Lecture #19

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31 Jul 2010
of 10
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
dividing uncontrollably.
- 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
called caspases.
! Precursor molecules
! Other caspases
! 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
acid residues.
- 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. Youll 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
executioner caspase.
- 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
their DNA.
- 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
- 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.