【作者】 姜玲；

【导师】 谢宇聪(Yu Chung Tse)；

【作者基本信息】 哈尔滨工业大学 ， 生物学， 2019， 硕士

【摘要】 核孔复合体由30多种核孔蛋白多拷贝组成,是一种八倍旋转对称的结构,且大部分核孔蛋白在线虫、植物和脊椎动物中是高度保守的。核孔复合体可根据其核孔蛋白的空间定位分成6个亚结构:在胞质和核内两边由胞质细丝和核篮蛋白形成的环状结构,由双层核膜的八个支架蛋白组成的中心框架,其两侧的胞质和核环部分以及包围它们一个中心孔,通过该中心孔发生核质运输,以便维持核膜结构及功能的完整。外周核孔蛋白主要调控mRNA的输出和通过核转运受体转运特定的分子,但很少有文献报道过有关位于核孔中心的支架蛋白调控某个蛋白的输入和输出,它们的特殊功能并没有得到人们的关注。几十年来有关核孔蛋白的研究越来越多,但大多数关注在核质运输及其在核膜上的功能,但对于核孔蛋白的其他功能所知甚少。最近的研究结果表明核孔蛋白不仅仅介导胞质和核内的运输,还可以参与其他功能,如保护基因组的完整性、调控DNA复制、基因表达和细胞分裂等。核孔蛋白功能的多样性促进我们去探究它们作为物理屏障之外的其他功能。另外,核孔蛋白在特定组织中的突变与各种的疾病相关,心房中NUP155突变可导致纤维化,这是一种可遗传的由心率不齐而致死的疾病,NUP133可调控胚胎神经谱系的发育,其突变可阻断神经干细胞分化形成神经元。然而有关核孔蛋白在其中的分子机制大多尚未可知。最近,在秀丽隐杆线虫早期胚胎中,我们的结果表明核孔蛋白NPP-3可以调控染色体在细胞前期的分布,在敲低NPP-3表达后,几乎所有的染色体都聚集在核膜边缘并形成环状结构。此外,该表型在晚期胚胎和位于性腺的卵细胞中也存在。大多文献报道敲低核孔蛋白一般会延迟细胞周期,影响纺锤体的旋转以及核质运输。例如,NPP-7和NPP-10会影响到染色体的凝缩和染色体分离。NPP-1,NPP-13和NPP-14可以调控纺锤体的旋转,但这些表型与NPP-3敲降的表型不同。NPP-3的敲低并不影响染色质的凝缩和染色体的分离以及和细胞核的大小。另外,NPP-3表达量的降低会导致细胞周期的延长,这对于秀丽隐杆线虫是致死的。通过前期实验,我们发现NPP-3敲低可影响部分位于核膜上的蛋白定位,例如NPP-3敲低可改变NPP-7和NPP-2的定位,这意味着NPP-3的敲低会破坏核膜的完整性,另外敲低NPP-7也会导致染色体聚集在核膜周围,并且该表型在早期和晚期胚胎里同时存在。通过相互敲低实验,敲低NPP-3可影响NPP-7的定位但NPP-7的敲低不改变NPP-3的定位,即证明NPP-7在NPP-3的下游调控染色体的空间分布。有文献曾报道过,当线虫胚胎处于24h的缺氧的环境中,其核内的所有染色体也会排列在核膜周围,其表型可在重新给氧后恢复。同时该文章发现在缺氧的时候,CDK-1会与在核膜边缘的染色体共定位;而正常情况下,CDK-1在间期的时候会位于细胞质和细胞核内,且核内多于胞质,在进入前期的时候,CDK-1的信号会在核内减弱,并且NPP-16的敲低也可以挽救一半的染色体排列在核膜周围的表型,但是文献并没有深入研究CDK-1和NPP-16参与调控染色体分布的机制,所以本论文也将探寻NPP-3是否也会影响到CDK-1的分布,以及NPP-16的敲低是否会挽救NPP-3敲低的表型。由于未发现相关文献报道染色体聚集在核膜周围的机制,我们只能自己探索,首先从已发现的NPP-3敲低的表型入手,NPP-3敲低改变了核膜的渗透性。通过文献查找,我们发现NPP-3敲低导致的渗透性改变与NPP-4和NPP-13敲低导致的渗透性改变基本一样,所以我们通过敲低NPP-4和NPP-13发现,NPP-4的敲低并不影响染色体的分布,即证明渗透性改变并不是染色体分布在核膜周围的原因。NPP-13的敲低也可以导致所有染色体聚集在核膜周围,并且在早期胚胎和晚期胚胎中存在。NPP-3和NPP-13同属于NUP93复合体,可以同时调控核孔复合体的功能,如某些特定蛋白的输出或者限制输出的分子的大小。所以我们也会从NPP-3的结构入手,探究NUP93复合体的敲低是否都可以调控染色体在细胞前期的空间分布。通过查找相关文献,我们发现只有位于核篮蛋白或者其附近的核孔蛋白才有机会与染色体进行互作。例如,Nup2/NPP-16蛋白的减数分裂自主区可以直接与染色体的特定序列结合并调控染色体的同源重组,另外在不同的组织中,某些核孔蛋白可以改变特定的染色体在核内的定位,进而调控组织特异性表达及功能。所以我们也会对于NPP-3是否通过改变核篮蛋白进而调控染色体定位进行进一步的探索。在线虫中,染色体聚集在核膜周围一般可以调控染色体的转录,聚集在核纤层的染色体上抑制其基因的表达,但是聚集在核孔复合体的染色体依旧保持一定的转录能力。所以我们也对核纤层的功能是否在NPP-3敲低后受到影响进行了探索。为了对NPP-3敲低导致染色体聚缩于边缘的机理有更深入的理解,我们利用早期线虫胚胎,去研究NPP-3/NUP205在染色体动态变化中的作用,为揭示核孔蛋白的新功能提供不同视角。由于核孔蛋白和染色体的空间定位差异较大,可能有其他蛋白去传导它们的功。,通过查找相关文献,我们推测NPP-3可能影响端粒锚定和纺锤体组装检验点来调控染色体在核内的定位。在第一次减数分裂前期,位于染色体两端的端粒会与KASH/SUN1蛋白结合,将染色体的两端锚定在核膜上,进而完成同源重组,这在线虫以及人类细胞中已经得到了验证,所以我们猜测是否NPP-3的敲低很可能影响了SUN-1蛋白和POT-1的功能,导致这一通路在有丝分离期被激活。在线虫中,着丝粒并不是像在脊椎动物中只有一个。着丝粒排列分布在染色体的两侧,动粒必须与每一个着丝点结合并组装纺锤体微丝,因此动粒也排列在染色体两侧。一旦动粒没有与纺锤体微丝结合,将会激活纺锤体组装检控点,将未结合微丝的染色体移动到核膜周围进行修复。我们推测NPP-3的敲低会导致纺锤体组装检控点的过度激活,将所有的染色体都移动到核膜边缘,所以我们可以将NPP-3与纺锤体组装检控点蛋白共同敲低来检测染色体在核膜周围聚集是否受到影响。另外,有文献报道Nup153可以调控纺锤体组装检控点的活性,而Nup153/NPP-7也属于核篮蛋白,我们前期的实验结果证明NPP-3的敲低会影响NPP-7在核膜上的定位,所以我们进一步将探索NPP-3,NPP-7和纺锤体组装检控点蛋白之间的关系。NPP-7对于细胞分裂和胚胎发育很重要。我们通过RNA干扰敲低其在胚胎的表达后,其父本细胞核中的染色质信号很弱,而且几乎没有染色质凝缩和染色体分离。我们采用稀释RNAi干扰菌浓度来削弱NPP-7敲低的影响,发现也会导致染色体聚集在核膜周围,并且在晚期胚胎中也有同样的表型,所以NPP-3和NPP-7是在同一通路上调节染色体的空间分布。我们的纺锤体组装检控点试验结果表明,同时敲除NPP-3和MAD-1,MDF-2,BUB-3可以拯救大部分有染色体异常分布的核;同时敲除NPP-3和其他不参与纺锤体组装检控点的动粒蛋白并不影响染色体分布,这证明NPP-3通过影响纺锤体组装检控点进而调控染色体的空间分布。我们下一步将探究NPP-3是如何调控纺锤体组装检控点。我们的实验结果表明NPP-3和NPP-7的敲低会延长CDK-1在核里面的定位,但由于CDK-1是一个细胞周期蛋白依赖性激酶,其敲低会严重影响细胞分裂,我们找不到第一次分裂的胚胎,大部分的胚胎都表现出在胚胎中只有一个大核的表型,所以我们还需要设计其他实验验证是否是过长时间的CDK-1在核内的定位导致染色体聚集在核膜周围。另外,CDK-1是蛋白激酶,可以对一些蛋白进行磷酸化,而纺锤体组装检控点蛋白的激活就是通过调节磷酸化的水平来激活纺锤体组装检控点。而CDK-1和PLK-1主要调控核膜的破裂,但并没有关于CDK-1和MDF-1的相互作用的报道,所以我们还将探究CDK-1与MDF-1的相互作用,进而完善我们的猜想模型。在本论文中,我们将会在不同的转基因线虫用RNA干扰相关基因的表达,并利用转盘式共聚焦荧光显微镜来获得并探索NPP-3在调节染色体运动中的功能。借助Crispr Cas9技术,我们将构建GFP::NPP-3和NPP-3::GFP的转基因线虫,来探究核孔蛋白NPP-3在细胞周期的动态变化。本论文将为我们将提供一种新的思路去理解染色体聚集在核膜周围的机制和其意义,初步探索NPP-3是调控染色体分布的分子机制,另外我们也希望该研究可以为进一步了解核孔蛋白缺陷导致的疾病机制提供理论依据,也为医学实验和临床应用提供理论基础。更多还原

