 So just following up on this very interesting talk, I mean, what did they die of? Then if you've shown at least that the quality control systems were still working fine, what did they die of? Yes. So what we can find in cerevisiae, in all cases where we can make conclusions, for example, if we prevent aggregate formation, the cells live longer. But what we observe is that actually this leads to a stress to the cell, because actually you have all these small seeds of misfolded proteins that float around in the cytoplasm, and that uses stress response. And the first thing that I mentioned before when I was asked, when you have this proto toxic stress in the cell, you actually down-regulate the diffusion barrier, you open the diffusion barrier, and now the circles start to leak out. So when you don't form an aggregate, you live longer not because the aggregate is killing you, but you live longer because the misfolded protein are treated like a stress that opens the diffusion barrier and leads to the release of aging factors to the progeny. Every single time where we have been able to prolongate the lifespan of a yeast model cell, the DNA circles were leaking into the blood. And each time that we very surgically mutated by single amino acids that lead to the detachment of the circles from the pores, to let them leak in the blood, the cell is long-lived. So far it looks like what they always die off is accumulation of DNA circles. Now how do the DNA circles kill the cell? Probably by the fact that they do change the nuclear pores. It's the model we are investigating now. So the circles, as they accumulate exponentially, pores accumulate with them exponentially as well. And so all mother cells are full of pores. But as I said, those pores are not classical pores. They are specialized pores with some subunits gone and others that have joined the pore. And those pores are very good at imports but very bad at exports. So they are specialized. So we think that this leads to an imbalance that actually kills the cell. Do you see any change in reactive oxygen species? We have not touched radioactive... No, not radioactive. We have not touched to ROS so far. Because the ROS data is completely ambiguous. If you prevent respiration, you are short-lived. You don't form ROS and you are short-lived. And you are in service. You can inhibit certain complexes in the respiration but still produce ROS. But in the mutants where people were showing that they were preventing ROS formation, these mutants were short-lived. It looks like a certain level of ROS is necessary and too much ROS is for sure deleterious. You take a mammalian cell, primary cell from the mouth, and you culture it, it's an S, right? After a while, stop dividing it's an S. Age, life span is short. The reason is because you grow them in atmospheric level of oxygen. If you decrease the atmospheric level to 2%, then they will live forever. It's an own observation. Yeah, sure. There are many things if you delete... For every fifth gene in the yeast genome, if you delete it, you will live short. Do you live short? Why? Because you are sick? No. If you grow a cell outside of the body in a petri dish with high oxygen concentration to which it is not made to live with and that you find them to live short, what is your price? Right? It's not because it's due to reactive oxygen species. Sure. And if you grow yeast cells in H2O2, they live short. Right. And also, if you take your ways... If you grow them at 42 degrees, they live short. Yes, scavenger away. Scavenger or frost away, they live shorter. Sure. But many of these things, living short is an easy phenotype, right? Yes. Almost like a bear. Do we have any questions for the small GTPase people? I'll ask them. I was curious for... Am I saying your name? Mayo. Mayo. Mayo. Mayo. Can you say a little bit more about Braggson? I was wondering, is it, first of all, is it a natural product or a synthetic chemical? And also, has there been any structure-activity relationship? Can you perhaps put a brasset probe on it? Yeah, well, it's not a natural product. And we did, indeed, a SAR study with that. I couldn't show that, obviously, because it's not published and we have some valorization issues as well. But we worked with analogs on one side and with mutant of the protein on the other side. So, we're pretty confident in the binding mode and what is important or not in the molecule. Do you think you could put, say, a fluorescent probe on it somehow and retain its activity? It might, let me see, it might be possible but you have to try it first, right? But we didn't try that yet. We have some other things going on. But after the phenotype takes place, like click chemistry kind of thing, so we're working on those kind of ideas. But we don't have a fluorescent component, for example, right now. Is it rapidly reversible? I was just wondering, because it's kind of an interesting binding mode. Well, I didn't do kinetics on that, but yeah, you treat your cells for half an hour, you just wash for half an hour. Meaning