08:02:07 So Good morning. Welcome to the second session of the 2021 more for Genesis seminar series of K dp.
08:02:14 I'm glad to see both new and familiar faces today.
08:02:18 Before we begin, I just like to say a couple of words about the serious are going more for Genesis. Genesis serious is to discuss and identify general principles and what for Genesis, which is the emergence of biological shape.
08:02:32 We would also like to translate problems from a biological language into physical terms and identify topics in which theory can help us make progress, and we hope to accomplish this by having speakers which worked on very different systems, different
08:02:53 different species, as well as speakers with different backgrounds both physics and biology. And as part of this effort. We're excited to have the Nell Devonport from Princeton with us today, And I would like to already think here for taking the time and
08:03:01 efforts will join us.
08:03:03 Another key issue is that the success of the seminar is dependent on discussion, and we encourage questions from the audience throughout the talk, so please feel free to unmute yourself when you have a question or you can also write the question in the
08:03:18 chat.
08:03:20 We would of course, step in and moderate if things get out of hand.
08:03:32 I would also like to encourage everyone to keep the camera on so that our speakers can feel less cybernetic and more humane. So thank you for that. And finally, I would like to thank the Kp for hosting the serious Mark Larson Boris for their support,
08:03:40 as well as our tech team David Alina and Craig and Big thanks to you, the audience for attending. And I will now hand off the stage to Noah would introduce our speaker for today.
08:03:51 Great, thank you so today we're super excited to have Danelle Devonport to know is an expert, particularly on plane herself polarity in tissue orphan Genesis, so how cells in a tissue and particular to the tissue quasi 2d can orient themselves in space.
08:04:11 Actually, first encountered Danielle's work or content Danelle in the literature, by her work for from her PhD studies at Cambridge on integrations the role of immigrants in the development of internal organs into safiullah.
08:04:30 But since then she went on did others, other interesting things both, both then and then at during from postdoc at Rockefeller and then went on to move to Princeton where she built her lab that focuses on Hello cells sense direction, how they communicate
08:04:49 that directional information to their neighbors, and how they maintain this orientation order, as they grow or recover from injury or experience other perturbations.
08:05:04 So, we'll hear today, I'll do a little bit of preamble for some of the physicists in the room, although I'm sure to know what will give a great introduction on this as well but PCP that's planar cell polarity orient each cell in a, in a planar tissue
08:05:20 in space, and we want to know how how cells are able to do this.
08:05:26 For instance, they could just sense some sort of morphine gradients and sort of long range gradient that the fuses through the tissue but, but, and then respond by by shuffling the distributions of their molecules at their boundaries, but these long range
08:05:41 gradients are typically very weak. And so a lot of the action in in the science here is looking at how cells communicate with one another to reinforce this information that they sense.
08:05:54 So for robustness these local interactions are very important, much like how interacting spins and the Heisenberg model can more robustly order than non interacting things in a week magnetic field.
08:06:10 So, yes, without much further ado, today we'll hear about a brand new work from to Nellis group that just came out, and an important ingredient in how cells established this plane yourself clarity.
08:06:23 So let's welcome Delta.
08:06:27 Thanks for the introduction. Noah, it's really great to be here and I really appreciate the invitation and chance to speak and share some of our recent work with you.
08:06:38 How's the volume Can you hear me okay.
08:06:41 Okay, perfect.
08:06:42 All right. So, um, I am a biologist.
08:06:48 Um, I don't know a lot of physics at all. So this will be a pretty molecular talk but I'm going to try to, you know, zoom out and you know place these things into the larger context, but please feel free to interrupt me if you want any clarification.
08:07:03 I'm not, I'm I don't have the chat function open on my laptop window right now just so I can see my slides so feel free to, if there is something in the chat you want to interrupt me and just say hey don't stop.
08:07:15 Okay, so very broadly I'm fascinated by how cells assemble into elaborate structures during embryonic development, and how those elaborate patterns and structures, ultimately give rise to the function of the tissue.
08:07:33 And here are just some lovely examples in the lung, the gut, kidney muscle brain of where such patterns can arise.
08:07:44 I think recent recent work in Oregon noise, where cells that are relatively undifferentiated so stem cells that are grown in a dish can be coaxed by simply you know throwing the right cocktail of signaling factors into the dish can instruct cells that
08:08:04 they have this incredible self organizing capacity to form structures that are really reminiscent of what the native organ would look like. So we've seen many brains.
08:08:15 Many kidneys many, many lungs and the guts.
08:08:20 And it's these organizers contend to contain all of the cell types that are native to a tissue.
08:08:27 And in an arrangement that also somewhat resembles the native tissue as well.
08:08:32 But as a developmental biologist I also look at organizations and think wow there's still so much missing. And, you know, Can we look at what's missing, to really understand how the larger scale, sort of global architecture of tissues and organs, what
08:08:48 information is needed to create that level of pattern. So one thing that's missing is tends to be the size and the scaling of these organizations relative to you know what the native scale would be, but also another thing that's often missing is the long
08:09:07 range coordinated polarization of the structures. And so the phenomenon that I'm referring to is playing herself clarity.
08:09:15 And that is the coordinated alignment of of either structure, or some cellular distribution of contents along the plane of a tissue and some really striking examples where you can find planar clarity in your body.
08:09:30 Include the airways, or their multi deleted cells, I'll be in a common direction, and that's to make sure that mucus and fluid are propelled out of your lungs rather than into your lungs.
08:09:41 You also find multi isolated cells that beat uniformly in the, in the brain, the ventricles that line the brain.
08:09:50 You also find planner polarized patterns in the inner ear. These are stereo cilia bundles that are critical for the mechanic transduction that allows us to hear.
08:10:04 And then the Collective Soul movements that shape tissues and n embryos during early development, can also be planar polarized where the global movement of cells is aligned within a particular plane.
08:10:21 So, one thing that's really quite striking is that and I'm using you know mammalian systems mostly as a, as my examples here but we find this across evolution is that there is a core set of molecules of genetic sort of module that can align all this whole
08:10:40 diversity of structures, whether it's so movements and Airways, there's a common machinery that can align very diverse structures and tissues.
08:10:52 And so this is known as the core players clarity pathway, it's going to be the focus of my talk today.
08:10:58 And mutations in mammals in components of the quarter planner self clarity pathway lead to really severe developmental defects, and all of these tissues that I'm showing here and a lot more.
08:11:09 So it leads to a loss of Hearing and Balance a shortened body access due to the failure of cells to converge and extend in a coordinated manner.
08:11:20 Probably the most notable severe defect in mice in invertebrates in general, is the complete failure to close the neural tube, which is shown here. This is a completely open neural tube in animals that lack Splinter Cell clarity, the failure to be cilia
08:11:38 in a common direction leads to fluid buildup in the brain, which is a condition known as hydrocephalus.
08:11:45 And the more people are looking at the more they're finding functions for planar polarity jeans and all sorts of tissues and organs.
08:11:52 So, our favorite model system.
08:11:58 Think I pushed advance on the slide and it's just a very large file.
08:12:09 There we go.
08:12:09 So our favorite model system to study planner clarity is the mammalian skin and prop just looking at the side you can tell why
08:12:20 the visual alignment of hairs across the mammalian skin is really striking.
08:12:28 There's this repeated so every one of these dark lines is an individual hair. And these structures can be aligned over really quite long distances. And you can see this is a great model for studying planar polarity one because it's right on the surface
08:12:43 it's genetically tractable, but also planar polarity is organized across multiple links scales.
