Chromosome Separation and Exit from Mitosis in Budding Yeast: Dependence on Growth Revealed by cAMP-Mediated Inhibition

Cell cycle progression of somatic cells depends on net mass accumulation. In Saccharomyces cerevisiae the cAMP-dependent kinases (PKAs) promote cytoplasmic growth and modulate the growth-regulated mechanism triggering the begin of DNA synthesis. By altering the cAMP signal in budding yeast cells we show here that mitotic events can also depend on growth. In fact, the hyperactivation of PKAs permanently inhibited both anaphase and exit from mitosis when cell growth was repressed. In S. cerevisiae the anaphase promoting complex (APC) triggers entry into anaphase by mediating the degradation of Pds1p. The cAMP pathway activation was lethal together with a partial impairment of the Cdc16p APC subunit, causing a preanaphase arrest, and conversely low PKA activity suppressed the lethality of cdc16-1 cells. Deregulated PKAs partially prevented the decrease of Pds1p intracellular levels concomitantly with the anaphase inhibition, and the PKA-dependent preanaphase arrest could be suppressed in pds1(-) cells. Thus, the cAMP pathway and APC functionally interact in S. cerevisiae and Pds1p is required for the cAMP-mediated inhibition of chromosome separation. Exit from mitosis requires APC, Cdc15p, and the polo-like Cdc5p kinase. PKA hyperactivation and a cdc15 mutation were synthetically lethal and brought to a telophase arrest. Finally, a low cAMP signal allowed cell division at a small cell size and suppressed the lethality of cdc15-2 or cdc5-1 cells. We propose that mitosis progression and the M/G1 phase transition specifically depend on cell growth through a mechanism modulated by PKAs and interacting with the APC/CDC15/CDC5 mitotic system. A possible functional antagonism between PKAs and the mitosis promoting factor is also discussed.     

Pds1p, an inhibitor of anaphase in budding yeast, plays a critical role in the APC and checkpoint pathway(s)

We report the isolation and characterization of pds1 mutants in Saccharomyces cerevisiae. The initial pds1-1 allele was identified by its inviability after transient exposure to microtubule inhibitors and its precocious dissociation of sister chromatids in the presence of these microtubule inhibitors. These findings suggest that pds1 mutants might be defective in anaphase arrest that normally is imposed by a spindle-damage checkpoint. To further examine a role for Pds1p in anaphase arrest, we compared the cell cycle arrest of pds1 mutants and PDS1 cells after: (a) the inactivation of Cdc16p or Cdc23p, two proteins that are required for the degradation of mitotic cyclins and are putative components of the yeast anaphase promoting complex (APC); (b) the inactivation of Cdc20p, another protein implicated in the degradation of mitotic cyclins; and (c) the inactivation of Cdc13 protein or gamma irradiation, two circumstances that induce a DNA- damage checkpoint. Under all these conditions, anaphase is inhibited in PDS1 cells but not in pds1 mutants. From these results we suggest that Pds1 protein is an anaphase inhibitor in PDS1 cells but not in pds1 mutants. From these results we suggest that Pds1 protein is an anaphase inhibitor that plays a critical role in the control of anaphase by both APC and checkpoints. We also show that pds1 mutants exit mitosis and initiate new rounds of cell division after gamma irradiation and Cdc13p inactivation but no after nocodazole-treatment or inactivation of Cdc16p, Cdc20p or Cdc23p function. Therefore, in the DNA-damage checkpoint, Pds1p is required for the inhibition of cytokinesis and DNA replication as well as anaphase. The role of Pds1 protein in anaphase inhibition and general cell cycle regulation is discussed.     

Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint

Cyclin A is a stable protein in S and G2 phases, but is destabilized when cells enter mitosis and is almost completely degraded before the metaphase to anaphase transition. Microinjection of antibodies against subunits of the anaphase-promoting complex/cyclosome (APC/C) or against human Cdc20 (fizzy) arrested cells at metaphase and stabilized both cyclins A and B1. Cyclin A was efficiently polyubiquitylated by Cdc20 or Cdh1-activated APC/C in vitro, but in contrast to cyclin B1, the proteolysis of cyclin A was not delayed by the spindle assembly checkpoint. The degradation of cyclin B1 was accelerated by inhibition of the spindle assembly checkpoint. These data suggest that the APC/C is activated as cells enter mitosis and immediately targets cyclin A for degradation, whereas the spindle assembly checkpoint delays the degradation of cyclin B1 until the metaphase to anaphase transition. The "destruction box" (D-box) of cyclin A is 10-20 residues longer than that of cyclin B. Overexpression of wild-type cyclin A delayed the metaphase to anaphase transition, whereas expression of cyclin A mutants lacking a D-box arrested cells in anaphase.     

Dual inhibition of sister chromatid separation at metaphase.

Separation of sister chromatids in anaphase is mediated by separase, an endopeptidase that cleaves the chromosomal cohesin SCC1. Separase is inhibited by securin, which is degraded at the metaphase-anaphase transition. Using Xenopus egg extracts, we demonstrate that high CDC2 activity inhibits anaphase but not securin degradation. We show that separase is kept inactive under these conditions by a mechanism independent of binding to securin. Mutation of a single phosphorylation site on separase relieves the inhibition and rescues chromatid separation in extracts with high CDC2 activity. Using quantitative mass spectrometry, we show that, in intact cells, there is complete phosphorylation of this site in metaphase and significant dephosphorylation in anaphase. We propose that separase activation at the metaphase-anaphase transition requires the removal of both securin and an inhibitory phosphate.     

Cell cycle–regulated membrane binding of NuMA contributes to efficient anaphase chromosome separation

Accurate and efficient separation of sister chromatids during anaphase is critical for faithful cell division. It has been proposed that cortical dynein–generated pulling forces on astral microtubules contribute to anaphase spindle elongation and chromosome separation. In mammalian cells, however, definitive evidence for the involvement of cortical dynein in chromosome separation is missing. It is believed that dynein is recruited and anchored at the cell cortex during mitosis by the α subunit of heterotrimeric G protein (Gα)/mammalian homologue of Drosophila Partner of Inscuteable/nuclear mitotic apparatus (NuMA) ternary complex. Here we uncover a Gα/LGN-independent lipid- and membrane-binding domain at the C-terminus of NuMA. We show that the membrane binding of NuMA is cell cycle regulated—it is inhibited during prophase and metaphase by cyclin-dependent kinase 1 (CDK1)–mediated phosphorylation and only occurs after anaphase onset when CDK1 activity is down-regulated. Further studies indicate that cell cycle–regulated membrane association of NuMA underlies anaphase-specific enhancement of cortical NuMA and dynein. By replacing endogenous NuMA with membrane-binding-deficient NuMA, we can specifically reduce the cortical accumulation of NuMA and dynein during anaphase and demonstrate that cortical NuMA and dynein contribute to efficient chromosome separation in mammalian cells.     

The budding yeast PP2ACdc55 protein phosphatase prevents the onset of anaphase in response to morphogenetic defects

Faithful chromosome transmission requires establishment of sister chromatid cohesion during S phase, followed by its removal at anaphase onset. Sister chromatids are tethered together by cohesin, which is displaced from chromosomes through cleavage of its Mcd1 subunit by the separase protease. Separase is in turn inhibited, up to this moment, by securin. Budding yeast cells respond to morphogenetic defects by a transient arrest in G2 with high securin levels and unseparated chromatids. We show that neither securin elimination nor forced cohesin cleavage is sufficient for anaphase in these conditions, suggesting that other factors contribute to cohesion maintainance in G2. We find that the protein phosphatase PP2A bound to its regulatory subunit Cdc55 plays a key role in this process, uncovering a new function for PP2A(Cdc55) in controlling a noncanonical pathway of chromatid cohesion removal.     

