小麦RAN1在酵母系统中影响微管的完整性和核质运输过程

真核生物的小G蛋白 Ran在进化过程中比较保守,它可直接参与细胞周期调控过程,它的缺失突变可以影响很多细胞生理进程。我们已经从小麦(Triticum aestivum L. cv. Jingdong No. 1) cDNA文库中克隆到一个新的RanGTPase的同源基因TaRAN1。在此基础上利用裂殖酵母模式系统研究了该基因的功能。研究结果表明,TaRAN1基因超表达可产生缺陷的纺锤体微管,这可能是导致我们以前观察到的异常染色体分离现象的原因。反义TaRAN1基因表达的酵母细胞,微管系统受到破坏。我们推测TaRAN1蛋白在细胞有丝分裂的纺锤体组装和维持微管系统的完整与稳定过程中起着重要作用。透射电镜观察实验结果显示, 超表达TaRAN1的酵母细胞具有异常的核膜结构,反义表达TaRAN1的酵母细胞有异常的液泡结构和紊乱的膜结构,由此推测, TaRAN1在整个核质运输事件中可能是必须的。     

RanGTPase激活蛋白RanGAP在嗜热四膜虫细胞中的定位及功能

RanGTPase激活蛋白（RanGTPase activating protein,RanGAP）和Ran相互作用,提高了Ran GTPase水解GTP的效率. RanGAP参与细胞内核质运输、纺锤体组装、核膜重建和异染色质的组装．生物进化过程中,不同生物的RanGAP表现出结构和功能的多样性．本研究从嗜热四膜虫大核基因组中鉴定出1个保守的RanGTPase激活蛋白基因RanGAP（TTHERM_00766430）．实时荧光定量PCR表明,RanGAP在四膜虫营养生长、饥饿和有性生殖过程中均有表达,且在有性生殖4～6 h表达水平最高．免疫荧光定位表明,在营养生长期、饥饿期及有性生殖的早期,RanGAP定位于细胞质中| 在有性生殖后期, RanGAP定位于凋亡的大核中．过表达RanGAP的细胞增殖速率下降,大核分裂和胞质缢缩异常, 产生无大核细胞．敲减RanGAP的细胞大核形态异常,细胞增殖速率下降,无丝分裂受到抑制,进而产生无大核细胞．RanGAP的过表达或敲除分别引起四膜虫RAN1,RanBP1和RCC1基因的表达下调或上调．结果表明,RanGAP通过Ran信号通路调控了嗜热四膜虫无性生殖过程中大核的无丝分裂,并可能参与了有性生殖过程中亲本大核的凋亡．     

小麦RAN1在酵母系统中影响微管的完整性和核质运输过程

真核生物的小G蛋白Ran在进化过程中比较保守,它可直接参与细胞周期调控过程,它的缺失突变可以影响很多细胞生理进程.我们已经从小麦(Triticum aestivum L.cv.Jingdong No.1)cDNA文库中克隆到一个新的RanGTPase的同源基因TaRAN1.在此基础上利用裂殖酵母模式系统研究了该基因的功能.研究结果表明,TaRAN1基因超表达可产生缺陷的纺锤体微管,这可能是导致我们以前观察到的异常染色体分离现象的原因.反义TaRAN1基因表达的酵母细胞,微管系统受到破坏.我们推测TaRAN1蛋白在细胞有丝分裂的纺锤体组装和维持微管系统的完整与稳定过程中起着重要作用.透射电镜观察实验结果显示,超表达TaRAN1的酵母细胞具有异常的核膜结构,反义表达TaRAN1的酵母细胞有异常的液泡结构和紊乱的膜结构,由此推测,TaRAN1在整个核质运输事件中可能是必须的.     

增强UV-B辐射对小麦TaRAN1蛋白分布与定位的影响

以增强UV-B(10.08 kJ.m-2.d-1)辐射后的小麦根尖细胞为材料,采用间接免疫荧光标记技术,利用激光共聚焦扫描显微镜,观察分析小麦根尖分裂期细胞Ran蛋白在分裂周期的分布及形态变化。研究结果显示,正常细胞中,Ran蛋白在细胞分裂间期主要定位于核膜周边,在后期定位于赤道板上和纺锤体上,末期又回到子细胞核膜周边;增强UV-B辐射处理后,在细胞分裂间期和前期有点状荧光分布在核膜的周围;中期和后期点状荧光分布在细胞质中;在末期部分点状荧光又回到核膜的周围,部分仍分散在核内,且出现落后染色体、染色体桥、不均等分裂等染色体畸变类型和异常分裂现象。     

