Hello. I'm Ron Vale, and in this talk I'd like to introduce you to molecular motor proteins, which are these fascinating protein machines that are featured in this animated video here. And these proteins are able to walk along a cytoskeletal track and transport a variety of different types of cargos inside of cells. What I'd like to do today is, first of all, tell you a little bit of a history of biological motility, and then I'll tell you about what these motor proteins do inside of cells, the various functions that they have, and then I'll you a little bit about how they work, how they're able to convert chemical energy into motion. And finally, I'll tell you some things about how these molecular motor proteins are relevant to human health and disease. So, first of all, biological motion is just a fundamental and very obvious attribute of all living organisms. For example, in terms of people, we're able to run, and a variety of animals are able to move and explore their environment. And this is due to the fact that we have muscles and they can contract, and theories about how muscles work are in fact very old, dating back to the ancient Greeks. A whole new world of life was evident with the invention of the microscope, and with that, also, a whole new world of motility. Leeuwenhoek, for example, when he looked at pond water under the microscope, saw a whole variety of different types of movements. And this is what he had to say about them: "The motion of most of them in the water was so swift, and so various, upwards, downwards, and roundabout, that I admit I could not but wonder at it." Indeed, all these types of biological motions are indeed very fascinating to Leeuwenhoek, and also fascinating even to school kids today. Now, not only do cells move, but material inside of cells can also undergo motion, and the first person that described this was Bonaventura Corti, where he described cytoplasmic streaming inside of plant cells. And this is shown here in this video, and what he described is shown here: "I know that the phenomenon that I announce is too strange to be believed at first... I saw two torrents inside each section... One of the torrents rose on one side and descended on the other, constantly... and this not once but thousands of times and for days, and for entire weeks." You can see that these early scientists were quite animated in their use of language to describe their scientific results, something that I think scientists have lost today. Now, microscopy continued to play a major role in our understanding of biological motility. And a pioneer in this was Shinya Inoue, who used more advanced types of microscopy, such as polarization microscopy, to observe the dynamics of living cells. And he did a lot to describe the process of cell division, which is shown here, where you can see the mitotic spindle, you can see the motion of the mitotic spindle, and also the movement of chromosomes here in this beautiful movie. Now, the next revelation, I would say a revolution, occurred with the development of fluorescent proteins. And now we can take that same object that you saw that Shinya Inoue described, where he had observed the mitotic spindle with natural contrast from the polarization microscope, but now we can tag specific proteins in the cell. Shown here are microtubules, which are tagged in red... with a red fluorescent protein, and the chromosomes with a green fluorescent protein, and if you look at this in a Drosophila embryo, you can see the chromosomes, the formation of the mitotic spindle, and the physical motion of these chromosomes here. And when people began to tag a lot of proteins in the cell, they found that they were not just static, but in fact in motion, and now we know that a whole variety of molecules inside of cells are actually undergoing active types of movement to be localized in specific destinations in the cell. Now, all of these types of biological movements that I've described are due to the actions of these special enzymes called molecular motor proteins, which interact with cytoskeletal tracks, and there are two main types of cytoskeletal tracks. One are the larger diameter microtubules, and the smaller diameter actin filaments. And these are the two major cytoskeletal filaments that act as tracks for these molecular motors. So, in the microtubules world, there are two main types of cytoskeletal motors. One are the dynein molecules, and I'm going to talk much more about these in my next two iBiology lectures, and kinesin, which will be a focus of this lecture. They move along microtubule tracks, which are shown in this movie over here, and you can see that these microtubules extend all throughout the cell, and they're also themselves quite dynamic. They can grow and shrink and change their position in cells as well. Now, these tracks, both microtubules and actin, are polar filaments. And the reason is that they're composed of a basic subunit, in this case tubulin, which polymerizes in a defined head-to-tail manner to create this polymer. So, because the polymerization is polarized, there is a distinct plus end of the microtubule and a distinct minus end. And the motors recognize this intrinsic polarity of the tracks. So, for example, the majority of kinesins will move in one direction, towards the plus end of the microtubules, whereas dyneins move in the opposite direction, towards the minus end. Now, these microtubules in cells are organized, also not randomly, but with a specific polarity. So many of the microtubules are nucleated and grow from this structure by the nucleus, which is called the centrosome. And this is where the minus ends of the microtubules are located, and they extend outward to the periphery, where the plus ends are localized. So, if a kinesin motor grabs hold of a cargo, it's going to move towards the plus end, and it's going to deliver that cargo from the interior of the cell to the periphery. Dynein, on the other hand, will move in the opposite direction, so it will deliver a