|Type||Kitchen Table Talk|
Amy Gladfelter joined Dewpoint scientists (virtually) on May 5, giving a beautiful talk as part of our Kitchen Table Talk series. Amy is a professor in the Biology Department at UNC, Chapel Hill, with an interest in how cells are organized in time and space. Amy studies both fungal and mammalian systems using quantitative live cell microscopy and a variety of computational, genetic, and biochemical approaches.
Amy’s work has been trail-blazing about the essential role that RNA plays in liquid-liquid phase separation. In her talk she highlights many fascinating aspects of the function of RNA in the architecture of biomolecular condensates. Enjoy!
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Mark Murcko (00:00):
We can go ahead and get started. So, Amy Gladfelter, great to have you giving us a seminar on your recent work. Obviously, you’ve been in the condensate game as long as just about anybody and have done some pioneering work and wonderful to have a chance to hear from you.
Amy Gladfelter (00:26):
Great. Thanks Mark. It’s really fantastic to be here. I’m just going to get my-
Amy Gladfelter (00:33):
screen set up.
Amy Gladfelter (00:38):
Okay. So today I’m really going to be focusing on an area of condensate biology that takes the perspective of RNA, which is a major feature of many condensates, and one of the main things we are thinking about in the condensate world. I often get asked how did I start thinking about condensates? What got me into this field and this business? And it really came out of a longstanding curiosity and interest in a very special kind of cell, which is a syncytial cell. I’ll be talking about a couple of different types of syncytia today…
Amy Gladfelter (01:15):
First, I’m going to talk about fungal syncytia, which… you can see an example of one here growing under the microscope. This is the form that many fungi take, both in the forest when they’re growing under a mushroom like this, but also as pathogens in hosts. What’s remarkable is these cells can achieve incredible size by virtue of having many copies of the genome within a common cytoplasm. But fungi are not the only syncytia. I’ll be also talking about muscle later in the talk today. But in fact, syncytia are actually found in many different contexts.
Amy Gladfelter (01:54):
We see syncytia occurring in a variety of different infections. So here’s an example of a plasmodium infection where there’s an obligate syncytial stage in the liver in the host. And in fact, many viruses also form syncytia as part of the progression through an infection, including SARS-CoV-2, is known to be able to form syncytia and uses this to potentially transmit between cells. A number of tumors also become syncytial. Here’s an example, just a diagram of a glioblastoma tumor, where becoming syncytial is associated with tumors really becoming more, stress-resistant and also resistant to different therapeutics.
Amy Gladfelter (02:39):
The idea here is essentially just like that fungus in the in the forest where a very large cell can make long range communication over large areas and integrate information over a lot of different areas. The same might be the case for tumor cells that achieve large sizes and can cover a lot more territory when they become syncytial. Then finally, I won’t talk much about this today but we have work in the lab looking at the syncytial trophoblast of the placenta, and that’s this dark blue cell in this diagram here. This is an amazing cell in that it’s performing functions as diverse as different tissues can even perform but it’s actually happening within a common cytoplasm.
Amy Gladfelter (03:21):
It’s really this general feature of syncytia that’s fascinated me for many, many years and drove me into this field. I was really led to this field by trying to understand how these very special large cells that contain many nuclei can perform such distinct functions at once. So in the case of the placenta, the syncytial trophoblast cell, you’re looking at endocrine function, excretion function, diverse metabolic functions, immunity all within a single cell. It was really in trying to understand how you could get compartmentalized function within a continuous cytosol that’s what’s led me to here today.
Amy Gladfelter (04:03):
And so, what we found in my group is that it is through the formation of condensed droplets, particularly of RNA and protein, that you can create functional neighborhoods or territories within a syncytia, that allows for this regionalized activity. And so what we think is special about condensates in the context of these large cells is it allows these cells to exist in these two states. One is this highly functionalized state where there’s discrete areas that are doing different things. But because of the nature of condensates, because they’re dynamic, they can assemble, they can disassemble, it allows these cells to also exist as super cells where they’re very large and actually maybe everyone is acting in unison in those cases. And that this ability to transform between these more unique cellular and more multicellular identities might be a real functional advantage of being in a syncytium and also exploiting condensates.
Amy Gladfelter (05:15):
How do condensates form? What I think is one of the most interesting features of this field is thinking about how these nanometer-scale interactions between the constituents that condense. RNA binding proteins is what I’ll be focusing on today, but obviously proteins that in general have some sort of disordered or low complexity sequences, but many of them also have the capacity to interact with nucleic acids such as RNA. RNA, generally, will have domains within it or features within it that allow it to interact with RNA-binding proteins.
