Tuesday, March 17, 2009
- If we go back to the question he raised in the first lecture: How do cells
control their shapes, their interactions, their numbers to build tissues,
organs & organisms?
- So far we’re talked about control of cell shape, cell interactions with
themselves or with the matrix & now he’d like to turn to the control of cell
numbers, both by controlling cell division & cell death & next week we’ll
talk more about mitosis & also the link to cancer b/c one of the key things
to cancer is the control of cell number.
- Next week we’ll discuss more about cancer, but during the lecture today,
he’ll also highlight molecules, link to the cell cycle that are also implicated
in cancer as well & so we’ll cover that again next week in more detail.
Cell duplicates and segregates its genome
- Here is the eucaryotic cell cycle illustrated in a basic way. Here we start
off with a single cell and then that cell divides. A key aspect of this is that
the material in the cell has to duplicate itself. If the cell is going to divide, a
lot of material has to be produced so the cell cycle is important to have cell
growth which is illustrated in this simple cartoon. You can see the increased
size of this cell, so that’s the increased growth of membranes, increased
growth of the cytoskeleton, for example, mitochondria, ER membranes,
Golgi membranes so there’s enough material to divide into 2 for the next
- The duplication and the segregation of the genome is critical because this
encodes all the instructions for everything that’s happening inside the cell
so if there is a mistake made in the duplication of this genome, during the
synthesis of the DNA, that could create mutations which would lead to
problems in the cell. If during mitosis, certain chromosomes were lost, then
of course that’s going to cause problems as well. So it’s critical for both
cells that are dividing & developing organisms that they synthesize &
segregate their DNA properly during division so that those cells maintain a
proper cell physiology & if they don’t, then that’s often associated with the
development of cancers in our bodies.
- Also for the proper passage of DNA from one generation to the another to
maintain the species, they need to maintain their DNA properly to be able
to pass on from generation to generation & this all comes down to the cell
cycle & the control of how the cell duplicates & segregates the genome
properly. - Here is the cell cycle in more detail, broken into specific phases. So we
can start right here – this is just after division has happened so this single
cell has just been born from a division & the first phase is called G1 or gap
phase 1 and it is a period of cell growth. Here is when the cell is partially
doubling its proteins & organelles so that it builds up in size getting ready
for a future division itself. So here it’s growing.
- The next phase is called S phase which is dedicated to the synthesis of
DNA so the replication of the genome. Here we have the replication of the
- This is then followed by a second gap phase, G2, and this is another
period of cell growth where the remaining proteins and organelles will
- Then if everything is in order the cell will then enter M phase where both
the nucleus & the entire cell will divide so 1 you’ll have a nuclear division,
then you’ll have a cell division to split all the material of the cell in 2.
- So next week we’ll talk about mitosis in much more detail. Today we’re
going to talk about the transition b/w these different phases.
- You can see from the fact that it has 1 phase after the other, this needs to
be temporally regulated & coordinated & there are many processes going
- So a key thing for this is the cell must ask itself, it must check whether
things are going okay during the process. Ex: Are there enough resources
for DNA replication? Is this initial growth phase going effectively? Are
there enough resources there for me, the cell, to replicate my DNA? Then
for example, has the DNA actually replicated before mitosis starts? So
before you start trying to split the DNA into two cells, the cell has to be
sure it has actually replicated the DNA, that it has 2 copies of the DNA to
separate into the 2 cells – if it doesn’t, then one of the cells is going to be
missing huge sections of the genome. So these questions are asked at
specific phases of the cell cycle.
- So if we start again at this growth phase, before the cell goes into the S
phase, the cell will ask itself is the environment favourable? Are there
enough resources present? Here is a key checkpoint right here, the
transition into the synthesis phase of the DNA so if there aren’t enough
resources present at that time, the cell will just stay in G1 phase, it will wait
until resources are available & then it will replicate its DNA when the
conditions are better.
- The other key point where there is a checkpoint is here for the entry of
mitosis. Here it will ask is all the DNA replicated? Are there 2 copies of the
genome to divide b/w the cells & it can also check again if the environment
is still favourable. Here is a growth phase right here, the cell can monitor
whether it’s growing properly, are there enough resources there? If there
aren’t then it can halt again at this stage & wait until the environment is
more favourable & then divide.
- Then there are other checkpoints during mitosis & we’ll touch on those
again next week. Protein kinases
- So the core molecules here are Cdks which are protein kinases & Cdks
stand for cyclin dependent kinase. These are protein kinases & they have
targets that control the cell cycle so they phosphorylate proteins that will
control different stages of the cell cycle.
