JM Lecture 57: Apoptosis

Question 1

How does necrosis differ from apoptosis?

Answer 1

necrosis- cells and organelles rupture inducing inflammatory response
apoptosis- cells shrink and condense, remnants are phagocytosed by neighboring cells or macrophages

Question 2

List the methods for measuring apoptosis.

Answer 2

1. stained with apo-orange to visualize morphology
2. DNA ladder on gel
3. flow cytometry
4. TUNEL
5. annexin V and phosphatidyl serine flip

Question 3

Describe the TUNEL assay.

Answer 3

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 fluorescent tag

Question 4

Describe how the annexin V method for assessing apoptotic cells works.

Answer 4

phosphatidyl serine is normally localized to the intracellular side of cells but flips during apoptosis (to notify macrophages)
Annexin V is a fluorescently labeled dye that will bind to phosphatidyl serine

Question 5

List some triggers of apoptosis.

Answer 5

glucocorticoids and antibody binding to cell surface (in T-cells)
ligands binding to specialized cell surface receptors
DNA damage (e.g. ionizing radiation, UV light, genotoxic drugs
oncogene activation
withdrawal from growth factors

Question 6

What are caspases?

Answer 6

zymogens
initiator caspases (caspase-8 and -9)- cleave and activate executioner caspases
executioner caspases (-2, -3, -6, -7)- cleave cellular substrates

Question 7

List some substrates for executioner caspases.

Answer 7

proteins involved in...
DNA degradation (CAD/ICAD)
DNA repair (PARP)
cytoskeleton (gelsolin)
nuclear structure (lamins)

Question 8

Go through the extrinsic pathway for apoptosis.

Answer 8

extrinsic signal + cell surface receptors –> initiation + caspase 8 –> execution –> death –> engulfment

Question 9

Go through the intrinsic pathway for apoptosis.

Answer 9

intrinsic signal + mitochondria bcl-2 family protein –> initiation + caspase 9 –> execution –> death –> engulfment

Question 10

List relevant ligand triggers for the extrinsic pathway.

Answer 10

Fas-Ligand
Tumor Necrosis Factor (TNF)
TRAIL

Question 11

What is the halmark of intrinsic apoptosis?

Answer 11

loss of mitochondrial transmembrane potential (diagnostic of the opening of permeability-transition pores)

Question 12

How is procaspase-9 activated?

Answer 12

when cytochrome C is released from the mitochondria and interacts with APAF-1 and dATP it forms a holoenzyme that can cleave procaspase-9

Question 13

What are the kinds of proteins in the Bcl-2 family?

Answer 13

anti-apoptotic Bcl2 (e.g. Bcl-2 and Bcl-XL)
pro-apoptotic BH123 (e.g. Bax and Bak)
pro-apoptotic BH3-only (e.g. Bad, Bim, Puma, Noxa, Bid)

Question 14

Describe the relationship between the three kinds of proteins in the Bcl-2 family.

Answer 14

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

Question 15

Why is apoptosis important for development?

Answer 15

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