 All right, so I must admit, I have to thank the organizers. And I think Navam and David must have played a key role in putting my name up for this discussion. So I'm going to try to keep it very simple. I wasn't sure whether this meeting is going to be filled with students and post-docs or is it just the senior scientists. So I'm going to try to present to you what we publish. And I really like to get to the point where I'll present stuff that's not in print yet. So, you know, this pathway of protein secretion that I have projected here on this slide is an old process. It sort of goes all the way back to the days when George Palade, for the first time in the 60s and in the early 70s, presented from his analysis the path taken by a protein, which is to be secreted. And the proteins that have to be secreted by the cells begin their life in the endoplasmic reticulum. They are then transported to the Golgi. And from Golgi they then make their way out of the cells. And his major thesis was that these transfers between compartments ensure the compartmental identity. So when you want to take something out of the ER, you take it selectively. You leave behind the residents and you take the cargo to the next station and you go on sorting these proteins. Till you get to the stage where you have determined that some of the proteins have to be secreted, the other ones might have other centers in the cell where they have to go. So that was in 1974. And for the last 40 years of so or 50, a lot of people have worked on this process to gain an insight into how cargos are sorted in the endoplasmic reticulum, how they're packed into a kind of vesicle that has been monocled or termed cop two vesicles. And these vesicles will bring the cargo into the Golgi. Now with the Golgi, the cargos are transferred in the forward direction, but proteins that need to be recycled are brought back by means of cop one vesicle. So cop one is a class of vesicle in fact that I had purified with Jim Rothman in 1989. And these vesicles were identified in 1992, hence the one versus two. So there is still a lot of doubt in the field, even after 40 years of work, whether the traffic in this forward direction is mediated by vesicles, or is it simply a process by which this cisterna of the Golgi matures? By mature I mean proteins that need to be returned are being extracted while material that needs to go forward keeps moving. And this becomes particularly important for molecules that are far too big to fit into the class of vesicles such as cop one and cop two. And these class of molecules are collagens, musins, chylomicrons, PLDL particles. And I'll talk about that today. So it has been said by a few that we have almost all the information that we need for this particular pathway of transport. And I tend to differ, I disagree completely. I think we just have the nuts and bolts of this process. Most of our understanding in how cells compartmentalize and how cells secrete many of the proteins, the signals, how you control the organization of the compartments when the cargos are being moved back and forth is completely unclear. And this is what my lab has been trying to address for the last many years. Now, my talk is going to depend and describe a lot about cop two vesicles which form at the ER. So I think there is a need for me to present our current understanding of what a cop two vesicle is and how it forms. So you need a set of about six polypeptides. And this is mostly the work of Randy Sheckman and Bill Bolch and colleagues. So what happens is a protein called SAR-1 which lives in the cytoplasm in a GDP bound form interacts with sec 12, which is a transmembrane protein of the endoplasmic reticulum. This will change GDP for GTP. And when that happens, this protein SAR-1 exposes an amphipathic helix which inserts into the outer leaflet. Thereby bringing this protein into the ER membrane. SAR-1 GTP then binds to a dimer which is made up of sec 23 and sec 24. Now there are different isophones, you needn't worry about that at this stage. Now this dimer has the potential or the ability to start collecting cargos. So sec 24 has the ability to bind to receptors which can then bind to soluble secretory cargos. Okay, so this is a way to collect material in the lumen of the endoplasmic reticulum and connect it to the coats, to the inner layer of the cop two coats. Now the binding of this dimer to SAR-1 and once the cargos are associated, then recruits another dimer which is made up of sec 13 and sec 31. So there's only one isoform of 13 but there are two of sec 31. Now this binding of this dimer to sec 23 and 24, what it does is it starts turning off this process or terminating this process. So sec 31 has the capacity to increase the GTP as activity of sec 23, thereby converting sec SAR-1 GTP back into GDP. And when GTP to GDP switch is made, that basically is the signal for the cells to terminate the production of this particular container. And so what happens is a cop two coated vesicle then emerges from the endoplasmic reticulum and it can be then targeted to the Golgi. Now these vesicles cop two shown here and cop one shown here. They've been purified, they've been studied extensively. Hello Tommy. They've been studied extensively for the last 30 or so years and we know a lot about them. In fact, almost all the components of these coated vesicles have a role in protein secretion. But there's a common feature here that is worth mentioning. And the feature is to do with the size of these vesicles. They are about 69 meters in diameter. And they look very homogeneous in size. Now this is fine for most of the proteins that are being secreted by the cells, especially if you happen to be saccharomyces cerevisiae. But we buy bypads and mammals and make a lot of proteins that just cannot fit into these vesicles. So for example, collagen. So there are 28 different types of collagen that you and I make. Now, and you need them for almost every cell cell interaction. Without the collagen, you will not have bones to begin with. You will not have skin. Now, the problem with the collagen is that they contain a very rigid triple helical region, which in the case of collagen seven, which is absolutely necessary for the formation of skin, can be up to 450 nanometers in length. And this is really like a rod. There is no force inside the cell that can bend this rod into a structure, small enough that could be encapsulated into a copter vesicle. So the question then becomes, how does a cell which need to secrete collagen, and believe it or not, that collagen composes 25% of your dry body weight. So these are the most abundant of the secretory cargos. So the