Session 7: Cell Division
Transcript of Part 2: Understanding Mitosis through Experimentation
00:00:09.24 Hello, my name is Dick McIntosh, and I am a professor 00:00:12.29 of Cell Biology at the University of Colorado 00:00:15.11 and this is the second of three lectures in which I am talking about 00:00:18.05 chromosome movement and cell division. In the first lecture, 00:00:22.20 we talked about background material, 00:00:24.11 that is the cells getting ready for division; 00:00:26.22 now we are going to look at experiments and the ways in which 00:00:30.04 they have informed us about the machinery that does 00:00:33.12 the division process. In the previous lecture I emphasized 00:00:37.23 the complexity of cells and the difficulties 00:00:40.27 that one encounters because they are so small. 00:00:42.22 This pair of factors really has a huge impact 00:00:47.22 on both the work that people have done to try to understand cell division 00:00:52.18 and on the results that many excellent scientists have gotten, 00:00:56.29 as they have pursued this challenging problem. 00:01:00.02 So what you'll hear about in this lecture is a range of 00:01:03.19 methods of experimentation, approaches that people have used to try to 00:01:09.04 perturb the spindle in informative ways. 00:01:11.18 And some of them included genetic approaches, 00:01:14.17 some have included mechanical approaches, 00:01:16.25 and some are biochemical, some are immunological, 00:01:20.09 using antibodies that can block functions, 00:01:22.14 and some are pharmacological, using drugs. 00:01:24.15 And I'll show examples of a variety of these things, 00:01:27.17 in order to give you a sense of the experimental richness 00:01:30.29 that is available to us in trying to understand this complicated process. 00:01:34.17 The difficulty is that experiments have been done on different organisms 00:01:40.06 and one cannot always compare information that comes from these different organisms 00:01:45.29 in a literal way because the mitotic processes in different cells may not be identical. 00:01:50.25 Further than that, they may respond differently to a 00:01:54.20 particular pharmaceutical agent or to a particular mutation, 00:01:58.00 so we have the complexity that is brought into this process of 00:02:01.20 biological variability. Of course there's the 00:02:04.08 biological variability of the experimenter as well. 00:02:07.06 So, by no means can I tell you at the end of this lecture how mitosis works, 00:02:13.11 but what we'll be able to do is see the relative importance of several aspects 00:02:17.22 of spindle function and the ways in which 00:02:19.29 they have impact on chromosomal movement. 00:02:22.11 So the jobs that the cell has to do in order to segregate chromosomes 00:02:27.13 accurately is to make the mitotic spindle. 00:02:30.27 It has to attach the chromosomes to the spindle 00:02:33.26 and organize them and then segregate them into the two distinct sets. 00:02:39.04 The approaches we are going to take in this lecture to these different problems are 00:02:44.13 quite different. The first one is simply to bow out because Ron Vale 00:02:49.01 has done a very nice lecture on spindle formation showing 00:02:53.07 some beautiful work from his lab that illuminates aspects 00:02:57.07 of this complicated phenomenon. So I am really not going to deal with it at all here. 00:03:01.28 On the other hand we will focus on the mechanical properties of the spindle in order to see 00:03:07.27 how they affect chromosome attachment and chromosome segregation. 00:03:12.23 Now I am going to start with spindle properties 00:03:15.27 and then go on to the elongation of the spindle 00:03:20.06 because that's a simpler problem than 00:03:22.16 the whole business of attaching chromosomes to the spindle and organizing 00:03:26.10 their segregation, but we'll get there. 00:03:28.24 I have included this beautiful slide, a text book slide, 00:03:33.18 the work of Bill Earnshaw using immuno-fluorescence with 00:03:36.03 red staining to show the microtubules and blue staining 00:03:39.04 to show the chromosomes because it displays 00:03:42.02 very beautifully the overall pattern of spindle formation 00:03:46.08 and function in a wide variety of vertebrate cells. 00:03:50.23 We are going to form this metaphase structure 00:03:53.20 by means of attaching the chromosomes 00:03:56.14 to the microtubules that will come out of the cytoplasm 00:04:00.10 into the nucleoplasm and then organize 00:04:02.18 the chromosomes to produce this structure. 00:04:05.02 Anaphase will occur, in two stages: Anaphase A, 00:04:09.06 which is that shortening of chromosomes to poles. 00:04:11.18 Anaphase B then is the elongation and then finally cytokinesis will come 00:04:17.02 and pinch that bundle of microtubules down 00:04:20.11 into a single shaft that I have called the mid body. 00:04:23.08 This process is fairly well conserved in its overview. 00:04:28.24 The details vary, but we can take this as the general process for mitotic action. 00:04:34.18 And I showed this slide in the previous lecture to emphasize 00:04:38.18 the simple symmetry of the spindle once it is formed. 