北京大学细胞生物学课件-11xibaozengzhijiqitk

1、Cell proliferation and its regulationSignificance: 1. For the growth and development of a multicellular organism, and for the generation of offspring; 2. Produce new organisms in unicellular species; 3. Renew the aging, apoptotic cells, and damaged tissue; So, cell proliferation is one of the most i

2、mportant characters for lifeChapter 11If damaged seriously by UV, mouse will die within several days.1. The cell cycleA. Overview of the cell cycleThe most basic function of the cell cycle is to duplicate accurately the vast amount of DNA in the chromosomes and then segregate the copies precisely in

3、to two genetically identical daughter cells.Cell cycle phases:Interphase: G1-S-G2M phase: Mitosis, Cytokinesis Different cell cycle lengthSome eukaryotic cell cycle timesThe greatest variation occurs in the duration of G1The shortest eukaryotic division cycles of all are the early embryonic cell cyc

4、les, no G1 and G2 Biochemical events of cell cycleG1 phase: Synthesize proteins (RNA) for the DNA replication. Uncondense chromatin.S phase: Synthesis of DNA and HistonesG2 phase: Synthesis of a few proteins (RNA)M phase: Mitosis and meiosis and cytokinesisTwo daughter cellsChromosome condenseMitoti

5、c spindleContractile ring Three categories of cells in vivoCycling cells Dividing continuouslyStem cells(2) G0 cells Do not divide normally, but divide when given an appropriate stimulus: liver cells, lymphocytes(3) Terminally Differentiated cells Highly specialized, have lost the ability to divide

6、until they die: muscle cells, red blood cells, nerve cells Embryo cellsCycling cellsG0 cellsTerminal cellsB. Synchronization of cell (for cell population)(1) Natural synchronizationEarly embryo in most invertebrates and a few vertebratesFruit fly embryo(2) Selected synchronization artificiallyIsolat

7、ion of cells in M phase Isolation of cells by centrifugation (3) Synchronization of cells induced by drugsBlocking DNA synthesis by TdR G1/STdRG1SG2MG1/SBlocking cells in metaphase by colchicineAffect the assembly of mitotic spindleSpecial cell cycles Early embryo (30min/cell cycle)No G1 and G2 phas

8、e, all replicons are activated, so S phaseis very short(2) Have little or no need to synthesize components other than DNA, cell division (3) No cell growth during cell cycleFrog Xenopus laevis2. Mitosis(1) ProphaseChromatins condense to form compacted mitotic chromosomesActivated M-Cdk phosphrylates

9、 condensin subunits,triggering the assembly of condensin complexes on DNA and condensation of the chromosome. The condensin can use energy of ATP hydtolysis to promote DNA coiling(in vitro).The sister chromatids are glued together by multisubunit protein complex called cohesins. Centrosome duplicate

10、d at S, and separate to form mitotic spindle at the beginning of prophaseAssembly of kinetochore at the end of prophaseGolgi, ER etc. disperse to form vesicles; kinetochore assemblySchematic representation of the kinetochore(2) PrometaphaseNuclear envelopes breakdown (Lamin phosphorylation)Spindle M

11、Ts capture chromosomes The pushing and pulling forces drive the chromosomes to the metaphase plate .MT behavior during formation of the metaphase plate. Initially,MT from opposite poles are different in length.Experimental demonstration of the importance of mechanical tension in metaphase checkpoint

12、 control.(3) MetaphaseAll chromosomes align at metaphase plate Microtubules are highly dynamic in the metaphase spindle.The events of Anaphase: Both anaphase A and anaphase B contribute to the movement of chromosome toward the spindle polesAnaphase A: The movement of the chromosomes toward the poles

13、; Kinetochore MT disaassenble at both ends during anaphase A.Anaphase B: The two spindle poles move farther apart. Both pushing and pulling forces contribute to anaphase B(4) AnaphaseAnaphase B: The two spindle poles move farther apart.The sliding of overlap MT at anaphase.MetaphaseLate anaphaseA mo

14、del explains the chromosome movement in anaphaseTwo alternative models of how the kinetochore may generate a poleward force on its chromosome during anaphase A.The control of Anaphase: SCF and APC activities during the cell cycleCdc20 and cdh1 are subunits to binding APC. APCcdc20 becomes activated

