Developmental Biology of a Simple Organism: Bacillus subtilis
Transcript of Part 1: Spore Formation in B. Subtilis
00:00:07.09 Hello, my name is Richard Losick and I'm a professor at Harvard University. 00:00:12.16 The title of my presentation is "Developmental biology of a simple organism." 00:00:17.25 We generally think of biological development in the context of complex multicellular organisms. 00:00:24.27 But, even the most primitive kinds of cells can also exhibit dramatic processes 00:00:30.25 of cellular differentiation and morphogenesis. I'm going to tell you one such example, 00:00:35.20 with the spore forming bacterium Bacillus subtilis. 00:00:39.22 Let me introduce Bacillus subtilis to you in the way it was introduced 00:00:43.17 to the academic community in 1877 by its discoverer Ferdinand Cohn. 00:00:50.29 Ferdinand Cohn published his findings in the Biology of Plants and he drew what he saw 00:00:59.02 in this marvelous plate that you see here. And what's apparent in the plate 00:01:04.22 is that he saw long chains of cells that had tiny ovoid bodies in them 00:01:10.07 which he recognized as bacterial spores. So this marked the discovery 00:01:14.17 of spore formation by a bacterium. 00:01:19.28 You should also know that this benign bacterium Bacillus subtilis has an evil cousin. 00:01:26.26 Itâ€™s the anthrax causing pathogen Bacillus anthracis which was discovered 00:01:31.29 by Robert Koch at about the same time as Cohn. And Cohn and Koch 00:01:37.17 agreed to publish their findings back to back in the Biology of Plants and, in fact, this cartoon 00:01:43.03 of drawings contains contributions of Koch for Bacillus anthracis 00:01:50.04 as well as for Cohn for Bacillus subtilis. 00:01:54.23 I've divided my presentation into three parts. The first part concerns the process, 00:02:02.05 the overall process by which B. subtilis makes a spore. 00:02:07.22 The second part is new research on multicellularity. We traditionally have thought 00:02:13.25 of Bacillus subtilis and many other bacteria as being solitary creatures 00:02:18.07 that go about their business as individual cells, but now we've come to appreciate that 00:02:23.14 Bacillus subtilis makes elaborate multicellular communities and that 00:02:28.01 spore formation takes place in these communities. 00:02:32.09 The last part is the topic of stochasticity and cell fate. 00:02:38.00 Itâ€™s generally believed that cell fate decisions 00:02:42.01 in developmental biology are highly determined. 00:02:45.11 And indeed they are. But increasingly we've begun to see examples in which 00:02:51.08 decisions about cell fate are made in a stochastic fashion 00:02:56.27 and that will be the topic of my last presentation 00:02:59.26 which will have four examples from Bacillus subtilis. 00:03:04.27 OK, so to begin this part of my talk: 00:03:09.07 Bacillus subtilis makes a spore. How does it do it? 00:03:12.12 So there are three main topics: The first is that spore formation involves two cells. 00:03:20.11 So this is really a tale of two cells. You'll see that two cells collaborate in making a single spore. 00:03:27.09 Having created two cells, each cell has its own fate, 00:03:31.12 follows its own distinct program of gene expression, 00:03:36.01 and this is governed by a series of transcription factors that act in a cell specific fashion. 00:03:43.02 The final part of my talk concerns how the two cells talk to each other. 00:03:49.11 The two cells are not completely independent, even though they follow 00:03:52.18 their own distinct programs of gene expression, but rather they talk back and forth 00:03:57.05 at each stage of development to keep the two processes in coordination with each other. 00:04:04.10 Here in cartoon form are the principle stages of spore formation. 00:04:09.15 Spore formation is triggered by nutrient limitation and that results in the cells entering a pathway 00:04:16.17 that involves the formation of two different cells. At the very start we have a single cell, 00:04:24.20 and I'll refer to that as the pre-divisional sporangium. 