question then becomes, how can a cell export something so big by using a vesicle that's only 60 nanometers in diameter? Now, similarly, cells of the liver and intestine secrete chylomicrons and very low density lipoproteins. These are basically lipid droplets coated with specific proteins, and they are secreted and their job is to scavenge or transfer cholesterol and triglycerides in circulation. And again, these can be huge structures, and they cannot be accommodated into a copter vesicles. Droplets, while they can be as big as 100 to 150 nanometers. But remember, you're secreting one droplet at any given time. You're secreting many of these. So the question then becomes, how can a structure that is only 60 nanometers contain many of these? Now, finally, to get to another point, we produce about a liter of mucine per day, which is absolutely necessary for the lining of your epithelium all the way from your nose to the very end, and its job is to protect the underlying tissue from pathogens. Now, there are 21 different kinds of mucine genes. Again, huge molecules. And you make a liter of mucine per day. And these are again far too big for to be transported by copter vesicles. So the question is, how are the cells transporting these molecules? Because they're certainly very, very important for our physiology. So over the number of years, I was running into 10 years. And I must admit, Alberto Luini is sitting here, so he should be credited for rekindling a field of biology. And it was Alberto's paper in 1998. In fact, I was visiting his lab at that time, which highlighted this issue of how the current understanding of that time, meaning cop too mediated vesicle transport, just couldn't explain how collagens are transported because they were just far too big. People had just simply ignored this issue and challenged people would just say, well, there must be some variants of cop too and cop one vesicles that can do this job. But it turns out that it is not so simple. So many years ago, we performed a genome-wide screen in I think 2005 or 2006 in MetaZones. And we had looked for new proteins that have not been assigned a function in the secretory pathway and we isolated lots of genes that were new in this business. And we call them Tango for transport and Golgi organization because I'm also fascinated by the structure of the Golgi and how it forms and breaks and is partitioned into daughter cells. So Tango one is the protein that I'm going to describe to you today very briefly. It's a very large protein. It is a protein of 1907 amino acids. Its N-terminus is in the lumen. Its C-terminus is in the cytoplasm. In the cytoplasm, it has a proline rich domain, two coil-coil domains. In the lumen, it has 900 amino acids which are not depicted to the scale here which are unstructured and they turn out to be very, very important for the overall process by which cargos are exposed. And again, if time permits, I will gladly discuss that. It has a coil-coil domain and an SS3-like domain at the very N-terminus. So we had shown that the SS3-like domain binds collagen and we had shown in 2009 that proline rich domain binds to sec 23. So this gave us the reason to believe that this protein must have a role in trafficking. And when Kota Seitho was in my lab, he was also able to show that Tango one, so this is endoplasmic reticulum. Tango one localizes to very discrete sites at the ER and these sites happen to be the ER exit site. So these are the places where cargo is collected and then this is where you generate transport carriers which will then move the carriers from the ER in the direction of the Golgi. So Tango one binds to sec 23, it binds to collagen and it localizes to the ER exit site so the thinking would be that it must have a role in the export of collagen. And the answer is yes it does. So if you knock down Tango one by S-I-R-N-A, now of course one does CRISPR, but this is 2009, 2010. There's about an 80% reduction in the amount of collagen. Here I'm showing you collagen type seven that is secreted compared to the control and there is a concomitant increase in the intracellular pool of collagen which is found arrested in the ER. This is just a quantitation of this process and this is just to show you the level of knockdown that we can achieve by this particular procedure. This told us that Tango one was required for collagen type seven export from the cells. Now soon thereafter I was very pleasantly surprised that friends of people that I had been in contact with, Andy Peterson and colleagues at Genintag, we were trying to generate a mouse knockout of Tango one but we are not very good mouse geneticists and we lost to our dear friends. They were able to create this mouse knockout. So Mia three is the same as Tango one. It's just a different name for it. So they were able to generate a mouse knockout for Tango one and what they found was that the mouse dies at birth. It dies at birth because it does not have any mineralized bones. So the bones are really like rubber and in fact many of the bones are missing and so it dies and it has this defect because it fails to secrete many of the collagen that looked at one, two, three, seven, nine and 11. So this therefore gave us the hope that A, Tango one has a role in collagen export and B even more importantly it is not just collagen type seven that we had studied but many of the collagen that most likely follow the same pathway. And this also gave us the confirmation that whatever we have been doing in tissue culture system has an in vivo physiological significance. Therefore time to dig deeper into the problem and that by deeper I mean how does Tango promote collagen export? And this is where we've made some very interesting findings. So it turns out as I told you earlier the proline rich domain binds to cop to coat sec 23 that's what we had shown before. I'm going to show you, well I'm going to show you the work of ours and the others that the SS3 like domain binds to cargo and I'm going to show you our work that this particular domain here is required for recruitment of membranes. The point being that the mechanism which Tango one generates a big transport carrier that you need is not simply by collecting membranes from the ER and creating a transport carrier. It's a completely different mechanism. So we had shown that this part binds to sec 23 but Jonathan Goldberg two years ago quietly published a paper while not quietly he published a paper in 2016 in PNAS in which he was able to co-crystallize parts of Tango's PRD with sec 23 and what he found was that these parts contain triplex of proliens and Tango one contains seven such triplex of proliens and it