|Type||Kitchen Table Talk|
Emily Sontag, from Marquette University, shared her lab’s work with the Condensates.com community on June 2, as part of our Kitchen Table Talk series. Emily’s expertise in both neurodegeneration and protein quality control (PQC) has developed out of her PhD training in the lab of Leslie Thompson at UC, Irvine and postdoctoral studies with Judith Frydman at Stanford. She recently opened her own lab at Marquette to explore how cells deal with misfolded proteins, studying this critical biology through a condensate lens. Her lab uses cutting-edge biophysical, structural, and imaging methods to understand the fascinating condensates involved in PQC. We loved that Emily also discussed how these PQC processes break down in neurodegeneration, as well as in aging and cancer. Enjoy her talk below.
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Mark Murcko (00:00):
Great to see you all. Our speaker today, as you know, is Emily Sontag, who comes to us from Marquette in Chicago [Editor’s note: Marquette is, in fact, in Milwaukee]. Emily’s doing amazing work that I think many of you may already know some of it, but it’ll just be so exciting to hear about her latest work. She’s been in the game now for a long time. She got her PhD at U Cal, Irvine and then did a long postdoc with Judith Frydman at Stanford, before taking up her position at Marquette. And what she’s been doing for quite some time now is detailed exploration of the protein quality control mechanisms that are used to handle stress response to unfolded proteins. It’s a very complex field. It’s been studied for decades. And what Emily is doing is really exciting because she’s exploring this in an entirely new way, through a condensate lens, using super-resolution microscopy and biophysical methods and structural methods, very cutting edge biochemical methods, really a very broad approach to studying this complicated process in an entirely new way, and in particular the applications of this protein quality control mechanism to neurodegenerative disease, although as you’ll see, it’s not limited to that, because this biology applies across all disease areas. So her title today is Sorting out the JUNQ, the spatial nature of protein quality control. Emily, take it away. Thank you.
Emily Sontag (01:32):
Wonderful. Thank you so much for that very kind introduction, Mark, and thank you to Jill and Mark and everyone for the invitation to present my work today. I’m really excited to get to introduce myself as a new faculty member and tell you a lot about the work that I started as a postdoc in Judith’s Lab at Stanford, but now what I’m planning to do continuing forward in my own lab at Marquette to examine the spatial protein quality control process and the formation of these protein quality control compartments…
Emily Sontag (01:58):
As we all are aware, maintaining a properly folded proteome is absolutely critical for cell health and survival. And if this process goes awry, that can lead to a number of different diseases, including neurodegenerative diseases, as Mark mentioned, like Alzheimer’s, Parkinson’s, Huntington’s and LS. These are all thought to arise from a breakdown in protein quality control, but interestingly, it’s not just neurodegenerative diseases. It’s also looking at things like general aging and cancer. In all of these, you see the formation of these large protein deposits in the cells, indicating that they’re no longer able to turn over the misfolded proteins, and they get sequestered into these inclusions. So what I really want to do is to understand the basic cell biology underlying this process of sorting them into the inclusions so that we can then start to create some novel avenues for therapeutics for all of these different disorders. All of the work I’ll be showing you today was done in budding yeast, and one common question that we always get is why study neurodegenerative diseases in yeast? They don’t have a brain. They don’t have neurons. And those are all valid points, but the yeast is really cool, and it enables us to do a lot of experiments that we wouldn’t have been able to do otherwise in a more straightforward fashion, given the genetics of the yeast and the ability to do the knockouts.
Emily Sontag (03:19):
So now that we have CRISPR coming online and doing CRISPR in some of the neurons, it will be really cool to follow some of these experiments forward into a more complicated mammalian system. Another advantage of using yeast is that all of these protein quality control processes are critical for the yeast to survive because they can’t control their environment. So they’ve established a really complex network to maintain protein homeostasis, or proteostasis network. And many components of this proteostasis network are conserved from yeast up into mammalian cells, allowing us to understand a lot of this basic cell biology that then will be applicable to higher order systems. So when I talk about these protein quality control compartments, what do I mean? When you have a protein that misfolds inside the cell, it will get rapidly transited into these highly dynamic compartments that we’ve termed Q-bodies. This is to be more analogous to a P-body, if you’re familiar with those. Q-bodies reside all over the cell, but we see them particularly associated with the ER. Now, if the stress leading to the protein misfolding is severe or prolonged in nature, or if you inhibit the proteasome, you’ll see the formation of an inclusion right next to the nucleus called the juxtanuclear quality control department, or JUNQ.
Emily Sontag (04:40):
If the protein itself becomes insoluble or is amyloid in nature, such as in the case of Alzheimer’s or Huntington’s for the neurodegenerative diseases, we actually see these proteins shuttle to a separate compartment called the insoluble protein deposit, or IPOD. The IPOD resides out in the cell periphery, and we see it more associated with vacuoles. So there are obviously a number of different pathways that these misfolded proteins can go on to get sequestered into these different compartments. This is what compartments actually look like in non-schematic form. So these are yeast cells that are expressing a temperature sensitive misfolded protein reporter that will misfold at 37 degrees and get sequestered into these compartments so we can track where the compartments are in the cell. So these green spots, you can see, form very quickly inside the cell. And you can see as you track these same cells over time, they sort of coalesce, condense and get cleared. And by 30 minutes, there’s really only a couple of dim puncta left. So that led us to then think that some of the function of sequestering these misfolded proteins is to help them be degraded potentially by the proteasome because when we inhibit the proteasome, we see the formation of the JUNQ compartment. So we thought these Q-bodies are sort of en route to the JUNQ to help clear through the proteasome.