00:04:42.07 The spindle is bipolar because it grows from two poles 00:04:46.10 and these microtubules interact with one another, 00:04:49.04 and some of them interact with the chromosomes 00:04:51.06 setting aside the particular microtubules 00:04:55.02 that are called kinetochore microtubules, 00:04:57.01 because those are the ones that interact with this 00:04:59.23 chromosomal specialization, the kinetochore. 00:05:02.15 When anaphase starts there is a severing 00:05:05.11 of the connection between sister chromatids, 00:05:08.04 and then the chromatids move apart and we are left behind 00:05:12.00 with this interzone spindle which can elongate in order 00:05:15.13 to increase the distance between the poles and achieve anaphase B. 00:05:23.10 Now the first question I want to address is that of 00:05:26.25 how do those two half spindles interact with one another 00:05:31.28 to form the two fold symmetric structure? 00:05:34.15 Because if you imagine structures that are simply forming microtubules 00:05:39.08 like a radial array that might grow out of a centrosome, 00:05:41.20 it isn't clear that those microtubules 00:05:44.23 would interact, but they do. And they do as the result of several protein factors. 00:05:50.16 One factor that has now been identified, actually comparatively recently, 00:05:54.09 is a microtubule binding protein called Ase1. 00:05:58.29 And Ase1 has the property that it is localized at the spindle here 00:06:02.09 this is showing you work from a fission yeast cell, which is this elongate structure. 00:06:07.24 Here are the microtubules shown in red, 00:06:10.18 and then here is this Ase1 protein shown in green 00:06:14.08 and the two of them overlapping at the bottom. 00:06:17.09 Ase1 is concentrated in the region where those two half spindles are interdigitating 00:06:22.27 and genetic work in pombe has shown that when 00:06:26.03 you delete the Ase1 gene, the spindle frequently falls apart 00:06:30.11 into two distinct halves, so this is a mechanical agent 00:06:35.03 that is helping to hold together interdigitating microtubules 00:06:38.13 at the middle of the spindle. 00:06:43.05 Ase1 is not the only factor that is important for this process however, 00:06:46.29 enzymes are also required to establish spindle bipolarity 00:06:51.03 and the central one in many cell types is this unusual looking molecule, 00:06:56.22 which is four single polypeptides that are identical, so it is a homo-tetramer, 00:07:02.24 and it assembles to form a bipolar structure in which there are two heads at each end. 00:07:08.28 And each of these heads is a motor enzyme 00:07:12.07 so this homo-tetrameric molecule of the kinesin family 00:07:18.07 is able to bind to microtubules and one form of binding is shown here, 00:07:23.16 where the microtubules are what we say is anti-parallel, 00:07:27.04 that is the plus end is here on this microtubule and pointing in the opposite direction there. 00:07:32.16 This kinesin-5 is a plus end directed motor, so it walks 00:07:38.23 towards the plus ends of the microtubules 00:07:40.21 and that means that it is going to rearrange the microtubules over time. 00:07:45.01 When it's interacting between two 00:07:47.23 parallel microtubules on the other hand, if it walks towards their plus end, 00:07:52.00 it doesn't cause the microtubules to slide, 00:07:54.00 but instead it moves relative to the microtubules, 00:07:57.03 up towards their plus ends. 00:07:59.00 And down at the bottom, is a diagram of a pole 00:08:02.01 and then an organized spindle, which represent the results 00:08:06.01 of the action of this kinesin-like protein 00:08:08.29 as it interacts with the microtubules that have come from two poles 00:08:12.22 and it pulls them together to form a bipolar structure in which there is 00:08:16.24 only a limited amount of overlap near the middle of the spindle. 00:08:25.09 The importance of this kinesin-5 has been demonstrated in several ways. 00:08:32.10 Genetically if you remove the gene for this motor 00:08:36.05 from yeast for example, what you find 00:08:40.07 is that the bipolar spindle simply will not form, 00:08:43.10 but that could be an indirect effect of some kind. 00:08:45.24 You could argue that without this motor you don't transport 00:08:48.17 some essential component to the middle of the spindle 00:08:51.21 and that is what is doing the linking. 00:08:53.23 A different kind of evidence 00:08:55.25 has been provided by pharmacology in which a number of different groups 00:08:59.11 have sought small molecules that will interact specifically with this kinesin-5 and inactivate it. 00:09:05.22 And one of the early ones is called Monastrol. 00:09:08.28 There's been quite a lot of experimentation done with it because 00:09:11.27 it seems to be quite specific when it acts in mammalian cells. 00:09:15.09 And what one finds is that a monastrol treated cell 00:09:19.17 going into mitosis forms a monopolar spindle. 00:09:23.07 There are two centrosomes in the middle here, but they can't separate. 00:09:27.07 They can't form independent units of astral microtubules which can interact because without 00:09:34.11 that motor there to push things apart the spindle just never forms. 00:09:39.22 So the activity of kinesin-5 is essential for forming a bipolar spindle. 