Building a machine at endoplasmic reticulum for collagen export
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Malhotra, Vivek
Secreted collagens compose 25% of our dry body weight and necessary for tissue organization, and skin and bone formation. But how are these bulky cargoes that are too big to fit into a conventional COPII vesicle exported from the ER? Our discovery of TANGO1 (Bard, Nature 2006; Saito, Cell 2009; Saito, Mol Biol Cell 2011; Santos, J Cell Biol 2016; Malhotra, Ann Rev Cell Dev Biol 2019), a ubiquitously expressed, ER-exit-site-resident, transmembrane protein has made the pathway of collagen secretion amenable to molecular analysis. TANGO1 acts as a scaffold to connect collagens in the lumen to COPII coats on the cytoplasmic side of ER. However, the growth of the collagen containing mega transport carrier is not simply by accretion of a larger COPII coated patch of ER membrane, but instead by rapid addition of premadesmall vesicles. This mode of transport carrier formation is fundamentally different from that used to produce small COPII vesicles. We have seen that TANGO1 rings the ER exit site and thus organizies a sub compartment within the ER (Nogueira, eLife 2014; Santos, eLife 2015; Raote, J Cell Biol 2017). We have now mapped all the components that work in concert along with the cargo to assemble TANGO1 into a ring (Raote et al., 2018. In review). Mathematical modelling, biochemistry and super resolution microscopy based analyses of this process will be discussed. TANGO1-family proteins (cyan) assembly into a ring at an ERES is mediated by interactions 1. with COPII (orange) 2. with triple helical collagen (purple),3.amongst the TANGO1 family proteins 4. with the NRZ tether (dark blue) which links TANGO1 to ERGIC membranes. TANGO1 acts as a lineactant, delaying the binding of the outer COPII coat and allowing for the formation of a mega-carrier.
 So, alright, 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 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 Polari 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 residence 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 are packed into a kind of vesicle that has been monocled or termed COP2 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 COP1 vesicles. So COP1 is a class of vesicles 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 COP1 and COP2 and these class of molecules are collagen musins, chylomicrons, PLDL particles and I'll talk about that today. So you know 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 COP2 vesicles which form at the ER. So I think there is a need for me to present our current understanding of what a COP2 vesicle is and how it forms. So you need a set of about six polypeptides. And this is mostly the work of Randy Schechtman and Bill Bolsch and colleagues. So what happens is a protein called SAR1 which lives in the cytoplasm in a GDP bound form interacts with Sec12 which is a transmembrane protein of the endoplasmic reticulum. This will change GDP for GTP and when that happens this protein SAR1 exposes an amphipathic helix which inserts into the outer leaflet thereby bringing this protein into the ER membrane. SAR1 GTP then binds to a dimer which is made up of Sec23 and Sec24. 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 Sec24 has the ability to bind to receptors which can then bind to soluble secretory cargos. So this is a way to collect material in the lumen of the endoplasmic reticulum and connect it to the inner layer of the COP2 codes. Now the binding of this dimer to SAR1 and once the cargos are associated then recruits another dimer which is made up of Sec13 and Sec31. So there is only one isoform of 13 but there are two of Sec31. Now this binding of this dimer to Sec23 and Sec24 what it does is it starts turning off this process or terminating this process. So Sec31 has the capacity to increase the GTPase activity of Sec23 thereby converting SAR1 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 COP2 coated vesicle then emerges from the endoplasmic reticulum and it can be then targeted to the Golgi. Now these vesicles COP2 shown here and COP1 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. So almost all the components of these coated vesicles have a role in protein secretion. But there is 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 or VCI 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 7 which is absolutely necessary for the formation of skin can be up to 415 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 coptovascular. 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 is only 60 nanometers in diameter. Now similarly cells of the liver and intestine secrete chylomicrons and very low density lip proteins. 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 coptovascular. So which are big droplets? Big as 100 to 150 nanometers. But remember you are not secreting one droplet at any given time. You are 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. And 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 coptovascals. So the question is how are the cells transporting these molecules because they are 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 copto mediated vesicle transport just couldn't explain how collagen 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 copto and copfone 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 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 called 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 Seito 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 sites. So these are 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 SIRNA, 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 7 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 knock down that we can achieve by this particular procedure. So this told us that Tango one was required for collagen type 7 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 them. We were able to generate a mouse knockout of Tango one but we are not very good mouse geneticist and we lost to our dear friends. They were able to create this mouse knockout. So Mia 3 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 1, 2, 3, 7, 9 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 7 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 by 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 well not quietly he published a paper in 2016 in PNAS in which he was able to co-crystallize parts of tangos 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 SS3 to collagen that we had proposed and reported is not direct but it is mediated through a protein called HSB 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 a as an acceptor. Now I should also mention that while HSB 47 is an acceptor for collagen type 7 and many others there are collagen that do not interact with HSB 47 but they interact with other chaperones and so our thinking is that this protein can interact with collagen through different molecules and not all of them have to work through HSB 47 but in principle the connection between collagen and Tango is mediated by