細(xì)胞生物學(xué)：chap8細(xì)胞核

1、Chapter 8 Cell NucleusHistoryRobert Brown1773-1858u The first discoverer is Felice Fontana (1730-1805). In 1781, he described the American eel skin epidermal cells of the nucleus is a circular structure .uScottish botanist Robert Brown is the first person to record the nucleus in 1831.Suggested the

2、nucleus played a key role in fertilization and development of the embryo in plants. Name (nucleus) derived from the Latin word for kernel/nut.Introduction of nucleusuThe nucleus is the most obvious organelle in any eukaryotic cell. uThe nucleus is the control center of the cell. Nucleus is the stora

3、ge of the genetic message of the cell in which the DNA replication, transcription occurs.1. The significance of nucleus There are three main types of instructions the nucleus does. 2. The evolutionary sense of nucleus uTo realize the leap from prokaryotic cells to eukaryotic cells.3. Structure of th

4、e nucleusThe Structure of the nucleus present a the periodic change with cell proliferation. nuclear envelope: two lipid membranes nucleolus: the center for rRNA synthesis, and the prominent structure. chromatin: the genetic material of the cell nuclear lamina: nuclear matrix:Interphase nucleus 核被膜核

5、被膜染色質(zhì)染色質(zhì)核仁核仁核基質(zhì)核基質(zhì)核纖層核纖層lGenerally, nucleus is spherical and centrally located in the cell. lIts volume is about 10% of that of the cell and its diameter is 5-10um(animal cell). lUsually each cell has a single nucleus, whereas some cells such as osteoclasts possess several nuclei. Still other cells

6、as red blood cells have extruded their nuclei. 4. Location,amount and shape of the nucleus110nuclear-cytoplasmic ratio(核質(zhì)比核質(zhì)比): 表示核質(zhì)體積關(guān)系表示核質(zhì)體積關(guān)系 NP=Vn/(VcVn) 一般胚胎細(xì)胞、腫瘤細(xì)胞核質(zhì)比大，衰老細(xì)一般胚胎細(xì)胞、腫瘤細(xì)胞核質(zhì)比大，衰老細(xì)胞、角質(zhì)化細(xì)胞核質(zhì)比小。胞、角質(zhì)化細(xì)胞核質(zhì)比小。HepG2細(xì)胞細(xì)胞 11人血細(xì)胞人血細(xì)胞小鼠精細(xì)胞小鼠精細(xì)胞 Liver cells have large, central nuclei (X500,Para

7、rosanilinetoluidine blue)大鼠骨骼大鼠骨骼肌細(xì)胞肌細(xì)胞112(1) A typical nonmitotic nucleus includes several major components.(2) Nuclear envelope and nuclear pore complex.(3) The components of chromatin and packaging of chromosome.(4) Nucleolus.Learning ObjectivesSection 1 nuclear membranenuclear membrane or nuclea

8、r envelopeuDouble-membrane nuclear envelope surrounds the nucleus.uTwo nuclear membrane at as a barrier that separates the contents of the nucleus from the cytoplasm. The nuclear membrane helps control the movement of macromolecules between the nucleus and the cytoplasm and assist in organizing the

9、chromatin.uThe nuclear membrane is perforated at various intervals by nuclear pores, which permit the communication between the cytoplasm and the nucleus.Part 1 chemical constitution of nuclear membrane n protein：6575 G6PD、NADH細(xì)胞色素細(xì)胞色素c還原酶、還原酶、NADH細(xì)胞色素細(xì)胞色素b5還原酶還原酶n lipid：卵磷脂、磷脂酰乙醇胺；膽固醇、三酰甘油卵磷脂、磷脂酰乙醇

10、胺；膽固醇、三酰甘油 n nucleic acid ：a fewProve that closely contact with RER chemical composition Outside the nuclear membrane surface with ribosomes.Part 2 Structure of nuclear membrane Two-membrane structurelThe nuclear envelope has two membranes, each with the typical unit membrane structure. lThey enclos

