 Okay, good afternoon everyone. So my name is Jamid Shah. I'm a PhD student in the lab of Professor Sandra City in Department of Cell Biology at University of Geneva. So our lab mainly works on epithelial cell-cell junctions. What makes epithelial cells special is the presence of the apical junctional complex that includes tight junctions and adjuncts junctions. And as you know that cell junctions, they are multi-molecular complexes that consist of transmembrane proteins, proteins that are found just under the cytoplasm, which we call as cytoplasmic plague proteins, which are connected to the cell cytoskeleton. So some of the proteins or in fact most of the proteins of the adren's junctions they're also found laterally and which we call them as lateral cell contacts, okay? The role of the cell-cell junctions is to protect the cells, to connect the cells to each other and to protect the cells and to maintain a permeability barrier between the outside and the inside. Now over the period of time, the bacterias have evolved to use these transmembrane proteins of the cell junctions as their receptor to attach first of all to the cells and to get the entry in the cells so that it can infect and basically kill the cells. But nothing really is known about the role of the proteins that are present at the cytoplasmic plague and how they can control the host-pathogen interactions. So this is an example to Staphylococcus aureus bacteria and in fact its antibiotic resistance strain that we call as MRSA. It's a growing threat to human health worldwide and it's responsible for many different kind of respiratory and skin infections. The reason they are so effective against us is because they they produce variety of virulence factors and one of the virulence factors is alpha toxin which is secreted by the bacteria as water soluble monomers. This monomers they can bind to Adam 10, which is the receptor. It's already known that it is the receptor for alpha toxin. It oligomerizes to form a first pre-pore complex and then it forms a fully functional pore causing imbalance of ions in the cells and eventually leading to cell that by apoptosis or necropotosis. So now our collaborators in Stanford they performed a screen using CRISPR library and HEP cells and they wanted to know that what apart from Adam 10 are the host factors required for alpha toxin cytotoxicity and most important thing for us that is they identify several of the proteins of the cell-cell junctions and the cytoplasmic plague proteins and one of them was placa-7. So they did a lot of experiments to validate the screen but the most important experiment that did was to look at the intracellular ATP levels upon the treatment of either the wild-tab cells the placa-7 knockout cells or Adam 10 knockout cells with the alpha toxin and as you can see here that the the initial drop of intracellular ATP between the wild-tab cells and placa-7 knockout cells is similar but somewhere at around three or four hours the placa-7 knockout cells they are recovering. Now it holds lot of significance because this says that there is no defect in the pore formation or no change in the kinetics of the pore formation. So how is that placa-7 able to regulate the susceptibility of the cells to toxin and here there is a control which is Adam 10 knockout and here you can see that there is no change in the ATP levels as it is already known that in the absence of Adam 10 there is no pore formation. They were able to also duplicate the results in vivo where you can see that they treat the ears of the wild-tab mice with a live bacteria and over the period of 14 days you see a lot of tissue damage. Well in the placa-7 knockout mice there was tissue damage but it was much less than the wild-tab and after after a few weeks they survived and it was all okay. So the my question or what I want to understand is how placa-7 is able to regulate the susceptibility of the cells to this toxin. So what is placa-7? So placa-7 as I mentioned before it's a protein that is present at the cytoplasmic plague of adren's junctions. As you can see that it has several domains and it has several interactors, but for this work it's important to remember that the N-terminal WW domain of placa-7 could interact with PDZ-11. This is we identified few years back and the pH domain is was identified to interact with aphidine. And it also involves in many cellular and pathophysiological processes. So now to understand the role of placa-7 in controlling the susceptibility of the cells to toxin we asked four questions. First question was what is the localization of edum-10 in epithelial cells and how the absence of placa-7 could affect the localization? So what we find here is that edum-10 was co-localized with placa-7 which is the marker of junctions and as well as ZO-1. But placa-7 was also found on the lateral surfaces. So this is important that you remember that there are two populations of edum-10, one at the junctions and one on the lateral surfaces. So now when you remove placa-7, so this is the scheme for the B result, so when you remove placa-7 what you see is only the zonular accumulation of edum-10 is absent in the knockout cells. But the lateral distribution of the edum-10 in placa-7 knockout cells is not affected at all. We also did some rescue experiments and we identified that the WW domain of placa-7 was partially able to rescue the junctional clustering of edum-10. And as I mentioned before that WW domain it