 Okay, well, this is what I get for saying it's a remote speaker today, but we have a pretty healthy audience online, and we have about a half dozen people in the room. So I'm going to go ahead and get us started. So welcome back everybody to the SMU physics department speaker series. Today we have a in the spirit of this being a hybrid event. We have Dr. Veneda Gordon joining us from UT Austin remotely, and so she'll be sharing her slides and everything they're up here on the main screen. She's going to see a camera view of the room. So hopefully when I run around with a microphone so you can ask questions, she'll be able to see who's basically speaking in that window. You will not be able to be heard unless you have the microphone and it's very unit directional. So make sure you hold it right up to your face and talk into it straight on. If you have a question during the event, I'm going to be sitting up front. So just say back to school or whatever, Steve, I have a question and I'll run over to you with the microphone and we'll we'll politely interrupt Dr. Gordon for her presentation. Okay, without further ado, I'm going to hand things over to our host today, Dr. Jodi Cooley to introduce our speaker. So take it away. Hello, everyone. It's a great pleasure that I have in welcoming Veneda to our seminar series. I first met Dr. Gordon several years ago. When we were both officers in the American physical society, I was an officer in the Texas section. I believe you were in the bio. I was in the bio. Yeah. Yeah. I'm not even sure what they call it on section, but we were at a leadership convention and as part of that sort of convention, what we do is we go a day early and we lobby Congress on behalf of the American physical society to try to improve the funding situation for physics and science in general. And so it was a really nice day that I got to spend with Veneda up on the hill. But let me tell you a little bit more about her credentials. Veneda Gordon is an associate professor in the Department of Physics at the University of Texas at Austin, where she has been on the faculty since 2010. Her research group studies biofilm forming bacterial systems with a view towards understanding how physics and biology interplay and how they impact disease course. She did undergraduate work at Vanderbilt University and graduate work at Harvard University as well as postdocs at the University of Enerboro and the University of Illinois, Urbana, Champaign. She likes doing science most of the time, but she also likes running, science fiction, singing, knitting, and spending time doing fun things with her family. And I kind of like this last point that she told us she sure wishes the pandemic were over. I think as we all do. Veneda, it's a pleasure to have you here today. I hope someday we can actually get you to come visit SMU in person. And I'm really interested to hear more about your talk or more about the topic of your talk. Thank you, Jodi. And thank you, Steve, both of you for inviting me and for that very nice introduction. I am going to share my slides and you should all be able to see my slides now and someone's going to yell at me if you can't, right? Yeah, no, we can see them. Can you hear me OK now that I can hear you? Yes, OK, so just from the very beginning of the talk, you should know that I really like being interrupted. I don't like to listen to myself talk for 45 minutes. I would far rather you interrupt with questions. If that leads us to not finish the whole talk, then that is OK with me because I've heard this talk before. So I'm not going to be sad if I don't get to hear the whole thing again. So please do interrupt with questions. And if Steve, if I if someone is raising their hand in the online Zoom meeting and I miss it, will you be able to to pick up on that? Yes, I'm on doing that, too. OK, good. So today I'm going to talk to you about bacterial mechanosensing of substrate stiffness during early biofilm formation. And to begin with, I want to say that the people who really deserve the credit for this are Leanne Wang, who was a postdoc in my lab. And you turn, Wang, who goes by Chad, Wang, who is a graduate student who's still in my lab right now. They are both really great scientists and great people. It's been super nice to have them be in my group. And they are contributing a lot more to this talk than I have. So. I gave this sort of a semi subtitle, which is a tale of two stages and two sensors. And this is meant to echo the title of a tale of two cities by Charles Dickens. I am not on the whole a giant fan of Charles Dickens, but I do like a tale of two cities. And if you're going to read one thing by Charles Dickens, I suggest this be it. And what I'm going to tell you about is two stages early in biofilm development when bacteria are attaching to the surface and how they respond to that surface to know to start making a biofilm. And I've just used a lot of words. You may not know what they mean. So let me go on and talk about what they mean. But before I do that, I want to step back and give you some context. So you carry out Excel cells that have a true nucleus, which are like the cells in our body or your dog's body or a tree or whatever. They are known to respond to the mechanics of their environment. This is known to be super important. This is a widespread field of study. And one of the things they do is mechanosensing, which is just sensing mechanics. And they are known to do that in several different ways. But one of the things they do it is by sensing the stiffness of whatever substrate they're attached to. This is known to be super important for controlling stem cell differentiation. So by changing only the substrate stiffness, you can change what type of cell a stem cell will differentiate into in some conditions. It's important for controlling whether or not the cell is as happy, like whether or not that things that should undergo program cell death or if it should keep growing or not. It controls things like migration on the surface, which is super important for things like wound healing and in controls, things like spreading. So this is it's well known that substrate stiffness really matters for you carry out excels. A lot less is known about how substrate stiffness matters for bacteria. And the particular way we're going to say, does this matter for bacteria is we're going to ask, how does it matter for biofilm formation? So a biofilm is a community of many bacteria that are bound together in a matrix. The matrix materials can be produced by the bacteria. Also, they can also uptake materials from their