tRNA核质动态分布与细胞命运

旺盛的细胞核、质间的物质运输(nuclear-cytoplasmic transport)是真核细胞代谢的基础．核质运输不仅将蛋白质运到目的地,还能通过在特定时间、地点结合靶分子,改变其在胞内的局部浓度,调控诸如有丝分裂等重要细胞活动．tRNA是细胞中最重要的大分子之一,合成于细胞核,在细胞质中参加蛋白质翻译．一直以来,学术界认为tRNA只是蛋白质合成的参与者,tRNA核质运输是tRNA跨越核膜进入细胞质是单向主动运输过程．然而,最近的研究成果在颠覆传统观念,tRNA不但能被转运出核,还能被逆向转运入核．2008年,新概念“tRNA核质动态分布”(tRNA nuclear-cytoplasmic dynamics)被提出,取代tRNA核质运输,描述tRNA在细胞核、质间的流动．在酿酒酵母中tRNA核质动态分布可以调控蛋白质翻译,锁定细胞周期．此领域内的最新研究正在改变着教科书中有关tRNA的传统论断．     

GTPase Ran及其生物学作用

GTPaseRan能连接并水解GTP,是许多代谢途径的重要调节物。GTPaseRan在真核细胞中一系列的生物过程,如DNA复制、RNA的转录和加工(或修饰)、核质转运、有丝分裂和减数分裂的开始和结束的控制、及其问纺锤体的组装、染色体的正确分配、核膜破裂和重组中,都起重要的作用。     

细胞核质转运受体Importin β家族与转运调控

核质转运是真核细胞的基本生命活动之一。Importinβ家族的蛋白质成员作为核质转运的受体,负责细胞内大部分蛋白质和核酸等生物大分子的跨核膜运输。同时,细胞通过多种方式对核质转运的过程进行精确调控,使底物能够在正确的时间与空间发挥功能,保证细胞增殖与分化的正常进行。核质转运的失调,则使得底物不能正常执行功能,导致个体发育的异常与疾病的发生。     

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。     

c-fos和c-myc在北方山溪鲵精子发生中的表达

用免疫组织化学方法检测原癌基因cf-os和c-myc蛋白在北方山溪鲵(Batrachuperus tibetanus)精子发生中的表达定位。结果显示,在精原细胞缓慢增殖期,8、9月,FOS阳性反应物出现在精原细胞的胞质及核膜外,10、11月,FOS在少量精原细胞的胞核中表达。在精原细胞快速增殖期,即翌年4月,FOS定位在精原细胞的胞质中;5月,FOS在大量的胞核中强阳性表达;6月,FOS定位于部分精母细胞核质和核膜下;7月,FOS在一些精子细胞的核质和核膜下表达。MYC在8、9月的部分精原细胞胞质中表达较弱,在101、1月阳性反应出现在个别精原细胞的核质中。翌年4月,MYC在精原细胞核周围的胞质中表达;5月在大量的精原细胞核膜下有强表达;6月,MYC在一些精母细胞核膜下表达;7月,MYC在部分精子细胞的核膜下弱表达。结果表明,北方山溪鲵的原癌基因cf-os和c-myc表达大强度在生精细胞发育中呈阶段性,表达的强度和细胞数量与细胞增殖的速度相一致。FOS和MYC在精原细胞内从胞质向胞核的转移与细胞快速增殖的时期相吻合。说明cf-os和c-myc对精原细胞有丝分裂有促进作用,并参与精母细胞成熟分裂的调控。     

RAN1基因过表达抑制嗜热四膜虫大核无丝分裂

Ran GTPase通过RanGTP/RanGDP循环的形式,参与调控多种细胞增殖方式：包括有丝分裂和减数分裂.敲减RAN1基因可导致嗜热四膜虫大核内微管组装紊乱,从而抑制大核无丝分裂.为进一步分析Ran1在无丝分裂中的功能,本研究将野生型Ran1以及模拟GTP（Ran1Q70L）和GDP（Ran1T25N）锁定形式的Ran1突变体在嗜热四膜虫中过量表达,均导致四膜虫细胞增殖速率下降,并引起大核无丝分裂异常,且这种核异常细胞比率与Ran1过表达量呈正相关.免疫荧光定位结果显示,过表达的HA-Ran1在整个细胞中弥散分布,破坏了正常的Ran1分布形式;而过表达的HA-Ran1Q70L明显集中在大核核膜和胞质中,HA-Ran1T25N则主要定位在大核和小核内,分别与Ran1GTP/Ran1GDP循环的辅助调节因子定位模式一致.以上结果表明,过表达Ran1及其突变体可能影响嗜热四膜虫细胞中正常的Ran1GTP/Ran1GDP循环,进而导致大核无 丝分裂异常.     

