 Thanks selecting me for a short talk. I'm a post-doc researcher in Institute Curie working with Patricia Becero. I'm working in a collaboration with Daniel Levi and Maxim Dom from Institute Curie. I'm going to introduce you my ongoing work on conformational dynamics related distribution on membrane. It's a cross talk between conformational dynamic of transmembrane protein and biophysical properties of the membrane, such as curvature or tensions. As we all know that cell membranes are two-dimensional pseudo-seed in which transmembrane proteins are embedded, peripheral membrane proteins. Some are transmembrane proteins, such as ABC transporters, are among the measure class of the transporters involved in transporter activity, lipid flippages activity. Mostly, they are involved in a multitude of resistance in a cancer cell, especially also they are resistant to the bacterial cell. I'm working with the bacterial ABC transporter, which has open conformation, ATP-driven conformational change induces or export the drug, which has open conformation, ATP binds, closes, and then the drug translocated outside the cell. So it has two shape. One is a conical, one is a cylindrical. What happens when protein goes in the conformational dynamics inside the membrane? It means that the transmembrane part, motion, is conveyed to my bilayer. So bilayer curvature is also changing upon dynamics. So what happens or what is a cross talk when the conformation changes happening to the membrane? And what happens when you have a certain membrane biophysical fixed parameter to the conformational dynamics? So it's both cross talks. Recently, in Daniel Levy group has shown that the open conformation, which is a conical, has a spherical arrangement. They form ring-like structure. Whereas when you inhibit by ortho vanadate, cycle gets arrested, ATP cycle, and you have a cylinder-shape protein, you usually end up getting a ring-like structure. So what happens here, that whenever there is a protein inclusion, which has a curvature in a conical shape, you have a membrane strain. Whereas when the protein is having a cylindrical shape, which has no curvature, they have no membrane strain. Usually, so the conformational change from conical, cylindrical, and I'm going to talk what is on the effect of the membrane. So a study, so a bit about the physics. Membrane is a flat membrane. You can define the bending of the membrane. When you have an insertion of the protein, then this spontaneous curvature induced by the protein comes into the play. And this bending energy, you can reduce as effective spontaneous curvature of protein derive the recruitment of the protein in the curved surface to minimize the bending energy of the membrane. And that's dependent on that's how the curved protein try to enrich into the curved membrane. And that's derive the membrane curvature to protein sortings of the curved molecules like conical transmembrane proteins or helical insertions. Our lab developed a tool to study how we can play around the membrane curvature and protein sorting. We grow a giant unilateral vesicles. This is a minimal in vitro reconstituted systems in purified minimal components where you have a lipid membrane. You reconstitute your protein. And you pull the tube, nanotube, with the optical tweezer. And you can play around. You have the almost flat surface. You have a curved surface. You can go from 100 nanometer to 10 nanometer. You can control the membrane tensions. You can play around with the force. So this is a very good system. And we can calculate the protein enrichment from flat to the curved surface. Our lab has shown that the KBAP, which is a conical shape, has a preference for the curved surface. Whereas the aquaporin, which is a cylindrical in shape, doesn't have any preference for the flat or curved surface. But what happens is that the protein has the curved shape, a conical, and it's going in a dynamic state. So I'm going to take open conformation, closed conformation, and in dynamic state. Let's see what happens. But first, I need to reconstitute my protein. We reconstituted open conformation. This is the most challenging task. And then we reconstituted the closed conformation. I want to remind here that when we do the reconstitutions by electroformation, we usually having a both leaflet or symmetric reconstitutions of the protein. In both cases, so I'm going to use this system, symmetric reconstituted system. Let's talk about the closed conformation, which we got in presence of orthovanadate. This is the fixed conformation. We reconstituted this protein. We pull the tube, and we started increasing the tension. You can see from the movie. When we increase the tension, we modulate the radius. And as we modulate the radius, you can see here that the protein is sorting. There is no protein. As you increasing the tension, you are modulating, decreasing the radius, and then the protein sorting started. So relative enrichment calculated. And then the radius, we calculated from the fluorescence. We can't calculate directly from the tension. There is a relation from tension to the radius. But we have the proteins, so the correlation doesn't go very well. And we plotted our protein enrichment versus curvature. And it depends on the protein density on the surface first. And also, protein enrichment has a curvature preference around 20 nanometer here. So lesser the protein density on the surface, easier to flow through the neck. So we have almost protein is flowing from flat surface to the curved surface, lipid is flowing from outside. So there is a mixing. And this allows the reduction in burning energy due to the spontaneous curvature that follows. And after fitting our curve with the model, we deduced the curvature of the protein around inverse of 6 nanometer. Initially, we presume that the protein is cylindrical, but it's not cylindrical. It has some spontaneous curvature. And this goes well with the crystallite structure of the other ABC transporter. And so here, what we propose is that the protein which are inside out are sorting out into the tube, which has a preferred