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Hello I am Ron Vale, professor at
University of California, San Francisco.
and investigator with the Howard Hughes Medical Institute.
And in the first two parts of my lecture
I talked about molecular machines and even studying
these machines at the single molecule level.
Now I am going to a very different scale
at the level of whole genomes
trying to understand very complex structures
made up of many proteins,
in this case the mitotic spindle here.
Now mitosis is a wonderful problem.
It is one of the oldest problems in cell biology,
and in fact, it dates back to
biologists from the 19th century
So here is actually and image by Flemming
of the 19th century. Remarkably accurate depiction
of the mitotic spindle,
showing the chromosomes and these fibers,
which we now know are microtubules.
And the problem of mitosis is also very important
for medicine as well.
First of all failures in the assembly
of the mitotic spindle can give rise
to chromosome misegregation
that results in aneuploid cells.
Aneuploid cells are often a precursor for cancers,
and uncontrolled cell division.
And in addition, there are many drugs used
in cancer chemotherapy that work
by targeting the mitotic spindle
as I'll describe to you at the end of this lecture.
So the questions that I would like to discuss with you
today are what are the molecules
and the molecular interactions
that are needed to build this remarkable assembly
of the mitotic spindle. Here we see microtubules
in green; the chromosomes in blue.
And the poles of the mitotic spindle
marked here in orange.
And what I'd like to primarily focus this lecture
on is the first step of mitosis.
The building of the mitotic spindle.
And here are tubulin-GFP
emanating from these centrosomes,
and at the onset of mitosis
what you'll see is the nuclear envelope break down
and these microtubules form into the mitotic spindle
and it's that formation of the mitotic spindle
that we'd like to understand
and I will discuss in this particular lecture.
And there are many questions that remain in this field,
even though it's been the subject of extensive study.
So first of all, there are lots of microtubules
that build...that build the spindle.
and we'd like to know how those microtubules are assembled.
There are specialized structures in the spindle:
the centrosome, found at the poles;
kinetochores, at chromosomes
that interact with the microtubules.
And we'd like to now how these structures are formed.
And we'd also like to know how
this characteristic football shape
of the spindle is formed and organized.
So there's lots of questions that we need to understand.
And the approach that I am going to share with you today,
the approach done in our lab to find new proteins
involved in creation of the mitotic spindle
using a whole genome RNAi screen
in Drosophila S2 cells.
And this work was executed by
a very talented group of people.
The leader of this project was Gohta Goshima
who has now started his own laboratory in Nagoya,
and Nico Stuurman, who's a senior research scientist in the lab
that developed a lot of the technology infrastructure
to make this happen.
And follow up work after the screen was performed by
Nan Zhang and a new student, Sarah Goodwin.
And also another key person for this work was Roy Wollman
working in Jon Scholey's lab
and Roy did a lot of the computational analysis
that I'll share with you later.
But as you'll see, it's possible to do a whole RNAi screen even
with a relatively small group of individuals.
So why do an RNAi screen?
Well, I would submit to you that you do this
to try to discover something new
and I'll even use this analogy of how early explorers
went out on expeditions to find new things in the world.
So this shows an early world map
where much of the world was very poorly defined here
There are coasts, large landmasses
that have yet to be defined.
This is very analogous in many
ways to 20th century biology
Even as a graduate student I had very little idea
of how big the genome was, what was in there
And at that time, which was even in the mid 1980s
you could still discover a big protein super family
like the kinesins. And a lot has changed
both in world geography and in biology.
World geography over centuries,
but in biology over a short span of even a couple decades.
And now the world looks more like this
in biology the genomes through all the sequencing effort
and work by thousands of investigators
we know quite a lot.
The genome has now become a finite entity.
We know a lot of the components
that are present in the genome.
But I do want to stress that there are still a lot
of undiscovered genes out there
that we need to understand.
And this RNAi screen that I'll share with you
is a good way of making new discoveries.
So, let me just tell you a little bit about RNAi.
It's a technique that allows you
to destroy mRNA corresponding to one gene
and as a result of the destruction of that mRNA,
result eventually over several days
the depletion of that particular protein.
And by this method,
you then can deplete a particular protein
and look at a particular outcome of the cell.
So the way this works is, at least in Drosophila
is that you can first make a double stranded RNA
corresponding to a particular gene
and you'd like to use a particular piece of that gene
that's unique for the gene
and not found in other genes in the genome.
