Regulation and coordination of intra-cellular trafficking pathways
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00:00
Computeranimation
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Vorlesung/Konferenz
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ComputeranimationVorlesung/Konferenz
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Transkript: Englisch(automatisch erzeugt)
00:16
The title for my talk is Regulation and Coordination of Intracellular Trafficking Pathways.
00:27
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.
00:45
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.
01:02
So, I'll start, my talk will 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 intracellular trafficking.
01:23
What's really good is that I don't need to give a really long introduction, because you've heard everything that I'm going to say in the introduction, you probably have heard throughout these days. So, the secretory pathway or the exocytic pathway takes proteins and membranes from the endoplasmic reticulum,
01:42
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 going to be full in a minute. So, this was talked in length on Tuesday.
02:04
You also probably know that traffic in every step is bi-directional, and there is crosstalk also between the endocytic and exocytic pathway. And everything between compartments is transported via vesicles,
02:24
between the ER and the Golgi, between the Golgi and the plasma membrane. From the present membrane to endosomes, endosomes to lysosomes. So, you also heard that inside compartments, Golgi, the sorting compartments,
02:40
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, takes the cargo, which can be either luminal, luminal is the inside of the compartment, or membranous,
03:06
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,
03:23
and you heard about these two. So, the vesicle first has two forms, 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,
03:41
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 complex, complexes that will help the fusion of this vesicle with the acceptor compartment.
04:04
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?
04:24
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.
04:43
They are very small, but they can cycle by themselves from the GDP bound form to the GDP bound form. From here to here, from GDP to GDP, they exchange the nucleotide, and from GDP to GDP, they hydrolyze the GDP. We, and I'll explain it later, when they're in the GDP bound form,
05:03
they are mostly off, in the GDP bound form, they are on. So, in yeast, there are 9 to 11 of them. The other two, we don't know yet much about them, so I'm not sure.
05:22
I'll explain in a second. And here are the major ones. So, even when we say 9, we have, for example, YPT1 in the beginning here, and YPT3, 1, 3, 2, which are sometimes called 31, 32, they are functional homologues.
05:40
And, for example, we don't know much, we know about YPT5, 1 and 2, 3, but we know mostly about 51, but they are involved in the same process. So, even when we say 9, it's actually less, and it's important for later. Does each one have a clear homologue in mammalian cells?
06:02
Yes, and this is shown here. So, thank you. So, there are about 70 mammalian human RABs. They are also regulating the human protein trafficking pathways. So, there are more, but here I'm showing for, oops.
06:21
So, I just wanted to point that they are a really large group of GTPases. Together with their friends, with their upstream regulators, and some of the downstream effectors, they form like a 200-protein group, which is about 1% of the human proteome.
06:40
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. RAB1, and I'll talk about the localization and function. He's going to get a heart attack, as you say. No, no, Bruno, no heart attacks here.
07:00
So, YPT-1, the best homologue is, the close homologue is RAB1. YPT-3132 is mostly RAB11. RAB6, actually the RAB6 homologue, YPT-6, is also a functional homologue. YPT-1 is also a functional homologue. RAB1 is a functional homologue. You can delete YPT-1, put RAB1, very highly conservative, can you see?
07:26
And the same is true about YPT-6 and RAP6. I don't know why. We haven't figured it out. It's not essential in yeast for viability, and we actually focused. Bruno talks about that on them.
07:42
We focused only 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.
08:00
So, it all started in yeast. All this family was discovered in yeast. First, why yeast? Because in yeast, 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.
08:25
Of course, in combination with molecular genetics. So, in phase one, we first looked at two YPTs really. YPT-1, which is essential for viability.
08:42
YPT-31 and 32, 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 minor other functions that we don't know yet. But for now, we usually delete one, and then the other one becomes essential.
09:04
So, this we did. How did we discover them? Somebody asked me. It's 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.
09:21
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. So, first we knew that it was a huge thing to see that, no, they are not at the plasma membrane.
