Chromosome Segregation In Mitosis Research Articles

NDP52 tunes cortical actin interaction with astral microtubules for accurate spindle orientation.

Oriented cell divisions are controlled by a conserved molecular cascade involving Gαi, LGN, and NuMA. Here, we show that NDP52 regulates spindle orientation via remodeling the polar cortical actin cytoskeleton. siRNA-mediated NDP52 suppression surprisingly revealed a ring-like compact subcortical F-actin architecture surrounding the spindle in prophase/prometaphase cells, which resulted in severe defects of astral microtubule growth and an aberrant spindle orientation. Remarkably, NDP52 recruited the actin assembly factor N-WASP and regulated the dynamics of the subcortical F-actin ring in mitotic cells. Mechanistically, NDP52 was found to bind to phosphatidic acid-containing vesicles, which absorbed cytoplasmic N-WASP to regulate local filamentous actin growth at the polar cortex. Our TIRFM analyses revealed that NDP52-containing vesicles anchored N-WASP and shortened the length of actin filaments in vitro. Based on these results we propose that NDP52-containing vesicles regulate cortical actin dynamics through N-WASP to accomplish a spatiotemporal regulation between astral microtubules and the actin network for proper spindle orientation and precise chromosome segregation. In this way, intracellular vesicles cooperate with microtubules and actin filaments to regulate proper mitotic progression. Since NDP52 is absent from yeast, we reason that metazoans have evolved an elaborate spindle positioning machinery to ensure accurate chromosome segregation in mitosis.

Aurora B-INCENP Localization at Centromeres/Inner Kinetochores Is Required for Chromosome Bi-orientation in Budding Yeast

SummaryFor proper chromosome segregation in mitosis, sister kinetochores must interact with microtubules from opposite spindle poles (chromosome bi-orientation) [1, 2]. To promote bi-orientation, Aurora B kinase disrupts aberrant kinetochore-microtubule interactions [3, 4, 5, 6]. It has long been debated how Aurora B halts this action when bi-orientation is established and tension is applied across sister kinetochores. A popular explanation for it is that, upon bi-orientation, sister kinetochores are pulled in opposite directions, stretching the outer kinetochores [7, 8] and moving Aurora B substrates away from Aurora-B-localizing sites at centromeres (spatial separation model) [3, 5, 9]. This model predicts that Aurora B localization at centromeres is required for bi-orientation. However, this notion was challenged by the observation that Bir1 (yeast survivin), which recruits Ipl1-Sli15 (yeast Aurora B-INCENP) to centromeres, can become dispensable for bi-orientation [10]. This raised the possibility that Aurora B localization at centromeres is dispensable for bi-orientation. Alternatively, there might be a Bir1-independent mechanism for recruiting Ipl1-Sli15 to centromeres or inner kinetochores [5, 9]. Here, we show that the COMA inner kinetochore sub-complex physically interacts with Sli15, recruits Ipl1-Sli15 to the inner kinetochore, and promotes chromosome bi-orientation, independently of Bir1, in budding yeast. Moreover, using an engineered recruitment of Ipl1-Sli15 to the inner kinetochore when both Bir1 and COMA are defective, we show that localization of Ipl1-Sli15 at centromeres or inner kinetochores is required for bi-orientation. Our results give important insight into how Aurora B disrupts kinetochore-microtubule interaction in a tension-dependent manner to promote chromosome bi-orientation.

