Molecular Motor Proteins
Transcript of Part 1: Molecular Motor Proteins
00:00:15.04 Hello. I'm Ron Vale, 00:00:16.25 and in this talk 00:00:18.09 I'd like to introduce you 00:00:19.21 to molecular motor proteins, 00:00:21.18 which are these fascinating protein machines 00:00:23.12 that are featured in this animated video here. 00:00:26.26 And these proteins are able 00:00:29.00 to walk along a cytoskeletal track 00:00:31.23 and transport a variety of different types of cargos 00:00:34.25 inside of cells. 00:00:36.28 What I'd like to do today 00:00:39.02 is, first of all, 00:00:40.17 tell you a little bit of a history 00:00:42.01 of biological motility, 00:00:44.07 and then I'll tell you about 00:00:46.03 what these motor proteins do inside of cells, 00:00:48.08 the various functions that they have, 00:00:50.22 and then I'll you a little bit about 00:00:52.06 how they work, 00:00:54.04 how they're able to convert chemical energy 00:00:56.05 into motion. 00:00:57.13 And finally, I'll tell you 00:00:59.06 some things about 00:01:00.28 how these molecular motor proteins 00:01:02.16 are relevant to human health 00:01:04.14 and disease. 00:01:06.15 So, first of all, 00:01:07.21 biological motion 00:01:08.28 is just a fundamental 00:01:10.12 and very obvious attribute 00:01:12.08 of all living organisms. 00:01:14.11 For example, 00:01:16.05 in terms of people, we're able to run, 00:01:18.01 and a variety of animals 00:01:19.28 are able to move 00:01:21.07 and explore their environment. 00:01:22.27 And this is due to the fact 00:01:24.09 that we have muscles 00:01:25.28 and they can contract, 00:01:28.11 and theories about how muscles work 00:01:31.01 are in fact very old, 00:01:32.23 dating back to the ancient Greeks. 00:01:34.25 A whole new world of life 00:01:36.29 was evident with the invention 00:01:39.05 of the microscope, 00:01:40.12 and with that, also, 00:01:42.04 a whole new world of motility. 00:01:44.16 Leeuwenhoek, for example, 00:01:46.16 when he looked at pond water 00:01:47.27 under the microscope, 00:01:48.29 saw a whole variety 00:01:50.11 of different types of movements. 00:01:52.00 And this is what he had to say about them: 00:01:54.07 "The motion of most of them in the water 00:01:56.13 was so swift, 00:01:57.21 and so various, 00:01:59.09 upwards, downwards, and roundabout, 00:02:01.01 that I admit I could not 00:02:02.24 but wonder at it." 00:02:04.20 Indeed, all these types of biological motions 00:02:07.01 are indeed very fascinating 00:02:10.04 to Leeuwenhoek, 00:02:11.24 and also fascinating 00:02:13.07 even to school kids today. 00:02:16.02 Now, not only do cells move, 00:02:18.10 but material inside of cells 00:02:19.29 can also undergo motion, 00:02:22.08 and the first person that described this 00:02:24.08 was Bonaventura Corti, 00:02:26.20 where he described cytoplasmic streaming 00:02:29.19 inside of plant cells. 00:02:32.02 And this is shown here in this video, 00:02:35.04 and what he described is shown here: 00:02:37.23 "I know that the phenomenon that I announce 00:02:39.29 is too strange to be believed at first... 00:02:42.16 I saw two torrents 00:02:44.11 inside each section... 00:02:45.29 One of the torrents rose on one side 00:02:48.18 and descended on the other, 00:02:49.25 constantly... 00:02:51.07 and this not once 00:02:53.00 but thousands of times 00:02:54.21 and for days, and for entire weeks." 00:02:57.02 You can see that these early scientists 00:02:59.24 were quite animated 00:03:01.08 in their use of language 00:03:02.21 to describe their scientific results, 00:03:04.11 something that I think scientists 00:03:06.07 have lost today. 00:03:08.13 Now, microscopy continued 00:03:11.19 to play a major role 00:03:13.09 in our understanding of biological motility. 00:03:16.11 And a pioneer in this 00:03:18.00 was Shinya Inoue, 00:03:19.13 who used more advanced types of microscopy, 00:03:23.01 such as polarization microscopy, 00:03:25.26 to observe the dynamics of living cells. 00:03:29.00 And he did a lot to describe 00:03:30.24 the process of cell division, 00:03:32.17 which is shown here, 00:03:34.02 where you can see the mitotic spindle, 00:03:36.00 you can see the motion of the mitotic spindle, 00:03:38.12 and also the movement of chromosomes 00:03:40.20 here in this beautiful movie. 00:03:43.16 Now, the next revelation, 00:03:45.26 I would say a revolution, 00:03:47.26 occurred with the development 00:03:49.18 of fluorescent proteins. 00:03:51.11 And now we can take that same object 00:03:53.24 that you saw that Shinya Inoue described, 00:03:56.06 where he had observed 00:03:58.05 the mitotic spindle 00:03:59.27 with natural contrast 00:04:01.22 from the polarization microscope, 00:04:03.16 but now we can tag 00:04:05.11 specific proteins in the cell. 00:04:07.12 Shown here are microtubules, 00:04:09.10 which are tagged in red... 00:04:12.03 with a red fluorescent protein, 00:04:14.00 and the chromosomes 00:04:15.17 with a green fluorescent protein, 00:04:17.14 and if you look at this 00:04:19.12 in a Drosophila embryo, 00:04:21.01 you can see the chromosomes, 00:04:23.04 the formation of the mitotic spindle, 00:04:25.28 and the physical motion 00:04:27.25 of these chromosomes here. 00:04:30.24 And when people 00:04:32.22 began to tag a lot of proteins in the cell, 00:04:34.14 they found that they were not just static, 00:04:37.03 but in fact in motion, 00:04:38.15 and now we know that a whole variety of molecules 00:04:41.04 inside of cells 00:04:42.25 are actually undergoing 00:04:44.14 active types of movement 00:04:46.02 to be localized 00:04:47.20 in specific destinations in the cell. 00:04:50.23 Now, all of these types of biological movements 00:04:53.05 that I've described 00:04:55.00 are due to the actions 00:04:56.28 of these special enzymes 00:04:58.21 called molecular motor proteins, 00:05:00.07 which interact with cytoskeletal tracks, 00:05:03.05 and there are two main types 00:05:04.27 of cytoskeletal tracks. 