Developmental Biology of a Simple Organism: Bacillus subtilis

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.

Part 2: New Research on Multicellularity

00:00:04.20 Hello, my name is Richard Losick and I'm a professor at Harvard University
00:00:10.11 and this is the second part of a three part presentation on
00:00:14.25 the developmental biology of a simple organism.
00:00:18.18 In this part, I'll be telling you about multicellularity in a bacterium,
00:00:23.09 the spore-forming bacterium Bacillus subtilis.
00:00:27.16 Bacteria have traditionally been thought of as solitary creatures
00:00:31.16 that go about their business on an individual basis
00:00:35.14 but increasingly we've come to recognize that bacteria can form
00:00:38.16 complex, multicellular communities known as biofilms.
00:00:42.21 I'm going to tell you about biofilm formation in Bacillus subtilis
00:00:48.28 where you'll see that the ability to produce a complex community
00:00:54.06 is linked to the process of sporulation.
00:01:00.05 The first topic that I'll cover is the formation of these communities
00:01:04.29 and a visualization of spore formation taking place in aerial structures.
00:01:11.11 Then I'll tell you about how these communities are held together,
00:01:16.04 what's the basis for their architecture?
00:01:18.05 And you'll see that there's an extracellular matrix
00:01:20.17 that glues the cells together, kind of the mortar for building these structures
00:01:25.18 and that the synthesis of this matrix is governed by
00:01:29.19 an intricate regulatory circuit that I'll describe.
00:01:32.24 And then finally, we'll look into a biofilm and we'll see that it resembles a tissue
00:01:40.11 with different kinds of cells in different places and with dynamic changes
00:01:44.22 in which different kinds of cells either grow or diminish
00:01:48.27 in their relative abundance in this tissue-like structure.
00:01:52.17 Well, that B. subtilis was capable of forming architecturally complex communities
00:01:59.29 was missed for many years for the following reason.
00:02:03.10 In the many years since this bacterium was discovered,
00:02:07.17 itâ€™s been worked on in the laboratory and, as a consequence,
00:02:11.20 over time, itâ€™s inadvertently become domesticated.
00:02:14.15 And so the standard laboratory form of bacteria don't form robust, multicellular communities.
00:02:22.18 Itâ€™s as if we've bred this out of the bacterium over time.
00:02:26.03 Here, for example, is a colony of B. subtilis on a plate
00:02:30.07 and you can see it forms a relatively unstructured colony.
00:02:33.23 And here is a culture...a standing culture of cells in which the bacteria
00:02:39.14 have collected as a very thin film at the surface of the culture.
00:02:44.08 But once again, not much architecture there.
00:02:47.03 But now if we go back to a wild strain of the bacterium
00:02:51.06 we see something that's dramatically different.
00:02:53.19 In the colony you can see a rich architecture with thick veins
00:02:58.13 of cells and other kinds of structures
00:03:01.06 and likewise, at the air liquid interface, a thick mat of cells known as a pellicle forms
00:03:09.03 that also has a distinctive architecture with all kinds of detailed features.
00:03:14.20 Let's look more closely at the biofilm structure and in particular
00:03:22.20 I'd like us to focus on the outer edge of the colony where you can see
00:03:27.17 that there are aerial structures that rise up from the surface.
00:03:30.24 I'm going to show you next one of these aerial structures in a cross-section view
00:03:36.22 with cells that harbor a fusion of the LacZ gene for beta-galactosidase
00:03:42.18 to a promoter that's under the control of a sporulation transcription factor.
00:03:47.26 So we'll be able to see where spore formation is taking place in this aerial structure
00:03:53.25 by staining the structure with a chromogenic dye
00:03:56.25 that turns blue when the beta-galactosidase product
00:04:00.19 of the reporter is produced.
00:04:02.21 And as you can see there's a striking, intense blue staining near the tip, the aerial tip
00:04:10.00 of this surface of this structure that rises from the surface.
00:04:14.11 And as I go on, I'll argue later that it might be appropriate to think of these structures
00:04:19.09 as fruiting body-like structures that perhaps have been selected in evolution as...