08:12:50 So here I'm showing you the global alignment of the structures and this can be on the order of centimeters two meters depending on what organism we're talking about.
08:13:02 And if we zoom in. Each, there are regions where these structures are locally aligned on the order of millimeters, two centimeters.
08:13:12 Now, each individual, if we zoom in again, each individual hair, which derives from a hair follicle is planner polarized itself. And this is a multicellular unit.
08:13:24 So each one of these dark structures here is comprised of thousands of cells at this developmental stage, and that group of thousands of cells, collectively orient toward one side, where the growing end is this larger bulbous end and the hair points out
08:13:39 towards the tail, so the growing end points toward the head and the hair part pros out towards the tail.
08:13:47 And this is on the order of, say, you know, hundred microns to a millimeter.
08:13:52 Now if we examine the cells that are in the interface the color space. So this is the interface of the epidermis or the part of your skin that isn't hairy.
08:14:03 This area is also clear and polarized into illustrate that first I'm just showing you what the individual cell membranes, with the overall cell membrane and nucleus looks like.
08:14:13 So the nucleus of labeled in blue, and the membrane is localized, or is shown in in white. And if you compare the distribution of just this membrane protein to a plane or sell clarity component, which is localized to the membrane.
08:14:30 You'll see that in this case I'm showing you a protein called cell Sir, it's enriched on the vertical boundaries of individual cells and depleted from more horizontal boundaries.
08:14:45 We can even zoom in further at an individual junction and show that these are also planner polarized. So one side of the junction contains a protein called frazzled, which is a player and a different player cell clarity protein, whereas the other side.
08:15:02 Contains bang.
08:15:04 So, every individual skin cell in your body has the ability to sense direction, and to localize components of the player cell clarity put pathway to one side or the other individual junctions and these a line across millions of cells.
08:15:26 So may I ask a question, please. Yes, you may. Yeah, this is David Nelson.
08:15:31 Hi. So the is the one they have hairs, the skin cells is the hair direction correlated with this a cemetery maybe you said that. I didn't say it but it absolutely is.
08:15:43 So, um, let me.
08:15:47 So the hair, always points toward the anterior, which is the Vang side of an individual cells I'm showing you the cell, cell junction so yes it is absolutely correlated with the distribution of these proteins and dependent on dependent on them to.
08:16:03 Thank you, and I'll show you that in a second.
08:16:07 Okay.
08:16:10 So, if we zoom in further we have to draw cartoons, because we really don't know the protein interactions, that, that, that generate this asymmetry along a cell, cell boundary along the membrane or the junction of a cell, cell boundary.
08:16:24 But we do know some of the key components, and I'll introduce them here. So, first, there is a coherent proteins and coherence or cell solid Asian proteins, and they interact, across the extracellular space via domains that are called could hear and repeats.
08:16:42 And each one of these bridges these coherent bridges is paired on opposite sides of the different protein. So frazzled is paired with cells are on one side, vein is paired with cells around the other.
08:16:55 And so we refer to this bridge is an asymmetric bridge.
08:16:59 But it's thought to be coupled physically across the extracellular space, through the adhesive interactions of the cells or protein. Now I've drawn the interaction with this like little loopy bit.
08:17:14 And I should say we know nothing about what the binding interfaces of this protein, I just draw it this way so I can fit it on the slide.
08:17:22 It is something we would like to know.
08:17:24 Alright, so, preferential interactions between frazzled and bang containing complexes are thought to couple polarity between adjacent cells.
08:17:38 And similarly, negative interactions between frazzled and Big Bang containing complexes within cells also helps to generate this asymmetry. So, by preferential interactions opposing complexes between cells and inhibitory interactions of these opposite
08:17:57 complexes within cells start to align polarity across the tissue in a sort of almost a domino effect across the tissue.
08:18:09 How the direction frazzled is, you know, biased toward, you know, one side of the cell across the entire tissue is something very interested in and it's been, you know, one of the hardest things to, to understand that the field of planar polarities of
08:18:37 identify what those kind of global long range cues might be all right.
08:18:30 Sure. So how should we think about this. Is it like a fixed issue. And then the protein going to different sides of the South, or are the cells also mobile can change their intuition change neighbors etc.
08:18:43 So I'm going to introduce that a little bit in a second. Um, I'm not really going to talk about the neighbor exchange. It's really different depending on the tissue, but in our system, the cells rearrange a lot like even under because they divide like
08:18:59 crazy this is a very proliferative tissue.
08:19:01 And so there is there's got to be some sort of dynamics that maintain the asymmetry once it's there. And we think that some of those dynamics could even play a role in establishing a cemetery to, I won't get into that so much today but I'll talk about
08:19:15 the movement of proteins and a little bit.
08:19:18 But one thing to keep in mind is that there are, you know, series of feedback loops in the system such as the elimination of any one polarity component leads to a loss of asymmetry of all the others.
08:19:29 So in this example I'm showing you what happens when we remove Vang from the system. Now Selzer frazzled, and the other planner clarity components I haven't shown yet will now uniformly just become distributed around us all.
08:19:46 And what that leads to in terms of the hair fall I can jump in with a question.
08:19:51 Just a quick follow up to the question you just answered.
08:19:56 When says to buy, how are these what are the factors inherited.
08:20:01 Oh my goodness. So, a whole like half of my lab works on that problem, and I'm not going to talk about it today.
08:20:10 But I'll tell you just briefly, that actually these proteins which are integral membrane proteins are removed from the cell surface during mitosis, through into psychosis.
08:20:22 So there were moved into membrane bicycles they're held within the cytoplasm where they're allowed to mix.
08:20:28 And then they're recycled back to the cell surface outside of confuses, so that's another like really fascinating component to the to this whole system and, yeah, I won't have time to talk about that today.
08:20:39 Super Thank you.
08:20:42 Alright. So, someone asked before about whether the weather the direction of the polarized distribution of clarity proteins correlates with the orientation of hair follicles, and it does.
08:20:58 So hair follicles point in the direction of the same vein localization within the cell towards the towards the anterior.
08:21:04 now we'll point completely random randomly, and here that shown by a color coding scheme where you know the color gives you is shows you what orientation the structures are pointing in.
08:21:30 And this is something that you see across the entire back skin so you've lost, you know the full global coordination of hair follicle alignment. In the absence of a planner so clarity component such as being.
08:21:45 Okay, so we want to really understand this process across these different biological scales. So how are the asymmetric PCP bridges assembled between cells, and how are those local asymmetries driving the collective polarized behavior that is the collective
08:22:02 of cells that point in the orient the hair follicle in one direction.
08:22:07 We're very interested, also in the global cues orient clarity across the tissue or organism.
08:22:14 And so today I'm going to talk about this first problem. If we have time maybe I'll talk a little bit about how local PCP symmetries drive collective behavior.
08:22:26 But we'll see. We'll see how far we get.
08:22:29 But maybe you'll get to this later, but when the cells divide the polarization is reserved. Typically, when the cells divide polarity is erased and then restored outside of can uses.
08:22:43 So it isn't it is erased via into psychosis, meaning so removal of those proteins from the membrane to the cytoplasm, and then recycling of those testicles back to the plasma membrane during static analysis where polarity is restored, or remembers the
08:22:59 polarity the surrounding cells surrounding cells provide a template for the budgetary. I think I don't think we nailed down yet.
08:23:10 Thanks.
08:23:13 Okay.
08:23:15 So, in order to generate polarity.