Ran GTPase: a master regulator of nuclear structure and function during the eukaryotic cell division cycle?

Ran is an abundant GTPase that is highly conserved in eukaryotic cells and has been implicated in many aspects of nuclear structure and function, especially determining the directionality of nucleocytoplasmic transport during interphase. However, cell-free systems have recently shown that Ran plays distinct roles in mitotic spindle assembly and nuclear envelope (NE) formation in vitro. During spindle assembly, Ran controls the formation of complexes with importins, the same effectors that control nucleocytoplasmic transport. Here, we review these advances and discuss a general model for Ran in the coordination of nuclear processes throughout the cell division cycle via common biochemical mechanisms.     

Mitotic functions of the Ran GTPase network: the importance of being in the right place at the right time

The Ran GTPase has important roles in nucleocytoplasmic transport, cell cycle progression, nuclear organization and nuclear envelope (NE) assembly. In this review, we discuss emerging evidence that implicate the Ran GTPase system in mitotic control in mammalian cells. Recent work indicates that members of the Ran network control two fundamental aspects of the mammalian mitotic apparatus: (i) centrosome and spindle pole function, and (ii) kinetochore function. It is also emerging that, after NE breakdown, specific Ran network components assemble in local combinations at crucial sites of the mitotic apparatus. In the light of these findings, the original notion that nucleotide-bound forms of the Ran GTPase are distributed along a unique "gradient" in mitotic cells should be re-examined. Available data also suggest that the Ran system is deregulated in certain cellular contexts: this may represent a favoring condition for the onset and propagation of mitotic errors that can predispose cells to become genetically unstable and facilitate neoplastic growth.     

Mitotic Functions of the Ran GTPase Network: the Importance of Being in the Right Place at the Right Time

The Ran GTPase has important roles in nucleocytoplasmic transport, cell cycle progression, nuclear organization and nuclear envelope (NE) assembly. In this review, we discuss emerging evidence that implicate the Ran GTPase system in mitotic control in mammalian cells. Recent work indicates that members of the Ran network control two fundamental aspects of the mammalian mitotic apparatus: (i) centrosome and spindle pole function, and (ii) kinetochore function. It is also emerging that, after NE breakdown, specific Ran network components assemble in local combinations at crucial sites of the mitotic apparatus. In the light of these findings, the original notion that nucleotide-bound forms of the Ran GTPase are distributed along a unique “gradient” in mitotic cells should be re-examined. Available data also suggest that the Ran system is deregulated in certain cellular contexts: this may represent a favoring condition for the onset and propagation of mitotic errors that can predispose cells to become genetically unstable and facilitate neoplastic growth.     

The Ran GTPase as a marker of chromosome position in spindle formation and nuclear envelope assembly

The small GTPase Ran is a key regulator of nucleocytoplasmic transport during interphase. The asymmetric distribution of the GTP-bound form of Ran across the nuclear envelope--that is, large quantities in the nucleus compared with small quantities in the cytoplasm--determines the directionality of many nuclear transport processes. Recent findings that Ran also functions in spindle formation and nuclear envelope assembly during mitosis suggest that Ran has a general role in chromatin-centred processes. Ran functions in these events as a signal for chromosome position.     

The defective nuclear lamina in Hutchinson-gilford progeria syndrome disrupts the nucleocytoplasmic Ran gradient and inhibits nuclear localization of Ubc9

