Thursday, March 19, 2009
- Today we continue our discussion about the control of cell numbers and today instead of talking about cell
proliferation we will talk about programmed cell death or apoptosis.
- On this slide up here, we have a few examples where programmed cell
death is used in a developmental context. On the top here, we can see how
programmed cell death can be used to sculpt fingers and toes during
development. Here, our bodies take the strategy of building the full
structure initially, so right in (A) we can see tissue between the digits of the
hand or foot. It builds the full structure and then uses apoptosis to
specifically kill off the cells in between the digits.
- Another example of this is seen during different stages of the life cycle
here in the frog. There may be a part of the body that is necessary at one
stage but then the organism may dispose of it at a later stage, the removal of
the tadpole tail for example.
- Even in adults as well the regulation of organ size, apoptosis plays a key
role in that. If organs become too large, the body can recognize that and use
apoptosis to trim down their size.
- In addition in adults, programmed cell death plays a key role in protecting
the body against dangerous cells.
- Here is one danger to a cell, right here is excessive production of the
protein Myc. As we learned about last day, Myc will drive forward the cell
cycle promoting S phase and the synthesis of DNA, promoting cell
division. If you have too much Myc, this is oncogenic, it is called an
oncogene, there is more potential for this cell to become cancerous and
- Programmed cell death can help control this. It goes through this pathway:
The cell can detect that there is too much Myc activity going on and then
this feeds into the pathway involving p53 so this detection of excessive
Myc production produces a protein called Arf. This then binds to the
protein Mdm2 and removes it from p53. So you remember that normally,
Mdm2 is actively degrading p53 but now that there is this signal that there
is a danger in the cell, now the p53 is no longer degraded so the p53 will
now act to arrest the cell cycle as we learned about last day and if things
aren’t rectified by arresting the cell cycle, the p53 will then induce
apoptosis. If the cell can’t get control of its cell division, then signals will
be sent out to kill off the cell since it is a danger to the body.
- You can think of this as well in response to DNA damage. We learned last
day that instead of excessive Myc production, that another danger to the
cell would be DNA damage so the cell does not want to go a round of DNA
synthesis, replicating that damaged DNA, replicating any mutations. The
same thing applies here, in that case as well, we learned last day how p53
will arrest the cell cycle to try and repair the damage but if the damage
can’t be repaired, then that cell will just be killed off by p53. So this is an
example of how programmed cell death can kill off dangerous cells.
- Apoptosis is a very regulated and stereotyped process and we contrast this
with necrosis. The cell death is not regulated by the cells themselves but
something outside has damaged them and killed them off accidentally.
- The bursting of the cell is shown in the slide, there is the outline of the cell
and its contents bursting out into the surrounding area. This material that is
normally kept inside the cell is now in the extracellular space. This can lead
to damaging inflammatory reaction, immune cells will come to deal with
this and there will be a lot of swelling in that part of the body. There will be a lot of energy going to dealing with this.
Shrinking the cell
Collapsing the cytoskeleton
Fragmenting the DNA
Cell removal by engulfment
- So apoptosis occurs differently, it is much more regulated. Apoptosis is
basically all about neatly packaging up all the compartments of the cell &
disposing of that cell in a clean way. There will be a general shrinking of
the cell, the cytoskeleton is collapsed that normally gives the cell its
structure so now it is broken down so it can now be collapsed down. The
DNA are huge molecules in the cell that has to be chopped up into pieces &
once the components of the cell is packaged & we can see it in the slide,
there’s an apoptotic cell and instead of spewing its contents out, we can see
all these compartments within the cell, compartmentalizing all of these
different breakdown products. Once it is in this state, it signals to
macrophages in the body which cleanly remove these cells by engulfing
them so they can be eaten up and taken up by other cells. Their contents are
never exposed to the extracellular space.
- This right here (bottom right slide) is just showing us in a tissue, there is a
phagocytic cell around the outside of the apoptotic cell being engulfed.
