Molecular Motor Proteins
Transcript of Part 3: Regulation of Mammalian Dynein
00:00:15.10 Hello. 00:00:16.07 I'm Ron Vale from UCSF, 00:00:18.20 and in this part of my iBiology talk 00:00:20.04 I'd like to discuss 00:00:21.23 the regulation of mammalian dynein, 00:00:24.21 research that's come from out of our lab 00:00:26.28 as well as others in the field. 00:00:29.26 So, we began 00:00:32.00 to do our research on dynein 00:00:34.15 using yeast cytoplasmic dynein 00:00:36.27 and there are many reasons for this. 00:00:39.01 It was easy to express 00:00:41.28 large quantities of yeast dynein 00:00:44.23 and we can also modify it 00:00:46.25 to create different types of recombinant protein, 00:00:49.13 and furthermore when we tested 00:00:51.13 yeast cytoplasmic dynein 00:00:53.13 for single molecule motility, 00:00:55.22 we got this type of fantastic motion 00:00:57.27 that you see here. 00:00:59.27 Again, all of these individual green spots 00:01:02.00 are single molecules of dynein 00:01:04.09 that are moving along microtubule tracks, 00:01:06.29 shown here in blue. 00:01:09.04 So, later we wanted 00:01:11.02 to extend our work 00:01:12.26 to mammalian dynein... 00:01:16.05 this was work that Rick McKenney did, 00:01:18.00 and we thought it was going to be 00:01:19.28 fairly straightforward, 00:01:21.07 we'd just do all of the same things 00:01:22.28 that we did with yeast 00:01:24.13 and apply it to dynein from a different species. 00:01:27.07 But unfortunately, 00:01:28.19 the initial results were rather disappointing. 00:01:31.26 So we first tried to make 00:01:33.24 recombinant human dynein, 00:01:35.24 very much like the type of recombinant dynein 00:01:38.03 that we made with yeast, 00:01:40.27 and the dyneins here are in red in this case, 00:01:43.14 but we saw very little motility. 00:01:45.17 They would bind to the microtubule 00:01:47.08 but did not move. 00:01:48.29 So we took, then, a step back 00:01:51.15 and decided to purify dynein 00:01:53.26 from a native tissue, from rat brain. 00:01:56.15 So this is the normal dynein from rat brain, 00:02:00.10 and again we purified it, 00:02:02.02 we fluorescently labeled it, 00:02:03.22 but again found fairly disappointing results. 00:02:06.12 We saw dynein binding to the microtubule, 00:02:08.19 but no movement. 00:02:11.05 However, these dyneins 00:02:13.14 weren't completely inactive 00:02:15.02 because we then tested them 00:02:17.04 in another kind of motility assay 00:02:19.17 and saw that they could produce movement, 00:02:22.24 and this is what's called a microtubule gliding assay. 00:02:24.28 In this case, the dynein 00:02:27.17 is coated and attached 00:02:29.21 onto a glass cover slip. 00:02:31.09 It then grabs hold of a microtubule 00:02:33.04 and then it can move it across the surface. 00:02:35.12 So, in this case, however, 00:02:37.04 this is not single molecule motility; 00:02:39.07 there are many dynein molecules 00:02:40.24 underneath that microtubule. 00:02:42.24 And these are the microtubules, here. 00:02:44.20 When you give it ATP, 00:02:46.24 they move very nicely across the glass. 00:02:49.20 So, the conundrum was: 00:02:51.21 Why didn't we see any single molecule motility, 00:02:55.02 processive motility of the dynein? 00:02:58.05 We saw it in yeast, 00:02:59.28 but we didn't see it in mammalian cells, 00:03:01.23 and we expected the mammalian dynein 00:03:04.00 also to be processive, 00:03:05.20 especially if it's going to carry cargo 00:03:08.07 for long distances inside of cells. 00:03:11.25 So when you get a negative result like this, 00:03:14.12 one often has to go back to the drawing board 00:03:16.23 and figure out, 00:03:18.09 well, maybe something 00:03:20.01 wasn't right about the experiment. 