周期性单轴拉伸力学刺激对哺乳动物细胞有丝分裂方向影响的研究

哺乳动物细胞的有丝分裂过程与细胞的增殖、分化以及生物体发育、组织器官形成、损伤组织的修复和疾病的发生有关．广泛存在的力学刺激能否对细胞有丝分裂方向产生影响,以及其影响有丝分裂定向的途径尚未完全阐明．采用小鼠成纤维细胞作为模型,研究周期性单轴拉伸力学刺激对细胞应力纤维排布和有丝分裂方向的影响．结果表明,周期性单轴拉伸诱导细胞有丝分裂与应力纤维垂直于拉伸方向排布．而阻断应力纤维的两种基本组成成分(微丝和肌球蛋白Ⅱ),会造成在周期性单轴拉伸条件下的应力纤维和有丝分裂方向重排．特别是,Y27632 (10 μmol/L) 和低浓度的ML7 (50 μmol/L)、Blebbistatin (50 μmol/L)可以诱导细胞有丝分裂与应力纤维平行于拉伸方向排布．统计结果表明,在不同实验条件下,应力纤维排布和有丝分裂方向均具有高度相关性．Western blot实验表明,肌球蛋白轻链磷酸化水平与周期性单轴拉伸刺激下的应力纤维排 布和有丝分裂方向密切相关．上述结果提示：周期性单轴拉伸力学刺激通过诱导应力纤维的排布,决定了细胞的有丝分裂方向．     

静态单轴拉伸应变刺激对细胞有丝分裂方向的影响

力学刺激对细胞发育具有重要意义,它如何对细胞分化及组织形态的发生产生影响是一个尚未完全阐明的问题.细胞的有丝分裂过程与细胞增殖、分化以及胚胎发育、组织器官形态形成和损伤组织的修复再生等特性密切相关,例如,细胞的有丝分裂方向就是影响细胞极性分化,乃至组织形态发生的因素之一.那么,力学刺激是否通过改变细胞有丝分裂方向从而影响细胞的分裂分化呢?以小鼠成骨细胞系MC3T3为模型,探讨了静态单轴拉伸应变刺激对细胞形态、应力纤维排布方向和有丝分裂方向的影响.结果显示,在4%及8%静态单轴拉伸应变条件下,48 h之内细胞形态发生明显变化,细胞呈梭状,长轴沿应变方向排列,细胞骨架微丝呈束状平行排列,方向与应变方向相关.统计学分析表明,4%应变刺激48 h后、8%应变6 h后、8%应变12 b后、8%应变24 h后,及8%应变48 h后,分别有49%,43%,54%,54%,和62%的细胞应力纤维排列方向与单轴拉伸应变方向的夹角在300以内,以及50%,48%,56%,53%和62%的细胞有丝分裂方向与单轴应变方向夹角在300以内.统计学分析表明,细胞形态、应力纤维排布及有丝分裂方向与拉伸方向相关,且应力纤维排列方向和有丝分裂方向之间呈现高的相关性,这种相关性在拉伸刺激48 h后表现很明显,由此推测,存在力学刺激影响细胞形态及细胞应力纤维排布方向,控制有丝分裂方向的机制.     

抑癌蛋白CYLD调控纺锤体定向

正细胞分裂方向的确定对于细胞命运的决定和组织的形成十分重要,此过程依赖于有丝分裂纺锤体相对于细胞皮层的精确旋转(即纺锤体定向)。纺锤体定向错误会导致发育异常以及肿瘤等疾病的发生,但有关的分子机制尚不清楚。在国家科技部973计划课题和国家自然科学基金的资助下,南开大学生命科学学院药物化学生物学国家重点实验室周军教授课题组的博士生杨云帆等人发现抑癌蛋白CYLD在纺锤体定向过程中发挥重要作用,并进一步证明CYLD通     

