Session 7: Cell Division

Transcript of Part 3: Moving Chromosomes to the Spindle Poles: the Mechanisms of Anaphase A

00:00:10.29	Hello, I am Dick McIntosh, professor of Cell Biology at the University of Colorado
00:00:14.24	in Boulder. This is the third of three lectures that I am going to give on the subject of chromosome
00:00:20.27	movement. And in this one, I am going to build on information which you've seen
00:00:26.06	from the previous lectures in order to talk about this single
00:00:30.05	problem of how do chromosomes approach the poles
00:00:33.13	during anaphase--the process called Anaphase A.
00:00:40.00	Anaphase A is an essential part of chromosome segregation
00:00:46.20	in most cells. And there's a wide range of evidence from different experimental methods
00:00:53.03	that people have applied, and if you've seen the previous lecture
00:00:55.21	you got a little flavor of just how broad
00:00:59.11	the experimental landscape is and people have used in order to try to understand mitotic processes.
00:01:06.02	And this account that I am going to give now is
00:01:09.27	quite a personal one in that it is going to based on recent work from our lab
00:01:13.29	and it's of course, a limited perspective.
00:01:16.09	Because any individual's approach to a scientific problem is going to look at only a part of it
00:01:22.09	because that is the way you can dig deeply enough in order to try to make some progress.
00:01:26.06	But, nonetheless, I hope that what I will be able to convey to you
00:01:31.12	is the ways in which you could use different
00:01:33.22	approaches to get pretty deep and maybe come close to understanding
00:01:38.16	a fundamental biological process.
00:01:40.06	So how do chromosomes approach the poles in anaphase A?
00:01:46.12	There have been two important hypotheses that have been very active
00:01:50.17	in this field for a long time. The Motor Hypothesis in which enzymes,
00:01:54.21	for example, dynein that we were discussing last time
00:01:57.18	or a kinesin could be involved in driving the chromosomes to the poles,
00:02:02.18	as a motile process just as those same motors move vesicles around.
00:02:06.11	in cells, for example. But it's clear when you look at mitosis
00:02:10.23	that microtubules must shorten during Anaphase A.
00:02:14.21	That is what it means to approach the poles, and this has given rise
00:02:17.26	to the Depolymerization Hypothesis, which originally was formulated by Gunnar Ostergren
00:02:25.04	back in the 1940's and 50's and then pioneered by Shinya Inoue
00:02:29.19	based on some beautiful work that he did with polarizing microscopy
00:02:33.07	and I was very much a motor hypothesis man for much of my career,
00:02:37.28	because it seemed just such an attractive way of thinking about this complex motile process.
00:02:43.24	But, what I am going to show you today is that I've switched sides, and I've come to believe
00:02:49.01	that depolymerization may well be at the root of chromosome movement.
00:02:54.04	Now, how do you test a hypothesis with a complicated process like mitosis?
00:03:02.11	The obvious way would be to inactivate a motor protein so that it will make problems
00:03:10.09	for the mitotic process and you'll be able to see
00:03:13.13	what are the ways in which chromosomes move or don't move
00:03:16.12	under circumstances where they no longer have this particular motor
00:03:19.21	function.  What's been seen by a number of people
00:03:22.25	who have taken this approach either genetically or pharmacologically
00:03:26.00	is that if you can get a spindle built, and you can get the chromosomes
00:03:29.17	there so that you can now study Anaphase A,
00:03:32.21	if you perturb the function of a motor, you mess up aspects of
00:03:40.01	spindle function. And this messing up can show up in several ways,
00:03:43.23	It can show up in the failure of the spindle to retain
00:03:46.23	its structural integrity
00:03:48.05	or it can show up in the fact that if you have a way of measuring how frequently a chromosome is lost,
00:03:55.25	then chromosomes are lost more frequently when a motor
00:03:57.14	is missing. The remarkable thing though is when you look at a lot of the data that is in the literature
00:04:02.25	many aspects of mitosis continue even when a given motor is perturbed.
00:04:09.00	They go a little slower, not all the chromosomes may segregate
00:04:13.04	properly, but many of them do.
00:04:15.19	And of course if you are interested in the importance of your own work,
00:04:19.10	what you want to do if you've made a perturbation is see it as causing a lot of trouble.
00:04:23.18	And so you emphasize the things that are not working.
00:04:25.28	But what I've been doing in my own mind, and in the work we are doing in our lab
00:04:30.14	is asking what is still going on even when motors are not there.
00:04:35.18	And the way in which we've done this is to turn to yeast cells
00:04:38.17	where it is possible to do gene deletions comparatively easily.
00:04:43.00	and prove to yourself that the entire piece of DNA is gone from
00:04:47.14	the cell. So there is no possibility that the motor which is the protein
00:04:51.07	product of that gene is contributing to the mitotic process.
00:04:54.08	that you are seeing. And then you can ask: What happens?
00:04:58.02	And we've done this in a fission yeast cell using
00:05:02.00	the typical molecular techniques to delete
00:05:05.20	Two kinesin like proteins, each of which is a minus end in its directed activity
00:05:11.26	and the dynein heavy chain, and so there are no minus end directed
00:05:16.27	motors left in this cell, and how do we know that?
