Session 6: Cytoskeletal Motor Proteins
Transcript of Part 2: The Mechanisms of Dynein Motility
00:00:15.09 Hello. 00:00:16.17 I'm Ron Vale from UCSF. 00:00:18.24 In part one of my iBiology talk, 00:00:20.20 I introduced biological motility 00:00:23.14 and I focused on the mechanisms 00:00:25.05 of kinesin and myosin, 00:00:26.13 and in this talk 00:00:27.25 I'd like to discuss 00:00:29.08 our more recent research 00:00:30.26 on the mechanism of dynein motility. 00:00:33.29 Now, this year, 2015, 00:00:37.18 is the first anniversary 00:00:39.12 of the discovery of dynein, 00:00:40.29 which was made by Ian Gibbons, 00:00:43.12 and Ian described 00:00:45.11 a new type of ATPase 00:00:47.13 from cilia 00:00:49.07 that was involved in powering its motility. 00:00:53.13 Now, even though dynein was discovered 00:00:55.21 twenty years before kinesin, 00:00:57.25 we know a lot more about kinesin motility 00:01:00.22 than we do about how dynein works. 00:01:03.16 And the reason for that is that 00:01:06.13 dynein is an incredibly complicated machine. 00:01:08.23 First of all, 00:01:10.15 it's a massive protein complex, 00:01:11.29 one of the largest in the cell. 00:01:14.11 It's about one and a half megaDaltons 00:01:16.22 in size. 00:01:17.28 Even the gene that encodes 00:01:20.18 the motor polypeptide 00:01:22.20 is very large. 00:01:24.25 The motor polypeptide 00:01:26.01 is about 500 kiloDaltons in size, 00:01:29.02 so it's one of the largest polypeptides 00:01:30.18 in the genome. 00:01:32.03 Even the motor domain itself 00:01:34.12 is massive, shown here. 00:01:36.16 It's about eight times larger 00:01:38.13 than the kinesin motor domain. 00:01:40.20 So simply because this motor 00:01:42.08 has been so big and complicated, 00:01:44.14 it has made it difficult to study. 00:01:46.15 It's been difficult to express it 00:01:48.29 and obtain pure protein, 00:01:51.07 difficult to manipulate it 00:01:53.10 by recombinant protein techniques, 00:01:56.01 and also non-trivial to get a structure 00:01:59.05 by X-ray crystallography. 00:02:01.01 So all of these have posed challenges. 00:02:04.18 And in 2007 00:02:06.17 I actually gave an iBiology talk, 00:02:08.25 which you can find in the archives, 00:02:11.02 and at that moment in time 00:02:12.26 we were primarily studying 00:02:15.02 the single molecule motility of yeast dynein, 00:02:18.25 but we knew very little 00:02:20.25 about the structure of dynein 00:02:22.18 in any great detail. 00:02:24.28 Well, a lot has changed 00:02:26.19 in those intervening years 00:02:29.06 and I'd like to share to share with you today 00:02:31.21 recent progress that's been made 00:02:33.28 on the dynein structure, 00:02:36.19 and now we do have atomic structures for dynein 00:02:38.25 and we're beginning to get 00:02:40.15 some insight into how this motor works. 00:02:44.09 So first of all, 00:02:45.14 let me just tell you about the different kinds of dyneins. 00:02:48.17 A major class of dynein 00:02:50.12 are the axonemal dyneins. 00:02:52.07 These are dyneins that power 00:02:54.04 the movement of cilia, 00:02:56.04 they also power the movement 00:02:58.21 of flagella from a sperm, for example, 00:03:01.16 and this just shows what the architecture 00:03:03.17 of the axoneme looks like. 00:03:05.27 It's composed of 00:03:09.14 nine unusual types of microtubules 00:03:11.13 that are called outer doublet microtubules, 00:03:15.04 and they have this particular structure here. 00:03:18.16 And on these outer doublet microtubules 00:03:21.20 sits the dynein molecule. 00:03:23.03 In fact there are two different kinds of dyneins 00:03:25.03 -- there's an outer arm dynein 00:03:26.08 and an inner arm dynein -- 00:03:28.07 and they sit statically 00:03:31.02 on one of the outer doublets, 00:03:33.16 the A tubule, 00:03:35.23 and then they reach across 00:03:37.03 to the neighboring B tubule 00:03:39.15 and they cause a relative sliding 00:03:41.21 between these adjacent microtubules. 00:03:44.20 Now, this sliding motion 00:03:46.11 of the two microtubules 00:03:49.01 in the cilia 00:03:50.27 gets converted into this sinusoidal 00:03:52.27 beating pattern 00:03:54.14 by a process that's still 00:03:56.10 very poorly understood. 00:03:59.00 Now, in addition to these axonemal dyneins, 00:04:01.12 there are cytoplasmic dyneins 00:04:02.27 that are more cargo-transporting motors. 00:04:06.15 So, one of these 00:04:07.28 is cytoplasmic dynein 2 00:04:09.11 and it's responsible 00:04:10.21 for a particular type of cargo transport 00:04:12.24 that actually occurs inside of cilia and flagella, 00:04:16.15 and it's responsible for transporting, 00:04:19.01 first of all, building blocks 00:04:21.00 up and down 00:04:23.10 for the maintenance of cilia and flagella, 00:04:24.23 and also some kinds of signaling molecules 00:04:26.20 are also present in cilia, 00:04:29.05 and they are trafficked 00:04:31.27 by motor proteins as well. 00:04:34.09 This shows an image of what 00:04:36.13 cargo transport looks like, 00:04:38.29 what intraflagellar transport looks like, 00:04:40.13 by fluorescently tagging 00:04:42.06 one of these cargo subunits. 