BIO3153 Cell Biology Caroline Petit-Turcotte

Cell Cycle (Apoptosis) Lecture 9

Shivan Desai

Apoptosis:
- Programmed cell death
- Number of cells is, in part, controlled by regulation
of cell death
o 1) If the cell isnt intact one does not want
the cell to divide and keep these mutations
and keep the process going
o 2) To avoid an inflammatory response caused
by the cell that exploded and their content spread hazardly to
the neighbouring cells
o 3) Avoids Mess
- Differs from necrosis: trauma or cytotoxicity leading to ATP and
Na+/K+-ATPase activity, followed by lysis
- Necrosis is triggered by a trauma, something unplanned during the
cells life span
o The sodium potassium pump does not work properly, no ion
exchange, membrane becomes permeable to everything, the cell
swells and lysis, active enzymes then are in the vicinity of the
healthy cells
- It is a very clean process
o It gets rid of the cells without impacting the neighbouring cells
o However, some neighbouring cells participate in taking up some
of the debris, gain more space and can
divide faster
- Importance in development
- Balance of cell division with death in adult tissues
- Some cells are selectively programmed to die at a
particular moment during its life time to let the
organism grow and develop
o There is a timing and a trigger component
o For an organisms development cells are programmed to die and
be born at a certain time
- Apoptosis is important to regulate how many cells will live and how
many will die and when they will die or live

Apoptosis can also occur because of a situation not related to

development such as a mutation
In order for apoptosis to occur, a cell must undergo significant
biochemical and morphological change
Initiation by intracellular or extracellular signal
o Stress, Damage, Hormones, Toxins, Hypoxia, Excessive
intracellular Ca2+, insufficient nutrients, etc.
Activation of a series of proteins involved in promoting apoptosis (or
inhibition of those that prevent it)
o During both pathways there is a series of biochemical events
that leads to the cell dying
o The cell always begins with these biochemical pathways as a
disposable cell which has no impact on the neighbouring tissues
and/or cells
Important intracellular proteins necessary for survival are cleaved
during apoptosis
Orderly disposal of dead cell

Structural Changes during Apoptosis:

- Begin by having condensation of chromatin in the nucleus and the
cytoplasm will shrink
- Nucleus becomes fragmented and DNA is cut into smaller portions
(called DNA Laddering)
o This is a very important hallmark of apoptosis
o Because as DNA becomes fragmented the ends of these
fragments can be used to determine how DNA is fragmented and
can be used to detect it and to confirm that it is a apoptosis
process
- Then the cells under goes this process and encapsulating with the
portions of the membrane (portions of the cell), called blebbing
o Blebbing consists of little vesicles that contain part of DNA and
organelles wrapped in membrane
o These blebs are called Apoptotic bodies and go through
phagocytosis
o Neighbouring cells absorbs these blebs and get rid of them
o Therefore, there is no free debris flying around such as proteins
or enzymes

Phagocytosis:
- Asymmetric distribution of plasma membrane is lost
- Negatively charged phosphatidylserine then becomes exposed on the
outside of cell
- The cell is then marked for phagocytosis by a macrophage
- The membrane of the bleb (apoptotic body) is made up of two bilipid
layers, however, they are not organized the same
- The negatively charged polar heads are directed
towards the inside of the cell membrane (cytosol)
o When the apoptotic body is formed these
switch and the negatively charged polar
heads are phosphatidylserine molecules and
become exposed to the extracellular
environment/ layer
Which triggers the reaction and the
little vesicles are detected and get rid
of by phagocytosis through other cells
Cell
-

Death Mutants:
Caenorhabditis elegans is a nematode
959 somatic cells
131 apoptose, always the same
Discovery of ced-3 gene that encodes proteins
similar to mammalian protease caspases
- ced-3 encodes ICE/Caspase 1; ced-4 encodes
Apaf-1; ced-9 encodes Bcl-2
- During the 1800s, a group started to study
elegans
o Easy accessible genetic material
o Quick to get a new generation (takes 3 days
to get a new nematode with a different
function after)
o Nematodes have 959 somatic cells
o When the nematode is developing and differentiating 131 cells
go through apoptosis and die. Always the same
o Mechanism which decides what cell will die:
Discovered the ced-3-gene that encodes proteins similar to
mammalian protease

The group mutated the gene and none of the 131 cells died
in the nematode
There are more of these genes such as ced- 3 and ced 4
etc