00:09:45.06 This is a reversible effect, because if you take out the drug, 00:09:49.07 the spindle will form and function in anaphase perfectly well. 00:09:52.01 And what you can see in this pair of images 00:09:55.01 is that if you take the monastrol poisoned spindles, which are monopolar, 00:10:00.05 and use a reagent that will tend to dissolve the microtubules, 00:10:04.08 calcium ions will do this, 00:10:06.02 then what happens is you are left simply with bundles of microtubules 00:10:10.10 that connect directly to kinetochores. 00:10:13.19 So, what we have in this structure is evidence that monastrol 00:10:18.07 is very important for the interdigitation and the interaction of the microtubules 00:10:22.19 that are coming from the two poles. 00:10:25.25 Surprisingly chromosomes are not required to have a bipolar spindle, 00:10:31.12 and this is a really rather remarkable genre of experimentation involving 00:10:35.24 micromanipulation, started really and developed 00:10:39.10 most fully by Bruce Nicklas, but his student Dahong Zhang 00:10:43.05 has done a wonderful experiment here in which he has used a micro needle 00:10:47.20 to reach into the cell and remove the chromosomes, 00:10:50.24 pulling them off into another region. 00:10:53.19 So we have a spindle forming with no chromosomes on it. 00:10:57.01 It is being seen in polarized light here, and here we get a change from bright to dark 00:11:02.29 as a result of this being a linearly polarized microscope 00:11:07.02 and this structure here is similar to what is shown down here with 00:11:11.07 antibodies to tubulin revealing where the microtubules are. 00:11:13.26 This structure is perfectly capable of going ahead 00:11:17.24 and helping the cell set up a cleavage furrow 00:11:21.12 even with no chromosomes and it retains its bipolarity 00:11:24.10 quite nicely as it functions with the chromosomes gone. 00:11:28.01 Now, when chromosomes are present the spindle is mechanically somewhat different, 00:11:35.27 and I am going farther afield in terms of biology 00:11:38.23 here in order to make this point. This is the spindle of a diatom, 00:11:43.06 which is a kind of alga, and it has the virtue that the interpolar spindle 00:11:48.29 is sufficiently well organized that it gives rise to a very birefringent object 00:11:56.02 that shows up in the polarizing microscope. 00:11:58.05 And Jeremy Pickett-Heaps and his students 00:12:00.27 used polarization optics to visualize this spindle and then 00:12:04.13 a micro-beam of ultraviolet light in order to irradiate a small portion of the spindle, 00:12:10.07 and they destroyed the microtubules in that area 00:12:13.11 with this perturbation. And what you can see in the row of images 00:12:17.05 across the bottom is that when the spindle is no longer symmetric in its strength, 00:12:22.10 it bends inward, revealing the fact that the poles are being pushed inwards 00:12:27.05 towards one another by the mitotic structure. 00:12:30.16 The chromosomes are apparently being pulled towards 00:12:33.28 the poles and they are pulling inwards on the poles 00:12:37.13 as this mechanical equilibrium is set up during the mitotic process. 00:12:42.10 Well, what is pushing out in order to prevent the poles from just collapsing? 00:12:48.06 And the answer probably is in large part this same kinesin-5 00:12:52.08 that we talked about before. Here are immuno-localization images 00:12:56.10 in which we can see kinesin-5 and tubulin and then the superposition 00:13:01.11 of those two shown in different colors. 00:13:04.03 And you can see here that kinesin-5 is plentiful in the spindle 00:13:08.22 and it is found in this mid-region 00:13:11.13 in the spindle where there are not even quite so many microtubules 00:13:14.12 and in that region its function of crosslinking anti-parallel microtubules 00:13:20.07 and walking towards the plus ends of the microtubules 00:13:23.02 which will tend to push apart is going to 00:13:25.00 provide a force that will tend to keep the poles from collapsing. 00:13:29.07 But as is so often is true in biology, things are not that simple. 00:13:33.26 Kinesin-5 is not the only motor in the spindle; 00:13:36.11 there is also a different kinesin, and in this case it is called kinesin-14. 00:13:42.07 which has the opposite polarity of motion. 00:13:45.05 it walks towards the minus ends and so in this image here 00:13:49.18 what you can see is that kinesin-14, represented in green, 00:13:55.19 is concentrated in the middle of the spindle 00:13:58.18 tubulin represented by the red staining, which is giving us purple towards the poles 00:14:04.24 is more concentrated towards the poles, 00:14:07.25 and so this intermediate region, the interzone, 00:14:11.18 even at metaphase, is a region where we find kinesin-14 00:14:15.22 that pushes the microtubules towards the middle 00:14:19.09 of the spindle and kinesin-5 which pushes them away. 00:14:22.07 Suggesting some kind of a dynamic equilibrium between them. 00:14:25.27 This is diagrammed here as a balance of forces 00:14:29.13 in which kinesin-5 is pushing outwards as it walks 00:14:34.04 towards the plus ends of the microtubules and the kinesin-14 is pulling inwards 00:14:39.28 as it walks towards the minus ends of the microtubules 00:14:42.13 and evidence for this kind of balance comes from genetic experiments 00:14:47.19 where if you delete the kinesin-5 then the spindle will tend to collapse. 