09:41
No, they are not in signal transduction 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,
10:01
we already knew that it's conserved from a yeast to humans, you know, the RAB1 and YPT-1, by also a combination of molecular genetics and cell biology. So, this is the minimalistic view of cells, exocytic pathway to plasma membrane, endocytic pathway.
10:22
And what I'm showing here, based on function, based on mutant phenotypes, we saw that they are in the exocytic pathway. YPT-1, a set of papers, we showed that it's in the early transport from ER to Golgi or Golgi-2.
10:41
And then the YPT-3-1 and 3-2 are late Golgi. Exactly what they regulate. Exactly. If you stop them, actually it's very fast. So, I'm saying they are essential. How do we work with them? It's a good question. We have temperature sensitive conditional mutants. We can use different conditional mutants. The easiest one to understand,
11:02
stop of trafficking, transport between. We can measure ER to Golgi transport. Exactly. We weigh the transport, but just transport, not the transport. Yeah, but we can see it's transport or no transport, but also we can see which step, by using cell biology, combination with cell biology. No, step is one of the two.
11:21
When I say step, it's one of the two. Yeah, YPT-1 is here. We block it, block from ER to Golgi. If we stop YPT-3-1-3-2, block from Golgi to plasma membrane. So, why you should stop it some day? You always have a strategy. What do you mean to stop it?
11:42
I'll explain why in a minute. So, because we saw it, we saw that YPT-1 is an entry, entre, so this is my French, also T from the Golgi. We call them the Golgi gatekeepers.
12:05
Okay. In phase two, and I'm getting to your question, don't worry. We wanted, so we knew that there are molecular switches. We knew that somehow they regulate vesicular trafficking, but we didn't know what's in between, okay?
12:22
So, the first question was how are they regulated? What's the upstream regulation of their abs? Who turns them on and off? So, these are the people who turn off the switches, like Georgiana here, turn on the switch.
12:41
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 GTPase activating factor to turn them off.
13:01
So, just to put it in perspective, I put here, this is one of the GIFs in cells, and by the way, they are very different between proteins, but for just YPT-1, you need a core trap, which is a complex of four different proteins, five subunits, but two are identical,
13:22
and this is the little YPT-1 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,
13:45
and of course they play a big function, a big role in what YPT-RABs do. So, in addition to this upstream regulation by Jeffs, as Bruno also suggested, these YPTs,
14:01
the way that they are attached to membranes is by having a lippy tail, so they have a lippy tail, and they cycle between the cytoplasm and the membrane. To be in the cytoplasm, because it's a lippy tail, they have to be buried in a france,
14:22
so there is a place where this lippy 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 lippy tail here, and then they can be seen by the Jeff. So, there are actually two for now,
14:40
we are thinking of as two upstream inputs to activate a RAB. One is to do the membrane attachment, and the second is to turn them on with the Jeff, with the activator. And once they are in their GTP-bound form on the membrane,
15:01
this is when they are on. What does it mean to be on? The way that we think about it right now is that the gap which turns from GTP to GDP is just required for them to be able to recycle back to the cytoplasm, which is also important, so they can function again.
15:23
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 happens downstream is
15:43
that when they are on, they interact with these proteins. So, they interact with these effectors, this means that they are on.
16:02
So, who are these effectors? These are the people who really do the work, unlike those activators which only turn them on and off. 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,
16:20
but now I just want to say, for every step you'll see a YPT-RAB, and this is a summary of work in many labs, YPT-RAB in every step, and its effector will be in red. Oh, and the other thing that I want to say, a single RAB can interact with multiple effectors.
16:44
So, the first step in the formation, remember, here is the YPT-RAB, and it's one of its effectors which interacts with the cargo receptor. Here in vesicle motility, we have a motor that can be either,
17:02
and Bruno already talked about myosin or kinesin, depending on which cytoskeletal route they want to go on. There 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 tethering factors,
17:22
but this is just one of their kind of effectors. And then finally, here the YPT-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
17:46
are recruited by these RABs. Does this answer your question? No, my question is why you need to regulate the tow? Why you just have some protein and transport them? Why do you stop normally? Okay, so let's continue to talk, okay?
