Apoptosis

Apoptosis, also known as programmed cell death, is a controlled and orderly process by which cells self-destruct in a regulated manner, typically in response to signals that indicate the cell is no longer needed or is damaged beyond repair. Unlike necrosis, which is a form of traumatic cell death resulting from acute injury, apoptosis is a beneficial and necessary process for development, immune function, and tissue homeostasis. For instance, during development, apoptosis sculpts tissues by eliminating cells between forming digits to prevent webbing.

The process of apoptosis involves several key features:

1. **Caspase Activation**: Caspases are a family of protease enzymes that play a central role in apoptosis. They are activated in a cascade, where initiator caspases activate executioner caspases, leading to the systematic dismantling of the cell.

2. **Cytochrome c Release**: In the intrinsic pathway of apoptosis, the release of cytochrome c from mitochondria into the cytosol triggers the formation of the apoptosome, a heptameric complex involving Apaf-1, which activates caspase-9 and initiates the caspase cascade.

3. **DNA Fragmentation**: Apoptosis is characterized by the fragmentation of DNA into nucleosome-sized fragments, creating a "ladder" pattern visible on a gel electrophoresis, due to the activity of enzymes like CAD (caspase-activated DNase).

4. **Membrane Blebbing and Formation of Apoptotic Bodies**: As the cell progresses through apoptosis, the plasma membrane forms protrusions called "blebs," and the cell breaks apart into membrane-bound fragments known as apoptotic bodies. These are then phagocytosed by immune cells, preventing inflammation.

5. **Detection Methods**: Apoptosis can be detected by assays such as TUNEL (which labels DNA fragments), Annexin V staining (which binds phosphatidylserine on the cell membrane), and assays for caspase activity.

### Common Clues Across Questions

1. **Caspase Cascade**: Activation of caspases, particularly through a cascade, is a hallmark of apoptosis. Caspases are proteases that cleave specific cellular proteins to dismantle the cell in a controlled way.

2. **Cytochrome c Release**: In the intrinsic pathway, cytochrome c is released from mitochondria, which leads to the formation of the apoptosome and initiates the caspase activation cascade.

3. **DNA Laddering**: DNA fragmentation into nucleosome-sized pieces results in a ladder pattern on a gel, a distinctive feature of apoptosis that is absent in necrosis.

4. **Membrane Blebbing**: The formation of membrane blebs and apoptotic bodies is a visible morphological feature of apoptosis.

5. **Intrinsic vs. Extrinsic Pathways**: Apoptosis can be triggered via the intrinsic (mitochondrial) pathway or the extrinsic (death receptor-mediated) pathway. The extrinsic pathway involves death receptors like Fas on the cell surface binding to their ligands.

6. **Assays**: Common assays for detecting apoptosis include TUNEL (labels DNA breaks), Annexin V (binds phosphatidylserine exposed on the outer cell membrane), and caspase activity assays.

### Related Quizbowl Facts

Fill in the blanks with key terms related to apoptosis:

1. During apoptosis, a family of cysteine proteases called ___1___ cleaves cellular proteins and orchestrates cell dismantling.

2. In the intrinsic pathway, mitochondria release ___2___ into the cytoplasm, which binds Apaf-1 to form the apoptosome.

3. DNA fragmentation, known as ___3___, results in a ladder pattern when visualized on an agarose gel.

4. ___4___ are visible protrusions on the cell membrane during apoptosis, leading to the formation of apoptotic bodies.

5. The ___5___ pathway of apoptosis is triggered by signals from within the cell, while the ___6___ pathway is activated by external death ligands binding to receptors.

6. The ___7___ assay labels DNA fragments to detect cells undergoing apoptosis by marking nicked DNA ends with labeled nucleotides.

