Cellular Organization of Complex Cell Structures
Transcript of Part 4: Formation of P Granules
00:00:01.07 Hello. My name is Tony Hyman. 00:00:03.13 I'm a director of Max Planck Institute in Dresden in Germany. 00:00:07.09 And I'd like to talk to you today about formation of a structure known as a P granule. 00:00:13.04 Before I start, on the right hand side, 00:00:17.24 is a picture of our institute in Germany that we built 00:00:21.06 when we went there about 10 years ago. 00:00:24.25 And, this was a new institute. It was put up in old East Germany 00:00:28.12 after the wall came down. 00:00:30.14 I was involved in designing this institute. 00:00:34.00 And, before we started building it, I climbed up next door in a house 00:00:39.13 and put a camera to time-lapse the building of this particular institute. 00:00:47.03 And that's shown in this movie. You can see that we can follow 00:00:51.19 the building of this Max Planck Institute through the different seasons. 00:00:56.00 It took about two years to build. 00:00:57.21 You can see that first, the wings of the building have gone up. 00:01:01.01 You can see the two separate wings of the building going up. 00:01:04.25 You can see them getting built from bottom to top. 00:01:07.20 And, you can see that sadly, the weather is not as good as it is here. 00:01:12.26 I'm in California. Sadly, there's a lot of snow and a lot of rain. 00:01:17.10 You can follow the changing conditions around a building. 00:01:19.22 You can see the intermediates in the process of building, 00:01:24.07 with these coverings over the structures once they're built. 00:01:27.26 And, we can then follow the process through to its completion. 00:01:33.14 And from that, we can get an understanding of how this building is put together. 00:01:38.07 I show that to remind you how important it is to get kinetic information 00:01:44.16 on how things are put together, and that's also true in any biological system. 00:01:50.23 If you simply look at the fixed, finished building, 00:01:54.15 it's very hard to work out how it's put together, and if you guessed, 00:01:58.07 you are likely to make mistakes. 00:02:01.28 And so, in biology, it's time lapse microscopy which has been absolutely key 00:02:06.25 for understanding how these different compartments are put together. 00:02:10.15 And getting kinetic information can really tell you a huge amount about 00:02:14.28 how the process is put together. 00:02:16.04 And I'd like to tell you today about how kinetic information, 00:02:19.21 and looking at the kinetics of a process, 00:02:21.25 told us so much about P granules that we couldn't get by looking at the static picture. 00:02:28.23 Coming back to our scale, 00:02:33.19 I've shown you some steps of organization of the cell 00:02:41.19 from individual tubulin molecules to microtubules to centrioles, 00:02:45.06 and there are many, many different protein complexes 00:02:47.07 that exist in this nanometer scale space. 00:02:50.18 Other protein complexes, such as ribosomes and nuclear pores, and 00:02:56.22 proteasomes exist around that scale. 00:02:59.09 But, there's a whole set of compartments 00:03:03.25 that exist at this scale, where actually we have much less idea how they're put together. 00:03:09.28 As I showed you from the first 3 segments of my talk, 00:03:13.29 we understand quite a lot about the rules by which we put together 00:03:16.19 even very complex things, such as a microtubule 00:03:19.08 or a centriole. We can understand the structure. 00:03:22.26 We can understand the mechanism by which the proteins are working. 00:03:25.16 But, at this level of scale, we understand much, much less. 00:03:30.14 And this is the problem I've brought up in my introductory segment, 00:03:34.01 which is, these are non-membranous bound compartments 00:03:38.18 which contain many different protein complexes, whose individual structures 00:03:43.15 we can understand -- the protein complexes. 00:03:44.20 But, we don't really understand the rules by which these protein complexes live together 00:03:49.27 to form these compartments. 00:03:51.19 Remember, I mentioned that they're extremely dynamic. 00:03:54.09 And so, that's essential for us in cell biology. 