Cell Cycle two

initiating M phase

So we just completed the S phase. We've divided up the, uh, or replicated the genome and gone through that G2 phase.

That's, uh, quality control. Everything is okay.

So now we're we want to initiate m phase and actually start segregating chromosomes and dividing the cell into two new cells.

So remember I told you that that the m CDK at least has a set of brakes that can be put on through phosphorylation.

It shows two phosphates being here, I don't know if that's the case or if they just wanted to make it look like I's.

What do I know? Right? So, um, so in any case, there are inhibitory phosphate or phosphates on here that are applied by that kinase.

We one that we saw a few slides back in the, uh, the previous lecture.

Right. Well, um, what removes these phosphates is a, a phosphatase called CDC 25 in this figure.

I think it's fun because it looks like Squidward winking at you. Right.

So CDC 25 removes these inhibitory phosphates, which activate M phase CDK complex here.

Now you'll notice something interesting And that is these things work in a positive feedback.

So CDK DC 25 rather is a target of M phase cyclin CDK complex.

So that may be another reason why we have that super burst of M phase activity.

Because activating a few M phase cyclin CDK complexes is going to initiate a whole

cascade of activating more M phase cyclin CDK complexes through the activation of CDC 25.

Okay, this is a side a slide.

That's a little bit of an aside. Okay. Um, and it may be something that you'd be aware of from the 3100 class,

but I want to make sure everybody knows about these, um, Two particular sets of proteins that condenses and the cohesion,

because at least the cohesion are going to be really relevant here when we start talking about the actual division of the nucleus.

So I have a problem with this figure, though, because the right side of the figure, these three are individual DNA molecules, okay.

Being wrapped into those condensed chromosomes.

So you can see condenses here in the blue are making DNA loops.

And then those condenses are come to come together to make a kind of a daisy of those loops.

And then those daisies are getting stacked together right.

To form the actual condensed chromosome. Then it's over here on the left where we have the sister chromatids.

So you know that the, um.

Replicated chromosomes get to come together in a in a bundle, right?

So this is one side of the replicated chromosome, and then it's happening again a second time on the other side, creating the sister chromatids.

The, uh, those sister chromatids are held together by large protein complexes that are called cohesin.

So that classic X-shaped chromosome, those two sister chromatids that may be linked here at the center,

are also held together by these cohesin proteins.

So these are going to be important later when it comes to the separation of those sister chromatids.

All right. Now into M phase. M phase is named, of course, for mitosis, the division of the nucleus.

But remember, the division of the nucleus is not the only thing that happens here.

Right. There is this final part, cytokinesis, which is the actual division of the cell.

So m phase represents both the division of the nucleus and the rest of the division of the cytoplasm.

M phase is divided into 12345 sub phases.

Right. Which are actually representative of mitosis.

Right. The last sub phase Telephus may happen simultaneously with cytokinesis.

And in fact, my little bracket here may go up as far as anaphase, right?

So once anaphase, uh, is well underway, the cell may start to divide too.

We'll see that here in a second. So we're going to look at a little bit of what's going on in each one of these um mitotic

sub phases and leading up to the ultimate separation of the entire cell into two.

So one of the main things that happens in prophase is the formation of something called the mitotic spindle during G1 and into G2,

the centrosome, the microtubule organizing center which each cell that's not dividing has one duplicates and becomes two at the end of G2.

These things begin. These replicated centrosomes begin to to separate from each other, and since they are both microtubule organizing centers,

they begin to build microtubules, as we saw originally in the non dividing cell.

Right. So this ultimately creates what's called a mitotic spindle as our.

Two centrosomes begin to move to either side of the cell.

What does this is in fact interaction of microtubules.

So this microtubules are interacting. And that essentially shoves our replicated centrosomes off to equal distance on either side of the cell.

Meanwhile. You can see the genetic material inside the nucleus starts to condense.

The generic brown dot up here represents the interphase nucleus, where we, you know,

most of the genetic material and proteins that are associated with it are dissolved.