00:03:21.19 Maybe the conditions for the assay 00:03:24.05 weren't quite right, 00:03:25.20 whatever buffers we were using 00:03:27.09 or other elements of the assay, 00:03:29.14 or maybe we're just 00:03:31.13 entirely missing some critical component 00:03:33.19 that dynein needs to move 00:03:35.12 at a single molecule level. 00:03:37.14 So, we began to test this latter possibility, 00:03:40.28 and a good candidate for that missing factor 00:03:43.21 was a protein complex 00:03:46.00 that was known as dynactin. 00:03:48.01 Now, dynactin was discovered 00:03:50.09 many years ago 00:03:51.28 by Schroer and colleagues 00:03:53.23 as a cofactor 00:03:55.21 that would stimulate organelle transport by dynein. 00:03:58.17 And subsequently 00:04:00.10 it's been found that dynactin 00:04:01.27 is required for many functions 00:04:03.21 of cytoplasmic dynein, 00:04:05.00 effectively a knockout of dynactin 00:04:07.14 produces a very similar phenotype 00:04:09.15 to knocking out dynein itself. 00:04:12.10 So it seems to be involved 00:04:13.29 in many kinds of dynein functions, 00:04:16.12 although the exact role of dynactin 00:04:18.25 has not been entirely clear. 00:04:21.19 So, this is what dynactin looks like 00:04:24.25 and it's a monstrous protein 00:04:26.18 of comparable size to dynein itself. 00:04:29.02 It has about 23 proteins 00:04:31.09 in one dynactin complex 00:04:33.07 and it's about a megaDalton in size. 00:04:36.02 And it has a very interesting 00:04:38.11 and unusual architecture. 00:04:40.02 The core of dynactin 00:04:41.19 is composed of a short filament 00:04:44.04 of actin-related proteins 00:04:45.23 that are capped 00:04:47.26 at both ends by a different group 00:04:51.03 of special capping proteins, 00:04:53.00 and in the middle 00:04:54.18 there's another group of proteins 00:04:56.22 that create a type of shoulder 00:04:59.24 on one part of the dynactin molecule, 00:05:03.05 and there's a long coiled-coil 00:05:06.20 that extends from this core structure, 00:05:09.06 called p150, 00:05:10.23 that also contains 00:05:12.13 a microtubule binding domain at the end. 00:05:14.29 Now, recently, there's also been 00:05:17.02 some exciting developments 00:05:19.19 because Andrew Carter's label 00:05:21.08 recently got a cryo-EM structure 00:05:24.10 of dynactin, 00:05:25.26 so we now know the organization 00:05:27.21 of all of these subunits 00:05:30.02 through their work 00:05:31.29 at near atomic resolution. 00:05:34.12 So... but the conundrum is that 00:05:37.23 for many years, although it was known 00:05:39.23 that dynein and dynactin 00:05:41.19 are involved in cellular activities together, 00:05:44.08 it was very hard to demonstrate 00:05:46.01 a biochemical interaction 00:05:47.27 between dynein and dynactin. 00:05:52.12 But this mystery was resolved 00:05:56.00 when another protein came on the scene 00:05:58.25 called Bicaudal-D or BicD. 00:06:01.25 Now, BicD is a much smaller protein 00:06:04.00 and it's composed of several coiled-coils, here. 00:06:07.13 It was originally discovered 00:06:10.24 in a Drosophila genetic screen 00:06:13.08 by Eric Wieschaus 00:06:15.11 and Christiane Nüsslein-Volhard, 00:06:18.00 and subsequently it was shown 00:06:20.13 that this big BicD is involved 00:06:22.24 in recruiting dynein 00:06:24.21 to membrane organelles. 00:06:28.02 And an important piece of work 00:06:29.24 was done in this paper 00:06:31.25 by Splinter and colleagues 00:06:33.25 where they showed that this BicD 00:06:35.19 can join dynein and dynactin, now, 00:06:38.13 into a stable interacting complex, 00:06:41.03 and this was shown in this sucrose gradient, 00:06:44.05 here, where all three of these components 00:06:46.12 are combined, 00:06:47.27 and they comigrate together 00:06:49.20 in a very large molecular weight complex... 