嗜热四膜虫染色体浓缩调节因子(RCC1)的定位及功能

真核细胞中染色体浓缩调节因子(regulator of chromosome condensation 1,RCC1)是RanGTPase唯一的鸟嘌呤核苷酸交换因子.染色质结合的RCC1和RanGTPase相互作用,催化细胞核内RanGDP向RanGTP的转化,进而调控了核质间的定向运送、有丝分裂期纺锤体的组装以及核膜的形成.本实验从原生生物嗜热四膜虫大核基因组中鉴定了1个新的RCC1(TTHERM_00530380)基因.该基因全长2 541 bp,包含2个内含子序列,开放阅读框为2 181 bp,编码726个氨基酸.实时荧光定量PCR表明,RCC1在四膜虫营养生长、饥饿以及有性生殖时期都有表达,且在有性生殖转录水平达到最高.免疫荧光定位分析表明,HA-RCC1在营养生长和饥饿时期,定位于大核和小核中;在有性生殖时期,定位于亲本大核、减数分裂的小核、新生成的大核和凋亡的大核中.过表达RCC1导致大核的无丝分裂异常,细胞增殖变慢,最终产生无大核的后代细胞.敲减RCC1导致了多小核的产生.结果表明,RCC1参与调控了四膜虫细胞核的分裂,RCC1的正常表达对核分裂以及细胞增殖起到重要的调控作用.     

嗜热四膜虫染色体浓缩调节因子(RCC1)的定位及功能分析

真核细胞中染色体浓缩调节因子（regulator of chromosome condensation 1, RCC1）是 RanGTPase 唯一的鸟嘌呤核苷酸交换因子. 染色质结合的RCC1和RanGTPase相互作用,催化细胞核内RanGDP向RanGTP的转化,进而调控了核质间的定向运送、有丝分裂期纺锤体的组装以及核膜的形成. 本实验从原生生物嗜热四膜虫大核基因组中鉴定了1个新的RCC1（TTHERM_00530380）基因. 该基因全长2 541 bp,包含2个内含子序列,开放阅读框为2 181 bp,编码726个氨基酸. 实时荧光定量PCR表明,RCC1在四膜虫营养生长、饥饿以及有性生殖时期都有表达,且在有性生殖转录水平达到最高. 免疫荧光定位分析表明, HA RCC1在营养生长和饥饿时期,定位于大核和小核中|在有性生殖时期,定位于亲本大核、减数分裂的小核、新生成的大核和凋亡的大核中. 过表达RCC1导致大核的无丝分裂异常, 细胞增殖变慢,最终产生无大核的后代细胞. 敲减RCC1导致了多小核的产生. 结果表明,RCC1参与调控了四膜虫细胞核的分裂, RCC1的正常表达对核分裂以及细胞增殖起到重要的调控作用.     

嗜热四膜虫胱硫醚γ-裂解酶Cgl1的表达、定位及功能分析

含硫氨基酸在不同的生物体中具有重要调节功能,转硫途径相关酶促进半胱氨酸的生成和硫化氢产生。本研究从嗜热四膜虫中鉴定一种胱硫醚γ-裂解酶（cystathionine γ-lyase 1,CGL1,TTHERM_00052400）基因。CGL1在营养生长期高水平表达,而在饥饿阶段和有性生殖期,维持在较低的表达水平。通过密码子优化,人工合成CGL1基因,构建重组表达质粒pGEX-CGL1,转化大肠杆菌BL21(DE3)。E.coli/pGEX-CGL1表达重组蛋白质GST-Cgl1,并通过亲和层析获得纯化。GST-Cgl1裂解胱硫醚产生半胱氨酸,也具有裂解半胱氨酸和同型半胱氨酸产生H2S的活性。进一步构建重组质粒pNEO4-3HA-CGL1和pSMC1hpNEO-CGL1,转化四膜虫细胞,获得带有HA标签和干扰CGL1的突变体细胞株。免疫荧光定位表明,HA-Cgl1生长期定位在亲本大核,饥饿期定位在细胞质,有性生殖前期定位在亲本大核,而在后期定位在胞质中。CGL1干扰的突变体细胞株在有性生殖过程中不能形成合子核,发育中的小核异常降解,产生仅有大核的异常单细胞。结果表明,嗜热四膜虫含有进化中保守的胱硫醚γ-裂解酶Cgl1。Cgl1具有产生和裂解半胱氨酸的活性。Cgl1定位在细胞质和细胞核中,参与了有性生殖过程细胞核的发育。     

