Cell Death 2

From CellBiology

Introduction

Neuron necrosis and apoptosis (tem)

Apoptosis nuclei tem.jpg

Apoptotic nucleus (tem)

This second lecture will focus upon programmed cell death, known as apoptosis. The previous lecture Cell Death 1 looked at mainly stress and pathological processes.


This single term "apoptosis" describes the way in which the majority of cells within our body are removed every day. While the morphological changes associated with this process are the same in all cells, the many different signaling pathways that "trigger" this process can be quite different.

HeLa cell Apoptosis Movie  
HeLa cells were induced to undergo apoptosis by exposure to 10 muM daunorubicin. Images were taken every minute over approx12 hours and were animated at 10 frames/second.
  • The first image represents healthy cells 2 h after treatment.
  • By 4 h, one of the cells begins to round up and detach from the substratum, and this is followed 30 min later by retraction of the neighbouring cell. Dynamic plasma membrane blebbing is then evident in both cells and this continues for several hours.
  • Later, membrane blebs become larger and more stable until, approx 8.5 hours after the cells were exposed to the pro-apoptotic drug, secondary necrosis begins.
  • During secondary necrosis, cells cease to bleb and large balloon-like swellings can be seen as cells lose plasma membrane integrity and release their contents into the surroundings.
  • Secondary necrosis is a highly undesirable endpoint and this is normally prevented in vivo through removal of apoptotic cells by phagocytes early in the process.

Rebecca C Taylor, Sean P Cullen, Seamus J Martin Apoptosis: controlled demolition at the cellular level. Nat. Rev. Mol. Cell Biol.: 2008, 9(3);231-41 PubMed 18073771

Hela Apoptosis Movie | Apoptosis Movie

Apoptosis Reviews  

Shigekazu Nagata, Masato Tanaka Programmed cell death and the immune system. Nat. Rev. Immunol.: 2017, 17(5);333-340 PubMed 28163302

Wenbin Zeng, Xiaobo Wang, Pengfei Xu, Gang Liu, Henry S Eden, Xiaoyuan Chen Molecular imaging of apoptosis: from micro to macro. Theranostics: 2015, 5(6);559-82 PubMed 25825597

Kongning Li, Deng Wu, Xi Chen, Ting Zhang, Lu Zhang, Ying Yi, Zhengqiang Miao, Nana Jin, Xiaoman Bi, Hongwei Wang, Jianzhen Xu, Dong Wang Current and emerging biomarkers of cell death in human disease. Biomed Res Int: 2014, 2014;690103 PubMed 24949464

Susan Elmore Apoptosis: a review of programmed cell death. Toxicol Pathol: 2007, 35(4);495-516 PubMed 17562483


The International Cell Death Society


Lecture Slides: 2017 Lecture PDF

Archive

MH - note that content listed below will not match exactly current lecture structure but has been selected as having similar content

Apoptosis Research Timeline

Apoptosis Research Timeline  
The timeline below shows key discoveries in our understanding of apoptosis.[1]

Apoptosis research timeline.jpg

Abbreviations

AICD, activation-induced cell death; ALPS, autoimmune lymphoproliferative syndrome; BCL-2, B cell lymphoma 2; BH3, BCL-2 homology 3; C. elegans, Caenorhabditis elegans; CED3, C. elegans homologue of mammalian caspases; CED9, C. elegans homologue of mammalian BCL-2; cGAMP, cyclic GMP–AMP; cGAS, cGAMP synthase; CTL, cytotoxic T lymphocyte; FASL, FAS ligand; FDA, US Food and Drug Administration; gld, generalized lymphoproliferative disease; lpr, lymphoproliferation; LPS, lipopolysaccharide; MLKL, mixed lineage kinase domain-like protein; PCD, programmed cell death; PtdSer, phosphatidylserine; RIPK, receptor-interacting protein kinase; SLE, systemic lupus erythematosus; STING, stimulator of interferon genes protein; TLR, Toll-like receptor; TNF, tumour necrosis factor; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling.

