 And welcome to the valedictory function of the biology orientation cum selection camp of 2018. This is the first part of the valedictory function where we traditionally have a lecture by a scientist and today we are very pleased to have among us Rup Malik from Tappan Institute of Fundamental Research. I will give a more detailed introduction of Rup Malik later in the formal function. I just welcome Prasam Malik to give his introduction. Thank you Anvesh for having me here. And before I begin I do want to congratulate you on the fantastic service that you are doing by coordinating these Olympiads. It's a lot of work and it's well appreciated. So I have no particular plan for this talk. It is kind of going to be a mix of, you know, like a story. But the theme will revolve around what we will see are kind of machines and some of them are shown here and I will try to tell you a little bit more about them as we go on. Now all of you would be aware of this movie. I'm sure most of you have seen it. There have been many, many versions of this and there's a new one Jurassic World coming in. And this is by Michael Crickton. But probably some of you may also have read about this novel written by the same author, Michael Crickton, where he talked about these solar powered nanobots, one of these dots is a nanobot, which can organize into many kinds of structures and, you know, and then they prey on, you know, various people and it gets quite horrific at the end. But when I have read both of these and I find this to be much more interesting, you know, than actually this movie, but it didn't get so much publicity anyway. But what I want to say is that small, you know, if you look at each of these individual things, is really where the action is in many situations in nature. And, yeah, so the business end is at very, very small dimensions and, you know, of course, this dimension can vary, but I'm talking about, you know, things happening on the scales of nanometers and microns and probably even less than that. And that's where we are going to be for most of the, you know, next one hour today at the size scale of molecules which exist at nanometers and, you know, of that scale. Now, many of you may be aware that the Nobel Prize in chemistry a few years ago was awarded to people, you know, these three gentlemen, they shared this Nobel Prize who, you know, made something which is shown as a cartoon here and using the chemistry which we will not go into right now at these size scales. And if you run this movie, you will see, I mean, it doesn't look so dramatic, but basically something like this is moving around and you are imaging it using an electron microscope because it is, of course, so small. But what people have been trying to do is that they have been trying to make very tiny machines which hopefully in the future we can, you know, probably use for clinical or other kinds of applications where you have to provide some fuel. In this case, you can, you know, shine light on and off and let us say these molecules here, let's not get into the details of that. They, depending on whether they see life or not, they will undergo a structural change. And when they do that, repeated cycles of on-off light will, you know, rotate these wheels and this thing will move around. Now, as we see all this, of course, they are inspired by nature, actually. And you know, so this, you know, when this wheel is turning there is some equivalent of a molecule here and these are benzene rings where if you shine light, this thing rotates in the way shown by this arrow and, you know, this, you know, rotational motion because of shining of light, changing of pH, changing voltage, various kinds of, you know, things can, you know, be harnessed to get this thing to move around like what you saw in the previous slide. Now, of course, this is motivated by what is happening to you as, you know, you watch me, you know, give this talk here because you know that in your eye, in your retina, you have these kinds of, you know, rodents in complexes where you have proteins bound to a molecule here in the membrane. This is a lipid membrane and we will talk a little bit more about lipids here later in the talk where you have this molecule called retinol, some version of this, which when you shine light, you know, it undergoes a sister transconformation and that is, it leads to a change in this entire complex and that is registered as, you know, signal as something you have seen. So, these machines already exist in nature and we are trying to make versions of this, but our success till now is, you know, of course, nowhere near what nature has already implemented. So, as we go on, I will move to what these natural machines are and how we can study them, how we can understand them, how strong are they, what do they do. And one of the things which I particularly very close to what, you know, I like to do is that, you know, you will immediately understand that these machines are on the size scales of nanometers and but, you know, me and you are on the size scale of meters, right. So, the kind of, you know, things that these machines do, they must add up somehow so that you can see things happening over the size scale of meters, like when I talk, you know, in my vocal cords, there are vibrations. That's why, you know, I can generate a sound and that is actually because of these kinds of machines. When I move my hand, that same thing is happening but one molecule doing something is not going to bring out so much sound and allow me to move my hand. So, how do these machines cooperate in large numbers to, you know, do things that you and me are very familiar with is something very close to my heart and maybe we will be able to talk about that. Now, as we talk about machines, I will kind of keep myself restricted because of the shortage of time to proteins. Now, DNA, you know, there are different kinds of molecules. When we are talking about nature, I'm talking about biology and this is the biology of Olympiad so let us stay at biological molecules. We will talk about proteins. You can also think about DNA as machines. You can think about other kind of structures and largely, you know, overwhelmingly largely the machines that exist in, you know, in your body are actually proteins, okay? And all of you know, I don't need to go through this in detail. That proteins consist of a chain of amino acids and these amino acids can be, you know, 20, 21 different types as you see here and some of them like water, some of them don't like water and whenever we talk of biology, we must remember that everything is happening underwater, okay? So now depending on the sequence here, some parts of this protein may wind up like this. Why? Because at a particular repeat frequency, there are amino acids, let's say, which don't like water and there are amino acids then also which