Emily Sontag (05:59):
What’s really important about these Q-bodies is that this experiment was actually done without proteasome inhibition. So this isn’t some alternative pathway that gets activated when the proteasome is blocked. This is part of the cell’s normal protein quality control response to misfolded proteins, indicating that maybe these inclusions aren’t something that we think of as just being a breakdown of the process. They’re something that’s much more intrinsic to the cell’s response. Here are some of the initial images describing the JUNQ, or the juxtanuclear quality control compartments. You can see it here residing just by the nucleus with two different misfolded protein markers. These do sequester soluble proteins, so if you do a photo bleaching experiment, it does recover. So there is actually material being transported back and forth in and out of the JUNQ in real time. We see it residing near the perinuclear. It’s really concentrated in molecular chaperones and 26S proteasomes, again, leading some evidence towards this hypothesis that the JUNQ is actually to help clear these misfolded proteins. It’s unclear if clearance is actively happening in the JUNQ, or if it just sequesters all of these components together, so that as soon as the stress is alleviated, all of the chaperones, the proteasomes, and the misfolded proteins are all very concentrated together to help facilitate this clearance process.
Emily Sontag (07:23):
Then here is the IPOD. So this is what we see being associated with amyloid proteins, like Huntington, in this case, a polyglutamine protein. Those reside out here in the cell periphery. And as I mentioned before, we see it more associated with vacuoles and potentially autophagic vesicles. And as opposed to the JUNQ where we saw a concentration of proteasomes, we see a lot of autophagy proteins associated with the IPOD. So we thought perhaps that the JUNQ was to help facilitate clearance through the proteasome, whereas the IPOD was maybe to facilitate it through autophagy. Now, I will say in the context of all of the experiments that I’ve done, I don’t actually see the IPOD ever being cleared from the cell. It seems to be much more static. And that corresponds to what we see with the photobleaching experiments, where if you photobleach the IPOD, there’s really no recovery. There’s not a lot of material coming in and out. It seems to be a more static compartment than the JUNQ, but it is possible that it is cleared through autophagy and that’s why autophagy proteins are there. It just isn’t functioning in the context of my experiments. Maybe I’m not going out long enough, or I don’t have the experimental parameters quite set right to see that autophagy. So it is still possible, but it’s just not something that I’ve personally seen in this system.
Emily Sontag (08:33):
And so I think this gets to a little bit of the question in the chat. So these compartments are actually conserved in mammalian cells. So here are three different HeLa cells. And what you can see here is the IPOD shown with Huntington the JUNQ marked with VHL. And as you can see, these are still separate compartments inside a HeLa cell, and this is also a HEK-293T cell. You can see, again, the JUNQ and the IPOD being separate. And so one of the questions that has actually already popped up in the chat, and it’s one I get frequently, is what’s the difference between a JUNQ and an aggresome? And that’s a great question and something that Judith and I have actually gone back and forth a lot with. And Rahul, who I actually saw is here. I saw him pop into the waiting room earlier. So Rahul Samant also we would have lots of discussion about what the difference is between a JUNQ and an aggresome. And we never really settled on a good definition. There are differences. So the JUNQ that we see here in mammalian cells does not have the vimentin cage that is stereotypical of what you see of an aggresome. So it does seem like they’re different, but sometimes when you’re just doing these fluorescent microscopy images, it’s going to be really hard to tell the difference between a JUNQ and an aggresome. So I don’t have a great answer for that question, unfortunately, but it is something that I feel like we do need to figure out some of the differences between each of these compartments and when we see them and in what context.
Emily Sontag (09:49):
Right around the time I was getting started in Judith’s Lab, there was actually the description of another one of these subcellular compartments inside the nucleus. The term is the intranuclear quality control compartment, or INQ. That really then led me to the question of what’s the relationship between spatial protein quality control in the nucleus and the cytoplasm? Do they work in the same way? Do they interact? That was really what I wanted to address with doing this project. I very quickly ran into a problem, though, that all of the misfolded protein reporters that we used were actually small enough to passively diffuse back and forth between the nucleus and the cytoplasm, making it impossible for me to tell exactly where it resided, exactly where it started, where did it move, if they were in both places and looked identical.
Emily Sontag (10:32):
So what I did to get around this was to tag my reporter proteins with either a nuclear localization signal to keep it inside the nucleus, or a nuclear export signal to keep it in the cytoplasm, allowing me then to see exactly how these compartments form and look for interactions between them. For most of the experiments that I will show you today, I used a temperature-sensitive luciferase mutant. When you shift to 37, this luciferase will become denatured and get sequestered into these different protein quality control compartments, allowing us to monitor them throughout degradation or aggregation, particularly in the instance when we block the proteasome with MG132. So what does this look like? Here is the microscopy image. So I actually, for these live cell imaging experiments have the microscope at 37. So the luciferase is completely soluble here when I put the sample on. And then as soon as I click go and when we start the microscopy, that’s going to induce the heat shock that leads to the protein unfolding and getting sequestered. So we can watch sequestration into these compartments happen in real time. So what you will see here is many puncta formed very, very quickly inside the nucleus. And then it gets coalesced into one large inclusion inside the nucleus. That likely corresponds to this intranuclear quality control compartment, or INQ.
Emily Sontag (11:49):
In the cytoplasm with NES protein, you can see, again, nice diffuse expression throughout the cytoplasm. And then when we induce the sequestration, see there are dozens of those teeny-tiny little purple puncta. And then they will coalesce and condensed down into two large inclusions inside the cell for a time. So one of those is the JUNQ, and the other is the IPOD, but I don’t have a secondary marker in this to confirm which is which, so at this point, I don’t know exactly which one is which. So now that I know that my exogenous protein localization tags aren’t really affecting things… We still see INQ, JUNQ and IPOD all forming, everything seems to be working as expected… I wanted to look at these in a little bit more detail to see exactly where they were residing in the cell. Because yeast are teeny-tiny, I ended up having to do super-resolution microscopy for this. So in this case, I did structured illumination microscopy, and I took basically what would be the final time point of those live cell imaging movies, so at two hours of heat shock. I then fixed and immunostained for nuclear pores. So that’s what’s shown with these gold spots here.