#### Answer Key for Practice

1. caspases

2. cytochrome c

3. laddering

4. blebs

5. intrinsic

6. extrinsic

7. TUNEL

These clues focus on the molecular and cellular mechanisms of **apoptosis**, or programmed cell death, an essential biological process that removes damaged, infected, or unnecessary cells to maintain health and development. Here’s a breakdown of each clue with mentions included:

1. **Cytochrome c release from mitochondria (45 mentions)**: This step is central to the **intrinsic pathway** of apoptosis, where **cytochrome c** is released from mitochondria into the cytoplasm, triggering the formation of the apoptosome and the subsequent activation of caspases.

2. **Activation of caspases (cysteine proteases) (41 mentions)**: **Caspases** are the main executioners of apoptosis. These proteases degrade cellular components, leading to cell dismantling in a controlled manner.

3. **Programmed cell death / Apoptosis (39 mentions)**: Apoptosis is a form of programmed cell death crucial for development, immune function, and preventing diseases like cancer. It contrasts with necrosis, which is uncontrolled cell death due to injury.

4. **DNA laddering as an indicator (32 mentions)**: During apoptosis, DNA is cleaved in a characteristic pattern, producing a “ladder” on gel electrophoresis, which serves as a hallmark of apoptotic DNA fragmentation.

5. **Membrane blebbing (30 mentions)**: This refers to the bubbling of the cell membrane as the cell dismantles during apoptosis, forming small protrusions that are characteristic of this form of cell death.

6. **Detection assays: TUNEL assay, Annexin V binding (26 mentions)**: **TUNEL** labels fragmented DNA, while **Annexin V** binds to phosphatidylserine, a marker on the cell surface during apoptosis. These assays are commonly used to detect apoptotic cells in research and clinical settings.

7. **Bcl-2 family proteins (e.g., BAX, BAK, Bcl-2) (24 mentions)**: The **Bcl-2 family** includes both pro-apoptotic proteins (like **BAX** and **BAK**) and anti-apoptotic proteins (**Bcl-2**), which regulate mitochondrial membrane permeability and cytochrome c release, making them key regulators of apoptosis.

8. **Intrinsic vs. extrinsic pathway (e.g., Fas ligand, Apaf-1) (20 mentions)**: The **intrinsic pathway** is triggered internally by signals like DNA damage, involving the mitochondria, while the **extrinsic pathway** is initiated by external signals (e.g., **Fas ligand**) binding to death receptors on the cell surface.

9. **C. elegans specific cell death events (exact cell count) (18 mentions)**: The nematode **C. elegans** has a precisely defined pattern of cell death, making it a model organism for studying apoptosis due to the predictability of which cells will undergo programmed cell death.

10. **p53 involvement in response to DNA damage (17 mentions)**: The **p53** protein is a tumor suppressor that can induce apoptosis in response to DNA damage, preventing the proliferation of potentially cancerous cells.

11. **Phosphatidylserine exposure on cell membrane (15 mentions)**: During apoptosis, **phosphatidylserine** flips from the inner to the outer leaflet of the plasma membrane, signaling phagocytic cells to engulf the dying cell.

12. **Cancer and failure of apoptosis (13 mentions)**: Apoptosis plays a critical role in preventing cancer by removing damaged cells. When apoptosis fails, cells with DNA damage can survive, leading to uncontrolled cell proliferation and tumor formation.

13. **Apoptotic bodies and cell fragmentation (12 mentions)**: As cells undergo apoptosis, they break into smaller membrane-bound fragments called apoptotic bodies, which are then cleared by phagocytes to prevent inflammation.

14. **Role in development, such as separation of fingers and toes (10 mentions)**: Apoptosis is essential for proper development, like the sculpting of fingers and toes during embryonic growth by removing cells in between them.

15. **DISC complex (Death-Inducing Signaling Complex) (8 mentions)**: In the extrinsic pathway, the **DISC** forms in response to death receptor activation, leading to the activation of caspase-8 and downstream executioner caspases, initiating apoptosis.

16. **Tumor Necrosis Factor (TNF) and Fas ligand binding to Fas receptors** - 8 mentions  

   - These molecules are crucial in the *extrinsic pathway* of apoptosis. When TNF or Fas ligand bind to their respective receptors on the cell surface, they trigger a cascade that ultimately activates downstream caspases, leading to cell death.

