Chapter 11 Cell proliferation and its regulation
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 characters for life
1A. Overview of the cell cycle
The 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 genetically identical daughter cells.
Cell cycle phases:
M phase: Mitosis, Cytokinesis
Different cell cycle length
Some eukaryotic cell cycle times
The greatest variation occurs in the duration of G1
The shortest eukaryotic division cycles of all are the early embryonic cell cycles, no G1 and G2
Biochemical events of cell cycle
G1 phase: Synthesize proteins (RNA) for the DNA replication. Uncondense chromatin.
S phase: Synthesis of DNA and Histones
G2 phase: Synthesis of a few proteins (RNA)
M phase: Mitosis and meiosis and cytokinesis
Chromosome condense, Mitotic spindle, Contractile ring
Two daughter cells
Three categories of cells in vivo
(1)Cycling cells: Dividing continuously—Stem 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 cells,red blood cells, nerve cells
Embryo cells can transform into Cycling cells, G0 cells and Terminal cells.
1B. Synchronization of cell (for cell population)
(1) Natural synchronization: Early embryo in most invertebrates and a few vertebrates
Fruit fly embryo
(2) Selected synchronization artificially Isolation of cells in M phase
Isolation of cells by centrifugation
(3) Synchronization of cells induced by drugs
locking DNA synthesis by TdR: G1/S—TdR
Blocking cells in metaphase by colchicine
Affect the assembly of mitotic spindle
Special cell cycles—Early embryo (30min/cell cycle)
(1) No G1 and G2 phase, 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 cycle
Chromosomes condense to form compacted mitotic chromosomes
Activated M-Cdk phosphrylates condensin subunits,triggering the assembly of condensin complexes on DNA a nd condensation of the chromosome. The condensin can use energy of ATP hydtolysis topromote DNA coiling(in vitro).
The sister chromatids are glued together by multisubunit protein complex called cohesins.
Centrosome duplicates at S, and separates to form mitotic spindle at the beginning of prophase
Golgi, ER etc. disperse to form vesicles; kinetochore assembly
Schematic representation of the kinetochore
Nuclear envelopes breakdown (Lamin phosphorylation)
Spindle MTs 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 control.
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 poles Anaphase A: The movement of the chromosomes toward the poles; 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
Anaphase B: The two spindle poles move farther apart.
A model explains the chromosome movement in anaphase:
Two alternative models of how the kinetochore may generate a poleward force on its chromosome during anaphase A.
The control of Anaphase: SCF and APC activityies during the cell cycle
Cdc20 and cdh1 are subunits to binding APC. APCcdc20 becomes activated 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 cohsin complex. Cohesin holds sister chromatids together. Destruction of cohesin triggers the separation of two chromatids.
The spindle-attachment checkpoint:
Anaphase is delayed until all chromosoms are positioned at the metaphase plate
Nuclear envelope reforms around individual chromosome
Golgi, ER reconstruct
3A. In animal cells
The MT of mitotic spindle determine the plane of animal cell division; Mitosis can occur without cytokinesis.
Contractile ring: Actin and myosinII in the contractile ring generate the force for cytokinesis
3B. In plant cells: The phragmoplast guides cytokinesis in higher plants.
The assembly of the cell plate begins in late anaphase and is guided by phragmoplast
The comparison of meiosis and mitosis
Two major contributions to the reassortment of genetic material that occurs in the production of gametes during meiosis.
Visible evidence of crossing over
Comparison of the mechanisms of chromosome alignment (at metaphase) and separation (at anaphase) in meiotic division I and meiotic division II.
Meiotic chromosome pairing culminates in the formation of the synaptonemal complex
A mature synaptonemal complex
Influence of Sry on gonad development.
The stages of oogenesis and spermatogenesis
5. The cell-cycle control system
A. The cell-cycle control system triggers the major processes of the cell cycle
B. The control system can arrest the cell cycle at specific checkpoints
C. The cell cycle control system is based on cyclically actived protein kinases---cyclin-dependent kinases (Cdks). Engine molecules for cell cycle
D. MPF (Maturation-promoting factor， Mitosis-promoting factor)
The prematurely condensed chromosome (PCC)
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.
MPF(M-Cdk): p34cdc2 AND cyclinB
Catalytic subunit transfers -P from ATP to Ser and Thr;
Regulatory subunit called cyclin
E. A simplified view of the core of the cell-cycle conreol system
Cdk associates successively 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 and are required for the initiation of DNA replication.
M-cyclins promote the events of mitosis.
The structural basis of Cdk activation
The 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) Phosphorylation of Cdk2 by CAK at a threonine residue in the T-loop, fully active.
Cdk activity can be suppressed both by inhibitory phosphorylation and by inhibitory proteins.
P27—Cdk 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 proteins), SCF serves as a ubiquitin ligase.The ubiquitylated CKI is then immediately recognized and degraded in a proteasome.
(B)M-cyclin ubiquitylation is performed by APC.
F. Intracellular control of cell-cycle events
S-phase Cyclin-Cdk complexes (S-Cdks) initiate DNA replication once per cycle
Evidence 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 is 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 mitosis
The activation of M-Cdk
The 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-chromatid separation
The sister chromatid separation is triggered by proteolysis
The triggering of sister-chromatid separation by the APC.
APC: anaphase-promoting complex
The destruction of Securin allows separase to cleave the cohesin complex.
(1)Mad2 is normally localized at the kinetochores of prometaphase and misaligned metaphase chromosomes.
Mad2 provides a “wait” signal that delays a cell’s 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 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.
Mad2 protein on unattached kinetochores.
Exit from mitosis requires the inactivation of M-Cdk
M-Cdk inactivation occurs mainly by ubiquitin-dependent proteolysis of M-cyclins
The G1 phase is a state of stable Cdk inactivity
The creation of a G1 phase by stable Cdk inhibition after mitosis.
The mechanisms cotrolling S-phase initiation in animal cells.
The Rb protein acts as a brake in mammalian G1 cells
The 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 activities
A simplified model of one way that mitogens stimulate cell division
DNA damage checkpoint: Cell-cycle progression is blocked by DNA damage and p53
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 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 cell-cycle control system
An 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 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, 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 together. 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.