 the title for my talk, is it okay, the first? Is regulation and coordination of interest in a lot of trafficking pathways. So halfway through my organizing this conference, the mathematicians who talked with me, said that they added, we added the phrase, where is the red? Ideas and concepts. So I decided to stick to it and just talk about ideas and concepts mostly and give some examples of data. And also to put it in the context as the organizer of the rest of the conference, okay? So I'll start my talk, we'll include introduction, regulation of secretion and regulation of recycling. So what's really good, so in the introduction I just want to bring you to what does it mean regulation of interstellar trafficking. What's really good is that I don't need to give a really long introduction because you've heard everything that I'm gonna say in the introduction, you probably heard throughout these days. So the secretory pathway or the exocytic pathway takes proteins and membranes from the endoplasmic reticulum through the Golgi apparatus, through secretory vesicles to the plasma membrane. In the endocytic pathway, stuff is taken either from the outside or from the membrane through a set of endosomes to the lysosome, which is the degradative compartment of the cell. It's all gonna be full in a minute. So this was talked in length on Tuesday. You also probably know that traffic in every step is bidirectional and there is cross talk also between the endocytic and exocytic pathway. And everything between compartments is transported via vesicles between the ER and the Golgi, between the Golgi and the plasma membrane, plasma membrane to endosomes, endosomes to lysosome. So you also heard that inside compartments, Golgi, the sorting compartments, Golgi and endosomes transport is done actually by maturation. And I'll talk about these two later. That's why I wanted to. So what is vesicular transport? A donor compartment, let's say ER, you take the cargo, which can be either luminal. Luminal is inside of the compartment or membranes. And then this vesicle will form. And then it needs to fuse with the right acceptor compartment. So now this is vesicular transport, just the overview. But if you look really at the sub steps, it's much more complicated and you heard about these two. So the vesicle first has two form and there will be coats and cargo receptors. And then it will have to move. And we heard about motors, either for actin or tubulin, the cytoskeleton. And then there will sometimes be membrane remodeling. And then you heard about tethers that will bring the vesicles close to the acceptor compartment. And then you will get the snare complexes that will help the fusion of this vesicle with the acceptor compartment. Okay? So if you want to think about it simply, about regulation, so if this is the cell and this is our vesicle, this is Paris, and the vesicle, the question is who are the traffic lights of the cell? Okay? So at least some of the major traffic lights are these GTPases. Which are called YPT in yeast and RAB in mammalian cells. So you heard about RAB6. And these are really what they do. They are molecular switches. They are very small, but they can cycle by themselves from the GDP bound form to the GTP bound form. From here to here, from GDP to GDP, they exchange the nucleotide. And from GDP to GDP, they hydrolyze the GTP. And I'll explain it later. When they're in the GDP bound form, they are mostly off in the GDP bound form, they are on. So in yeast, there are nine to 11 of them. The other two we don't know yet much about them. So I'm just, I'm not sure. I'm indicated in membrane. I'll explain in a second. So, and here are the major ones. So even when we say nine, we have, for example, YPT1 in the beginning here, and YPT3132, which are sometimes called 3132, they are functional homologues, okay? And for example, we don't know much. We know about YPT5, one and two, three, but we know mostly about 51, but they are involved in the same process. So even when we say nine, it's actually less. And it's important for later. Okay. Does each one have a clear homologon in million cells or? Yes, and this is shown here. So thank you. So there are 70, about 70 MMLI and human rubs. They are also regulating the human protein trafficking pathways. So there are more, but here I'm showing for, oops. So I just wanted to point that there are really large group of FGTPases together with their friends, with their ups and regulators, and some of the downstream effectors. They form like a 200 protein group, which is about 1% of the human protein. Yes. So for example, RAP6, here you put it between the ER and the Golgi? Yeah, RAP6 is not very clear what it does. And Bruno just told you, it's like somewhere here floating. RAP1, and Alec, talk about the localization and function. It's going to get a heart attack, can you say? No, no, no, Bruno, no heart attacks here. So YPT1, the best homologue is, the close homologue is RAP1, YPT3132 is mostly RAP11. RAP6, actually the RAP6 homologue, YPT6, is also a functional homologue. YPT1 is also a functional homologue. RAP1 is a functional homologue. You can delete YPT1, put RAP1, very highly conservative, can you say? And the same is true about YPT6 and RAP6. I don't know why, we haven't figured it out. It's not essential in yeast for viability. So, and we actually focused, Bruno talks about that. On them, we focused mostly here. That's why the Golgi is so big. So I just want to give you in the introduction some idea about how we even got into where we are now. So I'll call them the phases one, two, three. So it all started in yeast. All this family was discovered in yeast. First, why yeast? Because in yeast we can do, it's the same compartments. We do cell biology the same way. We can do biochemistry. But the genetics is what separates