Molecular motor proteins are fascinating enzymes that power much of the movement performed by living organisms. In the first part of this lecture, I will provide an overview of the motors that move along cytoskeletal tracks (kinesin and dynein which move along microtubules and myosin which moves along actin). The main focus of this lecture is on how motor proteins work. How does a nanoscale protein convert energy from ATP hydrolysis into unidirectional motion and force production? What tools do we have at our disposal to study them? The first part of the lecture will focus on these questions for kinesin (a microtubule-based motor) and myosin (an actin-based motor), since they have been the subject of extensive studies and good models for their mechanisms have emerged. I conclude by discussing the importance of understanding motor proteins for human disease, in particular illustrating a recent biotechnology effort from Cytokinetics, Inc. to develop drugs that activate cardiac myosins to improve cardiac contractility in patients suffering from heart failure. The first part of the lecture is directed to a general audience or a beginning graduate class.
In the second part of this lecture, I will discuss our laboratory’s current work on the mechanism of movement by dynein, a motor protein about which we still know very little. This is a research story in progress, where some advances have been made. However, much remains to be done in order to understand how this motor works.
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,
00:02:51.07 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,
00:31:06.23 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.
Last week we discussed how actin uses energy from polymerization to bias random thermal motion and produce directed motility. Transposing this idea to a kinesin motor on a microtubule track: how could it use energy from ATP hydrolysis to bias random thermal motion to produce directed motility?
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,
00:05:26.14 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,
00:23:48.17 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,
00:33:30.29 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
00:35:17.26 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.
Professor of the Department of Cellular and Molecular Pharmacology; Investigator in the Howard Hughes Medical Institute
University of California, San Francisco Continue Reading