general idea about apoptosis

1. BY : SAGARIKA MUKHERJEE
M.SC (SEM-IV)
VISVA BHARATI
E.mail-zoo.sagar2015@yahoo.in

2.  apoptosis, a Greek word that means “dropping off” or “falling off,” as
leaves from a tree.

3. 1.Programmed cell death is as needed for proper development as mitosis is.
• The resorption of tadpole tail
• Formation of toes and fingers in fetous
• Formation of synapses
2.Programmed cell death is needed to destroy cells that represent a threat to the
integrity of the organism.
• Cells infected with viruses
• Cells with DNA damage
• Cancer cells
3. Apoptosis in physiologic situations
• Programmed destruction during embryogenesis
• Involution of hormone dependent tissues
• Cell loss in proliferating cell populations
• Elimination of harmful self- reactive lymphocytes

5. • Cell shrinkage , cells became smaller in size, cytoplasm dense and cell organelle
tightly packed.
• Pyknosis ,characteristic feature of apoptotic cells, is actually chromatin
condensation.
• Extensive plasma membrane blebbing.
• Karyorrhexis is the destructive fragmentation of the nucleus of a dying cell
whereby its chromatin is distributed irregularly throughout the cytoplasm.
• Organelle intigrity maintained ,all are enclosed in plasma membrane.
• These bodies are subsequently phagocytosed by macrophages and degraded
within phagolysosomes.
Fig 1A .Photomicrograph of a section of
exocrine pancreas from a mouse. The
arrows indicate apoptotic cells.
Different Steps of apoptosis:beginning of apoptosis,bleb
formation,apoptotic body formation and phagocytosis.

6. N.B. The Extrinsic and Intrinsic Pathways are under the Canonical Apoptotic
Pathway and others are under Non-canonical Pathway.

7. • They have proteolytic activity.
• Caspases stands for Cystein dependent aspertate
specific protease.
• Caspases are so named because they contain key
cystein residue in the catalytic site and
selectively cleave protein at just C-terminal to
the aspertate residue.
• All caspases synthesizes in form of a
procaspases.Procaspase splits into large and small
subunit,that form heterodimer, two such
heterodimer assemble to form active tetramer
• Once activated,they activate other caspases and
resulting in an amplifying proteolytic cascade.
• Target of caspases are DNase, nuclear lamin,
cytoskeletal protein and golgi matrix protein.

8. Activated
Caspase
1 2 3 54
DNA, Nuclear lamin, cytosketal
protein,Golgi matrix protein
PROCASPASE
Activate
other
Caspases

9. Example:
• Fas receptor or Fas (a member of
TNF receptor family).Its ligand is
FasL (A homotrimeric protein
expressed in cytotoxic T
lymphocytes and NK cells)
• When Fas binds to its ligand
,FasL,an adaptor protein called Fas-
Associted Protein with Death
Domain(FADD) is recruited to bind
with Fas.
• This is followed by recruitment of
Procaspase-8
• The death domain of FADD binds
with the Procaspaser-8 and results
in the formation of Caspase-8.
• The activated caspase-8 molecule
then activate downstream
procaspases to induce apoptosis.
*Picture from : Molecular Biology of the Cell-Alberts 5th ed , Garland Science

10. Schematic representation of
the role of mitochondria in
apoptosis. Cytosolic
cytochrome c interacts with
Apaf-1,aloowing subsequent
caspase activation.

11. • Key molecules of this pathway are trans membrane pore forming
Perforin (secreted by cytotoxic T lymphocytes), two Serine protease
with substrate specificity similar to the caspase family of apoptotic
cysteine proteinases Granzyme A and Granzyme B.
• Granzyme A activate DNA nicking via DNAse. Nucleosome assembly
protein SET normally inhibits DNAse but here in this case DNAse
becomes activated because Granzyme A cleaves SET.As a result
cellular DNA degrades and apoptosis proceeds.
• Granzyme B can take part in apoptosis in several ways.
1. Granzyme B can cleave pro caspase 10 to activate caspase 10 which
in turn cleave ICAD (Programmed cell death activated DNAse).
2. They regulates specific cleavage of Bid (this cleavage is more readily
occurred than that of caspases), which induces cytochrome c release
from mitochondria , and promotes apoptosis by intrinsic pathway.
3. Granzyme B directly activates caspase 3 , then promotes apoptosis
by execution pathway .
*Ref- Apoptosis: A Review of Programmed Cell Death , Susan Elmore.

