VIDEO: Elvan Böke on Membraneless Organelles in Female Germ Cells: the Balbiani Body and the ELVAs

Elvan Böke (04:45):
So, actually, oocyte lifespan of female fertility is a very, very modern problem. Everyone knows that there is a decline in female fertility. There are some magic number ages, like 35, 38 being thrown around there. There is not a certain age. It depends on the woman, I will say, but it is certainly true that around the age of 40, the female fertility declines. And I’m saying it’s a very modern problem because these are actually graphs from the US, because I got the statistics by The New York Times. If you look at 1980, the age of first-time mothers in 1980 is at around 20 or 19, and if you look at the same stats for 2016, actually the graph keeps on shifting towards right. It says that age of first-time mothers are at the beginning of 30s.

Elvan Böke (05:40):
And Europe is much worse, i.e. where I’m based now in Catalonia. The age of first-time mothers, the average age of first-time mothers, is already 35. So, it’s already at the age that might be considered fertility starts its severe decline. So, we know very little about how oocytes can stay healthy for many decades or why their health declines with advanced age. And also, understanding how an oocyte can avoid aging as a little bit like flip the coin moment for many years can give clues for other aspects of aging research. So, to basically, again, put an analogy, if you think about a 21-year-old mother, we you think about, “Oh, such a young mother, she is.” But then, I’ll just like you to think the age your mother gave birth to you and then that very oocyte was that many years old. So, the oocyte that gave rise to me was 26 years old when I was born. Okay, so then we move on. So, my lab works with oocytes from three vertebrates-

Elvan Böke (06:39):
Sorry.

Anupa (06:39):
Xenopus. Xenopus, yeah. Yeah.

Elvan Böke (06:42):
Yeah. So, my lab works with three vertebrates. We worked with Xenopus, because honestly half of Xenopus, like this is-

Anupa (06:51):
Xenopus? What was that?

Elvan Böke (06:55):
Anupa, I can hear you, huh. So, one of it, half of ovary is like this and the other ovary of Xenopus is here. So, it’s basically like these giant ovaries. We work with mouse because it is genetically more amenable for manipulation and physiology. And of course, we work with humans and we mostly work with donated ovaries that is given to us by our collaborators in the hospitals around Barcelona. And what you see here is dormant oocytes, Xenopus, mouse, and human. And I always like showing this slide to kind of somehow show why we work with three species. This is actually scale comparison of the same stage oocytes on three stages, three organisms, sorry. So, this is Xenopus oocyte, mouse oocyte. So, you can put several mouse oocyte in one human oocyte, and the same applies to Xenopus. And this is the reason why we have to work with three species, to understand the questions that we’re asking.

Elvan Böke (07:56):
So, I told you already that oocytes can remain in the female for a while, and if we look at the main differences between a dormant resting oocyte and a newly activated, growing oocyte is that the major difference between these two guys is dormant oocytes have a Balbiani body in their cytoplasm. So, what is a Balbiani body? It was first discovered in 1800s by a Mr. Balbiani, and then it has been studied actually extensively by electron microscopic studies in 1960s or ’70s in humans. And there’s some research about it, like very recent research about it, too, but briefly what you see here is an electron microscope image of one human oocyte, here. And this is a giant nucleus of the human oocyte.

Elvan Böke (08:47):
And what you see here is the Balbiani body. So, basically, Balbiani body is a conglomeration of mitochondria, mostly… All these small, little circles you see are mitochondria… some membranes and, yeah, some unknown particles, let’s say, about [inaudible 00:09:06]. And if we think of other membranous organelles or other compartments we need in the cell, like ER, nucleus, or mitochondria, Balbiani body does not have a surrounding membrane. So, it makes it a condensate. And I asked during my postdoc a couple several years ago now, “What is the glue that keeps the Balbiani body together?” And I asked this question in frog oocytes, because as I showed you, frog oocytes are really large.

Elvan Böke (09:36):
And one of the things I did was basically I did a frog oocyte under the microscope, and what you see here, again, the oocyte, the nucleus, and this structure, actually. You can even see in a not fancy DIC scope is the Balbiani body. And I basically attacked the oocyte, let’s say, to isolate the Balbiani body. And this is really me doing the work with just normal forceps, like nothing fancy. As you can see here, I could isolate the Balbiani body. Let’s put it here. It remains together and this thing turned out to be very highly resistant to high salts and even to some extent SDS. And having isolated it, the first thing I did was to send it to mass spec and to figure out what is it made of.

Elvan Böke (10:26):
All right. And that basically led us to Velo1, and then later I showed that… Again, all this work is published, so I’m just going to go very fast here… that Velo1 actually localizes to the Balbiani body. And, moreover, it fills the gaps inside the Balbiani body. Then, I later showed that it practically doesn’t recover after photobleaching. So, it really forms a very stable matrix inside the Balbiani body. And I would like to emphasize here, this thing is 60 microns. So, if you are more used to working with human cell lines, this is like five HeLa cells in width. Just try to think about basically the scale of these things.

