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Hello, I am *** McIntosh, professor of Cell Biology at the University of Colorado
in Boulder. This is the third of three lectures that I am going to give on the subject of chromosome
movement. And in this one, I am going to build on information which you've seen
from the previous lectures in order to talk about this single
problem of how do chromosomes approach the poles
during anaphase--the process called Anaphase A.
Anaphase A is an essential part of chromosome segregation
in most cells. And there's a wide range of evidence from different experimental methods
that people have applied, and if you've seen the previous lecture
you got a little flavor of just how broad
the experimental landscape is and people have used in order to try to understand mitotic processes.
And this account that I am going to give now is
quite a personal one in that it is going to based on recent work from our lab
and it's of course, a limited perspective.
Because any individual's approach to a scientific problem is going to look at only a part of it
because that is the way you can dig deeply enough in order to try to make some progress.
But, nonetheless, I hope that what I will be able to convey to you
is the ways in which you could use different
approaches to get pretty deep and maybe come close to understanding
a fundamental biological process.
So how do chromosomes approach the poles in anaphase A?
There have been two important hypotheses that have been very active
in this field for a long time. The Motor Hypothesis in which enzymes,
for example, dynein that we were discussing last time
or a kinesin could be involved in driving the chromosomes to the poles,
as a motile process just as those same motors move vesicles around.
in cells, for example. But it's clear when you look at mitosis
that microtubules must shorten during Anaphase A.
That is what it means to approach the poles, and this has given rise
to the Depolymerization Hypothesis, which originally was formulated by Gunnar Ostergren
back in the 1940's and 50's and then pioneered by Shinya Inoue
based on some beautiful work that he did with polarizing microscopy
and I was very much a motor hypothesis man for much of my career,
because it seemed just such an attractive way of thinking about this complex motile process.
But, what I am going to show you today is that I've switched sides, and I've come to believe
that depolymerization may well be at the root of chromosome movement.
Now, how do you test a hypothesis with a complicated process like mitosis?
The obvious way would be to inactivate a motor protein so that it will make problems
for the mitotic process and you'll be able to see
what are the ways in which chromosomes move or don't move
under circumstances where they no longer have this particular motor
function. What's been seen by a number of people
who have taken this approach either genetically or pharmacologically
is that if you can get a spindle built, and you can get the chromosomes
there so that you can now study Anaphase A,
if you perturb the function of a motor, you mess up aspects of
spindle function. And this messing up can show up in several ways,
It can show up in the failure of the spindle to retain
its structural integrity
or it can show up in the fact that if you have a way of measuring how frequently a chromosome is lost,
then chromosomes are lost more frequently when a motor
is missing. The remarkable thing though is when you look at a lot of the data that is in the literature
many aspects of mitosis continue even when a given motor is perturbed.
They go a little slower, not all the chromosomes may segregate
properly, but many of them do.
And of course if you are interested in the importance of your own work,
what you want to do if you've made a perturbation is see it as causing a lot of trouble.
And so you emphasize the things that are not working.
But what I've been doing in my own mind, and in the work we are doing in our lab
is asking what is still going on even when motors are not there.
And the way in which we've done this is to turn to yeast cells
where it is possible to do gene deletions comparatively easily.
and prove to yourself that the entire piece of DNA is gone from
the cell. So there is no possibility that the motor which is the protein
product of that gene is contributing to the mitotic process.
that you are seeing. And then you can ask: What happens?
And we've done this in a fission yeast cell using
the typical molecular techniques to delete
Two kinesin like proteins, each of which is a minus end in its directed activity
and the dynein heavy chain, and so there are no minus end directed
motors left in this cell, and how do we know that?
Well, the genome of this organism is sequenced
and we know that, as well as we understand, motor function with microtubules, there is no motor left
in the cell, and yet, when I know show you
the motion of the poles and one chromosome,
which we've marked with a fluorescent tag near the kinetochore,
this time lapse will show the separation of the spindle poles,
and you can see the chromosome, which is here, and you are going
to see it migrate towards the spindle pole
and we can measure the speed at which it migrates towards the pole,
as it becomes attached to the spindle.
Now of course, it should bi-orient and come to the metaphase plate,
this particular chromosome was a little tardy in this,
and that's common with these chromosomes that lack several motors, but you could
see it did actually segregate correctly
and from this kind of raw data we've been able to
measure the speed of the final approach of a chromosome
to the pole in a variety of genotypes.