【Abstract】 Nuclear pore complexes(NPCs),composed of multiple copies of 30 different nucleoporins in vertebrates,persist in featuring an eightfold rotational symmetry structure.Most nucleoporins are highly conserved in C.elegans,plant and vertebrates.NPCs can b e divided into six subcomplexes according to their spatial distribution.Cytoplasmic filaments and the nuclear basket form ring structure at the sides of nuclear envelope(NE).NPCs maintain the integrity of NE and facilitate the transportation of molecules including mRNA and molecules by nuclear transport receptor(NTR)between cytosol and nucleus,which is mainly controlled by the peripheral nucleoporins.Up to now,the controlling mechanism of central nucleoporins remain poorly understood.Besides the classical function,NPCs have been reported to participate in modulating chromosome architecture,preserving genome integrity and also regulating DNA replication,gene expression and cell divis ion.In this study,we investigate the roles of NPCs in addition to its physical barrier function.Nucleoporins alteration has been found to be related to various diseases accompanied with their specific function in different tissues.For example,atrial fibrillation(AF),an inherited clinical arrhythmia leading to sudden cardiac death,is caused by NUP155 mutation in atrium.Moreover,NUP133 can regulate the neural lin eages during the embryonic development and the mutation of NUP133 can cause defect in di fferentiation from neural progenitors to terminally differentiated neurons.The molecular mechanisms of nucleoporins alteration in these abnormalities are still unclear.Recently,studies performed in Caenorhabditis elegans providing an advancement method to overcome obstacles in nuclear pore complexes research.Our recent study demonstrated that one of the nucleoporin members,NPP-3,regulated chromosome localization in prophase.We observed that after the depletion of NPP-3,almost all chromosomes gather ed in the nuclear periphery and formed a ring structure in prophase and also the late stage of embryos and oocyte s development.Previous studies reported that nucleoporins depletion could alter cell cycle,spindle rotation and cytoplasmic and nuclear trans port.For example,depletion of NPP-7 and NPP-10 can affect the DNA condensation and the chromosome segregation.Moreover,it has been found that NPP-1,NPP-13 and NPP-4 can regulate the rotation of spindle.However,these phenotypes are different to the N PP-3 depletion phenotype observed in our study.NPP-3 depletion did not affect the DNA condensation and the chromosome segregation but caused the longer time of cell cycle and this defect caused lethality in C.elegans.According to the prospective experi ments,we found NPP-3 depletion affected the localization of some proteins in the nuclear envelope.For example,NPP-3 depletion changes the localization of NPP-2 and NPP-7,which means NPP-3 depletion can disrupt the integrity of nuclear envelope.In addition,NPP-7 depletion also causes all chromosomes gathering in the nuclear periphery and the phenotype is existent in the early and