00:05:06.01 One are the larger diameter microtubules, 00:05:09.06 and the smaller diameter actin filaments. 00:05:13.06 And these are the two 00:05:15.04 major cytoskeletal filaments 00:05:16.18 that act as tracks 00:05:18.08 for these molecular motors. 00:05:19.26 So, in the microtubules world, 00:05:21.23 there are two main types 00:05:23.18 of cytoskeletal motors. 00:05:24.27 One are the dynein molecules, 00:05:26.09 and I'm going to talk much more about these 00:05:29.11 in my next two iBiology lectures, 00:05:31.10 and kinesin, 00:05:32.27 which will be a focus of this lecture. 00:05:34.22 They move along microtubule tracks, 00:05:36.20 which are shown in this movie over here, 00:05:38.27 and you can see that these microtubules 00:05:41.10 extend all throughout the cell, 00:05:43.02 and they're also themselves 00:05:45.11 quite dynamic. 00:05:46.19 They can grow and shrink 00:05:48.05 and change their position in cells as well. 00:05:51.20 Now, these tracks, 00:05:53.11 both microtubules and actin, 00:05:55.16 are polar filaments. 00:05:57.03 And the reason is that 00:05:58.28 they're composed of a basic subunit, 00:06:00.22 in this case tubulin, 00:06:03.04 which polymerizes 00:06:05.05 in a defined head-to-tail manner 00:06:06.19 to create this polymer. 00:06:08.19 So, because the polymerization 00:06:11.11 is polarized, 00:06:13.08 there is a distinct plus end of the microtubule 00:06:15.19 and a distinct minus end. 00:06:18.13 And the motors 00:06:20.11 recognize this intrinsic polarity of the tracks. 00:06:22.24 So, for example, 00:06:24.09 the majority of kinesins 00:06:25.28 will move in one direction, 00:06:27.13 towards the plus end of the microtubules, 00:06:29.06 whereas dyneins 00:06:30.20 move in the opposite direction, 00:06:32.20 towards the minus end. 00:06:34.10 Now, these microtubules in cells 00:06:36.22 are organized, 00:06:38.07 also not randomly, 00:06:39.28 but with a specific polarity. 00:06:41.14 So many of the microtubules 00:06:43.02 are nucleated and grow from this structure 00:06:45.14 by the nucleus, 00:06:46.23 which is called the centrosome. 00:06:48.16 And this is where the minus ends 00:06:50.18 of the microtubules are located, 00:06:52.08 and they extend outward to the periphery, 00:06:54.24 where the plus ends are localized. 00:06:57.15 So, if a kinesin motor 00:06:59.08 grabs hold of a cargo, 00:07:00.18 it's going to move towards the plus end, 00:07:02.20 and it's going to deliver that cargo 00:07:04.09 from the interior of the cell 00:07:05.28 to the periphery. 00:07:07.27 Dynein, on the other hand, 00:07:08.29 will move in the opposite direction, 00:07:10.21 so it will deliver a cargo 00:07:13.01 towards the cell interior. 00:07:15.16 Now, the other major class 00:07:17.29 of molecular motors 00:07:19.20 work on actin filaments, 00:07:21.05 and there's one major class of these motors 00:07:23.01 and those are the myosin motors. 00:07:26.01 Now, they move along actin, 00:07:28.06 which tends to have 00:07:29.27 a very different distribution than microtubules. 00:07:32.14 So, in this mitotic spindle, 00:07:34.22 for example, 00:07:36.14 the microtubules are in the center 00:07:38.04 and you can see the actin, in red, 00:07:39.26 all around the perimeter of the cell. 00:07:41.21 So actin, in general, 00:07:43.14 tends to be more cortical, 00:07:45.05 the microtubules more interior. 00:07:48.22 Now, when I say myosin 00:07:51.08 or kinesin or dynein, 00:07:52.23 I'm not just talking about 00:07:54.22 one molecular motor. 00:07:55.23 In fact, these are families 00:07:57.15 of related motor proteins. 00:08:00.11 In humans, for example, 00:08:01.21 there are 45 different kinesin genes, 00:08:05.07 there are 40 myosins, 00:08:06.21 and about 15 dyneins. 00:08:08.12 And there's some many different types of motors 00:08:10.27 because there are a whole variety 00:08:12.14 of motility functions 00:08:13.23 that are needed 00:08:15.13 in human cells, 00:08:17.03 and these different motors 00:08:18.18 carry out different types of transport 00:08:20.26 or force-generating functions. 00:08:23.26 Now, I'd like to tell you 00:08:25.16 a little bit about the anatomy of these motor proteins, 00:08:28.25 what makes motors proteins 00:08:30.21 in the same family similar 00:08:32.07 and also how they differ. 00:08:33.26 So, they have one end of the motor protein 00:08:35.28 which is the actual part 00:08:38.29 that moves along the track, 00:08:40.26 and another part that interacts with the cargo, 00:08:44.02 and I'll show you this in more detail 00:08:46.28 for one member of the kinesin family 00:08:48.17 called kinesin-1. 