00:04:43.29 Now, a kinesin motor 00:04:46.15 transports the cargo up to the tip, 00:04:48.17 to the plus end, 00:04:50.00 but dyneins are minus end-directed motors, 00:04:51.19 and so the dynein 00:04:55.08 transports cargo in the opposite direction, 00:04:56.24 from the tip towards the cell body. 00:04:59.02 Now, there's also a cytoplasmic dynein 1, 00:05:01.24 and that's responsible 00:05:03.09 for virtually all of the cargo transport 00:05:05.00 that occurs in the cytoplasm of cells 00:05:08.06 towards the minus end. 00:05:10.01 So kinesins, 00:05:11.23 as you remember from the first lecture, 00:05:13.03 move things out to the plus end, 00:05:14.22 and this one type of cytoplasmic dynein 00:05:16.08 carries out 00:05:17.28 virtually all of the transport in the opposite direction, 00:05:20.18 towards the minus end. 00:05:21.27 So, things that are transported 00:05:23.12 include membranes, 00:05:24.29 mRNAs, 00:05:26.14 protein complexes are transported, 00:05:28.02 as are viruses. 00:05:29.16 Here's just one example, here, 00:05:32.08 which is a melanocyte cell. 00:05:33.27 This is a skin cell 00:05:35.21 that carries pigments 00:05:37.25 which are melanosomes, 00:05:39.27 and in some organisms 00:05:41.22 like amphibians and fish 00:05:43.23 they can change the distribution 00:05:45.07 of their melanosomes, 00:05:47.14 so that when they're distributed outward 00:05:50.11 the skin color is dark, 00:05:52.18 when they're moved inward 00:05:54.07 the skin color turns lighter, 00:05:55.14 and this transport 00:05:57.11 towards the center 00:05:58.20 that you see here 00:05:59.27 is driven by cytoplasmic dynein. 00:06:02.12 Now, in the first iBiology talk, 00:06:04.29 I told you that myosin and kinesin, in fact, 00:06:08.17 are relatively similar to one another. 00:06:11.12 They're similar in structure 00:06:12.27 and they clearly evolved, 00:06:14.11 some time in evolution, 00:06:16.11 from a common ancestor. 00:06:18.28 But even though kinesin and dynein 00:06:22.02 both operate on microtubules, 00:06:24.05 they're not at all related to one another. 00:06:26.03 In fact, dyneins 00:06:27.27 emerged from a completely 00:06:29.29 different evolutionary lineage of ATPases 00:06:32.18 and they belong to a group of ATPases 00:06:34.26 that are called the AAA ATPases. 00:06:38.15 And in fact dynein 00:06:40.11 is a rather unusual member 00:06:41.27 of this AAA ATPase family. 00:06:44.01 Most of them are not traditional motors 00:06:46.00 that you think of in terms 00:06:47.16 of moving along a track. 00:06:49.05 Instead, they use ATP energy 00:06:51.24 to produce mechanical work 00:06:54.03 on molecules like proteins 00:06:56.00 to basically break them apart 00:06:57.27 and unfold them. 00:06:59.13 So, an example 00:07:01.16 that occurs in bacterial and eukaryotic proteolysis 00:07:06.05 is that there's AAA ATPases 00:07:09.07 that sit on top 00:07:11.07 of the proteolytic chamber 00:07:12.15 and their job is to take an incoming polypeptide 00:07:15.19 and basically unravel it 00:07:17.10 and stuff it through this hole 00:07:19.24 into the proteolytic chamber 00:07:21.28 so that the polypeptide can be degraded. 00:07:25.20 So, let me tell you 00:07:27.04 a few things are kind of 00:07:29.00 more universal 00:07:30.13 about these AAA ATPases. 00:07:32.12 First of all, most AAA ATPases 00:07:36.16 encode a relatively small protein 00:07:39.06 that has two domains, 00:07:40.13 a large domain and a small domain. 00:07:43.11 This is the basic ATP binding unit, 00:07:46.12 but this single subunit 00:07:47.25 is not the functional element 00:07:49.17 of how these proteins work. 00:07:51.22 They self-assemble, 00:07:53.23 oligomerize into a hexamer, 00:07:55.10 and it's this hexamer that's the active agent. 00:07:59.06 And in fact 00:08:01.10 adjacent subunits help one another 00:08:03.01 to hydrolyze the ATP, 00:08:05.02 and in the last example thing ring-like structure 00:08:08.09 is what actually unfolds the polypeptide 00:08:11.14 and stuffs the polypeptide 00:08:13.19 into this chamber that you see here. 00:08:15.27 Now, dynein again 00:08:17.11 is unusual in the fact 00:08:19.06 that it makes 00:08:21.15 a ring of AAA ATPases, 00:08:24.05 but it does so by placing 00:08:26.11 all the AAA domains 00:08:28.01 in one very, very long 00:08:29.29 polypeptide chain. 00:08:31.22 And this just shows the motor domain structure 00:08:35.06 of dynein 00:08:36.26 and shows the positions 00:08:38.18 of the six AAA domains. 00:08:41.05 And because they're in one polypeptide, 00:08:44.15 they've each evolved different amino acid sequences 00:08:46.26 over time 00:08:48.10 and have evolved different functions. 00:08:50.00 So AAA1, for example, 00:08:53.05 is the main ATPase site of dynein, 00:08:55.22 so this is what is really responsible 00:08:57.20 for driving motility, as I'll show you later. 00:09:00.08 AAA2 binds ATP, 00:09:02.09 but it doesn't seem to be... 00:09:05.14 bind it in a cyclic or hydrolytic manner. 00:09:08.18 AAA3 also plays an important role 00:09:10.22 that I'll come back to later 00:09:12.08 and it may be a mechanism 00:09:13.26 of regulating dynein. 00:09:15.14 AAA4 also hydrolyzes [ATP], 00:09:18.03 but it seems to play a very minor role 00:09:20.23 in dynein motility 00:09:22.06 and one that we don't completely understand. 00:09:25.07 So, I'd like to address this subject now 00:09:27.05 of how dynein can move along a microtubule, 00:09:30.17 and in addressing this problem 00:09:32.22 one has to tackle it 00:09:34.24 using different kinds of techniques 00:09:36.19 that are complementary. 00:09:38.04 So, one approach is to measure 00:09:40.09 the motility of dynein, 00:09:41.20 particularly at the single molecule level, 00:09:43.24 and this gives you all kinds of information 00:09:45.25 about the dynamics of dynein 00:09:47.10 and how it's stepping along the microtubule track. 