Fidelity Of Mitosis Research Articles

The Role of Mitotic Kinases and the RZZ Complex in Kinetochore-Microtubule Attachments: Doing the Right Link.

During mitosis, the interaction of kinetochores (KTs) with microtubules (MTs) drives chromosome congression to the spindle equator and supports the segregation of sister chromatids. Faithful genome partition critically relies on the ability of chromosomes to establish and maintain proper amphitelic end-on attachments, a configuration in which sister KTs are connected to robust MT fibers emanating from opposite spindle poles. Because the capture of spindle MTs by KTs is error prone, cells use mechanisms that sense and correct inaccurate KT-MT interactions before committing to segregate sister chromatids in anaphase. If left unresolved, these errors can result in the unequal distribution of chromosomes and lead to aneuploidy, a hallmark of cancer. In this review, we provide an overview of the molecular strategies that monitor the formation and fine-tuning of KT-MT attachments. We describe the complex network of proteins that operates at the KT-MT interface and discuss how AURORA B and PLK1 coordinate several concurrent events so that the stability of KT-MT attachments is precisely modulated throughout mitotic progression. We also outline updated knowledge on how the RZZ complex is regulated to ensure the formation of end-on attachments and the fidelity of mitosis.

Abstract 289: The key role of Mitostatin in the maintenance of genome stability

Abstract Genomic instability is a characteristic of most cancers and it refers to an increased tendency of alterations in the genome during the life cycle of cells. The fidelity of DNA replication is highly ensured by different checkpoints; the activation of spindle checkpoints prevents cells from premature entry into mitosis, avoiding incorrect chromosome segregation and aneuploidy, a typical feature of many cancers. Mitostatin, a novel protein, endowed with tumor suppressor activity, has been reported to bind centrosomal proteins Odf2 and ninein, and its depletion causes an alteration of the anchorage of microtubules to the centrosome. Since functional defects of centrosomes are associated to mitotic failure, Mitostatin may have a key role in guarding the fidelity of mitosis in cells.Here we show that the depletion of Mitostatin in cancer cells, synchronized by aphidicolin (G1/S) block and released into nocodazole-containing medium, leads to mitotic slippage and adaptation to the spindle checkpoint in the presence of a spindle inhibitor. Concomitantly, Mitostatin depletion promotes the early degradation of Mad2 and cyclin B1. Since the activated spindle checkpoint delays cell exit from mitosis by preventing cyclin B1 proteolysis, the cyclin B1 early degradation leads to mitotic checkpoint escape and resulting chromosome instability. In particular, we observed a premature sister-chromatid separation, chromosome bridges and mis-segregation in anaphase that are consistent with defective activation of the spindle checkpoint.In this study, we report for the first time that the depletion of Mitostatin induces an increase of numerical and structural chromosomal aberrations compared to control cells. These aberrations include aneuploidy (P=.0005), the formation of triradials (P=.0061) and broken chromosomes (P=.0066). Moreover, 3D nuclear telomere analysis using TeloView shows decreases in telomeric signals (P=.0061), in the total number of aggregates (P=.0027), and in total intensity (P=.018) and single intensity (P=.019) in cells depleted of Mitostatin. These findings suggest that telomere dysfunction is increased in the absence of Mitostatin; the decrease in telomere intensity is associated with ongoing proliferation (mitotic slippage), and the maintenance of telomeric aggregates is indicative of ongoing genomic instability. Taken together, these observations suggest that Mitostatin plays a critical role in guarding the fidelity of mitosis, enabling the optimal activation of the spindle checkpoint. Thus, low levels of Mitostatin found in certain human tumors may contribute to cellular transformation by promoting genomic instability. Citation Format: Angela Lauriola, Andrea Caporali, Sabine Mai, Domenico D'Arca. The key role of Mitostatin in the maintenance of genome stability [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 289. doi:10.1158/1538-7445.AM2017-289

Centromeric Cohesin: Molecular Glue and Much More.

Sister chromatid cohesion, mediated by the cohesin complex, is a prerequisite for faithful chromosome segregation during mitosis. Premature release of sister chromatid cohesion leads to random segregation of the genetic material and consequent aneuploidy. Multiple regulatory mechanisms ensure proper timing for cohesion establishment, concomitant with DNA replication, and cohesion release during the subsequent mitosis. Here we summarize the most important phases of the cohesin cycle and the coordination of cohesion release with the progression through mitosis. We further discuss recent evidence that has revealed additional functions for centromeric localization of cohesin in the fidelity of mitosis in metazoans. Beyond its well-established role as "molecular glue", centromeric cohesin complexes are now emerging as a scaffold for multiple fundamental processes during mitosis, including the formation of correct chromosome and kinetochore architecture, force balance with the mitotic spindle, and the association with key molecules that regulate mitotic fidelity, particularly at the chromosomal inner centromere. Centromeric chromatin may be thus seen as a dynamic place where cohesin ensures mitotic fidelity by multiple means.

