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。     

GTP酶Ran在细胞核运输、核分裂与重建中的作用

小分子的单体G蛋白Ran具有鸟苷三磷酸酶活性,其结合形式Ran-GTP作为区分间期细胞的核质和胞质的一个分子标记,并参与调控核质运输、指导纺锤体形成以及引导核膜解体与装配。现就Ran在真核细胞核质运输、有丝分裂纺锤体组装与核膜动力学中的功能作一综述。     

朱顶红生殖细胞分裂过程中微管超微结构的变化

用透射电镜的方法,对朱顶红（Ａｍ ａｒｙｌｌｉｓｖｉｔｔａｔａ Ａｉｔ．）花粉管中生殖细胞的分裂过程中微管分布和结构形态变化进行了观察,获得如下主要的结果：有丝分裂前期,微管的数量较分裂前减少并变短,靠近细胞核分布。分裂前中期,微管出现于原来的核区并与染色体发生联系,形成着丝点微管。分裂中期,染色体排列于赤道面上形成赤道板,微管构成纺锤体。分裂后期,染色体分成两群,被缩短的着丝点微管拉向两极。在纺锤体两极的微管汇聚。后期的晚期,当极的微管尚未消失时,在赤道区域出现丰富的成膜体微管,在成膜体中央,细胞板前体物聚集。分裂末期,极微管和着丝点微管消失,成膜体微管在新形成的核膜和细胞板间扩展并穿过细胞板     

甘蔗茎尖细胞有丝分裂过程中微管骨架的变化

利用改进的冰冻切片法结合间接免疫荧光标记技术对甘蔗茎尖细胞有丝分裂过程中微管骨架的变化进行了研究。结果表明, 在甘蔗茎尖细胞有丝分裂过程中存在4种循序变化的典型微管列阵,即周质微管、早前期微管带、纺锤体微管及成膜体微管。同时, 还观察到在各种典型微管列阵相互转变过程中存在各种微管列阵的过渡状态。甘蔗茎尖正在伸长的幼叶部位细胞的周质微管主要为与细胞伸长轴相垂直的横向周质微管; 茎尖幼叶部位伸长缓慢细胞的微管主要为纵向及斜向排列的周质微管,在甘蔗茎尖幼叶基部初生增粗分生组织处, 横向、斜向、纵向及随机排列的周质微管列阵均有分布。在少数分裂前期的细胞中, 发现细胞具有2条早前期微管带, 其具体功能还不清楚。表明甘蔗茎尖细胞微管列阵的变化与许多双子叶植物及部分单子叶植物具有共同的变化规律, 进一步证明微管骨架的周期性变化在植物中具有普遍性。     

川百合花粉管的生殖细胞分裂过程中微管骨架的分布变化

应用透射电镜辅以免疫荧光定位技术研究了川百合 (Lilium davidii Duch.)花粉管中生殖细胞分裂过程中染色体动态和微管分布的关系。在生殖细胞分裂前和有丝分裂前期 ,电镜观察一直未见微管结构 ,但免疫荧光图象显示生殖细胞中有微管蛋白存在。直到分裂的前中期—中期 ,染色体出现 ,它们沿花粉管的长轴前后排列 ,横向的着丝点对相应地一对对地纵向排列。这时 ,生殖细胞中才出现大量微管 ,它们分布于细胞周质区和染色体之间 ,并跨越染色体的整个长度。前中期—中期开始时 ,只有 1～ 2对着丝点从横向转为纵向 ,微管垂直插入着丝点形成着丝点微管 ,而非前人用免疫荧光方法观察到的微管与着丝点侧向联接的图象。随着横向的着丝点对逐渐转变成纵向的过程 ,着丝点微管数量逐渐增多 ,但不形成典型的纺锤体。分裂后期 ,染色体交错分离 ,微管的分布与前中期—中期的基本相同。晚后期 ,染色体呈明显的两群 ,除极区和细胞中央区有微管残余外 ,大部分微管消失。通过染色体长度的测量 ,间接证明了分裂后期 B的存在。分裂末期的晚期 ,核膜形成后 ,在两精核之间的区域 ,微管数量开始增多。此区可能代表用免疫荧光所观察到的微管重叠区。细胞板出现后 ,微管消失     

