Session 6: From Prokaryotes to Multicellular Organisms
Transcript of Part 1: Evolution of a dynamic cytoskeleton
00:08.0 Hello. I'm Julie Theriot from Stanford University, 00:11.1 and I am back for the third part of my iBio Seminars presentation. 00:15.0 Now, in the first two parts, 00:16.3 I focused on well-understood systems 00:18.3 with strong experimental evidence 00:20.2 supporting all of the conclusions that I was telling you. 00:22.3 For the third part, 00:24.1 I'm going to venture into the field of wild speculation, 00:27.1 and in fact I'm going to wildly speculate 00:28.3 about the biggest questions of all 00:31.1 in the context of biology, 00:33.1 which is how things evolve. 00:35.1 And in particular what I'd like to discuss 00:38.1 is some big picture ideas 00:40.2 about how the dynamic cytoskeleton 00:42.2 has evolved throughout the history of life on Earth 00:45.0 in order to give us such an incredibly diverse array 00:48.1 of cell types, of structures, 00:50.1 and of behaviors of cells. 00:52.2 And just to illustrate that, 00:54.1 here on the left I have an image 00:56.2 of one of the most fabulous of protozoa, 00:58.2 called Stentor, 00:59.3 which is an absolutely enormous cell 01:02.0 with, as you see, a great deal of very detailed subcellular specialization 01:04.1 in terms of its structure. 01:06.1 And then on the right, 01:08.0 a luridly colored electron micrograph 01:09.1 of some bacteria, 01:10.3 which are quite diverse in terms of their 01:13.3 molecular components, 01:15.2 but fairly similar and fairly simple 01:17.2 in terms of their shape. 01:20.0 Now, one of the things that's become very clear 01:22.0 in the modern era of sequencing 01:25.2 is confirming something that of course Darwin had 01:29.1 originally speculated about some time ago, 01:31.2 which is the proposition that 01:33.2 all of the organisms that are currently living on our planet 01:35.1 are descended from a common cellular ancestor 01:38.0 or a common pool of cellular ancestors. 01:41.1 And certainly, one of the most undeniable pieces of evidence 01:44.0 in support of that hypothesis 01:45.1 is the observation that there's 01:47.1 a tremendous number of molecular components 01:49.3 that are found highly conserved 01:51.2 throughout all branches of life. 01:54.1 This particular illustration 01:56.3 shows the sequence similarity among ribosomal RNA 02:00.1 for bacteria, 02:02.2 for archaea, 02:03.2 and for eucaryotes (eukaryotes). 02:05.1 And the fact that it is possible 02:07.1 to arrange all of the known living cells on Earth 02:09.2 in this sort of tree, 02:11.3 where everything looks related to everything else 02:13.2 is a very good indication that in fact 02:17.0 all of the ribosomal RNAs 02:18.2 that exist in cells on the modern Earth 02:20.2 all came from some common ancestor 02:22.3 back at some distant point in the past. 02:25.2 The common ancestor on this diagram 02:27.1 is indicated with the green circle, 02:28.3 and it's fondly called "LUCA", 02:30.1 which stands for the last universal common ancestor. 02:32.2 Now, this was not the first cell, 02:34.1 this was just the last cell 02:36.1 that gave rise to everything that's living today, 02:38.1 and it was already very complex. 02:40.0 It already had ribosomes, it already had DNA, 02:42.2 it already had most of the components of central metabolism, 02:45.1 including, for example, 02:46.2 all of the enzymes involved in glycolysis, 02:48.2 and it also had cytoskeletal components. 02:51.1 And we know this because 02:53.1 those common components 02:54.2 are found in every single branch 02:57.2 of currently living organisms. 02:59.3 Now, given that everybody started off 03:02.0 as part of the same family, 03:03.1 it is interesting, I think, 03:05.1 to speculate about why it is that 03:08.1 some parts of this tree of life 03:10.0 have ended up behaving so differently 03:12.1 from other parts of the tree of life, 03:14.0 and in particular I'd like to draw the distinction 03:16.3 between what I will loosely call the prokaryotes, 03:19.2 which are on the top part of the tree, 03:21.1 and that includes the bacteria and the archaea, 03:24.1 which are very distinct branches, 03:26.0 versus the eukaryotes on the bottom part of the tree. 03:28.3 Now, although bacteria and archaea 03:31.0 are no more closely related to each other 03:33.1 than they are to the eukaryotes, 03:34.3 they nonetheless are quite similar morphologically, 03:38.1 and in fact we didn't even know that the archaea 03:41.1 existed as a separate kingdom until sequencing data started coming along, 03:44.0 because if you just look down a microscope, 03:45.2 it's very hard to tell the difference 03:47.0 between a bacterium and an archaeon. 03:49.2 Whereas the morphology is very simple 03:51.3 in those prokaryotes, the metabolism is extremely diverse, 03:55.0 so almost any interesting chemistry 03:57.2 that's done in living organisms 03:59.1 was first invented by the prokaryotes. 04:02.1 So, what we got going for us down on the eukaryotic side of the tree? 04:05.2 Well, although our chemistry is not 04:07.3 necessarily as interesting or as complex 04:09.1 as what's going on with the little guys, 04:11.1 we have morphology. 04:12.2 We get very big, 04:13.2 we get very complicated, 04:15.0 and we get very multicellular. 04:16.2 So, within this context of everybody 04:18.3 being related to everybody else, 04:20.1 and in fact all of these different organisms 04:23.0 having something that looks like cytoskeletal proteins, 04:25.1 I think it becomes a very interesting question to ask, 04:28.0 well, what is it really then that makes eukaryotes different from prokaryotes? 04:31.0 Specifically, what is it that gives eukaryotes 04:33.0 the ability to have such extremely diverse 04:35.1 and complicated cellular morphologies, 04:37.0 based on cytoskeletal structures? 04:41.0 Just to, again, 04:42.3 give you a little bit of a taste 04:44.2 of the kind of diversity you find 04:46.1 even within a very closely related group of eukaryotes, 04:48.2 these are some illustrations 04:50.0 from one of the great microscopists of the 19th century, 04:52.3 just showing a group of fairly closely related 04:55.1 ciliates and flagellates. 04:56.2 Each of these are unicellular organisms... 04:58.1 they're not all drawn to scale with each other... 05:00.0 this is Stentor, 05:01.2 that I showed you a photograph of on the very first slide, 05:03.1 and a number of other familiar organisms, 05:06.0 including, for example, Paramecium, here, 05:08.3 and Giardia. 