Senior Scientist, Dewpoint Therapeutics
|Type||Kitchen Table Talk|
The Dewpoint scientists and Condensates.com community welcomed Lindsay Case for a Kitchen Table Talk on March 2. Lindsay joined the department of Biology at MIT in January of 2021. During her PhD at UNC Chapel Hill with Clare Waterman, Lindsay studied how force is transmitted to the extra cellular matrix through integrin focal adhesions. She extended this work to phase separation in membrane signaling during her postdoc at UT Southwestern with Mike Rosen, one of the founders of the condensate field.
Lindsay’s seminal contributions to the condensate field includes one of my favorite papers about phase separation, the 2019 Science paper showing that phase separation can lead to clustering of membrane receptors and is directly linked to function. Her paper was published in tandem with another Science paper from Jay Grove’s lab at Berkeley, and I had the privilege of coauthoring the preview for them. Lindsay recently published work in eLife that builds on these models and brought her back to integrin focal adhesions.
She shares some of this work in her talk, along with some stimulating preliminary data from her new lab. As you’ll notice by all the great questions after the talk, we all thoroughly enjoyed hearing about Lindsay’s work. And she kindly provided written answers to the ones she didn’t have time to answer during the show–you can find those here. I hope you enjoy her talk in the video below as much as we did.
Create an Account or Sign In to view the video.
Erik Martin (00:00):
It’s my pleasure to introduce Lindsay Case today to the Kitchen Table talk. I met Lindsay, I think, in 2016 at a Gordon Conference in the Swiss Alps along with, I think, several other people here, including Jill and Bede and several others. It was a, I guess, wonderful environment to do science and it’s been a great experience following Lindsay’s work since.
Erik Martin (00:26):
Lindsay did her PhD at UNC Chapel Hill with Clare Waterman, studying dynamics of actin and integrin focal adhesions, which has, as it turns out, been a wonderful background into the phase separation field as it’s developed over the last 10 years. So I guess that was a very fortuitous move to do her postdoc at UT Southwestern with Mike Rosen, who was one of the founders of the field with his 2012 Nature paper on multivalent assemblies.
Erik Martin (01:00):
Lindsay has had some seminal additions to this particular aspect of the field, I guess, starting with a 2019 Science paper that was published in tandem with a paper from Jay Grove’s lab at Berkeley, that I had the pleasure of writing the preview for. These were some of my favorite papers in the phase separation field. It was basically showing both how phase separation could lead to the assembly of all of these membrane receptors, and how this is directly linked to function.
Erik Martin (01:33):
She had a very recent eLife paper where it’s extended this type of modeling back to her work in her PhD working on integrin and adhesion complexes. She has also had the distinct pleasure of being one of the people to try and start a lab at the start of 2021, and is now an assistant professor at MIT. So I’m greatly looking forward to what she has to say and hope everyone else does as well.
Lindsay Case (02:00):
Well, thank you, Erik, for that introduction and also for the opportunity to speak here today. I’m really excited to share some of my more recent work with the condensate community.
Lindsay Case (02:13):
So today I’ll be sharing my work investigating membrane associated condensates and their role in regulating signal transduction. I’m sure this audience doesn’t need too much of an introduction into phase separation and condensates, but suffice it to say that over the last decade or so, we’ve really come to appreciate that there are many biological molecules capable of undergoing phase separation at physiological conditions…
Lindsay Case (02:40):
So phase separation occurs when it becomes energetically favorable for a solution to demix, forming two discrete phases, a condensed phase and a dilute phase. If both of these phases have liquid-like material properties, we can refer to this as liquid-liquid phase separation. And we find in biology that many different types of molecular interactions can promote this type of phase separation, including protein-protein interactions, protein-RNA interactions, and RNA-RNA interactions.
Lindsay Case (03:07):
While the molecules that can undergo phase separation are often chemically quite different, they often share certain molecular features, including multivalent binding domains, or weak multivalent binding motifs connected by flexible linkers. This multivalency can help to promote phase separation through the formation of higher-order oligomers.
Lindsay Case (03:29):
So the prevalence of phase separation has led to this new hypothesis that phase separation is a really important organizing principle in cells. Shown here is, a very commonly used cartoon to illustrate all of these membraneless compartments, which we now refer to as biomolecular condensates that can be found throughout the cell in both the nucleus and in the cytoplasm.
Lindsay Case (03:52):
But in my research during my postdoc with Mike Rosen at UT Southwestern, I was really interested in exploring condensates that actually can associate with or form on the plasma membrane. Me and others have observed that there are many different receptors that can be organized through a very similar phase separation mechanism.
Lindsay Case (04:13):
Also, although my research is really focused on these condensates that associate with the plasma membrane, I don’t think the plasma membrane is really unique in its ability to regulate or associate with condensates. We’re seeing more and more examples of lots of intracellular condensates, including protein-RNA condensates that can associate with a variety of intracellular membranes, including the ER, the golgi vesicles, autophagosomes, endosomes, and lysosomes.
Lindsay Case (04:41):
So I hope that many of the general principles that we are beginning to understand through studying condensates at the plasma membrane may also have important implications for intracellular membranes and intracellular condensates as well. So many condensates can associate with membranes, and in some cases it seems like the membranes may even help regulate the condensates as well.
Lindsay Case (05:08):
We have many examples of phase separation organizing receptors at the plasma membrane to form these membrane-associated condensates and so there’s growing number of these examples. We see the cell-cell adhesion receptor nephrin can cluster through phase separation driven by multivalent interactions, protein interactions between the receptor and cytosolic adapter proteins. Similarly, the Lat receptor in T cells, the Wnt receptor, claudins at tight junctions, receptor tyrosine kinases, NMDA receptors, and postsynaptic densities, and integrin receptors, which is what I’ll be telling you about today.
Lindsay Case (05:44):
So in all of these cases, interactions between the receptor and cytosolic adapter proteins can drive phase separation and promote clustering of receptors on the plasma membrane. So what I have been really fascinated with trying to understand is, how does phase separation regulate downstream signaling in these pathways? And if so, how does phase separation actually impact the regulation of signaling?
