Extracellular Matrix Physical Properties Alter Cell Mechanics, Morphology, and Proliferation

Alright, I think we'll get started, my name is Professor Ken Christensen, I'm the associate head of mechanical programs in the department of mechanical science and engineering, and I'd like to welcome you to the second installment of 2013-2014 busy owners. from the

mechanics

seminar series um mechanical discussions means investigations and

mechanics

and the notion of this series began in 1997 in the then department of theoretical and applied mechanics here on this campus at the time it was held as a two-day symposium one in fluid mechanics one in solid mechanics bringing eminent scholars in those particular areas to campus to make presentations discussing and observing cutting-edge aspects of these particular areas this year's discussion disease mechanics seminar series bringing scholars in the areas of biomechanics and mechanobiology and the idea is for them to visit our campus for two days today and tomorrow give a lecture on perspectives which we will hear here in a moment and they will interact with the faculty and staff and then they will give a more focused scientific talk on Friday, which I'll let you know what else will happen in Meanwhile, if you're interested, with that as background, I'd like to introduce you to Professor Tahir Saeed from the Mexican department, who will introduce today's speaker.

Thanks again, Paul asked me to keep it very brief and have more time for technical discussions. Do that, so uh graduated in philosophy from Oberlin College in 1976 and then teaches academics in medicine in chemistry, focusing on polymers, biopolymers for biological grade athletes and then joined Harvard medical school in 1982 and worked as a research program and then in 1999, 1999 and since then he has been there and as a professor of physiology and in the physics and astronomy departments at Houston, biochemistry and some extensive work on the mechanics of

cell

s, the quality of

cell

s in relation to the mechanical

properties

and the relationship with the existing relationships that we have. quite a few awards, scholarships and television leaks, very well known internationally as well, but what doesn't appear on his resume, I think I mention it, is that I think his works, these articles from the last two decades have actually inspired everyone. a generation at certain times.

I am one of those many missionaries. Many of the people he has spoken to today have already told me that they would be inspired by his previous work, as well as the discussions he has had today. Somehow some of the fundamentals of some mechanics are going to be needed and that's a big part of some of these words in the textbooks with biology, so the contributions are huge, so with that, hello, thank you very much. Can you hear? Good, great, thank you. for the kind introduction and also for the opportunity to return to this truly wonderful campus.

Great, it's my fourth or fifth time here. I really love it, so I want to tell you a couple of stories about what we've been working on. recently to try to see if mechanical signals I stole the laser pointer is not my pocket oh, sorry, um of how the

physical

properties

of the environment impact cell biology and to see to what extent that mimics, reproduces or

alter

s the way That chemical signaling works I want to show you a couple of stories about how changing the mechanical properties of the materials that cells attach to changes their own mechanical state, but also things like their

morphology

, their cytoskeletal assembly and their and it works things like

proliferation

, so the work that I'm going to show you is done by engineering postdocs and graduate students in the anon chopra lab, justin weiner and jeff byfield and a cell biologist maria murray and we will also, towards the end, show you some experiments done by a really fantastic physics student who will be visiting us at a tasha fulbright pagoda this summer, okay, here is the kind of summary of the three stories that I would like to tell you, one is to show the evidence accumulated in recent years suggests that the Physical properties of things like soft tissues and organs are equally important or at least vitally important to the eventual biological or physiological outcome of the cells and they can provide very important results. specific signals can be as strong and specific as chemical stimuli and part of the specificity by which

physical

properties can impact the cell depends on the fact that different cell types are surprisingly differently sensitive to physical signals with what I want say imposition of forces from the outside or encapsulating cells in rigid or soft materials or purely elastic or more viscous materials and one of the reasons why cells are so specific or so different from each other is that the mediation of this physical information comes to through specific transmembrane receptors exactly in parallel to the way that hormones and acute biochemical signals impact the cell by binding to some receptor.

What those receptors are for physical signals is much less well worked out, but that's not really surprising that people knew. That insulin affected glucose metabolism for decades before anyone knew what the insulin receptor was and here we are in a similar state in terms of physical signals, but what I do want to show you is a couple of examples of three different kinds of transfer membrane receptors that mediate this type of physical interrogation, are the best studied, a class of receptors, much of which has been done here, has been done by analyzing how integrins, the cellular

matrix

of the receptors, carry out this type of mechanical signaling, but also cell adhesions through cad.

The audiences are also equally important and I want to show you towards the end what happens if you take a completely different class of receptor receptors for a component of the

extracellular

matrix

called hyaluronic acid and show you that this changes a lot how mechanosensing works and the basis of these guys. One of the questions is always this, this notion, can physical stimuli be imitated? Are they different in kind from the way biological stimuli work? Do they activate the same pathways? And if they activate the same pathways, can we play with the responses? to physical signals by getting the biochemistry inside the cell, uh appropriate, okay, so just as a way of introduction, here are the kind of tools of the trade for what in our case are fairly simple measurements of tissue mechanics or the mechanics of a single cell.