Nobel Prize 2002

Physiology or Medicine 2002 - genetic regulation of organ development and programmed cell death

C.elegans cell death

  • Sydney Brenner (b 1927), Berkeley, CA, USA, established C. elegans as a novel experimental model organism. This provided a unique opportunity to link genetic analysis to cell division, differentiation and organ development – and to follow these processes under the microscope. Brenner's discoveries, carried out in Cambridge, UK, laid the foundation for this year's Prize.
  • John Sulston (b 1942), Cambridge, England, mapped a cell lineage where every cell division and differentiation could be followed in the development of a tissue in C. elegans. He showed that specific cells undergo programmed cell death as an integral part of the normal differentiation process, and he identified the first mutation of a gene participating in the cell death process.
  • Robert Horvitz (b 1947), Cambridge, MA, USA, has discovered and characterized key genes controlling cell death in C. elegans. He has shown how these genes interact with each other in the cell death process and that corresponding genes exist in humans.
Sydney Brenner Interview (2011) Crick-Jacobs Center, Salk Institute for Biological Sciences.


YouTube Link

H. Robert Horvitz (MIT/HHMI) - Discovering Programmed Cell Death (9:30)

YouTube Link

  • Red - mitochondria
  • Green - GFP, caspase sensor

(Henderson et al. Cell Death Diff. 2005 12:1240)


Regulator → Adaptor → Effector
C. elegans      Ced-9 → Ced-4 → Ced-3 → Death
Vertebrates      Bcl-2 → Apaf-1 → Caspase-9 → Caspase-3 → Death

Regulators can initiate or block apoptosis, the regulators shown block apoptosis.

Apoptosis?

Term used to describe "Programmed Cell Death" originally by Kerr, J. F., Wyllie, A. H. & Currie, A. R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239-257 (1972). PMID: 4561027

Greek, ptosis = falling, as in when leaves fall from a tree in autumn.

Apoptosis Signals

Some signal pathways (apoptosis is lower left)

A diverse group of signals can induce apoptosis

Selective process for deletion of cells
  • Superfluous
  • Infected
  • genetic errors
  • transformed cells

Process required for

  • Embryogenesis
  • Metamorphosis
  • Endocrine dependent tissue atrophy
  • Normal tissue turnover
  • Variety of pathologic conditions
Apoptosis - genetic errors

Apoptosis - genetic errors

Apoptosis Examples

Development

Limb development

Ossification

  • Death of chondrocytes

Nervous System

  • Death of neurons
  • Disruption of Brain Development


Links: Developmental Mechanism - Apoptosis | limb - FIG5 Development - TUNEL staining

BMP syndactyly.jpg

Apoptosis in digit development

Adult

Immune System

Apoptosis immune system.jpg

Mammary Gland
  • balances proliferation
  • occurs each menstrual cycle
  • occurs during "involution" when breast-feeding ceases
Mammary apoptosis

Apoptotic Cell Morphology

Apoptosis DNA ladder

The following cellular changes occur in sequence during apoptosis.

  • loss of cell membrane phospholipid asymmetry
  • Condensation of chromatin
  • Reduction in nuclear size JCB - Nucleus changes
  • Internucleosomal DNA cleavage TUNEL staining
    • DNA ladder
  • shrinkage of the cell
  • membrane blebbing
  • breakdown of the cell into membrane-bound apoptotic bodies
    • bodies then phagocytosed by other cells

A number of different experimental techniques have been developed to identify these changes. Apoptosis Methods

Two Main Pathways

The intrinsic and extrinsic pathways.[1]

Apoptosis pathways cartoon.jpg

Signal transduction pathways.png

Apoptosis Signal transduction pathways

Apoptosis cleaved caspase 3 fl.jpg

Apoptosis cleaved Caspase 3

Apoptosis- Caspase-3 activation.jpg

Caspase-3 activation

  • death-receptor pathway (extrinsic)
  • mitochondrial pathway (intrinsic)
    • may also be several alternate pathways

Both pathways

  • Converge on caspase-3 activation
  • branch into many pathways
  • leading to eventual cell death


Note that there is a second extrinsic pathway mediated through Granzyme B.

Caspase activation pathways  
Caspase activation pathways.jpg

Extrinsic pathway (route 1)

  • involves the binding of extracellular death ligands (such as FasL or tumour necrosis factor-alpha (TNFalpha)) to transmembrane death receptors.
  • Engagement of death receptors with their cognate ligands provokes the recruitment of adaptor proteins, such as the Fas-associated death domain protein (FADD), which in turn recruit and aggregate several molecules of caspase-8, thereby promoting its autoprocessing and activation.
  • Active caspase-8 then proteolytically processes and activates caspase-3 and -7, provoking further caspase activation events that culminate in substrate proteolysis and cell death. In some situations, extrinsic death signals can crosstalk with the intrinsic pathway through caspase-8-mediated proteolysis of the BH3-only protein BID (BH3-interacting domain death agonist).
  • Truncated BID (tBID) can promote mitochondrial cytochrome c release and assembly of the apoptosome (comprising approx7 molecules of apoptotic protease-activating factor-1 (APAF1) and the same number of caspase-9 homodimers).