11、e a flattened sac and are connected at the nuclear pore sites. structure of nuclear envelope Outer nuclear membrane Inner nuclear membrane Perinuclear space Nuclear pore comlex1. Outer nuclear membraneuThe outer nuclear membrane faces the cytoplasm. uIt is continuous with the rough endoplasmic retic

12、ulum. uIts cytoplasmic surface usually possesses ribosomes actively synthesizing transmembrane proteins.uIts cytoplasmic surface is surrounded by a thin loose meshwork of intermediate filaments, vimentin.u The inner nuclear membrane faces the nuclear contents. uIt is in close contact with the nuclea

13、r lamina, an interwoven meshwork of intermediate filaments, 80-100nm thick. uThe nuclear lamina help to organize and provide support to the bilayer nuclear membranes and the perinuclear chromatin. 2. Inner nuclear membraneuperinuclear space -the space between the outer and inner membranes 2040nm con

14、tinuous with RER space.3. Perinuclear space u Found by H.G.Callan S.G.Tomlin in 1949.u Named by M.L.Waston in 1959.u Nuclear pores are formed at sites where the inner and outer membranes of the nuclear envelope are joined, leaving a space filled with filamentous material.4. Nuclear pore lPore size i

15、s variable according to the cell types and tissue. In general 40 - 100 nmlNumbers of pores in given arealow in cells with slow metabolism and at times of low activity during cell cycle.high in cells after cell division and with higher activity of RNA transport and protein synthesis.mammalia actived

16、cell 30004000/one nucleus， xenopus laevis oocyte 60/m2, mature erythrocyte 1-3/m2 .(1)The overviews of nuclear pore complexNuclear pore density of numbers of the nuclear pores per nucleusNuclear pore complex (NPC)An electron micrograph obtained by freeze-fractured cell shows the two layers of the nu

17、clear envelope and nuclear pores. The fracture plane occurs partly between the two nuclear envelope membranes (left) but mostly just inside the envelope with the chromatin removed. The size and distribution of the nuclear pore complexes are clearly seen. X60,000.24 Cytoplasmic ring nucleoplasmic rin

18、g Spoke column subunit luminal subunit annular subunit Central plug (2) Structure of nuclear pore complexFish-trap model Goldberg put forward in 1992.Its thought to be composed of four elements: Cytoplasmic ringlBecause it face to cytoplasm, it was also called outer ring. There are 8 symmetric thick

19、 filaments distribute on the ring. It is suggested that these filaments may act as a staging area for the binding of the proteins that are to be transported into the nucleus. nuclear ring l It face to the inner of the nucleus, and was also called inner ring. Nuclear ring was more complex than outer

20、ring. There are also 8 filaments extend inside to 50-70nm. It formed a little ring on the bottom of the filaments which composed of 8 particles. Thus the whole nuclear ring looks like a fish trap. So some people called it muclear basket. Spoke l The spoke fixed on the nuclear membrane and face to th

21、e center of the nuclear pore. It is also symmetric and complex. In detail, spoke may part into three subunit. column subunit: Distribute at the edge of nuclear pore. It connect the outer and inner ring and support the whole nuclear pore. luminal subunit: The domain which linked the nuclear membrane

22、was called luminal subunit. It perforate the nuclear membrane and extend into the perinuclear cisterna. annular subunit is composed of 8 particles and formed a channel to exchange substances. Central plug lThe particle that lied in the center of the nuclear pore. It was also termed central granule.

23、The current understanding is that the transporter functions in transport of material into and out of the nucleus. lBut not all the nuclear pores could be seen this structure. So some scientists think that central plug is not a structure part of nuclear pore but a particle that just being transported

24、 through the nuclear pore. l35 nm in diameter, transporter l The NPC is a huge, supermolecular complex (125106). Depending on the species, NPCs contain anywhere from 30 to 50 or more different proteins, including transmembrane glycoproteins. Gp210: Structural transmembrane proteingp210 with N-Man ol

25、igosaccharide may play a role in anchoring-NPC in the membrane and promoting fusion of both inner and outer nuclear membranes.p62 (Nup155): functional protein of NPC two functional domain located on the nuclear surface and cytoplasmic face in form of central granules.Nup214,Nup358: cytoplasmic ring

