My name is Shinya Inoue.
I'm going to start off by showing you a movie that I took
and then explain what the movie means as we go along.
Here we're seeing a dividing pollen mother cell of an Easter lily.
And, the movie is showing structures that people had not seen inside living cells before
because scientists had been fixing cells in order to visualize those structures,
if they could see them. Here, we see the fibers pulling chromosomes apart,
and then new filaments appearing between the separating sets of chromosomes
and laying down the cell plate.
And, the significance of all this is why looking at the living cell,
we can not only tell where the fibers are
and what they're doing, but tell what the molecules are doing inside them.
Especially what I'd like to talk about is about how cells divide,
and what we found out about how molecules come together or fall apart,
and how this plays a very important role in terms of how cells divide.
So, how did this whole thing happen?
Well, it happened because I met Professor Katsuma Dan.
He was a very unusual teacher
because he had taken his PhD at the University of Pennsylvania
and had come back to Japan in 1937 with his American wife,
and then I was in the first class that he ever taught in Japan.
And, what he did was so different from the other Japanese high schools that I attended.
He even let us not do experiments that he had prescribed for us,
but let us try things that were of interest to us in the lab.
And so what I did was to try Lilly's iron wire model; of nerve conduction.
And this was such an interesting project that this really took me into biology.
And then a few years later, when I met KD at his Injean Dans; home,
he showed me this book which is on the right-hand side.
This was by W J Schmidt in Essen, Germany, who had a picture that's shown in the middle.
Those were of sea urchin eggs in which you see these bright or dark structures.
Schmidt thought that those were chromosomes when he wrote this book,
but in 1939, he revised his view and said those were actually mitotic spindles.
So, this is of interest to us, especially to Katsuma Dan,
because Dan thought that the spindle elongating
was what was responsible for what divided cells.
So, he said, "Let's try and repeat this experiment of Schmidt's."
And we tried once in the dark, but it didn't work.
After the war, we got back together in Misaki at the Marine Biological Laboratory,
after he recovered it from the occupation army.
And I built this microscope, which is a polarizing microscope,
somewhat different from what is used in mineralogy.
And this has a very good extinction polarizer and analyzer,
and a compensator, and so on.
Very bright light source, which makes it easier to see the various things inside living cells.
And so, using this, we were able to see images somewhat better than what W J Schmidt saw
and were able to follow what was going on inside dividing cells.
So, this is what started me on the whole quest for following living cells
and asking with the polarizing microscope, "What are molecules doing inside yourself?"
The microscope that is shown here is what I built when I came to Princeton in 1948
as a graduate student.
And with the microscope, I was able to show what part of the cell was birefringent.
Birefringence is a word I'll keep on using that means
it has different optical properties than the rest,
which you can see by using a polarized light microscope.
And by using polarized light, not only can you visualize a structure that is birefringent,
but birefringence tells us how molecules are lined up, how they change, and so on.
So, this is the special kind of microscope that I built for that purpose.
Now, with that, what I was able to do was to, first of all, show that in dividing cells,
there are actually fibers that are pulling chromosomes apart during cell division.
And this was important because when I first came, it was argued back and forth,
even though many people had studied cells after fixation,
that the cells may not have been quite happy or alive.
And so we needed to find a condition where the cells were alive
and still see the fibers that pulled the chromosomes,
and those were not visible except by using polarized light,
and the polarizing microscopes that were available
were not good enough to show the details,
so this is why I built the microscope.
Now, what can we see here? The bright structures are the birefringent spindle fibers
which had been thought to be present, but not necessarily proven.
And those are pulling the chromosomes apart,
and after the chromosomes are pulled apart, then you see some filaments,
which in the case of plant cells, lay down the cell plate,
and that divides the cell into two.
So, after seeing this, even the skeptics could not argue anymore
that these were artifacts of fixation,
but were really present inside the living cells.
That was fun enough as it was, but what was really interesting to me
was to find out that these birefringent structures
were not just there to move chromosomes and so on,
but had some very intriguing properties. They would come and go.
Here's a dividing sea urchin egg, which is about to divide,
and you see the birefringent spindle in the middle.
But now I'm going to drop the temperature, and raise it again, drop it, and raise it,
and as you see, this is a time-lapse movie, each time I drop the temperature,
then the birefringence just disappears.
What it means is that the molecules are not bound there together tightly,
but can fall apart very easily.
Again, dropping the temperature, and this doesn't affect what the cell does.
