 Okay, so I'm going to present you what the work we have done on the septin and had a few time, I'm going to focus on the problem of the curvature sensing. So septin, if you don't know, we are working on buddingy septin and they are located between the mother and the daughter cell during cytokinesis and you can see them here with fluorescent or here by electron microscopy. During the cell cycle, septin undergoes some modification like phosphorylation, simulation or de-simulation and from a structural point of view, they start from a hard gloss shape here that you can see there and they split in two rings with perpendicular direction during the cytokinesis by interacting with the actomyosine cortex rings. From a structural point of view, what we know about septin is that they assemble in vitro in high soil concentration as an octamer with a symmetry here, an octamer or something which is around 32 nanometers in length and by decreasing the soil concentration by interaction with membrane, septin organized as paired filament here and they polymerized to form longer filaments. So a goal and what we are interested in is we want to understand if the septin can modify the mechanical membrane properties of the membrane and also if septin are able to sense membrane curvature. So I'm going to start with the mechanical membrane properties. So what we are doing is, as I told you, we are working in vitro and what we are doing is we produce some giant unileveral vesicles which are vesicles with a lipid bilayer that we label in red. The side is typically 10 of micron and we incubated with this vesicle some GFP septin meaning green. So in the absence of septin you can see that this is a section of confocal microscopy. The vesicles are completely spherical and when we have the septin you see some yellow signal meaning that it's green plus red and in the absence of septin if we have the same osmotic pressure inside and outside the protein then we also have some spherical protein and all that in up in the low septin concentration. If we make an osmotic shock on this vesicle that means that we make the pressure inside an outside difference we can make an hyper osmotic shock that means water goes in on an up osmotic shock that water goes out of the vesicle and usually when we make this kind of shock or when we make a difference of 20% then the vesicle burst and in the presence of septin we see that the vesicle they are able to resist to very high shock and here you can see that they resist to a shock of 350% which is really huge. So we found here that the septin they rigidified the the lipid membrane. To go further what we've made is that we make some micropipette experiments so you have here micropipette here the vesicle still labeled in red for the lipids and we held this vesicle with a micropipette by imposing a difference of pressure and this difference of pressure is controlling the tension of the vesicle and what we are doing is that we keep this difference of pressure constant meaning the tension of the vesicle constant and we inject over time the septin on the vesicle and what we monitor is we monitor the tongue lengths which is the length that you can measure here and is directly related to the variation of the vesicle of the vesicle hurry are here and what you can see here is that over time that's mean increase that injecting septin concentration you can see that the vesicle become more and more red that's mean that there is more and more septin interacting with the vesicle and that's what you can see here with this red curd that you see that over time there is more and more septin and what was striking for us is that we keep the aspiration constant in this experiment but we see that the tongue lengths here is decreasing over time meaning that the variation of the the hurry are variation is decreasing of the vesicle sorry is decreasing over time so what this means exactly size wise this shape what corresponds to the shape this graph what do you mean which percentage of this actually so what we measure is we simply measure we know the radius of the vesicle we know the tongue length so then we just have the vesicle area it's very small decrease of hurry are fraction so if you want to see something like two percent of decrease of hurry are fraction you can clearly more visible see it here with the length of the tongue comparing to the to the vesicle so it's just to see some very small fraction area so and that's what I'm quantifying here we made that for different vesicle and we see that the septin reduced the apparent surface area of the vesicle from two to ten percent and I remind you that's all that is made as a constant volume that's mean the septin they can either change the mechanical property of the membrane and or reshaping the membrane so we know that they change the mechanical property of the membrane and that's mean that they also reshape the membrane and this is what we are going to focus now on so the the goal here is that I remind you that the septin they are located between the mother and the daughter cell here the constrictor time which is highly curved so we want to see if the septin are able to sense this membrane curvature so we again take a giant in illamela vesicle and this time we think we have we are working at higher septin concentration and you can see that the vesicle they are not run so this is spinning projection so it's not anymore a confocal but you can see that the vesicle totally not run anymore they are sort of bumpy and it's completely static it's not something which is moving and if we increase again the septin concentration then here it's 600 nanomolar you can see that we have this kind of spiky vesicle which appearing with with a distance between spike which seems to be repetitive and that's what we quantify here you can see that's the so the the wavelength between two spies is ranging from one two three four microns so it's probably depend on the on the vesicle size but if we look on the on the curvature here impose and on the lengths of this spike it seems that the septin they are imposing some spike of one micron one over one over one micron radius curvature so to go further in this so it's I forgot to say it seems then that septin able to sense micrometer curvature range so to go further we produce some p some wave is substrate so how we do that we take a pdms we stretch the pdms and then we make some oxidase oxidation and by relaxing the stress so this