Cell biology: nondisjunction, aneuploidy and tetraploidy

One simple, widely accepted mechanism for generating an aberrant chromosome number, or aneuploidy, is through nondisjunction--a chromosome distribution error that occurs during mitosis when both copies of a duplicated chromosome are deposited into one daughter cell and none into the other. Shi and King challenge this view, concluding that nondisjunction does not yield aneuploid cells directly, but instead gives rise to tetraploid cells that may subsequently become aneuploid through further division. Here we show that the direct result of chromosome nondisjunction is gain or loss of a single chromosome, which results in near-diploid aneuploidy, not tetraploidy. We suggest that chromatin trapped in the cytokinetic cleavage furrow is the more likely reason for furrow regression and tetraploidization.     

The reversibility of mitotic exit in vertebrate cells

A guiding hypothesis for cell-cycle regulation asserts that regulated proteolysis constrains the directionality of certain cell-cycle transitions. Here we test this hypothesis for mitotic exit, which is regulated by degradation of the cyclin-dependent kinase 1 (Cdk1) activator, cyclin B. Application of chemical Cdk1 inhibitors to cells in mitosis induces cytokinesis and other normal aspects of mitotic exit, including cyclin B degradation. However, chromatid segregation fails, resulting in entrapment of chromatin in the midbody. If cyclin B degradation is blocked with a proteasome inhibitor or by expression of non-degradable cyclin B, Cdk inhibitors will nonetheless induce mitotic exit and cytokinesis. However, if after mitotic exit, the Cdk1 inhibitor is washed free from cells in which cyclin B degradation is blocked, the cells can revert back to M phase. This reversal is characterized by chromosome recondensation, nuclear envelope breakdown, assembly of microtubules into a mitotic spindle, and in most cases, dissolution of the midbody, reopening of the cleavage furrow, and realignment of chromosomes at the metaphase plate. These findings demonstrate that proteasome-dependent degradation of cyclin B provides directionality for the M phase to G1 transition.     

A MAP kinase-dependent actin checkpoint ensures proper spindle orientation in fission yeast.

The accurate segregation of chromosomes at mitosis depends on a correctly assembled bipolar spindle that exerts balanced forces on each sister chromatid. The integrity of mitotic chromosome segregation is ensured by the spindle assembly checkpoint (SAC) that delays mitosis in response to defective spindle organisation or failure of chromosome attachment. Here we describe a distinct mitotic checkpoint in the fission yeast, Schizosaccharomyces pombe, that monitors the integrity of the actin cytoskeleton and delays sister chromatid separation, spindle elongation and cytokinesis until spindle poles have been properly oriented. This mitotic delay is imposed by a stress-activated mitogen-activated protein (MAP) kinase pathway but is independent of the anaphase-promoting complex (APC).     

A cytokinesis furrow is positioned by two consecutive signals

The position of the cytokinesis furrow in a cell determines the relative sizes of its two daughter cells as well as the distribution of their contents. In animal cells, the position of the cytokinesis furrow is specified by the position of the mitotic spindle. The cytokinesis furrow bisects the spindle midway between the microtubule asters, at the site of the microtubule-based midzone, producing two daughter cells. Experiments in some cell types have suggested that the midzone positions the furrow, but experiments in other cells have suggested that the asters position the furrow. One possibility is that different organisms and cell types use different mechanisms to position the cytokinesis furrow. An alternative possibility is that both asters and the midzone contribute to furrow positioning. Recent work in C. elegans has suggested that centrosome separation and the midzone are implicated in cytokinesis. Here we examine the relative contributions of different parts of the mitotic spindle to positioning of the cytokinesis furrow in the C. elegans zygote. By spatially separating the spindle midzone from one of the asters using an ultraviolet laser, we show that the cytokinesis furrow is first positioned by a signal determined by microtubule asters, and then by a second signal that is derived from the spindle midzone. Thus, the position of the cytokinesis furrow is specified by two consecutive furrowing activities.     

Cell division

In creating the mitotic spindle and the contractile ring, natural selection has engineered fascinating precision machines whose movements depend upon forces generated by ensembles of cytoskeletal proteins. These machines segregate chromosomes and divide the cell with high fidelity. Current research on the mechanisms and regulation of spindle morphogenesis, chromosome motility and cytokinesis emphasizes how ensembles of dynamic cytoskeletal polymers and multiple motors cooperate to generate the forces that guide the cell through mitosis and cytokinesis.     