And that doesn't mean that it's dissolved like a liquid where everything is mixed up.

In fact, there are hot spots for each chromosome that are that are inside the nucleus.

But right, that all that DNA is generally very, very diffuse at the beginning of n phase.

Prophase, we get the, uh, the start of the condensation of the of the genetic material into chromosomes, as we see right here.

Now you'll notice that the nucleus here is still intact.

But when we get to pro metaphase, that nucleus begins to fall apart.

So that allows the some of the microtubules from these replicated centrosomes access to those chromosomes.

So some of the microtubules of those replicated centrosomes will selectively stabilize each other.

Those microtubules ultimately become what are called inter polar microtubules.

These spread across the cell. Basically creating a kind of a band of microtubules across the cell.

They're going to be important ultimately for pushing the two sides of the cell apart.

These black dots here represent motor and other proteins.

But right once we have microtubules interacting with microtubules,

they selectively stabilize each other, which means we no longer have that dynamic instability.

And those microtubules potentially only grow.

So they only get longer until our two centrosomes are where we want them to be.

Some of the other spindle microtubules will bind to chromosomes.

So here we have the two replicated chromosomes,

sister chromatids that are brought together and held together by those cohesion and also held together at a central spot that's called the centromere.

The centromere is wrapped with proteins that make up what's called a kinetochore.

You can kind of think of the kinetochore as a belt around the waist of the chromosome, right?

That not only helps hold the chromosome two chromosomes together, but also gives a spot for the anchoring of kinetochore microtubules.

Okay. Sorry.

Gotta make a pause here. And maybe I will do that.

That seemed to work last time, right? Just a moment. Always something.

Okay. Good news. That turned out to be nothing.

So, you know, I tried to shut off notifications, but can't shut it off everywhere, apparently, unless I put it on under.

Um, do not disturb. And I don't want to do that either. So. Okay.

Meanwhile, back at the ranch. So these kinetochore proteins allow for the attachment of microtubules and also again selectively stabilize them.

So we don't get the dynamic collapse of these things, but only the growth.

The growth of those kinetochore microtubules, as they're called, are what shoves those chromosomes into the very center of the cell.

So in the end, we've got three sets of microtubules coming from those replicated centrosomes.

We have the Aster microtubules, which may be anchored into the opposite ends of the cell or remain unaffiliated.

So these may undergo continue undergoing dynamic instability.

In the pink, we've got the inner polar microtubules, which are spreading across the center of the cell but are associated with each other.

Right. Again, these black dots are motor or other proteins that are also associated with those microtubules.

And finally we have the kinetochore microtubules which are attaching to the kinetochores on the replicated chromosomes.

Oh excuse me. And you can see we have a, um, false color electron micrograph here of our microtubule or microtubule organizing center,

the centrosomes uh, and they inter polar microtubules.

And then the red dots are the kinetochores where the kinetochore microtubules are attaching to the blue replicated chromosomes.

So once we have all the chromosomes attached to microtubules,

and those microtubules have shoved our chromosomes equally to the very center of the cell, we create what's called a metaphase plate.

The amount of tension on those microtubules,

those kinetochore microtubules is actually a what defines the metaphase plate and is the M phase checkpoint.

So the kinetochore microtubules have to be experiencing equal tension, right.

In order for the cell to be convinced that all its chromosomes have been attached to microtubules,

and they are pushed to the exact center of the cell.

So metaphase doesn't last very long right? So very relatively rapidly we can get those are those chromosomes lined up across the center of the cell.

Now we move into anaphase. Okay. Anaphase begins with the cleavage of the coherence.

Remember those sister chromatids are being held together by this belts of cohesin complex proteins as we see down here at the bottom.

So this is what we see in metaphase right with the kinetochore microtubules attaching to the kinetochore.

Okay. Meanwhile in the cytoplasm.

We're activating through M phase.

Cyclin an, a group of enzymes called APC the anaphase promoting complex.

Anaphase promoting complexes. Target, among other things, is a pair of proteins called securing and separates.