00:04:27.25 Then that pre-divisional sporangium undergoes 00:04:31.00 a conspicuously asymmetric process of cell division in which a division septum is 00:04:37.25 formed near an extreme pole of the cell. That divides the developing cell into 00:04:43.26 two cells; a forespore cell, the smaller cell, and a larger mother cell. 00:04:49.13 The forespore is destined to become the spore, 00:04:52.18 whereas the mother cell nurtures the developing spore 00:04:56.09 and eventually liberates the mature spore. At this early stage of development, 00:05:02.14 the forespore and the mother cell lie side by side. But later in development, 00:05:07.04 in a remarkable cell biological process, the mother cell swallows up the forespore, 00:05:13.26 in a process that resembles phagocytosis in higher cells, 00:05:18.13 fully engulfing the forespore and pinching 00:05:21.07 it off as a free cell in the mother cell cytoplasm to create a cell within a cell. 00:05:26.22 So that inner cell will become the spore and the outer mother cell nurtures the spore and 00:05:32.24 then eventually will liberate the spore by lysing. 00:05:36.15 Here are fluorescence micrographs of cells at these various stages of sporulation. 00:05:42.25 The cells have been stained with a membrane dye 00:05:45.09 to highlight the features that I've been speaking about. 00:05:47.15 So, at the stage of asymmetric division, you can see that a polar septum is formed 00:05:53.21 at an extreme polar position in the sporangium. Next, the mother cell membrane 00:06:01.03 starts to migrate around the forespore, eventually fully engulfing it, 00:06:07.09 and then pinching it off as a free cell in the sporangium. 00:06:14.01 So, how does this process of asymmetric division take place? 00:06:18.17 Well, bacteria divide by means of a tubulin-like protein called FtsZ 00:06:27.01 which forms a cytokinetic ring known as the Z-ring. 00:06:30.09 Higher cells rely on actin, bacteria rely on tubulin. 00:06:35.12 Here is a fluorescence micrograph showing the Z-ring, 00:06:39.20 which has been tagged with the green fluorescence protein. 00:06:42.18 Itâ€™s in the center of the cells and itâ€™s at the future site of cell division in a vegetatively growing cell. 00:06:49.16 So, when cells are growing, a Z-ring forms in the middle 00:06:52.21 and itâ€™s then converted into a division septum 00:06:56.03 to give rise to two equal sized daughter cells. But when cells enter the pathway to sporulate, 00:07:02.25 what happens is that two Z-rings form. One near each pole of the sporangium. 00:07:09.08 And then only one of these two Z-rings gets converted into a division septum 00:07:15.17 whereas the other one is disassembled. 00:07:17.26 So that at the end of the process we have a single polar septum 00:07:22.01 that's created the two unequal size cells. 00:07:25.05 Now, immediately this raises interesting issues about how 00:07:29.10 each of these two cells acquire a chromosome. 00:07:31.28 The pre-divisional sporangium has two chromosomes and the challenge for the developing cell 00:07:38.29 is to ensure that the forespore and the mother cell each inherit a complete chromosome. 00:07:46.09 Sporulating B. subtilis goes about this process in a fascinating way 00:07:51.07 that differs from almost all other cell types that we know about. 00:07:55.14 Here is a cartoon that illustrates what the chromosomes look like in a growing cell. 00:08:02.29 They're in two masses referred to as nucleoids, 00:08:07.04 and each chromosome, of course, has an origin of replication 00:08:11.29 and the origins are at the outer edges of the two DNA masses. 00:08:16.01 When the cells enter the pathway to sporulate, those two DNA masses get remodeled into 00:08:22.17 a filament, known as the axial filament, that extends across the cell 00:08:27.21 from pole to pole with each of the two origins at the extreme opposite poles of the sporangium. 00:08:35.22 You can see this in this fluorescence micrograph, in which the DNA in this slide 00:08:43.07 is labeled in green and the outline of the cell in red. 00:08:47.20 In the growing cell you can see two distinct DNA masses 00:08:51.26 and in the cell that's begun to sporulate you can see that the DNA is 00:08:57.10 elongated and is extending all the way across the cell. 00:09:03.06 And the interpretive cartoon shows that the origins are at 00:09:06.21 the extreme opposite poles of this axial filament. 00:09:11.14 How does this happen? Well, this process of remodeling the chromosome into a filament and 00:09:18.27 anchoring it at the poles is mediated by two proteins. 00:09:22.06 They're called RacA and DivIVA. RacA binds to the DNA at multiple sites to collapse it 00:09:31.11 but especially around the origin to create a kind of Velcro that will stick it 00:09:37.04 to DivIVA, a protein which is anchored at the poles of the cell. 00:09:41.22 So you can see that in this cartoon. DivIVA is at opposite poles of the cell. 00:09:48.04 Here are the two DNA masses. 