00:08:51.23 And this globular domain at the end I just showed you 00:08:54.22 is the motor domain, 00:08:56.05 that's the part that walks along the track. 00:08:57.23 Many of these motor domains 00:08:59.06 also come together as dimers, 00:09:01.09 in some cases even tetramers, 00:09:03.09 and that dimerization 00:09:05.21 is mediated by a coiled-coil, 00:09:08.08 which is also interrupted 00:09:10.10 -- it has hinges in it -- 00:09:11.27 and that provides flexibility 00:09:13.10 for the motor protein to bend. 00:09:15.17 And at the very distal end 00:09:17.08 is the tail domain. 00:09:18.18 So, this may contain 00:09:20.11 a globular domain 00:09:22.02 belonging to the motor polypeptide. 00:09:23.17 It may also bind 00:09:25.04 other associated subunits 00:09:27.02 to make a larger complex, 00:09:29.22 and this tail domain 00:09:31.12 is what defines, usually, 00:09:33.07 what that motor protein will bind to in the cell, 00:09:35.28 what kind of cargo it will transport. 00:09:38.21 Now, there are other classes 00:09:41.19 of motors proteins 00:09:44.12 -- this just shows kinesin-2 and kinesin-3 -- 00:09:46.21 and all of these motor proteins 00:09:48.23 share in common 00:09:50.21 a very similar motor domain, 00:09:53.13 which is shown here. 00:09:55.18 But the rest of the protein 00:09:58.01 is really completely different. 00:09:59.15 So, the coiled-coils are different, 00:10:01.12 and the cargo binding domains 00:10:03.04 in fact have no sequence identity 00:10:05.11 between them. 00:10:06.28 And the reason is that 00:10:08.25 these non-motor domains, 00:10:10.06 again, are defining 00:10:11.21 unique types of cargos 00:10:13.05 that these motors are interacting 00:10:14.19 with inside of cells. 00:10:17.04 So, let me tell you a little bit 00:10:18.23 about the motor domain and what it does. 00:10:20.15 It's an enzyme 00:10:22.17 and it's an enzyme 00:10:24.02 that hydrolyzes ATP. 00:10:26.17 So, it first binds an ATP molecule 00:10:29.28 -- adenosine triphosphate -- 00:10:31.09 and then it hydrolyzes 00:10:33.07 a bond between the beta- and gamma-phosphate. 00:10:36.03 And after the hydrolysis 00:10:38.11 it then releases the products 00:10:39.26 in a sequential manner. 00:10:40.28 So, it first releases 00:10:42.09 a phosphate 00:10:44.06 then, next, the ADP is released, 00:10:47.08 and then it's able to rebind ATP 00:10:50.29 and start the cycle all over again. 00:10:53.22 And during one round of this ATP hydrolysis cycle 00:10:58.10 there are structural changes that occur in the motor 00:11:00.11 that I'll describe later 00:11:01.27 that allow it to take one step 00:11:03.17 along the track. 00:11:04.24 So, every time it undergoes this cycle 00:11:07.03 it steps forward, 00:11:09.13 and then multiple rounds of this ATP hydrolysis cycle 00:11:12.03 allow the motor 00:11:13.24 to move a long distance along the track. 00:11:16.01 Now, let me just say a few things 00:11:18.07 about how this motor 00:11:20.10 performs in comparison 00:11:22.18 to a motor 00:11:24.22 that you might be more familiar with, 00:11:26.05 like a car engine. 00:11:27.07 So, first of all, obviously 00:11:29.12 the kinesin motor is much smaller, 00:11:31.10 in fact that motor domain 00:11:32.24 is only 10 nanometers in size. 00:11:36.00 It uses a fuel, 00:11:37.17 as I told you in the last slide, 00:11:39.04 adenosine triphosphate, 00:11:41.15 in comparison to your car, 00:11:43.01 which uses hydrocarbons. 00:11:45.15 It moves at a few millimeters per hour, 00:11:48.07 which seems very slow. 00:11:50.22 However, you have to take into account 00:11:52.24 the size of the motor protein, 00:11:54.10 and if you measure 00:11:56.14 how far it moves 00:11:58.00 in terms of its own length, 00:12:00.02 you find in fact that it's moving 00:12:02.18 faster than your car is moving 00:12:04.20 on a highway. 00:12:06.12 It's also much better than your car 00:12:08.00 in terms of work efficiency, 00:12:10.12 which is essentially 00:12:13.01 how much of the chemical energy 00:12:14.18 that it can convert into productive work, 00:12:16.18 and these motor proteins 00:12:18.08 work at about 60% efficiency, 00:12:20.00 whereas your car engine 00:12:22.00 works at a much more pathetic 10-15%. 00:12:25.11 So, we indeed have a lot to learn 00:12:27.28 about how nature's own motors 00:12:29.23 are able to execute motility. 00:12:32.28 So, let me tell you know a little bit about 00:12:35.18 what the cytoskeletal motors do in cells, 00:12:37.18 and I'm just going to provide a few examples 00:12:39.16 because the number of different types of motility 00:12:42.24 that cells engage in 00:12:44.20 is really vast. 00:12:47.13 So, one thing that the motor proteins do 00:12:49.18 is to transport membrane organelles. 00:12:53.04 In some cases, 00:12:54.18 they're small organelles, 00:12:55.27 they're transport intermediates 00:12:57.15 that are traveling between the Golgi apparatus 00:13:00.04 and the plasma membrane, 00:13:01.15 or endosomes that are traveling 00:13:03.10 in the opposite direction, 00:13:05.04 from the plasma membrane to other organelles, 00:13:06.18 such as lysosomes. 00:13:07.24 And this just shows an example, 00:13:10.05 in living cells, 00:13:11.28 of some of these transport vesicles 00:13:13.27 moving along microtubule tracks. 