is this particular, it's these particular proliens that bind to sec 23. So he was able to narrow it down to the mechanism or the structural aspects of how Tango will interact with sec 23. So this is great confirmation for us and very valuable for further dissection of this process. And in the same year what Hans-Peter Buckinger found and reported that the binding of sec 32 collagen that we had proposed and reported is not direct but it is mediated through a protein called HSP 47. So this solves a problem again for us because as I said there are 28 different types of collagen. So it was difficult for us to envision how would this particular protein bind to all different collagen. This therefore acts as an acceptor. Now I should also mention that while HSP 47 is an acceptor for collagen type seven and many others there are collagen that do not interact with HSP 47 but they interact with other chaperones. And so our thinking is that this protein can interact with collagens through different molecules and not all of them have to work through HSP 47. But in principle the connection between collagens and Tango is mediated by a connector in between which is in the case of collagen type seven at least HSP 47. And you know all of them yet? We don't know all of them but we we know for a few of those. You know again it will become a detail. I mean I think the principle has been established. This one is very specific because you know how it works. Again I don't know how I'm going to run this time. You know the collagen primers start at one end and as they are folding these proteins start binding. They bind in fact Nagata Sa had previously supported the idea that these were chaperones for folding and that turns out to be not true. What they do is they bind to the fully folded part and as it is being zipped as it is being folded this binding increases. So I think this interaction brings to this particular molecule the part that is folded. If I have time I'll try to explain to you how this unstructured part and this one works together to only collect fully folded collagen for export. So this was again a great piece of confirmation for us. Now, so the question then becomes you have very big molecules and need to export them. So how do you do it? So one possibility is that in order to make a big transport carrier you need more membrane. So one possibility is that if you use a cop two code, so the idea is that if a cop two code needs to make a small vesicle it assembled into a small structure and then it pinches and you get a membrane that's about 60 to 90 nanometers in diameter. But if you want to make a big one all you do is you make a very big code. You instead of using a handkerchief to collect this much material you throw in a big towel. You know just collect big membranes. Would that be the way to generate a transport carrier? The alternative, so this is basically it, right? So if you want to make a vesicle of this size you have a code structure let's assume 100 nanometers by 100 nanometers, okay? But if you want to make a big structure to carry collagen the size of the code is huge. But I'm going to tell you that this is not how the structure is generated. The reason for that is that we looked at a patch of collagen. So this is looking at a patch of collagen which has not left the ER yet. And these gold particles are gold particles to show that we're looking at collagen. It is huge, okay? This is about almost reaching about 500 nanometers. Now we expect it, so red here is the same as this one is collagen and the green structures you see are cop two codes. It doesn't matter whether you set 23 or set 24, okay? So I expect you to see a complete area here covered by these green structures. So instead of seeing these punctate elements I expect you to see a sheet of code on top of these structures and we did not see that. So this made us think that perhaps there is a different way to generate this mega transport carrier. And Antonio Santos, when he was in my lab he made a very peculiar observation. He found that when collagens are about to leave the ER here again shown in red, these structures seem to be studded with membranes that contain a protein called Ergic 53. So what is Ergic 53? It is a compartment or it's a collection of membranes in between the ER and the Golgi. And their job seemed to be material coming from the ER comes as far as Ergic 53 compartment and then they go back, okay? And they go back mostly by cop one vesicles. What becomes of Ergic 53 is still kind of unclear. So you can call them Ergic 53 containing membranes or you can call them cop one vesicles that contain Ergic 53, it doesn't matter. So ordinarily we saw or Antonio saw that these membranes were tightly opposed to collagen patches. But if you look at cells from which Tango one has been deleted, these membranes are still there but they're not attached to patches of collagen. So this made us think that perhaps these membranes are being recruited by Tango and their fusion to these membranes or these patches is what is providing the extra membranes required for generating these big structures. So I'm going to come to that but I just want to highlight what is this particular region of Tango which recruits these membranes? So we were able to pin it down to a stretch of amino acids about 150 amino acids from here to somewhere in between to the middle of the Coil-Coil first of Tango one. And we call this domain tier which stands for tether for Ergic at ER. So what you can do is you can take these 150 amino acids of Tango one and put it at the mitochondria artificially. And when you do that the Ergic membranes simply go to the mitochondria. So instead of coming to the ER they simply are diverted to the mitochondria. So this told us that this part has the capacity to recruit Ergic membranes. And so we can now, this is not published but we can now keep on cutting this structure into smaller and smaller pieces and it turns out amino acids 1255 to 1296 of Tango one have the capacity. So this is about 50 amino acids. So we take this 50 amino acids and put them on mitochondria. Then Ergic membranes go to the mitochondria. If you put them on plasma membrane then Ergic membranes go to the plasma membrane. Ergic membrane is lipis plus protein. Yeah. If you know which protein being actually involved. I'm going to tell you that just you're too quick. It's a bit early for me, but yeah, I'm going to get to that point. Yeah, so I'm just trying to tell you that there is a mechanism built into Tango which has the capacity to recruit Ergic membranes. And we can narrow it down to 50 amino acids now. And we can use this 50 amino acids as a peptide to inhibit