00:05:20.01	Well, the genome of this organism is sequenced
00:05:22.03	and we know that, as well as we understand, motor function with microtubules, there is no motor left
00:05:28.16	in the cell, and yet, when I know show you
00:05:32.04	the motion of the poles and one chromosome,
00:05:35.10	which we've marked with a fluorescent tag near the kinetochore,
00:05:39.15	this time lapse will show  the separation of the spindle poles,
00:05:43.21	and you can see the chromosome, which is here, and you are going
00:05:46.11	to see it migrate towards the spindle pole
00:05:49.11	and we can measure the speed at which it migrates towards the pole,
00:05:52.20	as it becomes attached to the spindle.
00:05:55.05	Now of course, it should bi-orient and come to the metaphase plate,
00:05:59.07	this particular chromosome was a little tardy in this,
00:06:02.27	and that's common with these chromosomes that lack several motors, but you could
00:06:06.26	see it did actually segregate correctly
00:06:09.20	and from this kind of raw data we've been able to
00:06:13.00	measure the speed of the final approach of a chromosome
00:06:17.05	to the pole in a variety of genotypes.
00:06:20.05	Wildtype shown in yellow
00:06:21.18	and then one motor mutant after another and down here
00:06:26.04	in green we are seeing a deletion of all three minus end directed motors
00:06:32.05	and yet this final approach to the pole
00:06:34.27	is occurring at a speed which is no different from wildtype.
00:06:38.13	So these cells make mistakes. They are not healthy, and they would not
00:06:42.21	survive in the wild, and indeed we've measured the frequency of chromosome loss
00:06:46.27	and it's up by several, well even hundreds of fold.
00:06:52.12	So this is not a healthy organism
00:06:54.05	but it's an organism in which chromosome to pole motion occurs at a speed which is
00:06:59.20	undistinguishable from the normal wildtype chromosome movement.
00:07:04.20	That means these motors are not important for such motion.
00:07:08.12	They may be important for other things, like attaching
00:07:10.20	the chromosomes to the spindle, or
00:07:12.22	segregation or integrity of the spindle
00:07:15.01	poles, but not for this fundamental phenomenon of anaphase A.
00:07:19.27	So what...well, first of all we can say this is not simply due to fission yeast
00:07:25.20	idiosyncracies, it is also true in budding yeast,
00:07:28.23	where the Tanaka group has been able to demonstrate
00:07:31.11	this quite clearly. So, these motions must be caused
00:07:35.02	by some thing that is going on in this cell
00:07:38.08	which is not a minus end directed motor.
00:07:41.06	it could be some non microtubule component
00:07:43.21	of the spindle, but this doesn't seem very likely
00:07:45.24	because although there have been the identification of
00:07:49.21	actin in the spindle, it turns out that the actin is generally not
00:07:52.22	fibrous when it is in this spindle.
00:07:53.28	And there's been the identification of matrices in the spindle,
00:07:58.06	but these matrices are not yet known to have any kinetic function.
00:08:01.17	And it could be then that it is simply microtubule depolymerization,
00:08:05.20	which is itself a motor in some way.
00:08:08.19	How do you find out?
00:08:11.17	This is an implausible idea, and in order to convince anybody
00:08:16.14	we really need some strong experimental evidence.
00:08:19.03	The implausibility of this was described best to me
00:08:22.27	by the expert kineticist who studied myosin and other enzymes, Ed Taylor,
00:08:28.15	who was very skeptical about the disassembly hypothesis, and he
00:08:32.09	pointed out that if you were a rock climber suspended on a rope
00:08:36.08	from a cliff and you wanted to go up the cliff, you certainly wouldn't do it by
00:08:40.24	lopping off the rope to make it shorter.
00:08:42.19	And this analogy certainly casts doubt on the hypothesis.
00:08:47.28	But what I am going to show you is that it has some validity to it.
00:08:52.16	Microtubule polymerization can do mechanical work.
00:08:56.12	And this was shown very nicely in the laboratory of Hotani, many years ago
00:09:01.04	where they put soluble tubulin inside lipid vesicles
00:09:04.26	and induced it to polymerize
00:09:06.19	and the polymerization of tubulin drove these deformations of the lipid membrane
00:09:11.21	showing that polymerization could do work.
00:09:14.04	And indeed it is now well known that actin polymerization can do work
00:09:18.21	and you should look at the iBio seminar that deals with this very nicely
00:09:24.21	because it has a beautiful amount of detail, all shown by Julie Theriot.
00:09:30.23	So polymerization is easy to understand
00:09:34.22	as a motor, but what about depolymerization?
00:09:38.08	We designed an experimental system in which to look at this
00:09:41.17	in which we had an objective lense on the microscope, and a coverslip
00:09:44.25	we were looking at an object which was sort of our
00:09:48.21	in vitro manifestation of a spindle pole,
00:09:50.23	we happened to use a ciliated protozoan
00:09:54.20	that we lysed with a detergent to wash away the membrane and clean out the cytoplasm
00:09:59.05	but it left behind what is called a pellicle
00:10:01.22	which has about 500 basal bodies for flagella
00:10:05.24	and that structure will now nucleate large numbers of microtubules.
00:10:10.05	We used purified brain tubulin to flow it in and get this forest of microtubules
00:10:15.11	all of whose plus ends are pointing away
00:10:18.09	from the organizer, just as the spindle microtubule plus ends
00:10:21.22	are pointing away from the centrosome.
00:10:24.07	We could then flow in chromosomes that we had
00:10:27.13	partially purified from CHO cells and ask, "Do they bind?