割手密茎尖细胞有丝分裂过程中微管骨架的变化

利用冰冻切片法结合间接免疫荧光标记技术对割手密茎尖细胞有丝分裂过程中微管骨架的变化进行了研究。结果表明:在割手密茎尖细胞有丝分裂过程中存在4种循序变化的典型微管列阵,即周质微管、早前期微管带、纺锤体微管及成膜体微管。在割手密初生增粗分生组织细胞中观察到的大多数是周质微管列阵,很少观察到其它3种典型的微管列阵,这可能这是割手密茎较小的原因之一。     

小鼠孤雌胚早期发育过程中γ-微管蛋白的动态变化

微管蛋白是构成微管的主要蛋白,其中α、β亚单位形成异二聚体,而γ-微管蛋白在微管组装中起作用。为了研究小鼠早期孤雌胚中廿微管蛋白的动态变化,本实验采用了免疫荧光化学染色与激光共聚焦显微镜观察相结合的方法,在SrCl2激活的卵母细胞减数分裂以及早期孤雌胚有丝分裂过程中对γ-微管蛋白进行了定位观察。结果显示,SrCl2和细胞松弛素B（cytochalasin B,CB）诱导的第二次减数分裂中期（metaphase Ⅱ ofmeiosis,MII）小鼠卵母细胞恢复减数分裂,并且纺锤体始终与质膜平行,表明纺锤体旋转被抑制,但核分裂不受影响。减数分裂过程中γ-微管蛋白主要定位于中期纺锤体两极和后期分开的染色单体之间;孤雌活化两雌原核形成以后,γ-微管蛋白聚集在两雌原核周围。在早期孤雌胚有丝分裂间期无定形的γ-微管蛋白均匀分布于核;前中期γ-微管蛋白向两极移动,遍布于整个纺锤体区。有丝分裂中期、后期和末期廿微管蛋白的分布变化与减数分裂相似。结果表明,SrCl2和CB激活的MII卯母细胞产生杂合二倍体;γ-微管蛋白具有促微管负极帽形成和稳定微管的功能,从而促进纺锤体的形成;分裂后期和末期廿微管蛋白的重新分布可能是由纺锤体牵引同源染色体分离所诱导的：γ-微管蛋白负责两雌原核的迁移靠近。     

甘蔗茎尖细胞有丝分裂过程中微管骨架的变化

利用改进的冰冻切片法结合间接免疫荧光标记技术对甘蔗茎尖细胞有丝分裂过程中微管骨架的变化进行了研究。结果表明,在甘蔗茎尖细胞有丝分裂过程中存在4种循序变化的典型微管列阵,即周质微管、早前期微管带、纺锤体微管及成膜体微管。同时,还观察到在各种典型微管列阵相互转变过程中存在各种微管列阵的过渡状态。甘蔗茎尖正在伸长的幼叶部位细胞的周质微管主要为与细胞伸长轴相垂直的横向周质微管：茎尖幼叶部位伸长缓慢细胞的微管主要为纵向及斜向排列的周质微管,在甘蔗茎尖幼叶基部初生增粗分生组织处,横向、斜向、纵向及随机排列的周质微管列阵均有分布。在少数分裂前期的细胞中,发现细胞具有2条早前期微管带,其具体功能还不清楚。表明甘蔗茎尖细胞微管列阵的变化与许多双子叶植物及部分单子叶植物具有共同的变化规律,进一步证明微管骨架的周期性变化在植物中具有普遍性。     

