Cell Adhesion, Signaling and Cancer
Transcript of Part 2: Discovery and Characterization of a Focal Adhesion Protein Implicated in Tumor Progression
00:00:03.26 Hello, my name is Mary Beckerle, 00:00:05.24 and I'm a professor of Biology and Oncological Sciences at 00:00:08.26 Huntsman Cancer Institute at the University of Utah. 00:00:11.04 In this part of my presentation, I want 00:00:15.13 to describe to you some work in my laboratory 00:00:19.07 on the discovery and characterization of a focal adhesion 00:00:22.22 protein that is implicated in tumor progression. 00:00:28.01 As we've discussed in part one, cells are surrounded by 00:00:34.19 other cells and extracellular matrix 00:00:37.05 material and lots of soluble cues and factors that influence 00:00:42.01 their behavior in really dramatic and important ways. 00:00:45.13 And one really important type of informational cue comes from the information 00:00:55.10 that cells get from interacting with the extracellular matrix. 00:01:00.01 Extracellular matrix interaction occurs at specialized zones of the cell surface, 00:01:09.12 which in cultured cells are called focal adhesions. 00:01:12.11 These focal adhesions, as we discussed last time, 00:01:16.28 are areas that are rich in integrin adhesion receptors, 00:01:22.17 and they are playing a really important role 00:01:26.23 in bidirectional transmembrane communication. 00:01:32.21 The focal adhesions sense extracellular matrix, 00:01:36.12 but they also are involved in sensing cues that are present on cell surfaces. 00:01:41.03 And they also, as we will discuss in part three, 00:01:44.12 can sense physical stress such as mechanical stimulation. 00:01:51.22 The responses that cells launch as a result of the signaling through 00:01:58.20 these specialized regions of the membrane are really 00:02:01.13 very diverse and include control of cell growth or cell death, 00:02:05.26 cell motility, cytoskeletal organization, 00:02:09.27 and these signals can even result in changes in 00:02:14.05 gene expression, illustrating that there must be some mechanism by 00:02:18.01 which the molecules present at these adhesion sites 00:02:22.09 are able to communicate with the nucleus. 00:02:25.06 Integrins are the primary receptor for extracellular matrix 00:02:30.22 that are located at these specialized adhesion zones. 00:02:36.11 They are concentrated at these focal adhesions. 00:02:39.11 They mediate transmembrane bidirectional 00:02:41.29 signaling, and a really interesting challenge has been to try and understand 00:02:47.01 how they signal to effect so many really important cell behaviors. 00:02:52.08 Because unlike growth factor receptors, 00:02:54.22 they themselves don't have any intrinsic catalytic activity. 00:02:58.18 Rather they seem to operate by recruitment of a large collection of 00:03:04.11 cytoplasmic proteins to the cytoplasmic face 00:03:11.04 of the plasma membrane, and it is these proteins 00:03:13.27 which really facilitate integrin signaling function. 00:03:18.02 There are over 50 components that are present 00:03:22.04 with integrins at these focal adhesions, 00:03:25.28 and these are highly dynamic structures where proteins 00:03:30.01 are coming and going. There are both structural components 00:03:33.23 and catalytic constituents. Here you can see an example 00:03:37.09 of the evidence of one catalytic constituent, and that 00:03:42.03 is tyrosine kinases and their substrates 00:03:45.28 because here we are labeling the cell with an antibody 00:03:48.26 directed against phosphotyrosine. 00:03:50.29 And you can see that these bright patches here 00:03:55.25 where phosphotyrosine is accumulated represent 00:03:58.26 the focal adhesions where integrins would also be concentrated 00:04:03.10 and where you can see there is a very close connection with the ends 00:04:07.22 of the actin filaments, the stress fibers that terminate at these sites. 00:04:13.14 One protein that is one of the many proteins present at these focal adhesions 00:04:20.11 is a protein that was discovered in my laboratory, 00:04:23.02 several years ago, called zyxin. 00:04:25.15 This was a really interesting discovery process, I think, 00:04:31.03 and one that was very serendipitous. 00:04:33.01 In fact, I was making a lot of different antibodies against focal adhesion 00:04:38.05 constituents. At that time we only knew of three focal adhesion constituents, 00:04:42.15 and I was trying to look for new ones. 00:04:44.02 And I was making antibodies against some candidate proteins 00:04:47.24 and one of the rabbits, prior to immunization 00:04:50.12 actually was producing an antibody that already stained 00:04:55.04 focal adhesions of cultured cells. 00:04:56.22 And so I followed what this antibody was recognizing, 00:05:02.07 and it turned out it was recognizing a protein that was 00:05:05.04 previously not appreciated as a focal adhesion constituent. 00:05:07.29 And this protein now has the name zyxin, 00:05:12.04 which comes from the Greek root zeugos, which means to join together, 00:05:17.14 because it is at places where actin filaments are joined to the plasma membrane. 00:05:20.21 This is a very interesting protein that had a number of 00:05:25.20 unusual structural features: highly enriched in proline residues 00:05:30.12 and it also had 3 C-terminal double zinc finger motifs called 00:05:36.16 the LIM domain. The LIM domain was first identified in three transcription factors 00:05:42.25 Lin-11, Isl-1 and Mec-3. And these are all homeodomain proteins 00:05:49.11 that contained two N-terminal LIM domains. 00:05:54.14 In collaboration with Mike Summers and Dennis Winge 00:05:57.17 we solved the structure of the LIM domain, 00:05:59.19 and we determined that it is really a double zinc finger structure 00:06:04.10 with two sort of independent modules, each of which binds a zinc atom. 00:06:10.24 And surprisingly, although many zinc fingers are involved in direct 00:06:17.24 nucleic acid binding, it's quite clear from our analysis that 00:06:22.13 LIM domains serve as protein binding interfaces. 