00:09:50.13 But it's relatively low resolution information, 00:09:53.26 in other words it can't really see 00:09:55.28 the protein structure 00:09:57.12 and what it's doing. 00:09:58.22 On the other hand, 00:10:00.12 we can do X-ray crystallography 00:10:01.28 or do electron microscopy 00:10:04.27 and these give higher resolution information, 00:10:07.14 even down to atomic detail, 00:10:09.08 but they're static images, 00:10:11.00 so, you know, 00:10:12.18 we see the protein frozen in time 00:10:14.11 and it doesn't provide the information 00:10:16.02 on the dynamics. 00:10:17.13 So, somehow to piece together 00:10:19.21 the answer to this problem, 00:10:20.28 one has to use the information from both of these techniques 00:10:23.03 and try to work out a model 00:10:25.03 of how dynein might work. 00:10:28.05 So, let me tell you first about 00:10:30.04 in vitro motility assays. 00:10:32.02 This shows an in vitro motility assay 00:10:33.29 for yeast cytoplasmic dynein, 00:10:35.26 where we've labeled the dynein 00:10:37.14 with a fluorophore, 00:10:38.29 and you can see 00:10:40.13 these individual dynein molecules 00:10:41.29 moving beautifully 00:10:43.19 along these microtubule tracks. 00:10:45.02 It's processive movement, 00:10:46.29 meaning the dynein can take many steps 00:10:48.23 along the microtubule track 00:10:50.00 without letting go. 00:10:53.03 Now, we can also 00:10:56.05 measure this motility 00:10:57.14 with greater precision 00:10:59.09 if we use a computational approach. 00:11:02.01 Basically, each of these individual spots, 00:11:05.15 fluorescent spots of dynein that you see... 00:11:08.24 as they pass through the microscope, 00:11:10.25 the light spreads out 00:11:13.28 to what's know as a point spread function, 00:11:16.12 so they appear... 00:11:18.12 these single fluorophores 00:11:20.22 appear to have a diameter 00:11:22.05 of about 250 nanometers. 00:11:24.08 But if you collect enough photons, 00:11:26.21 you can describe that fluorescence, 00:11:28.29 that spread out fluorescence intensity profile, 00:11:33.22 and you can fit that intensity profile 00:11:35.22 with a Gaussian curve, 00:11:37.20 and the center of that Gaussian curve 00:11:39.14 defines kind of the midpoint 00:11:41.08 of where that fluorescent spot is. 00:11:44.15 Now, you can take successive 00:11:46.29 snapshots of dynein moving along the microtubule 00:11:49.18 and at each snapshot 00:11:51.10 you can mark the position of that centroid, 00:11:54.19 and that's what all these individual dots 00:11:58.01 are here, data points are, 00:11:59.16 and this is for a kinesin molecule, 00:12:01.16 but you can see for example, here, 00:12:03.19 the motor protein 00:12:05.05 was pretty much stationary on the track 00:12:07.07 and then it took a jump forward, 00:12:09.26 so it took an abrupt step forward 00:12:12.25 along the microtubule track, 00:12:14.21 and this kind of mechanism 00:12:16.04 allows you to get 00:12:18.20 a great deal of information 00:12:20.10 on the stepping behavior of the motor 00:12:22.06 on the microtubule. 00:12:24.08 And I should say that this general method 00:12:25.24 was first developed 00:12:27.28 by Ahmet Yildiz and Paul Selvin. 00:12:31.22 So, let me first describe to you 00:12:33.28 how kinesin steps along the track 00:12:36.18 for comparison with dynein. 00:12:37.25 So, kinesin always walks 00:12:39.16 in this hand-over-hand manner, 00:12:41.25 where the front motor domain... 00:12:44.24 these two motor domains are identical... 00:12:46.24 but the front one undergoes a conformational change 00:12:49.18 and that causes the displacement 00:12:52.10 of the partner head 00:12:54.02 from a rear site to a forward site. 00:12:58.00 And this is how kinesin 00:12:59.21 walks for long distances 00:13:01.12 in this kind of very regular hand-over-hand manner 00:13:06.16 where it's stepping from one tubulin subunit 00:13:08.18 to the next. 00:13:09.26 And you can even see this 00:13:11.15 if we label the two heads 00:13:14.22 with two different fluorescent dyes. 00:13:17.10 So, we marked the two heads 00:13:19.06 by a red color and a blue color 00:13:21.19 and now we plot 00:13:23.28 the position of these heads 00:13:25.07 as they're stepping along, 00:13:26.28 and you can see here, for example, 00:13:28.26 in this frame over here, 00:13:30.24 the red head is in front of the blue head, 00:13:33.00 just like this diagram, 00:13:35.03 but then the blue head 00:13:36.28 leapfrogs past the red head 00:13:38.29 and now the red head 00:13:41.04 leapfrogs past the blue head, 00:13:42.29 etc, etc, 00:13:44.17 and you can see how these two heads 00:13:46.07 are exchanging position 00:13:47.29 in a regular, alternating manner. 00:13:50.21 Now, dynein stepping 00:13:52.10 doesn't look anything like this, 00:13:54.26 so a similar experiment 00:13:57.16 of marking the two dynein heads 00:13:59.09 with two different dye colors 00:14:02.08 was done by Ahmet Yildiz 00:14:04.11 and Sam Reck-Peterson. 00:14:05.23 Both Ahmet and Sam were postdocs in the lab, 00:14:08.03 but the work that I'm showing you here 00:14:09.18 was done in their independent laboratories 00:14:14.11 at Berkeley and Harvard. 