00:13:16.16 But even very large organelles 00:13:18.15 also can move in cells. 00:13:20.07 Probably the biggest organelle of all 00:13:22.00 is the nucleus, 00:13:23.19 and this just shows an image of the nucleus 00:13:26.19 being transported 00:13:28.17 inside of a nerve cell, 00:13:30.11 which during development 00:13:32.14 is migrating towards the cortical region 00:13:34.25 of the brain, 00:13:36.09 and it has to transport the nucleus 00:13:38.23 during this migration process. 00:13:42.28 Now, here's another 00:13:44.17 beautiful example of organelle movement. 00:13:46.23 These are pigmented organelles 00:13:49.16 called melanosomes 00:13:50.24 that are found in special skin cells 00:13:52.27 called melanocytes, 00:13:55.00 and this is what gives skin its color. 00:13:56.23 And some organisms 00:13:58.16 such as amphibians 00:14:00.19 and also fish 00:14:02.05 can change the color of their skin 00:14:03.28 and they do that by moving these melanosomes. 00:14:07.29 When the melanosomes are dispersed like this 00:14:10.16 the skin color appears darker, 00:14:13.08 but when they're all concentrated 00:14:14.24 in the center 00:14:16.10 the skin takes on a lighter color, 00:14:18.08 and this distribution of organelles 00:14:19.28 occurs by motors. 00:14:21.18 Here are dynein motors 00:14:23.11 transporting all these melanosomes 00:14:24.29 towards the cell center 00:14:26.29 and kinesin motors 00:14:28.16 can transport these organelles 00:14:30.04 in the opposite direction, 00:14:31.23 and this is under hormonal control. 00:14:33.16 So, hormones interact with receptors 00:14:36.03 that control these motors 00:14:37.17 to change the distribution inside of cells. 00:14:40.18 But also, there are other objects 00:14:42.15 that are not membrane bound 00:14:43.27 that also can be transported. 00:14:45.10 For example, 00:14:46.25 there are many kinds of viruses 00:14:48.19 that have learned to pick up molecular motors 00:14:50.23 and transport themselves inside of cells. 00:14:53.13 This is an example of vaccinia virus 00:14:55.12 and all of these little particles, 00:14:57.12 these myriad little particles 00:14:58.29 that you see here, 00:15:00.12 are viruses 00:15:02.01 moving along microtubule tracks. 00:15:03.08 Another example being rabies, 00:15:05.01 which can transport itself 00:15:07.07 inside of nerve cells. 00:15:10.03 In addition, 00:15:12.02 mRNAs also can be localized 00:15:14.01 and transported in cells, 00:15:15.18 and that allows the mRNA 00:15:17.11 to be localized 00:15:19.03 in a particular region of the cell 00:15:20.21 where that mRNA 00:15:22.13 can be translated into a protein 00:15:24.25 and therefore the protein 00:15:26.13 is made where the protein is needed, 00:15:27.27 in a localized destination. 00:15:29.21 And this is an example 00:15:31.17 of one RNA called gurken 00:15:33.29 that is transported 00:15:35.13 in a Drosophila oocyte, 00:15:36.29 it's shown here in this orange color, 00:15:38.23 and you can see it moving 00:15:40.22 into this one anterior corner 00:15:43.00 of the oocyte 00:15:44.27 by active transport. 00:15:47.11 Now, in addition to transporting cargo, 00:15:50.05 motors can move the filaments themselves, 00:15:52.08 and this is how muscle contraction works. 00:15:55.08 And the basic unit of a muscle 00:15:57.11 is called a sarcomere 00:15:58.25 and it's a repeating unit 00:16:00.25 of actin and myosin. 00:16:03.00 In the sarcomere, 00:16:04.12 myosin is concentrated 00:16:06.02 in a filamentous form 00:16:08.04 in the center of the sarcomere 00:16:09.26 and it interacts 00:16:11.17 with interdigitating actin filaments 00:16:14.04 which are coming in from both sides 00:16:15.29 of the sarcomere. 00:16:17.21 So, myosin 00:16:19.12 wants to walk in one direction on this side, 00:16:22.28 towards the end of the actin, 00:16:24.16 and the other side of the myosin filament 00:16:26.06 is trying to walk in this direction 00:16:27.26 along the actin, 00:16:29.12 so when a muscle is activated, 00:16:31.00 which occurs with nerve stimulation, 00:16:33.11 calcium comes in 00:16:34.19 -- that's calcium shown here -- 00:16:36.02 and the myosin starts walking 00:16:37.15 and it starts bringing these actin filaments 00:16:40.21 closer together. 00:16:42.04 That shortens the sarcomere 00:16:43.19 and that's what causes your muscle to contract. 00:16:46.25 In this case, 00:16:48.06 the filaments and the motors 00:16:50.10 are very well organized in your muscle, 00:16:51.25 but motors 00:16:53.23 can also take a disordered 00:16:55.19 or almost random array of filaments 00:16:57.22 and create order amongst these filaments, 00:17:01.02 and this is what happens in the formation 00:17:03.24 of the meiotic spindle. 00:17:05.28 So, in this case, the microtubules start off random, 00:17:08.25 but then there are some motors, 00:17:10.22 such as shown in green here, 00:17:12.21 that are crossbridging filaments 00:17:15.02 and they're trying to move to the minus end. 00:17:17.07 So, they're moving to the minus end 00:17:19.10 and, as they do, 00:17:20.19 they collect all the minus ends 00:17:22.09 of the microtubules together. 00:17:23.20 There are other motors, 00:17:25.01 such as shown in orange, 00:17:26.08 that work on antiparallel filaments, 00:17:29.01 and they work to slide those filaments apart. 