Caspases:
- Identification of ced-3 gene led to the discovery of a homologous
family of proteins, called caspases, in mammals
- They are proteases that cleave essential proteins
- Involved in most changes observed during cell death
- This enzyme has a cysteine residue at its catalytic site and cleaves
other proteins at an aspartate site
- Caspases may also cleave each other, leading to their activation
- 2 main classes of caspases: Initiator (CASP2,8,9,10) and Executioner
(CASP3,6,7) caspases
- The group kept studying the genes and discovered that they are a
family of proteins called caspases
o They are enzymes, and they have a cysteine residue at the
catalytic site and they cleave other proteins at their aspartatic /
ate site
o There are two main classes of caspases:
Initiator caspases
Executioner caspases
- They are enzymes that cleave by proteolysis and they themselves are
activated by being cleaved
- Some caspases have the job of activating other caspases
- The machinery of the cell them puts together everything to make sure
the cells go through the apoptosis process
- Caspases cleave essential proteins:
o 1) Caspases being proteiosis can cleave of other caspases and
proteins in the cell
They can cleave certain kinases, for example: FAK (focal
adhesion kinase), these cleaved and non-active kinases
cannot function and perform their job
Fak allows the cell to stay adhered/ attached
o 2) Caspases can also cleave Lamins, which can no longer
maintain the cell structure of the nucleus and the nuclear lamina
breaks down and nucleus shrinks
This occurs when the DNA is being fragmented and
chopped into smaller pieces

Caspases undergo CAD (caspase activated DNAase)

cleavage which is the only CAD that grams the caspases
added activity and activates CAD
o 3) Caspases change cell shape, structure and size, cytoskeleton
They need to break the strong network made up of
filaments, membrane to make vesicles

Procaspase Activation:
- Caspases are first produced as procaspases
and are inactive
- These are activated by proteolytic interactions
(cleavage) with other active caspases
- They have a protein which keeps them inactive
and that portion needs to be cleaved off by a
proteolytic cleavage by another active caspase
- Once they are active they can perform their
job in the cascade depending on which
caspase is activated
The
-

Caspase Cascade:
An active caspase activates another caspase
Irreversible, all-or-none
Amplification
Result is intracellular proteins are cleaved
This is a domino effect because one caspase
activates another and the process continues which
is irreversible once it has begun
- Amplified process is where one signal can activate
many caspases and begin to work towards apoptosis
for the cell
- Caspases are responsible for the events that govern
apoptosis and what amplifies the signals in the cells

2 Major Apoptotic Pathways:

- 1) Extrinsic pathway:
o Procaspase activation triggered from outside the cell
o Cell death receptors
- 2) Intrinsic pathway:
o Procaspase activation triggered from inside the cell
o e.g. intracellular damage
o e.g. via Bcl-2 family of proteins (note: this may involve an
extracellular survival factor)
o Something on the inside of the cell triggers apoptosis and may or
may not involve survival factors
o Survival factors are outside the cell and bind the receptors
However, in the absence of survival factors it is the inside
of the cell that responses to the damage/ trigger
Extrinsic Pathway:
- Extracellular signal (Fas ligand) activates death receptor (Fas protein)
- Recruitment of adaptor proteins and procaspase activation
- Caspase cascade
- The signal is called the Fas Ligand
o Fas is a protein which is expressed on the surface of the cell and
this is mostly used in immunology
o Fas binds to death receptor on the cell surface
The binding triggers a conformation change within the cell
o Sub units of another protein called FADD which recruits the
procaspase and adaptor proteins which produces a DISC
Leads to the cleavage of initaitor caspase which renders it
active
o These caspases activate the extrinsic caspases
o Cell adherence decreases, and the executioner caspases are
going to cleave the tyrosine kinases that are responsible for cell
adhesion, lamins, CAD and will easily access the nucleus because
cytoskeleton is weak
- Executioner caspases cleave everything