00:14:16.17 So, what you can see here 00:14:18.09 is if we look at the position 00:14:20.02 of the blue head, here, 00:14:22.10 in step number 1, 00:14:24.11 it's taken a big step forward, 00:14:27.17 but in step number 2, 00:14:29.27 instead of the partner taking the step, 00:14:32.04 that same head now 00:14:34.04 has taken yet another step 00:14:35.19 along the microtubule. 00:14:37.06 That is step number 2. 00:14:39.08 And now, finally, 00:14:40.20 the rear head, in step number 3, 00:14:42.23 begins to catch up, 00:14:44.11 but it doesn't pass the blue head. 00:14:45.26 And now in step number 4 00:14:47.16 the blue head 00:14:49.02 still takes another step forward. 00:14:51.05 So, what you can see from this 00:14:52.26 is that the dynein 00:14:54.23 is exhibiting an inchworm pattern, 00:14:57.04 where the two heads 00:14:59.01 can maintain their front and rear position 00:15:01.25 and both step forward together. 00:15:04.08 And second of all 00:15:06.19 the two heads are not necessarily 00:15:08.06 exchanging roles in timing of stepping. 00:15:11.25 Here, for example, 00:15:13.06 the blue head took two successive steps 00:15:15.01 before the red head took a step. 00:15:17.14 So, this is just a very 00:15:19.14 different kind of motility, 00:15:20.27 an irregular motility 00:15:22.21 that's not present in kinesin. 00:15:24.22 Also, dynein can take 00:15:27.03 very different sized steps as well, 00:15:29.06 so for example, here, 00:15:30.16 here's a very large step of dynein 00:15:32.17 going forward, 00:15:35.16 but these steps here are smaller, 00:15:37.10 so the step size of dynein 00:15:39.06 is not as regular as kinesin. 00:15:41.17 Furthermore, if you look at this trace, 00:15:43.17 there are many times when dynein 00:15:45.17 is actually taking a step backward 00:15:47.25 before it takes a step forward, 00:15:49.14 and these backward steps 00:15:51.08 are fairly frequent for dynein 00:15:52.27 and very rarely seen for kinesin, 00:15:55.11 especially if kinesin 00:15:57.14 is not trying to work against the load. 00:15:59.29 So, let me just review 00:16:01.14 the things that I just told you. 00:16:02.21 Kinesin has a very regular step size, 00:16:05.10 this is the distance 00:16:06.21 between subunits on the microtubule track, 00:16:09.03 dynein more variable. 00:16:10.24 Kinesin has this hand-over-hand stepping. 00:16:14.28 Dynein can exhibit this as well, 00:16:16.02 but it also exhibits 00:16:17.18 this inchworm pattern. 00:16:20.00 The two heads of kinesin take turns moving; 00:16:21.23 that is not necessarily true with dynein. 00:16:25.03 And while backwards steps are rare for kinesin, 00:16:27.22 as I showed you they're quite frequent 00:16:29.14 for the dynein molecule. 00:16:31.13 So, now I'd like to go on 00:16:33.11 and discuss: 00:16:34.17 How is it that dynein 00:16:36.12 can actually take these steps along the microtubule track? 00:16:38.20 What is the structural basis for this movement? 00:16:42.02 Well, a first big breakthrough 00:16:44.22 in this problem 00:16:46.13 came from pioneering 00:16:48.03 electron microscopy studies 00:16:49.23 by Stan Burgess, 00:16:51.25 and this shows the images 00:16:53.14 that they got of dynein 00:16:55.04 in two different nucleotide states, 00:16:56.19 and from these EMs (electron micrographs) 00:16:58.10 you can see, for example, 00:16:59.23 the ring of these AAA ATPase domains, 00:17:02.10 but you can also see a couple appendages. 00:17:04.17 One is a long stalk 00:17:06.29 that comes out of dynein 00:17:08.11 that leads at the very tip 00:17:09.28 to its microtubule binding domain, 00:17:12.00 and there's another appendage 00:17:13.17 that you can see here as well. 00:17:14.21 This is something that they termed 00:17:16.11 the linker. 00:17:17.20 It's something that sits kind of across the ring 00:17:21.04 and then extends out of the ring. 00:17:23.15 And what they noticed 00:17:24.25 in these two different nucleotide conformations 00:17:27.13 is that the position of the linker 00:17:29.27 relative to the ring and to the stalk 00:17:33.05 can change. 00:17:34.22 So, here it's sitting... 00:17:37.02 it's emerging from the ring 00:17:39.01 far from the stalk 00:17:40.23 and here they're merging close together. 00:17:43.04 And they thought that this motion of the linker 00:17:46.09 may act kind of like a lever arm 00:17:48.11 or a mechanical element 00:17:50.13 similar to the lever arm of myosin, 00:17:53.29 so what they proposed 00:17:55.18 is that the motion of the linker 00:17:58.21 relative to the ring 00:18:00.24 might be able to generate 00:18:03.05 a force upon a microtubule 00:18:05.00 that would cause it to slide, 00:18:07.21 and I'll come back to this later. 00:18:10.06 So, of course 00:18:12.26 we had to get higher resolution information of dynein 00:18:15.27 and that had to be derived from X-ray crystallography, 00:18:19.28 and it was quite a struggle 00:18:22.07 to get a crystal structure of dynein 00:18:25.06 and in fact our lab 00:18:27.00 was able to get the first crystal structure 00:18:29.09 of dynein in a nucleotide-free state in 2011, 00:18:33.27 but shortly thereafter 00:18:35.13 a whole bunch of other 00:18:37.15 nucleotide conformations of dynein 00:18:39.20 were reported. 00:18:41.20 So, the group of Kon and colleagues 00:18:44.00 from Japan 00:18:46.19 reported a very nice structure 00:18:48.00 of Dictostelium cytoplasmic dynein with ADP, 00:18:52.12 and in the last year or two 00:18:55.21 our lab got a structure of dynein 00:18:57.18 with an ATP analogue called AMPPNP, 00:19:03.00 and Andrew Carter's lab 00:19:04.11 was able to get a structure 00:19:06.13 with ADP-vanadate, 00:19:07.14 which may be mimicking an ADP-Pi state. 