00:14:51.23 So we have a combination here of the mechanics 00:14:57.05 that is offered by the stability of the microtubules themselves 00:15:00.18 and motors in the middle that are pushing and pulling, 00:15:03.08 so we can regulate quite carefully what is going to happen 00:15:06.17 in this zone of overlap. I've often thought about this a little bit the way 00:15:10.22 of how you might think about how you do fine motor control. 00:15:13.16 You want to be able to push in both directions. 00:15:15.19 So if you hold something between thumb and fingers 00:15:18.26 and now you can manipulate it quite precisely like the violin bow or something. 00:15:22.28 And here the spindle is manipulating the interzone 00:15:26.21 microtubules by being able to both push and pull on them. 00:15:29.23 at the same time. But this is not all that is going on in this spindle, 00:15:34.28 there are also the dynamics of the microtubules themselves. 00:15:38.23 Microtubules, of course, can polymerize and de-polymerize and this cycle 00:15:44.00 has been well described by many people in many labs. 00:15:47.28 And I am not going to dwell on it here. 00:15:49.11 But polymerization involves the assembly of tubulin 00:15:53.09 that has GTP bound to it. The GTP is hydrolyzed, 00:15:57.01 and then when disassembly occurs these microtubule strands, 00:16:03.03 so called protofilaments seem to bend during the course of the disassembly process. 00:16:07.09 This kind of dynamics is going on in the spindle all the time, 00:16:11.24 and there is good evidence for this: evidence has come from photobleaching, 00:16:16.24 where you can see the spindle microtubules turn over quickly, 00:16:19.25 but the most remarkable evidence for it has come from using a fluorescent tubulin 00:16:24.17 in order to mark individual microtubules in the cell 00:16:27.20 and take advantage of very sensitive cameras 00:16:30.26 to be able to see this fluorescence even when there is so little there 00:16:34.03 that a microtubule is not uniformly labeled, 00:16:37.06 but it is heterogeneously labeled or it looks like speckles. 00:16:40.16 And this is called speckle imaging and it's been used by a number of investigators, 00:16:45.25 having been invented by Ted Salmon and Clare Waterman 00:16:49.01 as a way of looking at microtubule dynamics in living cells. 00:16:53.15 And here I am showing you some spindles that were imaged 00:16:56.12 with this method showing that the microtubules 00:16:59.08 of the spindle are continuously moving towards the pole 00:17:02.04 in both directions as if kinesin-5 is pushing them outwards from that zone of overlap 00:17:09.04 in the middle. but if that were true, the spindle should be elongating and it's not. 00:17:14.15 suggesting that there's control on the microtubule dynamics 00:17:18.13 and this dynamics comes from yet another microtubule motor, a kinesin-13 00:17:25.05 It has the behavior that it can help promote disassembly of microtubules, 00:17:30.26 one kinesin-13, anyway, is concentrated at the spindle poles. 00:17:36.19 and that means that it can help to chew up the microtubules 00:17:40.06 as they are pushed towards the pole, 00:17:42.06 allowing the spindle to treadmill away from the center without getting longer. 00:17:47.28 And indeed in this work from Sharp, you can see that as the microtubules 00:17:53.01 are being followed with speckle imaging 00:17:55.04 when the kinesin-13 has been inactivated by antibody injection 00:18:00.16 the motion towards the poles reflected here by this movement outwards 00:18:06.27 because this is a time axis and this is a space axis 00:18:10.24 and the slope of these lines reflects how fast these speckles are moving 00:18:16.04 The speckles are moving much more slowly when we impede 00:18:19.21 the activity of this disassembly motor at the poles. 00:18:22.29 So we not only have motors functioning as mechanical entities pushing, 00:18:28.01 we have motors functioning in the dynamics of spindle microtubules. 00:18:34.06 This can all be assembled in a diagram and I have taken 00:18:38.04 this very nice diagram from the Pollard and Earnshaw textbook 00:18:41.27 showing microtubules attached to chromosomes in the middle, 00:18:45.20 the addition of subunits in this region here, 00:18:49.18 and the motion of the microtubules towards the pole 00:18:51.21 in a process which Tim Mitchison called "flux". 00:18:54.17 We also have the overlapping microtubules in the middle here, 00:18:58.20 we are adding subunits on either side of this overlap region at the plus ends 00:19:04.22 of the microtubules and pushing the microtubules towards 00:19:08.03 the pole using the kinesin-5, but balancing 00:19:11.04 that action with the kinesin that is working in the opposite direction in the middle. 00:19:16.11 So this is a complicated structure that is sliding microtubules 00:19:20.21 towards the pole all the time that it appears simply to be sitting there in metaphase. 00:19:26.22 Does this really explain chromosome motion? 00:19:30.12 You can think of it in the way that if all the microtubules 00:19:33.17 in a metaphase cell were sitting there in flux, anaphase A could happen simply by 00:19:40.12 allowing the separation of the sister chromatids, 00:19:43.06 and then allowing each of the sister chromatids 00:19:46.08 to join the flux and go to the poles. Anaphase B could happen simply by 00:19:50.24 turning off the disassembly that is going on at the poles, 00:19:54.11 and this summary may be part of how mitosis really works. 