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APOPTOSIS: An overview
Sanjeev Sharma*, Aarti Bhardwaj$, Shalini Jain# and Hariom Yadav# *Animal Genetics and Breeding Division, #Animal Biochemistry Division, National Dairy Research Institute, Karnal , Haryana, India $College of Applied Education and Health Sciences, Meerut, U.P.
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INTRODUCTION Cell death by injury -Mechanical damage
-Exposure to toxic chemicals Cell death by suicide -Internal signals -External signals
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Apoptosis plays a major role from embryonic development to senescence.
Conted….. Apoptosis or programmed cell death, is carefully coordinated collapse of cell, protein degradation , DNA fragmentation followed by rapid engulfment of corpses by neighbouring cells (Tommi, 2002) Essential part of life for every multicellular organism from worms to humans. (Faddy et al.,1992) Apoptosis plays a major role from embryonic development to senescence.
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Why should a cell commit suicide?
Apoptosis is needed for proper development Examples: The resorption of the tadpole tail The formation of the fingers and toes of the fetus The sloughing off of the inner lining of the uterus The formation of the proper connections between neurons in the brain Apoptosis is needed to destroy cells Cells infected with viruses Cells of the immune system Cells with DNA damage Cancer cells
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What makes a cell decide to commit suicide?
Withdrawal of positive signals examples : growth factors for neurons Interleukin-2 (IL-2) Receipt of negative signals increased levels of oxidants within the cell damage to DNA by oxidants death activators : Tumor necrosis factor alpha (TNF-) Lymphotoxin (TNF-β) Fas ligand (FasL)
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History of cell death / apoptosis research
1800s Numerous observation of cell death 1908 Mechnikov wins Nobel prize (phagocytosis) Studies of metamorphosis Cell death in chick limb & exploration of NGF 1955 Beginning of studies of lysomes Necrosis & PCD described 1971 Term apoptosis coined 1977 Cell death genes in C. elegans DNA ladder observed & ced-3 identified Apoptosis genes identified, including bcl-2, fas/apo1 & p53, ced-3 sequenced (Richerd et.al., 2001)
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Necrosis When the cell membrane is damaged
Necrosis occurs when cells are exposed to extreme variance from physiological conditions (e.g., hypothermia, hypoxia) which may result in damage to the plasma membrane. Under physiological conditions direct damage to the plasma membrane is evoked by agents like complement and lytic viruses.
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Apoptosis Apoptosis is a mode of cell death that occurs under normal physiological with no damage to cell membrane conditions and the cell is an active participant in its own demise (“cellular suicide”). It is most often found during normal cell turnover and tissue homeostasis, embryogenesis, induction and maintenance of immune tolerance, development of the nervous system endocrine-dependent tissue atrophy.
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Necrosis vs. Apoptosis Necrosis Apoptosis Cellular swelling
Membranes are broken ATP is depleted Cell lyses, eliciting an inflammatory reaction DNA fragmentation is random, or smeared In vivo, whole areas of the tissue are affected Cellular condensation Membranes remain intact Requires ATP Cell is phagocytosed, no tissue reaction Ladder-like DNA fragmentation In vivo, individual cells appear affected
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NECROSIS Vs APOPTOSIS Wilde, 1999
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STAGES OF APOPTOSIS Induction of apoptosis related genes, signal transduction Sherman et al., 1997
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APOPTOSIS: Morphology
organelle reduction membrane blebbing & changes cell shrinkage mitochondrial leakage nuclear fragmentation chromatin condensation Hacker., 2000
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membrane blebbing & changes
mitochondrial leakage organelle reduction cell shrinkage nuclear fragmentation chromatin condensation APOPTOSIS: Morphological events
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Blebbing & Apoptotic bodies
The control retained over the cell membrane & cytoskeleton allows intact pieces of the cell to separate for recognition & phagocytosis by MFs Apoptotic body MF
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Caenorhabditis elegans