00:06:26.03 However, I should point out that this structure of 00:06:30.09 the LIM domain is very, very similar to the GATA-1 DNA binding domain, 00:06:35.09 so there remains a possibility that in addition to protein-protein interaction, 00:06:39.17 LIM domains may be able to associate with nucleic acids, 00:06:43.05 although this has not been demonstrated directly. 00:06:46.28 There are now a number of LIM domain proteins that have been identified- 00:06:52.12 more than 50 human proteins, and they are 00:06:57.04 functionally quite diverse. Some of them have 00:07:00.00 catalytic features, such as serving as kinases, 00:07:04.00 others are present exclusively in the nucleus and are known 00:07:08.01 to play a role in regulation of gene expression 00:07:11.28 and many others are found in association with the cytoplasm. 00:07:16.20 And what I'll tell you today is that this protein zyxin, 00:07:20.11 seems to be a multifunctional protein that resides 00:07:24.00 prominently at the focal adhesions, but also 00:07:26.27 has... where it seems to regulate cell motility 00:07:30.10 and perhaps even cell death, but it also has 00:07:36.05 the capacity to move into the nucleus, so it is an amazingly interesting protein 00:07:41.02 that is a candidate for participating in direct communication 00:07:44.21 between the cell surface and the nuclear compartment. 00:07:48.28 Zyxin also, we now have come to appreciate, 00:07:55.04 may be implicated in a particular type of cancer: Ewing's sarcoma. 00:08:00.11 I want to tell you a little bit about Ewing's sarcoma, and then 00:08:04.22 about the connections between this focal adhesion protein 00:08:08.28 and its properties and the progression 00:08:11.09 of Ewing's sarcoma that we now understand from pre-clinical model studies. 00:08:17.16 Ewing's sarcoma is a very devastating childhood and young adolescent 00:08:24.26 tumor. It's a small, round, blue cell tumor 00:08:28.22 that you can see histologically is very homogeneous 00:08:33.18 in terms of the way it appears at the cellular level. 00:08:37.23 It's a tumor of the bone, extremely rare, but very, very aggressive. 00:08:43.24 And although it is rare, because it impacts children and young people 00:08:48.13 there's been a fair amount of emphasis on trying to understand its cause 00:08:54.08 and develop better treatment strategies. 00:08:56.21 Right now, we are faced with the challenge that metastatic 00:09:01.01 Ewing's sarcoma is really a lethal disease. 00:09:05.12 Here you can see some data looking at survival 00:09:08.22 from Ewing's sarcoma if the disease was localized, 00:09:15.23 where there's a really good, greater than 80% survival rate, 00:09:20.13 versus if the tumor has spread. 00:09:25.23 And many, many cases of Ewing's sarcoma have micro-metastases 00:09:28.25 at the time of initial detection, and 00:09:31.02 you can see that once one has metastatic disease 00:09:35.04 there is a very, very poor prognosis. 00:09:38.00 Even more strikingly, I think is the fact that we really don't have effective therapies 00:09:45.06 to treat this disease. And here you can see 00:09:47.11 the outcomes for patients after relapse from their initial 00:09:54.00 disease, and those treated with the most aggressive therapy really 00:10:00.18 ultimately do not do much better than those that receive palliative care. 00:10:05.05 So unfortunately this is although a rare tumor, an extremely aggressive and 00:10:11.08 difficult to treat tumor. We understand now that 00:10:16.10 Ewing's sarcoma is caused by a reciprocal chromosome translocation, 00:10:21.00 and 11;22 translocation that results in the productive fusion of 00:10:26.13 a region of the EWS gene with a region of the FLI1 gene. 00:10:32.17 And here you can see one gets out of that fusion a chimeric protein, 00:10:39.00 the EWS-FLI1 chimera, which brings together a transactivation domain from EWS with 00:10:46.05 a DNA binding domain from FLI1. 00:10:50.17 This protein is believed to function as an aberrant 00:10:54.20 transcription factor that we know is responsible for 00:11:00.19 causation of Ewing's sarcoma. 00:11:02.21 In the lab we can demonstrate that EWS-FLI1 expression is sufficient to behave 00:11:11.17 as an oncoprotein, and it's sufficient to transform cells. 00:11:14.26 Here are on the left you can see cells grown in soft agar 00:11:19.10 when they are expressing FLI1, and you can't really see any colonies developing here. 00:11:23.26 But when those cells are expressing the EWS-FLI1 fusion protein, 00:11:30.27 you can see many colonies growing 00:11:34.16 in this soft agar cloning assay. 00:11:37.19 So these cells display anchorage independent cell growth 00:11:41.27 and behave as transformed cells by this classical assay. 00:11:47.17 So a number of groups have been really interested in trying to understand 00:11:52.05 what happens within cells when you express EWS-FLI1, 00:11:56.13 and there's been a recent report by Amsellem and colleagues 00:12:01.26 in which they explore what is the difference between gene expression 00:12:07.18 patterns in 3T3 cells and in 3T3 cells that are programmed to express the EWS-FLI1 00:12:15.14 oncoprotein. And their analysis identified several genes which were 00:12:22.14 misregulated or displayed altered expression patterns 00:12:26.21 when EWS-FLI1 was present. And one of these was 00:12:32.10 the focal adhesion protein zyxin that I mentioned 00:12:35.17 was discovered by my laboratory. 00:12:38.10 You can see here if we look at the level of protein in a 3T3 cell control 00:12:44.04 using an anti zyxin antibody, you see 00:12:46.17 this much protein, and a typical 3T3 cell expressing 00:12:51.04 EWS-FLI1 shows this dramatic reduction in the level of the zyxin protein. 