Dynein-dependent transport of spindle assembly checkpoint proteins off kinetochores toward spindle poles

A predominant mechanism of spindle assembly checkpoint (SAC) silencing is dynein-mediated transport of certain kinetochore proteins along microtubules. There are still conflicting data as to which SAC proteins are dynein cargoes. Using two ATP reduction assays, we found that the core SAC proteins Mad1, Mad2, Bub1, BubR1, and Bub3 redistributed from attached kinetochores to spindle poles, in a dynein-dependent manner. This redistribution still occurred in metaphase-arrested cells, at a time when the SAC should be satisfied and silenced. Unexpectedly, we found that a pool of Hec1 and Mis12 also relocalizes to spindle poles, suggesting KMN components as additional dynein cargoes. The potential significance of these results for SAC silencing is discussed.     

Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) Protein Is Involved in Centrosome Separation through the Regulation of NIMA (Never In Mitosis Gene A)-related Kinase 2 (NEK2) Protein Activity

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is overexpressed in most human cancers and has been described as being involved in the progression of several human malignancies via the inhibition of protein phosphatase 2A (PP2A) activity toward c-Myc. However, with the exception of this role, the cellular function of CIP2A remains poorly understood. On the basis of yeast two-hybrid and coimmunoprecipitation assays, we demonstrate here that NIMA (never in mitosis gene A)-related kinase 2 (NEK2) is a binding partner for CIP2A. CIP2A exhibited dynamic changes in distribution, including the cytoplasm and centrosome, depending on the cell cycle stage. When CIP2A was depleted, centrosome separation and the mitotic spindle dynamics were impaired, resulting in the activation of spindle assembly checkpoint signaling and, ultimately, extension of the cell division time. Our data imply that CIP2A strongly interacts with NEK2 during G₂/M phase, thereby enhancing NEK2 kinase activity to facilitate centrosome separation in a PP1- and PP2A-independent manner. In conclusion, CIP2A is involved in cell cycle progression through centrosome separation and mitotic spindle dynamics.     

Phosphorylation Regulates the p31Comet-Mitotic Arrest-deficient 2 (Mad2) Interaction to Promote Spindle Assembly Checkpoint (SAC) Activity

The spindle assembly checkpoint (SAC) ensures the faithful segregation of the genome during mitosis by ensuring that sister chromosomes form bipolar attachments with microtubules of the mitotic spindle. p31^Comet is an antagonist of the SAC effector Mad2 and promotes silencing of the SAC and mitotic progression. However, p31^Comet interacts with Mad2 throughout the cell cycle. We show that p31^Comet binds Mad2 solely in an inhibitory manner. We demonstrate that attenuating the affinity of p31^Comet for Mad2 by phosphorylation promotes SAC activity in mitosis. Specifically, phosphorylation of Ser-102 weakens p31^Comet-Mad2 binding and enhances p31^Comet-mediated bypass of the SAC. Our results provide the first evidence for regulation of p31^Comet and demonstrate a previously unknown event controlling SAC activity.     

Synthetic studies on mitotic kinesin Eg5 inhibitors: Synthesis and structure–activity relationships of novel 2,4,5-substituted-1,3,4-thiadiazoline derivatives

The 2,4,5-substituted-1,3,4-thiadiazoline derivative 1a has been identified as a new class of mitotic kinesin Eg5 inhibitor. With the aim of enhancement of the mitotic phase accumulation activity, structure optimization of side chains at the 2-, 4-, and 5-positions of the 1,3,4-thiadiazoline ring of 1a was performed. The introduction of sulfonylamino group at the side chain at the 5-position and bulky acyl group at the 2- and 4-position contributed to a significant increase in the mitotic phase accumulation activity and Eg5 inhibitory activity. As a result, a series of optically active compounds exhibited an increased antitumor activity in a human ovarian cancer xenograft mouse model that was induced by oral administration.     