Emily Sontag (12:54):
So this is a 3D reconstruction of the SIM data. And if we blow it up and spin it around, you can see that here’s this green spot up there nicely inside the nucleus in a little groove in between the nuclear pores and the DNA from the DAPI counterstain. So we do see INQ formation there just as expected. With the cytoplasmic compartments, again, with the nuclear pores shown here in gold, when we kind of spin it around, you can see the JUNQ here just outside the nucleus and the IPOD residing out here in the cell periphery, just as expected. What was interesting to us is we didn’t really see any trafficking of these back and forth between the nucleus and cytoplasm. The INQ and the JUNQ really seemed to stay as being separate compartments. And I wanted to confirm that without having those localization tags there, just to make sure that that wasn’t interfering with something, if I was preventing something from being imported. So I repeated the super-res microscopy using that Ubc9 temperature-sensitive reporter. So, again, it’s going to misfold at 37 and get sequestered into these compartments.
Emily Sontag (13:51):
And so what you can see here, again, with nuclear pores shown in gold, we have the INQ forming inside the nucleus and the JUNQ just outside nucleus. So they are in fact separate compartments. If we do a line intensity profile across the image, you can see them being separate compartments with the nuclear pores in between. So you have the peak here for the JUNQ, peak with the nuclear pores and then the peak with INQ. So we do see them as being separate compartments with the nuclear envelope residing between them. So now we’re going to start building this model of spatial protein quality control that I’ll update with each piece of data as we go along through the talk. But for right now, with our model, we have nuclear protein quality control resulting in the formation of intranuclear quality control compartment and cytoplasmic protein quality control leading to the formation of this juxtanuclear quality control compartment.
Emily Sontag (14:42):
I was also then really fortunate to have an amazing collaboration while I was in Judith’s lab with Carolyn Larabell, whose lab is at Lawrence Berkeley National Labs. So she was close by, but her lab does the soft x-ray tomography technique. So what this is is you take your sample and you flash freeze in liquid nitrogen. Then we’re able to do the fluorescence image to correlate our fluorescent tags to the x-ray tomograms. They then hit them with these soft x-rays, and those will be absorbed by things that are very carbon-rich, and they’re able to then annotate all of the subcellular structures without doing any fixing, slicing, staining, anything that introduces a lot of artifacts into the samples. So this will allow us then to observe our protein quality control compartments directly in their sort of endogenous environment and get a much better idea of their architecture and their exact localization in the cell and see what they’re interacting with, what’s nearby, to help give us an idea of what these are doing inside the cell. Because this is a relatively new technique and people aren’t as familiar with it, I wanted to walk you through a little bit of what this looks like. So this is a yeast cell seen by the soft x-ray. This is the tomogram. And so, as I mentioned, things that are carbon-rich really absorb the x-rays. So these really dark spots that you see here, those are lipid droplets, things that are membrane-bound. So this is the nucleus, really highlight well with a soft x-ray tomography technique.
Emily Sontag (16:05):
These [light-ish] spots are vacuoles. And so you can really start to even just without doing much annotate a lot of these subcellular features of the yeast. This yeast cell happens to be one that has the endogenous Hsp104. So this is the yeast disaggregase that we know localizes to both the JUNQ and the IPOD. And this is from the GFP collection, so this has endogenous Hsp104 tagged with GFP. And then we put mutant Huntington in as an IPOD marker to then be able to distinguish JUNQ from IPOD. So you can see our fluorescence overlays here on top of the x-ray tomogram. They then were able to generate these 3D reconstructions from the tomograms. So we have the nucleus shown here, is this big gold ball. You can see mitochondria forming this nice network throughout the cytoplasm in blue. Vacuoles are shown here in gray. And if I spin this around, what you can see here is the JUNQ forming here just outside the nucleus. And we have the IPOD over here in purple, I’ll come back around on this side, again, interacting with vacuoles as expected.
Emily Sontag (17:06):
One feature that I would like to highlight from this technique, though, is if you look, these actually look much smaller than what we would expect, given their florescence images over here. That’s a huge advantage to this technique, because by fluorescence microscopy, these fluorescent protein tags become hyper-fluorescent inside the protein inclusions and compartments, making them look much brighter and much larger than what they actually are. But because we’re mapping this fluorescence back onto the x-ray tomograms, we then get a much better idea of the exact size, shape and location of these compartments.
Emily Sontag (17:39):
This is what the x-ray tomography data looks like from the NES luciferase protein. And I think that’s where this technique really shines for this. So if I spin this around, you can, again, see nucleus shown in gold, vacuoles in gray, mitochondria forming this network here around it. And here are Q-bodies homing in and forming a JUNQ, as you can see happen here. So if I drop other organelles out of this, what you see is this actually looks like Q-bodies fusing together to form the JUNQ. And so you can see there’s much more structure to this JUNQ, where it actually looks like the fused Q-bodies coming together. So what we’re working now to determine is if these are actually fused Q-bodies, or if there’s some other cellular architecture underlying this. Are there microtubules, something that’s interacting with this to give it more of this shape? But this is something we never see by the fluorescence microscopy. This JUNQ always looks like the nice spherical object that we see by fluorescence microscopy. So this really indicates that there’s perhaps a lot more going on architecturally to these compartments than we had previously appreciated.
Emily Sontag (18:47):
Some other highlights from this soft x-ray tomography data that were a little unexpected and really exciting are that we actually see interactions of Q-bodies with the vacuoles. So you can see that here. This is the same cell just rotated 180 degrees. So you can see the backside of the vacuoles and see that they actually have Q-bodies as well. But these Q-bodies look like they’re being pulled into the vacuole. And you can actually see deformation of these vacuolar membranes around the Q-bodies. It’s like they’re being pulled in, indicating perhaps there’s much more of a method for direct clearance of Q-bodies through the vacuole, through direct import into the vacuole. And like I said, that was unexpected for us. We tend to think of the IPOD as being the one that associates with vacuoles, not so much Q-bodies. So this was something really exciting. And we’ll kind of come back to it a little bit at the end of the talk, what we think is going on in this process.