a connector in between which is in the case of collagen type 7 at least HSB 47. And you know all of them yeah? We don't know all of them but we know for a few of those. You know again it will become a detail I mean I think the principle is has been established through a specific. This one is very specific because you know how it works again I don't know how I'm going to run a time you know the collagen primers start at one end and as they are folding these proteins start binding they bind in fact Nagata 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 to code so the idea is that if a cop to code needs to make a small vesicle it assembles into a small structure and then it pinches okay 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 expected so red here is the same as this one is collagen and the green structures you see are copter codes it doesn't matter whether you use sec 23 or sec 24 okay so I expected to see a complete area here covered by these green structures so instead of seeing this punctate elements I expected 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 collagen are about to leave the ER here again shown in red these structures seem to be studied 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 seem 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 on 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 1 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 kind Tango 1 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 is 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 stretcher for amino acids about 150 amino acids from here to somewhere in between to the middle of the coil called first of Tango 1 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 1 and put it at the mitochondria artificially and when you do that the Ergic membrane 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 1 have the capacities it's 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. Remember it's lipis plus proteins. Yeah. You know which proteins 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 okay 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 okay. Now the idea then is that this part here can recruit Ergic membranes they fuse and their fusion and rapid built 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 snare 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 1 of course is necessary because it acts as the receptor for collagen okay. Syntaxin 5 is not a surprise because if you knock down this particular snare then there is no gull-cheaper say 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 gull-cheaper 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 Tango 1 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 SA3-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 will have a lumen and it will have a face that is on the size of that's the cytoplasm. So in the lumen of the ER this SA3-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 okay. Now this pushing is probably mediated by SA3 domain through HSP47 which might just be sort of walking on the collagen as it's assembling okay. 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 spatula? Is this a patch of membrane? 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 here okay. 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 Vivek 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. And 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. Now at this stage. Well I think tango is not stretching like N2C terminals it's basically assembling laterally into a ring like this. Just one. It's just a polymer. It's just give me a few. It's a polymer. It's a polymer. 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. 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. 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. 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. 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 the 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 has been placed into this container the SS3 domain dissociates from the collagen molecules. When this dissociation takes place the proline rich domain dissociates from SEC23 which allows SEC1331 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. 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 COP2 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 is another protein that has been identified it is called tali or it is also given the name mya2. 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 its export of collagen, tali in the cells that express it is required for the export of kylo-microns and VLDL which are again very big particles. So it is similar principle but there are slight differences. It is not the same but it is 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 is a polymer of three very related proteins. Now in certain cancer cells this protein mea 2 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 etc. 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. So is it possible that when collagen are being pushed through the function of tango and associates into a structure that is growing? 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 collagen have been transferred then you cut here 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. And the first cistern of the Golgi which then contains collagen just keeps moving forward and this is what cistern maturation is all about. 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 the cisterna can 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 collagen 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 one molecules in cells that secrete collagen. 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 one binds to one collagen trimer this allows us to guess that there could be 100 to 200 collagen trimers 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 the 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... So there are no vesicles coming. But here they are making it a fusion. Sure of course for every structure that has to fuse there will be fusion components. If you do an ER Golgi certification on cells that are active secreted in 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 life cell imaging and this is exactly what we are trying to do. You know we spend years purifying Golgi's 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 do you think would be the transfer? So 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 the electron microscopy. Electron microscopy. 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 onto the copper codes but they cannot come off. So we think we might be able to see these connectors. But you need a transport... Sorry, you said that there are like, 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. We are looking for someone who I would love to. I mean this is exactly what we would like to do. Why do you invoke the need for connectors 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-Golci events. But it isn't clear whether they are vesicles or they are 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. 