Cell Adhesion, Signaling and Cancer
Transcript of Part 3: Focal Adhesions as Stress Sensors
00:00:03.08 Hello, my name is Mary Beckerle, and I am a professor of Biology 00:00:06.07 and oncological sciences at Huntsman Cancer Institute 00:00:09.28 and the University of Utah. In this last segment, 00:00:13.07 I am going to talk with you about 00:00:14.19 some very interesting and exciting recent results from my laboratory, 00:00:19.08 in which we are trying to understand how cells sense and respond to mechanical cues. 00:00:25.14 We know that cells are really sensitive to a lot of different types of cues, 00:00:35.24 chemical cues, as well as mechanical cues, and what we are trying to understand 00:00:42.19 is how cells maintain a stable environment 00:00:48.01 and how organisms maintain a stable environment 00:00:49.21 in the face of all of this external simulation. 00:00:54.12 Now the concept of homeostasis, or maintaining a stable internal environment, 00:01:01.09 is one that physiologists have been aware of and have been working on 00:01:05.05 for many, many decades. 00:01:07.15 And basically there is in this concept of homeostasis 00:01:12.13 a model cassette for how a stimulus can impact 00:01:19.07 with a sensor and an effector pathway, 00:01:21.24 and then, via a combination of positive and negative feedback, 00:01:26.07 control the environment and maintain stability. 00:01:30.25 So how would this work when we are talking about 00:01:34.16 the exposure of cells and organisms to 00:01:37.19 significant mechanical stress? Well, we know that there are many 00:01:42.26 mechanosensitive organs in our body: bone, muscle, lung, the vasculature, 00:01:50.08 the heart. And if cells and organs don't respond 00:01:55.13 appropriately to mechanical signals, this can lead to very serious consequences 00:02:00.20 and serious disease. In the case of bone, for example, 00:02:05.06 which is a weight bearing organ, if there's not sufficient weight on the bones, 00:02:11.19 this can give rise to disuse osteoporosis. 00:02:15.01 In muscle, as illustrated schematically here, 00:02:19.21 there's a very, very dramatic response 00:02:22.22 to mechanical stimulation, where if muscle is exposed 00:02:27.29 to additional force or weight bearing, this can 00:02:33.14 give rise to significant hypertrophy or expansion of the muscle mass. 00:02:39.17 And in contrast, if muscle is not utilized due to 00:02:46.10 disease or disuse, it can atrophy. And one of the 00:02:51.02 ways in which both hypertrophy and atrophy happen 00:02:54.16 is by expansion of individual cells or the reduction in size of individual cells. 00:03:04.27 So how is it that muscle cells can respond to these mechanical 00:03:10.14 signals, communicate to the nucleus to either 00:03:16.00 activate programs of gene expression that lead to more muscle growth 00:03:19.05 or allow for a decrease in muscle mass when it is not required? 00:03:26.17 Similar response to mechanical stress is evident in the cardiovascular system. 00:03:32.26 Chronic high blood pressure is known to put an additional load on the heart. 00:03:39.01 And when that kind of condition exists, 00:03:43.02 you can see that you get this morphological 00:03:45.07 increase or hypertrophy of the heart. 00:03:48.27 And we now understand that this is not due to the 00:03:52.28 production of additional cardiac cells. 00:03:54.23 We are born with the number of cardiac muscle cells 00:03:57.07 that we are going to have when we die. 00:03:58.12 However, excessive mechanical load or enhanced mechanical load 00:04:03.19 causes the activation of cellular programs that lead to 00:04:09.04 expansion of the size of individual cardiac muscle cells, 00:04:13.27 and this ultimately leads to the expansion of the size of the heart. 00:04:17.17 So here you can see unstimulated or unstressed cardiomyocytes, 00:04:23.18 and here you can see corresponding cardiomyocytes 00:04:27.09 from a heart that has been put under stress. 