A very commonly used way of measuring in the bioengineering literature and elsewhere is to do with a piece of tissue or a piece of macroscopic material what polymer physicists do with their materials, scientists do with bread dough. gum and everything else putting it in the middle. two plates by applying a torque to one of the plates, either oscillating or static, and observing the deformation and from the relationship between the applied stress and the resulting deformation, calculate a shear modulus and I will talk a little more about this tomorrow , but it turns out that there is a very interesting interaction between performing these types of pure rheological measurements and applying constant uniaxial stresses in the orthogonal direction, so this is something that does not automatically happen with simple elastomers or polymeric materials, but in the case of biological materials this turns out to be a great thing the other types of tools of the trade instead of applying torque and shear stress on a macroscopic piece of material, it is also very easy to indent materials with a flat indenter, this is measuring the stiffness and the material exactly how it does.

What you would do if you pricked someone with your finger, you could more or less calculate the softness of what you're touching from the same thing that one of these vertical probes calculates how much force and how much force you apply. and how much indentation results and the kind of nanoscale variant of these uniaxial penetration devices is the atomic force microscope and the same principle applies and this is drawn roughly to the scale of the measurements of the cells that I will show you later. has been done by gluing a two micron sphere to the afm cantilever and gently pushing a few hundred nanometers on the top of the cell to see what its viscoelastic response is, so if you do something like that and take a very, very crude approximation and by wait, forget about time or strain dependence completely and just take a lot of fabrics and look at their elastic modulus; in this case, convert it to a shear modulus and the small strain limit on a time scale of about one second.

Many interesting comparisons. They come out between the tissues. I'll show you a very small subset of this, but it turns out that in normal physiology the elastic properties of normal healthy tissue from the same animal of roughly the same age is a remarkably well-controlled parameter, so I don't have error bars here, but here For example, there are the variations in measurements of many different aortas that are normal or diseased and you can see that the variation in the normal state is surprisingly small compared to the average, so the stiffness of the organs is not is. an accident of what the internal cells do is a very, very well controlled parameter for whatever reason and if you look at the development of the disease in many different contexts, for example, the development of fibrosis in the liver, the elastic modulus of a Normal human liver or normal animal liver is a few hundred pascals and increases by approximately an order of magnitude even quite early during the development of fibrosis.

The most studied example of this is probably the development of a malignant disease in the breast, where the stiffness of the breast tissue increases quite a bit even early in a fairly premalignant histological lesion, but becomes much stiffer as the tumor arises throughout. rule and of course this is why palpation works as a diagnostic method, but these types of stiffness differences arise in all types. of pathophysiological states and one of the examples that I highlight here is the work of the rickisoyen laboratory at penn in which we have observed the elastic moduli of the aorta taken from normal wild-type mice or from mice that are genetically depleted of apoe is a component of a lipoprotein complex, but in any case the loss of this particular protein predisposes these mice to atherosclerosis, but even before they have any signs of the disease, if you euthanize them before there is any type of histologically evident lesion in his blood.

The blood vessels are already stiffer to begin with, so this, as well as this hardening of the fibrotic liver, suggests that tissue hardening could be at least a contributing factor and I'll show you some evidence of this on the next slide, It is not simply a consequence of the disease, it could actually contribute to its development, but I do not want to exaggerate the fact that the stages of stiffness are not simply a pathological thing during normal embryonic development, there is a huge change in many soft tissues, TRUE? Embryos are much softer than the adults they generate and one of the most spectacular examples is the rigidity of the embryonic heart or the cardiac cushion from which the cardiac cells will eventually emerge is orders of magnitude softer than the adult heart it will generate. it is eventually generated and is a normal part of development and I will come back to this in a moment.

It's not true either. I'll talk a little more about this tomorrow. The rigidity of the tumors. Tumors are not always stiffer than the normal tissue they come from. It came up and one of our projects recently has been to look at the mechanical changes in brain tumors and, somewhat surprisingly, the elastic modulus of a removed glioma tissue is no different from that of a normal part of the brain, so it's not an automatic occurrence . that the tumor is stiffer than the tissue it comes from, but why that's something we'll maybe touch on tomorrow, okay, here's a little bit of the tools of the trade of how we try it.

I've been trying to measure. A lot of labs and I've been trying to measure the response to these mechanical signals in a controlled way and the big tools of the trade are these two things, almost all of the data that I'll show you in the next few slides. come from our attempts to basically use a method developed by Yulie Wong about a decade and a half ago in which you simply replace the glass coverslip that cell biologists have traditionally grown cells on and place a soft cushion on top of them that is about a hundred microns thick it is optically transparent as much as it can be made and in one case, in the simplest case, it is chemically inert to the cell, meaning that the cell, if you just placed it on this cushion, would not be able to bind to because it doesn't have anything sticky to hook on for the cell to adhere to, you covalently place on top of this thing whatever you want, fibronectin or collagen or some kind of matrix-mimicking ligand.

extracellular

or you could place a cellular adhesion fragment. molecule like a catalin to make the cell attach to this soft cushion like it attaches to a giant flat cell, but you can pretty much put whatever you want in there, the variation I'll show you is what happens if we substitute the inert gel that in in most cases it is a polyacrylamide for another semi-inert gel made of cross-linked hyaluronic acid another very flexible linear elastic hydrogel is very well studied it has the slight difference that it is a polyelectrolyte but it does not matter so much for that, but most of the Cells will not bind to this on their own or to polyacrylamide, so in order for the cells to stick we still have to put fibrin, actin, collagen or something like that, but the difference is that this turns out that the cells have receptors. for this thing even if they can't anchor to it, but now you can make all kinds of objects work and change the elastic modulus from maybe 50 pascal to 50,000 pascal and thus cover the entire range of stiffness of the tissues that I showed you on the slide above so that we can mimic things like embryonic tissue on the soft side to things like heart muscle and more rigid things, we can't get to the rigidity of cartilage, but we are on our way to the other system that I won't have time to tell you about, but in reality it is equally revealing and also very common.