Inhibition of autophagy in mitotic animal cells

In nutrient-deprived cells autophagy recycles cytoplasmic constituents by engulfing and degrading them in membrane-bound autophagic vacuoles. The regulation of autophagic vacuole formation is poorly understood, but here we show this process is under strict cell-cycle control in cultured animal cells. We found strong inhibition of autophagic vacuole accumulation in nocodazole-arrested pseudo-prometaphase cells, and also in metaphase and anaphase cells generated on release from the nocodazole arrest. Autophagic vacuoles reappeared after closure of the nuclear envelope in telophase/G1. Treatment with phosphoinositide 3(PI3)-kinase inhibitors wortmannin, LY294002 and 3-methyladenine (known to inhibit the autophagic response in interphase cells) rescued autophagy in mitotic cells without inducing reassembly of vesiculated ER and Golgi compartments. The autophagy induced in mitotic cells was inhibited by amino acids, and the resulting autophagosomes contained proteins LC3 and Lamp1, known to be associated with autophagosomes in interphase cells. The mitotic inhibition of autophagy was not relieved by rapamycin treatment or in PDK1–/– embryonic stem cells, by microinjection of inhibitory antibodies against the class III PI3 kinase VPS34, or in cell lines lacking the p85 regulatory subunits of class IA PI3 kinases. Our results show that autophagy is under strict mitotic control and indicate a novel role for phosphoinositide 3-kinases or other wortmannin/LY294002-sensitive kinases in mitotic membrane traffic regulation .     

Phosphorylation of histone H3 serine 10 in early mouse embryos: Active phosphorylation at late S phase and differential effects of ZM447439 on first two embryonic mitoses

Cell division in mammalian cells is regulated by Aurora kinases. The activity of Aurora A is indispensable for correct function of centrosomes and proper spindle formation, while Aurora B for chromosome biorientation and separation. Aurora B is also responsible for the phosphorylation of histone H3 serine 10 (H3S10Ph) from G2 to metaphase. Data concerning the Aurora B activity and H3S10Ph in embryonic cells are limited to primordial and maturing oocytes and advanced pronuclei in zygotes. In the present study we have analyzed H3S10Ph in 1- and 2-cell mouse embryos. We show that H3S10 remains phosphorylated at anaphase and telophase of the second meiotic division, as well as during the anaphase and telophase of the first and second embryonic mitoses. At late G1 H3S10 is dephosphorylated and subsequently phosphorylated de novo at late S phase of the first and second cell cycle. These results show that the H3S10 phosphorylation/dephosphorylation cycle in embryonic cells is different than in somatic cells. The behaviour of thymocyte G0 nuclei introduced into ovulated oocytes and early 1-cell parthenogenotes confirms that kinases responsible for de novo H3S10 phosphorylation, most probably Aurora B, are active until G1 of the first cell cycle of mouse embryo. The inhibition of Aurora kinases by ZM447439 caused abnormalities both in the first and second mitoses. However, the disturbances in each division differed, suggesting important differences in the control of these mitoses. In ZM447439-treated mitotic zygotes Mad2 protein remained continuously present on kinetochores, what confirmed that spindle checkpoint remained active.     

Growth regulation of a human mature B cell line, B104, by anti-IgM and anti-IgD antibodies

