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Learning Objectives
Describe apoptosis including its features and
importance
Understand TUNEL assays to assess apoptosis
Lecture Outline
Define apoptosis, describe its features, and
discuss its importance
Discuss caspases, their activation, and their
roles in apoptosis
The extrinsic apoptotic pathway
The intrinsic apoptotic pathway
Apoptosis and disease
Cancer prevention
Body Sculpting:
1 day later
Phagocytosis
of Apoptotic
Cell
Necrosing
Cell
Symptoms of Apoptosis
Cytoskeleton collapses; cells becomes more compact
Nuclear DNA breaks up into nucleosomal-sized fragments
Nuclear envelope disassembles and chromatin condenses
Phosphatidylserine flips from the inside to the outside of
the plasma membrane bilayer
Mitochondrial function is lost
Plasma membrane is altered (blebbing), allowing dying cell
to be engulfed by macrophages via phagocytosis
Having phosphatidylserine
on the outside of the cell
serves as an eat me
signal, which is why
phagocytes engulf it.
TUNEL assay of
apoptosis in a
developing chick
bud when the
digits are being
formed.
TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assays add
a fluorescent nucleotide to DNA ends. The DNA ends are extended by adding
fluorescently labeled dUTPs using the template-independent DNA polymerase
terminal deoxynucleotidyl transferase (TdT). Large numbers of labeled
10 DNA
fragments leads to bright fluorescent dots in apoptotic cells.
Lecture Outline
Define apoptosis, describe its features, and
discuss its importance
Discuss caspases, their activation, and their
roles in apoptosis
The extrinsic apoptotic pathway
The intrinsic apoptotic pathway
Apoptosis and disease
11
GPCR
Cytokine
Src
PI3K
Grb2/SOS
G-protein
Adenylyl
Cyclase
PLC
PKA
PKC
AKT
Ras
mdm2
Raf
p53
PTEN
Jak
R-Smad
Smad4
MAPK
Caspase 8
Caspase 3
Guanylyl
Cyclase
PKG
NO
Dsh
Mito
Caspase 9
ANP
Guanylyl
Cyclase
MEK
CREB
FADD
TGF
APC + GSK3
Gene Regulation
Cytochrome C
Apoptosis
-catenin
Cell Proliferation
NR
NUCLEUS
12
Wnt
Cells in culture
were treated
with Fas ligand
(FasL) to initiate
apoptosis at time
0.
After various
times, DNA was
isolated and
analyzed by
agarose gel
electrophoresis.
procaspase
(Val-Glu-Thr-Asp)
(Leu-Glu-Thr-Asp)
19
Lecture Outline
Define apoptosis, describe its features, and
discuss its importance
Discuss caspases, their activation, and their
roles in apoptosis
The extrinsic apoptotic pathway
The intrinsic apoptotic pathway
Apoptosis and disease
20
GPCR
Cytokine
Src
PI3K
Grb2/SOS
G-protein
Adenylyl
Cyclase
PLC
PKA
PKC
AKT
Ras
mdm2
Raf
p53
PTEN
Jak
R-Smad
Smad4
MAPK
Caspase 8
Caspase 3
Guanylyl
Cyclase
PKG
NO
Dsh
Mito
Caspase 9
ANP
Guanylyl
Cyclase
MEK
CREB
FADD
TGF
APC + GSK3
Gene Regulation
Cytochrome C
Apoptosis
-catenin
Cell Proliferation
NR
NUCLEUS
22
Wnt
23
25
Figure 18-5 Molecular Biology of the Cell ( Garland Science 2015)
26
Figure 18-5 Molecular Biology of the Cell ( Garland Science 2015)
28
Lecture Outline
Define apoptosis, describe its features, and
discuss its importance
Discuss caspases, their activation, and their
roles in apoptosis
The extrinsic apoptotic pathway
The intrinsic apoptotic pathway
Apoptosis and disease
30
GPCR
Cytokine
Src
PI3K
Grb2/SOS
G-protein
Adenylyl
Cyclase
PLC
PKA
PKC
AKT
Ras
mdm2
Raf
p53
PTEN
Jak
R-Smad
Smad4
MAPK
Caspase 8
Caspase 3
Guanylyl
Cyclase
PKG
NO
Dsh
Mito
Caspase 9
ANP
Guanylyl
Cyclase
MEK
CREB
FADD
TGF
APC + GSK3
Gene Regulation
Cytochrome C
Apoptosis
-catenin
Cell Proliferation
NR
NUCLEUS
31
Wnt
Released cytochrome c
binds to Apaf-1(apoptotic
protease activating factor
1), an adaptor protein
with a CARD domain
(caspase-associated
recruitment domain).
33
CytC
34
Structure 18: 571 (2010)
Mechanism of
activation is not
known. Activation is
probably through
cleavage by adjacent
Caspase 9 proteins
or intearction of
Caspase 9 and Apaf1.
35
38
Bax - cytosolic
39
Bax Bak
Anti-apoptotic Bcl2
proteins bind to and
inhibit the BH123 (Bax)
proteins, preventing
clustering, cytochrome
c release, and thus
apoptosis.
The Bcl2 proteins are
actually bound to the
mitochondrial surface,
although not shown
here.
41
42
Bcl2
Bax
Bad
43
Lecture Outline
Define apoptosis, describe its features, and
discuss its importance
Discuss caspases, their activation, and their
roles in apoptosis
The extrinsic apoptotic pathway
The intrinsic apoptotic pathway
Apoptosis and disease
45
47
Gilberts, Developmental Biology 9 edition, Figure 3.32
th
48
49
BAX is a proapoptotic
BH123 family
member
BAX
BAX
Figure 17-62 Molecular Biology of the Cell ( Garland Science 2015)
52
Bax
BH123
Puma
BH3-only
Noxa
BH3-only
53
54