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Hello, my name is Tony Hyman. I'm the director of Max Planck Institute
at Dresden, in Germany. And, for the second part of my talk,
I'd like to tell you about polymers: microtubules.
which are a fascinating part of the mitotic spindle,
which I've illustrated over here in this little cartoon.
If you remember, in the last talk, when we were discussing about scale in biological analysis,
microtubules are an organization of protein molecules, called tubulin, shown here.
And, tubulin molecules come together to organize these microtubule polymers.
Now, you can look at microtubules growing in cells,
and in this movie, you can see the ends of microtubules growing throughout our C. elegans embryo.
The ends of the microtubules are marked with a protein called EB1,
which is known to follow and recognize only the beginnings of microtubules,
growing from centrosomes.
Now, microtubules have interesting organization,
At the top, I've shown you dimers. We know the structure of the dimer in detail,
from a number of different structural techniques,
such as crystallography and also from electron microscopy.
And dimers form head to tail arrangements of protofilaments,
which I've shown down here using a technique called atomic force microscopy.
But, then these protofilaments associate side-to-side,
and form a tube. And, in vivo, there are about 13 protofilaments per microtubule.
And, in the bottom, you're seeing a microtubule by a technique known as vitreous ice,
where you can see the individual protofilaments.
The interesting thing about microtubules is that they grow from their ends.
So, you have a polymer, which is a tube,
and individual subunits come on to the ends, and they leave the ends,
and therefore you have an on rate of tubulin subunits,
and an off rate. And the growth of microtubules is defined by these different rates.
The other interesting thing about microtubules is that they have polarity.
So, you have a tubulin dimer, and the dimer is a heterodimer,
with two different subunits: alpha and beta.
And, those alpha-beta subunits set up a polarity in the microtubule,
with the beta subunit at the plus end.
So, the beta subunit marks the plus end of the microtubule,
and in the cell, the plus ends tend to be out in the periphery of the cell,
and the minus ends are concentrated at the centrosome.
So, a microtubule will nucleate from the centrosome, grow out through the cell,
with its plus ends leading.
So, it has dynamics, but it also has polarity.
Now, we can look at microtubules growing in vitro.
You can isolate tubulin from cells. One of the key places we isolate it from is brain
because there's a massive amount of tubulin in brain because it makes up all our neurons.
And then we can study microtubules growing in a test tube, as I've shown in this movie.
The big structure here is a centrosome, which we've also isolated from the cell.
We've isolated tubulin, and you can see it growing out along the coverslip,
simply from the tubulin molecules themselves.
So, in theory, microtubules do not require any other proteins to grow.
These are simple polymer systems.
But, microtubules in vivo... in the cell...
have very different behavior than microtubules in a test tube.
And the key difference is that, for any particular tubulin concentration,
microtubules grow faster in vivo than they do in a test tube.
They grow much faster.. sometimes 10x faster than you would expect.
The other thing is they tend to turn over more quickly in cells than they do in a test tube.
So, what I've illustrated here is an interesting behavior here known as dynamic instability,
where you can see the microtubule grows,
and at some stages it transitions to a shrinking state,
and then it starts growing again.
And what you can see is, both in vivo and in vitro,
microtubules are turning over by dynamic instability,
but they're much more dynamic in cells than they are in a test tube.
And that's something that's interested scientists for the last 25 years,
ever since the discovery of the different properties of microtubules
in vitro and those in vivo.
And we want to understand how microtubules are regulated in a cell, in an in vivo context
because that regulation is key to their activity in the cell.
Building a mitotic spindle, for instance,
requires that the activity of microtubules is regulated.
One of the questions you can ask, and we always have as biologists,
if you're interested in a problem like that... You look to your microtubules growing in a cell
and then you say to yourself, "I'm interested in that problem."
"How am I going to get at it?"
And the first thing you tend to ask yourself in biology is
How complicated is it?
Is this a solvable problem? Can I get at it?
And here's a review from Rebecca Heald, illustrating the numbers of different proteins
that are known to be involved in regulation of microtubules.
And you look at that, and it looks fairly terrifying.
There's so many different molecules involved in the different processes.
So, we decided to go and ask how complicated is the growth of microtubules
in the C. elegans embryo?
We just decided to focus on one particular problem, which is...
How many proteins are required to make the plus end of a microtubule
grow far through the cytoplasm?
If you remember, I said that it grows about 10 times faster in vivo than it does in vitro,
so you can ask how many proteins are required to do that.
Now, we did that by taking advantage of our genome-wide RNAi screen.
I mentioned this screen in the introduction, and this screen is an RNA interference screen,
where we can look for genes required for microtubule growth.
And to do that, we took our last set of 800 genes,
and we decided to screen subsets of those
for those that affected microtubule growth.