Roles for microtubule and microfilament cytoskeletons in animal cell cytokinesis

Microtubule and microfilament cytoskeletons play key roles in the whole process of cytokinesis. Although a number of hypotheses have been proposed to elucidate the mechanism of cytokinesis by microtubule and actin flament cytoskeletons, many reports are conflicting. In our study,combining the cytoskeletons drug treatments with the time-lapse video technology, we retested the key roles of microtubule and actin filament in cytokinesis. The results showed that depolymerization of microtubules by Nocodazole after the initiation of furrowing would not inhibit the furrow ingression, but obviously decrease the stiffness of daughter cells. Depolymerizing actin filaments by Cytochalasin B before metaphase would inhibit the initiation of furrowing but not chromosome segregation, resulting in the formation of binucleate cells; however, depolymerizing actin fillaments during anaphase would prevent furrowing and lead to the regress of established furrow, also resulting in the formation of binucleate cells. Further, depolymerizing microtubules and actin filaments simultaneously after metaphase would cause the quick regress of the furrow and the formation of binudeate cells. From these results we propose that a successful cytokinesis requires functions and coordination of both the microtubule and actin filament cytoskeletons.Microtubule cytoskeleton may function in the positioning and initiation of cleavage furrow, and the actin filament cytoskeleton may play key roles in the initiation and ingression of the furrow.     

Mad2蛋白在有HeLa细胞有丝分裂期的动态定位

准确的染色体分离依赖于有丝分裂过程的精确调控,包括有丝分裂的时间,及纺锤体检查点的正确调控等。通过动态观察有丝分裂染色体的运动可对上述研究进行精确定量。结果显示,我们利用逆转录病毒系统成功构建了稳定融合表达绿色荧光蛋白GFP-H2B的HeLa细胞系,结合细胞同步化方法,建立了一套利用活细胞荧光共聚焦显微镜观察HeLa细胞有丝分裂的实验体系。     

HeLa/GFP-H2B 细胞系的构建及对其有丝分裂进程的动态观察

准确的染色体分离依赖于有丝分裂过程的精确调控,包括有丝分裂的时间,及纺锤体检查点的正确调控等。通过动态观察有丝分裂染色体的运动可对上述研究进行精确定量。结果显示,利用逆转录病毒系统成功构建了稳定融合表达绿色荧光蛋白GFP—H2B的HeLa细胞系,结合细胞同步化方法,建立了一套利用活细胞荧光共聚焦显微镜观察HeLa细胞有丝分裂的实验体系。     

Mediation of meiotic and early mitotic chromosome segregation in Drosophila by a protein related to kinesin.

Contrary to the traditional view that microtubules pull chromosomes polewards during the anaphase stage of meiotic and mitotic cell divisions, new evidence suggests that the chromosome movements are driven by a motor located at the kinetochore. The process of chromosome segregation involves proper arrangement of kinetochores for spindle attachment, followed by spindle attachment and chromosome movement. Mechanisms in Drosophila for chromosome segregation in meiosis differ in males and females, implying the action of different gene products in the two sexes. A product encoded at the claret locus in Drosophila is required for normal chromosome segregation in meiosis in females and in early mitotic divisions of the embryo. Here we show that the predicted amino-acid sequence of this product is related to the heavy chain of kinesin. The conserved region corresponds to the kinesin motor domain and includes the ATP-binding site and a region that can bind microtubules. A second region contains a leucine repeat motif which may mediate protein-subunit interactions necessary for attachment of chromosomes to the spindle. The mutant phenotype of chromosome nondisjunction and loss, and its similarity to the kinesin ATP-binding domain, suggest that the product encoded at claret not only stabilizes chromosome attachments to the spindle, but may also be a motor that drives chromosome segregation in female meiosis.     