00:09:50.03 When RacA appears, it binds at diverse sites around the two chromosomes 00:09:55.23 to help collapse it, but forms a structure with many RacA molecules 00:10:01.20 at the origin regions. And as depicted in this cartoon, the chromosomes get stretched out 00:10:08.03 and adhere to the poles. Let me show that to you one more time. Here's the RacA protein, 00:10:13.24 and here's the process by which it gets pulled to opposite poles of the sporangium. 00:10:19.12 Now, finally, asymmetric division takes place. 00:10:22.20 But you'll immediately appreciate that when the division septum comes down, 00:10:27.16 because of its extreme polar placement, 00:10:30.08 only some of the chromosome destined to the forespore 00:10:34.05 will be in the little cell. So you can see this in these fluorescence micrographs. 00:10:39.19 Just after the division septum forms, only a little bit of DNA is in the forespore, 00:10:45.12 that which was trapped by virtue of being anchored at the pole. But then over time 00:10:50.20 the remainder of that chromosome gets pumped into the forespore compartment 00:10:55.20 until a complete chromosome is present in the forespore. This pumping 00:11:02.08 of a chromosome into the forespore is mediated by a molecular machine, 00:11:06.17 a protein called the DNA translocase, 00:11:09.17 which is located in the septum and uses energy from ATP 00:11:14.04 to pump the remaining portion of the chromosome into the forespore compartment. 00:11:19.18 So, most cells, or almost all cells that we know about separate their chromosomes prior 00:11:27.05 to cytokinesis. But, in sporulating cells, cytokinesis takes place before chromosome segregation. 00:11:34.10 We can visualize the DNA translocase that mediates this chromosome segregation by 00:11:39.24 tagging it with the green fluorescence protein shown in this cartoon. 00:11:44.00 So here's a sporangium, and you can see the polar septum 00:11:49.28 and this bright focus of green fluorescence 00:11:52.29 from the DNA translocase that's sitting in between 00:11:57.22 the mother cell and the forespore and is poised to pump DNA 00:12:01.10 across the septum into the small chamber 00:12:04.13 of the sporangium. So to review everything that I've said so far 00:12:10.24 in the very first stage of asymmetric division, the Z-protein is remodeled to form rings 00:12:20.16 at each pole of the sporangium. Then, one of these two Z-rings is converted into a division septum 00:12:26.21 and the other Z-ring is disassembled. 00:12:28.28 Next, chromosomes need to be segregated into the two cells. 00:12:36.20 And so while the Z-rings are forming, the two chromosomes are remodeled 00:12:41.21 into an axial filament by the RacA protein, which causes them to collapse 00:12:46.29 into elongated filament and anchors the origins at the poles, 00:12:51.04 where the DivIVA protein is present. 00:12:52.24 Then asymmetric division takes place, and the DNA translocase located in the division septum 00:12:59.11 pumps the remainder of the forespore chromosome into the small chamber of the sporangium. 00:13:05.25 So that when this process is complete we have two cells that lie side by side; 00:13:11.06 each has a complete chromosome. In the next stage of development, 00:13:17.21 the mother cell membrane migrates around the forespore 00:13:21.22 to fully engulf it and pinch it off as a free cell within a cell. So now the process of sporulation 00:13:30.02 is well underway and that inner cell will mature into a spore. This conversion of that inner cell 00:13:36.12 into a spore involves three principle, morphogenetic processes. 00:13:42.02 One, is the remodeling of the chromosome of the forespore into a doughnut-like structure, 00:13:47.15 in which state itâ€™s highly resistant to radiation. The second is the formation of a thick 00:13:53.20 layer of cell wall material called the cortex around the forespore. 00:13:58.12 And then a thick protein shell made up of 00:14:02.06 many different proteins that creates a protective shell 00:14:05.27 around the spore. This next cartoon illustrates these processes. 00:14:11.11 So you'll see the forespore chromosome being remodeled 00:14:14.19 into a doughnut. The white area is the cortex, 00:14:18.04 and the thick protein shell of coat proteins is created on the outside. 