00:03:58.04 If we're going to understand how the cytoplasm is organized, 00:04:00.06 we need to understand how compartments at this scale 00:04:04.26 are put together. 00:04:06.10 So, a more general question that we can ask is 00:04:09.27 how do we structure large, non-membranous bound organelles? 00:04:15.06 If we think about a problem like that, one thing we can do 00:04:20.16 is we can ask what can we learn from non-biological systems? 00:04:25.02 Because, actually, non-biological systems take up very complex patterns 00:04:30.00 as well, such as water. 00:04:32.09 Very simple... water goes between many different... gas, ice crystals... 00:04:38.04 And so simple, non-biological systems actually take up 00:04:40.27 quite complex structures. 00:04:43.04 And so, we can ask the following question then, 00:04:48.10 which is, what do non-biological structures have to do with biological assembly? 00:04:53.22 Do they give us a clue for how these complex compartments 00:04:57.17 are organized, which so far have been very difficult to understand. 00:05:01.27 And the work I'd like to talk to you about today 00:05:04.18 was primarily the work of Cliff Brangwynne 00:05:07.18 in collaboration with Frank Julicher, who is a colleague of mine 00:05:11.05 in the Max Planck Institute for Physics of Complex Systems, 00:05:13.21 also in Dresden. 00:05:14.25 The Max Planck is a society which consists of many different institutes, 00:05:18.25 some in physics, some in chemistry 00:05:22.06 some in humanities, some in biology, 00:05:24.00 and so we can then often talk to our colleagues in these other Max Plancks 00:05:28.29 in other subjects, and Frank Julicher is a theoretical physicist. 00:05:32.16 Christian Eckmann, also in our institute, a group leader who works on 00:05:38.05 the formation of the germ-line, and Carsten and Agata, 00:05:42.10 were also involved in this particular project. 00:05:44.13 And the question is, how do P granules form? 00:05:51.13 So, P granules are cytoplasmic hubs of proteins and RNAs, 00:05:55.23 and they were discovered in C. elegans all the way back in 1983, 00:05:59.00 actually, just before I started my PhD. 00:06:02.11 They are essential for forming the germ-line, and they tend to be very big. 00:06:10.01 You can see, they're right in this mesoscale that I was mentioning 00:06:14.02 of organization of compartments at this scale. 00:06:17.29 P granules are also complex. 00:06:24.23 So, here, there are many, many different proteins 00:06:27.18 in a P-granule. 00:06:29.20 And, there are RNA and RNA-binding proteins in P-granules. 00:06:34.06 And they're believed to be important for the totipotent state of the germ line. 00:06:38.14 In other words, maintaining the ability of the germ line to carry on to the next generation. 00:06:44.01 If you think about it, it's really amazing that all over millions of years of evolution, 00:06:48.04 of any particular organism, each time, the germ-line has to be able to deliver 00:06:54.10 a perfect copy of the previous generation onto the next generation. 00:06:59.08 There will be some slow mutation, but basically, unless it can do that, 00:07:02.22 they're not going to be able to propagate. 00:07:05.17 So, your cells in your body slowly age, and they slowly die away, 00:07:08.19 but the germ line stays totipotent. It does not age, 00:07:11.13 and that's why we can propagate ourselves. 00:07:15.01 So, these germ-line granules have been known to be important 00:07:20.02 for maintaining the totipotency of the germ-line. 00:07:22.07 So, how do they actually form, and how do they function? 00:07:25.06 Let's look at a movie of P granules. 00:07:30.04 And this is a particularly beautiful movie, where we've labeled P granules 00:07:33.09 with a GFP marker. So, we GFP-tagged one of the P granule 00:07:37.13 components, and then we're looking at them moving in the embryo. 00:07:43.06 Now, watch what happens. You'll see them all sweep down the embryo 00:07:47.15 towards the end. So, they start here, and they sweep down towards the end, 00:07:54.03 and they end up in one end of the embryo, 00:07:55.26 and they're inherited only by one cell. 00:07:59.02 And that's what classically is known as an asymmetric cell division, 00:08:02.11 where one cell is different than the other 00:08:05.15 because it inherits different amounts or different types of cytoplasm. 