Lindsay Case (06:11):
I think that signal transduction is a system in which phase separation has the potential to be quite important for regulation, because we know that many receptors require higher-order assembly–supramolecular complex assembly–for robust downstream signaling.
Lindsay Case (06:26):
So for example, in the case of receptor tyrosine kinases, ligand binding induces dimerization, and the dimer is the active form of the receptor. But for many receptor tyrosine kinases, even the formation of dimers or small oligomers is still not sufficient to activate robust downstream signaling. Rather, higher-order assembly, where tens or hundreds of receptors cluster together and recruit a specific subset of cytosolic adapter proteins and signaling molecules, is required. So the formation of these signaling clusters that can be hundreds of nanometers or microns in diameter, is required for downstream signaling.
Lindsay Case (07:02):
And we’ve known this for a really long time, but what we’ve begun to appreciate is that phase separation is a physical mechanism that can regulate this type of rapid, switch-like conversion from the dispersed receptor to a clustered receptor. And if phase separation regulates this transition to higher-order assembly, then it could potentially be really important for controlling downstream signaling.
Lindsay Case (07:24):
So the first study that we investigated this concept in was, studying the cell-cell adhesion receptor, nephrin. I’m just going to very briefly talk about this, but this really illustrates one of the approaches that we take to really study these types of membrane-associated condensates, and that is to try to biochemically reconstitute condensates on supported lipid bilayers.
Lindsay Case (07:48):
One thing I hope you can take away from what I’m sharing today is, how these types of somewhat simple biochemical reconstitutions can still be really valuable for beginning to understand these complex physiological condensates. So for reconstituting condensates on membranes, we can make supported lipid bilayers, attach the intracellular domain of the receptor to the bilayer and fluorescently label molecules to visualize them with TIRF microscopy. What we observe is, that when we combine nephrin with the adapter protein, Nck, and the actin regulatory protein, N-WASP, the multivalent interactions between these three proteins drive phase separation to form these micron-sized clusters on the bilayer.
Lindsay Case (08:32):
A former graduate student in the lab had done extensive experiments to show that these clusters are indeed forming through phase separation, but what I was really interested in understanding is, if these clusters can regulate downstream signaling. So N-WASP promotes Arp2/3-dependent actin polymerization, and we were able to reconstitute actin polymerization in this pathway. And what we observed is that we only see actin filaments nucleating from within the phase separated clusters. We never see filaments nucleating in these unclustered regions, despite the fact that there are actin filaments. Or, there are N-WASP molecules throughout the membrane, but only the N-WASP molecules in clusters nucleate filaments.
Lindsay Case (09:09):
Through our in vitro reconstitution, we were able to find that it was actually the dwell time of these molecules on the membrane that was important for regulating actin polymerization. So N-WASP and Arp2/3 stay associated with the membrane much longer inside of condensates. And this increased dwell time increases the probability of leading to an actual actin nucleation event.
Lindsay Case (09:33):
This was exciting because it suggests that, at least under some circumstances, phase separation can regulate or enhance downstream signaling in the case of nephrin and Arp2/3-dependent actin polymerization. But today, what I want to tell you about is more recent work that I’ve done investigating the role of phase separation in the assembly and regulation of integrin receptors.
Lindsay Case (09:54):
So integrins are adhesion receptors for the extracellular matrix and integrin-dependent signaling regulates many cellular processes. So integrins are really essential for mammalian cell biology and allow the cell to gather information about the environment. So integrin-dependent signaling regulates cell cycle progression, differentiation of stem cells, survival, and apoptosis, and cell movement, and cell migration.
Lindsay Case (10:22):
Integrins are heterodimers and they’re receptors for the extracellular matrix. So they bind to ligands like fibronectin and collagen, that is secreted in the extracellular environment. They have a very large extracellular domain that binds the ligand and very small cytosolic domains. The beta integrin cytosolic domain in particular, binds to two critical adapter proteins, cytosolic adapter proteins, talin and kindlin.
Lindsay Case (10:47):
Integrins regulate these various aspects of cell behavior through many different mechanisms. They directly regulate signaling molecules like kinases and Rho GTPases. They also physically link the actin cytoskeleton to the extracellular matrix and because of this, they’re important sites of force transduction. So the mechanical forces generated in the actin cytoskeleton are transmitted across integrin receptors, allowing the cell to exert force on its substrate.
Lindsay Case (11:16):
However, despite all of these critical functions, integrins themselves have no intrinsic catalytic activity. So they are actually a really classic example of a receptor that requires higher-order assembly for all downstream signaling. We know that integrins require this supramolecular assembly, where they recruit hundreds of cytosolic adapter proteins to form these signaling clusters that I will refer to us focal adhesions.
Lindsay Case (11:41):
So this is a cell with a focal adhesion protein, paxillin, labeled with GFP, and you can see that focal adhesions really rapidly form as these very small discrete spots at the protruding edge of the cell. The majority of these newly-formed focal adhesions turnover, but a subset undergo stabilization and undergo a process of compositional maturation and growth. So a mature focal adhesion can be quite stable and very large, several microns in size and can exist for several hours although the molecules within the adhesion are very dynamic and rapidly exchange with the cytoplasmic pool.
Lindsay Case (12:19):
We know that there are hundreds of different proteins that localize within focal adhesions, a handful I’ve illustrated in this cartoon. So we have the extracellular matrix, integrins, and then all of these cytosolic adapter proteins connecting to the actin cytoskeleton. Their composition varies a lot. So the composition of these complexes changes in response to different stimuli and they’re stoichiometrically undefined.
Lindsay Case (12:42):
Focal adhesions mediate integrin-dependent signaling through both chemical signal transduction and mechanical signal transduction. An example of chemical signal transduction is the phosphorylation that occurs, so it’s kinases like FAK and Src kinase localize within focal adhesions and phosphorylate their substrates within focal adhesions.