Elvan Böke (11:13):
So, the summary is that the Balbiani body that is in frogs is held together by this amyloid-like matrix… and we did lots of work–again, this is published–in dormant oocytes. And as the oocyte grows and matures into an egg, we also found that the Xvelo, the Velo1 protein is still present in the egg, but it’s soluble this time. So, basically, something happens to this amyloid-like protein to solubilize it. And this is one of the things that my lab is currently working on, like how do you make a physiological amyloid soluble? And also, very recently, we found that the amyloid-like nature of the Balbiani body, which we also probe here with a aggresome dye called Proteostat, is also conserved in humans. So, if you look at humans, human Balbiani body also stain with Proteostat.

Elvan Böke (12:07):
So, does anyone have any Balbiani body-related questions? Because I’m really going to switch gears here. No? Okay, perfect.

Avinash Patel (12:22):
Go ahead, none here.

Elvan Böke (12:22):
Then, I’m going to do new part which I’m more interested in. So, as I told you before, the offspring doesn’t inherit the age of their parents. And there are two main lines of research my lab does, to understand how do oocytes keep a young cytoplasm. The first part is the mitochondrial status and mitochondrial metabolism, and we have a couple of papers in revision right now about that. But then, today, what I’m going to tell a story is that proteostasis, meaning protein and homeostasis, how do oocytes deal about protein aggregates?

Elvan Böke (12:56):
And this is an interesting question because the only data out there comes from C. elegans and from a paper that was published somehow recently from Cynthia Kenyon’s lab. And in their paper, Bohnert and Kenyon show that oocytes in C. elegans accumulate protein aggregates throughout their lifespan, which is actually a fairly short couple of weeks in C. elegans, and they have inactive lysosomes. Just before fertilization by sperm signaling, they activate their lysosomes and clear all these protein aggregates and basically start with a fresh cytoplasm, let’s say, upon fertilization.

Elvan Böke (13:43):
Nothing is known about proteostasis in mammalian oocytes. So, this was our starting point two or three years ago, when my postdoc Gabri restarted. And we start asking questions like, what is going on in mammalian oocytes? First we checked the acidity or activity of lysosomes because here, as I said, the lysosomes were non-acidic, non-active in C. elegans upon until fertilization or just before the point of fertilization. But once we looked at the mouse oocytes, what we saw is actually lysosomes were completely acidic and thus active in mouse oocytes. So, that part of the C. elegans doesn’t apply to mammalians. We also showed that in humans later.

Elvan Böke (14:31):
So, lysosomes are active in all stages of oogenesis in mammals. Then we asked, “Do mammalian oocytes have protein aggregates?” And here is actually this very quick recap, because many people are, again, not used to looking at oocytes in oogenesis. This is how an immature oocyte looks like, and here, maybe what you should focus on is the nucleus here, right? So, this is the nucleus. It means that this oocyte hasn’t started meiosis yet. Then, it starts meiosis, so its nuclear envelope breaks down, and then when it’s basically finishes… So, basically, it gets arrested in metaphase II, and at that point, you see the first polar body.

Elvan Böke (15:15):
Upon fertilization, it excludes the second polar body and continues, like develop. So, basically, I would like you to pay attention whether an oocyte has a nucleus, meaning it’s an immature oocyte, or it does not have a nucleus and have a polar body. It means it’s an unfertilized egg, like it’s finished–like started now to finish the maturation process. Okay? So, when my postdoc, Gabriele started, he basically used this dye, Proteostat. In theory, it detects basically crowded environments in the cell, and as we asked whether oocytes have any compartments or protein aggregates. And we were surprised to find that oocytes have this five to 20 micron blobs, let’s say, in their cytoplasm. And there’s a nucleus, so it’s an immature oocyte.

Elvan Böke (16:05):
And then, Gabriele later just showed that actually these compartments overlap with LAMP1, a lysosome marker. Keep in mind that if this was a giant lysosome, it will just be like a membrane staining. The fact that it is completely crowded means it’s like several lysosomes inside one compartment. And I would like to, again, point out there are several other lysosomes inside the cytoplasm. So, not all lysosomes are in here, but there are cytoplasmic lysosomes and there are some lysosomes inside these compartments. Okay.

Elvan Böke (16:40):
And then, later, Gabriele actually went on to check with several different markers what these compartments have. And we checked an endosome marker, and they stained with endosome markers. They have early endosomes, they have autophagy markers, like aLC3B, and also, they have ubiquitin conjugates, meaning they have ubiquitinated proteins in them. So, in short, one day, I came to the lab, and Gabri came and told, “I named them! I named them!” And I was like, “What?” And he named them ELVAs, endo-lysosomal vesicular assemblies. So, it was actually completely out of my hands, but it also fits to the description. They are endosomal, lysosomal, and like lots of vesicular assemblies.

Elvan Böke (17:26):
And we continued characterizing these guys, and we actually collaborated with the Dresden’s EM Tomography unit. And what we found is that ELVAs are full with membranous organelles. They don’t have a surrounding membrane, but I’m just playing this EM tomography a couple of times. But you can see, for example, here’s an autophagosome, this double membrane here. It’s just merging with another body. There are classic autophagosomes with two membranes. There are lots of single membrane vesicles that could be endosomes or lysosomes. There are several, several multi-vesicular bodies. And every now and then, there are clathrin-coated vesicles, like that’s why it’s very dark.

