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1、细胞核与染色质,王飞,内容提要,基本结构及主要功能元件,1,核内分子生物学过程概述,2,核孔复合体及其介导的物质运输,3,核膜的崩解与组装,4,核仁及其他核内结构的组成与功能,5,细胞核基本结构,核被膜 核基质 染色质 核仁 核体,核被膜的结构组成,核被膜(nuclear envelope),核膜(nuclear membrane),外核膜 核糖体颗粒 与内质网相连 内核膜 无核糖体颗粒 LBR核周间隙 与内质网腔相通核孔 内外膜融合 孔膜区,核孔复合体(NPC),孔膜区域组装的复杂蛋白复合物 核质物质运输,核纤层蛋白(lamin A-C)纤维状网络结构结构支撑 调节基因表达 调节DNA损伤修
2、复,内核膜 NPC 核周染色质,核纤层(nuclear lamina),分裂期 核被膜的崩解与组装,核基质,核骨架蛋白质纤维网架结构体系真核生物细胞核起源于原核细胞入侵？融合？,细胞生物学：不同染色质形态的核内分布 核周异染色质袢环结构(loop)染色体区域化分布,染色质,核仁,核仁：染色体的核仁组织区 RNA 蛋白 核糖体工厂,核体：功能元件 RNP集中区域 非编码RNA 蛋白 RNA修饰 RNP组装 核糖体装配,核体,核内分子生物学过程,染色质结构调节DNA复制、修复、重组基因转录及其调控RNA 加工,短片：chromosome coil,In this animation well se
3、e the way our DNA is tightly packed up to fit into the nucleus of every cell.The process starts with assembly of a nucleosome,which is formed when eight separate histone protein subunits attach to the DNA molecule.The combined tight loop of DNA and protein is the nucleosome.Six nucleosomes are coile
4、d together and these then stack on top of each other.The end result is a fiber of packed nucleosomes known as chromatin.,短片：DNA replication,Using computer animation based on molecular research,we are able to picture how DNA is replicated in living cells.You are looking at an assembly line of amazing
5、 miniature biochemical machines that are pulling apart the DNA double helix and cranking out a copy of each strand.The DNA to be copied enters the production line from bottom left.The whirling blue molecular machine is called a helicase.It spins the DNA as fast as a jet engine as it unwinds the doub
6、le helix into two strands.One strand is copied continuously and can be seen spooling off to the right.Things are not so simple for the other strand because it must be copied backwards.It is drawn out repeatedly in loops,and copied one section at a time.The end result is two new DNA molecules.,短片：str
7、and exchange,Cross-strand exchange is an important intermediate structure in general DNA recombination.The characteristic junction is formed when two homologous double-stranded DNA molecules reciprocally exchange DNA strands.This junction can be visualized directly in the electron microscope.An impo
8、rtant aspect of recombination is that the junction point is not fixed after it has formed,but can migrate up and down the DNA as shown in this animation.In the cell,this junction is formed and stabilized by a specific protein,which can be seen here in the background.,短片：gene transcription,Transcript
9、ion is the process by which DNA is copied into RNA in the first step of gene expression.It begins with a bundle of factors assembling at the start of a gene,that is,a linear sequence of DNA instructions,here shown stretching away to the left.The assembled factors include an RNA polymerase,the blue m
