Tubulin conformation in microtubule-free cells ofVigna sinensis

Summary The primary leaf, epicotyl, and root cells ofVigna sinensis seedlings grown continuously in a 0.08% colchicine solution, become microtubule-free and polyploid. In meristematic root cells a tubulin transformation is detected 1–3 h after the treatment had begun. Tubulin strands are organized at the positions of the pre-existing microtubules. Frequently, the strands converge on or are organized in the cortical cytoplasmic zone where in normal cells the preprophase microtubule band (PMB) is assembled. In meristematic root cells subjected to a 6–12 h colchicine treatment, the tubulin strands become perinuclear, entering the cortical cytoplasm at regions close to the nucleus. One day after the onset of the treatment, tubulin generally forms a continuous reticulum of interconnected strands in all the organs examined. In most cells this reticulum surrounds the nucleus partly or totally or lies close to it, exhibiting variable configurations in different cells. After prolonged treatments, the organization of the tubulin reticulum changes further. Now this consists of crystal-like structures interconnected by thin strands.On thin sections of fixed tissue the tubulin strands consist of paracrystalline material. The distribution of this material in the affected cells coincides with that of tubulin reticulum visualized by immunofluorescence. In transverse planes each strand exhibits circular subunits arranged close to one another in a hexagonal pattern but in longitudinal ones variable images were observed. The paracrystalline material persists in root cells subjected to an 8-day continuous colchicine treatment. The immunolabeled strands seem to be composed of tubulin-colchicine complexes and not pure tubulin.     

Patterns of microtubule reappearance in root cells ofVigna sinensis recovering from a colchicine treatment

Summary The reticulum of paracrystalline tubulin strands, which is assembled in meristematic root cells ofVigna sinensis treated with a 0.08% colchicine solution, disaggregates and microtubules (Mts) reappear after a 10–14 h recovery of the seedlings from the drug. In recovering interphase cells, Mts reappear in the cortical cytoplasm. Initially, they are short and aligned in different directions but finally they elongate and usually become oriented transversely to the long cell axis.A single or a pair of preprophase Mt bands (PMBs) is organized in cells enclosing one or more nuclei. Simultaneously, Mts traverse the perinuclear cytoplasm. In recovering C-mitotic cells, Mt bundles emerge from the kinetochores. Initially, they exhibit diverse orientations. Afterwards, the C-chromosomes are aligned on ametaphase plate via kinetochore Mt bundles, which become parallel to one another. As time passes non-kinetochore Mts appear among the chromosomes and anaphase proceeds. In recovering cytokinetic cells, normal, abnormally curved or branched phragmoplasts are organized. The latter arise between the nuclei of multinucleate telophase cells or between the lobes of forming polyploid nuclei. In cells which were blocked at an advanced cytokinetic stage by colchicine, phragmoplasts return to the margins of the incomplete cell walls.The observations presented here suggest that in recovering colchicine-treated root cells the Mts and the tubulin reticulum are interchangeable. Although Mts appear in cytoplasmic sites where they are expected to be nucleated, the pattern of Mt reformation differs from that operating in normal and to a smaller extent from that functioning in cells recovering from other anti-Mt drugs.     

A spiral array of microtubules in the fertilized sea urchin egg cortex examined by indirect immunofluorescence and electron microscopy

A transient spiral system of fibers in the cortex of fertilized eggs of the sea urchin Strongylocentrotus purpuratus was examined with indirect immunofluorescence microscopy and found to contain tubulin. Electron microscopy identified the tubulin-containing bands as bundles of up to 40 or more microtubules. These cortical microtubules, which are initially radial, form a spiral array about the time of pronuclear fusion. This basket-like structure, at a depth of 10–15 μm below the cell surface, reaches a peak of development about 45 min after fertilization and disappears before the streak stage at 70 min, in a division cycle of slightly more than 2 h. Possible functions of the cortical microtubules, which appear to be independent of the interphase asters, are discussed.     