08:23:18 One thing I should say is the system starts off with a disordered or uniform distribution of clarity proteins, and they become increasingly a symmetrically distributed over time.
08:23:29 So the proteins need to redistribute within the cell.
08:23:33 And so,
08:23:39 that's right. I just want to show you what this looks like with actual data instead of schematics so early in mouse development and skin development, I should say, here's our favorite player cell clarity protein seltzer one and you can see it's distributed
08:23:54 pretty in a pretty disordered slash uniform way polarity starts to become a parent. Two days later, and two days later, it's very a symmetrically distributed.
08:24:05 We can put some numbers on the magnitude and orientation of that polarity and show that both of these things the magnitude and the collective alignment of polarity increases over developmental time.
08:24:20 So, how does this work.
08:24:23 Well, in order to generate a symmetry we think two things need to happen.
08:24:28 There needs to be selective stabilization of planners they'll come polarity components that vertical edges, I'm just going to call them vertical edges there the anterior posterior edges of the, of the dorsal backspin, but the way I'll show them on the
08:24:41 screen. I think it's easier to just refer to them as a vertical edges.
08:24:46 And then, there needs to be maybe removal or at least a destabilization of things at the horizontal edges. but then there also needs to be an unboxing of opposite Lee oriented PCP complexes at those vertical edges.
08:25:00 So I'll just show you what I mean by zooming in a little bit. Is that presumably they start off these asymmetric bridges, start off in mixed orientations, say we're first old is sometimes on this side and sometimes on the other.
08:25:12 And those needs to be unmixed into uniform orientation so all the Frizzell molecules are on one side and on the vein, or on the other side of the junction.
08:25:23 So in general, a lot of attention in the PCP field has been paid to the side of plastic components which I haven't talked about until now.
08:25:33 As far as their function in in doing this on mixing.
08:25:37 So disheveled interacts with frazzled and principal is not to interact with Dang, and the cytoplasm components have a ligament ization domains, which are thought to help promote the aggregation of like complexes alone, one side of the junction.
08:25:53 They also have inhibitory interactions, where the presence of prickle can induce the increased mobility and removal of state of Frizzell disheveled complex from from the opposite side, or within the same zone.
08:26:15 However, it's got to be true that there are inter cellular interactions that are important for this unboxing process. And we decided to focus on the cell so one protein, with its ability to interact across the extracellular space as possibly being important
08:26:32 for this establishment of a symmetry.
08:26:36 So let me tell you a little bit about this protein. So I mentioned it's a kid here in so immediate self validation. It's really big. It's got nine kid here and repeats and these are thought to mediate the homophobic confusion between cells.
08:26:50 It also has a bunch of laminate and EGF for Pete's a seven past trans membrane domain and alongside of plastic tail.
08:27:01 For for catering.
08:27:01 So really simply we wanted to know what the adhesive properties of this protein were because there was really not much known about it, and also how those adhesive properties contribute to cellular function and asymmetry.
08:27:13 So, first to figure out.
08:27:17 It's a piece of properties, we use a cell aggregation system in order to test whether the protein could actually mediate romantic occasion between cells.
08:27:28 So in this case, these are cells that grow and suspension.
08:27:32 They don't have any adhesion proteins cell, cell adhesion proteins of their own. And so, if you express within the cells, something neutral, like just GFP, you get a cell suspension of individual cells and in this case every.is a.
08:27:51 So, If we introduce into these k 562 cells. Cells are one tagged with GFP, you can see it mediate some aggregation between, and, resulting in these clusters and clumps of K 562 cells, suggesting that in fact yes this protein does on its own media itself
08:28:09 allocation.
08:28:11 If we remove the extracellular domain of this protein, it no longer mediates aggregation, suggesting that the character Pete's are involved in. In this process, and that's what we would expect.
08:28:27 So, from there we took advantage of a Lele, a specific mutation in the cells or one gene that had by been identified in a genetics screen for neural tube defects.
08:28:40 So this mouse is called a crash. And the reason it's called crash, is because when it's header zygote.
08:28:48 The animals actually have inner ear problems and they fail to maintain their balance of the crash into the walls of the cage.
08:28:56 But that in the homozygous phenotype is a complete failure to close the neural tube which looks a lot like a PCP defect. So what this is is a very severe loss of function Leland seltzer one, but it's just a single point mutation, and it changes a single
08:29:12 amino acid in the kid here and repeats itself. So it's located between the seventh and eighth could hear and repeat.
08:29:20 It just changes one amino acid, but causes this very very severe loss of function defect. So we thought, Okay, this is a mutation in what we figure is the adhesive domain, maybe this will give us an inroad to figure out how this protein might be involved
08:29:37 in. In a symmetry.
08:29:40 So first a little bit to investigate what this mutation really does today at Houston function. We used a couple of different cell systems. So in this case we're using epithelial cells and culture, so their cultured keratin affects the skin cells that
08:29:55 we that we isolate from from from our skin.
08:29:59 And we can introduce either the wild type version of this protein or the version with that crash in it.
08:30:08 And normally when two cells come together, that express the wild type version the protein localize is at the cell, cell junction where it's mediating homophobic adhesion.
08:30:18 However, when we introduced the crash version. It's very diffuse on the membrane and it looks to be as if it's not mediating occasion at all. Based on at least its enrichment at the cell, cell border relative to the rest of the edges.
08:30:32 However, what was a super surprising is that when we introduce this YouTube version into the K 562 suspension cell system. It mediated beautiful it cell aggregation, suggesting that in fact it does still mediate adhesion in trans, but that there, you
08:30:54 know, must be something sort of odd or different about it, and he said abilities.
08:30:54 So to look at that a little bit further.
08:30:58 We performed a cell mixing essay, with wild type and crash mutant expressing cells. So, as a control. If we have a green tagged seltzer molecule.
08:31:12 And those cells are mixed with cells that are expressed a red tagged cells are molecule, they will form mixed aggregates, so there are green cells and red cells together within the same aggregate.
08:31:22 But if we do the same experiment mixing cells that are contained the mutant version, the crash mutant version.
08:31:29 Now the cells sort out from one another. They mediate homophobic aggregation with other unmute themselves, but they won't meet it he did with the wild type partner.
08:31:38 So that suggests that although the crash Putin can still media it Asian and trans, there's something different about this binding interface that prevents it from interacting with the wild type protein.
08:31:50 Baby, it's a confirmation will change.
08:31:52 We don't know.
08:31:54 You can also appreciate this. This phenotype by looking at how the proteins, interact at mixed cell borders.
08:32:01 So in this case, the top cell expresses the green version of the protein, the bottom cell is expressing the red. And if they're both wild type, then everything cold localize is perfectly at a border.
08:32:15 However, if one of the cells expresses the mutant, as in this bottom cell here, neither the wild type or the mutant protein, get localized or recruited to that sell sell Porter.
08:32:28 So crash cannot interact and make homophobic interactions with the wild type 13.
08:32:33 Alright so what is wrong with this crash protein if it can still interact, if it can still meet it media it Asian and trans.
08:32:41 So we zoom in, and how to look at the distribution of a protein within these k 562 cells.
08:32:49 And one thing that was really striking is how enriched and aggregated.
08:32:54 The wild type protein is between neighboring cells so you can barely even see the circle.
08:33:01 That is the K five six to sell because all the protein is really you know enriched at the cell, cell boundary.
08:33:06 And we saw really district different distribution with the crash version of the protein.