it. And that's why we can do so much more faster, okay? Of course, in combination with molecular genetics. So in phase one, we first shown, we looked at two YPTs, really. YPT1, which is essential for viability. YPT3-1 and 3-2, which together are essential. The cell can have one or the other. So they are very similar in their functional homologues as of today. Maybe each one, they have some kind of other functions that we don't know yet, but for now, we usually delete one and then the other one becomes an essential. So this we did, how did we discover them? Somebody asked me, is by reverse genetics. Meaning that we saw, oh, this is an interesting protein. It has homology to us. It must be an oncogene and it is. But this took many years to see it. And then we went from the gene to mutant. We made mutations to phenotypes to see what's happening here. So the next thing was, or together, was to see in which transport it. So first we knew that it was a huge thing to see that no, they are not at the plasma membrane. No, they are not in signal reduction per se. So, but they are in trafficking. And then we wanted to know which transport step they, because by now we know that there is more than one, which transport step they regulate. And here again, combination of molecular genetics, genetics and cell biology. And the last thing in phase one, we already knew that it's conserved from AEs to humans, you know, the RAB1 and YPT1, by also a combination of molecular genetics and cell biology. So this is the minimalistic view of cells, exocytic pathway to plasma membrane andocytic pathway. Okay? And what we, what I'm showing here, based on function, based on mutant phenotypes, we saw that they are in the exocytic pathway. YPT1, a set of papers, we showed that it's in the early transport from ER to Golgi or Golgi2. And then the YPT3, 1 and 3, 2 are late Golgi. Exactly what they regulate, something like that. If you stop them, actually it's very fast. So I'm saying they're essential. How do we work with them? It's a good question. We have a temperature-sensitive conditional mutants. We can use different conditional mutants. The easiest one to understand, stop of trafficking transport between, we can measure ER to Golgi transport. Exactly, say we weigh the transport by just time for the auto-transfer, like that. Yeah, but we can see it's transport or not transport, but also we can see which step. By using cell biology, combination with cell biology. No, step's 1 and 2. When I say step, it's 1 and 2. Yeah, YPT1 is here. We block it, block from ER to Golgi. If we stop YPT3, 1, 3, 2, block from Golgi to plasma membrane. But why, but so, it brings, why you should stop them now? You always have a strategy. What do you mean to stop it directly? Or you've used no mutants, it's never stopped. I'll explain, okay? Why, okay? In a minute. So, because we saw it, we saw that YPT1 is an entry, entret, so this is my French, also T from the Golgi, we call them the Golgi gatekeepers, okay? Okay, in phase two, and I'm getting to your question, don't worry. So we knew that there are molecular switches. We knew that somehow they regulate vesticular trafficking, but we didn't know what's in between, okay? So the first question was, how are they regulated? What's the upstream regulation of the rubs? Who turns them on and off? So these are the people who turn off the switches, like Georgiana here, who turn on the switch. And who are they? So they are called GIFs or GIFs, so exchange of nucleotide guanine, nucleotide exchange factors to turn them on, and gaps for a GTPs activating factor to turn them off. So just to put it in perspective, I put here, this is one of the GIFs in cells, and by the way, they're very different between proteins, but it's for just YPT1, you need a core trap, which is a complex of four different proteins, five subunits, but two are identical, and this is the little YPT1 under them, that's the size. It's very small, it can interact with the GIF, it can interact with the factors, but not much more. That's why I want to argue that they are really regulators as opposed to doing something else in addition. So this is to remind you about Monday about protein complexes, and of course they play a big function, a big role in what YPT rubs do. So in addition to this after regulation by GIFs, they, as Bruno also suggested, these YPTs, the way that they are attached to membranes, they are, is by having a lepy tail, okay? So they have a lepy tail. So in the cycle between the cytoplasm and the membrane, okay? So to be in the cytoplasm, because it's a lepy tail, they have to be buried in a friend's, so there is a place where this lepy tail is inserted, and this is why they can be in the cytoplasm. It's called GDI, and then when they get on the membrane, they insert their lepy tail here, and then they can be seen by the GIF. So there are actually two for now, we are thinking of as two upstream inputs to activator rub. One is to do the membrane attachment, and the second is to turn them on with the GIF, with the activator, okay? And once they are in their GTP bound form on the membrane, this is when they are on, okay? What does it mean to be on? Oh, and the way that we think about it right now is that the gap, which turns from GTP to GTP is just required for them to be able to recycle back to the cytoplasm, which is also important, so they can function again. So we knew about the upstream regulators, regulation of YPT-1 and YPT-3-1 and 3-2, but now what happens downstream? So what happened downstream is that when they are on, they interact with these proteins, doesn't push, so I lost, oh, here, sorry. So they interact with these effectors, this means that they are on. Okay, so who are these effectors? These are the people who really do the work, unlike those activators which only turn them on