- One key step is that another protein called a cyclin has to bind to these
kinases to turn them on, so this is one way to turn on these molecules to
pass through the checkpoint.
- Diagram; We’ve got the cyclin present here, here is the Cdk – you need
both of these components, and more as we’ll see in a moment, to activate
At different stages of the cell cycle
- So to coordinate things in time during this cycle, there are different cyclin-
Cdk checkpoints at different stages of the cell cycle.
- So if we started off here at G1 right here, the cell is growing, things are
favourable, there are lots of resources available, if that’s the case the cell
will then signal to produce this S cyclin, the cyclin that will promote the
synthesis phase. This will then bind to the S-Cdk & this complex can then
phosphorylate machinery involved with DNA replication in the cell so the
cell can dedicate all of its machinery to replicating the DNA.
- Once that job is done, the cell wants to turn off that machinery since the
genome has been replicated, they don’t need all of those enzymes present
anymore to duplicate the DNA so now this cyclin is destroyed. So we have
a very specific cyclin-Cdk complex to promote a specific phase of the cell
cycle & then it’s destroyed after its job is done.
- Now once the cell then passes through S phase, it passes the G2 phase, it
grows again & then it monitors are there is enough resources still and has
the genome been fully replicated? So if both of those things are okay, then
it will produce a different cyclin called an M-cyclin which will interact with
the Cdk, producing a different cyclin-Cdk complex & this complex
promotes the machinery controlling mitosis which we’ll talk about in detail
- Once that is done, so this would be machinery like building the mitotic
spindles out of microtubules, once that’s job is done & the DNA has been
segregated into 2 cells, you don’t need that machinery anymore, so this
cyclin is destroyed, the activity of this cyclin-Cdk complex is turned off so
then this mitosis machinery is turned off.
- So this is a molecular cycle then where the cell can control whether it’s
activating DNA replication machinery or mitosis machinery depending on
which cyclins are available & these are monitoring the availability of
resources & whether the DNA has been duplicated & so on – these are the
checkpoints. - Here in this table it shows these different cyclin-Cdk complexes. Here we
can see the different phases of the cell cycle – the G1, the G1 to S, going
into S & here is the mitosis phase so it is focused around the synthesis
phase and the mitosis phase, the control of that DNA replication &
segregation into 2 cells.
- Now in vertebrates here, we can see the major cyclins that are associated
with these checkpoints, so cyclin D, E, A & B & here is the cyclin-
dependent kinase partner so we have all these different combinations, one
that is specific for each of these checkpoints.
- Notice that there is also combinatorial control here & that look at this one
right here, the same Cdk partner at both steps here but when it’s bound to
cyclin E it has one behaviour, whereas when it’s bound A, it has a different
behaviour, it regulates a different checkpoint. Now this combinatorial effect
is shown even more so in yeast so here is budding yeast & here are the
different cyclins that are expressed or that build up the different
checkpoints during the cell cycle, they are all binding to the exact same Cdk
partner, so it’s the same Cdk, the same cyclin dependent kinase, all through
the cell cycle in yeast & it’s just whether it’s in a combination with a
different cyclin that controls its downstream activity, whether it controls the
transition into S phase or the transition into M phase so there’s this key
combinatorial control bringing cyclin together with the Cdk.
- So this activation of the Cdk, this cyclin dependent kinase involves cyclin
as we’ve talking about but it also involves a phosphorylation step as well
which is highlighted here in this slide.
- Here we’ve got our Cdk, it is completely inactive, there’s no cyclin there
at all. Then this Cdk, once the cyclin is expressed at a specific stage in the
cell cycle, it will bind to the Cdk and now this Cdk will become partly
active but this cartoon highlights that there is a loop, a polypeptide loop,
within these Cdk that right at this stage here, this is blocking substrates
from going to the active site of this kinase so this kinase is about to
phosphorylate, for example, the machinery that is going to activate DNA
replication, but at this state right here all of those substrates that are in that
DNA replication machinery they can’t get into the active site of this kinase
b/c of this T loop. So to get this T loop out of the way, there is a Cdk
activating kinase that has to act, so this phosphorylates this T loop, opens
up the active site so now this kinase can phosphorylate its downstream
- So the full activation of the Cdk requires both cyclin binding & then also
this activating phosphorylation, the addition of this activating phosphate to
this T-loop by Cdk activating kinase. So once this is activated then, now it
can start to engage with the machinery that actually controls the structure of
the cell so that it can either drive DNA synthesis or drive mitosis. So the
Cdks regulate the machinery that directly replicate the cell.