Organization of microtubules in stabilized meristematic plant cells revealed by a rat monoclonal antibody reacting only with the tyrosinated form of alpha-tubulin

A rat monoclonal antibody against yeast tubulin (clone YL 1/2; Kilmartin et al., 1982) that reacts specifically with mammalian alpha-tubulin carrying a carboxyterminal tyrosine residue (Wehland et al., 1983) was used to localize microtubules in plant cells derived from onion root apices (Allium cepa L.). YL 1/2 reacted with all types of microtubular arrays known to occur in higher plant meristematic cells such as interphase cortical microtubules, pre-prophase bands, the mitotic spindle and phragmoplast microtubules. The specific labeling of microtubules in isolated cells from onion root tips by YL 1/2 indicates that plant cells like animal cells contain tubulin tyrosine ligase, the enzyme which posttranslationally modifies alpha-tubulin. This enzyme could be involved in the dynamic regulation of microtubular arrays in all eukaryotic cells.     

Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms

Seven monoclonal antibodies raised against tubulin from the axonemes of sea urchin sperm flagella recognize an acetylated form of alpha-tubulin present in the axoneme of a variety of organisms. The antigen was not detected among soluble, cytoplasmic alpha-tubulin isoforms from a variety of cells. The specificity of the antibodies was determined by in vitro acetylation of sea urchin and Chlamydomonas cytoplasmic tubulins in crude extracts. Of all the acetylated polypeptides in the extracts, only alpha-tubulin became antigenic. Among Chlamydomonas tubulin isoforms, the antibodies recognize only the axonemal alpha-tubulin isoform acetylated in vivo on the epsilon-amino group of lysine(s) (L'Hernault, S.W., and J.L. Rosenbaum, 1985, Biochemistry, 24:473-478). The antibodies do not recognize unmodified axonemal alpha-tubulin, unassembled alpha-tubulin present in a flagellar matrix-plus-membrane fraction, or soluble, cytoplasmic alpha-tubulin from Chlamydomonas cell bodies. The antigen was found in protein fractions that contained axonemal microtubules from a variety of sources, including cilia from sea urchin blastulae and Tetrahymena, sperm and testis from Drosophila, and human sperm. In contrast, the antigen was not detected in preparations of soluble, cytoplasmic tubulin, which would not have contained tubulin from stable microtubule arrays such as centrioles, from unfertilized sea urchin eggs, Drosophila embryos, and HeLa cells. Although the acetylated alpha-tubulin recognized by the antibodies is present in axonemes from a variety of sources and may be necessary for axoneme formation, it is not found exclusively in any one subset of morphologically distinct axonemal microtubules. The antigen was found in similar proportions in fractions from sea urchin sperm axonemes enriched for central pair or outer doublet B or outer doublet A microtubules. Therefore the acetylation of alpha-tubulin does not provide the mechanism that specifies the structure of any one class of axonemal microtubules. Preliminary evidence indicates that acetylated alpha-tubulin is not restricted to the axoneme. The antibodies described in this report may allow us to deduce the role of tubulin acetylation in the structure and function of microtubules in vivo.     

Colchicine-induced Paracrystals in Root Cells of Wheat (Triticum aestivum L.)

Tubulin conformations other than microtubules in the meristematiccells of wheat roots grown in the presence of 2 mM colchicinesolution were investigated by immunofluorescence and electronmicroscopy. In the affected cells microtubules disappeared andwere replaced by tubulin fluorescent strands that occurred inthe cortical cytoplasm. With increasing time of exposure tocolchicine the tubulin strands became better organized and occurredalso in the subcortical cytoplasm and finally they were restrictedto the area around the nucleus. In prophase and preprophasecells thick strands occupied the cortical cytoplasmic zone wherein normal cells a preprophase microtubule band (PMB) was expectedto be assembled. In the colchicine-treated cells electron microscopy revealedan accumulation of paracrystalline aggregates, which initiallyoccurred along the cell wall and later deeper in the cytoplasm,in the perinuclear regions and the cytoplasmic invaginationsof the nucleus. In transverse planes the paracrystalline strandsappear to consist of hexagonal subunits in a 'honeycomb' arrangement,while in longitudinal and oblique sections they exhibit variableimages. Since their distribution coincides with that of thetubulin strands visualized by immunofluorescence, they are consideredto be the same structure. Therefore, the paracrystals consistof, or at least contain, tubulin. They are most likely to bepolymers of tubulin-colchicine complexes.Copyright 1995, 1999Academic Press Wheat roots, colchicine, immunofluorescence, electron microscopy, tubulin paracrystals, Triticum aestivum L     

Visualization of assembled and disassembled microtubule protein by double label fluorescence microscopy