00:12:56.27 And here you can see this again by immunocytochemistry, 00:13:00.10 where in the control cells you can see zyxin nicely concentrated 00:13:04.18 at these focal adhesions, whereas in the 00:13:08.26 3T3 cells that have been transformed with EWS-FLI1, 00:13:13.19 there's both an altered morphology and a loss of zyxin at the focal adhesions. 00:13:20.10 Interestingly of course, when you put EWS-FLI1, a transcriptional regulator into cells, 00:13:29.03 you are expecting to have many, many dramatic changes occur 00:13:33.04 within those cells. So seeing that zyxin is down and it is 00:13:35.20 correlated with this change in morphology 00:13:37.10 is one thing, but knowing that it is responsible, and to what extent 00:13:41.24 it might be responsible for that change in morphology 00:13:43.28 is an open question that needs to be addressed directly. 00:13:48.01 And it was addressed by these investigators by taking these 00:13:52.05 3T3 cells that are transformed with EWS-FLI1, 00:13:57.14 which again show this really dramatic altered morphology 00:14:01.08 from this really well spread, fibroblast-like morphology 00:14:07.00 to a much more apparently motile and non-spread morphology. 00:14:17.06 And what they were able to do was to ask, okay, 00:14:19.13 in these cells, if we put back a wildtype zyxin, 00:14:23.11 what happens? And remarkably you can see this really dramatic phenotypic 00:14:29.13 reversion where the simple re-expression of this one gene, the zyxin 00:14:34.21 gene, causes a phenotypic reversion to something that looks 00:14:38.03 much more like the untransformed cells. 00:14:41.11 So this provided an indication that the zyxin protein 00:14:45.22 was not only reduced in expression, and in response to EWS-FLI1 00:14:51.20 transformation, but also might be responsible for some of the 00:14:56.13 transformed phenotypes that is associated with EWS-FLI1 expression. 00:15:02.23 So my laboratory has been interested in trying to understand what is the function of zyxin. 00:15:08.07 And we took an approach of generating a targeted disruption 00:15:12.20 of the zyxin gene in order to completely eliminate all expression of zyxin 00:15:17.27 and then be able to study the consequences of loss of zyxin 00:15:22.01 expression for cell function. 00:15:24.19 And here you can see that we have successfully generated cells 00:15:29.21 that lack zyxin. Here's wildtype cells, mouse embryo fibroblasts, 00:15:34.29 expressing zyxin. You can see nice focal adhesions 00:15:38.01 staining. And over here, you can see... you can't see. There is a cell here. 00:15:42.24 And it has been labeled with anti-zyxin antibody and there is no zyxin 00:15:46.28 protein. And this is confirmed by the Western blot here showing that 00:15:52.03 the targeted gene disruption was effective 00:15:55.04 and completely eliminates the zyxin protein. 00:15:58.06 Weâ€™ve now begun to characterize those cells 00:16:03.01 and trying to think about how the changes in those cells 00:16:06.20 might be relevant for this Ewing's sarcoma 00:16:11.16 phenotype that was observed by Amsellem and colleagues. 00:16:14.27 First thing that we looked at since we knew that zyxin was present at these 00:16:20.13 focal adhesions where actin filaments are tethered to the plasma membrane 00:16:24.05 is whether the actin cytoskeleton was disturbed in the zyxin null cells. 00:16:29.23 And here you can see that although the zyxin null cells 00:16:35.09 display actin stress fibers-so here are control cells 00:16:39.22 and then null cells-both display actin stress fibers, so we can't 00:16:44.15 conclude that zyxin is absolutely essential 00:16:47.24 for the establishment of these actin arrays in cells. 00:16:51.14 But if we perturb this system and challenge it with a Rho kinase inhibitor, 00:16:57.03 and remember now Rho signaling is important for building 00:17:02.07 of robust stress fiber arrays, and Rho acts in part through 00:17:06.21 the activity of the Rho kinase. So if we perturb Rho kinase signaling 00:17:13.12 with a Rho kinase inhibitor- if we treat wildtype cells with a Rho kinase inhibitor for short 00:17:20.06 periods of time under conditions where we don't see dramatic reduction in the stress fibers. 00:17:25.21 If we look at null cells, we see that the stress fibers are completely eliminated 00:17:31.22 when zyxin isn't there. And this would be 00:17:34.08 the ultimate outcome for the wildtype cells too 00:17:38.06 if we continued this treatment for a longer period of time. 00:17:41.11 So these results illustrated that although the actin stress fiber arrays are 00:17:49.10 present in both wildtype and zyxin null cells 00:17:52.14 that the arrays that are present in the zyxin null cells are less robust 00:17:57.02 and are more sensitive when stress fiber pathways are compromised. 00:18:03.10 And this is actually very interesting because there is a long history illustrating that 00:18:11.18 stress fiber content is inversely related to motility. That is that 00:18:15.13 the bigger and more robust the stress fibers are 00:18:19.05 the less motile the cells are. So you can imagine then that 00:18:23.19 if the zyxin null cells have reduced stress fiber content 00:18:28.29 or less robust stress fiber content 00:18:31.12 that one might anticipate that the cells would show enhanced motility. 00:18:36.05 And indeed that is exactly what we see. We've looked at 00:18:39.24 the zyxin null cells and evaluated their motility properties, 00:18:43.22 in a number of different ways. And here is just one experiment 00:18:47.12 where we looked in a monolayer wound assay. 00:18:51.17 We took wildtype and zyxin null cells, grew them 00:18:56.17 in a monolayer, and then scratched a wound in that monolayer, 00:19:01.16 measured that wound gap, and then monitored the recovery of that wound as the cells 00:19:09.29 migrated into the cleared area. 00:19:12.13 And this is just an endpoint analysis shown here, 00:19:16.11 but after eight hours you can see that the zyxin null...the wildtype 00:19:19.28 cells still have a pretty substantial gap in this wound, 00:19:23.05 whereas the null cells show complete recovery of the wound. 