高等哺乳动物LEM结构域蛋白家族的研究进展

在高等动物细胞开放式有丝分裂过程中,细胞核膜会发生高度有序的周期性去组装和装配的动态变化。近年的研究结果表明是LEM家族蛋白成员通过与BAF因子相互作用介导了内核膜、核纤层蛋白以及染色体之间的相互作用。LEM蛋白、核纤层蛋白以及BAF因子直接相互作用形成的三元复合体在结构与功能上是相互依赖的,在此结构与功能上组成的网络体系是形成细胞核的一些基本生物学过程的重要条件。该复合体在调控有丝分裂M期后期和末期染色体的正常分离、有丝分裂后核膜的重组装,细胞分裂间期细胞核及核膜形态维持,调控DNA复制和DNA损伤修复,调节基因表达和信号通路以及逆转录病毒感染等方面发挥着重要的生物学功能。并且LEM蛋白相关基因的异常对核纤层疾病和肿瘤的发生发展具有重要的影响。文章主要针对LEM蛋白家族成员的结构以及功能研究进展进行了详细的综述。     

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.     

一组多功能的错配修复蛋白

错配修复蛋白是DNA错配修复系统中主要功能蛋白质,主要参与DNA复制过程中对错配碱基的识别和修复.近年来研究表明错配修复蛋白还参与DNA损伤信号的传递、细胞周期的调控、减数分裂和有丝分裂等.错配修复蛋白缺陷会增加患肿瘤的危险性或者直接导致肿瘤;由于错配修复蛋白参与了DNA损伤信号传递、周期调控,错配修复蛋白缺陷还会导致细胞对相关抗癌药物产生耐受.     

PML与基因组稳定性

基因组稳定性同肿瘤的发生、发展密切相关,维护基因组稳定性对于细胞行使正常的生理功能是至关重要的.早幼粒细胞白血病蛋白PML(promyelocytic leukemia)主要借助分子中RBCC结构,同近50种有重要功能的蛋白相互作用而形成PML-NBs(PML nuclear bodies).PML-NBs是与核基质结合的、动态的、亚核多蛋白复合物,它作为区室化核结构(compartmentalized nuclear architecture)——染色质间区室(interchromatin compartment)的功能单位,满足了真核基因高层次表达调控模式的时空要求.最新的研究证明：PML是基因组稳定性“守门人”——p53分子的搭档分子,同样在基因组稳定性调控中发挥着重要的功能作用.它协同p53参与了DNA损伤反应所诱发的细胞凋亡,还可组织多种DNA修复分子参与DNA损伤修复,在DNA损伤反应中具有重要作用;此外,PML还通过调控aurora A的活性参与中心体复制检查点调控,借助调控survivin的表达参与有丝分裂纺锤体组装检查点调控,在染色体复制和细胞分裂中均显示了重要的调控作用.而当PML表达缺失或不足时则与多种肿瘤的发生、发展相关联,因此PML分子在维护基因组稳定性中具有重要功能作用,本文仅就相关的最新研究进展予以概述     

Polo样激酶1在细胞周期及细胞周期监测点中的功能

Plk1（Polo-like kinase 1）是一类从酵母到人类都高度保守的丝氨酸/苏氨酸蛋白激酶,是真核细胞有丝分裂的重要调控因子.Plk1随有丝分裂进程定位于不同位点,调节分裂期进入、纺锤体形成和胞质分裂等过程.Plk1能够与磷酸化的停靠蛋白结合,从而在不同空间被激活以满足其在细胞周期中的不同功能.Plk1还参与G2和M期DNA损伤监测点的调节,对于DNA损伤恢复后重新进入有丝分裂期是必须的.目前,Plk1的重要功能尤其是在DNA损伤监测点中发挥的重要功能正在被广泛研究.Plk1在多种恶性肿瘤中存在过表达且与肿瘤发生密切相关,对于Plk1功能的深入研究为以Plk1为靶的肿瘤治疗提供理论依据     