What you use is, instead of making a continuous hydrogel, you make little soft pillars made of PDM which has a very high elastic modulus out of scale of the stiffness of what you can make with hydrogels, but you can soften these little beds of nails effectively doing them. thin and tall and just by the deflection of the top of the pillars, the cells can even move them easily or move them strongly and it turns out that this is a spectacularly good imitation of how to make a soft material. I just want to take a minute to point out how surprising this is: why should a cell care if a pdms pillar is tall instead of short because the material that the proteins attach to is still almost infinitely rigid for the cell, so what each integrin or each adhesion molecule that lines the top of one?

These pillars and these polymeric fillers are typically a couple of microns in diameter, so hundreds of adhesion molecules bind there. They don't care about the local stiffness of the material they are actually attached to. What really matters to the cell is work. it has to do with deflecting the top of one pillar relative to another, so we're using these things to try to calculate the length scale uh uh the size of an object that a cell can measure, the stiffness of obviously can't, it can't detect or care that this is rigid, it only cares that the top of the pillar is flexible, but that's just an aside.

I want to show you mainly things about these hydrogels. Well, here is a relatively typical experiment using hepatic stellate cells. type that causes fibrosis in the liver to accelerate, so this is a type of cell that will spit out inflammatory signals from the extracellular matrix and really drive fibrotic disease, so if you isolate a hepatic stellate cell from a tissue that is normal or stiff, The environment it comes from turns out to be quite different, so, for example, if we measure the stiffness of a liver from a whole liver, the environment that these stellate cells will live in is a function of the stiffness, the The stiffness of the organ increases relatively quickly after In our case, we have caused fibrosis to occur by poisoning the animals with carbon tetrachloride, so the animals will be doomed to become fibrotic and if we sacrifice them and observe the stiffness of the liver, is increasing some. days after the initial poisoning, but if we look by histology, if we send sections of this tissue to a pathology laboratory and ask if any cells there appear fibrotic, the answer is no until a couple of weeks after the injury has occurred. , so stiffness actually precedes cell dysfunction or cell

morphology

changes that take place later, what we think is happening is that stiffness arises not because cells are changing their structures yet or are depositing more matrix , are changing because lysosol oxidases are activated in the matrix. that's already there, it's just more cross-linked and the cells will react later, the way they react is shown here in a sort of in vitro imitation of the liver hardening that I showed you before.

This is work done with Rebecca Wells' lab at Penn, so if you take a hepatic stellate cell from a healthy liver that has a very small elastic modulus and put it on one of these fibrin-actin-coated hydrogels, uh, that, but a hydrogel that has the same rigidity as a healthy liver will bind to these small cells. and they are metabolically happy, but they are not photogenic looking cultured cells, they are slightly brown because one of their functions is to store solid fat soluble vitamins, they are a little translucent but they don't spread much, but that doesn't mean they aren't healthy, they are actually perfectly healthy since we change the stiffness of the material underneath them, but we don't give them any chemical stimulus that we know, simply changing the elastic modulus underneath is enough to cause this mass. uh, it causes

proliferation

which I don't show you, but the cells become much more stellate, they make this very large cytoskeleton, they start to express extracellular matrix and they drive all the fibrotic pathways that will eventually lead to the disease, but we have This didn't trigger it chemically but simply playing with the same range of stiffness that normally occurs during fibrotic development.

Now I'm going to show you a couple of slides on a different type of block in cardiac myocytes to make it clear that these mechanical stiffnesses or responses are not the same for every cell type, in fact, they are not even always monotonous, and the most Dramatic cells that today have a very small window of rigidity in which something interesting happens in the muscle cells. Several groups have looked at skeletal muscle in the past. We have a collaboration with Joshua Precious Lab to look at neonatal rat ventricular myocytes, taken from the healthy beating heart and immediately placed in gels that are either much softer than the tissue they come from or much stiffer than the tissue they come from, the cells They will bind to these soft or rigid matrices perfectly fine and they are metabolically fine and they actually look pretty good, but they don't look like the cells in your heart should look like if you instead put them in chemically identical gels that have the same modulus. elastic of the tissue from which they come, they form immediately or not immediately, but spontaneously regenerate this elongated sarcomere that contains the shape they had in the tissue from which they come.

They came and started beating and functioning normally and you can achieve this not by modeling these asymmetrical shapes but by simply putting them into mechanically realistic things and letting the cell do its thing. In this case, we are placing them on gels that are coated with a ligand for integrins, so all the mechanical signaling and adhesion is done through integrins and this whole army of accessory proteins that link the transmembrane protein complex to the cytoskeleton, but if we do something very different, we substitute a cellular adhesion. molecule and catherine for the intermediate ligand something very similar happens, so in this case we are not saying that the cell is not able to activate any of its normal integrin or extracellular matrix receptors because there is nothing in the soft hydrogel to deal with he.