00:19:29.09 And a blind analysis of a large number of wounding assays 00:19:34.15 and quantitation of the velocity of cell migration revealed that 00:19:38.04 the zyxin null cells actually migrate about twice as fast as the wildtype cells, 00:19:46.26 consistent with the idea that the loss of zyxin could be involved in tumor progression 00:19:55.08 and motility. Zyxin null cells also seem to display migratory properties that are 00:20:02.11 uncoupled from extracellular matrix cues. 00:20:05.05 We talked a lot in the first part about how cells normally get 00:20:09.17 substantial signals from their environment and how that has an impact on 00:20:14.16 the signaling output and the behavior of the cells. 00:20:17.14 And in this experiment in which we've examined and compared 00:20:22.21 wildtype cells and zyxin null cells 00:20:24.08 for their migration properties in a Boyden chamber, a transwell migration assay, 00:20:30.27 we have some indication that the zyxin null cells are migrating 00:20:35.26 at sort of maximum speed regardless of any extracellular matrix cues. 00:20:41.23 And this is a really striking phenotype. 00:20:44.27 Here you can see in the open bars the wildtype 00:20:47.28 cells and what we've done here is to put cells on the top of this Boyden chamber 00:20:53.26 and then put different amounts of extracellular matrix protein, in this case fibronectin, 00:21:00.24 on the underside of that filter and allowed the cells to migrate through. 00:21:04.20 And what you can see is that wildtype cells don't migrate very well at all 00:21:10.00 when there is no fibronectin present, no sort of signal, 00:21:13.09 nothing to grab onto on the other side of that filter. 00:21:17.19 And as the fibronectin level increases, you can see that the 00:21:22.18 wildtype cells begin to migrate effectively 00:21:26.11 through the pores and onto the other side of the filter. 00:21:29.29 Interestingly the null cells in comparison, start out, even 00:21:34.25 with no extracellular matrix cues, migrating at maximal velocity 00:21:39.28 and seem unaffected by increasing fibronectin concentration. 00:21:45.25 So what this suggests is again consistently showing us 00:21:50.03 that the zyxin null cells are migrating more effectively than wildtype cells. 00:21:54.02 But also suggests that in the absence of zyxin 00:21:57.25 the cells are sort of almost primed for migration. 00:22:01.04 They appear to be behaving as if they have a signal 00:22:04.19 that there was matrix present when there really isn't any matrix present. 00:22:08.13 Now going back to the Ewing's sarcoma model system then, 00:22:13.13 what is the impact of the expression of EWS-FLI1 oncoprotein 00:22:21.05 in 3T3 cells vis-Ã -vis migration? 00:22:24.14 And does restoration of zyxin expression also suppress a migratory phenotype? 00:22:33.01 So here you can see work again from Amsellem and colleagues 00:22:37.05 where we are looking at migration of normal 3T3 cells 00:22:42.09 following sort of tracks of cells and measuring 00:22:46.08 net displacement. And here you can see when we put in 00:22:51.04 the EWS-FLI1 transgene into those cells in the blue bars, 00:22:56.23 we see a dramatic increase in motility, 00:23:00.27 consistent with what you'd expect for a transformed cell. 00:23:05.06 And interestingly... and we see down here 00:23:10.07 that zyxin levels have fallen there as I showed you before. 00:23:15.18 Now interestingly when you put zyxin back into these cells 00:23:19.10 what are the consequences for cell motility? 00:23:21.06 Well, you see a restoration of the more slow, wildtype motility. 00:23:27.06 So once again it appears that the changes in zyxin levels 00:23:32.27 are contributing very substantially to the motile phenotype of these cells. 00:23:38.06 And I think that this...some insight into how this 00:23:42.08 occurs may come from work of Yu-li Wang's lab, where he followed individual cells 00:23:50.07 that were plated on a polyacrylamide substratum 00:23:55.05 into which he embedded fluorescent latex beads 00:23:57.29 so he could really look at how much force 00:24:00.19 is generated at various adhesion points, 00:24:04.08 tethering points between the cell and the substratum. 00:24:10.23 And he noticed a very interesting thing, and that is that when you 00:24:17.03 compare the traction stress maps, where they are really measuring 00:24:20.21 the deflection of these beads with the location and intensity 00:24:24.28 of expression and localization of a GFP-zyxin, 00:24:29.24 what they observed is that where you have really, really high traction forces 00:24:35.11 at this leading edge here shown in this deeper red color, 00:24:38.00 you see that the area is really devoid of zyxin. 00:24:42.01 So, and then you can follow over time a single, the development of a single 00:24:48.18 focal adhesion and see that this focal adhesion starts out 00:24:54.05 with no zyxin and then the zyxin level goes up, up, up 00:24:58.14 and then plateaus. And strikingly, as the zyxin levels are going up here 00:25:03.13 the traction stress is going down. 00:25:06.20 So clearly there is an inverse relationship between 00:25:09.15 the presence of zyxin at these focal adhesions 00:25:12.21 and traction stress, and this could be a very interesting functional explanation 00:25:19.11 for why the cells that lack zyxin are more migratory, 00:25:24.25 that is they have the capacity to generate a greater or more sustained 00:25:29.05 traction stress. And that is something that we're all very interested in looking at 00:25:33.01 in more detail in the future. 00:25:35.00 So in summary of this part of the presentation then, 00:25:38.03 I described the Ewing's sarcoma as a lethal, devastating disease 00:25:46.02 of childhood, fortunately rare which is caused by 00:25:51.14 a reciprocal translocation that gives rise to the EWS-FLI1 oncoprotein. 