00:06:51.25 this top band is dynein, 00:06:53.18 this is the p150 subunit of dynactin, 00:06:57.11 and this lower band is this BicD. 00:07:00.18 So, they all comigrate together 00:07:03.09 on this sucrose gradient, 00:07:04.21 so we they're interacting 00:07:07.12 all together in one large complex, 00:07:09.08 and in fact with the recent cryo-EM work, 00:07:12.04 we actually have some idea 00:07:14.05 why and how BicD 00:07:15.29 can join dynein and dynactin together. 00:07:18.01 It basically... this is work from Andrew Carter's lab... 00:07:21.02 sits in between them. 00:07:22.21 It first of all... 00:07:24.01 the coiled-coil of BicD 00:07:25.21 falls along a groove 00:07:27.11 in the actin filament of dynactin, 00:07:29.14 but it also makes contact 00:07:31.12 with dynein as well, 00:07:32.28 so it kind of serves as a glue 00:07:34.17 to bring these two proteins together 00:07:36.12 and sits in the middle. 00:07:39.01 And also, work from Chowdury et al, 00:07:41.28 from another laboratory, 00:07:44.07 imaged this dynein-dynactin-BicD complex 00:07:48.07 on the microtubule, 00:07:50.03 and you can see the dynein motor domain here, 00:07:52.10 in this case j 00:07:54.21 ust kind of colored in yellow, 00:07:56.16 carrying this huge entourage behind it, 00:07:59.04 which is this complex of dynactin and BicD. 00:08:03.01 So, it's really an amazingly large 00:08:06.05 collection of proteins, here, 00:08:07.25 that are connected to the dynein motor domains. 00:08:11.06 Well, going back in time now 00:08:13.09 to 2013, 00:08:15.10 we were interested in whether this complex 00:08:18.26 would convert dynein 00:08:20.24 from that nonprocessive motor 00:08:22.13 that I showed you 00:08:24.02 to a processive motor. 00:08:25.24 So, Rick McKenney purified this complex. 00:08:29.01 He also tagged the BicD 00:08:32.02 with a GFP 00:08:33.28 and in this case we just used the N-terminus of BicD, 00:08:36.29 which is sufficient 00:08:39.14 to combine all these proteins together. 00:08:42.01 And to our great satisfaction 00:08:44.28 there was tremendously good processive movement 00:08:47.16 of this entire complex. 00:08:49.06 So here, all these, again, 00:08:51.04 all these individual green dots 00:08:53.12 are the BicD, 00:08:54.27 which are traveling along the microtubules 00:08:56.17 due to the interaction with dynein and dynactin. 00:08:59.10 In fact, this is one of the 00:09:02.04 most processive motors 00:09:04.09 that we've studied. 00:09:05.22 In fact, it's about five to ten times 00:09:07.19 more processive 00:09:09.20 than the kinesin motor. 00:09:11.12 So this converted this motor 00:09:13.08 from this very poor motor 00:09:15.19 for single molecule motility 00:09:17.14 into a real champion processive motor, here. 00:09:23.01 So, this same observation 00:09:25.08 was also made 00:09:29.02 by Andrew Carter's lab, independently, 00:09:31.08 specifically by Max Schlager. 00:09:35.00 So, we now also 00:09:38.03 wanted to be sure 00:09:40.19 that all these protein components 00:09:42.07 were indeed interacting 00:09:43.16 and traveling together on the microtubule, 00:09:45.22 so we then did this experiment 00:09:47.21 where we labeled each of the components 00:09:49.13 with a different fluorescent dye. 00:09:52.03 So, GFP was tagged 00:09:54.16 with a green dye, 00:09:56.00 BicD a blue dye, 00:09:57.19 and dynactin with a red dye, 00:09:59.20 and you can see when you superimpose 00:10:02.08 all of these colors 00:10:03.22 you get these really nice multicolored spots over here. 00:10:07.02 And I'll feature this for one spot in detail, 00:10:10.01 where you can see all three components 00:10:13.01 moving together along a microtubule. 