post-mitotic embryos.Our data demonstrated that NPP-7 is downstream of the NPP-3 to regulate the spatial organization of ch romosome.It has been reported that when the nematode embryo is put into hypoxia for 24 hours,all the chromosomes in the nucleus will also be aligned around the nuclear membrane,and the phenotype can be restored after re-oxygenation.At the same time,th e article found that CDK-1 will co-localize with the chromosome at the edge of the nuclear membrane in anoxia;CDK-1 is normally located in the cytoplasm and nucleus at the interphase,and more than in the nucleus.In the early stage,the signal of CDK-1 will be weakened in the nucleus,and NPP-16 depletion can rescue half of the phenotype of chromosomes alignment to the nuclear membrane,but the paper does not study CDK-1 in depth and the mechanism of NPP-16 in the regulation of chromosome distribution,so in my thesis I will also explore whether NPP-3 will also affect the distribution of CDK-1,and whether NPP-16 depletion will rescue the NPP-3 depletion phenotype.Since no relevant paper has reported the mechanism of chromosome aggregation around the nuc lear membrane,we can only explore it by ourselves.First,we start with the NPP-3 depletion phenotype that has been discovered.NPP-3 depletion changes the permeability of the nuclear membrane.Referred to papers,we found that the permeability change caused by NPP-3 depletion is basically the same as the permeability change caused by NPP-4 and NPP-13 depletion,so we depleted NPP-4 and NPP-13.NPP-4 depletion did not affect the distribution of chromosomes,which proves that the change in permeability is n ot the cause of chromosome distribution around the nuclear membrane.NPP-13 depletion can also cause all chromosomes gathering in the nuclear membrane and the phenotype is present in early and late embryos.NPP-3 and NPP-13 belong to the NUP93 complex and can simultaneously regulate the function of the nuclear pore complex,such as the output of certain specific proteins or the size of the output molecules.Therefore,we will start with the structure of NPP-3 and explore whether the depletion of NUP93 compl ex can regulate the spatial distribution of chromosomes in the early stage of cells.Nuclear basket proteins have been shown to interact with chromosomes.For example,the meiotic automatic region of Nup2/NPP-16 can directly bind to specific sequence of ch romosome and regulate the homologous pairing.In this study,we studied whether NPP-3 depletion could change the localization of nuclear basket proteins and regulate chromosome distribution.In addition,chromosome accumulating in the nuclear periphery regulate the level of gene transcription.Therefore,we also investigated the relationship between NPP-3 and nuclear lamina.In order to gain a clearer understanding of the function of NPP-3 in chromosome periphery,we used C.elegans as the research model to investigate the NPP-3/NUP205 role in the chromosome movement and provided the scientific basis for further analyzing the nuclear pore protein function.In addition,there is limited studies concerning the relationship between nucleoporins and chromosomes,in which our study could provide a novel perspective to uncover the functions of nucleoporins.For the spatial distribution of nucleoporins and chromosome,a candidate must be existed to link the