 Yes, reporting in progress. Okay. Here is the second part of a professor of knowledge lecture on biophysics. And let's continue. Okay. Here. So the second part. In the second part, I will start talking more about the outcomes of cell division that we discussed a little bit in the questions after the first part. So I mentioned at the beginning that chromosome segregation errors can cause aneuploidy just to make it more closer to you. This is how a segregation error looks. So these are the bridging microtubules of the methotic spindle. These are the kinetochores in pink. And this is one kinetochore that is lagging behind the others. And it may end up in a wrong cell, so that one cell gets both parts, both copies of this kinetochore, and the other one gets zero. So this is very bad. And then this kind of aneuploidy types look like this. This is a human cell. These are our chromosomes. As you know, we should have two copies of each chromosome, but this is a cell that had many errors over many generations before. So it has, for example, three copies of chromosome two and so on. This is what we mean by aneuploidy when a cell has a wrong number of chromosomes. And this is, as I mentioned before, very common in many types of cancer. And it's also thought to contribute to the appearance of cancer in the first place. But this is not very well understood. What is the interplay between aneuploidy and cancer, and how aneuploidy itself cannot lead to cancer? Okay, but I will start this by asking a question. Which chromosome is this one here? Is it some specific chromosome that makes mistakes more often than the others? Do some chromosomes make these mistakes more often? And this is something that we worked on recently. And in particular, we ask whether the chromosome location with respect to the mitotic spindle will affect its fate. By fate, I mean what will happen by the end of mitosis, whether it will segregate correctly or not correctly. So the idea is that it's not the same whether the chromosome matters, whether the chromosome is found in a certain regions of the cell behind the spindle pole. This is a spindle pole, or maybe here in the central part. This was the idea, and we wanted to test this. So it's known that different chromosomes, when the mitosis starts, they have different rules, how they come to the central part of the spindle. So some chromosomes that are found by accident or by some other reason, in the middle here in the region between the two center zones, they very quickly attach to microtubules and become aligned at the metaphase plate, which we have seen in the previous talk. Then the chromosomes that are somewhere here, they also kind of reach the spindle reasonably quickly. But it's not very well known what is happening with these chromosomes. These are called polar because they are behind the poles. So if you make this kind of line perpendicular to the spindle axis, then all the chromosomes that are behind this line, they are polar. And they are very interesting, very little is known about them. They are very interesting because if the chromosome is here, it has to somehow pass this region circumvent, bypass this region of the center zone to come to this place because this is the goal where the chromosome needs to go. So it's known that this kind of polar chromosome depends on certain motor proteins. In particular, the motor protein called dynein is moving this chromosome towards the pole. And it's also known that when the chromosome is somewhere here, the motor protein same P moves it towards the equatorial plane, sorry. But it's not known how the chromosome switches from here to here. And especially because this is a very crowded area with the loss of microtubules, how does the chromosome then come from here to here? So how to answer such a question? Well, the most direct way is to use microscopy and to observe all chromosomes and what they do. And this is what we decided to do. So we took a cell in which we have, it's a human cell, we labeled all kinetocores and centrosomes. And they are labeled all of them in green, but I'm showing you here in different colors the Z position. So the position along the Z axis or depth, meaning that those kinetocores that are more in orange color or yellow, they are down. And these more in blue and pink and red again, they are more up. So this is the Z position of the kinetocores. And we make this kind of movies. We film the cell from the beginning before my tosses even begins. And now you see the spindle forms. Now the chromosomes come to the metaphase plate, and eventually they settle it. And the track by software, all of these kinetocores with respect to the centrosomes, here's one centrosome on the other side was the other centrosome. And we can then track every kinetocore and see what it did. And then we wanted to analyze our polar chromosomes. And we defined three types of chromosomes in the following way. So when my tosses start, we have the two centrosomes. And then we call the chromosomes polar, those that are behind the centrosome. Then we have the central ones, they're in the central part between the two centrosomes. And then we have peripheral nonpolar. So they are in the periphery of the nucleus, but they are not polar, they are between the two centrosomes. And we measured the time that each of these groups, chromosomes in each of these