Wildtype shown in yellow
and then one motor mutant after another and down here
in green we are seeing a deletion of all three minus end directed motors
and yet this final approach to the pole
is occurring at a speed which is no different from wildtype.
So these cells make mistakes. They are not healthy, and they would not
survive in the wild, and indeed we've measured the frequency of chromosome loss
and it's up by several, well even hundreds of fold.
So this is not a healthy organism
but it's an organism in which chromosome to pole motion occurs at a speed which is
undistinguishable from the normal wildtype chromosome movement.
That means these motors are not important for such motion.
They may be important for other things, like attaching
the chromosomes to the spindle, or
segregation or integrity of the spindle
poles, but not for this fundamental phenomenon of anaphase A.
So what...well, first of all we can say this is not simply due to fission yeast
idiosyncracies, it is also true in budding yeast,
where the Tanaka group has been able to demonstrate
this quite clearly. So, these motions must be caused
by some thing that is going on in this cell
which is not a minus end directed motor.
it could be some non microtubule component
of the spindle, but this doesn't seem very likely
because although there have been the identification of
actin in the spindle, it turns out that the actin is generally not
fibrous when it is in this spindle.
And there's been the identification of matrices in the spindle,
but these matrices are not yet known to have any kinetic function.
And it could be then that it is simply microtubule depolymerization,
which is itself a motor in some way.
How do you find out?
This is an implausible idea, and in order to convince anybody
we really need some strong experimental evidence.
The implausibility of this was described best to me
by the expert kineticist who studied myosin and other enzymes, Ed Taylor,
who was very skeptical about the disassembly hypothesis, and he
pointed out that if you were a rock climber suspended on a rope
from a cliff and you wanted to go up the cliff, you certainly wouldn't do it by
lopping off the rope to make it shorter.
And this analogy certainly casts doubt on the hypothesis.
But what I am going to show you is that it has some validity to it.
Microtubule polymerization can do mechanical work.
And this was shown very nicely in the laboratory of Hotani, many years ago
where they put soluble tubulin inside lipid vesicles
and induced it to polymerize
and the polymerization of tubulin drove these deformations of the lipid membrane
showing that polymerization could do work.
And indeed it is now well known that actin polymerization can do work
and you should look at the iBio seminar that deals with this very nicely
because it has a beautiful amount of detail, all shown by Julie Theriot.
So polymerization is easy to understand
as a motor, but what about depolymerization?
We designed an experimental system in which to look at this
in which we had an objective lense on the microscope, and a coverslip
we were looking at an object which was sort of our
in vitro manifestation of a spindle pole,
we happened to use a ciliated protozoan
that we lysed with a detergent to wash away the membrane and clean out the cytoplasm
but it left behind what is called a pellicle
which has about 500 basal bodies for flagella
and that structure will now nucleate large numbers of microtubules.
We used purified brain tubulin to flow it in and get this forest of microtubules
all of whose plus ends are pointing away
from the organizer, just as the spindle microtubule plus ends
are pointing away from the centrosome.
We could then flow in chromosomes that we had
partially purified from CHO cells and ask, "Do they bind?
And if they bind, can we make them move?"
And in this movie taken by Vivian Lombillo taken when she was a graduate student
in the lab, you can see a pair of chromosomes that are caught in the microtubule
forest that has grown from this pellicle,
and as I run the movie, we will now flow in buffer
which contains no tubulin, and you can see the chromosome immediately wash down stream
in the flow of the buffer. This movie is real time, so
we are not exaggerating any speeds and the force of the flow
is substantial, and yet, when they come into focus,
you can see that the chromsomes are still attached.
This buffer contains no nucleotide triphosphate, no ATP, no GTP
and in fact it contains a pyrase, an enzyme that drops the concentration of nucleotide below nanomolar
and yet these chromosomes move into this structure here as the microtubules disassemble
at speeds which are even on the high side for physiological motion of chromosomes.
So this work demonstrates that microtubule depolymerization
without any ATP dependent motor activity
can move chromosomes in a test tube.
So we've seen that chromosomes can approach the poles
in vivo with no motors present in the cell
and they can approach this spindle pole here
when we have no motors present and fed a fuel that could move them.