08:33:11 So, even though it doesn't localize to the cell, cell boundary it's diffuse unexpressed all around the cell edges, suggesting, maybe a defect in the ability to aggregate or cluster.
08:33:27 Alright, so that led us to form the following hypothesis, so we knew from kid here in biology from classical coherence, that could hearings not only engage in transit interactions between cells in order to mediate occasion, but they also mediate.
08:33:43 They also undergo cysts interactions within the same style to formal lateral clusters. And that, that, that those interactions really contribute to the overall adhesive properties, such that they for more like Velcro kinds of interactions, you know, in,
08:33:58 in, in addition to their trip, in addition to their trans interactions. So we thought, ah, perhaps the crash mutation lies within an assist interaction domain which contributes to the overall adhesive properties and lateral organization within the memory.
08:34:14 So we did a bunch of cell biological experiments to test this idea.
08:34:19 The first was to just look at district, distribution, like does it cluster, and it certainly does. So if we examine the distribution of cells or one, the wild type version of it by super resolution microscopy where we can start to now resolved.
08:34:36 You know, smaller distances between molecules, we can see that it is distributed in a punk state organization along the junction.
08:34:45 We see this in keratin insights in vitro and we also see this organization in vivo risk and cleaner so clarity proteins are well known to form, you know, clustered aggregates and flies and so this was very much expected.
08:35:06 And now we have a raised hand Hello Henri. Is this a good time to ask her.
08:35:07 Hello, can you unmute yourself.
08:35:18 Okay, perhaps, perhaps there's some issue. So go ahead to No. Okay.
08:35:23 All right. So, using a different super resolution technique that allows us to gain even even higher resolution up to 20, up to 20 nanometers.
08:35:35 Just known as D storm. We characterized the, the SIS clustering ability of these structures, both in the wild type, and in the crash mutant.
08:35:46 And so if you image, these individual junction will areas by storm. You can see that the protein is organized in very very dense punk data that align kind of all across the junction.
08:36:02 And that's the crash Newton which is down on the bottom. Still organizes in kind of a clustered assembly, but they're much less dense and so down below, is the molecular density.
08:36:16 do or projection of these data here a couple examples of wild type versus a couple examples of punk data from the crash, and they have far fewer molecules per, per pumped up that are less dense, Lee associated even or less dense within the same area.
08:36:36 And if we quantify this over many different punk done many different junctions. The crash meeting has a severely lower molecular molecular count within a punk into their overall density within per pump data.
08:36:53 And then, that is, on average across the junction, the molecular density is also far reduced in the crash, suggesting a defect in the ability to cluster laterally at the membrane.
08:37:08 We figured that a defect in the ability to cluster may affect the mobility of the protein. So to to assess the mobility of the protein were, we performed a fluorescence recovery after photo bleaching experiment where we bleach GFP expressing seltzer injunctions.
08:37:28 And then monitor the recovery over time.
08:37:31 And one thing to note here is that the wild type protein is super stable.
08:37:38 You see very little recovery into the bleached area over the time period of the experiment that stability relies on the extracellular doing in the protein, because when we delete it.
08:37:50 In this delta inversion. On the right, we now get almost full recovery. Over the experimental time. And then the crash mutant, which has a defect in lateral clustering is somewhere in between, between the two, which again, kind of expected based on the
08:38:07 fact that we undergoes transit interactions that seems to have a problem with lateral clustering.
08:38:13 All right.
08:38:15 So if this is really due to a defect in the ability to interact insists. We had the idea that if we could forced diarization of the crash protein, maybe we could rescue some of these phenotypes.
08:38:28 So the experiment goes like this. There is a well characterized diarization domain, known as FKBP.
08:38:42 And you can use this to your protein of interest, and upon addition to what's called a homer Diamond Diamond razor, small molecule that can diffuse through the through the cell membrane.
08:38:50 Upon addition of this it will force diarization between any protein that carries this FKBP domain. So we've used it to the C terminus of the crash mutant protein and asked whether we could rescue, its distribution.
08:39:05 So here's what the crash crash distribution looks like in an ethanol control. So, it has the diarization domain on it but not the small molecule Dima riser added.
08:39:20 However, when we add that small diamonds are we now see recruitment and enrichment at the cell, cell Porter.
08:39:28 And not only that, we can rescue the interactions with the wild type protein.
08:39:33 So remember I showed you that experiment before where when the wild when there's a wild type expressing itself.
08:39:40 On one side, in a crash mute and sell on the other side.
08:39:44 Both proteins fail to enrich at the cell, cell boundary between them.
08:39:49 So that's the case when we add the FKB binding protein as well but we add the diamond riser the small molecule dime razor. Now we get really nice enrichment, and restoration of the ability of the crash meat and protein to now, interact with the wild type
08:40:04 13 and trans.
08:40:08 So, pretty good evidence for us this diarization defect, go ahead. Sorry, I just wanted to ask a quick question about the earlier experiment you showed with the non adhesive cells where you're doing the outer expressions, actually related to what the
08:40:21 results are showing here and maybe you're going to touch upon this but do those cells express any of the other PCP components you mentioned like do they express the angle, do they express it as a result.
08:40:40 Um, I don't know.
08:40:43 And this has been one of those things that we have been, we need to we need to go into those cells and profile all of the PCP protein expression so I don't, I don't know if they have to shovel critical thing for us.
08:40:56 It's certainly on the to do the to do this. Take you
08:41:01 know I'm sorry, can I just ask a quick question as well.
08:41:04 I'm sorry I ended my teaching early to come to this I'm sorry for being late.
08:41:10 In these punter, if you could do the, the, Duncan experiment which is going grab these pumped and do mass spec, do you think you would see a whole bunch of signaling things that are pointing up in the same counter or do you think that if you looked at
08:41:23 different signaling systems, you would kind of see each one with that punk take populations that are not overlapping. So which one is it do is your intuition, and my intuition is largely non overlapping.
08:41:37 And that comes from. If we call labels say these complex structures with, say, classical kid here in adherence junction components like he could hear in there in a step.
08:41:49 During an alternating pattern. If we look at the distribution is does visible coherence so another family of coherence that are again punk de adhesive structures, they are in there in between.
08:42:01 and largely non overlapping.
08:42:05 If we were looking at signal like signal zones like wind signaling or something like that that I'm not that I'm not so sure. But yeah, my intuition is that they need to be in separate compartments to do what they need to do it.
08:42:22 All right.
08:42:24 So, from here. We decided to move in vivo, because ultimately we want to understand how it he's been at this protein contributes to the a symmetric distribution and the development of PCP.
08:42:38 So remember I told you that the cells are one protein localize is preferentially to vertically oriented junctions, and is depleted from more horizontally oriented junctions.
08:42:48 And so this is the wild type distribution
08:42:52 that that single point mutation the crash mutation completely obliterates. The a symmetric distribution of this protein, it now becomes uniformly distributed along the cell edges.
08:43:06 Interestingly, and perhaps not surprisingly after, after what I've told you about the crash function is that the header is, I guess, so one copy one wild type copy one you can copy in the same animal also has a reduced, a symmetric distribution, which
08:43:22 lies somewhere in between.
08:43:24 and. Now, just to be clear, these are fixed samples that you you've raised the embryo.
08:43:31 In its normal state and then take it out, you fix the stain Yes, exactly. Yeah, so this is fixed in stained with the saucer antibody. So this experiment doesn't let us measure, you know, the turnover doesn't allow us to measure by crap.
08:43:48 We would need to generate an endogenous Lee tagged crashing mutant protein. In order to do that experiment. Yeah.
