How to Create a Body Axis
Transcript of Part 2: How to Polarize the Cytoplasm
00:00:13.09 Hello. 00:00:14.09 My name is Geraldine Seydoux. 00:00:15.09 I am a professor at Johns Hopkins University and an 00:00:18.23 Investigator with the Howard Hughes Medical Institute. 00:00:21.14 And this is the second part of my presentation on how embryos elaborate body axes and how 00:00:29.21 the single-cell zygote becomes polarized. 00:00:33.16 So, in this second part, I'm gonna focus on how the cytoplasm of the zygote becomes polarized. 00:00:40.25 So, in the first part, I described how the sperm in Caenorhabditis elegans polarizes 00:00:49.08 the PAR proteins at the membrane of the one-cell embryo. 00:00:54.13 It turns out that the PAR domains are controlling all of the different aspects of the polarity 00:01:02.24 of the one-cell embryo. 00:01:04.08 And that means that these proteins at the membrane direct what's going on in the cytoplasm. 00:01:09.12 And in the cytoplasm there are several proteins and organelles that become asymmetrically 00:01:16.18 distributed along the anterior-posterior axis. 00:01:19.22 And this happens because you want different molecules to end up in the anterior and posterior cell 00:01:27.13 that is formed at the first division. 00:01:30.05 So, in this presentation we're gonna focus on how do you generate these cytoplasmic asymmetries and, 00:01:37.08 in particular, we're gonna talk about how this protein here, MEX-5, forms an anterior-posterior 00:01:44.12 gradient in the cytoplasm. 00:01:46.07 So, here's a little movie showing you, again, how the PAR proteins become asymmetrically 00:01:52.16 segregated and, down below, how this MEX-5 protein becomes enriched on the anterior side. 00:02:00.12 Okay? 00:02:01.12 So, MEX-5 is an RNA binding protein, it's present throughout the cytoplasm, and, 00:02:08.28 as you can see, as the PAR proteins become asymmetrically segregated, MEX-5 responds and becomes enriched 00:02:18.25 on the anterior side. 00:02:20.12 Okay. 00:02:21.12 So, how does that work? 00:02:23.14 This was the project of a postdoc in my lab, Erik Griffin, and Erik was interested in figuring out, 00:02:32.27 how do you make this MEX-5 gradient? 00:02:37.02 And at first he considered three possibilities. 00:02:40.10 So, one idea, given that we can see that MEX-5 starts out uniformly distributed and then 00:02:46.16 becomes asymmetric, one possibility is that you just make more MEX-5 in the anterior side. 00:02:53.11 So, you could translate more MEX-5 protein specifically in the anterior cytoplasm and 00:02:59.04 that would give you a gradient. 00:03:00.17 That's one possibility. 00:03:01.26 Another possibility is that, in fact, you're doing the opposite. 00:03:05.18 You're actually degrading MEX-5 in the posterior cytoplasm, okay? 00:03:13.10 Another possibility is that you're doing neither one of those things, but instead you're just 00:03:18.02 taking the protein that was in the posterior and moving it towards the anterior side. 00:03:23.17 Okay? 00:03:24.17 So, you're redistributing... umm... redistributing existing protein. 00:03:28.25 So, to distinguish between these different models, Erik realized that these different models 00:03:35.20 actually make different predictions as to what happens to overall MEX-5 levels. 00:03:42.10 So, in the first case scenario, MEX-5 levels would increase, whereas if MEX-5 is being 00:03:52.24 degraded then the overall level should decrease. 00:03:56.04 But if you're just moving protein around, the MEX-5 levels should stay constant. 00:04:01.26 So, Erik had a feeling that he should really try to be able to study, carefully, what happens 00:04:08.20 to MEX-5 levels during this polarization. 00:04:13.02 He also realized that it would be very useful to be able to distinguish between the protein 00:04:18.16 that's already present at the beginning, before polarization, and the protein that might be 00:04:24.02 made during the polarization process. 00:04:26.25 So, to accomplish both of these goals, Erik created this umm... fusion version of MEX-5 00:04:36.15 where MEX-5 is fused to a fluorescent protein called Dendra that has very interesting capabilities. 00:04:45.24 This is a protein that, when it first folds, forms a fluorescent protein that fluoresces 00:04:53.01 green, okay? 00:04:55.03 But if you expose this protein to UV light, now it fluoresces in a different color, in 00:05:03.08 red, okay? 00:05:05.00 And so this is a way to basically label protein at the time that you expose the embryo to 00:05:11.20 UV light. 