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 important ch
2、aracters 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 into two genetic
3、ally identical daughter cells.vCell cycle phases:Interphase:G1-S-G2M phase:Mitosis,Cytokinesisv 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 cycles,no G1 and G
4、2v 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 condense Mitotic spindleContractile r
5、ingv Three categories of cells in vivo(1)Cycling 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 until they die:muscle ce
6、lls,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 artificiallyIsolation of cells in M phase Isolati
7、on 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)(1)No G1 and G2 phase,all replicons are activated,s
8、o 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)ProphasevChromatins condense to form compacted mitotic chromosomesActivated M-Cdk phosphrylates condensin subunits,triggering the ass
9、embly 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.v Centrosome duplicated at S,and separate to form mitotic sp
10、indle at the beginning of prophaseAssembly of kinetochore at the end of prophasevGolgi,ER etc.disperse to form vesicles;kinetochore assemblySchematic representation of the kinetochore(2)PrometaphasevNuclear envelopes breakdown(Lamin phosphorylation)vSpindle MTs capture chromosomes vThe pushing and p
11、ulling 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 control.(3)MetaphaseAll chromosomes align
12、 at metaphase plate Microtubules are highly dynamic in the metaphase spindle.vThe 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;Kinetochore MT disaassenble at both ends du
13、ring anaphase A.Anaphase B:The two spindle poles move farther apart.Both pushing and pulling forces contribute to anaphase B(4)AnaphasevAnaphase B:The two spindle poles move farther apart.The sliding of overlap MT at anaphase.MetaphaseLate anaphasevA model explains the chromosome movement in anaphas
14、eTwo alternative models of how the kinetochore may generate a poleward force on its chromosome during anaphase A.vThe control of Anaphase:SCF and APC activities during the cell cycleCdc20 and cdh1 are subunits to binding APC.APCcdc20 becomes activated at the metaphase/anaphase transition.Securin:ana
15、phase 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 two chromatids.Cohesin 细胞周期中细胞周期中SCF与与APC的活性。的活性。SCF和
16、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。The spindle checkpoint(mammalian cell in late pr
17、ometaphase labeled with anibodies against the spindle checkpoint protein Mad2 and tubulin).Mad2vThe 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，APC才能被激活，后期才能开始.(5)TelophaseChromosome unconde
18、nseNuclear 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.vContractile ring:Actin and myosinII in the contract
19、ile 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.MeiosisvThe comparison of meiosis and mitosisvTwo major contributions to the reassortment of genetic
20、 material that occurs in the production of gametes during meiosis.vComparison of the mechanisms of chromosome alignment(at metaphase)and separation(at anaphase)in meiotic division I and meiotic division II.vMeiotic chromosome pairing culminates in the formation of the synaptonemal complexvInfluence
21、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 arrest the cell cycle at specific checkpointsC.The cell cycle control system is based on cycli
22、cally 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 factor，Mitosis-promoting factor)Lab.of Toronto Univ and Yale Univ.maturation promoting factor,MPFPurdue U
23、niv.: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.；vThe prematurely condensed chromosome(PCC)MPF(M-Cdk):p34cdc2 cyclinBTwo subunits:Catalytic subunit transfers P from ATP to Ser and T
24、hr;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 successively with different cyclins to trigger the different events of the cycle.Cdk activity is usually terminat
25、ed by cyclin degradation.Tree of cyclins are required in all eucaryotic cells:1.G1/S-cyclins bind Cdks at the end of G1 and commit the cell to DNA replication.2.S-cyclins bind Cdks during S phase and are required for the initiation of DNA replication.3.M-cyclins promote the events of mitosis.vThe st
26、ructural 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 binding of cyclin causes the T-loop to move out of the active site,resulting in partial activition of Cdk2;(C