08:43:58 Last question.
08:44:04 Before you go to enable is actually about the the protein. Is this something similar to face separation of protein that it actually have a separate on the membrane.
08:44:11 Um, I guess, i. So, it says it clusters.
08:44:20 Yes, it's still somewhat mobile i mean i guess when I think about it like a liquid liquid phase separation, I think, of there still when you do a rap experiment there's still a lot of exchange with with molecules in the surrounding environment here right
08:44:41 actually clustering things to the point where they really lose their mobility and there's a lot less exchange with molecules from around. So then, for that reason I don't know if that fits one of the criteria at least a liquid liquid phase transition,
08:44:57 whether if it says, more like solid like transition.
08:45:00 I don't know,
08:45:03 has features that are that are reminiscent of it. And I should say that the cytoplasm it components to shoveled and prickle, they do went over expressed to like what very much looks like a phase transition good liquid liquid phase transition so components
08:45:18 of the of the of the pathway seem to be able to undergo a phase transition.
08:45:37 I have what
08:45:29 is what is perhaps a related question. If yellow prefers yellow across a junction and read prefers read.
08:45:47 So, so hang on. So just just to be clear, in this system normally yellow prefers read yesterday. Okay, so then can you repeat that doesn't happen.
08:45:59 It doesn't happen. Are there examples where that doesn't happen. Well sort of clustering across the junction in the spirit of the previous question. Um, so I'm going to show you that in the next slide or two.
08:46:08 Yes, right. Thank you know, sorry, maybe just one more question, you told us about the stability of cell sir at least the wild type. How about the turnover of fizzled, and, and, and we have not done that experiment, the, the prediction would be that the
08:46:32 turnover frazzled and bang is probably also enhanced in the crash mutant.
08:46:40 But we haven't actually measured that.
08:46:43 But, but the general assumption is that the result and bang is stable as sell sir as opposed to turning over fast and attaching to sort of what he says scaffold of cell Sir, I remain open minded I tend to think that like for little man kind of follow
08:47:01 seltzer and do whatever sounds or does.
08:47:03 But I don't think that that's not an entirely fair because we haven't looked at that in the context of dynamics. We've looked at that in the context of localization.
08:47:12 But yeah i'm open minded to the idea that maybe they've remained maybe they do remain somewhat mobile and attached to an adhesive it he's a complex but we haven't done this experiment, I guess in flies, where a lot of the experiments have been done for
08:47:42 Thank you.
08:47:42 Okay. So I just want to come back to this idea before I show you the phenotypes is that,
08:47:49 you know, there's the side.
08:47:52 In order to generate a symmetry, we need to stabilize protein selectively at vertical junctions, and clearly that is disrupted in the crash right because we now get spreading of the other proteins of seltzer, and any other proteins to those horizontal
08:48:08 edges. And given the increased mobility of the crash mutant protein that may be sufficient to sort of explain, you know, why those proteins, maybe diffuse or don't get removed from the vertical junctions.
08:48:20 But what about this and mixing process is that affected in the crash.
08:48:32 Do we get a defect in the partitioning of opposite Lee oriented complexes. So to look at that we went back to Super resolution imaging.
08:48:36 Here we're using structured illumination, which only gives about 120 nanometers resolutions and we just, just starts to separate or allows us to see the two sides of the junction.
08:48:51 And the reason we have to use this is that in vivo. The skin is is a little is thick it's multi layered, we're looking at a stage where it's not a lot of layers there's only two or three layers and so we can get it to the inner layer of interest.
08:49:05 But because storm which has the best resolution is a ultimately a wide field application, you really cannot have out of focus light and.
08:49:17 Yeah, it's really not amenable to to this kind of analysis. So, so sim was our way to go.
08:49:24 So this is an A representative sim image. We're standing now for frazzled and bang for seltzer again we see this pump a distribution along the junction and Here I'm showing you a vertical junction here.
08:49:38 And if we zoom in on some of these individual panto we can start to see separation of the two proteins, were vaping if when we can see separation Vang is consistently on the right side for a little is consistently on the left side of that junction.
08:49:54 However, the majority of you know the signal does seem, you know, is mostly overlapping what you see is that yellow haze in the middle.
08:50:02 Nevertheless, we thought this was enough resolution to be able to at least compare what the crash meeting with like, okay.
08:50:12 Okay. So before I show you the data I just wanted to tell you what I expected to see.
08:50:16 So, if you lose a symmetry and and PC, the asymmetric bridges are unable to become a mixed to me and one sentence.
08:50:30 I would expect the either the it all the punk to just look, yellow, right oh just a haze of overlapping
08:50:39 a haze of overlap, or that maybe we would see clusters that were a symmetric, but sometimes in the right orientation, and sometimes in the wrong orientation that those might flip along along the edge.
08:50:59 That is not what we thought all.
08:51:04 Instead, what we saw is that the frazzled and vain proteins seems to to laterally separate from one another and become alternating along the junction.
08:51:16 So here's an example. And, sorry, let me just show you that. Occasionally, we saw the expected result. And that is a symmetric punk de with frazzled on one side bang on the other, sometimes in the correct orientation and sometimes in the incorrect orientation,
08:51:33 we did see that sometimes. But by far the predominant phenotype was a lateral separation of frazzled and bang along the junction where you would have alternating clusters of frazzled or being alternating clusters of Frizzell alone along that junction,
08:51:51 which was very unexpected.
08:51:55 I should say that we can't resolve, whether these are what what David just asked about whether these are you know frazzled frazzled BANG BANG frazzled frazzled BANG BANG interactions all along the boundary, or whether it's like a heavy, heavy junction,
08:52:15 you know frazzled on one side, and maybe bang over here.
08:52:20 But still, the lateral separation of these proteins was was definitely not something that we were had considered.
08:52:27 So we can show this further by just, you know, doing this sort of line scan of fluorescence intensity and under normal conditions.
08:52:35 Every peak of frazzled coincides with a peak event along the junction.
08:52:43 Whereas in the crash mutant, we find that they're often alternating, so not only do they not co worker, but they're often an alternating domains.
08:52:54 And we can quantify this by just measuring with a Pearson's correlation the CO occurrence for Sultan bang along along the junction along the particular, and they're very highly correlated in wild type.
08:53:08 And that's reduced in the crash mean.
08:53:20 Now I just want to mention one last thing. And then, I think I'll summarize and wrap up, so we can have some questions at the end. Just want to mention this one last very intriguing thing is that ok and all the schematics I show that the horizontal functions
08:53:28 junctions are devoid of PCP proteins, that's not entirely true. They're there they're just they're at lower concentrations.
08:53:35 And if we examine by super resolution, the distribution of frazzled and bang along the horizontal junction. They show that same alternating lateral separation, that we saw at the vertical junction of a crash mutant, and probably not surprisingly in the
08:53:53 crash mutant horizontal junction also have this alternating frazzled and vain, distribution, and so there is no significant difference between the distribution or the CO occurrence for wild type or crash new injunction at those horizontal junctions.
08:54:09 And the reason I think that's interesting, is it suggests that maybe the.
08:54:18 Well, if we assume that we get this phenotype, because the crash malt liquor sorry the cells are molecule cannot undergoes this clustering.
08:54:28 It suggests that maybe this clustering could be something that is regulated along the PCP access, such that says clustering may occur sort of preferentially at vertical edges, but maybe not horizontal edges and we can speculate about that and talk about
08:54:44 that model, at the end.