00:04:32.11 And essentially what has happened here is that those muscle cells 00:04:35.17 are able to sense that increased mechanical load 00:04:39.24 and that signals them to activate and reactivate 00:04:43.12 the fetal program for gene expression 00:04:45.29 so that they build more contractile machinery. 00:04:49.01 This, of course, is initially adaptive and provides 00:04:52.06 for a mechanism by which the cardiac output 00:04:56.15 can be enhanced to compensate for the increased load on the heart, 00:05:03.16 but at some point it becomes maladaptive and leads to cardiomyopathy. 00:05:10.25 So where is the cell's stress sensor and how are these mechanical signals sensed, 00:05:19.11 and how are they responded to? 00:05:22.04 One of the really interesting ideas if you think about a cell 00:05:27.10 that is attached to the extracellular environment, 00:05:30.14 and we have, as we discussed before, these focal adhesions which are specialized 00:05:35.07 regions of the cell membrane that tether 00:05:37.28 the cell to the extracellular environment. 00:05:40.02 You can imagine that if this cell was being stretched, 00:05:43.25 that if you were pulling on this matrix here, 00:05:46.08 that these focal adhesions would really serve appropriately 00:05:50.01 as conduits for transmission 00:05:52.06 of that force or the signals that result 00:05:57.09 from that mechanical stimulation. 00:06:01.08 And so we were really interested in exploring this possibility: 00:06:05.29 first testing what is the normal cellular response to mechanical stretch? 00:06:12.28 One. And where does the stress sensor reside? 00:06:18.21 And three, what are some of the mechanisms by which cells compensate 00:06:23.28 and respond to these mechanical cues? 00:06:26.29 So with Masaaki Yoshigi, my bioengineering colleague, 00:06:31.18 who developed a very, very interesting stretch device, 00:06:36.21 we've taken cells and plated them on an elastic silicone 00:06:44.08 coverslip or sheet, and these cell can...these sheets are then clamped on 00:06:50.07 both ends, and we can expose these cells to cyclic stretch. 00:06:55.22 And we use 60 hertz and about 10 to 15% stretch. 00:07:01.16 And you can see that... this is my movie... 00:07:04.28 and you can see that you can stretch these cells, 00:07:10.07 and now we want to understand what are the consequences 00:07:12.07 of that stretch response. 00:07:15.09 So one of the first things that you see when you stretch cells in this way 00:07:20.28 is a really dramatic change in the actin cytoskeleton. 00:07:25.06 Here is an unstretched cell and you can see 00:07:28.13 the filamentous actin labeled with rhodamine phalloidin, 00:07:32.18 and you can see the actin stress fibers. However, if you stretch 00:07:36.09 these cells, and this particular cell was stretched, I believe, for one hour, 00:07:40.20 you see this really, really dramatic reinforcement of the actin cytoskeleton. 00:07:45.07 The stress fibers are much more robust, much more intense. 00:07:50.20 We developed a mechanism to actually quantitate the stress fiber thickness, 00:07:56.11 and when we do that we can see that even within thirty minutes of stretch 00:08:03.03 we get a really statistically significant enhancement 00:08:06.21 in the thickness of the actin filaments. 00:08:10.03 And this is sustained over a long period of time. 00:08:15.05 Another impact of cyclic uniaxial stretch on cells 00:08:23.27 is a re-orientation, and re-alignment of the actin cytoskeleton 00:08:28.12 perpendicular to the stretch vector. And these are 00:08:31.28 fibroblasts that we are using, and different cells respond in different ways. 00:08:35.15 But very robustly in fibroblasts you start out with 00:08:40.20 unstretched cells that are randomly 00:08:42.21 oriented. And then over time, progressing to about eight hours, 00:08:48.16 you can see that the cells have now begun to align perpendicular 00:08:53.00 to the stretch vector. 00:08:54.01 And again we can quantify this change, and you can see 00:08:57.27 we go from randomly oriented cells with 00:09:03.00 cells oriented in all different directions to 00:09:06.22 a cell population where there is a very high probability that they are going 00:09:11.05 to be aligned perpendicular to the stretch vector within eight hours. 