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.