Unformatted text preview: Overview of the Cell CycleNew organisms Development Maintenance The cell cycle control system checks and controls normal progression of the cycle and responds to extracellular cues Mitosis Time gap to DNA and organelle sense intra-cellular cues segregation & cell division Its all about the faithful replication and segregation of DNA molecules S-phase DNA replication Double protein & organelle mass The five stages of mitosis and the subsequent cytokinesis Time gap to sense extra-cellular cues can last hours, days or a lifetime condensation segregation decondensation START The cell cycle system can be dissected genetically in yeastseptum Yeast and human cell cycles are similar (except for nuclear envelope breakdown) Morphology of cell division cycle (cdc) mutant yeast cdc15 ts The behavior of temperature sensitive cdc mutant 2001 Nobel Prize to Paul Nurse, Lee Hartwell & Tim Hunt The cell cycle system can be analyzed biochemically in frog egg extractsA xenopus egg
Reconstitution of the simple cell cycle using Xenopus egg extracts Very rapid cell division (no G1 or G2… why?) The cell cycle can be studied in mammalian cell cultureMammalian cell lines are not normal-they have been immortalized… but are useful nevertheless Proliferating mammalian cells Monitoring cell division using DNA bindingfluorescent dyes and flow cytometry or FACS Rat fibroblasts Monitoring cell division cytologically (e.g. labeling methods)
Incorporation of radioactive thymidine (autoradiography) marks cells in S phase Incorporation of BrdU and immuno fluorescence Controlling the cell cycle through checkpoints at major eventsMajor mitotic events are triggered by a mitotic control system The control system can arrest the cell cycle at specific checkpoints Checkpoint components are not essential for progression and operate through negative signaling (arrest): if mutated, the cycle continues despite any major problems but can lead to disease The major mitotic events are controlled Checkpoints for progression
(accessory braking systems) Delay or proceed? The control system has: A clock A linear ordering system A mechanism to ensure vectoriality Binary switches Backup mechanisms Adaptability Delay or proceed? The two key components of the cell-cycle control system: cyclin and CDK
A simple embryonic cell cycle
Cyclin degradation makes the cycle vectorial
Cyclins controls CDK activity and direct CDK to specific targets Their abundance cycles… CDK activation triggers major cell cycle events via phosphorylation. Without cyclin, CDK is inactive A regular cell cycle Structural basis for CDK activation G1/S cyclins trigger progression through Start M-cyclins promote mitosis S-cyclins trigger DNA replication G1-cyclin controls G1/S activity Exposure of active site CDK activity is modulated in two ways- By inhibitory phosphorylation
(near active site) In the absence of Wee1 function cells enter mitosis prematurely at a small (wee) size By conformational distortion of the active site by CDK inhibitor proteins (CKI) (CKI) Cell cycle control depends on the timely cyclical proteolysis of key playersCDK activation: Removal of the p27 (CKI) inhibitor through degradation CDK inactivation: Down regulation of cyclin activity by degradation tag SCF is a ubiquitin ligase (E3) and its always active. Its substrates become available by phosphorylation. APC: Anaphase promoting complex is a ubiquitin ligase (E3) Its activity cycles and is regulated by subunit assembly Three mechanisms controlling the length of G1- Creation of G1 phase via prolonged inhibition of M-CDK activity I.
Cyclin degradation CDK inhibitor robustness M-cyclin degrad. continues II. M-Cdk Increases cyclin levels G1 is referred to as state of stable M-CDK inactivity Cdh1 I. Activation of CKI II. Activation of APC-Hct (APC-Cdh1) III. M-cyclin synthesis is down-regulated in G1 Control of G1 to S transition may lead to control of cancer Three positive feedback loops drive rapid onset of S phase- The E2F transcription factor stimulates expression of G1/S and S cyclins The Retinoblastoma (Rb) tumor suppressor protein keeps E2F inactive Inactivates p27 (Sic1) Inactivates Hct1/Cdh1 Rb was identified as an oncogene responsible for congenital cancer of the retina Intracellular control of cell cycle events: Prevention of DNA re-replication: ensuring only once per cell cycle
Evidence of a mechanism that inhibits DNA re-replication: cell fusion experiments
[Cdc6] up [Cdt1] up ORC is always bound at origins of DNA replication + Cdt1 Cdc6 & Cdt1 associate with ORC Cdc6/Cdt1 & ORC recruit Mcms S-Cdk targets Cdc6 for degradation & recruitment of preinitiation complex G2 nuclei become refractory (molecular memory) to prevent DNA re-replication
Cytoplasmic S factors drive G1 nuclei into M (DNA synthesis) Cytoplasmic S factors cannot drive G2 nuclei into M Cytoplasmic G1 factors and G2 factors are non-inductive towards each other Mcms are helicases Isolation of MPF… The activation of M-phase cyclin-CDK complexes: to enter mitosis or not?