Intrinsic pathway (route 2)

  • diverse stimuli that provoke cell stress or damage typically activate one or more members of the BH3-only protein family.
  • BH3-only proteins act as pathway-specific sensors for various stimuli and are regulated in distinct ways (Box 1).
  • BH3-only protein activation above a crucial threshold overcomes the inhibitory effect of the anti-apoptotic B-cell lymphoma-2 (BCL-2) family members and promotes the assembly of BAK–BAX oligomers within mitochondrial outer membranes.
  • These oligomers permit the efflux of intermembrane space proteins, such as cytochrome c, into the cytosol.
  • On release from mitochondria, cytochrome c can seed apoptosome assembly.
  • Active caspase-9 then propagates a proteolytic cascade of further caspase activation events.

Granzyme B-dependent route (route 3)

  • involves the delivery of this protease into the target cell through specialized granules that are released from cytotoxic T lymphocytes (CTL) or natural killer (NK) cells.
  • CTL and NK granules contain numerous granzymes as well as a pore-forming protein, perforin, which oligomerizes in the membranes of target cells to permit entry of the granzymes.
  • Granzyme B, similar to the caspases, also cleaves its substrates after Asp residues and can process BID as well as caspase-3 and -7 to initiate apoptosis.


BAD, BCL-2 antagonist of cell death; BAK, BCL-2-antagonist/killer-1; BAX, BCL-2-associated X protein; BID, BH3-interacting domain death agonist; BIK, BCL-2-interacting killer; BIM, BCL-2-like-11; BMF, BCL-2 modifying factor; HRK, harakiri (also known as death protein-5);


Rebecca C Taylor, Sean P Cullen, Seamus J Martin Apoptosis: controlled demolition at the cellular level. Nat. Rev. Mol. Cell Biol.: 2008, 9(3);231-41 PubMed 18073771


Caspases



Links: Apoptosis Signaling Overview

Death Receptor Pathway

TNF receptor model

Receptors

Cell surface receptors contain an intracellular death domain (DD)

  • belong to the tumor necrosis factor (TNF) super family
    • Fas (CD95/Apo1), TNF receptor 1 (p55), TRAMP (WSL-1/Apo3/DR3/LARD), TRAIL-R1 (DR4), TRAIL-R2 (DR5/Apo2/KILLER)
  • trigger apoptosis upon ligand binding

Ligand Binding

  • Fas Ligand (CD95 ligand) binds Fas
  • TNF and lymphotoxin a bind to TNFR1
  • TWEAK (Apo3 ligand) binds to TRAMP
  • TRAIL (Apo2 ligand) binds both TRAIL-R1 (Pan et al., 1997) and TRAIL-R2

Upon ligand binding receptor associates with an adaptor protein

  • Fas-associated death domain (FADD) directly or indirectly
    • through TNFR-associated death domain (TRADD)
  • FADD also interacts with pro-caspase-8
    • form a complex at the receptor called the death inducing signalling complex (DISC)

Death Inducing Signaling Complex (DISC)

  • DISC induces the activation of caspase-8
    • activates downstream effector caspases

Bid is also cleaved by pro-caspase 8 and translocates to the mitochondria to activate the intrinsic mitochondrial pathway, linking the two death pathways.

Decoy Receptors (DcR)

  • tumor necrosis factor (TNF) super family
  • inhibit death signaling by sequestration of ligand
    • DcR1, DcR2 and osteoprotegerin (OPG) bind to TRAIL
    • DcR3 binds Fas ligand

FLICE-like inhibitory protein (c-FLIP)

  • intracellular endogenous inhibitor
  • regulates by interacting with FADD
    • blocks apoptosis

Mitochondrial Pathway

MCB Movie

Apoptosis mitochondrial pathway
  • begins with permeabilisation of mitochondrial outer membrane
    • either permeability transition (PT) pore dependent or independent

Permeability Transition (PT) Pore

Structure formed from matrix, inner membrane, outer membrane proteins

  • matrix protein cyclophilin D
  • inner mitochondrial membrane protein adenine nucleotide translocator (ANT)
  • outer mitochondrial membrane protein voltage dependent anion channel (VDAC)