中等纤维在几种人体上皮细胞有丝分裂过程中的行为

本文用间接免疫荧光法和电镜术观察了分别来自人表皮(PcaSE-1)、复层上皮(CNE)和单层上皮(SPC-A-1)的3个上皮细胞系的细胞在有丝分裂过程中中等纤维的行为。结果表明,CNE细胞和SPC-A-1细胞表达两种不同类型的中等纤维系统:角蛋白纤维和波形纤维,而PcaSE-1细胞仅表达角蛋白纤维。当细胞进入有丝分裂时,PcaSE-1细胞的角蛋白纤维维持完整的形态且将有丝分裂纺锤体围绕在细胞中央。相反,在CNE细胞和SPC-A-1细胞中,在细胞有丝分裂时,角蛋白纤维解聚成无定形的胞质小体,然而它们的波形纤维始终保持完整的形态。我们认为(1)在分裂上皮细胞中,角蛋白纤维的解聚与细胞的恶性程度有关,而与间期上皮细胞中是否含有丰富的角蛋白纤维无明显关系。(2)在上皮细胞有丝分裂时,中等纤维可能参于纺锤体的定位和趋中。(3)在分裂CNE细胞中,波形纤维的可能功能是染色体的定位和定向。     

多纤毛细胞中心粒扩增的分子机制

中心粒是由九组三联体微管组成的圆筒状细胞器,主要存在于动物细胞中。中心粒在细胞中主要行使两大功能：一方面,中心粒是中心体的核心,而中心体是哺乳动物细胞的微管组织中心,在有丝分裂问期参与细胞迁移、胞内运输和形态维持等,而在有丝分裂期则作为纺锤体的极点参与纺锤体的形成,参与细胞分裂和遗传物质的分配;另一方面,细胞进入G0／G1期后,     

纺锤体组装检控点

细胞有丝分裂过程中,纺锤体组装检控点监控着染色体在赤道板的队列和向纺锤体两极的分离,确保动粒-微管的黏附和有丝分裂器的完整,使所有的染色体都置于赤道板并双极定向后才进入后期,保证遗传物质均等地分配给两个子代细胞。纺锤体组装检控点缺陷将导致非整倍体的出现,并与一些肿瘤的发生密切相关。现就近年来纺锤体组装检控点蛋白以及纺锤体组装检控点功能缺陷与肿瘤的关系方面的研究进展作一简要综述。     

6-溴异香兰素BVAN08诱发肝癌细胞HepG2纺锤体损伤和有丝分裂灾变死亡

研究香兰素衍生物中的6.溴异香兰素(BVAN08)对细胞纺锤体结构的影响及诱发灾变死亡的相关机制,为开发该化合物为新的抗癌药物提供理论依据.通过光学显微镜观察BvAN08作用后细胞形态学变化,流式细胞术检测细胞周期,纺锤体功能检测点实验和原位免疫荧光杂交实验分析细胞有丝分裂进程和纺锤体结构.western印记检测BVAN08作用后相关蛋白质的变化.结果表明20～60 μmol/L BVAN08作用后,HepG2细胞变圆不再贴壁生长、随后脱落死亡,具有浓度依赖性量效关系;明显诱导细胞G2/M期阻滞、导致细胞有丝分裂指数升高,并出现大量的非二倍体和多倍体细胞;破坏细胞纺锤体的结构,多中心体细胞显著增加;该化合物促使细胞周期转录调节因子FoxM1及其下游靶分子细胞周期蛋白B1和CdK1的降解、阻止有丝分裂过程而导致有丝分裂灾变死亡.研究揭示BVAN08通过破坏纺锤体结构、诱发M期阻滞,导致细胞有丝分裂灾变死亡,FoxM1失活可能参与其作用机制.     

