancer cell proliferation

Several sulfonamides have antitumor activities and are currently undergoing clinical evaluation for the treatment of cancer. In this study, we have elucidated the antiproliferative mechanism of action of five indole sulfonamides. The indole sulfonamides inhibited the polymerization of microtubule protein into microtubules in vitro. In addition, three representative derivatives, ER-68378 (2), ER-68384 (4) and ER-68394 (5), suppressed the dynamic instability behavior at the plus ends of individual steady-state microtubules in vitro. The analogues inhibited HeLa cell proliferation with half-maximal inhibitory concentrations in the range of 6-17 microM. The compounds blocked cell cycle progression at mitosis. At their lowest effective antimitotic concentrations, they depolymerized the spindle microtubules and disorganized the chromosomes but did not affect the microtubules in interphase cells. However, at relatively high concentrations, interphase microtubules were also depolymerized by these sulfonamides. Furthermore, all five compounds were found to induce apoptosis in the cells in association with the phosphorylation of bcl-2. The results suggest that the indole sulfonamides inhibit cell proliferation at mitosis by perturbing the assembly dynamics of spindle microtubules and that they can kill cancer cells by inducing apoptosis through the bcl-2-dependent pathway.     

Action of rotenone and related respiratory inhibitors on mammalian cell division. 1 Cell kinetics and biochemical aspects.

Inhibitors of mitochondrial respiration, phosphorylation inhibitors, and uncoupling agents have been reported to delay or inhibit mitosis in cultured mammalian cells. Although the molecular mechanism by which mitosis is delayed in the presence of most respiratory inhibitors presumably involves lowered ATP production for mitotic requirements, one respiratory inhibitor, rotenone, was determined to arrest mitosis by an unrelated mechanism. Cell cycle kinetics studies, oxygen consumption measurements, and viscosity assays indicate that rotenone arrests cultured mammalian cells in mitosis by inhibiting spindle microtubule assembly by a mechanism analogous with colchicine, Colecemid and related antimitotic drugs. Amytal, which blocks electron transport at the same site as does rotenone, failed to arrest cell progression at mitosis. Rotenone delayed cell progression in all phases of the cell cycle, apparently as a direct result of respiration inhibition. Thus, rotenone appears to exert a dual function on events of the cell cycle.     

Rotenone inhibition of tubulin self-assembly

Rotenone effectively inhibits the in vitro formation of microtubules from tubulin containing or lacking microtubule-associated proteins. In both cases a concentration of rotenone equal to that of tubulin present completely blocks assembly. The inhibition can be reversed by the addition of dimethylsulfoxide or by removing rotenone with charcoal.     

Microtubule disassembly and inhibition of mitosis by a novel synthetic pharmacophore

Microtubule drugs, which block cell cycle progression through mitosis, have seen widespread use in cancer chemotherapies. Although microtubules are subject to regulation by signal transduction mechanisms, their pharmacological modulation has so far relied on compounds that bind to the tubulin subunit. A new microtubule pharmacophore, diphenyleneiodonium, causing disassembly of the microtubule cytoskeleton is described here. Although this synthetic compound does not affect the assembly state of purified microtubules, it profoundly suppresses microtubule assembly in vivo, causes paclitaxel-stabilized microtubules to cluster around the centrosomes, and selectively disassembles dynamic microtubules. Similar to other microtubule drugs, this new pharmacophore blocks mitotic spindle assembly and mitotic cell division.     

Microtubule functions.

Microtubules, with intermediate filaments and microfilaments, are the components of the cell skeleton which determinates the shape of a cell. Microtubules are involved in different functions including the assembly of mitotic spindle, in dividing cells, or axon extension, in neurons. In the first case, microtubules are highly dynamic, while in the second case microtubules are quite stable, suggesting that microtubule with different physical properties (stability) are involved in different functions. Thus, to understand the mechanisms of microtubule functions it is very important to understand microtubule dynamics. Historically, tubulin, the main component of microtubules, was first characterized as the major component of the mitotic spindle that binds to colchicine. Afterwards, it was found that tubulin is particularly more abundant in brain than in other tissues. Therefore, the roles of microtubules in mitosis, and in neurons, have been more extensively analyzed and, in this review, these roles will be discussed.     