00:25:57.05 EWS-FLI1 expression in a model Ewing's sarcoma system 00:26:03.18 results in decreased zyxin, and our knockout studies 00:26:08.22 showed that actin stress fibers are less robust in zyxin null cells, 00:26:13.11 and that decreased zyxin is associated with enhanced cell motility. 00:26:17.23 So how is this enhanced motility and impact on the actin cytoskeleton achieved? 00:26:26.02 Well, we learned something about this actually 00:26:29.19 by a sort of surprising link by studying Listeria, 00:26:35.02 an intracellular pathogen that is really the subject of Julie Theriot's 00:26:39.12 iBioSeminar presentation, so you can learn much more about 00:26:43.26 Listeria by looking at her presentation. 00:26:46.00 But, studies of Listeria provided some important insight into zyxin function 00:26:53.20 and its mechanism of action. 00:26:54.23 Just briefly, Listeria is an intracellular bacterium 00:27:00.08 that invades its host and is taken up by phagocytosis. 00:27:04.12 It's a foodborne bacterium that can cause really 00:27:09.29 devastating disease, particularly in immunocompromised individuals. 00:27:13.25 But the cell biology of this organism is that it 00:27:16.25 gets into these phagosomes and manages to 00:27:20.22 escape from this phagosomal compartment 00:27:23.18 and gets released into the cytoplasm where it can replicate. And for the 00:27:27.08 purposes of our discussion, it harnesses 00:27:29.17 the host cell machinery for actin assembly, 00:27:32.02 growing this nice long actin comet tail, 00:27:36.23 which allows it to move within the host cell and actually generate these 00:27:43.07 microspike projections which enable it to invade neighboring cells. 00:27:47.21 And in this way, it completely is able to transmit itself from one 00:27:51.14 cell to the next without ever leaving the protection of the intracellular 00:27:56.25 environment. And from a variety of investigations 00:28:02.02 we now understand that there is a single protein present on the Listeria surface, 00:28:07.09 the ActA protein, which is both necessary and sufficient for pathogenicity of this bacterium. 00:28:13.22 And it's necessary and sufficient for the ability of these bacteria to generate 00:28:20.11 these actin comet tails and thus move within the host cell and be transmitted 00:28:26.03 from one cell to the next. The ActA protein 00:28:31.00 is a protein that is anchored in the bacterial surface via a membrane anchor 00:28:36.18 and then has a long protein sequence area which 00:28:43.07 is present in the host cytoplasm. 00:28:45.29 And genetic analysis via mutational studies and deletion studies 00:28:51.03 have shown that there are two really critical functional domains in ActA, 00:28:55.18 which are important for its ability to cooperate with host cell machinery to build 00:29:02.09 this actin comet tail on the surface of the bacterium. 00:29:06.02 One is the domain here which is critical for the nucleation of actin assembly. 00:29:09.21 And the other is a series of proline repeats here which are critical for acceleration 00:29:15.04 of actin assembly on the bacterial surface. 00:29:18.06 And so, in collaboration with Daniel Louvard's lab, we made a bunch of 00:29:23.09 antibodies against Listeria ActA with the idea of trying to look for what endogenous cellular 00:29:29.02 proteins ActA might be mimicking on the surface of the bacterium, in order to 00:29:34.24 recruit host machinery and stimulate the elaboration of this actin comet tail. 00:29:39.25 And when we did this we had the surprising result 00:29:45.18 that antibodies against Listeria ActA recognized 00:29:49.03 just a single eukaryotic protein, a single human protein, 00:29:53.27 at least in our experiments. And that protein turned out to be zyxin. 00:29:57.27 And interestingly, it turns out that 00:30:01.01 this really important proline rich domain in ActA, 00:30:04.01 which is critical for acceleration of actin assembly, 00:30:06.15 is almost completely conserved in zyxin, 00:30:10.10 giving rise to the idea that zyxin might be able to stimulate actin assembly. 00:30:16.07 And indeed zyxin shares with ActA the ability 00:30:20.07 to interact with proteins in the Ena/VASP family, 00:30:23.08 which are critical regulators of actin assembly 00:30:27.19 that recruit profilin and actin and by multiple mechanisms 00:30:32.26 enhance actin assembly. So, returning back to this Ewing's sarcoma model then, 00:30:41.13 we see that in model Ewing's sarcoma cells that are expressing the EWS-FLI1 00:30:47.03 transgene, that's associated with loss of zyxin, 00:30:54.14 decreased actin stress fibers, which are associated with loss of zyxin, 00:30:58.15 as is increased motility. And so, I think it's 00:31:03.16 going to be extremely interesting to explore further 00:31:06.21 what is the role of zyxin in human patient 00:31:10.10 samples, and evaluate whether or not loss of zyxin 00:31:14.23 is correlated with more aggressive disease. 00:31:18.05 Interestingly, in the original Amsellem experiments 00:31:23.10 they also, in addition to looking at motility, 00:31:26.02 looked at whether zyxin has an impact on anchorage independent 00:31:31.25 cell growth of this EWS-FLI1 transformed fibroblasts. 00:31:35.19 And they found that it had really quite a dramatic impact. 00:31:40.11 So here you can see again normal 3T3 cells 00:31:43.26 that are not transformed don't grow in soft agar, 00:31:49.06 and so you don't have any colonies. 00:31:51.21 If you express, if you transform those cells with a control 00:31:57.19 vector, you don't see any change. 00:32:00.15 If you express more zyxin in those cells you don't see any change. 