There may of course still be motor enzymes.
on the kinetochores, but this cannot be an ATP dependent motor activity.
so we interpret this as a disassembly dependent motility.
How could depolymerization of a fiber cause movement?
These images which are electron micrographs taken
of frozen hydrated microtubules by Eva and Eckhart Mandelkow
collaborating with Ron Milligan show what the ends of polymerizing microtubules
look like. They are quite blunt.
On the other hand depolymerizing microtubules show
this characteristic curl to the tip of the microtubule
where a strand of tubulin, a protofilament, is bending.
And this appears to be a characteristic event of the disassembly process.
Where does this come from?
Well it is related to the cycle of polymerization and depolymerization of tubulin.
Tubulin to polymerize has GTP bound and
the molecule is more or less straight and it adds onto the ends of the microtubule.
But the microtubule activates the GTPase activity of tubulin,
it is like a GTPase activating factor when you think about G proteins.
And so the majority of the tubulin in the microtubule is GDP tubulin.
And that irony is that GDP tubulin will not polymerize.
The GDP tubulin tends to fall apart.
and indeed, as it falls apart it shows this curvature
and the interpretation that has been put on this by a
number of investigators working on it
principally Eva Nogales, is that the tubulin molecule in the GTP bound state
tends to be more or less straight, but in the GDP bound state
it tends to bend, and this bending means that when GDP is in the wall of the microtuble
it's under strain as a result of interactions with neighboring tubulin molecules
that interact with it by non-covalent bonds.
So those interactions are keeping the molecule constrained and straight,
unless you are at an end without any GTP tubulin
on the end to provide straightness.
And now the curvature of these tubulin protofilaments is a relaxation of
tubulin GDP molecule to its minimum energy geometry.
What this means is that a microtubule in the course of depolymerization
is going to have a wave of conformational change.
Doug Koshland was the first person to point out that this conformational wave
might be a way of pushing on things
and it might help to pull chromosomes to the pole.
But of course, the cell to take advantage of it must find some way
to couple to this microtubule so it can grasp the microtubule
and experience the force from those bending protofilaments.
We need to understand what that coupler might be in order to see how this relaxation
of the tubulin molecule could be a power stroke that would drive chromosome movement.
The first indication as to what this might be
came from some more work done by Vivian with that experimental system that I showed you earlier
with the chromosomes moving in vitro.
She added antibodies to kinesin, first a general kinesin,
and then a kinesin that is specifically localized at kinetochores
so called centromere protein E.
And those antibodies caused a dramatic reduction
in the motion of the chromosomes in this depolymerization
dependent fashion, suggesting that a kinetochore motor
is important for depolymerization dependent movement even when no ATP is present.
But remember the caveat that I raised at the end of the last lecture
that even when you add antibodies that are monospecific
they may have indirect effects. So this doesn't really prove to us that this molecule
is a coupler or that it is working in this way.
It is strong suggestive evidence.
We've obtained other evidence, however,
that a microtubule dependent motor enzyme can
work as a coupler using a kinesin-8 from pombe cells.
And if that kinesin is attached to a bead and the bead is then allowed to interact with a microtubule
which isn't visible here, but it is shown in diagrammatic form over here
and we now induce the microtubules
to disassemble, the bead is pulled by microtubule disassembly
just as I have shown you in those other experiments
and these graphs show the rate and the trajectory.
Clearly this is quite a processive movement.
In the sense that the bead is following the disassembly microtubule end for quite a distance.
So a motor enzyme can serve as an ATP independent coupling factor
to bind a cargo to a disassembling microtubule.
Does it have to be a motor? No.
And one of the most remarkable discoveries in the mitosis field
recently has been the discovery of this complex called either Dam1 or DASH
depending on whose laboratory you happen to have been associated with.
The people who first discovered and named the Dam1 protein
and then gradually found more and more proteins that were part of a big protein complex
call it the Dam1 complex. 'veI collaborated with that lab,
so I'll use that name, but the name DASH is used
by many other labs for the same complex.
It's an unusual complex because it involves
ten different polypeptides all of which
assemble into a little football shaped object
and this is called the Dam1 complex.
And this complex polymerizes with others of its own kind
and those polymers form rings around microtubules.
Here are the rings forming just on the surface of a support
visualized in the electron microscope just by negative staining.
