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The cell Division and Apoptosis 2013-12-12 藥理所 陳韻雯 老師 •Cell Division 8N Cell 4N 2N Cell Cell Cell 1N Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Overview of Cell Cycle Phase of the cell cycle The cell cycle consists of Interphase – G1(gap1), S (Synthesis), G2 (Gap2) phase The mitotic phase – cell division h 3-4 DN n r, 4 612 hr ,2 n- 4n A DN A 6 nD ,2 r h - 12 NA Figure 17-4 Molecular Biology of the Cell (© Garland Science 2008) Interphase-G1 •G1 phase: START • Begins immediately after division • Commitment point beyond S phase • cell growth • New organelles formed • Ensure the ability of cell to replicate chromosome • End of G1, cell has doubled in size NA D n 1 6- r, 2 h 2 Interphase-S •S phase: 8 6s, hr NA nD 2-4 • The second part of interphase • Cells make a perfect copy of their DNA in its nucleus • This process is called REPLICATION • DNA is found in the chromatin in the nucleus • Each daughter cell must have a complete set of DNA to survive Interphase-G2 3- •Commitment point beyond M phase •The cell produces structures that it will use to divide into two new cells •Ensure the ability of cell to divide •Cell prepares to begin moiosis 4h r, 4 nD NA •G2 phase: M PHASE •M phase: •The division of a cell into two daughters •Consists of nuclear division, or mitosis, and cytoplasmic division, or cytokinesis. •Mitosis is itself divided into five stages •Together with cytokinesis are described in this panel Mitotic Division of an Animal Cell PROPHASE PROMETAPHASE METAPHASE Mitotic Division of an Animal Cell ANAPHASE TELOPHASE CYTOKINESIS Prophase-1st step in Mitosis • The chromatin fibers become more tightly coiled, condensing into discrete chromosomes observable with a light microscope. • The nucleoli disappear. • Each duplicated chromosome appears as two identical sister chromatids joined together. • The mitotic spindle begins to form. It is composed of the centrosomes and the microtubules that extend from them. The radial arrays of shorter microtubules that extend from the centrosomes are called asters (“stars”). • The centrosomes move away from each other, apparently propelled by the lengthening microtubules between them. Prophase Animal Cell Plant Cell Spindle fibers Centrioles Photographs from: http://www.bioweb.uncc.edu/biol1110/Stages.htm Prometaphase • Prometaphase starts abruptly with breakdown of the nuclear envelope. • Chromsomes can now attach to spindle microtubules via their kinetochore and undergo active movement nd Metaphase-2 step in Mitosis • Metaphase is the longest stage of mitosis, lasting about 20 minutes. • The centrosomes are now at opposite ends of the cell. •The chromosomes convene on the metaphase plate, an imaginary plane that is equidistant between the spindle’s two poles. The chromosomes’ centromeres lie on the metaphase plate. • For each chromosome, the kinetochores of the sister chromatids are attached to kinetochore microtubules coming from opposite poles. • The entire apparatus of microtubules is called the spindle because of its shape. Metaphase Animal Cell Plant Cell Photographs from: http://www.bioweb.uncc.edu/biol1110/Stages.htm The Mitotic Spindle • The spindle includes the centrosomes, the spindle microtubules, and the asters • The apparatus of microtubules controls chromosome movement during mitosis • The centrosome replicates, forming two centrosomes that migrate to opposite ends of the cell • Assembly of spindle microtubules begins in the centrosome, the microtubule organizing center • An aster (a radial array of short microtubules) extends from each centrosome The Mitotic Spindle • Some spindle microtubules attach to the kinetochores of chromosomes and move the chromosomes to the metaphase plate • In anaphase, sister chromatids separate and move along the kinetochore microtubules toward opposite ends of the cell Aster Microtubules Sister chromatids Chromosomes Centrosome Metaphase plate Kinetochores Centrosome 1 µm Overlapping nonkinetochore microtubules Kinetochore microtubules 0.5 µm Anaphase- 3rd step in Mitosis • Anaphase is the shortest stage of mitosis, lasting only a few minutes. • Anaphase begins when the two sister chromatids of each pair suddenly part. Each chromatid thus becomes a fullfledged chromosome. • The two liberated chromosomes begin moving toward opposite ends of the cell, as their kinetochore microtubules shorten. Because these microtubules are attached at the centromere region, the chromosomes move centromere first (at about 1 µm/min). • The cell elongates as the nonkinetochore microtubules lengthen. • By the end of anaphase, the two ends of the cell have equivalent—and complete—collections of chromosomes. Anaphase Animal Cell Plant Cell Photographs from: http://www.bioweb.uncc.edu/biol1110/Stages.htm Telophase- 4th step in Mitosis • Two daughter nuclei