00:10:16.20 And the reason why these colors 00:10:18.27 appear separated 00:10:20.08 is that the microscope is 00:10:23.01 first taking a snapshot in green 00:10:25.03 and then another one in red 00:10:26.14 and another one in blue, 00:10:28.06 and in between taking these 00:10:30.18 different colored snapshots 00:10:31.27 of course the dynein continues to move along the microtubule, 00:10:35.04 which is why these colors 00:10:36.22 appear to be separated, 00:10:39.07 but that's just due to the way 00:10:41.00 that these images were acquired. 00:10:43.20 So, the next thing we wanted to know was 00:10:46.08 whether this was some unique mechanism 00:10:48.16 of BicD, 00:10:50.17 or did this reflect 00:10:52.18 a more general mechanism 00:10:54.12 of how you activate dynein processivity 00:10:56.16 inside of cells. 00:11:01.02 So, to answer this question, 00:11:02.18 I need to give you a bit of more explanation 00:11:05.23 of BicD and how it interacts 00:11:08.11 with other molecules in the cell. 00:11:10.08 So, I just described to you 00:11:11.28 how the N-terminus of BicD 00:11:13.25 can join dynactin and dynein 00:11:15.21 into this one protein complex, 00:11:17.27 but BicD also has 00:11:20.00 another coiled-coil at the C-terminus, 00:11:23.16 and this C-terminal part of the coiled-coil 00:11:26.13 interacts with another protein, 00:11:28.11 which is a Rab GTPase, 00:11:31.08 and this Rab GTPase 00:11:33.02 is docked onto the surface 00:11:35.14 of a membrane vesicle. 00:11:37.16 So this BicD 00:11:39.14 really acts as an adapter protein. 00:11:41.06 At one end, it's connected to a cargo, 00:11:44.09 in this case this Rab6 protein 00:11:46.21 from the Golgi. 00:11:48.09 At the other end, 00:11:49.22 it's interacting 00:11:51.19 with the dynein motor and dynactin, 00:11:53.23 so it's really bridging 00:11:56.07 and bringing the dynein-dynactin complex 00:11:59.12 normally in the cell 00:12:01.10 to a specific cargo. 00:12:02.26 Now, in the dynein literature, 00:12:04.06 it was known that there were 00:12:05.27 other types of adapter proteins 00:12:07.14 that served other functions. 00:12:09.00 Some seemed to be linking dynein onto endosomes, 00:12:13.01 the Spindly protein 00:12:15.08 brings dynein to kinetochores... 00:12:17.10 so what we wanted to know was: 00:12:18.26 Do all these other adapter proteins 00:12:20.19 described in the literature... 00:12:22.09 could they also convert 00:12:24.18 dynein and dynactin 00:12:26.03 into a processive motor? 00:12:27.26 So we purified these other adapters 00:12:29.15 and tested them in the single molecule assay 00:12:32.01 and again found 00:12:34.06 this very gratifying, beautiful movement 00:12:37.11 where all of these adapter proteins 00:12:39.07 can form a complex with dynein-dynactin 00:12:43.26 and induce highly processive dynein motility. 00:12:48.03 Now, in addition to regulating dynein, 00:12:50.29 it also appears that this BicD 00:12:52.17 can regulate dynactin as well. 00:12:55.08 Remember that I said 00:12:57.19 at the end of this little antenna subunit here, 00:13:00.03 p150, 00:13:01.26 there's a microtubule binding domain. 00:13:04.01 Well, what we found with single molecules studies 00:13:06.12 was that if we looked at the microtubule binding 00:13:08.27 by dynactin alone, 00:13:11.15 or even dynactin with dynein, 00:13:14.03 we found that dynactin really 00:13:16.09 had very, very low binding 00:13:18.06 to the microtubule, 00:13:19.20 suggesting that this microtubule binding domain 00:13:22.12 is somehow repressed. 00:13:24.22 But when it's combined 00:13:26.27 with BicD and dynein, 00:13:30.01 the microtubule binding 00:13:32.29 of this dynactin component increases, 00:13:37.04 so, as I'll explain more later on in this talk... 00:13:39.21 so, it appears as though 00:13:41.19 this complex formation 00:13:43.17 also is able to convert 00:13:45.27 the microtubule binding activity of dynactin 00:13:49.00 from a repressed to an active form. 00:13:53.01 So, the general model that's emerging 00:13:55.11 from these in vitro studies 00:13:57.06 is that, perhaps, 00:13:59.09 in the cytoplasm, 00:14:00.24 when dynein is not attached to a cargo 00:14:03.05 and dynactin is not attached to a cargo, 00:14:05.24 these molecules are largely inactivated. 