and off, okay? So these are the people who really do the work. Who are they? They are all people who you already know. So I showed you this slide before, but now I just wanna say for every step, you'll see a YPT-Rab, and this is summary of work in many labs, YPT-Rab in every step, and its effector will be in red, okay? So remember, oh, and the other thing that I wanna say, a single rab can interact with multiple effectors, okay? So the first step in the formation, remember, here is the YPT-Rab, and it's one of its effector which interacts with the cargo receptor. Here, in vesicle motility, we have a motor that can be either, and Bruno already talked about myosinokinicin, depending on which cytoskeletal route they wanna go on. They can be membrane remodeling, and here are, again, a rab and its effector in vesicle docking. These are what the rabs are really famous for, and many people think of them just as sedering factors, but this is just one of their kind of effectors. And then finally, some, here the YPT and rab and its effector regulating also snare complex formation, so they also regulate vesicle fusion. So basically, all the machinery components that are required for moving a vesicle from one place to another are recruited by these rabs. Does this answer a question? No, my question is why you integrate to talk? Why you just have some protein to transport them? Well, why do you stop at normally? Okay, so let's continue to talk, okay? It's a conversation. Why do you say it? Why do you do it? I'll explain, it's coming, okay? So here we are with our peris and the vesicle and our traffic lights, and you would want, and this is partly the answer, that these traffic lights will talk with each other or will do some things so that to make the traffic go more smoothly, okay? So do YPT rabs coordinate trafficking at what levels? So now we are back to east again because there are only very few of them. We can cover the whole exocytic pathway with three rabs while in mammalian states you'll have to deal with 35. And if you think about all their interactors, the nets, the interaction nets are much smaller. So I think that this is again going back to east to try and understand this. So when I talk about regulation or coordination by YPT rabs, I'm going to talk about, first of all, coordination of multiple vesicular transports, sub steps. I told you that there are many sub steps. So if you just give the sale only the coat, but you don't, the vesicle, but you don't make sure that it also has the motor and the tethering factor, the traffic stops immediately, okay? So we have mutants that we can stop traffic. We cannot even measure it, less than one minute. It's done, stop, okay? The second one, which is a little higher regulation, is to integrate, there should be, there is a need to integrate different transport steps in the same pathway so that there will be one whole pathway smoothly going through Paris. And then also we found that they coordinate between different cellular processes and pathways, okay? So I'll try to give an example for each one of these. So we start with regulation in the secretory pathway, exocytic pathway, and I'll give you these two examples. One, coordination of sub steps, and I'll start with it, and then integration of whole pathways. So here is this example. So here is again, a vesicle forming from the donor compartment with all its friends. And if we look at formation, motility, docking and fusion, and if we look at Golgi and plasma membrane, we knew all the machinery components. We knew that YPT3132 is involved in the beginning. We knew that myosin works in motility, myosin five in this case. We knew that the tethering factor is called an exocyst. This is from work by Peter Novick's lab. And actually we knew also that there is a Jeff and this is another example of two YPTs that are needed for one transport step. The same way that you asked Bruno before, wrap six and maybe wrap eight, this is YPT3132 and sec four. This is important for just the beginning to bring sec four and then sec four takes it from there. And we also use the SNERS, here's an example, sec nine is a tether that is involved with the N. But we didn't know if there is any coordination between the sub steps, okay? So I'm gonna tell you that YPT3132 are required for vesicle formation. We showed this and then this is by using mutants, a electron microscopy. So then we see which step is, they stop it because mutants accumulate material from the step before the step in which they are blocked, okay? And then we showed by different, so whenever I say something, we showed it used by interaction, exhaustive interaction, first direct interaction and then to show, yeah, they interact in the cells, okay? And then to see what is the role of the interaction. So in this case, I'm just gonna tell you one little story. We showed that they interact, YPT3132 interacts with a myosin and we made an interaction because I think this is crucial, a interaction specific myomutant. Myomutant, because myosin five can do many things. But we made a mutant that just specifically cannot interact with YPT3132. It can interact and do jobs everywhere else, okay? And then- It's a mutation of a particular mutation of the gene, yeah? Yes, in the gene, but then it's also in the protein. It's produced by different. Yes, yes. So now we did the genetics, we replaced the myosin, we put it in cells and now we follow what happens by life cell microscopy and here what we do, we look at actin to just see. So this is budding yeast, just to remind you. Meaning that all the secretion is polarized, it's going through the bud. Actin is polarized to the bud. The vesicles we view here, just a marker for us, sec four in green and when you see them together, you get the yellow. So in wild type cells, you see them together, meaning