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.