environment. And when bacteria are in this biofilm, it makes them really resistant to the immune system and really resistant to antibiotics. So they are a real problem in medicine. So most chronic bacterial infections that don't respond to antibiotic therapy and can't be cleared by the patient's immune system are in the form of biofilms. And these biofilms can form in a lot of scenarios in the body. So they can form in soft tissue or really they can form in the mucus of the lungs, which is always classed with a soft tissue infection, although I always say is a mucus really tissue, but whatever. That's not the point. They can grow on implanted medical devices like like orthopedic implants. So here's someone whose hip implant is maybe having a bad day. They can grow on other scenarios. So the particular organism that we are going to look at is a big problem in eye infections, so you really don't want it to get it on your contact lenses. And they can grow on other types of materials here. You're seeing examples of dermal filters. Here's an example of a stiffer material that's not quite as stiff as an orthopedic implant. These are catheters and this axis here shows the kind of range of stiffnesses of materials that these bacteria can be exposed to and are known to be able to colonize in the body. So how do bacteria respond to these surface stiffnesses? And that hasn't really been known. But what has been known is that bacteria when they attach to the surface can sense that they're on a surface and can undergo a change in phenotype or a change in their gene expression that's controlled by the production of a signaling protein that makes them switch from being kind of a free swimming bacteria that can explore space freely to a bacteria that's making a lot of sticky stuff that will become a matrix so it will stick together in a biofilm with a lot of other bacteria. I should also say because this is a general audience, I'm just using the word bacteria. But if for those of you who are interested, the specific organism that we're studying here is called pseudomonas aeruginosa. It's an opportunistic human pathogen that forms biofilms in lungs and in chronic wounds and in the eye and on biofilms. It's a problem for people with cystic fibrosis and chronic obstructive pulmonary disease and diabetes, as well as people who just get really bad burn wounds or motorcycle accident wounds, things like that. If you don't want to remember the name of this organism, that is completely OK, too. Do not stress out about the organism name. So how are we going to test whether or not bacteria can sense and respond to the mechanics of the surface they're on? In other words, how are we going to test whether or not they're doing mechanosensing? So one thing we need is substrates that have the same chemistry, but different stiffnesses because we don't want to have chemical differences between substrates that could provide a confounding effect where we wouldn't know whether the bacteria was actually responding to chemistry versus responding to mechanics. And we if we want to know what the bacteria is using to do the mechanosensor, then we need to use genetic knockouts that are missing key mechanosensing proteins. Our proteins, we think, might be mechanosensiters. And the reason for that is if we see some behavior that seems to indicate that the bacteria are differentiating between surface mechanics and we knock out a sensor and that differentiation goes away, then that says that sensor probably has a role in sensing and responding to surface mechanics. But if we knock out a protein, we think might be a sensor and the bacteria still respond just the same to the different surface mechanics. So the differentiation is preserved. That suggests that that protein probably isn't an important sensor for whatever process we're looking at. I think I'd like to stop here because this is sort of the end of the background section of my talk and ask if there's any questions before I go into experiments. Yeah, any questions in the room? Oh, yeah, yeah, there's a question in the room. Let me bring the microphone over. OK, thanks, Lucas. Hi, can you hear me well? Yeah. OK. So I don't really have any background information on stiffness. So how exactly is stiffness measured? Because I saw on the previous slide it was measured in Pascal, which is pressure. So can you just give a little more background information on that? So you're right that Pascal's is the SI unit that we use for pressure. And when we use it as pressure, a pressure measurement, we think of it as a force per unit area. If you think about what that means, if so, you have a fraction that's forced on the top and area on the bottom. If you multiply both the top and the bottom of that fraction by link scale again, what you get is something that looks like energy per volume. Does that make sense? Sorry, he said yes. OK, so if you have an energy per volume, that is basically telling you the energy cost per unit volume of that material to produce some type of deformation. There's different types of deformation, right? So you might remember from your first semester mechanics class, maybe you talked about stretching, and that was one particular type of deformation. There's other types of deformation that maybe you didn't talk about in your introductory mechanics class, depending on who you had and how much time was in your semester. So there's shearing where you can displace one face of a material with respect to another. There's compression. Those are the main ones. You don't super need to worry about what type of deformation we're talking about here. We're mainly talking about about kind of a sheer deformation. But if you want to think of it as being like a young's modulus for stretching, I think that's close enough for what we care about. Does that answer your question? Or do you have any follow up questions? No, that helped. Thank you. Yeah. OK, any other questions from the room or? Oh, I'm sorry. Yeah, Pavel, go ahead. Yes, well, thank you. At some risk of detracting you from the main topic of your talk, I already find it fascinating that the bacteria can respond to the thickness and basically adjust its stickiness, if you like. Are you going to discuss how exactly it senses it and what exactly? Well, you mentioned that there is a protein released, but what exactly causes the release of this protein? And maybe say a few words of what happens at the chemical level, which changes this or triggers this response. OK, so in part, the answer to your question is not completely known. In part, I'm going to address it in my talk. But let me