groups, take to get aligned at the metaphase plate. And we found that indeed, as we hypothesized, that the polar ones would take more time to align, because they somehow need to get around this centrosome. They take more time than peripheral nonpolar and then central chromosomes. And I have to emphasize that the polar and peripheral nonpolar, we took these chromosomes in such a way that they all had the same on average, this group and the average of this group had the same distance to the metaphase plate. So it's not about the distance. It's not about that the polar ones have to cover a larger distance. These guys also have to cover a large distance. But the polar ones take more time because they are delayed in the region of the centrosome. They somehow spend more time in this region, because for some reason it's hard to pass the centrosome. And this is something that is unknown and that we are also currently studying. Okay. These, what I have shown you, the movie on the previous slide, this is a healthy cell. So it doesn't make errors because it has the checkpoint that checks that all the chromosomes have to be aligned. Everything has to be fine. Only then it will segregate the chromosomes. So this is why on healthy cells, we can measure only how much time it takes the chromosome to get aligned, but we cannot measure any errors in segregation because there are no errors. But we can do this trick. We can inhibit protein called MP1, which is an important protein of this checkpoint. So now we have a cell, when we treated it with this inhibitor, this cell doesn't have a properly working checkpoint. So it starts to segregate chromosomes, even though not all the chromosomes are properly attached, and it makes a lot of errors. And you can see the errors here. For example, these chromosomes that are behind, when you see the two groups of chromosomes separating in the middle, some chromosomes remain and there are potential errors. And also, you can see some chromosomes, like for example, there is a blue one that will come here. Here it is. These are two kinetics and they will move to the same pole. They will not separate one on one side and one on the other side. So these kinds of cells make a lot of errors. And now we can measure again whether it depends on our polar chromosomes. So first of all, we found that the number of errors correlates with the number of polar chromosomes. So the cells that had more chromosomes, more chromosomes behind the pole here in the beginning of metosis, they at the end of metosis made more errors. And also, when we looked at the error type, we found that the polar chromosomes in particular had a lot of errors, and these errors were either unaligned chromosomes, this is the one that stays at the pole all the time, or the lagging one that you saw that is lagging between the two regions. While the central chromosomes predominantly don't make any errors, meaning it's very important where you are in the beginning, if you're a chromosome, where you are in the beginning of metosis, this will determine how many errors there will be. Okay, this is just a scheme of these errors. So unaligned are here. This is the unaligned for the polar chromosomes and lagging chromosomes. These are the errors that these chromosomes make. Then we turn to cancer cells to see how this is in cancer cells. And this is an osteosarcoma cell, which often has these polar chromosomes. And we made this kind of analysis. We again made movies of these cells and followed these kinetics, especially the ones that stay at the pole. And we asked how many unaligned chromosomes come from the polar region? This is depicted here. So when you have cells in metaphase, and the majority of the chromosomes are here, sometimes we have an unaligned chromosome, this guy here, and we are asking how many of them come from the polar region behind the pole, and how many come from other regions. What we expect is 22 or so percent. If everything was random, then there will be 22% because this is the fraction of chromosomes from the polar region. And this is what we saw in the experiment. So much more than expected from a random case, we see more than 60% of these unaligned chromosomes come from the polar region. So the polar region is really a problematic region. These chromosomes typically end up unaligned here. And now let's see what happens with them with these unaligned chromosomes in anaphase. So we looked at errors in anaphase. Anaphase is the phase when the chromosomes are separating. And I'm showing you here three kinds of errors. One is unaligned, that is the chromosome that stays completely unaligned. It never went to the equatorial plane. It stays here, and both parts of the chromosome are retained in one cell, one daughter cell, and nothing is on the other side. Then we have the lagging chromosome. This is one lagging chromosome, which can then end up either here or there. And then we have a so-called chromosome bridge. This is a bridge of DNA. So this kind of error, this is an error because this DNA will break in the end. So you will have a broken DNA, but this is an error that is not related to microtubules in the spindle, but it's more related to the improperly replicated DNA in these kind of processes. And we asked, how many of these errors are preceded by an unaligned chromosome in metaphase? What we expect from random behavior, meaning