1、Chapter 9,Cell-Cycle and Cell Division,Overview of the cell cycle Regulation of the cell cycle Cell division,Cell-Cycle and Cell Division,根據(jù)增殖狀況，細(xì)胞分類三類,連續(xù)分裂細(xì)胞(cycling cell) 休眠細(xì)胞(Go細(xì)胞) 終末分化細(xì)胞 G0期細(xì)胞和終末分化細(xì)胞的界限有時難以劃 分，有的細(xì)胞過去認(rèn)為屬于終末分化細(xì)胞，目前 可能被認(rèn)為是G0期細(xì)胞。,Overview of the cell cycle,cell growth,DNA replicatio

2、n,distribution of the duplicated chromosomes,cell division,The cell cycle can be divided into two major phases based on cellular activities, interphase and M phase.,M phase includes mitosis and cytokinesis.,Interphase is the interval between divisionsis divided into G1, S and G2 phases.,so the cell

3、cycle includes four phases: G1, S, G2 and M phases.,Analysis of a large variety of cells has revealed great variability in the lengths of their cell cycle.,The four stages of cell cycle, G1 is the most variable. While the total time of S, G2 and M phases is relatively fixed.,It is the cell-cycle con

4、trol system that ensures correct progression through the cell cycle by regulating the cell-cycle machinery.,The progression of cells through the division cycle is regulated by extracellular signals from the environment and internal signals.,Cell cycle control Cell Cycle Checkpoints Cyclins Progressi

5、on regulatory of cell cycle,Regulation of The cell cycle,A major cell cycle regulatory point in many types of cells occurs late in G1 and controls progression from G1 to S.,In addition to serving as a decision point for monitoring extracellular signals, START is the point at which cell growth is coo

6、rdinated with DNA replication and cell division.,The proliferation of most animal cells is similarly regulated in the G1 phase of the cell cycle. In particular, a decision point in late G1, called the restriction point in animal cells.,The proliferation of these cells is triggered by platelet-derive

7、d growth factor, which is released from blood platelets during clotting and signals the proliferation of fibroblasts（纖維原細(xì)胞） in the vicinity of the injured tissue.,Cell Cycle Checkpoints,In most cells, the coordination between different phases of the cell cycle is dependent on a system of checkpoints

8、 and feedback controls that prevent entry into the next phase of the cell cycle until the events of the preceding phase have been completed.,At least, there are three major checkpoints exist within the cell cycle, G1/S checkpoint , G2/M checkpoint and M checkpoint .,The G1/S checkpoint monitors the

9、cell size achieved following the previous mitosis, and whether the DNA has been damaged.,The second checkpoint is the G2/M checkpoint, where physiological conditions in the cell are monitored prior to entering mitosis. It prevents the initiation of mitosis until DNA replication is completed.,The fin

10、al checkpoint, M checkpoint, occurs during mitosis. It monitors the alignment of chromosomes on the mitotic spindle, thus ensuring that a complete set of chromosomes is distributed accurately to the daughter cells.,蛋白激酶在細(xì)胞周期調(diào)控中的作用,細(xì)胞融合實驗 研究者：1970年，Colorado 大學(xué)的Potu Rao 和 Robert Johnson 研究思路 研究方法 使用的細(xì)

11、胞系 預(yù)測結(jié)果 早熟染色體凝集(premature chromosome condensation,PCC ）,G1期細(xì)胞與M期細(xì)胞融合,S期細(xì)胞與M期細(xì)胞融合,G2期細(xì)胞與M期細(xì)胞融合,Regulation of The cell cycle,Role of p53 in G1 arrest induced by DNA damage DNA damage, such as that resulting from irradiation, leads to rapid increases in p53 levels. The protein p53 then signals cell cyc

12、le arrest at the G1 checkpoint.,Induction of p21 by DNA damage DNA damage results in the elevation of intracellular levels of p53, which activates transcription of the gene encoding the Cdk inhibitor p21. In addition to inhibiting cell cycle progression by binding to Cdk/cyclin complexes, p21 may di

13、rectly inhibit DNA synthesis by interacting with PCNA (a subunit of DNA polymerase ),MPF的發(fā)現(xiàn) 成熟促進(jìn)因子(maturation promoting factor，MPF）,早期稱為M-期促進(jìn)因子(M-phase promoting factor, MPF)，是指M期細(xì)胞中存在的促進(jìn)細(xì)胞分裂的因子 研究思路 實驗設(shè)計 實驗材料的選擇 實驗方法 結(jié)果預(yù)測 實驗結(jié)果與分析 發(fā)現(xiàn) 推論,卵細(xì)胞提取物注射實驗,Cyclins,A series of experiments on the oocytes and e