18:00
It's a conversation. Why to 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
18:21
or will do some things so that to make the traffic go more smoothly. So do YPT-RAB 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,
18:44
while in mammalian states you have to deal with 35. And if you think about all their interactors, 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,
19:03
I'm going to talk about, first of all, coordination of multiple vesicular transport sub-steps. I told you that there are many sub-steps. So if you just give the cell only the coat, the vesicle, but you don't make sure that it also has the motor and the tethering factor,
19:23
the traffic stops immediately. So we have mutants that we can stop traffic, we cannot even measure it, less than one minute, it's done, stop. The second one, which is a little higher regulation,
19:40
is to integrate, 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.
20:03
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.
20:24
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,
20:40
we knew all the machinery components. We knew that YPT31-32 is involved in the beginning. We knew that myosin works in motility, myosin 5 in this case. We knew that the tethering factor is called an exocyst. This is from work by Peter Novick's lab.
21:01
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, rep 6 and maybe rep 8, this is YPT-32 and sec 4.
21:21
This is important for just the beginning to bring sec 4, and then sec 4 takes it from there. And we also knew the scenarios. Here is an example, sec 9 is a t-scenario that is involved at the end. But we didn't know if there is any coordination between the sub-steps.
21:45
I'm going to tell you that YPT-31 and 32 are required for basic information. We showed this. And then this is by using mutants, electron microscopy, so then we see which step is there.
22:03
They stop it because mutants accumulate material from the step before the step in which they are blocked. And then we showed by different... So whenever I say something, we showed it by interaction,
22:21
exhaustive interaction, first direct interaction, and then to show the interaction in the cells. And then to see what is the role of the interaction. So in this case, I'm just going to tell you one little story. We showed that they interact, YPT-31, 32 interacts with a myosin. And we made an interaction, because I think this is crucial,
22:45
an interaction-specific myomutant. Myomutant, because myosin-5 can do many things. But we made a mutant that just specifically cannot interact with YPT-31 and 32. It can interact and do jobs everywhere else.
23:01
Okay, and then... It's a mutation, a particular mutation of the gene, yeah? Yes, in the gene, but then it's also in the protein. It's still 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 live cell microscopy.
23:20
And here what we do, we look at actin to just see... So this is bading yeast, just to remind you, meaning that all the secretion is polarized. It's going through the bad. Actin is polarized to the bad. The vesicles we view here is just a marker for us. Sec4 in green. And when you see them together, you get the yellow.
23:41
So in wild-type cells, you see them together, meaning this is a polarized transport from the mother cell very efficiently to the bad. Now here is our, what I call a yeast mutant, which is YPT interaction-defective, myo-2 mutant.
24:01
It's only defective in this. It's there. And then you can see that all the vesicles are here, and we showed it also by electron microscopy. And the actin is in the right place, but the vesicles don't go to the bad. So there is no polarized secretion in this mutant. So what it means is that we already knew
24:24
that YPT3,1 and 3,2 are important for vesicle formation 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. How do you build a bad without polarized secretion?
24:43
So the beginning of the bad, probably it just can be random. No? You don't? Okay, we can talk. Mutants does not make any bad. Well, this is a myo-2 mutant that does make a bad. No, no, no, but if you inactivate the motor function...
25:01
Sure, sure, sure, sure, yes. Yeah. So maybe there are other things that the myo-2 does. I know there's another pathway. Is the excess still polarized properly? Because maybe... No, because the sec-4 is not there. Okay, we'll give it to you later. Thanks. So we showed that YPT3,1 and 3,2
25:23
actually couple vesicle formation and motility. A work published by Peter Novick also shows that YPT3,1 or 32 bring also sec-2, which is the J for the next rub that is going to be in this system. So actually it sets up the vesicle to a few sub-steps.
25:45
And I think maybe this now can answer your 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 going to form this.
