Cellular Organization of Complex Cell Structures
Transcript of Part 2: Building a Polymer: Microtubule Dynamics
00:00:01.11 Hello, my name is Tony Hyman. I'm the director of Max Planck Institute 00:00:05.14 at Dresden, in Germany. And, for the second part of my talk, 00:00:09.29 I'd like to tell you about polymers: microtubules. 00:00:13.21 which are a fascinating part of the mitotic spindle, 00:00:16.27 which I've illustrated over here in this little cartoon. 00:00:21.06 If you remember, in the last talk, when we were discussing about scale in biological analysis, 00:00:27.23 microtubules are an organization of protein molecules, called tubulin, shown here. 00:00:35.10 And, tubulin molecules come together to organize these microtubule polymers. 00:00:41.19 Now, you can look at microtubules growing in cells, 00:00:44.20 and in this movie, you can see the ends of microtubules growing throughout our C. elegans embryo. 00:00:49.25 The ends of the microtubules are marked with a protein called EB1, 00:00:54.02 which is known to follow and recognize only the beginnings of microtubules, 00:00:58.11 growing from centrosomes. 00:01:03.09 Now, microtubules have interesting organization, 00:01:07.17 At the top, I've shown you dimers. We know the structure of the dimer in detail, 00:01:12.08 from a number of different structural techniques, 00:01:14.14 such as crystallography and also from electron microscopy. 00:01:17.28 And dimers form head to tail arrangements of protofilaments, 00:01:23.01 which I've shown down here using a technique called atomic force microscopy. 00:01:27.05 But, then these protofilaments associate side-to-side, 00:01:32.11 and form a tube. And, in vivo, there are about 13 protofilaments per microtubule. 00:01:38.19 And, in the bottom, you're seeing a microtubule by a technique known as vitreous ice, 00:01:43.13 where you can see the individual protofilaments. 00:01:46.12 The interesting thing about microtubules is that they grow from their ends. 00:01:52.13 So, you have a polymer, which is a tube, 00:01:54.21 and individual subunits come on to the ends, and they leave the ends, 00:01:58.23 and therefore you have an on rate of tubulin subunits, 00:02:02.00 and an off rate. And the growth of microtubules is defined by these different rates. 00:02:07.21 The other interesting thing about microtubules is that they have polarity. 00:02:12.03 So, you have a tubulin dimer, and the dimer is a heterodimer, 00:02:16.09 with two different subunits: alpha and beta. 00:02:19.22 And, those alpha-beta subunits set up a polarity in the microtubule, 00:02:25.01 with the beta subunit at the plus end. 00:02:28.04 So, the beta subunit marks the plus end of the microtubule, 00:02:32.14 and in the cell, the plus ends tend to be out in the periphery of the cell, 00:02:38.11 and the minus ends are concentrated at the centrosome. 00:02:42.04 So, a microtubule will nucleate from the centrosome, grow out through the cell, 00:02:46.01 with its plus ends leading. 00:02:48.05 So, it has dynamics, but it also has polarity. 00:02:51.17 Now, we can look at microtubules growing in vitro. 00:02:56.00 You can isolate tubulin from cells. One of the key places we isolate it from is brain 00:03:00.19 because there's a massive amount of tubulin in brain because it makes up all our neurons. 00:03:06.05 And then we can study microtubules growing in a test tube, as I've shown in this movie. 00:03:11.26 The big structure here is a centrosome, which we've also isolated from the cell. 00:03:16.21 We've isolated tubulin, and you can see it growing out along the coverslip, 00:03:20.17 simply from the tubulin molecules themselves. 00:03:25.09 So, in theory, microtubules do not require any other proteins to grow. 00:03:30.19 These are simple polymer systems. 00:03:32.29 But, microtubules in vivo... in the cell... 00:03:38.21 have very different behavior than microtubules in a test tube. 00:03:43.11 And the key difference is that, for any particular tubulin concentration, 00:03:48.13 microtubules grow faster in vivo than they do in a test tube. 00:03:52.16 They grow much faster.. sometimes 10x faster than you would expect. 00:03:57.03 The other thing is they tend to turn over more quickly in cells than they do in a test tube. 00:04:02.02 So, what I've illustrated here is an interesting behavior here known as dynamic instability, 00:04:07.21 where you can see the microtubule grows, 00:04:09.14 and at some stages it transitions to a shrinking state, 00:04:13.23 and then it starts growing again. 