1090 cells 131 cells apoptosis decision to die engulfment degradation execution ced-3 ced-4 ced-1 ced-2 ced-5 ced-6 ced-7 ced-10 nuc-1 egl-1 ced-9 ces-2 ces-1
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Mitochondria/Cytochrome C
Apoptosis: Pathways “Extrinsic Pathway” Death Ligands Death Receptors Initiator Caspase 8 Effector Caspase 3 PCD “Intrinsic Pathway” DNA damage & p53 Initiator Caspase 9 Mitochondria/Cytochrome C
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MAJOR PLAYERS IN APOPTOSIS
Caspases Adaptor proteins TNF & TNFR family Bcl-2 family
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Ligand-induced cell death
Ligand Receptor FasL Fas (CD95) TNF TNF-R TRAIL DR4 (Trail-R)
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Ligand-induced cell death
“The death receptors” Ligand-induced trimerization FasL Trail TNF Death Domains Death Effectors Induced proximity of Caspase 8 Activation of Caspase 8
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APOPTOSIS: Signaling & Control pathways I
Externally driven Externally driven Apoptotic signals Activators of initiator enzymes p53 Cytochrome C Internally driven Initiator caspases 6, 8, 9,12 mitochondrion Execution caspases , 3, 7 Apoptosis events Activation
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APOPTOSIS: Signaling & Control pathways II
Externally driven Externally driven Inhibitors Apoptotic signals Activators of initiator enzymes p53 Cytochrome C Internally driven Bcl2 Survival factors External Initiator caspases 6, 8, 9,12 Internal Execution caspases , 3, 7 Inhibitors of apoptosis Apoptosis events Inhibition
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The mitochondrial pathway
Growth factor receptors DNA damage Fas Casp8 PI3K p53 Akt Bid casp3 BAD Bid Bax IAPs casp9 Bid Bcl2 Apaf1 ATP casp3 Bax Cyt.C Smac/ DIABLO H2O2 AIF Pollack etal., 2001
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REGULATION OF APOPTOSIS
Stimuli apoptosis selection of targets (Rich et al., 2000) Apoptosis by conflicting signals that scramble the normal status of cell (Canlon & Raff, 1999) Apoptotic stimulicytokines, death factors (FasL) (Tabibzadeh et al., 1999) DNA breaks p53 is activated arrest cell cycle or activate self destruction (Blaint & Vousden, 2001)
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Importance of Apoptosis
Important in normal physiology / development Development: Immune systems maturation, Morphogenesis, Neural development Adult: Immune privilege, DNA Damage and wound repair. Excess apoptosis Neurodegenerative diseases Deficient apoptosis Cancer Autoimmunity
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FUTURE PERSPECTIVES The biological roles of newly identified death receptors and ligands need to be studied Need to know whether defects in these ligands and receptors contribute to disease
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CONCLUSION an important process of cell death
can be initiated extrinsically through death ligands (e.g. TRAIL, FasL) activating initiator caspase 8 through induced proximity. can be initiated intrinsically through DNA damage (via cytochrome c) activating initiator caspase 9 through oligomerization. Initiator caspases 8 and 9 cleave and activate effector caspase 3, which leads to cell death.
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Programed Cell Death helps maintain Health
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DNA DAMAGE p53
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The bcl-2 family BH4 BH3 BH1 BH2 TM N C Receptor domain Raf-1 Membrane
calcineurin Membrane anchor Ligand domain Pore formation phosphorylation Group I Group II Group III Bcl-2 bax Bad bid bik Back
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P53 & Apoptosis If the damage is too extensive then p53
p53 first arrests cell growth between G1 S This allows for DNA repair during delay If the damage is too extensive then p53 induces gene activation leading to apoptosis (programmed cell death) BACK
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3 mechanisms of caspase activation
a. Proteolytic cleavage e.g. pro-caspase 3 b. Induced proximity, e.g. pro-caspase 8 c. Oligomerization, e.g. cyt c, Apaf-1 & caspase 9 Back
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CTL Virally infected cell Apoptosis signal to kill infected cells
Cytolytic lymphocyte/CTL (& natural killer lymphocyte) presents Fas ligand/CD178 on its surface to tell the infected cell to die Fas ligand CTL Virally infected cell Externally driven Apoptotic signals Cytochrome c Initiator caspases The immunological synapse holds the cells much tighter together than shown here Execution caspases Apoptosis events Fas/ CD95 is the ‘death receptor’
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