you just change the medium and half an hour later you fix the cell, do the microscope and the microscopy and it's almost completely fine. So, you can see that it's not as compact at the beginning but it's pretty good. You haven't tried to look in real time though, like this? No, no, no. I didn't. That will be interesting. There's lots of things to do, I think, in terms of visualization, real time, tough microscopy, this kind of approach. We have a question. I have a question for Nawa and Bruno. So, when you show the ER2-Galgie and the plasma membrane trafficking, which is a classic of pathway, but there are also some reports, ER2-Plasma membrane direct transfer and the bypass of the Galgie. So, I wonder how common this is and why you think certain cargo want to do this? So, what's the advantage? So, do you know about this? I mean, is this under certain conditions, in certain mutants, certain cargoes? Yeah, worse specialized conditions. So, I'm not sure that, I mean, what I know is this ER, for instance, plasma membrane contact site, I think they are used mainly to transport or to exchange lipids, right? I think it's not really used for protein transport. Yeah, I think there are a few reports of some proteins that can be also transported. But I'm not sure how strong the evidence is. I don't know. I think the evidence is very strong for lipids because, I mean, this protein that forms these contact sites, I mean, lipid transfer protein, but... I don't know. Do you know, Cathy? So far, there is not rab-GTPS involved in this. I think so. Paul Snare. Not yet. So, you were referring specifically to bypass membrane, in terms of contact sites. I don't think it's a lipids. Randy Scheckermann had a review article a couple years ago. So, they called it unconventional secretion. It's basically a protein secretion. Oh. By the way, now going through the Galgene. So, those are separate mechanisms? Right, I think it's very specialized. Yeah, so that's a separate process. And proteins are transferred, yeah. So, unconventional secretion mechanisms. And I guess, are the rubs involved? I mean, I think this process is very well known in neurons, right? I mean, you have a... There is two papers in E-Life recently, where you have a... You have a lot of anglicosylated proteins that bypass the Golgi that are transferred to the plasma membrane. I think, at least in the dendrite and axon, I think the paper says that it's due to, I mean, direct... I mean, transport from the ER to the recycling endosomes and then to the... I think it's a recent paper in cell or in neurons about... Oh, no, I... No, I can't just make a remark that YPT-11 in cerevisia seems to be associated with ER, plasma membrane contact sites at the butt cortex, but there is very little known about... It's a raw GDP, yes. So, it's as far from... I mean, sometimes we... Not about... With some YPTs... It's not even clear if they belong to the YPTs or to the next GPAs family. So, YPT-11 is, I think, one of them. But maybe, you know, now that if you say it, maybe they are involved in some membrane processes. It will be interesting. Tommy? I think I got confused in your talk. Yes, you're talking to me. Okay. Oh, you? Yeah, no. What is your price? Tell me. So, the constipation that these yeast cells are getting when they have the DNA attached to the neutral pore that is leading... It's not really constipation, but yeah. So, the entry goes, but the exit is difficult. Yeah. So, the way I understood it is that if you have this piece of DNA, this plasmid hook to the nuclear pore, that exacerbates the effects the cells get sick because they are not getting a proper balance of egress and ingress into the nucleus. Is that correct? Yes. So, that's the constipation effect. If you want. If you want. Okay. Yeah. Yeah. If you don't have that piece of DNA, then what you were saying is that you were connecting the fact that you were getting this trapping depending on the biophysics, let's say, the ceramide, etc. Is that the way you were thinking it? Mm-hmm. So, but under normal conditions, things do move correctly, right? So, these were consequences of your experimental setup of pushing the systems of the partition correctly or actually the nuclear pores? No. No, no. No, no. In a normal wild-type cells without pushing anything, you will see the pores accumulate in the aging mother cell and while she gives birth to daughter cells that have a normal number of pores. So, how do you count the nuclear pores? So, the way we have been counting nuclear pores so far is mainly by fluorescence intensity. But that's, okay, the nuclear membrane that is forming and the budding cell, maybe it's just simply sampling by concentration surface, I mean, density of nuclear pores. I know you will get lower number of nuclear pores simply because you have less nuclear membrane, right? No. So, we did... Yeah, yeah. Okay. I can give you more detail. I can give you more detail. If you let a wild-type yeast cells age in which you have labeled specific nucleoporins, some nucleoporins will accumulate