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-depp cells. R-depp cells are the best cells for studying collagen type 7 because they produce... These are the skin cells. So they produce carcinocytes, lots of collagen. So if we cannot see them there, then there is... R-depp. These are the cells of patients with epidermalysis bullosa. So they are producing gobs of collagen. So let me just... I mean, again, we can... Okay. With this, what you are losing is the directionality that is normally given by physical attractorism. 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, things... Other things would leak out. Yeah, calcium. Yeah, but the thing is that you could bring them back. I don't know. Calcium? No, calcium... Calcium, you might be able to pump it out. There are circa channels, the SPCA channels at 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. I think we have to figure 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... It 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... 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 vesicle. So, going there would be... I would need another lifetime. Now, let me just... 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 hypersecretion of collagen. So, this group at Myoclinic, in fact, has been able to, at least in a mouse model, what they do is they 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 by... 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 this is... 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, Christina and Antonio post-docs, they were the ones who showed that tango recruits ergic 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 Isheer 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-2 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 blue is tango, and this color, yellowish-brownish color, are the cop-2 coats. So the cop-2 coats are being corraled by tango, and we say that tango, whether it's a homomer or a heteromer, tango C-Tage-5. Because this is what creates the... 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-2 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-2 coats and therefore contain cop-2 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-2 in the middle, so there are interactions of tango to cop-2 components as shown here with these lines. And we know that this is through prolinege domain, and this is in sec-23 coats. So if you now knock down cop-2, you can do it two ways, right? You can express a tango that doesn't have a prolinege 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 prolinege domain to the cop-2 coats, but the lateral interactions which we think are being mediated by tango interacting with itself and tango interacting with c-tage-5 and tango interacting with tango-short. And in doing so, you end up getting these highly tessellated structures. Now, this is just to show you another example that if we now affect the cop-2 interaction, in this case we have expressed tango-1 without the prolinege domain, but you end up getting 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-2 tango and the binding of tango-2 itself and its associates is the process that is absolutely necessary to generate these structures. And this is the direction that we are going into try to understand how this structure is assembled at the ER. 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 the transcology, 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 exports 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 resort 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 coats. 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 ATPase. Yeah, Tommy. I might have missed 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, about over 1,000 amino acids, 900 are unstructured, one possibility is that it can only extend up to this distance. It cannot go like so and therefore dissociate. It's simply conformational change. And then... So going back to the translocom model, the pulse is constant. And then, anyway, from your pictures, it looks like it's on a translocation through some sort of a core. Actually, when we started to work on pathology many, many years ago, was to synchronize the folding of prokolog and indian-nupasana reticulum. This allowed us to generate large aggregates, indian-nupasana reticulum, very large, one micron by one micron. So when you release the block of secretion, it's the complicated to explain now, although they're so big, they go out easily. How does that fit with your models? Certainly not with a simple translocom through a core, 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 translocon as in the sense of a 9 nanometer pore. This might be much bigger. 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 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 molecules. So, to get fusion and to get this increase of area, you need to have a barrier somewhere. Otherwise, you will... So, basically, it means that... Ring is the barrier....is the barrier, right? Yes, the barrier. Call it a fence, call it a fence, call it a ring. But this is the... This is the ring. The tongue goes to the barrier, right? So, I can also show you images of basically the organization of the poorly enriched domains, etc. 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're... So much you can... Yeah. That was to me... Tom... Maybe here. Mark. Quick question. 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 just get it all? I mean, I was in Randy's lab for six months, two years ago, and I basically... We don't see that. They basically say that KLHL-12 is ubiquitin-hitting, Sec 31. But where? It's 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 2, 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, etc., etc., 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. Very quick question. So, back to this model, the connection between the R-Degolgi. 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 constipate? The ER should become constipated, because only there are a few limited number of regions where the Golgi is in contact with the ER. 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 would be a conduct 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, you know, 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, you know, Eve said, you know, 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. And you need to export even bigger, as you say, than what happens is these rings have the capacity to... It's like a fence. It fuses to the fence on the side, 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 a tango wand, but it collapses. So, it's very, very hard to work with. That's why we decided to go into PICCIA. And then we are trying to create in a PICCIA the ability to express. So, we can do it with tango wand. That's no problem. Will they generate bones? Will they generate bones? I don't know, but we are... So, there are four minimum... You need four proteins. That's a must. 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 Golgi and hug gets sorted, it's someone else's business. I think this would be sufficient. So, that's where we go. Thank you very much. Thank you.