An EBNA- human B lymphoma cell line, B104, was established. B104 cells express IgD as well as IgM on their surface, which is thought to be a basic characteristic of mature B cells. The growth of B104 cells was inhibited by treatment with a panel of anti-IgM antibodies. Cell cycle analyses revealed that the transition of B104 cells from the G2/M to the G0/G1 phase of the cell cycle was markedly inhibited by treatment with anti-IgM antibodies. Progression of B104 cells to the M phase of the cell cycle was found to be suppressed in the presence of anti-IgM antibodies. In contrast, both the entrance of G0/G1 phase cells into the S phase and the progression of S phase cells to the G2/M phase of the cell cycle did not seem to be inhibited significantly by treatment with anti-IgM antibodies. These results indicate that the mechanism of the inhibition of growth of B104 cells by anti-IgM antibodies is blockage of the transition from the G2 to the M phase of the cell cycle. In contrast to anti-IgM antibodies, anti-IgD antibodies could not cause growth inhibition of B104 cells at all. B cell growth factors such as IL-4 and IL-6 had no effect on the inhibition of growth of B104 cells by anti-IgM antibody. IFN-alpha and -beta, which have no B cell growth factor activity, did increase the number of cells that survived the treatment with anti-IgM antibodies. B104 is an excellent experimental model for the study of the mechanism of signal transduction through sIg as well as the functional difference between sIgM and sIgD.     

Arrest of 3T3 cells in G1 phase by low density lipoprotein

Low density lipoprotein (LDL) and high density lipoprotein (HDL) were purified from normal human serum by KBr density gradient centrifugation and gel filtration through Sepharose 4B. LDL reversibly inhibited proliferation of Swiss/3T3 cells, whereas HDL had no inhibitory effect on cell growth. The LDL-induced inhibition was LDL-dose dependent and was reversed by the addition of mevalonate, a product of the reaction of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34). These data suggest that a specific reduction in the activity of HMG-CoA reductase produced by the addition of LDL is the main cause of the inhibition of cell proliferation. Studies of the effect of LDL on the cell cycle showed that it inhibited the entry of cells arrested in G0/G1 into the S phase but that it did not affect the transition of cells at the G1/S boundary into the M phase. The cell cycle of 3T3 is arrested solely in G1 by LDL.     

Inhibition of cytokinesis and altered contractile ring morphology induced by cytochalasins in synchronized PtK2 cells

PtK2 cells and antigen affinity-purified antibodies to actin and tubulin were used to study the effects on mitosis of cytochalasin B (CB) and dihydrocytochalasin B (H₂CB). PtK2 cells were synchronized in S phase by a double thymidine block and CB or H₂CB was added at various concentrations at the time of release from the block. CB- and H₂CB-treated populations, and control populations not treated with either drug, progressed synchronously through G2 and into mitosis with similar time courses. By both phase contrast and immunofluorescence microscopy, CB- and H₂CB-treated cells appeared normal in terms of chromosome condensation, spindle formation and spindle dynamics throughout prophase, metaphase and early anaphase. At late anaphase, contractile ring staining with actin antibody was not normal. High actin antigenicity remained localized in the region of the contractile ring; however, it appeared atypically as a punctate line of fluorescence across the midzone. Although some degree of furrowing was often seen to occur, at suitable concentrations of CB or H₂CB only binucleate G1 cells formed. Scanning electron microscopy (SEM) of normal and CB- and H₂CB-treated cells verified that cleavage furrowing did not proceed normally in treated cells. Large numbers of microvilli and surface blebs occurred in the normally smooth furrow region in these treated populations. These results suggest that intact microfilament function is not necessary for progression from S phase into mitosis, for spindle formation or for chromosome movement. They indicate that CB and H₂CB lead to formation of binucleated cells by causing aberrant cleavage furrowing and inhibition of contractile ring microfilaments.     

Cdh1 is an antagonist of the spindle assembly checkpoint

The spindle assembly checkpoint (SAC) monitors unsatisfied connections of microtubules to kinetochores and prevents anaphase onset by inhibition of the ubiquitin ligase E3 anaphase-promoting complex or cyclosome (APC/C) in association with the activator Cdc20. Another APC/C activator, Cdh1, exists permanently throughout the cell cycle but it becomes active from telophase to G1. Here, we show that Cdh1 is partially active and mediates securin degradation even in SAC-active metaphase cells. Additionally, Cdh1 mediates Cdc20 degradation in metaphase, promoting formation of the APC/C-Cdh1. These results indicate that Cdh1 opposes the SAC and promotes anaphase transition.     