00:32:04.08 But look, as we saw in the original soft agar cloning experiment that I showed you 00:32:09.11 earlier, when you express EWS-FLI1 in those 3T3 cells, you see 00:32:15.12 a dramatic increase in the ability of those cells to grow in 00:32:19.14 soft agar. And you can suppress that ability by re-introducing zyxin into those 00:32:25.19 cells. So this was, in addition to the motility 00:32:29.18 phenotype, I think a very striking piece of data that 00:32:33.28 suggested that zyxin might also be influencing 00:32:37.21 either cell proliferation or apoptosis and contributing to this suppression 00:32:45.07 of anchorage independent cell growth. 00:32:48.15 Interestingly, I told you that it is very, very difficult to 00:32:54.11 treat Ewing's sarcoma, so there are many 00:32:56.11 many investigators really working hard to identify new strategies 00:33:01.17 for therapeutic intervention with this disease. 00:33:05.26 And one interesting new concept that several groups are working on 00:33:10.29 is the use of an antibody directed against a cell 00:33:15.21 surface component, CD99, which is highly expressed 00:33:19.29 on Ewing's sarcoma cells, human tumor cells. 00:33:24.21 And it turns out that if you take this anti CD99 antibody and 00:33:29.14 expose Ewing's sarcoma cells to this antibody, the clustering of that 00:33:36.10 cell surface marker somehow induces apoptosis of the Ewing's sarcoma cells 00:33:43.03 by a mechanism that at this point in time really isn't understood. 00:33:46.20 I mean we really don't understand yet what CD99's normal function is. 00:33:50.08 But because it is so highly enriched on Ewing's sarcoma cells 00:33:53.19 and because engagement of CD99 causes apoptosis, it's 00:33:58.25 really being thought of as a potential therapeutic intervention for Ewing's sarcoma. 00:34:05.10 And interestingly, in the course of trying to understand 00:34:09.18 how clustering and engagement of anti-CD99 00:34:13.23 with anti-CD99 antibodies causes apoptosis. 00:34:18.14 investigators found that zyxin is a critical componentin that apoptotic pathway. 00:34:27.21 And here you can see some flow cytometry analysis looking at 00:34:33.03 the ratio of viable cells to apoptotic or necrotic cells 00:34:36.15 before or after anti-CD99 treatment, and 00:34:44.14 with normal zyxin levels or suppressed zyxin levels. 00:34:48.18 And here you can see that in the normal cell population 00:34:53.06 76% of the cells are in the viable channel here. 00:35:00.01 And if you engage those cells with the anti-CD99 monoclonal antibody, 00:35:08.24 which is called 0662, you can see a shift from the viable channel to 00:35:14.26 the apoptotic channel consistent with the idea that 00:35:19.15 the monoclonal antibody can induce apoptotic signaling 00:35:22.23 in these cells. Now that doesn't shift at all when 00:35:29.04 you use a control antisense construct, 00:35:33.12 but if you use a construct that is specifically designed to knock down zyxin levels, 00:35:38.04 what you see is that you have a reduction in the apoptotic response 00:35:46.16 suggesting that zyxin is at least partially involved or partially required 00:35:52.00 for the ability of anti-CD99 to induce apoptosis. 00:35:56.09 And so, this leads to an interesting addition to the 00:36:04.03 prior thinking about zyxin function that in addition to 00:36:08.17 contributing to regulation of motility, 00:36:12.28 that it may actually be a pro-apoptotic factor under some circumstances 00:36:17.29 and loss of zyxin might contribute to increased survival. 00:36:21.16 So this is quite interesting in the sense that 00:36:24.06 we know that expression of EWS-FLI1 causes reduction in zyxin expression in this 00:36:30.26 3T3 model, and that would be then associated with 00:36:34.06 increased motility and increased survival based on the cell based studies. 00:36:39.26 And so I think a very important future goal is to really now 00:36:44.07 go and test whether zyxin expression level is an indicator of tumor stage 00:36:50.23 and in addition, whether or not it will predict therapeutic response. 00:36:56.18 For example if zyxin levels are extremely low, 00:36:59.22 that might give us the insight that those tumors 00:37:04.08 have greater metastatic potential because 00:37:08.07 of the increased motility associated with loss of zyxin. 00:37:10.29 Interestingly, many investigators are now starting to find zyxin down regulated 00:37:18.08 in a variety of tumors, and here's an example showing that it is down regulated 00:37:24.12 in invasive bladder cancer. Here's the superficial tumor where the brown staining 00:37:29.18 indicates there is still robust zyxin expression. 00:37:32.11 And in this invasive carcinoma you see 00:37:35.09 very little expression of zyxin, if any at all. 00:37:38.15 And these investigators did a very extensive analysis to look 00:37:42.26 at association of a number of potential biomarkers 00:37:48.14 with tumor stage and grade and found that expression levels for zyxin, 00:37:54.02 as well as a couple of other markers, were directly associated with tumor stage and grade. 00:37:59.09 So, this could be another important and valuable 00:38:03.18 insight that allows clinicians to be able to identify tumors 00:38:08.28 and diagnose them with a higher degree of resolution using 00:38:12.28 these types of biomarkers to further refine 00:38:16.06 the grade of the tumor. Now I mentioned in the beginning that focal adhesions also are 00:38:22.11 really important for communicating with the cell nucleus, 00:38:25.24 and that these signals that emanate from focal adhesions 00:38:30.26 also influence gene expression. And I think one of the biggest challenges 00:38:34.22 that we face as cell biologists is 00:38:38.04 knowing that that occurs, not really understanding what is the mechanism 00:38:42.13 of action by which these events that are happening at the cell surface 00:38:47.05 are communicated to the nucleus to directly regulate 00:38:51.01 gene expression. And I am sure there are going to be a number of mechanisms 00:38:57.01 involved here, but one thing that I like to point out is that the zyxin protein 00:39:02.25 may be a candidate for communicating with the cell nucleus. 