So, you remember our first screen was using Nomarski microscopy,
and we couldn't see microtubules.
It would have been too complicated for us, at the time,
to screen everything by fluorescent microscopy.
But, with our subset of genes, we can ask,
which ones of those are having their effects on the embryo
because they prevent the microtubules from growing properly?
And here's a movie where you can see the plus ends
of microtubules growing by EB1, as I mentioned.
And we can also track these microtubule ends automatically,
which helps a lot in terms of looking at the phenotype.
So, in essence, this is the outline of our screen now,
is we've taken the DIC screen -- the Nomarski screen --
and we've taken a number of genes, and we've got a set of genes here
required for cell division. So, we believe ... our hypothesis is
that any gene which affects microtubule growth is likely
to make the embryo not divide properly.
So, we take those genes and did some bioinformatics to subselect the genes
to reduce the amount of work we have to do,
and then we do our fluorescent secondary screen
using various different fluorescent markers,
and we look for the number of genes required for microtubule growth.
And when we did that, the results were really quite interesting,
because they actually showed that not many genes are required for a microtubule to grow.
If you have a look at this rather complicated bar chart here,
the white lines are showing the growth rate of microtubules.
So, on this particular axis, we have the growth rate of microtubules,
and you can see this layer here is about the growth rate of wild-type microtubules.
So, then you can say, let's go through the genes
and ask which genotypes no longer grow at wild-type rates?
And I've put those in the circle. You can see that there's a set of genes here -- two --
which are clearly required for microtubules to grow.
There's some other genes, which also affect microtubule growth, but we know
that those are required to actually make the tubulin dimer itself.
So, obviously, if you don't have enough tubulin, you're not going to grow.
We're not interested in those for this particular talk.
We actually want to know, when the tubulin is made,
what proteins are required to make the microtubules grow?
And here, all that work, we came up with two proteins that seem to be required for that --
TACC and Zyg9, which we happen to know are actually in a complex.
So, there's a complex of proteins which are required for the growth of microtubules.
Now, it turns out that this protein, which is in the middle here, Zyg9,
is part of a family of proteins. XMAP is one of the founder members in higher eukaryotes.
There's Stu2 in cerevisiae, and there's Dis1 in pombe.
And every organism studied so far ... every animal cell studied so far
has a member of this family.
And they have these very interesting domains in them, called TOG domains.
As you can see here, XMAP has 5 TOG domains, C. elegans has 3 TOG domains,
these yeasts have 2 TOG domains, but are thought to be in a dimer.
So, so far, what we've done is we've discovered then that actually, in an embryonic system,
controlling the growth rate of microtubules is quite simple.
You need these two proteins.
And that is the first part of any particular project in trying to work on any biological process.
We've done what's known as a genetic screen
using RNA interference to try and study the genes required for this process.
What is the catalog?
But then always comes the problem that any biologist then faces
is, what is the mechanism by which these proteins make the microtubules grow?
And so, how can one work on the mechanism of the activity of these different proteins?
It turns out, one of the key steps forward for us was to actually go
work on the protein in a different organism,
which was in Xenopus.
Now, biologists like to move around between different systems
to find the system which is most appropriate for the problem they're actually interested in.
And so, in this particular case, we use Xenopus because you can make extracts of cytoplasm
where you can take away the membranes.
Every time you work on a cell, you have the same problem, which is
how do I get components across the membrane?
The membrane of a cell has evolved over many millions of years
to exclude most things it doesn't like.
So, you're always fighting as a biologist to get things across the membrane.
Therefore, it's very helpful to be able to make cytoplasm extract
without membranes, and in Xenopus, you can actually make
very concentrated cytoplasm extracts in which most of the things actually...
many of the cell biology and cell division events we're interested in
actually still function.
So, that's shown here. We've got a couple of frogs. You take the eggs.
You crush the eggs in a centrifuge, and then you have a concentrated cytoplasm.
You can add centrosomes to that cytoplasm and watch microtubule growth.
And when we did that, we found
microtubules growing in the cytoplasm.
But, the interesting thing is we were then able to remove XMAP from the
extracts, so we can study the activity of XMAP in these extracts.
Over here, we have microtubules growing from a centrosome in the untreated extract,
and you can see lots of microtubules growing all over the cytoplasm.
But then what we can do with Xenopus, is we can make an antibody to the protein,
and we can deplete it from the extract,
and then you can see, you hardly have any microtubule growth at all.
So, both in Xenopus, and in C. elegans,
XMAP is a key protein required for microtubule growth.
So, then we'd like to understand how does XMAP make the microtubules grow?
And, to do that, the first thing you have to do,
is you have to make the protein in a test tube.
And then you can study it on its own.
And that's exactly what we did. We made XMAP in a test tube,
and we also were able to tag it with a GFP,
a green fluorescent protein, in the test tube,
so we could also look at the activity of the protein
as well as its localization.
