Apoptosis Study Guide

📖 Core Concepts Apoptosis – programmed cell death; tightly regulated, produces apoptotic bodies that are phagocytosed without inflammation. Intrinsic (mitochondrial) pathway – triggered by internal stress → mitochondrial outer‑membrane permeabilization (MOMP) → cytochrome c + SMAC release → apoptosome → caspase‑9 → executioner caspases. Extrinsic (death‑receptor) pathway – extracellular ligands (TNF, FasL) bind death receptors → DISC formation → caspase‑8 (or‑10) activation → direct activation of executioners (type I) or amplification via mitochondria (type II). Caspases – cysteine‑aspartate proteases. Initiators (caspase‑8, ‑9) are activated in multiprotein complexes; executioners (caspase‑3, ‑6, ‑7) cleave structural/repair proteins. Bcl‑2 family – balance of pro‑apoptotic (BAX, BAK, BID, BAD) vs anti‑apoptotic (Bcl‑2, Bcl‑XL) determines MOMP. SMAC/DIABLO – neutralizes IAPs, freeing caspases from inhibition. IAPs (Inhibitor of Apoptosis Proteins) – bind and block caspases; antagonized by SMAC. AIF (Apoptosis‑Inducing Factor) – mediates caspase‑independent DNA fragmentation after calpain cleavage. --- 📌 Must Remember Morphology: cell shrinkage, membrane blebbing, chromatin condensation, DNA “ladder” (≈180‑bp repeats). Key molecules: Intrinsic: BAX/BAK, Cytochrome c, Apaf‑1, ATP, Caspase‑9, SMAC, IAPs. Extrinsic: TNF‑α, TNFR1/2, TRADD, FADD, Caspase‑8/10, Fas (CD95). Executioner caspases = caspase‑3, ‑6, ‑7 → cleave most cellular proteins. Bcl‑2/Bcl‑XL ratio > 1 → survival; < 1 → apoptosis. Annexin V⁺ / PI⁻ = early apoptosis; Annexin V⁺ / PI⁺ = late apoptosis/secondary necrosis. Type I cells: caspase‑8 directly activates executioners. Type II cells: need mitochondrial amplification (tBid → BAX/BAK). --- 🔄 Key Processes Intrinsic Pathway Stress (DNA damage, hypoxia, Ca²⁺ overload, etc.) → activation of BH3‑only proteins (e.g., tBid). BAX/BAK insert → pores in outer mitochondrial membrane. Release of cytochrome c + SMAC into cytosol. Cytochrome c + Apaf‑1 + ATP → apoptosome (heptameric complex). Apoptosome recruits procaspase‑9 → caspase‑9 active. Caspase‑9 cleaves/activates caspase‑3/7 → execution phase. SMAC binds IAPs, neutralizing their inhibition of caspases. Extrinsic Pathway Ligand (TNF‑α, FasL) binds death receptor (TNFR1, Fas). Receptor trimerizes → recruitment of adaptor proteins (TRADD, FADD). Formation of DISC (death‑inducing signaling complex). Procaspase‑8/10 dimerizes → auto‑cleavage → active caspase‑8/10. Caspase‑8 → (a) direct activation of executioners (type I) or (b) cleavage of Bid → tBid → mitochondrial amplification (type II). Downstream convergence on executioner caspases → apoptosis. Caspase‑Independent Pathway (AIF) Calpain‑dependent cleavage of AIF from inner mitochondrial membrane. AIF translocates to nucleus → large‑scale DNA fragmentation (no ladder pattern). --- 🔍 Key Comparisons Intrinsic vs. Extrinsic Trigger: internal stress vs. external ligand. Key organelle: mitochondria vs. plasma membrane death receptors. Primary initiator caspase: caspase‑9 vs. caspase‑8. Type I vs. Type II Cells (extrinsic pathway) Type I: strong DISC → caspase‑8 directly activates executioners. Type II: weak DISC → requires tBid‑mediated mitochondrial amplification. BAX/BAK vs. Bcl‑2/Bcl‑XL Pro‑apoptotic: form pores → promote MOMP. Anti‑apoptotic: bind/sequester BAX/BAK → preserve membrane integrity. Caspase‑dependent vs. Caspase‑independent Caspase‑dependent: DNA laddering, sensitive to pan‑caspase inhibitors. Caspase‑independent (AIF): large DNA fragments, persists despite caspase inhibition. --- ⚠️ Common Misunderstandings “Apoptosis always involves DNA laddering.” False – caspase‑independent AIF‑mediated death gives a smear, not a ladder. “Necrosis = inflammation, apoptosis = no inflammation.” Generally true, but secondary necrosis can occur if apoptotic bodies aren’t cleared promptly. “All death‑receptor signaling kills the cell.” Some receptors (e.g., certain TNFR2 signals) can activate NF‑κB survival pathways. “Caspase‑8 only activates executioners.” In type II cells, caspase‑8 also cleaves Bid, linking to the intrinsic pathway. --- 🧠 Mental Models / Intuition “Two‑switch model” – Think of apoptosis as a circuit with stress (input) → switch (Bcl‑2 ratio) → power (MOMP) → alarm (cytochrome c) → execution (caspases). “Death‑receptor as a doorbell” – When the bell rings (ligand binds), the house either opens the front door (type I, direct caspase‑8) or calls the backup generator (type II, mitochondrial amplification). “IAPs as security guards” – SMAC is the “badge” that removes the guard, letting caspases proceed. --- 🚩 Exceptions & Edge Cases Viral Bcl‑2 homologs can block BAX/BAK, preventing MOMP even under strong stress. p53 mutations → loss of DNA‑damage‑induced intrinsic activation. Akt phosphorylation of BAD → BAD stays inactive, biasing toward survival despite pro‑apoptotic signals. Caspase inhibitors (viral or experimental) → cells may still die via AIF. --- 📍 When to Use Which | Situation | Best Indicator / Test | Reason | |-----------|-----------------------|--------| | Early apoptosis detection | Annexin V‑FITC (PS exposure) + PI‑negative | PS flips before membrane rupture. | | Distinguish necrosis vs. late apoptosis | Propidium iodide (PI) uptake | PI only enters cells with compromised membranes. | | Determine pathway activation | Western blot for cleaved caspase‑8 (extrinsic) vs. cleaved caspase‑9 (intrinsic) | Initiator caspases are pathway‑specific. | | Assess mitochondrial involvement | Cytochrome c release assay (subcellular fractionation) | Direct evidence of MOMP. | | Evaluate caspase‑independent death | AIF nuclear translocation + caspase inhibitor (z‑VAD‑fmk) | AIF moves to nucleus even when caspases blocked. | | Predict therapeutic response | Bcl‑2/BAX ratio (qPCR/Western) | High Bcl‑2 suggests resistance to intrinsic activators. | --- 👀 Patterns to Recognize “Ladder DNA + caspase‑3 activation” → classic intrinsic/extrinsic apoptosis. “Annexin V⁺ / PI⁺ without ladder → late apoptosis/secondary necrosis. “DISC formation + caspase‑8 cleavage but no caspase‑9 → type I extrinsic death. “Bid cleavage + cytochrome c release → type II extrinsic amplification. “SMAC release + IAP inhibition → checkpoint release allowing caspase cascade. --- 🗂️ Exam Traps Trap: “All cells undergoing apoptosis will show membrane blebbing.” Why wrong: Some cells die via caspase‑independent AIF pathways with minimal blebbing. Trap: “Caspase‑8 activation always means extrinsic pathway.” Why wrong: In type II cells, caspase‑8 activation is a bridge to the intrinsic pathway (via tBid). Trap: “Presence of DNA ladder proves apoptosis, not necrosis.” Why wrong: Certain necrotic processes (e.g., necroptosis with endonucleases) can mimic laddering; confirm with caspase activity. Trap: “High Bcl‑2 expression guarantees survival.” Why wrong: Overwhelming pro‑apoptotic signals (e.g., massive DNA damage) can still trigger apoptosis despite Bcl‑2. Trap: “Annexin V positivity alone confirms apoptosis.” Why wrong: Early PS exposure can occur in activated platelets or during some forms of necrosis; combine with PI negativity. ---