The Phosphatase PP1 Promotes Mitotic Slippage Through Mad3 Dephosphorylation

Accurate chromosome segregation in mitosis requires the bipolar attachment of kinetochores to spindle microtubules. A conserved surveillance mechanism, the Spindle Assembly Checkpoint (SAC), responds to lack of kinetochore-microtubule connections and delays the onset of anaphase until all chromosomes are bipolarly attached. SAC signalling fires at kinetochores and involves the formation of a soluble Mitotic Checkpoint Complex (MCC) made by Mad2, Mad3/BubR1, Bub3 and Cdc20, which inhibits the ubiquitin ligase activity of the Anaphase Promoting Complex (APC). The mitotic delay imposed by SAC, however, is not everlasting. After a variable time during which kinetochores have failed to establish bipolar connections, cells escape from the SAC-induced mitotic arrest through a process called mitotic slippage. Mitotic slippage occurs in the presence of SAC signalling at kinetochores, but whether and how MCC stability and APC inhibition are actively controlled during slippage is not known. The PP1 phosphatase has emerged as a key factor in SAC silencing once all kinetochores are bipolarly attached. PP1 turns off SAC signalling through dephosphorylation of the SAC scaffold Knl1/Blinkin at kinetochores. Here we show that in budding yeast PP1 is also required for mitotic slippage. However, its involvement in this process is not linked to kinetochores but rather to MCC stability. We identify S268 of Mad3 as a critical target of PP1 in this process. Mad3 S268 dephosphorylation destabilises the MCC, but in spite of that, does not affect the initial SAC-induced mitotic arrest. Conversely, it accelerates mitotic slippage and overcomes the slippage defect of PP1 mutants. Thus, slippage is not the mere consequence of incomplete APC inactivation that brings about mitotic exit, as originally proposed, but involves the exertive antagonism between kinases and phosphatases.

Dynamic acetylation of the kinetochore-associated protein HEC1 ensures accurate microtubule–kinetochore attachment

Faithful chromosome segregation during mitosis is critical for maintaining genome integrity in cell progeny and relies on accurate and robust kinetochore-microtubule attachments. The NDC80 complex, a tetramer comprising kinetochore protein HEC1 (HEC1), NDC80 kinetochore complex component NUF2 (NUF2), NDC80 kinetochore complex component SPC24 (SPC24), and SPC25, plays a critical role in kinetochore-microtubule attachment. Mounting evidence indicates that phosphorylation of HEC1 is important for regulating the binding of the NDC80 complex to microtubules. However, it remains unclear whether other post-translational modifications, such as acetylation, regulate NDC80-microtubule attachment during mitosis. Here, using pulldown assays with HeLa cell lysates and site-directed mutagenesis, we show that HEC1 is a bona fide substrate of the lysine acetyltransferase Tat-interacting protein, 60 kDa (TIP60) and that TIP60-mediated acetylation of HEC1 is essential for accurate chromosome segregation in mitosis. We demonstrate that TIP60 regulates the dynamic interactions between NDC80 and spindle microtubules during mitosis and observed that TIP60 acetylates HEC1 at two evolutionarily conserved residues, Lys-53 and Lys-59. Importantly, this acetylation weakened the phosphorylation of the N-terminal HEC1(1-80) region at Ser-55 and Ser-62, which is governed by Aurora B and regulates NDC80-microtubule dynamics, indicating functional cross-talk between these two post-translation modifications of HEC1. Moreover, the TIP60-mediated acetylation was specifically reversed by sirtuin 1 (SIRT1). Taken together, our results define a conserved signaling hierarchy, involving HEC1, TIP60, Aurora B, and SIRT1, that integrates dynamic HEC1 acetylation and phosphorylation for accurate kinetochore-microtubule attachment in the maintenance of genomic stability during mitosis.

Dissociation of membrane-chromatin contacts is required for proper chromosome segregation in mitosis.

The nuclear envelope (NE) aids in organizing the interphase genome by tethering chromatin to the nuclear periphery. During mitotic entry, NE–chromatin contacts are broken. Here, we report on the consequences of impaired NE removal from chromatin for cell division of human cells. Using a membrane–chromatin tether that cannot be dissociated when cells enter mitosis, we show that a failure in breaking membrane–chromatin interactions impairs mitotic chromatin organization, chromosome segregation and cytokinesis, and induces an aberrant NE morphology in postmitotic cells. In contrast, chromosome segregation and cell division proceed successfully when membrane attachment to chromatin is induced during metaphase, after chromosomes have been singularized and aligned at the metaphase plate. These results indicate that the separation of membranes and chromatin is critical during prometaphase to allow for proper chromosome compaction and segregation. We propose that one cause of these defects is the multivalency of membrane–chromatin interactions.

Distribution of Eg5 and TPX2 in mitosis: Insight from CRISPR tagged cells.