26、filaments31(3) Component of nuclear pore complex32lUnderlying the inner nuclear membrane is the nuclear lamina.lThe nuclear lamina is a dense (30 to 100 nm thick) fibrillar network inside the nucleus of a eukaryotic cell. It is composed of intermediate filaments and membrane associated proteins.5. n

27、uclear lamina(a) Bound to the inner membrane of the nuclear envelope is the nuclear lamina, a meshwork assembled from lamins (class V intermediate filament proteins). Nuclear pore complexes contain more than 30 core proteins (nucleoporins), span both membranes of the nuclear envelope, and regulate t

28、he bidirectional transfer of macromolecular complexes between the nucleus and cytoplasm.(b) Scanning EM of the inner nuclear membrane (nucleoplasmic face) showing portions of the nuclear lamina (NL) meshwork with many embedded nuclear pore complexes (NPC). The preparation is from an actively growing

29、 amphibian oocyte. Nuclei of these very large cells can be isolated manually, facilitating ultrastructural studies of the nuclear envelope. X100,000.(Used, with permission, of Dr M.W. Goldberg, Department of Biological and Biomedical Sciences, Durham University, UK.)35內(nèi)核膜內(nèi)核膜外核膜外核膜染色質(zhì)染色質(zhì)核纖層核纖層Composi

30、tion of nuclear lamina providing structural support to the nucleus by binding to inner nuclear membrane proteins Functional of nuclear lamina 3839Playing a role in nucleus assembly and disassembly before and after mitosisuBreakdown of the nuclear membraneStep 1: lamins are phosphorylated Step 2: the

31、 disassembly of the lamina Step 3: the nuclear envelope to break up into vesicles. uDephosphorylation allows the nucleus to reform.The B-type lamins remain bound to these vesicles lamins A and C are released as free dimers. 41Nuclear lamina disassembly in M phaseServe as a site of chromatin attachme

32、nt Chromatin organizationServe as a site of DNA replicationPart 3functions of nuclear membrane 1. acts as a barrier for gene expression in time and spaceThe function of the cytoplasm and nucleus can carry out respectively.2. Involved in protein synthesis3. Nuclear pore transportnThe nuclear pore com

33、plex（NPC） is an extremely large structure with a diameter of 120nm and an estimated molecular mass of 125 million daltons-about 30 times the size of a ribosome. 145u Due to the structural conformation of the subunits of the nuclear pore complex, there are several 9-11nm wide channels available for s

34、imple diffusion for ions and small molecules. u Substances larger than 11nm are unable to reach or leave the nucleus via simple diffusion; instead they are selectively transported via a receptor-mediated transport process.The characteristics of transport material through NPC below 5,000 MW are freel

35、y diffusable3000-4000 NPC/cell(mammalian); To import about 106 histone/3 mins. (DNA-sythesizing cell) = 100 histone/ min/NPC46 Passive transportpassively diffuseEach NPC contains one or more open aqueous channels: 9 nm in diameter and 15 nm longThe effective size of these channels has been determine

36、d by injecting various sizes of colloidal gold particles and examined by electron microscopy.10 nm in diameter60kd globular proteinAble to enter the nucleus MW 17000- 44000 Da The diameter of NPC can be adjusted （直徑（直徑9nm,可擴(kuò)張至可擴(kuò)張至26nm）;Transport process embodied in signal recognition and receptor-me

37、diated, and consumed energy;Two-way transportation （mRNA;組蛋白組蛋白,DNA復(fù)制酶等）復(fù)制酶等） 核定位信號(hào)，核定位信號(hào)，5m7G48 Active transport 149Molecules enter and exit the nucleus through nuclear pore complexBidirectional trafficNuclear protein transport mechanismbasic definition unuclear proteinunuclear localization signals