纺锤体组装检验点:染色体稳定性的守护神

有丝分裂是真核生物进行细胞增殖的基本方式,其根本目的是准确无误地将复制好的染色体平均分配到两个子细胞中。在细胞有丝分裂过程中,纺锤体组装检验点的作用是产生"等待"信号,直至所有的染色体都排列到赤道板上并建立正确的双极定向,以确保染色体的均等分配。在高等动物中,细胞的纺锤体组装检验点功能行使异常,染色体分离将出现错误,导致子代细胞的染色体数量不稳定,进而诱发肿瘤或导致其他疾病的发生。纺锤体组装检验点一直以来都是细胞生物学家研究的热点,然而其作用的分子基础和调控因素还不是十分明了,该文将对近年来关于纺锤体检验点的研究进展进行总结和探讨。     

γ-微管蛋白研究进展

概述了近年来对γ-微管蛋白复合体结构、分子机制以及功能的研究进展.γ-微管蛋白是真核生物体内一种重要的保守性功能蛋白,以γ-微管蛋白小复合体和γ-微管蛋白环式复合体两种形式存在.通过γ-微管蛋白复合体结合蛋白定位于微管组织中心,参与微管的晶核起始以及有丝分裂纺锤体的组装等细胞功能.     

细胞骨架与中心体的分离过程

中心体作为细胞微管组织中心,对于细胞的生理活动具有重要的调控作用.在G2期末和有丝分裂期开始阶段,复制之后的中心体需要向细胞核两端运动,到达形成双极纺锤体的位置.这一过程受到微管和微丝两个骨架系统的调控.在相关动力蛋白的驱动下,两种骨架相互配合,共同完成中心体的分离过程,从而保证细胞顺利进入有丝分裂期.本文分析和比较了两种骨架蛋门对下中心体分离过程中所发挥的作用.     

Many fingers on the mitotic trigger: post-translational regulation of the Cdc25C phosphatase

The mitotic inducer Cdc25 phosphatase controls the activation of Cdc2/cyclin B protein kinase and entry into mitosis in eukaryotic cells. Cdc25C is highly regulated by multiple post-translational modifications within its N-terminal regulatory domain and site-specific protein interactions. Phosphorylation of one inhibitory site targeted by multiple kinases determines the timing of Cdc25C activation and arrests cells in G2 in response to checkpoint, stress, developmental and extracellular signals. In mitosis, phosphorylation of several Ser/Thr residues and Pin1-catalysed peptidyl-proline isomerisation produces activation. Phosphorylation of one activating site is antagonistic to the proximal inhibitory site and maintains Cdc25C activity during mitosis. Phosphorylation and interacting proteins also modulate the nuclear import and export signals on Cdc25C, inducing dramatic changes in its localisation within the cell. Thus, the regulation of Cdc25C activity and localization integrates multiple signals that govern the decision to enter mitosis.     

Many Fingers on the Mitotic Trigger: Post-Translational Regulation of the Cdc25C Phosphatase

AbstractThe mitotic inducer Cdc25 phosphatase controls the activation of Cdc2/cyclin B protein kinase and entry into mitosis in eukaryotic cells. Cdc25C is highly regulated by multiple post-translational modifications within its N-terminal regulatory domain and site-specific protein interactions. Phosphorylation of one inhibitory site targeted by multiple kinases determines the timing of Cdc25C activation and arrests cells in G2 in response to checkpoint, stress, developmental and extracellular signals. In mitosis, phosphorylation of several Ser/Thr residues and Pin1-catalysed peptidyl-proline isomerisation produces activation. Phosphorylation of one activating site is antagonistic to the proximal inhibitory site and maintains Cdc25C activity during mitosis. Phosphorylation and interacting proteins also modulate the nuclear import and export signals on Cdc25C, inducing dramatic changes in its localisation within the cell. Thus, the regulation of Cdc25C activity and localisation integrates multiple signals that govern the decision to enter mitosis.     

Orientation of Mitotic Spindles during the 8- to 16-Cell Stage Transition in Mouse Embryos

Background

Asymmetric cell divisions are involved in the divergence of the first two lineages of the pre-implantation mouse embryo. They first take place after cell polarization (during compaction) at the 8-cell stage. It is thought that, in contrast to many species, spindle orientation is random, although there is no direct evidence for this.

Methodology/Principal Findings

Tubulin-GFP and live imaging with a spinning disk confocal microscope were used to directly study spindle orientation in whole embryos undergoing the 8- to 16-cell stage transition. This approach allowed us to determine that there is no predetermined cleavage pattern in 8-cell compacted mouse embryos and that mitotic spindle orientation in live embryo is only modulated by the extent of cell rounding up during mitosis.