Polar actomyosin contractility destabilizes the position of the cytokinetic furrow

Cytokinesis, the physical separation of daughter cells at the end of mitosis, requires precise regulation of the mechanical properties of the cell periphery. Although studies of cytokinetic mechanics mostly focus on the equatorial constriction ring, a contractile actomyosin cortex is also present at the poles of dividing cells. Whether polar forces influence cytokinetic cell shape and furrow positioning remains an open question. Here we demonstrate that the polar cortex makes cytokinesis inherently unstable. We show that limited asymmetric polar contractions occur during cytokinesis, and that perturbing the polar cortex leads to cell shape oscillations, resulting in furrow displacement and aneuploidy. A theoretical model based on a competition between cortex turnover and contraction dynamics accurately accounts for the oscillations. We further propose that membrane blebs, which commonly form at the poles of dividing cells and whose role in cytokinesis has long been enigmatic, stabilize cell shape by acting as valves releasing cortical contractility. Our findings reveal an inherent instability in the shape of the dividing cell and unveil a novel, spindle-independent mechanism ensuring the stability of cleavage furrow positioning.     

钙调素对肿瘤细胞周期的调节作用

利用钙调素拮抗剂三氟拉嗪(TFP)研究了钙调素对HeLa细胞周期进程的影响,TFP处理的细胞被阻抑在G_1/S,使S期群体及DNA合成下降,G_2期群体增加.有丝分裂(M)前期细胞减少,中期细胞增加.结果表明钙调素对G_1至S期.G_2至M期和M中期至M后期具有调节作用,钙调素通过细胞周期中上述3个位点对肿瘤细胞增殖进行调节.     

Determining the position of the cell division plane

Proper positioning of the cell division plane during mitosis is essential for determining the size and position of the two daughter cells--a critical step during development and cell differentiation. A bipolar microtubule array has been proposed to be a minimum requirement for furrow positioning in mammalian cells, with furrows forming at the site of microtubule plus-end overlap between the spindle poles. Observations in other species have suggested, however, that this may not be true. Here we show, by inducing mammalian tissue cells with monopolar spindles to enter anaphase, that furrow formation in cultured mammalian cells does not require a bipolar spindle. Unexpectedly, cytokinesis occurs at high frequency in monopolar cells. Division always occurs at a cortical position distal to the chromosomes. Analysis of microtubules during cytokinesis in cells with monopolar and bipolar spindles shows that a subpopulation of stable microtubules extends past chromosomes and binds to the cell cortex at the site of furrow formation. Our data are consistent with a model in which chromosomes supply microtubules with factors that promote microtubule stability and furrowing.     

Ser28磷酸化组蛋白H3在哺乳动物有丝分裂细胞中的动态分布

利用特异性抗Ser28磷酸化组蛋白H3抗体,应用间接免疫荧光标记技术,标记人乳腺癌细胞 (MCF-7)和小鼠成纤维细胞(NIH 3T3),用激光共聚焦显微术研究这两种哺乳动物细胞中Ser28磷酸化组蛋白 H3在有丝分裂过程中的动态分布,以研究Ser28磷酸化组蛋白在细胞有丝分裂过程中的作用.结果表明,Ser28 的磷酸化作用是这两种细胞有丝分裂期的特有事件.组蛋白H3的Ser28磷酸化信号首先出现在早期的核外周, Ser28磷酸化在中期达到高峰,并扩展到染色体的所有部分,后期和末期逐渐减退,随着胞质分裂的完成而消失. 实验结果表明,组蛋白H3 Ser28的磷酸化与有丝分裂染色体的凝集和解凝集过程有着时间和空间上的相关性. Ser28磷酸化使得组蛋白H3氨基末端的正电荷数降低,这可能是导致染色质变构凝集的原因之一.有丝分裂期 间组蛋白H3在Ser28位置磷酸化过程与Ser10相比有明显的差异,因此在动物细胞中,组蛋白H3氨基末端这 两个不同丝氨酸残基的磷酸化可能有不同的生物学功能.     

Protein kinase TTK interacts and co-localizes with CENP-E to the kinetochore of human cells

Spindle checkpoint is an important biochemical signaling cascade during mitosis which monitors the fidelity of chromosome segregation, and is mediated by protein kinases Mps1 and Bub1/BubR1. Our recent studies show that kinesin-related motor protein CENP-E interacts with BubR1 and participates in spindle checkpoint signaling. To elucidate the molecular mechanisms underlying spindle checkpoint signaling, we carried out proteomic dissection of human cell kinetochore and revealed protein kinase TTK, human homologue of yeast Mps1. Our studies show that TTK is localized to the kinetochore of human cells, and interacts with CENP-E, suggesting that TTK may play an important role in chromosome segregation during mitosis.     