Elvan Böke (18:16):
Right. So, we were more interested in these units because they basically have all of the degradation machinery inside the cell. So, next we checked whether they show any structural changes during oocyte maturation. And for that, what we did is that… Again, here is a reminder of different stages of maturation… is that what you see here at the beginning is we labeled the ELVAs with a membrane dye, and we let the oocyte mature in vitro. What you are going to see is the immature oocytes start with the nucleus, but throughout the movie, it’s going to go through nuclear envelope breakdown and going to arrest as a mature oocyte. The nucleus is gone now, and it’s going to pop up, the polar body, I guess, from here. Oh, from there. Okay.

Elvan Böke (19:13):
So, basically, in an immature oocyte, ELVAs are distributed throughout the cytoplasm, but almost immediately after nuclear envelope breakdown, almost immediately after the meiotic resumption, basically, they go to the cortex and they remain at the cortex throughout the oocyte maturation. Okay? So, Gabri quantified these things, obviously. This is basically as different maturation stages, like less mature to zygote. We found that the ELVAs corticalize through the oocyte development.

Elvan Böke (19:49):
And then, we also looked at when do they disappear? Do they stay around forever? And they disappear at 2-cell stage embryos. You can see in an immature oocyte, they are cytoplasmic. Then they go more cortical, and here’s the max z projections, and then zygote, they are still cortical and they start getting smaller, but they are still there. And in 2-cell embryos, they are barely there. It’s very hard to detect them anymore.

Elvan Böke (20:15):
So, we asked how their corticalization is regulated, and one clue came from the fact they had lots of actin inside them. And then, when we treat them with an actin poison, there’s also an accumulation of actin inside ELVAs. So, we incubated oocytes in the presence of DMSO. This is like a control that we just don’t let them mature; we just keep them as they are, immature, or in the presence of an actin poison, or in microtubule poison. But the long story short, this is how the control oocyte looks like. Basically, right after you see the nucleus disappear, ELVAs corticalize. But in the case of cytochalasin D, they are frozen, like they more or less stay at the same place. So, ELVA corticalization is actin-dependent. And in the nocodazole, although the oocyte very much hates it, ELVAs still corticalize in the presence of microtubule poisons. So, then we concluded that the corticalization is actin-dependent.

Elvan Böke (21:19):
Then we next asked the classic question, “What is the glue that holds ELVAs together,” because this is a very impressive 15, sometimes 20-micron conglomeration of different organelles. And for that, we actually had to go through it step by step. We, first, were wondering whether ELVAs are dynamic, because as you might have noticed in previous movies, they sometimes merge with each other. So, first, we FRAPed actually a couple of… We use a transgenic mouse, where the autophagic marker is labeled. But here, keep in mind, this market faces inside of the vesicle. So, basically, this is less mobile than what a structural protein would be.

Elvan Böke (21:59):
But then, if we FRAP our photobleached region, like in the middle, even after two hours, there is no recovery. And basically, this is also the movie that you can see there’s lots of autophagic vesicles going in and out of ELVAs, but the middle section stays in the dot in recovery. So, Gabri quantified it and we showed that basically there’s no recovery of ELVAs with this marker. And we enjoyed this because no recovery actually means we can isolate them. You know, if you remember from my first video file of Balbiani body, we are really, again, changing scales in the sense that Balbiani bodies are 60 microns. So, isolating them was easier. These guys are five to 20 microns, so it’s getting harder and harder to try to manually isolate them.

Elvan Böke (22:50):
But then we gave it a go, and this is brightfield images that you could see. Like here is actually where the ELVAs in the transgenic oocytes are marked. And once we just mechanicalized the oocytes, the ELVAs stayed together. And we were like, “Great, then we can actually FACS isolate them.” And that’s what we did. We FACS isolated them, and then we enriched for the ELVA fraction by using the GFP marker. And then, in the fold enrichment of… Then, we sent them to mass spec, and in the fold enrichment, actually you see that the first protein that came up was late endosome lumen, which we were happy about, because it just gives us lots of proteins about endosomes and lysosomes.

Elvan Böke (23:32):
And amongst the proteins we identified, there was a protein called RUFY1 that actually just striked us as very interesting. So, we thought RUFY1 is a good candidate to be the glue that holds ELVAs together because it has an interesting disordered domain as well as two coiled coil domains. It is very highly expressed in oocytes. So, we did this western blot, and we compared the oocytes to the fibroblasts, like cultured fibroblasts. And you can see here oocytes have really high amounts of this protein compared to just a somatic cell line. And it localizes to ELVAs. Then, we basically did the staining of RUFY1 and LAMP1. And also, there are a couple of papers in the literature that shows that if you overexpress RUFY1 in HeLa cells, it just shows a conglomeration of membranous organelles. And we were like, “Okay, let’s go for it.”