The mutant form of lamin A responsible for the premature aging disease Hutchinson-Gilford progeria syndrome (termed progerin) acts as a dominant negative protein that changes the structure of the nuclear lamina. How the perturbation of the nuclear lamina in progeria is transduced into cellular changes is undefined. Using patient fibroblasts and a variety of cell-based assays, we determined that progerin expression in Hutchinson-Gilford progeria syndrome inhibits the nucleocytoplasmic transport of several factors with key roles in nuclear function. We found that progerin reduces the nuclear/cytoplasmic concentration of the Ran GTPase and inhibits the nuclear localization of Ubc9, the sole E2 for SUMOylation, and of TPR, the nucleoporin that forms the basket on the nuclear side of the nuclear pore complex. Forcing the nuclear localization of Ubc9 in progerin-expressing cells rescues the Ran gradient and TPR import, indicating that these pathways are linked. Reducing nuclear SUMOylation decreases the nuclear mobility of the Ran nucleotide exchange factor RCC1 in vivo, and the addition of SUMO E1 and E2 promotes the dissociation of RCC1 and Ran from chromatin in vitro. Our data suggest that the cellular effects of progerin are transduced, at least in part, through reduced function of the Ran GTPase and SUMOylation pathways.     

Ran binds to chromatin by two distinct mechanisms

Ran GTPase plays important roles in nucleocytoplasmic transport in interphase and in both spindle formation and nuclear envelope (NE) assembly during mitosis. The latter functions rely on the presence of high local concentrations of GTP-bound Ran near mitotic chromatin. RanGTP localization has been proposed to result from the association of Ran's GDP/GTP exchange factor, RCC1, with chromatin, but Ran is shown here to bind directly to chromatin in two modes, either dependent or independent of RCC1, and, where bound, to increase the affinity of chromatin for NE membranes. We propose that the Ran binding capacity of chromatin contributes to localized spindle and NE assembly.     

A new role of ran GTPase.

Ran is a G protein similar to Ras, but it has no membrane binding site. RanGEF, RCC1, is on chromatin and RanGAP, RanGAP1/Rna1p is in cytoplasm. Ran, thus, shuttles between the nucleus and the cytoplasm to complete its GTPase cycle, carrying out nucleocytoplasmic transport of macromolecules. A majority of Ran binding proteins, thus far found, are required for this process. A recently found novel Ran-binding protein, RanBPM, however, is localized in the centrosome. Subsequently, four groups reported that RanGTP, but not RanGDP, can induce microtubule self-organization in Xenopus egg extracts where no nuclear membrane is present. Thus, Ran is suggested to have a new role beyond the nucleocytoplasmic transport of macromolecules. In both microtubule assembly and nucleocytoplasmic transport, chromosomal localization of RCC1 is important to carry out the functions of RanGTPase. In this regard, a future intriguing question is how RCC1 interacts with chromatin DNA.     

The nuclear GTPase Gsp1p can affect proper telomeric function through the Sir4 protein in Saccharomyces cerevisiae

The small Ras-like GTPase Ran/Gsp1p is a highly conserved nuclear protein required for the nucleocytoplasmic trafficking of macromolecules. Recent findings suggest that the Ran/Gsp1p pathway may have additional roles in several aspects of nuclear structure and function, including spindle assembly, nuclear envelope formation, nuclear pore complex assembly and RNA processing. Here, we provide evidence that Gsp1p can regulate telomeric function in Saccharomyces cerevisiae. We show that overexpression of PRP20, encoding the Gsp1p GDP/GTP nuclear exchange factor, specifically weakens telomeric silencing without detectably affecting nucleocytoplasmic transport. In addition to this silencing defect, we show that Rap1p and Sir3p delocalize from their normal telomeric foci. Interestingly, Gsp1p was found to interact genetically and physically with the telomeric component Sir4p. Taken together, these results suggest that the GSP1 pathway could regulate proper telomeric function in yeast through Sir4p.     

Mechanisms of receptor-mediated nuclear import and nuclear export

Nuclear transport of proteins and RNA occurs through the nuclear pore complex and is mediated by a superfamily of transport receptors known collectively as karyopherins. Karyopherins bind to their cargoes by recognition of specific nuclear localization signals or nuclear export signals. Transport through the nuclear pore complex is facilitated by transient interactions between the karyopherins and the nuclear pore complex. The interactions of karyopherins with their cargoes are regulated by the Ras-related GTPase Ran. Ran is assisted in this process by proteins that regulate its GTPase cycle and subcellular localization. In this review, we describe several of the major transport pathways that are conserved in higher and lower eukaryotes, with particular emphasis on the role of Ran. We highlight the latest advances in the structure and function of transport receptors and discuss recent examples of steroid hormone receptor import and regulation by signal transduction pathways. Understanding the molecular basis of nuclear transport may provide insight into human diseases by revealing how nucleocytoplasmic trafficking regulates protein activity.