15、at the metaphase/anaphase transition. Securin: anaphase inhibitor. The destruction of securin by proteasomes at the end of metaphase starts a train reaction that leads to the cleavage of the cohesin complex. Cohesin holds sister chromatids together. Destruction of cohesin triggers the separation of

16、two chromatids.Cohesin 细胞周期中SCF与APC的活性。SCF和APC是多亚基复合体，它们将底物蛋白泛素化，致使底物由蛋白酶体降解。（a）SCF主要在间期有活性，而APC主要在有丝分裂期有活性。APC有两种，它们的区别在于分别含Cdc20或Cdh1亚基，亚基改变APC识别的底物。有丝分裂中，APCCdc20比APCCdh1早激活。 （b） APCCdc20负责降解抑制后期的蛋白，如securin，它们的降解促使细胞由中期转至后期。APCCdh1负责蛋白泛素化（如 M-cyclin）,M-cyclin抑制细胞脱离有丝分裂，泛素化底物蛋白的降解促使细胞 M-G1期转换。 Th

17、e spindle checkpoint(mammalian cell in late prometaphase labeled with anibodies against the spindle checkpoint protein Mad2 and tubulin). Mad2The spindle-attachment checkpoint: Anaphase is delayed until all chromosoms are positioned at the metaphase plate已发现Mad2与Cdc20结合，抑制APC的激活;只有当Mad2分子从所有的染色体上失去后

18、，APC才能被激活，后期才能开始. (5) TelophaseChromosome uncondenseNuclear envelope reforms around individual chromosomeGolgi, ER reconstructNucleolus reassembleMitosis ends3. Cytokinesis(1) In animal cells The MT of mitotic spindle determine the plane of animal cell division; Mitosis can occur without cytokinesis

19、. Contractile ring: Actin and myosinII in the contractile ring generate the force for cytokinesis(2) In plant cells: The phragmoplast guides cytokinesis in higher plants; The assembly of the cell plate begins in late anaphase and is guided by phragmoplast4. MeiosisThe comparison of meiosis and mitos

20、isTwo major contributions to the reassortment of genetic material that occurs in the production of gametes during meiosis.Visible evidence of crossing overComparison of the mechanisms of chromosome alignment (at metaphase) and separation (at anaphase) in meiotic division I and meiotic division II.Me

21、iotic chromosome pairing culminates in the formation of the synaptonemal complexInfluence of Sry on gonad development.The stages of oogenesis and spermatogenesis 5. The cell-cycle control systemA. The cell-cycle control system triggers the major processes of the cell cycleB. The control system can a

22、rrest the cell cycle at specific checkpointsC. The cell cycle control system is based on cyclically actived protein kinases-cyclin-dependent kinases (Cdks). Engine molecules for cell cycle G1checkpoint: fission yeast cells: Start point；Animal cells: Restriction PointD. MPF (Maturation-promoting fact

23、or，Mitosis-promoting factor)Lab. of Toronto Univ and Yale Univ.maturation promoting factor,MPFPurdue Univ.:Behavior of MPF The fusion of M-phase HeLa cell with Ptk cell (G1、S、G2) inducing PCC. Exp. Demonstration that cells contain factors that stimulate entry into mitosis.；The prematurely condensed

24、chromosome (PCC)MPF(M-Cdk): p34cdc2 cyclinBTwo subunits:Catalytic subunit transfers P from ATP to Ser and Thr;Regulatory subunit called cyclin(Marine Biological Lab.at Woods Hole and UC BerkeleyUniv. of Oxford-yeast)E. A simplified view of the core of the cell-cycle conreol systemCdk associates succ

25、essively with different cyclins to trigger the different events of the cycle.Cdk activity is usually terminated by cyclin degradation.Tree of cyclins are required in all eucaryotic cells:G1/S-cyclins bind Cdks at the end of G1 and commit the cell to DNA replication.S-cyclins bind Cdks during S phase

26、 and are required for the initiation of DNA replication.M-cyclins promote the events of mitosis.The structural basis of Cdk activationThe human Cdk2 is shown in three states: (A) In the inactive, without cyclin bound, the active site is blocked by a region of the protein called the T-loop; (B) The b