Indirect immunofluorescence with rhodamine labelled antibodies and fluoresceinated colchicine (FC) are used to simultaneously localize microtubules and soluble tubulin in cultured ovarian granulosa cells. FC labelled tubulin is most concentrated in regions of the cell occupied by antitubulin stained microtubule bundles. Pretreatment of granulosa cells with colchicine results in a central accumulation of FC and antibody labelled tubulin that coincides with the disposition of 10-nm filament cables. In contrast, the microtubule disrupting agent nocodazole produces a diffuse tubulin distribution as detected with both FC and antibody probes. Taxol treatment, which enhances microtubule assembly, results in a striking concentration of microtubule bundles associated with the nucleus that avidly bind FC. These results suggest that disassembled tubulin is preferentially associated with cytoplasmic microtubules and possibly other formed elements of the cytoskeleton.     

Microtubule depolymerization in rat seminiferous epithelium is associated with diminished tyrosination of alpha-tubulin

In the testis, microtubule-disrupting agents cause breakdown of the Sertoli cell cytoskeleton and sloughing of germ cells with associated Sertoli cell fragments, although the mechanism underlying this event is not understood. In this study, we investigated the effects of carbendazim and colchicine on microtubule polymerization status and posttranslational modifications of tubulin in freshly isolated rat seminiferous tubules. Soluble and polymerized tubulin pools were separated and tubulin was quantified using a competitive ELISA. Carbendazim and colchicine caused extensive microtubule depolymerization, shifting the ratio of soluble to polymerized tubulin from 40%:60% to 78%:22%, and to 84%:16%, respectively. Total tubulin levels remained relatively constant after carbendazim treatment but decreased twofold after colchicine treatment. To determine if modifications to tubulin may be associated with polymerization status, tubulin pools were analyzed by immunoblotting. Acetylated alpha-tubulin and betaIII-tubulin distribution in tubulin pools was not affected by treatment. Tyrosinated alpha-tubulin (52 kDa) was localized in both tubulin pools and had decreased tyrosination in the microtubule pool after carbendazim treatment. A 47-kDa protein immunoreactive with both tyrosinated alpha-tubulin and general alpha-tubulin antibodies was found only in the microtubule pool. The 47-kDa protein (potentially an alpha-tubulin isoform) lost tyrosination, yet was still present in the microtubule pool based on detection with the general alpha-tubulin antibody, after carbendazim treatment. Similar effects were seen with colchicine, although loss of total tubulin protein was measured. Thus, decreased tyrosination of the microtubule pool of tubulin appears to be associated with depolymerization of microtubules.     

Local anesthetic-induced inhibition of collagen secretion in cultured cells under conditions where microtubules are not depolymerized by these agents

Tertiary amine local anesthetics previously have been shown to influence some microtubule-dependent cellular functions. Since several cell secretion processes, including secretion of collagen, have been shown to be inhibited by microtubule-disrupting drugs such as colchicine, we determined whether local anesthetics affect collagen secretion. Six local anesthetics inhibited collagen and non-collagen protein secretion (up to 98%) into the extracellular medium of 3T3 cells and human fibroblasts, an effect apparently independent of influences on proline transport and total protein synthesis. A combination of colchicine and cytochalasin B did not duplicate the effects of local anesthetics. The effects of subsaturating concentrations of colchicine and procaine on secretion were additive, suggesting that both drugs act on the secretory pathway at the level of microtubules, but other effects of the two types of drugs were strikingly different. In comparing the mechanisms of action of colchicine and local anesthetics, it was seen that, in contrast to colchicine, radioactive procaine and lidocaine were slowly transported into 3T3 cells, did not bind to the tubulin-containing TCA-insoluble fraction, and did not bind to purified tubulin in vitro. The fraction of cellular tubulin present as microtubules (47% in normal cells) was determined by measuring tubulin in stabilized, sedimentable microtubules compared to total tubulin, using a [3H]colchicine binding assay. Pretreatment of cells in the cold or with colchicine led to depolymerization of microtubules, but pretreatment with five local anesthetics tested did not. Therefore, in contrast to colchicine, local anesthetics in concentrations that inhibit secretion do not directly interact with or depolymerize microtubules. These drugs, however, do affect a microtubule-dependent process and may do so by detaching the microtubular system from the cell membrane.     

Microtubules in the fission yeast Schizosaccharomyces pombe contain only the tyrosinated form of alpha-tubulin.