Novel insights into mitotic chromosome condensation.

The fidelity of mitosis is essential for life, and successful completion of this process relies on drastic changes in chromosome organization at the onset of nuclear division. The mechanisms that govern chromosome compaction at every cell division cycle are still far from full comprehension, yet recent studies provide novel insights into this problem, challenging classical views on mitotic chromosome assembly. Here, we briefly introduce various models for chromosome assembly and known factors involved in the condensation process (e.g. condensin complexes and topoisomerase II). We will then focus on a few selected studies that have recently brought novel insights into the mysterious way chromosomes are condensed during nuclear division.

Abstract 3592: BaP induces chromosome missegregation through mitotic spindle defects in normal lung epithelial cells

Abstract Lung cancer remains the leading cause of cancer related deaths in the United States. Cancer results from an accumulation of multiple genetic events leading to genomic instability that is a crucial early event in carcinogenesis. Aneuploidy is the most frequently identified genomic abnormality in cancer. We have previously reported a significantly higher level of aneuploidy in smokers who developed lung cancer as compared to smoker controls. Benzo(a)pyrene [B(a)P], a polycyclic aromatic hydrocarbon, is a pulmonary carcinogen and one of 60 different carcinogens found in tobacco smoke. In this study, we hypothesize that B(a)P disrupts mitotic spindle assembly that impairs the fidelity of mitosis leading to chromosome missegregation and aneuploidy. To test our hypothesis, we use confocal imaging in combination with traditional molecular biology techniques to dissect the precise mechanism by which B[a]P induces chromosome missegregation and aneuploidy. Mitotic spindle defects in the form of chromosome lags and bridges, monopolar and multipolar spindles, were assessed in normal lung epithelial cells after treatment with 10μM B[a]P for 6 hrs. Spindle defects were assessed at 24, 48, 72 and 96 hours after treatment. Immunofluorescence revealed abnormal mitosis in the prometaphase through the telophase stage of mitosis. Our result show that B[a]P exposure induced abnormal spindles in the form of monopolar (14%) and multipolar (4%) spindles. High resolution confocal images further revealed that B[a]P exposure led to misoriented mitotic spindles with an abnormal spindle angle [-380] relative to normal plane [180]. Chromosome lagging at both metaphase (17%) and anaphase (9%) has also been recorded in response to B[a]P exposure, observations that result from defects in the mitotic apparatus and kinetochores. Furthermore, we assessed the extent of B[a]P induced genetic instability [in the form of double strand DNA breaks] using gama-H2AX in order to elucidate the underlying mechanism associated with accumulated genetic damage and chromosome missegregation. Our data indicate that B[a]P results in accumulation of mitotic abnormalities and gama-H2AX foci (22%) peaks at 48 hr compared to other time points. Taken together, our data show that B[a]P potently induces mitotic abnormalities by disrupting kinetochore microtubules. The induction of mitotic abnormalities by B[a]P leading to chromosome missegregation and aneuploidy, would unravel a novel targetable molecular mechanism of cigarette smoke induced lung tumorigenesis. Citation Format: Jose T. Thaiparambil, Randa El-Zein. BaP induces chromosome missegregation through mitotic spindle defects in normal lung epithelial cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3592.

Dual pathway spindle assembly increases both the speed and the fidelity of mitosis

SummaryRoughly half of all animal somatic cell spindles assemble by the classical prophase pathway, in which the centrosomes separate ahead of nuclear envelope breakdown (NEBD). The remainder assemble by the prometaphase pathway, in which the centrosomes separate following NEBD. Why cells use dual pathway spindle assembly is unclear. Here, by examining the timing of NEBD relative to the onset of Eg5-mEGFP loading to centrosomes, we show that a time window of 9.2 ± 2.9 min is available for Eg5-driven prophase centrosome separation ahead of NEBD, and that those cells that succeed in separating their centrosomes within this window subsequently show >3-fold fewer chromosome segregation errors and a somewhat faster mitosis. A longer time window would allow more cells to complete prophase centrosome separation and further reduce segregation errors, but at the expense of a slower mitosis. Our data reveal dual pathway mitosis in a new light, as a substantive strategy that increases both the speed and the fidelity of mitosis.

Caspase activity is not required for the mitotic checkpoint or mitotic slippage in human cells

Biochemical studies suggest that caspase activity is required for a functional mitotic checkpoint (MC) and mitotic slippage. To test this directly, we followed nontransformed human telomerase immortalized human retinal pigment epithelia (RPE-1) cells through mitosis after inhibiting or depleting selected caspases. We found that inhibiting caspases individually, in combination, or in toto did not affect the duration or fidelity of mitosis in otherwise untreated cells. When satisfaction of the MC was prevented with 500 nM nocodazole or 2.5 μM dimethylenastron (an Eg5 inhibitor), 92-100% of RPE-1 cells slipped from mitosis in the presence of pan-caspase inhibitors or after simultaneously depleting caspase-3 and -9, and they did so with the same kinetics (~21-22 h) as after treatment with nocodazole or Eg5 inhibitors alone. Surprisingly, inhibiting or depleting caspase-9 alone doubled the number of nocodazole-treated, but not Eg5-inhibited, cells that died in mitosis. In addition, inhibiting or depleting caspase-9 and -3 together accelerated the rate of slippage ~40% (to ~13-15 h). Finally, nocodazole-treated cells that recently slipped through mitosis in the presence or absence of pan-caspase inhibitors contained numerous BubR1 foci in their nuclei. From these data, we conclude that caspase activity is not required for a functional MC or for mitotic slippage.