00:09:15.13 So what we see then is with these fibroblast cells we have two 00:09:21.21 really dramatic and consistent responses to mechanical stimulation. 00:09:26.08 We have a cytoskeletal reinforcement, and we have a 00:09:32.17 cytoskeletal re-orientation. 00:09:34.09 And it's really been very unclear how these are related to each other. 00:09:39.11 You could imagine that since the reinforcement occurs 00:09:42.06 on a more rapid time course 00:09:43.29 that it could be required prior to re-orientation, 00:09:48.23 or alternatively in this other model, 00:09:51.29 you could envision that mechanical force independently stimulates 00:09:55.09 these two responses and that they are mechanistically distinct. 00:10:00.21 So we wanted to try and address how 00:10:05.06 the cells are responding to these mechanical cues, and what kinds of 00:10:09.00 changes are happening within the cells that give rise to 00:10:12.13 this cytoskeletal reinforcement and re-orientation. 00:10:16.18 And again, we thought about how the cells are sitting on their 00:10:21.11 extracellular substratum, and essentially when we stretch that 00:10:25.02 silicone membrane, we are pulling on the places where 00:10:30.15 cells are tethered to the matrix, and these are 00:10:32.21 focal adhesions. So we thought it would be very interesting 00:10:35.19 to look and see whether any 00:10:38.10 focal adhesion constituents changed their distribution, or altered it 00:10:42.00 in any way in response to stretch in order to 00:10:44.17 give us some insight into some of the molecular events 00:10:47.16 that occur when cells are exposed to this type of mechanical stimulation. 00:10:52.03 And we really did a survey of a large, large number of focal adhesion 00:10:56.25 constituents to explore their distribution before and 00:11:01.03 after stretch, and I'll just show you two examples 00:11:06.10 here this morning. One is the protein vinculin 00:11:10.22 which is a focal adhesion constituent that does not change 00:11:13.24 its distribution in response to stretch, and then I'll show you 00:11:16.29 a second one, the protein zyxin, which I talked to you about extensively in part two 00:11:22.04 of this series, which is a focal adhesion constituent 00:11:25.27 which really responds quite dramatically and rapidly 00:11:29.13 to mechanical stress. So here you can see 00:11:32.13 in the top two cells here an unstretched cell 00:11:37.22 that's been double labeled with antibodies 00:11:39.16 against vinculin on the left and zyxin over here on the right. 00:11:43.06 And you can see that these two proteins are nicely 00:11:46.26 co-localized at these focal adhesions. After stretch, you can 00:11:52.18 see that the vinculin is still found at these punctate 00:11:56.16 focal adhesions, but the zyxin is basically missing 00:12:00.21 from these focal adhesions and is now populating these long 00:12:03.18 actin stress fibers. And you can see that 00:12:07.27 these two proteins are behaving extremely differently 00:12:11.10 to the stretch stimulus in this overlay image 00:12:14.24 where in the unstretched cells you can see 00:12:19.06 the yellow merge indicating coincidence 00:12:21.21 of the zyxin and vinculin labeling, 00:12:23.25 showing that both proteins are prominently localized 00:12:27.04 at the focal adhesions, whereas in the stretched conditions you see red 00:12:32.09 here where the vinculin is remaining in the focal adhesions, 00:12:37.18 and the zyxin label has now traveled to a new distribution 00:12:41.24 in the cell to the stress fibers. 00:12:44.10 So this was a very, very striking observation 00:12:49.18 that really rapidly upon uniaxial cyclic stretch 00:12:53.21 there's a response within the focal adhesion 00:12:57.07 in which one of the constituents within the focal adhesion 00:13:00.04 rapidly is translocated from the focal adhesions to the actin stress fibers. 00:13:07.18 And we were very interested in this possibility. 