In silico study of kinetochore control, amplification, and inhibition effects in MCC assembly

Eukaryotic cells rely on a surveillance mechanism, the "Spindle Assembly Checkpoint"SACM in order to ensure accurate chromosome segregation by preventing anaphase initiation until all chromosomes are correctly attached to the mitotic spindle. In different organisms, a mitotic checkpoint complex (MCC) composed of Mad2, Bub3, BubR1/Mad3, and Cdc20 inhibits the anaphase promoting complex (APC/C) to initiate promotion into anaphase. The mechanism of MCC formation and its regulation by the kinetochore are unclear. Here, we constructed dynamical models of MCC formation involving different kinetochore control mechanisms including amplification as well as inhibition effects, and analysed their quantitative properties. In particular, in this system, fast and stable metaphase to anaphase transition can only be triggered when the kinetochore controls the Bub3:BubR1-related reactions; signal amplification and inhibition play a subordinate role. Furthermore, when introducing experimentally determined parameter values into the models analysed here, we found that effective MCC formation is not combined with complete Cdc20 sequestering. Instead, the MCC might bind and completely block the APC/C. The SACM might function by an MCC:APC/C complex rearrangement.     

A novel function of anaphase promoting complex subunit 10 in tumor progression in non-small cell lung cancer

The anaphase promoting complex/cyclosome (APC/C), a cell cycle-regulated E3 ubiquitin ligase, is responsible for the transition from metaphase to anaphase and the exit from mitosis. The anaphase promoting complex subunit 10 (APC10), a subunit of the APC/C, executes a vital function in substrate recognition. However, no research has reported the connection between APC10 and cancer until now. In this study, we uncovered a novel, unprecedented role of APC10 in tumor progression, which is independent of APC/C. First, aberrant increase of APC10 expression was validated in non-small cell lung cancer (NSCLC) cells and tissues, and the absence of APC10 repressed cell proliferation and migration. Of great interest, we found that APC10 inhibition induced cell cycle arrest at the G0/G1 phase and reduced the expression of the APC/C substrate, Cyclin B1; this finding is different from the conventional concept of the accumulation of Cyclin B1 and cell cycle arrest in metaphase. Further, APC10 was found to interact with glutaminase C (GAC), and the inhibition of APC10 weakened glutamine metabolism and induced excessive autophagy. Taken together, these findings identify a novel function of APC10 in the regulation of NSCLC tumorigenesis and point to the possibility of APC10 as a new target for cancer therapy.     

Probing the dynamics and functions of aurora B kinase in living cells during mitosis and cytokinesis

Aurora B is a protein kinase and a chromosomal passenger protein that undergoes dynamic redistribution during mitosis. We have probed the mechanism that regulates its localization with cells expressing green fluorescent protein (GFP)-tagged wild-type or mutant aurora B. Aurora B was found at centromeres at prophase and persisted until approximately 0.5 min after anaphase onset, when it redistributed to the spindle midzone and became concentrated at the equator along midzone microtubules. Depolymerization of microtubules inhibited the dissociation of aurora B from centromeres at early anaphase and caused the dispersion of aurora B from the spindle midzone at late anaphase; however, centromeric association during prometaphase was unaffected. Inhibition of CDK1 deactivation similarly caused aurora B to remain associated with centromeres during anaphase. In contrast, inhibition of the kinase activity of aurora B appeared to have no effect on its interactions with centromeres or initial relocation onto midzone microtubules. Instead, kinase-inactive aurora B caused abnormal mitosis and deactivation of the spindle checkpoint. In addition, midzone microtubule bundles became destabilized and aurora B dispersed from the equator. Our results suggest that microtubules, CDK1, and the kinase activity each play a distinct role in the dynamics and functions of aurora B in dividing cells.     