00:04:16.10 And what you can see is, both in vivo and in vitro, 00:04:19.14 microtubules are turning over by dynamic instability, 00:04:22.14 but they're much more dynamic in cells than they are in a test tube. 00:04:27.12 And that's something that's interested scientists for the last 25 years, 00:04:31.16 ever since the discovery of the different properties of microtubules 00:04:36.10 in vitro and those in vivo. 00:04:38.13 And we want to understand how microtubules are regulated in a cell, in an in vivo context 00:04:44.28 because that regulation is key to their activity in the cell. 00:04:48.03 Building a mitotic spindle, for instance, 00:04:50.11 requires that the activity of microtubules is regulated. 00:04:54.15 One of the questions you can ask, and we always have as biologists, 00:05:01.05 if you're interested in a problem like that... You look to your microtubules growing in a cell 00:05:05.00 and then you say to yourself, "I'm interested in that problem." 00:05:08.01 "How am I going to get at it?" 00:05:09.13 And the first thing you tend to ask yourself in biology is 00:05:12.00 How complicated is it? 00:05:13.23 Is this a solvable problem? Can I get at it? 00:05:15.24 And here's a review from Rebecca Heald, illustrating the numbers of different proteins 00:05:23.00 that are known to be involved in regulation of microtubules. 00:05:25.20 And you look at that, and it looks fairly terrifying. 00:05:28.17 There's so many different molecules involved in the different processes. 00:05:31.06 So, we decided to go and ask how complicated is the growth of microtubules 00:05:38.27 in the C. elegans embryo? 00:05:39.29 We just decided to focus on one particular problem, which is... 00:05:42.29 How many proteins are required to make the plus end of a microtubule 00:05:47.22 grow far through the cytoplasm? 00:05:49.15 If you remember, I said that it grows about 10 times faster in vivo than it does in vitro, 00:05:54.17 so you can ask how many proteins are required to do that. 00:05:58.07 Now, we did that by taking advantage of our genome-wide RNAi screen. 00:06:03.04 I mentioned this screen in the introduction, and this screen is an RNA interference screen, 00:06:10.29 where we can look for genes required for microtubule growth. 00:06:14.16 And to do that, we took our last set of 800 genes, 00:06:18.09 and we decided to screen subsets of those 00:06:22.03 for those that affected microtubule growth. 00:06:25.18 So, you remember our first screen was using Nomarski microscopy, 00:06:28.11 and we couldn't see microtubules. 00:06:29.28 It would have been too complicated for us, at the time, 00:06:32.09 to screen everything by fluorescent microscopy. 00:06:34.28 But, with our subset of genes, we can ask, 00:06:37.22 which ones of those are having their effects on the embryo 00:06:40.12 because they prevent the microtubules from growing properly? 00:06:44.00 And here's a movie where you can see the plus ends 00:06:47.20 of microtubules growing by EB1, as I mentioned. 00:06:50.22 And we can also track these microtubule ends automatically, 00:06:53.27 which helps a lot in terms of looking at the phenotype. 00:06:56.20 So, in essence, this is the outline of our screen now, 00:07:01.16 is we've taken the DIC screen -- the Nomarski screen -- 00:07:05.05 and we've taken a number of genes, and we've got a set of genes here 00:07:11.20 required for cell division. So, we believe ... our hypothesis is 00:07:16.10 that any gene which affects microtubule growth is likely 00:07:20.05 to make the embryo not divide properly. 00:07:23.22 So, we take those genes and did some bioinformatics to subselect the genes 00:07:31.01 to reduce the amount of work we have to do, 00:07:32.07 and then we do our fluorescent secondary screen 00:07:34.27 using various different fluorescent markers, 00:07:37.29 and we look for the number of genes required for microtubule growth. 00:07:41.29 And when we did that, the results were really quite interesting, 00:07:47.00 because they actually showed that not many genes are required for a microtubule to grow. 00:07:53.06 If you have a look at this rather complicated bar chart here, 00:07:57.03 the white lines are showing the growth rate of microtubules. 00:08:01.00 So, on this particular axis, we have the growth rate of microtubules, 00:08:04.24 and you can see this layer here is about the growth rate of wild-type microtubules. 00:08:09.27 So, then you can say, let's go through the genes 00:08:13.05 and ask which genotypes no longer grow at wild-type rates? 