基醫所 The Cell Cycle Chi-Wu Chiang, Ph.D. IMM, NCKU 1 1
Introduction to cell cycle and cell cycle checkpoints 2 2
Cell cycle A cell reproduces by performing an orderly sequence of events in which it duplicates its contents and then divides in two 3 3
The phases of cell cycle 4 4
What are the differences between embryonic and somatic cell cycle? 5 5
Comparison of the embryonic cell cycle and the somatic cell cycle Early embryonic cell cycle Somatic cell cycle 30 min M S 6h G2 M G1 12h S 6h 6 6
Yeast provides a good genetic system for study on cell cycle Mostly proliferating in a haploid state Easy for genetic manipulation Discoveries of Cdc (cell-division-cycle) genes via cdc mutants 7 7
How were yeast cdc mutants discovered? What are the major characteristics of the mutants? 8 8
Cdc mutants selected by temperature sensitive mutations A mutant that cannot complete the cell cycle, cannot be propagated Temperature-sensitive mutants Functioning in permissive condition (low temp., 23 ) and nonfunctioning in restrictive condition (high temp., 36 ) 9 9
Xenopus oocyte: a giant cell for study cell cycle biochemically 1 mm in diameter Carrying 100,000 times more cytoplasm than an average cell in the human body After fertilization occurs, the first division takes about 90 minutes, and the next 11 divisions occur at 30-mim intervals, producing about 4096 cells within 7 hours Each cycle is divided into S and M phase without detectable G1 or G2 phases 10 10
Studying the cell cycle in a cell-free system Observation of repeated nuclei decondense and DNA replication and mitosis In vitro Filled with cell-cycle control components 11 11
Measurements of cell cycle progression Propidium iodide (PI) staining BrdU staining (bromo deoxyuridine) EMBO 2003, 22:5459 PI staining 12 Oncogene 2001, 20:4507 12
What are the advantages of using BrdU staining in addition to PI staining? 13 13
The cell-cycle control system 14 14
The major components of the cell cycle control system Cell, 2008, 135 15 15
The evolutionary conserved cell cycle control system Cell 2008, 135 16 16
The cell cycle checkpoints G1 checkpoint (start checkpoint) G2 checkpoint Metaphase checkpoint At checkpoints, cell cycle can be arrested if previous events have not been completed At checkpoints, the cell cycle control system can be regulated by extracellular signals from the environment or other cells 17 17
What causes the responses of the cell cycle check points? What are the effects? 18 18
The regulation of cell cycle 19 19
The key regulators of cell cycle Cell, 2008, 135 20 20
Regulation of Cdks by reversible phosphorylation Human Cdk1 and also Cdc2 in yeast: Phosphorylation by Wee1 kinase at Tyr15 inhibits CDK activity Cdc25 phosphatase dephosphorylates the p-tyr15 and activates the Cdk 21 21
Cyclical proteolysis regulates cyclin-cdk activity Regulated by SCF complex in G1 and S phase SCF complex(ubiquitin ligase) Targets:G1/S cyclins, some CKIs (such as p27kip) 22 22
Cyclical proteolysis regulates cyclin-cdk activity Regulated by APC complex in M phase APC: anaphase promoting complex APC complex (Ubiquitin ligase) Target: M cyclin 23 23
The evolutionary conserved cell cycle regulators Cell, 2008, 135 24 24
E2F promotes cell cycle progression E2F increases the transcription of its own gene Increase in transcription of G1/S cyclin and S-cyclin genes Increases in G1/S cyclin and S-cyclin result in increases in G1/S-Cdk activity and S-Cdk activity Increases in G1/S-Cdk activity and S-Cdk activity result in more Rb phosphorylation and more release of E2F Increases in G1/S-Cdk activity and S-Cdk activity and inactivate p27 (CKI) and Hct-1 by phosphorylation 25 25
What triggers E2F synthesis? 26 26
Mitogens stimulate activation of Ras and a MAP kinase cascade to trigger cell division 27 27
Mechanisms of Myc-promoted cell cycle entry 28 28
What is the stage of cells that have influences on another cell to initiate DNA replication after fusion of cells? Can a cell at any stage become responsive and start DNA replication? 29 29
Start the DNA replication once per cycle Cell fusion experiments by R.T. Johnson and P. N. Rao, 1970 Only G1 cells are competent to initiate DNA replication S-Cdk triggers the initiation of DNA replication 30 30
What controls only one replication per cell cycle in normal cells? 31 31
Control of the initiation for DNA replication 32 Figure 17-23 Molecular Biology of the Cell ( Garland Science 2008) 32
Multiple roles of M-Cdk in mitosis Induce the assembly of mitotic spindle Ensure replicated chromosomes attach to the mitotic spindle Chromosome condensation Nuclear envelope breakdown Reorganization of the Golgi apparatus and endoplasmic reticulum 33 33
Mitosis Nocodazole treatment arrested 34 34
Mitosis, continued 35 35
Centrosome duplication begins at the time entering the S phase Inhibition of DNA replication blocks centrosome duplication Centriole s replication is cell-cycle dependent M-cdk and Aurora A help the centrosome separation and maturation Figure 17-31 Molecular Biology of the Cell ( Garland Science 2008) 36 36
The centrosome duplication cycle reveals asymmetrical characteristics 1: mother centriole 2: daughter centriole cilium Nat Cell Biol Rev 2011, 13:1154-1160 37 37
Sister chromatid separation is triggered by proteolysis Separase is required for sister chromatids separation Securin, can inactivate separase; degradation is promoted by APC APC: anaphase-promoting complex, a highly regulated ubiquitin ligase Cdc20 activates APC 38 38
The spindle-attachment checkpoint Ensure that all chromosomes are properly attached to the spindle before sister-chromatid separation occurs A sensor mechanism monitors the state of the kinetochore, the specialized region of the chromosome that attaches to microtubules of the spindle Improper attachment of kinetochore to the spindle sends out a negative signal to the cell-cycle control system, blocking Cdc20-APC activation and sister chromatid separation Several proteins, including Mad2, are recruited to unattached kinetochores. Mad2 binding results in Inhibition of Cdc20-APC and blocking securin destruction 39 39
Exit from mitosis and start of G 1 Complex changes at the end of mitosis The mitotic spindle must be dissembled Chromosomes decondensed The nuclear envelope reformed Inactivation of M-Cdk is required for exit from mitosis Cdc20-APC complex mediated ubiquitin-dependent proteolysis of M-cyclin 40 40
Suggested readings 1. Alberts B., Johnson A., Lewis J. et al (eds) (2002, 2008) Molecular Biology of the Cell, 4th & 5th ed. Chapter 17 2. Murray A. and Hunt T. (eds) (1993) The Cell Cycle: an introduction, 135-152 3. Bartek J. and Lukas J. (2003) Chk1 and Chk2 kinases in checkpoint control and cancer. Cancer Cell 3:421-429 41 41