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Effects of taxol treatment on the microtubular system and mitochondria of Tetrahymena
Cell Biology International 31 (2007) 724e732 www.elsevier.com/locate/cellbi
Effects of taxol treatment on the microtubular system and mitochondria of Tetrahymena P. Kova´cs a, G. Csaba a,*, E´va Pa´llinger b, Renate Czaker c a
Department of Genetics, Cell- and Immunobiology, Semmelweis University, Nagyva´rad te´r 4, POB 370, H-1445 Budapest, Hungary b Molecular Immunological Research Group, Hungarian Academy of Sciences, Budapest, Hungary c Center of Anatomy and Cell Biology, Medical University, Vienna, Austria Received 30 October 2006; revised 12 December 2006; accepted 10 January 2007
Abstract Complex investigation was done using immunocytochemical confocal microscopy, electron microscopy and flow cytometry on the effect of taxol to the microtubular arrangement and dynamics. The most interesting phenomenon was the rapid disappearance of transversal microtubule bands, while longitudinal microtubule bands remained and were submitted to the known effects of taxol. There was a broad variation in mitochondrial effect, some of them remained normal, while others swollen, desintegrated and their tubules disoriented. Treatment with 50 nM taxol significantly reduced the binding of anti a-tubulin antibody and a lesser degree anti-acetylated tubulin antibody. The difference between the transversal and longitudinal microtubules is emphasized by the results and the paper discusses the possibilities of indirect effects of taxol to the transversal microtubules (tubulin-GTP interaction, faster turnover, mitochondrial interaction). Polyglutamylation of tubulin has not a role in this difference. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Microtubules; Taxol; Tetrahymena; Transversal microtubule band
1. Introduction Microtubules are targets for many antimitotic agents, including taxol. These agents are able to suppress microtubule dynamics and they are useful probes to investigate the role of these polymers in vivo. Taxol is a complex diterpene isolated from the bark of Taxus brevifolia; it is an effective antitumor drug, a potent inhibitor of cell proliferation and arrests cells in mitosis by a stabilizing action on microtubules (Rowinsky and Donehower, 1995). The well-established action mechanism of taxol in the treatment of cancer is based on this stabilization of microtubules in tumor cells and induction of apoptosis. In addition greatly increasing the microtubule polymer mass in cells, taxol also induces microtubule ‘‘bundling’’ (Turner and Margolis,
* Corresponding author. Tel.: þ36 1 210 2950; fax: þ36 1 303 6968. E-mail address: [email protected] (G. Csaba).
1984). In contrast to other antitumor drugs such as colchicine and vinblastine, which bind to the tubulin dimer, taxol has a binding site on the microtubule polymer. Microtubules that are formed in the presence of taxol are resistant to depolymerization by cold, dilution and Ca2þ; resulting in the formation of stable bundles of microtubules (Schiff and Horwitz, 1980). In eukaryotic cells, tubulin heterogeneity is generated at the gene level and is considerably increased by several types of posttranslational modifications, as acetylation, polyglutamylation. In Tetrahymena, as in other ciliates and flagellates, the number of tubulin genes is reduced (one a- and two b-tubulin genes, and also a g-tubulin gene), in spite of the complexity of the microtubular cytoskeleton: Tetrahymena assembles 17 types of distinct microtubules, which are localized in cilia, cell cortex, nuclei and the endoplasm. These diverse microtubules have distinct morphologies, stabilities and associations with specific microtubule-associated proteins (Gaertig, 2000). Tubulin and the microtubules have e apart from cytoskeletal role e many important functions. Thus tubulin regulates
1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.01.004
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certain G-protein-mediated signaling pathways. This function based on the GTP-binding properties of tubulin. Signal transduction through Gq-regulated phospholipase C1 (PLC1) is activated by tubulin through a direct transfer of GTP from tubulin to Gq. Thus, tubulin, depending on local membrane concentration, may serve as a positive or negative regulator of phosphoinositide hydrolysis (Popova et al., 2002). Distribution of mitochondria as well as other intracellular organelles in mammalian cells is regulated by interphase microtubules. Moreover, the interphase microtubules may be essential for the regulation of mitochondrial biogenesis in mammalian cells. Numerous observations demonstrate the presence of specific MAP-binding sites on the outer membrane, suggesting an association between porin and the membrane domain involved in the cross-linkage between microtubules and mitochondria (Linden et al., 1989). Ultrastructural observations of the cortically-located mitochondria of Tetrahymena thermophila revealed associations not only between the mitochondria and certain cortical microtubule bands, but also between the mitochondria and epiplasm of the cortex. Most of the distal mitochondrial surface is close and parallel to the epiplasm; favorable views show bridge-like structures spanning the 20e40 nm gap between the mitochondrion and the epiplasm. Some studies have shown that the localization of mitochondria in the cortex appears to be determined by certain cortical microtubule bands (Aufderheide, 1980). The original concept of apoptosis stressed the morphological changes of the nucleus, condensation with the aggregation of chromatin, and the intactness of intracellular organelles including mitochondria. One of the cellular events that trigger the mitochondrial pathway of apoptosis is the disturbance of the dynamic formation of microtubules in the cell. This event can be triggered by a variety of microtubule-targeted, tubulinpolymerizing agents (MTPAs), which include paclitaxel (taxol) and several other anticancer drugs. Following intracellular uptake, MTPAs bind b-tubulin and promote tubulin polymerization, which interferes with the function of the mitotic spindle resulting in mitotic arrest at the metaphase-anaphase transition and subsequent induction of the mitochondrial pathway of apoptosis (Bhalla, 2003). Taxol early initiates an apoptotic signaling pathway associated with increases in the mitochondrial reducing potential, mitochondrial membrane potential, p53 expression, and Bax/Bcl-2 ratio (Pasquier et al., 2004). Anti-tubulin agents induce the release of cytochrome c from isolated mitochondria. Thus, tubulin is an inherent component of mitochondrial membranes, and it could play a role in apoptosis via interaction with permeability transition pore (Carre et al., 2002). Taxol exerts rapid effects on the cytosolic Ca2þ signal via opening of the mitochondrial permeability transition pore (Kidd et al., 2002). Both mammalian cells and unicellulars alter the activity of transcription of tubulin genes after treatments with microtubule-depolymerising and polymerising drugs: a gen-specific signal transduction participates between microtubules and tubulin genes (Gu et al., 1995). In our previous experiments we obtained data on the connection of microtubular and signaling systems in Tetrahymena
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(Kova´cs and Pinte´r, 2001; Kova´cs et al., 2000), and on the effect of drugs affecting microtubular assembly to different kinds of microtubules in this unicellular organism (Kova´cs and Csaba, 2005a). The aim of the present study is to obtain additional data: 1./ on the alterations of different microtubular structures of Tetrahymena; 2./ on the alteration of posttranslational modification (e.g. acetylation, polyglutamylation) of different tubular structures after taxol treatments; and 3./ on the functional connection of the microtubular system and mitochondria during the treatments with taxol. 2. Materials and methods 2.1. Chemicals Taxol; mouse monoclonal anti-a-tubulin, anti-polyglutamylated tubulin and anti-acetylated tubulin antibodies, FITC-labelled anti-mouse goat IgG, and tryptone were obtained from Sigma (St Louis, MO, USA). Yeast extract was obtained from Oxoid (Unipath, Basingstoke, Hampshire, UK). Agar 100 was purchased from Agar Scientific (UK). All other chemicals used were of analytical grade available from commercial sources.
