☠️ Lecture 15: Apoptosis

Fig. 19-32: An Overview of the DNA Damage Response System

Fig. 19-34: DNA Damage Checkpoints in the Cell Cycle

Cell Death and Regulation

Apoptosis Overview
 Programmed cell death that does not release cytosolic contents
 Uses intrinsic pathways to safely remove cells

Caspase Activation
 Caspases are proteases that degrade cellular proteins
 Activated during apoptosis to break down the cell in a controlled way

Physiological Role
 Essential for normal development
 Removes unwanted or damaged cells

Survival Signals
 Trophic factors bind to surface receptors
 Repress apoptosis to allow cell survival

Triggers of Apoptosis
 Deregulated cell cycle
 DNA damage

Clinical Relevance
 Genes regulating apoptosis are often mutated in cancers

Apoptosis in Development

Example: Embryonic Chicken Feet
 When apoptosis is blocked, webbing between digits is not removed
 Shows that programmed cell death is essential for shaping tissues during development

Cell Death Types – Necrosis vs Apoptosis

Necrosis
 Cell ruptures and releases contents
 Triggers inflammation and damages neighbouring cells

Apoptosis
 Cell membrane stays intact
 Cell dismantled into apoptotic bodies (vesicles containing cellular contents)
 Bodies are absorbed by macrophages and recycled
 No inflammation, controlled cell removal

Apoptosis Process – Key Steps

Mild Convolution
 Cell begins to change shape slightly

Chromatin Compaction and Margination
 Chromatin (DNA + proteins) condenses and moves toward the edges of the nucleus

Cytoplasm Condensation
 Cytoplasm becomes denser as cell shrinks

Nuclear Envelope Breakdown
 Nuclear membrane fragments to allow controlled dismantling

Blebbing
 Cell surface forms bubble-like protrusions

Phagocytosis
 Apoptotic bodies are recognized and eaten by phagocytic cells for recycling

DNA Fragmentation in Apoptosis

Apoptotic Stimuli
 Triggers activation of DNA nucleases that cleave DNA between nucleosomes

Chromatin Degradation
 Broken-down chromatin is packaged into apoptotic bodies

DNA Content
 Apoptotic bodies contain less than 2N DNA compared to normal living cells

Detection of Apoptosis – Apoptotic Bodies

Assay Principle
 Fragmented chromatin can be detected using various laboratory assays

Apoptotic Bodies
 Small vesicles containing degraded chromatin and cellular contents
 Formed during apoptosis and can be recognized and engulfed by phagocytic cells

Flow Cytometry – Detection of Apoptosis

Principle
 Measures the amount of stained DNA in each cell as an indirect measure of DNA content

Apoptotic Bodies
 Produced during apoptosis and contain less DNA than healthy cells
 Appear as a broad “lump” before the C1 peak in flow cytometry plots

Cell Cycle Peaks
 C1 – cells not dividing, diploid DNA (2N)
 C2 – cells that have duplicated DNA (4N) but have not divided
 Between C1 and C2 – cells synthesizing DNA (S phase)

Apoptosis Detection
 Apoptotic cells have DNA content less than 2N (C1)
 Low number of cells in this region indicates apoptosis level
 Every cell culture exhibits some baseline apoptosis

Comet Assay – Detection of DNA Fragmentation

Procedure
 Cells embedded in agarose
 Electric current passed through agarose
 DNA fragments move according to charge, similar to gel electrophoresis

DNA Movement
 Large DNA fragments (intact chromosomes) remain in place
 Fragmented DNA leaves the cell and forms a “comet-like” tail

Interpretation
 Presence of comet tails indicates apoptosis
 Healthy cells show minimal or no tail, apoptotic cells show prominent tail

C. elegans – Model for Cell Death

Overview
 Small transparent worm used to study apoptosis
 Consists of 1090 somatic cells produced during development

Cell Fate
 959 cells survive
 131 cells undergo programmed cell death (apoptosis)

C. elegans – Cell Lineage and Genetics

Complete Cell Lineage
 Mapped in 1983, showing fate of all 1090 somatic cells

CED Genes
 Cell death genes identified through genetic studies in C. elegans
 Regulate which cells undergo apoptosis and which survive

Cell Death Genes (CED) – Discovery in C. elegans

Mutant Screening
 R. Horvitz (Nobel Prize) isolated >4000 cell lineage (lin) mutants
 Mutants failed to induce apoptosis in some of the 131 cells

Conclusion
 Apoptotic cell death is genetically controlled
 Mutant genes responsible for apoptosis led to discovery of cell death (CED) genes

Key Mutants
 ced-1 mutant: apoptotic cells are not engulfed or digested (visible as arrows)
 ced-1/ced-3 double mutant: apoptotic cells do not appear
 ced-3: necessary for apoptosis, pro-apoptotic gene