00:10:33.11	And if they bind, can we make them move?"
00:10:34.17	And in this movie taken by Vivian Lombillo taken when she was a graduate student
00:10:39.09	in the lab, you can see a pair of chromosomes that are caught in the microtubule
00:10:45.05	forest that has grown from this pellicle,
00:10:47.22	and as I run the movie, we will now flow in buffer
00:10:51.02	which contains no tubulin, and you can see the chromosome immediately wash down stream
00:10:56.20	in the flow of the buffer.  This movie is real time, so
00:11:00.09	we are not exaggerating any speeds and the force of the flow
00:11:03.29	is substantial, and yet, when they come into focus,
00:11:06.18	you can see that the chromsomes are still attached.
00:11:09.08	This buffer contains no nucleotide triphosphate, no ATP, no GTP
00:11:14.20	and in fact it contains a pyrase, an enzyme that drops the concentration of nucleotide below nanomolar
00:11:22.11	and yet these chromosomes move into this structure here as the microtubules disassemble
00:11:28.29	at speeds which are even on the high side for physiological motion of chromosomes.
00:11:34.19	So this work demonstrates that microtubule depolymerization
00:11:39.06	without any ATP dependent motor activity
00:11:42.04	can move chromosomes in a test tube.
00:11:45.19	So we've seen that chromosomes can approach the poles
00:11:49.01	in vivo with no motors present in the cell
00:11:52.17	and they can approach this spindle pole here
00:11:55.22	when we have no motors present and fed a fuel that could move them.
00:12:02.05	There may of course still be motor enzymes.
00:12:04.09	on the kinetochores, but this cannot be an ATP dependent motor activity.
00:12:08.09	so we interpret this as a disassembly dependent motility.
00:12:13.13	How could depolymerization of a fiber cause movement?
00:12:17.24	These images which are electron micrographs taken
00:12:21.19	of frozen hydrated microtubules by Eva and Eckhart Mandelkow
00:12:27.14	collaborating with Ron Milligan show what the ends of polymerizing microtubules
00:12:32.10	look like. They are quite blunt.
00:12:35.01	On the other hand depolymerizing microtubules show
00:12:38.10	this characteristic curl to the tip of the microtubule
00:12:42.09	where a strand of tubulin, a protofilament, is bending.
00:12:47.11	And this appears to be  a characteristic event of the disassembly process.
00:12:52.14	Where does this come from?
00:12:54.07	Well it is related to the cycle of polymerization and depolymerization of tubulin.
00:12:59.26	Tubulin to polymerize has GTP bound and
00:13:04.00	the molecule is more or less straight and it adds onto the ends of the microtubule.
00:13:08.07	But the microtubule activates the GTPase activity of tubulin,
00:13:13.02	it is like a GTPase activating factor when you think about G proteins.
00:13:17.01	And so the majority of the tubulin in the microtubule is GDP tubulin.
00:13:23.02	And that irony is that GDP tubulin will not polymerize.
00:13:27.25	The GDP tubulin tends to fall apart.
00:13:31.06	and indeed, as it falls apart it shows this curvature
00:13:34.20	and the interpretation that has been put on this by a
00:13:37.07	number of investigators working on it
00:13:39.13	principally Eva Nogales, is that the tubulin molecule in the GTP bound state
00:13:45.27	tends to be more or less straight, but in the GDP bound state
00:13:49.18	it tends to bend, and this bending means that when GDP is in the wall of the microtuble
00:13:56.08	it's under strain as a result of interactions with neighboring tubulin molecules
00:14:01.23	that interact with it by non-covalent bonds.
00:14:05.21	So those interactions are keeping the molecule constrained and straight,
00:14:10.03	unless you are at an end without any GTP tubulin
00:14:13.13	on the end to provide straightness.
00:14:16.02	And now the curvature of these tubulin protofilaments is a relaxation of
00:14:21.21	tubulin GDP molecule to its minimum energy geometry.
00:14:27.02	What this means is that a microtubule in the course of depolymerization
00:14:33.17	is going to have a wave of conformational change.
00:14:38.11	Doug Koshland was the first person to point out that this conformational wave
00:14:43.27	might be a way of pushing on things
00:14:47.00	and it might help to pull chromosomes to the pole.
00:14:49.11	But of course, the cell to take advantage of it must find some way
00:14:53.17	to couple to this microtubule so it can grasp the microtubule
00:14:58.00	and experience the force from those bending protofilaments.
00:15:01.13	We need to understand what that coupler might be in order to see how this relaxation
00:15:07.23	of the tubulin molecule could be a power stroke that would drive chromosome movement.
00:15:12.03	The first indication as to what this might be
00:15:16.03	came from some more work done by Vivian with that experimental system that I showed you earlier
00:15:20.21	with the chromosomes moving in vitro.
00:15:23.09	She added antibodies to kinesin, first a general kinesin,
00:15:28.09	and then a kinesin that is specifically localized at kinetochores
00:15:31.17	so called centromere protein E.
00:15:33.28	And those antibodies caused a dramatic reduction
00:15:37.25	in the motion of the chromosomes in this depolymerization
00:15:42.08	dependent fashion, suggesting that a kinetochore motor
00:15:46.28	is important for depolymerization dependent movement even when no ATP is present.