错配修复蛋白Tmlh1维持嗜热四膜虫细胞核的稳定性

DNA错配修复(DNA mismatch repair,MMR)蛋白Mlh1和其它因子形成多种不同的复合物,在DNA复制后的MMR途径和减数分裂DNA重组中发挥重要作用。然而对Mlh1的功能并不完全清楚,进一步分析Mlh1在不同进化地位生物中的功能具有重要意义。嗜热四膜虫(Tetrahymena thermophila)含有不同的错配修复复合物,基因表达谱分析发现,MutL复合物中的TMLH1(TTHERM_00127000)在营养生长期和饥饿期低水平表达,在有性生殖减数分裂期表达水平显著上调。免疫荧光定位显示营养生长期,Tmlh1定位于生殖系小核和转录活跃的大核;有性生殖期,定位于功能性小核和亲本大核,但在凋亡的大核和小核中消失。在减数分裂和有丝分裂时期,Tmlh1和α-微管蛋白(α-tubulin)存在共定位;而有性生殖后期,Tmlh1与异染色质蛋白Pdd1共定位于DNA删除的异染色结构域。TMLH1敲除细胞增殖速率降低,DNA损伤修复抑制,导致有性生殖细胞配对率降低和微核形成。1 mmol/L甲基甲磺酸甲酯(methy methanesulfonate, MMS)处理下,ΔTMLH1细胞传代时间增加了4.53%±0.35%,而野生型细胞传代时间增加了0.60%±0.14%。TMLH1敲除突变细胞株小核上呈现强烈的γ-H2A.X的荧光信号。免疫共沉淀和蛋白质谱分析发现,Tmlh1同微管蛋白、错配修复因子MutS、同源重组修复关键因子Rad51,非同源末端修复因子Ku80因子等存在相互作用。这些结果表明,嗜热四膜虫错配修复蛋白Tmlh1通过多种途径参与DNA修复和基因组重排,从而维持四膜虫生长发育和有性生殖过程中细胞核的稳定性。     

错配修复蛋白Mlh3对四膜虫有性生殖是必需的

DNA错配修复(mismatch repair, MMR)是一种进化中保守的机制,它校正DNA复制过程中产生的错误,维持基因组的稳定性。MMR家族蛋白同时也参与多种DNA相关的生物学功能。本研究从嗜热四膜虫鉴定了一种新的错配修复蛋白MLH3基因,该基因预测编码 319 个氨基酸,在有性生殖期特异表达。免疫荧光定位表明,HA-Mlh3定位在有性生殖期减数分裂的小核和新发育的大核中。MLH3 敲除的突变体细胞株,在有性生殖发育期停滞在两大核和两小核阶段,新大核DNA复制受阻。γ-H2A.X 检测表明,新大核和小核有性生殖后期断裂的基因组不能正常修复,发育中的细胞裂解,不能形成有性生殖后代。结果表明,Mlh3参与四膜虫新大核发育过程基因组的断裂修复和复制,对四膜虫的有性生殖是必需的。     

绿色荧光蛋白表达载体pXS75－GFP的构建及在嗜热四膜虫中的应用

为获得能够用于构建嗜热四膜虫蛋白定位的载体,该研究将GFP基因与镉（Cd2＋）诱导的四膜虫金属硫蛋白基因（MTTl）启动子序列和终止子序列融合,获得表达载体pXS75-GFP。通过同源重组和抗性筛选,pXS75-GFP载体携带的目的基因整合入四膜虫MTTl位点,在cd2＋诱导下实现GFP融合蛋白的可控表达。将α－tubulin基因ATUl克隆JN－pXS75－GFP中,重组质粒pXS75－GFP－ATUl通过基因枪转化入四膜虫细胞,在巴龙霉素筛选下获得稳定的α-tubulin－GFP过表达细胞株。激光共聚焦显微镜观察α－tubulin．GFP的定位,结果显示,α－tubulin—GFP融合蛋白在四膜虫细胞中表达并分布于皮层上,表明pXS75．GFP载体可用于嗜热四膜虫功能蛋白的定位分析。     

嗜热四膜虫Ran结合蛋白1的表达对大小核分裂的影响

Ran是细胞内的一种具有GTP酶活性的功能蛋白,可以调节染色体稳定性、细胞核组建以及核质运输等多种细胞进程．Ran结合蛋白1（Ran-binding protein 1, Rbp1p ）是Ran的必要调控因子,促进Ran-GTP水解为Ran-GDP．本研究从嗜热四膜虫大核基因组中鉴定出1个保守的Ran结合蛋白基因RBP1(TTHERM_00158040, http://www.ciliate.org)．实时荧光定量PCR表明,RBP1在四膜虫营养生长和有性生殖过程中都有表达,且在有性生殖过程中表达水平提高．免疫荧光定位表明,在营养 生长期Rbp1p定位于细胞质中．过表达RBP1或敲减RBP1后,细胞生长速率下降,大核的无丝分裂异常,细胞分裂末期产生了无大核的异常细胞,同时过表达RBP1导致了多小核的产生．结果表明,Rbp1p影响四膜虫细胞核的分裂进程,它的正常表达对细胞增殖过程起到重要的调节作用．     