Lindsay Case (13:00):
But focal adhesions also regulate signaling through mechanical signal transduction. So these mechanical forces that are being propagated through this complex actually change the composition of the adhesion and can alter the conformation of many of the molecules within the adhesion. So this is a technique called traction force microscopy, which allows us to visualize the magnitude of force that a cell is exerting on its substrate. And you can see that focal adhesions are sites of high force and the forces that an adhesion experiences is dynamic and fluctuates. We know that these dynamic fluctuating forces can dramatically alter signaling such that focal adhesion signaling can completely change the fate of cells simply by changing the mechanical properties that a cell senses.
Lindsay Case (13:49):
So although focal adhesions are really critical for both this chemical and mechanical signal transduction, the mechanisms that regulate integrin clustering and initial supramolecular assembly are really poorly understood. So when I joined Mike’s lab as a postdoc, we thought that focal adhesions exhibited many characteristics that were familiar to us as being common in condensate, in these phase-separated condensates. They were these discreet micron-scale compartments, enriched with multivalent adapter proteins. They exhibit a wide range of molecular stoichiometries, and there are lots of examples that they exhibit liquid-like material properties. So there’s examples of focal adhesions fusing, and the molecules within them again, are very dynamic.
Lindsay Case (14:28):
So we decided to take a bottom-up approach to try to understand if phase separation could be playing a role in focal adhesion assembly. So the first question we asked was, were any focal adhesion proteins capable of phase separating in vitro? We purified candidate proteins and screened them. And we did find two sets of molecular interactions at focal adhesions that were sufficient to promote phase separation in vitro.
Lindsay Case (14:50):
The first set of molecules that underwent liquid-liquid phase separation were the adapter protein p130Cas, Nck and the actin regulatory protein, N-WASP. So Nck and N-WASP localize both at cell-cell adhesions, like nephrin and at integrin adhesions. We find that it’s the multivalent attractions between these proteins that promotes their phase separation. So when we combine these proteins in vitro, we observe liquid-like droplets forming.
Lindsay Case (15:18):
Similarly, we found that focal adhesion kinase was capable of forming droplets in vitro. Actually, of all the proteins we screened, FAK was the only protein capable of phase separating on its own. This is due primarily to interactions between the structured domains of FAK, which I’ll talk more about in a few slides, but we also found that paxillin was able to significantly enhance this phase separation and this was due to multivalent interactions between the paxillin LD motifs and the FAK C-terminus.
Lindsay Case (15:48):
So we have these two sets of interactions that are each sufficient to promote phase separation in vitro. However, p130Cas also directly interacts with both paxillin and FAK. And, none of these proteins that we observed can promote phase separation, directly bind to integrin receptors. Rather, we found that there was an additional adapter protein, kindlin that was required to couple the phase separation to the receptor. So we found that kindlin could bind directly to both p130Cas and paxillin and this was critical for targeting integrin to condensates.
Lindsay Case (16:22):
So we have this fairly complex network of interaction between these seven components and we really need a way to think about or understand the composition of condensates that have this more complex composition and complicated network of potential interactions. So one very simple framework we can use to describe condensate composition is this scaffold and client framework. It’s definitely oversimplification, but it can be really helpful for just thinking about condensate composition.
Lindsay Case (16:54):
So in this framework, we divide molecules or categorize molecules as either scaffolds or clients. Scaffolds are molecules that are required for condensate formation. So these are the molecules that drive phase separation. They will have molecular features that are common, that are important for phase separations, such as multivalency or intrinsically disordered regions, and they often tend to be very highly concentrated within the condensate.
Lindsay Case (17:21):
In contrast, clients are not required for phase separation. They don’t have features that drive phase separation, but rather they will partition into the condensed phase through molecular interactions, often by interacting directly with the scaffold. And they will often be less concentrated in the condensate.
Lindsay Case (17:38):
So with this framework, we can categorize our proteins. So we actually have five scaffolds, p130Cas, Nck and N-WASP are scaffolds for the Cas-dependent phase separation, FAK and paxillin are scaffolds for the FAK-dependent phase separation and kindlin and integrin in this interaction network are clients. They don’t directly undergo phase separation, but they can partition into the condensate.
Lindsay Case (18:00):
So what happens if we actually combine all of these proteins together? When we combine all seven of these proteins, we actually observe a single class of droplets that forms and all of these proteins uniformly co-localize in droplets. So we don’t observe like FAK droplets and Cas droplets, but rather all of these molecules perfectly colocalize in this single class of droplet. Furthermore, we can actually quantify the partition coefficient of all of these components into droplets and begin to better understand the relationship between all of these molecules.
Lindsay Case (18:33):
So what we observe is, that when all seven proteins are present, the partition coefficient of all components significantly increases. So if we compare droplets or condensates formed simply with the Cas-dependent phase separation, or just with the FAK-dependent separation with droplets with all seven components, we see that the partitioning significantly increases when both branches, both of these pathways are present. So we think that FAK- and p130Cas-dependent phase separation–these pathways phase separate synergistically because of these interactions between these critical scaffolds.
Lindsay Case (19:10):
We also observed that our five scaffold proteins have a much higher partition coefficient than the two client proteins, kindlin and integrin, which we think is to be expected. So just to illustrate this again, when we just combine p130Cas, Nck, N-WASP, kindlin and integrin to look at Cas-dependent condensates, we form condensates that very weekly partition integrin. If we just combine molecules to form FAK-dependent condensates, we form condensates that weakly partition integrin, but when all seven components are combined, we form condensates that more strongly partition integrin.
Lindsay Case (19:47):
So next, we are really interested in doing some more subtle perturbations to the phase separation in this system to test what happens if we only perturb either the Cas-dependent phase separation, or the FAK-dependent phase separation with more nuanced point mutations. Rather than completely removing some of the molecules, can we mutate these molecules to prevent their phase separation or reduce their phase separation?