17. **CED genes in C. elegans (e.g., CED-3, CED-4)** - 7 mentions  

   - These genes are essential in *programmed cell death* in *C. elegans*, a model organism. *CED-3* and *CED-4* proteins are homologous to caspases and Apaf-1 in mammals, playing a central role in apoptosis regulation.

18. **Apaf-1 (apoptotic protease activating factor 1)** - 7 mentions  

   - In the *intrinsic pathway*, Apaf-1 is a key protein that interacts with cytochrome c released from mitochondria to form the apoptosome, which activates *caspase-9* and promotes cell death.

19. **Detection via Annexin V (phosphatidylserine binding)** - 7 mentions  

   - Annexin V is commonly used to detect *apoptosis* because it binds to *phosphatidylserine*, a molecule that flips to the outer membrane surface in apoptotic cells, signaling phagocytes for cell clearance.

20. **CARD domains in apoptotic signaling** - 6 mentions  

   - *Caspase Activation and Recruitment Domains (CARDs)* are protein interaction motifs that help in caspase activation and signal transduction during apoptosis, especially in forming complexes like the apoptosome.

21. **BID and BAD proteins (pro-apoptotic Bcl-2 family members)** - 6 mentions  

   - These *Bcl-2 family* proteins promote apoptosis by facilitating mitochondrial membrane permeabilization, releasing apoptotic factors like cytochrome c into the cytosol.

22. **Activation of apoptosis by p53 in response to irreparable DNA damage** - 6 mentions  

   - The tumor suppressor *p53* induces apoptosis when DNA damage is beyond repair, acting as a safeguard against potential malignancies by activating pro-apoptotic genes.

23. **Difference from necrosis (apoptosis is orderly, necrosis is not)** - 6 mentions  

   - Apoptosis is a controlled and orderly process that avoids inflammatory responses, unlike *necrosis*, which results from cell injury and causes inflammation.

24. **Initiator and executioner caspases** - 5 mentions  

   - *Initiator caspases* (like caspase-8 and caspase-9) begin the apoptotic process, while *executioner caspases* (like caspase-3 and caspase-7) carry out the dismantling of cellular components.

25. **Mitochondrial outer membrane permeabilization (MOMP)** - 5 mentions  

   - MOMP is a key step in the *intrinsic pathway*, allowing the release of cytochrome c and other factors from mitochondria that lead to apoptosis.

26. **Role of apoptosis in immune function (e.g., lymphocyte regulation)** - 5 mentions  

   - Apoptosis helps maintain immune balance by eliminating autoreactive or unnecessary lymphocytes, preventing autoimmune responses.

27. **Inhibition by IAP (inhibitor of apoptosis proteins)** - 5 mentions  

   - *IAP proteins* prevent apoptosis by directly inhibiting caspases, ensuring cells only undergo apoptosis under the appropriate circumstances.

28. **Apoptosis triggered by oxidative stress or reactive oxygen species** - 5 mentions  

   - *Oxidative stress* can damage cellular components, often triggering apoptosis through mitochondrial pathways to prevent compromised cells from causing harm.

29. **Cleavage of PARP (Poly [ADP-ribose] polymerase) during apoptosis** - 5 mentions  

   - PARP cleavage by caspases is a hallmark of apoptosis, signaling the final stages of cellular disassembly and aiding in DNA fragmentation.

30. **Role in sculpting tissues during development (e.g., embryonic shaping)** - 4 mentions  

   - Apoptosis is essential in shaping tissues during development, like digit separation in the embryo, by selectively removing cells in precise patterns.

31. **Extrinsic and intrinsic apoptotic pathways** - 4 mentions  

   - Apoptosis can be activated by both *extrinsic* (death receptor-mediated) and *intrinsic* (mitochondrial) pathways, depending on internal or external cellular signals.

32. **Role of caspase-9 in the intrinsic pathway** - 4 mentions  

   - Caspase-9 is activated within the apoptosome in the intrinsic pathway, setting off a chain of caspase activations that leads to cell dismantling.