cargo towards the cell interior. Now, the other major class of molecular motors work on actin filaments, and there's one major class of these motors and those are the myosin motors. Now, they move along actin, which tends to have a very different distribution than microtubules. So, in this mitotic spindle, for example, the microtubules are in the center and you can see the actin, in red, all around the perimeter of the cell. So actin, in general, tends to be more cortical, the microtubules more interior. Now, when I say myosin or kinesin or dynein, I'm not just talking about one molecular motor. In fact, these are families of related motor proteins. In humans, for example, there are 45 different kinesin genes, there are 40 myosins, and about 15 dyneins. And there's some many different types of motors because there are a whole variety of motility functions that are needed in human cells, and these different motors carry out different types of transport or force-generating functions. Now, I'd like to tell you a little bit about the anatomy of these motor proteins, what makes motors proteins in the same family similar and also how they differ. So, they have one end of the motor protein which is the actual part that moves along the track, and another part that interacts with the cargo, and I'll show you this in more detail for one member of the kinesin family called kinesin-1. And this globular domain at the end I just showed you is the motor domain, that's the part that walks along the track. Many of these motor domains also come together as dimers, in some cases even tetramers, and that dimerization is mediated by a coiled-coil, which is also interrupted -- it has hinges in it -- and that provides flexibility for the motor protein to bend. And at the very distal end is the tail domain. So, this may contain a globular domain belonging to the motor polypeptide. It may also bind other associated subunits to make a larger complex, and this tail domain is what defines, usually, what that motor protein will bind to in the cell, what kind of cargo it will transport. Now, there are other classes of motors proteins -- this just shows kinesin-2 and kinesin-3 -- and all of these motor proteins share in common a very similar motor domain, which is shown here. But the rest of the protein is really completely different. So, the coiled-coils are different, and the cargo binding domains in fact have no sequence identity between them. And the reason is that these non-motor domains, again, are defining unique types of cargos that these motors are interacting with inside of cells. So, let me tell you a little bit about the motor domain and what it does. It's an enzyme and it's an enzyme that hydrolyzes ATP. So, it first binds an ATP molecule -- adenosine triphosphate -- and then it hydrolyzes a bond between the beta- and gamma-phosphate. And after the hydrolysis it then releases the products in a sequential manner. So, it first releases a phosphate then, next, the ADP is released, and then it's able to rebind ATP and start the cycle all over again. And during one round of this ATP hydrolysis cycle there are structural changes that occur in the motor that I'll describe later that allow it to take one step along the track. So, every time it undergoes this cycle it steps forward, and then multiple rounds of this ATP hydrolysis cycle allow the motor to move a long distance along the track. Now, let me just say a few things about how this motor performs in comparison to a motor that you might be more familiar with, like a car engine. So, first of all, obviously the kinesin motor is much smaller, in fact that motor domain is only 10 nanometers in size. It uses a fuel, as I told you in the last slide, adenosine triphosphate, in comparison to your car, which uses hydrocarbons. It moves at a few millimeters per hour, which seems very slow. However, you have to take into account the size of the motor protein, and if you measure how far it moves in terms of its own length, you find in fact that it's moving faster than your car is moving on a highway. It's also much better than your car in terms of work efficiency, which is essentially how much of the chemical energy that it can convert into productive work, and these motor proteins work at about 60% efficiency, whereas your car engine works at a much more pathetic 10-15%. So, we indeed have a lot to learn about how nature's own motors are able to execute motility. So, let me tell you know a little bit about what the cytoskeletal motors do in cells, and I'm just going to provide a few examples because the number of different types of motility that cells engage in is really vast. So, one thing that the motor proteins do is to transport membrane organelles. In some cases, they're small organelles, they're transport intermediates that are traveling between the Golgi apparatus and the plasma membrane, or endosomes that are traveling in the opposite direction, from the plasma membrane to other organelles, such as lysosomes. And this just shows an example, in living cells, of some of these transport vesicles moving along microtubule tracks. But even very large organelles also can move in cells. Probably the biggest organelle of all is the nucleus, and this just shows an image of the nucleus being transported inside of a nerve cell, which during development is migrating towards the cortical region of the brain, and it has to transport the nucleus during this migration process. Now, here's another beautiful example of organelle movement. These are pigmented organelles called melanosomes that are found in special skin cells called melanocytes, and this is what gives skin its color. And some organisms such as amphibians and also fish can change the color of their skin and they do that by moving these melanosomes. When the melanosomes are dispersed like this the skin color appears darker, but when they're all concentrated in the center the skin takes on a lighter color, and this distribution of organelles occurs by motors. Here are dynein