Opening the PT pore

  • triggers the dissipation of the proton gradient created by electron transport
    • uncoupling of oxidative phosphorylation
  • also causes water to enter the mitochondrial matrix
    • results in swelling of the intermembranal space
    • rupturing of the outer membrane
    • releasing apoptogenic proteins - cytochrome c, apoptosis inducing factor (AIF), endonuclease G

Apoptosome

Apoptosome formation
  • Formed by Cytochrome c, apoptosis protease activating factor (APAF-1) and pro-caspase 9 PNAS - Model of apoptosome formation
  • complex promotes the activation of caspase 9
    • activates effector caspases that collectively orchestrate the execution of apoptosis
  • AIF and endonuclease G - DNA fragmentation and chromosomal condensation


Other proteins released upon mitochondrial outer membrane permeabilisation

  • Smac/DIABLO - second mitochondria-derived activator of caspases/direct IAP-associated binding protein with low pI)
  • Omi/HtrA2 (high temperature requirement A2), which antagonize IAPs thereby promoting caspase activation

Bcl-2 Family

Apoptosis Bax regulation

PT pore independent mitochondrial membrane permeabilisation is regulated by Bcl-2 family members anchored to the mitochondria membrane by hydrophobic C-terminal

Anti-apoptotic members

  • Bcl-2 and Bcl-xL
  • Bcl-2 proteins protect integrity of mitochondria
    • prevent cytochrome C release
    • block caspase 9 activation

Pro-apoptotic members

  • two categories based on expression of Bcl-2 homology (BH) domains
  • Multi-domain proteins comprise BH domains 1-3 and include Bax, Bak, and Bok
    • form channels in outer mitochondrial membrane releasing intermembranal space apoptogenic proteins
  • BH3 only proteins consists of Bad, Bik, Bid, Puma, Bim, Bmf and Noxa.
    • activate multi-domain pro-apoptotic species
    • disrupt the function of anti-apoptotic Bcl-2 family members

Possible Bcl Mechanisms

  • Formation of a pore - cytochrome c and other intermembrane proteins escape
  • Heterodimerization - between pro- and anti-apoptotic family members
  • Direct regulation of caspases - by adaptor molecules
  • Interaction with mitochondrial proteins - generate a pore or to modulate mitochondrial homeostasis
  • Oligomerization- form a weakly selective ion channel

p53

Induced by pro-apoptotic stimuli activates apoptosis by intrinsic mitochondrial pathway

Normally p53 low levels

  • by murine double minute-2 (MDM2) or the human homologue (HDM2)
  • inhibit p53 transcription
  • promote p53 degradation (by proteosome)

Activation of p53

  • stabilization of p53 by post-translational modifications
    • disrupt interaction between p53 and MDM2
  • p53 drives expression of APAF-1 and pro-apoptotic Bcl-2 family members and transcriptional independent death pathways

Caspases

Apoptosome formation
  • cysteine proteases
  • central regulators of apoptosis
  • 14 different caspases identified
  • expressed as pro-enzymes
    • three domains NH2 terminal, a large subunit 20 kDa and a small subunit 10 kDa
  • activation by proteolytic processing between domains
    • allows association of large and small subunits
  • active caspases are a tetramer 2 heterodimers of large and small subunits

Initiator caspases

  • caspase 8, 9, 10 and 12
  • closely linked to pro-apoptotic signals
  • once caspases activated cleave and activate effector

Effector caspases

  • caspases 3, 6, and 7
  • cleave cytoskeletal and nuclear proteins
  • induce apoptosis

FasL and Fas-expressing Cells

Caspase Cleavage

  • leads to diverse results
  • depending on substrate and position of cleavage site
  • loss of biological activity eg lamin network
  • limited proteolysis by caspases result in a gain of biological activity eg Bcl-2 or Bcl-x, PAK2, Bid and CAD/ICAD

Caspase Activation Mechanisms

(a) proteolytic cleavage by an upstream caspase activation of downstream effector caspases induction of non-caspase proteases (granzyme B)

(b) Induced proximity aggregation of procaspase-8 molecules somehow results in cross-activation

(c) holoenzyme formation cytochrome c and ATP dependent oligomerization of Apaf-1 recruitment of procaspase-9 into apoptosome complex Activation of caspase-9 mediated by conformational change

Caspases and Cell Structures-Organelles

Caspases and cell structures-organelles.jpg

Caspases coordinate demolition of key cellular structures and organelles.