00:08:10.06 So, let's look at this movie. 00:08:11.04 You see how the P granules are sweeping down towards the posterior. 00:08:16.22 And, let's show you that again. 00:08:24.16 The P granules sweep down towards the posterior of the embryo, 00:08:27.21 and you can see how they seem to be moving with a cytoplasmic flow. 00:08:32.18 So, that was fascinating. You see this large-range flow of cytoplasmic granules 00:08:38.11 and you also see the P granules moving with them. 00:08:42.02 So, that led to my hypothesis that perhaps P granules are segregated by cytoplasmic flow. 00:08:47.00 However, we did an experiment which suggested that couldn't be all that was going on. 00:08:55.11 We did that by 3D particle tracking. 00:08:58.26 We track individual P granules as they're moving. 00:09:02.18 And, what that showed is that there is no net flux of P granules to the posterior of the embryo. 00:09:12.08 And that's because you can measure the P granules going one way and the other way. 00:09:18.09 And, this particular bar chart over here shows the average number crossing in the middle, 00:09:25.06 going from the anterior to the posterior and the posterior to the anterior. 00:09:31.02 And you can see the same number go both ways. 00:09:33.13 So, there's no net flux to the posterior. 00:09:36.20 So, it can't be that the flows are simply moving P granules 00:09:40.28 from one end of the embryo to the other. 00:09:42.27 So, how could they be being segregated? 00:09:45.04 Well, you can look at the fate of individual P granules. 00:09:49.25 Let's do time-lapse microscopy. 00:09:53.25 As I brought up in the introduction to this talk, let's not look at a static picture of a P granule. 00:09:57.04 Let's ask what is the life history of a particular P granule. 00:10:00.09 Maybe that will tell us something. 00:10:01.24 And, indeed it did. Because, this graph here shows the lifetime 00:10:09.17 of many P granules over the division of the embryo. 00:10:12.14 And this actually looks a little bit complex, but just bear with me. 00:10:14.19 On this axis, we have the intensity of different P granules. 00:10:18.26 By intensity, I mean the amount of fluorescent molecules in any particular P granule. 00:10:24.01 On this axis, we have the time. 00:10:29.10 So, you can see from -14 to 0, which is an arbitrary time point we set during cell division. 00:10:37.06 Now, look what happens. 00:10:38.26 Right out of fertilization, there are P granules 00:10:44.02 on the anterior (that are in blue) and the posterior (that are in red). 00:10:47.26 Can you see how there are lots of them in both sides? 00:10:50.23 And that's what you saw in the movie, at the beginning they're in the whole embryo. 00:10:54.02 But look what happens first. They all depolymerize. 00:10:57.14 Or, they shrink, let's say, until there are very few P granules left in the cytoplasm. 00:11:03.20 And then, an interesting thing begins to happen. 00:11:06.11 They start to grow in the posterior, but they don't grow in the anterior. 00:11:11.10 So, P represents the posterior, A represents the anterior. 00:11:15.26 You can always think of an embryo having a polarity axis, 00:11:20.00 and anterior-posterior is the basic polarity axis of any embryo that first starts. 00:11:23.12 You remember, you end up with three axes. 00:11:24.26 You have anterior-posterior, left-right, and dorsal-ventral. 00:11:28.06 That's... when you have a human... anterior-posterior, 00:11:30.07 you have left-right, and you have dorsal-ventral. 00:11:33.11 So, we have an anterior-posterior axis here, 00:11:36.16 and P granules are forming only in the posterior. 00:11:39.24 So, we learned they're dynamic, and also we learnt that the dynamics are different 00:11:45.29 in different parts of the embryo. 00:11:47.22 They dissolve at the anterior, and then they dissolve at the posterior, 00:11:52.27 but then they form again later on in the cell cycle. 00:11:58.16 We can look at that in a bit more detail 00:12:00.29 by graphing the intensity of P granule assembly. 00:12:03.29 This is a graph here to show you there is a gradient of P granule assembly. 00:12:07.23 So, this is on the X-axis, we've got the position along the anterior-posterior. 