They only connect through cell-to-cell contacts, and yet they can do something very similar if the elastic modulus is too low. Nothing, you get these little amorphous cells, if the elastic modulus is approximately correct, they don't do the job perfectly, but they are pretty. close, so everything is sick from a signaling point of view, things are very different if they are activated through cadherins instead of through integrins, but still this kind of elongated shape arises only if you get the appropriate elastic modulus, if obtained on the high side of the cells. They will continue to live but become fibroblasts, so this appears to be a very common signaling pattern for many types of cells, through a large class of receptors.

I'll show you another example to emphasize that this is not just a type. of a morphological phenotype of the cells, but many functions and many transcriptional programs also depend largely on these stiffness signals and one of the most interesting effects of playing with the rigidity of the matrix is ​​to change the cell cycle, so that in many primary cells the cell can be blocked you can block the cell cycle at various points in its uh uh either in g1 or g2 uh you can stop the cell cycle and the production of cyclin d that the cell needs to undergo cytokinesis and mitosis you can block it just putting it on a very soft substrate and not a pathologically soft substrate, but a substrate that is approximately the same as that of a healthy organ in which cell proliferation in an adult is relatively slow, so if you do that with stromal cells derived from human bone marrow and you put them in a matrix that is very soft like bone marrow in which these cells normally just sit but don't necessarily proliferate, they will remain in these materials for a long time and will not divide, so the incorporation of brdu is a measurement of entry to the s phase. the content of these soft materials is very low, if you increase the elastic modulus a little, they start to proliferate, they proliferate almost as much as if you put them on glass, practically everything will proliferate, as if the matrix were rigid enough. but we haven't done anything chemically and I'll show you here an example of how big this block of the cell cycle is.

You can reduce the amount of salt proliferation by changing the elastic modulus at least as efficiently as possible when producing them. confluent, so this rigidity in the influence of rigidity on the cell cycle is as important as contact inhibition for many types of cells, but these cells just to demonstrate that they are still viable cells we have not made them unhealthy or unable to work later. by growing them in a soft environment, Jasmine Weiner did this experiment using a baningo and wang method to make a little sandwich and what she did here is what I showed you here, they are incubated in a very soft substrate so they are dormant for a while and then the first day, take two of these types of small round cell populations and put a little hat on top of them and the second day, the hat is made of a hydrogel that is as soft as the place they were sitting on. or something that is very rigid and if you put on the top surface something that is very soft, the cells will not proliferate for a couple of days as long as we can keep them healthy, so this field of cells are the same cells that were here .

Two days ago they changed shape because they are adhesive at the top and bottom but they have not proliferated nor have they even moved. If, on the other hand, you put something rigid on top of them, you will see that these Things have changed very quickly, their shape has begun to proliferate and Eventually, in all cases, we can get them to become the type of cell we want by playing with the induction medium. In this case, we have converted them into osteocytes, the type of cell you want to see. a hard substrate even though they have been sitting on these soft materials for a long time, they do not lose the genetic information they need to become a differentiated cell, not only do they change their function but the cell also changes its own rigidity, so A peculiar aspect of many cells is that they will remodel their cytoskeletons or their cortical contraction to roughly match the rigidity of the material under which they are placed and bone marrow stem cells are particularly good at that, they will undergo a sort of what It seems like a biphasic change in its own internal rigidity, even without any differentiation in the fate of a particular cell, so it is just a means of induction, these cells will become rigid if we put them in rigid things, they will remain soft if we place them on soft things and how they sit on soft things as Disher's laboratory and many others have shown.

If grown in soft materials, these cell types like to differentiate into differentiated cells that are soft like fat, they don't want to become cartilage, which is a rigid cell. but if you pre-incubate them in rigid materials, they will be ready to function and respond to differentiation media. Oops, I was wrong about something, they will too. There is also a very interesting difference between normal and cancerous cell types. I'll talk. A little more on this tomorrow, but if you take a salt, a glial cell like an astrocyte that again changes its stiffness in response to the stiffness of the substrate in a range that is roughly relevant to the tissues of the central nervous system, if you takeglioma cells that arose from the same astrocyte precursors, but now they are transformed, they do it very differently, so they are much softer, they have much less mechano response, they still have mechano response, but they have lost a little bit of this control So let me conclude this first part by pointing out that soft tissues have a surprisingly well-controlled elastic modulus and that the modulus changes with disease status and development, but the cells within that tissue do not have a pre-programmed stiffness;

An individual cell can change its own stiffness over a very wide range in response to changing the mechanical properties of things around it, the way cells do this is very specific to the cell type. Different types of cells respond very differently and that response depends on the types of adhesion molecules that it uses to attach to the matrix or the material that it lives on and what we've been interested in lately and I'll show you some examples: we're trying to understand how this, two things, one, how the stiffness changes during the development of the disease, is it the cell that is changing and changing? the matrix or something else changes the matrix and the cells respond differently and the whole question of whether in a large complex composite material like a tissue how much is cell and how much is matrix remains an unresolved question, but the other question What What we're trying to understand is how the cell works, what are the molecular mechanisms that allow the cell to sense things like the rigidity of the matrix.