深度麻醉至脑死亡期间大鼠海马神经元活动的突变

全身麻醉若操作不当可能造成致命的中枢神经系统损伤,因此其安全性受到广泛关注.为了揭示麻醉不断加深的过程中神经元活动的变化规律,本文研究了大鼠在乌拉坦(urethane)深度麻醉至脑死亡期间海马区神经元兴奋性和信号传导功能的变化.利用微电极阵列记录和电刺激技术,在海马CA1区胞体层分别记录Schaffer侧支上正向刺激和海马白质上反向刺激诱发的群峰电位(population spike,PS).以PS的幅值和潜伏期为指标,分析海马神经元活动的变化.结果表明,随着乌拉坦血药浓度的增加,PS幅值逐渐减小,潜伏期逐渐延长,意味着乌拉坦抑制了神经元的兴奋性以及轴突传导和突触传递.特别是这些变化存在明显的转折点(即突变),将整个衰减过程分成慢变和快变2个阶段.快变期的剧烈衰减迅速导致脑死亡.而且,引起突变的决定性因素可能是乌拉坦的血药浓度,而非麻醉时间的长短.但是,当乌拉坦注射速率较慢时,延长的慢变期仍然会使神经元功能的受损加重.这些研究结果为动物实验的麻醉操作和临床麻醉的安全应用提供了重要的信息.     

陆地生态系统临界转换理论及其生态学机制研究进展

随着气候变化和人类活动对陆地生态系统双重扰动的不断加剧,越来越多的研究已经意识到生态系统结构和功能会发生难以预知的突变,并且恢复起来需要很长时间.开发判别典型生态系统临界转换的早期预警模型及理解其生态学机制成为生态学研究的热点.目前,基于跨越多个时空尺度的理论和实验研究,提出了多种预警陆地生态系统临界转换的理论框架和指...     

Furry Protein Promotes Aurora A-mediated Polo-like Kinase 1 Activation

Bipolar mitotic spindle organization is fundamental to faithful chromosome segregation. Furry (Fry) is an evolutionarily conserved protein implicated in cell division and morphology. In human cells, Fry localizes to centrosomes and spindle microtubules in early mitosis, and depletion of Fry causes multipolar spindle formation. However, it remains unknown how Fry controls bipolar spindle organization. This study demonstrates that Fry binds to polo-like kinase 1 (Plk1) through the polo-box domain of Plk1 in a manner dependent on the cyclin-dependent kinase 1-mediated Fry phosphorylation at Thr-2516. Fry also binds to Aurora A and promotes Plk1 activity by binding to the polo-box domain of Plk1 and by facilitating Aurora A-mediated Plk1 phosphorylation at Thr-210. Depletion of Fry causes centrosome and centriole splitting in mitotic spindles and reduces the kinase activity of Plk1 in mitotic cells and the accumulation of Thr-210-phosphorylated Plk1 at the spindle poles. Our results suggest that Fry plays a crucial role in the structural integrity of mitotic centrosomes and in the maintenance of spindle bipolarity by promoting Plk1 activity at the spindle poles in early mitosis.     

Naf1alpha is phosphorylated in mitotic phase and required to protect cells against apoptosis

Naf1α is an HIV Nef-associated factor expressed ubiquitously in human cells. Previously, we reported that Naf1α is phosphorylated with EGF through MEK/ERK2 pathway. In this study, we found an additional phosphorylation of Naf1α when cells are in mitotic phase (M phase) or arrested in M phase with anti-mitosis reagents, and disappeared when the cells exit from mitotic phase to G1 phase. Furthermore, we demonstrated that Naf1α plays an important role in preventing cells from apoptosis: over-expression of Naf1α in Saos-2 cells suppressed trichostatin A (TSA)-induced apoptosis either of random culture or of cell population synchronized in M phase. In addition, knock-down of Naf1α expression with small interfering RNA sensitized Saos-2 cells to TSA-induced apoptosis. Physiological significance of these findings is discussed in relation to protection of cells from the apoptosis induction.     

Drosophila parthenogenesis: a tool to decipher centrosomal vs acentrosomal spindle assembly pathways

Development of unfertilized eggs in the parthenogenetic strain K23-O-im of Drosophila mercatorum requires the stochastic interactions of self-assembled centrosomes with the female chromatin. In a portion of the unfertilized eggs that do not assemble centrosomes, microtubules organize a bipolar anastral mitotic spindle around the chromatin like the one formed during the first female meiosis, suggesting that similar pathways may be operative. In the cytoplasm of eggs in which centrosomes do form, monastral and biastral spindles are found. Analysis by laser scanning confocal microscopy suggests that these spindles are derived from the stochastic interaction of astral microtubules directly with kinetochore regions or indirectly with kinetochore microtubules. Our findings are consistent with the idea that mitotic spindle assembly requires both acentrosomal and centrosomal pathways, strengthening the hypothesis that astral microtubules can dictate the organization of the spindle by capturing kinetochore microtubules.