00:17:31.25 So, in this manner, 00:17:34.26 as shown in this movie 00:17:37.06 of the formation of a meiotic spindle 00:17:39.11 in a xenopus egg extract, 00:17:41.18 you can see that this initial random 00:17:44.09 organization of microtubules... 00:17:46.10 as it grows, 00:17:47.27 the motors act upon it 00:17:49.07 and they start elongating this spindle 00:17:52.13 and the minus ends all get organized 00:17:55.08 at this pole and it forms 00:17:57.00 this characteristic bipolar shape, 00:17:59.06 due to the action of these molecular motors. 00:18:03.04 So, I'd now like to turn to the subject of, 00:18:05.22 how do we study the mechanism of motility? 00:18:08.11 How do we understand 00:18:10.06 how these motors work? 00:18:11.18 Well, it's useful to look at the motility in cells, 00:18:15.15 but it's more powerful 00:18:17.20 to study these motors in a test tube, 00:18:20.04 where you really can be able to 00:18:22.09 dissect their mechanism. 00:18:24.01 So, we're able to use 00:18:25.17 in vitro motility assays, 00:18:27.10 where can take either purified motors out of cells, 00:18:30.03 or even express them in bacteria, 00:18:33.23 and study their motility 00:18:35.11 in a very controlled environment. 00:18:37.01 In fact, one can also do that 00:18:38.13 at single molecule level, 00:18:40.08 so one can study the actions 00:18:41.29 of even individual motors proteins 00:18:45.06 as they are being transported 00:18:47.04 on a filament. 00:18:48.07 So, I'll show you some examples, 00:18:50.11 first of in vitro motility assays. 00:18:52.01 Here's an example 00:18:53.25 where we have a plastic bead 00:18:56.13 to which one can attach a motor protein, 00:18:59.00 and then the motor will transport these beads 00:19:01.15 along a filament. 00:19:03.04 This is shown here in this movie, here. 00:19:05.02 These are inert plastic beads 00:19:06.23 being transported by kinesin 00:19:08.22 along a microtubule track. 00:19:11.25 Now, we can in fact 00:19:13.17 get rid of the whole bead entirely 00:19:17.07 and label the motor protein 00:19:18.17 with a fluorescent dye. 00:19:20.14 This is not really drawn to scale here, 00:19:23.02 in fact the dye is very small 00:19:25.09 relative to the motor, 00:19:27.05 but it provides a very bright signal 00:19:29.05 that we can track 00:19:30.29 the motor protein as it's moving, 00:19:32.09 and we can do that with a technique 00:19:33.22 called total internal reflection fluorescence, 00:19:36.23 which is also described 00:19:38.06 in videos in the iBiology microscopy course, 00:19:41.06 but this is what it looks like. 00:19:43.11 All of these green dots here 00:19:45.03 are individual motor proteins, 00:19:47.01 and you can see them 00:19:48.24 being moved along these microtubules here, 00:19:53.03 so you can track and 00:19:55.06 follow the details of this motion 00:19:57.00 at a single molecule level. 00:19:58.29 One other type of in vitro motility assay 00:20:01.10 is one where the motor proteins 00:20:02.29 are all coating a glass surface, 00:20:05.21 and in this case the motor proteins can't move, 00:20:08.20 but they grab hold of the filament 00:20:10.00 and then they start 00:20:11.15 transporting this filament 00:20:13.15 along the track, 00:20:15.04 and you'll see this in this video, here. 00:20:16.19 These are microtubules 00:20:18.15 and you can see them all sliding across 00:20:21.09 the glass surface, 00:20:22.29 driven by these molecular motor proteins. 00:20:25.19 Now, using these types 00:20:28.18 of in vitro assays, 00:20:30.04 you can actually measure 00:20:32.03 a lot of details of how these motors work. 00:20:34.17 For example, 00:20:36.14 using an optical trap, 00:20:38.05 again described in the iBiology microscopy course, 00:20:40.18 you can measure the forces 00:20:42.18 produced by individual motors. 00:20:45.10 The optical trap grabs hold of a bead 00:20:47.12 and tries to keep the bead in place. 00:20:49.18 On the other hand, 00:20:50.29 the motor is trying to move along the track 00:20:52.22 and trying to move the bead 00:20:54.14 out of the optical trap, 00:20:56.03 which is resisting, 00:20:57.09 and you can eventually measure the force 00:21:01.08 eventually when the motor stalls, 00:21:02.18 when it can't move the bead any farther, 00:21:05.08 and that's what's called the stall force, 00:21:07.11 and that's the maximal force 00:21:08.26 that the motor produces. 00:21:10.14 And it's really incredible 00:21:12.00 that one can measure these forces. 00:21:13.08 They're 1-7 picoNewtons, 00:21:14.28 which are very very small forces, 00:21:16.16 but they can be measured 00:21:18.27 quite accurately with an optical trap. 00:21:20.06 This is a lot of pioneering work 00:21:22.17 done in Steve Block 00:21:24.24 and Jim Spudich. 00:21:26.21 In addition, one can measure the steps 00:21:28.17 that are produced by motors. 00:21:30.03 So, you can track these single fluorophores 00:21:32.19 that I just showed you 00:21:34.10 with very high resolution, 00:21:36.12 and you can see where... 00:21:40.02 how that fluorescent dye is moving over time, 00:21:43.05 and you can see that that fluorescent dye 00:21:45.05 takes abrupt steps, 00:21:46.22 and these abrupt steps are when the motor 00:21:48.29 is actually moving from one subunit on the track 00:21:52.01 to the next subunit. 