00:13:12.11 It was the first example of a protein that was really 00:13:16.01 a mechanosensitive protein that responded in this dramatic way 00:13:21.07 to force, mechanical stimulation. And, as we discussed before, 00:13:27.08 this is the molecular architecture of the zyxin protein, 00:13:31.12 and we of course wanted to know now-we see this dramatic change 00:13:35.15 in zyxin's distribution-is the zyxin protein required 00:13:40.10 for any of the major morphological changes 00:13:43.26 that we see in response to stretch, either the actin reinforcement 00:13:47.17 or the re-orientation of the actin cytoskeleton. 00:13:51.22 So to address this question, we generated a zyxin null mouse, 00:13:57.13 and isolated mouse embryo fibroblasts that completely lack the zyxin protein 00:14:02.28 and then compared the behavior of those zyxin null cells with 00:14:08.03 wildtype cells that are stretched using this uniaxial cyclic stretch mechanism. 00:14:15.10 And here you can see on the left-hand side wildtype cells 00:14:19.25 unstretched and stretched, and just visually I think you can see how striking 00:14:24.23 it is that the cells are oriented in lots of different ways 00:14:28.14 prior to stretch, and then after stretch 00:14:31.12 they align perpendicular to the stretch vector, 00:14:33.21 and you can see this really quite dramatic reinforcement 00:14:37.23 or thickening of the actin stress fibers. 00:14:39.12 Over here in contrast, if we look at the zyxin null cells 00:14:43.21 you can see starting out that the cells are not aligned in any particular 00:14:48.18 way, and what you see after stretch is that they actually are aligned, 00:14:54.21 but you can see quite clearly that they have not 00:14:57.22 generated this really robust actin reinforcement response that 00:15:03.12 characterizes the response of the wildtype cells. 00:15:07.06 And we can see this quantitatively first down here 00:15:09.29 if we look at the alignment index you can see that there is no difference 00:15:14.01 really between the behavior of the wildtype cells and the zyxin null cells. 00:15:17.27 However, over here if we look at the actin 00:15:21.29 thickness index, you can see that the wildtype cells 00:15:27.08 in the closed circles really nicely reinforce their actin 00:15:32.13 cytoskeleton in response to stretch. 00:15:34.18 Whereas the zyxin null cells although they are re-aligning, 00:15:38.03 really don't dramatically increase the thickness of their actin filaments 00:15:41.18 in response to stretch. 00:15:44.29 So this told us really two very important things. First of all, 00:15:48.16 it told us that zyxin was required for the cytoskeletal reinforcement 00:15:56.12 that occurs in response to mechanical force, 00:15:59.04 and this was I think a very important new insight into 00:16:02.14 how cells respond to mechanical stimulation. 00:16:06.26 It also told us that zyxin was not responsible for cytoskeletal re-orientation, 00:16:13.16 and that then allowed us to distinguish between these two 00:16:18.19 possible models here and clearly illustrates 00:16:21.15 that re-orientation does not depend on reinforcement 00:16:25.11 and suggests that these two 00:16:29.09 responses are mechanistically distinct. 00:16:32.23 So thinking about...where is the sensor? 00:16:40.15 Obviously zyxin is at focal adhesions, so it seems reasonable to think 00:16:45.28 that the initial sensory device is housed within the focal adhesion 00:16:52.02 and one candidate for the molecular sensor, 00:16:56.13 or at least a molecule that really contributes 00:16:59.10 to the ability to sense these changes in mechanical stimulation 00:17:03.00 is the integrin adhesion receptor, 00:17:07.19 because clearly that is the transmembrane link between the 00:17:11.18 extracellular matrix and the actin cytoskeleton. 00:17:14.02 So it's positioned in sort of almost the perfect location 00:17:19.11 to be the conduit for sensing these mechanical changes. 00:17:23.10 So we looked to see whether or not integrin dependent 00:17:27.06 adhesion was required for the ability of zyxin 00:17:30.18 to move from the focal adhesions to 00:17:33.08 the actin cytoskeleton in response to stretch. 