10、olecule.Suddenly,RNA polymerase is let go,racing along the DNA to read the gene.As it unzips the double helix,it copies one of the two strands.The yellow chain snaking out of the top is the RNA,a copy of the genetic message.The nucleotide building blocks that are used to make the RNA enter through a
11、n intake hole in the polymerase.In the active site of the enzyme,they are then matched to the DNA,nucleotide by nucleotide,to copy the As,Cs,Ts and Gs of the gene.The only difference is that in the RNA copy,thymine is replaced with the closely related base uracil,commonly abbreviated“U”.You are watc
12、hing this process,called transcription,in real time.,核孔复合体-核质转运通道,跨内外核膜 亲水通道核孔直径 80-120nm 复合体直径120-150nm自由扩散 顺浓度梯度 有效直径 9-10nm 40-60KD 无需能量主动运输 信号肽 需能量 不去折叠双向同时运输 转运速度极快（500个/s）,短片：Rapid nuclear importation,Nuclear import and export can be directly visualized in living cells that express the green f
13、luorescent protein GFP fused to the gene regulatory protein NF-AT.NF-AT is normally localized in the cytosol,and excluded from the nucleus.But when the cytosolic calcium concentration is raised,NF-AT migrates to the nucleus.This is done here experimentally by adding an ionophore that allows calcium
14、to enter the cells from the medium.Upon removal of the ionophore,calcium levels return to normal and NF-AT is exported from the nucleus.Readdition of the ionophore triggers reimport of NF-AT.,核孔复合体的结构,30余种蛋白亚基 多拷贝组合 八边形对称结构,柱状亚基环带亚基腔内亚基环状亚基蛋白纤维核篮,核孔复合体的结构,核孔复合体转运的分子尺寸限制,无定形结构域(unstructured region)无序
15、屏障,主动运输与信号肽,蛋白分选：信号肽 核转运信号(NTS)核分选：核定位信号(NLS)核输出信号(NES)任何位置 完成分选后不剪切,核孔复合体转运的生物大分子,NTS/向导蛋白转入(Import)组蛋白 DNA聚合酶 RNA聚合酶 RNA加工相关蛋白 转出(Export)tRNA mRNA 核糖体前体颗粒 转入&转出 基因转录调控蛋白（转录因子）RNA向导蛋白,主动运输模型,通道：NPC 出入口：蛋白纤维 搬运工：核转运受体(nuclear transport receptor)辅助：FG-repeats Ran GTP,两类核转运受体(NTR)入核转运受体(NIR)识别NLS 蛋白入核
16、 出核转运受体(NER)识别NES 蛋白出核,直接结合：多种NTR 识别不同NTS 间接结合：适配蛋白(adaptor protein),无信号肽大分子“搭车”：RNA 蛋白质,核转运受体对靶蛋白的识别,GTPase：Ran,导航：控制转运方向 调控因子：Ran-GAP Ran-GEF,Ran控制的NTR分子开关,NPC主动运输的分子模型,RNA的出核转运,向导分子：具有NES的辅助蛋白质量检查：正确折叠和加工的RNA分子才能 获得出核资格,核膜的崩解与组装-骨架章交叉,发生于细胞分裂过程中触发点：磷酸化 p34cdc2 N端Ser22 C端Ser392 Lamin A 单体形式游离 Lami
17、n B 与核膜小泡结合,核膜的崩解与组装,核膜的崩解与组装,1 核孔复合体和核被膜片段如何找到染色体？2 游离到胞质中的核蛋白如何回到核内？3 如何避免不属于核内的胞质蛋白在核膜组装时混入核内？,找到染色体,前期核膜崩解后 游离于胞质内：核膜小泡(双层核膜+膜蛋白)NPC亚基蛋白+NIR FG repeats Lamin Ran Ran-GAP 末期染色体上：Ran-GEF Ran-GTP Cloud Lamin去磷酸化启动核膜重组：核膜小泡 Lamin NPC蛋白+NIR NPC+NIR Ran-GTP Cloud,核蛋白回归及非核蛋白屏蔽,NPC 组装完毕即开始工作：入核转运NLS不剪切的
18、生理意义：反复利用核膜组装紧贴染色体：屏蔽非核蛋白,无意义转运 核蛋白回归,核仁-Ribosome-producing factory,Unlike many of the major organelles in the cell,the nucleolus is not bound by a membrane,it is a large aggregate of macromolecules.染色质（rRNA genes）前体rRNA 成熟rRNA rRNA加工酶 向导RNA（snoRNP）核糖体蛋白 核糖体亚单位前体颗粒 核骨架,染色质的核仁组织区,核仁组织区袢环DNA 核仁主体结构,染色质的核仁组织区,核糖体加工过程,rRNA基因转录前体rRNA加工成熟rRNA组装核糖体亚单位前体颗粒出核,核仁的电镜结构,纤维中心：rRNA基因区域 rRNA基因转录致密纤维组分：snoRNP 前体rRNA加工颗粒组分：核糖体亚单位前体颗粒组装及出核转运,核体,了解最少的核内亚结构 高度动态性特殊蛋白或RNA聚集的区域 RNA工具箱Cajal body&GEMS 染色质间颗粒 snoRNP snRNP,核体-RNA工具箱,Thank You!,