begin to form in the cell. • Nuclear envelopes arise from the fragments of the parent cell’s nuclear envelope and other portions of the endomembrane system. • The chromosomes become less condensed. • Mitosis, the division of one nucleus into two genetically identical nuclei, is now complete. Telophase Animal Cell Plant Cell Photographs from: http://www.bioweb.uncc.edu/biol1110/Stages.htm Cytokinesis • Cleavage of cell into two halves – Animal cells Constriction belt of actin filaments – Plant cells Cell plate – Fungi and protists Mitosis occurs within the nucleus Cytokinesis In Animal And Plant Cells Cleavage furrow Contractile ring of microfilaments 100 µm Vesicles forming cell plate 1 µm Wall of patent cell Cell plate New cell wall Daughter cells (a) Cleavage of an animal cell (SEM) Daughter cells (b) Cell plate formation in a plant cell (SEM) Animal Mitosis -- Review Interphase Prophase Metaphase Anaphase Telophase Cytokinesis Plant Mitosis -- Review Interphase Prophase Metaphase Anaphase Telophase Cytokinesis Cell Cycle 28 Cell Cycle Control System The Cell Cycle Control System • The sequential events of the cell cycle are directed by a distinct cell cycle control system, which is similar to a clock • The cell cycle control system is regulated by both internal and external controls • The clock has specific checkpoints where the cell cycle stops until a go-ahead signal is received The Major Cell-Cycle Regulatory Protein Overview of the Cell -Cycle control system Figure 17-21 Molecular Biology of the Cell (© Garland Science 2008) Checkpoint control system • Checkpoints • cell cycle controlled by STOP & GO chemical signals at critical points • signals indicate if key cellular processes have been completed correctly G0 G1 checkpoint G1 (a) Cell receives a go-ahead signal. G1 (b) Cell does not receive a go-ahead signal. Checkpoint control system • 3 major checkpoints: • G1/S • can DNA synthesis begin? • G2/M • has DNA synthesis been completed correctly? • commitment to mitosis • spindle checkpoint • are all chromosomes attached to spindle? • can sister chromatids separate correctly? Spindle checkpoint G2 / M checkpoint Chromosomes attached at metaphase plate • Replication completed • DNA integrity Inactive Active Inactive Cdk / G2 cyclin (MPF) M Active APC C cytokinesis mitosis G2 G1 S MPF = Mitosis Promoting Factor APC = Anaphase Promoting Complex Cdk / G1 cyclin Active G1 / S checkpoint Inactive • Growth factors • Nutritional state of cell • Size of cell “Go-ahead” signals • Protein signals that promote cell growth & division • internal signals • “promoting factors” • external signals • “growth factors” • Primary mechanism of control • phosphorylation • kinase enzymes • either activates or inactivates cell signals Cell cycle signals inactivated Cdk • Cell cycle controls • cyclins • regulatory proteins • levels cycle in the cell • Cdk’s activated Cdk • cyclin-dependent kinases • phosphorylates cellular proteins • activates or inactivates proteins • Cdk-cyclin complex • triggers passage through different stages of cell cycle The Major Cyclins and Cdks The Cyclin-Cdk complexes: the major components of the cell cycle control system Regulation of cyclin-CDK activity • Three mechanisms for controlling cyclin-CDK activity: • (1). Regulation of the cyclin concentration of (mitotic) cyclins by ubiquitin-protein ligases (SCF and APC/C). • (2). Regulation of the kinase activity of CDKs by phosphorylation. • (3). Regulation of cyclin-CDK activity by CDK inhibitors (CKIs). (1) (2) (3) APC/C ubiquitin-protein ligases • Anaphase-promoting complex (APC/C) is a ubiquitin-protein ligase which is involved in regulating two transitions in cell cycle: • (A) from metaphase to anaphase- the APC/C catalyzes the destruction of securin, leading to the separation of sister chromatins and move to opposite of the spindle during anaphase. • (B) from anaphase to cytokinesis- the APC/C targets the S- and M-cyclins for destruction, leading to the loss of most CDK activity in anaphase.CDK inactivation allows phosphatases to dephosphorylate the many CDK substrates in the cell, as required for the completion of mitosis and cytokinesis. DNA damage and checkpoint Nature 2000;408:433-9 DNA damage arrests the cell cycle in G1 Figure 17-63 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008) Human DNA damage response pathway DNA damage Replication block Signals Common intermediate ATM or ATR Rad17/RFC Transducers Hus1/Rad1/Rad9 Chk1 BRCA1 Others Cell cycle Apoptosis arrest Sensors p53 DNA repair Effectors Checkpoints in Cell-cycle Regulation Checkpoint and Cancer Cancer & Cell Growth • Cancer is essentially a failure of cell division control • unrestrained, uncontrolled cell growth • What control is lost? • lose checkpoint stops • gene p53 plays a key role in G1/S restriction point • p53 protein halts cell division