Elvan Böke (24:32):
And then, we also overexpressed RUFY1 in HeLa cells, just to basically make sure that what we see is right. And it is true overexpression gives us these condensates in HeLa cells, and this is the wildtype. And what we did is basically, we either removed the intrinsically disordered region or the larger portion of the coiled coil domain, or its membrane-binding region, actually, that I didn’t explain before. As you can see here, full length, especially in some cells, forms these giant condensates. If you remove the IDR, they are much smaller in comparison but they still do form condensates. In the one the coiled coil domain is gone, the protein is practically soluble, and presence or absence of this lipid, phospholipid-binding domain is actually not really affecting much.

Elvan Böke (25:24):
All right. So, then we actually contacted Melina Schuh’s lab in Gottingen and we asked whether we could use their technique to force degradation of proteins, because if you are not familiar with this Trim-Away, what you can do is you can inject an antibody against a protein with an E3 ligase inside the oocytes, and your protein of interest gets degraded. And this is a very powerful technique. The only flip side is if your protein is very highly concentrated in a cell, which our protein is, you may not be able to degrade everything. But we just decided to give it a go and a postdoc in Melina’s lab is really talented.

Elvan Böke (26:12):
So, this is the mock injection that you will see, and this is the ELVAs that are attacked. And what you can see here, they are just going to do nothing much. They are just going to basically go around the cell and get brighter. And then, once we inject it with the RUFY1 antibody, if you follow the ELVAs, they are getting incredibly small over time. Okay? So, then, what Gabri did is actually he quantified these things, like he quantified the mock-injected and the RUFY1 antibody-injected oocytes. And then, he found out that not only their volume decreases quite a lot, also the number of ELVAs per oocyte decreases quite much. So, from this, we are somewhat confident to say that RUFY1 is the glue that holds ELVAs together.

Elvan Böke (27:11):
And then, we injected… actually sorry. I was going to skip this slide in my mind… RUFY1. Once we injected RUFY1 into oocytes, it goes to ELVAs, again, and forms these… forms you can see here, and then they also merge over time. And moreover, if we actually FRAP a RUFY1-GFP ELVA, it recovers, actually, slowly but there’s recovery. Keep in mind, the first actually FRAP that I showed you was an internal luminal autophagosome protein. But this is probably the structural protein that holds ELVAs together, so its internal recovery is faster. So, once we quantified, as you can see here, there is a medium-sized recovery, I would say, within three minutes compared to zero recovery of two hours from the previous example.

Elvan Böke (28:13):
So, from these, we basically… and over time, if we just check average ELVA number or average ELVA volume per oocyte, we saw that their number decreases and their volume increases with time, again, telling us that they merge over time and they just kind of form larger and larger condensates. But from this part, we just concluded RUFY1 forms biomolecular condensates in oocytes without going too much into detail, like what kind of condensates they are forming, because we are more interested in what is the function of ELVAs. And for here, basically, I will just take a step back, and we just will say we found it particularly interesting that ELVAs disappear at the time the zygotic transcription starts.

Elvan Böke (29:01):
So, there’s a lot of information that maternal RNA is degraded in mouse at around the 2-cell embryo stage, because zygotic RNA transcription starts here, and the common notion is that you don’t want the maternal RNA or maternal proteins to be around so much, because there is going to be new zygotic proteins, like zygotic RNA being around, and things might be incompatible. And as I said, ELVAs disappear at around this time, too. So, we ask whether ELVAs help ensure proteostasis in oocytes because they are full with degradative machinery, by disposing of unnecessary or potentially harmful maternal proteins. Okay?

Elvan Böke (29:46):
So, the first thing we did was to check whether lysosomes in ELVAs are active. And actually, if we look in the immature oocytes and use a LysoTracker, meaning that LysoTracker is a dye that only accumulates in acidic and, thus, active lysosome, that I told you at the beginning, although cytoplasmic lysosomes are active, the lysosomes in ELVAs are not active in immature oocytes. But upon oocyte maturation, like an egg, actually LysoTracker is very strong in the eggs. And once we quantified it, you will see that the LysoTracker intensity really increases over time over maturation in oocytes. And we also quantified the LysoTracker intensity in the cytoplasm, just to ensure that this is not like a dye accumulation, like random thing. And in the cytoplasm, the LysoTracker intensity do not change within the same period of time. So, this is basically the specific intensity increase.

Elvan Böke (30:56):
Then, we looked back to our FACS list after we isolated ELVAs, and we just looked whether there’s any interesting proteins that could be ELVA cargoes, not the structure of protein holding them together, but some proteins that can give us a clue for their specific function. And we were interested in these two proteins because they are considered, let’s say, maternal proteins. And if you look at their transcript levels, both of their levels are very high in oocyte. They get completely degraded–like gone–in two-cell, four-cell stage or embryos, because they are necessary to specify inner cell mass. So, they are really important for later stages in embryonic development, but that for some reason they are completely degraded, especially maternal product is degraded upon oocyte growth.

Elvan Böke (31:52):
So, we pick C-Kit, actually I like to work on it, because it’s a membrane protein. We have more tools to work on it, because one annoying thing to work with oocytes is that you always work with primary cells, meaning that I can’t come to the lab or my people can’t come to the lab and work with cell lines. They have to go downstairs to the animal facility, sacrifice, and then we’ll isolate oocytes and just start the whole day. So, the number of tools we can use is actually very important for us. So, what we had is actually we can label C-Kit live with an antibody and just follow it throughout oocyte maturation.