38、 ,NLS nuclear export signals, NESuImportin exportin (1) Nuclear import of karyophilic protein親核蛋白與核定位信號(hào)親核蛋白與核定位信號(hào)親核蛋白（親核蛋白（karyophilic protein）：）：指在細(xì)胞質(zhì)中合成后，指在細(xì)胞質(zhì)中合成后，需要或能夠通過(guò)核孔進(jìn)入細(xì)胞核內(nèi)發(fā)揮功能的一類蛋白質(zhì)。需要或能夠通過(guò)核孔進(jìn)入細(xì)胞核內(nèi)發(fā)揮功能的一類蛋白質(zhì)。核定位信號(hào)核定位信號(hào)(nuclear localization signal，NLS):存在存在于親核蛋白內(nèi)的一些短的氨基酸序列于親核蛋白內(nèi)的一些短的氨基酸序列片段

39、片段（含（含48個(gè)個(gè)aa序列）序列），富含堿性氨基酸殘基，如，富含堿性氨基酸殘基，如LysLys、ArgArg，此外還常含有，此外還常含有ProPro。NLSNLS的氨基酸殘基片段可以是一段連續(xù)的序列（的氨基酸殘基片段可以是一段連續(xù)的序列（T T抗原），也抗原），也可以分成兩段，兩段之間間隔約可以分成兩段，兩段之間間隔約1010個(gè)氨基酸殘基（核質(zhì)蛋個(gè)氨基酸殘基（核質(zhì)蛋白）白）。NLSNLS序列可存在于親核蛋白的不同部位，在指導(dǎo)完成核輸入序列可存在于親核蛋白的不同部位，在指導(dǎo)完成核輸入后并不被切除后并不被切除。NLSNLS只是親核蛋白入只是親核蛋白入核的必要條件而非核的必要條件而非充分條件。充分

40、條件。15354Nuclear Localization Signals(NLS)basic or classic NLS55核轉(zhuǎn)運(yùn)受體核轉(zhuǎn)運(yùn)受體(nuclear transport receptor) 位于核孔復(fù)合體上的一些可溶性的蛋白質(zhì)和位于核孔復(fù)合體上的一些可溶性的蛋白質(zhì)和RNA-RNA-蛋白質(zhì)復(fù)合物（蛋白質(zhì)復(fù)合物（RNPRNP），相對(duì)分子質(zhì)量在），相對(duì)分子質(zhì)量在9000090000130000130000之間，屬于酸性蛋白。在被轉(zhuǎn)運(yùn)的之間，屬于酸性蛋白。在被轉(zhuǎn)運(yùn)的親核蛋白上具有核轉(zhuǎn)運(yùn)受體的識(shí)別位點(diǎn)而實(shí)現(xiàn)跨親核蛋白上具有核轉(zhuǎn)運(yùn)受體的識(shí)別位點(diǎn)而實(shí)現(xiàn)跨核膜運(yùn)輸。而被轉(zhuǎn)運(yùn)親核蛋白上的核轉(zhuǎn)運(yùn)受體

41、識(shí)核膜運(yùn)輸。而被轉(zhuǎn)運(yùn)親核蛋白上的核轉(zhuǎn)運(yùn)受體識(shí)別位點(diǎn)就稱為別位點(diǎn)就稱為核定位信號(hào)。核定位信號(hào)。 包括：入核素包括：入核素+ +轉(zhuǎn)運(yùn)素（胞質(zhì)中）轉(zhuǎn)運(yùn)素（胞質(zhì)中） 出核素（胞核中）出核素（胞核中）56 e.g. nuclear import of nucleoplasmin(核質(zhì)蛋白核質(zhì)蛋白) MW 165103, a pentamer protein實(shí)驗(yàn)結(jié)果表明有某種親核的實(shí)驗(yàn)結(jié)果表明有某種親核的輸入信號(hào)存在于尾部片段。輸入信號(hào)存在于尾部片段。57Transport of large proteins into nucleus needs nuclear localization signal (

42、NLS)Imported many types of proteins DNA polymerase, RNA polymerase and other proteins are imported through nuclear pore.vNuclear import and export Nuclear import receptors bind NLS and Nucleoporins The Ran GTPase drives directional transport through NPCThe compartmentalization of Ran-GDP and Ran-GTP