this is a polarized transport from the mother cell very efficiently to the bud. Now here is our, what I call a yeast mutant, which is YPT interaction defective, myotomeutant. It's only defective in this, it's there. And then you can see that all the vesicles are here and we showed it by other, also by electron microscopy. But then, and the actin is in the right place, but the vesicles don't go to the bud. So there is no polarized secretion in this mutant. So what it means is that we already knew that YPT 3132 are important for vesicle formation for, because of interacting with other effectors, but now we show that it's also required, not just the formation, but to put on the right myosin to take them to the right place, okay? How do you build a bud without porous secretion? So the beginning of the bud probably, it just can be random, no? You don't? Okay, we can talk. Mutant does not make any bud. Well, this is a myotomeutant, it does make a bud. No, no, no, but if you inactivate the motor function. Sure, sure, sure, sure, yes. Yeah. So maybe there are other things that the myotomeutant does. No, there's another pathway. Is the excess still porous properly, because maybe. No, because the rub is rub, sec 4 is not there. Okay, we'll give it to the later. Thanks. So we showed that YPT 3132 actually couple vesicle formation and motility. Work published by Peter Novick also shows that YPT 31 or 32 bring also sec 2, which is the J for the next rub that is gonna be in this system. So actually it sets up the vesicle to a few sub steps. And I think maybe this now can answer a question that if you can start, the cargo is there, cargo coats are there, the coats are there, but the vesicle just will not, maybe it's not even gonna form this. Yeah, it will stop probably from recruiting anything. The cargo receptor included. I have a question, why? Of course it may happen. Why the cell, okay, so that's a different question. That's philosophical. You have to stop some type of transportation that are proteins. Okay, this is because in Paris, you know, there are intersection two dimensional. Oh God, can you imagine how Paris will look like if all these cars will be there without the traffic lights? It's not my subject because on two dimension it's poorly organized. Because the street cross, here they don't cross. Why you have to stop it? That's the question. And I don't know. It's absolutely wrong. Absolutely nothing to do with the cells. Two-dimensionality here, 3D, nothing in common. Why? What's the logic? Why do we have a red light? Can we? What is the function of a red light? But maybe the answer is that actually you stop only when you have a mutant. So you are in a situation. Exactly, but why normally you have a regulation and then you have a regulation? So the regulation is perhaps not to stop or put it on, it's just to make sure that the right vesicle attached to the right motor to go to the right destination. What is the right destination? In this case it's the plasma membrane. It's only one. A to B is only one destination. You don't choose. No, but it could go back. It could go back. It could go to endosomes. It could go anywhere. It could just stay in place like... Wait, wait, did you always go from A to B? Why do you always go from A to B? But there's many membranes. There's colg, er... Okay, so you choose which one. So you wanna choose plasma membrane. Which one goes to where? Yeah. But you never stop it. So there is no red light. Only there is direction. No, no. So the problem is, let's suppose I'm coming out of the plasmic reticulum and make a vesicle. So that vesicle, where would I like to take it? I would like to take it, for biological reasons, to the Golgi apparatus, right? But that vesicle could have actually gone directly to the plasma membrane. And that's a mistake. So they're not red light, just directions. Yeah. Okay, so there is no red light. They're not really red light. That's one level of revelation. Another level of revelation is the content. Which is not being discussed. Not all the guys, not all the carriers have always the same stuff. You also sort out, you segregate, right? And we're not discussing that level of complexity. Yeah, we're still normal. My point was there is no red light. There's this mutants make something very artificial. It never normally happens. So the problem, so, okay. So the other problem is, how do we biologists can study this, right? So historically, the way it has been done is to put perturbations, right? And one type of perturbation is like, shut down the pathway. You can shut it down super fast. You can shut it down slow. These are actually relatively slow. They are not super fast. Even though it's a temperature-sensitive mutant. In general, correct me on this. I don't think it's an instantaneous block. It takes a while, right? It takes less than a minute. I don't know if... No, I just said it depends on the mutant. But in, so, this is another level of complication. Oh, thank you. I understand, but I'm saying analogy. With the red light, it's completely wrong. Okay, fine. I agree. Okay, I take it back. No red lights. Because, no, the rate is important. Sometimes it goes fast. Sometimes it goes slow. How do you control the rate? Why do you make it slow? If you have some amount, it's... No, because the timing, it's responsive. Different carbons actually go different speeds. So there is a car that you wait, you wait, you wait on the spot, yeah? For some time. How does it? Yes. Why would you wait? What does the cell do it? And that's... Okay, can I just try to answer this question? This is a question of evolution, and it's a little philosophical, so can we leave it to the end and please forget about the red lights, okay? Just continue to listen. Okay. If you want, you don't have to. But there is a... There is an answer. It's