27、)Phosphorylation of Cdk2 by CAK at a threonine residue in the T-loop,fully active.vCdk activity can be suppressed both by inhibitory phosphorylation(pp586:14.6)and by inhibitory proteins(CKIs).P27Cdk inhibitor proteins(CKIs)vThe cell-cycle control system depends on cyclical proteolysis-The control o
28、f 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 proteins),SCF serves as a ubiquitin ligase.The ubiquitylated CKI is then immediately recognized and degraded in a pro
29、teasome.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-cyclinsF.Intracellular control of cell-cycle eventsvS-phase Cyclin-Cdk complexes(S-Cdks)initiate DNA replication once
30、per cycleEvidence from cell-fusion exp.For a replication block.vThe initiation of DNA replication once per cell cycle.ORC:origin recognition complex;Cdc6:regulatory protein,it is present at low levels during most of the cell cycle but increases transiently in early G1,where it is required for the bi
31、nding of a complex composed of a group of closely related proteins,the Mcm proteins.vThe activation of M-phase cyclin-Cdk complexes(M-Cdks)triggers entry into mitosisThe activation of M-CdkvThe DNA replication checkpoint:Entry into mitosis is blocked by incomplete DNA replication The experiments of
32、DNA replication checkpoint in the mammalian cells in culture were treated with caffeine and hydroxyurea.vThe spindle-attachment checkpoint:Unattached chromosomes block sister-chromatid separation v The sister chromatid separation is triggered by proteolysisThe triggering of sister-chromatid separati
33、on by the APC.APC:anaphase-promoting complexThe destruction of Securin allows separase to cleave the cohesin complex.Securin(后期抑制因子后期抑制因子)separasecohesin1.Mad2 is normally localized at the kinetochores of prometaphase and misaligned metaphase chromosomes.Mad2 provides a“wait”signal that delays a cel
34、ls 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 the APC,an event that is required for the metaphase-to-anaphase transition.It is only after the Mad2 is absent from all of
35、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 a biattached chromosome and aligned at the equator.vMad2 protein on unattached kinetochores.vExit from mitosis requires the
36、inactivation of M-CdkvM-Cdk inactivation occurs mainly by ubiquitin-dependent proteolysis of M-cyclinsvThe G1 phase is a state of stable Cdk inactivityThe creation of a G1 phase by stable Cdk inhibition after mitosis.vThe mechanisms cotrolling S-phase initiation in animal cells.vThe Rb protein acts
37、as a brake in mammalian G1 cellsvThe control of G1 progression and S-phase initiation is often disrupted in cancer cells,leading to unrestrained cell-cycle entry and cell proliferation.Mitogens stimulate G1-Cdk and G1/S-Cdk activitiesA simplified model of one way that mitogens stimulate cell divisio
38、nvDNA 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 S phase;2.One in late G2:provents entry into mitosis;P53:gene regulatory protein.DNA damage activates p53 by an indirec
39、t 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.vThe summary of cell-cycle control systemSumm
40、aryAn ordered sequence of cyclin-Cdk activities triggers most of the events of the cell cycle.During G1 phase,Cdk activity is reduced to a minimum by Cdk inhibitors(CKIs),cyclin proteolysis,and decreased cyclin gene transcription.When environmental conditions are favorable,G1-and G1/S-Cdks increase
41、in concentration,overcoming these inhibitory barriers in late G1 and triggering the activation of S-Cdk.The S-Cdk phosphorylates proteins at DNA replication origins,initiating DNA synthesis through a mechanism that ensures that the DNA is duplicated only once per cell cycle.Once S-phase is completed
42、,the activation of M-Cdk leads to the events of early mitosis,whereby the cell assembles a mitotic spindle and prepares for segregation of the duplicated chromosomes-which consist of sister chromatids glued together.Ananphase is triggered by the destruction of the proteins that hold the sisters toge
43、ther.The M-Cdk is then inactivated by cyclin proteolysis,which leads to cytokinesis and the end of M phase.Progression through the cell cycle is regulated precisely by various inhibitory mechanisms that arrest the cell cycle at specific checkpoints when events are not completed successfully,when DNA damage occurs,or when extracellular conditions are unfavorable.