12. Overview of Apoptotic Pathway
Extrinsic Pathway Intrinsic Pathway Granzyme Pathway
LIGAND
DEATH RECEPTOR
Adaptors (FADD)
DISC(Death inducing
signalling
compound)
Formation
Execution
Pathway
Mitochondrial Changes
Apoptosome Forms
Perforin
Granzyme B
Caspase 8
activation
Caspase 9
activation
Caspase 10
activation
Caspase 3
activation
Granzyme A
Cleave
SET
Complex
Dnase
activation &
DNA CLEAVAGE*Ref- Apoptosis: A Review of Programmed Cell Death , Susan Elmore.

13. Activation of Exicution Caspase (Caspase-3,6,7)
Cytoplasmic
Endonuclease
Protease
Degrade Nuclear Material
Degrade nuclear and
cytoplasmic proteins
CELL DEATH
The main Caspase which is
associated with execution
pathway is Caspase 3.
- Caspase 3 activate
endonuclease CAD (by
removing the inhibitor
molecule ICAD) which degrade
chromosomal DNA and causes
chromatin condensation.
- Caspase3 helps in cytoskeleton
reorganisation -disintegration.
- Caspase3 binds with Gelsolin,
which is the nucleus for actin
polymerisation & prevents
this.
- This molecule is the cause for
lipid asymmetry in plasma
membrane ,which promotes
phagocytosis of these cells

14. • The key regulator of apoptosis is the Bcl-2 family of
proteins.
• They are of two main types , Antiapoptotic Proteins ,
which includes Bcl-2, and Proapoptotic proteins , which
are again subdivided into two groups , Multidomain
proapoptotic protein (includes Bax ,Bak) and BH3-only
proteins.
• Other than this family proteins, there is another family
of proteins which are called IAP ,they directly interact
with caspases and suppress apoptosis by ubiquitination
and degradation of them.
B.Regulatory interaction between Bcl-2
family members.
B
B
C.Regulation in mitochondrial pathway of
Apoptosis
*Picture A from : Molecular Biology of the Cell-Alberts 5th ed , Garland Science
Picture B,C from: The Cell , A molecular approach , Fourth Edition , Geoffrey M.Cooper ;
Robert E.Hausman , ASM Press , Sinauer Associates , Inc.
A
C

15. 1. Plasma membrane assessment
2. DNA fragmentation
3. Detection of caspases, cleaved substrates, regulators and inhibitors
4. Mitochondrial inner membrane potential alteration
5. Detection of apoptosis in whole mounts
6. Mitochondrial assays.
Abnormalities in cell death regulation can be a significant component of
disease.Some conditions feature insufficient apoptosis whereas others feature
excessive apoptosis.
INSUFFICIENT APOPTOSIS
Different Types of cancer
EXCESSIVE APOPTOSIS
Neuro-degenerative disease
(Alzeimers Disease , Parkinsons
Disease). Auto-immune diseases,
Ischemia associated injury.

16. • Apoptosis is regarded as a carefully regulated energy dependent process,
characterized by specific morphological and biochemical features in which
caspase activation plays a central role.
• The importance of understanding the mechanistic machinery of apoptosis is
vital because programmed cell death is a component of both health and
disease, being initiated by various physiologic and pathologic stimuli.
• Understanding the mechanisms of apoptosis, and other variants of programmed
cell death, at the molecular level provides deeper insight into various disease
processes and may thus influence therapeutic strategy.