And some really excellent work both by the Westermann group where Nogales
has been doing the electron microscopy
in her lab and in the group that's at Cambridge, Massachussetts
under Steve Harrison has been...they've been providing
expert and excellent evidence about
the structure of this complex and the way in which
it interacts with microtubules.
We've collaborated with the Berkeley group
which included Westermann and his mentors, Georjana Barnes and David Drubin
and with them we've also been able to purify this complex and
label it with fluorescent dye, and allow it to interact with microtubules
in our in vitro system, where this is that pellicle
that you've been seeing
and these then are complexes of the Dam1 protein,
which are fluorescent. This is
what our Dam1 complex looks like when it surrounds the microtubules seen
in the electron microscope, but of course you can't do kinetic experiments
in the electron microscope. So we are going to do an experiment here
which we watch what happens to these Dam1 complexes
when we cause the microtubules to disassemble.
And here we will now bleach the little tip
that's on the end of the microtubule, and the microtubule
starts to depolymerize, and you can see that the Dam1 complex
is moved with the ends of the microtubule as the ends of the microtubule
shortens. So the Dam1 complex
is also a coupler that can take advantage of the structures
that are found at the end of the microtubule.
This coupler can actually pull a load, and
what we've done here is to put the Dam1 complex
onto a bead. And this bead now can be followed
as an object which is a load for the Dam1 complex
to move as it associates with the microtubule.
And again, when we induce microtubule disassembly, as the disassembly
reaches the bead, the bead will stop its Brownian movement
just back and forth and will start a progressive motion
towards the origin of the microtubules, which is that pellicle sitting over at the side.
Now this kind of work allowed us to determine that the Dam1 complex seems to
form a variety of structures, all of which can interact with the microtubules.
If we attach Dam1 to a bead, and do not have the Dam1 complex in solution,
but instead just allow the bead to bind to the microtubule,
we get a distribution of velocities that is shown here in red.
If we have Dam1 in solution, so that a complex can form that would make this ring
shaped structures that I showed you in the electron microscope,
what we see is that we still get bead movement, but the bead movement is slower,
as if the formation of that ring might actually retard the rate of disassembly of the microtubules.
So this looks likea process that needs detailed study,
and we've done enough experiments that I won't be able to describe them all to you.
by any means in the course of this short lecture,
but I want to show you the tool that has been the most important to us in
trying to do this kind of work. It is a standard light microscope, which has
a sensitive camera at its top.
and then over here it has a couple of lasers, one of which
is very strong and can be led through a device that allows you
to steer the laser beam and then up into the microscope
the other laser that is over there is just to help us align things.
So a laser beam is coming down through our objective lense
and this very bright light can be used in what is called an optical trap.
A device where you can grab a small object
that refracts light, like a bead of glass or plastic.
Over on this side, we have two other lasers, one green and one blue,
that we use for bleaching the fluorescence
of some parts of our specimen. And what
we are going to use is tricks in order to
be able to do experiments on beads and ask:
Can we monitor the force that is generated in this system?
So here's again our pellicle serving as a nucleator;
microtubules growing from purified tubulin.
The problem is that these microtubules have to be labile.
And that means that if we dilute the preparation of
tubulin by washing anything else in
we are going to cause them to disassemble, so we need to stabilize them.
And we do that by putting a cap on the
tip of them where we polymerize the tubulin
in an analog of GTP which does not hydrolyse, or hydrolyzes very slowly.
And the result is that we have now stable microtubules
so we can drop the tubulin concentration to zero and
now we can bring in beads coated with
something that will make them stick to the microtubule itself.
The way we do this is we use biotinylated tubulin
and beads coated with the protein avidin, so the
connection between the microtubule and the bead
is one of the strongest non-covalent interactions known in biology
and this is not going anywhere, it's certainly not
a motor and we can ask: when the microtubule disassembles,
what happens? And the way in which we do this experiment is
to turn on our laser trap so that this bright laser light is holding that
bead and we have a way of measuring the position of the center of the bead
very accurately and then we turn on our photobleaching laser
in order to inactivate the tubulin that is there, and the cap comes off, and the microtubule
will disassemble, and you may have noticed that I drew in just a little
bit of a movement there of that bead
as the disassembly went by.