like water so if you make such a structure like a spring, the amino acid side chains which like water can project out and the ones which don't like water can stay inside the spring where, you know, you have some exclusion of water inside this tube and similarly you can have, you know, you can have so these are like alpha helices. All of you are familiar with this term in protein size, right? So these are alpha helices, these are beta sheets and there can be other structures like this but the main point that I, because we were talking about machines, the importance of this is that you can actually, it actually works like a spring in many situations. There is a potential energy inside that, okay? And inside such a structure or inside these kind of structures and you can store energy and release it when required, okay? And so that is why this is a machine. So that is what you're all familiar with, let us say you think about a catapult, okay? You think of it as a machine, you know, you're going to pull this and you're going to store energy in it and then you release it and then, you know, you will shoot something from that. So, you know, that's what machines do many times, right? They store energy in some way and dissipate in some other way and that's something like that you can also do here. And of course, you know, I don't need to go into this. There are many kinds of more complicated structures inside proteins and it is not easy to understand how this kind of a machine would work, okay? How something will bend here and that will create something, do something here. So that's obviously not easy to understand but there are ways in which people are trying to do that and one of the things you can do is that if you want to understand, you know, what does this part of the machine do, you can take help of something wonderful called recombinant technology where you can use molecular biology to change some residues inside that, okay? And maybe, you know, disable this thing. So if this thing is not working, what happens to the whole machine? You can ask this kind of question, okay? So, by the way, you can interrupt me at any time, all right? I'm happy to answer. And this is one example. If you can think of it as a machine, again, remember that the sizes we are talking about are typically less than 100 nanometer, 10 nanometer, 20 nanometer. That is the kind of sizes we are talking about and this, everybody knows what this is, right? This is hemoglobin, either having oxygen or not having oxygen depending on where it is in your body, in your lungs or in some other part of your body and, you know, you can see that it's depending on oxygen is bound or not, this structure changes. So you can think of this as a machine which, you know, undergoes this kind of, you know, routine periodic transformations to do its job, which is to deliver oxygen where it is needed. Now, it is important when you think about biology to think about the physical aspects of things as well. And yes, of course, the chemical aspects. So I will try to, you know, do that a little bit. Now, if you were going to, what is it like to be a machine? Now, you know that if you have seen, have all of you seen something like a lathe machine? Have you heard of what a lathe machine is? In this workshop, you have this, you know, machine things, you have a large machine, okay? Now, these machines are much smaller, obviously. And if I am standing, if you think of me as a machine, whether the fan has been turned on or not, doesn't matter to me, okay? If for these machines, the kind of, you know, quote-unquote wind, thermal vibrations, all of these things matter a lot because at those size scales, these, you know, the thermal noise itself is of the order of the energy scales involved with these machines, okay? So you know that thermal noise is measured in terms of Boltzmann constant into temperature, right? And the typical energies and, you know, thermal energies you talk about as KBT, okay? Boltzmann constant into absolute temperature. And the typical energies at which, energy scales at which these molecules work are of the order of, you know, 5 to 10 KBT, sometimes actually even lesser. So they are not that much higher than the thermal noise around it. So it's quite remarkable that they can actually work, even being so close to the thermal, you know, thermal background. So obviously then you can ask many questions and I will come to specific examples of machines. Why should you, you know, as I told you, people got the Nobel Prize for building, trying to build these kinds of machines. Why does one want to build them? How does society benefit? These are, you know, questions of the future and I mean, I, okay, I don't have that quote here, but so to answer this, you know, I read a very interesting quote somebody had said, I forgot the name of the gentleman, but if you today ask why are we building some nano machines, then it would be like asking, you know, whatever, one thousand years ago when primitive man made the wheel, asking them why are you making the wheel. Okay, it is something like that. Okay, so now because somebody made a wheel, we have highways, we have Ferrari cars and you have bullet trains. Okay, so that's the kind of motivation why build them, but the implications are obvious. If you can program a machine like this to deliver a drug inside a body at a particular, you know, organ of interest, then, you know, that would be fantastic and people are actually trying to do that. Now, there can be different, now when we now start talking about machines, I want you to understand that we are talking about things that can generate force, okay, because that's what typically any machine has to do. They can generate force and to generate, why is it important to generate force? Let us just try to understand it at a very kind of, you know, crude level. You all have, you know, seen, you know, images of cells where you have a nucleus, you have, you know, the Golgi somewhere like this, you have the endoplasmic reticulum spread out and you have all kinds of, you know, vesicles here and there and you can have mitochondria. So there is a, when you see a typical picture of a cell in your books or wherever you have seen it, there is an organization here, okay, and this doesn't happen automatically because you constantly have to fight against entropy to get this state where, you know, you organize things into different compartments. So if you don't fight entropy, if you don't burn energy to fight entropy, it will all become a soup eventually, right, just like what happens to your bedroom, okay, if you don't maintain it, it becomes a mess. So that's what will happen here also. And there must be somebody who is constantly, you know, burning energy, okay, to keep all this organized like this and unless the cell is organized like this, at a cellular level, I and you are