00:19:10.09 And what we'd like to do 00:19:12.02 is kind of similar to what you see 00:19:14.00 in this image of the horse here, 00:19:16.00 where you could see different snapshots 00:19:17.25 of the horse 00:19:19.06 taken as it's executing a gallop 00:19:21.29 and from these different snapshots 00:19:23.14 you can see the different conformations 00:19:25.12 of the horse 00:19:26.21 and begin to piece together 00:19:27.29 how this horse 00:19:29.28 is able to execute motility, 00:19:33.08 and by the same principle 00:19:34.21 we're trying to use these snapshots of dynein 00:19:36.19 to understand 00:19:38.06 how it changes its conformation 00:19:39.20 in order to execute motion. 00:19:41.19 So, now I'd like to give you 00:19:43.22 kind of a tour of what we learned 00:19:46.12 about the crystal structures, 00:19:47.27 not just from our lab but from all the crystal structures 00:19:50.05 that have emerged from the field. 00:19:52.23 First of all, here's just an image of dynein 00:19:54.24 compared to kinesin, 00:19:56.07 and you can see how much bigger dynein is 00:19:58.19 compared to kinesin 00:20:00.10 and how much more complicated 00:20:02.11 a motor domain it is. 00:20:06.24 And here's the position of the different AAA domains 00:20:10.25 that I showed you before 00:20:12.15 in this linear diagram, 00:20:13.26 but here's how they map out 00:20:15.19 on the dynein motor protein, 00:20:17.11 and they're all color coded in the same way 00:20:19.28 that you see in this linear diagram, here. 00:20:23.28 So, I'll focus on 00:20:25.29 a few important components... 00:20:27.16 so, the first is AAA1. 00:20:29.24 So, this is, again, 00:20:31.14 the main hydrolytic site. 00:20:32.17 If you make a mutation in AAA1, 00:20:34.15 you completely knock out dynein motility, 00:20:36.28 and interestingly this AAA1 00:20:39.08 is actually the region 00:20:41.08 that's farthest away from the microtubule. 00:20:44.08 Now, the other domain that I... 00:20:46.17 AAA subunit that I mentioned 00:20:48.05 that's important is AAA3, 00:20:50.00 and this is its position over here. 00:20:52.22 As I said before, it also hydrolyzes ATP 00:20:55.00 and plays an important role 00:20:56.20 in the mechanism, 00:20:58.06 and I'll explain how it works later in this talk, 00:21:02.19 but if you prevent ATP hydrolysis by AAA3, 00:21:05.23 dynein isn't completely inactive, 00:21:08.19 but the velocity of movement goes 00:21:11.06 way down with a hydrolysis mutant. 00:21:14.26 So, here's now 00:21:16.23 an atomic resolution image of the linker 00:21:18.23 that I described before as a mechanical element, 00:21:21.16 and here it's shown 00:21:23.20 extending across the ring. 00:21:26.14 Here is the microtubule binding domain 00:21:28.29 that's a small domain 00:21:30.23 that interacts with the microtubule, 00:21:32.12 and in between the ring and the microtubule binding domain 00:21:35.15 lie these two coiled-coils. 00:21:38.12 One is called the stalk, 00:21:41.06 but there's a second coiled-coil called the buttress, 00:21:44.14 which in fact extends out of the ring 00:21:46.20 and makes an important interaction with the stalk 00:21:49.24 that I'll describe in a second. 00:21:53.11 So, one of the interesting things 00:21:54.27 that we want to know from this structure 00:21:57.15 is how information, 00:21:59.10 or conformational changes, 00:22:01.06 are propagated 00:22:03.11 to control various aspects of dynein function, 00:22:06.17 and this is a particularly fascinating question for dynein 00:22:10.02 because we know that when ATP binds 00:22:13.01 at the very top of this molecule over here 00:22:15.23 it has to relay a conformational change 00:22:18.12 all the way down to the microtubule binding domain, 00:22:23.02 which in fact causes this microtubule binding domain 00:22:25.26 to release from the microtubule 00:22:27.23 so it can step forward along the track. 00:22:30.14 So, how this propagation occurs 00:22:32.25 is a fascinating question, 00:22:34.11 especially over this long distance 00:22:36.02 of about 25 nanometers. 00:22:38.11 We also know that the ATP binding 00:22:41.10 must be transmitted also 00:22:43.12 to somehow change the conformation 00:22:45.25 of where this end of the linker 00:22:47.29 is going to be positioned on the ring. 00:22:50.25 So, I'd like to now share with you 00:22:53.23 some ideas of how we think 00:22:55.07 this long range conformational change works, 00:22:58.00 based upon this collection of new X-ray structures 00:23:00.23 that were obtained. 00:23:02.20 So first of all, 00:23:04.20 let me just tell you a hint that we had 00:23:06.22 from our first X-ray crystal structure 00:23:08.26 in the nucleotide-free state, 00:23:11.10 and this just shows the AAA ring, 00:23:13.15 just focusing on the large domains. 00:23:17.09 And the one thing that you notice here 00:23:19.08 is that this ring is not symmetric, 00:23:21.01 it's a very asymmetric structure 00:23:23.08 and there are a couple gaps in this ring 00:23:25.05 where the AAA domains are farther apart. 00:23:29.24 And this gap between AAA1 and AAA2 00:23:32.26 was particularly interesting 00:23:34.23 and also surprising, 00:23:36.10 because this is the region 00:23:38.03 where ATP binds 00:23:40.07 and drives motility, 00:23:42.04 but we know from other AAA proteins, 00:23:46.16 ATPases, 00:23:48.17 that for ATP to be hydrolyzed, 00:23:51.06 these two domains, AAA1 and AAA2, 00:23:53.26 have to come closer together 00:23:56.08 because there are residues 00:23:57.29 that contribute to the hydrolysis 00:23:59.12 both from AAA2 and AAA1. 