00:19:58.25 At least in some cells, but we have biological variability to deal with, 00:20:04.28 and here now I am going back to fission yeast. 00:20:08.00 and we are taking advantage of the ability to perturb fluorescence in a cell. 00:20:13.21 The fluorescence here is coming from tubulin 00:20:17.05 which is marked with a fluorescent dye, and in this live cell 00:20:20.19 you are seeing a series of time frames here of a normal spindle as it elongates 00:20:26.07 Here we have a spindle and we are photobleaching the fluorescence in the middle 00:20:30.27 that means we use a bright light in order to kill the fluorescence, 00:20:35.02 but the microtubules are still there. 00:20:37.04 And what you can see is that the fluorescence comes back, 00:20:41.09 on the other hand if we go a little later in mitosis, 00:20:43.11 and we photobleach in the middle, what you now see is that 00:20:47.03 that spot divides in two, and separates and moves towards the poles . 00:20:51.17 This is just what you would expect if you had those overlapping tubules in the middle 00:20:57.00 and they were sliding apart and you put a mark on them, those marks would move apart. 00:21:01.15 But we are not seeing flux. This is Anaphase B and it is the migration of the poles apart. 00:21:09.03 and there is no indication of dissolving the microtubules at the poles in this cell. 00:21:15.13 So flux does not seem to be a universal, and yet these cells 00:21:20.05 and many others where flux does not appear 00:21:22.07 to occur, divide chromosomes perfectly well. 00:21:25.07 So flux may be important, but it can't be the whole answer. 00:21:28.04 There is even more to the mechanics of this because we learned a long time ago 00:21:33.13 from some work that was done with beautiful micro-irradiation 00:21:37.12 again with ultraviolet light, and this has now been confirmed by laser irradiation 00:21:41.25 with some more recent experiments. In a number of fungi, if you have an un-irradiated cell 00:21:48.11 and you measure the rate of spindle elongation you get some value 00:21:52.27 and you would imagine now, if we irradiated the middle 00:21:56.00 and kill this region where I've been telling you kinesin-5 has been acting, 00:22:00.06 what would happen then is that the spindle then would either collapse 00:22:03.25 or would no longer elongate, or it would elongate more slowly. 00:22:07.05 But what happens in fact is that it elongates faster. 00:22:10.13 We have a situation here where the experimental evidence suggests 00:22:15.02 that that central bar in the spindle is helping to direct 00:22:19.18 chromosome movement, and helping to regulate it, 00:22:22.09 but it is not the driving force in these cells. 00:22:25.05 And down below are important control experiments where 00:22:28.05 you miss the spindle in the middle and have essentially no effect on the rate or the velocity 00:22:33.14 and irradiate outside the spindle and again. So this looks like a very 00:22:37.19 reliable result, and indeed as deeper work has gone on in the laboratory 00:22:42.13 of Gero Steinberg recently using genetics as well, 00:22:45.24 he has been able to show that the probable motor 00:22:49.16 for this in the cell he has been studying is a dynein 00:22:53.22 which is somehow anchored in the cortex 00:22:56.14 of the cell and is able to walk towards the minus ends of the microtubule 00:23:00.15 that grow out of astral arrays and help to pull the poles apart. 00:23:06.11 So it looks as if spindle elongation in at least some cells, 00:23:10.01 and this is probably true of fungi and others, 00:23:12.13 is a front wheel drive, not a back wheel drive. 00:23:15.17 Is the back wheel drive important? 00:23:18.05 Yes, because if the two spindles went off in different directions like this, 00:23:22.29 you might imagine that what would happen then 00:23:25.25 is that when cytokinesis occurred the two nuclei would wind up in the same daughter cell. 00:23:30.15 so it is important that the direction of motion be in opposite directions, 00:23:36.25 so that we can get these daughter nuclei into very distant regions in the cell. 00:23:43.12 So the pulling is mechanically important, 00:23:46.00 but for sure this middle region is important 00:23:48.24 in guiding and controlling even in those cells where it doesn't seem to be 00:23:53.06 the major driving force for the motility. 00:23:55.29 So now we are ready to start talking about 00:23:58.17 interacting with the chromosomes and how does the spindle 00:24:01.17 bind them. Over on the left here are two stages 00:24:06.12 from that fluorescence pair that I showed at the very beginning of this lecture 00:24:10.03 and as you can see we start out with the microtubules in the spindle that is forming 00:24:15.10 and the chromosomes scattered all around. 00:24:17.07 But they come to this very ordered arrangement of metaphase. 00:24:21.06 Here is an electron micrograph again from the laboratory of 00:24:25.15 Jeremy Pickett-Heaps looking at the early stages 00:24:29.04 of chromosome attachment to the spindle. 00:24:31.09 The important features here are that these are two chromatids. 