not going to be organized like this, okay. So, and where is that energy coming from? Who is doing that? That these are all important questions and how is this organization maintained and at least some of it, actually a lot of it is maintained by the kind of machines that I am interested in and I will come to that in a bit. But before that, this is one example of a machine. There can be two kinds of machines. You know that in your cars, you have rotary machines, right. You have rotary machines, your engine called internal combustion engine or nowadays they're electric engines. They basically rotate and there is a gear tooth, right. There is a gear tooth that is coupled to an axle and then you generate your wheel rotates and that's how you generate but the wheel generates reaction against the ground so you move forward, okay. You can convert rotary motion to linear motion. That's what you're doing all the time in your car, okay. And that's what this bacteria also does in a different way. It basically has this propeller like thing, they are called and they can, you know, so they have a small engine sitting there which I won't go into right now and that engine, it uses ATP. All of you know what ATP is, that is in triphosphate. It uses ATP actually to turn this thing round and round and when it turns, there is a reaction force against these helices when they are turning so you get translational motion and this bacteria, this is important for the bacteria to live. There can be no life without motion. If it just sits at one place, where is it going to get food from, right? So it has to move around. All of you have heard of chemotaxis, right? So this is how it kind of runs around here and now you can compare it to this machine here, okay. This is the propeller of a big ship and this also turns around so the ship moves but there is a vital difference between this machine and this machine which you should appreciate is that in this world where you are talking about the micron size scales or the nanometer size scales there is nothing like inertia, okay. So in a ship, you know that, you know, all of you must have heard of big, beeping horn from this. The ship needs a horn, right? Your car needs a horn, okay. It needs a horn. Why does it need a horn? Because it is not easy to stop with it, right? You have inertia. Even if you break, you will, you know, probably stop over those distance and imagine a big ship. It can take kilometers to stop, okay. So in your world where, you know, you are driving a car, you are yourself walking or you push your friend, you are very used to inertia, okay. That, if I, so for example, if I go to the pool and I jump into the pool, I dive into the pool, I don't actually need to swim. I can almost cover half the pool if it's not a very large pool. Half the pool just by doing nothing. That is because I have inertia, okay. But suppose this guy, this bacteria jumps into a pool. Nobody has ever seen it jumping into a pool, but suppose it jumps into a pool, it will go nowhere, okay. Because everything is dominated by viscosity, okay. So maybe if people should go back and look, there is something called the Reynolds number, okay. It is the ratio of, you know, viscous to inertial forces. Look that up into that or inertial to viscous. I forgot which way. But basically it quantifies whether you are going to be in the viscosity-dominated regime or you are going to be in the inertial domain. So whatever we are going to talk about now is going to be in the viscousity-dominated regime. It's called the low Reynolds number regime, okay. For you and me, the Reynolds number can be maybe 10, 100, something like that, okay. But for these guys, the Reynolds number is 10 is for minus 5. 10 is for minus 4 of that order. So, you know, it's very difficult to move around here. So the main point is that you need to constantly burn something and generate force to move. If you stop doing something, you can't move, okay. And that is exactly what these machines must do. They must constantly burn adenosine triphosphate or whatever other molecule, usually adenosine triphosphate to move around. Otherwise they can't go anywhere. They will just sit at one place. Maybe diffuse around, yes, but, okay. So that was about these rotary motors. Now what, you know, there are other kinds of motors which are linear motors, okay. Now when I do this, I am a linear motor, okay, because I am, you know, walking in a, more or less in a straight line. And you must have all seen this movie. I'm quite sure where this, this object is the machine here of our interest. And it, this, everything is immersed in water. Again, remember, this is happening inside a cell, okay. This is all immersed in water. And this distance from here to here is about 40, 50 nanometers, okay. So it's small again. And it is carrying something on its head, just like this guy here, okay. So this is this, this is this. But there's a difference that this guy walks in, you know, in air, obviously, like you and me. But this guy has to walk underwater. This guy, if somebody pushes it, you know, he can, he has inertia. He or she has no inertia, okay. So, now, so, now the important thing is that this, they are actually walking. They are not swimming here, okay. Although they are underwater. Why are they walking? Because they require this, you know, long filament which is underneath that, right. You can see that it is, you know, it must be generating reaction force against this filament, like what you see here, right. So, you know, it is basically doing something like this. And at, so this is an enzyme, okay. All of you know what enzymes are. This is an enzyme which has an enzymatic cycle which means which has a chemical cycle where it, you know, burns the molecule, you know, removes the phosphate group from somewhere, does something somewhere. And it has this, it goes through this chemical cycle but that chemical cycle is coupled to a mechanical cycle. Okay. So, therefore, it is a mechanochemical enzyme and that mechanical cycle, because it is coupled to the chemical cycle, also repeats, okay. So, what happens here is that every time it takes a step, one molecule of ATP is diffusing around, comes and binds here, okay. It gets chewed up, okay. It gets hydrolyzed and that energy is used to bend a spring here, okay. And when that spring bends, it stores energy here. It will release by pushing this in one particular direction and which direction I will come to that in a minute. And then you keep repeating that and, you know, that's what you are seeing here in this cartoon. Now, this is a cartoon, okay. It's not a real thing. Now, this is digressing a bit from this but what we have been extremely interested in is something which not many people are seem to be interested in but is that this blue surface that you see here, okay. That is not, you know, it's very cool. When you