核孔复合体的结构及其功能

核孔复合体(Nuclear pore complexes, NPCs)镶嵌在核膜上,是细胞核与细胞质之间的唯一通道。冷冻电子X射线断层扫描将环状NPCs分为三个环,分别称为胞质环、内环和核质环,胞质环上附有胞质纤丝,核质环上附有核篮。由于物种不同,NPCs由30～50多种不同的核孔蛋白(nucleoporins, Nups)组成,但结构和功能高度保守。根据其结构、氨基酸序列,NPCs定位和功能,Nups被分为跨膜Nups、屏障Nups、骨架Nups、胞质纤丝Nups和核篮Nups。相互间作用稳定、紧密连接的数个Nups可组成亚复合体。为了应对不同生理需要,NPCs处于高度动态变化中,间期和有丝分裂期均可通过组装和去组装改变核孔数量和功能。NPCs的主要功能是调控核质转运,小分子物质可自由扩散,大分子物质则需在核转位信号和转运载体的介导下以主动运输的方式进行转运。除了核质转运这一主要功能外,Nups还能以一个独立于转运的方式影响基因组功能。通过影响染色质结构和影响转录调控元件对靶基因的访问,Nups促进或抑制转录。在酵母,Nups介导的基因调控主要由位于NPCs中的Nups执行;在多细胞生物,不仅NPCs中的Nups,核质内游离的Nups也具有基因调控功能。此外,Nups还能通过参与形成染色质边界和形成转录记忆对基因进行调控。在增殖细胞, Nups通过与DNA修复机器相互作用,参与DNA损伤修复,保护基因组完整性。有丝分裂时,Nups协助核膜解体和中心体迁移,并通过作用于着丝粒来控制有丝分裂组件的空间定位与活性,稳定它们与微管之间的相互作用,保证纺锤体正常组装和染色体准确分离。总之,NPCs与生物分子的核质转运、基因表达和细胞周期密切相关,它的结构和功能的稳定是真核细胞生长、增殖、分化等生命活动的基本保证。     

Rho小G蛋白介导的细胞周期调控

Rho小G蛋白作为一个信号分子家族具有多样化的功能, 可以调节细胞骨架重排 、细胞迁移、细胞极性、基因表达、细胞周期调控等. Rho小G蛋白家族对细胞周期 调控的研究主要集中在其对于有丝分裂期细胞的调节作用,包括调节有丝分裂期前 期细胞趋圆化、后期染色体排列及收缩环的收缩作用.近期的研究显示,Rho小G蛋白及其效应分子对于细胞周期G1、S、G2期的调控主要是通过影响细胞周期的正调控因子细胞周期蛋白D1 (cyclin D1) 和负调控因子细胞周期蛋白依赖型激酶相互作用蛋白1及细胞周期蛋白依赖型激酶抑制蛋白27 (p21cip1/p27kip1) 进行的.本文总结了Rho小G蛋白及其效应分子在细胞周期调控,尤其是对G1/S期调控的研究进展,并简要阐述了Rho小G蛋白介导的细胞周期调控异常与癌症发生的关系.     

极光(aurora)激酶在细胞有丝分裂和肿瘤形成中的重要功能

极光激酶（aurora kinases）是负责调控细胞有丝分裂的一类重要的丝氨酸／苏氨酸激酶。在不同的模式生物中,极光激酶各家族成员的结构和功能都高度保守。近年来,随着极光激酶相关研究的不断深入,人们逐渐认识到极光激酶在细胞有丝分裂以及肿瘤形成中的重要功能。在细胞有丝分裂中,极光激酶参与了诸如中心体成熟分离、纺锤体组装和维持、染色体分离以及胞质分裂等多个事件。异常表达的极光激酶往往会导致细胞在有丝分裂的过程中出现大量的异常现象。此外,极光激酶还参与了肿瘤形成的过程,已经发现一些靶向作用于极光的小分子具有显著的抑癌作用。本文围绕哺乳动物的三种极光激酶,重点讨论了它们在细胞有丝分裂中的动态定位、生物学功能以及时空上的调节方式,并分析了异常表达的极光激酶参与肿瘤形成的可能途径,提出了肿瘤治疗的新思路。     