Conclusions

These results clearly demonstrate that spindle orientation is not controlled at the 8- to 16-cell transition, but influenced by cell bulging during mitosis, thus reinforcing the idea that pre-implantation development is highly regulative and not pre-patterned.     

睾丸酮对大鼠前列腺上皮细胞有丝分裂方向的影响

目的探讨丙酸睾丸酮（T）对大鼠前列腺上皮细胞染色体有丝分裂方向的影响及其差异基因表达。方法 SPF级SD雄性大鼠（110~130 g）20只,随机分为2组。深麻醉下对所有大鼠进行去势手术,恢复一周后,给药组大鼠皮下注射T,每只0.5 mg,每日1次,连续30d;对照组大鼠皮下注射橄榄油0.1 mL。取前列腺。通过免疫组化、HE染色观察结果。并做基因芯片检查差异基因表达谱。结果给药组大鼠前列腺发生形态学增生。前列腺腺腔扩张,腺上皮高度明显增高,前列腺增生。且前列腺上皮细胞染色体有丝分裂的方向与基底膜平行,而对照组前列腺上皮细胞有丝分裂的方向与基底膜垂直。AR免疫组化染色后发现给药组的前列腺上皮中,均可见到AR标记的阳性细胞,而对照组均为阴性。基因芯片和RT-PCR结果：促进细胞增殖的基因如雄激素受体相关蛋白（RAN）、TGM4和Wnt通道的WNT2等基因均上调,而抑制细胞增殖的基因如负调控Wnt通道的DKK3和促进细胞凋亡的Fas等基因下调。结论 T注射后改变了前列腺上皮细胞有丝分裂的方向,Wnt和AR信号转导通路参与了细胞增殖和有丝分裂方向改变。     

Cancer cell cannibalism: Multiple triggers emerge for entosis

Entosis is a form of epithelial cell engulfment and cannibalism prevalent in human cancer. Until recently, the only known trigger for entosis was loss of attachment to the extracellular matrix, as often occurs in the tumour microenvironment. However, two new studies now reveal that entosis can also occur among adherent epithelial cells, induced by mitosis or glucose starvation. Together, these findings point to the intriguing notion that certain hallmark properties of cancer cells, including anchorage independence, aberrant proliferation and metabolic stress, can converge on the induction of cell cannibalism, a phenomenon so frequently observed in tumours. In this review, we explore the molecular, cellular and biophysical mechanisms underlying entosis and discuss the impact of cell cannibalism on tumour biology.     

The extracellular matrix guides the orientation of the cell division axis

The cell division axis determines the future positions of daughter cells and is therefore critical for cell fate. The positioning of the division axis has been mostly studied in systems such as embryos or yeasts, in which cell shape is well defined. In these cases, cell shape anisotropy and cell polarity affect spindle orientation. It remains unclear whether cell geometry or cortical cues are determinants for spindle orientation in mammalian cultured cells. The cell environment is composed of an extracellular matrix (ECM), which is connected to the intracellular actin cytoskeleton via transmembrane proteins. We used micro-contact printing to control the spatial distribution of the ECM on the substrate and demonstrated that it has a role in determining the orientation of the division axis of HeLa cells. On the basis of our analysis of the average distributions of actin-binding proteins in interphase and mitosis, we propose that the ECM controls the location of actin dynamics at the membrane, and thus the segregation of cortical components in interphase. This segregation is further maintained on the cortex of mitotic cells and used for spindle orientation.     

LIM kinase-mediated cofilin phosphorylation during mitosis is required for precise spindle positioning