00:24:01.19 So we speculated, 00:24:03.22 although we just had one nucleotide state here, 00:24:06.01 that what may happen in dynein motility 00:24:08.15 is that in the nucleotide free state 00:24:10.09 there's a large gap, 00:24:11.18 but when ATP binds that gap closes, 00:24:14.12 and that closure then propagates 00:24:16.25 a conformational change around the ring 00:24:20.03 that gets transmitted to the microtubule binding domain 00:24:23.04 and also gets propagated to the linker 00:24:28.07 to change the linker conformational, 00:24:31.01 all, though, initiated by the binding of ATP 00:24:35.09 and the closure of this gap. 00:24:37.29 So, I'll show you that these general ideas 00:24:39.26 appear to be true, 00:24:42.09 and what you're seeing here 00:24:44.03 is a morph, 00:24:45.23 so we're going to slowly 00:24:47.25 go from one crystal structure, 00:24:49.24 which is the ADP structure 00:24:52.26 from the Kon et al lab 00:24:55.23 to another crystal structure, 00:24:58.00 which is ADP-vanadate, 00:24:59.24 which is more like an ATP state. 00:25:01.18 So, this is the conformational change 00:25:03.05 that presumably happens with ADP 00:25:06.06 is exchanged for ATP 00:25:08.07 in AAA1. 00:25:10.04 So, when ATP binds to AAA1, 00:25:14.17 you'll see a conformational change 00:25:17.07 and in this video I'm going to focus particularly 00:25:19.10 on these coiled-coils, 00:25:21.07 and how the conformational change 00:25:23.06 can be propagated from AAA1 00:25:25.09 all the way down to the microtubule. 00:25:27.16 So, here's the movie. 00:25:29.08 You can see the whole ring kind of distorting in shape, 00:25:32.22 and if you look at what happens here, 00:25:35.16 this orange coiled-coil, the buttress, 00:25:37.12 gets pulled away from the stalk, 00:25:40.21 so that creates tension on the stalk, 00:25:43.00 over here, 00:25:44.22 and that does something interesting 00:25:46.12 to the two helices that make up the stalk. 00:25:49.05 It causes a sliding motion to occur 00:25:53.03 so that the two helices 00:25:55.08 can move a short distance relative to one another, 00:25:59.08 but that sliding motion 00:26:01.12 gets propagated all the way down the coiled-coil, 00:26:04.13 all the way to the microtubule binding domain, 00:26:06.24 and causes a subtle change 00:26:08.22 in the microtubule binding domain structure 00:26:11.03 that changes its affinity for microtubules. 00:26:13.08 And in fact this kind of mechanism 00:26:15.19 was speculated many years ago 00:26:17.11 by Ian... in 2005 00:26:19.23 by Ian Gibbons and colleagues, 00:26:21.22 and now it looks like there's 00:26:23.28 good structural evidence for this 00:26:25.22 as well as other types of evidence 00:26:28.00 that has been obtained by other laboratories, 00:26:31.11 including Kon and Sutoh. 00:26:34.13 So, I now want to also 00:26:37.10 focus this same morph 00:26:39.03 between these two nucleotide states, 00:26:40.17 but with reference to the linker. 00:26:42.07 And you'll see that when ATP binds to AAA1, 00:26:46.14 you'll see the change in the AAA subunits, 00:26:49.27 and now we'll focus on what's happening in this linker, 00:26:52.29 and you can see it undergoes this large conformational change, 00:26:55.29 effectively going from a straight state 00:26:58.15 to this bent conformation. 00:27:00.23 So, earlier in this talk, 00:27:02.08 I described single molecule motility studies 00:27:04.14 that provide information 00:27:06.08 on how the dynein motor steps 00:27:08.00 along the microtubule track 00:27:09.12 and then I described X-ray crystallography 00:27:11.08 and EM studies 00:27:12.25 that provide information 00:27:14.18 on conformational changes 00:27:16.09 that occur in the dynein motor domain, 00:27:18.03 and now what I'd like to do 00:27:19.22 is to synthesize both pieces of information together 00:27:23.05 into a model that describes how dynein 00:27:26.00 is able to move along a microtubule. 00:27:28.28 And this model is presented 00:27:30.20 in the form of an animal 00:27:32.20 that's made by Graham Johnson. 00:27:34.22 Many parts of this animated model 00:27:37.18 are speculative at the present time 00:27:39.22 and no doubt, 00:27:41.20 as we get more information on dynein, 00:27:43.14 this model will change over the years. 00:27:47.06 But for right now it's useful 00:27:49.05 as a way of synthesizing data that's been gathered 00:27:52.26 by many different laboratories on dynein, 00:27:55.16 and also to generate models 00:27:57.13 for dynein motility 00:27:59.04 that can be tested in the future 00:28:00.27 by experimentation. 00:28:03.03 So first of all, let me show you 00:28:04.10 what you're going to see in this movie. 00:28:06.08 This image that you see here 00:28:08.04 of the dynein dimer 00:28:09.12 is derived from X-ray crystallographic data. 00:28:13.29 However, we don't know very much 00:28:15.23 about how the two dynein motor domains 00:28:17.21 are connected to one another 00:28:19.25 or how they're attached 00:28:21.17 onto a membrane cargo, for example. 00:28:23.28 So this part of the dynein molecule 00:28:26.17 is more stylistic and simple 00:28:28.07 because we simply don't have that structural information 00:28:30.19 right now. 00:28:32.12 Now, when I start playing this movie, 00:28:33.27 you can see the dynein jiggling back and forth. 