Elvan Böke (32:29):
What you see here is a C-Kit labeled and C-Kit is a membrane protein, and I think this is a max z projection. That’s the reason why it’s very dotty. And FM4 labels ELVAs. It’s a membrane marker. What you are going to see is an oocyte, immature oocyte, going to go through maturation. So, to play it more slowly, C-Kit is in the membrane at the beginning, and then we release the oocytes from their arrest. And then throughout that time the nucleus disappear, C-Kit starts going into the oocyte and specifically into ELVAs. And you’re going to see it is more and more concentrated in the ELVAs. And then, once actually the oocyte is mature, right here, like this is an egg, the C-Kit is almost entirely in ELVAs and there is none left in the plasma membrane.

Elvan Böke (33:21):
So, next we ask whether, is everything on the plasma membrane gets endocytosed unspecifically or this is specific for C-Kit? And for that, we used another protein, and this is the quantifications. So, we use another protein called Juno, and Juno is important for sperm entry into the oocyte. In short, it means you shouldn’t endocytose much of Juno because otherwise your fertilization is going to fail. But then, let’s see what’s going to happen with C-Kit. So, we label them at the same time, in the same oocyte, by using antibodies live and then we release the oocytes from their arrest.

Elvan Böke (34:04):
As you can see, after the oocyte nucleus disappears, more or less, C-Kit is almost entirely in ELVAs, and there’s like, you can see the background endocytosis of Juno, but almost all of Juno is still in the plasma membrane, if I just compare this, and they both started as plasma membrane proteins, to that. So, basically, the internalization of C-Kit is specific. And targeting of C-Kit is specific. That’s why it’s not like any plasma membrane protein… receptor in the plasma membrane is being internalized. Then, we also checked on what happens to the proteins targeted, sorry, to ELVAs, and they are degraded upon maturation.

Elvan Böke (34:45):
So, what you see is just levels of the C-Kit and this is IF in the ELVAs in an untreated immature oocyte. And if we treat during the maturation process this oocyte with the lysosome inhibitor, C-Kit is at much, much higher levels. So, it accumulates in the ELVAs, and this is the quantification of several experiments on several oocytes. Moreover, we also show the same thing for ubiquitinated proteins. In immature oocytes, if you’ll remember, I told you that ELVAs actually accumulate these ubiquitinated proteins, and in mature oocytes, we couldn’t see any ubiquitination inside ELVAs. And what Gabri did is basically he matured oocyte in the presence, again, of a lysosome inhibitor and then we, again, started seeing these experiments are done at the same time, like ubiquitinated proteins, meaning that also the ubiquitinated proteins gets degraded in ELVAs upon maturation.

Elvan Böke (35:50):
So, like an overall outlook or summary, ELVAs are novel membraneless compartments filled with membranous organelles. I guess, joke to me, they are like the Balbiani body in the sense that they are membraneous organelles in a membraneless compartment in an oocyte. And they are not degradative for the majority of the lifespan of the oocyte but become degradative upon maturation. So, it is more like I see them more as like a trashcan. So, you put some things in that you don’t want to cause trouble, but only upon maturation, you basically just trash the trashcan. And many proteins targeted to ELVAs are maternal proteins that needs to get degraded upon proper embryonic development.

Elvan Böke (36:36):
And here, I would like to stop and take any questions. Sorry, here we go. And this is majority of the work of Gabriele, my postdoc and I would like to thank all of my lab and collaborators.

Avinash Patel (36:55):
Great. Thank you so much, Elvan, for this, again, very inspiring talk, and this telling us about the novel ELVAs. I have some questions, but before that, maybe if we have a question in chat, Xiao, do you want to come on camera and ask the question yourself?

Xiao Yan (37:17):
Yeah. Hello, um yeah. I’m David. Sorry I haven’t registered.

Elvan Böke (37:23):
Yeah, hi, David.

David (37:23):
Yeah, my question was concerning the dynamics of those vesicle assemblies. How do they form? Do you think it’s the scaffold protein that forms condensates first and then you see membrane-bound organelles petition later on, or do you think the surfaces of those organelles are important, that maybe the scaffold protein wet the surfaces of those organelles and then glue them together subsequently? What do you think is the order?

Elvan Böke (37:49):
So, actually, to be honest, we are also checking that. It’s like a secondary priority, let’s say. But in the early oocytes, if you look at early stages before the obvious ELVAs are around, we see lots of lysosome aggregates. So, it might be that there are lots of membranous proteins that get together and then the structural protein starts holding them together, but I wouldn’t give you a clear answer on that. This is something we are looking into, but first we want to just finish this part of the story and get it out and start looking into how they are formed.

Avinash Patel (38:28):
Great. Thank you, David, for your question. Elliot, you have a question. Do you want to come on screen as well and ask the question yourself?

Elliot Dine (38:36):
Sure. Really cool talk, Elvan, I really enjoyed. And it’s really cool that you have this Trim-Away tool to block the formation of the super-organelle while keeping all the other organelles intact and functional. So, I’m wondering if you could use that to look at the function of ELVAs in general, like if you do Trim-Away for RUFY1, could you then look at if C-Kit is no longer degraded and other such things?