In the simplest case, you can imagine that there are a whole series of small molecular springs that arise all the way from the nuclear matrix to the nuclear membrane via linking complexes or something through the cytoskeleton to proteins on the intracellular side. from the transmembrane proteins to the extracellular matrix proteins which are also large flexible molecular springs and if you now pay attention to this whole set of springs you can imagine that the There are two scenarios: if the cell is relatively rigid and the outside world is very soft , if you pay attention to this whole chain, to this whole flow of molecules, the one that is going to unfold the most is the softest, so the outside world is softer, you could unfold something that exposes. a cryptic signal that will send a signal to something else in the cell and then drive things the way they were always driven through single transduction chemical pathways or it could attract an ion channel and again trigger basically biochemical signaling, but through an event initially physical may also be much more physically intact, it could be that you transmit stress from the outside world to something within the cytoskeleton or perhaps in the nuclear matrix itself and then drive things very differently, so the question is whether mechanical signaling is fundamentally different. of chemical signaling or it can be avoided if we knew what the biochemistry was in more detail, so this is what here are a couple of examples that I will skip.

I'll go through them relatively quickly because I don't want to dwell on There are too many molecular mechanisms, but there are two kinds of fairly commonly observed changes that cells undergo when they experience changes in the rigidity of what's around them, so there are things that happen quickly or sharply. and there are things that happen very slowly. and that they are adaptive and transcriptional, the things that happen very quickly are actually three types, one is that somewhere in the nuclear membrane there are changes in the activity of things like the p120 highway gap, which is a gap for , not surprisingly, one row, but other small gtp So if you inhibit this activity, you regulate the gtp activity of the pathway, you also upregulate some transcription factors that will appear to be specific to endothelial cells, but you go through many signaling pathways molecular that will activate things like rho-dependent kinase. that will activate mice and light chains that will increase contractility at the same time that stiffness also increases things like focal adhesion kinase phosphorylation that will drive a different class of small gtpases that will drive actin assembly for melapodial formation all kinds of changes cytoskeletal but that will also affect cyclization and control proliferation, so I already mentioned that stiffness changes a lot in the proliferative state and this is one of the pathways in which it could work and, you know, largely driven by the work of here but from other laboratories.

It is also equally plausible to think that the changes in stiffness that give rise to these changes in the contractility of the cell may deform the cytoskeleton in different ways or transmit force directly from the cytoskeleton through mediator protein complexes that bind to the nuclear membrane. which eventually binds to chromatin which could change the arrangement of the DNA within it and make transcription events different in response to physical signals. If this is the way things work, then it's going to be really difficult to reproduce this biochemically because there's really no biochemistry here, it's all, it's all mechanics until you get into the nucleus and start extracting the DNA, but it's clear that many of these things, in a sense, change nuclear activity, uh, at least transcription activity in one way, there are two. different pathways that this could work in, one is a more biochemical pathway where, as I always mentioned, things like mechanical signals will change the degree of actin polymerization and there are transcription regulators like mrtf uh or mal that are inhibited by cytoplasmic actin as you cause more actin to polymerize as the cytoskeleton reorganizes in response to stiffness, you lose these actin monomers, you lose the inhibition of the transcription factor that goes into the nucleus and does what it does.

On the contrary, it is more fundamentally mechanical, there is also a second class of nuclear transcription regulators called yaps and tabs that have attracted a lot of interest lately, in which tension in the cytoskeleton is somehow sensed directly by this transcriptional regulatory complex that now it enters the nucleus and causes many adaptive changes in the cells. they are characteristic of cells growing in rigid, perhaps fibrotic tissues, they may be cancerous and not in softer materials, so I want to come back to this point later, but I just want to give you an idea of ​​the kind of things that people have. have obtained from mechanical changes, but this is what we are most interested in, most of what people have done in vitro, including us and the experiments that I showed you at the beginning, is take a single cell or maybe a small island of cells and put them on soft hydrogels or pillars or something that has a specific protein that will bind to a specific transmembrane receptor so we can figure out what the molecular ingredients might be, but in real life this scenario is much more complicated, they are sending mechanical signals simultaneously.

From cell to cell, there are multiple types of transmembrane proteins that engage this extracellular matrix in that signal very differently, but in addition to things like integrins that can actually act as anchors, there are a large number of proteins that include receptors for this extracellular matrix. hyaluronic acid polysaccharide that I will talk about that also interacts with the extracellular matrix, they cannot act as physical anchors but they change the response and I want to show you a couple of examples of this that have been surprising. Motivated by this problem, I already showed you that if you take cultured cells and put them on very soft materials, you cannot create a functional myocyte, regardless of what we do chemically, we cannot create a cell that looks like this on a material which is as soft as this if we just engage them through integrity, the problem is that this is the rigidity of an adult heart where the sarcomeres commonly require a good amount of tension and a large amount of rigidity, but in the embryonic heart with the sarcomere The first form is as smooth as this, so how are those first myocytes formed?