00:28:36.18 This jiggling is due to 00:28:39.13 Brownian motion, 00:28:41.04 which is driven... thermally driven 00:28:43.16 collisions of water molecules with the dynein, 00:28:46.03 in fact this Brownian motion 00:28:47.22 is probably much more vigorous 00:28:49.23 than shown here in this animation. 00:28:51.27 Here are the different parts of the dynein. 00:28:53.19 Here's the ATPase ring, 00:28:55.14 the stalk that connects the ring 00:28:57.10 to the microtubule binding domain. 00:28:59.01 Here, colored in dark blue, 00:29:01.00 this is the strong binding state of dynein. 00:29:03.05 You'll see it transition 00:29:05.07 to a light blue color 00:29:07.23 when it undergoes a transition 00:29:09.18 to a weak binding conformation. 00:29:12.02 You'll see conformational changes 00:29:13.29 occurring in the linker 00:29:15.16 that I already described 00:29:17.04 and that transition will be shown 00:29:18.24 from a change in color 00:29:21.10 from this yellow state to a red state. 00:29:26.22 And when the microtubule binding domain 00:29:28.18 is detached 00:29:30.00 you'll see it also jiggling, 00:29:31.22 kind of moving randomly back and forth 00:29:33.10 along the microtubule. 00:29:34.26 That again is due to Brownian motion 00:29:38.07 and it probably helps this microtubule binding domain 00:29:41.06 execute a search for new binding sites 00:29:43.21 along the microtubule lattice. 00:29:46.09 So now, let's start this movie 00:29:48.19 and watch how dynein steps 00:29:50.21 along the microtubule. 00:29:52.05 And I'll show you the first step 00:29:53.20 and then we'll analyze it in greater detail 00:29:55.23 in the second step. 00:29:57.15 So, here this leading head 00:29:59.14 takes a step forward, 00:30:01.06 it's jiggling around 00:30:02.18 and now it redocks onto a microtubule binding site. 00:30:05.24 Now you'll see the rear head take a step. 00:30:08.05 It took a step forward 00:30:09.20 and you can see this linker 00:30:11.11 undergo a conformational change 00:30:13.06 from this yellow state to this red state, 00:30:16.22 and this conformational change 00:30:19.22 is accompanied, we think, 00:30:22.23 potentially, by a rotation of the ring, 00:30:26.11 and this rotation of the ring 00:30:28.19 can change the angle of the stalk, 00:30:31.12 pointing it and the microtubule binding domain 00:30:35.00 forward on the microtubule track, 00:30:37.12 which then allows this microtubule binding domain 00:30:39.26 to reattach to a tubulin subunit 00:30:42.27 farther towards the minus end of the microtubule. 00:30:45.25 So that's what you'll see in this next step. 00:30:48.07 It's going to redock, 00:30:50.05 right there, 00:30:52.12 and once it rebinds 00:30:54.19 that is accompanied by, we believe, 00:30:56.23 hydrolysis of ATP 00:30:58.13 and the release of phosphate from AAA1, 00:31:01.21 and that release of phosphate 00:31:03.24 causes this conformational change, 00:31:05.19 again, of the linker 00:31:07.17 from this bent red state 00:31:10.08 to this straighter yellow state, 00:31:12.06 and this conformational change, 00:31:13.29 we also think, 00:31:15.08 may produce a tug on the cargo 00:31:17.22 that advances the cargo forward along the track. 00:31:20.12 So, now let's see 00:31:23.02 these conformational changes again, 00:31:25.17 in this next sequence, 00:31:27.03 and you'll also see the different types of dynein 00:31:29.14 stepping in this next part of the movie. 00:31:32.23 So here, the leading head steps forward, 00:31:35.18 again, takes a big step forward. 00:31:37.27 It redocks, 00:31:39.13 but now it actually takes a step backward 00:31:42.12 along the microtubule track. 00:31:43.25 Here's the rear head, 00:31:45.23 it actually, by Brownian motion, 00:31:47.13 scoots around the other head 00:31:49.04 in this hand-over-hand motion. 00:31:51.06 It now takes another step forward 00:31:53.17 along the microtubule track, 00:31:55.04 and now its partner head 00:31:57.08 again undergoes a conformational change 00:32:00.15 and takes a step forward 00:32:02.20 along the track. 00:32:04.08 And we think by this 00:32:06.29 kind of process 00:32:08.07 the dynein molecule is able 00:32:10.23 to progressively move along on the track, 00:32:12.19 and now let's have another look 00:32:16.04 at this video 00:32:17.28 and see all these steps in action one more time. 00:33:24.19 Now let me come at the end 00:33:26.13 to this other AAA domain, 00:33:29.13 AAA3, 00:33:30.29 and let me tell you how we think that works. 00:33:32.20 As I said, 00:33:34.04 this also plays an important role in motility, 00:33:37.10 and in particular we know 00:33:38.23 if we block ATP hydrolysis, 00:33:40.17 the motor stops working. 00:33:43.06 And the mystery was why that was true, 00:33:46.07 because we know that 00:33:49.13 hydrolysis in AAA1 00:33:51.15 is sufficient 00:33:53.09 to do all the conformational changes 00:33:54.27 of the linker 00:33:56.13 and for dynein to take a step forward, 00:33:58.18 so the reason why AAA3 00:34:01.23 seemed to be important 00:34:03.08 wasn't that clear. 00:34:04.20 But an answer to this 00:34:06.11 came from structural studies 00:34:09.04 from our lab, 00:34:11.00 Gira Bhabha and Hui-Chun Cheng, 00:34:12.29 where they looked at 00:34:15.21 the conformation and the conformational changes of dynein, 00:34:19.09 not so much when there are different nucleotides 00:34:22.17 in AAA1, 00:34:24.07 but in two different nucleotide states 00:34:26.08 in AAA3. 