start with what was already known before I ever got into this field. So the bacteria that we're talking about have kind of little protein clusters on the surface and it's known that if they have those clusters, if you haven't knocked them out genetically, then when it attaches to the surface, those clusters will somehow give rise to a signaling molecule, which is called cyclic dye GMP. This cyclic dye GMP molecule changes the expression of a lot of different genes, including the genes that control the production of the sticky matrix material. So that signal level goes up. You could produce more sticky matrix material and and it starts to stick to the surface more strongly and then as more matrix material is produced, you know, it kind of starts to form a glob of other bacteria. More recently, but this is still not original to us. People learned that in addition to having these kind of clusters of proteins on the surface in order to respond to the surface, there was a protein called PIL Y1, which you will see later in this talk, that seems to be really important for activating signaling. So if you don't have this protein, they don't increase signal levels when they attach to the surface. They don't get the trigger that they need to form a biofilm. Now, this was already and it's also important to say, this is a protein that's embedded in the cell envelope. So the envelope of a bacterial cell consists of for this type of bacteria consists of a cell wall and a lipid membrane. And so this is embedded in that envelope. So whatever the envelope experiences, presumably this protein should be able to get some readout on that. Everything I've said up to now is not diagnostic that these bacteria are sensing the mechanics of the surface. You may think it's suggestive, or you may not. But there was not really consensus on how the bacteria know that they were on a surface. A few years ago, we and a few other people started publishing things saying that bacteria were responding to mechanical cues in a way that was linked to the development of these biofilms. The mechanical cue that we studied before was to do with friction with the surface as the bacteria moved along the surface and sheer flow of fluid over the surface. Which is, which are both mechanical cues, but not directly linked to the mechanics of the surface itself. There was another group which published a readout, not of biofilm formation per se, but of the activation of virulence factors, which are things that harm the host, and showed that stiffer substrates did better at activating the production of virulence factors. These hosts is harming materials. So that was a clue that bacteria could sense the mechanics of the cell substrate. What we published earlier was a clue that bacteria could sense mechanics from the environment in a way that was linked to biofilm development. And now we're trying to say, can we make a direct link between substrate mechanics and biofilm development? Does that make sense? Oh, very good. Yeah. Thank you so much. Yeah. So that's again, obviously the details are very well subtle, but it explains what is going on in broad, very broad terms, of course. Sure. Sure. No problem. Anybody else? Yeah. Any more questions online or in the room? Okay. I think you can go ahead. All right. So, so we're going to test whether or not bacteria or sensing mechanics by creating substrates that have the same chemistry, but different stiffness and then measuring the bacterial response when they're attached to these substrates. And to identify what's important for bacterial mechanic sensing, we're going to try some genetic knockouts that are missing key mechanic sensors. That's our plan. So we want a way to have substrates that have the same surface chemistry, for example, the same level of stickiness for the bacteria, but have different mechanics. And the way we're going to do this is we're going to use gels. These are, in our case, agarose and alginate gels. So for those of you kind of on the soft matter chemistry end of things, you will recognize that agarose and alginate gels are not exotic at all. These are gels you can, you can buy the materials to make them from Sigma Aldridge and they'll show up in your office later in the week. What we're going to do is to make these gels on glass cover slips. We're using glass cover slips because we're going to use a microscope to investigate what the bacteria do in response to attachment. And we're going to make the gels with two different thicknesses. We're going to make an ultra thin gel that's about five microns thick and a thick gel that's about 150 microns thick. And the reason we're doing this is because if you, you can do some analytical modeling and some approximations that are good in the limit that this gel is much thicker than just this gel and also that this glass cover slip is much thicker than any gel involved. And what you get is an estimation of the effective modulus of the composite material that's made up of the gel plus the cover slip. This shows you the estimated effective modulus plotted in orange for some constant bulk stiffness of the gel with different ratios of the thickness of the gel to the thickness of the glass. So for our experiments, the thickness of the glass was held constant. That's just a glass cover slip. That's whatever we buy from Corning. And the, the thickness of the gel was varied to change the effective composition of this, to change the effective modulus of this composite material. The mechanis sensors that we wanted to test on here are really two things. And the, these are plausible mechanis sensors to us because of our own previous work and because of the work of others. So one of the things that we thought might be really important for sensing the mechanics of a surface is the surface-associated protein, PIL-Y1. So PIL-Y1 sits in the envelope of the bacterium. And as I mentioned earlier, whatever the envelope feels that protein and principle should feel, it should be transmitted to the protein somehow. Other people before us showed that you need PIL-Y1 in order to activate the signaling that leads to biophome development. And the same other group pointed out that this protein PIL-Y1 has a weak structural resemblance to something called the von Willembrand factor. The von Willembrand factor is a like a structural factor motif, whatever you want to call it, that's found in eukaryotic proteins that has a mechanis sensitive function. It was first characterized and it's best known for its role in blood clotting. So the von Willembrand factor in, in its role in blood clotting when, when things switch from