05:10.3 And all of these cells, 05:12.2 to a first approximation, 05:14.0 have the same components. 05:15.1 If you look at the protein components of these cells, 05:17.2 there certainly are things that are a little bit different between them, 05:20.0 but the major components, 05:21.1 certainly the major structural components, 05:22.3 are pretty well conserved, 05:24.0 and yet somehow all these different organisms 05:25.3 are able to make drastically different cell shapes 05:28.0 with very different motile behaviors, 05:29.2 very different kinds of division behaviors, 05:31.1 simply by rearranging this common set 05:33.2 of molecular components. 05:37.0 Now, if we contrast that to the bacteria, 05:39.2 you certainly will hear 05:41.2 proponents of the prokaryotic side of the tree 05:45.0 claiming that there is a lot of morphological diversity among bacteria... 05:48.2 and to a small extent that's true. 05:50.1 So, if we look at this illustration 05:54.1 from a really enchanting review by Kevin Young that came out a few years ago, 05:57.3 this shows essentially the diversity 06:00.2 of the different kinds of shapes 06:02.1 that have been described for isolated bacterial species. 06:04.3 And here in the zoomed-in version, 06:07.1 we find a couple of the fairly familiar shapes, 06:09.2 so we see things like E. coli here, 06:11.3 which is a rod shape, 06:13.2 there are some crescent-shaped bacteria here like Caulobacter, 06:17.1 there are helical-shaped bacteria, 06:19.2 but they're all sort of fairly simple variations on a theme. 06:24.2 And then all of those guys are also quite small. 06:27.2 And then we zoom out to higher magnification... 06:29.1 there's really very few bacterial species 06:30.3 that get much bigger than just a few microns 06:33.1 in characteristic size. 06:34.2 One interesting exception 06:36.2 is illustrated here by this yellow dome 06:39.0 that stretches across this drawing, 06:40.3 and this is actually to scale 06:42.2 with the other bacteria here, 06:44.1 an illustration of an organism called Thiomargarita, 06:47.0 which is one of the largest bacteria know, 06:49.0 and these guys actually can be almost a millimeter across. 06:52.2 And this shows a photograph 06:54.3 of Thiomargarita next to a Drosophila 06:58.1 #NAME? 06:59.2 and you can see its absolutely enormous size. 07:02.0 But the trick here is that 07:04.1 even though the cell itself is very large, 07:06.0 its cytoplasm is very small. 07:07.2 Thiomargarita is basically 07:09.1 just a hollow shell of a very thin layer, 07:11.3 a couple of microns thick of cytoplasm, 07:13.3 that's stretched an enormous vacuole. 07:16.1 So, even when bacteria get to be pretty large 07:18.2 and, relative for bacteria, 07:20.0 morphologically complex, 07:21.2 they still have diffusion-limited dimensions. 07:24.1 And it is obviously observable that, 07:28.0 overall, if you take a random eukaryote 07:29.2 and compare it to a random prokaryote, 07:31.0 the eukaryotic cell is bigger 07:33.0 and is much more structurally complex. 07:35.2 So, this just pulls out a couple of those things 07:39.0 as examples. 07:40.1 Here's Paramecium compared to a bacterium to scale, 07:43.0 and then that bacterium blown up, 07:44.2 and here I've chose Vibrio cholerae. 07:46.2 And you can see, again, there is subcellular localization, 07:49.1 subcellular specialization 07:51.0 in the context of Vibrio cholerae. 07:52.1 It's got its flagellum growing out of one pole, for example, 07:55.1 so it somehow is able to know 07:57.1 which end is which 07:58.3 and know the difference between the poles and the sides, 08:00.1 but even at that level the complexity 08:02.2 is just vastly less than what you see in something like Paramecium. 08:06.1 So, if you think about, 08:08.1 what are all the observable differences 08:10.0 between eukaryotes and prokaryotes, 08:11.2 there's a pretty long list that is... 08:14.0 you know, there are some exceptions, 08:15.1 but this is reasonable typical, 08:17.0 reasonably representative of these different kinds of life. 08:19.3 Eukaryotes have, of course, 08:21.1 a membrane-enclosed nucleus, 08:22.3 that's the definition. 08:24.1 And so that means they have separated, 08:25.3 spatially and temporally, 08:27.1 the process of transcription 08:28.3 from the process of translation, 08:30.1 which gives rise to many options 08:32.1 for very complicated regulation 08:33.3 that basically can't be present 08:35.1 in bacteria or archaea. 08:38.0 Along with the membrane... 08:41.1 err, sorry, the nuclear membrane, 08:43.2 the eukaryotes of course also have lots of other internal membranes 08:45.2 making things like the Golgi apparatus 08:47.1 and the endoplasmic reticulum. 08:48.2 They typically have much, much larger genomes 08:51.1 than our bacterial friends, 08:53.0 with often with multiple chromosomes that can be very large. 08:56.2 As I mentioned, they have much larger cell size. 08:58.1 Instead of being characteristically 09:00.1 just a few microns long, 09:01.2 they're usually a few tens to even hundreds of microns in size, 09:05.1 and they have a very high degree of subcellular compartmentalization. 09:08.2 In addition, eukaryotes have endosymbionts, 09:11.3 so they have mitochondria and chloroplasts 09:13.1 that used to be bacteria 09:15.2 that have now been captured and domesticated 09:17.2 by the eukaryotic cells 09:19.1 in order to perform energy functions. 09:21.0 And then, finally, you know, 09:23.0 the reason we're even able to talk about this 09:25.0 is because eukaryotes make multicellular organisms. 09:27.1 Now again, 09:29.1 just as in the context of the shape, 09:30.3 it's not the case that bacteria 09:32.2 never make multicellular organisms, 09:34.0 it's just that they're not very complicated. 09:35.2 So, for example, 09:37.3 the best understood multicellular bacterial organism 09:39.3 is this thing called a stromatolite. 09:42.1 We see a bunch of stromatolies here, 09:43.3 growing in an ocean next to Australia, 09:45.2 and these are basically big piles of colonial cyanobacteria. 09:50.2 They grow and deposit 09:52.0 some extracellular matrix, 09:53.2 and then the next generation will grow on top of it, 09:55.1 making these very characteristic layer cake patterns 09:58.2 that are seen not only in the kinds of stromatolites 10:00.2 we have growing today 10:02.0 but also are found in stromatolites 10:03.2 that are several billion years old in the fossil record. 