Cell Biology - 4&5. Apoptosis

• Reasons cell death is needed

  • Fighting infectious agents
  • Getting rid of malfunctioning cells
  • Physiological processes (menstruation)
  • Immune system development
  • Needed during development
• 50-70 billion cells lost a day due to apoptosis
• Necrosis


  • Membrane damaged
  • Leakage of organelles and contents of cell
  • Inflammatory response
  • Necroptosis = programmed necrosis
• Stages of necrosis


  1. Chromatin clumps, organelles swell, flocculent (fluffy) mitochondria

  2. Membrane disintegrates and intracellular contents release

  3. Swelling results
• Apoptosis


  • Gets rid of webs between fingers and toes
  • No leakage
  • No inflammatory
  • Rapid clearance of apoptotic cells
• Stages of apoptosis


  1. Mild convolution + chromatin compaction and segregation

  2. Nuclear fragmentation + blebbing (small blobs of cell form)

  3. Phagocytosis eats blobs (apoptotic bodies)
• Roles of apoptosis


  • Development (Sculpts tissues, Regulates neuron number, Immune system function)
  • Means of regulation (Without trophic factors most cells will undergo apoptosis, Some triggered to undergo apoptosis by specific signals)
• Cell changes due to apoptosis

  • PS externalised (phosphatidylserine) on membrane
  • Blebbing (membrane)
  • Osmotic gradient maintained and membrane sealed (transglutaminase)
  • Nuclease shrinks and chromatin condenses
  • Endonuclease activates (cleaves DNA into fragments)
• Apoptotic cells cleared by phagocytes
• Assays to assess apoptosis

  • Annexin V binding assay
  • Visualising membrane blebbing in SEM
  • Nuclear condensation and fragmentation
  • DNA fragmentation
• Annexin V binding assay


  • Annexin V normally functions in cytosol (repairs membrane damage by binding to phosphatidyl serine on inside)
  • Can take Annexin V, add fluorophore to it (FITC (green))
  • It binds to outside presented phosphatidyl serine showing apoptotic cells
  • Membranes lit up green
• Mechanism of membrane blebbing

  1. Actin contracts due to myosin motor protein

  2. RhoA activates Rho Kinase-1 (ROCK1)

  3. Phosphorylates myosin light chain and inactivates MLC phosphatase

  4. Causes phosphatase to be maintained on light chain which causes contractility

  5. During apoptosis ROCK1 cleaved by caspases which keeps it constituently active causing constant activation of myosin

  6. Whole actin cytoskeleton contracts

  7. Decouples plasma membrane from cytoskeleton causing blebbing
• Nuclear condensation and fragmentation

  • Large nuclear structure condenses into black blobs which enter apoptotic bodies
  • Acridine orange stain binds to DNA and RNA which can show the condensed blobs in the apoptotic bodies
  • Lamina breakdown during apoptosis facilitates chromatin condensation and nuclear breakdown
  • During apoptosis, caspase associated DNA cleaves DNA between nucleosomes which leaves fragments of 180bp (length wrapped around nucleosomes)
• DNA fragmentation


  • Normal cell flow cytometry plot shows most cells in G1, nothing sub G1
  • Apoptotic cells have sub G1 fluorescence as well
  • DNA fragmented here, stain with propidium iodide to analyse fragment lengths
  • TUNEL assay (Terminal deoxynucleotidyl transferase (TdT) dUTP Nick End Labelling)
  • Apoptotic cells have nicks in DNA, TdT enzyme adds biotinylated nucleotides to free 3' ends of nicked DNA
  • Enzymes coupled to streptavidin bind to labelled bases, enzymes generate coloured product
  • Can only add if fragmented so not to normal DNA only to apoptotic cells
• Apoptosis in human disease

  • Cancer cells escape apoptosis
  • Neurodegeneration can be caused by too much apoptosis i.e. Parkinsons and dementia
  • Autoimmunity roles (Without apoptosis autoimmune diseases and impaired clearance can occur, Apoptosis prunes immune system during development to stop i attacking itself (autoimmune diseases), T killer cells induce apoptosis in cells, 90-95% T cells and B cells die in thymus and bone marrow respectively, killed because fail to produce antigen specific receptors, Self reactive receptors which recognise normal antigens (can cause autoimmune disease if not killed), Negative selection makes sure cells binding too strongly to antigens are killed, Positive is for ones that bind too weakly)
  • Cytotoxic T cells induce apoptosis in targets and kill them
  • If defective then infected cells become hosts for infectious agent
• Cytotoxic T cells


  • Recognises antigens on MHC complex
  • Perforin binds to target cells and makes pores in plasma membrane of target cells
  • Granzimes go through pores to activate caspase in target cells which destroys it
• Immune system cleans up junk (phagocytosis)
• Pathways of apoptosis


  • Intrinsic pathway (trophic factor withdrawal, DNA damage, stress signals one)
  • Extrinsic pathway (signalling through cell surface death receptor)
  • Final pathway and features are same
• Once initiated apoptosis is irreversible (needs tight control)
• Triggers for apoptosis