00:14:11.06 And that the activation that these molecules 00:14:14.06 require is linked to their simultaneous 00:14:17.04 binding to a cargo. 00:14:18.29 So, in this case there's a cargo molecule 00:14:22.03 with a receptor 00:14:25.19 that can bind to an adapter protein, 00:14:27.22 in some cases we think it actually 00:14:29.18 can activate that adapter protein, 00:14:31.25 and that adapter protein 00:14:33.25 then brings dynein and dynactin 00:14:36.09 onto the surface of the cargo, 00:14:38.12 and also activates it 00:14:40.15 so that the motor is now active 00:14:42.02 and can transport that cargo 00:14:43.29 along the microtubule track. 00:14:46.10 So, I showed you, now, 00:14:49.06 how the cargo 00:14:51.13 can regulate dynein-dynactin motility. 00:14:54.02 What about the microtubules themselves? 00:14:56.25 Are they just kind of inert tracks? 00:14:59.08 Or do they have a regulatory function as well? 00:15:03.09 Well, it's known that microtubules 00:15:05.18 actually have many different types 00:15:07.16 of post-translational modifications. 00:15:11.09 These modifications are described here 00:15:14.06 and they often occur 00:15:16.11 on the very C-terminus, 00:15:20.16 these somewhat disordered peptides 00:15:22.26 that emerge from the surface of the microtubule. 00:15:25.22 And I'd like to focus on one of these 00:15:29.10 post-translational modifications, 00:15:31.14 which involves 00:15:33.21 the very C-terminal tyrosine 00:15:35.25 of the α-tubulin subunit. 00:15:38.12 So, it turns out that 00:15:40.11 the C-terminus has this tyrosine residue, 00:15:42.29 but in cells there's an enzyme, 00:15:44.27 a carboxy-peptidase, 00:15:46.28 that can specifically remove 00:15:49.10 that tyrosine residue, 00:15:51.21 and in fact, post-translationally again, 00:15:53.19 there's an enzyme, 00:15:55.16 a tubulin-tyrosine ligase, 00:15:58.09 that can shift the reaction in the opposite direction 00:16:00.19 and re-add that tyrosine 00:16:03.04 to the end of α -tubulin. 00:16:05.21 So, this modification has been known for many years, 00:16:08.15 it's not clear exactly 00:16:10.10 what it's doing in cells, 00:16:12.00 but cell biological studies have shown that 00:16:17.08 these types of microtubule tracks 00:16:19.05 in fact can be separate, 00:16:21.06 even inside of one cell. 00:16:23.06 So, for example, this just shows 00:16:25.20 an image of a fibroblast 00:16:27.24 which is migrating, it's polarized, 00:16:29.18 it's extending and moving in one direction, 00:16:32.13 and it was found that 00:16:35.01 the microtubules that are extending 00:16:37.18 in the direction of movement 00:16:39.23 are preferentially detyrosinated. 00:16:43.28 So it's been tempting to speculate 00:16:46.10 that these different types of microtubule tracks 00:16:50.08 that are tyrosinated and detyrosinated 00:16:52.27 may serve and interact differently 00:16:55.25 with different types of molecular motor proteins 00:16:58.14 in cells, 00:17:00.16 although evidence for that hasn't been entirely clear. 