that if unaligned chromosomes do not influence the errors, is something like 40%. And these are the results. Now, for unaligned, we have 100% because by definition, these are the chromosomes that are unaligned in anaphase, and they are preceded by being unaligned in metaphase. So this is no surprise. This is just a definition of the unaligned chromosome. Now let's look at the chromosome bridge. Chromosome bridge is very close. The measured value is very close, not statistically different from the expected from a random case. And this is because the chromatin bridge is not related to microtubules and alignment and so on. It's just a matter of unreplicated DNA. So we expect this one to not be related to unaligned chromosomes. And then the interesting part is this one, the lagging chromosome. These guys here that lag behind the others, they are often, much more often than expected, they are preceded by an unaligned chromosome in metaphase. So somehow this unaligned chromosome in metaphase ends up with one part lagging and one part properly segmenting. So altogether this means that the chromosome location behind the pole makes the chromosome delay their congression. Congregation is when they move to the metaphase plate and this contributes to the high frequency of segregation errors of these polar chromosomes. So the chromosome location determines this fate. This is just a conclusion and we call this a danger zone. So behind the pole there is this danger zone. If a chromosome is here, it will have a more chance to mis-segregate, meaning make an error in segregation than the chromosome in this region. And this is very important because different, biologically, medically, it's very important because different cancers have different, very specific chromosomes multiplied or sometimes not two copies, but only one copy of a chromosome. So for example, it's typically in several types of cancer, chromosome 7, for example, is present in more copies. And this can be related to the position of the chromosomes before the spindle forms because chromosomes have their territories in the nucleus before the division and this may then affect their position where they are located before mitosis. It affects whether they will make an error or not. This is what we have shown and this may affect during cancer development how certain cancers will end up with certain surplus of certain chromosomes and not some other chromosomes. Okay, but now let's go back to biophysics. How does this polar chromosome here bypass the center zone and get to here? We saw now that there is something problematic happening here. It has to somehow move across this center zone region to come to the center of fire. So how is this happening? Well, again, when we ask a question, how is something happening in the cell? The best thing is to do microscopy to just see what is happening there. And now I have to explain what we are seeing here. It's a fireworks, but the fireworks is the big white spot is the center zone or the spindle pole. The small white spot that are shooting out of the center zones are protein EB3 and protein EB3 marks the tips of a growing microtubules. So each of these spots is one growing microtubules. So you are seeing how microtubules grow from the center zone and the blue guys are the kinetochores on the chromosome. Okay, so this is our pole. This is a polar chromosome because it's behind the pole, these two kinetochores here. And we see microtubules shooting. What is very interesting in this movie, I will show you on the next slide because maybe it's hard to see in this fireworks movie. So if we take single frames from this movie, actually we take several frames and we superimpose them on each other, but that's not so important now. What is important is we can get a trace of individual microtubules, these gray lines. And now look at this. Here's the center zone. Here are our kinetochores. This microtubule is here. 30 seconds later, it's here. Some more seconds later, it's here. If we superimpose these positions of a microtubule, we get the first one, second one, and the third one. And the center zone is here. Do you see what this microtubule is doing? It's making a so-called pivoting motion. It's rotating around the center zone. It looks like the center zone is some kind of pivot point or a hinge, and the microtubule is moving like this. Now, how is this possible? What is this pivoting movement? Well, we have seen pivoting before. My lab has found pivoting in yeast. What I have shown you before was human cells. Now we go to yeast. In particular fission, yeast doesn't matter. It's some kind of yeast. Yeast is great because yeast cells are very small and they have very few microtubules. So human cell has several thousand new measurements show that the spindle in a human cell has 6,000 microtubules. This guy has altogether 20 microtubules. So it's great to work on yeast because you can see each individual microtubule. And what we did some time ago, it's almost 10 years ago, is that we saw these microtubules in yeast and we discovered that they can pivot. And you can see this here. This is the ingreen microtubules. You can see the mitotic spindle of the yeast. It's this line, this bundle here. And then look at these guys going up here and here. You see that they are changing their angle. This microtubule here is changing its angle