13 cell cycle - Cell Division Cell Death • The number of...

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Unformatted text preview: Cell Division Cell Death • The number of cells in multicellular organism is regulated by controlling both the rate of cell division and the rate of cell rate rate death Cancer is a potentially lethal disease caused by imbalanced growth, death, and senescence of cells 1 • Objectives 1. To know how extracellular growth signals are transmitted into cell and affect cellular division 2. To study the cell cycle in eukaryotic cell 3. To understand the regulatory mechanisms by which the cell proceeds through the different phases of cell cycle 2 Cell Cycle 3 Growth Control • Extracellular signaling proteins (growth factors); can be stimulatory or inhibitory stimulatory inhibitory - Control / coordinate rate of cell growth / division among cells in multicellular multicellular organisms - Most are mitogens (stimulate cells to (stimulate proceed from G1 phase into S phase) 4 Stimulatory Growth Factors • Stimulate cell proliferation: A protein produced by blood platelets to stimulate proliferation of connective tissue / smooth muscle cells (in wound repair / (in tissue regeneration) Stimulates tissue growth during embryonic development 5 A. Platelet-derived growth factor (PDGF) B. Epidermal growth factor (EGF) - (Genes) (Lipids) ? Growth factors 6 Ras-MAPK/ERK Pathway • Binding of a growth factor to a receptor on cell surface, causing cell to pass through G1 checkpoint into S phase 2 1 Phosphate (P) 4 3 adaptor protein 5 6 7 8 7 I. RTK and G-protein Signaling 1. 2. 3. Binding of growth factor to plasma membrane receptor Activation of receptor tyrosine kinase (RTK) Binding of adaptor protein Grb2 and guanine exchange factor Sos to the phosphorylated tyrosines Activation of plasma membrane monomeric GTPase Ras (binding of GTP and release of GDP) Activated Ras activates protein kinase Raf Grb2 Sos 4. 5. 5. 8 (Sos) Sos) Guanine exchange factor GTPase-activating protein GTPase- 9 II. Mitogen-activated Protein Kinases (MAPKs) • A family of serine / threonine protein kinases, involved in cell proliferation, cell differentiation, cell movement, cell and cell death and • Organized hierarchically into three-tiered modules: three - MAPKs are phosphorylated and activated by MAPKphosphorylated kinases (MAPKKs); MAPKKs are phosphorylated and phosphorylated activated by MAPKK-kinases (MAPKKKs); MAPKKKs are activated by interaction with a family of small GTPases and / or other protein kinases, connecting the protein MAPK module to cell surface receptors or external stimuli 10 11 The Three Known MAPK Cascades G protein-coupled receptor (GPCR); proteinExtracellular signal-regulated kinases (ERKs); MAPK/ERK kinase (MEK) signal- 12 6. Activated Raf → phosphorylates protein kinase protein MEK → phosphorylates mitogen-activated mitogen protein kinases MAPK/ERK 7. Activated MAPK phosphorylates and activates transcription factors (e.g., Jun and Ets) 13 8. Activated Jun and Ets → transcription of early response genes (e.g., Myc, Fos, etc.) 9. Transcription of delayed response genes (e.g., E2F, cyclins and Cdks) and 10. Formation of cyclin-Cdk complexes → phosphorylation of Rb → G1 → S 14 MAPK/ERK kinase (MEK) Extracellular signal-regulated kinase (ERK) signal- MAPK phosphatase (MKP) 15 Eukaryotic Cell Cycle Eukaryotic • A series of physiological stages that a eukaryotic cell has to pass through orderly orderly before it can divide into two daughter cells - Different phases: G1 (Gap 1), S (Synthesis), G2 (Gap 2), and M (Mitosis) - Overall length of cell cycle (generation time) 16 Cell Cycle 17 How to measure cell cycle? Flow cytometer • A machine consisting of laser, optics, mechanics, and laser optics mechanics electronics that can measure simultaneously a number of electronics physical and biological properties of a cell population Relative size, granularity, levels and states of macromolecules (DNA, RNA, proteins), etc. - 18 Cells stained with DNA-binding dye (e.g., propidium iodide) (e.g., Laser beam (e.g., 488 nm) A jet of single cells in special fluid (connected to computer for analysis) (conn Signal detectors Electromagnets 5 or more signals can be measured simultaneously in each cell http://www.wehi.edu.au/cytometry/flowintrol.html 19 Cell Cycle Analysis by Flow Cytometer Cells in G1 phase (if DNA content = X) Number of cells (DNA content >X and <2X) Cells in S phase Cells in G2 and M phases (DNA content = ~2X) Relative amount of DNA per cell (Signal from DNA-binding dye) 20 Control System - Cell Cycle Checkpoints • A series of control points in cell cycle to determine whether or not the cell should proceed to the next stage (checkpoints) • At least three checkpoints: 21 I. G1 Checkpoint / Restriction