Apoptotic Pathway – C. elegans vs Mammals

Pathway Overview
 Apoptosis in C. elegans is more straightforward than in mammals
 All C. elegans cell death genes (CED) have analogous genes in mammals

Significance of C. elegans
 Very few cells in the nematode, so cell lineage and apoptosis are easy to track
 Allows precise study of genetic control of cell death
 Important model organism for understanding conserved apoptotic mechanisms

Caspases – Key Classes and Functions

Definition
 Caspases = Cysteinyl Aspartate-Specific Proteinases (enzymes that cleave proteins at specific sites containing aspartate)

Initiation Caspases
 Process and activate effector caspases
 Caspase-9: intrinsic (internal signal) pathway
 Caspase-8 and Caspase-10: extrinsic (external signal) pathway

Effector (Executioner) Caspases
 Cleave specific cellular proteins to dismantle the cell
 Caspase-3, Caspase-6, Caspase-7
 Activate nucleases that degrade DNA and chromatin
 Essential for completing apoptosis

Intrinsic (Mitochondrial) Apoptosis Pathway

Activation
 Triggered by release of cytochrome c (Cyt C) from mitochondria

Regulation by Bcl-2 Family Proteins
 Pro-apoptotic proteins: Bak, Bax, Boc (promote Cyt C release)
 Pro-survival proteins: Bcl-2, Bcl-xl (prevent Cyt C release)
 All share Bcl-2 Homology (BH) domains: BH1, BH2, BH3, BH4

BH3-Only Proteins
 Bad, Bim, Puma
 Regulate pro-survival proteins by inhibiting them, tipping balance toward apoptosis

Bcl-2 Family Proteins – Mitochondrial Control

Function
 Control transport through outer mitochondrial membrane

Pro-Survival Proteins
 Close pores in outer mitochondrial membrane
 Suppress release of cytochrome c

Pro-Apoptotic Proteins
 Open channels in outer mitochondrial membrane
 Promote release of cytochrome c into cytosol

Cell Death Pathways – Vertebrates

Intrinsic Pathway
 Activated by mitochondrial proteins (Bak, Bax, Bcl-2, Cytochrome C)
 Triggered by DNA damage, cell stress, loss of adhesion, or lack of trophic signals

Extrinsic Pathway
 Activated through cell surface death receptors
 Triggered by direct contact with other cells signaling apoptosis

Intrinsic Cell Death Pathway – Additional Notes

Integrins
 Transmembrane proteins that link the extracellular matrix (ECM) to the intracellular cytoskeleton
 Provide survival signals; loss of integrin-mediated attachment can trigger intrinsic apoptosis

Intrinsic Death Pathway – Pro- and Anti-Apoptotic Proteins

Pro-Apoptotic Proteins
 Bak and Bax can oligomerize (bind together) to form pores in the mitochondrial membrane
 This allows cytochrome C release, triggering apoptosis

Pro-Survival Proteins
 Bcl-2 and Bcl-XL bind to Bak or Bax and prevent pore formation

BH3-Only Proteins
 Bad, Bim, and Puma displace Bcl-2 from Bak or Bax
 This allows Bak/Bax to form pores and activate the intrinsic apoptosis pathway

Intrinsic Death Pathway – Cyt c and Caspase Activation

Loss of Pro-Survival Proteins
 Without Bcl-2 or Bcl-XL, Bak and Bax oligomerize and form pores in the outer mitochondrial membrane

Cytochrome C Release
 Cytochrome C exits the mitochondria and binds Apaf-1

Caspase Activation
 Apaf-1 activates initiator caspase-9
 Caspase-9 activates effector (executioner) caspases such as caspase-3
 Leads to systematic degradation of cellular proteins and DNA, completing apoptosis

Extrinsic (Death Receptor Mediated) Apoptosis Pathway

Death Receptors
 Located on immune cells, hepatocytes, and structural cells (e.g., cartilage)
 Trigger apoptosis when bound by external death signals

Caspase Activation
 Activates initiator caspase-8
 Caspase-8 activates effector caspases 6 and 7
 Leads to systematic breakdown of cellular proteins and DNA

Cross-Talk with Intrinsic Pathway
 Via Bid, the extrinsic pathway can activate the intrinsic (mitochondrial) apoptosis pathway

Cell Death and Its Regulation

Intrinsic Apoptosis Pathways
 Genetically programmed pathways that initiate cell death from within the cell
 Crucial for normal development

Caspase Activation
 Cellular caspase proteases cleave cellular proteins to dismantle the cell

Extrinsic Apoptosis Pathway
 Death signals from other cells can trigger apoptosis via surface receptors

Triggers
 Disruption of the cell cycle, such as DNA damage, can also initiate apoptosis