simply, for example, in the cell cycle, you want to have different rates of trafficking, depending on where you are and the rest of the... So really, you have excess of some production, so you don't have... If you have excess, excess, it's in some kind of... And you keep it. You don't transport it, and you wait for what? That can happen too. For example, if you don't need a certain receptor or the plasma membrane, it might still be synthesized, but it does not go to the plasma membrane until you need it at the plasma membrane. Yes. Okay, okay. This is the answer. At least we're waiting for it. In that sense, it is a red. Okay, at least we're waiting for it. I'll take it, but it's really not, because regulated secretion at the last step is different kind of regulation, but I'll take it for now. Yes. At least it's another function of the red sort of quality controller. Think of it more like a production line, like if you're making cars, you don't want to send a car out without the steering wheel. Make sure that everything's correct. So this is exactly what is shown here. It's like it has the motor and it has the next protein that will take it to the right place. Okay? So maybe the red lights are not good and I'm sorry that I confused you. Okay, so what about, so I just wanted to show you and give you a flavor of this one type of regulation. The other one is to look at the whole pathway. And this, we go back to the two GTPases, YPT1, YPT31 and 32. And the question here is, oh, sorry, first I wanted to say that we showed it by function, okay? I told you we showed it by mutants. More recently, we had to go back and show it by clear localization because there was in the field, there was confusion, whereas this protein and which compartment, it's also very difficult to decide which compartment, which are the compartmental markers in the Golgi. So what we did is to using a life-sale microscopy with different colors. And we first wanted, we had at least two markers for the early Golgi. We had two markers for the late Golgi that they co-localized with each other. And then we made the YPT1 or YPT31 in green and we looked at the co-localization. We confirmed everything that we saw by life-sale also by immunofluorescence microscopy. So the answer was that now when we did it very thoroughly that they actually localized to opposite sides of the Golgi. YPT1 is starting early, very, very, very low level at late in the late compartments. YPT31 is the opposite. So, and they both, about 25% of them, and it will just become a little interesting. They actually co-localize with each other and they co-localize in a compartment that is a regular, is marked by 6-7. And now just treat them. I know that Kathy Jackson likes the 6-7. It's a Rf, a Jeff, it's another GPA, but for now they are only serving and some people like Cup 1 and CHC1, but for now they are only serving us as markers, okay? So we also did some three-color IF and with all the combinations and we could show that, again, this is the point that I was trying to make earlier, YPT31 to localize about 25% of them, but 95% of this localization happens only on the 6-7 compartment, okay? So now if we make a Golgi map with the YPTs, we have Cup 1 early, we have 6-7 and CHC1 late, but we also saw some co-localization of early and late Golgi markers and we called it a transitional compartment. I'm not calling it cis-medial trans because we didn't show that they really correlate with the enzymatic activities for cis-medial trans, but that's why we called them early, transitional and late, transitional tells you, it's fast, it just comes and goes. So the transitional compartment is also, so YPT1 is on the early and transitional, 31 is in transitional and late, okay? So they co-localize it, the transitional compartment. So now I want to just branch off for a minute to Golgi's cisternal progression or cisternal maturation. We talked about it, this is what people think, how transport occurs through the Golgi. So the cargo stays in each compartment and what happened is the compartment matures and this is within the same compartment, okay? So in 2006, two groups showed in yeast. I think they provided a really good evidence for that you can see cisternal progression. The way that they did it is by looking at shifting from cup one, which is the early Golgi to six seven, which for us it's a transitional late, okay? But until then, until we came along, there was no genetic evidence that this is actually regulated. And I want to go back to what Alberto Luini said yesterday, you do not see a regulation until you just do something to it, yes? I mean, because is there a regulation or not or does it just happen, okay? So we wanted to ask this question, do YPTs, do the YPTs which are in the Golgi regulate the cisternal maturation? How do we do it? We have mutations that can affect the YPT either to be super active or to be not active, okay? So now we look to see what happens to the dynamics of the cisternal maturation that they showed, the two other groups showed, but now when we look at with these YPT mutations and we look both by collocalization, doing life cell and IF in immunofluorescence and looking at dynamics, okay? So, but we also added one more thing. We actually had now a third marker. So we looked at cup one to six seven, which is what they looked at. And we also looked at what happened with six seven to CHC one, okay? So we determined the effect of what, when we, I'll show you hyperactivation, but we also show inactivation of these YPTs on this both Golgi cisternal, Golgi proteins collocalization, snapshots, but also dynamics. And I'll show you just the dynamics. So when we look at cup one to six seven, so we watch it by looking at the dynamics of these. And I'm not gonna ask you to look at this, but just to understand what we