00:12:12.28 Ok, the X is down here. 00:12:15.14 And on the other one, we've got the intensity. 00:12:17.19 And the dotted line is the line above which the net growth tends to be more than 0. 00:12:26.21 So, they don't ... they're tending to grow. 00:12:29.15 So, each point is an average of many different P granules 00:12:33.26 that either tend to grow or tend to shrink, but the net... 00:12:36.18 what we call the net assembly goes over this point, about 0.6 of the cell axis. 00:12:44.19 We can actually do a nice little dynamic graph, 00:12:48.10 where we took graphs during the different points of the cell cycle 00:12:50.24 and look at the growth, and you see that the growth and shrinkage changes 00:12:58.15 across the embryo, through time. 00:13:00.09 You see the way the graph... they're all shrinking at the beginning 00:13:04.16 And now, as you move through the cell cycle, some at the posterior are now tending to grow. 00:13:11.11 So, P granule assembly/disassembly changes with time. 00:13:16.20 It changes spatially, and that's why P granules form at the posterior. 00:13:22.02 So, we can then conclude from what we've done so far 00:13:26.07 that P granules separate by a gradient of assembly/disassembly, 00:13:30.10 where dissolution is favored at the anterior and formation is favored at the posterior. 00:13:36.13 So, then you can ask a question, right? As biologists, it's always improtant to think of a question. 00:13:42.24 And the question that came out of that study was 00:13:46.14 Why do P granules form at the posterior? 00:13:50.22 Because that's why and how they segregate. 00:13:53.21 If we understand that, we can understand how these granules 00:13:56.21 end up in the posterior of the embryo. 00:13:59.22 The breakthrough in this project came in Woods Hole. 00:14:06.07 So, the Woods Hole is a very old marine station 00:14:10.08 that every year runs summer courses, and I can highly recommend 00:14:13.23 these summer courses to anybody that's interested 00:14:16.12 in trying to understand problems in cell physiology, 00:14:19.05 and there are also other courses in embryology. 00:14:21.04 And every year, I taught that course for five years. 00:14:25.03 Every year, one would go for the summer for a couple of weeks. 00:14:27.13 You'd get 10 very motivated students who want to do something interesting, 00:14:32.03 and you have to think up different projects for them to have a go at. 00:14:35.08 And one year, Cliff, who had actually been a student in the course, 00:14:42.13 decided to come and teach the course and look at in more detail 00:14:51.04 the details of P granule assembly. 00:14:53.00 What we discovered is that P granules have characteristics of liquid-like drops. 00:14:59.13 Let me show you some movies which illustrate what I mean by that. 00:15:04.06 So, look at this little P granule here, and watch it approaching this big one 00:15:10.03 And watch it getting sucked up and fusing. 00:15:21.27 Like two drops coming together. 00:15:24.03 Here's another P granule. Look at the two of them coming together and fusing 00:15:28.08 like two drops. 00:15:35.19 See them just squeezing together and making one droplet, just like you had two droplets of liquid. 00:15:41.13 So, from that, we thought, yeah they seem to have liquid-like properties. 00:15:47.09 And so, we want to do other experiments, like dissect them out of their 00:15:52.18 endogenous context, put a sort of flow of buffer, and now what you can see, 00:15:57.04 they seem to be dripping, just like the honey dripping off your spoon in your morning breakfast. 00:16:03.07 So, look at them dripping. Just imagine that you had your honey on a spoon 00:16:08.13 dripping off. They're dripping off the sides of the nuclei, just like 00:16:12.27 a very viscous liquid. 00:16:15.05 And a number of a different things we did suggested that indeed 00:16:20.23 they behave like liquid drops. First of all, they're spherical. 00:16:23.15 It turns out, that it's quite hard to be a sphere unless you have something called surface tension, 00:16:27.26 which is a property of liquids. 00:16:30.10 You can look it up in Wikipedia and find out more about surface tension there, 00:16:34.19 but you can see it's a key property of liquids. 00:16:36.08 The other thing they do is they tend to wet when attached to the nucleus. 