00:05:13.09 Whatever protein is there is now going to become red, okay? 00:05:16.27 Any new protein that's made later will still be green. 00:05:21.06 So, if you follow the embryos, just looking at the red fluorescence, you can look at protein 00:05:27.25 that was existing at the time that you started the experiment. 00:05:31.20 Okay? 00:05:32.20 So, this is called a photoactivatable fluorescent protein. 00:05:36.08 And it's very useful to follow proteins over time. 00:05:42.04 So, when he did this experiment, just exposing the whole embryo to fluorescent... to UV light, 00:05:50.23 he saw that this red MEX-5 protein was able to redistribute into a gradient during the 00:05:59.07 polarization process of the... of the zygote. 00:06:04.13 And so, from this, he concluded that you don't need any new synthesis -- whatever protein 00:06:09.04 is already there, it knows where to go. 00:06:11.17 So, that was interesting. 00:06:13.13 Next, he measured very carefully the amount of protein that existed at the beginning, 00:06:20.17 before polarization, and at the end, after polarization. 00:06:25.00 And what he found is that, basically, nothing changes. 00:06:28.05 The total amount of protein doesn't change. 00:06:32.01 What changes is that the amount in the anterior goes up and the amount in the posterior 00:06:36.01 goes down. 00:06:37.09 And that is consistent with some kind of redistribution, okay? 00:06:41.24 And then another experiment that he did that that confirmed that, in fact, the protein 00:06:45.27 was moving around somehow is this one, where he labeled... instead of labeling all of the 00:06:53.07 protein, as was done in this first experiment, over here, he labeled only the protein that 00:06:57.26 was on the posterior side at the beginning of the experiment. 00:07:01.19 And then he watched, what happens to this protein over time? 00:07:04.02 And he found that, yes, it did accumulate on the anterior side. 00:07:09.04 So, somehow, protein is moving around in the embryo and knowing to go to the anterior side. 00:07:16.08 So, how could this happen? 00:07:19.14 So, this really suggested that this middle option is the right one. 00:07:26.26 How is this happening? 00:07:28.07 So, then Erik decided to do another experiment, where, instead of illuminating large parts 00:07:36.00 of the embryo with the UV, he decided to illuminate the embryo in just two areas: 00:07:44.02 one stripe over here and another stripe over here. 00:07:48.13 Okay? 00:07:49.13 So, these two areas were illuminated at the same time, and then you can see what happens 00:07:55.01 to the protein in these two stripes over time. 00:07:58.01 So, over just six seconds, that's what I'm showing you here. 00:08:02.01 And so... it's a little bit hard to... to really understand what's happening when you 00:08:05.14 just look at the embryos like this, so what he did is he created a kymograph from these pictures. 00:08:13.04 And a kymograph is basically just taking one slice, one line across the embryo and... 00:08:22.02 as shown here, and then showing how that line changes over time, here. 00:08:26.13 Okay? 00:08:27.13 So, he could see what happens to the fluorescent protein that's very high in the middle of 00:08:33.04 that line at the beginning of the experiment, and what happens to it over time. 00:08:37.08 So, each of these little lit-up dots represent protein that's now diffusing away from the 00:08:46.00 original spot where that protein was when it was first activated, photoactivated to 00:08:52.03 fluoresce in the... in the red channel. 00:08:54.04 So, what you might notice is that protein is diffusing. 00:08:59.01 And it's diffusing in both directions, in both... towards the anterior, which would 00:09:04.05 be over here, and towards the posterior. 00:09:06.17 So, there doesn't appear to have any directed movement. 00:09:10.23 The protein molecules are just diffusing randomly. 00:09:14.15 Okay... so that was a little puzzling, because we know that, overall, there's more protein 00:09:20.15 ending up in the anterior. 00:09:21.24 So, how can that be if the protein is just moving around randomly? 00:09:26.27 Another thing that you might notice is that these two areas look different from one another. 00:09:32.20 This one stays brighter in this area, in the middle, whereas this one is getting diffuse faster. 