flowing this way to flowing out, like you've got a wound, it kind of causes this von Willembrand factor domain to unzip and that starts to trigger the blood clotting cascade. There are some other cases where the von Willembrand factor is known to play a mechanis sensitive role in eukaryotic cells. But the interesting thing that this other group pointed out is that this PIL-Y1 protein has some resemblance to the PIL-Y1 factor, a weak resemblance to the von Willembrand factor. So if it has this structural resemblance to the von Willembrand factor and that factor is known to be important for mechanis sensitive responses, it might make sense that this protein might respond to surface adhesion through sensing some mechano, mechanical cue. The second thing that we wanted to test were these appendages that come off the bacteria, that's what's shown here in this, this kind of fluorescence image are these things called type 4 PIL-Y or I might just call them PIL-Y if that's okay with you. These are small submicroscopic appendages of the bacterium that reach out, grab onto surfaces and then pull the bacterium forward. So think of it like you're pulling yourself forward with a bunch of grappling hooks or you're dragging yourself along the ground by gripping the ground and pulling forward. And both the we and another group had seen indications before that these type 4 PIL-Y were not only important for responding to the surface but they seem to have a different response to different mechanical cues. So these seemed like the most plausible candidate mechano sensors that might respond to surface mechanics. Should I pause here? Are there any questions? Yeah, any questions online or in the room? So I mean essentially this gives us a sense of how it is from a broad perspective bacteria can interact with a surface either through this surface associated protein or through these appendages. Is that right? Yes. Okay. And it's my contention which I have not proven at all. Oh, I see. Okay. That I haven't proven this yet because we've only studied one species of bacteria so far. But my contention is that we can pretty much understand all bacterial mechanosensing through this kind of two process framework. Okay. So if NSF funds my grant I can tell you more about this. If any of you review my grant remember that we can learn more about this. NSF we know you're listening. That's right. So the first thing we're going to measure is the accumulation of bacteria on a surface. So we have a suspension of bacteria. They were grown up in culture. Sorry, in a liquid culture. And now we're exposing them to the surface and we're just going to measure how many bacteria accumulate on that surface over an hour. So after an hour of incubating with a surface, we see that bacteria accumulate more on stiff surfaces than on soft. So in both of these, this is agarose in panel A and alginate in panel B. In both of these we see significantly more accumulation on the ultrathin gel, which is the stiffer effective composite than we do on the thick one. And that's consistent for that's what we call the wild type. So this is the bacterium that we haven't messed with. For the bacterium that's missing pili altogether, so it doesn't make these grappling hook appendages at all. We have ones that don't make the retraction motor that allows the pilus to pull back and do its function. All of these accumulate more on the ultrathin gel than on the thick gel. And if we look at the ratio of accumulation on the ultrathin to the thick gel, they're all about the same. So they're all three, three and a half times more on the thin gel. Except for the bacteria that are missing pil Y1. This NS means not significant. So these are not statistically significantly different from each other and neither are these. So what that's telling you is that bacteria preferentially accumulate on the stiff surface unless they lack pil Y1. And once they lack pil Y1, this preferential accumulation on the stiff surface goes away. That's significant in a couple of ways. One is it showing some sort of response to a mechanical property of the surface. And the second is it's showing that that response requires pil Y1 in order to happen. So from this, what we're going to say is that pil Y1, this envelope protein plays a key role in sensing substrate stiffness during attachment. So how might that be? How might that work? So to answer this question, we turn to finite element modeling. And this finite element modeling is done in Abacus. And what we're measuring is what happens to the bacterium when it hears to us of substrate? So the bacterium we treat as an inflated pressure vessel. So it's inflated by its own turgor pressure. That's because there's a bunch of stuff inside the bacterium, which kind of acts to provide an osmotic pressure. You have a thin wall, which is like the cell envelope of the bacteria. And then it attaches to some substrate. And we can vary the stiffness of the substrate to mimic the stiff or the soft substrate in our experiments. What we see here, and I'd like you to look primarily at this first part of this graph where we're looking at the contact zone, is that inside the contact zone, we see a greater change in the circumferential stress, the stress going around the bacteria, like you're going around the cylinder. When it's attached to the stiff substrate, then when it's on the soft. And these points here, they're more spread out. So you can see we did this measurement a lot of times. Here we still have the same number of measurements, but they're more clustered. You can see this here as well. This IAM and OM correspond to the inner and outer membrane of the bacteria. And we're looking at what's called the peptidoglycan cell wall between them. And we're looking at how that stress changes, the stress in the cell membrane changes upon adhesion. And in both cases, there's a greater change upon adhering to the stiff or substrate than the soft. We also measured the contact pressure and find a greater contact pressure on the stiff substrate than the soft. So also I want to point out, this is an equilibrium physics idea. The idea of something deforming in response to adhesion until everything's kind of equilibrated, that's very equilibrium physics. And one of the things that makes this project really appealing to me is the idea that we might be able to use very basic ideas from equilibrium physics to understand a biological process. If the PIL Y1 protein is embedded in the cell envelope, which is bracketed by these membranes, and they're experiencing much greater changes in stress when the bacterium adheres to a stiff