00:14:23.10 This, then, matures into a spore, a golf ball-like spore. The mother cell, having done her job 00:14:32.05 lyses and liberates the mature spore, which can remain inert 00:14:36.16 for many years, but on a moment's notice, when 00:14:40.05 good conditions return, it can crack open like an egg, and give rise to a cell 00:14:46.09 that can resume vegetative growth and binary fission. 00:14:53.19 OK, so we've seen now how during sporulation, two cells are formed. 00:14:59.16 And the key point is that each of these two cells needs to follow its own distinct program 00:15:06.23 of gene expression. These two cells have their own pathways of cellular differentiation, 00:15:11.29 the forespore and the mother cell. 00:15:13.10 These pathways of differentiation are driven by transcription factors 00:15:18.19 that act in a cell specific manner and that's the topic I want to talk about now. 00:15:24.08 So, in this cartoon I've indicated the five principle 00:15:31.15 transcription factors that drive the process of sporulation. 00:15:35.25 The first one is known as Spo0A and itâ€™s the master regulator for sporulation. 00:15:41.24 Itâ€™s the protein which becomes activated in response to nutrient limitation 00:15:47.03 and causes the cell to enter this pathway and causes all the events 00:15:52.07 that I've been talking about to unfold, including asymmetric division. 00:15:56.04 After asymmetric division takes place, 00:15:58.13 then a transcription factor appears that's called sigmaF. 00:16:03.19 SigmaF is a member of a family of regulatory proteins 00:16:08.13 in bacteria known as RNA polymerase sigma factors that work by 00:16:12.14 binding to RNA polymerase and directing it to particular kinds of promoter... 00:16:17.17 promoter sequences in the chromosome. 00:16:20.13 The first of these sigma factors, sigmaF, becomes activated in the forespore compartment. 00:16:26.28 Then, a sigma factor called sigmaE gets activated in the mother cell compartment. 00:16:33.01 Then, after engulfment takes place, sigmaF gets replaced in the forespore 00:16:38.02 by a transcription factor called sigmaG. 00:16:40.23 And then lastly, in the mother cell, the final transcription factor 00:16:46.23 in the developmental program, sigmaK, appears. 00:16:50.00 That these transcription factors act in a cell specific manner 00:16:55.01 can be seen in the inset on the right. 00:16:58.09 Here I show you single sporangia that harbor a fusion of the gene for the 00:17:05.00 green fluorescence protein joined to a promoter under the control of sigmaE 00:17:09.26 in the top example or a promoter controlled by sigmaG in the bottom example. 00:17:14.16 You can see in the upper case fluorescence is restricted to the mother cell, 00:17:19.26 the compartment in which sigmaE is active. 00:17:22.14 Whereas in the bottom example, we have the opposite pattern. 00:17:26.09 The fluorescence is accumulated in the forespore compartment 00:17:29.09 where sigmaG is active. 00:17:31.22 So each of these four sigma factors act in a cell specific fashion. 00:17:36.05 And each one of them has presented a puzzle as to the molecular mechanisms 00:17:40.27 that cause it to become activated in a specific cell type. 00:17:45.06 We've, over the years, helped to unravel these mysteries 00:17:51.10 and figure out how these four transcription factors are activated. 00:17:54.23 And let me tell you just one story about what we know about the activation of sigmaF. 00:18:00.22 So, sigmaF as you've seen is activated in the forespore 00:18:05.14 but itâ€™s actually synthesized and present in the pre-divisional sporangium. 00:18:10.01 Itâ€™s not active in the pre-divisional sporangium because itâ€™s held inactive 00:18:16.11 in the pre-divisional sporangium by an antagonistic protein called AB. 00:18:21.09 AB is a so-called anti-sigma factor that binds to sigmaF and holds it in an inert state. 00:18:28.28 AB holds sigmaF inert in the pre-divisional cell and also 00:18:34.09 after asymmetric division in the mother cell. 00:18:37.06 But in the forespore, sigmaF manages to escape from AB and 00:18:41.27 becomes active in directing gene expression. 00:18:44.29 How does it escape? 00:18:46.12 Well, its escape is mediated by another protein that we call AA. 00:18:51.08 AA is an anti-anti-sigma factor that reacts with a complex of AB and sigmaF 00:18:59.20 to discharge free and active sigmaF. 00:19:03.23 AA itself is regulated by phosphorylation. Itâ€™s a phospho-protein and 00:19:10.01 in its phosphorylated state it is inactive and in its dephosphorylated state 00:19:15.28 itâ€™s active and capable of triggering the activation of sigmaF. 