Tubulin assembly protein: immunochemical and immunofluorescent studies on its function and distribution in microtubules and cultured cells.

Cytoplasmic microtubule assembly from tubulin monomers requires an accessory protein or proteins present is isolated microtubules. These proteins have been designated "tau" factors. One such factor, tubulin assembly protein (TAP), has been purified to homogeneity from calf brain microtubules. A precipitating, monospecific antibody against the protein has been prepared. The antibody has been used to investigate the mechanism of TAP action in microtubule assembly and the distribution of TAP in cellular microtubules. Immunochemical, immunofluorescent and electron microscopic studies indicate that TAP functions stoichiometrically by binding physically to tubulin to form a complex active in microtubule assembly. TAP is an elongation protein which is required throughout the growth of a microtubule and which is actually present along the entire microtubule. Immunofluorescence microscopy has been used to demonstrate that TAP is distributed throughout the cytoplasmic microtubule network of cultured human, hamster and rat cells-both normal and virally transformed. Immunofluorescence of cells in mitosis shows that TAP is present in the mitotic spindle. These results demonstrate the biological importance of tubulin assembly protein and suggest that it or immunologically related "tau" proteins represent ubiquitous cofactors in cytoplasmic microtubule assembly.     

Cytoplasmic dynein-mediated assembly of pericentrin and gamma tubulin onto centrosomes

Centrosome assembly is important for mitotic spindle formation and if defective may contribute to genomic instability in cancer. Here we show that in somatic cells centrosome assembly of two proteins involved in microtubule nucleation, pericentrin and gamma tubulin, is inhibited in the absence of microtubules. A more potent inhibitory effect on centrosome assembly of these proteins is observed after specific disruption of the microtubule motor cytoplasmic dynein by microinjection of dynein antibodies or by overexpression of the dynamitin subunit of the dynein binding complex dynactin. Consistent with these observations is the ability of pericentrin to cosediment with taxol-stabilized microtubules in a dynein- and dynactin-dependent manner. Centrosomes in cells with reduced levels of pericentrin and gamma tubulin have a diminished capacity to nucleate microtubules. In living cells expressing a green fluorescent protein-pericentrin fusion protein, green fluorescent protein particles containing endogenous pericentrin and gamma tubulin move along microtubules at speeds of dynein and dock at centrosomes. In Xenopus extracts where gamma tubulin assembly onto centrioles can occur without microtubules, we find that assembly is enhanced in the presence of microtubules and inhibited by dynein antibodies. From these studies we conclude that pericentrin and gamma tubulin are novel dynein cargoes that can be transported to centrosomes on microtubules and whose assembly contributes to microtubule nucleation.     

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

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

Microtubule regulation in mitosis: tubulin phosphorylation by the cyclin-dependent kinase Cdk1

The activation of the cyclin-dependent kinase Cdk1 at the transition from interphase to mitosis induces important changes in microtubule dynamics. Cdk1 phosphorylates a number of microtubule- or tubulin-binding proteins but, hitherto, tubulin itself has not been detected as a Cdk1 substrate. Here we show that Cdk1 phosphorylates beta-tubulin both in vitro and in vivo. Phosphorylation occurs on Ser172 of beta-tubulin, a site that is well conserved in evolution. Using a phosphopeptide antibody, we find that a fraction of the cell tubulin is phosphorylated during mitosis, and this tubulin phosphorylation is inhibited by the Cdk1 inhibitor roscovitine. In mitotic cells, phosphorylated tubulin is excluded from microtubules, being present in the soluble tubulin fraction. Consistent with this distribution in cells, the incorporation of Cdk1-phosphorylated tubulin into growing microtubules is impaired in vitro. Additionally, EGFP-beta3-tubulin(S172D/E) mutants that mimic phosphorylated tubulin are unable to incorporate into microtubules when expressed in cells. Modeling shows that the presence of a phosphoserine at position 172 may impair both GTP binding to beta-tubulin and interactions between tubulin dimers. These data indicate that phosphorylation of tubulin by Cdk1 could be involved in the regulation of microtubule dynamics during mitosis.     