00:09:40.01 Okay? 00:09:41.01 And that is not because the laser was different in those two areas -- the laser photoactivated 00:09:45.26 the same amount of protein in those two areas -- but what happened is that, by the very 00:09:51.05 first frame, you can see that the protein in this area is diffusing faster out of the 00:09:58.03 original photoactivated area, whereas the protein in this area is staying around in 00:10:03.17 this area longer. 00:10:04.24 So, that tells us that the rate of diffusion of the protein is different in these different 00:10:11.18 parts of the cytoplasm. 00:10:13.03 So, Erik then decided to redo this experiment by sampling many, many different points along 00:10:22.02 the anterior-posterior axis. 00:10:23.23 So, in this graph, the whole length of the embryo is shown down here, okay, from the 00:10:31.20 anterior over here all the way to the posterior. 00:10:36.03 And at each of these points, Erik measured how fast the protein diffuses. 00:10:41.26 Okay? 00:10:43.01 And you can see, in the blue line here, before polarization, the protein is diffusing very slowly -- 00:10:51.02 less than one micron squared per second. 00:10:53.20 Okay. 00:10:54.20 So, it seems to be maybe bound to something. 00:10:57.04 It's not moving very fast. 00:10:59.08 But during the polarization process, something very interesting happened -- the protein speeds up. 00:11:05.06 But it speeds up only in the posterior cytoplasm. 00:11:08.13 So, just to summarize what I've shown you here, we start out at the beginning of the process, 00:11:15.01 before polarization, with a MEX-5 protein that's very sluggish in the cytoplasm. 00:11:21.02 And then, during the polarization process, MEX-5 becomes fast, but only on one side of 00:11:27.25 the cytoplasm. 00:11:29.03 Okay? 00:11:30.03 And this happens at the same time that we see this concentration of protein in the anterior. 00:11:36.12 Okay. 00:11:37.22 So, we knew of another little tidbit. 00:11:41.22 And that is that we knew that PAR-1 is very important to create this MEX-5 gradient. 00:11:47.24 And this was work from Jim Priess' lab, who showed that PAR-1... remember, this is one 00:11:53.18 of the kinases that are part of the PAR group of polarity regulators. 00:11:59.11 And PAR-1 is enriched in the posterior side of the embryo. 00:12:04.09 And what Jim Priess' lab had shown is that PAR-1 phosphorylates MEX-5. 00:12:09.09 And this is important for MEX-5 to become asymmetric. 00:12:12.20 So, we wondered, what exactly is PAR-1 doing to MEX-5 diffusion in the embryo? 00:12:21.00 So, here's an experiment where Erik measured the diffusion rate of MEX-5 in both the anterior 00:12:28.03 and the posterior cytoplasm. 00:12:30.13 So, in wild-type, you get two different values, because it's slower in the anterior 00:12:35.26 and faster in the posterior. 00:12:37.25 And then, what happens if you get rid of PAR-1? 00:12:40.16 You can get rid of PAR-1 by getting rid of it using an RNAi treatment or by using a nice 00:12:47.24 PAR-1 allele that was generated and characterized by Ken Kemphues. 00:12:53.13 In both of these cases, we now see that PAR-1 stays... 00:12:56.22 I mean, sorry, that MEX-5 stays very sluggish. 00:13:01.08 So, if PAR-1 is not around to phosphorylate MEX-5, MEX-5 stays slow in both the anterior 00:13:07.26 and posterior cytoplasm. 00:13:09.06 Okay. 00:13:10.06 So, that suggests that PAR-1 is somehow required to speed up MEX-5. 00:13:15.06 And Erik got a very nice confirmation of this result using a different PAR-1 allele, 00:13:22.00 this b274 allele. 00:13:24.24 This is a PAR-1 allele that actually is a premature stop codon in the par-1 gene, and 00:13:31.20 it creates a truncated PAR-1 protein that, now, is not able to attach to the membrane, 00:13:37.04 and this PAR-1 protein is uniformly distributed throughout. 00:13:41.03 So, now you have the kinase everywhere. 00:13:43.28 And when you do that, amazingly, what you see is that MEX-5 becomes fast everywhere, 00:13:50.19 both in the anterior and in the posterior cytoplasm. 00:13:54.05 So, this kind of experiment said that PAR-1 is both necessary and sufficient to 00:14:01.05 speed up the diffusion of MEX-5 in the cytoplasm. 00:14:05.15 Alright. 00:14:06.17 The next experiment is we wondered whether this phosphorylation of MEX-5 by PAR-1 is... 00:14:13.28 might be reversible. 00:14:15.19 Could it be that MEX-5 is actually cycling between being phosphorylated and unphosphorylated? 00:14:20.08 And so, to test this idea, we took MEX-5 and PAR-1 kinase in a test tube, put them... 00:14:28.16 the two together and looked at MEX-5 phosphorylation using autoradiography, as shown here. 00:14:35.21 So, the dark signal here shows you that MEX-5 has been phosphorylated by PAR-1. 