Now the work I'm going to talk to you about has been done together with Joe Howard,
who's a close collaborator of mine, and most of the work from the last
10 years on microtubules have been done together with Joe,
who's a keen cricket fan.
And, we'd like to look at the role of XMAP in controlling the growth rate of microtubules.
Now, in order to do that, we have to look at microtubule growth
in a test tube, and we want to look particularly at the growth of the plus ends.
And we can monitor that in the test tube using fluorescence microscopy.
You can see the red segment marks the minus end,
and the green segment marks the plus end.
And you can see the green segment growing from the red minus segment.
Now, what you'll notice is the red segment is stable.
It's not growing and shrinking. And you can ask yourself, how is that?
That's key to our assay. By stabilizing the minus end, we can isolate the plus end growth
and look at how that's regulated.
Now I just want to go into, a little bit for you, about how we go about
stabilizing the minus end, because it's interesting both to think about the assay,
but also it gives us a little bit more understanding of microtubule and tubulin biology itself.
So, what we're doing in this instance is we're making polarity-marked microtubules.
So what we do is we take brightly labeled tubulin, here.
And, we've labeled tubulin in a test tube with a rhodamine dye...
chemically attached rhodamine to tubulin.
Then, we warm it up, and we make microtubules.
The next thing we do is we take dimly-labeled tubulin,
and we grow that from the seeds, and when we do that,
we end up with the dimly labeled tubulin growing from the seeds,
We warm it for another 15 minutes, and then we have these polarity marked microtubules,
with a bright minus end down here and a dim end that's grown off the end of it.
Now, you notice what I said here is that the seeds are stable.
So, how do we make them stable?
Well, there are a number of ways, but the most important and interesting way,
is to modulate the GTP-hydrolysis cycle of the tubulin itself.
So, it turns out that a tubulin dimer has two molecules of GTP:
alpha has a GTP molecule, and beta has a GTP molecule.
But, when tubulin polymerizes into a microtubule,
only the beta hydrolyzes GTP to GDP.
Now, there are analogs of GTP which can affect this cycle.
So, the cycle shown here, where the tubulin dimer comes on
to the end of the microtubule and docks.
When it docks, that completes the hydrolysis pocket in the beta subunit,
so the GTP now hydrolyzes.
So, we think that, mainly it's just the end of the microtubule that has a un-hydrolyzed GTP.
So, what happens if we block the hydrolysis of GTP?
Well, we can do that using analogs of GTP, as I mentioned.
There are a number of different ways of making analogs of GTP.
If you remember your high-school chemistry, you have guanosine,
and you have free phosphates
at the end of any nucleotide. And each one has an alpha-oxygen bond between
the different phosphate groups.
Now, what it turns out we were able to do, is you can modify GTP,
so that the alpha-beta oxygen is a carbon.
And you can see the name of that molecule above: GMPCPP,
or guanalyl alpha-beta-methylene diphosphonate.
And it turned out that this molecule was very, very good
at mimicking the GTP state of tubulin,
and when the tubulin goes into microtubules, what we discovered
is that GMPCPP is no longer hydrolyzed, and so it allows you to ask
what is the effect of preventing GTP hydrolysis
on the dynamics of microtubules?
And when we did that, you get this very interesting result,
which is that, if you look at a GTP microtubule, it grows and shrinks,
and then grows again, as you can see on this graph of microtubule length against time.
But, GMPCPP microtubules grew at the same rate as GTP-tubulin,
but they never transitioned to shrinking.
And, that confirmed old observations with other nucleotides
that the role of GTP hydrolysis in microtubules
is to destabilize them.
You don't need GTP hydrolysis of microtubules to grow,
but you do need GTP hydrolysis for microtubules to shrink.
So now, what we do, of course, is make our seeds using GMPCPP, which is stable.
And that way, we have the following assay, with a GMPCPP seed,
and the tubulin growing from the end of that stable seed.
So, now we have our assay. How are we going to analyze the role of XMAP?
Well, you have to use a special kind of microscopy to do this,
which is total internal reflection (TIRF) microscopy.
And Joe Howard's lab developed ways to do this to look at
the dynamics of microtubules using Total Internal Reflection Microscopy,
which is a way to just look at molecules which are very close to the surface of the coverslip.
Now, if you take your growing microtubule,
and then you take labeled XMAP and add it to the test tube,
what you see is XMAP has very interesting behavior. It's processive.
Or, it surfs at the end of the microtubules.
So, if you have a look at this figure here,
you can see that the XMAP at the end of the microtubule stays with the end
as it grows. It likes to be at plus ends,
and it likes to stay with them as they're growing.
So, you can then begin to ask, what are the dynamic properties
of XMAP at the ends of microtubules
by looking at single molecules of GFP-XMAP.