It's perfectly happy and keeps on going. Drop the temperature, raise again.
So, we can keep on doing this experiment over and over again.
This tells us that the molecules that make up these fibers are in a dynamic equilibrium
with something... they can either fall apart or be put together again,
and we'll see more of this in the next slide.
Here, what we see is the effect of colchicine.
It's a well-known drug, known from Egyptian tombs, even,
which was used for treating gout.
But more recently, it's been used for collecting chromosomes,
because one can collect metaphase chromosomes and use this for diagnosing
how chromosomes... whether they are normal or not.
Now, what happened with colchicine is, when I applied colchicine to living cells...
In this case, it's a oocyte -- a cell that forms an egg -- of a marine worm, Chaetopterus.
These are my favorite material because the cell stays in metaphase
unless you stimulate it to go further.
So, it has a metaphase spindle built in.
But, then when you apply colchicine, then as you see in the lower row,
the birefringence gradually disappears in a few minutes.
And then, if you use a lower concentration, then as you see in the upper row,
then not only does the birefringence disappear,
but the spindle gets shorter and shorter and shorter,
and at the same time, it pulls the chromosomes to the cell surface.
So, from this, I concluded that colchicine, just like cold,
is one of the agents that makes the spindle material fall apart.
But what's interesting is that, as the molecules are falling apart,
they can generate force for pulling. This is a very strange concept
that you can generate pulling force that will pull chromosomes
and the inner spindle pole to the cell surface.
And then if you take the colchicine away,
the reverse happens -- the molecules come back together,
and it pushes the chromosomes and the inner pole towards the middle.
So, this gave rise to the whole concept that molecules that are falling apart can actually
generate pulling forces.
Now, this seems so strange. It took 20 years until, in the test tube,
this was proven to be correct.
One other graduate student working with us, Howard Fuhr;, did another experiment.
He used a small spot of ultraviolet light, as you see in the top, second to the left panel...
a bright spot of ultraviolet light.
When you shine a bright microbeam of ultraviolet light on the spindle
and watch the birefringence, then you see that spot itself loses birefringence,
and you develop an area of reduced birefringence -- Arb as you see there.
What was really surprising is that this Arb then gradually migrates to the spindle pole
and then disappears, which means that there must be some
movement of the spindle material
from the chromosomes towards the spindle pole.
And while this is going on, the part between the chromosomes and the Arb,
and between the Arb and the pole,
the birefringence doesn't change
which meant that there must be a microtubule organizing center
both at the chromosomes and at the spindle poles.
So, this was quite a revolution... a revelation.
And, when we put all of this together,
then as Ted and I put together the summary article later on,
the spindle has organizing centers both at the spindle poles
and right at the chromosome kinetochore.
And then those are both responsible for lining up microtubule material, tubulin,
but the tubulin is constantly flowing from the chromosomes towards the spindle pole.
And as Tim Mitchison and others showed later on, this occurs very, very rapidly.
But, in spite of this, then if we apply cold or colchicine,
or hydrostatic pressure, that this whole equilibrium is shifted towards depolymerization,
so in spite of all this flow and so on taking place, we get a shorter spindle
and the microtubules fall apart and form tubulin. Again, this is completely reversible.
So, we have a dynamic equilibrium between microtubules
which are flowing from the chromosomes towards the pole,
and then which can be made to fall apart or come back together,
so this forms one of the major current concepts of how spindles work.
Of course, there are motor protein molecules,
in addition to this, which also play important roles.
And, as people have found out recently, even at the kinetochore itself,
on the chromosome, there are 40 different protein molecules
that are organizing the spindle microtubules.
So, the whole story gets more and more complex
and is not as simple as I portrayed it initially.
But, nevertheless, the whole general scheme seems to hold.
And finally, as a summary reflection, polarized light and...
I didn't get a chance to talk about video microscopy,
but both of these combined together have allowed us to probe the dynamic behavior
of cell architecture and the structural molecules which are far smaller
than the resolution limit of the light microscope.
And we can do so directly and non-destructively in living cells.
Of course, there are non-destructive methods these days.
But, these are especially powerful approaches,
and still I believe that we've only scratched the surface.
Now, I look forward to further developments based on key insights
into the interaction of polarized light,
(which is an electromagnetic wave) with matter,
and the broader biological explanation through which distinct living cells
which may not be ordinarily used can teach us the finer mechanisms
underlying the mysteries of nature and of life itself.
So, this is my summary reflection. Thank you very much for listening.