增强UV-B辐射和He-Ne激光对小麦原生质体微管骨架的影响

以小麦叶片原生质体为材料,采用间接免疫荧光定位法标记其微管系统,并利用激光共聚焦扫描显微系统进行观察。研究了低剂量He-Ne激光(5mW.mm-2)、增强UV-B辐射(10.08kJ.m-2.d-1)及二者的复合处理对小麦幼苗叶肉细胞中微管骨架的影响。结果表明,增强UV-B辐射后,小麦叶片细胞中微管骨架发生解聚,呈短棒状或点状分布,微管束弥散且荧光强度减弱;而增强UV-B辐射后再施以He-Ne激光处理,小麦叶肉细胞微管骨架有部分断裂,但较单独UV-B处理组的损伤程度轻,说明低剂量的He-Ne激光可以部分修复增强UV-B辐射对微管骨架的损伤,且对微管的聚合有促进作用。     

酵母pac—1基因的克隆、序列分析和在大肠杆菌中的高效表达及活性测定

粟酒裂殖酵母菌 (Ｓｃｈｉｚｏｓａｃｃｈａｒｍｙｃｅｓｐｏｍｂｅ)的 ｐａｃ 1基因于 1991年Ｙｕｉｃｈｉｌｉｎｏ等首次报道。在温度敏感型的酵母突变体中 ,ｐａｃ 1基因是一个多拷贝阻遏子 ,它使酵母在限制温度培养条件下的减数分裂不受调控 ,对酵母的生长过程起着重要的调节作用。该基因有一个编码 36 4个氨基酸的开放阅读框 ,编码产物的羧基端与大肠杆菌核糖核酸酶Ⅲ有2 5 %的同源性。通过酶活性测定 ,已经确定它是一类依赖ｄｓＲＮＡ的核糖核酸酶 ,具有降解ｄｓＲＮＡ的功能[1,2 ] 。由于大多数植物病毒为ＲＮＡ病毒 ,因此无论它的基…     

The mammalian Sec6/8 complex interacts with Ca(2+) signaling complexes and regulates their activity

The localization of various Ca(2+) transport and signaling proteins in secretory cells is highly restricted, resulting in polarized agonist-stimulated Ca(2+) waves. In the present work, we examined the possible roles of the Sec6/8 complex or the exocyst in polarized Ca(2+) signaling in pancreatic acinar cells. Immunolocalization by confocal microscopy showed that the Sec6/8 complex is excluded from tight junctions and secretory granules in these cells. The Sec6/8 complex was found in at least two cellular compartments, part of the complex showed similar, but not identical, localization with the Golgi apparatus and part of the complex associated with Ca(2+) signaling proteins next to the plasma membrane at the apical pole. Accordingly, immunoprecipitation (IP) of Sec8 did not coimmunoprecipitate betaCOP, Golgi 58K protein, or mannosidase II, all Golgi-resident proteins. By contrast, IP of Sec8 coimmunoprecipitates Sec6, type 3 inositol 1,4,5-trisphosphate receptors (IP(3)R3), and the Gbetagamma subunit of G proteins from pancreatic acinar cell extracts. Furthermore, the anti-Sec8 antibodies coimmunoprecipitate actin, Sec6, the plasma membrane Ca(2+) pump, the G protein subunits Galphaq and Gbetagamma, the beta1 isoform of phospholipase C, and the ER resident IP(3)R1 from brain microsomal extracts. Antibodies against the various signaling and Ca(2+) transport proteins coimmunoprecipitate Sec8 and the other signaling proteins. Dissociation of actin filaments in the immunoprecipitate had no effect on the interaction between Sec6 and Sec8, but released the actin and dissociated the interaction between the Sec6/8 complex and Ca(2+) signaling proteins. Hence, the interaction between the Sec6/8 and Ca(2+) signaling complexes is likely mediated by the actin cytoskeleton. The anti-Sec6 and anti-Sec8 antibodies inhibited Ca(2+) signaling at a step upstream of Ca(2+) release by IP(3). Disruption of the actin cytoskeleton with latrunculin B in intact cells resulted in partial translocation of Sec6 and Sec8 from membranes to the cytosol and interfered with propagation of agonist-evoked Ca(2+) waves. Our results suggest that the Sec6/8 complex has multiple roles in secretory cells including governing the polarized expression of Ca(2+) signaling complexes and regulation of their activity.     