substrate than to a soft, that might provide a way that the bacteria are able to sense the mechanics of the substrate. There's another thing that we can measure, which is an indirect confirmation of the modeling that we just did. So I just showed you modeling that said that we expect more membrane stress when the bacterium adheres to a stiff substrate than to a soft substrate. If we want to read out for membrane stress, we somehow need something that responds to membrane stress. And one of the things that we can do here is use something called a mechanic-sensitive ion channel. These are naturally occurring ion channels that are in these bacteria. They do anything to put them in there. They're pretty widespread in common. And what they do is when the membrane tension is high, they open up to allow more stuff to pass through the membrane. You can think of these as safety valves, because what the cell really, really doesn't want to do is explode, right? An exploded cell is a dead cell. So if the tension in the membrane is starting to get high so that you're in the risk of lysing, then it could be a really good strategy to open up holes in the membrane to let osmolites go out so that you can reduce the tension in the membrane and then you can close it back up and keep all your valuable stuff inside you again. So these mechanic-sensitive ion channels are open when the membrane is tensed and closed when it's less tense. And what we find is that they're more active or more open when the membrane is on stiff surfaces than on soft. This is two histograms overlaid with each other. We are using a fluorescent indicator that tells us how much intercellular sodium has been able to enter the cell and that these channels open up to allow the passage of sodium when the membrane tension is high. When on the thick gel, you see in orange that most of the cells have a very low fluorescence indicating not a lot of membrane channel activity. For the thin gel, you see the histogram in blue indicating a higher peak. You have more active membrane channels, more open membrane channels. This is another type of analysis of the same data where we're just looking at some of the key and values instead of a histogram of distributions of fluorescence. And they're both telling us that we get more open membrane channels on the stiff gel, which is consistent with greater membrane tension being induced on the stiff gel. This is what we would expect from our finite element modeling and it's what we think probably links to the differential response of the PIL-Y1 protein. We might also, you know what, I'm going to stop and ask for questions here before I go into the next step. What are your questions at this point? Any questions online or in the room? Let me ask a question here, Vernita. So as you're talking about this, of course I'm being the classic physicist who's trying to think of electrical circuit and mechanical analogies. And so as I'm looking at this, I'm thinking, oh gosh, PIL-Y1 appears to be some kind of piezoelectric material that maybe signals to the bacterium in some way to either go ahead and adhere to the surface or don't or form a biofilm or don't with your friends. Is that sort of biochemical, biophysical trigger the relationship between pressure and then the response of the opening channels? Is there a direct link that you can draw through these proteins through some kind of electrical analogy? I certainly cannot draw such an analogy. Whether or not such an analogy can be drawn, I don't know. I think though, so there's two types of analogy. One is kind of like a modeling-ish analogy where you say I'm going to model this process as this thing I know about. And those can often prove very powerful and insightful. There's another type of analogy where you say this thing acts like this thing. And that I think is more dicey here because whatever's going on with the PIL-Y1 protein and we don't know what's going on on a molecular level, something has to be inducing some sort of deformation in the protein, which is then somehow transduced into different signaling levels. I don't know how this protein is being deformed and I don't know how that deformation is being transduced. I wish I did. I'm sorry. No, I'm sorry. It's not like you can just like, okay, we're going to grab this protein at both ends and start twisting it and pulling on it and then measure its electrical response, essentially, to that. Well, there are people who do those types of measurements on proteins. We are in my group completely not equipped to do stuff like that. But I think, I mean, if someone did this, it'd be great and I'd be happy to work with someone on doing something like this. I don't think it's going to be an electrical response. I think the piezoelectric analogy in terms of a modeling approach is very appealing because that could give us an insight into how the components of the system might conceptually connect to each other. But my suspicion is that if we ever do figure out how this protein is deforming, we're going to see that it's really more changes in its structure and possibly in what chemical groups are exposed that somehow leads to this change in signaling inside the cell. That's really neat. I love it when things are not simple. So that's cool. Okay, we have a hand up from Pavel. Go ahead, Pavel. Yeah, so just to follow up on this very interesting discussion. Well, of course, when we talk about piezoelectric materials, we assume that they are just dialectics, quite simple materials. But this is a much more complex system. So how much do the observations that you make depend on the conditions and other very kind of control parameters which will definitely modify the response? So do you mean like the culture and conditions? Yes, but of course, while there are these parameters, again, like temperature or pressure, what else, but are there any other conditions that will affect how the bacteria respond? So the first answer to your question is we've not explicitly tested that. However, it's well well known that bacteria behavior depends on culture and conditions. So if I change the temperature in which we're growing these bacteria, changing the temperature, it won't change how fast they grow and how quickly they respond to things. If I gave them a different growth medium, that would change how quickly they grew and it could also induce additional changes in their metabolism. I've got no reason to believe that the fundamental principles that we're saying, like preferential accumulation on stiff surfaces over soft surfaces related to greater membrane stress transduced by COI-1, I don't