interacts with the protein PDC-11 and also helps its recruitment at the junctions. We wondered that PDC-11 might also be involved and in fact PDC-11 was also identified in the screen. So what you see here is that when you treat the wild-tab cells or the knockout for placa-7 and PDC-11 with alpha toxin and look at the cell that by propanium iodide staining and fax, you see that both placa-7 and PDC-11 knockout cells they are more or less behave similar and they are more resistant compared to the wild-tab cells to alpha toxin. Again, we tried to look at the intracellular ATP levels and PDC-11 knockout that behave similar to placa-7 when they are treated with alpha toxin. And also I don't show here, but the phenocopy also the localization of edum-10 are similar to that of placa-7. So now we ask other question. So here I don't explain a lot of basic introduction things, but what is already known from the literature is that tetraspinins there are around 33 of tetraspinin proteins of which there are six proteins which are classified as tetraspinin C8 cell family and all of them are known to interact with edum-10, promote its maturation and surface delivery. So we thought that the question was that what brings edum-10 to cell-cell junctions. So we did a lot of screening and we identified that tetraspinin-33 is actually localized exactly at apical zonular junctions at adren's junctions co-localizing with placa-7. And this localization of tetraspinins is also dependent on placa-7 as well as PDC-11. So if you remove placa-7 or PDC-11 they are more cytoplasmic and I don't show here the XZ staining but it's more lateral and not zonular as you can see for the wild type conditions. So next we ask the third question which is what happens to the toxin pores when you treat the cells with alpha toxin. So similar to edum-10 tetraspinin it's again the repetitive things but what we find is that also alpha toxin pores they are clustered at the zonular junctions, ZO1 being the marker and they are distributed on the lateral surfaces of the cells. But in the absence of placa-7 or PDC-11 the lateral distribution of pores is still present but only the zonular accumulation of the pores is absent. So this leads, this becomes more clear that why in placa-7 knockout cells you still see effect of toxin but it is able to recover. So next we move to hap cells with the use in the screen and they are very easy to use, they require very less toxin. So we prefer to use this instead of epithelial cells and they form small cell-cell contact sites instead of zonular junctions. So we treated these hap cells with toxin and then we wanted to know how stable is the toxin pores that is present at cell-cell contacts. So we treat them for either 5 minutes and 30 minutes, we can see that at 30 minutes there is significant amount of pore formation in the wild tab cells but now what we do is we remove the toxin from the media. So we let the cells recover for either 2 hours or 4 hours and then what we found that the amount of toxin which was clustered at the cell-cell contact sites of these cells was more or less similar for 2 hours and dropped only a little bit at 4 hours. Now if you compare placa-7 or pdc-11 knockout cells we see that there is little formation of the pores at the cell-cell contact sites in placa-7 but then as soon as you remove toxin from the media and let them recover they are not stable at all. So they go away from the cell-cell contact sites but remember that they are still present on the other cell surfaces but they are just not clustered. So the fourth question and the final question we asked that how all these proteins adom-10, placa-7, pdc-11 and tetraspanins they are interconnected to each other. So I just give you like a small piece of data from lot of biochemistry we did. So this is a proximity ligation assay. So in simple terms that if the two proteins are in proximity you see a signal in the form of red dots. So this is the control placa-7 and pdc-11 they are in proximity at the junctions so you see a nice signal at the junctions. In the absence of pdc-11 you do not see it so it is a negative control. So we find that all these proteins for example placa-7 and adom-10 and also placa-7 and tetraspanin-33 or adom-10 or tetraspanin and tetraspanin-33 they are all in close proximity as we can detect the signals in the epithelial cells at the junctions but in the pdc-11 knockout cells you do not see the signals anymore. So that is quite interesting that pdc-11 somehow promotes the interaction between these proteins and that is exactly what we find in the CoIP experiments which I do not show here and the pull down which is here. So here if you can just look in this like last three lanes what we find that the C-terminal of tetraspanin-33 could bind to the N-terminal amino acids of placa-7 but more interestingly when you put the third protein in the lysate to see the interaction is significantly increased but it does not this N-terminal region of placa-7 does not interact with adom-10 but when we when we make the bigger N-terminal like with 500 amino acids we see that it interacts very weakly with adom-10. We also did other pull down experiments showing that pdc-11 neither binds to tetraspanin-33 or adom-10. We also show that afadin can bind very strongly to the C-terminal of adom-10 and third stabilizing the whole complex. So if I have to sum up everything what I have said in a slide so this is the model we propose that at apical at