14、arly embryos of frogs revealed the existence of factors trigger DNA replication and promote a cell into mitosis or meiosis, and showed that entry of a cell into M phase is initiated by a protein kinase called MPF (maturation promoting factor or M phase-promoting factor ).,MPF is a dimeric kinase, co

15、ntaining a catalytic subunit (是絲氨酸/蘇氨酸型蛋白激酶) and a regulatory subunit (cyclin).,Regulatory subunit triggers the onset of mitosis, while the catalytic subunit transfers phosphate groups from ATP to specific serine(Ser) and threonine (Thr)residues of specific protein substrates.,The regulatory subunit

16、s concentration rises and falls in a predictable pattern as the cell cycle progresses, so called “cyclin”.,The cyclin that helps drive cells into M phase is called M-cyclin, cyclin A, cyclin B etc. Whereas, the cyclin that becomes active toward the end of G1 phase and is responsible for driving the

17、cell into S phase is called G1-cyclin, for example, cyclin C and cyclin D.,Cyclin-Dependent Kinase and its Inhibitors,Progression regulatory of cell cycle,Progression through the fission yeast cell cycle requires the phosphorylation and dephosphorylation of critical cdc2 residues,有絲分裂的退出：周期蛋白B的降解 細(xì)胞

18、周期蛋白基因的cDNA分析 N端破壞框(destruction box),遍在蛋白與周期蛋白的降解 多遍在蛋白化作用(polyubiquitination) 遍在蛋白活化酶(ubiquitin-activating enzyme, E1) 遍在蛋白綴合酶(ubiquitin-conjugating enzyme, E2) 遍在蛋白連接酶(ubiquitin ligase, E3) 蛋白酶體的降解作用 促后期復(fù)合物 (anaphase-promoting complex, APC),Polyubiquitination,APC的活性調(diào)節(jié)控制周期蛋白B的降解 當(dāng)MPF的活性在有絲分裂中期達(dá)到最高峰

19、時, 它將APC磷酸化并將其激活； 接著發(fā)生周期蛋白B遍在蛋白多聚化, 引起周期蛋白B的降解； 由于周期蛋白B是MPF的一個必需亞基, 它的降解勢必導(dǎo)致MPF的失活； 在G1期的后期,APC失活,使得周期蛋白B的濃度升高,同時提高M(jìn)PF的活性, 以便進(jìn)入下一個有絲分裂期。,Regulation of mitotic cyclin levels in cycling cells,真核生物細(xì)胞周期調(diào)控的一般模型 三類周期蛋白-CDK復(fù)合物: G1期周期蛋白-CDK復(fù)合物 S期周期蛋白-CDK復(fù)合物 有絲分裂周期蛋白-CDK復(fù)合物 三個關(guān)鍵的過渡 G1期S期 中期后期 后期末期及胞質(zhì)分裂期過渡,MP

20、F的作用機制,促進(jìn)染色體凝集 H1組蛋白磷酸化 H3組蛋白磷酸化 核纖層磷酸化 核被膜裝配 細(xì)胞相關(guān)的酶與蛋白質(zhì)磷酸化,Targets of MPF MPF induces multiple nuclear and cytoplasmic changes at the onset of M phase, both by activating other protein kinases and by phosphorylating proteins such as condensins and the nuclear lamins.,Histone HI is a substrate for t

21、he Cdc2 protein kinase and is phosphorylated during mitosis of most cells, consistent with its phosphorylation playing a role in mitotic chromosome condensation. However, recent experiments have shown that phosphorylation of histone H1 is not required for chromosome condensation, so the potential ro

22、le of H1 phosphorylation is unclear. In contrast, phosphorylation of histone H3 has been found to be required for condensation of mitotic chromosomes, although the mechanism by which H3 phosphorylation affects chromosome condensation remains to be elucidated.,Chromosome condensation,核纖層的結(jié)構(gòu),MPF與核纖層解體

23、,MPF激活肌球蛋白促進(jìn)胞質(zhì)分裂,Cell division,Mitosis meiosis,Mitosis,Mitosis is a process of nuclear division in which replicated DNA molecules of each chromosome are faithfully partitioned into two nuclei.,Mitosis is conventionally divided into six stages, prophase, prometaphase, metaphase, anaphase, telophase a

24、nd cytokinesis.,Prophase Prometaphase Metaphase Anaphase Telophase Cytokinesis,Prophase,the replicated chromosomes condense the mitotic spindle begins to assemble the dissolution of nuclear envelope the partitioning of cytoplasmic organelles,Formation of the Mitotic Spindle,The Dissolution of Nuclea

