Mechanism of Cell Death: Apoptosis, Autophagy, and Necrosis Flashcards
Apoptosis
The cell itself senses that there is something wrong with it and it decides to kill itself
Autophagy
Catabolic process where cell physically eats itself.
Necrosis
Premature death of cell by external factors accident or murder
Normal Functions of Apoptosis
- Normal development: cell death needed for tissue sculpting
- Immune system- removal of B and T cells after infection
- Maintain homeostasis: balance between proliferation and cell death
- Eliminating cells damaged by UV, chemical toxins, or viral infections
Inhibiting apoptosis results in:
- Syndactily: finger webbing
- Abnormal brain development
- Tail resorption
Morphological markers of Apoptosis
- Electron dense nuclei
- Nuclear fragmentation
- Intact cell membrane but there is blebbing due to actin cytoskeleton degradation
- Disorganized cytoplasmic organelles.
- Large clear vacuoles
- Loss of cell-cell adhesion
- Apoptotic bodies (contain DNA)
- Chromatin condensation (DAPI)
- Cell shrinkage
Phosphotidyl Serine
- In early stages of apoptosis , phosphotidyl serine which is normally on cytoplasmic side flips to the outside of the cell.
- Can be stained with Anexin V antibody coupled with fluorophor
- Can use flow cytometry to see number of cells undergoing apoptosis by using column that contains Anexin 5 antibody to phosphotidyl serine
TUNEL assay
- Normal DNA is not nicked
- DNA that is in process of getting degraded has nicks
*Terminal transferase is used to add labeled dUTP to
the 3’end of the DNA fragments.
TUNEL assay Advantages and Disadvatages
Advantages
*High sensitivity so can detect fewer than 100 cells that are undergoing cell death
*Fast: can be completed in 3 hours
*High reproducibility with good precision
Disadvatages
*We don’t know minimum number of strand breaks necessary for detection so you may miss early stages of apoptosis
*Necrotic cells also have nicks so can’t really tell the difference
*Detergent used to permeabilize cells can make cell fragile
DNA laddering
If you take DNA from apoptosis and smear it, you get laddering with fragments of 180-185 bp due to DNA being cleaved by caspaces at the internucleosomal linker region
Initiator Caspaces
- 2,8,9,10
- Give order to have the effector capsules kill the cell
Effector Caspaces
- 3,6,7
- Caspaces kill the cell
Caspace activation
- Initiator caspaces are cleaved in two spots and become activated.
- Initiator caspaces cleave effector caspaces in 2 spots which activate them.
- Effector caspaces cleave:
*nuclear lamins
*inhibitors of DNAses
*actin
*Inactive caspace= 33 kd *Active caspace= 17 kd
How does caspace activation result in DNA fragmentation?
-Executioner caspaces 3 and 7 cleave DFF45 and DFF40 dimer (DNA fragmentation factor)
*In dimer form, they are inactive.
- DFF45 is released from DFF40 when executioner caspaces 3 and 7 cleave
- DFF40 oligomerizes and in that form, it is an active DNAse
- Oligomeric DFF40 cleaves DNA at the intranucleosomal area
- DNA fragments of 180-185 bp typically resulting in a DNA ladder on agarose gel
Intrinsic Apoptotic pathway
- Some signal tells cell to kill itself
- DNA damage is sensed by p53 -P53 up-regulates PUMA/NOXA
- PUMA/NOXA activate BAX and BAK (pro-apoptotic)
- BAK and BAX antagonize BLC-2 and bind to mitochondria making them leak and causing release of cytochrome C
- Cytochrome C activates APAF1 -APAF1 (apoptosis peptidase activating factor) form apoptosome
- Apoptosome activates initiator caspase 9
- Initiator capsace activates effector capsaces 3,6,7
- Caspaces 3,6,7 result in apoptosis