43、.鳥嘌呤核苷酸交換因子RanGEFGTPase激活蛋白R(shí)anGAPA model for how GTP hydrolysis by Ran provides directionality for nuclear transport親核蛋白質(zhì)從細(xì)胞質(zhì)向細(xì)胞核轉(zhuǎn)運(yùn)的過(guò)程示意圖親核蛋白質(zhì)從細(xì)胞質(zhì)向細(xì)胞核轉(zhuǎn)運(yùn)的過(guò)程示意圖（參考（參考G.Karp，2002）61(2) Nuclear export of RNA and ribosomal subunit The export of mRNAs, rRNAs and tRNAs from the nucleus to the cytoplasm is

44、a critical step in gene expression in eukaryitic cells. The export of RNAs through NPC is also active, energy-dependent process that requires the Ran-binding protein.Nuclear export works like nuclear import, but in reverse hnRNP proteins contain a nuclear-export signal (NES) Nuclear Export Signal (N

45、ES,核輸出信號(hào)核輸出信號(hào))：RNA分分子的出核轉(zhuǎn)運(yùn)需要蛋白分子的幫助，這些蛋白因子本身子的出核轉(zhuǎn)運(yùn)需要蛋白分子的幫助，這些蛋白因子本身含有出核信號(hào)。含有出核信號(hào)。 入核轉(zhuǎn)運(yùn)與出核轉(zhuǎn)運(yùn)之間有某種聯(lián)系，它們需要某入核轉(zhuǎn)運(yùn)與出核轉(zhuǎn)運(yùn)之間有某種聯(lián)系，它們需要某些共同的因子些共同的因子(Ran-GDP and Ran-GTP)。63*RNA聚合酶聚合酶I 轉(zhuǎn)錄的轉(zhuǎn)錄的rRNA分子：以分子：以RNP的形式離開(kāi)細(xì)胞核，的形式離開(kāi)細(xì)胞核，需要能量；需要能量；*RNA聚合酶聚合酶III 轉(zhuǎn)錄的轉(zhuǎn)錄的5s rRNA與與tRNA的核輸出由蛋白質(zhì)介導(dǎo)；的核輸出由蛋白質(zhì)介導(dǎo)；*RNA 聚合酶聚合酶II 轉(zhuǎn)錄的轉(zhuǎn)錄的h

46、nRNA，在核內(nèi)進(jìn)行，在核內(nèi)進(jìn)行5端加帽和端加帽和3端附端附加多聚加多聚A序列以及剪接等加工過(guò)程，然后形成成熟的序列以及剪接等加工過(guò)程，然后形成成熟的mRNA出出核，核，5端的端的m7GpppG“帽子帽子”結(jié)構(gòu)對(duì)結(jié)構(gòu)對(duì)mRNA的出核轉(zhuǎn)運(yùn)是必要的出核轉(zhuǎn)運(yùn)是必要的；的；*mRNA的出核轉(zhuǎn)運(yùn)過(guò)程是的出核轉(zhuǎn)運(yùn)過(guò)程是有極性有極性的，其的，其5端在前，端在前，3端在后。端在后。*細(xì)胞核中既有正調(diào)控信號(hào)保證細(xì)胞核中既有正調(diào)控信號(hào)保證mRNA的出核轉(zhuǎn)運(yùn)，也有負(fù)調(diào)控的出核轉(zhuǎn)運(yùn)，也有負(fù)調(diào)控信號(hào)防止信號(hào)防止mRNA的前體被錯(cuò)誤地運(yùn)輸，后者與剪接體有關(guān)。的前體被錯(cuò)誤地運(yùn)輸，后者與剪接體有關(guān)。65Transport o

47、f mRNA through a poreStructure of the nucleus:nuclear envelopechromatinnucleolusnuclear matrix68Section 2 Chromatin and Chromosome1.Walther Flemming named chromatin (strongly absorbed basophilic dyes) in 1879. Flemming surmised for the first time that all cell nuclei came from another predecessor nu

48、cleus2.Wilhelm von Waldeyer-Hartz Named the chromosomes (meaning coloured body) in 1888HistoryChromatin:What is chromatin and chromosome? Chromosome: Part 1 chemical constitution of chromatin and chromosomeHistones Nonhistones731. DNA is the carrier of genetic information The type of chromosome DNA