to... And we can show they are associated with a pore and these pores associate to very high levels such that at the end of her lifespan, a yeast mother cell can have more than 10 times more pores than a young cell in a wild-type. So, we go from 100 pores to 1000 pores or even more. We have seen cells that are even more. And their symmetry is maintained. The daughters are born with normal number of pores. If we prevent the any-circle accumulation, there are mutants that prevent the any-circle accumulation, then you don't accumulate pores. If you detach the pores, the circles from the pores, then you don't accumulate pores. If you promote circle formation, then you accumulate pores. And each time, the lifespan of the cell corrode extremely well with the pore content. The cells that have a lot of pores die early. The cells that accumulate pores much slower live much longer. What we know is that the circles, when it is at the pore, it recruits an acetyltransferase, SAGA, and SAGA acetylates, and this is something that happens in a normal cell cycle, in interface. And SAGA acetylates a number of nucleoporins, such now we can mimic acetylation. If we mimic acetylation on these nucleoporins, we accumulate pores without circles, and the cells die earlier as a short lifespan. Those nucleoporins that we are playing with are involved in controlling the import versus export. In all cases, what we see is an increased import and a decreased export. That's the deal. Does that clarify? That's a joke. That's a joke. David. I'm wondering, forgive my ignorance on this, but for RAV proteins and ARF proteins, what are the, in the most primitive eukaryotes, are they present? And what insights, if you look at the very, very simple eukaryotes, can you get about what's the most fundamental mechanism of RAV proteins? It seems like now there's maybe a diversity of function, when this family formed, is there some insight from looking at the evolution? Maybe not to repeat the question for posterity there. The question that we cannot answer, if we know about evolution of the RAV family. So from what I remember, there will be... I'm trying to be an ARF. ARF, basically there can be one. RAVs, there is sort of like the minimal set with and without duplication, so there could be less than a service. Does Giardia have RAVs in ARF? Who says... Just one thing. So the first eukaryotic common ancestor had ARFs and RAVs. It had very few ARFs, maybe just one. I'm not sure exactly how many, but very few and more RAVs. So it seems to be a conserved feature to have few ARFs and more RAVs. And this goes way back to the first eukaryote, the last common ancestor of eukaryotes. Are they in archaea or in prokaryotes? Well, so there's been a discovery, there's a discovery of a very recent, very recently of an archaebacterium that's the closest to eukaryotic ancestor that has been identified. And so there are ARF-related and RAV-related proteins in that archaeum, that archaebacterium. So that's actually a very interesting path to follow. Yeah, that would be very interesting to follow up. They are different than the eukaryotic ARFs and RAVs that we have, but clearly they're signatures that make them, that put them into that family. So yeah, we don't know anything about this organism because it's only been identified at the genome level. So what would be very interesting is to see what its membrane compartments are like and that will await its identification. We only know of its genome through deep sequencing, through its identification. So Bruno, sorry. Just to finish on this question, I'm talking about RAV-GTPS, so this highly conserved throughout evolution. And I think so, now we have genomic data on many, many species. And I think there is, I mean, you need five or six, I mean, there is a minimal set of five or six RAV or YPT that are enough to sustain the secretary and probably endocytic pathway. So this is RAV1 or YPT1. I think YPT3, YPT6, YPT5, and maybe another one, NSEC4. NSEC4. NSEC4. NSEC4. I'm not sure about seven, you need? Yeah, maybe yeah, YPT7. Think about why not in bacteria probably? Because they don't have intercellar compartments. They don't need this machinery, but there will be some in bacteria that are parasites in human cells. They actually have sometimes RAV or RAV-interacting proteins that they took and you can see transferred horizontally from hosts many, many years ago. So not snares as such, no. There are long and domain proteins, so the long and domain is present and that is present in some snares as such. That does suggest exactly that, yes. Well, there's many questions, yes. Stop this misconception of people saying grabbers are involved only in fusion by saying they are involved in any all aspects of circular trafficking. Not just fusion. Formation, too. And in the east, another, is there examples where one transport step could have more than one YPT involved? Yes. So one that I showed is, for example, going from trans-Golgi vesicles to the plasma membrane actually uses two RAVs, YPT3-1 and 3-2 or 3-2 and