00:17:37.12 And the way we manipulated the adhesion of the cells 00:17:40.19 is to plate cells on polylysine, which allows the cells to spread, but they 00:17:45.21 do not require integrin dependent adhesion. 00:17:48.20 And here you can see that in unstretched cells plated on polylysine 00:17:53.06 you see zyxin in these concentrated adhesive structures, 00:17:57.29 and when we stretch these cells, 00:18:01.15 in contrast with cells plated on a fibronectin or collagen matrix 00:18:06.11 that engages the integrins, 00:18:08.12 where zyxin would move to the actin cytoskeleton, what we see is that the zyxin 00:18:11.28 protein really does not change its distribution in response to stretch. 00:18:17.27 So this experiment illustrated that zyxin depends on integrin 00:18:24.03 engagement in signaling in order to 00:18:25.29 sense the cues that are coming when 00:18:30.02 cells are exposed to mechanical stimulation. 00:18:33.03 So we see now that cells respond to mechanical stress by enhancing 00:18:40.03 their cytoskeletons, reinforcing their cytoskeletons 00:18:43.18 in a very dramatic way, and in a way that depends on integrin dependent adhesion 00:18:48.19 and the focal adhesion protein zyxin. 00:18:52.13 And we know that cytoskeletal reinforcement is actually really 00:18:59.21 regulated in general by the small GTPase Rho, which is 00:19:06.05 required for the ability of cells to build stress fibers. And so we were interested 00:19:10.18 in whether or not there was a role for Rho in this cytoskeletal 00:19:15.23 reinforcement process, or in the reorientation process, 00:19:20.11 but basically, what is the role of Rho in the response to mechanical stress? 00:19:24.09 And we tested this by exploring the impact of 00:19:28.11 inhibiting one of the downstream effectors of Rho, the Rho-kinase, 00:19:31.13 and analyzing how cells responded to mechanical stimulation 00:19:35.22 when Rho-kinase was inhibited. 00:19:37.25 And here you can see again on the top panel control cells 00:19:41.23 either exposed to...unstretched or exposed to stretch, 00:19:46.10 and once again, the very dramatic 00:19:48.22 reinforcement of the actin cytoskeleton 00:19:51.06 and realignment of the cells perpendicular 00:19:54.24 to the stretch vector. And the surprising thing 00:19:57.26 that we observed when we inhibited the Rho signaling pathway, in particular 00:20:01.26 Rho-kinase, is that not unexpectedly, the unstretched cells 00:20:08.16 didn't have very robust actin cytoskeletons, 00:20:11.06 but surprisingly, when we stretched those cells they actually 00:20:15.18 responded many of them, by building these actin filament 00:20:21.14 bundles that interestingly were not oriented perpendicular to the stretch vector. 00:20:27.16 So these results I think were both striking and surprising. 00:20:32.23 And suggests that actually the Rho signaling pathway may be important for 00:20:38.09 the cell's ability to reorient the cytoskeleton in response to stretch 00:20:45.17 but does not appear to be critical 00:20:48.03 for the cytoskeletal reinforcement. 00:20:51.19 So what are the mechanisms by which the cytoskeletal reinforcement 00:20:57.04 might occur? We see this thickening of the actin stress fibers. This could occur 00:21:01.17 by recruitment of filamentous actin from the 00:21:05.29 cytosol into the actin stress fibers. 00:21:08.16 It could occur by increasing actin bundling along the 00:21:12.17 length of the stress fibers, stabilizing preexisting 00:21:16.29 actin filaments against depolymerization 00:21:20.08 or perhaps stimulation of de novo actin assembly 00:21:24.27 and polymerization. And we wanted to explore this possibility 00:21:30.16 and assess which other factors might collaborate with zyxin 00:21:35.06 in this cytoskeletal reinforcement process. 