Apoptosis- Lecture 57 Flashcards
Describe the defining features of apoptosis.
cell membrane intact
dead cells phagocytosed and eliminated quickly
organism is unaware
Describe the defining features of necrosis.
cell membrane disrupts
induces inflammatory response
What are the methods for measuring apoptosis?
- stained with apo-orange to visualize morphology
- DNA ladder on gel
- flow cytometry
- TUNEL
- annexin V and phosphatidyl serine flip
Describe the TUNEL assay.
breaks in DNA found during apoptosis can be labeled using bacterial enzyme terminal deoxynucleotidyl transferase
biotinylated dUTP is used in the reaction and can be readily detected using a streptavidin with a fluorescent tag
Describe how the annexin V method for assessing apoptotic cells works.
phosphatidyl serine is normally localized to the intracellular side but flips during apoptosis (to notify macrophages)
Annexin V is a fluorescently labled dye that will bind to phosphatidyl serine
Name the types of caspases.
initiator (8 and 9)
executioner (2, 3, 6, and 7)
What are the substrates for executioner caspases?
proteins involved in DNA degradation (CAD/ICAD), DNA repair (PARP), the cytoskeleton (gelsolin, adn nuclear structure (laminins)
all of these lead to apoptosis of the cell
Describe the intrinsic pathway of apoptosis.
signal (mitochondria bcl-2 family) –> initiation (caspase 9 is activated by holoenzyme composed of cytochrome C, APAF-1 and dATP) –> execution –> death –> engulfment
Describe the extrinsic pathway of apoptosis.
signal (ligand induced trimerization of cell surface receptor) –> initiation (caspase 8 is activated by induced proximity with the cell surface receptor bringing together many procaspase 8 molecules) –> execution –> death –> engulfment
What are the relevant ligand triggers for the extrinsic apoptosis pathway?
Fas-ligand, Tumor Necrosis Factor (TNF), and TRAIL
What are the kinds of proteins in the Bcl-2 family?
anti-apoptotic Bcl2 (eg. Bcl-2 and Bcl-XL)
pro-apoptotic BH123 (eg. Bax and Bak)
pro-apoptotic BH3-only (eg. Bad, Bim, Bid, Puma, Noxa)
Describe the relationship between the three kinds of proteins in the Bcl-2 family.
Bid (BIH3 protein) activates Bax and Bak directly, leading to conformational change allowing them to insert into the mitochondrial membrane and create a pore for the release of Cytochrome C
Bcl-2 binds Bax or Bak and inhibits formation of the pore
PUMA and Noxa (BH3 only proteins) block Bcl-2 binding to Bax or Bak and allow for the pore formation
Why is apoptosis important for development?
formation of digits
hollowing out of solid structures to form lumina
deleting unneeded structures (vestigial structures)
control of cell numbers
elimination of abnormal, misplaced, non-functional, or harmful cells