collagen secretion, to be quite honest with you. It's very simple. And it turns out the first coil-coil domain that I showed you is not just one contiguous coil-coil domain. It is made up of three coil-coil domains. And the tier domain is this part here which is made up of these many amino acids. So this tells us that coil-coil domain I initially thought would be a rod-like structure, but it is made up of three bits that can bend. Now, the idea then is that this part here can recruit Ergic membranes. They fuse and their fusion and rapid build into the structure that is growing out is what creates a mega-transport carrier. But is there any evidence that these membranes fuse to the ER? And the answer is yes. So fusion requires these specific proteins called snares. They have to assemble at the site where a vesicle is fusing with the target membrane. And we know a lot about these proteins, so I'm just going to simply run through it. So it turns out there are three T snares and one V snares. V is for vesicle and T is for the target membrane. So we were able to show that the T snares syntax in 18, USC1 and BNP1 were required for export of collagen from the endoplasmic reticulum. Tango one, of course, is necessary because it acts as the receptor for collagen. Syntax in five is not a surprise because if you knock down this particular snare, then there is no Golgi per se, and therefore the end product would be a defect in secretion, okay? Now, if there are T snares, there must also be a V snare. There are many V snares that work at the ER Golgi junction, but the point to remember here is that YKT6, this one, if you remove that from the system, then collagen export from the endoplasmic reticulum is blocked, whereas knockdown of BNP1 and sec22B has no effect, okay? So this told us that the membranes that are being recruited to the ER exit site via the function of Tango1 do fuse and it is their fusion that is necessary for the export mechanism. Now, I have to sort of go through this model building a little bit in order to explain the data. So what we propose is the following, that in the lumen of the endoplasmic reticulum, the SS3-like domain through HSP47, lumen is the inside of the ER, inside of the ER. It's a container, right? ER is a membrane-bounded compartment, so it'll have a lumen and it'll have a face that is on the site, that's the cytoplasm. So in the lumen of the ER, this SS3-like domain binds to collagen and this binding we propose initiates the interaction of the proline which domain to sec23, and this is how this reaction starts. Now, at this stage, we propose, and this is fictional, and this is something that we are trying to address now in the lab, that there is a mechanism by which collagen is being pushed into a structure that is growing on the cytoplasmic side. Now, this pushing is probably mediated by SS3 domain through HSP47, which might just be sort of walking on the collagen as it's assembling. This also relies on collagen having a binding partner here because if you have a container that is growing and this part is anchored and this is not, it's going to do this, so it helps if it is anchored. And we don't know what this anchoring mechanism is, but we think it might be that it is anchored to integrins as they are leaving the ER. Now, based on the fact that there are two coil-coil domains and the coil-coil domains can extend up to about 40 to 50 nanometers, we propose that a bud can be created through this interaction alone, which would be about 40 to 50 nanometers, but still not sufficient to grow a structure, to commensurate with the size of the collagen. Now... What is your green stuff? Is this a patch of membrane? This, these are copter coats. This is the membrane. But the membrane which has been added? Well, it's coming to that. I'm going to, up to this stage, we think there is no membrane added yet. Now, this also, we propose that if this is true, then tango should be a ring. So if you imagine this in three-dimension, then tango would just basically circle the ring, circle this structure as a ring, perhaps at the neck of the transport carrier that is coming out of the ER. And one day, lo and behold, Ishir, who really is probably one of my best postdocs ever in the last 30 years, comes up to me and he says, Veevek, I have data, tango one assembles into a ring, at the neck of a structure that is coming out of the ER. And by a ring, he means this. So this here in green is tango and red in the middle, as you see, are copter coats. So it turns out that if you were to look at is, if this is the ER, this is the structure that's coming out, this is the lumen, this is where the collagen is and it's dripping on my palm, the liquid, the collagen would be inside. Tango is a ring here, okay? And this is the membrane and the coat, the cops would be in the middle. So this is quite fascinating because what it tells us is that tango has a capacity to form a ring, assemble into a ring, and the cops are in the middle and the structure is going to grow in this form here. And this is about 300 nanometers, but we have data now that this can be made to shrink and expand based on the dynamics of tango, okay? Now, at this stick. This will be, yeah, so it's a certain amount of stretch, how long does this? Well, I think tango is not stretching like N2C terminus. It's basically assembling laterally into a ring like this. Just one, just one, it's just monomer. It's, just give me a few, it's a polymer. It's a polymer, you know. But it's not a homopolymer, it gets complicated. So at this stage, we propose that these membranes, ergic 53 containing membranes are being recruited, which will fuse here through the action of the snare mediated pathway, okay? Now, as these membranes fuse, you had asked earlier, someone had asked earlier, how does this tier domain recruit or what does it bind on these particular membranes here? So most recently, this is a paper of us that just got accepted this morning. What we have found is that if this is a ring of tango, the red dots that you see here are in fact a set of tethers. There are three proteins, they're called the NRZ complex. It's a detail, but basically tango through tier domain recruits these tethers and these tethers then recruit the ergic membranes, okay? And it really is quite spectacular because we find these tethers usually either on one side of the ring or at most at the opposite pole. So I think what might be going on is that one half of tango ring and another half of tango ring somehow is brought together and it's at these nucleation sites, at the sites where the tethers are and they do like this and you end up with a full ring. So you have a complex