motors transporting all these melanosomes towards the cell center and kinesin motors can transport these organelles in the opposite direction, and this is under hormonal control. So, hormones interact with receptors that control these motors to change the distribution inside of cells. But also, there are other objects that are not membrane bound that also can be transported. For example, there are many kinds of viruses that have learned to pick up molecular motors and transport themselves inside of cells. This is an example of vaccinia virus and all of these little particles, these myriad little particles that you see here, are viruses moving along microtubule tracks. Another example being rabies, which can transport itself inside of nerve cells. In addition, mRNAs also can be localized and transported in cells, and that allows the mRNA to be localized in a particular region of the cell where that mRNA can be translated into a protein and therefore the protein is made where the protein is needed, in a localized destination. And this is an example of one RNA called gurken that is transported in a Drosophila oocyte, it's shown here in this orange color, and you can see it moving into this one anterior corner of the oocyte by active transport. Now, in addition to transporting cargo, motors can move the filaments themselves, and this is how muscle contraction works. And the basic unit of a muscle is called a sarcomere and it's a repeating unit of actin and myosin. In the sarcomere, myosin is concentrated in a filamentous form in the center of the sarcomere and it interacts with interdigitating actin filaments which are coming in from both sides of the sarcomere. So, myosin wants to walk in one direction on this side, towards the end of the actin, and the other side of the myosin filament is trying to walk in this direction along the actin, so when a muscle is activated, which occurs with nerve stimulation, calcium comes in -- that's calcium shown here -- and the myosin starts walking and it starts bringing these actin filaments closer together. That shortens the sarcomere and that's what causes your muscle to contract. In this case, the filaments and the motors are very well organized in your muscle, but motors can also take a disordered or almost random array of filaments and create order amongst these filaments, and this is what happens in the formation of the meiotic spindle. So, in this case, the microtubules start off random, but then there are some motors, such as shown in green here, that are crossbridging filaments and they're trying to move to the minus end. So, they're moving to the minus end and, as they do, they collect all the minus ends of the microtubules together. There are other motors, such as shown in orange, that work on antiparallel filaments, and they work to slide those filaments apart. So, in this manner, as shown in this movie of the formation of a meiotic spindle in a xenopus egg extract, you can see that this initial random organization of microtubules... as it grows, the motors act upon it and they start elongating this spindle and the minus ends all get organized at this pole and it forms this characteristic bipolar shape, due to the action of these molecular motors. So, I'd now like to turn to the subject of, how do we study the mechanism of motility? How do we understand how these motors work? Well, it's useful to look at the motility in cells, but it's more powerful to study these motors in a test tube, where you really can be able to dissect their mechanism. So, we're able to use in vitro motility assays, where can take either purified motors out of cells, or even express them in bacteria, and study their motility in a very controlled environment. In fact, one can also do that at single molecule level, so one can study the actions of even individual motors proteins as they are being transported on a filament. So, I'll show you some examples, first of in vitro motility assays. Here's an example where we have a plastic bead to which one can attach a motor protein, and then the motor will transport these beads along a filament. This is shown here in this movie, here. These are inert plastic beads being transported by kinesin along a microtubule track. Now, we can in fact get rid of the whole bead entirely and label the motor protein with a fluorescent dye. This is not really drawn to scale here, in fact the dye is very small relative to the motor, but it provides a very bright signal that we can track the motor protein as it's moving, and we can do that with a technique called total internal reflection fluorescence, which is also described in videos in the iBiology microscopy course, but this is what it looks like. All of these green dots here are individual motor proteins, and you can see them being moved along these microtubules here, so you can track and follow the details of this motion at a single molecule level. One other type of in vitro motility assay is one where the motor proteins are all coating a glass surface, and in this case the motor proteins can't move, but they grab hold of the filament and then they start transporting this filament along the track, and you'll see this in this video, here. These are microtubules and you can see them all sliding across the glass surface, driven by these molecular motor proteins. Now, using these types of in vitro assays, you can actually measure a lot of details of how these motors work. For example, using an optical trap, again described in the iBiology microscopy course, you can measure the forces produced by individual motors. The optical trap grabs hold of a bead and tries to keep the bead in place. On the other hand, the motor is trying to move along the track and trying to move the bead out of the optical trap, which is resisting, and you can eventually measure the force eventually when the motor stalls, when it can't move the bead any farther, and that's what's called the stall force, and that's the maximal force that the motor produces. And it's really incredible