Emily Sontag (19:38):
I mentioned this a little bit too, but we always see mitochondria nearby with these compartments forming almost like a cage around where we see these protein quality control compartments forming. So you can see that here with this endogenous Hsp104 with the JUNQ. Here you can see it with different Q-bodies kind of dotted along the mitochondria as they’re moving around the cell. And I was able to confirm this doing our confocal microscopy. So we have our purple inclusions here shown, and then we have the mitochondria almost wrapped around these inclusions, giving them like a big bear hug. So we see a really strong interaction between these mitochondria and the protein quality control compartments. I’ve started trying to tease this apart, and we haven’t figured out exactly what role mitochondria are playing in the spatial protein quality control process, but this is something that’s really exciting and something that I’m actively working on, particularly this summer, in the lab.
Emily Sontag (20:28):
So updating our model of spacial protein quality control, again, we have the INQ and the JUNQ forming separately, but also that we see the interaction between all three cytoplasmic protein quality control compartments and mitochondria going on in the cell. And further, we see a novel interaction between Q-bodies and vacuoles where we see this deformation and perhaps import of Q-bodies into vacuole for clearance.
Emily Sontag (20:53):
Next question. Now that we know that the INQ and the JUNQ are separate compartments, then, was to say what relationship is there in between these two compartments? So in order to do that, I’m going back to the time-lapse imaging, like what I showed you at the very beginning of the talk. But in this case, I’m going to co-express the NLS and NES luciferase in the same cell so we can see if the INQ and the JUNQ actually do interact. Again, this is what this looks like on the microscope. So we have the cytoplasmic diffusely purple, and then we have the LS in the nucleus. We induce the temperature shock. You can see, again, these bright puncta form in the nucleus and the cytoplasm. If you watch this little purple dot right here, though, what’s going to look like it happens is it’s going to look like it gets imported into the nucleus and is co-localizing with INQ.
Emily Sontag (21:41):
And that’s something I told you from the super-res that we didn’t see. And that’s actually not what’s happening here. It’s actually that it’s going up out of plane of the microscope, and it’s going to fall back off right about now on the other side. And you can see there’s also a purple one up out of plane here. But what you do see is this sort of tethering of these two together, where it seems like they’re actually moving together. And you can see that happening here as well in this cell, where the INQ and the JUNQ really seem to be homed in together and tethered together. And this is maybe a little more obvious when we do the particle tracking experiment. So we actually just track these. This is the INQ shown here in green and the JUNQ shown in purple. And you can watch them move through the cell. And so you’ll watch them home in together and then, like I said, almost co-migrate through the cell like they’re tethered together. So you’ll see them start just moving up and around the nucleus on either side together. So you can see them there, like they’re just hanging out together, moving around. So this was fascinating and something that we’re spending a lot of time trying to tease out exactly what’s going on with this homing process.
Emily Sontag (22:41):
Obviously, wanting to look at this homed in compartments and a little bit more detail, I returned back to our super-resolution microscopy experiment. So we have the INQ shown here in green, JUNQ in purple, and the nuclear pore is highlighted in gold. And I’ll spin this around, and you can see they are separate compartments here. And if we drop the JUNQ out, you can see there’s actually a row of nuclear pores that runs there in between the two compartments. So we do see the membranes still intact between the two. So they are still separate compartments, just homed in on either side of the nuclear membrane.
Emily Sontag (23:16):
Our next big question is what’s driving this homing signal? Why is this happening? And one of the first things we thought to look at then was nuclear pores because that’s something that allows communication back and forth between the nucleus and the cytoplasm. So we wanted to see what role the nuclear pores were playing in this. In order to do this, I did the co-expression experiment, this time in nuclear pore mutant yeast strain, where the nuclear pores remain clustered on one side of the nucleus. So it’s almost like a little half moon cap of nuclear pores sitting on one side of the nucleus. This will allow us then to see where the INQ and the JUNQ reside in relation to nuclear pores to see if they’re perhaps transmitting a signal homing these two compartments together.
Emily Sontag (23:57):
So this is what this looks like. Again, we have the nuclear portion in gold, and then these are three different nuclear pore mutant cells. So you can kind of get a good idea for the different types of phenotypes that we see when we do these experiments. So when you have just the JUNQ in the cell, so when you only have the cytoplasmic compartment, you can see the nuclear pores clustered together, and the JUNQ always resides next to those clustered nuclear pores. However, when you only have the INQ here, you see that the INQ can be anywhere in relation to this clustered nuclear pores. You see the clustered nuclear pores here in gold. The INQ here is on the opposite side of the cell. Here it’s tucked in underneath, and here it’s just off to the side from those clustered nuclear pores. So we don’t really have any firm localization between the nuclear pores and the INQ.
Emily Sontag (24:45):
However, if we express both the INQ and the JUNQ in these cells, you can see that they do still home in together, and they home in right at the edge of these clustered nuclear pores, indicating that perhaps nuclear pores are playing a role in transmitting the signal. And it does seem that perhaps it’s initiating from the cytoplasmic side, as the JUNQ is always recruited to the nuclear pores. But until we have the JUNQ there to recruit the INQ, we don’t see the INQ going to the nuclear pores. So perhaps that signal, then, is coming from the cytoplasmic side.