27、inding of cyclin causes the T-loop to move out of the active site, resulting in partial activition of Cdk2; (C) Phosphorylation of Cdk2 by CAK at a threonine residue in the T-loop, fully active.Cdk activity can be suppressed both by inhibitory phosphorylation (pp586:14.6) and by inhibitory proteins

28、(CKIs).P27Cdk inhibitor proteins(CKIs)The cell-cycle control system depends on cyclical proteolysis-The control of proteolysis by SCF and APC during the cell cycle.(A)The phosphorylation of a target protein(CKI),allows the CKI to be recognized by SCF (constitutive active), E1and E2(two additional pr

29、oteins), SCF serves as a ubiquitin ligase.The ubiquitylated CKI is then immediately recognized and degraded in a proteasome.SCF mediate the destruction of G1-cyclins, Cdk inhibitor,and other cell cycle proteins.(B)M-cyclin ubiquitylation is performed by APC.In G1 and S phaseIn metaphase/anaphase M-c

30、yclinsF. Intracellular control of cell-cycle eventsS-phase Cyclin-Cdk complexes (S-Cdks) initiate DNA replication once per cycleEvidence from cell-fusion exp. For a replication block. The initiation of DNA replication once per cell cycle.ORC: origin recognition complex;Cdc6: regulatory protein, it i

31、s present at low levels during most of the cell cycle but increases transiently in early G1,where it is required for the binding of a complex composed of a group of closely related proteins,the Mcm proteins.The activation of M-phase cyclin-Cdk complexes (M-Cdks) triggers entry into mitosisThe activa

32、tion of M-CdkThe DNA replication checkpoint: Entry into mitosis is blocked by incomplete DNA replication The experiments of DNA replication checkpoint in the mammalian cells in culture were treated with caffeine and hydroxyurea.The spindle-attachment checkpoint: Unattached chromosomes block sister-c

33、hromatid separation The sister chromatid separation is triggered by proteolysisThe triggering of sister-chromatid separation by the APC.APC: anaphase-promoting complexThe destruction of Securin allows separase to cleave the cohesin complex.Securin(后期抑制因子 )separasecohesinMad2 is normally localized at

34、 the kinetochores of prometaphase and misaligned metaphase chromosomes. Mad2 provides a “wait” signal that delays a cells progression into anaphase. The cell that possess mutant Mad2 fail to arrest at metaphase when their chromosomes are misaligned. Mad2 bind to Cdc20, inhibiting its activation of t

35、he APC, an event that is required for the metaphase-to-anaphase transition. It is only after the Mad2 is absent from all of the chromosomes that APC activation can occur and anaphase can begin.2. The cell that contains Monoattached chromosome delays the onset of anaphase until the chromosome becomes

36、 a biattached chromosome and aligned at the equator.Mad2 protein on unattached kinetochores.Exit from mitosis requires the inactivation of M-CdkM-Cdk inactivation occurs mainly by ubiquitin-dependent proteolysis of M-cyclinsThe G1 phase is a state of stable Cdk inactivityThe creation of a G1 phase b

37、y stable Cdk inhibition after mitosis.The mechanisms cotrolling S-phase initiation in animal cells.The Rb protein acts as a brake in mammalian G1 cellsThe control of G1 progression and S-phase initiation is often disrupted in cancer cells,leading to unrestrained cell-cycle entry and cell proliferati

38、on.Mitogens stimulate G1-Cdk and G1/S-Cdk activitiesA simplified model of one way that mitogens stimulate cell divisionDNA damage checkpoint: Cell-cycle progression is blocked by DNA damage and p53How DNA damage arrests the cell cycle in G1.Two such checkpoints:1.One in late G1: provents entry into

39、S phase;2.One in late G2: provents entry into mitosis;P53: gene regulatory protein.DNA damage activates p53 by an indirect mechanism. Mdm2 acts as a ubiquitin ligase that targets p53 for destruction by proteasomes. Phosphrylated p53 reduce its binding to Mdm2.P21(CKI protein) binds to G1/S-Cdk and S-Cdk and inhibits their activities, thereby helping to block entry into S phase.The summary of c