Cohesin Rec8 is required for reductional chromosome segregation at meiosis.

When cells exit from mitotic cell division, their sister chromatids lose cohesion and separate to opposite poles of the dividing cell, resulting in equational chromosome segregation. In contrast, the reductional segregation of the first stage of meiotic cell division (meiosis I) requires that sister chromatids remain associated through their centromeres and move together to the same pole. Centromeric cohesion is lost as cells exit from meiosis II and sister chromatids can then separate. The fission yeast cohesin protein Rec8 is specific to and required for meiosis. Here we show that Rec8 appears in the centromeres and adjacent chromosome arms during the pre-meiotic S phase. Centromeric Rec8 persists throughout meiosis I and disappears at anaphase of meiosis II. When the rec8 gene is deleted, sister chromatids separate at meiosis I, resulting in equational rather than reductional chromosome segregation. We propose that the persistence of Rec8 at centromeres during meiosis I maintains sister-chromatid cohesion, and that its presence in the centromere-adjacent regions orients the kinetochores so that sister chromatids move to the same pole. This results in the reductional pattern of chromosome segregation necessary to reduce a diploid zygote to haploid gametes.     

Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells

A long-standing hypothesis on tumorigenesis is that cell division failure, generating genetically unstable tetraploid cells, facilitates the development of aneuploid malignancies. Here we test this idea by transiently blocking cytokinesis in p53-null (p53-/-) mouse mammary epithelial cells (MMECs), enabling the isolation of diploid and tetraploid cultures. The tetraploid cells had an increase in the frequency of whole-chromosome mis-segregation and chromosomal rearrangements. Only the tetraploid cells were transformed in vitro after exposure to a carcinogen. Furthermore, in the absence of carcinogen, only the tetraploid cells gave rise to malignant mammary epithelial cancers when transplanted subcutaneously into nude mice. These tumours all contained numerous non-reciprocal translocations and an 8-30-fold amplification of a chromosomal region containing a cluster of matrix metalloproteinase (MMP) genes. MMP overexpression is linked to mammary tumours in humans and animal models. Thus, tetraploidy enhances the frequency of chromosomal alterations and promotes tumour development in p53-/- MMECs.     

AIKs相关的色氨酸-苏氨酸激酶家族

综述AIKs相关激酶在不同物种中的分布,及其在染色体分离及胞质分裂过程中的关键作用。     

Movement and segregation of kinetochores experimentally detached from mammalian chromosomes

The kinetochore is a specialized structure at the centromere of eukaryotic chromosomes that attaches chromosomes to the mitotic spindle. Recently, several lines of evidence have suggested that kinetochores may have more than a passive role in the movement of chromosomes during mitosis and meiosis. Kinetochores seem to attract and 'capture' microtubules that grow from the spindle poles and microtubules may lengthen or shorten by the addition or subtraction of tubulin subunits at their kinetochore-associated ends. An attractive hypothesis is that kinetochores function as 'self-contained engines running on a microtubule track'. Here, we show that kinetochores can be experimentally detached from chromosomes when caffeine is applied to Chinese hamster ovary cells that are arrested in the G1/S phase of the cell cycle. The detached kinetochore fragments can still interact with spindle microtubules and complete all the mitotic movements in the absence of other chromosomal components. As these cells enter mitosis before DNA synthesis is completed, chromosome replication need not be a prerequisite for the pairing, alignment and segregation of kinetochores.     

细胞骨架在植物细胞周期进程中的动态变化及其调控?

细胞周期,即细胞生长与分裂的周期,是生命得以世代繁衍而生生不息的基础.真核细胞有丝分裂周期进程调控的分子机制高度保守.其间,微管和微丝骨架进行有规律的动态变化,顺次组成各种细胞生长和分裂装置,主动参与细胞周期进程的调节.然而,高等植物细胞周期不同时相分别有着与动物细胞不完全相同的、独特的细胞骨架列阵.而这些列阵的产生和维持直接依赖于众多细胞骨架结合蛋白以及上游信号分子的调控.本文重点综述了植物细胞周期进程中微管和微丝骨架的动态变化规律以及参与植物细胞骨架动态和有丝分裂装置组装调控的细胞骨架结合蛋白的最新研究进展,同时对细胞骨架在植物细胞周期进程中研究进行总结和展望.