The state of tubulin tyrosination in the fission yeast Schizosaccharomyces pombe was investigated using a combination of indirect immunofluorescence microscopy and Western blotting. Antibodies specific for the tyrosinated form of alpha-tubulin stained all microtubule arrays in wild type cells and recognised the two alpha-tubulin polypeptides in Western blots of cell extracts enriched for tubulin by DEAE-Sephadex chromatography. Antisera that specifically recognised the detyrosinated, glu, form, on the other hand, gave consistently negative results, both in cells undergoing rapid exponential growth and in those allowed to accumulate in stationary phase. Neither the "ageing" of microtubules, by arresting cells at different points (late G1 or G2/M) in the cell division cycle, nor stabilising them, using D2O, lead to any detectable tubulin detryrosination. These results suggest that S. pombe lacks the carboxypeptidase that carries out the tubulin detyrosination reaction. This is the first report of an organism that possesses the correct C-terminal alpha-tubulin sequence yet fails to carry out this post-translational modification. The implication of this novel finding for the biological role of these events is discussed.     

Modulation of tubulin mRNA levels by interferon in human lymphoblastoid cells.

Blot hybridization with labeled tubulin cDNA showed that treatment of Ramos cells, a human cell line of lymphoblastoid origin, with either alpha or beta interferon (IFN) induced a marked increase in the amount of tubulin mRNA sequences. The level of tubulin mRNA sequences increased rapidly after exposure of cells to IFN-alpha and reached a maximum after 1 h of treatment, which was four times the control level. Treatment with IFN-beta induced a maximal increase after 4 h; the amount of tubulin mRNA sequences was seven times higher than the control level. The mRNA extracted from IFN-treated and nontreated cells was translated in vitro in a reticulocyte lysate cell-free system containing [35S]methionine. Electrophoretic analysis of the labeled cell-free products showed an increase in the amount of translatable tubulin mRNA that parallels the time course of induction of tubulin mRNA sequences. Two-dimensional gel electrophoresis of the labeled protein products directed by mRNA indicates that IFN caused a more pronounced increase in the level of alpha-tubulin than beta-tubulin mRNA. Treatment with colchicine, which disrupts the cell microtubules, caused a marked decrease in the tubulin mRNA content. Concomitant treatment of the cells with colchicine and IFN abolished the interferon-dependent induction of tubulin mRNA.     

Colchicine-induced polyploidization depends on tubulin polymerization in c-metaphase cells

The microtubule cytoskeleton plays a crucial role in the cell cycle and in mitosis. Colchicine is a microtubule-depolymerizing agent that has long been used to induce chromosome individualization in cells arrested at metaphase and also in the induction of polyploid plants. Although attempts have been made to explain the processes and mechanisms underlying polyploidy induction, the role of the cytoskeleton still remains largely unknown. Through immunodetection of alpha-tubulin, different concentrations (0.5 or 5 mM) of colchicine were found to produce opposite effects in the organization of the cytoskeleton in rye (Secale cereale L.). A low concentration (0.5 mM) induced depolymerization of the microtubular cytoskeleton in all phases of the cell cycle. In contrast, a high concentration (5 mM) was found to induce the polymerization of new tubulin-containing structures in c-metaphase cells. Furthermore, both treatments also showed contrasting effects in the induction of polyploid cells. Flow cytometric analysis and quantitative assessments of nucleolus-organizing regions revealed that only the high-concentration colchicine treatment was effective in the formation of polyploid cells. Our studies indicate that spindle disruption alone is insufficient for the induction of polyploid cells. The absence of any tubulin structures in plants treated with colchicine at the low concentration induced cell anomalies, such as the occurrence of nuclei with irregular shape and/or (additional) micronuclei, 12 h after recovery, pointing to a direct effect on cell viability. In contrast, the almost insignificant level of cell anomalies in the high-concentration treatment suggests that the presence of new tubulin-containing structures allows the reconstitution of 4C nuclei and their progression into the cell cycle.     