nucleoporins and chromosome.Referring to studies working o n the chromosome and nuclear envelope,we speculate that NPP-3 may affect the telomere anchoring and the spindle assembly checkpoint to regulate the chromosome localization in the nuclei.During the meiosis I prophase,telomere at the both sides of chromosome can bind to the KASH/SUN1 protein in the nuclear envelope for homologous pairing,which has been proved in C.elegans and human cells.We speculate whether depletion of NPP-3 can affect the function of SUN-1 and POT-1 to activate this pathway in mitosis.Centromere in the C.elegans is different from the vertebrates.In C.elegans,centromeres align at the both sides of chromosome and anchored by the kinetochore to completer spindle microtubules assembly.If this process is not accurate,the spindle assembly checkpoint will active and make the unattached kinetochore chromosomes move to nuclear periphery for repairing.Therefore,we wonder whether depletion of NPP-3 can overactive the spindle assembly checkpoint to regulate chromosome pe riphery.So we will deplete NPP-3 and spindle checkpoint proteins to observe the chromosome dynamics.Nup153 is reported to regulate the activity of spindle assembly checkpoint.Overall,we conclude that NPP-3 and NPP-7 function in the same pathway to regu late the organization of chromosomes.So we will further explore the relationship between NPP-3,NPP-7 and spindle assembly checkpoint proteins.Our spindle assembly checkpoint results showed that co-depletion of NPP-3 and MAD-1,MDF-2,and BUB-3 can rescue most of the nucleus with chromosomal abnormalities;depletion of NPP-3 and other non-related spindle assembly checkpoint kinesin does not affect the chromosomal distribution,which proves that NPP-3 regulates the spatial distribution of chromosomes by affecting the spindle assembly checkpoint.Our next step is to explore how NPP-3 regulates spindle assembly checkpoints.Our results suggest that depletion of NPP-3 and NPP-7 prolongs the localization of CDK-1 in the nucleus,but since CDK-1 is a cyclin-dependent kinase,its depletion will severely affect cell division.The first cell division embryo could not be found.Most of the embryos showed a phenotype with only one large nucleus in the embryo,so we also need to design other experiments to verify wheth er the duration of CDK-1 is the cause of chromosomes accumulating around the nuclear membrane.In addition,CDK-1 is a protein kinase that phosphorylates some proteins,and the activation of the spindle assembly checkpoint is controlled by the level of phosphorylation of spindle assembly checkpoint proteins.While CDK-1 and PLK-1 mainly regulate nuclear membrane rupture,but there is no report on the interaction between CDK-1 and MDF-1,we will also explore the interaction between CDK-1 and MDF-1,and then improve Our guess model.For the further studies,we can use different RNA inference in transgenic strains and the spinning disk confocal microscope to investigate NPP-3 function in chromosome movement.We will design transgenic strains of GFP::NPP-3 and NPP-3::GFP by Crisper Cas9 to investigate the dynamics of NPP-3 in the cell cycle.Then,we can offer a clearer understanding of chromosome periphery and the meaning of this behavior.This study provides theoretical support for medical practice and basis fo r further application.更多还原