  • Effector caspases (such as caspase-3, -6 and -7 in mammals) orchestrate the dismantling of diverse cell structures through cleavage of specific substrates.
  • Cleavage of ICAD (inhibitor of caspase-activated DNase) releases CAD (caspase-activated DNase), which can then catalyse inter-nucleosomal DNA cleavage.
  • Caspase-mediated cleavage of nuclear lamins weakens the nuclear lamina, allowing nuclear fragmentation, and nuclear envelope proteins are also proteolysed.
  • Proteolysis of proteins at focal adhesion sites and cell–cell adhesion sites allows cell detachment and retraction. Caspase activity is required for the exposure of phosphatidylserine (PS) and other phagocytic signals on the cell surface.
  • Proteolysis of the Rho effector ROCK1 leads to contraction of the actin cytoskeleton and plasma membrane blebbing as well as nuclear fragmentation, whereas cleavage of tubulins and microtubule-associated and motor proteins leads to changes in the microtubule cytoskeleton that may contribute to apoptotic body formation (not shown).
  • Caspases also cleave the Golgi-stacking protein GRASP65 and other Golgi proteins, causing fragmentation of the Golgi apparatus.
  • Proteolysis of the mammalian sterile-20 kinase MST1 results in translocation of a catalytically active fragment of this kinase to the nucleus where it phosphorylates histone H2B to provoke chromatin condensation.
  • Cellular functions such as translation disrupted through caspase-mediated proteolysis of multiple translation initiation factors (eIFs). ER, endoplasmic reticulum; MLC, myosin light chain.

Apoptosis Inhibitors

  • directly inhibit caspases or prevent activation

Akt

  • serine/threonine protein kinase
  • important anti-apoptotic factor
  • inhibits pro-apoptotic Bcl-2 family member, Bad
  • directly inhibits caspase 9

TNF-alpha

  • Tumour Necrosis Factor -alpha
  • both anti-apoptotic and pro-apoptotic effect
  • activate the transcription factor NF-kB
  • which then induces expression of IAP
    • an inhibitor of caspases 3, 7,and 9

Neurotrophins

  • regulate apoptosis through protein kinase cascades
  • phosphoinositide 3-kinase/Akt
  • mitogen-activated protein kinase pathways


Final Stages of Apoptosis

  • The membrane enclosed cell fragments are phagocytosed by macrophages and other cells.

Apoptosis - macrophage.jpg

References

Textbooks

Molecular Biology of the Cell

Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter New York and London: Garland Science; c2002

Molecular Cell Biology

Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. New York: W. H. Freeman & Co.; c1999

The Cell- A Molecular Approach

Cooper, Geoffrey M. Sunderland (MA): Sinauer Associates, Inc.; c2000

Search Online Textbooks

PubMed

Reviews

  • Morphological and cytochemical determination of cell death by apoptosis. Taatjes DJ, Sobel BE, Budd RC. Histochem Cell Biol. 2008 Jan;129(1):33-43. Epub 2007 Nov 14. Review. PMID: 18000678
  • Frisch SM, Ruoslahti E. Integrins and anoikis. Curr Opin Cell Biol. 1997 Oct;9(5):701-6. Review. PMID: 9330874

Articles

  • Kerr, J. F., Wyllie, A. H. & Currie, A. R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239-257 (1972). PMID: 4561027

Search Entrez

Acronyms

  • AIF apoptosis-inducing factor
  • CARD caspase recruitment domain
  • DISC death-inducing signaling complex
  • DED death effector domain
  • FADD Fas-associated death domain protein
  • FADD-DD FADD-death domain
  • ICE interleukin-1β-converting enzyme (-like) protease
  • PARP polyADP ribose polymerase
  • TNF tumor necrosis factor
  • TRADD TNF receptor-associated death domain protein
  • TRAIL TNF-related apoptosis inducing ligand
  • BCL-2

External Links


2017 Course Content

Moodle

Lectures: Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cytoskeleton Introduction | Cytoskeleton - Microfilaments | Cytoskeleton - Microtubules | Cytoskeleton - Intermediate Filaments | Cell Mitochondria | Cell Junctions | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells 1 | Stem Cells 2 | Development | 2017 Revision

2017 Laboratories: Introduction to Lab | Fixation and Staining |


2017 Projects: Group 1 - Delta | Group 2 - Duct | Group 3 - Beta | Group 4 - Alpha

Dr Mark Hill 2015, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G
  1. 1.0 1.1 Shigekazu Nagata, Masato Tanaka Programmed cell death and the immune system. Nat. Rev. Immunol.: 2017, 17(5);333-340 PubMed 28163302