Amy Gladfelter (05:53):
And so our current understanding of this is that it’s essentially a series of weak multivalent interactions that can drive these molecules together at the nanometer scale. But the many, many of these molecules interacting together of course, will nucleate and ultimately drive the demixing process leading to the formation of a condensate. Obviously, what we detect are these mesoscale droplets. What’s really interesting to me is thinking about how the strength and the spacing and the information in these nanometer-scale interactions ultimately inform the mesoscale physical properties and ultimately function of the condensates.
Amy Gladfelter (06:42):
And so it really is the placement and the strength of the interactions at the small scale that are going to determine features such as viscosity, surface tension, the ability to fuse, the size of the droplets, sphericity of the droplets, all of these things that we can actually measure and that we think are probably important for function. What I’ll really want to be focusing on today is thinking about the RNA component of this. And so there’s been a tremendous amount of interest and focus on the protein component. But what about the RNA?
Amy Gladfelter (07:15):
So, RNA is… everyone knows, is a very common constituent of many condensates in the cytoplasm and also in the nucleus. And so what I think makes it especially powerful for condensate formation is the variety of different features that it has. One feature that I think has been most thought about the most in the field is that it is a uniform negative charge. And so obviously this charge is going to be the amount of charges dependent on the length, but I think this is really only the beginning. And so there’s a tremendous amount of more information within an RNA molecule other than just charge. So, multivalency is a feature of many specific RNA-protein interactions where a given RNA will actually have multiple in general binding sites for an RNA-binding protein. And these are generally low affinity interactions.
Amy Gladfelter (08:07):
And so having multiple ones helps facilitate that interaction with the protein. We know that RNAs can be highly variable in size, just to the different sizes they’re encoded but also through alternative splicing, which I’ll be talking about in the later part of my talk. But one thing I think that’s potentially not widely appreciated is how large RNA is. RNA molecules can be on the order of hundred times as large as a protein. This is really important for thinking about their potential roles as scaffolds and also thinking about their mobility and the idea that they may be actually quite long lived constituents of condensates.
Amy Gladfelter (08:52):
So, we’ve thought a lot about how the structure of RNA can influence condensate specificity. I’ll talk about that published work in a few minutes. I’m just showing you here some pieces of RNAs that we’ve studied. What’s remarkable is that RNA can take on sequence-encoded structures like this. There’s a whole ensemble of different sorts of structures that any given sequence, can take on. And then these secondary structures obviously give rise to tertiary and then ultimately even quaternary structures that are sequence encoded. Then finally there are specific mechanical properties. There’s specific flexibilities depending on the degree of disorder within an RNA sequence and order within an RNA sequence. So properties that we think about often for say features of the cytoskeleton, such as persistence length or the stiffness of the polymer, I think can ultimately become really relevant for thinking about what RNA may be contributing to condensate properties.
Amy Gladfelter (09:59):
So, I’ll talk today really about some published work that we’ve done on this fundamental question of what is the basis of molecular identity and how these features of RNA are critical, we think, for specifying the composition of a condensate. We spend a lot of time talking about or thinking about cell biological context of condensates. How are they scaled? How has their position controlled? Won’t be talking about that so much today. Instead, I want to spend a fair amount of time dwelling on the question of how RNA may contribute to the material state of condensates. But to begin with, let’s think about the molecular identity. I’ll very quickly introduce you to some of the molecules that we think about in a model fungal system. So this is a filamentous fungus that is a syncytium.
Amy Gladfelter (10:54):
The problems that I was fascinated with in the system are shown here in this video where one feature is the nuclei divide asynchronously even though they’re all bathed in the common cytoplasm. This suggested to me that there was some compartmentalization or organization of the cytosol. This was when I was a postdoc, and so it was many years actually before anyone had thought about a condensate, but I was imagining a general insulator that kept cell cycle components from freely diffusing. The other feature in the cell that’s so interesting is the way it grows. It grows by this break, branching morphogenesis. So all growth is directed at tips. This is a feature, it’s very similar to how neurons develop and neurons grow. I think a lot of the problems that syncytia face by being very large and having to compartmentalize their cytosol are also faced in large neuronal cells.
Amy Gladfelter (11:53):
So for each of these branches to come out, basically the cytosol has to be regionalized or specified in some way, again suggesting there might be organization in the cytosol to break symmetry for growth. The molecules that we now know that are important for both the cell cycle and also cell polarity is first a protein called Whi3. This is a poly-glutamine tract containing RNA-binding protein that condenses into liquid-like condensates at sites of polarized growth and in the periphery of nuclei, which are black holes in this movie here.