00:14:41.02 So, after Erik did that experiment, he added to the MEX-5/PAR-1 mixture embryonic extract, 00:14:50.08 just cytoplasmic extract from C elegans embryos. 00:14:54.06 And then he saw a really remarkable effect of the embryonic extracts, that... 00:14:59.27 that over time this embryonic extract was able to take away the phosphorylation from MEX-5. 00:15:08.10 And so, it seems that this phosphorylation that PAR-1 does to MEX-5 is actually reversible. 00:15:15.02 It's short-lived, it doesn't last for very long. 00:15:17.23 And, in fact, Erik was able to discover the phosphatase that is responsible for removing 00:15:23.12 this phosphate. 00:15:25.00 And it's present throughout the cytoplasm in the zygote. 00:15:28.07 Okay, so PAR-1 phosphorylates MEX-5 but MEX-5 has a way to quickly get rid of this phosphorylation. 00:15:34.22 So, keep that in mind. 00:15:35.22 The next interesting observation that Erik made is he looked at the size of the complexes 00:15:44.24 that MEX-5 existed in in the cytoplasm. 00:15:47.27 He did this simply by running a whole-worm extract, with MEX-5 labeled here with Dendra, 00:15:57.17 so we can see where it runs in the extract. 00:16:00.20 And he passed this extract over a sucrose gradient so as to separate light complexes 00:16:08.18 from heavy complexes. 00:16:10.28 Okay? 00:16:12.03 And what he saw is that MEX-5 actually exists in both light complexes and heavy complexes. 00:16:20.17 So that suggested to us that, maybe, that's how MEX-5 is slow -- maybe it's slow when 00:16:26.26 it's big and it's fast when it's in those smaller complexes. 00:16:32.04 So, putting these observations together led us to this hypothesis for how a MEX-5 gradient 00:16:41.04 might form under the influence of this PAR-1 kinase. 00:16:45.24 We imagined that, at the beginning of the phos... of the polarization process, 00:16:51.17 MEX-5 exists in these sluggish, large complexes that cannot move very fast into the cytoplasm. 00:16:58.20 They might actually be tethered to something in the cytoplasm, 00:17:02.24 and so that keeps them in place. 00:17:05.22 When PAR-1 becomes asymmetric, and it is enriched on one side of the embryo, it can phosphorylate 00:17:15.06 MEX-5, preferentially, on that side. 00:17:17.21 And we imagine that, when MEX-5 becomes phosphorylated, it now breaks away from these large complexes 00:17:25.00 and exists in smaller complexes, which are faster diffusing. 00:17:29.24 Now these smaller complexes can diffuse everywhere, in all directions, so they do so, all throughout 00:17:36.24 the cytoplasm. 00:17:38.03 But if... remember that the phosphorylation by PAR-1 is short-lived. 00:17:44.18 There is a phosphatase present throughout the cytoplasm that can remove this phosphorylation 00:17:50.14 from MEX-5. 00:17:52.02 And when that happens, MEX-5 is going to return into these larger complexes. 00:17:58.14 And if this dephosphorylation event happens in this part of the cytoplasm, where there's 00:18:04.11 no PAR-1, then the larger complexes will stay there longer. 00:18:11.05 And so, just following this kind of thinking, you can imagine how, by creating this diffusion gradient, 00:18:20.15 you end up with a concentration gradient where you have more MEX-5 in these 00:18:24.22 slower complexes in the anterior side of the cytoplasm. 00:18:29.18 So, this was just a hypothetical model that we came up with based on the amount of data 00:18:36.26 that we had. 00:18:37.26 But a... a model is really only useful in... if it actually predicts something that you 00:18:44.06 can then experimentally test. 00:18:46.16 And this model really predicted that MEX-5 should exist into two species. 00:18:52.20 So, I already showed you that it looked like, on a sucrose gradient, MEX-5 did exist in 00:18:58.19 both light and heavy species. 00:19:00.26 But we wanted to be able to see those directly in the embryo. 00:19:05.06 Could we detect a fast MEX-5 and a slow MEX-5, okay? 00:19:11.11 And so, for this, Erik turned to a different technology called fluorescence correlation spectroscopy, 00:19:19.14 which is a microscopy technology that allows you to monitor the diffusion rate 00:19:27.05 of individual molecules in small volumes of cytoplasm that you can sample with your microscope. 00:19:33.26 Okay? 00:19:34.26 And so, by following the fluctuation in fluorescence in this small volume, you can, using mathematics, 00:19:43.01 deduce what kind of molecules are traversing this small volume, and how many different 00:19:51.20 species are there, and how fast are they diffusing? 