植物核质运输与其先天免疫研究进展

植物为了抵御病原菌的侵染而进化出一套独特的先天免疫系统,它主要通过定位在细胞膜或细胞质上的受体介导并激活下游抗病基因表达而实现,但在这些信号传递过程中,细胞质的信号向核传递需要核质运输相关元件的参与。虽然目前只有个别核质运输的信号元件被证实参与了植物的先天免疫信号传递过程,但越来越多的研究表明核质运输是连接抗病基因表达和信号识别受体的一个主要方式。研究发现,病原菌的效应因子也可以利用植物核质运输机制侵入到宿主细胞核内,调控敏感基因的表达,干扰植物的免疫反应。该文对近年来国内外有关植物的核质运输机制、各层次免疫反应需要核质运输作用、核质运输相关蛋白在免疫反应中的作用等方面对核质运输参与植物先天免疫反应研究的研究进展进行综述,并指出该领域未来研究的主要内容和方向。     

Microtubules Deform the Nuclear Membrane and Disrupt Nucleocytoplasmic Transport in Tau-Mediated Frontotemporal Dementia

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Fission yeast Ryh1 GTPase activates TOR Complex 2 in response to glucose

The Target Of Rapamycin (TOR) is an evolutionarily conserved protein kinase that forms 2 distinct protein complexes referred to as TOR complex 1 (TORC1) and 2 (TORC2). Recent extensive studies have demonstrated that TORC1 is under the control of the small GTPases Rheb and Rag that funnel multiple input signals including those derived from nutritional sources; however, information is scarce as to the regulation of TORC2. A previous study using the model system provided by the fission yeast Schizosaccharomyces pombe identified Ryh1, a Rab-family GTPase, as an activator of TORC2. Here, we show that the nucleotide-binding state of Ryh1 is regulated in response to glucose, mediating this major nutrient signal to TORC2. In glucose-rich growth media, the GTP-bound form of Ryh1 induces TORC2-dependent phosphorylation of Gad8, a downstream target of TORC2 in fission yeast. Upon glucose deprivation, Ryh1 becomes inactive, which turns off the TORC2-Gad8 pathway. During glucose starvation, however, Gad8 phosphorylation by TORC2 gradually recovers independently of Ryh1, implying an additional TORC2 activator that is regulated negatively by glucose. The paired positive and negative regulatory mechanisms may allow fine-tuning of the TORC2-Gad8 pathway, which is essential for growth under glucose-limited environment.     

Insight into Actin Organization and Function in Cytokinesis from Analysis of Fission Yeast Mutants

Actin is a key cytoskeletal protein with multiple roles in cellular processes such as polarized growth, cytokinesis, endocytosis, and cell migration. Actin is present in all eukaryotes as highly dynamic filamentous structures, such as linear cables and branched filaments. Detailed investigation of the molecular role of actin in various processes has been hampered due to the multifunctionality of the protein and the lack of alleles defective in specific processes. The actin cytoskeleton of the fission yeast, Schizosaccharomyces pombe, has been extensively characterized and contains structures analogous to those in other cell types. In this study, primarily with the view to uncover actin function in cytokinesis, we generated a large bank of fission yeast actin mutants that affect the organization of distinct actin structures and/or discrete physiological functions of actin. Our screen identified 17 mutants with specific defects in cytokinesis. Some of these cytokinesis mutants helped in dissecting the function of specific actin structures during ring assembly. Further genetic analysis of some of these actin mutants revealed multiple genetic interactions with mutants previously known to affect the actomyosin ring assembly. We also characterize a mutant allele of actin that is suppressed upon overexpression of Cdc8p-tropomyosin, underscoring the utility of this mutant bank. Another 22 mutant alleles, defective in polarized growth and/or other functions of actin obtained from this screen, are also described in this article. This mutant bank should be a valuable resource to study the physiological and biochemical functions of actin.     