Shrinking the cell
Collapsing the cytoskeleton
Fragmenting the DNA
Cell removal by engulfment
- How are all of these processes here triggered? So this involves molecules
Amplified cascade of caspase activity
- So the basic enzymatic activity of caspases is that they’re proteases. They
are cysteine proteases and they cleave target proteins at specific aspartic
- These proteases are normally kept off so they’re expressed and
synthesized as procaspase precursor molecules that don’t have this activity.
These procaspases are then cleaved and activated by other caspases. Once
this prodomain is cleaved off, these proteases become activated and then
they start attacking different protein components of the cell to start
digesting the protein complexes. All of this then is part of an amplified
cascade of caspase activity.
- So here if we look at an individual caspase molecule, this is what it looks
like right here. Here is one single polypeptide chain right here, & this is the
inactive procaspase. Right here, this grey trapezoid is the prodomain that
must be cleaved off in order to activate it. Right now, these molecules
would be present in the cell but held in an inactive state by the prodomains
but they’re ready to be activated at any time if the cell needs to be killed
- So this cleavage involves 2 cut sites so on this one molecule, they will be
cut to remove the prodomains and then cut at another site. This then creates
two different pieces from this single polypeptide and those two pieces, one of the pieces is released, the prodomain is shed but then the two remaining
pieces then interact with each other (dark green and green). Then you have
these two created from this one polypeptide and then these guys then come
together as pairs.
- The active protein is actually a tetramer. Here we have these 2 proteolitic
fragments from one caspase here & 2 proteolitic fragments from the
caspase. The active caspase then has two large and two small subunits.
- Here is what this amplification cascade will look like then. You’ll have
some caspase up at the top, here you have an initiator caspase up there. The
first thing that this initiator caspase acts on is another layer of caspases. So
you have initial activation up here and then this activates a whole series of
caspases downstream so the signal will then start to spread. One caspase
leads to the activation of many and then these can be referred to as the
- These caspases are also proteases but their targets will be other cellular
components that need to be broken down during apoptosis. They will cleave
cytosolic proteins for example, as well as cytoskeletal regulators & start
breaking down the cytoskeleton, they will start breaking down the nucleus,
they may cleave the nuclear lamin, they will also cleave inhibitors of
DNases so now if you remove an inhibitor of a DNase, it will be active &
start chopping up the DNA. Now the cell can fragment its own DNA.
- One way to monitor if a cell is undergoing apoptosis is by monitoring the
structure of its DNA.
- So right in the slide, we’re detecting fragmented DNA using agarose gel
electrophoresis after induction of apoptosis. So at time 0 right here,
apoptosis has not been induced and then we’re looking at the number of
hours after apoptosis has been triggered in these cells. Here we have our
DNA so it is negatively charged and it will run towards the positive
electrode down at the bottom and the agarose gel will cause resistance,
resisting that migration so the larger molecules will be held back and stay at
the top. Then you can see over time as apoptosis is induced, now there are
all of these smaller faster migrating products that begin to accumulate. Here
we can see the fragmentation of the genome because some of these
executioner caspases have digested an inhibitor of a DNase and we have
breakdown of the DNA. Here we can see that in a gel that is happening
here. We can also detect this in tissues as well.
- For example in this case we talked about on the first slide where we have
cells in between the digits of the developing hand, we believe these cells are
undergoing apoptosis since they disappear. What is one way we can test
whether they’re going under apoptosis? We can see if they’re fragmenting
- This takes advantage of a technique called TUNEL labelling. This detects
fragmented DNA in cells and tissues by TUNEL labelling after these cells
have triggered apoptosis. TUNEL stands for terminal deoxynucleotidyl
transferase mediated dUTP Nick End Labelling (DON’T MEMORIZE
THAT FOR THE EXAM).