26:01
Yeah, it will stop probably from recruiting anything, the cargo receptor included. This is my question, why? Why the cell, okay, so that's a different question, that's philosophical. Why the cell have to stop some type of transportation that approaches, it might be the spoiler. Because in Paris, you know, there are intersections, two-dimensional... Oh God, can you imagine how Paris will look like
26:22
if all these cars will be there without the traffic lights? Two-dimensional geometry, okay? It's not by something, because in two dimension, it's poorly organized, because... This is three-dimensional. Three cross, here they don't cross. Why you have to stop it? That's the question. I don't know. Absolutely wrong. Absolutely nothing to do with the cells. Two-dimensional geometry here, 3D,
26:42
nothing in common. Why? What's the logic? Why do we have red light? Can we... What is the function of red light? Maybe the answer is that actually you stop only when you have a mutant, so you are... But why normally you have a regulation, negative regulation? So the regulation is perhaps not to stop or put it on.
27:02
It's just to make sure that the right physical attach the right motor to go to the right destination. What is the right destination? In this case, it's the plasma membrane. But 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...
27:25
But there's many membranes, there's Golgi, ER... Okay, okay, so choose which Golgi... So you want to choose plasma... Which one goes to where? So there is no red light, only this direction. No. So the problem is, let's suppose I'm coming out of the endoplasmic reticulum
27:41
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. So there's not red light, just directions. Okay, so there is no red light.
28:00
There's not really red light. That's one level of regulation. Another level of regulation is the content, which is not being discussed here. 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. We still know, but my point was there is no red light.
28:21
Mutants, for example, are very artificial. 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.
28:42
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 it takes a while. No, I just said it depends on the mutant.
29:00
So this is another level of complication. No, I understand, but I'm saying analogy with red lights, computer. 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? To make it slow. You can have some amount.
29:21
No, because it's the timing. Different carbons actually go different speeds. So there is a current and you wait, wait, wait, don't transport it. How does it? Why? Why would you wait? Why would the cell do it? 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?
29:43
And please forget about the red lights, okay? Just continue to listen, okay? If you want, you don't have to. But there is an answer. For example, in the cell cycle, you want to have different rates of trafficking depending on where you are and the right structure.
30:01
So you may have excess of some production, so you don't have it. If you have excess, excess is something that 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.
30:21
Okay, okay, okay. This is the answer. This is what we're waiting for. In that sense, it is a red light. Okay, this is what we're waiting for. I'll take it, but it's really not, because regulated secretion at the last step is a different kind of regulation, but I'll take it for now. Listen to another function of the red, sort of quality controller. It's more like a production line, like if you're making cars,
30:42
you don't want to send a car out without the steering wheel. It makes sure that everything is correct. So this is exactly what is shown here. It has the motor, and it has the next protein that will take it to the right place.
31:03
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, YPT-1, YPT-31, and YPT-32.
31:26
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,
31:43
where is 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 live cell microscopy with different colors, and we first wanted, we had at least two markers for the early Golgi.
32:08
We had two markers for the late Golgi that they colocalized with each other, and then we made the YPT-1 or YPT-31 in green, and we looked at the colocalization.
32:21
We confirmed everything that we saw by live cell, 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. YPT-1 is starting early, very, very, very low level at late,
32:42
in the late compartments, YPT-31 is the opposite. So, and they both, about 25% of them, and it will just become a little interesting, they actually colocalize with each other, and they colocalize in a compartment that is marked by Sec-7.
33:04
And now just treat them, I know that Kathy Jackson likes the Sec-7, it's a Jeff, it's another GPAs, but for now they are only serving, and some people like CAP-1 and CHC-1,
33:22
but for now they are only serving us as markers. 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, YPT-31, 32 localize about 25% of them, but 95% of this localization happens only on the Sec-7 compartment.
33:47
So now if we make a Golgi map with the YPTs, we have CAP-1 early, we have Sec-7 and CHC-1 late, but we also saw some colocalization of early and late Golgi markers,
34:04
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 enzymatic activities for cis-medial trans, but that's why we call them early, transitional and late. Transitional tells you it's fast, it just comes and goes.