Elvan Böke (39:04):
That’s the dream experiment, Elliot. But we are trying to do it, but it turns out to be an incredibly hard experiment to perform, because basically once the injected… Shiya from Melina Schuh’s lab, the postdoc working on the project, told us that he had to inject lots of Trim-Away and lots of components of Trim-Away just so… because RUFY1 was very much concentrated. So, he got 50% decrease in maturation efficiency even in the control oocytes, because you have to inject lots of E3 ligase and everything. So, the oocytes don’t like it.

Elvan Böke (39:41):
Then, you have to mature them, you have to in vitro fertilize them, and you have to basically do all of them until the blastocyst stage. So, if we think about there’s 50, 40% efficiency in every stage, then it really becomes like… reaches the border of impossibility in terms of technical challenges to do it. But we are trying. To be honest, Gabri is still sweating on it, whether we would be able to get some oocytes. But then we also would like to work with larger numbers that we can just quantify, because every oocyte is different. So, if we said, “Oh, one oocyte did that,” it doesn’t mean much for me. So, if we want to say, “Oh, we managed to image,” I don’t know, “20 blastocysts coming from 10 animals,” then it’s really becomes a super big technical challenge. But yeah, that is a dream experiment. Let’s put it that way. Yup.

Avinash Patel (40:37):
Great. Thank you, Elliot. Before just going to Alex… Alex your question is next… quick question about the Trim from my side. So, Elvan, is this now like very restricted still to injection or are there some novel advancements that this Trim technology can be applied to cells and culture and stuff without needing microinjection?

Elvan Böke (40:59):
So, I think in the original paper, they electroporated them into it, like electroporated somatic cells with Trim-Away. And that is a possibility, I will say, but for oocytes, oocytes are not… To my knowledge, you can’t re-transfect them, at least efficiently, let’s say, because we tried every possible suggestion, let’s say, in the literature several years ago when I started my lab, and they are not efficient; they are really not worth it.

Avinash Patel (41:28):
Okay, great. Thank you. Alex. Alex, you want to come on screen as well?

Alex Holehouse (41:33):
Sure. Hello, hello. Beautiful talk. So, with the-

Elvan Böke (41:37):
[It means you’re] following the development of the project. But go ahead.

Alex Holehouse (41:40):
I know, it’s great. It’s great. With the cortical rearrangement, where you see upon meiosis I this kind of redistribution of ELVAs to the cortex, is that something that is being, from an active process, are their motors that are driving that or is this just something that happens, essentially, right? Is everything getting pushed to the side during meiosis I that’s being… there’s just a lot being rearranged? I just don’t know.

Elvan Böke (42:01):
It is active. No, it’s a great question, and it’s active, because it’s a great question of you are coming from… I’m not sure if you know the work, because there’s also something called cytoplasmic streaming in oocytes. So, it is true. Many things are pushed to the cortex. So, it’s a thing, let’s say.

Alex Holehouse (42:16):
Right.

Elvan Böke (42:17):
But then we also know that there are several different wraps in ELVAs that can generate their own power.

Alex Holehouse (42:23):
Oh, okay.

Elvan Böke (42:24):
So, basically, we think that ELVA corticalization is an active process. But we did check on it. We did check whether it is the cytoplasmic streaming that they are just happening to be there, but no, the answer is no, it’s active.

Alex Holehouse (42:38):
Very cool. Awesome. Thank you.

Avinash Patel (42:41):
Great. Thank you, Alex. Shruti, do you want to come on screen as well and ask your questions? Shruti, you around or you are on the kitchen table? I don’t know. Oh, yeah, you’re there. Yeah. Do you want to unmute yourself?

Shruti Jha (43:04):
Hello?

Avinash Patel (43:04):
Yeah, we can’t hear you. But maybe- … in the interest of time, I can just read out the question. So, Shruti asks, “Can this mechanism of cytoplasmic renewal be used in somatic or stem cells to get rid of pathogenic aggregates or renew their age?” So, can this mechanism, let’s say I think what she’s trying to get at is, can it be transferred to somatic cells?

Elvan Böke (43:30):
In theory, it could. Or I think it could also totally work, for example, looking at lysosome conglomeration or RUFY1 conglomeration in different cell types, because no one… Many people observe there’s some lysosomal conglomeration in oocytes before, but they thought they were giant lysosomes, because no one really paid attention to it, really, in the field. So, if we actually scan through papers, we see many papers just labeling ELVAs saying they’re lysosome, like a large compartment. Now, we’re like, “Okay, it’s not.” But, yeah.

Avinash Patel (44:04):
Yeah, please. I think, Shruti, wasn’t that you?

Shruti Jha (44:11):
Hello?

Avinash Patel (44:11):
Yeah. We can hardly hear you though.

Elvan Böke (44:17):
Okay, the microphone is being passed.

Shruti Jha (44:19):
Hello?

Elvan Böke (44:19):
Hello.

Avinash Patel (44:20):
Much better.

