Lecture 20 - Apoptosis Flashcards
What is apoptosis often referred to as?
Programmed cell death or cell suicide
How is apoptosis cell death?
Via an active cellular response to extracellular or intracellular signals
What was studied to look at apoptosis as a developmental process?
- Salamander tail
- Removal of interdigital cells during digit formation
- Development of visual system and other neural circuits - neurons that fail to connect undergo apoptosis
- Regulation of tissue size by cell competition
- C. elegans development - the ced genes
What did Sidney Brenner develop?
C. elegans as a model system - early 60s
What did John Sulston’s lineae map reveal?
That many cells undergo programmed cell death - early 70s
What did Robert Horvitz screen for?
Genes that when mutated allow these cells to survive - ced genes - early 80s
How is apoptosis different than necrosis?
Apoptosis involves the activation of a cell death pathway while necrosis is a passive process resulting from damage to the cell
What are some features of apoptotic cells?
- Cytoskeleton collapse
- Nuclear envelope breaks down
- Chromatin condenses and breaks into fragments
- Cell surface blebbing
- Apoptotic bodies
- Apoptotic cells signal to phagocytic cells to be phagocytosed
What are some features of necrotic cells?
Swell, burst, and their cellular contents illicit an inflammatory response
How were apoptotic cells identified?
By incubation in Acridine Orange
- TUNEL labeling (label DNA ends with Terminal deoxynucleotidyl transferase): apoptotic cells have fragmented chromosomes
What apoptosis assays were done?
1) Plasma membrane loses activity
- Uptake of cell impermeable DNA dyes - Acridine orange
2) Chromatin breakage
- Ladder of DNA seen by gel electrophoresis
- TUNEL labeling
3) Membrane flipping
- Labeled Annexin5 - Phosphatidylserine flips to outside of membrane (acts as a signal for macrophages)
4) Mitochondrial activity is lost
- Positively charged fluorescent dyes that accumulate in active mitochondria - rhodamine 123
5) Pro-caspase cleavage
- Antibodies against cleaved caspases
What are caspases?
Proteases that mediate apoptosis made as pro-caspases
What keeps caspases inactive?
Pro-domain
What may mediate procaspase cleavage?
Another caspase
What are the classes of caspases?
1) Initiator (activator) caspases - Cleave other caspases
2) Effector (executioner) caspases - Cleave specific cellular targets to cause apoptosis
What does initiator caspase activation lead to?
Rapid amplification of a caspase cascade
What does caspase activation lead to?
Rapid and irreversible initiation of apoptosis
Activation of caspases: Intrinsic pathway
- Cellular response to various stresses, DNA damage, and depletion of survival factors
- BH-domain family of proteins regulate release of proteins from mitochondrial intermembrane space to activate procaspases
- Inhibitor caspases activated by apoptosome
Activation of caspases: Extrinsic pathway
- Apoptosis induced via a signal from specialized cells - e.g., cytotoxic T-lymphocytes
- Cell surface “death receptors” - e.g., Fas, TNF, TRAIL respond to TNF-related ligands
- Adaptor proteins interact with receptors and activate procaspases
How is the intrinsic pathway of apoptosis regulated by the BH-Domain family of domains?
- Proapoptotic Bax and other BH1,2,3 proteins from mitochondrial channels that promote the release of cytochrome c and other proteins from the intermembrane space of mitochondria
- Cytochrome c promotes the formation of apoptosome that in turn stimulates activator caspases
What induces the intrinsic pathway of apoptosis?
- Various stresses acting through p53
- External signals
What are the BH-domain proteins?
- Anti-apoptotic Bcl2 protein
- Pro-apoptotic BH123 protein
- Pro-apoptotic BH3-only protein
What do all Bcl2-related proteins (BH-domain proteins) have?
1 or more Bcl2 homology domains (BH1-4)
What do the anti-apoptotic Bcl2 proteins do?
Inhibit BH123 pore formation