Securing is an inhibitory protein that keeps separates from being active until we're ready for it to be active.

APC has a enzyme that will add ubiquitin to securing, and it also may contain a protease that breaks down.

Securing and APC also has a kinase that will activate Cyprus, so it does two jobs.

When separate is active. It is now a protease that targets coherence.

Once the cohesion have been broken, as we see in the the right side of the figure,

the tension on those kinetochore microtubules rips our replicated chromosomes apart, or rather the system chromatids apart.

So remember each one of these is identical to each other.

And so now our two B cell has its own copy of each chromosome.

This separation of the Sister chromatids here is a part of what's called anaphase A,

but at the same time, the inter polar microtubules are starting to push away from each other.

Right. This is so-called anaphase B.

So we're beginning to elongate the cell as those chromosomes are being pulled to their own little space and either side of the,

um, the replicating cell. So at anaphase, we are really starting to kind of push the two sides of the cell apart.

Right. And so you can see now why cytokinesis might really start about now.

Right. Because as we've gotten those chromosomes into two different sides,

we can now start pinching down on that elongated cell membrane and and start actually pinching off the two cells.

Now, remember in, uh, excuse me, in pro Metaphase, we went through a process of breaking down the nucleus.

How did that happen? Right. Well, good old m fe cycling and other kind or.

Excuse me, m phase, cyclin CDK complex and the other, uh, some other kinases target, uh,

the proteins that are present in the nuclear membrane, in particular proteins of the nuclear pores, and also the layman's.

Okay, the cytoskeletal components, the intermediate filaments that keep the nucleus in its round shape when those lemons are phosphorylated.

They separate from each other and as you can see here, sometimes take some nuclear membrane along for the ride.

So this breaks down the nucleus and frees up those replicated chromosomes, which allows the kinetochore microtubules to attach to them.

When we get into tela phase, though, the very last part of Em phase,

their phosphatases become active and those phosphatases start removing the phosphates from our nuclear components, in particular the nuclear.

Lemons. Those lemons come together into intermediate filaments and allow our nuclear membranes to start reforming.

The chromosomes themselves may be a a site where the layman's are congregate,

so you'll notice how some of them are clustered around the the chromosomes.

So the chromosomes themselves may be drawing the nuclear membrane around them.

And so reforming their own nucleus. So it's not like, you know,

the nucleus is wrapping around the the chromosomes so much as the chromosomes are drawing the nucleus in and creating a, a cover for themselves.

So that's what we see happening here, right?

Eventually we get the complete fusion of the nuclear membrane again around each set of chromosomes and two new nuclei, which occur in in T phase.

Meanwhile, those inner polar microtubules keep pushing the two sides of the cell apart.

At the center point of those inner polar microtubules where the motor proteins and other proteins are are found.

We start building actin filaments in the form of what's called the contractile ring.

And of course, the job of the contractile ring is to work like that.

Okay, dynamo in protein. When we were making vesicles,

we're bringing the sides of the cell together and attempting to get within the the repulsion limit of the phospholipid membranes.

So the dark band here in the scanning electron micrograph here is the contractile ring.

And you can see that the contractile ring is almost completely squished down on the remaining inner polar microtubules.

So those inner polar microtubules have not completely, you know, disassociated.

Okay. That contractile ring is squishing down on it, attempting to get it to complete the process.

So the I we can bring the edges of the membrane together and cause the the two new cells to pinch off completely as we see here.

Okay. So that's how you make more individual cells.

Just in a nutshell. What about what if what about if you had two Many cells and wanted to get rid of some

guy or a cell has basically become nonfunctional or was functioning improperly.

And you potentially want to wanted to cause that cell to disappear.

Right. Well, the way that this is done is through a process called apoptosis.

Okay. Apoptosis regulates cell numbers by causing a programed cell death.

So we've got two possibilities here for getting rid of cells.

One is necrosis where you see doesn't look very nice kind of messy right.

Where the cell essentially ruptures and its cytoplasmic contents get released into the extracellular matrix.

