M11L1 - Apoptosis

Why does regulated cell death occur?

• Normal part of cell life cycle

• It’s an eqm where you gain and lose as a constant process

• Helps in preventing cancer i guess by not having too little cell loss

Apoptosis in embryo hand/feet development

• Even in early development, apoptosis takes place 

• In week 6, skin forms webbing between the digits 

• By week 11, the webbing disappears due to apoptosis

Proof of Apoptosis in Embryo Webbing (Mouse Paw)

• Mouse paw embryo stained with a dye to detect apoptosis

• Shown as yellow dots seen mostly where the webbing is

• As webbing disappears, the bright spots do too

• This is done through TUNEL assay

TUNEL Assay in Apoptosis Detection

• Takes advantage of the nicks present in apoptosis cell’s DNA

• dUTP can be incorporated into the nicks by enzymes

  • Enzyme: Terminal Deoxynucleotidyl Transferase

• Flourescently-labelled dUPT can specifically detect cells in apoptosis

Apoptosis in Frog Metamorphosis

• Tadpoles undergo metamorphosis to become a frog

• The tail disappears as the cells are induced to undergo apoptosis 

• This is stimulated by the increase of the thyroid hormone in blood

Apoptosis in Human Nervous System Development

• Half of the cells originally produced are required in normal brain development 

• The other half undergo apoptosis 

  • Cells that haven’t achieved synaptic connections

  • Cells with faulty connections

  • Cells not having made contact with a target cell

• Matches the number of nerve cells with the number of target cells

Inappropriate Cell Death Diseases

• Alzheimers: Neurons in hippocampus and cerebral cortex die

• Huntingtons: Neurons in striatum die 

• Parkinsons: Dopamine neurons in substantia nigra die

• Duchenne Muscular Dystrophy: Muscle cells die

What are the 2 ways in which cells die

1. Necrosis 

2. Apoptosis 

Necrosis

• Cell death through damage to exterior 

• Cells swell and release contents to surrounding tissue 

• Can lead to infection

Apoptosis

• Programmed cell death that is regulated

• Cells suicide in response to stress/damage or as a part of normal development 

• The debris isn’t released to damage cells nearby

• The debris is contained and recycled 

Apoptotic Pathway

• Cell Execution: Kill the cell

• Engulfment: Get rid of the body 

• Clearance: Destroying the evidence

Ultrastructural Features of Apoptosis (7)

• Chromatin compacts and condenses 

• Nuclear envelope breaks down

• Nucleus contents are fragmented and the DNA / proteins are degraded 

• Cytoplasm undergoes condensation as cellular components aggregate 

• Mitochondria is permeabilized and released into the cytosol 

• Cell membrane moves and changes shape to create blebs (protrusions)

• Cell fragments create compartments with debris which will be phagocytized and recycled

C. elegans Apoptosis Model

• They’re studied very much in detail 

• 947 somatic cells have been identified in the adult worm

• The lineage of them all is traced to a single cell undergoing rounds of division 

• 131 cells undergo apoptosis 

Apoptosis Genes Identified by C. elegans model

• Done by assay for identifying mutations in genes 

• These genes are called “cell death genes” (ceds)

• Mutation in ced-1: Allows apoptosis but not the associated phagocytosis 

• Mutation in ced-3: No apoptosis observed 

• Four essential genes:

  • ced-3

  • ced-4

  • ced-9

  • egl-1

Mammalian Apoptotic Pathway

• EGL-1 Homologs: Bid and Bim

• CED-9 Homolog: Bcl-2

  • Bcl-2 controls Bak and Bax

• CED-4/3 form a complex called the caspase holoenzyme 

  • Protease targeting many different proteins for degradation

• CED-3/4 mutations prevent death

• ced-9 mutations make all cells die

  • Inhibits activation of caspase holoenzyme

  • Inhibits apoptosis in this was

  • EGL-1 signals apoptosis by inhibiting CED-9

Caspase Holoenzyme

• Apoptosome in mammalian cell

• Contains direct homologues of C. elegans proteins

• Apaf 1 = CED-4

• Caspase-9 = CED-3

• Protease activity of caspase holoenzyme leads to protein degradation and cell death

Activation of Caspase Holoenzyme (C. elegans)

• In C. elegans

• CED-9 inhibits apoptosis by binding to CED-4 dimers

  • Keeps them inactive 

• EGL-1 binding to CED-9 releases CED-4

• CED-4 then join with CED-3 to form caspase holoenzyme 

• This leads to degradation of cytosolic and nuclear proteins 

Mammalian CED-9 homologue

• It’s Bcl-2 

• Normally anchored to outer membrane of mitochondria 

• Alters permeability of it 

  • It maintains low permeability when present 

  • When inactive, it forms pores associated with apoptosis 

Bad: Apoptosis Signaling Pathway with Cytochrome C.

• Mammalian cell apoptotic signal is called Bad

• It’s inactive while phosphorylated and bound to 14-3-3 

  • 14-3-3 is a cytosolic adaptor protein

• Signalling pathways allow dephosphorylation of Bad

• It then releases from 14-3-3

• It then binds to Bcl-2 on mitochondria

• This activated Bcl-2 to allow for Bax to be activated 

• Bax aggregated into clusters in the membrane to make pores 

• Pores increase membrane permeability 

• Allows release of mitochondrial proteins into cytosol

• This includes cytochrome C which is essential in forming mammalian apoptosome

Trophic Factors in Apoptosis Prevention

• Trophic factors prevent apoptosis to keep the cell alive

• They initiate a kinase cascade leading to phosphorylation of the Bad protein 

• When trophic factors are removed, Bad can be dephosphorylated