00:04:25.22 for the purposes of dispersal of spores and I'll further suggest
00:04:30.18 that sporulation in the context of this community
00:04:35.00 depends on the very formation of the biofilm.
00:04:40.11 Let's look even more closely at this aerial structure
00:04:43.00 by scanning EM...by scanning electron microscopy.
00:04:48.02 And what you can see is that the biofilm consists of long chains of cells
00:04:52.17 that are cemented in parallel fashion to each other.
00:04:56.22 Long chains of cells, each one is glued to each other.
00:05:00.22 The glue that holds all these chains of cells together is known as the extracellular matrix.
00:05:08.13 The bacteria export a matrix material that cements the chains of cells together
00:05:15.09 so that the architecture can be built. This matrix consists of a polysaccharide
00:05:21.16 or a so-called exo-polysaccharide and a specific protein.
00:05:26.12 The polysaccharide is produced by enzymes encoded
00:05:30.28 within a large operon known as the eps operon,
00:05:34.06 for exo-polysaccharide operon. And the protein is encoded
00:05:38.15 with an operon that's referred to as the tasA operon.
00:05:42.06 Let's consider how these two operons are turned on under the right circumstances
00:05:50.28 that lead to the formation of the multicellular community.
00:05:53.12 This is mediated by an elaborate regulatory network that involves fully six regulatory proteins.
00:06:01.06 So on the right are the two operons, the one for polysaccharide synthesis
00:06:06.12 and the other for the protein and circled are the six regulatory proteins
00:06:13.07 that govern the expression of these two target operons.
00:06:18.01 This looks bewilderingly complex but, as I hope to show you
00:06:23.00 at its heart it has a simple logic.
00:06:25.24 So to simplify things, first of all let me get rid of the regulatory protein on the right
00:06:31.10 and let's just focus on the remaining five proteins;
00:06:35.00 SinI, SinR, AbbA and AbrB and Spo0A.
00:06:42.06 SinR and AbrB are repressor proteins, they each cross-repress both target operons.
00:06:50.08 So SinR represses the polysaccharide operon and the protein operon.
00:06:55.02 Likewise, AbrB contributes to the repression of the polysaccharide operon and the protein operon.
00:07:01.06 So two different repressors help to hold both of these matrix operons off.
00:07:07.01 In order for the operons to be de-repressed,
00:07:10.18 we need to antagonize the action of these two repressors.
00:07:14.27 So let me first convert the names of those two proteins into repressors,
00:07:20.00 the red repressor and the blue repressor, to make things look simple.
00:07:23.09 And now let's consider the SinI and AbbA protein. What are they doing?
00:07:27.25 Well, SinI is an anti-repressor that binds to its respective repressor to inactivate it.
00:07:36.08 Likewise, AbbA is an anti-repressor that binds to its repressor to inactivate it.
00:07:42.12 So let's convert SinI and AbbA into anti-repressor and anti-repressor.
00:07:50.21 So now you can see that there's a simple logic here
00:07:53.20 in which two parallel pathways of repression and anti-repression govern the expression
00:08:01.23 of the two operons that are responsible for matrix production.
00:08:06.28 This redundancy probably helps to ensure that the matrix genes are held totally silenced
00:08:13.22 until the right time and the right place for a community to form.
00:08:18.07 How does this whole system get going?
00:08:20.28 Well that's the role of the master regulator Spo0A.
00:08:24.12 Spo0A is a master regulator both for sporulation
00:08:29.16 as we saw in my previous presentation on sporulation
00:08:34.17 and itâ€™s also the principle regulatory protein
00:08:37.26 that's responsible for triggering biofilm formation.
00:08:41.14 Spo0A turns on the gene for both anti-repressors:
00:08:46.03 the SinI anti-repressor and the AbbA anti-repressor.
00:08:50.13 So, when Spo0A becomes activated, that leads to the production of two anti-repressors.
00:08:57.03 The two anti-repressors then bind to and inactivate their respective repressors,
00:09:01.22 and that finally leads to de-repression of the two operons that are responsible
00:09:08.02 for matrix production.
00:09:12.03 OK, now let's look in more detail at the biofilm.