of tethers here and you have a complex of tethers here which then recruit ergic membranes. And so this is a picture that we don't, nobody in my lab wants to do microscopy but we've been blessed with an absolutely wonderful microscopy facility. So by using super resolution microscopy, we've finally been able to capture images of a ring of tango, ergic membranes and the tether. So this just shows you how this whole complex is brought together. So these are the membranes that would be fusing in this domain to allow this structure to grow in size. Now, once these membranes start fusing, this structure is going to grow and it grows to a size which is large enough to encapsulate collagen. Once the collagen's have been placed into this container, the SS3 domain dissociates from the collagen molecules. When this dissociation takes place, the proline rich domain dissociates from sec 23, which allows sec 1331 to bind here. This binding initiates this GTP hydrolysis cycle that has been suggested long time ago which would then in principle at least cut the membranes here to now create a big transport carrier as shown in this reaction, okay? So this therefore is a working hypothesis of how we believe tango can promote the capture of specific cargoes and allow the cells to create, by recruiting other components, a mega transport carrier from the ER. So it is in principle different from how a cop two vesicle is generated. But it is not going to be as simple as we have been saying because it turns out that tango has other family members. So this is a full length tango. There's another protein that has been identified. It's called Tali or it is also given the name Mia too. So this protein turns out to be present only in liver and intestine. And we have shown that just like tango is required for the capture and export of collagen, Tali in the cells that express it is required for the export of chylomicrons and VLDL, which are again very big particles. So it's similar principle, but there are slight differences, okay? It's not the same, but it's a related protein. They have the same domain structure, meaning it has two coil-coil domains in the cytoplasm. It has a proline rich domain. It has an SS3 domain. But Tali lacks this luminal coil-coil domain. Now it turns out that Tali is spliced to give rise to two proteins. A protein called C-Tage-5, which is expressed in every cell type. So C-Tage-5 and tango-short form a complex. And there is also a splicing of tango-1 to generate a protein called tango-short. I should have written it here, but it is not. So the ring of tango that I showed you is composed of tango-1, C-Tage-5, and tango-short. So it's a polymer of three very related proteins. Now in certain cancer cells, this protein MiA2, which is basically a part of tango and Tali where you cleave here. So you have the luminal part, which has the capacity to bind collagen. This is secreted. And when it's secreted, there are groups who are working on this. Their thinking is that this can sequester collagen in the extracellular matrix. And by sequestering collagen, what it does is it prevents the assembly of ECM. And because it prevents the assembly of ECM, you have the possibility of promoting metastasis. We are not working on this. So this is just to show you that there is tango-1, which has the capacity to bind to cargos. There is tango-short, which is just like tango-1, but it cannot bind the cargo. And there is C-Tage-5, which is again related to tango, but it cannot bind the cargo. The three of these proteins are usually found in a complex. And a lot has been known about which part binds where, et cetera, et cetera, et cetera. So those details are available. Each one of them is required for this exit of collagen? Well, this tango is absolutely necessary. If you don't have these ones, there is a defect. And I think it's simply because what they might be doing is to provide the proline rich domain, which has the capacity to interact with Sec 23. So instead of just providing monovalent interactions, what you're doing is you're increasing the valency. So you increase the affinity. Now here it gets a bit complicated. So I've told you that you generate a transport carrier of this kind, big structure that leaves the ER and takes collagen to the Golgi. But you know, no one has been able to visualize these big collagen-containing carriers, except one report. And if the time permits, I'll be happy to go over what that means, okay? So is it possible that when collagens are being pushed through the function of tango and associates into a structure that is growing, that what happens is this structure here at this end fuses to the Golgi cisterna. And if it were to fuse to the Golgi cisterna prior to the fission here, what you will end up with is a kind of conduit, a tunnel between the ER and the Golgi, and the collagen would basically simply go across the channel. It's like Calais to England. The trains are going across. And there is no transport carrier per se. Once the collagens have been transferred, then you cut here, okay? And once that structure is cut, it's simply absorbed into the first cistern of the Golgi. So this basically means that there is no specific transport carrier per se. What you do is you create a tunnel for a short term, which allows cells to push these kinds of big molecules from here to here now. And the first cisterna of the Golgi, which then contains collagens, just keeps moving forward. And this is what cistern maturation is all about. That this structure again doesn't need to then pick collagen into a big transport carrier, move it to the next one, next one, so on and so forth. So what happens is, as the collagen leaves the ER, it is already in a cisterna of the Golgi. And this cisterna simply continues to mature because there is no other tango-like molecule in the secretory pathway. They're all at the level of the endoplasmic reticulum. So this also helps us understand how there is no further sorting of the molecule once it has left the endoplasmic reticulum. So we are really keen on this model and we are testing this extensively. Now, you might ask how many collagens would be secreted if this was the case. We've been able to do some calculation with Matthias Mann and Ben Glick put together. So it turns out there are about 40,000 tango-1 molecules in cells that secrete collagens. Their number of ER exit sites is an estimate, but it's between 200 to 400 in mammalian cells. So from this, we calculate that there are about 100 to 200 tango molecules per ER exit site. And since each tango-1 binds to one collagen trimer, this allows us to guess that there could be 100 to 200 collagen trimmers