10:06.2 And in all that time, 10:08.1 look how far eukaryotes have come, 10:09.3 but bacteria are basically still making the same structures. 10:12.2 Eukaryotes have been able to make things like Redwood trees, 10:15.2 they're been able to make things like coral reefs, 10:17.2 with this extraordinarily morphological diversification. 10:21.1 Okay, so this very fundamental question of, 10:23.3 what is the difference between eukaryotes and prokaryotes 10:26.0 that gives us such extraordinary morphological complexity, 10:30.1 is a question that I would say, today, 10:31.2 we don't know the answer to, 10:33.1 but a few decades ago 10:34.3 we thought we did know the answer to it, 10:36.1 and before 1990 or so 10:38.2 the nearly universally accepted explanation 10:41.2 for why there's such big morphological differences 10:43.1 came down to the eukaryotic cytoskeleton. 10:45.2 If we go through all of those characteristics I described 10:49.1 about what's special about eukaryotes 10:50.2 as compared to bacteria or archaea, 10:52.0 all of those things can be attributed 10:54.0 to functions of the cytoskeleton. 10:55.2 So, for example, 10:57.1 the existence of the membrane-enclosed nucleus 10:59.1 and all of the complex internal membrane systems 11:01.1 can be ascribed as being due to 11:03.3 intracellular membrane transport 11:05.3 with motor proteins, for example, 11:07.2 pulling vesicles along microtubules. 11:10.1 Nuclear lamins, 11:11.3 which are essentially a form of intermediate filaments, 11:13.2 stabilize the nuclear membrane. 11:16.2 The expanded genome in eukaryotes 11:18.3 of course divided by the extraordinary machine 11:21.1 of the mitotic spindle, 11:22.2 which with all of its self-organized microtubules, 11:25.2 and its massive amount of flux 11:28.2 and force generated by motor proteins 11:31.0 such as kinesins and dyneins, 11:32.2 is able to divide a lot of chromosomes, 11:34.2 and it's no big deal for a eukaryote 11:36.2 to generate an extra chromosome, 11:38.0 split a chromosome in half, 11:39.2 vastly expand the size of its genome, 11:41.2 because the mitotic spindle 11:43.1 is able to handle a lot of capacity 11:46.0 in terms of segregating chromosomes. 11:48.2 The much larger cell size of eukaryotes 11:51.1 can be attributed to the fact 11:53.0 that they're no longer diffusion-limited. 11:54.2 Because of the direct action of motor proteins, 11:56.2 you can actually have transport and mixing 11:58.2 within the cytoplasmic compartment. 12:01.2 Similarly, the subcellular compartmentalization, 12:03.3 the existence of endosymbionts, 12:05.2 and even the ability to have cells come together 12:08.2 and make multicellular organisms 12:09.3 with very complex extracellular matrices, 12:12.2 largely involves coupling between microtubules, actin, intermediate filaments, 12:17.3 and other components within the cell 12:21.2 or from one cell to another. 12:22.3 Endosymbionts, for example, 12:25.0 are thought to be the product of a phagocytic event, 12:27.2 where an ancient eukaryotic ancestor 12:29.2 ate a cyanobacterium 12:31.2 and turned it into a chloroplast. 12:35.1 Now, the reason I say we don't know the answer today, 12:37.0 even though we did know the answer in 1990, 12:39.1 was because it became clear in the 1990s 12:41.3 that bacteria actually do have a cytoskeleton 12:44.1 that in many ways is remarkably similar to that 12:46.3 of eukaryotes. 12:48.1 And so the first real nail in this coffin 12:50.1 was the discovery of 12:52.1 a tubulin homologue in bacteria 12:53.3 that goes by the name of FtsZ. 12:56.1 FtsZ had been originally identified genetically 12:59.2 as being a protein that was required for cell division, 13:01.2 and some beautiful work in the 1990s, 13:03.2 combining biochemistry and cell biology of this protein, 13:07.0 has established that it really acts as 13:09.2 a legitimate cytoskeletal protein in bacteria 13:11.2 that's involved in cell division. 13:13.1 It forms a ring right around the middle of a bacterium 13:15.2 that's about to divide, 13:17.0 and as the bacterium divides, 13:18.1 that ring actually becomes smaller. 13:20.2 Furthermore, the purified protein 13:22.1 is able to self-assemble into filaments 13:24.0 in a manner that depends on the presence of hydrolyzable GTP, 13:27.2 which again is a very cytoskeletal behavior. 13:30.1 And any remaining doubt 13:32.1 had to be squashed 13:34.0 when the crystal structures came out 13:35.2 for both tubulin and FtsZ 13:38.1 at about the same time, 13:39.2 back to back in the same issue of Nature, 13:41.3 and it was clear that the structure of one 13:43.3 could be almost perfectly superimposed 13:45.3 on the structure of the other. 13:47.2 So, it's clear that these are not only sort of analogous proteins, 13:50.1 but in fact true homologues, 13:51.3 in the sense that they are descended from a common ancestor, 13:53.3 and by far the simplest explanation for that 13:56.1 is that the LUCA, the last universal common ancestor, 13:59.2 also had a protein like tubulin or FtsZ. 14:04.1 Okay, so bacteria have something like tubulin... 14:06.2 then it turned out a couple of years later, 14:08.2 bacteria also have something like actin, 14:11.1 and just like with FtsZ, 14:13.1 these proteins 14:15.0 -- they go by several names, there's MreB and Mbl 14:17.1 and a few other family members -- 14:18.3 were originally described as being genes that are necessary 14:21.0 for some aspect of bacterial shape, 14:23.1 a fundamental cytoskeletal-type function. 14:26.0 And so, for example in Bacillus subtilis, 14:28.0 disruption of any of these actin homologues 14:30.0 causes really abnormal, overall, 14:32.2 types of cell shapes. 14:34.3 And these proteins, when purified, 14:36.3 can assemble into filaments, 14:38.1 again like FtsZ or like any of the eukaryotic cytoskeletal proteins. 14:42.2 Now, one thing that's I think been particularly amusing 14:45.2 in the context of the bacterial actin homologues, 14:48.1 has been the proliferation 14:50.1 of such a large number of them 14:52.0 that appear to have slightly different specialized functions. 14:54.3 So, for example, 14:56.1 one of my favorite examples 14:58.0 is in magnetotactic bacteria 14:59.2 that are able to orient themselves with respect to the Earth's magnetic field. 15:03.0 They do so my lining up these little crystals of magnetite 15:05.3 along a structural element inside the cell 15:10.1 that is in fact made up of an actin-like filament. 