25、r Envelope and the Partitioning of Cytoplasmic Organelles,The nuclear membranes are progressively fragmented, initially into flattened, saclike vesicles that surround the condensing chromatin, and eventually into a population of small spherical vesicles that disperse throughout the mitotic cell.,Pro

26、metaphase,The disassembly of the nuclear envelope, which breaks up into small membrane vesicles and dispersed into cytoplasm, marks the start of the second phase of mitosis, prometaphase.,nuclear lamina disassembles mitotic spindle assembly is completed the chromosomes are moved into the center of t

27、he cell,Metaphase,Once all of the chromosomes has become aligned at the spindle equator, halfway between the two spindle poles, thereby forming the metaphase plate (equatorial plate) .,equatorial plate,three types microtubules of the metaphase spindle,Anaphase,Anaphase can be divided into two stages

28、, anaphase A and anaphase B, and they occur more or less simultaneously. In anaphase A, the kinetochore microtubules are shortened by depolymerization, and the attached chromosomes move poleward. In anaphase B, the polar microtubules are gradually elongated, and the two spindle poles move farther ap

29、art.,Anaphase A,Anaphase B,Telophase,Daughter cells return to the interphase condition. Kinetochore microtubules disappear, polar microtubules sequentially elongate. The nuclear envelope reassembles around each group of chromosomes to form the two daughter nuclei.,Cytokinesis,Cytokinesis in plant ce

30、ll,有絲分裂的機理,紡錘體微管類型及形成 紡錘體又稱為有絲分裂器 (mitotic apparatus) Kinetochore microtubules Polar microtubules Astral microtubules 中心粒 中心粒確定分裂極 形成紡錘體,紡錘體微管的類型,中心粒的復(fù)制周期,染色體分離的兩個階段：后期A與后期B 染色體分離的力 拉力：由動粒微管去裝配產(chǎn)生 推力：由極微管的聚合所產(chǎn)生 后期可分為兩個階段 后期A 后期B 力產(chǎn)生的機制： 后期A，微管去聚合假說 后期B，紡錘體微管滑動假說,Anaphase A and Anaphase B,Re-formation

31、 of the Interphase Nucleus,During the completion of mitosis (telophase), two new nuclei form around the separated sets of daughter chromosomes. Chromosome decondensation and reassembly of the nuclear envelope appear to be signaled by inactivation of Cdc2, which was responsible for initiating mitosis

32、 by phosphorylating cellular target proteins, including the lamins, histone H3, and condensins. The progression from metaphase to anaphase involves the activation of a ubiquitin-mediated proteolysis system that inactivates Cdc2 by degrading its regulatory subunit, cyclin B. Inactivation of Cdc2 lead

33、s to the dephosphorylation of the proteins that were phosphorylated at the initiation of mitosis, resulting in exit from mitosis and the re-formation of interphase nuclei.,Re-formation of the Interphase Nucleus,細(xì)胞周期同步化,自然同步化，如有一種粘菌的變形體plasmodia， 某些受精卵早期卵裂 人工選擇同步化 藥物誘導(dǎo)法 條件依賴性突變株在細(xì)胞周期同步化中的應(yīng)用： 將與細(xì)胞周期調(diào)控

34、有關(guān)的條件依賴性突變株轉(zhuǎn)移 到限定條件下培養(yǎng)，所有細(xì)胞便被同步化在細(xì)胞 周期中某一特定時期。,人工選擇同步化,有絲分裂選擇法：用于單層貼壁生長細(xì)胞。優(yōu)點是細(xì) 胞未經(jīng)任何藥物處理，細(xì)胞同步化效率高。缺點是 分離的細(xì)胞數(shù)量少。 密度梯度離心法：根據(jù)不同時期的細(xì)胞在體積和重量 上存在差別進(jìn)行分離。優(yōu)點是方法 簡單省時，效率高， 成本低。缺點是對大多數(shù)種類的細(xì)胞并不適用。,藥物誘導(dǎo)法, DNA合成阻斷法 G1/S-TdR雙阻斷法：最終將 細(xì)胞群阻斷于G1/S交界處。優(yōu)點是同步化效率高， 幾乎適合于所有體外培養(yǎng)的細(xì)胞體系。缺點是誘 導(dǎo)過程可造成細(xì)胞非均衡生長 分裂中期阻斷法：通過抑制微管聚合來抑制細(xì)胞