Amy Gladfelter (12:35):
So the poly-glutamine tract at the protein is essential for these droplets to form. Here are just some stills of the condensates. When we delete the poly-glutamine tract, you no longer see anywhere near the level of condensate formation. This has major consequences on these two features of the cell. One is when we eliminate either the protein or the poly-glutamine tract, the nuclei no longer divide asynchronously. Instead, you see these tracts of synchronous division because the cytoplasm we think has become more homogeneous in this area. So the cell cycle is being shared amongst nuclei. Then when we look at cell polarity, we can see the normal cells put out many, many different side branches to create these beautiful mycelial shapes. Whereas, mutants lacking the ability to form condensates make these very straight hyphae with very few new sites of cell polarity.
Amy Gladfelter (13:30):
So we’ve spent a lot of time trying to understand the mechanisms of phase separation for this protein. I think one of the most powerful and interesting features of the Whi3 protein is that it really will not condense with any particular, any old RNA. It has real specificity for particular RNAs that are important in nuclear division and cell polarity. The protein on its own does not undergo a demixing under physiological salt and physiological protein concentrations but it robustly demixes and the presence of cyclin RNA or polarity RNAs. This has really allowed us to ask some very fundamental questions about how liquid droplets could have distinct molecular identities. Because you should imagine this is the case for all condensates. They should just fuse and mix together into one gigantic droplet. And that doesn’t happen. We know that there’s a myriad of different condensates of many compositions in the cell.
Amy Gladfelter (14:25):
And so in this system, we have one protein, the Whi3 protein, that can localize different RNAs. I’ll show you that here in the next image. In fact, it localizes, it’s important for localizing. These are actually the RNAs that it’s regulating. It’s important for localizing polarity RNAs and RNAs important for the cell cycle. And it’s forming distinct assemblies with these different RNA. So it’s same protein but different RNA components. And so we are very interested to understand how a single protein could be in these functionally distinct assemblies. And so in published work, we were able to show that in fact, we can reconstitute this identity, even in vitro, with very minimal components. So if we mixed polarity RNA and cyclin RNA with the Whi3 protein, we could see that in fact they form distinct condensates, even with no other information in the cell.
Amy Gladfelter (15:21):
And so this told us that the RNA was really important for this. So, we did a simple experiment early on in this study where we melted the cyclin RNA. So we didn’t change the sequence at all. We simply melted it, which would eliminate all of the secondary structure in the RNA. What we could see is this actually allowed these two RNAs to now co-condense and mix together. And so we went on and did a lot of structural studies to look at sequences in the RNA that we thought might be somehow imparting specificity. Ultimately after doing a lot of structural analysis, we essentially had a hypothesis that RNAs are able to sort, depending on their ability to undergo intermolecular interactions. And so that specific sequences within cyclin and cell cycle RNAs or polarity RNAs, were allowing those RNAs to undergo a complimentary-based pairing and ultimately specify their location within either the same droplet or keep them actually in separate droplets.
Amy Gladfelter (16:31):
We’ve done a number of experiments to test this hypothesis, both in vitro and also in cells. But here’s just an example of one of the approaches using the structural studies that we had, we knew what sequences of the cyclin RNA might be able to interact with the formin RNA if it was available. But the idea is, in a normal cell, those RNA sequences are actually tied up in secondary higher order structures. And so we did the following experiment where we melted the RNA. I already showed you this, where they mixed together. But now when we add oligo nucleotides to these sequences where they might be able to interact, we can actually block that interaction and restore the segregation, suggesting that there really are RNA sequences that can determine whether RNAs are together or apart.
Amy Gladfelter (17:20):
What we’ve thought… what we’re thinking now is this is a really important feature of sorting into droplets is that the secondary structure is essentially licensing, which RNAs might be able to interact with one another and which RNAs are not able, don’t have an affinity for one another, and they end up segregated into distinct structures. This work I think has implications and lots of applications within the field, because I think there’s more and more cases where we’re seeing RNA-RNA based interactions being relevant for granule formation. So we know from beautiful work from Ruth Lehmann’s lab, that in fact there appears to be RNA high degree of organization of specific RNAs within these germ granules. This has been postulated to occur through RNA-RNA interactions that sort the RNs within the condensate and Ankur Jain has shown beautifully that RNA assemblies, exclusive RNA assemblies as can occur from triplet containing RNAs. And Roy Parker’s lab has shown important roles for nonspecific RNA self-assembly in seeding stress granules. We think this is probably the tip of the iceberg in terms of thinking about how RNA-RNA interactions are relevant.