08:54:49 So,
08:54:49 what we observe in a crash mutant is potentially the formation of symmetric bridges and alternating clusters along the edge.
08:55:03 And that somehow sis interaction and clustering of cells are one overcomes this tendency of, of the proteins to form, alternating clusters, perhaps through a confirmation will change, which would be really interesting.
08:55:22 And that somehow that system interaction and clustering itself, or one promotes the formation of the asymmetric bridge, helping to align them on the same orientation.
08:55:35 I should say that paper is no longer in revision, and it's now.
08:55:39 It's now published.
08:55:41 Okay, so I'm going to stop there. I had a little bit more, just in case I was going to go through this really fast, but I think, in order to just tell one story.
08:55:56 I think I'll stop there.
08:55:57 But let me, let me just very quickly. Acknowledge the people who did the work.
08:56:05 Where are you, here we are.
08:56:09 So, today I told you a story that was pioneered by a postdoc in my lab serious daily.
08:56:15 This is Sarah here.
08:56:18 And she has recently started her own group at Penn State.
08:56:22 We're all very excited about because job market was tough, in encoded times but she just left to start her own lab last month.
08:56:30 So yeah, I want to acknowledge her contribution to this work.
08:56:34 And with that and we'll go back to the model, and take any questions.
08:56:39 So thanks for your attention.
08:56:44 Hi. I have two questions. One is.
08:56:50 So do you think that this asymmetric bridges are due to.
08:56:58 It has to be within each cell right so you could, like, you can't swap across across different cells. Right. That's right. Okay.
08:57:09 And then my other question is are a bigger picture question which is, is unknown what decides what is that that result is, enter ending is posterior because it could swap.
08:57:22 Theoretically, using the same mechanisms Yes.
08:57:25 So it is not known what makes result in bang interior Yeah, yeah, yeah.
08:57:33 at least not not in this, not in our system. So, maybe the best understood, is the fly wing. And in that case, it's thought that there is a directed transport along micro tables of frazzled towards one side of the cell.
08:57:53 And that's enough to give you this little bias.
08:57:56 And then that the thing that oriented the micro tutorials.
08:58:02 Is the fat DAX the system,
08:58:05 which is, you know, expressing the gradient and overlays The, the entire wing.
08:58:12 Whether biased, you know, directed transport occurs in the system.
08:58:19 We don't really know, but but there. There are obviously micro tutorials oriented in one direction at the time this is happening.
08:58:28 If a bunch of questions I might just ask moderate so that we don't all jump in at once I think Boris was next.
08:58:49 Then I'll thank you so much for a wonderful talk and
08:58:43 I have to two questions so one is still about the picture that's on the screen.
08:58:51 So normally, let's call it the yellow and red. The wild type of anti correlated to you saying that they actually correlated, but in the data. It looked like they were just uncorrelated.
08:59:07 Right. There are three possibilities right so if it's uncorrelated and sometimes it will look like, you know, read as against read.
08:59:15 So in the data when you quantify it. Do you really have the evidence of.
08:59:20 Right, so then the simplest hypothesis would be that that anti correlated in the wild type, and the anti correlation is lost.
08:59:29 Right. but in that case, I would expect
08:59:34 everything to just be sort of mixed right the resolution is only 120 nanometers.
08:59:41 So, that suggests, very, you know, a lot of molecules that are the same.
08:59:57 And another patch of a bunch of molecules that are the same, but the two things one is correlations or anti correlations across belief. Yeah.
09:00:00 And another one is clumping correlations within.
09:00:13 And you suddenly have the evidence that red correlates with red in the stadium reflect. Yes, but I completely agree with you, we cannot say whether that is basically a homophobic or symmetric bridge, because we can't resolve the two sides absolutely so
09:00:24 this is my question. My next question my other question.
09:00:30 They'll all be.
09:00:33 You told us that salsa and crash so the wild type and crash have different densities.
09:00:41 Hmm.
09:00:43 Do you see different densities of bang, and, and,
09:00:52 and for result in the, in the crash mutant in this mutant.
09:00:59 So, we haven't quantified it as density per punk de in Sim, so all of the density quantification we did by storm and care to the sites, and never looked at frazzled and bang and in that context, but I guess by just looking at, you know, without quantifying,
09:01:23 it looks to see that seemed like the density is pretty similar.
09:01:29 But yeah, we haven't we haven't actually measured that. But then if.
09:01:34 But you told us that.
09:01:37 Once it's a classmate it's a cluster so then I would say that if salsa is a crash. Okay Newton salsa is at lower density that should be at lower that's right right and that's why I say, I don't know if we can measure this bye bye sim and the way that
09:01:50 we can with storm right i mean there you can count like individual molecules.
09:01:56 can with storm right i mean there you can count like individual molecules. I think that's a hard, it's harder to know with this other type of imaging. Yeah.
09:02:02 Thank you very much.
09:02:04 Jocelyn, you had a question.
09:02:06 Yeah, thanks, you know, thanks to you then after the talk.
09:02:10 Two quick questions.
09:02:12 First, when you reach.
09:02:15 He kept areas. Yes Do you see you have this quick recovery in some cases, does it does it come uniformly or does it is it is it molecules which are already bound and that migrate along the membrane, can you see that, I think, I think, Jennifer Dana and
09:02:33 could see something like that. In one paper. All right, so I would have to ask Sarah, I don't remember if she can really see sort of lateral, you know, filling of the space from from those like lateral positions in word or whether it comes up.
09:02:51 All simultaneously presumably from like new molecules, being exercised toast into, into that region, I'm not sure.
09:02:58 I have to go back and look at the data, I'm sure the data will tell us but I don't remember.
09:03:03 Maybe I emailed you about it. And then another quick question.
09:03:09 That was.
09:03:18 Sorry.
09:03:12 That's escapes thing just now.
09:03:15 Oh yes, in at day 12.
09:03:36 You've shown us snapshots, they travel, they 14 they drive it was looking like it's in a gated, and on TV. Yeah, something mechanical happening at the same time, there is there is we actually have a paper that we published about that from 2016.
09:03:41 And there is a huge stretch mechanical stretch of the skin along along the spine, along the medial lateral axis of the embryo.
09:03:55 I think it's because of like you know whatever the more underlying more for genetic events of the spine are. That's really really stretched the skin, and that corn coincides with the very first.
09:04:08 The very first signs of asymmetry in the system. And so, yeah, that paper was trying to dissect the link between that cell shape change.
09:04:18 And the asymmetry so for example if we take the skin off and we stretch it in the different direction in the opposite direction we then get a corresponding rotation in.
09:04:27 In the polarity to some extent.
09:04:30 Okay.
09:04:32 Interesting.
09:04:32 Thank you, David. The Penske.
09:04:36 Can you unmute yourself. Thank you. Hey yeah, I'm sorry my bandwidth is kind of bad, I'm not going to turn on the video, but, um, I was wondering if you knew anything about what sets the size, the pump, for example, are there that are individual comfortable,
09:04:54 or, or smaller.
09:04:57 We don't know
09:05:03 the size of the puncture is different in crash, but not by a ton.
09:05:09 Um, so I feel like maybe that's not the best mutant.
09:05:14 The biggest difference is that like the, the density of molecules within those those.
09:05:23 Um, maybe, maybe some of the cytoplasm what components, would be necessary for this pump take distributions that would be the first place I would look maybe dishevelled mutant are critical mutant.
09:05:34 There are multiple there three to shovels in mice and they're like four prickles in in mice and so it's not clear which one is the major player in the scan and so that's kind of why we haven't gone that direction.