The mitotic spindle is a dynamic bipolar structure that mediates chromosome segregation in mitosis. In most organisms, spindle formation requires the action of kinesin-5 motor proteins that generate outward force on antiparallel microtubules to establish spindle bipolarity. Previous work has shown that Eg5 and TPX2, a spindle microtubule-associated protein that suppresses Eg5 motor activity, are enriched on parallel microtubules near spindle poles. This distribution is inconsistent with the requirement for Eg5-dependent force production during mitosis. To investigate this, we used CRISPR/Cas9 gene editing to tag Eg5 and TPX2 with EGFP and quantify protein distribution throughout mitosis. The results show that at metaphase both Eg5-EGFP and TPX2-EGFP are enriched toward spindle poles, but only TPX2-EGFP is enriched relative to microtubules. Eg5-EGFP and TPX2-EGFP show distinct localization patterns in anaphase, with Eg5-EGFP relocalizing to the midzone earlier than TPX2-EGFP. Analysis of spindles oriented at 90° to the coverslip confirmed that Eg5-EGFP was present on bridge microtubules in metaphase and anaphase; in contrast, TPX2 was not enriched, or enriched at later times, on these microtubules. Overall, TPX2 was present at 3.6X the level of Eg5 on the spindle and Eg5 was locally enriched at the prophase centrosome (~7×) compared to the whole cell. Our results show that using cells with fluorescent tags at the endogenous locus can provide novel insight into protein distribution during mitosis.

Rpb9-deficient cells are defective in DNA damage response and require histone H3 acetylation for survival

Rpb9 is a non-essential subunit of RNA polymerase II that is involved in DNA transcription and repair. In budding yeast, deletion of RPB9 causes several phenotypes such as slow growth and temperature sensitivity. We found that simultaneous mutation of multiple N-terminal lysines within histone H3 was lethal in rpb9Δ cells. Our results indicate that hypoacetylation of H3 leads to inefficient repair of DNA double-strand breaks, while activation of the DNA damage checkpoint regulators γH2A and Rad53 is suppressed in Rpb9-deficient cells. Combination of H3 hypoacetylation with the loss of Rpb9 leads to genomic instability, aberrant segregation of chromosomes in mitosis, and eventually to cell death. These results indicate that H3 acetylation becomes essential for efficient DNA repair and cell survival if a DNA damage checkpoint is defective.

Role of ubiquitylation of components of mitotic checkpoint complex in their dissociation from anaphase-promoting complex/cyclosome

The mitotic checkpoint system ensures the fidelity of chromosome segregation in mitosis by preventing premature initiation of anaphase until correct bipolar attachment of chromosomes to the mitotic spindle is reached. It promotes the assembly of a mitotic checkpoint complex (MCC), composed of BubR1, Bub3, Cdc20, and Mad2, which inhibits the activity of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. When the checkpoint is satisfied, anaphase is initiated by the disassembly of MCC. Previous studies indicated that the dissociation of APC/C-bound MCC requires ubiquitylation and suggested that the target of ubiquitylation is the Cdc20 component of MCC. However, it remained unknown how ubiquitylation causes the release of MCC from APC/C and its disassembly and whether ubiquitylation of additional proteins is involved in this process. We find that ubiquitylation causes the dissociation of BubR1 from Cdc20 in MCC and suggest that this may lead to the release of MCC components from APC/C. BubR1 in MCC is ubiquitylated by APC/C, although to a lesser degree than Cdc20. The extent of BubR1 ubiquitylation was markedly increased in recombinant MCC that contained a lysine-less mutant of Cdc20. Mutation of lysine residues to arginines in the N-terminal region of BubR1 partially inhibited its ubiquitylation and slowed down the release of MCC from APC/C, provided that Cdc20 ubiquitylation was also blocked. It is suggested that ubiquitylation of both Cdc20 and BubR1 may be involved in their dissociation from each other and in the release of MCC components from APC/C.

ATR Promotes Faithful Chromosome Segregation in Mitosis

Abstract ATR localized to centromeres to prevent lagging chromosomes independent of its role in replication stress.

Manipulating Cohesin Levels in Live Mouse Oocytes.