34:24
So the transitional compartment is also, so YPT-1 is on the early and transitional, 31 is in transitional and late. So they colocalize the transitional compartment.
34:41
So now I want to just branch off for a minute to Golgi's external progression or external maturation. We talked about this is what people think, how transport occurs through the Golgi. So the cargo stays in each compartment, and what happens is the compartment matures,
35:00
and this is within the same compartment. So in 2006, two groups showed in yeast, I think they provided a really good evidence that you can see external progression. The way that they did it is by looking at shifting from CUP-1,
35:21
which is the early Golgi, to CEC-7, which for us it's a transitional late. 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 regulation until you just do something to it.
35:41
Is there a regulation or not, or does it just happen? So we wanted to ask this question, do the YPTs which are in the Golgi regulate the external maturation? How do we do it? We have mutations that can affect the YPT,
36:00
either to be super active or to be not active. So now we look to see what happens to the dynamics of the external maturation that they showed, the two other groups showed. But now when we look at with these YPT mutations, and we look both by co-localization,
36:21
doing live cell and IF in immunofluorescence, and looking at dynamics. But we also added one more thing. We actually had now a third marker. So we looked at CUP-1 to CEC-7, which is what they looked at. And we also looked at what happened with CEC-7 to CHC-1.
36:40
So we determined the effect of, I'll show you hyperactivation, but we also show inactivation of these YPTs on both Golgi proteins co-localization snapshots, but also dynamics. And I'll show you just the dynamics.
37:02
So when we look at CUP-1 to CEC-7, so we watch it by looking at the dynamics of these. And I'm not going to ask you to look at this, but just to understand what we're doing. And then we have the chymographs of tracings of these CUP-1 to CEC-7.
37:23
And this is in wild-type cells. And we have numbers. We are looking at what happens to CUP-1 appearance versus CEC-7. So CUP-1 always appears about 15 to 20 seconds before CEC-7.
37:46
In Golgi. In the Golgi, in wild-type cells. But now when we hyperactivate the YPT-1 or YPT-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.
38:02
It's just always bound to GTP. Or it looks like it has the structure that it's always bound to GTP. So what we see here is that when we overactivate YPT-1, but not YPT-31, it goes faster.
38:21
So this step goes faster. With YPT-31 and YPT-32, it's similar to the wild-type. So this is our regulation of whatever was in the field, CUP-1 to CEC-7. But then we also looked at what happens to CEC-7 to CHC-1. We did the same experiment.
38:41
We looked at the dynamics. And then we do the kymographs and the quantification. And now what happens is that YPT-1 does not affect it, but YPT-31 makes it go faster from 10, 12 seconds to 4 seconds. So the effect is two or threefold, which is significant.
39:06
So YPT-1 activation now makes the CEC-7 go to CHC-1 faster. So together, we can see it here. Together, YPT-1 makes this step go faster.
39:23
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,
39:40
we show that, first of all, this is the first genetic evidence for systemic progression. Second, we actually divided it into two different steps. And YPT-1 regulates the first step, and YPT-31 regulates the second step. One question, sorry. Yes. So you're showing some percentages
40:01
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.
40:22
20% reaching to the tip, to the top, and 50%. We are looking at the initial rate. And what happened to the other events? Then they are together, sort of, no? Why? We are looking, it's not event. We are measuring 20% increase.
40:42
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 2 seconds. That's the average? Yes. Plus minus the standard deviation. But there are events that are happening either faster or slower, right?
41:03
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 2 or 4 plus minus 1, percentage of,
41:23
I said percentage, sorry, it's increase of the red light, the red marker to, 100% is the maximum. Of what? Of intensity of the light.
41:40
So it was the percentage of events, right? Light being red or green? No, in which unit? Is it doubling or what? 20%, meaning? It's fifth of the maximum. 20? Okay, it's bad. I understand.
42:01
I should have written what the percentages are. No, the answer is as I understand. 100% of intensity measured whatever in the amount of dye. And then 20%, you mark 20% of this intensity and you say this 20% level is reached in 12 seconds.