00:17:04.17 So we decided to test that, 00:17:06.14 whether the [tyrosine of the C-terminus] 00:17:08.22 makes a difference or not 00:17:11.24 for the processive motility 00:17:13.17 of dynein and dynactin 00:17:15.17 connected to this BicD. 00:17:17.07 So we made, first of all, 00:17:19.02 recombinant tubulin 00:17:20.23 that was absolutely identical, 00:17:22.14 except for one difference: 00:17:24.04 whether the α-tubulin had the tyrosine 00:17:26.19 or not. 00:17:28.01 And second of all, then, 00:17:30.04 we labeled those two different types of microtubules 00:17:32.22 with different fluorescent dyes, 00:17:34.18 so the tyrosinated microtubules 00:17:36.26 you'll see, in the movie that I'll show you, 00:17:38.18 are labeled with a red dye, 00:17:40.18 whereas we labeled 00:17:42.25 the detyrosinated tubulin 00:17:45.12 with a blue dye. 00:17:47.00 And then we just combined these two microtubule types 00:17:49.07 together in the same cover slip 00:17:52.05 and then examined how they would interact 00:17:54.12 with different molecules. 00:17:56.29 So, first of all, 00:17:59.02 we tested just the p150 domain of dynactin, 00:18:02.03 so this is not the whole dynactin molecule, 00:18:04.05 it's basically 00:18:07.00 the microtubule binding portion of dynactin. 00:18:12.01 And this was actually known earlier 00:18:14.16 and we replicated the results, 00:18:16.09 and you can see see all these dynactin molecules 00:18:18.24 found in green 00:18:20.22 decorating these red microtubule tracks, 00:18:23.29 but these blue microtubule tracks 00:18:26.01 that don't have the tyrosine 00:18:27.21 are devoid of the p150 00:18:31.05 microtubule binding region. 00:18:33.22 We tested this also for dynein, 00:18:36.06 again labeled in green, 00:18:38.02 and remember that dynein on its own 00:18:39.21 does not really move along microtubules, 00:18:41.26 so we're just examining here 00:18:44.08 its microtubule binding properties 00:18:46.10 in this static image. 00:18:48.03 Well, you find here that 00:18:50.08 the dynein doesn't really have much of a preference 00:18:52.20 between either of these tracks; 00:18:54.08 it binds to both. 00:18:55.23 So, the question is, 00:18:57.11 if we look at this whole complex together, 00:18:59.17 would it look more like dynein 00:19:01.07 or would it look more like the dynactin molecule? 00:19:05.03 So we did this, combined the microtubules, 00:19:06.26 and did a motility assay, 00:19:08.19 and the result is quite striking. 00:19:10.08 I think you can see that there are 00:19:12.22 lots of these individual dynein-dynactin molecules 00:19:15.02 moving along the tyrosinated microtubules, 00:19:18.12 but there's very little motility 00:19:20.19 occurring on these blue microtubules 00:19:23.06 without the tyrosine. 00:19:25.13 So the motility 00:19:28.02 seems to behave much more 00:19:29.24 like the p150 binding properties 00:19:31.21 than the dynein. 00:19:34.06 So, another question is: 00:19:37.15 What is the mechanism of how 00:19:39.20 this p150-dynactin 00:19:41.27 is influencing dynein motility? 00:19:43.20 Is it necessary just to 00:19:45.12 initiate the first encounter of the motor with the track, 00:19:49.14 or is it continuously needed 00:19:52.10 to hold on to the track 00:19:53.29 during processive motility? 00:19:55.17 So we wanted to distinguish 00:19:57.03 between these two possibilities 00:19:58.18 with the following type of experiment. 00:20:01.19 We actually made a chimeric microtubule 00:20:04.26 by fusing a tyrosinated microtubule, 00:20:07.12 again in red, 00:20:08.23 with a detyrosinated microtubule 00:20:11.04 in blue, 00:20:12.18 but in this case it's a continuous track, here. 00:20:15.21 And we wanted to know... 00:20:17.03 we know the dynein-dynactin 00:20:18.20 will first bind to this tyrosinated track 00:20:21.18 and then move along it, 00:20:23.24 but what happens when it reaches the junction? 00:20:26.02 Is it going to fall off 00:20:28.01 or is it going to continue along the track? 00:20:30.22 And the result is shown here 00:20:32.27 in a real movie 00:20:34.01 and you can follow these spots 00:20:35.21 that are first binding to this red portion, 00:20:38.15 and you can see that they 00:20:41.00 move continuously from the red portion 00:20:43.10 into this blue region. 