 So, alright, 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 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 Polari 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 residence 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 are packed into a kind of vesicle that has been  monocled or termed COP2 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 COP1 vesicles.  So COP1 is a class of vesicles 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 COP1 and COP2 and these class of molecules are collagen  musins, chylomicrons, PLDL particles and I'll talk about that today.  So you know 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 COP2 vesicles which form at the  ER.  So I think there is a need for me to present our current understanding of what a COP2 vesicle  is and how it forms.  So you need a set of about six polypeptides.  And this is mostly the work of Randy Schechtman and Bill Bolsch and colleagues.  So what happens is a protein called SAR1 which lives in the cytoplasm in a GDP bound form  interacts with Sec12 which is a transmembrane protein of the endoplasmic reticulum.  This will change GDP for GTP and when that happens this protein SAR1 exposes an amphipathic  helix which inserts into the outer leaflet thereby bringing this protein into the ER  membrane.  SAR1 GTP then binds to a dimer which is made up of Sec23 and Sec24.  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 Sec24 has the ability to bind to receptors which can then bind to soluble secretory cargos.  So this is a way to collect material in the lumen of the endoplasmic reticulum and connect  it to the inner layer of the COP2 codes.  Now the binding of this dimer to SAR1 and once the cargos are associated then recruits  another dimer which is made up of Sec13 and Sec31.  So there is only one isoform of 13 but there are two of Sec31.  Now this binding of this dimer to Sec23 and Sec24 what it does is it starts turning off  this process or terminating this process.  So Sec31 has the capacity to increase the GTPase activity of Sec23 thereby converting  SAR1 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 COP2 coated vesicle then emerges from the endoplasmic reticulum  and it can be then targeted to the Golgi.  Now these vesicles COP2 shown here and COP1 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.  So almost all the components of these coated vesicles have a role in protein secretion.  But there is 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 or VCI 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 7 which is absolutely necessary for the formation of skin can be  up to 415 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 coptovascular.  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 is only 60 nanometers in diameter.  Now similarly cells of the liver and intestine secrete chylomicrons and very low density  lip proteins.  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 coptovascular.  So which are big droplets?  Big as 100 to 150 nanometers.  But remember you are not secreting one droplet at any given time.  You are 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.  And 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 coptovascals.  So the question is how are the cells transporting these molecules because they are 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 copto mediated vesicle transport just couldn't explain how  collagen 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 copto and copfone 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 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 called 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 Seito 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 sites.  So these are 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 SIRNA, 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 7 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  knock down that we can achieve by this particular procedure.  So this told us that Tango one was required for collagen type 7 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 them.  We were able to generate a mouse knockout of Tango one but we are not very good mouse  geneticist and we lost to our dear friends.  They were able to create this mouse knockout.  So Mia 3 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 1, 2, 3, 7, 9 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 7 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 by 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 well not quietly he published a paper in 2016 in PNAS in which he was able  to co-crystallize parts of tangos 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 SS3  to collagen that we had proposed and reported is not direct but it is mediated through a  protein called HSB 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 a as an acceptor.  Now I should also mention that while HSB 47 is an acceptor for collagen type 7 and many  others there are collagen that do not interact with HSB 47 but they interact with other  chaperones and so our thinking is that this protein can interact with collagen through  different molecules and not all of them have to work through HSB 47 but in principle the  connection between collagen and Tango is mediated by a connector in between which is in the case  of collagen type 7 at least HSB 47.  And you know all of them yeah?  We don't know all of them but we know for a few of those.  You know again it will become a detail I mean I think the principle is has been established  through a specific.  This one is very specific because you know how it works again I don't know how I'm going  to run a time you know the collagen primers start at one end and as they are folding these  proteins start binding they bind in fact Nagata 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 to code so the idea is that if a cop to code  needs to make a small vesicle it assembles into a small structure and then it pinches  okay 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 expected so red here is the same as this one is collagen and the green structures  you see are copter codes it doesn't matter whether you use sec 23 or sec 24 okay so I  expected to see a complete area here covered by these green structures so instead of seeing  this punctate elements I expected 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 collagen are about to leave the ER here again shown in  red these structures seem to be studied 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 seem 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 on 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 1 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 kind Tango 1 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 is 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 stretcher for amino acids about 150 amino acids from here to somewhere in between to  the middle of the coil called first of Tango 1 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 1 and put it at the mitochondria artificially and when you do that the Ergic membrane 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 1 have  the capacities it's 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.  