00:21:55.01 So, the next thing that we want to know 00:21:56.28 in order to understand how motors work 00:21:58.28 is what they look like. 00:22:00.10 What are their structures? 00:22:01.18 And for this we need higher resolution techniques 00:22:03.28 like X-ray crystallography 00:22:05.28 and electron microscopy, 00:22:08.23 and we don't just want one snapshot of the motor. 00:22:12.09 We want snapshots of the motor 00:22:14.05 as it goes through its whole ATPase cycle. 00:22:17.04 It's very much like in the old days 00:22:19.19 when they tried to understand 00:22:21.05 how a horse moves, 00:22:22.14 they made high speed cinematography 00:22:24.04 and got various snapshots 00:22:25.28 of the horse in action, 00:22:28.16 and that's what we'd like to do 00:22:29.28 with the motor protein. 00:22:31.01 We want different snapshots 00:22:32.19 of what that motor looks like 00:22:33.29 at different stages of this ATPase cycle. 00:22:37.21 Now, one thing that the structure 00:22:40.13 did tell us right away, 00:22:41.19 when we got the structure of kinesin, 00:22:43.25 is whether the motor protein myosin and kinesin 00:22:47.10 would operate by a similar type of 00:22:49.25 structural mechanism or not. 00:22:51.18 Before the structure was available for kinesin, 00:22:55.03 we actually thought that they were completely 00:22:57.22 different types of motors, 00:22:58.23 and there were several obvious reasons. 00:23:00.19 One is that kinesin works on microtubules, 00:23:02.21 while myosin works on actin. 00:23:04.05 The myosin motor domain 00:23:06.02 is also over 2-fold larger 00:23:07.21 than that of kinesin, 00:23:10.10 and if you asked a computer 00:23:11.25 to line up the sequences 00:23:13.13 the computer said that 00:23:14.27 there wasn't any real clear amino acid identity 00:23:17.13 on alignment. 00:23:19.10 However, when we got the structure, 00:23:21.14 we found a big surprise, 00:23:23.18 and that is that there are parts of the kinesin 00:23:25.18 and myosin motors 00:23:26.28 that are very similar to one another structurally. 00:23:29.16 In fact, these molecular motors, 00:23:31.11 even though one works on microtubules 00:23:32.26 and the other one works on actin, 00:23:34.15 they must have evolved 00:23:35.28 from a common ancestor 00:23:37.12 at some point during evolution. 00:23:39.12 And the part of these motors 00:23:41.10 that is most common 00:23:43.28 is this central core here, 00:23:45.06 which is featured in blue. 00:23:46.15 And this does the basic chemistry, 00:23:49.03 this is the part that binds the nucleotide, 00:23:50.26 it hydrolyzes it, 00:23:52.16 and it also undergoes 00:23:54.03 very small structural changes 00:23:55.22 when it's in different nucleotide states, 00:23:58.17 for example, between ADP and ATP. 00:24:01.12 And that mechanism is also very similar 00:24:03.28 between kinesin and myosin. 00:24:06.09 These motors then also 00:24:09.03 have a similar what's called 00:24:10.20 a relay helix, 00:24:12.09 which I'm showing here in green, 00:24:14.15 and it relays information 00:24:16.09 from this nucleotide binding site 00:24:17.24 to a mechanical element 00:24:19.22 that I'll describe in a second, 00:24:21.08 and it does that by sliding back and forth 00:24:23.19 between the nucleotide side 00:24:25.19 and this mechanical element. 00:24:27.24 So, these are the mechanical elements 00:24:30.01 of myosin and kinesin 00:24:32.19 and, indeed, those look completely different 00:24:34.20 from one another 00:24:36.10 and they work differently, as I'll show you shortly, 00:24:37.22 but you can see that these mechanical elements 00:24:39.28 are positioned 00:24:41.26 in the same relative place 00:24:43.23 relative to this common enzymatic core here. 00:24:48.15 So these two different motors 00:24:50.08 evolved different kinds of mechanical elements, 00:24:51.29 but they kind of hooked them up 00:24:53.28 to the same basic enzyme 00:24:56.21 and sensing unit. 00:24:58.07 So now, 00:25:00.22 let me tell you a little bit about 00:25:02.11 what we've learned from the structure of these motors 00:25:04.08 in different nucleotide states 00:25:05.25 and how that explains 00:25:07.15 the motion of these motors. 00:25:09.18 So, what you're seeing here is an animation, 00:25:11.19 but it's based upon 00:25:13.13 real structural data from muscle myosin. 00:25:18.02 And this is the actin filament here. 00:25:20.12 This is the... 00:25:22.01 this large yellow element is the mechanical element 00:25:23.17 that I just showed you, 00:25:25.12 and now let's see how it works. 00:25:27.17 So, when it binds to actin, 00:25:30.02 actin causes this phosphate group 00:25:32.04 to come off the active site 00:25:33.13 and that causes a large rotation 00:25:35.12 of that big lever arm-like unit, 00:25:39.00 causing about a 10 nanometer displacement 00:25:40.13 of the actin filament. 00:25:42.01 ATP then comes in, 00:25:43.12 that dissociates the myosin, 00:25:46.12 and then the hydrolysis recocks that lever arm 00:25:49.03 for another round. 00:25:50.07 So here it is again. 00:25:51.23 Phosphate release, 00:25:53.12 that big swing causing the motion, 00:25:55.00 the release, and the recocking. 