中心粒周蛋白相关疾病发病机制的新进展

中心粒周蛋白(pericentrin,PCNT)是一种高度保守的广泛存在于动物和人体组织细胞中的蛋白,是中心粒周围物质(pericentriolar material,PCM)的组成成分之一。PCNT在细胞的周期进程和信号传导中发挥关键作用,包括参与调控中心体的结构和功能,参与有丝分裂期纺锤体形成和微管成核等,其异常表达与Ⅱ型骨发育不良性原发性侏儒症、糖代谢异常、恶性肿瘤、精神类、唐氏综合征、纤毛类等多种疾病的发生有关。近期有研究表明,其对胰岛B细胞的胰岛素分泌也有一定的影响。本文对PCNT的结构与功能及其相关疾病的发病机制的研究进展作一综述。     

SRp38基因在小鼠视网膜细胞中的表达以及对GluR-B小基因可变剪接的调控

SR蛋白在前体mRNA可变剪接调控中发挥重要作用.SRp38作为一种新近发现的具有神经及生殖组织特异性的SR蛋白,能够调控一些在神经组织中起重要作用的基因(如GluR-B,Trk-C,NCAML1等)的前体mRNA可变剪接,同时还可以在有丝分裂M期及热休克时抑制前体mRNA剪接的发生.利用Western blot以及免疫组织化学方法研究了SRp38蛋白在小鼠视网膜中的表达以及分布情况,结果显示,SRp38蛋白在视网膜中的表达具有区域特异性,在外网层、内核层、内网层以及节细胞层中均有表达,而在外核层无表达.对分离培养的小鼠视网膜细胞进行免疫双标记分析的结果表明,SRp38蛋白在视杆-双极细胞的胞体、轴突、树突中表达.通过瞬时共转染以及RT_PCR分析,发现在R28细胞中,SRp38过表达可以促进GluR-B小基囚Flip亚型的剪接.结果提示SRp38蛋白可能通过调控小鼠视网膜内前体mRNA可变剪接、进而在小鼠视网膜功能中发挥重要作用.     

The centrosome in normal and transformed cells

The centrosome is a unique organelle that functions as the microtubule organizing center in most animal cells. During cell division, the centrosomes form the poles of the bipolar mitotic spindle. In addition, the centrosomes are also needed for cytokinesis. Each mammalian somatic cell typically contains one centrosome, which is duplicated in coordination with DNA replication. Just like the chromosomes, the centrosome is precisely reproduced once and only once during each cell cycle. However, it remains a mystery how this protein-based structure undergoes accurate duplication in a semiconservative manner. Intriguingly, amplification of the centrosome has been found in numerous forms of cancers. Cells with multiple centrosomes tend to form multipolar spindles, which result in abnormal chromosome segregation during mitosis. It has therefore been postulated that centrosome aberration may compromise the fidelity of cell division and cause chromosome instability. Here we review the current understanding of how the centrosome is assembled and duplicated. We also discuss the possible mechanisms by which centrosome abnormality contributes to the development of malignant phenotype.     

Regulated assembly of the mitotic spindle: a perspective from two ends

Chromosome segregration and cell division requires the regulated assembly of the mitotic spindle apparatus. This mitotic spindle is composed of condensed chromosomes attached to a dynamic array of microtubules. The microtubule array is nucleated by centrosomes and organized by associated structural and motor proteins. Mechanical linkages between sister chromatids and microtubules are critical for spindle assembly and chromosome segregation. Defects in either chromosome or centrosome segregation can lead to aneuploidy and are correlated with cancer progression. In this review, we discuss current models of how centrosomes and chromosomes organize the spindle for their equal distribution to each daughter cell.     