42:24
Then you mark 50% level on this whatever. It's just two measurements of the same common ground. 20% level of this intensity is reached in 10 seconds. This is what is the idea. Okay, thank you. When you get 20% and when you get 50%.
42:43
And you measure intensity in which number? This I never know, I don't know. In which number you measure intensity? Intensity means number of molecules. So your top signal, is that 10 molecules, 50 molecules? I don't know. And then each one? No, no, I think that intensity is measured in...
43:03
By how you feel, how you... All the molecules are, whatever, each molecule maybe does it, emits the fluorescence in a different way, but the same molecule is the same. You have one Golgi and got, let's say, 10 fluorescent molecules, right? The next Golgi have also 10 or had 20?
43:23
No, they all, so the answer to your question, they all had about the same intensity top here. About. There was no, no. So this is fluorescent marker. This is what I, Nisha, to your, the answer to your question, this is fluorescent marker and the intensity of the signal
43:41
is the intensity of the measured fluorescent marker. There will be no lunch today, because I... For official, the intensity, how often, which one, number? This is what I guess, this is what I understand. The number also will get you everything. If you do it wrong, you completely miss number. This is another question, let us continue.
44:00
You change the scale to the regular number. You change the scale, how you measure, you go play, you have the way, you have systems. It depends on the scale. This scale or this scale, right? The convenient scale to complete your looking. The scale is linear. The scale is linear. Okay, why don't we continue to talk afterwards?
44:24
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 on maturation,
44:41
and we are trying now to look at the Jeffs for YPT-1, TRAP-1, 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 Golgi's system on maturation.
45:07
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, but fast by jumping over slides. So I want to talk about autophagy.
45:20
So if you look at my simple model of the cell, I want 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, etc. But there is another one, which is autophagy,
45:41
that brings stuff from somewhere, from all these compartments, to the lysosome. And lysosomes, as we know, they actually spit back all the block, building blocks, and then they can be built again to get back into the ER, for example.
46:00
So the question is, is the coordination between trafficking and recycling? And I'm going to jump over this. Oops. I'll 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.
46:21
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.
46:43
So, okay, I will jump over this. Okay, so I want to talk about YPT-1, because we also showed that YPT-3 and YPT-2 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
47:01
going from ER to the Golgi, it turns out that it's also required for autophagy. So the way that 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
47:20
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 under stress. And here is an example of how we look at this pathway by GFP-ATG8, which is LC3 in mammalian cells.
47:44
And what happens is that in this pathway, an autophagosome is formed. And an autophagosome is, this is to remind me to say that it's a double membrane organelle. And it takes cargo,
48:01
so the autophagosome forms around 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,
48:22
growth conditions. And we have in yeast, for example, this was one available cargo that we were following. And both under stress or in normal autophagy, the process starts by formation of PAS,
48:42
which is the pre-autophagosomal pathway. And PAS is a combination, a complex of about 30 proteins, which are called ATGs, and membrane. And USOMI got the Nobel Prize just for showing this first step, how this is all starting.
49:04
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
49:20
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. Recycling? Yes. In the stomach.
49:41
Stomach or the skull? It's illegal. Auto-irrigable. Auto-irrigable. 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
50:02
that had no 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 nitrogen. Then another group came up with a subunit of TRAP,
50:22
also has a role specifically in this process. And then we did a yeast two hybrid screen, and to our surprise, maybe we shouldn't have been so surprised, we got ATG11, one of the ATGs that the stomach discovered in his screen for ATG mutants. And we got it by yeast two hybrid.
50:42
And so I'm not going to talk much about it, I'll just tell you that we showed that while TRAP1 is required for the role, so now I'm talking about the activators. The activator TRAP1 with the YPT-1 takes cargo from the ER to the plasma membrane,
51:05
while two other TRAPs, TRAP3 and TRAP4, which have similar core but different specific subunits, take it to autophagy. Okay, same YPT-1. And at least one JEF. So YPT-1 is required for cell viability
51:23
because of its role in secretion. It's required for autophagy under stress. No YPT-1, no stress. These TRAPs, one JEF is enough. Sorry, one JEF is enough, so you need to delete both in order to see the full effect.