Post-translational tubulin modifications in spermatogeneous cells of the pteridophyteCeratopteris richardii

Summary Acetylation and tyrosinization are post-translational modifications of tubulin generally associated, respectively, with highly stable or dynamic microtubule arrays in animals and protists. Little is known of these modifications in land plants, however. We examined the presence and distribution of post-translational tubulin modifications in developing spermatogenous cells of the pteridophyteCeratopteris richardii by immunofluorescence and immunogold, utilizing antibodies specific for acetylated and tyrosinated tubulin. Acetylated tubulin is found in mid to late stage spermatogenous cells in stable microtubule configurations: the spline, flagella, and basal bodies. Tyrosinated tubulin, a modification associated with dynamic microtubule arrays, is also present in these structures as well as all other microtubules in the cell. The lamellar strip of the multilayered structure, a body previously described as tubulin-containing, was not labelled by any of the tubulin antibodies or antiserum. Treatment of cultures with the microtubule stabilizer taxol results in the appearance of new arrays of microtubules, including bundles in the cytoplasm. Only those new taxol-induced microtubule arrays present in mid to late stage cells (i.e., those with other normally acetylated tubulin arrays) have acetylated domains. Younger spermatogenous cells had similar microtubule bundles but no acetylated tubulin. Tyrosinated tubulin was found in all these taxol-stabilized arrays. These data indicate that, although these pteridophyte cells have the ability to acetylate tubulin, that this ability is limited to stages after the final spermatogenous cell mitosis and is limited to the highly stable spline and flagella microtubules.Abbreviations LS lamellar strip of multilayered structure - MTOC microtubule organizing center     

alpha-Tubulin limits its own synthesis: evidence for a mechanism involving translational repression

A Chinese hamster alpha-tubulin cDNA was modified to encode an 11-amino acid carboxyl-terminal extension containing the immunodominant epitope from influenza hemagglutinin antigen (to create HA alpha 1-tubulin) and was cloned into a vector for expression in mammalian cells. 12 stable CHO cell lines expressing this HA alpha 1-tubulin were isolated and characterized. HA alpha 1-tubulin incorporated into all classes of microtubules, assembled to the same extent as the endogenous tubulin, and did not perturb the growth of the cells in which it was expressed. However, overexpression of HA alpha 1-tubulin strongly repressed the synthesis of endogenous alpha-tubulin while having little or no effect on the synthesis of beta-tubulin. Treatment of transfected cells with sodium butyrate to induce even greater expression of HA alpha 1-tubulin led to a further decrease in synthesis of endogenous alpha-tubulin that was fully reversible upon removal of the inducer. Decreased synthesis of alpha-tubulin in transfected cells did not result from decreased levels of alpha-tubulin mRNA, as demonstrated by ribonuclease protection assays. On the other hand, colchicine, a drug previously shown to destabilize the tubulin message, caused a clear reduction in both protein synthesis and mRNA levels for transfected HA alpha 1- tubulin and endogenous alpha-tubulin, thus indicating that the decreased alpha-tubulin synthesis observed as a result of HA alpha 1- tubulin overexpression is distinct from the previously described autoregulation of tubulin. The results are consistent with a mechanism in which free alpha-tubulin inhibits the translation of its own message as a way of ensuring stoichiometric synthesis of alpha- and beta- tubulin.     

Distinct localization and cell cycle dependence of COOH terminally tyrosinolated alpha-tubulin in the microtubules of Trypanosoma brucei brucei

alpha-Tubulin can be posttranslationally modified in that its COOH-terminal amino acid residue, tyrosine, can be selectively removed and replaced again. This reaction cycle involves two enzymes, tubulin carboxypeptidase and tubulin tyrosine ligase. The functional significance of this unusual modification is unclear. The present study demonstrates that posttranslational tyrosinolation of alpha-tubulin does occur in the parasitic hemoflagellate Trypanosoma brucei brucei and that posttranslational tyrosinolation can be detected in both alpha-tubulin isoforms found in this organism. Trypanosomes contain a number of microtubular structures: the flagellar axoneme; the subpellicular layer of singlet microtubules which are closely associated with the cell membrane; the basal bodies; and a cytoplasmic pool of soluble tubulin. Tyrosinolated alpha-tubulin is present in all these populations. However, immunofluorescence studies demonstrate a distinct localization of tyrosinolated alpha-tubulin within individual microtubules and organelles. This localization is subject to a temporal modulation that correlates strongly with progress of a cell through the cell cycle. Our results indicate that the presence of tyrosinolated alpha-tubulin is a marker for newly formed microtubules.     