see this movie, you just watch this motor doing thing, okay. But to us what is cooler is actually this blue surface here which consists of a lipid membrane, all right. And that lipid membrane is the surface on which all of these motors spend their lives everywhere inside the surface, okay. How does this lipid membrane organize these motors? What are the mechanical properties of these lipid membrane? These are questions which we are very interested in and maybe if I have the time, I will come to that. Are there any questions at this stage? No, everything is clear, okay. So, now, okay, please. Yeah, so in this, I have no answer to that because this happened a long time back in evolution but ATP, that is actually not true. There are many machines which have evolved around ATP also like, you know, you know, tubulin polymerizers. That is also like a machine, okay. But ATP repeats often and often because probably of its energetic properties because, you know, this is also true for ATP to some extent because what you are doing in all these molecules is that you are packing three phosphate groups and when you are doing that, you are putting in a lot of negative charge essentially. And to do that, you have to spend energy. This is not specific to ATP again, okay. And that energy is used to generate force later. So, I have no answer if you ask me exactly why is ATP used and not, I think, but ATP and ATP are both used in different ways. But this is an evolutionary question. Okay, all right. So, now here is a cartoon of a cell and all of you have heard about what microtubules are. All of you, okay. So, these are microtubules and you can see a real version of this in this fluorescent image. This is a cell taken from underneath the skin of a frog, okay. And this is a, you know, melanocyte and you can see this beautiful radial structure. It looks like, you know, some supernova explosion or something. But these are all these microtubules, these straight lines here. And these microtubules have one end which is so-called plus end and the other end which is usually somewhere here which is called the minus end and they have an asymmetry, okay. So, this plus and minus is the fast-growing end and is the slow-growing end. But if you really look, if I go back, if you look, you know, here, closely, they are polymers, okay. They are made about of this protein called tubulin. You take one tubulin, take another tubulin, take another tubulin and join them like this. But importantly, there are two kinds of tubulins, alpha and beta, which are so alpha, beta, alpha, beta, and then there are protofilament. This is a multilane highway. And these motors can recognize which is alpha and beta, okay. And that is how there are some which will move towards the beta end of the microtubule and some which will move towards the alpha end and this is going to be important for what I'm going to tell you in a bit. So, there is an asymmetry in this system which is alpha is not beta and they are arranged one after the other and that's how, you know, this kind of organization arises inside the cell because you have these filaments, right. So, you have these filaments and you have to imagine alpha, beta, alpha, beta, alpha, beta along this, okay. And these motors can recognize them. And so, now, if you, if I show you, if I run this movie, so let me run this movie first, right. So, this is, so these dots are herpes virus particles which are, when I run this movie, you will see this movie is sped up about ten times or so. They are inside and the axon of a neuron and they are moving in this, they're going to be moving in this manner, okay. So, some are going this way, some are going that way and there is no apparent order in this, okay. This is true for almost any kind of virus, any kind of bacteria that infects you that this motion is driven by the kind of protein which I showed you here and they are called motor proteins, okay. Almost all kinds of viral bacterial, all kinds of pathogenic infections, they can hijack these motors and that is how, you know, that is important for their life cycle and I will come to that in a bit. Now, when you see this movie running, this is actually, the whole thing is ten minutes, but you will see all these black dots come to the center and that's what you are seeing here also. All the black dots are at the center here, okay. So, each of those black dots here is a ball of, little ball of ink, okay. It's called a pigment granule and these cells are underneath your skin and your brain can send signals, cyclic AMP or the other hormone I forgot. Basically, there are two kinds of signals by which you can either get all the ink balls to come to the center or you can use them to go to the periphery and that is how you, you know, you can, you can change and you probably know that the chameleon uses this in a very nice way to change its colors, okay, when it has to camouflage itself and this motion actually is, when I run this again, if you look carefully, each of these granules is not just starting here and going to the center. Each of them is moving in a back and forth, back and forth manner, but there is a net direction, that's why everything comes to the center which we are trying to, we and many others are trying to understand. Now, if you look here, imagine this as one of these ink granules, here it is a mitochondria, but these things are true for both ingranules and mitochondria. What happens is that typically you have these kind of motor proteins, like I told you about kinesin, the one which I showed you in that movie is called kinesin. Kinesin comes from the word kain, kain in Greek means to move, okay, so kinesin is shown here in red and there's a little arrow which shows you that kinesin generates force in this direction towards the plus sign of the microtubule which is typically towards the periphery of the cell and the dynein generates force inwards which is towards the minus end of the microtubule and quite remarkably almost all kinds of things which are moving around like this inside the cell, they have both kinds of motors attached to them and so they both generate force and that's why this thing moves in a back and forth manner, but eventually the cell can control whether dynein will dominate or kinesin will dominate and in this case dynein dominated and that's why you saw everything going to the center at the end, okay. I can also give a different kind of form on and this movie will exactly run in reverse and everything will go towards the periphery that is when kinesin is dominating so there is a lot of interest in trying to understand how this opposing activity of these two kinds of motors is controlled and that is also of interest to us and remember that this opposing activity is important for generating this spatial organization, okay. The Golgi is here because it is bound to a microtubule which