2.2. Tetrahymena cultures In the experiments, T. pyriformis GL strain was tested in the logarithmic phase of growth. The cells were cultivated at 28 C in 0.1 per cent yeast extract containing 1 per cent tryptone medium. Before the experiments the cells were washed with fresh culture medium and were resuspended at a concentration of 5 104 cells ml1.
2.3. Confocal scanning laser microscopic (CSLM) analysis of Tetrahymena cells labelled with monoclonal anti-a-tubulin, anti-polyglutamylated tubulin and anti-acetylated tubulin To localize tubulin-containing structures, taxol (50 nM) treated (1 h) and untreated (control) cells were fixed in 4% paraformaldehyde dissolved in PBS, pH 7.2. After washing with wash buffer (WB; ¼ 0.1% BSA in 20 mM TriseHCl; 0.9% NaCl; 0.05% Tween 20, pH 8.2) the cells were incubated with monoclonal anti-a-tubulin, anti-polyglutamylated tubulin and anti-acetylated tubulin antibodies diluted 1:500 with antibody [AB] buffer (1% BSA in 20 mM TriseHCl; 0.9% NaCl; 0.05% Tween 20, pH 8.2) for 45 min at room temperature. (To determine the rapidity of action of taxol-treatments on the transverse microtubular bundles we decorated the cells with anti-acetylated tubulin-antibody also after tenth and twentieth minutes of the taxol treatments.) After three washings with WB the anti-tubulin antibody treated cells were incubated with FITC-labelled anti-mouse goat IgG (diluted to 1:500 with AB buffer) for 45 min at room temperature. After this incubation the cells were washed four times with WB, and were mounted onto microscopic slides. The mounted cells were analyzed in a Bio-Rad MRC 1024 confocal scanning laser microscope (CSLM) equipped with a krypton/argon mixed gas laser as a light source. Excitation was provided by the 480 nm line from the laser.
2.4. Electron microscopy Tetrahymena specimens (control and treated with10 and 50 nM Taxol for 1 h) were fixed in 1.2% glutaraldehyde containing 0.1 M phosphate buffer (pH 7.0) for 1 h at room temperature, washed three times in the same buffer and then enclosed in 4% 50 C hot low melting agarose. Post fixation of cooled down cell blocks was performed with 1% OsO4 diluted with 0.1 M phosphate puffer (pH 7.0) for 1 h at room temperature. Dehydration was carried out by a graded series of ethanol and embedding in Agar 100 (Agar Scientific, UK) was done according to the manufacturer’s protocol. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a JEOL 1200EX electron microscope.
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2.5. Flow cytometric analysis Samples of cells were fixed with paraformaldehyde solution (dissolved in PBS, pH 7.2) for 5 min, and then washed twice in wash buffer. To block nonspecific binding of antibodies the cells were treated with blocking buffer (1% BSA in PBS) for 30 min at room temperature. Aliquots from cell suspensions were transferred into tubes, and primary antibodies were added for 30 min at room temperature. Negative controls were carried out with PBS containing 10 mg/ml BSA instead of primary antibodies. After washing four times with wash buffer to remove excess primary antibody the cells were incubated with secondary antibody (FITC-labeled monoclonal anti-mouse IgG developed in goat; diluted 1:50 with AB buffer) for 30 min at room temperature. For controlling the specificity, autofluorescence of the cells and aspecificity of the secondary antibodies were detected. The measurement was done in a FACSCalibur flow cytometer (Beckton Dickinson, San Jose, USA), using 25 000 cells for each measurement. For the measurement and analysis CellQuest Pro program was used. The numerical comparison of detected values was done by the comparison of percentual changes of geometric mean channel values to the appropriate control groups by using Origin program and Student’s t-test. All experiments were done in triplicate.
3. Results 3.1. Confocal scanning laser microscopic (CSLM) analysis of Tetrahymena cells labelled with monoclonal anti-a-tubulin, anti-polyglutaminated tubulin and anti-acetylated tubulin In the control (untreated) cells the transverse microtubule (TM) and longitudinal microtubule bands (LM), the oral
apparatus, deep fibers, basal bodies (BB), and the pore of contractile vacuoles (usually 2 per cells, rarely 3) are strongly labelled with anti-acetylated tubulin antibody (Fig. 1). Similar structures were labeled with anti-a-tubulin antibody (Fig. 2). Using anti-polyglutamylated antibody the cilia (mostly the tip of cilia) and the oral field were strongly labeled (Fig. 3). The use of taxol to alter microtubule assembly/disassembly dynamics resulted in some alterations of the different microtubular systems of Tetrahymena; these alterations seem to be different in the certain microtubular systems. After taxol (50 nM) treatments the labeling of the transversal microtubule-bands and the labeling of oral apparatus with anti-acetylated tubulin antibody disappear, sometimes only the remnants of transversal microtrubule-bands are visible (Fig. 4). Similar alteration is visible in the case of anti-a-tubulin antibody labeling, except the oral field, which is to a certain degree labeled (Fig. 5). This treatment does not alter the labeling-pattern with antipolyglutamylated antibody. The action of taxol on the transversal microtubule bands is relatively quick: after the tenth minute of treatments only the remnants of these structures are visible, and after twenty minutes they are totally disappeared (Fig. 6). 3.2. EM results Taxol caused severe ultrastructural damages, affecting a number of organelles especially mitochondria and, to a lesser degree the microtubular systems.