抗沉默因子Asf1调控嗜热四膜虫细胞核的稳定性

组蛋白H3/H4的分子伴侣Asf1(anti-silencing factor 1),参与依赖DNA复制及不依赖DNA复制的核小体装配,同时参与转录调控、基因沉默以及DNA损伤修复等过程. 在不同生物中,Asf1具有功能的保守性和多样性．嗜热四膜虫ASF1（TTHERM_00442300）基因编码的蛋白质含有保守的N端结构域和酸性的C端结构域．N端结构域同源序列进化树分析表明,Asf1进化与物种进化一致．实时荧光定量PCR表明,ASF1在四膜虫营养生长、饥饿及有性生殖时期均有表达,且在有性生殖4～6 h转录水平达到最高．免疫荧光定位分析表明,HA-Asf1在营养生长时期以及有性生长时期定位于功能大核和小核中,而在凋亡的大核中信号消失．过表达ASF1导致大核及小核变大,抑制细胞增殖．敲减ASF1后会导致大核形态异常,小核缺失．结果表明,ASF1表达对细胞核的形态和结构维持发挥重要的调控作用．     

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．     

过表达周期蛋白Cyc28影响四膜虫的有性生殖进程北大核心CSCD

真核生物的细胞周期通过连续的激活和失活特定的周期蛋白/周期蛋白依赖性激酶复合物活性进行调控。嗜热四膜虫含有34种周期蛋白,有性生殖期特异表达的周期蛋白Cyc2和Cyc17在四膜虫小核减数分裂中发挥重要功能。本研究从嗜热四膜虫中鉴定出一种新的周期蛋白CYC28(TTHERM_00082190)基因,预测编码266个氨基酸。实时荧光定量PCR表明,CYC28在有性生殖时期特异表达,且在4 h表达水平最高。通过同源重组构建获得MTT1启动子调控下的HA-CYC28突变体细胞。免疫荧光定位表明,HA-Cyc28定位在细胞质和凋亡的亲本大核中。分别构建CYC28敲除突变株和RNA干扰细胞株,对CYC28敲减突变体细胞的分析发现,营养生长和有性生殖期突变细胞发育正常。然而,过表达株Cyc28突变体引起原核染色体排列异常,原核不能完成有丝分裂形成配子核,有性生殖进程终止。结果表明,Cyc28参与细胞的有性生殖进程,它的正常表达和降解对原核有丝分裂的完成是必需的。     

微管蛋白酪氨酸连接酶类似物12通过干扰微管蛋白酪氨酸硝基化促进Hep-2细胞生长

硝基化酪氨酸与酪氨酸在结构上相似,它在病理情况下会出现,并在细胞内与微管蛋白结合,从而阻碍微管的正常功能. 硝基化酪氨酸在肿瘤中的作用,目前研究甚少.本文利用头颈鳞癌Hep-2细胞株,研究微管蛋白酪氨酸连接酶类似物12（tubulin tyrosine ligase like 12,TTLL12）和硝基化酪氨酸对头颈鳞癌Hep-2生长的影响,通过Western 印迹试验和MTT试验发现,随着硝基化酪氨酸的浓度升高,细胞内生成的硝基化酪氨酸微管蛋白含量也增高,同时细胞生长受抑制的程度显著增高; 对建立的TTLL12高表达细胞株加入硝基化酪氨酸培养,结果显示,TTLL12高表达细胞株内的硝基化酪氨酸微管蛋白含量明显低于对照组细胞;对照组细胞的生长明显受到抑制,而高表达细胞株的生长无明显改变,两者的细胞生长有显著性差异（P＜0.05）.本研究结果提示,TTLL12可通过阻碍硝基化酪氨酸与微管蛋白的结合,使头颈鳞癌Hep-2细胞逃避硝基化酪氨酸的打击. 对这一调控机制的进一步研究,必将有助于控制肿瘤细胞的生长,为治疗肿瘤寻找到新的治疗靶点.     