00:15:52.26	But remember the caveat that I raised at the end of the last lecture
00:15:57.25	that even when you add antibodies that are monospecific
00:16:01.19	they may have indirect effects. So this doesn't really prove to us that this molecule
00:16:06.24	is a coupler or that it is working in this way.
00:16:09.24	It is strong suggestive evidence.
00:16:11.19	We've obtained other evidence, however,
00:16:14.06	that a microtubule dependent motor enzyme can
00:16:18.06	work as a coupler using a kinesin-8 from pombe cells.
00:16:23.03	And if that kinesin is attached to a bead and the bead is then allowed to interact with a microtubule
00:16:32.05	which isn't visible here, but it is shown in diagrammatic form over here
00:16:35.25	and we now induce the microtubules
00:16:37.26	to disassemble, the bead is pulled by microtubule disassembly
00:16:42.01	just as I have shown you in those other experiments
00:16:44.29	and these graphs show the rate and the trajectory.
00:16:48.05	Clearly this is quite a processive movement.
00:16:50.26	In the sense that the bead is following the disassembly microtubule end for quite a distance.
00:16:56.03	So a motor enzyme can serve as an ATP independent coupling factor
00:17:01.29	to bind a cargo to a disassembling microtubule.
00:17:05.25	Does it have to be a motor? No.
00:17:09.02	And one of the most remarkable discoveries in the mitosis field
00:17:12.09	recently has been the discovery of this complex called either Dam1 or DASH
00:17:19.23	depending on whose laboratory you happen to have been associated with.
00:17:23.24	The people who first discovered and named the Dam1 protein
00:17:27.04	and then gradually found more and more proteins that were part of a big protein complex
00:17:31.26	call it the Dam1 complex. 'veI collaborated with that lab,
00:17:35.19	so I'll use that name, but the name DASH is used
00:17:38.29	by many other labs for the same complex.
00:17:40.29	It's an unusual complex because it involves
00:17:44.21	ten different polypeptides all of which
00:17:47.17	assemble into a little football shaped object
00:17:50.13	and this is called the Dam1 complex.
00:17:52.24	And this complex polymerizes with others of its own kind
00:17:56.27	and those polymers form rings around microtubules.
00:18:00.22	Here are the rings forming just on the surface of a support
00:18:04.17	visualized in the electron microscope just by negative staining.
00:18:07.06	And some really excellent work both by the Westermann group where Nogales
00:18:13.20	has been doing the electron microscopy
00:18:15.15	in her lab and in the group that's at Cambridge, Massachussetts
00:18:20.07	under Steve Harrison has been...they've been providing
00:18:23.19	expert and excellent evidence about
00:18:26.20	the structure of this complex and the way in which
00:18:29.06	it interacts with microtubules.
00:18:31.02	We've collaborated with the Berkeley group
00:18:34.09	which included Westermann and his mentors, Georjana Barnes and David Drubin
00:18:40.00	and with them we've also been able to purify this complex and
00:18:43.28	label it with fluorescent dye, and allow it to interact with microtubules
00:18:49.13	in our in vitro system, where this is that pellicle
00:18:52.01	that you've been seeing
00:18:53.02	and these then are complexes of the Dam1 protein,
00:18:56.09	which are fluorescent. This is
00:18:58.18	what our Dam1 complex looks like when it surrounds the microtubules seen
00:19:02.02	in the electron microscope, but of course you can't do kinetic experiments
00:19:05.19	in the electron microscope. So we are going to do an experiment here
00:19:09.17	which we watch what happens to these Dam1 complexes
00:19:13.01	when we cause the microtubules to disassemble.
00:19:15.19	And here we will now bleach the little tip
00:19:18.19	that's on the end of the microtubule, and the microtubule
00:19:20.25	starts to depolymerize, and you can see that the Dam1 complex
00:19:25.05	is moved with the ends of the microtubule as the ends of the microtubule
00:19:29.09	shortens. So the Dam1 complex
00:19:32.14	is also a coupler that can take advantage of the structures
00:19:38.02	that are found at the end of the microtubule.
00:19:40.17	This coupler can actually pull a load, and
00:19:45.21	what we've done here is to put the Dam1 complex
00:19:48.16	onto a bead. And this bead now can be followed
00:19:54.05	as an object which is a load for the Dam1 complex
00:19:57.12	to move as it associates with the microtubule.
00:20:00.25	And again, when we induce microtubule disassembly, as the disassembly
00:20:04.23	reaches the bead, the bead will stop its Brownian movement
00:20:09.04	just back and forth and will start a progressive motion
00:20:12.18	towards the origin of the microtubules, which is that pellicle sitting over at the side.
00:20:17.24	Now this kind of work allowed us to determine that the Dam1 complex seems  to
00:20:24.18	form a variety of structures, all of which can interact with the microtubules.
00:20:28.23	If we attach Dam1 to a bead, and do not have the Dam1 complex in solution,
00:20:36.00	but instead just allow the bead to bind to the microtubule,
00:20:39.27	we get a distribution of velocities that is shown here in red.
00:20:43.07	If we have Dam1 in solution, so that a complex can form that would make this ring
00:20:50.10	shaped structures that I showed you in the electron microscope,
00:20:52.29	what we see is that we still get bead movement, but the bead movement is slower,
00:20:57.15	as if the formation of that ring might actually retard the rate of disassembly of the microtubules.
00:21:05.06	So this looks likea process that needs detailed study,
00:21:08.14	and we've done enough experiments that I won't be able to describe them all to you.
