Lecture 20 - Death & Development - programmed cell death Flashcards
(33 cards)
Why is death important in development?
- metamorphorsis (loss of tail tadpoles)
- Elimination of cells
- removal of excess cells
- sculpting
What is the difference between apoptosis & necrosis?
- Apoptosis is a ‘planned’ event
- Necrosis is normally a consequence of an event (trauma/lack of blood supply)
What occurs during Apoptosis?
- cell shrinkage/DNA fragmentation
- membrane blebbing
- formation apoptotic bodies/cell fragmentation
- NO INFLAMMATORY RESPONSE
What occurs during Necrosis?
- cell swelling
- membrane breakdown
- cell disintegration
- inflammatory response
What are the 3 phases of Apoptosis?
Specification phase - cell instructed to undergo apoptosis
Killing phase - apoptotic programme activated
Execution phase - cell dismantled & engulfed
This prevents an inflammatory response
What are the central enactors of Apoptosis?
- Caspases & proteolysis
CASPASE PROTEASES - cysteine in active site cleaves proteins after aspartic acid residue
-target nuclear lamins causing nuclear envelope breakdown
- components of the cytoskeleton
- cell adhesion proteins
- activate endonuclease
Not all caspases are involved in apoptosis
Continuously synthesised/present in cell
Cell is primed for apoptosis & this must be kept in check.
What is the process of Caspase activation?
Synthesised as inactive PROCASPASES.
The initiator caspases are in an inactive monomer form. These then form an inactive dimer. This leads to an active tetramer.
Autoproteolysis occurs when activated - activated initiator caspases then acts on executioner caspases by cutting them.
What is Caspase-activated DNAse?
- Caspase activated DNAse (CAD)
- Function as dimer, but folding of monomer requires a chaperone - inhibitor of CAD (ICAD)
- ICAD binds CAD & prevents CAD:CAD dimerization
- Caspases cleave ICAD, allowing CAD dimers –> DNA fragmentation
What is the extrinsic pathway - Triggering Apoptosis - events upstream of caspases?
- Death receptors found at plasma membrane
- Bind to death ligands (TRAIL, TNF)
- Receptors internalize & bind Adaptor protein (e.g. FADD)
- Adaptor binds to initiator caspase (bringing them into close proximity) allowing autoproteolysis
Dimerization of initiator caspases = Death-Inducing Signaling Complex (DISC)
What is the intrinsic pathway - Triggering Apoptosis - events upstream of caspases?
- Signal triggers mitochondrial cytochrome c release into cytosol
- Cyt c activates APAF1 (apoptotic protease activating factor - 1)
- Apaf1 oligomerises (Apaf1 form complex) & recruits initiator Procaspases
- Activated inhibitor caspases activate executioner caspases.
Cytochrome C doesn’t leak into the cytoplasm normally, as there are proteins in the mitochondrial membrane. Some of these proteins initiate apoptosis, whilst some stop it.
What are the pro & anti-apoptotic factors that regulate mitochondrial membrane permeability - involved in triggering apoptosis?
BLC-2 family of pro & anti Apoptotic proteins
Healthy:
BLC-2 bind BOX/BAK in mitochondrial membrane
Apoptotic signal:
BH3 domain displaces BCL-2 - BAX/BAK oligomerize & form pore permeabilizing membrane.
This leads to a pore forming in the mitochondrial membrane, which allows leakage of cytochrome C.
What is an overview of key events?
Extrinsic:
- External signal - e.g. FAS-L, TNF
- Receptor binding
- DISC assembly
- Activation initiator caspases
- Executioner caspase cascade
- Proteolysis key targets
Intrinsic:
- Low signal - e.g. low oxygen/DNA damage
- Mitochondrial disruption
- Cytochrome C release
- Activation initiator caspases
- Executioner caspase cascade
- Proteolysis
What is apoptosis in C. elegans development?
Some of the cells produced during early embryogenesis were then killed later in development - not all cells contribute to the adult body plan - some are killed.
- 20% of developed neural cells undergo apoptosis in C. elegans
What are mutants in the killing phase?
Dominant egl-1 mutants are defective in egg laying
- All Hermaphrodite-specific neurons (HSN) undergo apoptosis which they shouldn’t do - leading to eggs-laying behaviour failing.
Loss of function in ced-3 or ced-4 leads to cell survival.
ced-1 mutant - dying cells persist longer than usual. Easily visible.
ced-1 ced-3 mutant. No dying cells visible.
ced-4 mutants similar phenotype - no dying cells.
What is Ced-1 involved in?
Clearing dead cells away. This made it easy to identify genes involved in earlier part of the pathway.
What is Ced-3 & Ced-4 required for?
must be required to kill cells (apoptosis)
How are Ced-3 & Ced-4 controlled?
Dominant ced-9 mutants resembled ced-3/4 mutant
- extra cells found indicating cell survival
- ced-9 inhibits apoptosis
Eg-1 mutant lacks HSN cells
- Ced-9 rescues HSN c
cell death in Egl1/Ced-9 double mutant
Something must control Ced3/4, as they promote apoptosis. Identified another mutant through a mutagenesis experiment - Ced-9
ced-9 —I ced-3/4 —> cell death
Describe different Ced’s (as seen in the KILLING PHASE)
Pro-apoptotic:
Egl-1
Ced-3
Ced-4
Anti-apoptotic:
Ced-9
What does activation of Ced-9 lead to?
Ced-9:
on = cell survival
off = cell death
Explain the components of the killing phase
Promotes cell death:
Egl-1 - BH3 motif
Ced-4 - ApaF1
Ced3 - CASPASE
Inhibits cell death:
Ced-9 - similar to human proto-oncogene blc-2
Egl-1 –I ced-9 –I ced-4 –> ced-3
What is the molecular explanation for dominant vs recessive alleles?
Recessive might have a premature stop codon (truncated version) - leading to inactive version. Dominant allele often act upon proteins key for degradation.
Describe apoptosis during sculpting
Inter digit webbing in mammalian limb development
What are caspases?
proteases that initiate the breakdown of cells.
Where are anti-apoptotic markers in digits?
Bcl-2 expression is detected in developing digits (e.g. arrow) but not inter-digit tissue
Expression of something in an area doesn’t mean it is significant, as correlation isn’t causation.