49、sequence unique sequence：約占人類基因組的約占人類基因組的 60 65 repetitive sequence： middle repetitive sequence ：拷貝數(shù)拷貝數(shù)101 104, 20 30 在物種進(jìn)化過(guò)程中是基因組中可移動(dòng)的遺傳元件，在物種進(jìn)化過(guò)程中是基因組中可移動(dòng)的遺傳元件， 并且影響基因表達(dá)。并且影響基因表達(dá)。 highly repetitive sequence ：拷貝數(shù)大于拷貝數(shù)大于 105，10 主要分布在染色體著絲粒、端粒部位主要分布在染色體著絲粒、端粒部位74u Replication origin sequenceu Centrom

50、ere sequenceu Telomere sequenceThree important DNA sequences of chromatinThe histones are small proteins containing a high proportion of basic amino acids with positive charge that facilitate binding to the negatively charged DNA molecule. Histones are Rich in Lysine and ArginineThere are two classe

51、s of histones:Nucleosomal histones H2A, H2B, H3, and H4 which are highly conserved.H1-like Histone VariableuHistones are bound with DNA in a non-specific way.uThese proteins can inhibit the replication and transcription of DNA. 2. HistonesStructural Organization of the Core Histonesl This DNA-associ

52、ated proteins bind DNA in a specific way, therefore named sequence specific DNA binding protein, which can form several specific structures and may also involved in the regulation of DNA activities.l acidic protein Species and tissue specificity l Functions: structure, regulation,enzyme (phosphoryla

53、tion)3. NonhistonesNonhistonesPart 2 Euchromatin and HeterochromatinEuchromatin: scattered throughout the nucleus and not visible with light microscope, which is the active form of chromatin where the genetic material of the DNA molecules is being transcribed into RNA.1. Euchromatinuit is visible un

54、der light microscope, which located mostly at the periphery of the nucleus but also forms irregular clumps throughout the nucleus. It is condensed inactive form which means it is not active in RNA synthesis. It can be divided into two types: Constitutive heterochromatin Facultative heterochromatin D

55、ark-staining, condensed chromatin; No transcriptional activity; There are position effects; in a typical mammalian cell, approximately 10% of the genome is packaged into heterochromatins forming CEN and TEL Divided into two classes: Constitutive & facultativeCompacted state at all time: Centrome

56、reInactivated at certain phase of life heterochromatin(1) Constitutive heterochromatin Except for replication, this type of heterochromatin is always in a condensed inactive form during the whole cell cycle.(2) Facultative heterochromatin Barr Body（ X Chromosome ）: microscopic study of interphase nu

57、clei of cells from female displays a very tightly coiled clump of chromatin, the sex chromatin (Barr Body), the inactive counterpart of the two X Chromosomes. It is a facultative heterochromatin, which is one of the two X chromosomes becoming condensed randomly at embryonic 16 days of female.X chrom

58、osome inactivation in mammalsXYDosage compensationXXXPart 3Chromatin packaging into chromosomes88 Each human cell contains about 1.74m of DNA within nucleus if stretched end-to-end, yet the nucleus of a human cell itself is only about 6 um in diameter. Compaction ratio=nearly 10000-fold. From DNA to

59、 chromosome needs four-step packaging. The basic Unit of ChromatinThe first step of condensation, from 2 nm to 11nm. 1. Nucleosome structure of nucleosomelnucleosome core particle Core DNA (146bp，1.75 turns ) a histone octamer ( two molecules each of H2A, H2B, H3, and H4)lLinker segment DNA(60bp) ：l

60、inker DNA extending to the next “bead” H1 histone is bound to the DNA as it enters each nucleosome core particle. l diameter: 11nm, packaging of DNA shortens its sixfold. 核心：核心： (H2A,H2BH3,H4)2 構(gòu)成八聚體構(gòu)成八聚體DNA繞八聚體繞八聚體1.75周（周（146bp)核心核心顆粒顆粒DNA雙鏈片斷（約雙鏈片斷（約60bp)， H1組蛋白與該組蛋白與該DNA鏈結(jié)合鏈結(jié)合連接部連接部9293Evidence: (1)