Fig. 1. Binding of anti acetylated-tubulin antibody to the Tetrahymena. Confocal microscopic pictures. Asterisk ¼ oral apparatus; arrow ¼ deep fiber; dotted arrow ¼ contractile vacuole pores; dotted arrows with circle ¼ transversal microtubule bands; thin arrows ¼ basal bodies; semicolon arrows ¼ longitudinal microtubule band. A and B ¼ same cell, optical sections at different levels. Magnification: A and B ¼ 1100; C ¼ 1500; D ¼ 2000.
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Fig. 2. Binding of anti a-tubulin antibody to the Tetrahymena. Confocal microscopic pictures. Same cell, optical sections at different levels. Asterisk ¼ oral apparatus; arrow ¼ deep fiber; dotted arrows ¼ contractile vacuole pores. Magnification: 900.
The somatic cortex in the untreated control sample of Tetrahymena was characterized by intact pellicular microtubular systems. Closely associated with the epiplasm were the supraepiplasmic microtubules constituting the so-called longitudinal microtubule bundle and the laterally directed subepiplasmic microtubules which form the so-called transverse microtubule bundle. The cortical ridges between the kineties contained the majority of the typical tubular mitochondria positioned in close association with the epiplasm and the pellicular microtubular systems (Fig. 7). Taxol (10 nM) treatment initiated the beginning of moderate alterations in the cortical region of Tetrahymena which, of course, were not found in all individuals. Thus, while a part of the individuals obviously appeared to be unaffected by the drug (Fig. 8A), the alterations in the other part primarily concentrated on the cortical mitochondria which successively began to
swell. (Fig. 8B) However, the longitudinal as well as the transverse microtubule bundles appeared unaltered (Fig. 8A). Taxol (50 nM) treatment was characterized by a marked increase of alterations which apparently affected in a first step the cortical mitochondria and in a second step the transverse microtubule bundles. However, in this group, too, individuals were found which exhibited a normally looking ultrastructure. What concerns now, is the structure of the mitochondria, it could be observed that the swelling proceeded. Thereby individuals were to observe in which only the most cortical mitochondria were affected while those beneath appeared unaltered. This situation corresponded to that in 10 nM taxol in so far as the longitudinal and transverse microtubule bundles showed a more or less regular pattern (Fig. 9A). The persistence of both these microtubule bundles could also be observed in individuals with markedly swollen mitochondria
Fig. 3. Binding of anti polyglutamilated-tubulin antibody to the Tetrahymena. Confocal microscopic pictures. Same cell, optical sections at different levels. Arrow ¼ oral apparatus. Magnification: 1200.
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Fig. 4. Binding of anti acetylated-tubulin antibodies to the taxol (50 nM for 1 h) treated Tetrahymena. Confocal microscopic pictures. The transversal microtubule bands disappeared. Arrow ¼ deep fiber; dotted arrow ¼ nucleus (stained with daunorubicin); rods ¼ basal bodies. Magnification: 1700.
provided that some intact ones were still present (Fig. 9B). However, in those individuals in which the greatest part of the swollen mitochondria began to disintegrate, the transverse microtubule bundles tended to disappear. In this stage the
mitochondria exhibited extremely reduced and peripherally placed, disoriented mitochondrial tubules The walls of the mitochondria became attenuated and lost their integrity by rupturing (Fig. 9C).
Fig. 5. Binding of anti a-tubulin antibodies to the taxol (50 nM for 1 h) treated Tetrahymena. Confocal microscopic pictures. The transversal microtubule bands disappeared. Asterisk ¼ oral apparatus; arrows ¼ basal bodies. Magnification: A and D ¼ 1700; B and C ¼ 900.
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Fig. 6. Binding of anti acetylated-tubulin antibodies to the taxol (50 nM, 10 min) treated (C and D), 20 min treated (E and F) and control (untreated) Tetrahymena (A and B). Confocal microscopic pictures. After 10 min treatments only remnants of transversal microtubule bands are visible, while after 20 min treatments totally disappeared. Asterisk ¼ oral apparatus; arrow ¼ contractile vacuole pores. Magnification: A, E and F ¼ 900; C and D ¼ 1200; B ¼ 2000.
3.3. Flow cytometric analysis Treatments with 50 nM taxol reduced the binding of anti a-tubulin-antibody significantly. These treatments decreased also the binding of the anti-acetylated tubulin-antibodies significantly, but to a lesser degree than in the case of anti a-tubulin-antibody. The binding of anti-polyglutamylated tubulin- and anti-g-tubulin antibodies were practically unchanged (Fig. 10).
induced alterations cause ramified modifications in numerous cellular events. In addition some of our present results are distinct from that reported in the literature. Although taxol induces several above mentioned alterations, the exact
4. Discussion The biological systems are very complex, the different structures are often in close morphological and functional connections. Taking into consideration this fact, sometimes it is very difficult to determine which structure is the first target of the applied treatment. This serious problem has arisen also in our present work. Microtubular structures e in addition to the cytoskeletal functions e have effects among others to the mitochondria, on the production of high energy nucleotide molecules, on the apoptotic events and on the signaling: these
Fig. 7. Electron microscopic picture of the cortical region of control (untreated) Tetrahymena. B ¼ basal body; M ¼ mitochondria; c ¼ cilia; arrow ¼ longitudinal section of transversal microtubule band; arrowhead ¼ cross section of longitudinal microtubule bands.
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Fig. 8. A and B. Electron microscopic pictures of 10 nM taxol (for 1 h) treated Tetrahymena. On the cellular organelles only moderate alterations are visible e compared to the controls. M ¼ mitochondria; c ¼ cilia; R ¼ rough endoplasmic reticulum; arrow ¼ transversal microtubule band; arrowhead ¼ cross section of lingitudinal microtubule band.