The cohesin complex is essential for chromosome segregation in mitosis and meiosis. Cohesin is a tripartite protein complex that holds sister chromatids together from DNA replication until anaphase. In mammals, meiotic DNA replication occurs in oogonia of embryos and chromosome segregation occurs in oocytes of sexually mature females. Sister chromatid cohesion establishment and chromosome segregation are thus separated by months in the mouse and decades in the human. The meiotic cohesin complex that maintains sister chromatid cohesion must therefore hold replicated sisters together for a long time in oocytes. Remarkably, this is achieved by establishing cohesion exclusively in prenatal oocytes. Meiotic cohesion in females ismaintained without detectable turnover and cohesinis therefore thought to be a long-lived protein complex. Nevertheless, the lifespan of cohesin molecules is limited as chromosomal cohesin levelsdecline with maternal age. The age-related loss of cohesin and weakened cohesion correlate with an age-related increase in chromosome missegregation of meiosis I oocytes that results in aneuploid eggs. Therefore, loss of chromosomal cohesin has been proposed to be a leading cause of the maternal age effect. To better understand cohesin deterioration in oocytes, it is crucial to gain insights into mammalian cohesion establishment and maintenance mechanisms by manipulating cohesin in live oocytes.This chapter describes techniques that address the manipulation of meiotic cohesin levels in mouse oocytes. First, we describe how cohesin can be efficiently removed from meiotic chromosomes by injecting mRNA encoding TEV protease in live oocytes expressing cohesin with engineered TEV recognition sites, followed by imaging. Secondly, we describe how cohesin expression can be induced during different stages of oocyte development using genetically modified mouse strains. In particular, we describe how to determine the deletion timing of germline-specific Cre recombinases using β-galactosidase staining of fetal ovaries. Lastly, we provide guidance on how to quantify cohesin levels on metaphase I chromosome spreads.

Hydrogen Peroxide Prolongs Mitotic Arrest in a Dose Dependent Manner and Independently of the Spindle Assembly Checkpoint Activity in Saccharomyces cerevisiae

Oxidative stress and chromosome missegregation are important factors that are linked to aneuploidy. A major reason for chromosome missegragation is the inappropriate activity of the spindle assembly checkpoint (SAC), a conserved surveillance mechanism that monitors the state of kinetochore-microtubule attachments to ensure equal chromosome segregation in mitosis. SAC-activation induces a prolonged mitotic arrest. Mitosis is considered the most vulnerable cell cycle phase to several external signals, therefore increasing the time cells spent in this phase via mitotic arrest induction by SAC-activating agents is favorable for cancer therapy. Cancer cells also display elevated oxidative stress due to abnormally high production of reactive oxygen species (ROS). However, the effect of increased oxidative stress on the duration of mitotic arrest remains largely unknown. In this study, we investigated the effect of H2O2-induced oxidative stress on the mitotic arrest induced by a SAC-activating agent (nocodazole) in Saccharomyces cerevisiae. Our data suggest that oxidative stress prolongs SAC-activation induced mitotic arrest in a dose dependent manner. We, in addition, investigated the effect of H2O2 treatment on the mitotic arrest induced independently of SAC-activation by using a conditional mutant (cdc23) and showed that the effect of H2O2-induced oxidative stress on mitotic arrest is independent of the SAC activity.

UBASH3B-mediated silencing of the mitotic checkpoint: Therapeutic perspectives in cancer

ABSTRACTDefects in mitosis can lead to aneuploidy, which is a common feature of human cancers. Spindle Assembly Checkpoint (SAC) controls fidelity of chromosome segregation in mitosis to prevent aneuploidy. The ubiquitin receptor protein Ubiquitin Associated and SH3 Domain Containing B (UBASH3B) was recently found to control SAC silencing and faithful chromosome segregation by relocalizing Aurora B kinase to the mitotic microtubules. Accordingly, loss and gain of function of UBASH3B have strong effects on mitotic progression. Downregulation of UBASH3B prevents SAC satisfaction leading to inhibition of chromosome segregation, mitotic arrest, and cell death. In contrast, increased cellular levels of UBASH3B trigger premature and uncontrolled chromosome segregation. Interestingly, elevated levels of UBASH3B were found in aggressive tumors. Therefore, we raised the question whether the oncogenic potential of UBASH3B is linked to its role in chromosome segregation. Here we show that in cancer cells expressing high levels of UBASH3B and SAC proteins, downregulation of UBASH3B, can further potentiate SAC response inducing mitotic arrest and cell death. Moreover, data mining approaches identified a correlation between mRNA levels of UBASH3B and SAC components in a set of primary patient tumors including kidney and liver carcinomas. Thus, inhibition of UBASH3B may offer an attractive therapeutic perspective for cancers.