Amy Gladfelter (18:42):
So there’s a tremendous number of questions I think on RNA based specificity in the condensate world. We don’t know where in the cell RNA-RNA interactions are initiated. Is this happening in the nucleus after transcription? Is it happening in the cytoplasm? Essentially how might that be regulated? Are these RNA complexes essentially nucleating condensates, are they very important seeding events for assemblies? Is that a general principle? What is the degree to which specific RNA-RNA interactions are being actively remodeled by helicases in a controlled manner? Finally, does the composition, do different RNAs actually lead to distinct physical properties of droplets? So is the RNA sequence also imparting a physical identity to the droplet? And so this is really the question I’m going to talk about now for a few minutes. So it’s well appreciated that condensate physical properties can vary widely and so these are all examples of RNA-rich granules that vary in terms of their viscosity over orders of magnitude.
Amy Gladfelter (19:53):
And in our own work, we’ve shown that the physical properties of droplets can vary depending on the RNA sequence in a simple reconstitution system, the protein and RNA system I already described. And so here are measurements we did using microrheology where we’re just tracking the diffusion or the mobility I should say, of microspheres within condensates to estimate the material state of them. And what we could see is that RNAs encoding polarity components are much less viscous than cell cycle encoded components and droplets. One thing that’s really interesting about this is you might think, “Oh, maybe that’s just a length dependence.” What’s really interesting is that the polarity RNA is actually much longer than the cell cycle RNA and yet it makes actually a less viscous condensate. And so we think it’s something much more interesting and complicated than just simply a length dependence of the condensate properties.
Amy Gladfelter (20:57):
And so we’re spending a lot of time thinking right now of RNA as a structural scaffold for droplet assembly. We think that this is a very attractive scaffold for a number of reasons, some of which I’ll be able to share with you today. I’m just reminding you here of the great size disparity between proteins and RNAs within the droplets. The ability of RNA is to really form large system-spanning networks through their ability to have these stable intermolecular base-pairing interactions. And so how can we imagine RNA as a scaffold? I think you can imagine two general classes potentially of RNA scaffolds.
Amy Gladfelter (21:40):
So, one would involve nonspecific RNA interactions. And this might be akin to what the Parker lab is postulating for stress granules where essentially you might have heterogeneous assemblies of many different RNAs, different compositions. Because there’s not necessarily sequence encoded specific interactions, these may be more dynamic or more labile partly because there’s probably a tremendous amount of helicase activity that may inhibit these under normal circumstances, these nonspecific interactions. However, it seems quite clear that there’s evidence that these sorts of interactions can be induced by stress when translation is paused and RNA sequence becomes available to become tangled.
Amy Gladfelter (22:27):
Now what I’m thinking much more about are sequence encoded RNA interactions. And so what’s really special about these interactions and making a scaffold is that you can actually create a mesh of a particular length scale, because there’s actually sequence encoded interactions that could in a sense specify the exact properties. I think this could impart elastic properties. This can also of course be tunable and regulable by helicases. But I think where we’re seeing examples of this that tends to be in developmental regulation cases where there’s a high degree of organization and regulation that is required by the condensate.
Amy Gladfelter (23:10):
So here’s some example of work we’re doing to look at the importance of RNA-RNA interactions in the material properties and scaffolding functions. So, based on some structural work and predictions that we’ve done we find that there’s a large area of predicted interaction just between two molecules of the cyclin Cln3, this is one of the cell cycle components I was talking about earlier. So there’s a large degree of potential homologous interaction here. We’ve generated mutants in this that substantially disrupt the pairing and we think substantially change the higher order structure of even just this pairwise interaction. What’s really remarkable is theses sequences are largely similar. There’s just a very small handful of substitutions. But when we look at condensates made from either wild type or these pairing mutants, we see a dramatic effect on droplet size, the assembly rates, they ripen much slower, we see very different fusion rates and really different rates of hardening. This is based on relatively small changes in the sequence that are having big effects on the overall droplet properties.
Amy Gladfelter (24:40):
So, that’s sort of an example of thinking about RNA-RNA interactions. I think another key piece of RNA acting as a scaffold and potentially being set up as a scaffold are protein-RNA interactions. So this is where we start thinking about the valence of the RNA and the number of binding sites that it has for a protein within a condensate. And here too, we’ve seen that small differences have a big effect. So, if we look at an RNA that has four potential binding sites for the Whi3 protein, we get condensates forming. If we add a single other binding site so that we have now five binding sites, we see a dramatic change in the size of the droplets that form. And I should say, this is the native number of sequences for the structure.
Amy Gladfelter (25:31):
So taking one away… and I should say this is also relevant, we think that it’s positioned at the distinct location from these other sites. And so it’s not just having a certain number of binding sites but we think it’s also the position of these binding sites that’s really relevant for again, determining the material state of the condensate. So we have a large battery of wild fungi that we study that have varying different sequences. And we’ve collected these fungi from many different climates. This is part of a study that we haven’t published yet but we’re really excited about because we think we’re able to look at essentially adaptive sequence changes that are really relevant for allowing the fungus to adapt to growth at different temperatures. One of the things that’s so interesting is across our collection of wild isolates that we’ve collected from a lot of different climates, what we’re seeing is that the targets of Whi3 actually increase in their valence as the temperature, the ambient or average temperature in a climate, drops. So if we see sequences that are from a more Northern climates, they tend to have many more, Whi3 binding sites.