00:24:35.19 You can imagine their arms extending 00:24:37.10 way off, because we are looking at a thin slice through the chromosome. 00:24:41.02 But the good thing about this slice is 00:24:43.10 it shows us this specialization which is the kinetochore 00:24:46.05 on each of the two chromatids and it shows us 00:24:49.16 microtubules in two kinds of interactions with the chromosome. 00:24:53.05 One it is going grazing right by the kinetochore, 00:24:56.20 and in the other it is making a sort of butt end connection. 00:25:00.03 Both of these kinds of connections turn out to be important 00:25:03.10 for the process of attachment of chromosomes to the mitotic spindle. 00:25:07.16 This process has been studied experimentally in the lab of Conly Rieder. 00:25:12.19 And he set up a wonderful experimental system 00:25:15.22 where he was working with these newt cells 00:25:17.29 which we saw in the movie of the first lecture, 00:25:20.22 and they have very big chromosomes as you are seeing here. 00:25:24.13 And Rieder injected this cell with fluorescent tubulin, and you can see a single microtubule 00:25:30.20 growing here, and as that microtubule continues to grow, 00:25:34.27 because these are two frames in different times, 00:25:37.26 it actually makes contact with the chromosome shown here 00:25:42.00 as a dark ghost because there is soluble tubulin around in the background. 00:25:45.10 And this is a graph showing chromosome movement 00:25:48.15 which is diddling around without much happening 00:25:51.20 until the microtubule makes contact, and then off it goes. 00:25:55.25 So this is direct evidence for the importance of microtubule contact 00:26:00.29 with a chromosome being essential for the initial motions of the chromosome. 00:26:06.26 They've gone ahead and done electron microscopy 00:26:09.11 on one of these chromosomes that has just made contact, 00:26:12.06 and as you can see here the microtubule, diagrammed as this line, 00:26:17.10 is passing right by the kinetochore. 00:26:19.11 This appears to be one of these grazing contacts that I showed you 00:26:22.21 in that first electron micrograph of a chromosome and a spindle. 00:26:25.25 And what's going on apparently is that there are mechanical contacts 00:26:30.11 that allow the kinetochore now to motor over the surface of the microtubule 00:26:35.04 and it goes towards the pole. 00:26:36.28 The pole is where the minus end of the microtubule 00:26:40.02 resides, so this is a minus end directed movement. 00:26:43.20 Dynein has that directionality of motility and many people believe 00:26:49.02 that this interaction that we find early on in chromosomes attachment is 00:26:53.18 dynein mediated at least in some cells. 00:26:56.04 Dynein is indeed found in the spindle, and is localized at the kinetochores, 00:27:02.23 either on an isolated chromosome as shown here, 00:27:05.04 or on chromosomes as they go into mitosis in pro-metaphase. 00:27:10.14 Now this kind of evidence from antibody localization is very suggestive. 00:27:16.21 And since dynein is a minus end directed motor, you could imagine 00:27:20.23 that the dynein which is here and here is going to be 00:27:23.23 involved in pulling these chromosomes apart, 00:27:27.00 and it could be a part of the very important machinery for chromosome segregation. 00:27:35.21 The question that I want to get at, though, 00:27:37.02 before talking about the mechanics of chromosome segregation 00:27:39.26 is how do chromosomes form stable attachments to the spindle. 00:27:44.18 And the reason I focus on this, is if you look at this first sentence 00:27:48.26 which I've outlined here in red, this is a really critical point, 00:27:55.02 because the central problem of mitosis is attaching sister kinetochores to sister poles. 00:28:00.01 Once you have achieved that, all you have to do is pull the chromosomes apart 00:28:05.00 And so, how does a kinetochore know how to grab 00:28:10.23 a hold of microtubules that are coming all from 00:28:13.09 one pole or coming all from another pole 00:28:15.13 and setting up so that the pair of kinetochores is interacting with a pair of poles? 00:28:22.10 Now microtubules come at the chromosomes from both poles, 00:28:28.07 and so how does the kinetochore know which ones it should bind too? 00:28:34.14 Experiments suggest that kinetochores will bind any microtubule, 00:28:39.15 either its wall or its plus end, and they have a fairly high affinity for the plus end. 00:28:45.18 And they can't choose between east pole or west pole, 00:28:49.03 but what makes the decision is that the attachment 00:28:54.00 is going to form and become stable only when the kinetochore 00:28:57.23 microtubule junction is under tension, 00:29:00.04 that is when this kinetochore is being pulled one way, 00:29:03.06 and its sister is being pulled in the other way. 00:29:07.00 And any other form of attachment is not stable. 00:29:09.16 The evidence for this comes from some beautiful experimental work by Bruce Nicklas 00:29:14.19 whom I mentioned before as one of the master micromanipulators. 00:29:18.17 This is a grasshopper spermatocyte. 