This is potentially a problem because the proteins and other substances inside the cell, um, aren't necessarily used to train the immune system,

so anything that's inside the cell and shouldn't be outside the immune system doesn't worry about.

But if we get a situation where some of the cell contents get spilled out into the, um, uh,

into the extracellular matrix, we can get a massive immune response, which we don't necessarily want.

Okay. That's where apoptosis comes in to play.

So instead of the cell rupturing and releasing its contents out into the extracellular fluid, right.

It very neatly packs itself up and actively signals the um innate immune system.

So cells like the um Macrophages and other types of cells to come in and gobble this cell up, which is what we see here.

Okay. So these dark blobs right here. Probably the nucleus that has been sectioned out.

Okay. All these other big blobs, likely lysosomes that are stuffed full of things that we would want to get rid of anyways.

But notice the cell membrane is left intact here.

Okay. Which means the cell is not activating the immune system except in a way that it would want to control.

So eventually the the immune system cell would show up, gobble our apoptotic cell up and complete the breakdown and recycling of its components.

So back in cell signaling we talked about a protein called BCL two.

BCL two is made constantly if the cell is receiving a survival signal.

So if it gets, say, like a growth factor signal,

which is usually the survival factor signal that we're talking about the activation of the receptor for that growth factor,

I will result in the production of BCL two.

BCL two I is an apoptosis blocker.

Right. And remember if we want to block BCL two we activate bad.

So bad is potentially not good right.

One of the things that BCL two prevents is the activation of enzymes called CAS bases.

CAS bases are fairly generic proteases that start to tear down proteins in the.

In the cell. We only want to activate these CAS bases when we're ready to.

So there is a cascade of cascade bases and other proteins that activate each other and progress the process of a pertussis.

So, for example, just like with the CDK, the cell is always making this so-called initiator CAS base.

This initiator CAS base is a protein that has multiple subunits that are not necessarily in the proper order.

What initiates the um. activation of this initiator CAS base.

Use the word initiate twice. Okay. Is the presence of an adapter.

So this adapter is probably inhibited by BCL 2 in 1 way or another, which means the initiator CAS base will never get together with the adapter.

Right. Never get rearranged in such a way to be active.

Now, this initiator CAS base doesn't necessarily act on other cell proteins.

It acts specifically on what's called an executioner CAS base.

The executioner CAS base is the one we only want to activate when we need to.

And you can see not only do we have to bring two of these together, but we have to affect a rearrangement.

That's one of the things that the initiator CAS base does is cause a rearrangement of the, uh,

executioner caste based protein and bring these two sides of the protein together in such a way that it becomes active.

Right. So now, after a multi-step process, have we turned on something that will actually start breaking down certain protein structures in the cell?

So these executioner cast spaces are kind of soluble and will move throughout this, uh, throughout the cytoplasm.

Right. Some CAS bases though, become uh, um, exist in big clusters that like sit in one spot of the cell.

So that's what we see here. Um, this CAS Bass Pro CAS base nine is dependent on a separate activator protein.

And that activator protein, though, is one that we've come across before,

and that is cytochrome c, that little protein that is at the end of the electron transport chain.

Cytochrome c is normally locked up inside the mitochondria, but in the presence of a cytochrome c transporter called Bax or back,

okay, we get cytochrome c out into the cytoplasm, which we don't want, right.

Because it functions as a coactivator with the this pro cast base nine.

Right. Which right turns this into a structure that generally tears down a it works to tear down particular organelles at a site.

Interesting thing about Bax and Bak are that they are part of the BCL two family proteins.

So on one hand you've got BCL two, the OG that inhibits a ptosis.

And then we've got Bax and Bak that are BCL two family proteins that initiate apoptosis.

So another weirdness about how we've taken one basic sequence of a protein and turned it into something that can do multiple jobs.

Okay, that's the end of that. Finally. Right.

I haven't seen anybody say that they've had any trouble. So I'm hopeful that everything is going all right with the exam.

So I'll hear about it later I'm sure. Okay.