think that should be affected by these types of conditions, but I certainly think that the speed of the response and the magnitude of the response will be affected by conditions. Okay, thank you. Sure. Any other questions? I don't see anything in the room or online, so I think you can go ahead and get going again. All right. So one of the things that we don't believe about bacteria is that we don't believe that they have precognition. Precognition means being able to tell the future, knowing things before they've happened. We don't think bacteria have that power. So that means that whatever is causing bacteria to differentially accumulate on these surfaces of different stiffnesses must happen after they've already encountered the surface. They don't have any way of knowing what stiffness of surface they're about to encounter until they encounter it. So the initial encounter rate of bacteria with the surface should be the same. And what we wondered is, well, how does that lead to differential accumulation on these surfaces of different stiffnesses? And one really plausible answer is, maybe they're equally likely to initially counter and initially stick to the surface, but then there's a difference in how likely they are to detach from the surface and go away. Because if you think about, like, if things are attaching and things are going away, one way to change the amount that accumulates is to change the amount of stuff that's going away. And if they change their likelihood of detachment after they've encountered the surface, then that doesn't require the bacteria to know the future, because now they're in contact with the surface and they can change their behavior to change their likelihood of detachment. So measuring detachment is a really tedious thing to do. And my postdoc, in fact, did wind up measuring detachment rates, and I'll spoiler alert, you get higher detachment rates from thick gels, which are soft, than from ultra-thin gels, which are stiff. However, something more insightful, I think, comes into measuring a behavior that leads to detachment. And this behavior that can lead to detachment is called spinning. These bacteria have a polar flagellum that I didn't talk about very much before because it's not super-duper relevant for this mechanosensing talk, but they have a flagellum that sticks off the end of the bacterium. And when they're in liquid suspension, this lets them swim. If the flagellum is stuck down to a surface, it can keep spinning like it's trying to make the bacteria swim. And what that does is make the bacteria spin in place. I have to go back to my spinning movement. Okay. And a group that I was a part of as a postdoc showed that bacteria that were spinning were more likely to detach from the surface. So that means we can measure spinning motility as a proxy for the probability of detachment. And we did this for bacteria on the ultra-thin gel up here, bacteria for the thick gel down here. Remember, ultra-thin is stiff, thick is soft. And then we can track these bacteria using automated tracking. And we can measure what their spinning speeds are. What we find is that bacteria that are wild-type that contain the PIL-Y1 protein are more likely to be spinning and are more likely to be spinning faster on soft surfaces than on stiff. So again, the orange is the thick gel, which is a soft composite. This orange histogram shows the likelihood of spinning. You see there's a long distribution out to high-spinning speeds. Whereas the bacteria on the thin gel, which is the stiff gel, are more likely to be clustered down here at these low-spinning speeds. This is connected to a greater likelihood of detaching from thick surfaces, from thick gel, soft surfaces, which is something else my postdoc measured directly is actually happening. But I think more importantly, or maybe of equal importance, this is an active response. So if the bacteria are spinning with different speeds and different likelihoods on these two types of gels, that is because they are controlling their spinning speed in a way that responds to the gel stiffness. When you go to bacteria that don't have PIL-Y1, their distribution and spinning speeds become indistinguishable. These are statistically indistinguishable from each other. So the active response to gel stiffness that leads to greater detachment from soft surfaces goes away if you don't have this envelope protein PIL-Y1. And these are the measurements that my postdoc did where she measured the actual number of detachment events and the probability of detachment, so the number of detachment events divided by the number of cells imaged and found that it was statistically significantly different for the wild type on the different types of gels but not for the bacteria missing PIL-Y1 on the different types of gels. So this shows that the difference in accumulation on these two surfaces arises from an active bacterial response to the surface that changes the likelihood of detaching from the surface. So what we've said so far is that when we have a mechanical input, which is attachment initially to stiffer soft surfaces, we get different cell membrane deformations and stresses, and we measure that in two ways by using finite element modeling and by measuring the activity of these mechanosensitive ion channels. And then we also have measured certain mechano responses. So we see that the bacteria on the soft gel are more likely to be spinning and they spin faster. As a result or a proxy with that, more detachment happens on soft gel surfaces and that gives rise to less bacteria being accumulated on soft surfaces. And all this is somehow mechanotransduced through PIL-Y1 because if I don't have PIL-Y1, all the things in this blue box go away. But clearly they're still present for the initial input. Then we have the bacteria population on the surface and it just grows over the next few hours. When we did these experiments at this point after the first hour, we removed all the free-swimming bacteria and just added fresh media. So there's no more bacteria coming in from above to attach to the substrate. It's just whatever's there on the substrate and then we're going to say what happens now. So when bacteria enter a new environment, they undergo something called a lag phase. And that's when they kind of pause and the bacterial population doesn't really grow for a while because the bacteria are making whatever changeovers and metabolism they need to make to adapt to their new environment. And how long this pause is depends on how big a change it is from the first environment to the second one. And also just how kind