curvature from the inside. Let's take an example of the open conformation. Here, I pull the tube, and I wait it for 15 minutes. I'm not changing any tension. I'm not modulating my radius. Protein itself modulating, it enriches to the curved surface once you provide. And it remodulates the radius. And it reaches up to 30 nanometer. So this almost enrichment to the tube is almost 30 times. And it automatically modulates from 100 nanometer to 30 nanometer. Always it reaches 30 nanometer. You can see here, sometime you have hues, clusters, and phase segregation. There might be. So here, the protein is sorting which are outside to the leaf light. Here, the protein is not sorting from inside. And here, our curvature for this one is 30 nanometer. From the previous cryo-EM image, they came up with the radius of 15 nanometer. So you have to keep in mind that this radius which we are calculating, which is this transmoment domain and interplay of the lipid bilayer, this is coming from the extracellular domain also. So we have to keep in mind. There might be protein-protein interaction. We are not ruling out because we have a hues. We don't know is it a cluster or not. And there is a crowding effect because we have almost 50 time enrichment in the tube. Now let's take an example of ABC transporter dynamics where, in presence of ATP, you can see when it's open, there is one curvature. When it's closed, then you have another curvature. So the sign of the curvature, membrane curvature, is changing upon the ATP cycling. So in this experiment, what I did it, I pulled the tube in open conformation. There is a protein enrichment. And then I added the ATP on the tube from here. And then what we observed that the protein which is enriched in the tube, they just went back to the flat surface. And this decreases with the time. And then it scores a steady state. So in dynamic state, our protein, you have to remember that we have a symmetric reconstitutions. So when I add the ATP, only outside molecules are in a dynamic state. Inside molecules are open always. And when I add, usually, when they are in the tube, outside protein is in the wrong side. It doesn't have a preferred curvature. So they move to the side. And the good part is it's impossible to check in this giant enamel or vesicles the activity of the protein. So here we've shown that the protein is active first. In conclusion, I want to say that the ABC transporter has a dynamic which is the closed conformation has a membrane curvature around inverse of 90 nanometer. It has a conical shape. It's not a cylindrical shape. Oppo form modulates the membrane curvature by itself. And it reaches almost to 30 nanometer. My protein is active. And it's in dynamic state because of the flexibility and negative curvature preference. They move out to the surface. And this is so BMRA, in my experiment, I propose that the BMRA stay longer in a post-hydrotic conformation during the cyclic state. And our observation is orthogonal to the rest of the experiments. They propose that the ABC transporter usually stays in open conformation longer time. Thanks to Patricia Vasero, to our groups, and the funding agencies. And also, I want to mention to Daniel, Susan, who is preparing the protein liposome, and also the other collaborator, Maxim Dam. Thank you very much. So what would happen if you drive the ATP hydrolysis inside of the GV by, let's say, optical methods, right? And KH ATP. It's in Azure, adding the ATP from inside rather than from outside. So one is a technical part. When I'm going to add the ATP inside, then the protein which are going to cycle into the inner leaflet of the bilayer, in that case, they have the wrong preference of the curvature. So usually, they will just, so. In this case, if they will start pumping out some, there is a physics also, there is a, they will cluster first. That's for sure, because there are two, not all the protein are going to be synchronized in one conformation. So probably one conformation will derive, like population of the conformation will derive to have a cluster. So we will have a two sort of clusters. But again, it's in a dynamic state, so it's very hard to say what's going to happen. Frankly. I think there was never an ELECA. We can have two quick questions. All right, ELECA. Is there any evidence that in the cell, you have some sorting based on either conformational state of the protein? OK, in vivo observations, there are some of the observations where you have the curved membranes, like you have some of the transporters there, and they are the curved membranes. So they usually, but as such, there is no direct evidence that the transporters are clustering. It's just a theoretical model proposed in 86. And that says that if the protein are curved, they will cluster. So that's a logical conclusion in physics that if they are curved, they will cluster. Do you have a question? Oh, yeah, so around the same line. So what is the implication for in vivo? Because there is not what's the density of this protein in normal cells, and there will be other normal cells. So what can you think about what's the physiological importance of this? If there is an immediate. So when the cell is in dynamic state, you have a lot of curved surfaces, like whenever the cell is dividing, or you have the producer of the cells, or. So usually, I'm not correlating my concentration with the in vivo, but the sorting at the curved surface is in sense that it needs a transport, because these ABC transporters are the lipid flippuses also. So they usually would cluster at the neck, or the curved membrane. And depending on the scenario, they want to have export of the molecules, or metabolites, or anything. They will do that functions. Or even in the curved membrane, people propose that the lipid need to be flipped. They do these functions properly. But I'm not correlating the concentration in vitro No, but it's hard, because in the cell there will be also not just one protein in the membrane, but with other proteins. But each one will want to have its own curvature. So, yeah. Thank you. OK, thank you very much. See you later.