Apoptosis Flashcards
Describe the roles of apoptosis
Harmful cells (e.g. cells with viral infection, DNA damage).
- Developmentally defective cells (e.g. B lymphocytes expressing antibodies against self antigens).- lack of self tolerance
- Excess / unnecessary cells:- important in morphogenesis
(embryonic development: brain to eliminate excess neurons; liver regeneration; sculpting of digits and organs). - Obsolete cells (e.g. mammary epithelium at the end of lactation).- involution- returns to its dormant state
- Exploitation - Chemotherapeutic killing of cells.
What technique can be used to detect dying, apoptotic cells
The TUNEL technique
Define necrosis
Necrosis - unregulated cell death associated with trauma, cellular disruption and an inflammatory response
Define apoptosis
Apoptosis (programmed cell death) - regulated cell death; controlled disassembly of cellular contents without disruption; no inflammatory response
What is important to remember about the different types of cell death
There is a spectrum- and some types have characteristics of both necrosis and apoptosis.
Outline the process of necrosis
Plasma membrane becomes permeable
Cell swelling and rupture of cellular membranes
Release of proteases leading to autodigestion and dissolution of the cell- everything becomes dysregulates as the cell contents are no longer contained within the plasma membrane.
Localised inflammation
Basically the cell explodes.
Describe what happens upon trauma to the cell in necrosis
Trauma to the intestinal epithelium:
Cells and organelles swell
Chromatin condenses
Membrane compromised- fluid rushes in
Dissolution of cellular structures (proteases) Cell lysis (fluid rushing in) Invasion from phagocytic cells and inflammation Proliferation of neighbouring cells to restore the integrity of the epithelium.
Describe the latent phase of apoptosis
Latent phase – death pathways are activated, but cells
appear morphologically the same
Changes are at a molecular level- the cell’s programming is getting ready to apoptose.
Describe the excitation phase of apoptosis
Loss of microvilli and intercellular junctions (so impermeability of the cell is compromised)
Cell shrinkage (unlike the swelling of necrosis).
Loss of plasma membrane asymmetry
(phosphatidylserine lipid appears in outer leaflet)
Chromatin and nuclear condensation
DNA fragmentation
Formation of membrane blebs
Fragmentation into membrane-enclosed apoptotic
bodies
What is the charge of phosphatidylserine
Negative
What is a key feature of apoptosis that distinguishes it from necrosis
Plasma membrane remains intact – no inflammation
Outline the process of apoptosis
Faulty mitosis/excessive DNA damage- usually localised to one cell:
Latent phase
Microvilli contract
intercellular junctions break (impermeability compromised)
Chromatin begins to condense
Cell shrinks- neighbouring cells close around to restore impermeability
Chromatin condenses around nuclear periphery
cell blebs violently
chromatin condensation continunes
cell fragments into membrane-enclosed apoptotic bodies
apoptotic cell bodies phagoyctosed by neighbouring cells and roving macrophages
Describe the DNA modifications that occur during apoptosis
DNA ladders – fragmentation
(agarose gel)- DNA initially intact- too big to fit into channels- but as it condenses- we see fragmentations on the DNA ladder
“TUNEL” assay
DNA fragmentation leads to more “ends” which are labelled by adding an extra fluorescently-tagged base
Describe apoptosis like-PCD
Apoptosis-like PCD - some, but not all, features of apoptosis. Display of phagocytic recognition molecules before plasma membrane lysis
Describe necrosis like-PCD
Necrosis-like PCD - Variable features of apoptosis before cell lysis; “Aborted apoptosis”
What does the fact that there are other types of cell death (other than necrosis and apoptosis) suggest
o The fact that these other forms exist suggests a GRADED response of cell death.
Summarise the mechanisms of apoptotic cell death
o Caspase cascade – the executioners.
o Death response – death receptors and mitochondria- sequential activation of proteins to drive intracellular response
o Bcl-2 family -regulators
o Stopping the death programme.
What are caspases
Cysteine-dependent aspartate-directed proteases (so their cleavage site is between cysteine and aspartame)
Executioners of apoptosis
Activated by proteolysis (secreted as dimers or auto-folded- need to be cleaved by another protease to activate their
Cascade of activation- need both an initiator and effector capsase
State the initiator caspases
2,9,10 and 8
State the effector caspases
3,6 and 7
Describe the structure of initiator caspases
§ 2 subunits – p20 and P10 (P20 is larger)
§ They have an extra targeting subunit:
· CARD – Caspase Recruitment Domain ( 2 and 9)
· DED – Death Effector Domain ( 10 and 8)
o These direct them to a location.
Describe the structure of effector caspases
P20 and P10
What are the key differences between initiator caspases and effector caspases
Initiator caspases: contain N-terminal CARD (CAspase Recruitment Domain) or DED (Death Effector Domain) for homotypic protein-protein interactions (i.e caspase 8 can only interact with caspase 8)
Effector caspases do not contain protein-protein interaction domains.
o Initiator caspases – 2, 9, 8, 10 – TRIGGER APOPTOSIS.
o Effector caspases – 3, 6, 7 – CARRY OUT APOPTOSIS PROCESS.
What structures do caspases recognise
o A cysteine residue in the active site is required for their activity.
o They cut proteins after their aspartate residue.