the second environment is to the bacteria. So we can look at how long this pause is and the length of this pause is here on the y-axis it's the number of bacteria that we count on the x-axis. It's the incubation time or the time we've had them on the surface. And for the time that the bacteria population isn't consistently increasing, we can say, okay, that's the lag phase. That's the pause phase. And what we see is that the duration of the lag phase when it's transitioning from the free swimming state to the biofilm state because now it's attached to a surface is different for different substrate stiffnesses. So the lag phase is a lot longer for the wild type bacteria, the bacteria we haven't messed with. When it's on the stiff substrate then it is when it's on the soft substrate. And then we get that by looking at these measurements of bacterial population and saying the lag phase ends when we start seeing steady growth of bacteria. If we knock out pill Y1 so we don't have this protein that we've already said is important for early surface discrimination we don't see any difference in the duration of the lag phase. But we also see something else. As these bacteria enter what's called exponential growth or you might call this the biofilm growth phase so we see a steadily increasing bacterial population we can take this slope and we can use it to measure the growth rate of the bacteria or the doubling time for the bacteria. And what we find is that for the wild type bacteria that we haven't messed with they grow faster on the soft gel than on the stiff gel. And that's also true for bacteria that are missing pill Y1. So their lag phase is the same but if you look at their difference in their growth rates the difference in their growth rates is still there it's still statistically significant and it follows the same trend that we saw for the wild type that has pill Y1. So pill Y1 even though it's been super important for everything I've said up to now does not seem to be the most important mechanic sensor for controlling this differential growth rate based on surface mechanics. However, if we look at bacteria that are lacking the pilus retraction motor so if you remember the pilus are these grappling hook appendages that reach out and grab a surface pull the bacteria forward like that like Spider-Man crawling up a building. This would be like if Spider-Man can reach out but he cannot pull his arms back, right? These are missing that retraction motor and bacteria that are missing their retraction motor still have differential lag phases because pill Y1 is still doing its job but once they start growing their growth rates are statistically indistinguishable. And so this is the second stage in the second sensor that I talked about early in this talk when I did a PR thing for Charles Dickens. This is a second stage of biofilm development where you're no longer just accumulating on the surface but the bacterial population on the surface is actually starting to reproduce and increase in its own right and the rate at which that happens also depends on surface stiffness and it seems to depend on this pill T sensor which wasn't super important early on but seems to be played an important role here. So from this we learn two things. One is that pill Y1, this famous envelope protein impacts the duration of the lag phase but the type IV P-Li are the pilus motor controls the growth rate of the bacteria. This is our second sensor in the second stage. We also very quickly I put this in mainly in case there were any biologists in the audience. We wanted to understand the connection between these different sensor activities and the biological phenomena we were measuring in terms of cyclic dye GMP signaling. And what we find is that far bacteria that have pill Y1 we see much stronger cyclic dye GMP signaling levels on the stiff gel that lasts a lot longer. If you don't have P-Li you don't see any difference between the two gels. If you have pill T you see an, sorry if you have pill Y1 but you don't have pill T so you don't have this pilus retraction motor but you do have the envelope protein. You see an initial strong response upon surface attachment that looks very similar to the baseline wild type but then you see a much more rapid decay which may be associated with these undifferentiable growth rates for pill T on the different surface stiffnesses. So this is what you should learn. Pseudomonas originosa senses surface stiffness initially using pill Y1 probably because of stresses in the bacterial envelope and later using type IV P-Li this is how biofilms get started and we want to know if we can use this knowledge to enter the development of harmful biofilms part of the step toward doing that is to work toward generalizing this knowledge to other important biofilm forming species of bacteria which is something we want to work on next. I am really happy to have had Leon and Chad to work on this project and I'm really happy to have had the other people in my group doing other things. They have made my life a lot better and if any of you I have no idea if there's any students in the audience because I can't see anybody's face but if any of you are students and you're interested in joining our group I think we have a lot to offer. You get close mentoring in a very tight-knit group that works well together. You get to be a part of a supportive community that's bigger than just my group but smaller than the whole physics department because my group is a part of the center for nonlinear dynamics and we have a whole support infrastructure within that group and you get to work on things that will improve people's lives. So if you're interested in graduate school or a post talk I'd like you to email me. That's my email address there and with that if there's any remaining questions I'll take them. Thank you very much. Let's thank the speaker. Okay, questions online. We had a lot of discussion during the talk which was very nicely prompted by you, Vanita. I don't like just talking to myself for an hour. It's not very fun. No, you handle that very effectively. Yeah, Pavel, go ahead. Yeah, well, I'm just to go to the last slide. So again, well, help me understand. So, well, how do you extend this knowledge to the other biofilms? And again, just to give us an idea what is so special about this specific culture that you use? It's that somehow you learn a lot about the other cultures and maybe this is a very naive question. So I think you have two questions, right? One is what's so special about this organism that we used and maybe why did we choose to use this organism? And