00:08:17.16 And I've put those in the circle. You can see that there's a set of genes here -- two -- 00:08:23.08 which are clearly required for microtubules to grow. 00:08:27.20 There's some other genes, which also affect microtubule growth, but we know 00:08:31.13 that those are required to actually make the tubulin dimer itself. 00:08:36.00 So, obviously, if you don't have enough tubulin, you're not going to grow. 00:08:39.12 We're not interested in those for this particular talk. 00:08:43.02 We actually want to know, when the tubulin is made, 00:08:45.19 what proteins are required to make the microtubules grow? 00:08:47.25 And here, all that work, we came up with two proteins that seem to be required for that -- 00:08:52.13 TACC and Zyg9, which we happen to know are actually in a complex. 00:08:58.19 So, there's a complex of proteins which are required for the growth of microtubules. 00:09:03.10 Now, it turns out that this protein, which is in the middle here, Zyg9, 00:09:07.29 is part of a family of proteins. XMAP is one of the founder members in higher eukaryotes. 00:09:15.00 There's Stu2 in cerevisiae, and there's Dis1 in pombe. 00:09:20.04 And every organism studied so far ... every animal cell studied so far 00:09:24.02 has a member of this family. 00:09:27.10 And they have these very interesting domains in them, called TOG domains. 00:09:30.14 As you can see here, XMAP has 5 TOG domains, C. elegans has 3 TOG domains, 00:09:36.05 these yeasts have 2 TOG domains, but are thought to be in a dimer. 00:09:40.02 So, so far, what we've done is we've discovered then that actually, in an embryonic system, 00:09:47.10 controlling the growth rate of microtubules is quite simple. 00:09:52.05 You need these two proteins. 00:09:54.04 And that is the first part of any particular project in trying to work on any biological process. 00:10:01.04 We've done what's known as a genetic screen 00:10:03.18 using RNA interference to try and study the genes required for this process. 00:10:08.20 What is the catalog? 00:10:09.12 But then always comes the problem that any biologist then faces 00:10:12.27 is, what is the mechanism by which these proteins make the microtubules grow? 00:10:18.09 And so, how can one work on the mechanism of the activity of these different proteins? 00:10:25.16 It turns out, one of the key steps forward for us was to actually go 00:10:32.11 work on the protein in a different organism, 00:10:35.02 which was in Xenopus. 00:10:37.28 Now, biologists like to move around between different systems 00:10:41.09 to find the system which is most appropriate for the problem they're actually interested in. 00:10:45.08 And so, in this particular case, we use Xenopus because you can make extracts of cytoplasm 00:10:53.00 where you can take away the membranes. 00:10:56.05 Every time you work on a cell, you have the same problem, which is 00:11:00.04 how do I get components across the membrane? 00:11:02.17 The membrane of a cell has evolved over many millions of years 00:11:05.24 to exclude most things it doesn't like. 00:11:08.01 So, you're always fighting as a biologist to get things across the membrane. 00:11:10.29 Therefore, it's very helpful to be able to make cytoplasm extract 00:11:16.11 without membranes, and in Xenopus, you can actually make 00:11:21.13 very concentrated cytoplasm extracts in which most of the things actually... 00:11:26.08 many of the cell biology and cell division events we're interested in 00:11:30.28 actually still function. 00:11:33.12 So, that's shown here. We've got a couple of frogs. You take the eggs. 00:11:36.20 You crush the eggs in a centrifuge, and then you have a concentrated cytoplasm. 00:11:40.24 You can add centrosomes to that cytoplasm and watch microtubule growth. 00:11:45.08 And when we did that, we found 00:11:48.20 microtubules growing in the cytoplasm. 00:11:50.16 But, the interesting thing is we were then able to remove XMAP from the 00:11:55.01 extracts, so we can study the activity of XMAP in these extracts. 00:12:00.24 Over here, we have microtubules growing from a centrosome in the untreated extract, 00:12:08.07 and you can see lots of microtubules growing all over the cytoplasm. 00:12:12.25 But then what we can do with Xenopus, is we can make an antibody to the protein, 00:12:15.15 and we can deplete it from the extract, 00:12:17.19 and then you can see, you hardly have any microtubule growth at all. 00:12:21.01 So, both in Xenopus, and in C. elegans, 00:12:24.27 XMAP is a key protein required for microtubule growth. 