Centromere position in budding yeast: evidence for anaphase A.

Although general features of chromosome movement during the cell cycle are conserved among all eukaryotic cells, particular aspects vary between organisms. Understanding the basis for these variations should provide significant insight into the mechanism of chromosome movement. In this context, establishing the types of chromosome movement in the budding yeast Saccharomyces cerevisiae is important since the complexes that mediate chromosome movement (microtubule organizing centers, spindles, and kinetochores) appear much simpler in this organism than in many other eukaryotic cells. We have used fluorescence in situ hybridization to begin an analysis of chromosome movement in budding yeast. Our results demonstrate that the position of yeast centromeres changes as a function of the cell cycle in a manner similar to other eukaryotes. Centromeres are skewed to the side of the nucleus containing the spindle pole in G1; away from the poles in mid-M and clustered near the poles in anaphase and telophase. The change in position of the centromeres relative to the spindle poles supports the existence of anaphase A in budding yeast. In addition, an anaphase A-like activity independent of anaphase B was demonstrated by following the change in centromere position in telophase-arrested cells upon depolymerization and subsequent repolymerization of microtubules. The roles of anaphase A activity and G1 centromere positioning in the segregation of budding yeast chromosomes are discussed. The fluorescence in situ hybridization methodology and experimental strategies described in this study provide powerful new tools to analyze mutants defective in specific kinesin-like molecules, spindle components, and centromere factors, thereby elucidating the mechanism of chromosome movement.     

Postmitotic reconstruction of nucleoli in culture cells with UV-microbeam photoinactivated centrosome.

Ultraviolet microirradiation of one of the poles of the mitotic spindle of PK cells was performed 1 min after the onset of the anaphase. Formation of the nucleolus in the telophase and G1 period was studied by vital observation, electron microscopy and indirect immunofluorescence using antibodies against B23 protein. Sister cells with nonirradiated centrosomes and cells with partially irradiated cytoplasm were used as controls. During the first hour after the anaphase, the nuclei in both sister cells were identical and contained numerous small dense particles with granular ultrastructure. B23 protein detected in the mitotic poles and at the chromosome surface in the anaphase was dispersed in the cytoplasm in both cells in the early G1 period. Later, control cells did not display any difference from intact cells: nucleoli of a typical structure were formed, B23 protein appeared in the karyoplasm and was then accumulated in the nucleoli and disappeared from the cytoplasm and karyoplasm. Nucleoli in cells with irradiated centrosomes did not achieve the normal size and contained a significantly lower amount of granular component. B23 protein was dispersed in the karyoplasm and was not accumulated in the nucleoli. Nucleoli in cells with irradiated centrosomes contained small dense particles for at least 24 h. Telophase cells where microtubule formation had been inhibited by nocodazole formed normal nucleoli. It shows that the effects observed in cells with irradiated centrosomes are not due to the absence of the microtubule radial system. We conclude that UV microirradiation of the mitotic centrosome disturbs the postmitotic reconstruction of nucleoli probably because of the photodestruction of B23 protein accumulated in the mitotic pole.     

Effect of ouabain on growth regulation by serum components in Balb/c-3T3 cells: inhibition of entry into S phase by decreased protein synthesis

The effect of inhibition of the cell membrane Na+-K+ pump on the Balb/c-3T3 cell growth cycle was studied. Inhibition of the Na+-K+ pump resulted in a dose-dependent reduction of intracellular K+ concentration ((K+)i). However, inhibition of protein synthesis in Go/G1 and of subsequent entry into S phase occurred only after (K+)i fell below a critical threshold (50-60 mmoles/liter). Thus, when the (K+)i falls below a critical threshold, protein synthesis is inhibited, preventing cells from entering the S phase. The platelet-derived growth factor (PDGF) induces cells to become "competent" to traverse the cell cycle; the platelet-poor plasma component of serum allows competent cells to progress through G0/G1 and enter S phase. Inhibition of the Na+-K+ pump did not prevent the induction of competence by PDGF, but it did reversibly inhibit plasma-mediated events in early G0/G1. Similarly, cycloheximide inhibited plasma-mediated events but did not prevent PDGF-induced competence. Thus, protein synthesis may not be required for induction of competence; alternatively, the induction of the competent state may occur in these cells after removal of PDGF and protein synthesis inhibitor. Protein synthesis is required for subsequent plasma-mediated events in G0/G1.     