That's the kind of event we are looking for
in order to take motors completely out of the equation
and ask: can microtubule disassembly do work?
And the answer is yes.
Here is a cartoon of what we think is going on
with the bead drawn very small relative to the microtubule,
and you can see the bending protofilament that could be
exerting a force on the bead.
This is a trace that we get from a very sensitive device
called a quadrant photo detector which is allowing us
to determine the position of the center of the
bead extremely accurately to within a nanometer or so.
And all of this wiggling you see
here is the thermal noise that the bead
is oscillating as a result of interactions with molecules
in solution. But as the disassembly occurs,
the bead is pushed a little bit toward the minus ends of the microtubule, and then released.
and now it is simply in the center of the trap, and the microtubule has
disappeared. Now if this really is
a force being generated in this way, you could imagine that we are pulling
here on the center of the bead, with our trap, and the radius of the bead then
is like a lever arm. And the smaller the bead, the less our mechanical advantage
and the bigger the force that we should generate, and indeed
here you can see with a 2 micron bead
versus a 1 micron bead versus a half micron bead
we get bigger forces as the bead gets smaller,
suggesting that this really is the bending of the protofilament
pushing on the bead to give us the force that we are seeing.
How much force? Not very much.
with this non-physiological system. But, we also presume that we are only interacting
with one side of a microtubule, and if you think
about the Dam1 complex, which surrounds the microtubule
as a ring, one could imagine that it would experience force from all the bending protofilaments
at once, and would give you significantly more force.
So we have naturally gone ahead and tried to attach the beads
to Dam1 complex where here this is a real micrograph of
the Dam1 complex on a microtubule, but this is just a cartoon
with representations of the antibodies that we have bound to the bead.
And we are using antibodies that interact with the Dam1 complex,
and give us quite a tight bond, and now we can ask, "When disassembly comes by, what do we see?"
And the answer is a longer and much stronger force.
This force is now about six times as great as the force that we observed
when we were only sampling one side of the microtubule.
And so it really looks as if a ring
is surrounding the microtubule and
sampling the action of all the protofilaments
and producing a force, which, once we've made corrections
for the bead diameter, looks as if it would be 20, 30, even 40 piconewtons,
which is an unusual unit of force for most of you,
but a kinesin molecule or a dynein molecule develops somewhere around 5 piconewtons.
So a microtubule interacting with a ring
is really powerful. It is sort of like a bulldozer
and it's no wonder that you can delete motors from the cell
and chromosomes will continue to move, if this is the process that
is really doing it. So how do these things work?
Well, here are the two hypotheses that are central to the way people are thinking about this movement
at this point. On the left you are seeing
a Brownian movement, a random walk by diffusion of a ring which is modeled here in these accurate simulations
done by my colleague, Fazly Ataullakhanov and his students in Moscow
and they are allowing the diffusion that would occur with an object of
the size of the ring and loose binding
and this diffusion still can give rise to processive movement
because the diffusion is biased
by the disassembly of the microtubule.
Over here we are showing a different model
And this is one in which the ring is presumed to bind
tightly to the wall of the microtubule, noncovalently,
but still tightly, so that as these protofilaments
bend they have to force the ring from one position to the next position.
to the next, and you can see kind of stall as it is going
and then it will go ahead and go farther again.
This is then a situation where a forced walk
is causing the migration of the ring.
Now intuitively you might think that this biased diffusion is a more efficient
system because you are not having to expend so much force in overcoming the binding
energy between the ring and the microtubule.
But this has problems, because polymerization and depolymerization
of microtubules are molecular events which occur
with random fluctuations, and so every now and then
the depolymerization pauses and the ends of the microtubule presumably
go straight for a little while, and if there were a load on this ring, it might just pull right off
the end of the microtubule which would be a terrible catastrophe
if you were trying to move chromosomes by microtubule disassembly.
So intuitively we favor this tight binding model
but indeed there is quite a bit of evidence that
that is the case.
So is the Dam1 ring then the answer
for how you couple chromosomes to microtubules?
Umm, in budding yeast, Dam1 is in the spindle, it binds to chromatin.
It is essential for proper chromosome segregation
and this says that the Dam1 complex is an excellent candidate
for the coupler in budding yeast.
Interestingly, in fission yeast, it is no longer essential
in cells that are otherwise wildtype.