Lindsay Case (20:09):
So, one way we can do this is by preventing the phosphorylation p130Cas. So in order for these three proteins to undergo phase separation, the SH2 domain of Nck has to interact with the phosphotyrosines on p130Cas. So if we combine these three proteins with phosphorylated Cas, we see–and this is measuring solution turbidity that we do lots of experiments to confirm with–that droplets form when Cas is phosphorylated, but not when we add in unphosphorylated Cas, wild type Cas, or unphosphorylated Cas where all 15 tyrosines have been mutated to phenylalanine.
Lindsay Case (20:46):
So what we wanted to know is, what happens if now we introduce this Cas protein that can’t promote phase separation into our seven component system? So in this system, we’ve essentially turned down the Cas-dependent phase separation, such that Cas, Nck N-WASP are now really behaving more like clients than like scaffolds. What we find is that all components still partition into droplets when we do this, but we see that when Cas is not phosphorylated, the phase separation is significantly less. So we see less droplets, the droplets are smaller, and if we measure the partition coefficient of paxillin, this is significantly decreased when Cas is not driving phase separation.
Lindsay Case (21:25):
The other reason we did this experiment is because this is an experiment we can mimic in cells. So this is a way we can compare our really simple reconstitution to the cellular focal adhesions. When we introduce this, we take Cas-null fibroblasts, and we reintroduce either wild type Cas, which can be phosphorylated or this mutant that cannot be phosphorylated, we see that this mutant that cannot be phosphorylated, which has turned down the Cas phase separation, has less focal adhesions. And paxillin partitioning in those adhesions and cells is decreased, similarly to what we’ve measured in vitro.
Lindsay Case (22:00):
So we can take the same approach and try to now perturb the FAK phase separation. Can we turn down FAK phase separation in our seven component system? We have found that FAK phase separation is driven by oligomerization. And it requires two sets of intermolecular interactions of FAK, the FERM domain of FAK dimerises, and then the C-terminus of FAK interacts with the N-terminus, and both of those interactions seem to be required for droplet formation in vitro.
Lindsay Case (22:27):
We can introduce a point mutation that disrupts the FERM dimerization interface, and this completely prevents phase separation in vitro. So while type FAK has high solution turbidity and forms droplets, whereas this mutant FAK does not undergo phase separation.
Lindsay Case (22:43):
So when we introduce this mutant FAK into our seven component system, we’ve turned down the FAK phase separation such that FAK and paxillin are now more like clients than like scaffolds. What we observe is that similar to what we observe with the Cas perturbation, that phase separation is reduced and paxillin partitioning is reduced. And when we mimic this perturbation in cells, we take FAK-null cells and re-express wild type FAK, or the mutant FAK, we see when we’ve turned down FAK phase separation in cells, there’s less focal adhesions and the paxillin partitioning in those adhesions is reduced.
Lindsay Case (23:17):
So we think that even though we have a very simple seven-component system it’s still complex enough to recapitulate a lot of the effects that these mutations have on phase separation and the effects these mutations have on composition in vitro. So we’re seeing very similar trends in our in vitro reconstitution to what we’ve observed in cells.
Lindsay Case (23:41):
However, there’s a really important component missing from this reconstitution and that is the membrane. I’ve mentioned integrins are receptors that are found on the plasma membrane, focal adhesions are condensates that associate with the membrane. So next, we really wanted to try to understand what the membrane is doing in this system.
Lindsay Case (23:59):
First, we just turned to a supported lipid bilayer reconstitution, where we attach the integrin receptor to bilayers and we fluorescent label integrins, so integrin on its own is uniformly distributed on the bilayer. What we find is if we add this FAK-dependent set of molecules to drive FAK-dependent phase separation or Cas-dependent phase separation, this is sufficient to start to induce some clustering on membranes. But when we combine all seven components, we see significant increase in the amount of clustering observed.
Lindsay Case (24:30):
So this is quantified here. So actually the synergistic effect of these two pathways is much more dramatic on the supported lipid bilayer, than in solution. We see that when we have both the Cas- and the FAK-dependent pathways, we see a significant increase in the amount of clusters that we’ve quantified on bilayers.
Lindsay Case (24:49):
So we were confident that this phase separation that we observe is sufficient to induce integrin clustering in vitro. But when we looked at these integrin clusters in the early days, we were very dissatisfied because integrins were not very dense inside of these clusters. So if we compare these integrin clusters to some of the earlier membrane-associated condensates we had reconstituted in the Rosen lab, Lat condensates, and nephrin condensates, integrins are not really very bright. These are not dramatic, bright clusters. They’re very weak clusters. So integrin’s only about twofold more concentrated in the cluster than in the unclustered regions.
Lindsay Case (25:33):
So for a while, we thought maybe we’re missing some critical component that if we just add some extra component, integrin clusters will become more like these Lat and nephrin clusters. But eventually we actually realized that… We were able to compare the density of integrin in our reconstituted clusters to the density of integrins that had been measured in focal adhesions in cells, and we were surprised that actually we were recapitulating the cellular density of integrins in clusters. So we actually think that this is the physiological density of integrins.
Lindsay Case (26:02):
And upon further reflection, we actually think that this makes a lot of sense because integrin, as I’ve mentioned is not a scaffold for phase separation, it’s a client. Whereas both Lat and nephrin are really scaffolds for phase separation. Lat and nephrin are both multivalent proteins that interact with multivalent cytosolic adapter proteins to drive phase separation. So, I think it’s actually to be expected that a client receptor is not going to be as concentrated as a scaffold receptor.
Lindsay Case (26:30):
But this led us to another, I think, important question that we were grappling with, which, if the receptor is a client, do you even need the membrane at all? Is the membrane important for phase separation when the receptor is a client? I’ve just shown you that we can form droplets in solution with these proteins in the absence of the membrane. So what is the role of the membrane in this type of system?