mechanism of its complex action in Tetrahymena is not yet known. Tetrahymena assembles 17 types of distinct microtubules, which are localized in cilia, cell cortex, nuclei, and the endoplasm. These diverse microtubules have distinct morphologies, stabilities, and associations with specific microtubule-associated proteins. It is unlikely that the unique properties of individual microtubules are derived by the utilization of diverse tubulin genes, because Tetrahymena expresses only a single isotype of a- and two isotypes of b-tubulin. However, Tetrahymena tubulins are modified secondarily by a post-translational mechanisms. Each microtubule organelle type displays an unique set of secondary tubulin modifications. Both highprecision targeting of molecular motors to individual organelles as well as organelle-specific tubulin modifications contribute to the creation of diverse microtubules in a single cytoplasm of Tetrahymena (Gaertig, 2000). It has been suggested that posttranslational modifications contribute to microtubule
stability indirectly by generating epitopes to bind stabilizing microtubule-associated proteins (Boucher et al., 1994). It was demonstrated in our previous experiments that two types of cytoplasmic microtubule bands (longitudinal and transversal) react differently to drugs affecting microtubular structures (Kova´cs and Pinte´r, 2001; Kova´cs and Csaba, 2005a). E.g. in nocodazole or colchicine treated cells longitudinal microtubules became thinner without any change of transversal ones. After taxol treatment LM also became thinner, however TM disappeared. Vinblastine made TM thinner, while it was ineffective to LM. These observations render probable that in contrast to the identity in morphology and main protein composition LM and TM are very different in their reaction and binding ability. Though in Tetrahymena we were the first who observed the different effect of taxol to LM and TM; in heliozoan axonemes Hausmann et al. observed the loss of intermicrotubule links under the effect of taxol (Hausmann et al., 1983). In our most recent experiments fluorescent labelled taxol (Arregui et al., 2002; Lecke et al., 2002) was used and the results further justified this idea: taxol does not label transversal MTs, while it stains longitudinal ones (Kova´cs and Csaba, 2005b) in fixed and living preparations. This points to the indirect disappearing effect of taxol to TMs, while the effect to LMs is caused by a direct binding. However, how can be imagined this indirect mechanism? Under the effect of taxol treatment TMs disappear quickly: after ten minutes the sign of destructions are visible, and after twenty minutes TMs totally disappear. Besides the CSLM pictures also the EM-pictures show this TM-degradation. A possibility for disturbing TM-s is the hindrance of the tubulin e GTP interaction. GTP bound to b-tubulin is hydrolyzed after addition of tubulin dimers to the microtubule ends, which gives rise the dinamic microtubule behaviors: the ‘‘treadmilling’’ (net addition of tubulin at one microtubule end and the net loss of tubulin at the opposite end) (Margolis and Wilson, 1978), and the dynamic instability, is a stochastic switching between shortening and growing phases at both ends of individual microtubules (Mitchison, 1988). Growing and shortening behavior may be due to a stochastic gain and loss of a stabilizing cap at both microtubule ends, which is thought to consist of a short region of GTP- or GDP-Pi-liganded tubulin at the ends of the microtubules (Carlier and Pantaloni, 1981). Taxol does not inhibit the binding of GTP to the tubulin or the hydrolysis of GTP. Thus taxol does not appear to directly affect gain and loss of the stabilzing GTP- (or GDPPi) cap at the microtubule ends (Schiff and Horwitz, 1981). Since taxol does not inhibit these phenomena, an other possibility is the disturbing effect of the normal mitochondrial functions, which leads to the insufficient GTP-synthesis, and this inhibits the TM-recovery e because of their supposed rapid turnover. Some results indicate that taxol act on the different structures sometimes independently from the microtubules. Taxol act directly on mitochondria isolated from human neuroblastoma cells (Andre et al., 2002); exerts rapid effects on the cytosolic Ca2þ signal via the opening of the mitochondrial permeability transition pore (Kidd et al., 2002). In
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Fig. 9. Electron microscopic pictures of 50 nM taxol (for 1 h) treated Tetrahymena. In certain cells (B and C) the mitochondria (M) are swollen and disintegrated, and also the transversal microtubule bands are destroyed (C, arrowhead). But other individuals exhibit a normal looking structures (A). Arrow and arrowhead ¼ microtubules; c ¼ cilia; B ¼ basal body; R ¼ rough endoplasmic reticulum; E ¼ exocytotic mucocyst.
Tetrahymena taxol influences tubulin genes as well as chaperonin genes (Casalou et al., 2001). In Trichomonas it influences hydrogenosomes, which had abnormal size and shape and autophage vacuoles appeared containing large amount of microtubules (Madeiro da Costa and Benchimol, 2004). These alterations also could influence the behavior of TMs, however the mechanisms are not known. Palmitoylation or polyglutamylation does not explain the difference in the taxol-effect. The effect of taxol on mitochondria seems to be especially important. In mammalian models this effect also can be observed (Andre et al., 2000; Andre et al., 2002), and there is an interrelation between the microtubular and mitochondial alterations. In taxol-treated cells the mitochondria are associated with taxol-stabilized microtubule bundles (Soltys and Gupta, 1992). It is supposed that microtubules have a role in mitochondrial biogenesis and -which is more interesting- two subpopulations of mitochondria were detected in taxol-treated cells (Karbowsky et al., 2001). This latter observation could explain why can be found normal and seriously harmed mitochondria in the same cell in our experiments. Treatment of mammalian cells with microtubule-depolymerizing and microtubule-polymerizing drugs causes decreases and increases in tubulin mRNA, respectively (Cleveland, 1989). In striking contrast to the case with mammalian cells, perturbation of microtubules in Tetrahymena thermophila by microtubule- depolymerizing or -polymerizing drugs increases the level of the single alpha-tubulin gene message by increasing transcription. T. thermophila has distinct, gene-specific mechanisms for modulating tubulin gene expression
depending on whether ciliary or cytoplasmic microtubules are involved (Stargell et al., 1992). This observation is distinct from that we report in the present experiments. On the basis of our flow cytometric measurements after taxol treatments the Tetrahymena cells are decorated to a lesser degree with antia-tubulin antibodies than the control (untreated) ones. However it is possible that taxol increases the transcription of tubulin mRNA: in this case the activity of translation of tubulin mRNAs are inhibited, for this phenomenon it is responsible presumably the impaired mitochondrial functions.
Fig. 10. Flow cytometric analysis of a-tubulin, acetylated tubulin and polyglutamylated tubulin of control (untreated) and taxol (50 nM, for 1 h) treated Tetrahymena. S ¼ p < 0,01 to the controls.
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An other effect of taxol-treatment in Tetrahymena the decreased acetylation of tubulin. After our FACS measurements the amount of acetylated-tubulin is significantly lower than in the controls. This decreasing thought to the disappearing of the TM-s and the decreased labeling of oral apparatus. These treatments does not affect the another tubulin-modification, the polyglutamylation. Acknowledgements The author thank Ms. M. Steiner and Ms. E. Vanyek-Zavadil for their skilled technical assistance. This work was supported by the National Research Fund (OTKA-T-037303), Hungary.