15:14.0 Now, this is not the same actin-like filament 15:16.2 that is contributing to cell shape, 15:18.0 it's actually a copy of the gene 15:20.1 that was historically duplicated, 15:22.1 and then diverged for this specialized function. 15:25.0 So, bacteria not only have actin, 15:26.2 they have a bunch of different actins, 15:28.0 but they can use them both to determine their overall shape 15:30.2 and to determine the distribution 15:33.0 of intracellular organelles. 15:35.2 So now I think we're set up to ask ourselves 15:37.2 a slightly different question. 15:38.2 It's clear that bacteria do have a cytoskeleton, 15:41.1 so the existence of the cytoskeleton 15:42.3 cannot be the thing 15:45.0 that distinguishes eukaryotes from prokaryotes. 15:46.2 So, we might ask then, well, 15:48.0 if bacteria do have a cytoskeleton, 15:49.2 then why don't they do something more interesting with it? 15:51.1 And by that I mean in this very eukaryotic-centric world view, 15:54.2 why don't they have morphological complexity 15:57.0 and why don't they make 15:59.0 big, fancy multicellular organisms? 16:01.0 So, here we get to the point of wild speculation, 16:03.2 and my specific hypothesis is that 16:07.2 the thing that all of the cytoskeletons have in common 16:10.0 is the ability to perform large-scale cell organization 16:13.1 based on self-assembly of helical filaments 16:15.2 that are highly dynamic. 16:17.0 That's true among all the different branches of life. 16:21.3 I am proposing that was is special about eukaryotes 16:24.2 is not the cytoskeletal filaments per se, 16:26.3 but rather two specific classes 16:29.0 of cytoskeletal associated proteins: 16:31.1 the nucleators and the molecular motors. 16:35.1 And I'll go into some detail about why I think these are the things 16:38.2 most likely to really be different, 16:40.0 but so far there is no strong positive evidence 16:43.0 that prokaryotes have 16:45.2 any classical molecular motor proteins 16:47.0 or any regulated nucleators for cytoskeletal filaments, 16:50.0 which raises a second question. 16:52.1 You know, if the prokaryotes don't have them, 16:53.3 why don't they have them? 16:55.0 That's something even I'm not going to be able to speculate about, 16:57.3 but as far as why I think these two things 17:00.0 are really the key to this morphological difference, 17:02.1 I'll give you some of the information 17:04.2 that has led me to that proposition, 17:06.2 and suggest a few very specific ways 17:10.0 that these ideas could be tested. 17:13.2 Okay, so the overall basis for this hypothesis, 17:17.2 that you need nucleators and molecular motor proteins 17:19.2 in order to get morphological diversity, 17:22.2 comes from a series of observations 17:24.2 about the kinds of structures 17:26.2 that can be made by cytoskeletal filaments 17:28.0 in bacteria versus in eukaryotes. 17:32.1 In particular, 17:34.1 any self-assembling helical filament 17:37.0 can make either a structure with a single filament 17:40.1 or it can make a bundle or a raft of filaments, 17:44.1 where the filaments are oriented in random directions 17:46.1 with respect to each other. 17:47.2 All that requires is helical self-assembly, 17:49.2 which as we'll see is a very universal feature 17:52.0 of proteins that are able to interact with themselves. 17:56.1 On the other hand, the kinds of structures that really require localized nucleation 18:00.1 or localized motor activity 18:01.2 are illustrated here on the bottom. 18:03.0 These include things like microtubule asters, 18:04.3 which are familiar from the organization 18:07.0 of overall organelles inside of a cell. 18:10.2 These include things like parallel bundles, 18:12.1 which we find inside of eukaryotes flagella, 18:13.3 or we find in muscle... 18:16.0 you know, the sarcomeres that are able to give large-scale muscle contraction... 18:19.3 and also things that have bipolar morphology, 18:23.0 like this mitotic spindle shown here. 18:25.1 We know enough about how these different structures are formed 18:27.1 that I feel fairly confident in stating 18:30.3 that the easiest way to make these kinds of structures 18:32.3 is either by localizing nucleation of filament growth, 18:36.1 or having molecular motor proteins 18:38.2 that are able to sort of filaments of different orientations, 18:41.1 or some combination of both things. 18:43.2 And what I will assert is that 18:46.2 no structures of the type shown under here in class B 18:49.2 have yet been found in bacteria, 18:51.3 specifically have yet been found in the cytoplasm of bacteria, 18:54.2 with perhaps one or two interesting exceptions 18:57.0 that I think actually sort of prove this rule. 18:59.3 So, if nucleators and molecular motor proteins 19:02.1 essentially enable you to take your 19:05.0 helical, self-polymerizing, self-assembling 19:07.2 cytoskeletal filaments 19:08.2 and make large structures out of them, 19:10.2 you might ask, well, 19:12.1 what evidence is there that eukaryotes can do this 19:14.2 but prokaryotes cannot? 19:16.2 So, I'd like to now again take a step back 19:19.0 and say, if we are willing to accept the premise 19:21.3 that something about the activity 19:23.2 of the eukaryotic cytoskeleton 19:24.3 is the key difference, 19:26.2 then the question is, what is so special 19:28.1 about the eukaryotic cytoskeleton? 19:30.0 And if you look at the most abundant 19:32.0 and most studied eukaryotic cytoskeletal proteins 19:34.3 -- we have tubulin making microtubules, 19:36.2 actin monomers making actin filaments, 19:39.2 and then intermediate filaments 19:40.3 made up of their own subunits -- 19:42.1 and what you immediately see, 19:44.0 looking at all these things, 19:45.1 is that they're all basically helical self-assembled structures, 19:49.3 but I would argue that helicity 19:52.2 of a self-assembled structure 19:54.1 is not something that's particularly difficult to evolve. 19:56.2 And in fact there is a very strong argument 19:58.2 made first by Crane 20:01.1 and then by Pauling, back in the 1950s, 20:03.1 that pretty much any protein 20:05.1 that has any tendency to associate with itself 20:08.1 is more likely to form a helix than it is to form anything else. 20:12.0 And the argument is pretty simple: 20:13.3 if you have a globular protein 20:16.2 where every aspect of that protein's surface 20:18.0 has slightly different physical attributes 20:20.2 -- slightly different charge distribution, 20:22.1 slightly different shape -- 20:24.2 and that protein has some tendency 20:28.0 to interact with a second copy of itself 20:30.0 in some particular orientation 20:31.2 where, you know, part A of one protein 20:33.2 binds to part B of another protein... 