Lindsay Case (26:51):
So to look at this, we did the following experiment: We took empty bilayers that just had lipids and no protein, no integrins, and compared them to bilayers where we’ve attached integrin to the bilayer. To these bilayers, we add our cytosolic adapter proteins, kindlin, paxillin, FAK, Cas, Nck and N-WASP, but this time we’re going to fluorescently label one of the cytosolic proteins and then visualize these bilayers with TIRF microscopy over time.
Lindsay Case (27:18):
So what we observe is that if we have an empty bilayer, we do see some droplets forming in solution and settling on the bilayer over time, but there’s significantly more condensates or droplets visible on the bilayers that have integrin receptors. If we look over time, we see that early on, there’s a significant difference between the integrin bilayers and the empty bilayers, but over this 10 minute experiment, that difference decreases. So over time, there are lots of droplets nucleating, we think in solution and settling on the membrane.
Lindsay Case (27:50):
However, this concentration of proteins that we’ve been using up to this point is pretty high. It’s not really the physiological concentrations of these proteins. So we decided to repeat this experiment with the cellular concentrations of these proteins. When we do this, we see a much starker contrast where on empty bilayers, we see really no condensates forming, whereas the integrin-containing bilayers do have condensates. If we look over the course of a 10-minute experiment, we see that even at 10 minutes, there’s this really stark difference between the empty bilayers and the bilayers containing the receptor.
Lindsay Case (28:27):
So we think that client receptors can still promote condensate nucleation on membranes, likely by increasing the local concentrations of these cytosolic scaffolds. So even though the receptor is not driving phase separation, it is still binding to these cytosolic proteins that are driving phase separation and that local recruitment, local concentration, of these cytosolic proteins onto the membrane, we think can possibly promote condensate nucleation.
Lindsay Case (28:53):
I think this is really interesting, and this is something I want to continue to better understand, because we know there are lots of other factors that can locally concentrate molecules on membranes, including post-translational modifications–like prenylation, direct interactions between proteins and lipids–like phosphoinositides and pH domains, and proteins that recognize membrane curvature–like bar domains. So it’s possible that all of these factors that might locally concentrate a cytosolic protein could potentially influence condensate nucleation if those proteins are capable of undergoing phase separation.
Lindsay Case (29:32):
Finally, I think this has important implications for disease. So with these proteins, the normal cytosolic concentration of these proteins requires membrane for nucleation, but we’ve found that increasing the protein concentration can decouple the phase separation from the membrane. And we know that both FAK and p130Cas are really commonly over-expressed in cancer and their over-expression is correlated with poor prognosis. So it’s intriguing to think that perhaps that is related to their ability to phase separate.
Lindsay Case (30:01):
So finally, our in vitro work led us to a new hypothesis that Cas and FAK should synergistically promote focal adhesion formation and we wanted to test that in cells. So to do that, we took wild type fibroblasts or Cas-null fibroblasts and used siRNA to knock down FAK. So this allows us to generate cell lines that either express both proteins, only express FAK, only express Cas, or don’t express either. We can plate cells on fibronectin, which is the ligand for integrin, allow focal adhesions to form and quantify the number of focal adhesions formed over time.
Lindsay Case (30:38):
When we do this experiment, we do see that there is this synergistic relationship between p130Cas and FAK, such that cells expressing both Cas and FAK form normal amounts of focal adhesions. Knocking out one of the proteins reduces the number of focal adhesions and knocking out both of the proteins significantly reduces the number of focal adhesions compared to just losing one of the proteins alone. If we look at a 20-minute timepoint, I think the phenotype becomes even more dramatic, where if you don’t have p130Cas or FAK, we really see very few focal adhesions forming over time.
Lindsay Case (31:11):
So I think that phase separation can provide an intracellular trigger for integrin clustering and focal adhesion formation. I think focal adhesions are a condensate that bridges the integrin receptors and the actin cytoskeleton, and these two adapter proteins, p130 and Cas, promote focal adhesion formation through their shared ability to undergo phase separation. So even though these proteins don’t have any structural features in common, they have this shared ability to phase separate and that is why they have this redundant function in focal adhesion assembly.
Lindsay Case (31:45):
So moving forward, in my lab at MIT, one of the strategies we’re taking is to continue to use focal adhesions as a model system for understanding these types of membrane-associated condensates. And focal adhesions have several features that make them, I think, really interesting model systems for understanding a lot of general questions about condensate biology.
Lindsay Case (32:06):
First of all, they have a really well understood biology, including a lot of proteomics information. So many groups have done proteomic studies cataloging how the composition of focal adhesions changes under different circumstances. They’re important sites of enzymatic activities, so kinases and Rho GTPases are specifically modifying their substrates within focal adhesions. I think there’s a system in which the viscoelastic material property of the condensate is likely to be really physiologically important because of these mechanical forces that have to propagate through the material.
Lindsay Case (32:39):
They are also a system that has interesting higher-order self organization. So mature focal adhesions in cells are actually not isotropic, but exhibit a layered organization. So it’s intriguing, to understand if phase separation potentially plays a role. So we’ve seen in three dimensional systems how these multiphase condensates can form, but how that might happen on a membrane in a more two dimensional context hasn’t really been explored. And membranes have an inherent polarity, right? You have the membrane on one side and the cytosol on the other. So I think understanding any potential role for phase separation in driving or organizing these molecules is interesting.
Lindsay Case (33:17):
Focal adhesions are also a system in which there are a lot of known important protein-lipid interactions. So in the seven proteins that I’ve studied so far, three of them directly interact with phosphoinositides, PIP2 particularly. So understanding how direct interactions between these proteins and the membrane might also influence phase separation is something I’m interested in.
Lindsay Case (33:37):
Finally, in addition to these interesting mechanistic features, also integrin-dependent signaling is a really important therapeutic target for disease. And integrins are misregulated in many diseases, including inflammation, arthritis, heart disease, cancer. Lots of different bad things can happen when integrins don’t function properly.