have dynein on it and there is a lot of dynein activity on the Golgi which is keeping it here. The ER is spread out, these mitochondria whatever they want because you know where they are needed, let's say for example a neuron is growing in a child's brain, okay. That neuron grows and it has to branch to build the entire network and at that branch you have, mitochondria has to be taken there, the energy has to be supplied there to build that branch and that's actually what these motors are able to do. So I have about 15-20 minutes more, right. Yeah, so I think I have one time. So now we have also been trying to ask this question and I will tell you a little bit about what we do. Now I will, what you are seeing here is a cultured cell, in this case a dicustelium cell, okay. Dicustelium is an amoeba but you can basically do this experiment with any other cell also and when I run this movie you will see a lot of things moving around inside this, okay. And these are actually small plastic beads roughly one micron size so what we do is that we can grow these cells on a cover slip, on a microscope cover slip, they will grow happily and multiply and then there is medium around that, water basically with some nutrients and you can purchase these beads from, you know, various places and you add them to this medium and these beads settle down after a while and now these cells you have these kinds of cells in your immune system also. They are your first line of defense against all kinds of infections. These are called macrophage cells dendritic cells, you know various cells of that family their job is to eat up whatever nasty things are coming in, okay and they eat up bacteria, pathogens diesel fumes, diesel particles all these kinds of things in your lungs or in your other parts of your body and those when something comes from outside and is taken in inside the cell it is enclosed in a lipid membrane okay, so this is so imagine something good or bad, who knows comes in this is what will happen here this object is here and then eventually it will come here okay, with this object still sitting inside and this now quickly gets onto these microtubules why, because this now if we zoom in here there is a lipid membrane here okay, it consists of phospholipids, all of you know it is very difficult to draw this, basically a bilayer membrane and to this these motor proteins, these tinnins and the tinnins will attach, okay how they attach and all that, that I can get, I do not want to get into that but when that happens these guys start moving inside okay and you see this happening, now what you can also do is that you can take these cells you can burst them open okay, by applying mechanical shear or by changing the medium and when you do that and you can take these objects and put them on a single microtubule so on this, in this picture this is now outside a cell this is inside a cell, I put them on a microtubule and they move like this okay, and what you are seeing is real time, I have not sped it up at all so this is happening at so these velocities are typically one micron per second, one micron per second may seem small to you but inside a cell it is pretty fast okay, because a cell is about maybe 30, 40 microns, so you know you can go from one end to the other I mean it does not work like that but in fairly short time so right, so imagine these kinesin motors or the dinin motors attached to each of these objects and you know they are doing their thing and you know they are moving in this case we only had kinesin motors therefore they were all moving in one direction but you can also do experiments where they are both and then you will see this kind of motion now the important thing to appreciate is the electron microscope image they really exist, they do not just exist in cartoons, they are real you can actually see them in an electron microscope and they actually walk on stepping stones here okay, and these stepping stones why are they stepping stones because this peak cannot just land anywhere on this polymer it has a particular place where the local charges are correct for charge matching shape matching between these two and so they will just land on stepping stones and these stepping stones are eight nanometers apart from each other okay, so eight, eight, eight, eight and that is how it moves here now this is important to okay, so let me just go back here so let us talk a little bit about the kind of energies involved in this as I told you that the amount of energy available from an ATP molecule adenosine triphosphate is a certain amount of energy is available and that energy is approximately 100 piconewton nanometer now this is, all of you will know that this is a unit of force into distance that is the unit of work it is the unit of energy you can convert this into joule or whatever you want piconewton nanometer okay, why do I use piconewton nanometer and I can also write this in terms of Newton meter but because the relevant forces are of the order of piconewton pico is 10s power minus 12 minus 12 okay and nanometer is 10s power minus 9 meter okay, now let us try to understand what kind of forces put these motors generate okay, so the amount of energy you are putting into the molecule, the amount of maximum amount of work suppose it was 100 percent efficient the maximum amount of work it would be equal to this it cannot do more than that because that is how much you are giving it okay, now what are the dimensions of these motors okay, they are of the order of nanometer tens of nanometer 10 nanometer, so they can certainly never move than 10 nanometer, if you are yourself one meter long how can you move one kilometer so they are one so so this number here is of the distance moved is typically of the order of 10 nanometer I told you the distance between these stepping stones is 8 nanometer that is approximately 10 nanometer then the forces are going to be of the order of 10 piconewton right, 10 into 10 will be 100 and this is actually 25 times thermal energy there approximately that much okay, so and they are not going to be 100 percent efficient obviously but they are remarkably efficient so probably you know that the one of the most efficient cars that man has made is the Honda Accord okay, so and that has an efficiency of about 10 percent you know whatever fuel it burns or how far it goes 10 percent is the efficiency of a Honda Accord these you can calculate the efficiency and I will show you how work can do that these are about 40-50 percent efficient in fact some of the rotary motors are about 80 percent efficient okay, so that is quite remarkable so now then you can also you can measure the kind of forces that they generate and by measuring the forces these piconewton and by measuring the displacement you can calculate how strong they are, how efficient they are etc etc etc and you can actually do