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Effects of taxol treatment on the microtubular system and mitochondria of Tetrahymena P. Kova´cs a, G. Csaba a,*, E´va Pa´llinger b, Renate Czaker c a
Department of Genetics, Cell- and Immunobiology, Semmelweis University, Nagyva´rad te´r 4, POB 370, H-1445 Budapest, Hungary b Molecular Immunological Research Group, Hungarian Academy of Sciences, Budapest, Hungary c Center of Anatomy and Cell Biology, Medical University, Vienna, Austria Received 30 October 2006; revised 12 December 2006; accepted 10 January 2007
Abstract Complex investigation was done using immunocytochemical confocal microscopy, electron microscopy and flow cytometry on the effect of taxol to the microtubular arrangement and dynamics. The most interesting phenomenon was the rapid disappearance of transversal microtubule bands, while longitudinal microtubule bands remained and were submitted to the known effects of taxol. There was a broad variation in mitochondrial effect, some of them remained normal, while others swollen, desintegrated and their tubules disoriented. Treatment with 50 nM taxol significantly reduced the binding of anti a-tubulin antibody and a lesser degree anti-acetylated tubulin antibody. The difference between the transversal and longitudinal microtubules is emphasized by the results and the paper discusses the possibilities of indirect effects of taxol to the transversal microtubules (tubulin-GTP interaction, faster turnover, mitochondrial interaction). Polyglutamylation of tubulin has not a role in this difference. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Microtubules; Taxol; Tetrahymena; Transversal microtubule band
1. Introduction Microtubules are targets for many antimitotic agents, including taxol. These agents are able to suppress microtubule dynamics and they are useful probes to investigate the role of these polymers in vivo. Taxol is a complex diterpene isolated from the bark of Taxus brevifolia; it is an effective antitumor drug, a potent inhibitor of cell proliferation and arrests cells in mitosis by a stabilizing action on microtubules (Rowinsky and Donehower, 1995). The well-established action mechanism of taxol in the treatment of cancer is based on this stabilization of microtubules in tumor cells and induction of apoptosis. In addition greatly increasing the microtubule polymer mass in cells, taxol also induces microtubule ‘‘bundling’’ (Turner and Margolis,
* Corresponding author. Tel.: þ36 1 210 2950; fax: þ36 1 303 6968. E-mail address: [email protected] (G. Csaba).
1984). In contrast to other antitumor drugs such as colchicine and vinblastine, which bind to the tubulin dimer, taxol has a binding site on the microtubule polymer. Microtubules that are formed in the presence of taxol are resistant to depolymerization by cold, dilution and Ca2þ; resulting in the formation of stable bundles of microtubules (Schiff and Horwitz, 1980). In eukaryotic cells, tubulin heterogeneity is generated at the gene level and is considerably increased by several types of posttranslational modifications, as acetylation, polyglutamylation. In Tetrahymena, as in other ciliates and flagellates, the number of tubulin genes is reduced (one a- and two b-tubulin genes, and also a g-tubulin gene), in spite of the complexity of the microtubular cytoskeleton: Tetrahymena assembles 17 types of distinct microtubules, which are localized in cilia, cell cortex, nuclei and the endoplasm. These diverse microtubules have distinct morphologies, stabilities and associations with specific microtubule-associated proteins (Gaertig, 2000). Tubulin and the microtubules have e apart from cytoskeletal role e many important functions. Thus tubulin regulates
1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.01.004
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certain G-protein-mediated signaling pathways. This function based on the GTP-binding properties of tubulin. Signal transduction through Gq-regulated phospholipase C1 (PLC1) is activated by tubulin through a direct transfer of GTP from tubulin to Gq. Thus, tubulin, depending on local membrane concentration, may serve as a positive or negative regulator of phosphoinositide hydrolysis (Popova et al., 2002). Distribution of mitochondria as well as other intracellular organelles in mammalian cells is regulated by interphase microtubules. Moreover, the interphase microtubules may be essential for the regulation of mitochondrial biogenesis in mammalian cells. Numerous observations demonstrate the presence of specific MAP-binding sites on the outer membrane, suggesting an association between porin and the membrane domain involved in the cross-linkage between microtubules and mitochondria (Linden et al., 1989). Ultrastructural observations of the cortically-located mitochondria of Tetrahymena thermophila revealed associations not only between the mitochondria and certain cortical microtubule bands, but also between the mitochondria and epiplasm of the cortex. Most of the distal mitochondrial surface is close and parallel to the epiplasm; favorable views show bridge-like structures spanning the 20e40 nm gap between the mitochondrion and the epiplasm. Some studies have shown that the localization of mitochondria in the cortex appears to be determined by certain cortical microtubule bands (Aufderheide, 1980). The original concept of apoptosis stressed the morphological changes of the nucleus, condensation with the aggregation of chromatin, and the intactness of intracellular organelles including mitochondria. One of the cellular events that trigger the mitochondrial pathway of apoptosis is the disturbance of the dynamic formation of microtubules in the cell. This event can be triggered by a variety of microtubule-targeted, tubulinpolymerizing agents (MTPAs), which include paclitaxel (taxol) and several other anticancer drugs. Following intracellular uptake, MTPAs bind b-tubulin and promote tubulin polymerization, which interferes with the function of the mitotic spindle resulting in mitotic arrest at the metaphase-anaphase transition and subsequent induction of the mitochondrial pathway of apoptosis (Bhalla, 2003). Taxol early initiates an apoptotic signaling pathway associated with increases in the mitochondrial reducing potential, mitochondrial membrane potential, p53 expression, and Bax/Bcl-2 ratio (Pasquier et al., 2004). Anti-tubulin agents induce the release of cytochrome c from isolated mitochondria. Thus, tubulin is an inherent component of mitochondrial membranes, and it could play a role in apoptosis via interaction with permeability transition pore (Carre et al., 2002). Taxol exerts rapid effects on the cytosolic Ca2þ signal via opening of the mitochondrial permeability transition pore (Kidd et al., 2002). Both mammalian cells and unicellulars alter the activity of transcription of tubulin genes after treatments with microtubule-depolymerising and polymerising drugs: a gen-specific signal transduction participates between microtubules and tubulin genes (Gu et al., 1995). In our previous experiments we obtained data on the connection of microtubular and signaling systems in Tetrahymena
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(Kova´cs and Pinte´r, 2001; Kova´cs et al., 2000), and on the effect of drugs affecting microtubular assembly to different kinds of microtubules in this unicellular organism (Kova´cs and Csaba, 2005a). The aim of the present study is to obtain additional data: 1./ on the alterations of different microtubular structures of Tetrahymena; 2./ on the alteration of posttranslational modification (e.g. acetylation, polyglutamylation) of different tubular structures after taxol treatments; and 3./ on the functional connection of the microtubular system and mitochondria during the treatments with taxol. 2. Materials and methods 2.1. Chemicals Taxol; mouse monoclonal anti-a-tubulin, anti-polyglutamylated tubulin and anti-acetylated tubulin antibodies, FITC-labelled anti-mouse goat IgG, and tryptone were obtained from Sigma (St Louis, MO, USA). Yeast extract was obtained from Oxoid (Unipath, Basingstoke, Hampshire, UK). Agar 100 was purchased from Agar Scientific (UK). All other chemicals used were of analytical grade available from commercial sources.