sec-4. So they share this step. Okay. But they share... The idea is that one goes half way and then the other one takes off. Yes. Or you mean if they are parallel? Is that really true or is that just true for certain types of granules and then YPT3-1 could take all the way to the... could bypass sec-4 for certain cargo also? No. Over expression of YPT3-1 does not surprise sec-4. Okay. Still, whatever sec-4 transports could be could be very important. Yes. And lead to lethality. But that doesn't necessarily mean that YPT3-1 could still take some... All the way, no? Yes. Well, it doesn't interact with also with the specific effectors of sec-4. So I don't think so. Yes. So this in the endoscopic clavicle vesicles we see after the vesicle buds from the membrane we get two waves of two different RAVs coming in, one is RAV-5 and the other one is RAV-35. And they have different... They have different effects in different lipids. They have different effectors. And they can... Even though they come in the same vesicle where you trap them, right? They come in waves that are... They're not synchronized. They're slightly shifted in time, right? So... I think the RAV-5 is, I think, very important for homotypic fusion. RAV-35, I'm not exactly sure. Maybe it's more direct. Is it? No, this is endocytic clavicle vesicle. It's not recycling yet. The trust paper on RAV-35 was he made up, I can't wish... No, no, by the way, I don't mean to... Sorry. Go ahead, yeah. No, what I was trying to say is if you actually track on the vesicle the RAV, right? They're coming, but that vesicle will actually go to a RAV-5 still, right? So it's... Are they mutually exclusive? No, they're not, no. Yeah, that's the point. No, but they're looking at different... They're not looking at the same factors. Well, of course. No, but I think that... No, I mean, the very first paper on RAV-35, the current biology in 206, I mean, so we measure the internalization of transferring, okay? And, you see, I mean, the message that RAV-35 was involved in a very fast... I mean, very fast recycling of transferring even between... and before RAV-4. So we don't know if these vesicles don't form, I mean, reach the endosomes and then recycle. Is that what I wanted to say? So I would like to counter that, right? So those experiments are based on, let's say, dominant mutants, right? Right? No, in S-H-R-N-A. At that time, we used S-H-R-N-A, yeah. All right. So either you do that or you do a depletion by RNA-R, right? So these are slow effects. It takes a while until you build it. I'm trying to make the point that you just watch the arrival now of the proteins without perturbation in the system, right? As I said, in the endocytic corridors, the two proteins are coming, right? To all the corridors. Right? And it's regardless whether you're transferring or not. So there's no discrimination between transferring and the GF receptor or whatever that, they don't care, right? The sorting happens later, right? So I think what I'm trying to also bring into discussion is that we have the experiment that we do where we do the slower perturbations and we get the read on the concepts of that as compared to now the ability to see what's going on without the perturbation and there are some differences, right? But this is not incompatible with the fact that RAP certified could be involved in fast recycling, right? I mean RAP certified associated with the vesicle and then... No. I just said that I have no issues with that. That's fine. I was just saying that the vesicle that pinches from the membrane and came in, it has to decide what to do, right? I mean, is it going to recycle or is it going to be endosome? This vesicle, this very, very first vesicle is going to heal in one first compartment and then there's going to be a sorting, right? And now I'm going to recycle I'm going to go a bit deeper, right? But that very first vesicle doesn't know yet what to do and in spite of that is collecting the two RAPs, right? So... We were discussing a little bit about just to follow up on this if you don't mind. As we were discussing at lunch, I think the likelihood is in the million cells, given the complexity of the trafficking is that you always have you always going to have more than one round simultaneously in the same vesicles or most of those because what they're creating is domains and those domains create different functionalities and you're probably going to need different functionalities sometimes simultaneously or with different kinetics that they're forming and removing and disappearing and etc so it's all this combinatorial scheme where the RAPs will be I think that's the most likely scenario and that's entirely consistent with you seeing more than one RAP coming in even at the I just want to confirm this is not this is not over expression yes shh So I just want to add to the conversation what could be the role of cargo because we saw of the talk of Bruno you had RAP6 and that could move to the plasma membrane to the focal adhesions to the molar zones and we saw