Amy Gladfelter (26:49):
So the valency is increasing as a function of temperature. So we think the valence and the RNA is actually probably a really critical additional component here. One of the things that’s interesting that we’re trying to do now is start trying to look at the intersection between RNA-RNA interactions and valence. And so, one of the interesting features, these wild isolates is this warm weather isolate. These are cyclin sequences from a isolate from Florida. It has this ability to undergo pairing here and we think this is actually helping it to form larger and larger condensates at higher temperatures. Whereas, if we take an isolate from Wisconsin, in fact these RNAs appear to be disrupted in this pairing area. There’s quite a bit of variation within the sequence in this area. This is actually changing the droplets ability to form at higher temperatures.
Amy Gladfelter (27:46):
And so what we’re imagining is that maybe there’s increased valency in these colder climates that’s compensating for the lack of intermolecular pairing that you see in the warmer climates and that’s missing in these colder climates. And so the point I want you to take from this is that I think there is a tremendous capacity for variation within the RNA sequence, in that there’s essentially a balance between valency and RNA-RNA interactions that’s able to give you essentially different strengths of interaction that maybe tunable in terms of both the material properties and also the environmental responsiveness of the condensates.
Amy Gladfelter (28:30):
And so a lot of our work is really trying to imagine and come up with ways to actually measure the long range RNA interactions that we think are important for giving RNA this potential scaffolding property for droplet assembly. What I’d really like to propose is there’s been a lot of emphasis in thinking about proteins as clients and scaffolds and condensates. But I think we really need to think about RNA similarly in these classes, and that RNA may be playing a critical structural scaffolding role and there may be other RNAs that are clients within condensates.
Amy Gladfelter (29:11):
In the last few minutes here, I’m just going to talk a little bit about… I think a really important feature of RNA that’s also probably been a little bit underappreciated and the condensate world. That is the potential of alternative splicing that occurs throughout higher eukaryotes that can alter essentially all the features of RNA. It can influence the ultimate charge in being driven through a complex coacervation by affecting the length, the number of binding sites depending on which exon are included. Obviously, it can affect the size and the structure. All of these properties that are sequence encoded are going to be influenced by alternative splicing. I think everybody appreciates this but there’s a tremendous potential for variation here within a single gene to essentially create both high degrees of variation at the RNA sequence level but also influence ultimately the encoded protein, which is what so often thought about. But I think it’s really important to think about both of these features in the context of condensate biology because it’s appreciated that many RNA-binding proteins bind to their own RNA.
Amy Gladfelter (30:29):
So alternative splicing can have potentially very potent effects on ultimate condensate properties by influencing both RNA species and the protein species that might be present. I’m not talking here about the condensates involved in splicing, that’s a problem for another day and probably another speaker. But instead what I’m talking about here is the effects of alternative splicing. And so what’s got us thinking about alternative splicing as an important problem is our broader interest, again, in syncntial cells. And so as I’ve already talked a lot about fungal cells, skeletal muscle cells are another really important multinucleated cell where it’s known that nuclei can essentially be autonomous or independent from one another. And particularly nuclei that are in the vicinity of neuromuscular junctions express different RNAs. And in a process of a myotube formation, that there’s tremendous amount of nuclear to nuclear heterogeneity.
Amy Gladfelter (31:40):
And so this smells a lot like the nuclear asynchrony that we saw in fungi. And so this got us starting to think about myotube formation and whether there might be spatially distinct cytosolic territories in these multinucleated cells. What drew our attention to splicing was this protein called FXR1 or fragile X related protein one. FXR1 is a large gene and it’s highly expressed in muscle and in myoblasts and also in myotubes. So the way myotubes form is through myoblast differentiating, changing shape and then fusing to create multinucleated myotubes, which will then ultimately go on and give rise to muscle tissue. It’s important to note that this happens in development but it’s also happening in repair and is really relevant for muscle regeneration as well. FXR1 undergoes a major shift in its isoforms that are present between the myoblast state and the myotube state.