exported per ER exit site, which is this. So this gives us an estimate of how much collagen, because these, as I said to you, these are the most abundant secretory cargos. They have to be secreted in huge amounts and very fast. So this might be... So, but you still have a problem going through the Golgi spine, but because you have these tunnels and then maturation, how do they get out of the Golgi to the plasma membrane? Well, the same cisterna. When you get to the last one, it has nothing but just collagen. Oh, so it fuses as a cisterna, not as a... Not as a specific... So there are no vesicles coming... But you don't make any of the fusions, the other ones, some proteins... Sure, of course. For every structure that has to fuse, there will be fusion components, yes. Tommy. If you do an ER Golgi purification on cells that are active secretion collagen, do you now pull out Golgi apparatus on the ER fraction? It's very hard to do that, Tommy, but the best is to do this lifestyle imaging, and this is exactly what we are trying to do. You know, we spend years purifying Golgi, and you can never, ever get, first of all, a pure Golgi. There's always some Golgi in the ER fraction, and there is always some ER in the Golgi fraction. So that wouldn't work. What else do you think would be the transfer...? So in chondrocytes, in chondrocytes, the export of collagen from the ER to the outside is like in five minutes. It's the fastest. So if you would lock this cell... We are trying to find that way to lock it. Electromycrotherapy. Yeah. Electromycrotherapy. You would trap the intermediate. So we are trying to do that. We are using tangos, bits and pieces, where you allow it to latch on to the copter codes, but they cannot come off, so we think we might be able to see these connectors. But you need a transport. You need a... Sorry, it's just that you said that there are, like, let's say, I don't know, how many hundreds of events per minute, right? So if I would just snapshot the cell and do a 3D EM, I should see the connectors. Do it. I would... We are looking for someone who... Who I would love to. I mean, this is exactly what we would like to do. That is, why you invoke the need for a connector as opposed to creating a vesicle that then fuses? We haven't seen it, David. I mean, you know, we've been searching for years, and so has the whole field to see. There are vesicles that have been seen in post-Golgi events, but it isn't clear whether they're vesicles or they're just cisterna. I mean, Alberto might have more on this if he's going to talk about it, but we have never been able to see something that separated from the ER and is en route to going to the Golgi. We have seen collagens that are separated from the ER in big structures, but they are not going to the Golgi. They are going to the lysosomes for degradation. Maybe there is another question behind this one. The key to those experiments will be to have the right cells and cells that are actually actively secreting collagen because a lot of the models are cells that are probably not very active and probably that's why you haven't seen it. So dermal fibrovas... So we do it in R-deb cells. R-deb cells are the best cells for studying collagen type 7 because they produce... These are the skin cells. So they produce keratinocytes, lots of collagen. So if we cannot see them there, then there is R-deb. These are the cells of patients with epidermalysis bullosa. So they're producing gobs of collagen. So let me just... I mean, again, we can... With this, what you're losing is the directionality that is normally given by a viscular transom. Well... How do you see the directionality to be given here? Well, yeah, there are issues, I agree. I mean, one possibility is that this binding and pushing mechanism is what is responsible for the directionality. I mean, I was thinking you might ask, well, other things would leak out. Yeah, calcium. Yeah, but the thing is that you could bring them back. I don't know. No, calcium... Calcium, you might be able to pump it out. But there are CERCA channels, the SPCA channels of the Golgi, they can take care of that if the need be. I don't know, I mean, I... Do you think your disorder domain is importantly making some sense? Yeah, so, okay, God, you guys are... I will need more time. So it turns out that Tango1 has 900 and 8 amino acids that are unstructured. They bind to collagen weekly, but there comes a time when the binding becomes very tight. So we think the SH3 domain binds to HSP47 and as collagen is assembling, the unfolded part is bound to the unstructured part. And this unstructured part simply separates into undergoes phase separation. We're doing this with Tony, and this is responsible for the directionality and pushing. So it's all coming together, but we're not quite there yet. That's why I'm just trying to present to get your feedback. Anyways, so unstructured part. And the lipids? The lipids, no. Lipids, I don't think I will get into the lipids. We have no way of knowing. I mean, we still don't know the lipid composition of a cop-1 or a cop-2 bicycle. So going there would be, I would need another lifetime. Now let me just... So I just want to come to a few more things before I leave. So, you know, we are good at doing, I think we are reasonably good at doing what we do in the lab, figure out Tango bits and pieces, but, you know, we are also getting very excited about the fact that ourselves and the others are trying to see if we can use inhibitors of Tango to control fibrosis, which is hyperecretion of collagen. So this group at Myoclinic, in fact, has been able to, at least in a mouse model, what they do is that you can induce liver fibrosis by using bleomycin. So what they do is they knock out Tango-1 in the liver and then treat the mouse with bleomycin. It doesn't develop a fibrotic tissue. So there is a way to do this, and we are collaborating with people where we can deliver RNAi and CRISPR tools for Tango to direct them directly to liver to see if we can control in mice, not using this approach, but better approaches. So there is a potential, or perhaps a possibility, that we might be able to attack the issue of fibrosis for which there is absolutely nothing you can do at this stage. Now, this is the lab. But Fred Bard started this whole screen of Tango genes. He is here now. Kota Saitho is the one who decided to work on Tango-1 from all the 74 tangos that we had, and I'm thankful to him. He is at Tokyo University, and as of last week, he's now been made a full professor at Akita University. He's probably one of the youngest professors there. Patrick, Cristina, and Antonio, post-docs, they were the ones who