with respect to the spindle. So it's doing this. And what we found is that this kind of motion helps the microtubule to capture the kinetocore, to find the chromosome in the kinetocore because it's kind of swiping through space. Also, later, we found that this motion is important to make the spindle in yeast. In yeast, this is one spindle pole. This is the other. And I hope in this movie you can appreciate this kind of movement. The microtubules start like this and they go like this and they make a spindle. Later, another lab has identified the molecules that are allowing for this pivoting. So you can see this molecule here is important to be flexible enough to make this pivoting. And if you make it shorter, then the pivoting will not happen again, because this will be too stiff. This link will be too short and too stiff. So again, comes our Nenad Pavlin, our great collaborator with whom we are making so many models. This was one of our early models. And in this case, we said we see this pivoting and we asked, can this pivoting help really the microtubules to capture the kinetocores? How fast is this capture process? And we can make a very simple model. So in our model, we have the spindle pole body, the spindle pole. Here is a microtubule. This is a model for yeast. Here is the nucleus. And here is the kinetocore on the chromosome, which needs to be captured. The microtubule is performing angular diffusion. So it's moving like this in a purely diffusing manner. The kinetocore itself is also diffusing. And we are just asking, how long will it take for the microtubule and kinetocore to meet? So it's just one kind of a first- passage problem. And this is what the model predicts. So if we solve this model and calculate the fraction of lost kinetocores, these are the ones that are not yet captured by microtubule, over time, it goes like this. With the parameters from the measured cell, we start with 100 percent of all kinetocores being lost. And over time, they are being captured. And after, let's say, 10 minutes, we have still 30 percent still lost kinetocores. This is a direct prediction of the model. There is no fitting parameters, because we have measured the diffusion of the microtubule of the kinetocore, the size of the microtubule, the size of the kinetocores. So we have all the parameters that we need for the model, we measure them in our experiment. So this is a direct prediction from the model. And then we measure in experiments how our kinetocores are being captured, how fast, and these are the experimental data. And they fit very nicely with the model. There is some discrepancy at later times, but at later times, biologically, many things happen, the spindle grows and so on. But overall, this is a really nice agreement with the direct prediction of the model. And what it means, if we include pivoting in this system, so our model is based on pivoting, the typical time to capture about half of the kinetocores is about three minutes. And the old model, the prevailing model in the field, which does not include pivoting, it is called search and capture model, which just includes growth and shrinkage of the microtubules, it would take for the same parameters about 100 minutes. So this shows you how pivoting, this pivoting motion of the microtubules can increase 30 times, can accelerate the capture, the process of the capture that microtubules need to capture the chromosomes. The pivoting is a very efficient way for the microtubules to capture something. So this was our work on the kinetocore capture. Then later we extended this model on spindle assembly and you see a simulation based on the same idea. So microtubules start and they just diffuse, they perform angular diffusion. When they need, motor proteins bind and motor proteins move towards the pole, this pole and this pole, and this is why the motor proteins make the microtubules align into a spindle. And this is what we propose, how the spindle assembles in yeast. And microtubule pivoting, so this motion is very important here. Okay, this was a yeast, but what about human cells? Nobody has studied this yet in human cells, but there are some indications that pivoting may happen. For example, there are beautiful experiments with the microneedle, where another lab put the microneedle here in the spindle and they pull like this. And after the pull, you'll get a structure like this. So you see that the angle of the microtubule with respect to the spindle axis changed. It increased by this pull, meaning microtubule can pivot if you pull it around the spindle pole. It will not get pulled out, but rather it will pivot. Also, our experiments on spindle compression, if you just take the spindle and compress it from above, the angle of the microtubule increases. This angle here increases compared to here. And the same if you put the cells on cold, then many microtubules disassemble and the remaining spindle has a much higher angle of these microtubules at the pole. So this all means if you perturb the spindle in different ways, microtubules can pivot around the pole. And we thought that this pivoting is the crucial process that brings the chromosome from here to here. Now, how does this happen? Is it really happening, what is driving this process? So we quantified it by quantifying the angle that the kinetochores make with the spindle axis. And we see that