Amy Gladfelter (33:00):
And so specifically myoblasts are characterized by these shorter isoforms, the A and B isoforms. Whereas, during the process of differentiation, we see an inclusion of these two additional exons, 15 and 16 in the myotube form. So what’s important about 15 and 16 which hopefully everybody can appreciate, is that FXR1 contains a very large intrinsically disordered domain at the C-terminus. It has an RGG motif that we think is relevant for RNA binding as well as two additional RNA binding motifs KH1 and KH2. The inclusion of 15 and 16 alters the sequence and the length of the C-terminal disordered sequence. So because of this developmentally controlled splicing that changes the disordered sequence, we were very interested in whether this could be a protein that’s important for patterning and setting up territories of cytoplasm within the myotube.
Amy Gladfelter (34:04):
While we were studying this, we were really emboldened and excited to hear about and a genetic study on FXR1, identifying recessive mutations in a muscle-specific isoform of FXR1. Essentially, a variety of different mutations that either frameshift or eliminate splicing of 15 and inclusion of 15 and 16. So essentially leads to a truncated or altered form of the protein, so that you don’t get that extended differentiated form of FXR1. This is a recessive disease. What was really interesting in this literature and these are in mature muscle cells. You can actually see organized assemblies of FXR1 in the skeletal muscle in patient mutations. You interestingly see a disorganization of this. You also actually see things that sure scream to be condensates, the larger droplets that form. You see a much more heterogeneous size distribution compared to the wild type, or in some cases a diminishment of condensates.
Amy Gladfelter (35:17):
So, importantly here we see splicing associated with the disruption or the alteration of an IDR. So this is work that we recently published and is in the JCB, if you want to read more about it. But essentially what we were able to show is that in myoblasts, here’s just a scheme looking at this disorder tendency so that you can appreciate the myoblast form has a much shorter intrinsically disordered sequence compared to the myotube isoform here, which has this extended disordered sequence. This is an endogenous protein, how it’s localizing within cultured C2C12 cells. You can see they’re syncytial. These are very early on in their differentiation, so they have a mixture of these isoforms. But you can start to see that in fact there are condensates that form and we’re able to show that they can fuse and they appear to be dynamic. This is just a pelleting assay showing essentially that before differentiation the protein is mostly soluble. But as the cells differentiate, you can see more and more of it assembling in the pellet, consistent with some sort of higher-order assembly and maybe a more solid- or gel-like core.
Amy Gladfelter (36:37):
We did a number of different experiments with this protein. This is just an example of trying to understand essentially how FXR1 behaves. And so we made a number of different mutants in RNA binding domains. And what was very clear to us is that the FXR1 protein requires RNA binding in order to undergo phase separation. Here’s a mutant that is lacking both the RGG and the two KH domains. And you can see that the protein is no longer visible in puncta of any sort and appears to be in this soluble state. But one of the things that was most surprising to us from this is when we looked at this last variant here which is a shortened form of it. And it’s essentially very close to the A isoform, so the myoblast form of it. So it’s missing a large chunk of the IDD. And so recall that… in fact, we hypothesized that maybe the IDR was getting longer as a function of splicing in order to promote phase separation.
Amy Gladfelter (37:43):
But it turns out in shortening this, we actually got even larger condensates and more condensates at lower concentrations of the protein, suggesting that it’s really not such a simple story as just a longer IDR is more and better for phase separation. But this is a much more complex relationship between the sequence and the condensate state. And so we’re still really trying to understand this at this point but we know that FXR1 combined to its own transcript and that this facilitates phase separation. How we’re imagining this right now is that alternative splicing of FXR1 is likely relevant, both at the level of the protein and the level of the RNA. And so here in myoblasts we have a smaller IDR, a smaller FXR1 RNA and this is actually associated with more liquid-like assemblies we think early on in development.
Amy Gladfelter (38:50):
But now as we differentiate more into myotube, the IDR is expanded. This also actually increases phosphorylation sites in the IDR. And I didn’t have a chance to talk about that. We have a little bit of this in the paper. This also expands the length of FXR1 RNA. And that these two features are actually associated with more gel-like or solid-like assemblies. The longer IDR and RNA are actually associated with less dynamic assemblies of FXR1.
Amy Gladfelter (39:23):
We don’t fully understand the implication of this. We think that this is regulating the translation of FXR1. But that’s really future work to be done. But I think the lesson in this is simple predictions just based on sequence, are really very difficult to necessarily truly predict well. I’ll just leave you with this key, big… I don’t know that it’s a big idea but this key idea, that RNA we think is a really critical structural component for droplet assembly and that the sequence of RNA has really important properties both to specify which RNAs end up in a condensate. But ultimately the network of RNA that can form that is important for these larger mesoscale physical properties that I think we all envision playing an important functional role in condensate biology.
Amy Gladfelter (40:27):
I just want to thank my lab for making this… just a phenomenal job. And being so excited to come to work when we’re allowed to come to work. And this work was primarily done by a current postdoc in the lab Ben Stormo and Christine Roden. The lab is made up of people who are really span the spectrum of biologists to biophysicists to actually theoreticians. It’s been a really privilege to train all of these young scientists. I also want to thank my great collaborators and my funding sources and thank you for giving me a chance to share these ideas.