oxidation they make two layer with different mechanical properties and by simply relaxing the stress then we obtain this kind of wavy pattern this is made in collaboration with the team of joe cabral in the imperium college london and we are able to to trigger the to to to trigger the the wavelengths and the amplitude of the substrate in the micrometer range which is exactly the range that we are interested in so after what we are doing is that we put some lipid bilayer on top of the on on top of the substrate and then we incubate it with the septin you can see here a low magnification images where you can find the the wrinkle surface that we have here and this is kind of defect that we have when we make the when we make the substrate and if we zoom in you can see that you have some septin filament here aligning and this is someone fused vesicle which remain when we make the lipid bilayer if we zoom in again you can see that the septin they are not aligned they are aligned parallel to the hill on positive curvature and this is clearly visible with this greenish color and on negative curvature meaning in the valley they seem to align perpendicular so that's mean that the septin they doesn't like positive curvature but they're more like negative curvature in the micrometer range this is what is quantifying here with the alignments on the top meaning on positive curvature and on the bottom meaning negative curvature which are orthogonal to each other moreover if we focus again on this kind of defect that i show you we clearly see that the septin they are aligned parallel to the hill and when there is a crossover they don't not go in the valley they continue on the positive so that's mean they really don't like positive curvature and this is also what we quantify here with again this reddish here and greenish color here and if we go more in the valley then we can clearly see that the septin they are aligned parallel to the parallel to the valley meaning that they really like this curvature so we have been able to quantify we have been able to quantify the the density of the septin depending on the curvature and we have been able to see that so for the lipids so this has not been made by same but by fluorescent microscopy but you can see that for the lipids there is no sensing of the curvature this is what we expect the lipid they are uniformly distributed on the on the surface but the septin they are really present some difference density depending on the surface and we can find them that they have a higher density for a curvature of two micrometers which is the top of the hill where they are aligned where they arrange parallel to each other so has a conclusion I show you that septin have a curvature preferences with this wave is substrate this curvature preference is problem is for sure related to the give to the deformation on the give that I observed this is exactly on the same range so the question is how that something which is 32 nanometer can sense on hand use some micrometers scale curvature we have a theory for that but unfortunately I really don't have time to talk about that now so has a conclusion I show you that septin they can deform the membrane they can affect the mechanical property of the membrane they can sense and feel some micrometer size curvature and to finish I would like to thank Alexan Bebe who is the PhD student to perform this work he is co-supervised by Aurélie Bertin and me hold the bathroom team the collaborator Dr. Okabral of Manuela Nanyani who make the wrinkle surface the electron microscopy for the scanning electron experiments and the Nikon Center for the fluorescent microscopy thank you we have time for questions so can you connect your surface association of your protein with what happens in cells in the orientation of the septin so yeah so what we what we found what well also this so it's more the theory would give us that but what we have is in a theory explain what we have in cell with these all glasses shape and the splitting because when it's pleated it's because it's too narrow so this is too narrow for the septin so when it's too narrow for the septin that's mean with the theory we have and we can discuss about that later if you want that's mean they we think they prefer to bundle and that's mean that then it would be a bundle and I think I have the images somewhere at least I hope I keep it yesterday I don't know I remove it if you look at the images of the septin in vivo it seems that they are if it's a monomer filament they are parallel parallel and what's in bundle of filament they are perpendicular so it's really because we think that it's when it's too curved they bundle and then they like this curvature and then could explain what we what we have yes yes they have there well they undergo some some that's what I said at the beginning they undergo some phosphorylation simulation and so on but already I think just the curvature make them I think moving because they they they they when it's too narrow for them then they they bundle and then they they switch probably so if I if I remember septins are gtp binding proteins yeah gtp is yeah but but they don't in fact we don't know exactly what's why is why is there still some gtp because all that is made in the absence of gtp if you had some gtp the no not that we know at least it's pretty clear now that the gtp is involved in the assembly of the complex and particularly to form different types coming to that and this is related to thomas question do you know if you have only modern ones or do you have you know septins and so what yeah so we have at the beginning we have the the rods and we make it so this is really important that we make them polymerized on the membrane with the filaments if you make the filament first and make them the polymerized polymerized yeah it's going to be a little different so you think this is not being driven by polymerization of septins into a higher-order structure once again so the effects that you see are the binding it's because they polymerized and then it's because of these long filaments so it's not the cause of kind of discern between the cause and the effect yeah i mean is it well it's probably related is it because septins have to assemble into all of them thus therefore they change the properties well they can assemble on what they want but then if you when it's curved then they this is what we have it is perfect timing so thank you very much stéphanie