if it detects damaged DNA p53 is the Cell Cycle Enforcer • options: • stimulates repair enzymes to fix DNA • forces cell into G0 resting stage • keeps cell in G1 arrest • causes apoptosis of damaged cell • ALL cancers have to shut down p53 activity p53 discovered at Stony Brook by Dr. Arnold Levine p53 — master regulator gene NORMAL p53 p53 allows cells with repaired DNA to divide. p53 protein DNA repair enzyme p53 protein Step 1 Step 2 Step 3 DNA damage is caused by heat, radiation, or chemicals. Cell division stops, and p53 triggers enzymes to repair damaged region. p53 triggers the destruction of cells damaged beyond repair. ABNORMAL p53 abnormal p53 protein Step 1 Step 2 DNA damage is caused by heat, radiation, or chemicals. The p53 protein fails to stop cell division and repair DNA. Cell divides without repair to damaged DNA. cancer cell Step 3 Damaged cells continue to divide. If other damage accumulates, the cell can turn cancerous. Development of Cancer • Cancer develops only after a cell experiences ~6 key mutations (“hits”) • unlimited growth • turn on growth promoter genes • ignore checkpoints • turn off tumor suppressor genes (p53) • escape apoptosis • turn off suicide genes • immortality = unlimited divisions • turn on chromosome maintenance genes • promotes blood vessel growth • turn on blood vessel growth genes • overcome anchor & density dependence • turn off touch-sensor gene It’s like an out of control car! What causes these “hits”? • Mutations in cells can be triggered by UV radiation chemical exposure radiation exposure heat cigarette smoke pollution age genetics •Apoptosis are born, Cells Cells are born, livelive for for a given a given period of time period and then die of time and then dieBowen, 1998 Bowen, 1998 APOPTOSIS --- Physiological cell death --- Cell suicide --- Cell deletion --- Programmed cell death Cell Death • The body is very good at maintaining a constant number of cells. So there has to exist mechanisms for ensuring other cells in the body are removed, when appropriate. • Two forms • Apoptosis - suicide - programmed cell death • Necrosis - killing - decay and destruction Apoptosis – Programmed Cell Death Apoptosis – Programmed Cell Death Necrosis vs. Apoptosis • Apoptosis – programmed cell death • Necrosis – un-programmed cell death Apoptotic cells are biochemically recognizable Characteristic biochemical changes in cells undergoing apoptosis 1. Chromosomal DNA cleaved into fragments 2. Change in the plasma membrane – phosphatidylserine (PS) in the outer leaflet 3. Loss of electrical potential across the inner membrane of the mitochondria 4. Relocation of cytochrome c from the intermembrane space of the mitochondria to the cytosol Cleavage of chromosomal DNA into a characteristic ladder of fragments Apoptosis Signaling What is a Caspase? • Single chain of pro-enzymes • Contains an N-terminal domain, a small subunit and a large subunit (similar to a ribosome) • Apoptotic stimulus Activation Substrate Cleavage Enzyme • Proteins which degrade other proteins are employed by apoptosis - caspases • Made as inactive precursors - procaspases • These are activated by other proteins when the right signal is received • One caspase cleaves the lamin proteins resulting in the irreversible breakdown of the nuclear membrane. 3 Types of Caspases • Inflammatory Caspases: -1, -4, and -5 • Initiator Caspases: -2, -8, -9, and -10 • Long N-terminal domain • Interact with effector caspases • Effector Caspases: -3, -6, and -7 • Little to no N-terminal domain • Initiate cell death Two Caspase Pathways Extrinsic Pathway Death Ligand Death Receptors Caspases Cell Death Cell-surface death receptors activate the extrinsic pathway of apoptosis The extrinsic pathway of apoptosis activated through Fas death receptors Two Caspase Pathways Intrinsic Pathway Mitochondria Cytochrome C Apoptosome Complex Caspases Cell Death The intrinsic pathway of apoptosis depends on mitochondria Apoptosis depends on an intracellular proteolytic cascade that is mediated by caspases Procaspase activation during apoptosis Detection of cell cycle by Flow Cytometry • Cell Cycle Analysis by Propidium Iodide (PI) Staining 2n 4n Detection of Apoptosis by Flow Cytometry • Early stage • Mid stage • Late stage Annexin V/7-AAD(PI) TUNEL assay < Go/G1 DNA content Annexin V: An Early Marker of Apoptosis One of the earliest indications of apoptosis is the translocation of the membrane phospholipid phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane. Once exposed to the extracellular environment, binding sites on PS become available for Annexin V, a 35-36 kDa, 2+ Ca -dependent, phospholipid binding protein with a high affinity for PS. • TUNEL(Terminal deoxynucleotidyl transferase-mediated dUTP nick-endlabeling) TUNEL assay Sub-G0/G1 • Apoptosis by Propidium Iodide (PI) Staining 2n 4n Any Questions??