Molecular Motor Proteins

Transcript of Part 1: Molecular Motor Proteins

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