Lindsay Case (33:58):
So to conclude for the last two minutes of my talk, I want to give you a really quick sneak peek on some of the early work we’re doing in my lab. This is investigating this question of enzymatic activity and how it might be regulated within a condensate. So we’ve identified that FAK, this kinase, focal adhesion kinase can form droplets. It can undergo phase separation. So we really want to understand what the relationship between its phase separation and its kinase activity is.
Lindsay Case (34:25):
To address this, we’re first really addressing this very simple question or relatively simple question of FAK autosphosphorylation. So like most kinases, FAK autophosphorylation is the first step in activation of all FAK-dependent signaling. FAK autophosphorylates on this tyrosine 397, which is in this linker outside of the kinase domain. And we are really interested in understanding the relationship between phase separation and autophosphorylation.
Lindsay Case (34:53):
So Nick Lea is my first grad student to join the lab and he’s been tackling this problem. To do this, the first thing we did was just use our knowledge of FAK phase separation to take a one micromolar concentration of FAK and have it either not phase separate or phase separate to different degrees.
Lindsay Case (35:11):
So we know that FAK phase separation requires both the interaction of the FERM domain dimerization and an interaction between the N-terminus and the C-terminus, which is electrostatic in nature. So if we mutate the dimer interface, we prevent phase separation. Likely we can still perhaps have this electrostatic interaction, but there’s not the dimerization and therefore we don’t observe droplets at this concentration.
Lindsay Case (35:34):
Similarly, if we increase the salt in the solution, we also no longer see droplets. We do know that the dimer can still form, but the higher-order oligomer likely is disrupted. And we know that if we add paxillin to FAK, this enhances its phase separation due to the multivalent interactions between paxillin and FAK.
Lindsay Case (35:56):
So what Nick has done first of all, was just to do a simple Western Blot assay to quantify autophosphorylation over time. So we can take fully unphosphorylated FAK, add ATP and quantify autophosphorylation over a 10 minute period.
Lindsay Case (36:12):
And what he found is that when we don’t observe phase separation, there’s really a very moderate, slow rate of autophosphorylation over time. However, under conditions where droplets form, we see a significant increase in the rate of autophosporylation. And if we further enhance phase separation by adding paxillin, we see a further increase in the rate of autophosphorylation.
Lindsay Case (36:32):
So we’re really excited by this preliminary data. We’re currently trying to use the system. We have a single protein that undergoes phase separation and is both an enzyme and a substrate. So it’s a simple one-component system with which we can try to understand mechanistically, how localization within a condensate might actually enhance phosphorylation.
Lindsay Case (36:54):
So to conclude, I’ve show you today that condensates can organize and control diverse signaling molecules at the plasma membrane. We’ve seen that phase separation can promote actin nucleation downstream of the nephrin receptor. We also have very preliminary data that perhaps phase separation can regulate kinase activity within droplets. And Jay Grove’s lab has shown that phase separation can promote GEF activity to regulate Ras, to enhance Ras activation.
Lindsay Case (37:28):
So I think that one thing that I hope you take away from this talk is that phase separation can drive receptor clustering through many different types of interactions. Although some of the early examples of both Lat and nephrin phase separation were quite similar, those receptors all had these multivalent phosphotyrosine domains that could bind to multivalent SH2, SH3 domain adapter proteins. Those aren’t the only types of interactions that drive phase separation, and those aren’t the only types of interactions that can regulate receptor clustering at the membrane.
Lindsay Case (37:56):
We also find that receptors that are clients can still promote condensate nucleation on membranes and likely membranes perhaps can regulate condensate nucleation more broadly by locally concentrating cytosolic molecules on membranes.
Lindsay Case (38:14):
And I hope you can see how in vitro reconstitution of physiological condensates can be a useful tool for understanding these types of multicomponent condensates that might have pretty complex interaction networks. We can still start to tease apart how different molecules and different types of interactions regulate condensate formation and function.
Lindsay Case (38:35):
One final thought I want to leave you with is, that there’s likely to be a really complicated interplay between condensates and other cellular structures. We know condensates can be found throughout the cell, but there are lots of other structures and surfaces that can be found throughout the cell, including membranes, many numerous intercellular membranes, and the cytoskeleton as well. And there are examples of condensates associating with both the cytoskeleton and membranes.
Lindsay Case (38:59):
And I think a really exciting challenge in cell biology is really trying to understand how all of these things regulate each other. So it’s likely to be pretty complicated. Condensates might nucleate on membranes, but the condensates might also regulate the membranes and vice versa. So I think this is a really important, complicated problem to understand for the field moving forward.
Lindsay Case (39:23):
And with that, I will conclude. I’d like to thank my lab members. So all of the people who have joined my lab as brave pioneers, joining a new lab during a pandemic, particularly Nick, whose work, I briefly mentioned, the Rosen lab and Mike for being a fantastic postdoc environment and a fantastic postdoc mentor, and Steve Hanks and Larisa for sharing reagents that led to some of the cell experiments. And with that, I would love to take questions.
Erik Martin (39:55):
Awesome. Thank you for that great talk, Lindsay. I think your final slide is very appropriate because we have a couple very interesting questions in the chat specifically related to the interplay between these condensate and the membrane. So I think first up, I’d like to ask Simon to ask his question.
Simon Dujardin (40:16):
Yeah. It was a very, very interesting and terrific talk. So yeah, I guess I wonder how the composition of the lipid bilayer would influence the phase separation, especially like the concentration and local concentration of cholesterol or things like that. And did you guys try to replicate some pseudo lipid raft and see if that would increase or decrease the formation of phase separation?
Lindsay Case (40:56):
Yeah, so we have not done that, and that is definitely a question I’m actively interested in pursuing. So our bilayers, the lipid composition is very artificial. It’s just phosphatidyl choline and nickel lipids to attach the proteins. I know there are groups also working on this as well, specifically the question of lipid rafts. So I think, yes, likely this is likely to be really important, but we directly have not done these experiments yet. But that’s something I’m really interested in understanding more quantitatively. And I think the bilayer system is a great system to really specifically control lipid composition in order to understand that.