Thanks. Thanks, Amy. It’s great. Wonderful stuff. I’m sure we have a lot of questions but I’ll ask one while people are gathering. First of all, it’s great for somebody like me as an old protein guy to hear a talk like yours, which is just trying to get us all to think about the role of RNA differently. It’s interesting when you rattle off all that wonderful slide you have, you showed a couple of times all the different factors that matter, charge multivalency, the size of the RNA.
Amy Gladfelter (41:48):
The structure, right. The dynamic nature of it and all of that. And so, each of those is in itself complicated to study.
Amy Gladfelter (41:59):
And so it’s interesting to think about, in any given situation, which of those factors will be the most relevant? Or is it always… probably is, probably some mix of all of them.
Amy Gladfelter (42:14):
Yeah, I mean, in our hands, we often see mixtures, right? But you’re right, it’s hard to disentangle all these. I think this is true, honestly for protein biology also-
Amy Gladfelter (42:27):
… in the condensate world, right? If you shorten it, you’re eliminating sequence but you’re also potentially changing other physiochemical properties. Right? For sure, small sequence changes can change the structure. They potentially are changing the valence. We’ve seen major, major, major effects on length. Really, I think length is a really important feature that is critical. I think a lot of times what one is going to need to do for studying RNA is to move between simplified models of the RNAs of interest, right? So sort of toy molecules. I think they’re dangerous in some ways because they’re simple and they’re not necessarily have the complexity, but so that you can try to isolate one or two of these features at a time, and then move it back into the more complicated molecule that you can actually experimentally probe with a little bit more knowledge and simplicity.
Yeah. Exactly. Ann, did you have a question?
Ann Kwong (43:41):
Yes. Amy, I know your talk was about RNA, but I’m curious about something you mentioned in the beginning in your[inaudible 00:43:50]
Amy Gladfelter (43:54):
Oh, I just lost you.
I’m sorry. Can you hear me?
Amy Gladfelter (43:58):
I just… I can hear you now. Yes.
Okay. Sorry. I’m a virologist by training. And you mentioned viruses that cost syncytia, right? Like in herpes simplex virus, there’s these mutants that cause syncytia and there are glycoproteins. It really seems to be driven by… there’s several different glycoproteins that can cause syncytia. Are there any rules or basic principles that can be generalized from these single glycoproteins and viral systems that seem to cause syncytia and allow the fusion of these large syncytia fusion across the entire cell, just in itself.
Amy Gladfelter (44:45):
Yeah, I mean[crosstalk 00:44:48]
… amazing organisms that you’re describing that…
Amy Gladfelter (44:53):
I think there’s something… I needed a lot of ways to think about the problem you are addressing. I think one is the mechanism of fusion and how you become a syncytium. And whether those glycoproteins, there could actually be some phase separation-mediated processes that are helping promote fusion actually, right? I think that’s possible. Although, I don’t know of anyone who’s shown that at this point.
Yeah. I haven’t seen that.
Amy Gladfelter (45:17):
But we do have a project in the lab actually looking at how membranes may facilitate protein-driven based separation, where there maybe not be as much RNA present in those membrane fusion events. And so these could be protein driven. And I think glycosylation is certainly on the table as a very important post-translational modification that I think will be really relevant for phase separated condensates. But I don’t think anybody’s shown that for cell fusion at this point. But it wouldn’t surprise me. Then the second part is once you’re a syncytium, and there’s this massive state of what is that bringing these cells functionally. For a virus, it really may be a means to very stealthily expand, right?
But you’re not exposed to the external environment.
Amy Gladfelter (46:09):
You’re not exposed, right? But I think there could also be other advantages to this in terms of just how viral replication is controlled spatially. It’s something that we’ve really not studied ourselves at this point. But I’ve been thinking more and more about it in light of SARS-CoV-2 actually.
Yeah. It’s interesting because some viruses can often do both. They can do both. They can spread by fusion and in some situations…and then can spread by lytic infection. And there’s obviously advantages to go both, one way or the other.
Amy Gladfelter (46:49):
One way or the other. Yeah, absolutely. Absolutely. Yeah.
Okay. Thanks. That’s interesting.
Amy Gladfelter (46:56):
Diana Mitrea (46:59):
That was a great talk, Amy. I really enjoyed it.
Amy Gladfelter (47:01):
Amy Gladfelter (47:03):
Hi. So, Amy, you talked about helicases and how by changing the structure they modulate the valency and so on. I was wondering if you can comment a little bit about post-transcriptional modifications and how those contribute to real time.