00:29:20.22 Chromosomes are shown here, and this is meiosis I 00:29:24.17 so each of these is actually a bivalent chromosome, 00:29:27.16 which makes them large and easy to work with 00:29:29.25 and there is a kinetochore over down here and a kinetochore up here. 00:29:33.16 These chromosomes are big enough and these cells are tough enough 00:29:37.04 that Nicklas has been able to manipulate them 00:29:39.18 by taking a microneedle and reaching into the cell 00:29:42.28 and interacting with the chromosome. 00:29:44.28 And I am going to show you a movie that displays this now. 00:29:47.23 There is the needle. it is coming in and interacting with this chromosome. 00:29:51.01 And the chromosome then of course starts to try to reorient, 00:29:54.01 but Nicklas comes back in and knocks it back. 00:29:56.18 And it tries to get up there again, and now he is pulling hard on that chromosome, 00:30:00.21 and pulls off that attachment, so now this chromosome is sitting there 00:30:05.04 with no spindle attachment. 00:30:07.12 What's it going to do? Well, it sort of uncoils because it probably has a little elasticity to it, 00:30:13.21 and by chance it is the other kinetochore that interacts with the spindle and now is drawn back 00:30:19.04 up towards the pole, and so what you are seeing now 00:30:22.03 is the process of congression to the metaphase plate of a bi-oriented chromosome. 00:30:28.17 in which sisters have found sister poles. 00:30:31.19 And this is known to be a stable arrangement. 00:30:35.14 How is that known, again from the work of Nicklas, now not showing movies, 00:30:40.12 but instead stills from movies. 00:30:43.01 We start here with the manipulation experiment, 00:30:45.11 and what is going on is that Bruce is going to take a chromosome 00:30:49.24 and pull it out of the spindle, and you see it down here at the bottom. 00:30:54.24 This chromosome now would re-orient just like the one we were looking at before, 00:30:59.18 but he does something different now. 00:31:00.26 He takes his microneedle and inserts it right 00:31:03.27 into that chromosome and pulls in this direction. It's diagrammed over here. 00:31:08.15 So now the chromosome is trying to make attachments with one pole down here, 00:31:14.28 and this is obviously an inappropriate attachment 00:31:18.22 because this would bring both of those two halves of the chromosome 00:31:22.00 down here to one pole, a non-disjunction. 00:31:25.10 The needle is there behaving as if it were a sister kinetochore interacting with the other pole. 00:31:31.08 And what these data show you in time is that this chromosome is stable in its mal-orientation 00:31:37.27 for a factor of ten or twenty times as long 00:31:41.11 as it would normally take a chromosomes to re-orient. 00:31:43.21 when you remove this needle, within seconds it reorients 00:31:48.01 joins the metaphase plate, and the cell goes into anaphase, 00:31:51.03 which demonstrates that his manipulation was not damaging to the cell. 00:31:55.05 So when tension is being exerted on a microtubule-chromosome interaction 00:32:01.17 it gives you a stable attachment, so what this means is that 00:32:06.18 it is only when a chromosome is a mechanical entity 00:32:10.03 is attached to opposite poles, being pulled 00:32:12.24 in opposite directions that it is under tension 00:32:15.22 and is therefore going to be subjected to these forces 00:32:19.17 that will give you the tension that gives you stability. 00:32:21.29 So accurate chromosome segregation is a selective process. 00:32:26.08 It is choosing microtubules that give tension. 00:32:30.00 It isn't organizing things so perfectly that only the 00:32:34.00 right microtubule-kinetochore connections formed. 00:32:37.18 So what generates the tension at the kinetochores? 00:32:41.09 As I've described, dynein, it could be one of the possibilities. 00:32:45.05 However, we have done antibody injection experiments 00:32:49.00 with antibodies that block dynein's motility in vitro 00:32:51.28 and they did not affect the attachment of chromosomes to the spindle. 00:32:56.20 So I don't think it is that. 00:32:59.10 Another piece of evidence is that indeed most of the dynein 00:33:02.25 leaves the kinetochore shortly after the chromosomes attach to spindle microtubules. 00:33:08.09 This means that the dynein is not their in high concentration. 00:33:12.20 in order to develop a lot of tension during much of metaphase, 00:33:16.07 and you could imagine that it isn't able to do the tension when it is not there. 00:33:21.04 On the other hand, you don't know how much dynein 00:33:23.23 you need to get the tension you are after. 00:33:26.02 So are there direct tests? 00:33:29.00 You could use dynein mutations for example, 00:33:31.05 and this is not currently possible for practical reasons, really. 00:33:35.21 Dynein is a huge protein with a very big heavy chain. 00:33:38.12 present in two copies and a large number of lighter chains 00:33:42.03 and no one has yet been able to make a temperature 00:33:46.12 sensitive mutant which would allow you to do 00:33:48.10 a temperature shift and get a quick effect. 00:33:50.20 And drug experiments always have their problems, 00:33:53.09 and so if you want to learn more about dynein 00:33:56.16 and how it is functioning, do look at the seminar by Ron Vale 00:33:59.05 which will tell you a lot about it, 00:34:01.08 but we still don't have the tools we really need 00:34:04.17 to do experiments for evaluating dynein's role in the kinetochore. 