00:12:27.14 So, then we'd like to understand how does XMAP make the microtubules grow? 00:12:32.24 And, to do that, the first thing you have to do, 00:12:35.27 is you have to make the protein in a test tube. 00:12:38.20 And then you can study it on its own. 00:12:41.04 And that's exactly what we did. We made XMAP in a test tube, 00:12:44.27 and we also were able to tag it with a GFP, 00:12:47.27 a green fluorescent protein, in the test tube, 00:12:50.19 so we could also look at the activity of the protein 00:12:53.15 as well as its localization. 00:12:55.24 Now the work I'm going to talk to you about has been done together with Joe Howard, 00:12:59.29 who's a close collaborator of mine, and most of the work from the last 00:13:02.25 10 years on microtubules have been done together with Joe, 00:13:06.26 who's a keen cricket fan. 00:13:08.20 And, we'd like to look at the role of XMAP in controlling the growth rate of microtubules. 00:13:15.22 Now, in order to do that, we have to look at microtubule growth 00:13:19.22 in a test tube, and we want to look particularly at the growth of the plus ends. 00:13:23.20 And we can monitor that in the test tube using fluorescence microscopy. 00:13:29.05 You can see the red segment marks the minus end, 00:13:32.00 and the green segment marks the plus end. 00:13:34.10 And you can see the green segment growing from the red minus segment. 00:13:40.23 Now, what you'll notice is the red segment is stable. 00:13:43.25 It's not growing and shrinking. And you can ask yourself, how is that? 00:13:48.01 That's key to our assay. By stabilizing the minus end, we can isolate the plus end growth 00:13:52.25 and look at how that's regulated. 00:13:55.18 Now I just want to go into, a little bit for you, about how we go about 00:13:58.02 stabilizing the minus end, because it's interesting both to think about the assay, 00:14:01.05 but also it gives us a little bit more understanding of microtubule and tubulin biology itself. 00:14:08.09 So, what we're doing in this instance is we're making polarity-marked microtubules. 00:14:15.08 So what we do is we take brightly labeled tubulin, here. 00:14:18.27 And, we've labeled tubulin in a test tube with a rhodamine dye... 00:14:22.22 chemically attached rhodamine to tubulin. 00:14:26.00 Then, we warm it up, and we make microtubules. 00:14:29.13 The next thing we do is we take dimly-labeled tubulin, 00:14:34.01 and we grow that from the seeds, and when we do that, 00:14:37.21 we end up with the dimly labeled tubulin growing from the seeds, 00:14:41.10 We warm it for another 15 minutes, and then we have these polarity marked microtubules, 00:14:46.12 with a bright minus end down here and a dim end that's grown off the end of it. 00:14:52.05 Now, you notice what I said here is that the seeds are stable. 00:14:55.16 So, how do we make them stable? 00:14:57.12 Well, there are a number of ways, but the most important and interesting way, 00:15:02.17 is to modulate the GTP-hydrolysis cycle of the tubulin itself. 00:15:07.18 So, it turns out that a tubulin dimer has two molecules of GTP: 00:15:12.05 alpha has a GTP molecule, and beta has a GTP molecule. 00:15:15.11 But, when tubulin polymerizes into a microtubule, 00:15:19.26 only the beta hydrolyzes GTP to GDP. 00:15:24.16 Now, there are analogs of GTP which can affect this cycle. 00:15:32.01 So, the cycle shown here, where the tubulin dimer comes on 00:15:36.02 to the end of the microtubule and docks. 00:15:37.17 When it docks, that completes the hydrolysis pocket in the beta subunit, 00:15:42.13 so the GTP now hydrolyzes. 00:15:43.25 So, we think that, mainly it's just the end of the microtubule that has a un-hydrolyzed GTP. 00:15:50.09 So, what happens if we block the hydrolysis of GTP? 00:15:54.13 Well, we can do that using analogs of GTP, as I mentioned. 00:15:59.20 There are a number of different ways of making analogs of GTP. 00:16:02.13 If you remember your high-school chemistry, you have guanosine, 00:16:07.16 and you have free phosphates 00:16:08.25 at the end of any nucleotide. And each one has an alpha-oxygen bond between 00:16:16.18 the different phosphate groups. 00:16:20.00 Now, what it turns out we were able to do, is you can modify GTP, 00:16:26.09 so that the alpha-beta oxygen is a carbon. 00:16:30.17 And you can see the name of that molecule above: GMPCPP, 00:16:34.01 or guanalyl alpha-beta-methylene diphosphonate. 00:16:37.15 And it turned out that this molecule was very, very good 00:16:43.14 at mimicking the GTP state of tubulin, 00:16:48.13 and when the tubulin goes into microtubules, what we discovered 00:16:51.16 is that GMPCPP is no longer hydrolyzed, and so it allows you to ask 00:16:59.14 what is the effect of preventing GTP hydrolysis 00:17:03.02 on the dynamics of microtubules? 