00:09:17.08 We've just considered in some detail the regulatory pathway that's responsible
00:09:23.13 for turning on the synthesis of the matrix.
00:09:26.25 So some of the cells in the biofilm, at least, are responsible for matrix production.
00:09:32.01 But now let's consider what other kinds of cells might be present in the biofilm.
00:09:36.29 As we saw earlier, spore formation is also taking place in the biofilm.
00:09:42.03 And there are also vegetative cells that are capable
00:09:44.24 of motility that are present in the biofilm.
00:09:47.17 So, what we're going to do is take one of these biofilms, cut it in half,
00:09:52.17 and then look at it from the side by confocal microscopy using three different fluorescence reporters:
00:10:02.28 one to a gene under sporulation control; another under the control of the matrix producing pathway;
00:10:12.03 and a third under the control of genes involved in motility.
00:10:17.09 So each of these reporters represent three distinct kinds of cells:
00:10:22.28 sporulating cells; matrix producing cells; and motile cells, motile, vegetative cells.
00:10:29.10 OK, so if you look now at the bottom you can see that matrix producing cells in red
00:10:39.03 and sporulating cells in green have distinct locations.
00:10:43.09 They occupy distinct positions in the biofilm. The sporulating cells are near the top
00:10:48.26 as we saw in that fruiting body-like structure
00:10:51.25 in the light micrograph that I showed you at the beginning.
00:10:56.08 And the matrix-producing cells are underneath.
00:10:59.22 Now let's consider the position of motile cells.
00:11:03.01 The sporulating cells for comparison, once again, are near the extreme top and the matrix...
00:11:09.27 the motile cells are near the extreme bottom.
00:11:12.22 So motile cells, matrix cells and sporulating cells occupy three different regions of the biofilm;
00:11:20.23 with the motile cells near the bottom, the matrix-producing cells in the middle, and
00:11:25.11 the spore-forming cells near the top. So we can begin to think of the biofilm as
00:11:31.03 a kind of tissue that's composed of different kinds of cells that
00:11:35.29 occupy different positions in that tissue.
00:11:40.04 But this is a dynamic tissue because the relative proportion
00:11:43.27 of these different kinds of cells changes over time.
00:11:47.10 And I can illustrate that for you with a simple experiment
00:11:51.27 in which we take these biofilms harboring these fluorescent reporters
00:11:56.16 and separate all the cells from one another, disassemble the matrix
00:12:00.08 so that the cells are separated and then we use a fluorescence activated cell sorter
00:12:05.22 to measure the relative proportion of the three cell types over time.
00:12:10.20 Let's first consider sporulating cells. So the arrow marks increasing time up to 72 hours,
00:12:19.21 and the axis that runs from left to right represents increasing fluorescence.
00:12:25.23 As you can see, sporulating cells appear as a distinct population of high fluorescence
00:12:33.22 only at about 48 hours into the process.
00:12:37.00 If we look instead at matrix-producing cells, a peak of matrix producing cells
00:12:42.01 appears between 12 and 24 hours and then diminishes somewhat over time.
00:12:48.03 Then, finally, let's consider the motile cells.
00:12:52.16 The motile cells are most abundant at the beginning
00:12:56.02 and then they gradually diminish over time and become less and less
00:13:00.20 abundant by 72 hours into the process.
00:13:05.20 So gene expression is dynamic in the biofilm.
00:13:09.27 Cells occupy distinct positions.
00:13:12.11 Cells of different types and their relative abundance in the biofilm changes over time.
00:13:19.11 Now, finally, let me come to what I think might be the most interesting finding
00:13:25.00 concerning having more than one cell type.
00:13:27.15 As we've seen, spores are produced near the top of aerial structures.
00:13:31.29 And itâ€™s appealing to imagine that perhaps these are primitive fruiting body-like structures
00:13:38.27 that perhaps have a role in spore dispersal.
00:13:43.12 Well, is the process of spore formation not only associated with
00:13:49.26 multicellularity, is it actually dependent upon it?