Now if you start deleting mitotic motors
then the Dam1 complex becomes essential
but what we have here is a situation where in just going from
one ascomycete fungus to another,
we've moved from essential to contributory.
Another difference between these two spindles
is that in budding yeast there is one microtubule per kinetochore
whereas in fission yeast there are 2 to 4 microtubules
per kinetochore, so maybe, the Dam1 complex
is absolutely essential when you need to regulate the disassembly of microtubules
so tightly that you don't let the microtubule end get away from the kinetochore
but when you have more microtubules
and you have others that you can rely on, it is no longer an essential process.
Even more disturbing in terms of thinking about the generality of the Dam1 complex
is that outside the fungi, this protein has not yet been found.
Now this doesn't say that other rings are not going to be found, and a number of
possibilities have been detected becasue the Dam1 complex is so appealing
as a way of doing this job well that many scientists feel that rings must be the answer
and they are trying to find the components of the rings
in other cell types.
We've taken a different approach, which is
to go and look at the kinetochore-microtubule connection
with the best structural tools
that are available, and ask, "What do we find?"
And the way we've done this is to use
electron tomography. Now this is an electron micrograph
of a chromosome and a spindle fiber, although it is very unprepossessing
and the reason for it is that it is one of series of
images from a tilt series where we now have a thick sample
about three or four hundred nanometers thick
in the electron microscope tipping back and forth
and we are collecting images at 1 degree intervals from +70 to -70 degrees
and then we go ahead and tip around the orthogonal axis
in order to collect a large number of
views of this three dimensional object.
These can then be combined by a variety of mathematical approaches
to create what is called a tomogram, or a 3 dimensional reconstruction of all the material
that was in that region that we were imaging.
Chromatin down here. Kinetochore here.
Microtubules there and you can see flared ends
on the tips of many of the microtubules in this array.
We can then use software to pull out a single
slice from our three dimensional reconstruction
that contains the axes of one or more
microtubules, so that we can see just exactly what these ends look like.
These ends are in some way attached to the chromosome,
and what we want to know is how.
And by just taking a descriptive approach,
we can try to get insight, and tomography allows a completly novel way of looking at this
and what I am going to show you now is a series
of images in which I took a plane
that contained the microtubule axis and then
I am going to rotate that plane around the microtubule
axis, so that we can visualize a single microtubule from multiple orientaitons.
And you can see flaring protofilaments come and go.
There is one waving way up here, here is one coming down.
All of these are images at different orientations.
We can then extract this structural information
as a series of graphics, which allow us, then, to see
a representation of the protofilaments and their
flare at the kinetochore end of one kinetochore microtubule.
This has allowed us both to quantify
exactly the shape of these flares to within
the precision of our methods for preserving
this sample, and also to ask, "What is connected
to those flares?" Well, in the first instance, when we
look at the flares of the protofilaments from kinetochore microtubules
here, and non kinetochore microtubules here
and then compare them with depolymerizing and polymerizing
microtubules in vitro, imaged in that study that I showed you before
by the Mandelkows and Ron Milligan.
What you can see is that there is a tremendous range in the structure of the protofilament
of both kinetochore and non-kinetochore microtubules
They are sort of in between assembling and disassembling.
And this was quite hard to understand; however, what we've
done is to focus in on the bending protofilaments which are
in an intermediate group, and what we can find
is that many of them have little strands that connect from
the protofilament itself up into the region of the chromatin.
And then in the lower half of this slide, what I've done is to
put graphic objects down to show you
what I think are both the protofilaments themselves
and these little fibrils that are connected to the protofilamants and also connected
up into the chromatin. And we are calling these little fibrils
kinetochore fibrils, for obvious reasons.
The trouble is that this imaging is right at the limit
of the methodology, both are ability to preserve the sample
well because we are looking at a whole cell here, which has been prepared for electron microscopy
and the imaging resolution of the methods
that we are using-tomography of these thick samples.
It would be very nice if we could average things up in order to try to see
whether there is an averaged structure for the fibril.
And Katya Grishchuk in the lab had the insight that if we were able to
take the intermediate classes of protofilaments,
yes, in between polymerizing and depolymerizing
and look at these only, we might see something special
because these are not simply depolymerizing
and so we took those, sorted objectively, simply on the basis of their slope near the microtubule
wall. We could then take forty or fifty
such protofilaments from the original image data and average them all.