2.2. Tetrahymena cultures In the experiments, T. pyriformis GL strain was tested in the logarithmic phase of growth. The cells were cultivated at 28 C in 0.1 per cent yeast extract containing 1 per cent tryptone medium. Before the experiments the cells were washed with fresh culture medium and were resuspended at a concentration of 5 104 cells ml1.
2.3. Confocal scanning laser microscopic (CSLM) analysis of Tetrahymena cells labelled with monoclonal anti-a-tubulin, anti-polyglutamylated tubulin and anti-acetylated tubulin To localize tubulin-containing structures, taxol (50 nM) treated (1 h) and untreated (control) cells were fixed in 4% paraformaldehyde dissolved in PBS, pH 7.2. After washing with wash buffer (WB; ¼ 0.1% BSA in 20 mM TriseHCl; 0.9% NaCl; 0.05% Tween 20, pH 8.2) the cells were incubated with monoclonal anti-a-tubulin, anti-polyglutamylated tubulin and anti-acetylated tubulin antibodies diluted 1:500 with antibody [AB] buffer (1% BSA in 20 mM TriseHCl; 0.9% NaCl; 0.05% Tween 20, pH 8.2) for 45 min at room temperature. (To determine the rapidity of action of taxol-treatments on the transverse microtubular bundles we decorated the cells with anti-acetylated tubulin-antibody also after tenth and twentieth minutes of the taxol treatments.) After three washings with WB the anti-tubulin antibody treated cells were incubated with FITC-labelled anti-mouse goat IgG (diluted to 1:500 with AB buffer) for 45 min at room temperature. After this incubation the cells were washed four times with WB, and were mounted onto microscopic slides. The mounted cells were analyzed in a Bio-Rad MRC 1024 confocal scanning laser microscope (CSLM) equipped with a krypton/argon mixed gas laser as a light source. Excitation was provided by the 480 nm line from the laser.
2.4. Electron microscopy Tetrahymena specimens (control and treated with10 and 50 nM Taxol for 1 h) were fixed in 1.2% glutaraldehyde containing 0.1 M phosphate buffer (pH 7.0) for 1 h at room temperature, washed three times in the same buffer and then enclosed in 4% 50 C hot low melting agarose. Post fixation of cooled down cell blocks was performed with 1% OsO4 diluted with 0.1 M phosphate puffer (pH 7.0) for 1 h at room temperature. Dehydration was carried out by a graded series of ethanol and embedding in Agar 100 (Agar Scientific, UK) was done according to the manufacturer’s protocol. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a JEOL 1200EX electron microscope.
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2.5. Flow cytometric analysis Samples of cells were fixed with paraformaldehyde solution (dissolved in PBS, pH 7.2) for 5 min, and then washed twice in wash buffer. To block nonspecific binding of antibodies the cells were treated with blocking buffer (1% BSA in PBS) for 30 min at room temperature. Aliquots from cell suspensions were transferred into tubes, and primary antibodies were added for 30 min at room temperature. Negative controls were carried out with PBS containing 10 mg/ml BSA instead of primary antibodies. After washing four times with wash buffer to remove excess primary antibody the cells were incubated with secondary antibody (FITC-labeled monoclonal anti-mouse IgG developed in goat; diluted 1:50 with AB buffer) for 30 min at room temperature. For controlling the specificity, autofluorescence of the cells and aspecificity of the secondary antibodies were detected. The measurement was done in a FACSCalibur flow cytometer (Beckton Dickinson, San Jose, USA), using 25 000 cells for each measurement. For the measurement and analysis CellQuest Pro program was used. The numerical comparison of detected values was done by the comparison of percentual changes of geometric mean channel values to the appropriate control groups by using Origin program and Student’s t-test. All experiments were done in triplicate.
3. Results 3.1. Confocal scanning laser microscopic (CSLM) analysis of Tetrahymena cells labelled with monoclonal anti-a-tubulin, anti-polyglutaminated tubulin and anti-acetylated tubulin In the control (untreated) cells the transverse microtubule (TM) and longitudinal microtubule bands (LM), the oral
apparatus, deep fibers, basal bodies (BB), and the pore of contractile vacuoles (usually 2 per cells, rarely 3) are strongly labelled with anti-acetylated tubulin antibody (Fig. 1). Similar structures were labeled with anti-a-tubulin antibody (Fig. 2). Using anti-polyglutamylated antibody the cilia (mostly the tip of cilia) and the oral field were strongly labeled (Fig. 3). The use of taxol to alter microtubule assembly/disassembly dynamics resulted in some alterations of the different microtubular systems of Tetrahymena; these alterations seem to be different in the certain microtubular systems. After taxol (50 nM) treatments the labeling of the transversal microtubule-bands and the labeling of oral apparatus with anti-acetylated tubulin antibody disappear, sometimes only the remnants of transversal microtrubule-bands are visible (Fig. 4). Similar alteration is visible in the case of anti-a-tubulin antibody labeling, except the oral field, which is to a certain degree labeled (Fig. 5). This treatment does not alter the labeling-pattern with antipolyglutamylated antibody. The action of taxol on the transversal microtubule bands is relatively quick: after the tenth minute of treatments only the remnants of these structures are visible, and after twenty minutes they are totally disappeared (Fig. 6). 3.2. EM results Taxol caused severe ultrastructural damages, affecting a number of organelles especially mitochondria and, to a lesser degree the microtubular systems.