Remember it's lipis plus proteins.  Yeah.  You know which proteins 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 okay 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 okay.  Now the idea then is that this part here can recruit Ergic membranes they fuse and their  fusion and rapid built 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 snare 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 1 of course is necessary because it acts as the receptor for collagen okay.  Syntaxin 5 is not a surprise because if you knock down this particular snare then there  is no gull-cheaper say 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 gull-cheaper 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 Tango 1 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  SA3-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 will have a lumen and it  will have a face that is on the size of that's the cytoplasm.  So in the lumen of the ER this SA3-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 okay.  Now this pushing is probably mediated by SA3 domain through HSP47 which might just be  sort of walking on the collagen as it's assembling okay.  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 spatula?  Is this a patch of membrane?  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 here okay.  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 Vivek 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.  And 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.  Now at this stage.  Well I think tango is not stretching like N2C terminals it's basically assembling laterally  into a ring like this.  Just one.  It's just a polymer.  It's just give me a few.  It's a polymer.  It's a polymer.  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.  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.  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.  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.  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 the 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 has been placed into this container the SS3 domain dissociates from  the collagen molecules.  When this dissociation takes place the proline rich domain dissociates from SEC23 which allows  SEC1331 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.  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 COP2 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 is another protein that has been identified it is called tali or it is also given the  name mya2.  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 its export of  collagen, tali in the cells that express it is required for the export of kylo-microns  and VLDL which are again very big particles.  So it is similar principle but there are slight differences.  It is not the same but it is 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 is a polymer of three very related proteins.  Now in certain cancer cells this protein mea 2 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 etc.  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.  So is it possible that when collagen are being pushed through the function of tango  and associates into a structure that is growing?  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 collagen have been transferred then you cut here 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.  And the first cistern of the Golgi which then contains collagen just keeps moving forward  and this is what cistern maturation is all about.  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 the cisterna can 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 collagen 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 one molecules in cells that secrete collagen.  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 one binds to one collagen trimer this allows us to guess that there could  be 100 to 200 collagen trimers 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 the 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...  So there are no vesicles coming.  But here they are making it a fusion.  Sure of course for every structure that has to fuse there will be fusion components.  If you do an ER Golgi certification on cells that are active secreted in 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 life cell imaging and this is  exactly what we are trying to do.  You know we spend years purifying Golgi's 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 do you think would be the transfer?  So 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 the electron microscopy.  Electron microscopy.  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 onto the copper codes but they cannot come off.  So we think we might be able to see these connectors.  But you need a transport...  Sorry, you said that there are like, 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.  We are looking for someone who I would love to.  I mean this is exactly what we would like to do.  Why do you invoke the need for connectors 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-Golci events.  But it isn't clear whether they are vesicles or they are 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.  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-depp cells.  R-depp cells are the best cells for studying collagen type 7 because they produce...  These are the skin cells.  So they produce carcinocytes, lots of collagen.  So if we cannot see them there, then there is...  R-depp.  These are the cells of patients with epidermalysis bullosa.  So they are producing gobs of collagen.  So let me just...  I mean, again, we can...  Okay.  With this, what you are losing is the directionality that is normally given by physical attractorism.  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, things...  Other things would leak out.  Yeah, calcium.  Yeah, but the thing is that you could bring them back.  I don't know.  Calcium?  