over time, this angle goes down, it starts with about 120 degrees like on this drawing and goes down to about 50. This is when the kinetochores sit on the spindle. At the same time, spindle length is increasing. So this is important. The increase in length could drive this pivoting motion. But we first needed to test the motor proteins that I have mentioned before. There are two motor proteins, SEMPE and dinane, which is working together with this spindle, which are known to be involved in the process of alignment of polar chromosomes. And when we depleted each of them, we didn't see any difference in the angle change. So the angle change roughly followed the same pattern in these depletions. So the blue and green curves as in the white one for untreated cells and also the spindle length also more or less increased in the same manner. So these proteins are important for many different things, but not for this spindle length increase and angle decrease. So we thought that maybe it's the elongation of the spindle itself driving the pivoting motion. This hypothesis predicts that the reversal of spindle elongation, so if we would make the spindle collapse instead of elongate, this should reverse the pivoting. And to test this, we use the inhibitor of the egg five motor, which induces spindle shortening. And what we saw in this case, that while in untreated cells, the angle goes down, indeed in these cells, the angle goes up. And in that the spindle length goes down, shortens, and here the spindle elongates. And finally, we had the perturbation in which we can induce, sorry, a rigor binding of this motor, rigor binding means the motor just binds and there is no elongation and no shortening of the spindle, because the motor binds firmly and cannot detach. And in this case, the angle doesn't change and the spindle length doesn't change. So we propose it's the spindle length. If the spindle elongates, the pivoting will go towards the inside. And if the spindle shortens, the pivoting is directly towards the outside. And now we made a very simple model in which we assume that the resistance to pivoting around the spindle pole is very small, and the resistance to chromosome motion is very large. So chromosome basically stays, doesn't move much in the viscosatoplasm because it's big. And then we can calculate for a certain spindle length change, how much would the angle change. This is a very simple geometrical calculation. And these are our different treatments and untreated cells. They all lie close to this curve. And also you can see here when the spindle length decreases, the angle is increasing. And when the spindle length increases, then the angle goes down. So we are very happy with this thing. And we conclude from here that it's the spindle elongation that makes the chromosome move from the backside to the front side of the spindle. It's basically the chromosome moves not so much in the center some moves the most. And this just puts the chromosome there in the vicinity of the spindle body microchip. Okay, now the chromosome is here. How does it get to the midplane? We have seen in the first part of my talk how the chromosomes need to be here at the midplane. But how do they get from here to here? This is a centering problem. This is a problem of how something becomes centered between the two, center something else. And many people have worked on this for many years, including a lot of physicists. So a lot of theoretical models have been made. And this is a very, very interesting area. So I will tell you briefly about the main ideas of this field. So first you can get the centering of a chromosome by length dependent pushing forces, for three reasons. First of all, if you think of a centrosome, then if a chromosome is close to the centrosome, there will be more microtubules interacting with it and growing microtubules can push. So this chromosome, there will be many microtubules pushing it away from the centrosome than this one. So if you have two centrosomes, then in the middle, this centrosome will push it away from itself, the other centrosome from itself, so there will be in the middle. And microtubule density follows approximately this kind of relationship with the distance, with the distance squared from the centrosome. Then the microtubules are not all of the equal length, because as you remember from the first part, the microtubules grow and shrink. And if you have growing and shrinking microtubules, and randomly switching between growth and shrinkage, you get an exponential distribution of the microtubule length. So the microtubule number and the distance from the centrosome or microtubule lengths have exponential and exponential relationship, meaning that there are many, many small ones pushing a lot from the centrosome if the chromosome is close and very few long ones that are pushing just a little bit. And finally also there is a dependence of the force on the length. So the critical Euler force for buckling is smaller for a long microtubule than for a short one. So the short microtubule will be able to push the chromosome away from the centrosome more than the small one. So when you put all these three things together, you can get nice centering mechanism that can center the chromosomes between two centrosomes. But this is not the whole story. There is a length-dependent microtubule dynamics and