were doing. And then we have the chemographs of tracings of these cup one to six seven. And this is in wild type cells. And we have numbers. We are looking at what happens to cup one appearance versus six seven. So cup one always appears about 15 to 20 seconds before six seven, okay? In Golgi. In the Golgi, in wild type cells. But now when we hyperactivate the YPT one, or IPT 31, we wanted to see what happens to them. So what we see, say it again. There is a mutation that we can make it super active. It's just always bound to GTP, okay? Or it looks like it has the structure that it's always bound to GTP, okay? So what we see here is that when we overactivate YPT one, but not YPT 31, it goes faster. So this step goes faster, okay? With 31, 32, it's similar to the wild type, okay? So this is our regulation of whatever was in the field, cup one to six seven. But then we also looked at what happens to six seven to CHC one. We did the same experiment. We look at the dynamics. And then we do the chemographs and the quantification. And now what happens is that YPT one does not affect it, but YPT 31 makes it go faster from 10, 12 seconds to four seconds. The effect is two or three-fold, okay? Which is significant, okay? So YPT one activation now makes the six seven go to CHC one faster. So together we can say, we can see it here. YPT one makes this step go farther, faster. YPT 31 makes this step go farther if we make the other mutant, the activating mutant, it has the effect that we expect. So in conclusion to this part of our experiment, we show that first of all, this is the first genetic evidence for systemal progression. Second, we actually divided it to two different steps and YPT one regulates the first step and YPT 31 regulates the second step. One question, sorry. Yes. So you're showing some percentages and some fraction of things going faster and slower, but what happened to the other percentage of events? In your previous slide, there was something that said 20% or 50%. No, no, no, sorry, sorry. I didn't explain it, sorry. 20% reaching the tip to the top and 50%. We are looking at the initial rate. Yeah, and what happened to the other events? Then they are together, sort of, no? What? We are looking, we are looking, we are only, it's not event. We are measuring 20% increase. I'll put it on the slide next time. Okay, let me rephrase this. Every time you see this, you have exactly the same behavior or every tracing is a little bit different. No, here is, it takes 12 seconds plus minus two seconds. That's the average. Yes. Plus minus the standard deviation. But there are events that are happening either faster or slower, right? Yes. So those, what's happening? I mean, they're not. So I don't know, I mean, we can talk about quantification and if this is enough to say that if it's 12 plus minus two or four plus minus one. Percentage of, I said percentage, sorry. It's, it's increase of the red light, the red marker to aim. In what scale? Percentage of what? On, a hundred percent is the maximum. Of what? Percentage of what? Of intensity of the light. How much of the what? It's just intensity of the light. So it was the percentage of events, right? Light being. Red or green? No, in which units? Is it the green or what? 20% meaning? It's fifth of the, of the maximum. 20? Okay, it's, it's bad. I, I, I understand. I should have written what the, what the percentages are. Aim. No, the, the, the answer is, as I understand, 100% of intensity measured whatever in the amount of dye. And in, when, and then 20% use, you mark 20% of this intensity and you say, this 20% level is reached in 12 seconds. Then you mark 50% level or this whatever. It's just two measurements of the same common ground. The same level of this intensity is reached in 10 seconds. This is what, what is the idea. Yes. Okay, I, thank you. I'll, I'll. When you get 20% and when you get 50%. This is the. And you measure intensity in which number? This I, I don't know. I don't know. So what? Which number you measure intensity? Intensity means number of molecules. Yeah, yeah. So your, your top signal is that 10 molecules, 50 molecules. I don't know. You don't know. And then each, each one. No, no, I think that intensity is measured in. No, wait, wait, wait, wait, wait, wait, wait, wait, wait. How do you feel? How do you feel? I think all the molecules are, are a, whatever each molecule maybe does it em, emits the fluorescence in a different way. But the same molecule is the same. You have one Golgi and God, let's say 10 fluorescent molecules. The next Golgi have also 10 or have 20. No, they all, so the answer to your question, they all had about the same intensity top here. About. No, there was no, no. So this is fluorescent marker, marker. Yes. This is what I, Misha, to your answer to your question, this is fluorescent marker and the intensity of the signal is the intensity of the measured fluorescent marker. There will be no lunch today. Because I. Propagation of the intensity, how often? Yes, yes. Which one? Number. This is what I guess. This is what I understand. Another, you're also going to get it. If you do it wrong, you completely need to stop. Why? This is another question. Let us continue. If you change the scale, you'll get a really different number. If you change the scale, how much? You'll go 20, have to, you have to say something. It depends on the scale. This scale, this scale, right? If nonlinear scale, you'll get a really different scale. The scale is linear. The scale is linear. Okay, why don't we continue to talk afterwards? Thank you. So the question that we are asking now, this is for Alberto, is so we know that the YPTs are important for this system of maturation and we are trying now to look at the Jeffs for YPT one, Trap One, Jeff for YPT 31, 32 to go back in the, to see if they