That double stranded RNA is then taken up by cells
There's there...then there's a special enzyme called dicer
that chops this RNA into 21 base pair fragments
which then interact with a specialized protein
complex called the RISC complex
that then unravels these siRNAs.
It then base pairs them with an mRNA in the cell
and after base pairing results
in the degradation of that mRNA
and then, as I said, over a period of days
results in the depletion of the protein.
So, we used ...we've been using siRNA or, sorry, RNAi
in this Drosophila S2 cell line
and we've chosen this cell line for a number of reasons.
First of all, it's very easy to culture.
You can even grow it at room temperature.
You also get very efficient RNAi
knockdown in these particular cells.
And we worked out methods
that make these cells very good
for microscopy, which is critical for the screen
that I am going to tell you about.
And then, if you want, you can also follow up
with genetic manipulations in flies.
So the screen that we did was a visual screen.
What we wanted to do was to knockdown mRNA
corresponding to each gene
in the Drosophila genome,
of which there are over 14,000 genes,
and then look at the effects on the
morphology of the mitotic spindle.
And so we did this by imaging
the spindle after RNAi of each gene.
And there is a lot of information that you
can get from these images.
You can see the overall shape of the spindle.
You can measure the length.
You can see if the chromosomes are aligned properly.
You can measure a very special protein
that defines the poles,
called gamma tubulin that I'll tell you more about.
And you can see if they're are two poles
as there should be in a normal spindle
or if the poles are split or fragmented.
So there's a tremendous amount of information that you can get
from direct imaging. So we knocked down
all...each of the over 14,000 genes in the fly genome.
And in this screen we analyzed over 4 million spindles.
So let me just tell you a little bit about the technical details
of how this screen was done.
So first of all, you need to develop an RNAi library
against the entire fly genome.
And this takes a lot of careful bioinformatics
to design double stranded RNAs that are unique for particular genes...
for each particular gene, so you don't get cross knockdown of other genes
that knockdown multiple genes for example.
So this bioinformatic effort was done by
Nico Stuurman in the lab, in the lab
and in fact now this library is available at
reasonable cost from a company called Open Biosystems.
And then what we did is we treated these Drosophila cells
with double stranded RNAi corresponding to each gene
so we arrayed these S2 cells in 96- well plates,
each with a unique double stranded RNA.
And then to cover the whole genome,
one had to use nearly 150 such 96-well plates.
We also did double RNAi with another molecule
called the anaphase promoting factor.
And this is a protein that's involved in
the transition from metaphase to anaphase.
By knocking down this protein,
more of the cells arrested in this metaphase spindle
which was the structure that we wanted to investigate.
Now, after we did all this RNAi,
we had to then collect all the images of these mitotic spindles.
And this was done with a high throughput microscope
where we first transferred the cells
to glass bottom 96-well plates
and then put them into this robotic microscope
where you could place all these plates in this robot here
that will grab and place these plates then
on the microscope stage automatically.
The microscope will go around and automatically focus on
each well and then it will take a number of different images,
again totally automatically,
around the well of these cells.
So we get a tremendous amount of image data
and what we image here are the DNA.
We image microtubules.
We image this special protein gamma tubulin
that I'll tell you more about later
and another marker called phospho-histone,
which is used as a marker to know that the cell is in mitosis.
So out of this we get a tremendous amount of image data,
far too much for any person to manually go through.
So here is where computation proved very valuable.
and this was co-written by Roy Wollman,
who wrote MATLAB code to find these
mitotic spindles automatically in these images.
And after the computer found these images,
we did two things with them.
First of all, this computer code
arrayed all these mitotic spindles
into this big gallery that you see here.
And then the next day after the computer
collected all of these images
then Gohta Goshima could wake up in the morning,
come into the lab
and open up these files
with all these galleries of these mitotic spindles.
So this is, for example, two hundred mitotic spindles
found from one well of one RNAi knockdown.
And he could look at these images and just visually assess
whether there was any defect in the shape
or architecture of these mitotic spindles.
In addition to this human based analysis,
Roy also wrote code that would segment this mitotic spindle
and then analyze a number of features
of the spindle that a computer could analyze
such as the intensity of the stain,
or the length of the spindle,
or the number of poles.
So the combination of a human looking at the data
and a computer analyzing this quantitatively
proved to be a very effective way
of analyzing a whole genome RNAi screen.
So the result of the screen is that we found that there were
about 200 genes that when knocked down,
the protein levels were knocked down,
produced some kind of defect in the metaphase mitotic spindle,
and a lot of these different phenotypes
are shown in this gallery here.