00:17:06.12 And when we did that, you get this very interesting result, 00:17:09.20 which is that, if you look at a GTP microtubule, it grows and shrinks, 00:17:14.09 and then grows again, as you can see on this graph of microtubule length against time. 00:17:18.04 But, GMPCPP microtubules grew at the same rate as GTP-tubulin, 00:17:23.04 but they never transitioned to shrinking. 00:17:26.20 And, that confirmed old observations with other nucleotides 00:17:32.16 that the role of GTP hydrolysis in microtubules 00:17:36.07 is to destabilize them. 00:17:37.27 You don't need GTP hydrolysis of microtubules to grow, 00:17:40.11 but you do need GTP hydrolysis for microtubules to shrink. 00:17:44.23 So now, what we do, of course, is make our seeds using GMPCPP, which is stable. 00:17:53.01 And that way, we have the following assay, with a GMPCPP seed, 00:17:56.16 and the tubulin growing from the end of that stable seed. 00:18:01.22 So, now we have our assay. How are we going to analyze the role of XMAP? 00:18:06.24 Well, you have to use a special kind of microscopy to do this, 00:18:12.15 which is total internal reflection (TIRF) microscopy. 00:18:15.25 And Joe Howard's lab developed ways to do this to look at 00:18:20.06 the dynamics of microtubules using Total Internal Reflection Microscopy, 00:18:24.15 which is a way to just look at molecules which are very close to the surface of the coverslip. 00:18:30.16 Now, if you take your growing microtubule, 00:18:33.26 and then you take labeled XMAP and add it to the test tube, 00:18:39.05 what you see is XMAP has very interesting behavior. It's processive. 00:18:44.16 Or, it surfs at the end of the microtubules. 00:18:47.08 So, if you have a look at this figure here, 00:18:51.08 you can see that the XMAP at the end of the microtubule stays with the end 00:18:55.18 as it grows. It likes to be at plus ends, 00:18:58.14 and it likes to stay with them as they're growing. 00:19:01.26 So, you can then begin to ask, what are the dynamic properties 00:19:09.19 of XMAP at the ends of microtubules 00:19:13.24 by looking at single molecules of GFP-XMAP. 00:19:16.19 You can do single molecule techniques using TIRF. 00:19:19.20 And you can begin to ask questions like, 00:19:21.03 we know the XMAP is responsible for microtubules growing fast, 00:19:26.27 and so, how do the individual XMAP molecules behave 00:19:31.11 when the microtubules are growing? 00:19:32.22 If we do an experiment like that, 00:19:36.03 you can actually see the ends of the microtubules as they're growing, 00:19:40.28 with the GFP-XMAP, so when we use this assay, 00:19:44.13 we can look at GFP molecules growing at the ends of the microtubules. 00:19:49.19 And then we can ask, how long do individual molecules stay 00:19:54.12 at the ends of microtubules before dissociating? 00:19:56.15 And what we discovered was that, on average, an XMAP molecule stays about 4 seconds 00:20:03.02 at the end of a microtubule, which is about 25 tubulin dimers. 00:20:07.03 So, somehow, an XMAP is staying at the end of a microtubule, 00:20:10.25 and it's helping tubulin to get on to the end of the microtubule. 00:20:14.24 And how can that work? How can the XMAP molecule stay at the end of the microtubule 00:20:20.21 and help the tubulin add on at a faster rate, 00:20:24.14 which is required for the microtubules to grow faster? 00:20:26.27 One of the clues for this was that TOG domains bind tubulin. 00:20:30.29 Now, you remember that I told you at the beginning of the talk that XMAP 00:20:34.21 is a molecule with many different of these TOG repeats. 00:20:37.16 And so, Steve Harrison's lab solved the structure of a TOG domain 00:20:42.09 and was able to show that the TOG domains bind tubulin. 00:20:45.27 And, in fact, we were able to show that an XMAP binds one tubulin dimer, on average. 00:20:53.22 So, then you can ask, how is it that XMAP, by sitting at the ends of the microtubules, 00:21:00.01 helps these tubulin molecules get on to the ends of the microtubule? 00:21:06.01 One of the things we considered is that XMAP acts like an enzyme, 00:21:11.09 to catalyze the addition of tubulin molecules to the end of the microtubule. 00:21:17.18 And there are two things that should happen 00:21:20.03 if an enzyme is working in this particular case... 00:21:24.08 if XMAP is working as an enzyme. 00:21:25.17 The first thing is that it should also be able to make microtubules depolymerize 00:21:32.29 if there's no tubulin there. 