00:20:45.00 So this result indicates that 00:20:48.01 the dynactin 00:20:50.00 is needed to initiate the motility, 00:20:51.15 but once it starts 00:20:53.17 it can then function 00:20:55.13 without p150 interaction 00:20:58.18 with the tyrosinated tubulin. 00:21:02.13 So, in summary, 00:21:03.29 these experiments provided 00:21:05.18 some clues as to 00:21:07.11 how dynactin might be able 00:21:09.13 to activate dynein motility. 00:21:11.15 So, as I just described, 00:21:13.21 this microtubule binding domain 00:21:15.09 at p150 00:21:17.03 seems to play, initially, 00:21:18.25 an important role 00:21:20.22 in delivering dynein and dynactin 00:21:23.01 to tyrosinated microtubules. 00:21:25.22 But once this interaction is made 00:21:27.23 and the dynein starts going, 00:21:29.27 the motility doesn't seem to continue 00:21:32.14 to need that microtubule binding interaction. 00:21:36.14 So... but we also think 00:21:38.21 that dynactin is still needed 00:21:41.01 for the processive motion, 00:21:42.24 for the continued processive motion, 00:21:44.11 and that may occur 00:21:46.05 through some allosteric activation 00:21:49.03 of dynein by dynactin. 00:21:51.14 And there's some hints 00:21:53.02 of this from the beautiful cryo-EM work 00:21:56.26 that came from Andrew Carter's lab, 00:21:58.19 but we still really don't understand 00:22:01.29 very much of the details 00:22:03.29 about that allosteric activation mechanism. 00:22:07.06 In addition, there are plenty of remaining 00:22:09.23 open questions in this field. 00:22:12.26 So, what I showed you was some 00:22:15.22 in vitro experiments 00:22:18.08 showing that the tyrosinated state of a microtubule 00:22:22.08 plays an important role in dynein transport in vitro, 00:22:25.03 but what about in living cells? 00:22:27.12 Are tracks that are detyrosinated... 00:22:32.26 are they poor tracks for dynein motility 00:22:35.08 and perhaps select 00:22:37.18 kinesin-based motility? 00:22:39.14 So, we don't know the answers 00:22:41.29 to these questions 00:22:43.14 about how this cycle of tyrosination and detyrosination 00:22:47.07 is used to regulate 00:22:49.27 microtubule-based transport 00:22:51.18 between dynein and kinesin motors 00:22:54.01 in living cells. 00:22:56.10 We also know that in living cells 00:22:58.03 there are other types of dynein regulators, 00:23:01.05 ones that I haven't had a chance 00:23:03.07 to describe in this talk, 00:23:04.22 but other examples of dynein regulators 00:23:06.26 include this protein called LisI, 00:23:11.09 which was found as a mutation 00:23:13.18 in a disease called lissencephaly. 00:23:15.22 There's also another factor called NudE/L. 00:23:19.29 And what we don't know is 00:23:21.24 how all of these factors are working together 00:23:24.02 as a system to regulate dynactin motility in cells, 00:23:27.22 so there's still a tremendous amount of work to do 00:23:31.11 to understand the regulation 00:23:33.25 of dynein in cells. 00:23:36.20 And I'd like to just 00:23:39.02 with acknowledging the people 00:23:41.01 that did the work that I just described. 00:23:43.15 I already mentioned Rick McKenney, 00:23:46.06 who initiated this whole project. 00:23:48.13 He's now about to start 00:23:50.19 his own lab at the UC Davis. 00:23:53.02 Walter Huynh is a graduate student 00:23:55.00 that did a lot of the work 00:23:57.00 on the other dynein-dynactin 00:23:58.27 adapter proteins. 00:24:01.01 And Minhaj Sirajuddin, 00:24:04.13 a former postdoc in the lab, 00:24:06.26 he now has his own lab at the NCBS in Bangalore, 00:24:10.11 and he did a lot of the work 00:24:12.21 on the tubulin that I described, 00:24:14.20 and pioneered a system 00:24:16.10 for making recombinant tubulin. 00:24:18.13 And with that I'd also like to thank you 00:24:20.16 for your attention.