Molecular mechanisms of microtubular organelle assembly in Tetrahymena

Thanks to recent technological advances, the ciliate Tetrahymena thermophila has emerged as an attractive model organism for studies on the assembly of microtubular organelles in a single cell. Tetrahymena assembles 17 types of distinct microtubules, which are localized in cilia, cell cortex, nuclei, and the endoplasm. These diverse microtubules have distinct morphologies, stabilities, and associations with specific Microtubule-Associated Proteins. For example, kinesin-111, a microtubular motor protein, is required for assembly of cilia and is preferentially targeted to microtubules of actively assembled, immature cilia. It is unlikely that the unique properties of individual microtubules are derived from the utilization of diverse tubulin genes, because Tetrahymena expresses only a single isotype of alpha- and two isotypes of 1-tubulin. However, Tetrahymena tubulins are modified secondarily by a host of posttranslational mechanisms. Each microtubule organelle type displays a unique set of secondary tubulin modifications. The results of systematic in vivo mutational analyses of modification sites indicate a divergence in significance among post-translational mechanisms affecting either alpha- or beta-tubulin. Both acetylation and polyglycylation of alpha-tubulin are not essential and their complete elimination does not change the cell's phenotype in an appreciable way. However, the multiple polyglycylation sites on 1-tubulin are essential for survival, and their partial elimination dramatically affects cell motility, growth and morphology. Thus, both high-precision targeting of molecular motors to individual organelles as well as organelle-specific tubulin modifications contribute to the creation of diverse microtubules in a single cytoplasm of Tetrahymena.     

Identification of the Tubulin Gene Family and Sequence Determination of One β-Tubulin Gene in a Cold-Poikilotherm Protozoan, the Antarctic Ciliate Euplotes focardii

ABSTRACT. Four different tubulin genes were identified in the somatic nucleus (macronucleus) of Euplotes focardii , a strictly coldadapted, Antarctic ciliate: one of 1,800 bp for α-tubulin and three of 2,150, 1,900, and 1,600 bp, respectively, for β -tubulin. Preliminarily analysed for restriction fragment length polymorphisms, these genes showed remarkable differences in organisation from tubulin genes of other ciliates which live in temperate areas and were analysed in parallel with E. focardii. The complete coding sequence of the 1,600 bp β -tubulin gene was then determined and shown to contain unique structural features of potential importance for E. focardii microtubule organization and activity. Of eight unique substitutions detected, seven were concentrated in the large amino terminal domain of the molecule that directly interacts with the carboxy terminal region of α-tubulin for heterodimer formation. Sequence analysis of the cloned gene revealed, in addition, a potential new exception in the use of the genetic code by ciliates. A TAG codon was aligned in correspondence with Trp-21 which is strictly conserved in every tubulin sequence so far determined.     

Involvement of 14-nm Filament-Forming Protein and Tubulin in Gametic Pronuclear Behavior during Conjugation in Tetrahymena

We have studied in detail the immunofluorescence localizations of Tetrahymena 14-nm filament-forming protein (49-kDa protein) in relation to tubulin in conjugating wild-type Tetrahymena thermophila (B strain) pairs and in pairs between B strain and star strains with defective micronuclei. The results suggest that germ nuclear behavior during conjugation may involve the following cytoskeletal structures: (1) during meiosis, microtubule structures are involved in micronuclear elongation and meiotic division; (2) at the postmeiotic stage, 49-kDa protein network structures that are formed independently of the existence of pronuclei are involved in the selection and the survival of one of four meiotic products; (3) during the third prezygotic division, gametic pronuclear transfer, and zygote formation, a cytoskeletal structure in which the 49-kDa protein colocalizes with microtubules and which is dependent on the existence of a normal gametic pronucleus is involved in gametic pronuclear behavior, and (4) during the postzygotic divisions, the microtubules are involved in nuclear behavior.     

Anti-microtubule ‘plinabulin’ chemical probe KPU-244-B3 labeled both α- and β-tubulin

Plinabulin (1, NPI-2358), a potent microtubule-targeting agent derived from the natural diketopiperazine ‘phenylahistin’ with a colchicine-like tubulin depolymerization activity, is an anticancer agent undergoing Phase II clinical trials in four countries including the United States. In order to understand the precise binding mode of plinabulin with tubulin, a new bioactive biotin-tagged photoaffinity probe 4 (KPU-244-B3) was designed and synthesized. Probe 4 showed significant binding affinity to tubulin in a binding assay, and selectively bound to tubulin in an HT-1080 cell lysate without photo-irradiation. In a tubulin photoaffinity labeling study, probe 4 labeled both α- and β-tubulin subunits and these interactions were competitively inhibited by plinabulin during photo-irradiation. These results suggest that plinabulin binds in the boundary region between α- and β-tubulin near the colchicine binding site, and not inside the colchicine binding cavity.