00:16:40.15 So, if you think about a drop, and you take a drop of any liquid, 00:16:44.07 like a viscous liquid, and you put it on a surface, 00:16:47.11 it will tend to wet onto the surface. 00:16:49.23 Unless, you take steps to stop it wetting, like putting some kind of a surface on it. 00:16:55.20 And the other thing, they can form and fuse into larger spheres. 00:16:59.11 And all of these were characteristics that, the state of P granules 00:17:03.06 that is in, is a liquid-like state. 00:17:05.21 Now, what does that mean to be a liquid? 00:17:09.22 It's very confusing to talk about that, first of all. And what does it mean to be liquid? 00:17:14.02 Well, what does it mean to be a solid? Let's first think about a solid. 00:17:17.22 If you're a solid, you can come back over and over again, 00:17:22.20 and the amino acids or the atoms will always be in the same place that they were before. 00:17:28.02 You can do a crystal, of course, but you can take a microtubule 00:17:32.03 or a centriole, and you do any kind of structural biology, the components 00:17:36.00 will always be in the same place time and time again. 00:17:39.23 A liquid, that's not true. 00:17:42.06 The internal contents of a liquid are moving around very quickly. 00:17:46.12 So, over the time scales we're interested in -- 00:17:49.09 minutes -- the contents of these liquids continuously rearrange, 00:17:54.22 whereas the solids would not. 00:17:57.12 And, the best analogy that I know of to think about what the difference is 00:18:01.11 let's think about a school. When the kids are in the classroom, they're all 00:18:05.07 sitting at their chairs, and so you know, time and time again, 00:18:09.28 that Johnny will be in this chair and Jackie will be in this other chair. 00:18:15.11 So, that is something you can then predict where they're going to be. 00:18:18.08 The liquid-like state is more like recess, 00:18:20.27 where the kids are running around. You know they're in the playground somewhere, 00:18:24.02 but you can never predict exactly where they're going to be. 00:18:26.25 And that's what we call liquid-like state. 00:18:29.21 That, of course, also defines the issue that time scale is very important, 00:18:32.27 because, of course, if we looked at the playground over milliseconds, 00:18:36.13 the kids would also always be in the same place. 00:18:38.24 So, we're talking about the time scales in many, many minutes, 00:18:41.24 things are no longer in the same place. 00:18:43.16 So, that's a problem for us, of course, 00:18:45.04 as biologists, because all of the techniques that have been built up 00:18:48.16 over the last 50 years rely on molecules being in the same place over and over again. 00:18:54.16 They rely on series of interactions, and they rely on the ability to do averaging 00:18:58.00 to understand the molecular arrangements. 00:19:00.00 So, that's one issue is we can't use standard techniques to think about how liquids form. 00:19:03.26 Another interesting thing about liquids 00:19:07.28 is they undergo phase transitions. 00:19:11.01 Now, what is a phase transition? 00:19:13.04 Well, there's some very simple examples of phase transitions out in nature. 00:19:18.11 For instance, let's think about water vapor. 00:19:24.20 Let's say you did the following experiment, 00:19:28.00 and you can do this in your own home. 00:19:29.22 You take water vapor, and you have it in a hot chamber. 00:19:33.09 If you cool it down, the water vapor will condense into little droplets. 00:19:39.19 And that's because the saturation point of water changes at the temperature. 00:19:47.05 So, at the cold temperature, it becomes saturated, 00:19:50.11 and it condenses down into water droplets. 00:19:52.06 And that's exactly like you would see, for instance, on a cold day. 00:19:55.25 When you breathe out, your air... your warm air condenses onto the 00:20:04.01 cold air that's outside. 00:20:06.07 Phase transitions are interesting because they're a simple way of concentrating complex mixtures 00:20:11.10 of reactants. And, one way, for example of that, for instance, 00:20:15.11 is if you do an alcohol / chloroform separation. 00:20:17.22 The alcohol goes in the water layer because it has hydroxyl groups ... simple alcohols. 