00:39:07.00 And indeed a student in my lab recognized a number of years ago 00:39:11.12 that the zyxin protein has sequence that looks very similar to 00:39:16.16 a nuclear export signal, showing these very highly ordered leucine residues 00:39:22.11 that are in similar positions to nuclear export signals 00:39:26.13 in the Rev protein of HIV1 as well as IkB. 00:39:31.17 So we looked to see whether if we deleted that leucine rich region of zyxin if it 00:39:37.16 had an impact on the subcellular distribution of zyxin, 00:39:41.10 and here you can see expression of wildtype zyxin in cells. And at steady state 00:39:46.18 you really see the zyxin concentrated in these focal adhesions, 00:39:50.03 and very little zyxin if any detectable by 00:39:52.13 immunocytochemistry within the nucleus. 00:39:54.11 But, if we delete just these 17 amino acids from zyxin, this leucine rich region, 00:40:00.06 and now ask where is that protein concentrated, 00:40:03.23 what we see is this really dramatic shift in the subcellular distribution of zyxin with 00:40:09.07 the protein migrating really substantially from these focal adhesions and accumulating 00:40:14.28 in the nuclear compartment. 00:40:17.26 So to test further whether or not this 00:40:22.06 sequence of zyxin really functions as a nuclear export signal, 00:40:27.05 we did a classic experiment where we took that zyxin sequence 00:40:31.26 and hooked it onto a carrier protein, in this case I am showing you 00:40:36.28 here GST, glutathione S-transferase, and asked 00:40:42.03 whether or not if we inject normal GST or GST tagged with this zyxin sequence 00:40:49.07 directly into cell nuclei, whether the zyxin sequence will support 00:40:54.06 the nuclear export of that GST protein. 00:40:57.27 GST is too large to diffuse through nuclear pores, so if you inject 00:41:02.06 it into the nucleus in the absence of a functional nuclear export signal, 00:41:06.00 it will be retained in the nucleus. 00:41:07.17 And that's exactly what you see with the unmodified GST. 00:41:12.10 You microinject it into the nucleus, and this is a fluorescently tagged version of GST, 00:41:17.05 put it into the nucleus, and it stays there. 00:41:20.09 But if you take just, in this case, amino acids 319 to 335 of zyxin 00:41:26.07 and attach that to the GST through genetic engineering, 00:41:31.05 express that protein, label it, and inject that protein into the nucleus, 00:41:34.29 you see that very, very rapidly that protein winds up in the cytoplasm, 00:41:41.09 illustrating that that short sequence of zyxin has the capacity 00:41:45.03 to serve as a nuclear export signal 00:41:46.26 and transport GST from the nucleus to the cytoplasm. 00:41:50.24 So, this was very exciting because it really suggested that the zyxin protein had a nuclear export signal 00:42:00.21 and perhaps was present in both the focal adhesions 00:42:04.29 and the nuclear compartment and could be part of the 00:42:07.18 machinery that really functionally linked the cell surfaceto the nucleus. 00:42:12.15 However, of course, when you are cutting pieces of proteins up 00:42:15.14 and putting them onto other proteins, things can happen that are unanticipated, 00:42:20.21 and so we really wanted to have a direct mechanism to look at whether zyxin ever 00:42:26.14 goes into the nucleus. And this was also important 00:42:28.20 because, as I pointed out, at steady state we really don't see zyxin 00:42:31.25 in the nucleus, so we developed an assay to report 00:42:36.17 whether zyxin goes into the nucleus. 00:42:39.05 And, I think, this is an experiment that David Nix in my lab 00:42:43.08 did when he was a graduate student, 00:42:44.12 and I think it is really amazing that this experiment worked. 00:42:47.25 He decided to test whether or not zyxin goes into the nucleus 00:42:53.21 by injecting a cocktail of two different antibodies 00:42:57.17 into, directly into the nuclei of individual cells. 00:43:02.07 And the two antibodies were an anti-zyxin monoclonal antibody 00:43:06.23 and a matched control antibody that didn't recognize any cellular proteins. 00:43:11.25 And once again these antibodies, which were labeled with different color fluorochromes, 00:43:18.00 are too large to diffuse out through nuclear pores. 00:43:22.00 So you would expect them to be retained in the 00:43:23.28 nucleus unless they are specifically exported 00:43:27.21 by binding to another protein partner. 00:43:31.08 So here we start out, we inject these two antibodies into cell nuclei. 00:43:36.24 Zyxin is, as I mentioned, concentrated in the cytoplasm, 00:43:39.20 and if zyxin didn't shuttle into the nucleus, you would expect 00:43:44.22 over time for the antibodies to remain trapped in the nucleus 00:43:48.02 and for the zyxin to remain in the cytoplasm. 00:43:51.23 In contrast, if there is shuttling going on and zyxin 00:43:55.22 is moving from the focal adhesions into the nucleus and back 00:43:59.26 out again, then we might expect one of two things. 00:44:03.08 We might expect zyxin to itself become trapped in the nucleus 00:44:09.09 over time along with its antibody partner. 00:44:13.05 Or, alternatively, we might imagine that zyxin could migrate into the nucleus, 00:44:18.17 bind the specific anti-zyxin antibody, 00:44:21.22 and because the zyxin harbors a nuclear export signal, 00:44:25.17 it could actually extract that zyxin partner from the nucleus 00:44:31.25 and facilitate its nuclear export. And surprisingly, 00:44:35.16 this is exactly what we saw. 00:44:38.17 So here you can see a cell into which we have injected 00:44:43.07 control antibody in A and anti-zyxin antibody together into the nucleus of the cell 00:44:49.24 in B. And this is a very early time point after injection, and you can see 00:44:54.06 that both antibodies are prominently concentrated within 00:44:58.07 the nucleus of the cell. 00:45:00.08 And if we wait over time, and then look, what we see (and this is a different cell, of course) 00:45:05.26 the control antibody marks the site of injection. It is retained in the nucleus. 