then also consequently the pulling force, which I will explain now. So it's known that there are certain motors, for example, some of them are company's mate, they can move or they can bind everywhere along the microtubule and they move processively, meaning they move all the way to the end of the microtubule. And this is why a long microtubule will accumulate more motors near the kinetocler than a short one. And these motors have been shown to do different things. They can either increase the depolymerization rate so that the long microtubule, because it accumulates more motors, will depolymerize faster, which will then make a stronger pulling force on this chromosome in the direction to the right than to the left. Or they can undergo catastrophe frequency more often so they can switch from growing to shrinking more often, which will again move the chromosome from here towards the center or they can pause, which is similar to switching to shrinkage. In any case, overall, these motors suppress the growth of a long microtubule. So this means that the long microtubule will stop growing and the short one will not stop growing so the chromosome will move towards the middle. So this is known to work very well in the cells. And then we thought that there could be another player here. And this is our bridging. Because of our bridging microtubule that we discovered, we thought because this bridging microtubule is passing close to the kinetocler microtubules, it can exert force on kinetocler microtubules. And this way, we would get length dependent pulling forces, not just at the microtubule end, but along the length of the microtubule. So the longer one will have more motors, the longer microtubule will accumulate more motors and this is why this side will pull with a stronger force to the right than the short one to the left. This would be the length dependent pulling forces exerted along the length of the kinetocler fiber. And this is something that we proposed that was not, this was not existing in the in the models before we proposed this recently. This is still very controversial and exciting in the field. And I'm just reminding you why we proposed this. This is because we found this bridging fiber here between the kinetocler fibers. So there is this microtubule going all along the length of the kinetocler fibers and can probably accumulate motors there and generate some force, which we will see later. But there is yet another thing that I have to tell you that I haven't told you so far which also needs to be taken account. And this is a very cool thing in the spindle and it's called forward flux. So the spindle is constantly undergoing flux towards the pole. You can see this here, here we have labeled with this blue spot, we have labeled a certain part, we have photo activated the laser. It's like putting a mark on the spindle. And you see that this mark is moving towards the pole. So the spindle is all the time the microtubules are undergoing flux towards the pole. They are disassembling here and assembling here. And if we want to understand how the chromosomes get centered here, we have to take into account this kind of thing. And this kind of thing has been known for a long time since 89. But nobody has considered it in the models for chromosome centering until now. And we thought that we have to include these important things, the forward flux and the bridging fiber to understand the chromosome centering. And this is our model again by our then a paving and his group. And I will show you now the animation made on this model. And if you understand this animation, then you will understand everything about this part. So our hypothesis is that microtubule flux drives chromosome alignment. Let me show you the animation. So first let me tell you this is our model, very simple. Here are the kinetochores and the chromosome is this elastic spirit. Then we have the red and blue are microtubules. Red ones are kinetochore microtubules and the blue ones are bridging microtubules. You see that all of them move towards the edges. This is the polar flux. This is the flux that they are undergoing. In our model, the flux is driven by the motor proteins. Motor proteins are these white guys that are walking. And you see that these white guys are pushing every pair of antiparallel microtubules apart. Antiparallel means that you can see these arrows when the arrows are pointing in different directions then it's antiparallel. So they can move blue versus blue or this red versus this blue or this red versus this blue. Anytime the microtubules these motors have the property that they find antiparallel microtubules and slide them apart. This is how we think the flux is generated. And this is all we have in the model. And this model automatically puts the chromosome in the center. Why? Because the flux of the longer K-fider is faster in the model than the flux of the short K-fider. And if you have faster flux of the long one, it will move the chromosome in the direction of the long one, meaning to the center. And why is the flux of the longer faster? It's because the overlap of the longer is longer. This overlap with the bridging microtubules is longer on the side of the longer K-fider. So this overlap is longer than this overlap. So this is why this model centers the chromosome. And we think that this is an important factor, that