Shruti Jha (44:21):
Yeah, I just was thinking about some contrary concept that could it be possible that there are some kind of mechanism of transgenerational inheritance kind of thing? Because we only think of everything needs to cleared out, all the mRNA needs to be cleared out. But could it be that there are some mechanisms that wants to preserve some of the previous generation stuff that might be kind of useful?

Elvan Böke (44:51):
We entertained that idea, I have to say. But them being degradative, I would say, goes against that idea.

Shruti Jha (44:57):
Yeah.

Elvan Böke (44:58):
Because we, actually, at the beginning, we weren’t sure whether it was for storage or degradation, because if you think of it, they could’ve been for storage too. But then, the fact that they start degrading at maturation, I would say, probably is against that inheritance idea.

Shruti Jha (45:16):
Yeah, but-

Elvan Böke (45:16):
Only small, little compartments of pieces of stuff are somehow needs to be inherited, rather than full protein.

Shruti Jha (45:27):
But do you think it could be possible, like something to look for? The concept that everything needs to be cleared out. Yeah, it’s definitely a contraryl concept, but do you think it could be something possible?

Elvan Böke (45:42):
I think you are coming and going out, but what I can tell you is basically like in Xenopus, for example, and in zebrafish too, there’s something called the germplasm, meaning that one part of the oocyte cytoplasm in its entirety is necessary and inherited for the next generation of germ cells, and if that next generation of germ cells doesn’t have this transgenerational inheritance, they can’t form germ cells, right? But then, in humans and in mammals, actually, it was shown that is not the case. So, there’s no key feature you need to inherit to become germ cells. You just need to go through some certain migratory pattern and be exposed to some transcriptional factors.

Shruti Jha (46:27):
Oh. Thank you.

Avinash Patel (46:30):
I guess, just before we go to Sinem, kind of a follow up question to what Shruti was kind of, I think, trying to ask is that can you imagine using this for therapeutic purposes, right, somehow trying to instigate that in a neuron where we also want to get rid of aggregated proteins, but you can have a triggered… way to trigger it in a neuron, and that then just goes away and starts… you know, you can have it and you can activate it when you need it, right?

Elvan Böke (46:58):
Well, I will need to think deeply about it, just how to engineer it. That’s what I say, because one thing that we are curious of is to see whether we can find some sort of a code that targets things to ELVAs, right? Like why didn’t Juno get targeted? Why did C-Kit get targeted? How does DDX3 get targeted? Is there some sort of molecular signature? But that will be basically building it up from scratch step by step, I say. Like, if we can find a signature and then maybe we can just think about building up more onwards on that.

Avinash Patel (47:33):
Great. I think… Thank you, Elvan. And next, Sinem. Sinem, do you want to come on screen as well and ask a question?

Sinem Saka (47:39):
Sure. Hi, Elvan, great talk.

Elvan Böke (47:41):
Hey.

Sinem Saka (47:42):
So, I was wondering where does ER, Golgi, multi-vesicular bodies, other parts of the endolysosomal pathway and membrane trafficking fall in this whole picture and with respect to ELVAs?

Elvan Böke (47:53):
There’s no ER, there’s no Golgi…

Sinem Saka (47:56):
Nothing?

Elvan Böke (47:58):
No, no, just name them, because we’ve gone through literally all the lists of what we can think of. But basically, there’s no ER, there’s no Golgi, and yeah. But there’s…

Sinem Saka (48:08):
So, that’s a general feature of the oocytes.

Elvan Böke (48:11):
No, no. So, basically, there’s no ER, there’s no Golgi in ELVAs.

Sinem Saka (48:15):
I see. Okay.

Elvan Böke (48:15):
No, no, no. Otherwise, really, ER is everywhere. For example, if we were to stain ER in this picture, it’s just really occupying everything, I will say. But let me see whether I have any slides for that. I don’t think so. Do I? Oh, there you go. Here we go. So, this is what ER looks like in the oocytes. So, it’s really everywhere, but it does not localize with ELVAs, like it just basically goes in and out, but majority of it doesn’t localize. There’s no mitochondria; mitochondria is completely excluded. Also, the Golgi is not in ELVAs.

Sinem Saka (49:00):
So, that may be also suggests like a particular endosomal route is used for trafficking things to ELVAs, because retrograde transport and other things are completely skipped in general.

Elvan Böke (49:11):
Yeah. Exactly, exactly. Yeah.

Sinem Saka (49:14):
Cool. Great, thanks.

Avinash Patel (49:16):
Great, thank you, Sinem. Giulio, Giulio, if I pronounced it right.

Giulio Chiesa (49:22):
Hi. Hi. Yeah, it’s Giulio. I was wondering if you have an idea if when your oocytes age, just like women of like 30, 40 years old have ELVAs with different properties, like more condensed or more rigid than younger women, I mean, female or any species.

Elvan Böke (49:48):
Giulio, you are completely onto something. I don’t want to answer this question before I know the answer, though. But we are currently working on that right now.

Giulio Chiesa (50:00):
Okay. Okay.

Elvan Böke (50:00):
Yeah. Let’s put it that way. We are totally on the same thing. But the data is not super solid right now, I don’t want to announce it and it go the other way later.