much more you can learn a lot of biology from this so our interest is of course in the physics behind this but our really main interest in understanding the biology what does it do to your body and how do things matter so what you can do here and I will not go into the details of this is that you can take these so remember I showed you these beads which are moving around inside says they were called phagosomes okay, maybe I should write so when this thing has gone in this object is called a phagosome any kind of bacteria that goes into your body goes into a phagosome okay and it is very important clinically to understand how the cell deals with this and that's what we are trying to understand because many kinds of pathogens can avoid what these motors do to them and let's say hope I have time to go into that anyway what you can do is that you can take these phagosomes for example and you can pull them out of the cell and they move on that microtubule I showed you so here is a cover slip and you have stuck a microtubule on that and from underneath you can shoot a laser like this and you put a lens here which is typically the objective lens of your microscope so you will focus this laser light somewhere and this here so this is called a diffraction limited spot because this is diffraction limited optics and this spot is typically roughly 500 nanometer to a micron of that weight okay and in this diffraction limited spot you can hold objects which are micron size objects okay and these beads that I told you have one micron diameter you can hold them over there now and that's what you are seeing here now what happens is that this laser light which is coming in from beneath is a constant stream stream of photons light consists of photons and each of these photons goes in here and when it sees this material which has a certain refractive index the photon will deviate from its path in the light will bend like a diffraction okay and when the photon moves in a different direction its momentum changes because the direction is changing because of this momentum change there is an because of Newton's law there is an equal and opposite force okay and that force is like a small kick that each photon gives to this particle as it passes by okay and without going into more detail I don't have the time for that you can show that this particle gets trapped in a in a field of light in a gradient of light so remember that there is more light here focus the light there and this you can actually show that this particle is trapped in something called a harmonic potential have you all heard know what a harmonic potential is it's basically imagine taking a spring a simple spring and you pull it okay pulling it a little bit is easy pulling it more becomes difficult and difficult and difficult okay and if you see that so I am not writing it here okay so basically the amount of force is proportional to how much you pull it out within some regimes okay that's called the harmonic regime and that yeah here it is the proportionality constant is going to be you know some spring constant there should be a minus sign here because it's in the opposite direction but basically this is a harmonic spring and you can when you pull it you have to now imagine that if I tie you with a spring to this wall okay and I ask you to move forward initially it is easy to move but as you go further and further because this distance is increasing the opposing force increase the restoring force increases and it will become difficult for me to go exactly that is happening to this motor here okay as it tries to move now more and more away it can't move beyond a certain point because the restoring force is more than the maximum force this guy can exert and then this detaches here and then you fall back to the center again and this will repeat and repeat and this is the kind of data that you get from these kinds of experiments where you have time on this axis this typically you know this could be about 10 seconds 5 seconds or so and this you have distance on the other side this distance from the center of the trap how far you have gone and imagine a spring every time this is happening maybe I have a movie here yeah there this is a real experiment where this is one of those beads which is being so imagine remember the laser is like this coming in like this okay and it is focused here and it is being pulled out by the motor which you cannot see in this case and when you do that it goes out and then it will fall back okay so you can do this kind of experiments and you can measure this displacement very precisely and this is the data time versus distance this distance is this distance okay and you get these kinds of plots and here you can see if this is distance and this is time the slope is a velocity right and this slows down obviously because you have to do more work now against larger force and but you have to imagine a spring you are stretching a spring every time and you can see here this movement now is in steps you can see these steps clearly and these are 8 nanometer steps okay so these are the kind of experiments you do but in reality this is what the surface of that phagosome looks like okay there are not just motors there are going to be you know many other things as in biology all the time and to make matters more complicated you have both dinin motors and kinesin motors which want to pull in opposite directions existing on the surface of each phagosome and varying numbers of that we don't even know what those numbers are okay so to understand this complicated system is one thing that we do and what we were shown a 5 minutes so what we had shown if I go 5 minutes more is it okay yeah what we had shown a while back is that on each phagosome which was moving in that manner you have this kind of an arrangement where you have many weak dinin motors in competition and this is actually a tug of war we could show that okay and I will show you this here in competition against a single kinesin motor this is like Superman here okay so this guy exerts about 5-6 piconewtons of force and these exert significantly lesser force one piconewton force but then this if you have more dinin this is a force balanced tug of war okay nature has not made a strong dinin okay it has made multiple weak dinin opposed to a strong kinesin and who knows why but an intelligent guest is because this process can now be controlled okay you can add one dinin or remove one dinin and tilt the balance in one favor if you have two strong kinesins there will be not much opportunity for change but we can only speculate on that okay and if I run this movie if it runs you will see so okay so let me just this is happening here these two particles where one before this movie started and they have now been stretched and if you look carefully there is a tube connecting them okay so these are in