00:19:20.04 This conversion from the phospho-form to the dephospho-form is mediated by 00:19:25.23 a phosphatase called E. So E converts AA-phosphate to AA. 00:19:33.07 And then AA reacts with AB-sigmaF to discharge 00:19:38.08 free and active sigmaF in the forespore compartment. 00:19:41.09 How does this happen just in the forespore? 00:19:44.04 Well, we don't fully know the answer to this question. 00:19:46.21 But undoubtedly, an important clue is the discovery that the E phosphatase 00:19:53.09 is itself situated in the septum that divides the two cells from each other. 00:19:58.08 So here we've tagged the E phosphatase with the green fluorescence protein. 00:20:03.08 You can see that in the left fluorescence panel. 00:20:05.27 And in the right fluorescence panel we've stained the sporangium membranes with 00:20:11.05 a red membrane dye. And you can see that the E protein is located right in the septum. 00:20:17.07 Somehow, it acts preferentially or exclusively on the forespore side of the septum 00:20:23.12 to cause sigma F to be activated in that compartment and not the mother cell compartment. 00:20:29.13 So let me bring everything I've said up until now together; 00:20:33.01 AA, AB, activation of sigmaF, using a field of cells 00:20:39.27 in which each cell harbors a fusion of the gene 00:20:44.04 for the green fluorescence protein to the promoter 00:20:46.21 to a promoter under the control of sigmaF. 00:20:49.14 So as this movie begins you can see that all of the cells are dark; 00:20:54.10 they have no fluorescence. Then asymmetric division takes place. 00:20:57.28 And then sigmaF becomes activated and you can see the bright green foci appearing 00:21:02.16 massively in the cells in this field as they begin to sporulate. 00:21:08.03 Then those bright green foci get converted into opaque phase bright bodies 00:21:14.10 as sporulation is completed. Let's look at this one last time. 00:21:18.05 Here's the field of cells, then massively, green fluorescence appears 00:21:22.28 near one end of each of those sporangia. 00:21:26.12 And then those green foci become phase bright bodies that represent the maturing spores. 00:21:35.03 OK, let's now come to the last topic. 00:21:38.25 I may have left you with the impression up to now that after asymmetric division 00:21:43.18 the forespore and the mother cell each march to their own drummer, 00:21:48.02 independently follow their own independent programs of gene expression. 00:21:52.05 But nothing could be further from the truth, 00:21:54.10 because the two cells are having a conversation with each other 00:21:57.29 at each stage of development. They talk back and forth 00:22:01.06 to each other so as to coordinate the progress of development in one cell 00:22:06.13 to the progress of development in the other cell. 00:22:09.03 So, to begin, as we've seen sigmaF is activated in the forespore compartment. 00:22:16.01 But sigmaE does not appear until sigmaF is active. 00:22:21.14 SigmaF sends a signal across the membranes that separate the two cells 00:22:27.24 that leads to the activation of sigmaE in the mother cell. 00:22:31.04 Once sigmaE is activated in the mother cell, it in turn sends another signal 00:22:36.13 that leads to the activation of sigmaG in the forespore cell, 00:22:41.16 the now engulfed forespore compartment. 00:22:43.22 Once sigmaG is activated, it in turn sends a signal back to the mother cell 00:22:52.05 that allows the final transcription factor in this sequence, sigmaK, 00:22:57.12 to appear. So the two cells are talking to each other 00:23:00.27 in a two-way conversation: from forespore to mother cell, 00:23:05.04 to mother cell to forespore, to forespore to mother cell. 00:23:09.06 Let's listen in on one of these conversations 00:23:12.26 and see just how the language of one of these conversations between the two cells 00:23:18.14 the very last one, in which sigmaG tells the mother cell to activate sigmaK. 00:23:23.11 The way this works is as follows: 00:23:26.10 sigmaK is initially synthesized as an inactive pro-protein. 00:23:31.12 That is, the primary gene product has an N-terminal extension 00:23:36.00 of about 20 amino acids that renders pro-sigmaK inactive. 00:23:41.25 In order for it to be active a protease needs to chop off that N-terminal extension 00:23:48.11 to generate the mature and active form of the transcription factor. 