00:21:38.02 And you may recall from part two of my presentation that I talked with you 00:21:44.24 about the fact that the zyxin protein has a structural and functional similarity 00:21:51.06 to the Listeria ActA protein which is critical for enabling actin 00:21:57.29 filament assembly on the surface of this intracellular 00:22:00.19 pathogen, Listeria. And zyxin in particular has 00:22:06.04 four of these ActA repeats which are proline rich 00:22:09.04 repeats which are responsible for docking Ena/VASP proteins which are 00:22:15.13 clearly implicated in the regulation of actin assembly and 00:22:18.29 which contribute to enhancement of actin assembly. 00:22:22.05 So as an initial step to explore whether or not Ena/VASP proteins 00:22:26.28 might be involved as well as zyxin 00:22:29.18 in this cytoskeletal reinforcement in response to mechanical stimulation, 00:22:34.18 we explored whether or not Ena/VASP proteins like 00:22:40.01 zyxin move from the focal adhesions to the actin cytoskeleton 00:22:44.12 in response to stretch. So we first wanted to explore whether 00:22:49.13 or not the VASP protein responds to mechanical stress 00:22:53.16 by mobilizing from focal adhesions 00:22:56.22 to the actin stress fibers. And we did this by looking at wildtype cells, 00:23:04.08 and here you can see that VASP is normally localized 00:23:07.16 in focal adhesions of unstretched cells 00:23:10.10 where it's co-localized with the zyxin protein. 00:23:13.11 And then when you stretch those cells, 00:23:16.15 quite strikingly, and very similar to what we observed for zyxin, 00:23:20.00 the VASP protein moves to these actin filament arrays. 00:23:25.19 So like zyxin, VASP is mobilizing from focal adhesions to the actin stress fibers 00:23:30.22 very dramatically in response to this uniaxial cyclic stretch. 00:23:37.00 So since we know that zyxin interacts directly with VASP, we looked to see 00:23:43.09 whether or not this VASP redistribution 00:23:46.12 in response to stretch depends on the zyxin protein. 00:23:50.04 And so here we have examined the behavior of VASP 00:23:55.05 in zyxin null cells that have been 00:23:57.07 stretched. And what you can see is that already in the 00:24:01.19 unstretched cells VASP is absent from the focal adhesions 00:24:06.18 illustrating that VASP depends on zyxin to accumulate at these 00:24:11.21 focal adhesion sites. And likewise, after stretch, we don't see any accumulation 00:24:17.12 of VASP on the actin stress fibers in the zyxin null cells 00:24:22.03 compared to wildtype cells, 00:24:23.18 consistent with the idea that VASP really depends on zyxin 00:24:29.09 for both of these subcellular localizations. 00:24:32.13 Both localization to the focal adhesions in unstretched cells 00:24:36.02 and localization to the actin stress fibers in stretched cells. 00:24:41.04 And we can demonstrate this directly by re-introducing into the zyxin null cells 00:24:46.29 a GFP tagged zyxin, and under those conditions, we can see that VASP 00:24:54.09 now shows restored localization in the focal adhesions 00:24:59.05 and to the stress fibers upon stretch. 00:25:02.04 So clearly VASP is responding coordinately with zyxin 00:25:08.29 in response to stretch, and actually depends on the zyxin protein for its ability 00:25:16.05 to mobilize from focal adhesions to actin stress fibers in response to stretch. 00:25:22.13 A really interesting question that is not resolved 00:25:25.06 is whether or not the Ena/VASP proteins are absolutely essential 00:25:29.28 for this cytoskeletal reinforcement response. 00:25:33.01 And so we are hoping to approach that in two ways, 00:25:36.04 first to examine the response of Ena/VASP null cells 00:25:42.04 to uniaxial mechanical stretch and to see 00:25:46.08 whether or not these cells that lack Ena/VASP family members 00:25:50.10 will reinforce their actin cytoskeletons as wildtype cells do. 00:25:54.17 And secondly, since we have been able to show that we can reintroduce 00:25:58.03 a wildtype zyxin protein into zyxin null cells and reconstitute 00:26:04.08 a normal stretch response, we are developing mutated forms of zyxin 00:26:10.14 that lack functional ActA repeats. 