33. **Flipping of membrane phospholipids (e.g., phosphatidylserine exposure)** - 4 mentions  

   - The externalization of *phosphatidylserine* serves as an “eat me” signal for phagocytes to engulf apoptotic cells without triggering inflammation.

34. **Regulation of apoptosis by the PI3K/Akt pathway** - 4 mentions  

   - The *PI3K/Akt pathway* is a survival signaling pathway that inhibits apoptosis by activating anti-apoptotic proteins, thus balancing cell survival and death.

35. **Use of flow cytometry in apoptosis detection** - 4 mentions  

   - Flow cytometry is commonly employed to quantify apoptotic cells by detecting markers like Annexin V or DNA fragmentation patterns.

36. **Blebs and cellular fragments recognized and phagocytosed** - 4 mentions  

   - During apoptosis, cells form *blebs* and break into fragments that are easily phagocytosed, allowing for non-inflammatory cell removal.

37. **Role of apoptosis in response to infections (e.g., viral proteins inhibiting apoptosis)** - 4 mentions  

   - Apoptosis serves as a defense mechanism to eliminate infected cells, although some viruses have evolved proteins to block apoptotic pathways, aiding their persistence.

38. **DAPI staining and DNA fragmentation** - 4 mentions  

   - *DAPI staining* helps visualize DNA fragmentation, a hallmark of apoptosis, by binding to DNA and highlighting nuclear changes under fluorescence.

39. **DISC formation in extrinsic apoptosis** - 3 mentions  

   - The *Death-Inducing Signaling Complex (DISC)* forms in response to extrinsic apoptotic signals and is key to activating initiator caspases in this pathway.

40. **Role of caspase-8 in extrinsic pathway** - 3 mentions  

   - Caspase-8 is the main initiator caspase in the extrinsic pathway, linking receptor activation to downstream apoptosis signaling.

41. **Apoptosis induction by external signals (e.g., Fas ligand and TNF-alpha)** - 3 mentions  

   - External signals, such as Fas ligand and TNF-alpha, engage death receptors, initiating apoptosis through the extrinsic pathway.

42. **Loss of mitochondrial membrane potential** - 3 mentions  

   - Mitochondrial dysfunction, marked by a loss in membrane potential, is a critical step in the intrinsic pathway that leads to cytochrome c release and apoptosis.

43. **Involvement of SMAC/DIABLO in inhibiting IAPs** - 3 mentions  

   - *SMAC/DIABLO* proteins released from mitochondria bind to and inhibit IAPs, facilitating caspase activation and promoting apoptosis.

44. **Involvement of XIAP as an IAP in regulating caspases** - 3 mentions  

   - *XIAP* is a key inhibitor of caspases, tightly regulating apoptosis by preventing premature cell death.

45. **Engulfment of apoptotic cells by phagocytes** - 3 mentions  

   - Phagocytes recognize apoptotic cells and engulf them, ensuring they are removed cleanly without an inflammatory response.

46. **Induction by hypoxia or nutrient deprivation** - 3 mentions  

   - Cells under stress from low oxygen (hypoxia) or lack of nutrients may undergo apoptosis as a means to prevent damaged cells from accumulating.

47. **Involvement of mitochondrial cardiolipin in apoptosis** - 3 mentions  

   - Cardiolipin, a mitochondrial phospholipid, can facilitate apoptosis by helping recruit cytochrome c to the outer mitochondrial membrane for release.

48. **DNA condensation and chromatin margination** - 3 mentions  

   - DNA undergoes condensation and chromatin migrates to the nuclear periphery, a characteristic feature of cells undergoing apoptosis.

49. **TdT-mediated dUTP Nick-End Labeling (TUNEL assay) detects DNA breaks** - 3 mentions  

   - The *TUNEL assay* is a technique for identifying apoptotic cells by labeling DNA strand breaks, a common indicator of apoptosis. 

These components collectively illustrate apoptosis as a regulated cellular process essential for development, disease prevention, and tissue homeostasis.