  • Cell membrane - death receptors (extrinsic)
  • Cytoplasm - caspases (initiator and effector caspases)
  • Mitochondria - cytochrome C released and apoptosome forms
  • Nucleus - endonuclease activity
• C. elegans


  • Cells divide lots and lots to make worm
  • Very well understood, 1031 cells, 959 hemaphrodites (can trace all back to first cell)
  • Born with more but apoptosis needed to kill some (hence difference between 959 and 1031)
  • Cells that would've formed neurons are ones destroyed mostly because when death prevented those cells go on to form neurons
• Apoptosis in C. elegans embryo

  3 phases, specification (instruction to die), killing, and excution (engulfment)
• Apoptosis regulators in C. elegans

  • Ced-9 (regulator)
  • Ced-4 (adapter)
  • Ced-3 (effector)
• Apoptosis regulators in vertebrates

  • Bcl-2 (regulator)
  • Apaf-1 (adapter)
  • Casp9 and Casp3 (effectors)
• Process so conserved between human and worm that Bcl2 could complement Ced-9 lacking worm mutants
• Caspases


  • Normally inactive (pro-caspases)
  • Activated by death receptors (TNF, Fas) and cytochrome C release
  • Cleave proteins after Asp (D) residues
  • Substrates: Regulator of Dnase (CAD) which cleaves DNA, nuclear lamina (release nuclear membrane), ROCK1 (induces blebbing)
  • 14 in humans (Initiator: 8, 9, 2, 10; Effector: 3, 6, 7; Rest involved in inflammation)
  • Multi domain proteins - cleavage and conformational changes causes activation
• Caspase 3 activation


  1. Linear large and small subunit cut into 2

  2. Different conformation of binding of subunits which activates it

  3. Linear to stacked form for inactivation to activation (active has small, large, large, small stacked form)

  4. 12 stranded beta sheet when active

  5. His and Cys contributed by large subunit
• 100s of caspase targets
• Cytotoxic T (CTL) induces apoptosis

  Releases perforin making pores in target cell, granzymes secreted and activate caspases in cells
• Death receptors (extrinsic)


  • FasL/Fas (Fas is TNF receptor family member, Intracellular component titled death domain, Death domain = ligand receptor interaction (FasL and Fas (L for ligand, without is receptor))
  • TNF (TNF binds and trimerizes TNFR, TRADD adaptor binds, 2 path options: 1. FADD recruited by TRADD, 2. TRADD induces new gene transcription by recruiting RIP and TRAF2 (NFkB and Jun induce gene transcription))
• Bcl2 family proteins


  • BH3 domain are part of what makes Bcl2 proteins, some are pro and some are anti apoptotic
  • Pro apoptotic: Bax, Bak
  • Pro survival: Bcl-2, Mcl-1, A1
  • Balance of pro apoptotic or pro survival when signal is received causes survival or death
• Mitochondrial apoptosis pathway


  1. Cytochrome c leaks from mitochondria

  2. Apaf-1/cytochrome C complex activates caspase

  3. i-CAD cleaved, CAD enters nucleus and cleaves DNA

  4. Bcl-2 binds to mitochondrial blocking leaks and swelling and stops cell death

  5. Pro apoptotic ones cause more release of cytochrome C, pro survival cause less

  6. Pro apoptotic versions have hidden helices and area activates by Bcl2 family ones (only with BH3 domain)

  7. Helix of BH3 one triggers site in pro apoptotic one, causes oligomerisation due to causing helixes of pro apoptotic to project out and forms large complex (which forms a hole) on mitochondrial membrane

  8. Favours permeability of membrane

  9. Pro survival ones limit formation of large oligomer so smaller hole

  10. Cytochrome C binds to Apaf-1, forms complex and that forms hetramer (7 bits), Complex binds to caspase-9 initiator which activates 3 and 7 (effectors)
• Extrinsic apoptosis pathway


  1. Caspase 8 activated by FADD

  2. BID is a pro apoptotic Bcl2 family protein, is a substrate of caspase 8

  3. BID activated by caspase 8 permeabilises mitochondrial membrane

  4. Key caspase in both extrinsic and intrinsic pathway is caspase 3
• PI 3-kinase signalling in cell survival

  • PI-3 is intracellular enzyme which changes lipid composition of plasma membrane
  • Phosphorylates PIP2 to make PIP3 which activates Akt (aka protein kinase B, PKB)
  • Enhances growth, enhances many cellular functions that keep cell living
  • Bad (pro apoptotic BH3 only protein) used to trigger mitochondrial membrane permeabilization, is also an Akt substrate
  • If cell is happy and surrounded by growth factors (which cause PI-3 activation) and Bad phosphorylated by Akt which causes it to be grabbed by 14-3-3 protein which prevents it working
  • Unphosphorylated Bad can't be grabbed so it causes mitochondrial permeability increase
• Removal of apoptotic fragments


  • Once cell is blebbing is engulfed by phagocytes
  • Essentially is PS flipping presents target for phagocyte to bind to
  • Recruitment/attraction signals made to signal to phagocyte theres an apotosing cell nearby
  • Eat my signals cause movement of phagocyte down concentration gradient until finds PS presenting apoptotic cell
  • Cell then consumed and degraded