this kind of effect is important in this spindle. This is completely new. No one has proposed the role of bridging microtubules or of flux in this particular case to center the chromosome. So let's see whether our model is true. It seems very logical. Once Nenad Pavin and his group proposed this, it seemed very logical. But nobody has measured this before that the flux of one K-fider would be faster than the other one. In this spindle field, we always measure flux as one number per spindle. Every microtubule is fluxing at the same rate. But this model predicts that the bridging microtubules are faster than K-fibers. And the long red one is faster than the short red one. So let's see whether this is the case. Now, to do this, we experimentally needed to develop a new method to be able to see individual microtubules. And we did this by very small concentration of this certable indigene that I just talked before. And you can see these white spots. So first, let me tell you blue spots are kinetocores. And the white spots are spots on individual microtubules. So they are so rare, so sparse in space that each spot is a spot on an individual microtubule. And we can follow it. And we can follow it in this way. If we find the white spot passing by the kinetocores, we say this is a spot in the bridging fiber. And if we find a spot appearing near the kinetocore, we say this is a spot in the kinetocore fiber. And now we can measure everything. Let's remind ourselves what is the unique prediction of our model. It is that the longer kinetocore fiber, the darker red one has faster flux than the short one. And we can just measure this now. So we measure the kinetocore fiber length and the kinetocore fiber flux velocity. And we indeed found the correlation that the longer kinetocore fibers flux at the faster rate in the shorter one. So this is really cool. Nobody has measured something like this before. And people still don't believe us, let's say, because it's very new. And anyway, this shows you that this difference exists. And if a long one fluxes faster than the short one, this automatically has a centering effect because it will move the chromosome in the direction of the long one, which is towards the center. So this is the core of our centering mechanism. Okay, let's see some other predictions of this. The second prediction of this model is that the bridging fiber fluxes faster than kinetocore fibers no matter long short. And we measure this, the bridging fiber indeed covers a larger distance over time than kinetocore microchip. And then what we did is we also made some theoretical predictions for when we changed different parameters in the model. It's not so important what these parameters are now, but let's say this, I can tell you this is the parameter describing the velocity or the maximum velocity of the bridging fiber sliding. And this parameter is the parameter that determines the forces at the kinetocores and how fast the microchip will be polymerized here. In any case, here we changed this parameter and we got this kind of curve to describe how the kinetocore fiber flux depends on the bridging fiber flux. And this is the other curve. In all cases, always the bridging fiber flux is faster than the kinetocore fiber. So we are always in this triangle here. And now we experimentalists made a lot of experiments. We treated the cells with different treatments in which we deplete certain proteins. So each of these colored dots is depletion of one protein. And we performed experiments on many cells for each colored dot. And we get this kind of thing. So in all our treatments, we are in the lower triangle. So the bridging fiber is faster than the kinetocore fiber. And we also have data points that lie on the blue curve and data points that lie on the orange curve, which means that probably in these treatments, we mainly changed this parameter. Whereas in these treatments, we mainly changed this parameter. And untreated cells are here in white. And then the final prediction is that the ratio of the k fiber flux and the bridging fiber flux is the most important determinant of how well the chromosome will be centered. So here we plot the distance from the equator versus this ratio. And this is a theoretical curve. And when we plot our treatments that we did experimentally, we see that these treatments lie very nicely on this curve with untreated cells here in white. So this means that when we have a smaller k fiber to bridging fiber flux ratio, for example here, we have better alignment of chromosome because the distance is smaller. This is because in this case, the kinetocore fiber is allowed to slide with respect to the bridging fiber. And when we have this ratio close to one, so they are moving together, they're not sliding with respect to each other, then there is worse alignment because kinetocore fiber cannot slide with respect to the bridging fiber and cannot center the chromosome. So overall, I have shown you now that there is a new type of centering mechanism, which is based on length-dependent pulling forces. And just a summary to understand how this works, which is just a different representation of what I have shown you before. So if you imagine a chromosome being misaligned, not in the center, and if you imagine making marks here, here, here, and here, after some time, the bridging microtubules, these