00:25:56.27 And it's millions and millions 00:25:59.10 of these small displacements 00:26:01.09 by myosin 00:26:02.29 that collectively result 00:26:04.26 in the contraction of your muscle 00:26:08.24 and the shortening of that sarcomere. 00:26:11.20 Now, myosin is made to work 00:26:13.27 in large numbers, 00:26:15.11 but kinesin has a different problem. 00:26:17.23 It has to transport, potentially, 00:26:19.28 these very small organelles that I showed you, 00:26:23.08 and there's some indication 00:26:25.07 that some of that transport 00:26:26.25 is generated by single motor proteins. 00:26:29.16 So kinesin has to have... 00:26:32.09 can't release from the track. 00:26:33.16 It has to keep holding on to the track 00:26:35.17 as it moves, 00:26:37.12 something that's called processive motility. 00:26:39.15 So let me show you how this works. 00:26:41.16 So, in red here, 00:26:43.01 that is the mechanical element of kinesin, 00:26:45.13 which is called the neck linker, 00:26:46.26 and I'll show you how that changes its structure 00:26:49.12 during the motility cycle. 00:26:50.23 First, the kinesin comes on to the microtubule 00:26:54.03 and the microtubule binding kicks off a bound ADP, 00:26:58.06 ATP can rebind, 00:26:58.29 and that ATP binding 00:27:00.12 causes this mechanical element 00:27:02.22 to zipper up along the blue core. 00:27:05.21 And that, as you'll see, 00:27:07.13 displaces the partner head. 00:27:08.29 So, here is the zippering, 00:27:10.11 there is the movement of the partner head 00:27:12.00 from a rear site to a forward site, 00:27:14.04 and that zippering of the neck linker 00:27:16.25 helps to move 00:27:19.13 the two motor domains 00:27:20.25 in this hand-over-hand mechanism. 00:27:22.01 So, here is the zippering 00:27:23.13 and there's the partner head moving 00:27:24.29 from the rear site to a forward site. 00:27:26.29 So, this motor protein 00:27:28.20 has learned to 00:27:30.26 kind of walk in a coordinated manner, 00:27:32.10 where the two motor domains 00:27:36.09 are moving in a leap frog manner 00:27:38.15 along the microtubule. 00:27:42.04 Now, evolution also has learned 00:27:47.01 to develop different kinds of mechanical elements, 00:27:48.26 even within a superfamily, 00:27:50.17 for different purposes. 00:27:52.14 So, here are two different classes of kinesin motors. 00:27:55.24 This is the one I just showed you in the animation, 00:27:58.08 but here is another type of 00:28:00.25 member of the kinesin superfamily, 00:28:02.19 called kinesin 14, 00:28:04.05 and it's learned to walk 00:28:05.15 in the opposite direction of kinesin, 00:28:07.29 toward the minus end of the microtubule. 00:28:10.11 And it's evolved 00:28:11.28 a completely different mechanical element, 00:28:13.18 in fact 00:28:16.03 it's a rigid coiled-coil structure 00:28:18.11 and we've learned how this mechanical element works. 00:28:22.17 When it's in its nucleotide free state, 00:28:24.13 this lever arm, 00:28:27.29 which works much more like myosin, 00:28:30.03 is pointing to the plus end, 00:28:32.13 but when ATP binds 00:28:34.07 that lever arm swings 00:28:35.22 and it swings its cargo 00:28:37.28 and produces motion 00:28:40.02 towards the minus end of the microtubule. 00:28:42.18 So, evolution has learned 00:28:44.27 to take this basic element 00:28:46.23 of this enzymatic core 00:28:48.07 and couple it to onto different mechanical elements 00:28:51.08 to create different types of motility. 00:28:53.00 In fact, we now know so much 00:28:55.19 about the mechanism of how these motors work 00:28:58.01 that we can start to engineer 00:29:00.14 new kind of motor proteins with different functions, 00:29:03.06 and I'll just give you one example of this. 00:29:04.23 This is work from Zev Bryant's lab 00:29:06.27 and it's a pretty amazing result, 00:29:09.06 where they took that same kinesin 14 motor 00:29:11.16 that I just showed you 00:29:13.00 and they added on to that 00:29:15.08 a domain 00:29:17.22 that changes its structure 00:29:19.01 in response to blue light. 00:29:22.11 And I can't give you all the details of it, 00:29:23.26 it's found in this paper here, 00:29:25.25 but they could design this motor 00:29:28.18 such that when the light is switched on 00:29:31.27 the conformational change 00:29:34.26 that's produced upon ATP binding 00:29:36.13 switches the direction 00:29:38.20 of that mechanical element 00:29:40.05 so that it creates movement 00:29:41.29 in the opposite direction of the natural motor. 00:29:44.26 And these are microtubules moving on glass, 00:29:47.00 they're marked so you can see the direction, 00:29:49.25 and you'll see that they move in one direction in regular light, 00:29:54.25 but when you shine blue light on it 00:29:56.21 the motor completely reverses its direction of travel. 00:30:01.06 So this just shows and illustrates that, 00:30:03.04 you know, 00:30:04.06 we can actually begin to engineer these motor proteins 00:30:06.01 ourselves now. 00:30:08.25 So finally, I'd like to end with a discussion 00:30:10.26 of a little bit of how these motor proteins 00:30:12.24 are relevant for medicine. 00:30:15.02 Now, we now know that 00:30:17.28 many different diseases 00:30:19.20 are caused by mutations 00:30:22.12 in molecular motors 00:30:24.11 or in proteins associated with these motors. 