Simon Dujardin (41:37):
And just by curiosity, how do you do the membranes? Are these things like GUVs or things like that?
Lindsay Case (41:44):
No. So they’re flat. I mean, yeah. So there’s pros and cons. They’re flat, so they’re supported. So there’s a glass cover slip and you make vesicles and the vesicles fuse and form a bilayer on the glass. So there is this sort of artificial, solid glass surface immediately adjacent to the membrane that could potentially change perhaps some of the fluidity of the membrane and it also makes the membrane not possible to deform. It’s very flat, which is not very physiological. So there’s other GUV systems and other membrane systems for different techniques you can use to ask different questions in vitro I think, yeah.
Simon Dujardin (42:36):
I guess I have a third question. Can I go? Sorry. So what would you expect if you put like a protein that has nothing to do with it, some other protein that phase separates, let’s say FUS or something like that. Would it also go inside your droplets or not?
Lindsay Case (42:39):
Inside our droplets if it’s on… I don’t think so. I didn’t experience-
Simon Dujardin (42:44):
Would it phase separate basically? Would any protein that has phase …
Lindsay Case (42:48):
Go into those droplets.
Simon Dujardin (42:48):
… separation properties would separate with your scaffold basically?
Lindsay Case (42:55):
Yeah, I don’t think so, but I haven’t actually done that. I did an experiment where… So FAK actually has an IDR and we did a bunch of experiments where we were trying to like swap the FAK IDR for different IDRs to see if the IDR was important and that didn’t ever change anything. So we did swap in the FUS IDR for the FAK IDR and that didn’t influence its phase separation at all. I think these molecules all specifically interact with each other so I suspect that’s why they’re all colocalizing, but yeah, we could try that. And then yeah, or even if you had like two different receptors, both phase separating on the same bilayer like would they colocalize or not? I suspect it requires specific interactions, but we haven’t ruled out the possibility that other things could non-specifically interact.
Simon Dujardin (43:45):
Okay. Cool. Thank you so much.
Erik Martin (43:46):
Great. I think we have one more question focusing on the membrane side of things. I like to ask Karthik to unmute and ask his question.
Karthik Balakrishnan (43:57):
Hi, great talk. Thank you for it. I had a question regarding the phospholipids in the membrane itself, like for example, PIP2 or something like that, because usually neurons receptors, at least in synaptic vesicles are attracted to these PIP2 enriched regions in the membrane. So do you think that could be an integral role that could influence the phase separation of these receptors at the membrane?
Lindsay Case (44:23):
Yeah, I definitely think so. I mean, again, we don’t have any experiments investigating this yet, but I think in the system I’ve shown you like half of the proteins bind specifically to PIP2. So I would expect if we include PIP2 in the bilayer that would probably shift the phase diagram somehow. I predict you would need lower concentrations of proteins to cluster if you had the lipids there, but we haven’t done those experiments yet, but that’s something I’m really interested in understanding more.
Karthik Balakrishnan (44:52):
Okay. Just to follow up on that, is there any intra- or extracellular cues or signals that these receptors would move towards laterally on the plasma membrane after insertion that could trigger the coming together and phase separating property of these receptors?
Lindsay Case (45:16):
I don’t know. Like the only thing I know that… so like cytoskeleton can actively move receptors around on the membrane, but I don’t know of any specific examples doing what you described, but I think all of that’s probably possible also, but we don’t really, I can’t think of examples. Yeah.
Karthik Balakrishnan (45:37):
Just coming from, again from a neuroscience perspective, receptors always move in and out of synapses from the synaptic region to the extrasynaptic region or in the actual …
Lindsay Case (45:48):
Is this through diffusion though, like they just diffuse away or …
Karthik Balakrishnan (45:50):
Yeah, lateral diffusion, basically, based on local neurotransmitter concentration. So I was just wondering–
Lindsay Case (45:57):
I think that’s interesting, but I don’t know in our system, if that happens. Yeah. And our receptor, like integrins are immobilized once they bind their ligand in cells. So they’re not really, but they do still diffuse often. So there is diffusion. Yeah.
Karthik Balakrishnan (46:13):
Great. Thank you.
Erik Martin (46:17):
Awesome. Next up, I’d like to ask Shruti to unmute and ask her question.
Shruti Jha (46:24):
Hi Lindsay. Hey, it was really, really great talk. I was wondering, you showed this slide where the concentration of client was dramatically increased when two different clusters or condensates were fused. So why do you think this happens? Is it because there’s new kind of pockets formed for these clients? Or there were things that were not accessible before that got accessible when two different things fused? What might be happening?
Lindsay Case (46:59):
Yeah. I suspect that there’s just more binding sites for the client because we’ve now added more protein. So kindlin combined to both p130Cas and paxillin. So if we have both of those proteins present, there’s more binding sites, but it could be. We haven’t really explored that specifically. So is there like cooperativity in the binding, or even how, what domains it’s binding? We don’t really know. We just know that it can bind the full length protein. So I think we don’t fully understand which it could be, but the simplest explanation would be, there’s just more binding sites.
Shruti Jha (47:36):
And could this also be like a pathogenic situation in cell when there are two different kinds of condensates that require different scaffold, but also have common scaffold, but due to some misregulation, you have coexpression and then you suddenly have increased participation or unspecific participation?
Lindsay Case (48:00):
Yeah. I think that’s a really interesting… Well, I mean, this is slightly not what you asked, but yeah, I think that’s possible. And I think a really interesting aspect is a lot, especially at receptors on the membranes. A lot of receptors share the same adapter proteins. So like Nck binds to so many different receptors. And how does that allow for some sort of… Is there a limited pool of that protein and all the receptors are competing for the same pool of adapter protein? Or are they sharing? Or does sharing this adapter protein cause them to interact with each other? I think that’s a really interesting question, but I don’t know the answer. Yeah.
Shruti Jha (48:38):
Erik Martin (48:41):
Excellent. So next up, I’d like to ask Anthony to unmute and ask this question.