00:34:08.20 And on top of that, this diagram, which I have taken from a very nice review article 00:34:13.06 from the lab of Tim Yens shows how incredibly 00:34:16.08 complicated the biochemistry of a kinetochore is. 00:34:20.08 And we probably still have proteins that we do not yet identify, 00:34:23.26 certainly those whose function we don't understand. 00:34:27.02 So our problem is that you can imagine having a probe that interacts with dynein 00:34:32.01 or some other component here, and then there would be 00:34:34.08 indirect effects which would lead to the falling off of other essential components, 00:34:39.22 and what you'd observe is not due to what you initially perturbed, 00:34:42.25 but an indirect perturbation caused by a chain reaction. 00:34:48.05 So understanding the function of these proteins 00:34:52.04 at the kinetochore is a very hard job. 00:34:55.15 It's an important one, and indeed there are a lot of people 00:34:59.03 now working, trying to characterize all of the protein molecules that associate 00:35:03.29 with the kinetochore and trying to understand the phenotypes 00:35:07.22 of the deletion or inactivation of any one of them. 00:35:10.28 But there are always this problem of indirect effects 00:35:14.06 and it applies not only to mutants but also 00:35:16.28 to antibodies, and drug perturbation. 00:35:19.08 Many kinetochore proteins are modified during the course 00:35:23.21 of their function, by phosphorylation for example, 00:35:26.04 and what this means is that you are going to be looking at a moving target 00:35:30.28 because the function and action of the protein of interest 00:35:33.26 may change with time. So understanding the roles of all these proteins 00:35:38.11 is going to take the combined efforts of many people 00:35:40.24 using the full armamentarium of modern biology. 00:35:44.18 It's a wonderful problem and one that really deserves attention from many people. 00:35:49.03 What I want to finish up with though, now, 00:35:52.03 is a talk about one more problem, 00:35:54.22 which is the question of how do proteins get to the metaphase plate. 00:36:00.10 We've been talking about attachment, and attachment to two sister kinetochores 00:36:04.27 and if both kinetochores are being pulled to the pole you could imagine 00:36:08.13 that that creates a mechanical equilibrium, 00:36:10.27 but what pushes the chromosome to the midplane of the spindle? 00:36:14.21 And here coming back to experimental work done by Conly Rieder, we have a beautiful 00:36:19.19 example of what is certainly a part of this process 00:36:22.09 in many cells. What Rieder has done is to use a micro beam and to sever 00:36:27.19 the chromosomes at two places so the region 00:36:31.12 that has the spindle attachment point is there, and these 00:36:34.14 arms are now fragments without chromosome attachment points, 00:36:38.18 so called acentric fragments. And over the course of time, 00:36:42.13 one can see their behavior. They are pushed away from the pole 00:36:45.20 and eliminated from this monopolar situation. 00:36:49.29 What this suggests is that each pole is pushing 00:36:52.29 on all of the objects of the spindle, even as the kinetochores are being pulled towards the pole. 00:36:59.14 And it suggests that what we may have here is a situation 00:37:02.27 where chromosome mechanics is a balance between pole directed forces acting on kinetochores 00:37:09.27 and pushing forces acting on the body of the chromosome as a whole. 00:37:16.01 This is probably part of the story, and in some cells 00:37:19.15 it may be much of the story of understanding pro-metaphase. 00:37:23.04 because you can see here what we have is a pair of fibers 00:37:27.11 that are attaching to the chromosomes 00:37:29.02 and they are pulling towards the poles, and then I'm diagramming 00:37:32.26 the pushing forces that are pushing away from the poles, 00:37:36.16 and you want to know of course, 00:37:38.00 where do these pushing forces come from? 00:37:40.02 The best evidence at the moment is that they come from another 00:37:44.03 motor protein, a kind of kinesin that is often called a chromo-kinesin 00:37:48.02 because it binds to chromosomes and it interacts with microtubules 00:37:52.00 and it walks in the plus end direction. So it 00:37:55.11 may be pushing the chromosomes away from the poles, contributing to this force 00:38:00.17 that aligns the chromosomes at the metaphase plate. 00:38:04.10 Mitosis still provides lots of problems for interested biologists. 00:38:10.10 We really want to know the biochemical basis of each of the spindle functions. 00:38:14.10 The chromosome attachment, the congression to the metaphase plate, 00:38:18.00 the regulation of anaphase onset, and the mechanism of chromosome to pole motion. 00:38:23.01 Each of these is a complex cellular event involving 00:38:26.27 many, many proteins working together and it will take a consortium of people interested 00:38:32.08 in individual molecules and people interested in processes to work it out. 00:38:37.05 And what I'll talk about in the next lecture 00:38:39.04 is how our lab is approaching this kind of complexity 00:38:42.19 in order to try to understand a specific subset 00:38:46.09 of the problems: the motion of chromosomes to the poles.