Fig. 1. Binding of anti acetylated-tubulin antibody to the Tetrahymena. Confocal microscopic pictures. Asterisk ¼ oral apparatus; arrow ¼ deep fiber; dotted arrow ¼ contractile vacuole pores; dotted arrows with circle ¼ transversal microtubule bands; thin arrows ¼ basal bodies; semicolon arrows ¼ longitudinal microtubule band. A and B ¼ same cell, optical sections at different levels. Magnification: A and B ¼ 1100; C ¼ 1500; D ¼ 2000.
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Fig. 2. Binding of anti a-tubulin antibody to the Tetrahymena. Confocal microscopic pictures. Same cell, optical sections at different levels. Asterisk ¼ oral apparatus; arrow ¼ deep fiber; dotted arrows ¼ contractile vacuole pores. Magnification: 900.
The somatic cortex in the untreated control sample of Tetrahymena was characterized by intact pellicular microtubular systems. Closely associated with the epiplasm were the supraepiplasmic microtubules constituting the so-called longitudinal microtubule bundle and the laterally directed subepiplasmic microtubules which form the so-called transverse microtubule bundle. The cortical ridges between the kineties contained the majority of the typical tubular mitochondria positioned in close association with the epiplasm and the pellicular microtubular systems (Fig. 7). Taxol (10 nM) treatment initiated the beginning of moderate alterations in the cortical region of Tetrahymena which, of course, were not found in all individuals. Thus, while a part of the individuals obviously appeared to be unaffected by the drug (Fig. 8A), the alterations in the other part primarily concentrated on the cortical mitochondria which successively began to
swell. (Fig. 8B) However, the longitudinal as well as the transverse microtubule bundles appeared unaltered (Fig. 8A). Taxol (50 nM) treatment was characterized by a marked increase of alterations which apparently affected in a first step the cortical mitochondria and in a second step the transverse microtubule bundles. However, in this group, too, individuals were found which exhibited a normally looking ultrastructure. What concerns now, is the structure of the mitochondria, it could be observed that the swelling proceeded. Thereby individuals were to observe in which only the most cortical mitochondria were affected while those beneath appeared unaltered. This situation corresponded to that in 10 nM taxol in so far as the longitudinal and transverse microtubule bundles showed a more or less regular pattern (Fig. 9A). The persistence of both these microtubule bundles could also be observed in individuals with markedly swollen mitochondria
Fig. 3. Binding of anti polyglutamilated-tubulin antibody to the Tetrahymena. Confocal microscopic pictures. Same cell, optical sections at different levels. Arrow ¼ oral apparatus. Magnification: 1200.
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Fig. 4. Binding of anti acetylated-tubulin antibodies to the taxol (50 nM for 1 h) treated Tetrahymena. Confocal microscopic pictures. The transversal microtubule bands disappeared. Arrow ¼ deep fiber; dotted arrow ¼ nucleus (stained with daunorubicin); rods ¼ basal bodies. Magnification: 1700.
provided that some intact ones were still present (Fig. 9B). However, in those individuals in which the greatest part of the swollen mitochondria began to disintegrate, the transverse microtubule bundles tended to disappear. In this stage the
mitochondria exhibited extremely reduced and peripherally placed, disoriented mitochondrial tubules The walls of the mitochondria became attenuated and lost their integrity by rupturing (Fig. 9C).
Fig. 5. Binding of anti a-tubulin antibodies to the taxol (50 nM for 1 h) treated Tetrahymena. Confocal microscopic pictures. The transversal microtubule bands disappeared. Asterisk ¼ oral apparatus; arrows ¼ basal bodies. Magnification: A and D ¼ 1700; B and C ¼ 900.
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Fig. 6. Binding of anti acetylated-tubulin antibodies to the taxol (50 nM, 10 min) treated (C and D), 20 min treated (E and F) and control (untreated) Tetrahymena (A and B). Confocal microscopic pictures. After 10 min treatments only remnants of transversal microtubule bands are visible, while after 20 min treatments totally disappeared. Asterisk ¼ oral apparatus; arrow ¼ contractile vacuole pores. Magnification: A, E and F ¼ 900; C and D ¼ 1200; B ¼ 2000.
3.3. Flow cytometric analysis Treatments with 50 nM taxol reduced the binding of anti a-tubulin-antibody significantly. These treatments decreased also the binding of the anti-acetylated tubulin-antibodies significantly, but to a lesser degree than in the case of anti a-tubulin-antibody. The binding of anti-polyglutamylated tubulin- and anti-g-tubulin antibodies were practically unchanged (Fig. 10).
induced alterations cause ramified modifications in numerous cellular events. In addition some of our present results are distinct from that reported in the literature. Although taxol induces several above mentioned alterations, the exact
4. Discussion The biological systems are very complex, the different structures are often in close morphological and functional connections. Taking into consideration this fact, sometimes it is very difficult to determine which structure is the first target of the applied treatment. This serious problem has arisen also in our present work. Microtubular structures e in addition to the cytoskeletal functions e have effects among others to the mitochondria, on the production of high energy nucleotide molecules, on the apoptotic events and on the signaling: these
Fig. 7. Electron microscopic picture of the cortical region of control (untreated) Tetrahymena. B ¼ basal body; M ¼ mitochondria; c ¼ cilia; arrow ¼ longitudinal section of transversal microtubule band; arrowhead ¼ cross section of longitudinal microtubule bands.
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Fig. 8. A and B. Electron microscopic pictures of 10 nM taxol (for 1 h) treated Tetrahymena. On the cellular organelles only moderate alterations are visible e compared to the controls. M ¼ mitochondria; c ¼ cilia; R ¼ rough endoplasmic reticulum; arrow ¼ transversal microtubule band; arrowhead ¼ cross section of lingitudinal microtubule band.