Cyclin A/Cdk1 modulates Plk1 activity in prometaphase to regulate kinetochore-microtubule attachment stability.

The fidelity of chromosome segregation in mitosis is safeguarded by the precise regulation of kinetochore microtubule (k-MT) attachment stability. Previously, we demonstrated that Cyclin A/Cdk1 destabilizes k-MT attachments to promote faithful chromosome segregation. Here, we use quantitative phosphoproteomics to identify 156 Cyclin A/Cdk1 substrates in prometaphase. One Cyclin A/Cdk1 substrate is myosin phosphatase targeting subunit 1 (MYPT1), and we show that MYPT1 localization to kinetochores depends on Cyclin A/Cdk1 activity and that MYPT1 destabilizes k-MT attachments by negatively regulating Plk1 at kinetochores. Thus, Cyclin A/Cdk1 phosphorylation primes MYPT1 for Plk1 binding. Interestingly, priming of PBIP1 by Plk1 itself (self-priming) increased in MYPT1-depleted cells showing that MYPT1 provides a molecular link between the processes of Cdk1-dependent priming and self-priming of Plk1 substrates. These data demonstrate cross-regulation between Cyclin A/Cdk1-dependent and Plk1-dependent phosphorylation of substrates during mitosis to ensure efficient correction of k-MT attachment errors necessary for high mitotic fidelity.

A kinase-dependent role for Haspin in antagonizing Wapl and protecting mitotic centromere cohesion.

Sister-chromatid cohesion mediated by the cohesin complex is fundamental for precise chromosome segregation in mitosis. Through binding the cohesin subunit Pds5, Wapl releases the bulk of cohesin from chromosome arms in prophase, whereas centromeric cohesin is protected from Wapl until anaphase onset. Strong centromere cohesion requires centromeric localization of the mitotic histone kinase Haspin, which is dependent on the interaction of its non-catalytic N-terminus with Pds5B. It remains unclear how Haspin fully blocks the Wapl-Pds5B interaction at centromeres. Here, we show that the C-terminal kinase domain of Haspin (Haspin-KD) binds and phosphorylates the YSR motif of Wapl (Wapl-YSR), thereby directly inhibiting the YSR motif-dependent interaction of Wapl with Pds5B. Cells expressing a Wapl-binding-deficient mutant of Haspin or treated with Haspin inhibitors show centromeric cohesion defects. Phospho-mimetic mutation in Wapl-YSR prevents Wapl from binding Pds5B and releasing cohesin. Forced targeting Haspin-KD to centromeres partly bypasses the need for Haspin-Pds5B interaction in cohesion protection. Taken together, these results indicate a kinase-dependent role for Haspin in antagonizing Wapl and protecting centromeric cohesion in mitosis.

Mitosis-specific acetylation tunes Ran effector binding for chromosome segregation.

Stable transmission of genetic information during cell division requires faithful mitotic spindle assembly and chromosome segregation. The Ran GTPase plays a key role in mitotic spindle assembly. However, how the generation of a chemical gradient of Ran-GTP at the spindle is coupled to mitotic post-translational modifications has never been characterized. Here, we solved the complex structure of Ran with the nucleotide release factor Mog1 and delineated a novel mitosis-specific acetylation-regulated Ran–Mog1 interaction during chromosome segregation. Our structure-guided functional analyses revealed that Mog1 competes with RCC1 for Ran binding in a GTP/GDP-dependent manner. Biochemical characterization demonstrated that Mog1-bound Ran prevents RCC1 binding and subsequent GTP loading. Surprisingly, Ran is a bona fide substrate of TIP60, and the acetylation of Lys134 by TIP60 liberates Mog1 from Ran binding during mitosis. Importantly, this acetylation-elicited switch of Ran binding to RCC1 promotes high level of Ran-GTP, which is essential for chromosome alignment. These results establish a previously uncharacterized regulatory mechanism in which TIP60 provides a homeostatic control of Ran-GTP level by tuning Ran effector binding for chromosome segregation in mitosis.