00:13:53.06 And we can ask that question by looking at sporulation gene expression
00:13:58.16 or the process of sporulation both in a wild-type biofilm
00:14:03.12 and a biofilm that's mutant for matrix production.
00:14:07.09 Actually, a mutant that can't make a normal biofilm.
00:14:11.01 And then we can use the fluorescence activated cell sorter to measure the
00:14:15.13 proportion of sporulating cells in the wild-type and in the mutant.
00:14:20.04 And that's shown in this slide here. You can see that in the wild-type case in blue
00:14:25.09 there's a distinct peak of cells, a distinct sub-population of cells
00:14:31.09 that are undergoing sporulation, that are expressing sporulation genes.
00:14:35.28 But when we look at the expression of the same sporulation gene in a matrix mutant,
00:14:42.13 well, that peak almost completely disappears.
00:14:44.20 In other words, in the context of the biofilm, spore formation
00:14:49.15 is substantially dependent on the formation of an architecturally complex community,
00:14:55.26 on the production of the matrix and the formation of these structures.
00:14:59.22 So, that plus the earlier result that making a biofilm, like sporulation,
00:15:06.27 are both under the control of the same master regulator, Spo0A
00:15:11.04 leads us to speculate that this has an important biological significance.
00:15:16.09 And that in nature, spores are formed not in the form of individual cells on their own
00:15:22.04 at least not all the time but are sometimes produced in the context of
00:15:26.07 complex multicellular communities in which sporulation
00:15:29.15 is itself coupled to the process of assembling the community.
00:15:34.19 Lastly, let me point to the future on an important challenge that awaits us
00:15:41.03 in the years ahead. Even though we think we understand quite a bit
00:15:46.19 about the regulatory network that governs the production of the matrix
00:15:50.23 we're a long way from understanding how matrix-producing cells assemble
00:15:57.15 macroscopically into the elaborate architectures that I've been showing you.
00:16:01.19 Well, this issue comes into even sharper relief when you consider the following finding.
00:16:08.24 My collaborators and I have been collecting wild strains of Bacillus from around the world.
00:16:14.03 And the striking finding is that frequently these different wild strains
00:16:19.11 each exhibit their own distinctive architecture.
00:16:23.10 You can see that in this slide.
00:16:25.11 Here is a collection of different, very closely related strains of Bacillus subtilis
00:16:31.27 yet each one exhibits its own distinctive architecture.
00:16:36.02 Consider this one at the top or this one over here, or this one down near the lower left.
00:16:43.08 They have their own distinct morphotype.
00:16:46.01 Surely, this must be dictated by genetic differences between these two strains,
00:16:52.22 unknown genetic differences and in principle it should be possible to identify
00:16:57.25 the genetic differences between these strains that give rise to these different morphotypes
00:17:02.27 and thereby obtain a clue into the larger challenge of understanding
00:17:08.07 how morphogenesis is controlled, how a multicellular community with a distinctive
00:17:14.06 architecture is created by the expression of genes involved in the formation of the biofilm.
00:17:23.19 Finally, itâ€™s important for me to emphasize that all of the work that I've told you about
00:17:29.18 has been a wonderful collaboration between my laboratory
00:17:33.02 and that of my good friend Roberto Kolter at the Harvard Medical School
00:17:36.20 on the Cambridge side of the Charles River.
00:17:42.10 The individuals that have been involved in this story
00:17:46.09 that I've told you are Dan Kearns, Win Chai, Frances Chu and Anna McLoon.
00:17:51.09 And on the Harvard Medical School side of the Charles River in the Kolter lab
00:17:57.22 the individuals who have driven this project forward are Steve Branda, Dani Lopez,
00:18:01.29 Claudio Aguilar, Hera Vlamakis and Ashlee Earl.
00:18:06.23 Thank you very much.

Part 3: Stochasticity and Cell Fate

00:00:06.06 Hello, my name is Richard Losick
00:00:08.19 and this is the third part and last part of my presentation on
00:00:14.03 developmental biology of a simple organism.
00:00:17.10 This last part is devoted to the topic of stochasticity and cell fate.
00:00:23.04 I grew up in an era in which it was believed that development, biological development
00:00:30.20 is orchestrated by highly deterministic processes.