showed that Tango recruits urgent membranes to collagen patches. So this really is a new way of thinking how big transport carriers are generated not simply by acquisition of membrane by coats, but by addition of membranes. And Ishir, Maria, and Felix. Felix is a physicist in the lab. And they have figured out how Tango assembled into rings. Now, I have a few minutes left, so I just want to run through a few experiments. So now we know what part of Tango interact with cop-to-coats, what parts of Tango interact with C-Tage-5, and Tango short, and tier domain, et cetera, et cetera. So we start asking a very simple question. How does Tango assemble into a ring? So, again, by modeling this process, what we are proposing is that Tango should be, so this is blueish Tango, and this color, yellowish-brownish color, are the cop-to-coats. So the cop-to-coats are being corraled by Tango, and we say that Tango, whether it's a Homo mer or a hetero mer, Tango C-Tage-5. Okay, fine. So we say that it forms a filament. So it's a filament in the membrane. Now, the filament wets the edges of the cop-to-coats. So it acts literally as a line-actant, and by doing so, what it's doing is it's controlling the dimensions. So ordinarily what you would have is the filament will bind or will wet the rims of the cop-to-coats and therefore contain cop-to-coats in the middle. Now, if you want to make a bigger structure, what we are saying is that these filaments can fuse with other filaments in the vicinity and you end up getting a bigger structure, okay? Now, how do we know that? So in order for Tango to form a ring, it has cop-to in the middle, so there are interactions of Tango to cop-to components as shown here with these lines. And we know that this is through proline rich domain and this is insect 23 coats. So if you now knock down cop-to, you can do it two ways, right? You can express the Tango that doesn't have a proline rich domain or you can remove sec 23 and you come to the same conclusion. What happens is you don't form rings. What you end up getting is a structure that is highly tessellated. So basically what we have done is we've reduced the interaction of proline rich domain to the cop-to-coats but the lateral interactions which we think are being mediated by Tango interacting with itself and Tango interacting with CTAGE-5 and Tango interacting with Tango short. And in doing so you end up getting these highly tessellated structures, okay? Now this is just to show you another example that if we now affect the cop-to interaction in this case we have expressed Tango-1 without the proline rich domain but you end up getting at these sort of long, stringy elements. There are little rings sometimes but the rings seem to be fused and it is depicted in this pictorial form here. So I think what this is allowing us to test is finally, I'm not going to go through the whole process. So if you, for example, remove the ability of Tango to bind the collagens, it's a complete block and ring assembly. So it appears that the binding of the cargo to Tango and the binding of Tango to itself and its associates is the process that is absolutely necessary to generate these structures. And this is the direction that we are going in to try to understand how this structure is assembled at the ER. Nah, that's about it. I think I'm done. I'm going to stop here and gladly answer any questions. Thank you very much. I wonder what happens at a transcology, a more downstream. Do you think any special mechanism will operate there? Well, we've tried very, very hard. All the Tango-like particles that we found for experts of chylomicrons and a completely new one for nuisance. They all seem to be at the ER. There's nothing at the level of the Golgi. So my feeling is once you leave the ER, you don't need to sort these elements again. The structure that contains these big particles simply keeps moving forward. So there isn't a need to re-sort them into specific vesicles. This is a simplest answer. I mean, there might be other components that do it, but we haven't found it. And to be quite honest with you, I think if we could just solve at the level of the ER, I think that would do it for me. So in terms of the force and the movement of the collagen, isn't this reminiscent of the translocation of unfolded peptides across the ER and the HSBs? And Tango can somehow provide some force that will... So you're right. Yeah, you're right. So there is the unstructured part. And when you do an IP of Tango, so we can purify Tango. In fact, David is sitting here. We can now express Tango in Pichia pastores. It's taken us a long time. We're trying to engineer to answer some of these questions to see if we can express Tango in Pichia. And what does it do? So we can express it. It took almost three years. It goes and forms a ring around cop two codes. That's all we can do thus far. Ultimately, I would like to be able to see if we can secrete collagen. So we're doing bioengineering in a way. When we look at the components that bind to Tango in the lumen, they seem to be collagen and collagen-like molecules and chaperones. They bind to a lot of chaperones. So we think it is not just HSB 47 and that's it. They are switching chaperones. When they switch these chaperones, we don't know, we would like to be able to test when do these chaperones come into play? At what stage? But it has not been that particularly easy. So we can knock down these chaperones and we see an effect. But that doesn't give us the mechanism. So we are trying to figure out in vitro, I don't think we'll be able to do it. That's why we went into Pichia pastores. We are doing that with Ben Glick. But yeah, I mean, what you're saying, it's reminiscent of creating a translocom type structure, which is using tango and its force to move. We're also looking for an ATPase in the lumen of the ER to help perform this function. Yeah, they should be doing it, but still you need ATP. Yeah, Tommy. No, there is a question. I might miss it, but what do you think about the dissociation of the collagen from the chaperone, like after the pushing, do you need to best... Well, there are two ways of doing it. One is you make life very difficult and you invoke another protein. The alternative is very simple. The binding of SS3-like domains to their targets is weak affinity, very weak affinity. So if a molecule which is about 900 amino acids, 10, about over a thousand amino acids, 900 are unstructured. One possibility is that it can only extend up to this distance and it cannot go like so, and therefore dissociate. This is simply conformational change. And you're coming? Charlie, so going back to the translocom model that