The DNA replication protein Cdc6 inhibits the microtubule-organizing activity of the centrosome

The centrosome serves as a major microtubule-organizing center (MTOC). The Cdc6 protein is a component of the pre-replicative complex and a licensing factor for the initiation of chromosome replication and localizes to centrosomes during the S and G2 phases of the cfell cycle of human cells. This cell cycle-dependent localization of Cdc6 to the centrosome motivated us to investigate whether Cdc6 negatively regulates MTOC activity and to determine the integral proteins that comprise the pericentriolar material (PCM). Time-lapse live-cell imaging of microtubule regrowth revealed that Cdc6 depletion increased microtubule nucleation at the centrosomes and that expression of Cdc6 in Cdc6-depleted cells reversed this effect. This increase and decrease in microtubule nucleation correlated with the centrosomal intensities of PCM proteins such as γ-tubulin, pericentrin, CDK5 regulatory subunit-associated protein 2 (CDK5RAP2), and centrosomal protein 192 (Cep192). The regulation of microtubule nucleation and the recruitment of PCM proteins to the centrosome required Cdc6 ATPase activity, as well as a centrosomal localization of Cdc6. These results suggest a novel function for Cdc6 in coordinating centrosome assembly and function.

Abstract 4848: The functional synergism and pro-metastatic role of FOXM1 and CENPF in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide and metastasis is regarded as the major cause of HCC-associated lethality. In this study, we have analysed the TCGA whole-transcriptome sequencing data of paired human HCC samples (n=50), and identified Forkhead Box M1 (FOXM1) and Centromere Protein F (CENPF) to be the top-listing upregulated genes. Interestingly, both of them are the essential components in cell-cycle progression. FOXM1 encodes for the cell-cycle-dependent transcription factor that regulates genes for DNA replication and mitosis, while CENPF encodes for the centromere protein that is required for kinetochore function and chromosome segregation in mitosis. We hypothesized that the upregulation of FOXM1-CENPF signaling axis may drive hepatocarcinogenesis. In our human HCC cohort (n=34), FOXM1 and CENPF were shown to be upregulated compared with the non-tumorous liver tissues, and their mRNA expressions were positively correlated (p<0.0001). Co-upregulation of FOXM1 and CENPF in patient samples was demonstrated to be positively correlated with the absence of tumor encapsulation (p=0.035) in our clinico-pathological correlation analysis, whereas the samples without their co-upregulation was less likely to be correlated with venous invasion (p =0.045). This indicated that FOXM1 and CENPF are likely to be associated with HCC metastasis synergistically. Stable single and co-knockdown clones of FOXM1 and CENPF in four HCC cell lines (BEL7402, SMMC-7721, MHCC-97L, Huh7) were established using short-hairpin (sh) RNA approach for subsequent functional characterization. Both single and co-knockdown of FOXM1 and CENPF cells have shown reduced proliferation rate (p<0.05) in cell proliferation assay. Moreover, all single and co-knockdown of FOXM1 and CENPF cell lines were found to have a at least 2-fold reduction in migration and invasion rate (p<0.0001) in transwell cell migration and invasion assay. These suggested that FOXM1 and CENPF are critical for HCC cell proliferation, migration and invasion. To assess the translational significance of targeting FOXM1 and CENPF, seven HCC cell lines (BEL7402, SMMC-7721, MHCC-97L, Huh7, Hep3B, HepG2, PLC/PRF/5) were challenged with thiostrepton and zoledronic acid, which are inhibitors against FOXM1 and CENPF, respectively. Intriguingly, both individual and combined treatments of these inhibitors effectively inhibited HCC cell growth. It was also demonstrated that such inhibition acts via suppressing the respective endogenous transcript and protein expression of FOXM1 and CENPF. Taken together, our study has demonstrated that FOXM1 and CENPF play a critical oncogenic role in HCC and they may function as an attractive molecular therapeutic target. This study is supported in part by Health and Medical Research Fund (03142836). Citation Format: Wai Ling Macrina Lam, Lo Kong Chan, Daniel Wai-Hung Ho, Charles Shing Kam, Irene Oi-Lin Ng. The functional synergism and pro-metastatic role of FOXM1 and CENPF in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4848. doi:10.1158/1538-7445.AM2017-4848

Toxic effects of environmental pollutants: Comparative investigation using Allium cepa L. and Lactuca sativa L.