If you need tension to get them or stiffness to get them to form, because they are forming in a very soft world and that's where hyaluronic acid comes into play. This is the most common, it's the most prominent component of the extracellular matrices in the early stages of development, when there's not a lot of collagen fibers, there's not a lot of fibrinectin, but this is the world that embryonic cells originally grow in. It is also a matrix component that is strongly upregulated in cancer, it is upregulated in wound healing, so cells will synthesize and secrete this large polysaccharide and at the same time regulate its receptors.

And it turns out that hyaluronic acid is capable of doing this, so if we replace the poly with the inert polyacrylamide gel in which we have glued fibrinectin as an anchor to a myocyte, in which case the cells look like this, all we have done is to replace the inert polyacrylamide with an almost inert hyaluronic acid gel as what forms the soft matrix and again decorate with fibrinectin and now you have these massive myocytes so full of sarcomeres that they are beating, they are really beautiful cells, they do much less work during the contractile cycle because they don't pull as hard, but they have all the contractile machinery they need and they are adapted to a much gentler world and this is not unique to myocytes, so cardiac fibroblasts do the same thing that I showed you before.

This is what I just showed you. small cells. Small myocytes in a soft polyacrylamide gel. Beautiful elongated myocytes in h8 plus fibrinectin. This is what they do. They look like glass fibroblasts that do the same thing. I think what we're most familiar with are these beautiful fibroblast stress fibers on rigid materials that disappear when you put them on soft materials, but if you put them on very soft materials that have a combination of integrin ligands plus something that will bind to hyaluronic acid, you regenerate these huge stress fibers even though they're sitting on something very, very soft, it's also true for mesenchymal stem cells, exactly the same phenomenon works with endothelial cells and, in particular, it's not just the actin.

The cytoskeleton and the large stress fibers that reappear, these large focal adhesions, in this case it's a vinculin stain that's typically characteristic of the edges, the focal edges of a cell that's experiencing a lot of stress, like in a very rigid material. , these things too. reappear even though the cell is sitting on something much softer than it needs to generate tension, so we performed a couple of control experiments to make sure we aren't fooling ourselves into thinking these gels are as soft as we think . and because it's possible that the cell underneath that's on top of the gel is rearranging the matrix and depositing its own little rigid material, so here's an afm experiment where we puncture the hyaluronic acid gel in several places on this little hydrogel. lined with a grate and there is a cell there.

I hope you can see the outline of the cell. The red dots are places where we push with the afm to measure the elastic modulus of the cell, so here we show you how rigid the cell is around the gel. It turns out that in this case it is about 600 pascals, the cell on top is a little softer but it is close in the same region and now we gently interrupt by calcium chelation, take the cell out of the matrix and look under the cell. What was it that was actually touching the cell when we made the measurement?

And it's as smooth as ever, so the cell clearly isn't fooling us by placing some sort of rigid platform beneath it that it sits on. She's actually sitting on something. soft, then what hyaluronic acid does is change the response of the cell to change the stiffness set point where things start to form, for example a large adhesive sticky area or the appearance of large stress fibers that usually occur in very, very rigid materials if the just the signage comes from immigrants and it happens with much smaller stiffness if you put this additional layer of signage on top of it, I'm going to skip it, uh, I'll show you this slide, I'll skip the next one, um, one.

One of the things that has been intriguing is the signals that come from these two receptors that come from the integrins and that come from the hyaluronic acid receptors. They are not independent signals that can come from spatially different parts of the cells because if we make the same sandwich. trial that I showed you before, but they cover the cells with a very soft gel coated with fibronectin where they are around an amorphous if all they needed was hyaluronic acid somewhere for them to now spread, we should be able to achieve this by applying the fibronectin on one side and by placing hyaluronic acid on the top surface of the cell, if we make the cell not spread, if instead we put hyaluronic acid plus fibronectin on top, just aswe did before, now it spreads like crazy, so it ignores the smooth, round information of the cells. comes from the bottom, it's now dominated by the stress fiber information that extends out and we created from the top of the cell and the slide that I'll jump to later just points out in more detail that this is largely integrin.

Depending on the type of cellular integrand, this trick of propagation from mesenchymal cells works only through fibronectin receptors. If we try the same trick with collagen, we cover this gel with hyaluronic acid plus collagen, it doesn't work, cells have receptors for collagen. they activate collagen but they don't activate the spread response uh so this doesn't seem to be completely mechanically driven. This is a way of chemically inducing the same response that that rigid rigidity and mechanics normally develop and it's a sharp response that the cells know immediately when they've touched the substrate that it's soft or hard or that it has ha or just integrins, so here, For example, the fibroblasts of our embryo spread on rigid or soft materials depending on time and t zero, here is the moment when we see one cell out of three. -The dimensional suspension touches the surface of the gel and if you do it in a rigid matrix like or on glass, for example, there are usually a couple of periods of one or two minutes in which the cell adheres, you realize what is going on and then immediately starts to spread it in a way that it looks like the area increases roughly linearly for a while over time and if you put it on a very soft material the first few minutes are very similar but then instead of spreading nice and regularly, it kind of oscillates back and forth, it extends the bumps, it pulls them back and it never gets any bigger, so if you put them on very, very soft things that have this hyaluronic acid trick, they start to spread immediately, so this is not something regulated by transcription, it is an immediate response. that is indicated through one of these acute pathways that I showed you at the beginning.