was discussed in a way from your pictures, it looks like a translocom, a translocation through some sort of a fork. Actually, when we started to work on pathology and manning many years ago, was to synchronize the folding of prokolog and indian depository reticulum. This allows us to generate large aggregates, indian depository reticulum, very large, one micron by one micron. And they go out when you release the block of secretions to begin to explain now, although they're so big, they go out easily. How does that fit with your models, yeah? Certainly not with a simple translocom through a fork because that would have to be huge. Well, why not? I mean, the thing is that, you know, we're not saying that this is translocom as in the sense of a nine nanometer pore. This might be much bigger. So I don't know, we should do it in chondrocytes the way that you guys did it, where you accumulate so much collagen and then you release it all in one shot. We haven't done that because as is, they are so big to get them out. I expect that when you have such a massive efflux, there must be major reorganization. It would be terrific to know what happens to the ER exit site. We haven't looked at that, but it would be worth, it would be worth doing it. I had a question myself. So basically on your model, where you think that to enlarge this kind of carrier, you need fusion of all this molecule. So to get fusion and to get this increase of area, you need to have a barrier somewhere. Otherwise you will. So basically, so it means that- The ring is the barrier. The nine-acton is a barrier, right? It's a barrier, yeah. Call it a fence, call it a fence, call it a ring. But this is the- This is the ring. The tango or tango is the barrier, right? So you don't, I can also show you images of, basically the organization of the proline rich domains, et cetera, et cetera, et cetera. So they all seem to be, ordinarily tango starts off like this and then it opens like this as the tunnel is growing. And it just remains here. And then it keeps moving down. So we have all of those images. But those are images. We'd like to be able to do more than that. But I think what you're doing is you're making a fence, a line, and everything happens within that area and this restricts and then you grow. So what you're gonna do? Yeah. That would still mean- Tom, maybe here. Mark. Quick question, just- So Misha Rappa has found that the cop, one of the cop proteins or several of the cop proteins needs to be ubiquitin modified in order to package collagen. Where does that fit into this story? How does that, do you incorporate that at all? Or do you just get- I mean, I was in Randy's lab for six months, two years ago. And I basically, we don't say that. They basically say that KLHL 12 is ubiquitin hitting sec 31, but where, six years ago that paper was published, right? I would like to know where that thing is getting ubiquitinated. And there's a paper that's going to come out, shortly it's not my paper, where the authors of this new paper claim that this whole KLHL pathway, it's found on containers that contain cop two, but they're not going to the Golgi. They're going to lysosomes for degradation. So we had not seen any effect of KLHL 12, et cetera, et cetera in our pathway. We don't see its role in secretion, but I don't want to just, I just decided not to go there. So very quick question. So back to this model, the connection with the ER, the Golgi. If you're getting lots of collagens trying to come out from all the ER exit sites, most of them should be constipated. Who should constipated? The ER should become constipated because only there are few limited number of regions where the Golgi is in contact with the ER, right? So most of the sites would be not associated with Golgi. So all those sites should be constipated and you should have all the collagens stuck if there will be a contact kind of approach. But is there any evidence that you need to give some losing things? That's why I didn't go into medicine because I just don't think I could answer that. I really don't know. I mean, I think I'm just basically presenting you what I think what I don't have is not there. I mean, there are lots of questions that one could challenge. I mean, there's a recent paper from Maria Leptin's lab that a protein called dumpy, which is the largest protein known to anyone. It's dumpy. It's in flies, Drosophila. It is even bigger than collagen and it requires tango one, Drosophila tango one for its exit from the ER. So this is where most of the work is going to go. People are going to say, this cargo also requires tango, et cetera, et cetera, et cetera. The difficult questions are, in my opinion, whether there is a tunnel or whether there really is a sort of transport carrier. Number two, the directionality. Somebody, Eve said, how do you move it in that direction? I think this is very crucial. And then this business of how do you control how much collagen goes out? Because it depends on the type. So we think when there is small amount of collagen, you have rings of about 300 nanometers. When you need to export even bigger, as you say, then what happens is these rings have the capacity to, it's like a fence. It fuses to the fence on the site and what you do is you create a bigger structure. And so you can push even more out. But those things at the microscopy level are very difficult. And at the biochemical level, all you're looking for are increase in the number of molecules. And this is not at the surface. We're looking at something that happens inside the cell, which makes it a little difficult, I think. But so I think in my opinion, these are the two or three questions that we are trying to address. And we thought of doing everything in vitro. We were able to purify tango on, but it collapses. So it's very, very hard to work with. That's why we decided to go into piquea. And then we are trying to create in a piquea the ability to express. So we can do it with tango on, that's no problem. Will they generate bones? Will they generate bones? I don't know, but we are, so there are four minimal, you need four proteins, that's a must. No, no, no, no, no. I'm not trying to make a bony yeast, but what I'm trying to do is I'm trying to ask, can we get collagen to leave the ER with the following set of proteins? Three enzymes, collagen type four, which is the easiest collagen to work with, and tango. That's all we care. What happens once it comes out, whether it goes to the goal gene, how it gets sorted, it's someone else's business. I think this would be sufficient. So that's where we go anyways. Okay, so I think we should go for the coffee break. Thank you.