Amy Gladfelter (47:25):
Yeah. I think this is something I should have put on that slide, that I neglected to put on my… all the wonderful things of RNA. It should be there, right? So post-transcriptional modifications are for sure probably a major modulator that… The reason it wasn’t on my slide is because it’s basically m6A is one of the main ones…This has been linked to condensate biology, right? I think it’s just because, it’s a bit of a new world of mapping all the post-transcriptional modifications that are on RNAs. It’s just a few steps behind thinking about this in terms of protein modifications. But I think we should envision post-transcriptional modifications exactly the way we’re seeing effects on post-translational modifications on proteins, right?
Amy Gladfelter (48:26):
This is a great way to modify a shape, charge, conformations, binding proteins. It’s essentially critical. It was an absence on my slide, of all the wonderful things. I didn’t have room for all the amazing things that RNA can contribute to condensates. That’s again very early days, but I mean, I think m6A is the poster child for now, where that’s actually probably a modification happening within a phase separated assembly. But I think that is very, very early days. And partly it’s hard. It’s hard to make in vitro modified RNAs where you have site-specific modification for example, right? Like these are really hard experiments right now. To nail really what a specific modification may do at a specific residue, right.
Amy Gladfelter (49:25):
But it’s something we’re just beginning. We’re actually starting to try to map post-transcriptional modifications that are on the model fungal RNAs that we’re studying. Because we think in a fungal system this’ll be simpler. There’ll certainly be less, different kinds of modifications so that the code should be a little less complex. I’m hoping in the future that we have more insight into that, but that’s a hard problem.
I think you probably said it and I may have missed it too. Do the cyclin and polarity RNAs ever co-localize in vivo?
Amy Gladfelter (50:00):
Yeah. We haven’t found them but we think they must actually, because there is a capacity for them to mix. And so we haven’t exhaustively looked under different environmental conditions, but my hunch is that there probably is some either developmental context or stress-induced context where it would make sense for them to actually co-assemble. There actually maybe a context of that. And whether that promotes their translation or maybe inhibits their translation, I’m not sure, of course at this point. But I do think there’s a reason there’s some capacity there, because there are fairly reasonable tracts of homology that can enable them to interact, if they’re available. So potentially a stress-induced helicase or something could potentially expose that and allow them to then co-condense and coordinate maybe better nuclear division with growth.
One of the… go ahead please.
Avinash Patel (51:05):
Please go ahead Avi.
Amy Gladfelter (51:06):
Hi, Amy. Very nice talk. Really exciting. The effects are one story, right? So I just was wondering about the things that you presented towards the end of the story, right? That the changing material properties associated with the development of myoblast to myotubes. So have you also looked at this phenomenon in dynamic living, myotubes and… if I remember the cartoon that you showed, it seemed like these condensates assemble around the nucleus. Could that be a way, a mechanism that they make sure that there is an isolation or segregation in the cytoplasm, and the property actually does help them in the isolated or how they move around[crosstalk 00:51:56]
Amy Gladfelter (51:57):
Yeah. We really haven’t pushed the imaging to these later stages of development mostly because it’s really hard to image them at that point for us, because we’re not muscle specialists. We’re not using primary cells and they start to peel off and they become really difficult to work with. Unfortunately, we didn’t really in the end nail that model that this matters for nuclear functionalization. So, that remains a possibility. At this point I don’t… and sadly I don’t have evidence for that yet.
Other questions? Yeah. One thing about helicases that’s interesting is that unfortunately there are not a lot of good tool compounds yet. That that makes it a little harder to try to tease apart the role that they play. Of course, there are other… you could use CRISPR and other knock-out, knock-down methods instead. But from a pharmacology standpoint, there’s not too much available yet.
Amy Gladfelter (53:16):
Why do you think that is Mark? Is it because just people haven’t looked or is it because there’s something about their structure[crosstalk 00:53:26]
Yeah. They’re difficult targets. People have tried actually quite a bit. There’s a lot of examples in the literature and they often end up being quite challenging to make good, small, simple, selective drug-like molecules. I think there’ll be… I think there’s going to be a new wave of people trying to go after helicases again. I’m seeing more evidence of that in the literature. So hopefully in this next round of research efforts, we’ll have some better tools.
Amy Gladfelter (53:58):
Yeah. It’s clearly a very important area.
Amy Gladfelter (54:03):
That’s good. Any other questions? If not, Amy, thanks again, wonderful lecture as always. Thanks for sharing your thoughts.
Amy Gladfelter (54:13):
My pleasure. Thanks for listening.
It’s great stuff.
Thank you so much. That was great.
Thank you so much.
Amy Gladfelter (54:23):
Take care. See you. Bye. Bye.