00:21:11.16	by any means in the course of this short lecture,
00:21:14.07	but I want to show you the tool that has been the most important to us in
00:21:17.25	trying to do this kind of work.  It is a standard light microscope, which has
00:21:22.29	a sensitive camera at its top.
00:21:25.11	and then over here it has a couple of lasers, one of which
00:21:29.06	is very strong and can be led through a device that allows you
00:21:33.11	to steer the laser beam and then up into the microscope
00:21:36.14	the other laser that is over there is just to help us align things.
00:21:39.26	So a laser beam is coming down through our objective lense
00:21:43.07	and this very bright light can be used in what is called an optical trap.
00:21:47.02	A device where you can grab a small object
00:21:50.19	that refracts light, like a bead of glass or plastic.
00:21:54.00	Over on this side, we have two other lasers, one green and one blue,
00:21:58.22	that we use for bleaching the fluorescence
00:22:01.20	of some parts of our specimen. And what
00:22:04.11	we are going to use is tricks in order to
00:22:06.19	be able to do experiments on beads and ask:
00:22:11.00	Can we monitor the force that is generated in this system?
00:22:13.18	So here's again our pellicle serving as a nucleator;
00:22:17.13	microtubules growing from purified tubulin.
00:22:19.16	The problem is that these microtubules have to be labile.
00:22:22.27	And that means that if we dilute the preparation of
00:22:26.21	tubulin by washing anything else in
00:22:29.08	we are going to cause them to disassemble, so we need to stabilize them.
00:22:32.25	And we do that by putting a cap on the
00:22:35.08	tip of them where we polymerize the tubulin
00:22:38.02	in an analog of GTP which does not hydrolyse, or hydrolyzes very slowly.
00:22:42.05	And the result is that we have now stable microtubules
00:22:46.04	so we can drop the tubulin concentration to zero and
00:22:49.26	now we can bring in beads coated with
00:22:52.26	something that will make them stick to the microtubule itself.
00:22:55.28	The way we do this is we use biotinylated tubulin
00:22:59.01	and beads coated with the protein avidin, so the
00:23:01.27	connection between the microtubule and the bead
00:23:05.02	is one of the strongest non-covalent interactions known in biology
00:23:08.25	and this is not going anywhere, it's certainly not
00:23:11.25	a motor and we can ask: when the microtubule disassembles,
00:23:15.25	what happens? And the way in which we do this experiment is
00:23:19.02	to turn on our laser trap so that this bright laser light is holding that
00:23:24.14	bead and we have a way of measuring the position of the center of the bead
00:23:29.04	very accurately and then we turn on our photobleaching laser
00:23:32.15	in order to inactivate the tubulin that is there, and the cap comes off, and the microtubule
00:23:38.27	will disassemble, and you may have noticed that I drew in just a little
00:23:42.13	bit of a movement there of that bead
00:23:44.17	as the disassembly went by.
00:23:46.24	That's the kind of event we are looking for
00:23:48.11	in order to take motors completely out of the equation
00:23:51.26	and ask: can microtubule disassembly do work?
00:23:55.02	And the answer is yes.
00:23:57.13	Here is a cartoon of what we think is going on
00:24:00.14	with the bead drawn very small relative to the microtubule,
00:24:03.28	and you can see the bending protofilament that could be
00:24:06.08	exerting a force on the bead.
00:24:09.04	This is a trace that  we get from a very sensitive device
00:24:11.24	called a quadrant photo detector which is allowing us
00:24:14.22	to determine the position of the center of the
00:24:16.26	bead extremely accurately to within a nanometer or so.
00:24:20.15	And all of this wiggling you see
00:24:22.04	here is the thermal noise that the bead
00:24:24.23	is oscillating as a result of interactions with molecules
00:24:28.05	in solution.  But as the disassembly occurs,
00:24:31.00	the bead is pushed a little bit toward the minus ends of the microtubule, and then released.
00:24:35.09	and now it is simply in the center of the trap, and the microtubule has
00:24:39.14	disappeared. Now if this really is
00:24:43.04	a force being generated in this way, you could imagine that we are pulling
00:24:48.15	here on the center of the bead, with our trap, and the radius of the bead then
00:24:53.00	is like a lever arm. And the smaller the bead, the less our mechanical advantage
00:24:58.18	and the bigger the force that we should generate, and indeed
00:25:01.25	here you can see with a 2 micron bead
00:25:03.25	versus a 1 micron bead versus a half micron bead
00:25:06.17	we get bigger forces as the bead gets smaller,
00:25:09.25	suggesting that this really is the bending of the protofilament
00:25:13.09	pushing on the bead to give us the force that we are seeing.
00:25:16.07	How much force? Not very much.
00:25:18.27	with this non-physiological system. But, we also presume that we are only interacting
00:25:24.27	with one side of a microtubule, and if you think
00:25:28.04	about the Dam1 complex, which surrounds the microtubule
00:25:31.16	as a ring, one could imagine that it would experience force from all the bending protofilaments
00:25:36.10	at once, and would give you significantly more force.
00:25:39.24	So we have naturally gone ahead and tried to attach the beads
00:25:43.07	to Dam1 complex where here this is a real micrograph of
00:25:47.22	the Dam1 complex on a microtubule, but this is just a cartoon
00:25:51.11	with representations of the antibodies that we have bound to the bead.