51:41
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, the... But again, why under stress? What is the logic of that? Because it's recycling.
52:02
If you want to conserve a material under stress, you don't have any more nitrogen. 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.
52:22
Make sense? Okay. So what we showed, and the way that we think about it is, we're thinking about YPT-GTPase modules. So the module has a JEF, a specific JEF for the specific step,
52:43
a YPT-RUB, a GTPase, and at least one effector. So in this case, we have a module of TRAP3, specific subunits TRS85, YPT-1, and at least one specific JEF, ATG11.
53:02
So the way that we showed it is by showing interactions, collocalization, and function. And I'm going to jump over the collocalization, and maybe I'll just show you fast the function. So how do we study function in this case?
53:22
We are lucky because What kind of stress do you use? We starve them for nitrogen. In our cells, mammalian cells, you have to starve for Amino acids. Amino acids, thank you. So we have specific mutants,
53:40
which are only defective in autophagy, not in growth. TRS85 doesn't have anything to do with secretion, so we can delete it and see just the effect on this JEF on autophagy. Same YPT-1-1, the mutant that we later showed,
54:01
that it disrupts this mutation. It's a one amino acid change that disrupts the interaction of YPT-1 with ATG11. It does not affect other functions of YPT-1. And we have, of course, ATG11, which is, by definition, isolated and specific for autophagy. So we use all these mutants to ask
54:22
what is the effect? So, again, I don't have time to talk about results, but I'm just going to tell you that in YPT-1 or TRS85 or ATG11, there is no pass. So pass can be seen by co-localization of two ATGs
54:41
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 co-localization. They don't come together. So it's true also, as I said, for the whole module.
55:02
So the whole module, if we look at function, it's required for assembly of pass. It's the first step of autophagy. And until now, there was no shown regulation on it. So here we have what I call one,
55:21
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-1 and effector 1, effector US-1. And we have another
55:43
module and another process where the same YPT with a different Jeff, with a different effector now, goes through 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.
56:02
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 this system also has to have a way to recruit the right effector
56:20
to the right module. So the YPT in one module 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.
56:48
I'll just say it in two minutes. I'm not even going to show you that. Thank you. No, no February. I wanted to stop, I'll stop. So I just wanted to say,
57:03
so I wanted to tell you that we discovered a new autophagy pathway during normal growth and it's actually quality control of ER phagy. Quality control of the ER by autophagy
57:20
of the ER, the plasma 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
57:41
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.
58:02
Okay, so we pass through this. This is all the evidence. Both and so we actually showed the stop, the block by three ways. One was by
58:21
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 assay that shows that the ER is under stress. There is an overexpression of a protein, one protein, and it actually
58:40
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
59:00
control of the ER. Without overexpression of proteins, we have like two residents of ER. 61 is a translocon agent G1 and these are components of getting the vesicle to the Golgi. They do not go to this
59:20
pathway to ER phage. But these do. And about 20 to 50 percent of these proteins, depending on the protein, go there all the time. We just don't see it if we don't block it. And then if we overexpress a single protein, the cells are happy. There is no stress on the cells. There is no
59:41
induction of general autophagy. But we just have the ER is getting too blocked, then we get 95 percent of these proteins and these go now to the ER phage. So here are the other
01:00:00
what happens when you get, when proteins get from the ER? So if they are native, they'll go to secretion, secretion through the Golgi to the plasma membrane. If they are misfolded heavily, they'll go through ERAD to the proteasome. There is also a micro ERFAG process, which micro ERFAG means that they do not use the normal, the ATGs,
01:00:27
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
01:00:42
who participates and who regulates and the questions what you are 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
01:01:05
just to connect to human disease, I told you that RAB1 is the mammalian, the human homolog of YPT-1, and it's actually involved both in cancer and neurodegenerative disease. This is my connection to Friday, and thanks to everybody who helped me.
01:01:22
In my lab and 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...