Emily Sontag (25:17):
When I went back and looked at the soft x-ray tomography data, what I noticed is that the site where this JUNQ is forming happens to be next to sites of nuclear vacuolar junctions. So in yeast, nuclear vacuolar junctions are comprised primarily of two different proteins, Nvj1 on the nuclear side and Vac8 on the vacuolar side. So I wanted to see, then, if we had any interaction between these nuclear vacuolar junctions and our Q-bodies. So we went back to our time lapse microscopy experiments, in this case using endogenous Nvj1 tagged with GFP and looking for interactions between this green nuclear vacuolar junction and our Q-bodies. And what you’ll see in the time lapse is this sort of transient interaction between green and purple dots. And you can see what happens is they just almost like kiss off of one another. And I know that video happens really fast. I’ve slowed it down several times. And then they end up just sort of next to each other. So it’s a little hard to see, but here’s the green dot, and here’s the purple dot. And I don’t really ever see true co-localization of these puncta, but that maybe makes a little sense, given what we see in the soft x-ray tomography data, where we don’t see them actually inside the nuclear vacuolar junction. They’re just off to the side. So perhaps it makes sense that we don’t see co-localization localization if they do reside next to each other.
Emily Sontag (26:33):
But we do have evidence the nuclear vacuolar junctions are playing a role in clearance and sequestration of these proteins, because if we delete either of the proteins, so Nvj1 or Vac8, we see a large increase in both nuclear and cytoplasmic aggregation, indicating that these misfolded proteins aren’t as readily cleared and therefore are accumulating in the cell, and more cells are developing these inclusions when you delete these proteins.
Emily Sontag (26:58):
Interestingly, when we do the co-expression experiment, where we have the NLS and NES luciferase in the same cell and an NVJ deletion stream, what happens is you get the formation of the INQ and the JUNQ, but if you watch, this INQ will actually start budding out of the nucleus co-localized with the cytoplasmic NES protein. You can see this one here actually ends up quite far away from the nucleus. So it’s almost like they’re budding out of the nucleus. So what we think is happening normally is when you have the nuclear vacuolar junctions intact, the INQ and the JUNQ are recruited to the site and bud out from there into the vacuole for clearance to the vacuole. But if you break those nuclear vacuolar junctions, the budding process is still happening, but it’s not going into the vacuole anymore. So then we see it residing out in the soft periphery, perhaps for clearance through an alternative mechanism or just terminal sequestration until things can be reset in the cell.
Emily Sontag (27:53):
Then wanted to look at what was involved in this budding process. This was obviously something that’s super cool, and I wanted to look into a lot more. So one thing that we looked at was this ESCRT family of proteins. So the ESCRT proteins are membrane remodeling proteins. They’re known for binding to membranes and then creating these bud-like structures out of the membranes that then go on to lead to vesicle formation. This can be for cargo transport or endosome formation. And these are particularly critical for the formation of multivesicular bodies, which we know help to clear misfolded ubiquitinated proteins. So this was a really cool pathway family of proteins for us to look at being involved in this process. One particular component of this is the Vps4 proteins. And this is a triple A ATPase that’s involved in sort of the final step, that’s where it’s sort of membrane scission and dismantling of the ESCRT machinery, leading to vesicle release from the membrane. So I wanted to see if Vps4 was perhaps playing a role in this budding process and pulling these things out of the nucleus.
Emily Sontag (29:01):
So our hypothesis, then, would be that in these vps4-deletion strains, we would still get bud formation, but you wouldn’t get sort of release of the INQ out into the cytoplasm. And so when we actually just quantify the percentage of cells with inclusions in a vps4-deletion strain, you can see, again, an increase in the number of cells that have both nuclear and cytoplasmic inclusions, leading us to confirm that Vps4 is playing a role in clearance of these misfolded proteins. And then when we look in our microscopy data, what you can see here is that we have the nuclear pores shown in gold, and you have a cytoplasmic INQ here, co-localizes with JUNQ, and it’s still residing here next to the nucleus. We don’t see it getting pulled away. And here’s this happening in another cell down here, where you have the cytoplasmic INQ co-localizing with JUNQ right up next to the nuclear membrane. So it’s almost like we are still getting budding, but we’re not getting that release then out of the nuclear envelope.
Emily Sontag (29:59):
Now that we have evidence that ESCRT is playing a role in this, we really then wanted to see if we can identify more of the components to see if we can figure out what is leading to this homing mechanism, what’s bringing the INQ and the JUNQ to this site? And one interesting candidate for this is the Chm7 proteins. So this is the east homolog of Chm7, which many of you might’ve heard of. This is an ESCRT protein that’s been implicated in autophagic processes and in a lot of different neurodegenerative diseases. Chm7 is a nuclear envelope specific ESCRT protein. And it’s been shown that Chm7 is actually required for survival for yeast strains that develop these large nuclear herniations that look very much like the buds that we’re seeing. And this really cool paper came out right as I was working on this, fortunately, from Patrick Lusk’s lab at Yale, where he found that Chm7 will actually bind to ESCRTs and lead to Vps4 recruitment to that ESCRT site and help in clearance of damaged nuclear pores. So this was obviously a really cool candidate protein for us to look at and see if it was involved in this process.
Emily Sontag (31:08):
And this paper from Patrick Lusk’s lab actually went on and it showed that when you have a hyperactive form of Chm7, you can see this here in these correlated light- and electron- micrographs, you have this large spot of the activated hyperactive Chm7. And when you look at it in the EM tomogram, localizes just below these buds right at sites where these constrictions are happening. So the constrictions are shown with the black arrowheads, and the white is indicating the buds. So this looks very much like what we’re seeing with the budding of the INQ out of the nucleus. And these down here are the 3D reconstructions of sort of the Z dimension, so you can see what it looks like in Z with these buds coming out of the nuclear envelope.