Promoting DNA replication
To start mitosis
- So here we have our Cdk over here, if this is a cyclin Cdk complex that is
driving the transition into S phase, to synthesize the DNA, this kinase, its
phosphorylation inhibits an inhibitor of the origin recognition complex. So
if this complex is no longer inhibited then DNA replication can occur so it’s
directly engaging with the machinery that is replicating the DNA & it’s
turning that machinery on.
- Here is this is a cyclin-Cdk complex that’s promoting transition into M
phase or mitosis, this cyclin will phosphorylate components associated with
mitosis so this will phosphorylate a lamin, the intermediate filament that’s associated with the nuclear envelope & this will lead to breakdown of the
nuclear envelope so the chromosomes can be separated to the 2 separate
cells. They’ll also regulate & phosphorylate proteins that are required for
the condensation of chromosomes into small packages so they can be
separated into separate cells & they also phosphorylate microtubule
regulators & this will create this very specialized structure, the mitotic
spindle, to separate the chromosomes & these aspects of mitosis we’ll talk
about in much more detail next week.
- The key thing here is this is what is downstream of these regulators. The
actual machinery that is controlling what the cell is doing, right at the level
of microtubules, at the level of DNA & so on.
- So how are these guys regulated here? So what turns Cdks on and off?
The one key thing is the availability of cyclins. Another thing is the
presence or absence of proteins that can inhibit the Cdks & also there are
phosphorylation events that can inhibit the Cdks. So let’s walk through
these 3 examples.
- We’ve seen that the production of a cyclin can promote the activation of a
second cyclin Cdk complex and then the destruction of this will then turn
that cyclin Cdk complex off.
- So as we’ll see in a couple of examples later, the production of these
cyclins is often controlled by transcription so the cyclin gene will be
transcribed & translated when conditions in the cell are good for
progressing through that stage of the cell cycle so that will produce that
cyclin here, activate the complex & the destruction mechanism typical of
the cyclins is illustrated in the next slide.
The completion of mitosis
- So this is an example right here about the destruction of a cycle by the
anaphase promoting complex & so the APC targets M-cyclin, so the cyclin
of mitosis & this allows the completion of mitosis. So anaphase is a later
phase of mitosis, when the cell recognizes that it’s progressed through
mitosis, it will activate this complex to start completing the mitosis process.
- Now the regulation here involves regulated degradation of the M cyclin &
this is through, here we can start off with the inactive APC, here’s our
cyclin Cdk complex right here so this one needs to work on this one.
- The 1 step is the activation of the APC, then once the APC is activated, it
will recruit ubiquitination enzymes to the complex & add the small
molecule ubiquitin onto the cyclin molecule so you’ll have this chain of
ubiquitin on this cyclin & that chain then is a recognition site for the
proteasome so proteins that are polyubiquitinated in this way are recognized
by the proteasome, they are targeted to this structure for destruction so here
we can see that the ubiquitination of these cyclins will target the cyclin for
degradation at the proteasome.
- So this is the process that occurs up here at mitosis & a similar process
will occur during the S phases as well with the regulated degradation of
these cyclins. - Now more control is needed on top of this.
- Here is one way – this is by the cyclin, cdk inhibitor proteins, so in this
case, we have our active cyclin cdk complex, here it has its activating
phosphate group on it so it is all ready to go and activate downstream
targets. But if this protein p27 is present, it will bind to this complex and
hold it in an inactive state. So now there are two things that have to happen
to progress through the cell cycle when p27 is active. You have to receive a
signal to activate the complex and the cell also has to receive a signal to
- The cell cycle is critical that it is done properly and so the cell builds in
these multiple layers of regulation. Two things must be done for passage
through the cell cycle.
- Another example of that is regulation by phosphorylation.
- Here we have our active cyclin cdk complex but this complex, even
though it has the activating phosphate on the bottom site, another kinase
called Wee1 can phosphorylate it at another site and inactivate it. Now we
have an inactivating phosphate. Here even though this one is all ready to
go, if it gets phosphorylated by Wee1, it cannot activate downstream targets
and can’t promote the cell cycle.
- The cell needs to express or activate the protein Cdc25 which is a
phosphatase which specifically removes this inhibitory phosp