the second is how would we extend this knowledge to other types of bacteria? So this organism, Pseudomonasurigenosa, is a really nice, if you're a physicist and you want to work on biofilm development, this is a really nice organism because it has a lot of genetic stuff already sorted out for you. So there's a lot of mutants that already exist or if they don't already exist, people know how to make them and you can get people to help you out with that, which is really important because this is not a thing that I have expertise in doing. I don't know how to knock out genes and how to complement the backend and how to construct a plasmid that will report some gene activity. But for Pseudomonas, these things exist. And so we can put them together and we can use them to ask these more physical questions. It's also a nice organism for us to work with because it has important human health impact. And so if you're doing your initial model system, it's nice if your model system studies can reveal things that are going to matter in their own right, regardless of whether or not they're generalizable. So that's why we chose this organism. How we plan to generalize this to other organisms, at least initially, is to zero in on the stuff I was saying about the bacterial envelope and the importance of envelope proteins. Because what we think we've extracted from the first two-thirds of my talk is that there's a linkage between biofilm development that's triggered by surface attachment and mediated by envelope proteins and the equilibrium mechanical deformation of the bacterial envelope in response to adhesion. And so what we want to do now is to screen other biofilm-forming organisms to see whether they also have this differential response to surface mechanics. So I showed you several types of differential responses and we'll look at those, see whether they respond differently to surface mechanics. And if they do, that's going to be a strong hint for us that they are using envelope proteins to sense that surface and respond and then we'll start narrowing on what envelope proteins those could be. Okay, well, thank you very much. I really enjoyed this seminar a lot. Thank you. Thank you. The highest compliment anyone can pay or receive. Jin, you have a question. Go ahead. Okay, I think my question might be a little bit irrelevant, but I'm just wondering like, do the bacteria talk to each other? They do. It's not irrelevant. I didn't talk about it in this talk, but yes. There are methods for interbacterial communication. One of them is called quorum sensing. That's when bacteria release a signaling molecule called auto-inducers. Unlike the signaling molecule I focused on in this talk, this goes outside the cell. And then bacteria kind of sense what is its concentration. When they sense a high enough concentration, they're like, oh, I've got a lot of friends with me. Let's change and engage in cooperative group behaviors. So that's one type of talking they do. They also do what might be a less focused or less purposeful type of talking, where just by the process of living, they take in different growth materials and then they give off metabolites like waste products. The presence of those metabolites can alter the nearby environment in a way that alters the behavior of nearby bacteria or the response of nearby bacteria to antibiotics or whatever. So it can change the growth rate of nearby bacteria. It can change how susceptible they are to antibiotics and so on. That's an interesting area that a lot of people are working on. And 10 years ago, I thought I was going to do a lot of work in that area and that just seems not to be how it's worked out for me. So I've only peripherally touched on that. Okay, thank you. I'm going to exercise my prerogative to ask a question. So, Jing, I don't think, I mean, obviously, I needed and think your question was silly either. And in fact, I had a similar question in mind. So let me just follow up on Jing's question. So you mentioned that detecting surface stiffness is not a precognitive phenomenon. In other words, the bacterium has to come in contact with the surface to know, yes, I either want to remain here or no, maybe I don't want to remain here. I want to leave this surface. So as regards interbacterial signaling, is anyone looked at whether or not a first wave of bacteria coming in contact with a surface that's perhaps not as hospitable to forming a film or forming a colony can then signal to other bacteria in their neighborhood, don't bother, like don't come down here, go stick to another surface someplace else. Is anything like that happen? I don't know of anyone who's shown that. People have shown, not us, but other people have shown that transient attachments can prompt changes in behavior later on. So like these bacteria that come in and attach and that they fly off, like they maybe are primed to respond to the surface a little bit differently when they come to it the next time. But that's not the same thing that you're asking, because you're asking about if like a different set of cells came in, would they somehow know to respond differently? I don't think anyone's looked at that. I mean, I guess I know this is stretching the analogy with something this small and really this basic, but in other words, is there some kind of swarm behavior here from chemical signaling? Can the first wave basically leave a chemical trail behind that says don't bother? And then the second wave just doesn't go there, essentially. Not that I know of. That doesn't mean it doesn't happen, but it means I don't know of it. Yeah, no, I'm just, it occurred to me as you were talking that it was when you said precognition, because actually things can signal to other things don't bother, right? But obviously that's a really complex, and that may be overly complex behavior for something this simple. Okay, any other questions? I don't see any more hands, new hands online. And I don't see anybody in the room. So I think we've gotten all the questions out, Vernita. Thank you very much for giving this talk. I'm going to quickly go to my adverts. Yeah, go ahead. Advert side. This will be up on YouTube. So I know that they're this, this event was broadcast to the pre-health group at SMU and to the biology department. And I did get at least a couple of inquiries from biology folks about the passcode for this. They may, they may have had conflicts, but I know that people watch this on YouTube afterward, because the views always go way up after we post the video. So this is a perfect place to end on advertising. So let's thank Vernita one more time.