Session 6: Cytoskeletal 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.

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