00:21:34.25 And that is a classic feature of all enzymes you work on. 00:21:38.23 They go in one direction if they have substate there, 00:21:42.09 but if you take away the substrate, they'll go in the other direction. 00:21:45.05 So, synthetic enzymes often turn into degradative enzymes 00:21:48.12 if you take away the substrate. 00:21:51.08 And so, that should be the same for microtubules. 00:21:53.14 If XMAP is acting as a catalyst, if we take away tubulin, 00:21:59.03 one might expect it to start depolymerizing microtubules. 00:22:02.17 And that's exactly what we found. 00:22:06.21 If you add XMAP to microtubules in the absence of tubulin, 00:22:09.11 then microtubules start to shrink, 00:22:13.11 and this had first been noticed by the Mitchison lab in 2003. 00:22:17.25 The second thing is, the critical concentration of growth should not change. 00:22:25.15 And I bring this up, just to explain what we mean by the critical concentration 00:22:28.25 of the growth for microtubules ends, because you sometimes hear this term, 00:22:33.16 and it's sometimes quite confusing to understand what it means. 00:22:36.26 I remember when I first heard about it, 00:22:38.04 I had a lot of trouble trying to understand what this actually meant. 00:22:41.00 And the way to think about it, is to come back and look at our microtubule, 00:22:46.04 and think that tubulin has an off rate and an on rate. 00:22:50.19 The off rate is the rate at which tubulin molecules come off, 00:22:56.12 and the on rate is the rate at which tubulin molecules go on. 00:22:58.27 Now, if you reduce the tubulin concentration, you reduce the on rate, 00:23:04.17 until eventually the on rate and the off rate are matched, 00:23:08.00 and that's essentially the critical concentration for growth. 00:23:09.27 Just above that concentration, the microtubules will now begin to grow. 00:23:15.07 And so, we can come back and ask, what is the effect of XMAP on the critical concentration, 00:23:22.05 because for a catalyst, if you raise the off rate, you'll also raise the on rate, 00:23:26.13 and therefore the critical concentration will not change, 00:23:29.12 and that's exactly what we found here. 00:23:30.24 You can see the critical concentration of growth, 00:23:33.05 you can see the point where it goes above 0, 00:23:35.13 is exactly the same point. 00:23:37.08 So, therefore, what we conclude from these experiments, 00:23:41.17 is that XMAP acts as a polymerase, as an enzyme. 00:23:44.25 And I think the key experiment we did to show this is to show, if you take away tubulin, 00:23:49.17 microtubules shrink. If you add back a little bit of tubulin, 00:23:52.22 microtubules just begin to grow, and if we add more, they start to grow even faster. 00:23:57.09 So, the cycle of microtubule growth appears to be modulated 00:24:00.02 by the amount of this XMAP protein in the cycle. 00:24:04.12 So, we think then that XMAP acts as a polymerase. 00:24:10.18 And, I took you through this story to illustrate a number of different things. 00:24:15.29 At the beginning I showed you how we can use genetic screens 00:24:19.10 to get at the complexity of any particular system. 00:24:23.07 But then, I dived down into a little bit more detail to say that, once you get that molecule, 00:24:29.07 that's not enough. You then need to actually go and work on the mechanism 00:24:33.07 by which it's having its effects. 00:24:34.29 And that's what the goal we all have is, in the end, 00:24:37.16 to try and work on a mechanism 00:24:39.11 by which these individual proteins and their protein complexes 00:24:42.28 affect their particular activity. 00:24:48.01 And, if you remember at the beginning, 00:24:49.11 I said that microtubules are these very interesting complexes 00:24:51.21 of proteins, which grow and shrink in the cell. 00:24:55.18 And you can see how the interaction between these protein complexes 00:24:59.13 and other protein complexes 00:25:01.02 modulates other activity in order for the correct biology to happen. 00:25:06.17 I'd like to thank... there's two people mentioned who have been key to this work: 00:25:12.07 There's Gary Brouhard and Jeff Stear, 00:25:14.03 who were key to this particular experiment, and I think it's a classic example of teamwork, 00:25:20.05 where the two of them worked together, and I think it's very important to remember 00:25:23.11 that these complex sorts of experiments 00:25:25.07 we've been discussing about XMAP and microtubules 00:25:28.13 depend very much on this sort of teamwork, 00:25:31.14 of people working together for a common goal.