Lecture 12: Apoptosis
• Morphological features of apoptosis
• Contribution of C. elegans to understanding apoptosis
• Cytochrome C, Apaf-1 and Caspase activation
• Bcl-2 Family proteins
• Intrinsic vs extrinsic activation of apoptosis
• Death receptors
Ultrastructural features of apoptosis
Ultrastructural features can only be observed by electron microscopy
Programmed cell death (apoptosis) is a key and unique cellular process
• ensures normal embryonic development
• proper tissue homeostasis in adult animals
• Active cellular process that is morphologically distinct from necrosis (where cells swell and
• Electron microscopy is able to identify morphological changes associated with apoptosis.
- Big black blobs which correspond to DNA and pieces of Nucleus.
- Dramatic morphological changes associated with chromatin condensing,
- Condensation of the cytoplasm.
- Nuclei starts to break apart and form apoptotic bodies.
• Similar to breaking down an old building – don’t want to scatter things all over the place..
- Cell controls the collapse by sequestering intracellular cellular components into
membrane-bound apoptotic bodies.
- If the cell just spewed out all the intracellular debris all over the place, it could be
damaging to the neighbouring cells.
Could interact with other cells and inappropriately activating things or release
• Apoptotic bodies will be recognized by phagocytes that will break the compartments down.
• As opposed to necrosis – adverse physicological stress due to heat or a chemical bath –
causes cells to swell and burst. uncontrolled destruction.
Fragmentation of Nuclei and DNA
• DNA itself is cut up through activation of Dnase at interchromosomal units.
- Histone-wrapped nucleosomes have 200 bp between nucleosomes and this is where it
- If you run apoptotic cells through gel electrophoresis, it creates a DNA ladder – distinct
Page 1 of6 - Anti-apoptotic factors will supress that DNA ladder
- Trophic factor withdrawal – take away growth factors that will trigger apoptosis
How is apoptosis controlled?
C. Elegans was the model organism used to try and understand apoptosis.
• Small multicellular organism.
• Two sexes hermaphrodites and male.
- Hermaphrodite: 959 cells, Male: 1031 cells
• Can discern individual cells with microscope because it is transparent. 959 cells.
• Fully sequenced so it easy to genetically manipulate organism.
Mutations in the ced-3 gene block apoptosis in C. elegans
• Out of 1,090 newborn cells, 131 cells die during development, resulting in a nematode with 959
• Dead cells are highly refractile and can be detected by DIC microscopy as projections
• Horvitz screened for worms that — after mutagenesis of their genome — contained 'un-dead'
• Identified mutations in two genes, ced-3 and ced-4 (called ced for cell death abnormal)
- Mutations in those two genes result in no apoptosis (1090 cells remain) causing some
• Also found ced-9 which prevented death in cells that needed to survive.
• Horvitz was able to show that during development we make many more cells than we need and
we selectively call them off in a specific way.
Mammalian proteins analogous/similar to C. elegans Ced genes
Ced3 = Caspase 9 Ced4 = Apaf-1
Ced9 = Bcl-2
Caspases play key roles in apoptotic pathways
• Ced3 was isolated, sequenced and it coded for a caspase protein.
- Caspases: cysteine-dependent aspartate-directed proteases, an enzyme the
proteolytically cleaves other proteins i.e. DNAse
• Dnase used to periodically cleave DNA in apoptotic cell is constitutively expressed but inhibited
by ICAD inhibitor protein.
• Caspases cleave the ICAD and frees DNAse for DNA fragmentation.
• Lamins when cleaved result in destruction of the nuclear outer membrane.
• Cytoskeletal proteins when cleaved, no longer contribute to the shape of the cell
morphological changes in the shape of the cell.
• Ced3 is the C elegans equilvalent to caspase 9 in mammals.
Types of Caspases
1) Initiator caspases -cleave inactive pro-forms of effector caspases, there by activating them i.e.
Page 2 of6 2) Effector (executioner) caspases- cleave other protein substrates within the cell, to trigger the
apoptotic process – directly act on all those types of proteins directing the processes i.e.
Apoptosome: 1.4 megadalton wheel of death
1. Apaf-1 : Apoptotic protease activating factor 1
2. Cytochrome c (released from mitochondria into cytosol) – found in the IMS.
3. Dimer of Caspase 9
• In absence of Cytochrome C, Apaf-1 exists as a monomer
• After binding of Cytochrome C Apaf-1 forms a disk shaped heptamer – recruits Caspase 9 which
then forms a dimer – activation of initiator caspase
• Caspase 9 then activates effector caspases i.e capsase 3 that go around and cleave other
BCl-2 family members
All have BH domains and transmembrane region.
Bcl-2 & Bcl-xl – BH domains (1-4): sit on top of Bax and Bak and inhibit pore formation.
Bax & Bak – BH domains (1- 3): make up IMS pores
Bim, Puma, Bad & Bid – BH3 only domain: Regulate Bcl2 activity
• The first mammalian apoptotic gene to be cloned was bcl-2 and was isolated from a
human B-cell lymphoma (expressed high levels of protein),
- Bcl-2: inhibits apoptosis (Bclxl is an isoform)
- BH domains: Bcl-2 homology domains relate ~20 proteins in mammals to each other
- All the related proteins are either pro-survival or pro-apoptotic – similar structures but
DNA damage can lead to increased expression of pro-apoptotic genes
DNA damage can trigger the recruitment and activation of the kinase ATM
• ATM (DNA damage recognition protein) can phosphorylate and activate p53
- p53 (transcription factor) during DNA damage increase expression of proteins
associated with DNA damage and gets degraded regularly (unstable),
- When ATM is bound to damaged DNA, its kinase activity is activated.
Page 3 of6 - High level of p53 activation means there is