that one can measure these forces. They're 1-7 picoNewtons, which are very very small forces, but they can be measured quite accurately with an optical trap. This is a lot of pioneering work done in Steve Block and Jim Spudich. In addition, one can measure the steps that are produced by motors. So, you can track these single fluorophores that I just showed you with very high resolution, and you can see where... how that fluorescent dye is moving over time, and you can see that that fluorescent dye takes abrupt steps, and these abrupt steps are when the motor is actually moving from one subunit on the track to the next subunit. So, the next thing that we want to know in order to understand how motors work is what they look like. What are their structures? And for this we need higher resolution techniques like X-ray crystallography and electron microscopy, and we don't just want one snapshot of the motor. We want snapshots of the motor as it goes through its whole ATPase cycle. It's very much like in the old days when they tried to understand how a horse moves, they made high speed cinematography and got various snapshots of the horse in action, and that's what we'd like to do with the motor protein. We want different snapshots of what that motor looks like at different stages of this ATPase cycle. Now, one thing that the structure did tell us right away, when we got the structure of kinesin, is whether the motor protein myosin and kinesin would operate by a similar type of structural mechanism or not. Before the structure was available for kinesin, we actually thought that they were completely different types of motors, and there were several obvious reasons. One is that kinesin works on microtubules, while myosin works on actin. The myosin motor domain is also over 2-fold larger than that of kinesin, and if you asked a computer to line up the sequences the computer said that there wasn't any real clear amino acid identity on alignment. However, when we got the structure, we found a big surprise, and that is that there are parts of the kinesin and myosin motors that are very similar to one another structurally. In fact, these molecular motors, even though one works on microtubules and the other one works on actin, they must have evolved from a common ancestor at some point during evolution. And the part of these motors that is most common is this central core here, which is featured in blue. And this does the basic chemistry, this is the part that binds the nucleotide, it hydrolyzes it, and it also undergoes very small structural changes when it's in different nucleotide states, for example, between ADP and ATP. And that mechanism is also very similar between kinesin and myosin. These motors then also have a similar what's called a relay helix, which I'm showing here in green, and it relays information from this nucleotide binding site to a mechanical element that I'll describe in a second, and it does that by sliding back and forth between the nucleotide side and this mechanical element. So, these are the mechanical elements of myosin and kinesin and, indeed, those look completely different from one another and they work differently, as I'll show you shortly, but you can see that these mechanical elements are positioned in the same relative place relative to this common enzymatic core here. So these two different motors evolved different kinds of mechanical elements, but they kind of hooked them up to the same basic enzyme and sensing unit. So now, let me tell you a little bit about what we've learned from the structure of these motors in different nucleotide states and how that explains the motion of these motors. So, what you're seeing here is an animation, but it's based upon real structural data from muscle myosin. And this is the actin filament here. This is the... this large yellow element is the mechanical element that I just showed you, and now let's see how it works. So, when it binds to actin, actin causes this phosphate group to come off the active site and that causes a large rotation of that big lever arm-like unit, causing about a 10 nanometer displacement of the actin filament. ATP then comes in, that dissociates the myosin, and then the hydrolysis recocks that lever arm for another round. So here it is again. Phosphate release, that big swing causing the motion, the release, and the recocking. And it's millions and millions of these small displacements by myosin that collectively result in the contraction of your muscle and the shortening of that sarcomere. Now, myosin is made to work in large numbers, but kinesin has a different problem. It has to transport, potentially, these very small organelles that I showed you, and there's some indication that some of that transport is generated by single motor proteins. So kinesin has to have... can't release from the track. It has to keep holding on to the track as it moves, something that's called processive motility. So let me show you how this works. So, in red here, that is the mechanical element of kinesin, which is called the neck linker, and I'll show you how that changes its structure during the motility cycle. First, the kinesin comes on to the microtubule and the microtubule binding kicks off a bound ADP, ATP can rebind, and that ATP binding causes this mechanical element to zipper up along the blue core. And that, as you'll see, displaces the partner head. So, here is the zippering, there is the movement of the partner head from a rear site to a forward site, and that zippering of the neck linker helps to move the two motor domains in this hand-over-hand mechanism. So, here is the zippering and there's the partner head moving from the rear site to a forward site. So, this motor protein has learned to kind of walk in a coordinated manner, where the two motor domains are moving in a leap frog manner along the microtubule. Now, evolution also has learned to develop different kinds of mechanical elements, even within a superfamily, for different purposes. So, here are two