also, first of all, to clarify that they really work in these steps and also to see what they do to go GCC steriline maturation. So the last thing that I wanted to tell you about, and I'll do it, I'll try to do it fast, not fast by talking fast, by fast by jumping over slides. So I wanna talk about autophagy. So if you look at my simple model of the cell, I wanted to add to it two recycling groups. So there is one recycling group that everybody knows which is plasma membrane recycling and we talked about recycling endosomes, et cetera. But there is another one which is autophagy that brings stuff from somewhere from all these compartments to the lysosome and lysosomes, as we know, they actually split back all the block, building blocks and then they can be built again to get back into the ER, for example. So the question is, is there coordination between trafficking and recycling? And I'm gonna jump over these, oops. I just say, how can there be one rub? And this is a question in the field that a field can take a vesicle to two different places. And the answer is, I told you that each rub can recruit different effectors, so each rub can recruit a, sorry. For example, if it will recruit effectors one, two, three, this vesicle will go to the Golgi. If it will recruit for other effectors, it will go to the lysosome. So, okay, we'll jump over this. Okay, so I wanna talk about YPT-1 because we also show that YPT-3132 are required for this loop, not just going through the Golgi, but also for this loop. Here is YPT-1, which we showed required for going from ER to the Golgi. It turns out that it's also required for autophagy. So the way that we show it, we showed it, is by using a mutant to show a role and also identifying a recycling-specific effector, an effector that doesn't have nothing to do with its role in secretion, but it is required for autophagy. So first, let me just remind you what is autophagy. It's the pathway that usually people study it under stress. And here is an example of how we look at this pathway by GFP-ATG-8, which is LC-3 in mammalian cells. And what happens is that in this pathway, an autophagosome is formed. And an autophagosome, this is to remind me to say that it's a double membrane organelle, okay? And it takes cargo, which, so the autophagosome forms around a cytoplasmic things. They can be either proteins, protein aggregates, or even whole compartments. And then they take them to the lysosome for recycling. There is also autophagy under normal conditions, growth conditions. And we have in yeast, for example, this was one available cargo that we were following. And both under stress or a normal autophagy, the process starts by formation of PAS, which is the pre-autophagosomal pathway. And PATH is a combination, a complex of about 30 proteins, which are called ATGs, and membrane, okay? And SOMI got the Nobel Prize just for showing this first step, how this is all starting. But none of these ATGs are required for the rest of the pathway. So what is the rest of the pathway? What's beyond the ATG complex? It's a membrane process. So it must have all these other machinery components that we all know. So just to tell you what is the YPT-1 connection to autophagy, how did we even get to it? We had a mutant. It's a recycling process. It is a recycling. Yes. It's stomach. It's stomach, I would say. It's iri, autoiri. Organic. Auto-phagy, okay? So that's what I was trying to say. It can happen under stress or even under normal condition. So we had a mutant in YPT-1. This is actually a very early mutant that had no effect, not major effect on secretion, but definitely it didn't allow cells to grow under stress. And stress in yeast is you just starve them, in this case for starvation, for nitrogen. Then another group came up with a subunit of TROP, also has a role specifically in this process. And then we did a yeast to hybrid screen and to our surprise, maybe we shouldn't be, have been so surprised, we got ATG-11, one of the ATGs that Osumic discovered in his screen for ATG mutants. And we got it by yeast to hybrid. And we, so I'm not gonna talk much about it. I'll just tell you that we showed that while TROP-1 is required for the role. So now I'm talking about the activators. The activator of TROP-1 takes with the YPT-1, takes cargo to the Golgi from the ER to the Golgi to the plasma membrane, while two other TROPs, TROP-3 and TROP-4, which have similar core, but different specific subunits, take it to autophagy, okay? Same YPT-1. So, and at least one Jeff. So YPT-1 is required for cell viability because of its role in secretion. It's required for stress, for autophagy under stress. No YPT-1, no stress. These TROPs, one Jeff is enough, sorry, one Jeff is enough so you need to delete both in order to see the full effect, okay? Now what about the effectors? Here again, we have YPT-1 going from the ER, a vesicle goes from the ER to the Golgi, we know what is the effector. And two, two, the- But again, why under stress, for a problem of autophagy, what is the logical effect? Because it's recycling. If you want to conserve a material under stress, you don't have any more nitrogen. You cannot, the cell cannot make any more amino acids. So it needs to eat its own proteins to make these proteins that are really essential for stress, not just extra things that are not essential now. Make sense? Okay. So what we showed, and the way that we think about it is, we're thinking about YPT-GTPAs modules. So the module has a Jeff, a specific Jeff for the specific step, a YPT-Rab, a DGTPAs, and then at least one effector, okay? So in this case, we have a module of trap three, specific subunities TRS-85, YPT-1, and at least one specific Jeff, ATG-11. So the way that we showed it is by showing interactions, collocalization, and function. And I'm gonna jump over the