00:20:23.27 And so, the interesting thing is to find out what rules can be used in biological systems. 00:20:31.11 Because the simple rules can lead to large scale organizations. 00:20:34.02 A great way of concentrating, say, 50 different components 00:20:37.24 in a complex thing is the P granule. 00:20:41.19 And that will be an essential thing for the future will be to describe these rules at a molecular level. 00:20:47.04 We can ask then, why P granules actually form at the posterior. 00:20:53.16 If we think this is a phase transition, why is it they actually undergo this phase transition 00:20:58.15 at the posterior of the embryo? 00:21:00.04 And it turns out, there's a set of underlying biochemical asymmetries 00:21:04.25 which are really beautiful 00:21:06.12 and beautiful problems to study, also from a physics point of view. 00:21:09.23 You have the cortex set up into two domains. 00:21:12.11 Very interesting problem of establishing cortical asymmetry. 00:21:15.27 This cortical asymmetry then contributes to form 00:21:19.14 soluble downstream gradients of this protein called MEX-5. 00:21:25.18 And MEX-5 is thought to inhibit the formation of P granules at the anterior. 00:21:30.20 So, how does that work? Well, let's go back and look at our dynamic system, now, 00:21:34.20 and ask how MEX-5 inhibits the formation of P granules. 00:21:38.10 So here's a blow up of MEX-5. You can see a bigger picture, and you can see 00:21:43.27 the gradient of MEX-5. It's high in the anterior and low in the posterior. 00:21:47.29 And the P granules will form where MEX-5 is low. 00:21:52.25 Now, let's look at the intensity of fluorescence. And what you see from this graph 00:21:59.10 is very interesting, which is you can see in blue 00:22:02.14 the intensity of P granules -- the gradient that I showed you earlier in the talk. 00:22:06.12 But, look at MEX-5 -- it's opposite to the P granule gradient. 00:22:09.06 So, that's very interesting. Because they have opposite gradients, it might suggest 00:22:12.20 to us that indeed, P granules grow more slowly in the anterior 00:22:16.23 because the concentration of MEX-5 is high. 00:22:20.00 If we make a mutant experiment, where we RNAi MEX-5 or remove MEX-5 from the cell, 00:22:29.09 you can see something very interesting happen, which is 00:22:31.00 when you do an experiment where you remove the function of MEX-5 00:22:35.06 from the cell. You see the wild-type that I've showed you before, 00:22:38.20 where you have a gradient of assembly across the embryo. 00:22:41.03 Now, look at the red line, which is a MEX-5 mutant. 00:22:44.09 They're uniformly dynamic across the whole embryo 00:22:49.02 which is they're growing... the net tendency to grow is too slow for them to actually grow 00:22:53.21 anywhere in the embryo. They're growing a bit slower in the posterior than the wild-type, 00:22:59.15 so there's no net increase in amount of P granules, but look at the 00:23:02.04 anterior. Look at the anterior over here. 00:23:06.01 They're actually tending to grow more than the wild-type. 00:23:11.06 So, that suggests that somehow, MEX-5 suppresses growth of P granules here, 00:23:16.27 in wild-type. You take it away, and now it allows it to grow more fast. 00:23:22.07 But that somehow prevents the growth of P granules in the posterior around here. 00:23:28.03 How could that work? 00:23:31.00 We don't really understand that, but let's come back and give you some ideas 00:23:36.11 based on analogy of phase transitions in liquids. 00:23:39.23 Let's go back to our original experiment, and this is an experiment you could again do 00:23:43.15 in any classroom, which is to let segregate water 00:23:47.20 by phase transition. 00:23:49.16 So, take a water vapor and cool it down. 00:23:52.11 And, it condenses all over the slide. But, we can segregate water vapor 00:23:56.29 to one end of that chamber by putting a gradient of temperature. 00:24:00.09 So, instead of doing this experiment, we do a different experiment, 00:24:03.13 which is we make a chamber with hot at one end and cold at the other end. 00:24:09.00 And when we do that, what happens is that all the water vapor ends up at one end of the chamber 00:24:16.14 down here. And that's because water tends to condense on the cold end, 00:24:20.26 and then, you reduce the amount of soluble water vapor... there's water vapor 00:24:29.15 around the condensed out water. 