00:19:55.11 So, doing these kinds of experiments, Erik found that in the anterior cytoplasm there 00:20:02.10 in fact exist two different types of MEX-5 molecules: a very sluggish MEX-5 molecule 00:20:12.26 that is really not diffusing very much at all, and then a faster one, okay? 00:20:18.24 So, that's the two molecules that exist in the anterior cytoplasm. 00:20:23.13 And, in fact, remember that we had measured the average diffusion behavior of MEX-5 using 00:20:31.20 the Dendra experiment that I presented at the beginning of this presentation. 00:20:37.14 And that average number actually uhh... fits with an average that we can get from computing 00:20:44.17 the average of these two numbers and also taking into account how many of these two 00:20:52.06 types of molecules there are. 00:20:53.22 It turns out that there's a lot more of these very slow molecules in the anterior cytoplasm. 00:20:59.10 And that's why that makes the overall population average quite slow in the anterior cytoplasm. 00:21:06.04 What about in the posterior cytoplasm? 00:21:08.01 Well, there, Erik found that those two species exist as well. 00:21:12.27 There's also a very slow one and a very fast one, just like in the anterior cytoplasm. 00:21:18.05 The difference, however, is in the proportion of those two species. 00:21:23.08 In the posterior cytoplasm, the fast species is more abundant compared to what it was in 00:21:30.17 the anterior. 00:21:31.17 There's actually equal amounts of the fast and the slow in the posterior. 00:21:35.28 Okay? 00:21:37.02 So, now, this experiment is really confirming our model that MEX-5 exists in two species: 00:21:45.11 slow and fast. 00:21:46.17 And what differs between the anterior and the posterior cytoplasm is the proportion 00:21:50.24 of those two species. 00:21:53.23 So, now, we are getting close to a molecular model for how this gradient forms. 00:22:00.26 We know that we have two MEX-5 species, a slow and a fast one, and that these two... 00:22:06.01 the interconversion between these two species depends on this PAR-1 kinase, which creates 00:22:12.22 the fast species, but the fast species can revert back to the slow species through the 00:22:19.03 action of this phosphatase that is present throughout the cytoplasm. 00:22:24.06 So, based on this information, we thought that maybe we would be very close to actually 00:22:31.04 being able to model, using mathematical formulas, the MEX-5 gradient. 00:22:38.20 Okay? 00:22:39.19 So, if this explains everything about how the gradient forms, just by inputting 00:22:44.24 these values into a mathematical model, we should be able to recreate the MEX-5 gradient. 00:22:51.02 So, for this, we had to team up with David Odde, a computational biologist, who took 00:22:57.23 our experimentally determined values, together with the size of the C elegans embryo, 00:23:04.18 and putting PAR-1 kinase in the posterior. 00:23:08.18 And, with all of this information, David was able to recreate, in a computer, 00:23:16.04 the MEX-5 gradient. 00:23:17.23 So, here... umm... the black line represents the total MEX-5, which forms a gradient across 00:23:24.20 the anterior-posterior axis, and then the red and the green lines represent the fast 00:23:31.11 and the... and the slow MEX-5. 00:23:34.23 And you can see that, in the ant... the posterior cytoplasm, there's equal levels of those 00:23:39.01 two species, where... whereas in the anterior cytoplasm, there's more of the slow species. 00:23:45.00 So, this type of analysis is very satisfying, because it suggests that maybe we can explain 00:23:52.14 this MEX-5 concentration gradient just by imagining that the PAR-1 kinase can change 00:24:02.12 the diffusion rate of MEX-5. 00:24:05.10 Okay? 00:24:06.13 So, here we have a gradient that's formed in the cytoplasm without having any directed movement. 00:24:14.26 All we have is a local, reversible phosphorylation that induces a local change in diffusion rate. 00:24:23.03 And that is sufficient to create a gradient. 00:24:25.28 So, if you're interested in learning a little bit more about the MEX-5 gradient, and getting 00:24:32.04 more of a direct feel for how such a gradient might form, you might be interested in this 00:24:38.24 video from David Odde, who collaborated with a dance company to bring the MEX-5 gradient alive. 00:24:49.09 So, thank you again for following this presentation, and I hope to see you another time. 00:24:58.05 Bye.