Mammalian CLASP1 and CLASP2 Cooperate to Ensure Mitotic Fidelity by Regulating Spindle and Kinetochore Function

CLASPs are widely conserved microtubule plus-end-tracking proteins with essential roles in the local regulation of microtubule dynamics. In yeast, Drosophila, and Xenopus, a single CLASP orthologue is present, which is required for mitotic spindle assembly by regulating microtubule dynamics at the kinetochore. In mammals, however, only CLASP1 has been directly implicated in cell division, despite the existence of a second paralogue, CLASP2, whose mitotic roles remain unknown. Here, we show that CLASP2 localization at kinetochores, centrosomes, and spindle throughout mitosis is remarkably similar to CLASP1, both showing fast microtubule-independent turnover rates. Strikingly, primary fibroblasts from Clasp2 knockout mice show numerous spindle and chromosome segregation defects that can be partially rescued by ectopic expression of Clasp1 or Clasp2. Moreover, chromosome segregation rates during anaphase A and B are slower in Clasp2 knockout cells, which is consistent with a role of CLASP2 in the regulation of kinetochore and spindle function. Noteworthy, cell viability/proliferation and spindle checkpoint function were not impaired in Clasp2 knockout cells, but the fidelity of mitosis was strongly compromised, leading to severe chromosomal instability in adult cells. Together, our data support that the partial redundancy of CLASPs during mitosis acts as a possible mechanism to prevent aneuploidy in mammals.

Cell Biology: Ran, Mitosis and the Cancer Connection

The small GTPase Ran has been shown to regulate HURP, a protein that interacts with several mitotic spindle assembly factors. This discovery sheds new light on the role of Ran in the fidelity of mitosis and in cancer.

Inhibition of S/G2 Phase CDK4 Reduces Mitotic Fidelity

Cyclin-dependent kinase 4 (CDK4)/cyclin D has a key role in regulating progression through late G(1) into S phase of the cell cycle. CDK4-cyclin D complexes then persist through the latter phases of the cell cycle, although little is known about their potential roles. We have developed small molecule inhibitors that are highly selective for CDK4 and have used these to define a role for CDK4-cyclin D in G(2) phase. The addition of the CDK4 inhibitor or small interfering RNA knockdown of cyclin D3, the cyclin D partner, delayed progression through G(2) phase and mitosis. The G(2) phase delay was independent of ATM/ATR and p38 MAPK but associated with elevated Wee1. The mitotic delay was because of failure of chromosomes to migrate to the metaphase plate. However, cells eventually exited mitosis, with a resultant increase in cells with multiple or micronuclei. Inhibiting CDK4 delayed the expression of the chromosomal passenger proteins survivin and borealin, although this was unlikely to account for the mitotic phenotype. These data provide evidence for a novel function for CDK4-cyclin D3 activity in S and G(2) phase that is critical for G(2)/M progression and the fidelity of mitosis.

Key Events of the Cell Cycle: Regulation and Organization

The review surveys the studies of molecular genetic mechanisms of the cell cycle control on various eukaryotic models. The major cell cycle phenomena are considered: (1) checkpoints and their role in preserving DNA integrity and fidelity of mitosis, (2) the cell oscillator model, and (3) the role of cyclins in timing of cell division and coordination of mitotic events. The main classes of regulatory proteins involved in the cell cycle are discussed in detail.

Minireview: branded from the start-distinct oncogenic initiating events may determine tumor fate in the thyroid.

Thyroid follicular neoplasms commonly have aneuploidy, presumably due to chromosomal instability. This property is associated with a greater malignant potential and worse prognosis. Recently, there has been considerable progress in our understanding of mechanisms that may account for chromosomal instability in cancer cells. Many tumors with chromosomal instability have abnormalities in the cell cycle checkpoint that monitors the fidelity of mitosis. Mutations of Bub1 or BubR1, genes coding for kinases involved in mitotic spindle assembly checkpoint signaling, are found in a small subset of aneuploid tumors. Other components of protein complexes responsible for attachment of kinetochores to microtubules, or for cohesion between sister chromatids, may also be subject to alterations during tumor progression. Here, we also discuss the evidence that certain oncogenic events, such as Ras mutations, may predispose cells to chromosomal instability by favoring inappropriate posttranslational changes in mitotic checkpoint components through activation of upstream kinases during tumor initiation or progression.