guys down here will slide apart. So this mark will move here and this mark will move by an equal distance here. Now this one has a longer overlap with the bridging microtubules, so this one will move less than this one because the elastic spring here is preventing the sliding of this one to be equal to this one, so it has to be slower. So this mark is going to move until here. And now most importantly, the mark on the short microtubule, because it has a very short overlap with the bridging microtubule, will move only a little bit. So this one will move a lot until here, and this one will move a little bit until here. And this is the reason why the chromosome will move towards the center. So this is this new concept alignment by length-dependent pulling forces. And I am at the summary, so I have shown you that it's bad to be in this danger zone behind the centrosome. And if you are here, then you have, if you are a chromosome, then you have a higher chance to mis-segregate the endoselvination. How these chromosomes are rescued from here to get to here, it's by microtubule pivoting, which itself is driven by single elongation. And finally, once they are here, one important mechanism that puts them in the center is this length-dependent flux of kinetocore fibers, where longer one fluxes faster and brings the chromosome to the cell center. And at the very end, I want to acknowledge now a subset of my PhD students and postdocs that contributed to this part of the talk. I'm really lucky to have a wonderful lab, we have a great atmosphere in the lab, and we have lots of fun, lots of hard times also, as always, when you are a PhD student or a postdoc or a PI. But mostly, we are really enjoying what he is doing, and it's so important to have great collaborators. We are collaborating with experimentalists, this is for the first part of this talk, and again, our Nenad Pavin and Dijs from his group become the developing models. And I think, ah, I just have a slide about the conference that we are organizing. It's called the Mythotics Findle from Living and Syntatic Systems to Theory. So there will be a lot of work, like I have shown you today, from many different labs, the most important labs in the field, including our labs. And this will be in Dubrovnik in Croatia in April next year. So, yeah, follow us and come to this meeting, apply to this meeting. I'm not sure that we will have an online option, but if you can come to Dubrovnik, it will be great. And thanks for listening to this second part. I'll stop the share now. Okay, I'll take questions now. I guess I will share again if I need some slides, but I would like first to thank Eva for this. Of course, you can come to Dubrovnik in April. What is the deadline for application for the conference? Maybe one month before. We are very generously deadlines. I think it's going about two months. So, in February, January will be the deadline for abstracts to be selected for a talk, and in March or so will be the deadline for registration. You can follow us, me or Nana on Twitter. We will be tweeting about this. And also, there will be, let me just share my screen again so that I can show this again. There is a website for this, which is not written here, but if you Google my thought extended Dubrovnik Croatia 2023, it will come up. There is a website already. Great. So, do we have any questions regarding the second part of Professor Tolic's talk online in chat? There is one. Question. Okay, I can read it. Is it possible to regard chromosome attached on fiber as a sliding bead without friction? Sliding bead without friction. That's a good question. We are thinking a lot about this friction, and I think, without friction, it couldn't be because it's actually, so for this last part of my talk, the chromosome is attached like this to the fiber, and there is actually a really high friction here. So, the friction is important. And this is the parameter that was in orange on some graph. It was exactly about this friction. So, I think without friction, it wouldn't work, but it's a very interesting point that we can explore in the model what would happen exactly without friction or at least with very small friction. But experimentally, we know from in vitro experiments, meaning when people put a chromosome and the fiber without a cell just on a glass slide in some watery-like environment, and then it was visible that there is a high friction there. So, friction is definitely there, but the model is here powerful because it can explore the role of the friction when you have a higher and low friction. Thanks for the question. Okay, since everybody here are tired, they are here from more than 10 hours, I guess. No, no, they're not tired of you, of course. So, if you have any questions, you may send email to Eva and ask and contact her. And I guess if you have any idea of collaboration or together work with her lab, you can also send her the proposals, and yeah, that will be also okay. Absolutely. Yeah, please feel free to write me an email and I will try to answer the best I can. This is also valid for those who are online. So, yes, let's thank speaker again. I would like to close today's session, school, and yeah. We have to start recording in just a second. Thank you all. I just want to thank you for the invitation and for the great discussion that was especially between the two.