Point • At late G1 late Sufficient nutrients are present; a certain cell size is attained; presence of growth-factors States temporarily at G1; passes from G1 to S; or states indefinitely at G0 22 - II. G2 Checkpoint • At boundary between G2 and M phases phases Proper completion of DNA synthesis before mitosis can be initiated 23 III. Spindle Assembly Checkpoint • Between metaphase metaphase and anaphase in and anaphase in mitosis To make sure the 2 chromatids have attached to opposite spindle poles To guarantee each daughter cells will receive a complete set of chromosomes 24 - - Why does cell cycle need a control system? • To safeguard the events associated with each phase are carried out at appropriate time and in at in appropriate sequence To make sure each phase has been properly properly completed before the next phase is initiated To respond to external conditions that indicate external the need for cell growth and division 25 • • Cyclin-dependent Kinases (Cdks) 26 G1 Checkpoint / Restriction Point Checkpoint • Regulations: 1. Different cyclins are synthesized and synthesized and degraded during different phases 2. Different cyclins and Cdks interact in different combinations at different phases different 3. Activity of cyclin-Cdk complexes is controlled by phosphorylation (protein phosphorylation kinases) 4. Presence of different Cdk inhibitors (CDI) 27 (G1 cyclin) cyclin) (G1/S cyclin) (S cyclin) (M cyclin) G1 checkpoint 28 29 30 • Activated cyclin-Cdk complexes phosphorylate several target proteins; trigger entry in S phase - e.g., Retinoblastoma protein (Rb); a molecule that controls expression of genes for passage through G1 checkpoint into S phase 31 32 - Phosphorylated Rb molecules → lose ability to bind to E2F → activates transcription of genes for enzymes and other proteins → entry into S phase → initiates DNA replication 33 G2 Checkpoint • Controlled by M cyclin-Cdk complex (mitosis-promoting factor MPF) - Level of the Cdk remains relatively constant; concentration of M cyclin increases during G2 and M phases due to an increase of M-cyclin gene transcription - Critical threshold is reached at the end of G2 → M cyclin binds to and activates its Cdk 34 (M cyclin) G2 checkpoint 35 • Even after M cyclin has bound to Cdk, the resulting complex is still inactive: 1. Inhibiting kinase (Wee1) phosphorylates Cdk at specific amino acids (inactive MPF) (inactive 2. Activating kinase (CAK) phosphorylates the Cdk at another specific amino acid (inactive MPF) 36 37 3. Activating phosphatase (Cdc25) activates the inactive Cdk by de-phosphorylating the inhibiting phosphate (active MPF) (active 38 Functions of active MPF: 1. Phosphorylates several chromosomal proteins (e.g., histone H1) to trigger (e.g., chromosome condensation 2. Phosphorylates lamin proteins of nuclear lamina to destruct the latter and to destabilize and destruct nuclear envelope 3. Phosphorylates microtubule-associated proteins to facilitate mitotic spindle formation 39 Spindle Assembly Checkpoint • Neither a new cyclin nor a new Cdk is involved • Active MPF → phosphorylates / activates a large protein complex (anaphase promoting complex APC) → joins selected proteins to ubiquitin (a small protein with 76 amino acids) → the “labeled” proteins will be degraded degraded 40 Anaphase Promoting Complex (APC) • “Mitotic terminator” • Substrate-targeting: to ubiquinate proteins and target them for destruction by a proteasome (ubiquitin ligase) • A dozen subunits; one of which plays a key role in determining which proteins to be ubiquinated: 41 APC Ubiquitin-activating enzyme (E1); Ubiquitin-conjugating enzyme (E2); Ubiquitin ligase (E3) UbiquitinUbiquitin- 42 A. Cdc20 APC • Metaphase → anaphase • Destruction of anaphase inhibitor (securin) → activation of separase → cleavage of cohesin complex (a “protein glue” that holds sister chromatids together) → sister chromatids separate → anaphase begins 43 (APCCdc20) 44 Regulation of APCCdc20: • Negative signaling; details not clear Chromosomal kinetochores (protein complex at centromere of sister chromatids) remain unattached to spindle microtubules → a signaling protein (Mad2) is localized → binds to Cdc20 → APC is inhibited All kinetochores attached to spindle → all Mad2 molecules disappear → APC is not inhibited → onset of anaphase → completion of mitosis 45 - Centromere Kinetochore 46 47 B. Cdh1 APC • M phase → G1 phase • Destruction of mitotic cyclins → loss of MPF activity → chromosome decondensation; reformation of nuclear reformation envelope → exits from mitosis 48 ...
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This note was uploaded on 09/26/2010 for the course BIO BIO2110 taught by Professor Kwam during the Spring '10 term at 한동대학교.

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