Mechanisms of cellular adhesion : II. The interplay between adhesion, the cytoskeleton and morphology in substrate-attached cells

cAMP/theophylline exaggerates cell shape—whether the fibroblastic morphology of controls or the epithelioid shape of colchicine-treated cells. The ultrastructural basis is that cAMP/theophylline increases the number and linearity of microtubules and microfilament bundles, although where also treated with colchicine, the cells adopt a well-spread shape maintained by microfilament bundles alone. Since interference reflection microscopy shows that colchicine promotes the marked alignment of focal contacts (which terminate microfilament bundles) it is concluded that microtubules encourage angular cell form and modify the pattern of adhesions by influencing the directionality of microfilament bundle formation although they are inessential for the maintenance of the spread form or adhesion per se.     

Microtubules and morphogenesis in ordinary epidermal cells ofVigna sinensis leaves

Summary Undifferentiated ordinary epidermal cells (ECs) ofVigna sinensis leaves possess straight anticlinal walls and cortical microtubules (Mts) scattered along them. At an early stage of EC differentiation cortical Mts adjacent to the above walls form bundles normal to the leaf plane, loosely interconnected through the cortical cytoplasm of the internal periclinal wall. At the upper ends of the Mt bundles, Mts fan out towards the external periclinal wall and form radial arrays. Mt bundles and radial arrays exhibit strict alternate disposition between neighbouring ECs. An identical reticulum of cellulose microfibril (CM) bundles is deposited outside the Mt bundles. Local wall pads rise at the junctions of anticlinal walls with the external periclinal one, where the CM bundles terminate. They display radial CMs fanning towards the external periclinal wall. The CM bundles and radial CM systems prevent local cell bulging, but allow it in the intervening wall areas. In particular, the radial CM systems dictate the pattern of EC waviness by favouring local tangential expansion of external periclinal wall. As a result, ECs obtain an undulate appearance. Constrictions in one EC correspond with protrusions of adjacent ECs. ECs affected by colchicine entirely lose their Mts and do not develop wavy walls, an observation substantiating the role of cortical Mts in EC morphogenesis.Abbreviations CM cellulose microfibril - DTT dithiothreitol - EC epidermal cell - MSB microtubule stabilizing buffer - Mt microtubule - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride     

Identification of Saccharomyces cerevisiae Tub1 alpha-tubulin as a potential target for NKH-7, a cytotoxic 1-naphthol derivative compound

In a screening for small-molecule compounds that alleviate the deleterious effects of external CaCl(2) on zds1 Delta strain yeast, we found 2-((1-(hydroxymethyl) cyclohexyl) methyl) naphthalen-1-ol (NKH-7) to be an active compound. NKH-7 also inhibited cell growth at higher concentrations. To identify its target in growth inhibition, we isolated NKH-7-resistant mutants and selected those mutants that exhibited dominant or semi-dominant resistance specifically to NKH-7. By gene cloning, a TUB1 mutant gene encoding alpha-tubulin with a Ser248Pro mutation was identified. Deletion of the TUB3 gene, a minor gene encoding alpha-tubulin, led to supersensitivity to NKH-7. Cellular tubulin-containing arrays as visualized by green fluorescent protein (GFP)-labeled alpha-tubulin diminished rapidly on exposure to the inhibitor. The mutation was situated proximal to the alpha-beta interface of alpha-tubulin in microtubule protofilaments, suggesting the possibility that NKH-7 affects the hydrolysis of GTP bound to beta-tubulin. A functional connection perhaps exists between the tubulin inhibition and Ca(2+)-dependent cell-cycle regulation.     

Aluminum-induced rapid changes in the microtubular cytoskeleton of tobacco cell lines

Aluminum (Al) is a major factor that limits plant growth in acid soils. It causes a cessation of root growth and changes in root morphology suggesting a role of the root cytoskeleton as a target of Al-toxicity. Here we report a rapid effect of Al on the microtubular cytoskeleton of the suspension tobacco cell lines BY-2 and VBI-0. Viability studies showed that the cells were more sensitive to Al during exponential phase as compared to stationary cells. During the first hours of exposure, Al induced the formation of additional bundles of cortical microtubules (cMTs), whereas the thickness of the individual bundles decreased. Prolonged exposure resulted in disorientation of cMTs. These changes of cMTs preceded the decrease of cell viability by several hours and were accompanied by an increase in the levels of alpha-tubulin (in its tyrosinated form) and elements of the tubulin-folding chaperone CCT. These findings suggest that the microtubular cytoskeleton is one of the early targets of Al toxicity.