CBP-Mediated Acetylation of Importin α Mediates Calcium-Dependent Nucleocytoplasmic Transport of Selective Proteins in Drosophila Neurons

For proper function of proteins, their subcellular localization needs to be monitored and regulated in response to the changes in cellular demands. In this regard, dysregulation in the nucleocytoplasmic transport (NCT) of proteins is closely associated with the pathogenesis of various neurodegenerative diseases. However, it remains unclear whether there exists an intrinsic regulatory pathway(s) that controls NCT of proteins either in a commonly shared manner or in a target-selectively different manner. To dissect between these possibilities, in the current study, we investigated the molecular mechanism regulating NCT of truncated ataxin-3 (ATXN3) proteins of which genetic mutation leads to a type of polyglutamine (polyQ) diseases, in comparison with that of TDP-43. In Drosophila dendritic arborization (da) neurons, we observed dynamic changes in the subcellular localization of truncated ATXN3 proteins between the nucleus and the cytosol during development. Moreover, ectopic neuronal toxicity was induced by truncated ATXN3 proteins upon their nuclear accumulation. Consistent with a previous study showing intracellular calcium-dependent NCT of TDP-43, NCT of ATXN3 was also regulated by intracellular calcium level and involves Importin α3 (Imp α3). Interestingly, NCT of ATXN3, but not TDP-43, was primarily mediated by CBP. We further showed that acetyltransferase activity of CBP is important for NCT of ATXN3, which may acetylate Imp α3 to regulate NCT of ATXN3. These findings demonstrate that CBP-dependent acetylation of Imp α3 is crucial for intracellular calcium-dependent NCT of ATXN3 proteins, different from that of TDP-43, in Drosophila neurons.     

细胞核质转运及其受体在植物抗病防御反应中的调控作用

植物病害严重威胁全球粮食生产,研究植物对病原菌防御机制和病原菌对寄主作物的侵染过程和分子机制,有助于改良植物种源使其获得持久抗性。近年来, 日渐增多的研究表明, 一些抗病蛋白需要转移到细胞核内才能启动免疫反应,进而发挥抗病防御作用,而细胞核质转运受体是实现这些抗病蛋白核质转运必不可少的“载体”。因此,细胞核质转运及转运...     

Reprogramming Cdr2-Dependent Geometry-Based Cell Size Control in Fission Yeast

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Two Fission Yeast Rab7 Homologs, Ypt7 and Ypt71, Play Antagonistic Roles in the Regulation of Vacuolar Morphology

Small guanine triphosphatases (GTPases) of the Rab family are key regulators of membrane trafficking events between the various subcellular compartments in eukaryotic cells. Rab7 is a conserved protein required in the late endocytic pathway and in lysosome biogenesis. A Schizosaccharomyces pombe ( S. pombe ) homolog of Rab7, Ypt7, is necessary for trafficking from the endosome to the vacuole and for homotypic vacuole fusion. Here, we identified and characterized a second fission yeast Rab7 homolog, Ypt71. Ypt71 is localized to the vacuolar membrane. Cells deleted for ypt71 ⁺ exhibit normal growth rates and morphology. Interestingly, a ypt71 null mutant contains large vacuoles in contrast with the small fragmented vacuoles found in the ypt7 null mutant. Furthermore, the ypt71 mutation does not enhance or alleviate the temperature sensitivity or vacuole fusion defect of ypt7 Δ cells. Like ypt7 Δ cells, overexpression of ypt71 ⁺ caused fragmentation of vacuoles and inhibits vacuole fusion under hypotonic conditions. Thus, the two S. pombe Rab7 homologs act antagonistically in regulating vacuolar morphology. Analysis of a chimeric Ypt7/Ypt71 protein showed that Rab7-directed vacuole dynamics, fusion versus fission, largely depends on the medial region of the protein, including a part of RabSF3/α3-L7.