00:25:56.24	And we are using antibodies that interact with the Dam1 complex,
00:26:00.21	and give us quite a tight bond, and now we can ask, "When disassembly comes by, what do we see?"
00:26:06.08	And the answer is a longer and much stronger force.
00:26:09.24	This force is now about six times as great as the force that we observed
00:26:14.07	when we were only sampling one side of the microtubule.
00:26:17.11	And so it really looks as if a ring
00:26:19.28	is surrounding the microtubule and
00:26:22.19	sampling the action of all the protofilaments
00:26:25.04	and producing a force, which, once we've made corrections
00:26:28.00	for the bead diameter, looks as if it would be 20, 30, even 40 piconewtons,
00:26:33.18	which is an unusual unit of force for most of you,
00:26:36.23	but a kinesin molecule or a dynein molecule develops somewhere around 5 piconewtons.
00:26:42.04	So a microtubule interacting with a ring
00:26:45.13	is really powerful. It is sort of like a bulldozer
00:26:48.20	and it's no wonder that you can delete motors from the cell
00:26:51.17	and chromosomes will continue to move, if this is the process that
00:26:55.04	is really doing it. So how do these things work?
00:26:58.28	Well, here are the two hypotheses that are central to the way people are thinking about this movement
00:27:04.09	at this point.  On the left you are seeing
00:27:06.23	a Brownian movement, a random walk by diffusion of a ring which is modeled here in these accurate simulations
00:27:15.15	done by my colleague, Fazly Ataullakhanov and his students in Moscow
00:27:20.02	and they are allowing the diffusion that would occur with an object of
00:27:25.28	the size of the ring and loose binding
00:27:27.14	and this diffusion still can give rise to processive movement
00:27:32.16	because the diffusion is biased
00:27:34.22	by the disassembly of the microtubule.
00:27:37.05	Over here we are showing a different model
00:27:40.05	And this is one in which the ring is presumed to bind
00:27:43.17	tightly to the wall of the microtubule, noncovalently,
00:27:47.03	but still tightly, so that as these protofilaments
00:27:49.29	bend they have to force the ring from one position to the next position.
00:27:54.04	to the next, and you can see kind of stall as it is going
00:27:57.10	and then it will go ahead and go farther again.
00:27:59.24	This is then a situation where a forced walk
00:28:04.00	is causing the migration of the ring.
00:28:06.19	Now intuitively you might think that this biased diffusion is a more efficient
00:28:11.15	system because you are not having to expend so much force in overcoming the binding
00:28:16.02	energy between the ring and the microtubule.
00:28:17.27	But this has problems, because polymerization and depolymerization
00:28:23.29	of microtubules are molecular events which occur
00:28:26.17	with random fluctuations, and so every now and then
00:28:29.24	the depolymerization pauses and the ends of the microtubule presumably
00:28:35.10	go straight for a little while, and if there were a load on this ring, it might just pull right off
00:28:40.10	the end of the microtubule which would be a terrible catastrophe
00:28:44.13	if you were trying to move chromosomes by microtubule disassembly.
00:28:49.23	So intuitively we favor this tight binding model
00:28:53.23	but indeed there is quite a bit of evidence that
00:28:56.12	that is the case.
00:28:58.25	So is the Dam1 ring then the answer
00:29:01.24	for how you couple chromosomes to microtubules?
00:29:04.11	Umm, in budding yeast, Dam1 is in the spindle, it binds to chromatin.
00:29:11.04	It is essential for proper chromosome segregation
00:29:13.14	and this says that the Dam1 complex is an excellent candidate
00:29:18.23	for the coupler in budding yeast.
00:29:22.05	Interestingly, in fission yeast, it is no longer essential
00:29:27.03	in cells that are otherwise wildtype.
00:29:29.12	Now if you start deleting mitotic motors
00:29:32.01	then the Dam1 complex becomes essential
00:29:34.29	but what we have here is a situation where in just going from
00:29:37.27	one ascomycete fungus to another,
00:29:41.11	we've moved from essential to contributory.
00:29:44.19	Another difference between these two spindles
00:29:48.01	is that in budding yeast there is one microtubule per kinetochore
00:29:52.06	whereas in fission yeast there are 2 to 4 microtubules
00:29:55.23	per kinetochore, so maybe, the Dam1 complex
00:29:58.27	is absolutely essential when you need to regulate the disassembly of microtubules
00:30:04.02	so tightly that you don't let the microtubule end get away from the kinetochore
00:30:08.10	but when you have more microtubules
00:30:11.07	and you have others that you can rely on, it is no longer an essential process.
00:30:15.11	Even more disturbing in terms of thinking about the generality of the Dam1 complex
00:30:20.18	is that outside the fungi, this protein has not yet been found.
00:30:23.24	Now this doesn't say that other rings are not going to be found, and a number of
00:30:28.12	possibilities have been detected becasue the Dam1 complex is so appealing
00:30:33.07	as a way of doing this job well that many scientists feel that rings must be the answer
00:30:38.23	and they are trying to find the components of the rings
00:30:41.26	in other cell types.
00:30:43.12	We've taken a different approach, which is
00:30:45.09	to go and look at the kinetochore-microtubule connection
00:30:48.19	with the best structural tools
00:30:50.11	that are available, and ask, "What do we find?"