this case these are we have taken cells and we have just burst them open cells are taking in nutrients all the time okay from the outside medium and these nutrients are liquid media and they are enclosed in a small particle which has a membrane around it it basically food which is going to be degraded and digested they are called endosomes okay and just like this is a phagosome if I phagosome when you take in a solid part if I take in liquid nutrients they are called endosomes okay yeah so this you will see there is a tug of war happening you can see this membrane is stretched here there one gave up and the other will again pull so this you can see this and sometimes and then finally it will go in one direction there so this you can see this happening and this again these are about half a micron or so each of them in fact okay anyway so okay so cutting a long story short what we I can never remember what slide I have made so I will move on to the board a little bit now and okay the question that we have been interested in is that now let us imagine that this board is a cell okay it is a macrophage cell in your body it is part of a immune system and suppose somebody comes in from outside it could be a bacteria it could be a mycobacteria mycobacterium tuberculosis all of you know let's say a particle of mycobacterium comes in from outside it will form an imagination like this here it will come here this membrane here will bend like this okay and here is your bad guy eventually it will be taken in okay because there is active force generated all of you know that this you have heard of actin right so this cortex this is the cortex of the cell you have actin here and there are motor proteins in that actin which pull and push those actin and this eventually it breaks up and you have something enclosed in a bilayer membrane here okay and this is these are phospholipids here and now there are let's say the nucleus of the cell is here okay and this is the plus end of the microtubule because it's towards the periphery and this is the minus end towards the nucleus and on this you have these let me draw different kind what is d d k k k k means a kinesin and d means a dinin okay so you have both these kinds of motor attached to this and this object will therefore will I don't have the movie here it will move in a back and forth manner now what is quite remarkable actually is that this back and forth motion happens for approximately 10 minutes in most kind of sense in point after 10 minutes pass this back and forth motion stops and this object now will go very rapidly towards the nucleus of the cell where you have other things sitting which are called yeah maybe I'll draw again anyway I hope you can see so I will move away okay so you have things called lysosomes have all of you heard of lysosomes lysosomes are you know the bad place in the cell where you get which are acidic and where you you get degraded so these cells want to take these pathogens to the lysosomes where they have to be degraded that's how you know they will digest all these nasty things and now so there is some kind of a switch happening after some time after which the dynein motors predominates okay and you rapidly take this thing here now this switch is very important to understand because many pathogens can actually prevent this switch okay and by preventing that switch they don't go here and they survive and they multiply okay so we have been very interested in this switch and what we were able to show I will not go into details okay it's a very unusual mechanism by which this switch happens okay how could this switch happen usually you would think that okay we will add 20 more dyneins to this okay and then you will you know obviously go this way or we will remove the kinesis now that doesn't happen and you can actually show mathematically why that does not happen I won't go into that right now there is a simple surface area argument because this contact area between the molecule and this is limited okay increasing the dyneins or kinesis you would require 10 to 20 fold more to do anything because the surface area is limited rather what happens is that as you move around in this manner you start accumulating cholesterol on this membrane okay where does this cholesterol come from cholesterol comes from sorry cholesterol comes from other organelles which are also moving around at the same time and they stick with each other and so this object after 10 minutes has lots of cholesterol on it and this cholesterol is a hydrophobic molecule so in this lipid membrane there is when the cholesterol increases beyond a certain amount that cholesterol is like imagine like having water drops sorry oil drops on water okay that oil coalesces and you start getting levels of cholesterol on this and these are called lipid drafts many of you may have heard and the dynein motor actually likes this cholesterol okay so you get lots of dynein motors accumulating this cholesterol and because this region is similar to the contact area between these two if this rotates in a particular direction remember there are other molecules here all of these dynein motors together because they are right at that place okay and they can take this here now I will end by just you know bringing up one point is that what I am telling you here is that this dynein motor if you put lots of them together they seem to be able to work well together that's how this thing is taken to the lysosome this is actually not true for kinesin okay kinesin if you put lots of kinesin motors together and we and others have done that you cannot you know get much more out of this you know five kinesins will practically do what maybe one or one and a half kinesin done but that's not true for dynein okay this dynein motor if you take n motors up to a certain limit of course then there the amount of force is generated the time over which they can generate this force is n times the number is proportional to n okay and we were able to show that nicely and we have provided some evidence for why this happens how can this motor this the dynein motor work well in large teams it seems like that every motor has an automatic gear mechanism inside it by which it can vary its velocity depending on how hard you pull it back and so if you have a team of four dyneins going the one at the front when it sees a load from behind maybe by kinesin or by something else it can rapidly shift to lower gears and slow down and wait for the ones which are the back which are in higher gear which are coming faster to bunch together mechanically and that's how they can because when they're close together they can share mechanical load better and that maybe one reason by which you know this kind of a gear having a gear like ability allows them to work in large teams and it is also intriguing that many processes in nature which require