00:24:32.16 Now, there's a process known as diffusive flux, 00:24:35.16 which is a basic property of diffusion, which always tends to equalize the concentration 00:24:40.23 of fast-diffusing components. 00:24:43.06 And so then, of course, the concentration of water vapor is equalized. 00:24:47.22 Anything on the cold side also goes more into water droplets, 00:24:53.18 there's diffusive flux, so slowly all of the water vapor disappears 00:24:56.20 from the chamber and ends up in the condensed water vapor. 00:25:01.21 So, there we are, we've used phase transitions and a gradient of temperature 00:25:06.08 to segregate a water vapor. 00:25:08.02 Now, let's come back and think about it for P granules. 00:25:10.13 What we think is that P granules condense at the posterior for similar reasons. 00:25:16.16 Not because there's a temperature gradient, of course, 00:25:18.28 because there is no temperature change -- they're growing at uniform temperature. 00:25:21.29 But, rather, because there's a saturation point that's established 00:25:26.13 by gradient polarity proteins, such as MEX-5. 00:25:28.29 So, you change the tendency of the components to saturate out at the posterior, 00:25:35.21 but they are still happy to stay as individual components at the anterior. 00:25:39.17 And we have evidence for that when we look at the embryo 00:25:43.03 because we actually look at the amount of soluble components. 00:25:46.20 On the left hand side, I've got green, which shows the concentration 00:25:50.19 of individual P granule components that are not in P granules. 00:25:54.03 And look at how, as the P granules form, 00:25:57.10 they're being depleted from the cytoplasm, so they all end up at the P granules. 00:26:03.00 And, presumably, as P granule components form at the posterior, 00:26:06.20 diffusive flux equalizes the concentration. 00:26:09.18 If you do photobleaching studies, these P granules turn over very, very fast. 00:26:14.00 They're diffusing fast enough, and then slowly they all end up in the posterior. 00:26:18.12 And so, if you come back and ask the final question, 00:26:23.11 Why do P granules form at the posterior? 00:26:25.05 What we would say from that is that P granules segregate 00:26:29.18 because the diffusion rate of the liquid phase 00:26:32.27 is slower than the diffusion rate of the dissolved phase. 00:26:35.13 The individual components, which are not in P granules diffuse very fast, 00:26:38.29 and they can diffuse over the embryo by diffusive flux. 00:26:42.03 The P granules diffuse very slowly, 00:26:44.22 so once they form, they tend to stay where they are 00:26:47.13 and therefore you end up with most of the P granules components in the P granules 00:26:52.17 at the posterior, where the embryo wants them. 00:26:55.21 And I bring this up to illustrate how we can use thinking about physical chemistry... 00:27:02.05 all that physical chemistry you've done at university 00:27:05.26 can be used to think about these complex problems of biological assembly. 00:27:10.10 The work I've discussed you today is just the beginning, 00:27:13.25 and we're obviously short on molecular mechanism, 00:27:17.05 but it shows how powerful these ideas are of physical chemistry 00:27:21.21 for organizing the cytoplasm of the cell 00:27:24.21 at this 100 nanometer, 1 micron to 2 micron scale, 00:27:29.11 which has been so difficult to understand. 00:27:32.23 And, I hope in the future, that these sorts of ways of thinking about it will be very useful 00:27:38.02 for thinking about other organization of other compartments in cells. 00:27:43.12 It also shows why it's worth concentrating on chemistry in university. 00:27:49.09 And, what I'd actually like to say as well 00:27:53.00 is that this idea of using physical chemistry to think about cells 00:27:59.11 is not new at all. If we go back to E. B. Wilson, 00:28:04.09 E. B. Wilson wrote a very famous book called The Cell, Development, and Heredity. 00:28:10.11 The final volume is in the 20s. 00:28:13.01 It summarized all the knowledge of the great cell biologists of the turn of the century. 