20:36.0 if the protein is going to make a dimer, 20:37.2 you know, those two things that interact with each other 20:40.2 have to be distributed symmetrically on the protein. 20:42.2 But if the part A and part B 20:44.2 are in any orientation 20:46.2 other than just directly opposite each other, 20:49.0 then essentially what's going to happen 20:50.2 is those two subunits will come together 20:52.2 and there will be an interaction between the two 20:55.1 that will give a dimer that's got, 20:57.1 you know, some slightly off-center asymmetrical structure 21:00.1 where, if part A and part B are engaged here, 21:03.1 this subunit still has a part B available 21:05.1 and this subunit still have a part A available. 21:08.1 Now, if you think that through, as these guys did, 21:10.1 what you see is that you can then 21:12.1 add another subunit on this side, 21:13.3 you can add another subunit on that side, 21:15.2 and you're able to make helices 21:17.1 out of this very simple binary interaction rule. 21:20.2 And in this really creative 21:23.0 and beautiful illustration 21:24.1 done in this original paper 21:26.3 using little matchboxes, 21:28.0 you can see you can actually build all sorts of different kinds of helices 21:30.2 just by slightly changing 21:32.2 the orientation of the interaction between those two subunits. 21:36.1 So, making a helix is not a big deal, 21:39.0 and in fact a really great piece of evidence 21:41.3 to show that that's true 21:44.0 is the effect of the sickle cell mutation on hemoglobin. 21:47.0 Now, hemoglobin is highly selected to be very, very soluble 21:50.2 #NAME? 21:52.2 can get up to on the order of 300 mg/mL -- 21:55.2 but just one mutation in hemoglobin 21:58.1 can give it the tendency to precipitate. 22:00.3 And when it precipitates 22:03.0 it forms these incredible looking 22:05.1 helical filaments that look, really, 22:07.2 almost exactly like microtubules. 22:09.2 If I told you this was a microtubule, 22:11.0 you would probably even believe me, 22:12.1 but it's actually a polymer of sickle cell hemoglobin. 22:15.2 So, when you take a very soluble protein 22:18.1 and you make something go just a little bit wrong 22:20.1 with one part of its surface 22:21.3 so that it has a very minor tendency to self-associate, 22:24.1 the consequence is you get a helix. 22:27.2 Okay, so forming a helix 22:29.1 is not a special property... 22:31.1 what is it about cytoskeletal filaments 22:32.2 that gives them their ability to give us morphology? 22:35.2 Well, one other important thing 22:37.3 that is true of all of the eukaryotic cytoskeletal filaments 22:40.3 is that they have this very interesting dichotomy 22:44.3 that they have to be stable in order to have physical strength, 22:48.0 but they also have to be unstable 22:50.1 in order to allow the cell to change shape 22:52.1 or to move or to rearrange its elements 22:54.2 as it's dividing. 22:56.0 And at least for actin 22:58.1 and for tubulin, 23:00.0 part of the reason that they're able to 23:02.0 exhibit this dichotomy 23:04.0 is because those subunits are also able to bind and hydrolyze nucleotide -- 23:07.2 ATP in the case of actin, 23:09.1 GTP in the case of tubulin. 23:11.2 And as you see in this illustration here, 23:13.3 the fact that you can have 23:15.3 different conformational states of a protein 23:17.1 based on the identity of the nucleotide that's bound to it, 23:21.1 can set up a situation where you can have one conformational state, 23:24.3 in this case illustrated as being the thing binding ATP, 23:27.3 which is a conformation appropriate 23:30.2 for forming one of these helical filaments, 23:32.2 and you can have an alternative conformational state, 23:35.1 which is shown here in this case 23:36.3 as being the nucleotide free state, 23:39.0 but it could also be the ADP state, 23:40.2 that will tend to disassemble. 23:42.2 And so having that switch 23:44.2 between an assembly-competent form 23:46.2 and an assembly-incompetent form 23:49.0 can enable the dynamics 23:51.0 that are characteristic of eukaryotic cytoskeletal filaments, 23:53.1 and in fact it's very interesting 23:55.1 that actin itself 23:57.1 is a close structural relative of hexokinase, 24:00.0 the glycolytic enzyme which is best characterized 24:02.0 with respect to its very large conformational change 24:04.1 on binding and hydrolysis of substrate. 24:09.1 And in fact it's true 24:11.3 that if you look at the cytoskeletal elements of eukaryotic cell, 24:15.0 they're all highly dynamic, 24:16.2 and this is something that's been appreciated 24:18.1 for many decades in the context of microtubules, 24:20.2 actin, and intermediate filaments... 24:22.1 all of them turn over very fast inside of cells. 24:24.2 And in fact for things that require 24:28.2 the cytoskeletal filaments, like for example forming the mitotic spindle, 24:31.1 if you inhibit either the assembly 24:34.2 or the disassembly of the microtubules 24:37.3 in a mitotic spindle, 24:39.1 the net effect is the same in both cases, 24:41.1 which is that the cell fails to divide. 24:43.3 So it's not just the helical structure, 24:45.2 but also the turnover is clearly key. 24:47.3 Okay, well let's turn back to the prokaryotes -- 24:51.2 do they have dynamics associated with their cytoskeletal filaments? 24:54.3 Well, actually, as it turns out, they do. 24:56.2 So, as I mentioned FtsZ, 24:58.2 that's a close structural homolog of tubulin, 25:00.1 also binds and hydrolyzes GTP, 25:02.3 and in vivo turns over very, very rapidly, 25:05.2 as can be seen in this photobleaching study. 25:08.2 So, here there are two cells 25:10.1 that have assembled FtsZ rings. 