A four step regulation and a feedback loop
-Induces mitotic spindle assembly -Ensures chromosome attachment to spindle -Induces nuclear envelope breakdown -Induces actin cytoskeleton depolymer -Induces ER and Golgi reorganization M-Cyclin [up] during G2 (+ polo kinase = inhibited) 2 1 4
Exponential activation 3 The “incomplete DNA replication” checkpoint- DNA synthesis checkpoint inhibitor: Chemical inhibitor of DNA synthesis: > Incomplete DNA replication normally sends a signal that activates a kinase that inhibits Cdc25 (see above) Protein degradation by APC is required to exit mitosis- sister chromatid separation
Experimental proof (in frog egg extracts) Cohesin Degradation required for Degradation required also M>A transition. for progression of No degradation = no transition anaphase Before anaphase Initiates anaphase The Anaphase Promoting Complex (APC) triggers sister chromatid separation and thereby initiates anaphase The cohesins keep the chromosomes bound while under tension by microtubule pulling DNA damage checkpoints at G1 and G2 can halt the cell cycleMechanism of inhibition after X-ray damage Overview of the cell cycle control system In G1, cells use p53 (see diagram) Mdm2 is a ubiquitin ligase In G2, damaged DNA also activates kinases (polo) that phosphorylate and inactivate the Cdc25 phosphatase, which activates cyclin-Cdk Mutations of p53 occurs in nearly half of all human cancers… why? Radiation and cancer kill in opposite ways Programmed cell death- Apoptosis
Apoptotic cells labeled … a day later The apoptotic caspase cascade its irreversible Apoptosis during metamorphosis Hey, loose that tail Caspases cleave at aspartic acid Morphology of Cell Death An amplifying proteolysis cascade Necrosis (Bursting) Apoptosis (a tidy death) Altered cell surface… Apoptotic trigger from the outside… the tale of the killer lymphocyte
Adaptor proteins trigger the cascade by aggregating (initiator) caspases The ‘death’ receptor Some stressed or damaged cells commit suicide by expressing both: Fas ligand and receptor Apoptotic trigger from the inside… a mitochondrial sabotage
Cellular suicide The Bcl2 family of proteins help regulate activation of pro-caspases: Some block release of cytochrome c from mitochondria others enhance it. IAPs (inhibitors of apoptosis) inhibit caspases or bind to them to prevent activation Extracellular Control of Cell Division, Cell Growth and ApoptosisThe factors that control growth are: Mitogens- stimulate cell division by relieving an inhibitor Growth factors- stimulate cell growth (mass) via protein synthesis Survival factors- suppress apoptosis Most cells in our bodies are in Go (a variant of G1) Mitogen activated cell division through Ras, myc and G1 cyclins (lose the ‘G1 brakes’)
PDGF, EGF Body and organ size are tightly controlled via an equilibrium of cell growth, cell division, and cell death The Myc cascade
1 2 3 If Myc overdose… Ras and myc are oncogenes; They can cause cancer if mutated p21 (CDKI) synthesis The Fountain of Youth?
Cells are programmed to divide a fixed number of times. Governed by telomere length Overcoming cell senescence by overexpression of telomerase…
Telomerase (inactive in somatic cells) Control of cell proliferation through positive signals: growth factors and survival factors
Direct stimulation by growth factors Inhibition of apoptotic program by cell contact with target: survival factors Promote ribosome protein synthesis and ribosome activity Overview of the Cell Cycle and its control system. Control of cell division and growth. Apoptosis. Reading covered in this lecture: Alberts et al Chapter 17 pgs 1053-1071; 1101-1112; & Chapter 18 pgs 1115-1124 Reading covered in next lecture: Alberts et al Chapter 17 pgs 1071-1101 ...
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