00:34:27.23 So, in particular, 00:34:29.18 comparing when ADP in bound in AAA3 00:34:32.04 versus when ATP is bound in AAA3. 00:34:35.22 And I'll show you, 00:34:37.21 when AAA3 is in these different nucleotide states, 00:34:40.25 what happens to the conformational change 00:34:44.14 that occurs when ATP binds to AAA1. 00:34:46.29 So, the first is the movie you just saw. 00:34:49.28 That is the conformational change 00:34:52.27 that I showed you 00:34:54.29 where the linker undergoes 00:34:56.20 this large conformational change 00:34:58.08 and the whole ring changes its structure. 00:35:00.24 But, if we now 00:35:03.22 load AAA1 with ATP, 00:35:05.29 but now there's ATP in AAA3 as well, 00:35:09.24 what you'll see is a very different picture. 00:35:13.21 The conformation of this side of the ring 00:35:16.13 changes, 00:35:17.26 you can see a dramatic conformational change there, 00:35:19.20 but the conformational change 00:35:21.22 stops at about AAA4 00:35:23.29 and doesn't get propagated 00:35:25.20 around the rest of the ring, 00:35:27.08 and never causes a conformational change 00:35:29.04 in the linker 00:35:31.00 or in the stalk domain. 00:35:32.24 So AAA3 00:35:35.01 is in effect blocking the conformational change 00:35:37.25 and preventing it from propagating 00:35:40.02 throughout the ring. 00:35:41.13 So the way I like to think about this 00:35:43.11 is that AAA3 00:35:45.20 seems to be like a gate 00:35:47.15 that controls the propagation 00:35:49.08 of conformational change 00:35:51.11 throughout the dynein ring. 00:35:53.03 AAA1 is the trigger, 00:35:55.03 so in this image of dominos here, 00:35:57.26 it's what initially kicks off the chain reaction 00:36:01.08 that moves from one AAA domain 00:36:03.11 to the next, 00:36:04.25 and eventually can move all the way down 00:36:06.26 through the ring to AAA6 00:36:09.00 and cause this massive conformational change. 00:36:12.18 But if AAA3 00:36:14.22 has ATP in this site, 00:36:17.12 it actually acts 00:36:19.23 to block the propagation. 00:36:21.18 It's almost as if I have 00:36:23.29 a finger holding this domino down 00:36:26.15 and preventing the propagation 00:36:28.19 from going any further. 00:36:30.24 So the blocking of the conformational change, 00:36:33.17 or the release to allow it, 00:36:35.29 seems to be the primary activity 00:36:38.25 of AAA3. 00:36:41.09 So, that gives an update 00:36:43.04 of what we've learned about dynein 00:36:45.04 in the last few years, 00:36:46.28 but I must say we're still 00:36:48.19 very much at the beginning 00:36:50.04 and there are a tremendous number of unknown questions. 00:36:52.20 So, I illustrated some atomic structures 00:36:55.25 and conformational changes that occur, 00:36:58.22 but we don't really know 00:37:01.10 how those structural changes relate 00:37:03.12 to the stepping of dynein on the microtubule. 00:37:06.22 What would be particularly nice 00:37:08.08 is instead of just getting static images of dynein, 00:37:11.10 we can actually monitor and measure 00:37:14.09 dynein structural changes 00:37:16.01 while it's in the act of motility. 00:37:17.25 And there are ways of doing this, 00:37:19.28 for example techniques such as single molecule FRET, 00:37:22.21 which act as probes 00:37:24.17 to measure certain conformational changes 00:37:26.13 that occur in a protein, 00:37:28.02 and perhaps those kind of techniques 00:37:29.29 can be applied to dynein 00:37:31.17 so we can actually see 00:37:33.08 dynein stepping 00:37:35.03 and simultaneously measure 00:37:36.27 conformational changes. 00:37:38.06 I also gave you structural information 00:37:40.10 on the role of AAA3, 00:37:43.00 showing that it can block 00:37:44.26 a conformational change of dynein 00:37:47.12 and thereby prevent its motility. 00:37:50.06 But we don't really understand 00:37:52.00 how and why 00:37:54.10 AAA3 does this. 00:37:56.21 How does the cell 00:37:58.20 use this control mechanism 00:38:00.03 to regulate dynein motility? 00:38:01.21 How does it actually control 00:38:03.22 whether AAA3 00:38:05.13 has an ATP or an ADP in the active site? 00:38:08.17 So, we have no idea 00:38:10.20 on this issue right now, 00:38:13.28 and this is obviously 00:38:16.04 going to be important for understanding 00:38:17.23 what the real purpose of AAA3 is 00:38:20.22 in dynein cell biology. 00:38:24.00 So, with that, 00:38:25.16 I'd like to thank the many people 00:38:26.27 that contributed to this work. 00:38:28.10 First of all, 00:38:29.20 people that were in the lab previously, 00:38:32.25 a fantastic group of individuals 00:38:35.19 that helped launch the dynein project 00:38:37.18 in the lab 00:38:39.05 -- Sam, Ahmet, Andrew, and Arne -- 00:38:43.16 now have all gone off to their own labs 00:38:45.11 and are very successful, 00:38:47.17 and I've discussed a lot of their work 00:38:49.17 from their independent labs in this talk. 00:38:51.09 And Carol Cho was a graduate student 00:38:54.06 who has now gone on to Korea. 00:38:55.28 And the more recent work 00:38:58.02 is the work of Gira Bhabha 00:39:00.24 and Hui-Chun Cheng. 00:39:02.28 Gira is still in the lab 00:39:04.11 and Hui-Chun has moved 00:39:06.12 to her own lab in Taiwan. 00:39:07.29 And with that I'd like to thank you for your attention, 00:39:11.18 and in my third iBiology talk 00:39:13.20 I will discuss the regulation 00:39:16.03 of mammalian cytoplasmic dynein.