25:12.2 Here, one half of one of these rings 25:14.1 has been bleached out, 25:15.2 and you can see, actually within just a few seconds, 25:18.1 the ring is able to come back. 25:20.1 And this particular experiment 25:23.2 also had a really, really nice direct demonstration 25:28.2 that those dynamics are associated with nucleotide hydrolysis, 25:31.3 which was they were able to compare the dynamics of wild type FtsZ protein 25:34.1 with an FtsZ protein 25:36.1 that had a point mutation that slowed down GTP hydrolysis, 25:39.0 and looking at the turnover of the wild type protein 25:41.2 versus the turnover of the slower protein, 25:45.1 in vivo, 25:46.1 they were able to see that there's this direct correlation 25:48.1 between how fast the filaments turn over 25:50.2 and their ability to hydrolyze GTP. 25:53.2 Okay, so the prokaryotic filaments, 25:55.1 they do the same kinds of dynamics 25:57.1 as the eukaryotic ones do, 25:58.2 so it's not the helical structure, 26:00.0 it's not the dynamics in terms of turnover, 26:01.2 and it's not even fancy dynamics. 26:03.3 So, microtubules, for example, 26:05.1 famously do this dynamic instability, 26:07.1 where even in a uniform chemical environment 26:10.1 an individual microtubule end will grow for a while 26:12.0 and then shrink for a while, 26:13.1 and then grow for a while and then shrink for a while, 26:15.3 and it's not at all obvious why this should be possible. 26:18.1 This is something that people have been studying for many years 26:21.1 and we have a pretty good overall chemical understanding 26:23.1 of how this can happen, 26:25.0 but it's definitely dependent on 26:28.1 the ability of the structures to hydrolyze nucleotide 26:30.2 and change their conformational states. 26:33.2 So, this illustration up top 26:36.0 shows the classic direct demonstration 26:37.3 of this dynamic instability on microtubules, 26:39.3 it was done in 1986, 26:41.2 and then almost 20 years later 26:44.1 another group was able to show with one of the bacterial actin homologs, 26:47.0 this one called ParM, 26:49.0 that they were able to see exactly the same kinds of behaviors 26:51.0 of these filaments growing for a while 26:52.2 and then shrinking for a while, 26:54.1 even when the chemical environment is uniform. 26:57.0 Okay, so it's not the helicity, 26:58.2 it's not the ability to hydrolyze nucleotide, 27:00.3 it's not the ability to turnover rapidly in vivo, 27:03.1 and it's not even the ability to do fancy things 27:06.1 like dynamic instability. 27:08.2 What it is, then, that's different? 27:10.1 Well, looking at bacterial cells, 27:12.1 it's clear that the way they're able 27:14.2 to get some sort of overall cellular organization 27:16.1 definitely depends 27:18.1 on this tendency of proteins 27:19.3 to make helical filaments. 27:21.1 And in fact if you look at bacteria 27:22.2 and you look at different aspects of their structures, 27:24.2 what you see is the helices are all over the place, 27:27.2 and you can make lots of different kinds of helices, 27:29.2 ranging from different things like the overall helical pattern of a spirochete 27:32.3 to the twisting helices associated with bacterial flagella, 27:36.2 to even the much more, sort of uniform helix 27:42.0 associated with something like a bacterial pilus. 27:45.2 So, it's clear it's not just the classical cytoskeletal proteins in bacteria 27:49.2 that are able to do this helical self-assembly trick, 27:51.1 but in fact lots of other proteins as well. 27:55.2 And looking specifically at the cytoskeletal proteins, 27:59.1 I think one interesting example 28:01.1 is the bacterium Caulobacter crescentus, 28:03.0 which is this very cute little banana shape 28:05.1 with structural differentiation on its two ends. 28:08.1 It's got not just actin and tubulin, 28:10.3 but it also has something that looks very much like 28:13.3 an intermediate filament, 28:14.3 and each of those things assumes slightly different helical patterns 28:17.2 inside of a growing cell. 28:19.1 And mutations in either the actin homolog, 28:21.1 the tubulin homolog, 28:22.2 or the thing that looks like an intermediate filament 28:24.1 will affect the overall shape of the cell 28:26.2 in very different ways. 28:28.1 So, lots of helical organization, 28:30.3 lots of function associated with helical organization, 28:33.2 but still no great morphological diversification. 28:37.3 Now, like everything else in biology, 28:39.3 there's going to be some exceptions, 28:41.1 and there are some bacteria that have really, 28:42.3 particularly spectacular shapes, 28:44.0 and this just shows a couple of my favorites. 28:45.2 This is a star-shaped bacteria called Stella humosa. 28:48.3 It's flat, but it has either five or six points 28:51.1 depending on what stage of its cell cycle it's at. 28:54.2 This is another weird-looking flat prokaryote 28:58.1 that in this case is an archaeon. 29:00.2 This is called Haloquadratu walsbyii 29:02.3 and it was first cultured in pure form 29:05.1 only a few years ago, 29:06.3 and this is actually flat like a floor tile, 29:08.3 and when it divides 29:11.0 it divides across the middle and then again like that, 29:13.0 so you have one floor tile going into four smaller ones. 29:16.1 And then there are things like this, 29:17.2 this is Epulopiscium fishelsonii, 29:19.2 which competes with Thiomargarita 29:21.1 as being one of the biggest bacteria known, 29:22.3 and this is absolutely huge, 29:24.1 it can be half a micron long, 29:26.1 and it has many copies of its genome 29:28.0 that are actually distributed 29:29.2 in a very regular pattern 29:31.1 throughout the entire cell, 29:32.2 and it moves its genome around depending on what time of day it is. 29:35.1 So, I think for things like this, 29:36.3 there's gotta be something else going on 29:39.0 besides just our usual bacterial helical self-organization, 29:41.2 and I think it's an interesting challenge 29:43.1 to now try to go into these organisms 29:45.1 and find out what it is that's determining their ability 29:47.2 to have these very specific morphologies. 29:51.2 Okay, but getting back to the main thread, 29:53.2 I've been arguing that 29:56.1 you can have simple helical structures 29:58.1 without motor proteins and nucleators, 30:00.0 and in order to have more complicated things 30:02.1 that make eukaryotes what they are, 30:03.2 we need to have either nucleators or motors. 30:06.0 So, this raises the question, 30:07.3 well, why don't bacteria have them? 30:09.0 Where did they come from in eukaryotes? 30:10.1 And why does it seem like it's so hard for the bacteria 30:12.0 to pick them up? 30:14.1 Well, for the nucleators, 30:15.2 this is a particularly interesting problem 30:17.2 because the way that eukaryotic cells tend to nucleate 30:22.1 their cytoskeletal proteins 30:23.3 is very often by taking the subunit, 30:27.0 copying that gene, 30:28.2 and then having it diverge a little bit 30:30.3 to have it assume a specialized nucleation function. 