So first of all, I should say that
we knew going into the screen that there were
49 genes that we knew produced
mitotic spindle defects in the cells.
And through this blind whole genome screen
we found 45 of them
which is really quite good.
In addition to those 49 known genes,
of this list, many of the genes that gave spindle defects
are components of multi-subunit complexes
like ribosomes, spliceosomes,
proteasomes, and RNA polymerase.
Interestingly, actually the knockdown of these
different large protein complexes
produced quite interesting and, in fact,
unique spindle phenotypes from one another.
In addition, we found about 60 knockdowns
that...of unknown or unexpected genes that were previously
not known to have any role in the mitotic spindle.
And this is the list that
we spent a lot of time investigating.
Now I should say that all of this
data is publicly accessible.
It's on a website here, and you can actually scroll through
and find any gene that we analyzed in the genome
and all the details of the images and phenotypes
that we found for that particular gene.
So what can one learn from a screen?
Well, of course what you get is a big list of.. a big list of genes.
And you can arrange these genes, as many people do,
into a pie chart where you separate them
into cytoskeletal or signaling proteins,
and, indeed this list is somewhat useful.
It gives you kind of an overview
of the most important proteins
involved in spindle assembly.
But, I would argue that this list
itself really does not, on its own
provide insight into how the mitotic spindle really works.
And to do that you have to go beyond this list
of genes and do more experiments.
And what you are trying to find out of a screen
is some unexpected result, not just the big list of genes,
but some unique finding here that can give you
some new insight into how this spindle is formed.
And to track down an unexpected result, as I said,
you have to do a lot more work.
And we had...we then did a bunch of secondary assays.
If we found a novel protein, we wanted to know
where's it located in the cell?
for which we had to do GFP tagging.
To better understand the phenotype,
instead of having a fixed image of the cell,
as we did in our whole genome screen,
we wanted to analyze these phenotypes
by doing time-lapse imaging
and in addition, we also then custom designed a number
of very specific other experiments
to try to understand the phenotype
using additional RNAi knockdown approaches,
or adding drugs, or doing other kinds
of localization experiments to better understand
the mechanism of how these genes worked.
So, we did find a number of interesting results from the screen.
far too much for me to talk
about in this short lecture.
So I just would like to focus on one story
that came out of this screen
and that is how you make
microtubules to build a spindle.
So let me just tell you how the conventional view
that you'll read about in the textbook
of how these microtubules form.
So there are two main ideas.
One is that these microtubules emanate
from centrosomes, here,
and another is that microtubules are
nucleated at the chromosomes themselves.
So this first mechanism, chromosome..centrosome
nucleated microtubule nucleation
was really pioneered by Kirschner
and Mitchison in the 1980s.
And it, indeed, is a very important part
of building a mitotic spindle.
These microtubules grow out of these centrosomes
and these microtubules are growing
and trying to grab hold of chromosomes
and eventually some of these microtubules
make connections with chromosomes
and these microtubules become
stabilized into the mitotic spindle.
And indeed, you can see this by using a special tag
that just binds to the very tip of a microtubule;
a special protein called EB1
that just tracks along the microtubule plus end
which is here tagged with GFP.
And this is a very good mechanism
for following new microtubule nucleation.
So here if we look more carefully,
in this blow up here, we can
see the centrosome over here
and you can see these little comets of EB1-GFP
kind of emanating out of this central area,
and all of these little comets that you see growing
are new microtubules that are nucleating,
extending out, and trying to reach chromosomes.
Now we know the main microtubule nucleator there.
It is a complex that is called
the gamma tubulin ring complex,
which has been studied extensively.
It forms this ring-like structure here.
And from this multi-subunit complex,
this forms a nucleating base from which
a microtubule can be nucleated and extend.
This gamma tubulin ring complex
then docks onto this centrosome
at the very interior of the centrosome is a pair of centrioles.
There's a lot of proteins coating the centrioles,
and some of these proteins then are responsible
for docking the gamma tubulin ring complex.
One of those key docking proteins
is a protein called centrosomin
and then this centrosome can then nucleate
this spiny array of microtubules.
However, we also know
that spindles will form without centrosomes.
So they are not essential. For example,
we know that plant cells and germ cells in animals
don't have centrosomes,
and yet they form mitotic spindles.
And even Khodjakoc and Reider have shown
you can ablate the centrosome
in normal animal somatic cells
and the mitotic spindle will still form.