35、分裂器的形成，將細(xì)胞阻斷在細(xì)胞分裂中期。優(yōu)點 是操作簡便，效率高。缺點是這些藥物的毒性相對 較大,特異的細(xì)胞周期,特異的細(xì)胞周期是指那些特殊的細(xì)胞所具 有的與標(biāo)準(zhǔn)的細(xì)胞周期相比有著鮮明特點的細(xì) 胞周期。 爪蟾早期胚胎細(xì)胞的細(xì)胞周期 酵母細(xì)胞的細(xì)胞周期 植物細(xì)胞的細(xì)胞周期 細(xì)菌的細(xì)胞周期,爪蟾早期胚胎細(xì)胞的細(xì)胞周期,細(xì)胞分裂快,無G1期, G2期非常短,S期也短(所有復(fù) 制子都激活), 以至認(rèn)為僅含有S期和M期 無需臨時合成其它物質(zhì) 子細(xì)胞在G1、G2期并不生長，越分裂體積越小 細(xì)胞周期調(diào)控因子和調(diào)節(jié)機制與一般體細(xì)胞標(biāo)準(zhǔn)的 細(xì)胞周期基本是一致的,酵母細(xì)胞的細(xì)胞周期,酵母細(xì)胞的細(xì)胞周期與標(biāo)準(zhǔn)的細(xì)胞

36、周期在時相和 調(diào)控方面相似 酵母細(xì)胞周期明顯特點:首先，酵母細(xì)胞周期持 續(xù)時間較短；細(xì)胞分裂過程屬于封閉式，即在細(xì) 胞分裂時核膜不解聚；紡錘體位于細(xì)胞核內(nèi)；在 一定環(huán)境下，也進(jìn)行有性繁殖,植物細(xì)胞的細(xì)胞周期,植物細(xì)胞的細(xì)胞周期與動物細(xì)胞的標(biāo)準(zhǔn)細(xì)胞周期 非常相似，含有G1期、S期、G2期和M期四個時期。 植物細(xì)胞不含中心體，但在細(xì)胞分裂時可以正常 組裝紡錘體。 植物細(xì)胞以形成中間板的形式進(jìn)行胞質(zhì)分裂,細(xì)菌的細(xì)胞周期, 慢生長細(xì)菌細(xì)胞周期過程與真核細(xì)胞周期過程有 一定相似之處。其DNA復(fù)制之前的準(zhǔn)備時間與G1期 類似。分裂之前的準(zhǔn)備時間與G2期類似。再加上S 期和M期，細(xì)菌的細(xì)胞周期也基本具備四個

37、時期 細(xì)菌在快速生長情況下，如何協(xié)調(diào)快速分裂和最 基本的DNA復(fù)制速度之間的矛盾,Meiosis,Meiosis is a special kind of cell division, which only occurs at some stage of the sexual cell reproduction. Meiosis involves a single round of DNA replication that duplicates the chromosomes, followed by two successive cell divisions, called meiosisa

38、nd meiosis.,Meiosis,It can be factitiously divided into six stages, prophase, prometaphase, metaphase, anaphase, telophaseand cytokinesis. Prophaseincludes leptotene, zygotene, pachytene, diplotene and diakinesis five stages.,leptotene,The chromosomes condense and become gradually visible in the lig

39、ht microscope, and revealed to be composed of paired chromatids. Near the end of leptotene, there is a dramatic reorganization so that the telomeres become localized at the inner surface of the nuclear envelope at one side of the nucleus.,zygotene,Electron micrographs indicate that chromosome synaps

40、is is accompanied by the formation of a complex structure called the synaptonemal complex (SC). The SC is a ladderlike structure composed of three parallel bars with many transverse filaments connecting the central element with the two lateral elements.,邊側(cè)成份：蛋白質(zhì)和DNA,中央?yún)^(qū),重組節(jié),L-C纖維,pachytene,The end o

41、f synapsis marks the end of zygotene and the beginning of pachytene. During this period, the homologues are held closely together along their length by the SC . Recombination nodules form in this period.,diplotene,Diplotene is recognized by the dissolution of the SC and the tendency of the homologou

42、s chromosomes of the bivalents to pull away somewhat from each other.,diakinesis,During diakinesis the meiotic spindle is assembled, and the chromosomes are prepared for separation. Diakinesis ends with the disappearance of the nucleolus, the breakdown of the nuclear envelope, the terminalization of the crossover, and the movement of the tetrads to the metaphase plate.,Genetic Recombination During Meiosis,在減數(shù)分裂過程中發(fā)生了兩種方式的遺傳重組 同源染色體的部分交換（基因重組） 染色體分離時的自由組合（染色體組重組）。,同源染色體間的交換重組