30:33.1 And we've seen this in two cases, 30:35.1 completely independent cases, 30:36.2 for actin nucleation and for tubulin nucleation. 30:39.1 So, actin nucleation can be performed 30:41.2 by this complex that's called the Arp2/3 complex, 30:44.1 where Arp stands for actin-related protein, 30:46.3 because there are two different proteins 30:48.3 from different genes, 30:50.1 both structural homologs of actin, 30:52.1 that come together to make this regulated nucleation complex. 30:56.1 In the case of microtubules, 30:58.0 the nucleation is often carried out 30:59.3 by what's called the gamma-tubulin ring complex. 31:02.1 Now, the lattice of the microtubule 31:03.3 is made up with alpha-tubulin and beta-tubulin. 31:06.0 Gamma-tubulin is specialized only for nucleation function. 31:10.2 Why can't bacteria do this? 31:12.1 Well, I think they clearly could if they wanted to, 31:14.3 because we do see, as I mentioned, 31:17.2 among the actin homologs in bacteria, 31:19.1 an individual cell may have 31:21.2 several kinds of actin homologs 31:23.1 that are all present within that same genome 31:25.1 that have all been evolved for slightly different functions. 31:28.1 And yet, so far, as far as we know, 31:31.1 none of them have been specialized specifically 31:33.1 for filament nucleation. 31:35.3 I don't know why they don't want to, 31:38.0 but it seems like they could, 31:39.2 and yet they don't. 31:43.1 Turning now to the molecular motor proteins, 31:46.0 this is something where it might be a little more clear 31:48.2 why bacteria don't have them. 31:50.1 If we think about where the really good molecular motor proteins come from in eukaryotes... 31:54.3 there are of course three different classes, 31:56.3 there's the myosins, the kinesin, and the dyneins, 31:58.1 that all hydrolyze nucleotide 32:00.0 in order to undergo a conformational change, 32:01.3 and of these the kinesin and the dyneins move on microtubules. 32:06.2 The myosins move on actin filaments. 32:08.1 Now, these different motor proteins 32:10.0 had all been purified, 32:11.1 had all been studied biochemically, 32:12.2 and sequenced, 32:14.1 and then eventually their structures were determined, 32:16.0 and at the point when the structures were determined, 32:18.0 there was this huge surprise, 32:19.3 and this, again, was back in the 1990s, 32:22.0 where it was found that 32:25.1 the fundamental of the catalytic core of myosin 32:27.1 was almost identical 32:29.1 to the structure of the catalytic core of kinesin. 32:31.3 Even though myosin walks on actin filaments 32:33.2 and kinesin walks on microtubules, 32:35.2 they still seem to be homologs 32:37.2 derived from a common ancestor. 32:39.2 And in both cases, the fundamental way that ATP hydrolysis 32:42.2 at the core of the motor protein 32:44.2 results in the conformational change 32:46.0 that gives you a step 32:49.0 is very similar at the heart of the motor protein, 32:50.3 even though all the details about how it couples to its filaments 32:53.2 are quite different between the two. 32:56.1 So, this gives rise to, you know, 32:58.2 another field ripe for speculation 32:59.3 about where did the motor proteins come from. 33:01.2 Well, if kinesins and myosins are related to each other, 33:04.1 it seems like there must have been some sort of motor precursor, 33:07.2 and we don't know what its substrate was, 33:09.2 if it was actin or microtubules, 33:10.2 or if it might have been something else. 33:12.2 We don't know what the complement of motors was 33:15.2 in the earliest thing that became a eukaryote, 33:18.1 and whether this might have been something 33:20.1 that actually drove it to separation 33:23.1 from the prokaryotic branches of the tree 33:25.1 in terms of morphological diversification. 33:28.1 One thing we do know, though, 33:30.3 is that those motor proteins, kinesin and myosin, 33:33.1 are both derived from a particular branch of 33:37.2 the superfamily of proteins that's called P-loop NTPases, 33:40.1 that includes a lot of ATPases and GTPases. 33:43.1 And this fairly complicated diagram 33:45.2 is a summary of sequence diversification 33:49.1 among many, many different family members 33:51.2 of this large protein superfamily 33:53.2 across the entire tree of life. 33:56.1 And what this group, 33:58.1 this is Eugene Koonin's group that's done this, 34:00.0 has attempted to do 34:02.2 is look at what the distribution of what all these protein types 34:05.1 in currently living cells 34:06.3 and then project backwards in time 34:08.2 to see when they might have evolved. 34:10.3 And so there's some classes of proteins 34:12.3 that you see here with these pink lines 34:14.2 that are present in bacteria, in archaea, and in eukaryotes, 34:17.1 and so they must have been present in LUCA, 34:19.0 in the last universal common ancestor. 34:21.1 But there's one particular protein family, right here, 34:24.1 that's shown in brown, 34:26.0 that has both the myosins and kinesins in it, 34:30.0 and then also has a whole lot of other GTPases 34:32.1 that we associate with specifically eukaryotic functions 34:34.3 -- Ras, the Rab proteins that are involved in membrane trafficking, 34:38.3 the Rho proteins that are involved 34:40.2 in large-scale organization of cell polarity -- 34:44.0 all of those things come from 34:46.1 the same relatively narrow branch of the P-loop NTPases. 34:49.2 So, one possible explanation 34:53.2 for why eukaryotes and prokaryotes are different from one another 34:55.3 is because the proteins that are best poised 34:58.1 to become stepper motors 35:00.2 of the kind that are familiar from eukaryotes 35:02.2 happen to be in this branch of proteins 35:05.1 that didn't evolve until the eukaryotic ancestor 35:07.1 had already split off. 35:09.3 It's very tempting to speculate 35:11.1 that this particular class of proteins 35:12.2 that includes not only the myosins and kinesins, 35:15.3 but also all of these regulatory GTPases, 35:19.0 might be part of what makes eukaryotes what we are. 35:22.2 Now, I'd like to point out, of course, 35:24.3 there's plenty of other protein families 35:26.2 that are not shared between eukaryotes and prokaryotes 35:28.1 -- about 50 other classes of proteins 35:30.1 have been identified to date -- 35:31.1 however, I think, mechanistically, 35:33.0 this is a particularly intriguing group 35:36.1 for generating specifically morphological diversification 35:37.3 at the level of cells 35:40.1 and at the level of whole organisms. 