The effects of colchicine and dinitrophenol on the in vivo rates of anaphase A and B in the diatom Surirella

The rates of chromosome-to-pole movement (anaphase A) and pole-pole separation (anaphase B) in vivo were measured in the pennate diatom Surirella, using differential interference contrast (DIC) light microscopy. In control cells, the rate of anaphase A is 1.6 +/- 0.6 micron/min, the rate of anaphase B is 2.3 +/- 0.3 micron/min, and the extent of anaphase B is 26.7 +/- 9.7% of metaphase spindle length. Colchicine was added to metaphase cells in order to inhibit any further addition of microtubule (MT) subunits onto the spindle. Colchicine, which does not break down the well-ordered Surirella central spindle, caused no significant change in the rate of anaphase A (1.3 +/- 0.3 micron/min) while it significantly decreased both the rate of anaphase B (1.2 +/- 0.4 micron/min) and the extent of anaphase B (14.8 +/- 8.3% of metaphase spindle length). Surirella cells were also treated with the metabolic inhibitor 2-4-dinitrophenol (DNP) in order to test the effects of energy depletion on anaphase. When DNP was added early in anaphase A, prior to the completion of sister chromosome separation, anaphase A was inhibited. When DNP was added after initiation of sister chromosome separation, anaphase A continued to completion, although at a lower rate than control cells (0.5 +/- 0.2 micron/min). Anaphase B was completely inhibited by DNP, but upon recovery from DNP resumed at a normal rate (2.2 +/- 0.5 micron/min) and progressed to a slightly larger than normal extent (44.0 +/- 13.0% of metaphase length).(ABSTRACT TRUNCATED AT 250 WORDS)     

Aluminium effects on microtubule organization in dividing root-tip cells of Triticum turgidum. I. Mitotic cells

The effects of aluminium (Al) on dividing root-tip cells of Triticum turgidum were investigated with tubulin immunolabelling and electron microscopy. Aluminium affects the mechanisms controlling the organization of microtubule (MT) cytoskeleton, as well as tubulin polymerization, and induces the following aberrations in mitotic cells. (1) It delays the MT disassembly during mitosis, resulting in the persistence of preprophase MT bands in the late prophase cells, the presence of prophase spindles in prometaphase cells, and a disturbance in the shortening of kinetochore MT bundles in anaphase cells. (2) It interferes with the self-organization process of MTs into bipolar systems, inhibiting the formation of prophase and metaphase spindles. (3) Aluminium induces the formation of atypical MT arrays, which in the immunofluorescent specimens appear as ring-like tubulin aggregations in the cortical cytoplasm of the preprophase/prophase cells and as endoplasmic tubulin bundles in prophase and metaphase/anaphase cells; abnormal preprophase MT bands are assembled, consisting of atypical cortical and endoplasmic MT bundles, the latter clearly lining the nuclear envelope on the preprophase MT band plane. (4) It disorders the chromosome movements carried out by the mitotic spindle. In addition, after prolonged Al treatments chromatin condensation is inhibited. The outcome is greatly disturbed organization and function of the mitotic apparatus, as well as inhibition of cells from entering mitosis. This study shows that the MT cytoskeleton is a target site of Al toxicity in mitotic root-tip cells of T. turgidum . The possible mechanisms by which Al exerts its toxicity on MT organization and function are discussed.