The interaction of astral microtubules with cortical actin networks is essential for the correct orientation of the mitotic spindle; however, little is known about how the cortical actin organization is regulated during mitosis. LIM kinase-1 (LIMK1) regulates actin dynamics by phosphorylating and inactivating cofilin, an actin-depolymerizing protein. LIMK1 activity increases during mitosis. Here we show that mitotic LIMK1 activation is critical for accurate spindle orientation in mammalian cells. Knockdown of LIMK1 suppressed a mitosis-specific increase in cofilin phosphorylation and caused unusual cofilin localization in the cell cortex in metaphase, instability of cortical actin organization and astral microtubules, irregular rotation and misorientation of the spindle, and a delay in anaphase onset. Similar results were obtained by treating the cells with a LIMK1 in hibitor peptide or latrunculin A or by overexpressing a non-phosphorylatable cofilin(S3A) mutant. Furthermore, localization of LGN (a protein containing the repetitive Leu-Gly-Asn tripeptide motifs), an important regulator of spindle orientation, in the crescent-shaped cortical regions was perturbed in LIMK1 knockdown cells. Our results suggest that LIMK1-mediated cofilin phosphorylation is required for accurate spindle orientation by stabilizing cortical actin networks during mitosis.     

The interphase microtubule aster is a determinant of asymmetric division orientation in Drosophila neuroblasts

The mechanisms that maintain the orientation of cortical polarity and asymmetric division unchanged in consecutive mitoses in Drosophila melanogaster neuroblasts (NBs) are unknown. By studying the effect of transient microtubule depolymerization and centrosome mutant conditions, we have found that such orientation memory requires both the centrosome-organized interphase aster and centrosome-independent functions. We have also found that the span of such memory is limited to the last mitosis. Furthermore, the orientation of the NB axis of polarity can be reset to any angle with respect to the surrounding tissue and is, therefore, cell autonomous.     

Fibronectin receptors are functional on mitotic Chinese hamster ovary cells.

In this paper, evidence is provided indicating that the blockade of presynchronized CHO 15B cells in prometaphase by nocodazole is fully reversible and efficient enough to allow us to analyze the function of the integrin receptors. Flow cytometry analysis using a specific antibody raised against the fibronectin receptor, and binding studies of the radiolabeled fibronectin on the cell membrane, indicated a stable number of receptors at the cell surface during mitosis. Furthermore, in the mean time, only a slight increase in the Kd value of the fibronectin-receptor interaction was detected. A binding assay designed to test the affinity of the receptor for its extracellular ligand in an insoluble form was used. No difference was observed between mitotic and interphasic cells. Taken together, these results indicate that the rounding up of the cells observed during mitosis is not due to a loss of the receptor affinity for its extracellular ligand.     

Activation of extracellular signal-regulated kinase (ERK) in G2 phase delays mitotic entry through p21CIP1

Extracellular signal-regulated kinase activity is essential for mediating cell cycle progression from G(1) phase to S phase (DNA synthesis). In contrast, the role of extracellular signal-regulated kinase during G(2) phase and mitosis (M phase) is largely undefined. Previous studies have suggested that inhibition of basal extracellular signal-regulated kinase activity delays G(2)- and M-phase progression. In the current investigation, we have examined the consequence of activating the extracellular signal-regulated kinase pathway during G(2) phase on subsequent progression through mitosis. Using synchronized HeLa cells, we show that activation of the extracellular signal-regulated kinase pathway with phorbol 12-myristate 13-acetate or epidermal growth factor during G(2) phase causes a rapid cell cycle arrest in G(2) as measured by flow cytometry, mitotic indices and cyclin B1 expression. This G(2)-phase arrest was reversed by pre-treatment with bisindolylmaleimide or U0126, which are selective inhibitors of protein kinase C proteins or the extracellular signal-regulated kinase activators, MEK1/2, respectively. The extracellular signal-regulated kinase-mediated delay in M-phase entry appeared to involve de novo synthesis of the cyclin-dependent kinase inhibitor, p21(CIP1), during G(2) through a p53-independent mechanism. To establish a function for the increased expression of p21(CIP1) and delayed cell cycle progression, we show that extracellular signal-regulated kinase activation in G(2)-phase cells results in an increased number of cells containing chromosome aberrations characteristic of genomic instability. The presence of chromosome aberrations following extracellular signal-regulated kinase activation during G(2)-phase was further augmented in cells lacking p21(CIP1). These findings suggest that p21(CIP1) mediated inhibition of cell cycle progression during G(2)/M phase protects against inappropriate activation of signalling pathways, which may cause excessive chromosome damage and be detrimental to cell survival.