35:43.1 Now, I don't mean to say that bacteria don't have motors; 35:45.2 bacteria have amazing motors. 35:47.1 Bacteria have the flagellar rotor, 35:49.2 which is a very complicated structure, 35:52.0 it's much more complicated even than dynein, 35:54.1 that's able to spin at 200 Hertz 35:56.0 and is made up of more than 40 different gene products. 36:00.1 Bacteria also have extremely strong motors 36:02.2 like the motor that drives twitching, 36:05.3 by retraction of these type IV pili, 36:08.1 and this is something where a single motor 36:10.1 interacting with a filament 36:12.2 that's extended from the... 36:14.1 that's extended through the bacteria envelope 36:17.1 is able to generate up to 100 picoNewtons of force, 36:20.0 just from movement 36:23.0 of a single extended filament structure. 36:26.0 So, the motors in bacteria can be very powerful, 36:28.1 very efficient, 36:29.3 and very complicated, 36:31.2 and yet all of them seem to do things on the surface of the bacterium. 36:34.2 None of them seem to do anything in the cytoplasm. 36:37.1 So somehow this question of morphological diversification, 36:40.1 about how you get complex intracellular structures, 36:43.1 seems to be at least correlated 36:45.1 with the presence of linear stepper motors for cytoskeletal proteins 36:50.2 in eukaryotes, 36:51.2 and no real equivalent in prokaryotes. 36:54.0 And, you know, again, I can speculate wildly 36:55.3 about why there seems to be this difference, 36:59.0 but nevertheless this is a fairly compelling correlation. 37:02.3 It doesn't come down to complexity or anything like that, 37:05.1 but really only down to the presence or absence 37:07.1 of this one particular protein family. 37:11.1 Okay, so this is, overall, the hypothesis, again, 37:14.0 that we need nucleators and molecular motors 37:15.3 in order to drive things like the mitotic spindle, 37:18.2 or like microtubule asters, 37:19.2 or like parallel bundles of cytoskeletal filaments 37:23.1 that give eukaryotes morphological complexity, 37:25.1 and I've made the claim 37:28.2 that no structures of this kind, again, 37:30.0 with just very minor exceptions, 37:32.0 have yet been found in the cytoplasmic compartment in bacteria. 37:37.3 I'm focused, of course, 37:39.1 on the role of the cytoskeleton in all of this, 37:41.1 but it is worth think about maybe other kinds of explanations 37:43.2 for the difference, 37:45.0 and one thing that might really be connected 37:46.2 is the fact that it's been observed in bacteria, 37:48.2 where of course the chromosome 37:52.0 is just embedded in the cytoplasm 37:53.3 and there's no nuclear membrane separating the chromosome 37:56.2 from the cytoplasm, 37:57.3 in the cases where it's been well documented, 37:59.1 the bacterial chromosome is actually highly organized. 38:02.1 And one of the first hints of that came from these studies in Caulobacter, 38:05.1 showing that a circular bacterial chromosome 38:08.1 that was marked with different fluorophores 38:11.2 at specific locations would actually organize itself 38:14.1 in basically every cell within a bacterial colony 38:17.2 such that those markers on the chromosome 38:19.1 are all found in the same order. 38:20.3 In other words, the bacterial chromosome 38:22.1 is packed into the cell 38:24.1 in a highly structured, highly regular way 38:26.2 that can provide spatial information 38:29.1 for targeting other kinds of subcellular structures, 38:31.2 for example, to the poles 38:33.2 or to the nascent septal zone. 38:36.1 So, you know, one possible way 38:39.1 to bring these two ideas together 38:41.2 is that at some point in the development of the eukaryotic ancestor 38:43.3 something happened 38:46.0 that either enabled the nuclear envelope to come in 38:49.1 or somehow otherwise separated the chromosome 38:51.3 from the cytoplasm, 38:53.1 and if bacterial cells and archaeal cells 38:55.1 primarily rely on their chromosome 38:57.1 as being their major organizing principle 38:59.0 as far as where to put things in the cytoplasm, 39:00.3 then when that separation happened, 39:03.2 the little filaments that were left out in the cytoplasm, 39:06.0 that in bacteria had only been doing 39:08.0 fairly trivial things 39:09.2 of figuring out where to divide 39:11.1 and how to determine the overall shape 39:13.1 of where to lay down the cell wall, 39:14.2 those guys were left by themselves out in the cytoplasm. 39:16.1 They had no landmarks, 39:17.3 they had no chromosome to tell them where anything was, 39:19.1 and so they somehow had to figure out 39:21.2 how to make larger-scale structures 39:23.2 like asters, like bundles, like spindles, 39:26.2 that could help them organize themselves 39:29.1 in the cytoplasm, 39:30.2 in the absence of information from the chromosome. 39:34.0 Okay, like I said, this was wild speculation, 39:36.0 but one of the things that I think is fun about this 39:38.3 is that it's wild speculation that is absolutely disprovable. 39:41.1 So I'd like to give you a personal challenge 39:43.2 to look for and try to find 39:46.2 either a cytoskeletal stepper motor 39:48.1 that walks on a bacterial cytoskeletal filament 39:50.2 in a way analogous to what kinesin or myosin 39:53.1 does in eukaryotic cells, 39:55.0 or else to find a regulated nucleator 39:57.2 of any of these bacterial cytoskeletal filaments. 40:00.0 A lot of very smart people have been looking in various ways 40:02.1 for at least the past 20 years 40:05.0 and, as far as I'm aware, 40:06.3 nobody has yet found any proteins in one of these classes. 40:08.2 Now, that certainly doesn't mean they're not out there, 40:10.2 but it does mean that if either of these things were found, 40:13.0 that would be absolutely definitive proof that my big, crazy hypothesis 40:16.2 is wrong. 40:18.0 So, please try to prove me wrong, 40:20.1 and if you do find one of these classes of proteins, 40:22.1 a bacterial cytoskeletal stepper motor 40:24.1 or a bacterial regulated nucleator, 40:26.0 please send me an email and let me know. 40:28.0 I would love to hear about it. 40:29.2 And if you do, I personally will send you 40:32.0 a large bouquet of flowers 40:33.2 and also hearty congratulations 40:35.1 for taking another step forward 40:37.1 to trying to understand one of these biggest questions 40:39.1 about why cells on Earth look so different 40:41.1 from one another. 40:42.2 Thank you.