large forces actually use dynein although dynein is a weaker motor than kinesin it's probably because it can work well in teams but we'll have to see that I will stop there and I guess I'm on time and I would like to acknowledge these people some of the names are missing who actually do all the hard work while we come and talk Questions? Sir here you explained that before the switch it gets towards the nucleus so sir for coming towards the nucleus the dynein should be more yeah so that's what it doesn't quickly come to the nucleus it spends some time going back and forth where they are both equal activity okay but then after a certain time this cholesterol accumulates and the dynein clusters and that's when things change so your previous slide said that vesicular transport is like a coin tossing experiment yeah I did not have the time to go into that if you want I can discuss it this will require a little more time I can discuss with you so till now there are only this is the first instance where we are showing that these kind of liquid drops are there on phygozomes nobody has said that before probably they are also formed on endozomes because there are many things similar between these two but other organelles I don't they are normally spoken in the context of the plasma moment right in terms of intercellular organelles in these kind of domains hardly anything is known so we had a hard time convincing people that this exists in intercellular organelles also but in this case there has to be something specific to be added why should it form only on these because if this is a random event then for any organelle they would tend to land up with more dynein no no because the liquid the liquid content is very different from other organelles it is not used with anything in the picture you just showed lysosomes very close vicinity to the nucleus this kind of arrangement would really be existing in the cell or imagine lysosomes you can this I have exaggerated here but you will see lysosomes in there but the large majority is close to the nucleus it is not sitting at the periphery they usually don't show it this way in the pictures no this is a drawing it is not anything it is a very interesting scenario if this happens to be true what you are proposing and what I have done that basically the dynein concentrates with the help of cholesterol can be one of the primary factors to target lysosomes interesting in the sense because then it can be a very well mechanism for innate not specific to a pathosome but a general mechanism for a cell to take care of all pathogenic infections it is in that case altering that would become very extremely in terms of our experiments are not done with a real pathogen they are just done with any a benign inert plastic it is happening on that others have shown that this kind of an accumulation of not directly but a particular protein which has affinity cholesterol you can see that forming on the surface of leshmenia particles in fact what I did not go into that I could show in this we published that that leshmenia actually disrupts these clusters we could show that implication is very profound in terms of treatment of a pathosomal you will see because then you can have a very generalized treatment against all the infections in fact it is known and this is not so much informant that people who go to hospitals and get hospital infections many people who take statins who have low cholesterol are more statistically more prone to hospital infections whether it is because of this or not of course for this switch to happen you said one option was removal of the kinesin but you said that is not possible the kinesin is actually not removed here but why that happens is why kinesin is not removed so there are two ways of doing this switch one is you remove kinesin the other is you add dye now turns out that the surface area of this if I assume it to be a sphere it need not be exact sphere but it will go as the square of the dimension but and you can calculate this contact area it is about 3 to 4 percent of the total surface area now suppose I randomly throw suppose I have to increase the number of dyes but when I increase it what fraction will come into this area it is going to be about 4 percent a very small fraction so because it is goes as the square so therefore to now have 6 more dyes on that 4 percent or rather 6 will go there right so it is not a feasible mechanism to do this more feasible mechanism is to change the geometrical organization of this so in fact between this and this there is no difference that we can detect between kinesin and dynein numbers but so you know exactly how this has happened back in evolution I cannot say I can just argue on the basis of is anything known about how the pathogens can prevent that effect from happening this I mean prevent dynein from accumulating so that is what I was saying since you asked I will say a little bit more on that what we did is that we were specifically interested in a pathogen called Lysmenia Lysmenia says is kala azar very dangerous disease but what we showed is that suppose you have this pagozoam on which you have this cholesterol patches and you have these motors here what we could do is that we could purify a particular lipid from this pathogen we did not purify it somebody else and this you can never remember the name of that lipid but it does not matter okay lipid L and this lipid has lots of phosphate groups on it okay lipophosphoglycans yeah this has it is glycolipid it has lots of phosphate groups on it each phosphate group brings in negative charge your fourth mind it brings in negative charge and this lipid also has a part which kind of injects it into this here it likes cholesterol so now you can imagine when Lysmenia infects you will have I mean over and above dinin you will have lots of these lipids stuck here okay and people believe there is no hard evidence for this is that when these lipids get stuck here they bring in a huge amount of negative charge into this region and that elapses statically destabilizes this region we could actually show that when you have this lipid these clusters are visibly distributed like this okay we could show that so that maybe and these things don't move okay so that maybe one mechanism by which Lysmenia avoids you know going here now then again these pathogens they are not one trick ponies they do many things to disrupt the successful ones do many things any other questions okay thank you if not let's thank Prasam Malik again so there is a tea break now and we'll start the formal function at 11.30 I have one announcement to make for the students when you check out of the hostel so a security guard will come to your room and check the room before you can leave the key so please allow time for that and be patient while they do this thank you okay let's go and have tea outside