Studies that help understand the mechanisms of action of environmental pollutants are extremely important in environmental toxicology. In this context, assays using plants as models stand out for their simplicity and low performance cost. Among the plants used for this purpose, Allium cepa L. is the model most commonly applied for cytogenotoxic tests, while Lactuca sativa L., already widely used in phytotoxic investigations, has been gaining prominence in cytotoxic analyses. The present study aimed to compare the responses of A. cepa and L. sativa via macroscopic (root growth) and microscopic analyses (cell cycle and DNA fragmentation via TdT-mediated deoxy-uracil nick and labeling (TUNEL) and comet assays) after exposure of their roots to environmental pollutants with known cytogenotoxic mechanisms. Both species presented sensitive and efficient response to the applied tests after exposure to the DNA-alkylating agent Methyl Methanesulfonate (MMS), the heavy metal Cadmium, the aluminum industry waste Spent Potliner (SPL) and the herbicide Atrazine. However, they differed regarding the responses to the evaluated endpoints. Overall, A. cepa was more efficient in detecting clastogenic changes, arising from DNA breakage, while L. sativa rather detected aneugenic alterations, related to chromosome segregation in mitosis. In the tests applied to verify DNA fragmentation (comet and TUNEL assays), A. cepa presented higher sensitivity. In conclusion, both models are efficient to evaluate toxicological risks of environmental pollutants.

Centromere Dynamics in Male and Female Germ Cells.

In sexually reproducing organisms the germ line is the cellular lineage that gives rise to gametes. All germ cells originate from germline stem cells that divide asymmetrically to generate gonial pre-cursors, which are amplified in number by mitotic divisions, undergo meiosis and eventually differentiate into mature gametes (haploid eggs and sperm). Information transmitted with gametes is inherited by offspring, and potentially by subsequent generations, instructing in organismal development and beyond. Meiosis comprises one round of DNA replication, followed by two rounds of chromosome segregation; homologous chromosomes segregate in the first division (meiosis I) and sister chromatids segregate in the second division (meiosis II). Important mechanistic features of meiosis occur in substages of prophase I and are critical for genetic recombination, including pairing and synapsis of homologous chromosomes (at leptotene and zygotene), crossing-over (at pachytene), and the appearance of chiasmata (at diplotene/diakinesis). Another unique feature of meiosis is the altered centromere/kinetochore geometry at metaphase I, such that sister kinetochores face the same spindle pole (mono-orientation) and stay together at anaphase I. This chapter reviews centromere dynamics in germ cells, focusing on centromere function and assembly in meiotic cell cycles, as well as centromere inheritance in zygotes. Centromeres are functionally defined by the presence of the histone H3 variant CENP-A, the epigenetic determinant of centromere identity. In most eukaryotes, it is well established that CENP-A function is essential for chromosome segregation in mitosis. CENP-A function in meiosis is less well understood and emerging insights into the differential regulation of meiotic and mitotic CENP-A are discussed.

Mechanisms mitigating problems associated with multiple kinetochores on one microtubule in early mitosis.

ABSTRACTProper chromosome segregation in mitosis relies on correct kinetochore interaction with spindle microtubules. In early mitosis, each kinetochore usually interacts with the lateral side of each microtubule and is subsequently tethered at the microtubule end. However, since eukaryotic cells carry multiple chromosomes, multiple kinetochores could occasionally interact with a single microtubule. The consequence of this is unknown. Here, we find that, although two kinetochores (two pairs of sister kinetochores) can interact with the lateral side of one microtubule, only one kinetochore can form a sustained attachment to the microtubule end in budding yeast (Saccharomyces cerevisiae). This leads to detachment of the other kinetochore from the microtubule end (or a location in its proximity). Intriguingly, in this context, kinetochore sliding along a microtubule towards a spindle pole delays and diminishes discernible kinetochore detachment. This effect expedites collection of the entire set of kinetochores to a spindle pole. We propose that cells are equipped with the kinetochore-sliding mechanism to mitigate problems associated with multiple kinetochores on one microtubule in early mitosis.