00:30:26.22 So, for example, one disease, 00:30:29.03 which is called familial hypertrophic cardiomyopathy, 00:30:33.09 is caused by mutations 00:30:35.18 in cardiac myosin, 00:30:37.03 and this is a disease 00:30:39.18 that is often associated with sudden death in athletes, 00:30:42.21 and this is because they have 00:30:46.06 this enlarged heart 00:30:47.24 due to this mutation in this myosin. 00:30:50.10 Mutations of myosin 00:30:52.01 also are associated with deafness. 00:30:54.10 Mutations in dynein 00:30:56.00 give rise to diseases called 00:30:57.20 ciliary dyskinesias, 00:30:59.00 which have problems with ciliary function 00:31:01.11 such as respiratory dysfunction, 00:31:04.22 sterility, 00:31:06.23 and other problems as well. 00:31:08.18 And mutations in kinesin motors 00:31:10.12 are associated with neurodegenerative diseases. 00:31:14.18 Now, the exciting thing is that 00:31:17.00 we can also modulate motor protein function 00:31:20.04 to potentially ameliorate certain diseases, 00:31:24.21 and I'll give you one example of this 00:31:27.13 in the case of a disease called heart failure, 00:31:30.17 where the heart fails to contract vigorously enough 00:31:34.29 to properly pump out [blood], 00:31:38.10 and I showed you that's due to the function of myosin... 00:31:41.20 cardiac myosin molecules. 00:31:45.20 An exciting project 00:31:47.12 that was taken on by a company 00:31:49.03 that I cofounded 00:31:50.17 called Cytokinetics 00:31:51.28 and led actually by my first graduate student, 00:31:53.14 Fady Malik, 00:31:55.06 was to develop a small molecule 00:31:57.14 that would activate cardiac myosin 00:32:00.06 and actually make it perform better 00:32:02.01 to try to improve the contractility 00:32:04.11 of the motors 00:32:05.28 in this failing heart 00:32:07.09 and make the heart 00:32:09.24 able to pump out blood more effectively. 00:32:11.24 And the drug that came out of 00:32:13.23 a lot of work 00:32:15.20 is shown here, 00:32:16.27 Omecamtiv mercarbil, 00:32:18.27 and we know exactly the biochemical mechanism 00:32:21.04 of this drug. 00:32:22.15 It stimulates this one step 00:32:25.04 where phosphate is being released 00:32:27.17 by myosin 00:32:29.03 and the force producing step occurs, 00:32:31.19 and this drug stimulates this step 00:32:33.17 so it facilitates entry of myosin 00:32:36.12 into the force-generating state, 00:32:38.07 and this is what increases the contractility of the heart. 00:32:42.15 So that's what's shown here... 00:32:43.29 this is an echocardiogram. 00:32:46.09 You're looking at the left atrium, 00:32:49.18 the left ventricle, and the mitral valve, 00:32:51.16 and this is in a patient with heart failure, 00:32:53.15 so you can see that this valve 00:32:55.12 is kind of fluttering a bit 00:32:57.15 and that's due to the poor contractility 00:32:59.20 of the heart 00:33:01.16 in this patient with heart failure. 00:33:03.07 But now, after this patient is given this drug, 00:33:06.18 you can see what happens in this video. 00:33:08.29 Now you can see that this valve 00:33:11.13 is popping up 00:33:13.16 much more briskly 00:33:15.01 and that's due to the increased contractility of the heart, 00:33:17.29 and this drug is now 00:33:20.10 in phase 2 clinical trials, 00:33:23.18 and we'll see if this actually helps these patients 00:33:25.26 with less mortality, 00:33:27.24 less hospitalization, and so forth. 00:33:32.06 So it's an exciting time 00:33:33.22 and we'll see what happens 00:33:35.13 with this drug here. 00:33:37.19 So, in this introduction 00:33:39.23 I've shown you many things that we know 00:33:42.03 about molecular motor proteins, 00:33:43.25 but I want to assure you that 00:33:45.07 there are many open questions and problems to solve. 00:33:48.23 So, first of all, 00:33:50.09 I described to you that there 00:33:52.16 are tens of motor proteins inside of cells, 00:33:55.12 and all these motor proteins 00:33:57.20 get hooked up to hundreds 00:33:59.14 of different kinds of cargos, 00:34:01.14 and we still don't know... 00:34:03.00 we know in a few cases how this occurs, 00:34:04.24 but we don't really know the general rules 00:34:06.25 of what links all these motors 00:34:09.03 onto the correct cargos 00:34:10.19 to execute transport functions. 00:34:14.03 I also showed you how motor proteins 00:34:16.11 can be tuned 00:34:18.02 to create different types of biophysical properties, 00:34:20.02 like directionality, 00:34:21.18 like velocity, 00:34:23.19 but we still don't really understand 00:34:25.10 how those biophysical properties 00:34:28.03 have been tuned by evolution 00:34:30.17 to create very certain types 00:34:32.22 of in vivo performance 00:34:34.09 and cellular outcomes. 00:34:36.17 And lastly, I gave you one example 00:34:38.11 of how we can use a drug 00:34:40.23 to modulate a motor 00:34:42.14 to treat a particular disease, 00:34:44.02 but there are probably other ways 00:34:45.28 that we can modulate motor function 00:34:48.11 to treat other kinds of human diseases as well, 00:34:51.11 and that will be a challenge for the future. 00:34:54.18 So, with this, 00:34:56.00 I'd like to thank you, 00:34:59.02 and in the next lecture in the series 00:35:00.21 I'll tell you more 00:35:02.16 about our recent research on the dynein motor. 00:35:06.04 Thank you.