A tale of too many centrosomes

Having the correct number of centrosomes is crucial for proper chromosome segregation during cell division and for the prevention of aneuploidy, a hallmark of many cancer cells. Several recent studies (Basto et al., 2008; Kwon et al., 2008; Yang et al., 2008) reveal the importance of mechanisms that protect against the consequences of harboring too many centrosomes.     

Chromosomes take an active role in spindle assembly

The assembly of a bipolar spindle is essential for the accurate segregation of replicated chromosomes during cell division. Do chromosomes rely solely on other cellular components to regulate the assembly of the bipolar spindle or are they masters of their own fate? In the Zhang and Nicklas⁽¹⁾ study reviewed here, micromanipulation techniques and video microscopy were used to demonstrate the different roles that chromosome arms, kinetochores and centrosomes play in bipolar spindle assembly.     

Centrosome duplication: of rules and licenses

Most microtubule arrays in animal cells, including the bipolar spindle required for cell division, are organized by centrosomes. Thus, strict control of centrosome numbers is crucial for accurate chromosome segregation. Each centrosome comprises two centrioles, which need to be duplicated exactly once in every cell cycle. Recent work has begun to illuminate the mechanisms that regulate centriole duplication. First, genetic and structural studies concur to delineate a centriole assembly pathway in Caenorhabditis elegans. Second, the protease Separase, previously known to trigger sister chromatid separation, has been implicated in a licensing mechanism that restricts centrosome duplication to a single occurrence per cell cycle. Finally, Plk4 (also called Sak), a member of the Polo kinase family, has been identified as a novel positive regulator of centriole formation.     

LIN-5 is a novel component of the spindle apparatus required for chromosome segregation and cleavage plane specification in Caenorhabditis elegans

Successful divisions of eukaryotic cells require accurate and coordinated cycles of DNA replication, spindle formation, chromosome segregation, and cytoplasmic cleavage. The Caenorhabditis elegans gene lin-5 is essential for multiple aspects of cell division. Cells in lin-5 null mutants enter mitosis at the normal time and form bipolar spindles, but fail chromosome alignment at the metaphase plate, sister chromatid separation, and cytokinesis. Despite these defects, cells exit from mitosis without delay and progress through subsequent rounds of DNA replication, centrosome duplication, and abortive mitoses. In addition, early embryos that lack lin-5 function show defects in spindle positioning and cleavage plane specification. The lin-5 gene encodes a novel protein with a central coiled-coil domain. This protein localizes to the spindle apparatus in a cell cycle- and microtubule-dependent manner. The LIN-5 protein is located at the centrosomes throughout mitosis, at the kinetochore microtubules in metaphase cells, and at the spindle during meiosis. Our results show that LIN-5 is a novel component of the spindle apparatus required for chromosome and spindle movements, cytoplasmic cleavage, and correct alternation of the S and M phases of the cell cycle.     

Aberrant spindle dynamics and cytokinesis in Dictyostelium discoideum cells that lack glycogen synthase kinase 3

Eukaryotic cell division requires the co-ordinated assembly and disassembly of the mitotic spindle, accurate chromosome segregation and temporal control of cytokinesis to generate two daughter cells. While the absolute details of these processes differ between organisms, there are evolutionarily conserved core components common to all eukaryotic cells, whose identification will reveal the key processes that control cell division. Glycogen synthase kinase 3 (GSK-3) is a major protein kinase found throughout the eukaryotes and regulates many processes, including cell differentiation, growth, motility and apoptosis. In animals, GSK-3 associates with mitotic spindles and its inhibition causes mis-regulation of chromosome segregation. Two suppressor screens in yeast point to a more general effect of GSK-3 on cell division, however the direct role of GSK-3 in control of mitosis has not been explored outside the animal kingdom. Here we report that the Dictyostelium discoideum GSK-3 orthologue, GskA, associates with the mitotic spindle during cell division, as seen for its mammalian counterparts. Dictyostelium possesses only a single GSK-3 gene that can be deleted to eliminate all GSK-3 activity. We found that gskA-null mutants failed to elongate their mitotic spindle and were unable to divide in shaking culture, but have no chromosome segregation defect. These results suggest further conservation for the role of GSK-3 in the regulation of spindle dynamics during mitosis, but also reveal differences in the mechanisms ensuring accurate chromosome segregation.     