Giulio Chiesa (50:04):
I’m glad that I wasn’t completely off track then.

Elvan Böke (50:09):
No, no, no, no. You are totally on so quick. Yeah.

Giulio Chiesa (50:09):
Cool, cool, cool. And I’m glad to know that. Thank you.

Elvan Böke (50:18):
Okay.

Avinash Patel (50:19):
Great. Any further questions? I just want to ask maybe in the… on sitting on the kitchen table who might not have had a chance and want-

Jill Bouchard (50:30):
Looks like no.

Avinash Patel (50:33):
Whilst they are still thinking, I think one other question I have, Elvan, is so what’s the interplay between Xvelo and, yeah, sorry not… the Balbiani and the ELVAs? Have you looked into that? Do they crosstalk? What’s going on? Why does one take mitochondria? Why doesn’t one take mitochondria, kind of stuff? What gives them the specificity?

Elvan Böke (50:57):
The short answer is no, there’s not so much connection between them, let’s say. But it is also a very tough question because mouse oocytes don’t have a Balbiani body, it turns out, right? So, we have to either follow it up on humans. Practically, we have to follow it up on humans, actually.

Avinash Patel (51:28):
Ah, okay.

Elvan Böke (51:29):
Now that I’m thinking about it. And it is like it becomes very challenging, like how the link can be established. Because basically, mouse has some lysosome aggregates early on in the stages, like we can follow them, no problem and everything, but then I don’t know how that’s going to be relevant in the Balbiani body context.

Avinash Patel (51:49):
And a follow up question is that you look back, and so, there are definitely lysosomes that are active in the background, right, which do exist, so you just think that they are just not enough or the activity is not enough with those free lysosomes and it has to be activated by this kinase, C-Kit, in order to do that? And is that-

Elvan Böke (52:09):
So, that’s the question, right? So, it looks like, basically, the C-Kit [inaudible 00:52:18] is targeted to ELVAs at a very certain time. So, I can just… one explanation would be maybe just that the maturation step, that you need a much larger capacity for degradation, because you would need to degrade many, many things. So, that’s where ELVAs kick in and they just basically this extra boost to mature proteins, to, sorry, degrade proteins, or… Actually, that is the main thing, thinking about it, because you’re right, because there are active lysosomes in the cytoplasm, but you can [crosstalk 00:52:51] everything, they are just go into the ELVAs, not to the cytoplasmic ones.

Avinash Patel (52:55):
Yeah. So, one of the things that you also mentioned is this RNA, right, the RNA pool goes down and it goes up. I think the DDX3, you didn’t talk about much about that, but would that be involved in that kind of RNA homeostasis?

Elvan Böke (53:16):
It is basically, DDX3 is mostly involved in, again, inner cell mass specification for later things. So, yeah, honestly DDX, right, is involved in some RNA process. So, it might be. It might be.

Avinash Patel (53:26):
Okay. Great, okay. We are almost at the top of the hour. Any further questions.

Diana Mitrea (53:35):
Yeah, can you hear me?

Avinash Patel (53:37):
Yes, Diana.

Diana Mitrea (53:37):
Hi, this is Diana. Beautiful talk. I was just wondering if you have any insights in potential genetic or transcriptomics data out there that connects some of the proteins that you found in ELVAs with perhaps infertility?

Elvan Böke (54:00):
I mean, C-Kit, because if you don’t have C-Kit, you are totally… You can’t form an embryo, right? So likely, basically, you don’t have an inner cell mass and you die. And DDX3 knockout is, I think also, it’s just like you can’t form a viable embryo. So, it’s not infertility, these things actually. Many of the things that we work with is more like… it’s just like embryonic death, let’s say. So, it just goes before then. And for example, RUFY, not RUFY1, but RUFY3, a very close family homolog, again, is embryonic lethal. So, yeah.

Jill Bouchard (54:38):
Beautiful talk, Elvan. Thank you so much.

Avinash Patel (54:43):
Thank you.

Elvan Böke (54:43):
Thanks.

Avinash Patel (54:44):
So, I think if that’s it, then thank you so much, Elvan, for this wonderful talk.

Elvan Böke (54:49):
No, thank you for the feedback, because as I said, it’s just like we are just putting it together now. So, it’s just good to have all the feedback now.

Avinash Patel (54:56):
Looking forward to-

Jill Bouchard (54:56):
Global round of applause for you.

Elvan Böke (54:58):
Thank you.

Avinash Patel (55:01):
And I think the recording would be soon available as well, So, people, do go back and if you want to listen to the talk again and reach out to Elvan for further feedback or questions, I guess.

Elvan Böke (55:13):
Right, I guess just send me an email please, yeah.

Avinash Patel (55:14):
Yeah.

Elvan Böke (55:15):
Perfect.

Jill Bouchard (55:16):
Sounds good. We can include it with the recording on condensates.com. And anybody left, I hope you enjoyed our show and join us again in November. Good to see you all and thanks again, Elvan.

Elvan Böke (55:29):
Good, thank you. Bye-bye. Ciao.

Avinash Patel (55:30):
Bye-bye.

Jill Bouchard (55:30):
Bye.

Elvan Böke (55:30):
Bye. Bye.