00:45:11.28 But look over here, the anti-zyxin antibody which was co-injected into the nucleus of the cell 00:45:18.18 is completely missing from this nucleus 00:45:21.05 and has now begun to populate these focal adhesions. 00:45:25.17 So this was a very, very striking result 00:45:28.13 which illustrated to us that protein that was in the cytoplasm, 00:45:32.06 zyxin protein in the cytoplasm could move into the nucleus. 00:45:35.08 And that protein once dwelling in the nucleus 00:45:38.22 could come out of the nucleus and go back to the focal adhesions, 00:45:42.15 really, clearly suggesting a mechanism for communication 00:45:46.15 between these two cellular compartments. 00:45:49.17 And we can now show using leptomycin, an agent that inhibits 00:45:56.15 nuclear export, that indeedif we treat cells with leptomycin to inhibit 00:46:02.01 nuclear export that zyxin begins to accumulate in the nuclear compartment. 00:46:08.25 And if we look at large populations of cells, 00:46:10.16 we see this occurs in an asynchronous fashion 00:46:13.16 illustrating that there must be some physiological signals 00:46:16.17 that stimulate the release of zyxin from the focal adhesions 00:46:21.24 and the import of the protein into the nucleus. 00:46:25.21 And of course, a really important unanswered question 00:46:29.26 is what those signals might be. 00:46:32.26 So what could be the functional significance of nuclear shuttling of zyxin? 00:46:39.00 Well, I think there are two general possibilities. One is that the zyxin protein 00:46:43.20 could itself serve as a nuclear factor. You could imagine that the zyxin 00:46:48.15 protein is sort of retained at focal adhesions 00:46:51.14 where it's awaiting some sort of signal 00:46:55.11 or information from the extracellular environment 00:46:59.02 and under certain conditions would be released, 00:47:02.20 allowed to move into the nuclear compartment, and perhaps even 00:47:06.05 participate directly in regulating gene expression or some other nuclear function. 00:47:11.00 And this is a really intriguing idea, although to date 00:47:18.16 there's not really any direct evidence for a role for zyxin 00:47:24.14 within the nucleus, and particularly not in regulation of gene expression. 00:47:29.19 Though, I'll point out that the zyxin protein, remind you that the zyxin protein 00:47:34.16 has these LIM domains, which are found also in many transcriptional regulators, 00:47:40.22 so there remains, I think, an intriguing possibility that zyxin 00:47:44.09 could be directly influencing some nuclear activity. 00:47:47.21 The other alternative is a more general one that suggests that zyxin is, again, 00:47:55.10 sitting at these focal adhesions here, and then in response 00:48:00.18 again, to some signal, perhaps it could move into the nucleus 00:48:04.10 and recover a protein out of the nucleus, 00:48:09.13 returning it to the cytoplasm, and thus downregulating 00:48:11.28 the activity of that factor. Or alternatively 00:48:15.01 it could be a chaperone that really is designed 00:48:20.24 to retain a nuclear factor in the cytoplasm. 00:48:24.24 So for example, zyxin could be sitting at the focal adhesion 00:48:28.20 holding onto the protein that has a nuclear localization signal. 00:48:31.17 When that protein is bound to zyxin, if it ever went into the nucleus it would be carried 00:48:36.07 rapidly out because of the NES on zyxin. 00:48:38.11 But under certain conditions you could imagine that that protein would be 00:48:42.09 specifically released from zyxin, allowed to move into the nucleus 00:48:46.07 to activate some nuclear function. 00:48:48.29 So again, we now understand that this zyxin protein 00:48:52.15 clearly shuttles between the focal adhesions, 00:48:54.19 these really important signaling zones that are sensitive to extracellular cues. 00:48:59.22 It shuttles between those zones and the nuclear compartment. 00:49:04.27 And I think the next challenge is to try and identify what the 00:49:11.00 specific signals are that stimulate the release of zyxin 00:49:16.09 into the nucleus, and what its specific function there might be. 00:49:20.00 Interestingly, it is now appreciated, just in the last 00:49:24.10 several years, that many LIM domain proteins 00:49:27.19 have this very interesting sort of duality 00:49:31.14 in terms of their subcellular distribution. 00:49:33.24 All of these proteins listed here are proteins that reside both on 00:49:41.08 the actin cytoskeleton or in the focal adhesions, and also have 00:49:45.24 the capacity to move into the nucleus. 00:49:48.03 So I think there's a class of these molecules 00:49:50.25 that are going to be playing a really interesting 00:49:53.17 role in regulation of nuclear function 00:49:56.13 and in communication between the cytoskeleton and 00:50:00.14 the nucleus, real candidates for intracellular communication. 00:50:03.26 So I'll close there and just acknowledge some of the people in my lab 00:50:07.17 that participated in the work I described. 00:50:11.08 I think it's a very exciting time in which we are appreciating 00:50:16.00 much more the important roles of the proteins 00:50:19.24 that are present at these specialized adhesive zones, 00:50:24.09 and beginning to see not only how these proteins 00:50:27.14 influence cell behavior such as motility, perhaps 00:50:31.05 apoptotic signaling, perhaps communication with the nucleus, 00:50:35.22 but also are beginning to be able to put these pathways 00:50:40.24 into a broader context and appreciate how disturbance 00:50:44.17 of the functions of these molecules in these pathways 00:50:47.05 may impact on human disease.