mechanism of its complex action in Tetrahymena is not yet known. Tetrahymena assembles 17 types of distinct microtubules, which are localized in cilia, cell cortex, nuclei, and the endoplasm. These diverse microtubules have distinct morphologies, stabilities, and associations with specific microtubule-associated proteins. It is unlikely that the unique properties of individual microtubules are derived by the utilization of diverse tubulin genes, because Tetrahymena expresses only a single isotype of a- and two isotypes of b-tubulin. However, Tetrahymena tubulins are modified secondarily by a post-translational mechanisms. Each microtubule organelle type displays an unique set of secondary tubulin modifications. Both highprecision targeting of molecular motors to individual organelles as well as organelle-specific tubulin modifications contribute to the creation of diverse microtubules in a single cytoplasm of Tetrahymena (Gaertig, 2000). It has been suggested that posttranslational modifications contribute to microtubule
stability indirectly by generating epitopes to bind stabilizing microtubule-associated proteins (Boucher et al., 1994). It was demonstrated in our previous experiments that two types of cytoplasmic microtubule bands (longitudinal and transversal) react differently to drugs affecting microtubular structures (Kova´cs and Pinte´r, 2001; Kova´cs and Csaba, 2005a). E.g. in nocodazole or colchicine treated cells longitudinal microtubules became thinner without any change of transversal ones. After taxol treatment LM also became thinner, however TM disappeared. Vinblastine made TM thinner, while it was ineffective to LM. These observations render probable that in contrast to the identity in morphology and main protein composition LM and TM are very different in their reaction and binding ability. Though in Tetrahymena we were the first who observed the different effect of taxol to LM and TM; in heliozoan axonemes Hausmann et al. observed the loss of intermicrotubule links under the effect of taxol (Hausmann et al., 1983). In our most recent experiments fluorescent labelled taxol (Arregui et al., 2002; Lecke et al., 2002) was used and the results further justified this idea: taxol does not label transversal MTs, while it stains longitudinal ones (Kova´cs and Csaba, 2005b) in fixed and living preparations. This points to the indirect disappearing effect of taxol to TMs, while the effect to LMs is caused by a direct binding. However, how can be imagined this indirect mechanism? Under the effect of taxol treatment TMs disappear quickly: after ten minutes the sign of destructions are visible, and after twenty minutes TMs totally disappear. Besides the CSLM pictures also the EM-pictures show this TM-degradation. A possibility for disturbing TM-s is the hindrance of the tubulin e GTP interaction. GTP bound to b-tubulin is hydrolyzed after addition of tubulin dimers to the microtubule ends, which gives rise the dinamic microtubule behaviors: the ‘‘treadmilling’’ (net addition of tubulin at one microtubule end and the net loss of tubulin at the opposite end) (Margolis and Wilson, 1978), and the dynamic instability, is a stochastic switching between shortening and growing phases at both ends of individual microtubules (Mitchison, 1988). Growing and shortening behavior may be due to a stochastic gain and loss of a stabilizing cap at both microtubule ends, which is thought to consist of a short region of GTP- or GDP-Pi-liganded tubulin at the ends of the microtubules (Carlier and Pantaloni, 1981). Taxol does not inhibit the binding of GTP to the tubulin or the hydrolysis of GTP. Thus taxol does not appear to directly affect gain and loss of the stabilzing GTP- (or GDPPi) cap at the microtubule ends (Schiff and Horwitz, 1981). Since taxol does not inhibit these phenomena, an other possibility is the disturbing effect of the normal mitochondrial functions, which leads to the insufficient GTP-synthesis, and this inhibits the TM-recovery e because of their supposed rapid turnover. Some results indicate that taxol act on the different structures sometimes independently from the microtubules. Taxol act directly on mitochondria isolated from human neuroblastoma cells (Andre et al., 2002); exerts rapid effects on the cytosolic Ca2þ signal via the opening of the mitochondrial permeability transition pore (Kidd et al., 2002). In
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Fig. 9. Electron microscopic pictures of 50 nM taxol (for 1 h) treated Tetrahymena. In certain cells (B and C) the mitochondria (M) are swollen and disintegrated, and also the transversal microtubule bands are destroyed (C, arrowhead). But other individuals exhibit a normal looking structures (A). Arrow and arrowhead ¼ microtubules; c ¼ cilia; B ¼ basal body; R ¼ rough endoplasmic reticulum; E ¼ exocytotic mucocyst.
Tetrahymena taxol influences tubulin genes as well as chaperonin genes (Casalou et al., 2001). In Trichomonas it influences hydrogenosomes, which had abnormal size and shape and autophage vacuoles appeared containing large amount of microtubules (Madeiro da Costa and Benchimol, 2004). These alterations also could influence the behavior of TMs, however the mechanisms are not known. Palmitoylation or polyglutamylation does not explain the difference in the taxol-effect. The effect of taxol on mitochondria seems to be especially important. In mammalian models this effect also can be observed (Andre et al., 2000; Andre et al., 2002), and there is an interrelation between the microtubular and mitochondial alterations. In taxol-treated cells the mitochondria are associated with taxol-stabilized microtubule bundles (Soltys and Gupta, 1992). It is supposed that microtubules have a role in mitochondrial biogenesis and -which is more interesting- two subpopulations of mitochondria were detected in taxol-treated cells (Karbowsky et al., 2001). This latter observation could explain why can be found normal and seriously harmed mitochondria in the same cell in our experiments. Treatment of mammalian cells with microtubule-depolymerizing and microtubule-polymerizing drugs causes decreases and increases in tubulin mRNA, respectively (Cleveland, 1989). In striking contrast to the case with mammalian cells, perturbation of microtubules in Tetrahymena thermophila by microtubule- depolymerizing or -polymerizing drugs increases the level of the single alpha-tubulin gene message by increasing transcription. T. thermophila has distinct, gene-specific mechanisms for modulating tubulin gene expression
depending on whether ciliary or cytoplasmic microtubules are involved (Stargell et al., 1992). This observation is distinct from that we report in the present experiments. On the basis of our flow cytometric measurements after taxol treatments the Tetrahymena cells are decorated to a lesser degree with antia-tubulin antibodies than the control (untreated) ones. However it is possible that taxol increases the transcription of tubulin mRNA: in this case the activity of translation of tubulin mRNAs are inhibited, for this phenomenon it is responsible presumably the impaired mitochondrial functions.
Fig. 10. Flow cytometric analysis of a-tubulin, acetylated tubulin and polyglutamylated tubulin of control (untreated) and taxol (50 nM, for 1 h) treated Tetrahymena. S ¼ p < 0,01 to the controls.
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An other effect of taxol-treatment in Tetrahymena the decreased acetylation of tubulin. After our FACS measurements the amount of acetylated-tubulin is significantly lower than in the controls. This decreasing thought to the disappearing of the TM-s and the decreased labeling of oral apparatus. These treatments does not affect the another tubulin-modification, the polyglutamylation. Acknowledgements The author thank Ms. M. Steiner and Ms. E. Vanyek-Zavadil for their skilled technical assistance. This work was supported by the National Research Fund (OTKA-T-037303), Hungary.
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