Intrinsic Pathway:
- An intracellular death signal initiates caspase
cascade and cell death
- Initiated by e.g. DNA damage or loss of survival
factor
- Bcl-2 protein family
o Bcl-2 inhibits apoptosis
o Bax and Bak act on mitochondria and release
cytochrome c
- Balance of these determines fate (i.e. life or death) of cell
- Triggered from signal inside cell because of a damage to DNA, toxins
etc.
- An intracellular death signal initiates caspase cascade and cell death
- Need a family of proteins called Bcl-2 which is responsible to inhibiting
apoptosis and they are responsible for the bax and bak protein which
interact with the mitochondria and allow it to release cytochrome c
o Calcium is in mitochondria and the cytochrome c resides
between the inner and outer membrane of mitochondria
o Cytochrome is released from mitochondria by punching a hole in
the outer layer of the mitochondria and the pore is triggered
from the mitochondria because the internal concentration of
calcium increases and so mitochondria punches a hole to kick
the cytochrome c
- Apoptotic signal triggers dephosphorylation of Bad, which activates
Bax or Bak to form aggregates in outer mitochondrial membrane, AND
inactivates Bcl2
- Induces cytochrome c release from intermembrane space
- When bad becomes phosphorylated it becomes active and activates
Bax and Bak
o Also inactivates Bcl-2 and kicks it out by activating bax and bak
o Cytochrome c is then released out of the mitochondria to get the
cascade started

Formation of apoptosome
Caspase cascade
Cytochrome c joins Apaf1 (apoptotic protease activating factor) which
is triggered by P53 and Apaf1 is readily available to greet cytochrome c
o This forms a different compound called CARD caspase
recruitment domain
o CARD will be able to interact with the caspases and recruits them
o Therefore, cytochrome + Apaf1 = CARD complex recruits
caspases
o This is because they are inactive as procaspases and they are
ready to become protiolytically cleaved and form a structure
called Apoptosome
o Each card domain interacts with an initiator caspase and bind
together to form this apoptosome structure
o The apoptosome then activates the executioner caspases

Some mechanisms of the intrinsic pathway:

o Left side:
Survival factor receptor binds with activated PKB ( Protein
kinase B) which then activates Bcl-2 and put the brakes on
apoptosis
o Middle:
Another way to trigger this event is by having damaged
DNA
P53 then becomes stable because it is phosphorylated

It then triggers Bax and Apaf1

The middle and right side pathway leads to forming a
apoptosome. And our cascade of executioner caspase is
being activated and your intercellular proteins such as
DNA, lamins, cytoskeleton and kinases lead to the cell
being swallowed up by phagocytosis
o Right side:
If no survival factor, you do not activate PKB and Bcl-2 is
not active but you are able to dephosphorylate bad
Bad then allows activation of Bax and Apaf1

Executioner Caspase:
- Regardless of path taken, once executioner caspase cascade is
activated it undergoes the process
- Both pathways have it that when you activate the caspases cascade
your intracellular structures are broken down by the executioner
caspases
o Cell becomes disorganized and proteins are cleaved
o Both ways are clean and orderly disposal of the cell
- Regardless of the trigger the result is the same

Apoptosis in the Developing Nervous System:

- A growth factor, but also a survival factor
- Released by target cells
- Bax is an important mediator*
- Advantages of apoptosis are that the nervous system as it develops
produced a large amount of cells which all seek targets.
- An organism produces huge amount of neurons and cells
because:
o It is more efficient to have more of something then to
have less and not be able to function
o Neurons are special because only a few of them are able
to divide in special conditions
o To ensure that all of your required targets are set you produce
way too many neurons
o Some neurons are told from extracellular factors that their
service is done
o These factors include survival factors and growth factors
o In the absence of receiving growth factors from the target for the
cell, the cell will no longer grow/ reach the target and go through
apoptosis
o Target cells release the growth factors to reach the target
o This is an intrinsic pathways because the growth factor within the
cell doesnt allow the target to communicate with the neuron
- Mutations in genes encoding important proteins in the apoptotic
pathway have drastic consequences
o Mutations in genes hinders the encoding of proteins in the
apoptotic pathways and causes the cell to face consequences
o Such as cells will not be removed
o Cell cycle will have a hard time functioning and cell will keep
dividing
- In developing mouse embryo, forebrain protrusion and excessive
mitosis and differentiation result

Apoptosis:
- Cell death is characterized by the blebbing of the plasma membrane
and the fragmentation of the nuclei
- Suddenly, cells weaken attachment to the substrate that they had
been growing on and shrivel up leading to lysis
- The neighbouring cells have more space and their cytoskeleton is
going to grow and they will experience elongation of microtubules and
microfilaments and it will reorganize its intermediate filaments
- The mechanism of apoptosis involves very tight steps:
o 1) Sudden release of cytochrome c from the mitochondria into
the cytosol
o 2) In normal cell, phosphotidelserine is found only on the face of
the cytosolic membrane
But during apoptosis, it becomes exposed to the
extracellular space
o 3) This helps neighbouring cells to detect the dying cell and be
consumes from the process of phagocytises
o 4) The membrane of the dying cell becomes permeable to small
molecules