Cell Death Notes

Cell Death

Overview of Apoptosis

• Organisms can trigger apoptosis for a clean, orderly cell death.
• Apoptosis is programmed cell death.
• Role in Development:
  • Sculpting digits.
  • Metamorphosis.
• Functions:
  • Get rid of cells that are of no use (e.g., certain immune cells).
  • Tissue maintenance.
  • Recognition of damage.

Apoptosis vs. Necrosis

• Apoptosis:
  • Cells shrink, bleb, and are engulfed.
  • Cellular contents do not spill out into surrounding tissue.
  • Clean, non-harmful cell death.
• Necrosis:
  • Cells swell, burst, and spill contents into tissue.
  • Triggers inflammation.

Identifying Apoptotic Cells

• Imaging
• Agarose Gel Electrophoresis: Used to observe DNA fragmentation over time.
• Flow Cytometry: Used to quantify the amount of DNA per cell.
  • Cells undergoing apoptosis show a reduction in DNA content.

Hallmarks of Apoptotic Cells

• DNA condenses and fragments; the nuclear envelope breaks down.
• The cell shrinks and creates blebs, eventually fragmenting.
• The cytoskeleton disassembles, while organelles are preserved.
• Neighboring cells (e.g., macrophages) engulf the dead cell.

Caspases and Apoptosis

• Apoptosis is carried out by caspases, which are proteases (enzymes that cleave other proteins).
• Caspases exist as inactive procaspases in normal, healthy cells.
• Procaspases must be activated to trigger apoptosis.

Caspase Activation

• Caspases are activated by the cleavage of key domains.
• Cleavage leads to active site rearrangement.
• Activated caspases form larger complexes.

Procaspases vs. Active Caspases

• Procaspase: Inactive, full-size protein.
• Active Caspase: Cleaved, assembled caspases.

Initiator vs. Executioner Caspases

• Initiator caspases:
  • Cleave and activate executioners.
• Executioner caspases:
  • Cleave and destroy cellular proteins.
  • Can also cleave and activate more executioners, amplifying the death cascade.

Examples of Caspases

• Initiator caspases:
  • Caspase 8
  • Caspase 9
• Executioner caspases:
  • Caspase 3
  • Caspase 6
  • Caspase 7

Apoptotic Pathways

• Two main pathways activate apoptosis:
  • Intrinsic apoptotic pathway
  • Extrinsic apoptotic pathway

Intrinsic Apoptotic Pathway

• Cell triggers its own death internally.
• Involves mitochondrial membrane proteins.
• Can be triggered by developmental processes or when a cell senses damage.

Extrinsic Apoptotic Pathway

• Cell receives an extracellular signal.
• The signal binds to a death receptor to initiate a signaling cascade that leads to apoptosis.

Regulation of Intrinsic Apoptosis by Bcl2, Bax, and Bak

• Bcl2 (anti-apoptosis) inhibits Bax and Bak (pro-apoptosis).
• Multiple pathways can activate Bax and Bak, including DNA damage.
• Bcl2 \quad Bak \quad Bax
• Open pore; Bax and Bak activated; Bcl2 inactivated.

Cytochrome c and Intrinsic Apoptosis

• Cytochrome c is found in mitochondria in the intermembrane space.
• Cytochrome c can pass through the pore formed by Bax and Bak.
• When Bax/Bak form a pore, cytochrome c moves into the cytosol down its concentration gradient via passive transport (no energy required).

Apoptosome Assembly

• Released cytochrome c helps assemble the apoptosome.
  • Involves adaptor proteins and procaspase-9.
  • Adaptor proteins and cytochrome c recruit and activate procaspase-9, initiating a caspase cascade leading to apoptosis.

Extrinsic Apoptosis Pathway and Death Receptors

• An example of an extracellular death signal: Fas on killer lymphocytes.
• Fas binds the Fas death receptor on target cells.
• Fas binding brings together 3 receptor molecules for activation.

DISC: Death-Inducing Signaling Complex

• Activated Fas death receptor recruits adaptor proteins and procaspase 8.
• This assembly is called the DISC (Death-Inducing Signaling Complex).
• DISC activates caspase 8, which then activates executioner caspases.

Timing of Apoptosis

• The time from initial apoptosis signal to cell death can be hours to days.
  • Depends on cell type, tissue vs cell culture, type of death signal.
• Once cytochrome c is released:
  • Executioner caspases are activated within 10 minutes.
  • Cells begin blebbing in the hours that follow (often within an hour).

Cell Signaling and Regulation of Cell Cycle and Cell Death

• Survival factors suppress intrinsic apoptosis.
• Mitogens stimulate cell division by promoting progression through the phases of the cell cycle.
• Growth factors stimulate the growth of cells.

Survival Signals and Apoptosis

• Cells must receive survival signals to prevent intrinsic apoptosis.
• Survival signals lead to the expression of Bcl2, an inhibitor of apoptosis (IAP).
• Bcl2 inhibits apoptosis by inhibiting Bax and Bak.

Signaling and Cell Survival: Role of Bad and Akt

• Bad is an allosteric inhibitor of Bcl2.
• Akt phosphorylates and inactivates Bad, releasing Bcl2.
• Active Bcl2 promotes cell survival by inhibiting Bax and Bak.

Genetics and Apoptosis

• Likelihood of fraternal twins can run in families, connected to the mother.
• Two genes have been linked to an increased likelihood of fraternal twins: FSHB and SMAD3.
• SMAD3 regulates an inhibitor of apoptosis.
• Involves TGFB, RII, P-RI, IRS-1 P, XIAP, and Cyclin D1, which relate to survival, proliferation, and tumorigenesis.

Defects in Apoptosis and Developmental Disorders

• Defects in apoptosis can cause developmental disorders like syndactyly.
• Syndactyly is an inherited genetic disorder and one of the most common birth defects.
• Apoptosis is supposed to remove digit webbing between gestational weeks 6 to 8.

DNA Damage and Apoptosis

• p53 is activated when cells sense DNA damage.
• p53 activates the expression of p21.
• p21 is a G1/S-Cdk and S-Cdk inhibitor.
• DNA damage can pause the cell cycle at G1.
• p53 activates apoptosis in response to DNA damage; defects in p53 can lead to cancer.
• Network includes: ATM, ATR, Chk1/Chk2, MDM2, Bax, Bak, Bcl-2, Cytochrome c, APAF1, Caspase 9, Caspase 3, p21, Rb, E2F. Results in cell cycle arrest or apoptosis.