09:05:50 See Benjamin assignments.
09:05:56 I thought was a really cool talk. Sorry, I have a rather basic question, what's, what's PCP for in the skin, and of course they want to ask you know what's the phenotype of the mutant.
09:06:06 Ah, let me just maybe you might miss the first little bit.
09:06:13 So, here.
09:06:17 PCP is to align all of the hairs, or scales or feathers, depending on what kind of organism, it is but for for mice, it's to align all the hair follicles, in the same direction, so it's to orient their growth towards the anterior so that the hair points
09:06:34 towards the post area for talking about the back dorsal back skin, and the phenotype is a complete randomization of of hair follicle growth in the question mutant in the crash mutant or vain mutant or physical mutant Yeah, all of them comes.
09:06:54 David Nelson you have a question.
09:06:56 Yeah, thank you. from the also for the lovely talk.
09:07:01 If we zoom out a little bit and look at fingerprints.
09:07:06 There's a local.
09:07:12 It's I wouldn't call it polarity it's more of a two fold a cemetery, or the millimeter scale undulations of fingerprints correlated with the kind of cell polarization of skin that you've been talking about, are they just independent.
09:07:48 And so I think finger prints are thought to be due to buckling that happens due to the growth patterns of the skin. Wow.
09:07:41 Um, and I don't know if that has any correlation to the, to the polarity in this system at all. I wouldn't say, No, there's no, there's no hairs on my fingertips so I can't.
09:07:56 But if there were here so my fingertips I could tell you
09:08:01 what as you say there is a polarization there, even without the hair follicles are there can be. That's right, that's right.
09:08:07 And so, I don't know what the relationship of those polarization patterns and for example if they swirl it all at the at the fingertips which, you know, would be a bit like a world you know except that it's at the know the ultimate positioning within
09:08:23 a tissue right it's at the tip of the tissue where you tend to get things like worlds like at the top of your head. That's right. Um, so yeah I guess I would maybe predict that there would be swirly patterns of clarity to now what comes first and what
09:08:36 comes second. I don't know, I'm not really sure what's causing effect there.
09:08:40 One other thing I wanted to say, Oh, I'm in the fly when they're ridges.
09:08:45 And they're somewhat reminiscent of a fingerprints, and the ridges are related to the clarity, the clarity.
09:08:59 Yeah. Thank you.
09:09:03 Denise. I hope I'm not butchering your name.
09:09:07 That's That's perfect. Thank you so much again for the awesome talk is very very beautiful looking data as well.
09:09:13 I kind of had a question about this.
09:09:17 So I'm still a little puzzled. I understand that self sooth obviously very important for this polarity establishment But fundamentally, it also seems like there needs to, like, fizzled and bangle on like the opposing junctions of neighboring cells also
09:09:33 seem to have have to have some rule it so that like you get alignment. It can't just be mutual inhibition.
09:09:41 Really, and I don't want to, at all think that like this explains everything explains, you know, sure, tiny bit.
09:09:47 I guess looking at the data. My question was, could it just be that there's like, and this, I apologize, because I can't propose any good experiments because I don't know enough molecular biology to like propose experiments for this but could the social
09:10:04 physical complex, and the social bangle complex somehow have a fundamental difference that's reflected in this could hear in domain of seltzer like code that interaction effect that domain specifically.
09:10:18 That's what's your breaking maybe with the crush.
09:10:23 Right, so that is our absolute favorite hypothesis so we if we go back and just look at these molecular interactions I've drawn seltzer as a, as a perfectly symmetric on either side of that junction, but there's evidence from just offload that Flamingo
09:10:40 can have different forms depending on whether it's paired with frazzled, or whether it's paired with bang. And so this woman go, sorry it's the Flamingo okay sorry, let me go with some certain place.
09:10:55 So there's evidence implies that Flamingo depending on whether it's paired with frazzled, or whether it's paired with bang, that it behaves differently, right, it behaves differently in the genetic context.
09:11:08 And so,
09:11:11 you know, an idea is that maybe Frizzell promotes a confrontational change in the seltzer or in that case, the Flamingo protein that would somehow, and that could that confrontational change could act through its homophobic binding interface, such that,
09:11:28 that now favors interaction with say the vein bound form on on the other side. So that would be a really nice model for sort of symmetry breaking that would actually induce as a confirmation will change in Salesforce, that it now favors pairing with the
09:11:48 main form.
09:11:50 form. I guess this is also why I was asking this question earlier about the, the suspension suspended cells. Yeah, wondering if they had for his older bangle because if they don't you could imagine potentially making different colored lines.
09:12:06 With that, like, so that is that we're doing right now is to try to use this.
09:12:10 The fact that this cells separate when there's something different about the music that he said interface.
09:12:17 The fact that sells me the aggregate but they sort out from one another. We think that sorting out kind of is reflecting something of a confirmation change right like we've got something that's different about the binding interface they'll sort out, and
09:12:29 then maybe we can use that experimental system, and that behavior of sorting out, ask okay well like, what if we put frazzled in there, does that, you know, mediate sell aggregation only with the van paired, does it make it sorted out from a sales are
09:12:44 expressing the loan, sell so we're hoping that that system could maybe be used to detect this like, you know, possible confirmation will change, but ultimately like we're starting to get into like biochemical questions here that will ultimately have to
09:12:58 be resolved as biochemistry and not my expertise.
09:13:03 resolved with biochemistry and that's not my expertise.
09:13:05 Thank you so much. Sorry my computer crashed for a moment. And I missed the beginning of, of your explanation here. But, and maybe you're the explained it, but I was struck that the in the beginning when you were introducing this.
09:13:20 You didn't tell us that there was no notion that Frizzell then bang interact directly with a result is a receptor.
09:13:28 Yeah, as the view of this changed.
09:13:32 Whatever last 15 years when I learned about this 15 years ago they were interacting. Yeah. So if you express in as two souls and just awful frazzled and bang and mix them together, you will see accumulation at this also interface suggesting that they
09:13:52 can interact.
09:13:54 You can also attach vaping to like a bead and put frazzled extracellular domain, and it'll bind. And so there is some evidence that frazzled combined directly to bang.
09:14:09 The reason we like to put seltzer in between is.
09:14:15 It's not clear whether they the extracellular space can accommodate this interaction, I mean bang does not mean it basically does has hardly any extracellular domain at all.
09:14:26 It just has these tiny little loops that are about 20 amino acids, long,
09:14:33 and also that, because when you get rid of Flamingo in the, in the fly system, you know, frazzled and bang are basically just like gone diffuse you know they're barely even getting to the junction and so you at least need seltzer there to stabilize them
09:14:49 at the membrane and hold them there. But it might even be the case that it's is the bridge.
09:15:03 you need, sort of more than occasion you need polarity. So you need this hetero demonization of something.
09:15:13 Right.
09:15:14 And you already have this sort of very obvious Natural History memorization. Yeah, right, like why are you here you should get rid of.
09:15:24 So I. And, you know, they're all sorts of plausible reasons why frazzled Can you know as ligand receptor cannot really stabilize and interface cannot really fine.
09:15:37 But, so need strong adhesion conceivably also strong clustering as you were telling us.
09:15:47 In order to then stabilize the hetero Dima, but but this hetero dimer interaction is of course as you were explaining is essential in order.
09:16:08 And, And what confuses me is that somehow this idea that there is direct interaction
09:16:17 plays a role it has disappeared.