And this is such an average for a non-kinetochore microtubule.
This is a metaphase kinetochore microtubule
from that intermediate group, and I think you can
see now there is a very respectable fiber that is averaged
up out of these fifty or so data sets.
This is also found in anaphase, where the flare is slightly longer.
on this protofilament, whereas the ram's horn groups-the ones that have the big curvature
characteristic of depolymerization
don't have such filaments associated with them that
will average up in this way. So what this suggests is that
if you choose your protofilaments by an objective criterion
which suggests that they are under some kind of stress or strain
that is keeping them from going to be just like depolymerization
and then they are not polymerizing, you can
then find protofilaments that are there.
And our interpretation of this is that
these kinetochore fibrils are exerting force on
the bending protofilaments so that as the protofilaments try to bend, they stretch this fiber
and exert tension on the chromosome itself.
So this is a different kind of coupling,
one that does not involve rings,
one that need not be a motor, but that could simply be a static link of some kind.
Here is a drawing in which I have represented
protofilaments based on the tracings that I have
done of the microtubules themselves,
Protofilaments, sorry, kinetochore fibrils
that connect the protofilaments up into the chromatin
and this now is a simulation done by Grishchuk, Ataullakhanov and his students
and it is showing that even with a load
of about 40 piconewtons pulling in this way,
if you have tightly binding kinetochore fibrils,
that interact with polymeric tubulin, so that they will
stick to these bending protofilaments, but be released
as soon as tubulin falls off from the end,
you can make a processive motor that works perfectly well.
And indeed, some of the details of this motor that are now
described in the Cell paper that came out this year
give us confidence that this has advantageous properties
that may be even better than a ring for serving as a coupling
even though it is an improbable idea.
Structurally then fibrils are found in many places, but
we'd like to know what they are made from.
If we don't know the protein composition of these
structures, it is very difficult to do experiments that will
tell us definitive things about mechanism.
We have a number of ideas of what they could be
of course, because we have seen
that kinetochore related motors, like kinesin 7's and 8's
are both fibrous in their structure, and they can do the
coupling job. There are also non-motor proteins,
which are fibrous, that are localized
to the kinetochore, and NDC80 is perhaps the most attractive of these
because it is found across all cells where it has been sought,
and in every cell where such experiments have been done
and if you delete this motor, the chromosomes simply cannot attach to the spindle.
So NDC80 is an important fibrous molecule involved in
attaching chromosomes to spindle fibers.
It could well be part of this attachment machinery,
however, the evidence that now exists about NDC80 shows that it binds to the outside of the microtubule
and the way I have shown you the fibrils,
with the bending of the protofilaments, it almost looks as if the fibril attaches to the inside of the microtubule.
There are very few studies that have identified proteins that would bind to the inside
of a microtubule, so maybe NDC80 also has this function, or there may
be a new class of protein not yet identified
which can fulfill this function.
Or, alternatively, our resolution may not be good enough to give
the straight answer as to where the fibrils join the protofilaments.
And they may come around onto the far side and bind.
Certainly there are other fibrous proteins in the spindle. There are many
of them in the kinetochore, and so some of these might serve as connectors.
The molecular nature of this coupling is really something one wants to understand.
But it really is not yet known.
Evidence from localization of proteins
from genetic disruptions of particular components
from biochemistry, all of these different
kinds of experiments suggest that there are multiple factors
that are inolved in coupling chromosomes to microtubules.
Some more important than others, perhaps, like NDC80
but it may be that what we visualized in the electron microscope
is actually a little molecular zoo.
and there are multiple kinds of connections made by different components.
The fact is that this is a wonderful set of unsolved problems,
and it's a marvelous study or problem for future work.
It's the kind of thing where one hopes that many laboratories will come to partake
in this kind of research, contribute to
the knowledge that we have, because our group has been small.
We are enthusiastic about our work.
We have enjoyed the kinds of things we've done
as you can see from Katya's enthusiasm.
Fazly Ataullakhanov, the mathematician who has been responsible
for the supervision of these three mathematicians,
who are now becoming cell biologists as well
and we've had a wonderful time doing this work
but there's lots to be done, and we hope you and others will come and join us in this research.
Thank you. Goodbye.