Xu Zhaoyang

Department of cell biology

Basicl medicine colledge
Zhengzhou university

Apoptosis:
Death comes for
the Cell

From Ingmar Bergman’s The Seventh Seal

 “Death is a punishment to some,
to others a gift and to many a favor.”
– Seneca

■ Roman playwright Seneca’s keen assessment

of death resonates with modern concepts of
apoptosis, or programmed cell death.
Although suicide by a living unit may seem
contradictory to survival, apoptosis allows a
cell to sacrifice itself for the benefit of a
greater living unit.
 The Nobel Prize in
Physiology or Medicine 2002
■ The Nobel Assembly at Karolinska Institutet has
awarded the Nobel Prize in Physiology or Medicine
jointly to Sydney Brenner, Robert Horvitz and John
Sulston for their discoveries concerning "genetic
regulation of organ development and programmed cell
death".
■ By using the nematode Caenorhabditis elegans as a
model system, the Laureates have identified key genes
regulating these processes. They have also shown that
corresponding genes exist in higher species, including
man.
 The Nobel Prize in
Physiology or Medicine 2002

Sydney Brenner, born 1927, Robert Horvitz, born 1947, John Sulston, born 1942,
La Jolla, CA, USA. Cambridge, MA, USA. Cambridge, England.
 Types of Cell death
■ Cells die by one of two mechanisms
■ – necrosis or apoptosis
■ • Two physiologically different processes
■ – Necrosis – death by injury
■ – Apoptosis – death by suicide
■ • Apoptosis and necrosis have different
characteristics
 Features of Apoptosis
Vs Necrosis
Apoptosis Necrosis
• Chromatin condensation • Nuclear swelling
• Cell Shrinkage • Cell Swelling
• Preservation of Organelles • Disruption of Organelles
and cell membranes • Rupture of cell and release
• Rapid engulfment by of cellular contents
neighboring cells • Inflammatory response
preventing inflammation
• Biochemical Hallmark:
DNA FRAGMENTATION
 Detection of apoptotic cells

• Microscopy
– Cells have classic features (eg. small darkly stained
nuclei)
– Detection of free 3’ ends of DNA by TUNEL assay
(terminal deoxytransferase-mediated dUTP-biotin nick
end labeling)
• Gel electrophoresis
– Detect DNA ladder of 180 bp intervals caused by
inter-nucleosomal DNA cleavage
 Morphologic changes
during apoptosis

■ Membranes become irregular

■ Chromatin becomes condensed and
segregated
■ Condensation of cytoplasm
■ DNA is fragmented
■ Cell is fragmented and phagocytose
 Morphological Features of Apoptosis

Transmission electron Micrograph Scanning electron Micrograph

Cell undergoing apoptosis

The membrane of the cell

blebs as DNA is condensed
and destroyed. The cell
then shrinks and packages
itself into an easily-
movable material for
macrophages (the body’s
cleanup crew, indicated by
the black arrow).
Removal of apoptotic cell
by phagocytosis
Phagocytosis tags and
receptors
Removal of cell corpses
The molecular mechanism
of apoptosis
■ Triggers of apoptosis

■ Genes implicated in apoptosis

■ Molecular mechanism
 Triggers of apoptosis

• Programmed cell death in which many more

cells are produced than survive (e.g. development
of lymphocytes)
• Toxic stimuli (viruses, chemicals, ionizing
radiation)
• Extracellular signals (Fas, p75 NGF-R, TNF)
• DNA damage (p53)
Genes implicated in apoptosis
■ Ced

■ Bcl-2

■ Caspases(Cystein aspartate-specific protease)

■ Fas/APO-1
 Why study
Caenorhabditis Elegans ?
■ Reproduce very rapidly (3 week life span). Easy to induce
mutations. Capable of reproducing as Hermaphrodites.
■ Simple organism with only 1090 somatic cells, with 131 of
1090 somatic cells normally undergo PCD. Death of these
cells is not required for viability.
■ Development is invariant and has been mapped such that
the fates of all cells are known.
■ Special optics can be used to observe abnormal deaths in
living organisms.
 Bcl-2 Background
■ Juxtaposed next to the Immunoglobulin heavy
chain locus.
■ > 50 Kb intron may facilitate recombination
■ At the time was a unique sequence with limited
sequence homology.
 Bcl-2 family members

• A very large family with 19 members identified

• All have the BH3 domain (Bcl-2 Homology)
– BH-3 is the pro-apoptotic domain exposed on
activation
 The function of Bcl-2

Act as dimers=either hetero or homodimers

– Pro-apoptotic dimers (Bax) increase

mitochondrial permeability

– Anti-apoptotic members (Bcl-2, Bcl-XL)

form dimers with pro-apoptotic members to
inactivate them
The Bcl-2 Family
 Bcl-2: Proposed
Mechanisms of Action

Binds and inhibits Proapoptotic Family Members

• Regulates ion flux across the Mitochondria and
stabilizes the membrane potential (PTP)
• Regulates cytochrome C release.
• Binds and inactivates APAF1
• ROS inhibition
• Many others: Ca Homeostasis, RAF1 interaction
• Regulates VDAC and thus ATP/ADP ratio
 General features of Caspases
■ Human ICE (interleukin-1βprocessing enzyme,
1992) and C. elegans ced-3 gene (1993) were
the first identified caspases; So far, 11 members
from human, 4 from C. elegans and 7 from
Drosophila have been identified with obvious
conservation in evolution.
■ Apoptosis substrates are numerous (~40 and
rising) and include PARP, DFF(ICAD), BID.
■ Synthesized as inactive zymogens and activated
by proteolysis (after Asp).
 The function of Caspase

■ Active cystein residue in the catalytic site.

Specificity in cleavage after an Asp residue of
the substrate proteins to carry out cell death
program;
– Caspases –8 and –9 are “initiator” caspases
– Caspases –3 is the “effector” caspase
– Caspase activation requires a stimulus
 Proteolytic targets of
effector caspases
• Cytoskeletal regulatory proteins– Actin
• Nuclear Lamins
• Poly ADP-ribose polymerase (PARP)–
PARP activity depletes ATP, thus cleavage
of PARP may maintain store of ATP to
drive apoptosis
• DNA-fragmentation factor (DFF)
The regulation of capases:IAPs
 The molecular mechanism
of apoptosis
• Intrinsic/ Mitochondrial Apoptosis
– Regulated by Mitochondria
– Cytochrome c release
• Extrinsic/ Death Receptor Apoptosis
– Activated by ligation of Death Receptors
– Fas, TNF alpha
• These pathways intersect at the effector caspases.
Intrinsic/Mitochondrial Pathway
Cyto C+pro-caspase9+Apaf 1
=apoptosome
Extrinsic/ Death Receptor apoptosis

FasL+Fas +FADD+pro-caspase8

death-inducing signaling complex （ DISC ）

Sufficient caspase8 insufficient caspase8

activate caspase 3,6,7 Bid proteolyses tBid enter

mitchondria

apoptosis
CtyC release
Extrinsic/Death Receptor pathway
 Example Question
Compare the formation of the Death Initiation Signaling
Complex (DISC) of the extrinsic pathway to the formation
of the apoptosome of the intrinsic pathway.
– What signal initiates the formation of each (an
aggregation step)?
– Where are the complexes formed in the cell?
– What adaptor proteins mediate the formation of
eachcomplex?
– What are the initiator and effector caspases for each?
– How are the caspases activated? What do they do?
 The relation between
apoptosis and medicine

Development

Diseases
Death and the mouse’s paw

Dark fluorescence indicates apoptotic cells.