in the talk of NAVA that you had YPT1 that could go to the Golty or to the POS so is there any connection between what is in the fascicle that could determine the recruitment of RAPs and that determines the recruitment of effectors so thank you for asking this question this is exactly what we are thinking when I talked about the module I think the cargo is the one that will determine which Jeff will come which Rob will come which effector because otherwise that's our model and we are working on it and we have some evidence that it's true So how do you how do you make a carrier in the secretory pathway that only has cargo X and not cargo Y well just to answer your question together with Judith we are not doing it in the secretory pathway because it's easier to do it with autophagy and we have two different types of autophagy depending on the cargo and these are all not stress not under stress selective which now require go to different organelles and yes what but I think the problem in the normal secretory pathway let's say you are dealing with smaller carriers I don't understand how you you will end up having a carrier that has only one type of cargo so you can go here or there no she's so that's the problem I think you have as you know you have a few examples of a direct interaction between Rob and the cargo for instance between Rob 11 and better energy receptor and this drive probably the recycling of better energy receptor to the plasma membrane so this is known for this I think there is also the example of Rob 21 is work by Johanna Ivesca and I think it's involve Rob 21 for recycling of a pool of integrins so it's not about that you are doing the sorting no we we talk about that and also actually there was a report I think from most of a long time ago that Rob 3 was directly interacting with the poly poly for for transition for this because I think when I was working on the hops I thought okay it's in the vessel and it's there but I realized that it's a too static picture it's probably a continuum of recruiting reps or hops or tatters and in the end like Miguel was saying it will form domains or depending on the cargo one of these factors that is recruiting may kind of win and then you get that this physical recruits these effector proteins and it makes a kind of decision then okay so I should go left or right but I can imagine that cargo could be very determining in that and also sorting is not 100% so you may not come cargo to the wrong side but I think the majority of cargo may then influence what is happening it's just okay a question for Yves Barre he's have you found some other proteins a part of re3 which I will raise on his link to memory on aging yes so proteins with this type of domains there is a full study that has been done from the lab and the yeast genome contains at least 150 by the threshold they used and actually if you are losing a little not much per bit the threshold it goes rapidly to 250 proteins and actually we needed to lose a little bit their criteria to get to re3 top so there are way more and we know of at least 5 more proteins that are actually aggregating in response to pheromone and that are involved in the phenotype that you saw so it's not just re3 re3 is a key one but it's not alone and we are now characterizing a few more that respond to and that are necessary for stress memory describe long time ago by pastor actually acquired termotorrent and we have some hints about also some proteins with this type of domains involved in toxin response on the protein aggregation so protein aggregation has been a classical hallmark of aging cells but generally nobody knows what are in those aggregates we know in terms of memory that the candle and linguist lab had worked on at least the protein that aggregates in response to stimulation in the synapse aggregating response to stimulation of the synapse and is required for long term potentiation of the synapse so directly involved in the memory and there is the same protein in drosophila is involved in the memory of courtship our males flies have to learn courtship they do this by protein that aggregate with a similar aggregation domain and that protein happens to be the closest homolog to we3 we have a question here some of the questions from the computer person to be so you said the cell has to distinguish DNA cell from non-cell compared to very long strings and there are a lot of computer aggregates for this for other kinds of systems being used for example one which can be used to compare to signatures cryptographic signatures of other two strings you know so we think so far from all what we have been able to do it's completely sequence independent so we don't think that they are recognizing any of the sequence so what we think they recognize is in cerevisia it's whether they have a centrum or not we simply need to add these 150 nucleotides to any piece of DNA and now it will condense during mitosis and if we in Pompeii it's simply the history the fact that you saw that it wasn't in the chromosome was sufficient