The role of stathmin in the regulation of the cell cycle

Stathmin is the founding member of a family of proteins that play critically important roles in the regulation of the microtubule cytoskeleton. Stathmin regulates microtubule dynamics by promoting depolymerization of microtubules and/or preventing polymerization of tubulin heterodimers. Upon entry into mitosis, microtubules polymerize to form the mitotic spindle, a cellular structure that is essential for accurate chromosome segregation and cell division. The microtubule-depolymerizing activity of stathmin is switched off at the onset of mitosis by phosphorylation to allow microtubule polymerization and assembly of the mitotic spindle. Phosphorylated stathmin has to be reactivated by dephosphorylation before cells exit mitosis and enter a new interphase. Interfering with stathmin function by forced expression or inhibition of expression results in reduced cellular proliferation and accumulation of cells in the G2/M phases of the cell cycle. Forced expression of stathmin leads to abnormalities in or a total lack of mitotic spindle assembly and arrest of cells in the early stages of mitosis. On the other hand, inhibition of stathmin expression leads to accumulation of cells in the G2/M phases and is associated with severe mitotic spindle abnormalities and difficulty in the exit from mitosis. Thus, stathmin is critically important not only for the formation of a normal mitotic spindle upon entry into mitosis but also for the regulation of the function of the mitotic spindle in the later stages of mitosis and for the timely exit from mitosis. In this review, we summarize the early studies that led to the identification of the important mitotic function of stathmin and discuss the present understanding of its role in the regulation of microtubules dynamics during cell-cycle progression. We also describe briefly other less mature avenues of investigation which suggest that stathmin may participate in other important biological functions and speculate about the future directions that research in this rapidly developing field may take.     

Microtubule dynamics in the chromosomal spindle fiber: analysis by fluorescence and high-resolution polarization microscopy

We describe preliminary results from two studies exploring the dynamics of microtubule assembly and organization within chromosomal spindle fibers. In the first study, we microinjected fluorescently labeled tubulin into mitotic PtK1 cells and measured fluorescence redistribution after photobleaching (FRAP) to determine the assembly dynamics of the microtubules within the chromosomal fibers in metaphase cells depleted of nonkinetochore microtubules by cooling to 23-24 degrees C. FRAP measurements showed that the tubulin throughout at least 72% of the microtubules within the chromosomal fibers exchanges with the cellular tubulin pool with a half-time of 77 sec. There was no observable poleward flux of subunits. If the assembly of the kinetochore microtubules is governed by dynamic instability, our results indicate that the half-life of microtubule attachment to the kinetochore is less than several min at 23-24 degrees C. In the second study, we used high-resolution polarization microscopy to observe microtubule dynamics during mitosis in newt lung epithelial cells. We obtained evidence from 150-nm-thick optical sections that microtubules throughout the spindle laterally associate for several sec into "rods" composed of a few microtubules. These transient lateral associations between microtubules appeared to produce the clustering of nonkinetochore and kinetochore microtubules into the chromosomal fibers. Our results indicate that the chromosomal fiber is a dynamic structure, because microtubule assembly is transient, lateral interactions between microtubules are transient, and the attachment of the kinetochores to microtubules may also be transient.     

The interaction of triethyl lead with tubulin and microtubules

The impact of triethyl lead chloride was studied on: (i) the in vitro assembly and disassembly of microtubules from porcine brain by turbidometry and electron microscopy, (ii) the microtubule system of living mammalian cells using immunofluorescence microscopy, (iii) cell motility and chemotaxis employing the methods of phagokinetic track formation and the Boyden chamber assay, respectively, and (iv) thiol groups of the protein tubulin by their titration in the presence and absence of the organic lead compound. Triethyl lead chloride inhibited microtubule assembly and depolymerized preformed microtubules in vitro and in living cells. Random motility of cells was not markedly inhibited by triethyl lead chloride, whereas chemotaxis (directed cellular movement) was strongly inhibited. Triethyl lead chloride was found to interact with 2 thiol groups of the tubulin dimer. The interaction of triethyl lead chloride with the tubulin/microtubule system in vivo likely causes aneuploidy and is at least partly responsible for the cytotoxicity of the drug.