No, calcium...  Calcium, you might be able to pump it out.  There are circa channels, the SPCA channels at 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.  I think we have to figure 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...  It 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...  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 vesicle.  So, going there would be...  I would need another lifetime.  Now, let me just...  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 hypersecretion of collagen.  So, this group at Myoclinic, in fact, has been able to, at least in a mouse model,  what they do is they 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 by... 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 this is... 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, Christina and Antonio post-docs, they were the ones who showed that tango recruits  ergic 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 Isheer 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-2 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 blue is tango, and this color, yellowish-brownish color, are the cop-2 coats.  So the cop-2 coats are being corraled by tango, and we say that tango,  whether it's a homomer or a heteromer, tango C-Tage-5.  Because this is what creates the...  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-2 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-2 coats  and therefore contain cop-2 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-2 in the middle,  so there are interactions of tango to cop-2 components as shown here with these lines.  And we know that this is through prolinege domain, and this is in sec-23 coats.  So if you now knock down cop-2, you can do it two ways, right?  You can express a tango that doesn't have a prolinege 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 prolinege domain to the cop-2 coats,  but the lateral interactions which we think are being mediated by tango interacting with itself  and tango interacting with c-tage-5 and tango interacting with tango-short.  And in doing so, you end up getting these highly tessellated structures.  Now, this is just to show you another example that if we now affect the cop-2 interaction,  in this case we have expressed tango-1 without the prolinege domain,  but you end up getting 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-2 tango and the binding of tango-2 itself and its associates  is the process that is absolutely necessary to generate these structures.  And this is the direction that we are going into try to understand how this structure is assembled at the ER.  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 the transcology, 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 exports 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 resort 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 coats.  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 ATPase.  Yeah, Tommy.  I might have missed 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, about over 1,000 amino acids, 900 are unstructured,  one possibility is that it can only extend up to this distance.  It cannot go like so and therefore dissociate.  It's simply conformational change.  And then...  So going back to the translocom model, the pulse is constant.  And then, anyway, from your pictures, it looks like it's on a translocation through some sort of a core.  Actually, when we started to work on pathology many, many years ago,  was to synchronize the folding of prokolog and indian-nupasana reticulum.  This allowed us to generate large aggregates, indian-nupasana reticulum, very large,  one micron by one micron.  So when you release the block of secretion, it's the complicated to explain now,  although they're so big, they go out easily.  How does that fit with your models?  Certainly not with a simple translocom through a core, 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 translocon as in the sense of a 9 nanometer pore.  This might be much bigger.  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 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 molecules.  So, to get fusion and to get this increase of area, you need to have a barrier somewhere.  Otherwise, you will...  So, basically, it means that...  Ring is the barrier. ...is the barrier, right?  Yes, the barrier.  Call it a fence, call it a fence, call it a ring.  But this is the...  This is the ring.  The tongue goes to the barrier, right?  So, I can also show you images of basically the organization of the poorly enriched domains, etc.  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're...  So much you can...  Yeah.  That was to me...  Tom...  Maybe here.  Mark.  Quick question.  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 just get it all?  I mean, I was in Randy's lab for six months, two years ago, and I basically...  We don't see that.  They basically say that KLHL-12 is ubiquitin-hitting, Sec 31.  But where?  It's 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 2, 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, etc., etc., 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.  Very quick question.  So, back to this model, the connection between the R-Degolgi.  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 constipate?  The ER should become constipated, because only there are a few limited number of regions where the Golgi is in contact with the ER.  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 would be a conduct 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, you know, 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, you know, Eve said, you know, 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.  And you need to export even bigger, as you say, than what happens is these rings have the capacity to...  It's like a fence.  It fuses to the fence on the side, 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 a tango wand, but it collapses.  So, it's very, very hard to work with.  That's why we decided to go into PICCIA.  And then we are trying to create in a PICCIA the ability to express.  So, we can do it with tango wand. That's no problem.  Will they generate bones?  Will they generate bones? I don't know, but we are...  So, there are four minimum... You need four proteins. That's a must.  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 Golgi and hug gets sorted, it's someone else's business.  I think this would be sufficient.  So, that's where we go.  Thank you very much.  Thank you.
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