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Lecture 12

Lecture 12 - Apoptosis.docx

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Western University
Biology 2382B
Cumming/ Damjanovski

Lecture 12: Apoptosis Learning objectives • 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 burst) Morphological changes • 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. Controlled destruction • 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 toxins. • 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 is cuts. - If you run apoptotic cells through gel electrophoresis, it creates a DNA ladder – distinct bands. Page 1 of6 - Anti-apoptotic factors will supress that DNA ladder - Trophic factor withdrawal – take away growth factors that will trigger apoptosis overtime. 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 cells exactly • 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' cells • 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 problems. • 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. Caspase 9 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. Caspase 3 The Apoptosome Apoptosome: 1.4 megadalton wheel of death Composed of: 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 proteins. BCl-2 family members Overall structures: All have BH domains and transmembrane region. Pro-survival: Bcl-2 & Bcl-xl – BH domains (1-4): sit on top of Bax and Bak and inhibit pore formation. Pro-apoptotic: 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 different effects. 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
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