different classes of kinesin motors. This is the one I just showed you in the animation, but here is another type of member of the kinesin superfamily, called kinesin 14, and it's learned to walk in the opposite direction of kinesin, toward the minus end of the microtubule. And it's evolved a completely different mechanical element, in fact it's a rigid coiled-coil structure and we've learned how this mechanical element works. When it's in its nucleotide free state, this lever arm, which works much more like myosin, is pointing to the plus end, but when ATP binds that lever arm swings and it swings its cargo and produces motion towards the minus end of the microtubule. So, evolution has learned to take this basic element of this enzymatic core and couple it to onto different mechanical elements to create different types of motility. In fact, we now know so much about the mechanism of how these motors work that we can start to engineer new kind of motor proteins with different functions, and I'll just give you one example of this. This is work from Zev Bryant's lab and it's a pretty amazing result, where they took that same kinesin 14 motor that I just showed you and they added on to that a domain that changes its structure in response to blue light. And I can't give you all the details of it, it's found in this paper here, but they could design this motor such that when the light is switched on the conformational change that's produced upon ATP binding switches the direction of that mechanical element so that it creates movement in the opposite direction of the natural motor. And these are microtubules moving on glass, they're marked so you can see the direction, and you'll see that they move in one direction in regular light, but when you shine blue light on it the motor completely reverses its direction of travel. So this just shows and illustrates that, you know, we can actually begin to engineer these motor proteins ourselves now. So finally, I'd like to end with a discussion of a little bit of how these motor proteins are relevant for medicine. Now, we now know that many different diseases are caused by mutations in molecular motors or in proteins associated with these motors. So, for example, one disease, which is called familial hypertrophic cardiomyopathy, is caused by mutations in cardiac myosin, and this is a disease that is often associated with sudden death in athletes, and this is because they have this enlarged heart due to this mutation in this myosin. Mutations of myosin also are associated with deafness. Mutations in dynein give rise to diseases called ciliary dyskinesias, which have problems with ciliary function such as respiratory dysfunction, sterility, and other problems as well. And mutations in kinesin motors are associated with neurodegenerative diseases. Now, the exciting thing is that we can also modulate motor protein function to potentially ameliorate certain diseases, and I'll give you one example of this in the case of a disease called heart failure, where the heart fails to contract vigorously enough to properly pump out [blood], and I showed you that's due to the function of myosin... cardiac myosin molecules. An exciting project that was taken on by a company that I cofounded called Cytokinetics and led actually by my first graduate student, Fady Malik, was to develop a small molecule that would activate cardiac myosin and actually make it perform better to try to improve the contractility of the motors in this failing heart and make the heart able to pump out blood more effectively. And the drug that came out of a lot of work is shown here, Omecamtiv mercarbil, and we know exactly the biochemical mechanism of this drug. It stimulates this one step where phosphate is being released by myosin and the force producing step occurs, and this drug stimulates this step so it facilitates entry of myosin into the force-generating state, and this is what increases the contractility of the heart. So that's what's shown here... this is an echocardiogram. You're looking at the left atrium, the left ventricle, and the mitral valve, and this is in a patient with heart failure, so you can see that this valve is kind of fluttering a bit and that's due to the poor contractility of the heart in this patient with heart failure. But now, after this patient is given this drug, you can see what happens in this video. Now you can see that this valve is popping up much more briskly and that's due to the increased contractility of the heart, and this drug is now in phase 2 clinical trials, and we'll see if this actually helps these patients with less mortality, less hospitalization, and so forth. So it's an exciting time and we'll see what happens with this drug here. So, in this introduction I've shown you many things that we know about molecular motor proteins, but I want to assure you that there are many open questions and problems to solve. So, first of all, I described to you that there are tens of motor proteins inside of cells, and all these motor proteins get hooked up to hundreds of different kinds of cargos, and we still don't know... we know in a few cases how this occurs, but we don't really know the general rules of what links all these motors onto the correct cargos to execute transport functions. I also showed you how motor proteins can be tuned to create different types of biophysical properties, like directionality, like velocity, but we still don't really understand how those biophysical properties have been tuned by evolution to create very certain types of in vivo performance and cellular outcomes. And lastly, I gave you one example of how we can use a drug to modulate a motor to treat a particular disease, but there are probably other ways that we can modulate motor function to treat other kinds of human diseases as well, and that will be a challenge for the future. So, with this, I'd like to thank you, and in the next lecture in the series I'll tell you more about our recent research on the dynein motor. Thank you.