So we know something about how
that centrosome independent
mechanism occurs, and that's through nucleation
of microtubules around chromatin or DNA.
This was really beautifully
demonstrated by an experiment
by Rebecca Heald and Eric Karsenti
where they just put in beads coated with DNA
and put it into a mitotic extract from frog eggs.
And these DNA coated beads,
not even normal chromosomes,
would nucleate microtubules, and these microtubules would
reorganize themselves into a spindle-like structure.
And this is also shown in these S2 cells here.
I am showing you the centrosomes here.
Here is the nucleus, and you'll notice
right at nuclear envelope breakdown
that the... right over here
you can see chromosomes, these dark bodies,
and then you can see these flashes
of new microtubule growth
right around these chromosomes,
and then these chromatin mediated microtubules then
are then integrated into the body of the spindle.
So, however, the question is
are these the only two mechanisms
that are responsible for nucleating microtubules?
And here we did this experiment
that produced a surprising result,
which suggested that there may be
an additional mechanism.
What you're seeing here is a spindle
without any functional centrosomes.
So we knocked out the centrosomes completely
and what you can see here is that there are
no centrosomes in the cell
but most of the microtubule nucleation
is not happening from the chromosomes,
but in fact is happening throughout the body
of the spindle over here.
In fact, including at the poles,
and these new microtubule comets
are kind of moving out from these pole regions
out to the chromosomes.
So this suggested that there may be some
other kind of mechanism at play,
something that we really did not completely understand,
but which we got some insight
into from this big whole genome RNAi screen.
And that's when we looked at genes that effected
the localization of gamma tubulin,
this critical nucleator of microtubule assembly.
So here is normal gamma tubulin staining
shown over here, and this is superimposition
with DNA and microtubules,
but most of the gamma tubulin
is found at the centrosome here.
But there's also gamma tubulin that's found
in the body of the spindle itself.
Dimmer, but nonetheless still present.
Well, from the screen, we found a known gene, centrosomin,
that knocked out gamma
tubulin localization to the pole
but still preserved this gamma tubulin
localization to the body of the spindle.
So this was a known gene,
but then we found 4 totally unknown genes
which we called dim gamma tubulin genes
that produced the exact opposite effect.
They still allowed gamma tubulin to localize
to the spindle poles at the centrosome
but they completely knocked out
the localization of gamma tubulin
to the poles... to the..
knocked out gamma tubulin
localization to the spindle.
And this suggested that maybe this
gamma tubulin localization to the spindle
was actually specific. It involved
a specific set of docking factors.
Perhaps these new proteins that we found.
And if this was the case,
that these new proteins are involved
in docking gamma tubulin to spindle microtubules
then we would expect that these novel genes
that we found would be localized
to the spindle themselves.
And indeed, when we tag these proteins with GFP
we found that these new proteins
were localized to the spindle.
If we knocked out the microtubules
with a drug called colchicine
that localization of these Dgts went away.
So these new proteins, which we call Dgts
are in the right spot in the spindle to be
specific gamma tubulin docking factors.
Well, now we can ask this question:
Is spindle localization of gamma
tubulin by these Dgts important?
Because now we had a very specific tool.
We had ways of knocking out
gamma tubulin localization
at the centrosome.
That we can do by eliminating centrosomin.
Or we can knock out gamma tubulin
localization to the spindle
with these new proteins. And the results
here were quite dramatic. Here is a normal spindle.
We are looking at GFP-tubulin in green
and chromosomes here in red.
And you can see this is a wild type spindle,
perfectly normal.
The chromosomes are aligned normally.
And in this next video,
we are going to look at a cell that has
been depleted of one of these Dgts.
It is unable to localize gamma tubulin to the spindle.
And what you see here is that
the spindle is very much perturbed.
The chromosomes are misaligned.
The spindle microtubules are very weak.
And this is a very abnormal spindle indeed,
and quite different from this other wild type cell.
So let me just summarize what we learned here.
We learned that there are
actually two different pathways
for localizing the key microtubule
nucleator, gamma tubulin.
There is one pathway that localizes
gamma tubulin to the centrosome
and through the screen
and work from other labs as well
we know that a whole pathway of
components from the centriole
to other components in the pericentriolar material
that dock gamma tubulin to this pole structure.
But there is a separate set of components
including these novel Dgts that dock
gamma tubulin onto the spindle.
And surprisingly, the spindle
localization of gamma tubulin
appears to be more important
than the centrosome,
at least in Drosophila.