You can do single molecule techniques using TIRF.
And you can begin to ask questions like,
we know the XMAP is responsible for microtubules growing fast,
and so, how do the individual XMAP molecules behave
when the microtubules are growing?
If we do an experiment like that,
you can actually see the ends of the microtubules as they're growing,
with the GFP-XMAP, so when we use this assay,
we can look at GFP molecules growing at the ends of the microtubules.
And then we can ask, how long do individual molecules stay
at the ends of microtubules before dissociating?
And what we discovered was that, on average, an XMAP molecule stays about 4 seconds
at the end of a microtubule, which is about 25 tubulin dimers.
So, somehow, an XMAP is staying at the end of a microtubule,
and it's helping tubulin to get on to the end of the microtubule.
And how can that work? How can the XMAP molecule stay at the end of the microtubule
and help the tubulin add on at a faster rate,
which is required for the microtubules to grow faster?
One of the clues for this was that TOG domains bind tubulin.
Now, you remember that I told you at the beginning of the talk that XMAP
is a molecule with many different of these TOG repeats.
And so, Steve Harrison's lab solved the structure of a TOG domain
and was able to show that the TOG domains bind tubulin.
And, in fact, we were able to show that an XMAP binds one tubulin dimer, on average.
So, then you can ask, how is it that XMAP, by sitting at the ends of the microtubules,
helps these tubulin molecules get on to the ends of the microtubule?
One of the things we considered is that XMAP acts like an enzyme,
to catalyze the addition of tubulin molecules to the end of the microtubule.
And there are two things that should happen
if an enzyme is working in this particular case...
if XMAP is working as an enzyme.
The first thing is that it should also be able to make microtubules depolymerize
if there's no tubulin there.
And that is a classic feature of all enzymes you work on.
They go in one direction if they have substate there,
but if you take away the substrate, they'll go in the other direction.
So, synthetic enzymes often turn into degradative enzymes
if you take away the substrate.
And so, that should be the same for microtubules.
If XMAP is acting as a catalyst, if we take away tubulin,
one might expect it to start depolymerizing microtubules.
And that's exactly what we found.
If you add XMAP to microtubules in the absence of tubulin,
then microtubules start to shrink,
and this had first been noticed by the Mitchison lab in 2003.
The second thing is, the critical concentration of growth should not change.
And I bring this up, just to explain what we mean by the critical concentration
of the growth for microtubules ends, because you sometimes hear this term,
and it's sometimes quite confusing to understand what it means.
I remember when I first heard about it,
I had a lot of trouble trying to understand what this actually meant.
And the way to think about it, is to come back and look at our microtubule,
and think that tubulin has an off rate and an on rate.
The off rate is the rate at which tubulin molecules come off,
and the on rate is the rate at which tubulin molecules go on.
Now, if you reduce the tubulin concentration, you reduce the on rate,
until eventually the on rate and the off rate are matched,
and that's essentially the critical concentration for growth.
Just above that concentration, the microtubules will now begin to grow.
And so, we can come back and ask, what is the effect of XMAP on the critical concentration,
because for a catalyst, if you raise the off rate, you'll also raise the on rate,
and therefore the critical concentration will not change,
and that's exactly what we found here.
You can see the critical concentration of growth,
you can see the point where it goes above 0,
is exactly the same point.
So, therefore, what we conclude from these experiments,
is that XMAP acts as a polymerase, as an enzyme.
And I think the key experiment we did to show this is to show, if you take away tubulin,
microtubules shrink. If you add back a little bit of tubulin,
microtubules just begin to grow, and if we add more, they start to grow even faster.
So, the cycle of microtubule growth appears to be modulated
by the amount of this XMAP protein in the cycle.
So, we think then that XMAP acts as a polymerase.
And, I took you through this story to illustrate a number of different things.
At the beginning I showed you how we can use genetic screens
to get at the complexity of any particular system.
But then, I dived down into a little bit more detail to say that, once you get that molecule,
that's not enough. You then need to actually go and work on the mechanism
by which it's having its effects.
And that's what the goal we all have is, in the end,
to try and work on a mechanism
by which these individual proteins and their protein complexes
affect their particular activity.
And, if you remember at the beginning,
I said that microtubules are these very interesting complexes
of proteins, which grow and shrink in the cell.
And you can see how the interaction between these protein complexes
and other protein complexes
modulates other activity in order for the correct biology to happen.
I'd like to thank... there's two people mentioned who have been key to this work:
There's Gary Brouhard and Jeff Stear,
who were key to this particular experiment, and I think it's a classic example of teamwork,
where the two of them worked together, and I think it's very important to remember
that these complex sorts of experiments
we've been discussing about XMAP and microtubules
depend very much on this sort of teamwork,
of people working together for a common goal.