- But we can see what this is doing. Here is the enzyme that is a transferase
so it is transferring a nucleotide onto the terminus of a DNA fragment. So
when this DNA gets cut up into pieces, now there are going to be many
ends of DNA exposed. Now this enzyme can add a nucleotide onto those
DNA ends. Those ends will only be in apoptotic cells and not in normal
cells. The nucleotide that they add here can be labelled with something we
can detect under the microscope. Here this labelled nucleotide is then added
to these nicks in the DNA and then we can probe the tissue and see which cells have fragmented DNA, which of them have these exposed DNA ends.
So all these little dots right here are either nuclei or whole cells which
contain this fragmented DNA and so specifically we can see a region there
in the slide is undergoing apoptosis but cells in other regions are not.
- These are the downstream effects of this caspase signalling. We see
fragmentation of cytosolic proteins, cleavage of nuclear proteins and here
we can see cleavage of the DNA. We’re breaking down all the parts of the
- What is upstream of this? What starts to activate this whole cascade in the
- So these caspase cascades can be activated either by extrinsic or intrinsic
signals, either from signals outside the cell or from signals generated within
the cells itself.
- The example here is an extrinsic signal, and here we’re triggering
apoptosis from outside via death receptors. Here this killer lymphocyte will
be destroying this target cell right here. So this cell right there, it may be
recognizing that it has been infected by a virus so one of the body’s
defences against that is to kill off that cell before the virus can replicate
itself inside that cell and spread to neighbouring cells. If this cell knows that
it is infected with a virus, it will send out a signal telling the immune
system to come and destroy it by apoptosis. So it expresses what is called
the Fas death receptor on its surface so this tags it to be destroyed by the
- Here we have the killer lymphocyte, it expresses the ligand for this death
receptor. Now this ligand will bind to this death receptor and engage it here
and start to build a complex below. On the cytoplasmic side of this death
receptor, the first thing that is recruited is an adaptor protein, called Fadd so
this is the Fas Associated Death Domain adaptor protein. It is called the
death domain because one of its two domains is called the death domain
and then this red part here is called the death effector domain. This death
domain gets recruited to the cytoplasmic tail of the death receptor right here
and then it draws in a key molecule, the top caspase in the caspase cascade.
Here is our procaspase right here that will be at the top of that caspase
cascade. It has a very specific additional protein attached to it. This is the
death effector domain, it is the same one as earlier. This protein domain
interacts with itself and will recruit the procaspase to this complex, the one
that is attached here.
- Now we have our receptor engaged with a ligand, that drew in Fadd, that
drew in the top caspase of the caspase cascade. So now this thing is called
DISC or the death inducing signalling complex. - Once this is all recruited in here, now the difference between these
caspases here and in the cytoplasm is that they’re clustered together. This
appears to play a key role in activating this top caspase because remember
right now that it has this prodomain on it that is preventing it from having
proteolitic activity but when they’re drawn together, they do have some
small amount of proteolitic activity and if they’re drawn in close proximity
like right here, now they can cleave one another because they’re right next
to each other.
- Now they will cleave off their prodomain, they leave this DISC complex
behind and now can activate the executioner caspases downstream which
can then attack all the different parts of the cell to trigger apoptosis here.
- The main concept here is that there is an outside signal that is triggering
the clustering of the top caspase in the hierarchy so the clustering of that top
caspase activates its proteolitic activities, it cleaves each other and then
trigger downstream caspases.
- A similar idea occurs with intrinsic signals as well.
- In this example, the cell is triggering apoptosis from within and this
involves the release of electron carrier protein cytochrome c from damaged
mitochondria after there has been cell stress.
- If we start on the left, here is the mitochondria. If the cell is in normal
condition the cytochrome c (red dots) would be inside this space. With
apoptotic stimulus, the cytochrome c is released from the mitochondria and
binds to a cytoplasmic protein called Apaf1. This triggers a conformational
change in Apaf1 exposing this CARD domain and with the hydrolysis of
ATP and the exchange of ADP