the cell-cell junction so epithelial cells placa-7 recruits pdc-11 and the recruitment of pdc-11 somehow change the conformation of placa-7 so that now we can bind to very strongly to tetraspanin-33 C-terminal and also binds to adom-10 although weakly to the region which overlaps with afadin. Afadin this protein was also identified in the screen and and we show that afadin could bind very strongly with the C-terminal of adom-10 third stabilizing the whole complex. So this is the case what you see at the junctions and we suspect that at the lateral cell-cell surfaces this complex is not present so the pores that are formed they are not very stable or they are able to endocytose or they are able to remove quickly and that is the reason that we see recovering the knockout cells of placa-7 or pdc-11 but the pores that are formed here at the cell junctions they are very stable due to the presence maybe of cytoskeleton or the stable complex the cells are not able to get rid of this pores and this leads to cell that over the period of time. So with that I would end my talk I would like to acknowledge my boss Sandra for giving me opportunity to work in a lab also the people who are involved in this project the external collaborators mainly the screen was performed in the lab for Eric who works in Paris as well he is a guy of tetraspanency discovered tetraspanence and also the organizers for giving me opportunity to speak here and thank you for your attention. So to test your idea of this stability if you take your cells with a toxin that are making the monolayer yeah and you break the junction maybe by putting EDTA do you now get the loss of the toxicity maybe by endocytosis of the pores? So the thing is when we want to look at epithelial cells we have to administer the toxin basolaterally so we have to put because Edumton as you see it's at the junctions and distributed on the lateral surfaces and there is a tight junctions on top so the toxin cannot penetrate the tight junctions so we have to administer this basolaterally. That's okay but you have your model system where you have the layer of cells put the toxin it's accumulating so yeah that so that is that is if you now put EDTA to break so yeah so what I don't show here is that junctions formation of the junctions is very important and it has already been shown before that if the junctions are not formed properly there is that effect there is no effect of toxin so for example what we show is that this effect is also dependent on confluency of the cells so if I put on like the cells are not confluent enough there is very little effect of toxin but but when they are very well polarized and very confluent they die in like four or five hours compared to 24 hours in the cells which are not confluent and also if I treat the cells for example the HAP cells which are not connected we are like single cells there is no effect of toxin whatsoever so the junction formation is must for the toxin to act. It's not to work it's just here stability the toxin is acting all the time because you are losing ATP yeah so you're removing this so I did not look at the ATP but I don't see any morphological change so yeah the toxin is still working but you don't see cell death so this is my observation that if it dies or not so maybe if I look at ATP for example it would be like recovering for example. Okay so last question. Is it specific to epithelial cells and whether ecadverine is required for this? This is very difficult to test if ecadrine is required or not because if you remove ecadrine for example the whole complex is destabilized so it's it would be very difficult but and in the screen you know if you look if you remove ecadrine the effect is like they find in the scene for example Ncadrine and effect this effect is much less than placa 7 so I think ecadrine would be involved but indirectly and it might work in fact by removing placa 7 from the junctions so this could be. Is there any specific for epithelial cells? No it's not really specific for epithelial cells it just because HAP cells are not epithelial cells they are myoblastic origin as long as it has this complex in the cells it's enough. All right by measuring ATP you're actually measuring the metabolic state of the cell but I don't quite understand how they recover regenerating ATP if the cells are dying they're dying they cannot recover. I think they this has to do with the threshold limits of apoptosis because I only measure ATP but at the same time there is calcium efflux there are many things ATP is going down because the cells are first of all not healthy and they are like leaking from the pores. Yeah but if they are leaking they cannot recover they die. They in the case of volatile cells they die but in the knockout if imagine a situation that the pores are now removed and there are no pores anymore the cells are not well of course but over the period of time so that's why they don't recover 100 percent but over the period of time like if I wait for two three days they will be normal and dividing so there has it's there has some effect of ATP like toxin treatment but it takes time and they recover and you can see very well in this like mice model they also do for pneumonia so they treat the cells with bacteria the bacteria dies of pneumonia in the bulk type but if you treat the knockout cells the bacteria are happy I mean sorry the mouths are happy and they don't have pneumonia anymore. Thank you very much.