I'm going to skip this and show you that this combination of mechanical signals and appropriate chemical signaling also overrides this mechanical block of the cell cycle that I showed you. If you look at something like cells proliferating after touching a surface, what I've shown you before is that if you touch a cell with a very soft material and you don't do anything in particular to it, it will just stay there, the cells will stay. they will stay alive they will not proliferate if you put them on something more rigid like tissue culture plastic they will proliferate very well, but if you put them on these hyaluronic acid gels, they will proliferate again immediately even if they are on something soft, so I think what this tells us Sorry, we can somehow avoid the rigidity of the materials by doing the same thing. correct biochemistry, so at least in this case the mechanical signal is no different from biochemical signaling: it is transduced relatively early into a biochemical signal that we can reproduce by something else and with an example of our attempt to show that by mapping the pulling force these cells are able to produce these massive bundles of actin that look like stress fibers without applying tension to the cell and the way we've done it is a neat trick that someone other than Chopra learned to do, but It turns out that the chemistry The manufacturing of these hyaluronic acid gels is very susceptible to micropatterning, so what we have done is micropatterned, not the hydrogel, but rather micropatterned a glass surface with small dots of fluorescent fibrin actin so that the single molecule that the cell can produce In contact with that, we design a glass surface with one micron fluorescent fibrinectin beads spaced about 10 microns apart and then this is reversed from the way we actually do it and then we pour the gel on top and during the gelation chemistry. the cross-linked debris will capture the fluorescent fibronectin and bring it to the top of the gel and we can now have dots of one-micron adhesive proteins in an otherwise inert hydrogel, spaced however far apart we want.

This works much better than trying to modify the gel itself. but we put a cell on top of it and this is what it looks like, so we can print grids of these one-micron dots from a gel that has a stiffness of less than a kilopascal and put a fiber burst in here that wouldn't normally produce fibers of tension until the elastic modulus was 50 times greater, but here it produces them immediately, so we get big bundles of actin and we get stuck in the only place where the cell can adhere is in these little red dots and we have been trying to ask Well, how much force does the cell exert?

The cell is applied to these, to these dots and this is a pretty easy thing to do because here is another kind of more contrasty image of the square array of dots that we printed there and you'll see that the cell makes a lot of nice adhesions to these. points that have this kind of curvature that looks like tension is applied to them, but since we know the elastic modulus of the gel and the height of the gel and how big these things are, we can simply measure the displacement of the gel that the cell feels for the displacements of the dots that quote the cells away from the square pattern that they're placed in and it just keeps hitting this inappropriately um we draw the little circles where we know the cell has touched it instead of removing it from the cell through trypsinization we just don't look at it optically anymore, we just get rid of the green channel and you just look at the red channel where the dots are and you'll see that some of the dots are actually kind of warped a little bit by the integrins that have touched it but if you look in the displacement of the circular dots or those at the edge of the cell that were at the end of one of these actin bundles have moved remarkably little from the pattern in which they are stamped and, therefore, you can calculate From the displacement on the top surface of the gel and the elastic modulus of the gel you can calculate the amount of force that is applied on it, the displacements are always sub-microns in a gel that is perhaps 100 microns thick, so we can roughly calculate the force that's applied there and it's a remarkably small force, so if you calculate the stress on one of these little points it's less than 50 pascals per point. that's about a square micron, meaning less than 50 piconewtons of force on a contact area that has hundreds of adhesion molecules, so a remarkably small force is transduced into the lattice, and yet we're making all the ingredients that are typical of a stressed cell, so I'll conclude that I'll skip the signaling that we don't really understand, we're just beginning to try to understand it.

I'll tell you what the joke is, but what it looks like is the Substrate mechanics is a very strong control of cell structure and proliferation of functions, many functions and has been particularly exaggerated when signaling passes only through a single class. of receptors through integrins or caterins, if that is the dominant binding site, the mechanics seem to be overwhelming. Importantly, however, if you start playing with other chemical signals, particularly hyaluronic acid receptor signals, along with integrin signaling, you can do a lot to change the set point of the mechanical signals that a cell needs. to perform some function in specific cells of these cells.

Very soft gels can create very large adhesion sites and act in bundles, the sort of thing normally associated with tension, but the cell itself does not apply tension, at least not to the substrate beneath, how this works, I don't know. we understand at all. We are starting to fish out some of the ingredients you are using, it appears to be particularly receptive to signaling through three alpha v beta integrins, other types of integrands do not have as strong an effect, it works through at least two ha cd44 receptors and cd168 and many of the common pathways, uh, the common pathways that I showed you before, the phyla kinase pathway, but also phosphonous oxide synthesis and probably many other biochemical signals have not yet been determined.

Okay, thank you very much for your attention. A little overwhelmed, not sure. Where to start? Do you have any speculation you can give regarding this? I know there are a lot of signaling pathways that you're looking at, but the fact that they're making these vocal adhesions in the soft gels, yeah, it's a It doesn't surprise us, I think there are two things we're trying to pursue: one is that the receptors Hyaluronic acid interacts with a class of cytoskeletal proteins called erm proteins, so as we introduce things that aren't usually part of the big focus. adhesion, but they still have an important role to play in stabilizing the actin cytoskeleton so that that pathway can act synergistically with whatever the integrin is doing.