00:26:13.15 And we will be able to reconstitute our zyxin null cells 00:26:16.11 with a zyxin molecule that is functional except for 00:26:20.21 its ability to interact with Ena/VASP family members 00:26:24.02 and explore in those cells whether or not the cells respond 00:26:29.03 appropriately to uniaxial cyclic stretch. 00:26:32.10 So in conclusion then what we have been able to show is that the zyxin protein 00:26:40.16 is really, really important for the cells' ability to respond to mechanical stimulation 00:26:49.00 and in particular, to reinforce 00:26:52.09 the actin cytoskeleton in response to a mechanical stress. 00:26:58.16 This mechanical stress activates these two very important responses: 00:27:03.29 the cytoskeletal reinforcement response and a reorientation response. 00:27:09.01 The integrin proteins and zyxin 00:27:12.11 are important in this cytoskeletal reinforcement pathway and likely 00:27:18.17 the Ena/VASP family members will be as well 00:27:21.10 because they facilitate zyxin's ability to stimulate and promote actin assembly. 00:27:27.25 And we've shown that they move with zyxin in a coordinated fashion 00:27:32.08 in response to mechanical force. Interestingly, 00:27:36.19 this second response to mechanical force, the cytoskeletal re-orientation 00:27:43.21 we were able to show is independent of zyxin 00:27:47.04 but rather seems to be dependent on the activity of Rho kinase. 00:27:52.28 And we believe that one of the things that is happening 00:27:56.16 is that zyxin and VASP are recruited to the actin cytoskeleton in response to 00:28:03.13 stretch and that allows, because of their properties 00:28:07.27 in inducing actin assembly, that allows for reinforcement 00:28:12.23 of these actin stress fibers. 00:28:14.28 There are many, many more questions that remain to be addressed here 00:28:20.11 in this extremely interesting 00:28:23.01 research area. Again, I think that this 00:28:26.23 whole area of how cells respond to mechanical cues 00:28:30.07 is really a relatively new frontier in 00:28:32.19 cell biology that has important biomedical implications 00:28:36.25 since the inappropriate cellular and tissue response to stretch 00:28:46.18 or mechanical stress can lead to a variety of different types of 00:28:50.05 pathological situations. And we hope that 00:28:55.18 we are going to be able to understand more about exactly what 00:28:58.27 is happening in these situations by understanding what are the signals 00:29:03.03 that emanate from these integrin rich adhesions sites 00:29:07.16 to zyxin and its partners to stimulate their redistribution and 00:29:11.19 perhaps their change in activity, which leads to this important 00:29:15.27 cytoskeletal reinforcement response. 00:29:18.28 So again, we've got zyxin and its partners playing critical roles 00:29:24.19 in the cellular response to mechanical stress, 00:29:27.15 and I think these results really focus our attention on these 00:29:32.21 adhesion plaques, or focal adhesions, 00:29:35.06 as domains that are important domains in the ability to sense 00:29:40.21 mechanical cues, and really this leads me back 00:29:45.20 to one of my introductory comments 00:29:47.15 about homeostasis, and you begin to think about the ability 00:29:52.15 of cells to have tension homeostasis and maintain 00:29:56.29 a balance of force at these adhesion sites. Under normal 00:30:01.18 conditions you would imagine that there is a balance achieved with 00:30:05.27 the extracellular matrix and the actin stress fibers, 00:30:09.12 and if you actually pull on the extracellular matrix 00:30:16.17 or build up a lot more extracellular matrix which impinges on the cell 00:30:20.16 then the cell responds to that perhaps in a way that is similar to 00:30:24.10 what occurs with uniaxial cyclic stretch 00:30:26.29 by reinforcing the actin cytoskeleton. 00:30:30.06 And I think this is telling us that cells are really 00:30:33.18 able to sense this delicate balance 00:30:36.04 here and achieve a balance of forces 00:30:39.25 at these important sensory zones for the cell 00:30:44.10 So thank you very much, and 00:30:46.18 I look forward to being able to provide you an update on this 00:30:50.14 interesting research project in the future.