collocalization, and maybe I'll just show you fast the function. So how do we study function in this case? We are lucky because the- What kind of stress you're probably in use? What kind of stress? We start them for nitrogen. We start them, okay. In our cells, mammalian cells, you have to start for- Amino acids. Amino acids, thank you. So we have specific mutants which are only defective in autophagy, not in growth, okay? TRS-85 doesn't have anything to do with secretion, so we can delete it and see just the effect on this Jeff on autophagy. Same YPT-1-1, the mutant that we later showed, that it disrupts this mutation. It's a one amino acid change that disrupts the interaction of YPT-1 with ATG-11. It does not affect other functions of YPT-1. And we have, of course, ATG-11, which is by definition was isolated as specific for autophagy. So we use all these mutants to ask, what is the effect? So again, I don't have time to, I think, talk about results, but I'm just gonna tell you that in YPT-1 or TRS-85 or ATG-11, there is no pass. So pass can be seen by collocalization of two ATGs or more, if you have more. All the ATGs go to pass because I told you it's a protein complex, but in YPT-1-1, there is no pass. There is no collocalization. They don't come together, okay? So, okay. So it's true also, as I said, for the whole module. So the whole module, if we look at function, it's required for assembly of pass, okay? It's the first step of autophagy. And until now, there was no way, there was no shown regulation on it. Okay, so here we have what I call one, this is math for you, one to two to two. One YPT-Rab, two different modules, two processes. So YPT-1 with the first module is essential for cell viability, for secretion, with trap one and effector, one effector US-1. And we have another module and another process, where the same YPT with a different Jeff, with a different effector now goes to a different process. So now what we have to, when we think about, I told you in the beginning that there are two upstream inputs to activate a YPT. One is member attachment, the other one is exchange, the activation by Jeff, but I would like to add that there are actually three inputs. If you think about the possibilities, you should have also the system also has to have a way to recruit the right effector to the right module. So the YPT in one module can interact, cannot just interact freely with swimming effectors. It has to work in a module. It's a big question, how does it happen? Okay. I think that I should stop. I'll just say it in two minutes, I'm not even gonna show you the results. Thank you. No, no favor, favor is in my field. I need to stop, I'll stop. So I just wanted to say, so I wanted to tell you that we discovered a new autophagy pathway during normal growth, and it's actually quality control of ER phagee, quality control of the ER by autophagy of the ER, and the plasmic reticulum. So it's a selective autophagy pathway, and the cargo for this pathway are membrane proteins. Now, when we first discovered it, when we overexpressed, made too much of a protein, but when we found it, once we found it, we went back and looked at other ER membranes, residents, and it turns out that they, not all of them, some of them, and I'll show you who, at least two groups, some go to this pathway and some don't. Okay, so we pass through this. This is all the evidence, both. So we actually showed the stop, the block by three ways. One was by lifestyle microscopy, then we also looked by electron microscopy to see what is accumulating, and it's ER, and ER, resident proteins are there, and we also showed it that it's really ER by looking at UPR. It's an acid that shows that the ER is under stress. There is an overexpression of a protein, one protein, and it actually makes the ER stressed. So while other mutants that accumulate some structures with some of the cargo that we are looking at don't do that, okay? So this is just two more slides. So it's a new quality control of the ER. Without overexpression of proteins, we have two residents of ER, 61 is a translocone, agent G1, and these are components of getting the vesicle to the Golgi, they do not go to this pathway, to ER phagee, but these do, and about 20 to 50% of these proteins, depending on the protein, go there all the time. We just don't see it if we don't block it, okay? And then if we overexpressed a single protein, the cells are happy, there is no stress on the cells, there is no induction of general autophagy, but we just have, the ER is getting too blocked, then we get 95% of these proteins and these go now to the ER phagee, okay? So here are the other ER phagee, what happens when proteins get from the ER? So if they are native, they'll go to secretion, secretion to the Golgi to the plasma membrane. If they are misfolded heavily, they'll go through IRAD to the proteosome. There is also a micro ER phagee process, which micro ER phagee means that they do not use the normal, the ATGs, so there are ways to get to the lysosome without it, and now we added this new process which takes extra membrane proteins by this autophagy process to the lysosome and we know who participates and who regulates and the questions that you're asking now in the lab, which are more the basic questions is what I told you about the input, the three input, how does this work, if there is coordination, and just to connect to human disease, I told you the TRUB1 is the mammalian, the human homolog of YPT1, and it's actually involved both in cancer and all the generative disease. This is my connection to Friday, and thanks to everybody who helped me in my lab in beyond and stop. Sorry that I took over, we don't have to have questions. We can go have lunch and we can talk in the evening, unless you want questions. I just, I'm sorry that I... Thank you.