Dissection of cell division processes in the one cell stage Caenorhabditis elegans embryo by mutational analysis

To identify novel components required for cell division processes in complex eukaryotes, we have undertaken an extensive mutational analysis in the one cell stage Caenorhabditis elegans embryo. The large size and optical properties of this cell permit observation of cell division processes with great detail in live specimens by simple differential interference contrast (DIC) microscopy. We have screened an extensive collection of maternal-effect embryonic lethal mutations on chromosome III with time-lapse DIC video microscopy. Using this assay, we have identified 48 mutations in 34 loci which are required for specific cell division processes in the one cell stage embryo. We show that mutations fall into distinct phenotypic classes which correspond, among others, to the processes of pronuclear migration, rotation of centrosomes and associated pronuclei, spindle assembly, chromosome segregation, anaphase spindle positioning, and cytokinesis. We have further analyzed pronuclear migration mutants by indirect immunofluorescence microscopy using antibodies against tubulin and ZYG-9, a centrosomal marker. This analysis revealed that two pronuclear migration loci are required for generating normal microtubule arrays and four for centrosome separation. All 34 loci have been mapped by deficiencies to distinct regions of chromosome III, thus paving the way for their rapid molecular characterization. Our work contributes to establishing the one cell stage C. elegans embryo as a powerful metazoan model system for dissecting cell division processes.     

Generation and Characterization of a Tissue‐Specific Centrosome Indicator Mouse Line

Centrosomes are major microtubule organizing centers (MTOCs) that play an important role in chromosome segregation during cell division. Centrosomes provide a stable anchor for microtubules, constituting the centers of the spindle poles in mitotic cells, and determining the orientation of cell division. However, visualization of centrosomes is challenging because of their small size. Especially in mouse tissues, it has been extremely challenging to observe centrosomes belonging to a specific cell type of interest among multiple comingled cell types. To overcome this obstacle, we generated a tissue‐specific centrosome indicator. In this mouse line, a construct containing a floxed neomyocin resistance gene with a triplicate polyA sequence followed by an EGFP‐Centrin1 fusion cassette was knocked into the Rosa locus. Upon Cre‐mediated excision, EGFP‐Centrin1 was expressed under the control of the Rosa locus. Experiments utilizing mouse embryo fibroblasts (MEFs) demonstrated the feasibility of real‐time imaging, and showed that EGFP‐Centrin1 expression mirrored the endogenous centrosome cycle, undergoing precisely one round of duplication through the cell cycle. Moreover, experiments using embryo and adult mouse tissues demonstrated that EGFP‐Centrin1 specifically mirrors the localization of endogenous centrosomes. genesis 54:286–296, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.     

Fission yeast cells undergo nuclear division in the absence of spindle microtubules

Mitosis in eukaryotic cells employs spindle microtubules to drive accurate chromosome segregation at cell division. Cells lacking spindle microtubules arrest in mitosis due to a spindle checkpoint that delays mitotic progression until all chromosomes have achieved stable bipolar attachment to spindle microtubules. In fission yeast, mitosis occurs within an intact nuclear membrane with the mitotic spindle elongating between the spindle pole bodies. We show here that in fission yeast interference with mitotic spindle formation delays mitosis only briefly and cells proceed to an unusual nuclear division process we term nuclear fission, during which cells perform some chromosome segregation and efficiently enter S-phase of the next cell cycle. Nuclear fission is blocked if spindle pole body maturation or sister chromatid separation cannot take place or if actin polymerization is inhibited. We suggest that this process exhibits vestiges of a primitive nuclear division process independent of spindle microtubules, possibly reflecting an evolutionary intermediate state between bacterial and Archeal chromosome segregation where the nucleoid divides without a spindle and a microtubule spindle-based eukaryotic mitosis.