Because we can knock out
gamma tubulin localization
to the centrosome and the spindle forms just fine,
and chromosomes will segregate.
But if we knock out gamma tubulin localization
to the spindle as I already showed you,
we get these very abnormal spindles.
So, all of this work has led to kind of
at least a hypothesis...a new hypothesis
of how you build the mitotic spindle.
I emphasize this is a very new study.
It's a new hypothesis,
and we'll have to see how it translates in the next few years.
But the idea is that the well-known mechanisms
of the centrosome and chromatin
constitute the important way of getting the new microtubule
started to build the spindle.
You can nucleate new microtubule growth
at centrosomes or at chromosomes.
And that allows you to quickly build
microtubules to make the spindle.
But after the spindle forms, possibly one of the
more important mechanisms for maintaining the spindle
is that the spindle has a way of self propagating itself.
It has a way of bringing the microtubule nucleator,
via these Dgts,
onto the spindle microtubules themselves,
and this complex generates new microtubules
that helps to maintain microtubules in the spindle
and interactions with the chromosomes.
So as I mentioned at the very beginning of this lecture
understanding the mechanism
of spindle assembly
also has a lot of important medical implications.
Many of the drugs that we currently use to treat cancer
work by interfering with mitotic spindle function.
Some of these like the TAXOL, Vincristine,
Vinblastine, Vinorelbine
all work by effecting microtubules
and interfering with mitotic assembly.
And they are very effective anti-cancer drugs.
They block cell division of tumor cells
and they interfere with tumor growth.
However, these agents here not only
effect tubulin in dividing cells
but they effect tubulin in all cells of the body.
In addition to effecting other dividing cells
like bone marrow,
there are also a number of neurotoxic effects of these agents
because they interfere with microtubule
transport in neurons as well.
So maybe the future is to try to develop
more strategic, specific ways of effecting the mitotic spindle
to stop cell division.
And there are a number of other potential
targets besides the microtubules,
and ones that are much more specific to dividing cells.
An example of this is a special kinesin motor
called kinesin, a special class of kinesin,
called kinesin 5. And these kinesins
are only used in mitosis. They have
no function in nerve cells or other cells.
Their specific role is to help build a bipolar mitotic spindle.
And we know from studies in many organisms
that if you interfere with the function of these kinesin 5
motors, you get a very abnormal spindle indeed.
You get what is called the monopolar spindle
where instead of having microtubules coming from two poles,
they are coming from a single pole
and microtubules are spreading out in this radial array
and the chromosomes instead of aligning in the center of the spindle
are in fact aligned as a ring,
such as shown here in blue.
And this ring of chromosomes simply cannot divide equally
to two daughter cells.
And this results in a massive failure in mitosis.
Now, Cytokinetics, again I should say this is a company,
a small biotech company in South San Francisco
in which I am a co-founder, shareholder
and also on the SAB.
took a strategy to develop inhibitors
against this specific kinesin to try
to develop a new strategy for blocking tumor growth.
And indeed through in vitro assays,
developing small molecules inhibitors
to get this motor using biochemical
tools and high throughput assays.
Refining these small molecules.
Then testing them in animal tumor models.
They developed an inhibitor which has been called Ispinesib
which is now in clinical trials in humans.
And you can see in this tissue biopsy
from a tumor from a patient
after treatment with this drug. This shows a stain
of the DNA and you can see that there is
this characteristic ring like pattern
of the DNA, indicating that this anti-kinesin drug
is working in people just as it is in vitro and in animal models.
And currently these drugs against mitotic kinesins
are now in phase II clinical trials
They have shown activity for patients with breast cancer
and over the next couple years we will see
how these mitotic kinesin inhibitors develop
and whether they will join the armament of anti-cancer therapies.
And beyond the...so in addition to these mitotic kinesin inhibitors
there are likely other candidates, new targets
involving the mitotic spindle that might be next generation
strategies for cancer chemotherapy.
Maybe these Dgts that I described in this lecture
that so profoundly effect the spindle at least in fly cells
might be candidates to look for in human cells
and human cancers to see if they might be interesting targets
for new cancer chemo-therapeutics.
So at least I have hopefully illustrated that
the connection between the study of basic molecular machines,
at the level of even single molecule assays,
cell biological studies such as I have shown you here
with whole genome RNAi, not only provides insight
into mechanism but also gives us
new routes of thinking for new kinds of medical therapy.