00:30:52.24	And the way we've done this is to use
00:30:55.04	electron tomography.  Now this is an electron micrograph
00:30:58.26	of a chromosome and a spindle fiber, although it is very unprepossessing
00:31:03.07	and the reason for it is that it is one of series of
00:31:06.01	images from a tilt series where we now have a thick sample
00:31:10.20	about three or four hundred nanometers thick
00:31:12.26	in the electron microscope tipping back and forth
00:31:15.27	and we are collecting images at 1 degree intervals from +70 to -70 degrees
00:31:21.17	and then we go ahead and tip around the orthogonal axis
00:31:25.01	in order to collect a large number of
00:31:27.09	views of this three dimensional object.
00:31:30.15	These can then be combined by a variety of mathematical approaches
00:31:34.13	to create what is called a tomogram, or a 3 dimensional reconstruction of all the material
00:31:40.10	that was in that region that we were imaging.
00:31:43.15	Chromatin down here. Kinetochore here.
00:31:45.20	Microtubules there and you can see flared ends
00:31:49.03	on the tips of many of the microtubules in this array.
00:31:52.15	We can then use software to pull out a single
00:31:55.29	slice from our three dimensional reconstruction
00:31:58.03	that contains the axes of one or more
00:32:00.24	microtubules, so that we can see just exactly what these ends look like.
00:32:05.25	These ends are in some way attached to the chromosome,
00:32:11.01	and what we want to know is how.
00:32:14.00	And by just taking a descriptive approach,
00:32:16.23	we can try to get insight, and tomography allows a completly novel way of looking at this
00:32:22.12	and what I am going to show you now is a series
00:32:24.23	of images in which I took a plane
00:32:26.27	that contained the microtubule axis and then
00:32:29.24	I am going to rotate that plane around the microtubule
00:32:32.16	axis, so that we can visualize a single microtubule from multiple orientaitons.
00:32:38.23	And you can see flaring protofilaments come and go.
00:32:42.02	There is one waving way up here, here is one coming down.
00:32:46.01	All of these are images at different orientations.
00:32:47.29	We can then extract this structural information
00:32:51.27	as a series of graphics, which allow us, then, to see
00:32:55.22	a representation of the protofilaments and their
00:32:58.25	flare at the kinetochore end of one kinetochore microtubule.
00:33:03.29	This has allowed us both to quantify
00:33:06.28	exactly the shape of these flares to within
00:33:09.29	the precision of our methods for preserving
00:33:12.17	this sample, and also to ask, "What is connected
00:33:16.05	to those flares?" Well, in the first instance, when we
00:33:20.15	look at the flares of the protofilaments from kinetochore microtubules
00:33:24.17	here, and non kinetochore microtubules here
00:33:28.15	and then compare them with depolymerizing and polymerizing
00:33:32.01	microtubules in vitro, imaged in that study that I showed you before
00:33:37.14	by the Mandelkows and Ron Milligan.
00:33:40.02	What you can see is that there is a tremendous range in the structure of the protofilament
00:33:44.21	of both kinetochore and non-kinetochore microtubules
00:33:48.00	They are sort of in between assembling and disassembling.
00:33:52.09	And this was quite hard to understand; however, what we've
00:33:56.00	done is to focus in on the bending protofilaments which are
00:34:02.27	in an intermediate group, and what we can find
00:34:05.23	is that many of them have little strands that connect from
00:34:09.29	the protofilament itself up into the region of the chromatin.
00:34:13.10	And then in the lower half of this slide, what I've done is to
00:34:16.06	put graphic objects down to show you
00:34:19.29	what I think are both the protofilaments themselves
00:34:22.22	and these little fibrils that are connected to the protofilamants and also connected
00:34:28.06	up into the chromatin. And we are calling these little fibrils
00:34:31.19	kinetochore fibrils, for obvious reasons.
00:34:34.21	The trouble is that this imaging is right at the limit
00:34:39.16	of the methodology, both are ability to preserve the sample
00:34:42.17	well because we are looking at a whole cell here, which has been prepared for electron microscopy
00:34:47.07	and the imaging resolution of the methods
00:34:50.02	that we are using-tomography of these thick samples.
00:34:53.19	It would be very nice if we could average things up in order to try to see
00:34:58.04	whether there is an averaged structure for the fibril.
00:35:00.25	And Katya Grishchuk in the lab had the insight that if we were able to
00:35:06.05	take the intermediate classes of protofilaments,
00:35:10.16	yes, in between polymerizing and depolymerizing
00:35:14.08	and look at these only, we might see something special
00:35:18.13	because these are not simply depolymerizing
00:35:21.06	and so we took those, sorted objectively, simply on the basis of their slope near the microtubule
00:35:27.12	wall.  We could then take forty or fifty
00:35:30.19	such protofilaments from the original image data and average them all.
00:35:36.00	And this is such an average for a non-kinetochore microtubule.
00:35:38.22	This is a metaphase kinetochore microtubule
00:35:41.18	from that intermediate group, and I think you can
00:35:43.14	see now there is a very respectable fiber that is averaged
00:35:47.14	up out of these fifty or so data sets.
00:35:49.28	This is also found in anaphase, where the flare is slightly longer.
00:35:53.29	on this protofilament, whereas the ram's horn groups-the ones that have the big curvature
00:36:00.17	characteristic of depolymerization
00:36:01.25	don't have such filaments associated with them that
00:36:05.24	will average up in this way.  So what this suggests is that
00:36:09.28	if you choose your protofilaments by an objective criterion
00:36:12.28	which suggests that they are under some kind of stress or strain
00:36:19.10	that is keeping them from going to be just like depolymerization
00:36:23.07	and then they are not polymerizing, you can
00:36:25.03	then find protofilaments that are there.