Emily Sontag (31:55):
So then that led us to wonder if this active Chm7 is what’s recruiting the INQ and the JUNQ to this site and leading to downstream activation of Vps4 for budding and release of these compartments into the vacuole. When I looked at the localization of the active Chm7 in relation to the INQ and the JUNQ, you can see here in these sets when I blow it up, here’s the Chm7, these teal dots, and we do see it localized right next to the INQ and the JUNQ, right on the other side of the nuclear membrane. So we do see INQ and JUNQ homing into where this active Chm7 is localized. Then when we delete Chm7, we break homing. So you see that the INQ and the JUNQ are no longer homed. They’re actually almost on the opposite side of the nucleus at that point. So Chm7 really seems to be involved in this INQ/JUNQ homing, potentially then for recruitment of ESCRT machinery and downstream budding into the vacuole for clearance.
Emily Sontag (32:55):
So coming back to our model of spatial protein quality control, we have the INQ and the JUNQ form will home in, and the JUNQ will recruit the INQ through nuclear pores or some protein near nuclear pores, perhaps the Chm7. We see this happening near nuclear vacuolar junctions. And again, this Chm7 and Vps4 may be playing a role in this recruitment and clearance through the vacuole. So this whole area would just get budded out and engulfed into the vacuole. Going back to the earlier data, we see interactions between mitochondria and the Q-bodies, JUNQ and IPOD in the cytoplasm and this really cool phenomenon where we see the Q-bodies almost being directly imported into the vacuole. And so we think maybe the ESCRT components could be playing a role in this direct import of Q-bodies into the vacuole through this other side as well. So perhaps some of these ESCRT proteins, multivesicular bodies are really like pulling all of these misfolded proteins in directly into the vacuole. It could also be import through some like, chaperone mediator autophagy mechanisms or things like that as well. So those are all active areas that we’re looking into for sort of alternative clearance and direct import into the vacuole for clearance of these protein quality control compartments.
Emily Sontag (34:07):
That leads me now to what I want to do. So my future directions for my own lab are to really start to understand what some of these subcellular organelles are doing, how they’re engaging with these protein quality control compartments, and what they’re doing in the cell. We see these really cool interactions between mitochondria, ER, vacuoles and particularly organelle-organelle contact sites, so nuclear-vacuolar junctions, ER-mitochondrial junctions, things where there’s a lot of membranes present, and see what role these organelles are playing in this protein quality control process, all with the goal, then, of understanding how this process fails in the context of neurodegenerative disease associated proteins.
Emily Sontag (34:46):
So, see, we know where Huntington goes. Where do some of these other neurodegenerative disease associated genes get sorted? And does it work through the same process that we see with our regular reporter proteins? Or do these proteins that cause neurodegenerative disease go through a separate sorting process? And really understanding how that works is going to be critical to understanding this underlying cell biology for these diseases. Because being a new faculty member I’m allowed to get a little more aspirational in where I see the field going and where I would like to go, I also wanted to point out that spacial protein quality control has been shown for a very long time to be implicated in just general aging. So as cells and as individuals get older, we see a large accumulation of misfolded proteins happening in the cells. So are these spatial sequestration processes breaking down with age? Or are there some other defects that’s happening that’s preventing proteins from being cleared in the proper way? And one really interesting avenue for this is actually to look in cancer. And so it was shown almost 10 years ago that p53 will form oligomers and actually amyloid inclusions inside the cell. And there are a number of different tumor suppressor proteins that will undergo this oligomerization and eventual amyloid formation.
Emily Sontag (36:01):
And there’s a paper from Frederic Rousseau and Joost Schymkowitz on my archive right now looking at PTEN. And they were really sort of my entry into thinking about this, because they have a joint lab together, and they came to Stanford a few years ago and gave a talk, and I was fortunate enough to get to go to lunch with them, and we just had a fascinating discussion about how these different tumor suppressor proteins could be going through these different spatial protein quality control processes inside the cell. And that’s something eventually, obviously not within the the next five or probably even 10 years, that we would get to, but I really would like to see the field start to address how some of these other diseases are using these same protein quality control machineries because we tend to think of it in the case of neurodegeneration where these things go awry and the cells dies. But perhaps these same processes are being hijacked for hyperproliferation of cells in the case of cancer. So it’s not just cell death, but also cell proliferation that would be effected by these protein quality control processes.
Emily Sontag (36:58):
So coming back to what I’ve shown you, to summarize everything. You see there are three different cytoplasmic protein quality control compartments, the Q-bodies, the JUNQ and the IPOD, as well as the formation of a subnuclear protein called a control compartment, INQ. We see the INQ and the JUNQ home in to a specific location on either side of the nuclear envelope near sites of nuclear-vacuolar junctions. And these nuclear vacuolar junctions may represent a novel protein quality control site in the cell. Further, we see a role for ESCRT machinery in the clearance of these misfolded protein compartments.
Emily Sontag (37:34):
So with that, I would like to give just an enormous thank you to Judith for her support through this postdoctoral project. It was something that we like to joke about, that it’s a very difficult project to get published because every time we answer one question, we open up 10 more about what is this budding? What is this homing? What is going on? So it’s very difficult to get published, but it’s been an awesome project for a postdoc to do to launch my own lab, because there are so many amazing places that we can take this research for me moving forward. And Judith was enormously supportive of me throughout my entire time in her lab, as well as the other members of Judith’s lab. Obviously, this project was a lot of work, and so having really talented, smart people nearby to help out, to ask questions, and to really lend their expertise was phenomenal for this project.