We still need integrins to get these large focal adhesions, but whatever is doing the ha is on top of that. The other thing it does that is much less understood is that it stimulates the synthesis of phosphonoketides, so especially the synthesis of pip2 from pip and pip3 from pip2 converges, and those phosphonoketides are also big drivers of assembly of actin and they will activate things. like paline, they will interact with vinculin, so many focal adhesion complexes are also attracted to those lipids, so we wonder if lipid synthesis isn't part of what causes these adhesions to form.

No, no, it is much more difficult to do. such a good square ray, but they're going to stick with polyacrylamide, yeah, so far I think we haven't seen, we haven't seen cell types, at least primary cells that don't have some kind of enthusiastic response to hyaluronic acid, but it just works if you combine it with the appropriate integrin, so it depends and one of the things that Tasha Pagoda worked on that I might talk about a little bit tomorrow is we've looked at three different human glioma cells, they all will. respond to hyaluronic acid because hyaluronic acid is also very prominent in brain tumors and many other tumors, but these cells express different subsets of integrins and depending on the type of integrand, some will activate with h8 plus laminin and will not care about collagen and the others are exactly the opposite, so the receptors that the different tissue cells express are that spectrum is going to be really important for how they perceive mechanically, I think when you have these very soft shells, yeah, as far as I know .

In these small patterned ha gels we see two things well in the patterned hhl we see very little out-of-plane movement because we also see very little in-plane movement, but if we take a cell that pulls harder like a mesenchymal stem cell in a polyacrylamide gel coated with fibronectin, there the out-of-plane movements can be quite large, so it looks less like the cell, the edge of the cell, if you contract strongly, the edge of the cell rises and the center of the cell is pushing down or is it pushing down or is it just passively having to go somewhere and those movements are in the same order as the transverse movements, they are smaller but they are still surprisingly large and if we put cells in something like a collagen gel or a fibrin gel where we can put little fluorescent nanobeads on the fiber, then things go very badly because I think the architecture of the networks is very strange and they have this normal negative stress when you put them in shear.

As the cell pulls like this, the beads actually go out of focus, you know, even in a not very focused microscope, so there's a lot of downward movement as you pull these fibrous gels, which you don't I understand, but in the aha, things that we also don't see anything up and down, to a certain extent, yes, so we know that if we just take a polyacrylamide gel and contaminate it with hyaluronic acid polymers, then more ha we can put into that. matrix, the more the matrix doesn't get much stiffer, but the cells start to spread out more, what we can't do is get the ha density big enough if we don't do anything but reticular, hey, the density is high, the space between adjacent h's change is quite small, uh, 20 nanometers, maybe we can't put that much stuff in the polyacrylamide layer, but qualitatively, something like that happens.

Do you see the same kind of effects with hydrogen? So yeah, we don't see it. with alginate um and I think years ago there was a mit or bu paper that had used alginate instead of polyacrylamide for chondrocyte development and they showed very beautifully that the cells needed to get 10 to 20 kilopascal of alginate before they started. spread and resemble a chondrocyte, so it appears to be specific for glycosaminoglycans for which cells have receptors. I think we see global effects. One of the things that we havenoticed is that if we put cells. a collagen coated gel on the bottom and a fibronectin gel a day later on the top, the cell will detach from the collagen and simply move to the top surface, so that seems to be what is happening on one side of the cell. it communicates to the adhesion receptors on the other side of the cell um no, I think it's not and well, we have cells from cd44 knockout animals and they, uh, they, they do mechanical sensing well, they have other defects.

They can't polarize, but they can still spread, uh, but they also don't completely lose this business, so there are others, there are at least six receivers for aj, so it's not just that one, but yeah, I guess. The other thing we don't know and we're trying to look at now, although these focal adhesions are very large, we don't know if they are stable, so they could be very large and rotate much more rapidly than the previous ones. on the glass they are so in a snapshot they look similar, but what their dynamics is, we don't know yet, um, but I don't think so, but I guess both signals are rigidity or h8 plus integrating activates the same path and once you activate that path you are destined to have big action errors and vocal adhesions, so my question is, for the hills you see, this one is ready, is it just the presence of h.a inducer? extends or comes also with or is combined with a detailed stress increase because, as I mean credited with so many stress fibers, if it wasn't a polyacrylamide for a particular pascal, there was a lot more deformation, yes, yes, so, what do we do?

What we know is that if we take one of these cells that are spread on these gels and we treat it with glebostatin or something like that or even cytokinesis, it changes its shape much more slowly than a cell that is spread on a rigid substrate. So I think we probably still need ectomy activity to create any of these structures and we know that there is actin in these actin bundles. What we don't know is how much of that actin is actually activated, so we don't have. It wasn't tested whether it's activatedACTOMYOSIN or just myosin, so that's all we know, we know you can't completely avoid the pull, you need some contractility, this is not, it's not exactly like that, no It's just a drop of water on a surface, there's something more involved than that, but in a way that you can imagine, you could get them to spread in this fantastic way, not by putting more contraction, but by simply relaxing the cell cortex and letting the adhesion energy gain, so that's it.

Unfortunately that's all that's all we know about that.