00:23:52.20 You can see that in this Western blot experiment. 00:23:58.00 So this is an experiment in which all the proteins from the sporulating cell were 00:24:01.17 separated on a gel and then sigmaK and pro-sigmaK were visualized 00:24:07.12 with antibodies to the protein. And as you can see in a wild-type sporulating cell 00:24:13.29 most of the sigmaK is in the form of the mature and active protein 00:24:18.27 and relatively little in the larger pro-protein form. 00:24:22.29 This conversion of pro- to mature depends on the action of sigmaG. 00:24:28.19 And you can see this key point if we use a mutant of sigmaG. 00:24:34.24 When sigmaG is mutant there is no conversion and if you look at the Western blot 00:24:39.25 analysis on the far right of the mutant, now you can see that all of the protein 00:24:45.12 is in the pro-form and little or none is in the form of the mature sigma factor. 00:24:50.10 Somehow, the activation of sigmaK in one cell, depends on genetic events 00:24:56.15 taking place in the adjacent cell. How does this work? 00:25:00.26 Well, the protease is a membrane protein and it mediates the cleavage of the pro-sequence. 00:25:09.13 But initially itâ€™s held inactive by two other membrane proteins 00:25:15.03 that are inhibitory and together hold the protein in an inactive complex. 00:25:20.22 In order for the protease to become activated, 00:25:24.28 the protease needs to escape from this inhibition. 00:25:28.21 And that event is caused by a signaling protein 00:25:34.04 that's produced in the forespore compartment under the control of sigmaG. 00:25:39.12 So sigmaG turns on the gene for a signaling protein, 00:25:42.15 that signaling protein is secreted across the membrane 00:25:46.16 of the forespore where it interacts with a complex of proteins 00:25:51.27 that includes the protease and its inhibitory proteins 00:25:55.07 and reverses the inhibition so that now cleavage of 00:25:59.18 pro-sigmaK to the mature form of the transcription factor can take place. 00:26:04.23 This protease turns out to be especially interesting on two counts. 00:26:10.13 First, we infer that its active site is located in the membrane. 00:26:15.16 The blue bars represent the inferred catalytic regions 00:26:20.18 of the protease and itâ€™s inferred that the N-terminal extension on sigmaK 00:26:25.25 inserts into a cavity, in the membrane, created by the protease. 00:26:30.25 Well, this kind of membrane cleavage leading to gene... activation of gene expression 00:26:38.17 is a fore-runner of something that's widespread in biology 00:26:42.19 and is referred to as regulated inter-membrane proteolysis. 00:26:46.11 And interestingly, this very example of it in a bacterium 00:26:51.26 is conserved all the way up to mammals. 00:26:54.09 Mammals have a protease with homologous features, in particular the catalytic center, 00:27:01.15 to the bacterial protease. In the case of mammals, 00:27:06.03 the protease activates a transcription factor that's bound to the membrane 00:27:11.07 and cleavage by the protease releases it from the membrane 00:27:15.02 and so it can migrate to the nucleus and activate gene expression, 00:27:19.22 in this case, genes involved in cholesterol metabolism. 00:27:22.22 But, the overall principle is the same. 00:27:27.21 Two transcription factors are in inactive states that require proteolysis to be activated 00:27:33.22 so that they can turn on genes. 00:27:36.21 And the similar proteases, conserved over eons of evolution, mediate both processes. 00:27:44.09 So finally, let me point you in the direction of future research: 00:27:51.00 what I see as a principle challenge for the future. 00:27:55.13 We've talked about the cell biology of sporulation, and we've talked about 00:28:00.27 the orchestrated expression of genes under the control 00:28:05.15 of a series of cell-specific transcription factors. 00:28:08.20 These transcription factors are activating over 500 genes 00:28:14.26 in a cell-specific and temporal fashion. And itâ€™s the products of those genes 00:28:21.18 that mediate the morphogenesis that culminates in the spore. 00:28:25.27 So the final challenge is to understand how the myriad proteins 00:28:31.15 produced under the control of sigmaF, E, G and K mediate morphogenesis, drive 00:28:39.07 the assembly of the spore into this remarkable dormant structure that can resist 00:28:45.18 the ravages of time and insults of the environment in such a robust manner. 00:28:52.04 Thank you.