Anthony Vega (48:48):
Lindsay Case (48:49):
Anthony Vega (48:49):
Good to see you. That was a very cool talk. So I had a question about the experiments in which you were looking at different conditions and the ability for focal adhesion formation. I was wondering whether you had looked at whether there were differences in the amount of nascent versus mature focal adhesions, and if not, what do you expect might be happening?
Lindsay Case (49:15):
So we weren’t really able to do careful analysis of that because we didn’t want to over-express proteins when we were quantifying adhesion formation because changing the concentration is going to change phase separation. So we were relying on fixation and immunostaining of endogenous protein. So we didn’t have live cell imaging.
Anthony Vega (49:34):
Lindsay Case (49:35):
But we did try to really try to mostly focus on nascent adhesions, more specifically than focusing on maturation. So we did things like plating cells only for five minutes where you really only just formed adhesions and they haven’t been able to mature. And we see that the effect, it does reduce nascent adhesions, but it may also affect maturation. We haven’t been able to really look at that yet.
Anthony Vega (49:59):
Okay. All right. Thanks.
Erik Martin (50:03):
Awesome. It looks like we have maybe time for one more question before we have to stop at 11:00. I’d like to ask Diana at our own Kitchen Table to unmute, and ask her a question.
Diana Mitrea (50:17):
Sure. Lindsay, it was a beautiful talk. So my question was actually along the lines of Shruti’s in the sense that, so one of the discriminatory factors by which you define whether a protein is a client or a scaffold is a partition coefficient. So it seemed that in that experiment that you showed, at a different blend of protein concentrations, integrin was potentially, had the partition coefficient similar to the protein you were calling a scaffold. So I’m wondering, do you think that, what are your thoughts about the changes in the network composition and potentially swapping roles of co-scaffolds between what previously have been a client depending on how you modulate the concentration?
Lindsay Case (51:14):
Yeah, so I will say integrin, it’s never really as concentrated as the scaffolds. The partition coefficient is never higher than three. So I guess the scaffold and the integrin images were not contrast-adjusted to match. So that might have been misleading. But we do see situations where you can mutate the scaffold and it becomes kind of like a client. And we see this in cells too, like when we make the mutation in FAK that prevents its phase separation, we see the partitioning and focal adhesions dramatically decreases. So I think that… And we know for example, that p130Cas, its conversion from client to scaffold is dependent on phosphorylation. So that’s something that could be dynamically regulated. So I think that’s a really, I mean, I don’t fully… I think that probably cells do. Like that is something that’s regulated so it’s probably pretty complicated.
Lindsay Case (52:12):
But yeah, I think these types of reconstitutions can be really helpful for starting to understand when you just change one component, how does it affect all of the other components? But yeah, and I think that probably there is probably some temporal relationship in vivo where FAK perhaps phase separates first, and that recruits Cas, and Cas phosphorylated, and then that can further promote condensation. But we weren’t able to really test that hypothesis.
Diana Mitrea (52:45):
Yeah, because I mean, going back to the comment that you made about the different, one of the proteins being upregulated and cancer would be interesting if we could understand how the changes in the blends and the condensate affect speific pathways.
Lindsay Case (53:00):
Right. And you’re upregulating one and not the other, that’s going to totally throw off the network in some direction. Yeah. And it could have been like, yeah, changing the composition could completely change the function of the condensate.
Diana Mitrea (53:13):
Yeah. Thank you.
Erik Martin (53:18):
Awesome. Yeah. I guess that would ties in with some of the original work in the science paper too, which is really cool.
Lindsay Case (53:24):
Erik Martin (53:28):
Yeah. So I’d like to thank everyone for coming. It was a beautiful talk and a great conversation afterwards. And I’ll turn it back over to Jill for some final comments.
Jill Bouchard (53:37):
Yeah. Lindsay, first of all, thank you for sharing with the community. This was wonderful. And clearly there are a ton more questions that we’ll be shipping over to you by email, and we’ll publish with your-
Lindsay Case (53:49):
Jill Bouchard (53:49):
… video when we put it up online. So thank you for agreeing to do that. And if anybody has lingering questions, just email us at email@example.com and we’ll filter those right back to Lindsay too. So I just want to thank everyone for joining again today. Thank you, Lindsay, again, and we’ll see you next month.
Lindsay Case (54:08):
Jill Bouchard (54:11):
Question from Avinash Patel: in cells, were the condensates normalised against the number of membrane protrusions (filopodias)?
Lindsay’s Response: No. We did not normalize to number of protrusions or cell area. We simply calculated the number of condensates per cell at specific time points…
Question from Jeremy Schmit: Do you think the membrane enhancement is a nucleation effect? That the proteins are supersaturated but in the metastable regime?
Lindsay’s Response: We suspect the membrane enhancement is most likely due to nucleation, but we haven’t been able to directly test this experimentally. Would love to hear ideas for experiments that could differentiate between possible mechanisms!
Question from Xiaolei Su: Beautiful talk, Lindsay! Does FAK autophosphorylate in trans or in cis? Is there a pY reporter you can use to visualize FAK phosphorylation in live?
Lindsay’s Response: FAK predominantly autophosphorylates predominantly in trans, although there is some low-level baseline phosphorylation in cis (both in cells and in vitro). There are several FRET-based FAK reporters that have been developed, and we are trying to decide which may be reliable and robust reporters for autophosphorylation. We are also working on developing new ways to visualize FAK phosphorylation in vitro, so stay tuned!
Question from Erik Martin: Do you think that the phase separation mechanisms could help specifically localize FAs due to the fact that a small increase in local concentration of integrins would be greatly favored as a site for droplet formation?
Lindsay’s Response: I suspect this is very possible! The ligand for the integrin receptor are polymers that also have multivalency, so ligand binding to the extracellular domain of integrin could also locally initiate integrin clustering, which would than favor droplet formation on the intracellular surface of the membrane at that site. We haven’t directly tested this idea in cells yet.