00:28:19.08 This was the really hot field in those days. 00:28:22.19 And, he summarized, in this classic textbook, basically everything we knew, 00:28:28.01 which was the Bible for those of us who have started to work on these problems 80 years later. 00:28:33.26 Much of it, for instance, was in German, and inaccessible to many people. 00:28:39.03 But, of course, he was also an active scientist 00:28:41.24 and here's a paper that he published in Science in 1899, 00:28:44.14 entitled "Cytoplasm is a colloidal liquid emulsion." 00:28:49.01 So, these ideas of physical chemistry existed 100 years ago. 00:28:53.08 The ideas of colloids and physical chemistry were very topical then, 00:28:58.24 and people thought they might be useful for understanding how the cytoplasm works. 00:29:03.08 Now, the problem was, there were no molecules then, and so the field didn't really advance, 00:29:08.02 as often happens. Fields get to a certain stage and then they stop, and no advance can be made. 00:29:12.15 And if we look at a sort of short history of 20th century biology, 00:29:16.19 you can think about it, it's in the first half of the 20th century, 00:29:20.13 and the end of the 19th century, people thought about the physical chemistry of the cytoplasm, 00:29:24.25 with no knowledge of the molecules. 00:29:26.15 Then, there came the idea that we have to understand these problems at a molecular basis. 00:29:34.05 And this great challenge to catalog and understand the molecules of the cell started, 00:29:39.08 which had the DNA sequencing, as I mentioned in my introduction, 00:29:42.17 then RNA interference has been a way to really do high-throughput analysis 00:29:49.09 of molecular function in complex eukaryotes. 00:29:52.20 There are other high-throughput genetic techniques in more simple systems. 00:29:56.10 Proteomics defined the way that they're organized into complexes. 00:30:00.27 That triumph of humanity will surely be seen, looking back in a couple of years, 00:30:07.03 what a triumph that was in 30 or 40 years to really catalog the molecules in the cell. 00:30:11.26 Of course, it's not told us anything... 00:30:13.16 Well, it's told us something, but it hasn't told us a lot 00:30:16.09 about how the cell is actually organized. 00:30:19.02 And, I would contend that it makes sense now to go back with our knowledge of molecular biology 00:30:27.18 and the catalog and use the physical chemistry ideas 00:30:31.19 and join these two ideas together to try and understand how the cell is organized 00:30:38.13 in order to perform its myriad and complex functions. 00:30:43.10 Everything stems from chemistry. Originally, we were chemistry. 00:30:47.18 The initial formation of life came out, almost certainly, of 00:30:51.05 formation of complex chemical building blocks, 00:30:54.18 so it makes sense to use chemistry to think about how the cell is organized 00:30:59.27 because that would have been its original basis. 00:31:02.11 And I'll finish by summarizing the three topics I talked to you about today. 00:31:10.15 And just to show, as I mentioned at the beginning of the talk, 00:31:14.23 that you've got to use different ways to think about things 00:31:20.09 at different length scales and different organizations. 00:31:22.18 So, when we think about microtubules, we think about them as polymers. 00:31:26.13 They grow, and they shrink. 00:31:30.27 When we think about centrioles, we think about more this virus-like mechanism, 00:31:36.00 where you step in different stages of assembly. 00:31:40.02 Because... think about them as molecular complexes, but they have the intermediate states. 00:31:45.00 And then finally, this other level we talked about is liquid-like states, 00:31:48.04 where we have to think different kinds of theories. 00:31:50.17 You have to think about chemistry of liquids to understand how they work. 00:31:55.02 And, that illustrates, I think, what's so wonderful about being a biologist 00:32:00.04 in the modern era, that we can take all these different techniques, 00:32:03.27 and all these different theories. 00:32:05.24 We can bring physics, we can bring chemistry, 00:32:08.13 and try and use these to understand these myriad, complex problems 00:32:12.29 that we're confronted with every day we look at the cell.