09:16:21 Direct your action of frazzled and bang, bang, so I'm just trying to understand what's the sort of really what what's the evidence that they do not interact because I think logically.
09:16:33 It presents several major puzzle, then you have to say, well, that they don't interact, but they modify, they make sort of at least naively symmetric cells interaction asymmetric but you already have a symmetry that that's what puzzles.
09:16:49 Right. So, and maybe that's why I start to
09:16:57 maybe ignore the possibility that they interact directly because we know that the Flamingo cells are just its presence and its ability to add here, and mediate adhesion is so essential for the formation of a symmetry, that I feel like that alone tells
09:17:19 you that, you know, the interaction between frazzled and bang that header, typical interaction across boundaries isn't enough. So that's not to say it doesn't, it's not there it's not happening it doesn't play a role but it's absolutely not sufficient.
09:17:33 You need this seltzer molecule to be involved, and whether you know I think for a long time, it was just thought that it just holding stuff in place stabilizing things, but I think this data shows that no it's more.
09:17:48 It's, you know, it's more active in promoting the formation of the asymmetric bridge, and almost counteracting either a Hemi bridge or, or, or symmetric bridge think we need better resolution as you as you pointed out before we can conclude that.
09:18:07 So yeah, I think that that interaction could definitely be happening, the personal bank interaction is just not sufficient to explain everything.
09:18:16 Thank you. Thank you.
09:18:18 If tomorrow questions Dinah lucky.
09:18:22 I wanted to ask about something that you said at the very end of your talk that I found very interesting. So at the end you were saying that. So, you were, you're making the comparison between the wild type versus the versus the mutant, and you were comparing
09:18:38 that to the patterning you actually see in the horizontal interfaces. And so, first of all, the question was, Am I understanding correctly that you could say you're working hypothesis that the difference between what type versus mutant is maybe the same
09:18:52 as the difference of vertical versus horizontal in your, in your what type. Yes, absolutely. Okay, maybe sell services clustering happens at vertical borders and my dad regulated to occur at vertical borders and not at horizontal borders, this is super
09:19:11 speculative right okay well evidence for this at all. But, oh, yeah, that's exactly the idea, if, if that is the case so I work in often up but. So we have the same like we always make the same cell drawings as you have in the beginning, where we have
09:19:26 these like perfect hexagons right and we have you know perfectly vertical interfaces and like perfectly horizontally interfaces. But of course, the reality is that in many cases we have kind of like intermediate behavior.
09:19:37 And you were saying that in your in vivo system the cells are quite dynamic because of you know cell division and whatnot. So I'm guessing that you probably have a number of cells that are deforming right in response to, you know, division.
09:19:51 And so as a result of those deformations you would probably have some interfaces that change orientation over time from being perfectly vertical to going somewhere into the horizontal direction.
09:20:02 So I'm wondering whether you had already done the experiment where you take one of those vertical interfaces and track it over time as it becomes more horizontal.
09:20:12 And then what happens to the patterning right so if you actually assume that the patterning changes from like vertical type patterning to the horizontal type patterning.
09:20:22 You know that that would also offer inside and obviously into the changes that happened between what type versus the mutant right.
09:20:30 Yes.
09:20:32 So, this is not exactly the right slide to show you that that does happen.
09:20:38 This slide was just really to show you a new tool that we built that allows us to to do exactly what you're what you're asking that is to track vertical boundaries over time.
09:20:48 So, this is indigenously tagged result with three copies of GFP in mouse.
09:20:58 Yes, so it's inserted into the endogenous Lucas and it's homozygous it's viable and fertile and happy and doesn't have any phenotypes the distribution is polarized like we would imagine and you can.
09:21:09 This is a six hour movie just showing that like it doesn't bleach and it maintains clarity over time. But if you zoom in to individual clusters themselves, you know, and watch them over time over these period.
09:21:22 Yeah, their neighbor junctions are shrinking cells are rearranging cells are dividing. It's super dynamic and so the little local chip there tons of local changes in polarity, where borders gain and lose clarity constantly, but if you, you know the average
09:21:40 polarity is maintained over the tissue over the entire time. So I think this is going to be really fun and interesting to to watch the dynamics and just, you know, just successfully made the tools for seltzer, and for bang we've got them all homozygous
09:21:55 now so that'll be the future finally came out finally in mouth so we can do this. Yeah.
09:22:04 Great. And we have one final question from one. I have one question, just because you are discussing here about the global architecture of their symmetric business in skin cells so my question is, how much robust is against like any local perturbations
09:22:22 like, if any, if it is that some sort of local partnerships happen, will it maintain its shape, or a symmetric shape, or you have find some interesting by incorporating some perturbations in there.
09:22:36 Thank you.
09:22:38 So I would say the average polarity is very robust in that there, you know, a lot of divisions and some rearrangements that are occurring across the tissue.
09:22:48 And, on average, the polarity is maintained. So that to me means that, you know, if, if a junction loses the polarity it can regain it over time.
09:23:03 Larger product so that's like division, you know differentiation things like that that are happening all the time in the skin. Other perturbations like a wound.
09:23:08 That's, that's much more severe that causes shifts in polarity that become propagated over summer slow distances.
09:23:19 And it's not clear, to what extent they will restore after, you know, after the moon closes and heels, so a big perturbation like cutting this and you know messing with the ability of cells to communicate with each other.
09:23:32 This system is not super robust to that but it's, but it is two things like division and sorry majors.
09:23:39 Thank you. Thank you.
09:24:01 Well, we have one more. We have one more question and then I think we should close it out so it's great that we have so much discussion but we'll close it off.
09:23:57 I'm sorry you.
09:23:58 Thank you so much for taking another question. Um, so, it was very nice plots were we were talking about anti correlation or lack thereof the bread minus the greens to is nice weekly things.
09:24:10 Um, I think the question I have is could you explain the degree of asymmetry simply as a function of density meaning imagine I did read. Mine is green, as a function of red plus green.
09:24:22 So red plus blue green kind of giving an overall sense of how much stuff is that at that location along the memory and read mine is green, being the kind of asymmetry locally.
09:24:34 If you just took all your data and plotted read mine is green, but as a function of red plus green.
09:24:42 Could Do you think you could explain all the symmetry purely in terms of density variations.
09:24:47 So is it just for noise because idiot theorists like myself write these equations and that's actually the input we put in. No jokes that's the input we basically say that as a function of total amount of stuff, once there's enough stuff, the interactions
09:25:03 will make the system Polaroid's right basically depends on density, that's the crucial thing that we always say, Yeah, so being able to measure the density.
09:25:13 And I think you're the. You're the first person that could make that measurement because you can see the split.
09:25:18 You can see the red line is green that's the part that we will always missing. I think that's a really neat idea, I would love to play around with that, I certainly need to talk to Sarah, about what the, you know what the data coming from the sim
09:25:39 really looks like, you know, because I could imagine with storm for sure absolutely, but but maybe even, you're right, maybe even the separation of red and green in symbol would allow us to do that too.
09:25:52 That's a fun idea I think you
09:25:58 guys I think this was our last question, um, thank you again for a great great great talk and thank you everybody for taking participating discussion.
09:26:09 We hope to see you again next month.
09:26:12 Next month we have a seminar on March 24 and we'll have Marina particular from Harvard.
09:26:20 And if you want to see the top again or recommended to other people, it's going to be recorded and on the KITP website.
09:26:28 So, so that's it again. See you soon. Thanks everybody had a lot of fun. Thanks for all your questions. Yeah.