Emily Sontag (38:22):
I also would like to acknowledge Jon Mulholland from the Stanford Cell Sciences Imaging Facility. He’s the one who taught me how to do the super-resolution microscopy, which on yeast, given how small they are, was not trivial. So thank you to him for helping me get started with that. Another enormous thank you to Carolyn Larabell and her lab for their collaboration to do the soft x-ray tomography. I really feel like that technique helped open our eyes to a lot of alternative possibilities for these protein quality control compartments, and those experiments are a lot of work, to do all of the data acquisition and then analysis. And so having me be able to go there and work with them, it was a huge opportunity. So a big thank you to Carolyn, Jerry and Jian-Hua for their collaboration there, as well as funding for my postdoc from an F32 fellowship from the Ellison Foundation. And then a quick shout-out to people from Marquette. So I have my first graduate student who just joined the lab, Sarah Rolli, and two really talented undergraduates who worked with me this last semester, Rebecca and Alyssa, to help me get things kind of up and running, as well as my startup funds. So with that, I am happy to take any questions.
Mark Murcko (39:25):
Thank you, Emily. Really exciting stuff. I see exactly the challenge that you just described about each thing you learn raises 10 more questions. I totally get it. I’m sympathetic about the publication challenge. But it’s such important work. This is really going to help us to understand something that’s just so fundamental. There’s a lot of questions coming in on the chat. You saw the first one, which you answered on the fly. But I think it’s a good question about resolution of the tomography. It is far higher resolution, really-
Emily Sontag (40:08):
Yeah, it’s really high resolution. One of the sort of limits to resolution for that, though, is the ability to distinguish each organelle, sort of, from its surroundings. So it has to have something to distinguish it. So things like ER, you really think you would be able to see pop from this technique, and we can’t really distinguish ER, even rough ER, out from the surrounding cytoplasm. So we do see a really good resolution of the things we can see well. So we do see around 10s of nanometers to maybe around 100 nanometers, depending on the sample prep. I did see in there, they asked about doing the fluorescence concurrently. So what we did was to flash-freeze the samples and then put them in the microscope. And it has a fluorescence detector, so we would do the fluorescence imaging and then swing those out of the way and then do the x-ray. So we didn’t manipulate the sample in any way in between the two, but they weren’t kind of acquired right after one another.
Mark Murcko (41:02):
Exactly. And then there’s a question about how to… I’m sure you can see it there… considering different ways of studying partitioning into preexisting condensates.
Emily Sontag (41:14):
Yeah, that is something that we hadn’t really done previously, but it’s something that obviously needs to be done. Particularly given the way that those Q-bodies look like they’re fusing together into the JUNQ definitely looks like something where it’s like the condensates sort of fusing together. But we haven’t done anything to show that Ubc9 or luciferase will actually go through this process. So that’s something we would have to start to work characterizing from the ground up to make sure that they can actually form condensates and they work the same way that many of the other proteins that we see forming condensates do.
Mark Murcko (41:43):
Yep. And then Charlotte has a really interesting question. I don’t know, Charlotte, if you’re still on, if you want to amplify this a little bit, but it’s a very interesting direction to take the conversation.
Charlotte Fare (41:53):
Hi. Can you hear me okay?
Emily Sontag (41:55):
Yeah, I can.
Charlotte Fare (41:56):
Okay, great. So I was wondering if you think that the INQ in JUNQ co-localization is being directly sort of positioned by the nuclear pore. Or do you think that there’s some sort of other either on the nuclear membrane itself or the connection with the ER some structure that’s changing in the presence of the nuclear pore that’s helping to position the two?
Emily Sontag (42:19):
Yeah, so that’s a fascinating question, and it’s really difficult to answer, but I feel like there’s some hints there because of that Chm7 pathway. So Chm7 itself has actually been shown to clear aberrant nuclear pores. So it kind of triggers this cascade to clear aberrant nuclear pores. So we know that Chm7 localizes nearby nuclear pores. We know it’s in the nuclear membrane. So that’s kind of one that we’re thinking about is kind of directing this homing signal. But what I’m not sure, then, is I mean, really trying to dig into Chm7 now and understand exactly how it works, is I don’t know that Chm7 itself crosses the nuclear membrane and would be able to actually transmit that signal. So I don’t know if it’s something where it’s all part of that ESCRT complex, and there are parts on either side of the nuclear envelope that can recruit those misfolded proteins there, but my guess is it’s going to be something with this ESCRT-mediated clearance of aberrant nuclear pores. So that’s why we see misfolded protein quality control compartments and nuclear pores kind of all in the same place there.
Mark Murcko (43:17):
Beautiful. So Kamran, you had a question about whether the components are only coming from the nucleus. You had another interesting question about a particular heat shock protein. Do you want to amplify that a little bit?
Kamran Rizzolo (43:29):
Yes, thank you. Emily, great talk. Really just looking at the model that you’re describing, which is super cool, are the components here only coming from the nucleus? Are they only proteins that are being misfolded that are being fed through the nuclear pores just going straight into these bodies, just thinking of your model that if you meet that requirement, that attachment? Or are there components coming from the cytoplasm as well?
Emily Sontag (44:02):
I think given that we see the Vps4 and the Chm7 also playing a role in–When we delete those components, we see an increase in cytoplasmic aggregation as well–I think there probably is a cytoplasmic component to that. So I just almost picture this entire area there where the INQ and the JUNQ have homed in, and that entire area sort of gets engulfed into the vacuole through that, and then potentially aberrant nuclear pores, everything just kind of gets sucked into this clearance. It’s almost like a bulk turnover of that entire area of the cytoplasm. But that’s something that we’re really going to have to work to try to tease out exactly where those components are coming from that are getting cleared.
Kamran Rizzolo (44:36):
Gotcha. And then is there any evidence that Hsp104 teases apart, takes some components out and gives them a chance to fold as well?
Emily Sontag (44:47):
Yeah, so possibly for the JUNQ, particularly because when we do the FRAP experiment, we do see some recovery there. So it’s possible that JUNQ, they are able to do that. We do see Hsp104 localizing to both the JUNQ and the IPOD. And we have a really cool project that we’re doing in collaboration with Wah Chiu’s lab at Stanford looking at Hsp104 by cryo-EM and seeing kind of how it’s involved and then going back and then doing some of the biochemical and biophysical analysis to see what role Hsp104 is playing in that. So I’m hoping within the next year or so we’ll have an answer to that question, but it does seem like preliminarily Hsp104 is definitely involved in this process.