00:36:27.17	And our interpretation of this is that
00:36:30.07	these kinetochore fibrils are exerting force on
00:36:34.15	the bending protofilaments so that as the protofilaments try to bend, they stretch this fiber
00:36:40.26	and exert tension on the chromosome itself.
00:36:43.03	So this is a different kind of coupling,
00:36:46.07	one that does not involve rings,
00:36:48.10	one that need not be a motor, but that could simply be a static link of some kind.
00:36:53.14	Here is a drawing in which I have represented
00:36:55.26	protofilaments based on the tracings that I have
00:36:58.07	done of the microtubules themselves,
00:37:00.06	Protofilaments, sorry, kinetochore fibrils
00:37:03.15	that connect the protofilaments up into the chromatin
00:37:06.19	and this now is a simulation done by Grishchuk, Ataullakhanov and his students
00:37:12.23	and it is showing that even with a load
00:37:15.20	of about 40 piconewtons pulling in this way,
00:37:19.05	if you have tightly binding kinetochore fibrils,
00:37:22.13	that interact with polymeric tubulin, so that they will
00:37:26.19	stick to these bending protofilaments, but be released
00:37:29.14	as soon as tubulin falls off from the end,
00:37:32.26	you can make a processive motor that works perfectly well.
00:37:36.05	And indeed, some of the details of this motor that are now
00:37:39.05	described in the Cell paper that came out this year
00:37:41.27	give us confidence that this has advantageous properties
00:37:46.18	that may be even better than a ring for serving as a coupling
00:37:50.04	even though it is an improbable idea.
00:37:54.04	Structurally then fibrils are found in many places, but
00:37:59.22	we'd like to know what they are made from.
00:38:01.26	If we don't know the protein composition of these
00:38:03.24	structures, it is very difficult to do experiments that will
00:38:07.07	tell us definitive things about mechanism.
00:38:09.06	We have a number of ideas of what they could be
00:38:12.06	of course, because we have seen
00:38:13.21	that kinetochore related motors, like kinesin 7's and 8's
00:38:19.05	are both fibrous in their structure, and they can do the
00:38:22.23	coupling job. There are also non-motor proteins,
00:38:26.17	which are fibrous, that are localized
00:38:28.19	to the kinetochore, and NDC80 is perhaps the most attractive of these
00:38:32.23	because it is found across all cells where it has been sought,
00:38:38.02	and in every cell where such experiments have been done
00:38:41.09	and if you delete this motor, the chromosomes simply cannot attach to the spindle.
00:38:45.19	So NDC80 is an important fibrous molecule involved in
00:38:49.26	attaching chromosomes to spindle fibers.
00:38:52.16	It could well be part of this attachment machinery,
00:38:56.29	however, the evidence that now exists about NDC80 shows that it binds to the outside of the microtubule
00:39:03.01	and the way I have shown you the fibrils,
00:39:05.17	with the bending of the protofilaments, it almost looks as if the fibril attaches to the inside of the microtubule.
00:39:12.16	There are very few studies that have identified proteins that would bind to the inside
00:39:17.15	of a microtubule, so maybe NDC80 also has this function, or there may
00:39:24.06	be a new class of protein not yet identified
00:39:27.06	which can fulfill this function.
00:39:29.09	Or, alternatively, our resolution may not be good enough to give
00:39:32.23	the straight answer as to where the fibrils join the protofilaments.
00:39:35.26	And they may come around onto the far side and bind.
00:39:39.29	Certainly there are other fibrous proteins in the spindle.  There are many
00:39:43.15	of them in the kinetochore, and so some of these might serve as connectors.
00:39:48.08	The molecular nature of this coupling is really something one wants to understand.
00:39:52.28	But it really is not yet known.
00:39:56.14	Evidence from localization of proteins
00:39:58.14	from genetic disruptions of particular components
00:40:02.08	from biochemistry, all of these different
00:40:05.01	kinds of experiments suggest that there are multiple factors
00:40:09.01	that are inolved in coupling chromosomes to microtubules.
00:40:13.00	Some more important than others, perhaps, like NDC80
00:40:16.02	but it may be that what we visualized in the electron microscope
00:40:19.16	is actually a little molecular zoo.
00:40:22.06	and there are multiple kinds of connections made by different components.
00:40:26.04	The fact is that this is a wonderful set of unsolved problems,
00:40:30.15	and it's a marvelous study or problem for future work.
00:40:35.04	It's the kind of thing where one hopes that many laboratories will come to partake
00:40:40.21	in this kind of research, contribute to
00:40:43.12	the knowledge that we have, because our group has been small.
00:40:47.00	We are enthusiastic about our work.
00:40:49.23	We have enjoyed the kinds of things we've done
00:40:50.27	as you can see from Katya's enthusiasm.
00:40:53.14	Fazly Ataullakhanov, the mathematician who has been responsible
00:40:56.21	for the supervision of these three mathematicians,
00:41:00.21	who are now becoming cell biologists as well
00:41:02.21	and we've had a wonderful time doing this work
00:41:05.13	but there's lots to be done, and we hope you and others will come and join us in this research.
00:41:10.27	Thank you. Goodbye.