Kamran Rizzolo (45:25):
Cool. Thank you.
Mark Murcko (45:27):
So, Rahul, you had a good question, if you want to amplify that a little bit.
Rahul Samant (45:33):
Hello. How’s it going?
Emily Sontag (45:35):
Rahul Samant (45:38):
Good to see that data again. I haven’t seen any of the ESCRT stuff. That’s super cool. So I had a question that I’m sure you’ve thought about a lot before, but I had a thought when I saw previously the INQ in your asymmetric Nup mutant that the INQ doesn’t actually home anymore. Did that have any functional consequence to the yeast? I know they were generally not very happy to start with, but did they not clear the INQ anymore? Were they less fit? And I guess do you think… When we’re talking about all of these disease-causing nuclear aggregates and they actually interfere with Nups, but is there any evidence that not homing to the Nups for intranuclear aggregates actually affects pathogenesis or anything like that?
Emily Sontag (46:29):
Yeah, that is something that I am absolutely working on at the moment. So we didn’t do any of sort of the followup with looking at clearance in that mutant strain with the clustered nuclear reporters. I didn’t look at clearance or toxicity or anything. So that’s something that I’m following up on now. I actually have an undergrad who’s going to do that project this summer, and then obviously, looking into tying that to disease state, because, like you mentioned, neurodegenerative diseases are really tied into these nuclear pores, and nuclear pores are seen in Huntington’s inclusions, for example. So there definitely seems to be a role for all of this process together, then, in moving forward into these neurodegenerative disease associated proteins. So it would be really cool, then, if having the INQ no longer homing to those nuclear pores in that deletion strain then would affect toxicity of those.
Rahul Samant (47:16):
It’s difficult. Keep going. It’s awesome.
Emily Sontag (47:18):
Yeah, thanks, Rahul.
Mark Murcko (47:21):
Great. Let’s go to Kriti.
Kriti Chaplot (47:21):
Hi. I wanted to ask whether you have looked at any RNA protein complexes which might be interacting with INQ or JUNQ. Are they any substrates that need to be cleared away? Because a lot of neurodegenerative diseases have RNA processing, which is impacted. So do they interact with any of these complexes?
Emily Sontag (47:52):
Yeah, at this point, no, we haven’t looked at that. That’s still a really interesting area to take this forward into. We haven’t looked at any of the sort of RNA-protein complexes or stress granules or anything to see how that plays a role into any of this process. So that’s absolutely something that would be cool to do.
Kriti Chaplot (48:08):
Yeah, thank you.
Emily Sontag (48:09):
Yeah, no problem. Great question.
Kriti Chaplot (48:11):
Mark Murcko (48:15):
So, Edgar, you had a question about super resolution.
Edgar Boczek (48:18):
Yeah, thanks, Mark. Hey, Emily. Great talk. Thank you. I was wondering if you are planning to use super resolution at the early stage of formation of these bodies. So maybe you are aware of this paper, which I find pretty cool, from Ibrahim Cisse, who looked at alpha-synuclein and basically found that it kind of forms liquid droplets before it starts to jellify. And yeah, I was just wondering if you want to look at [inaudible 00:48:48].
Emily Sontag (48:50):
Yeah, unfortunately, it’s probably not something I could do anymore. I don’t have access to the super-res scope now that I’m at Marquette. But I do know there are some people who may be interested in following up on this in Judith’s lab, and we have talked about timing, because I only really did the terminal time point for that super-resolution microscopy because we were really interested in sort of where it was going towards the end. So that’s what I focused on, but absolutely looking at the timing of these things in more detail would be a fascinating way to do that. I think I would probably do it first through doing the structured elimination microscopy to get an idea before we would go and do something like the soft x-ray tomography, just because those soft x-ray tomography experiments were loads of work. I think it took us over a year to collect and analyze that data set. So obviously we’re getting better at it and getting faster, but I think we would probably start with doing the SIM data just because I was able to do that in a couple of days.
Mark Murcko (49:39):
That’s great. So we’ve got one additional question from Kamran about quiescence. Another interesting topic.
Kamran Rizzolo (49:47):
Yeah, thanks, Mark. So, Emily, I used to work with proteasome storage granules, and these are just kind of the exports of the proteasome components into the cytoplasm, but it happens in quiescence. And they form these condensate-looking structures. So obviously, just thinking, is there any known interaction between these bodies and these proteasome storage granules? Because we don’t really know the function of the PS genes, right?
Emily Sontag (50:15):
Yeah, that’s something actually that Rahul and I discussed over many, many months and many journal clubs and discussing your papers and everything. It was something we would really like to look into, but yeah, at this point we haven’t done it. And I haven’t even looked at quiescence in yeast. It’s something we talked about moving forward for doing some of these aging projects because in yeast we can do both chronological and replicative aging studies and start to look at quiescence and start to see what happens to these different protein quality control compartments during aging, during quiescence and start to understand what happens then to them. Do they still get cleared? Do they move? Do they interact with other things? I think that would be a really cool thing to do.
Kamran Rizzolo (50:53):
Emily Sontag (50:54):
Mark Murcko (50:57):
Well, thank you, Emily. I think that’s all the questions that we had coming in. I’m sure there’ll be more later. People always follow up later with all the speakers. So don’t be surprised if you get questions from people over the next few days.
Emily Sontag (51:07):
Sure. Wonderful. I look forward to it.
Mark Murcko (51:10):
Wonderful talk. Really appreciate it.
Emily Sontag (51:16):
Thank you so much, again, for the opportunity. Thanks, everyone.
Mark Murcko (51:17):
Sure. And thanks, all the participants, for all the questions. Thank you all.