00:00:34.23 And indeed, that's largely the case. That's true in most cases.
00:00:39.23 But, increasingly, we're seeing that there are examples, especially in the microbial world
00:00:45.01 of cell fate decisions that are stochastic.
00:00:51.18 I'm going to tell you about a series of such examples from the spore forming
00:00:56.28 bacterium Bacillus subtilis. Let me begin with a famous quote
00:01:02.00 from Albert Einstein, the father of modern physics.
00:01:05.20 Einstein famously said that "I, at any rate, am convinced
00:01:10.10 that He does not play dice."
00:01:12.08 Thereby rejecting Heisenberg and his Uncertainty Principle.
00:01:17.21 Well, in biology, as I've said, most decisions are, indeed, highly deterministic.
00:01:26.01 But, itâ€™s also the case that some decisions, some cell fate decisions,
00:01:32.12 are, in fact, done by a role of the dice, as I'll explain.
00:01:37.21 So, I'm going to give you four examples from Bacillus subtilis
00:01:40.28 which, so far, appears to be the champion of stochasticity in the microbial world.
00:01:46.05 The four examples are: growth and competence, swimming versus chaining,
00:01:51.25 eating versus being eaten, and community versus individuality.
00:01:59.25 What do I mean by growth versus competence?
00:02:03.03 Well, B. subtilis can switch into an alternative state,
00:02:07.19 the state of competence, in which it stops growing and instead, acquires
00:02:12.29 the ability to take up DNA from its environment
00:02:15.20 which it can recombine into the chromosome.
00:02:21.13 Why would B. subtilis do such a thing if this means stopping to grow?
00:02:25.04 Well, what one imagines is that producing cells
00:02:30.13 that are able...that are on the prowl for
00:02:33.00 new genetic sequences improves the fitness of B. subtilis by allowing
00:02:38.21 it to uptake new genetic information that may help it cope
00:02:43.23 with changed circumstances in the future. So there must be a fitness benefit
00:02:48.15 to the bacterium for temporarily entering this non-growing state
00:02:52.21 so that it can be always on the look out for
00:02:55.28 potential new genetic sequences that can be useful to it.
00:03:00.16 Entry into this state of competence is controlled by a transcription factor
00:03:05.11 called ComK and as you'll see, the synthesis of ComK
00:03:10.22 is governed by a noise-driven stochastic switch. So in other words,
00:03:15.11 when cells are under conditions in which they're capable of entering the
00:03:20.08 competent state, only some of them do and the ones that decide
00:03:23.12 to do so, do so in a stochastic fashion.
00:03:26.17 Let me illustrate this to you with a beautiful experiment
00:03:31.02 from David Dubnau, who, along with others in the field are
00:03:34.06 responsible for our understanding of stochasticity in the competent state.
00:03:43.00 So what I'm going to show you is a field of cells in which all of the cells
00:03:46.10 harbor a fusion of the gene for the green fluorescence protein
00:03:50.09 to a promoter under the control of ComK.
00:03:54.09 And what you can immediately see is that only a subset of the cells
00:03:58.11 are brightly green. That is, most cells are off for ComK
00:04:03.25 and some cells are on for ComK.
00:04:07.23 These cells were grown and held in a homogeneous environment.
00:04:12.03 All of them, in principle, are capable of becoming competent,
00:04:16.25 of activating ComK but only some of them do so
00:04:20.15 and they do so in a random fashion, independently of
00:04:26.07 what the neighboring cells are doing.
00:04:28.25 How does this work?
00:04:30.07 So at the heart of this system is the following circuitry.
00:04:35.12 The comK gene, of course, encodes the ComK regulatory protein
00:04:40.04 which can bind to and activate the transcription of about 100 target genes
00:04:44.14 that define the state of competence.
00:04:48.03 But ComK also bind to the promoter for its own gene
00:04:52.18 in which case it sets up a positive feedback loop
00:04:56.18 which can stimulate transcription of its own genes.
00:04:59.26 So when ComK binds there, that leads to more transcription of comK
00:05:04.03 that results in yet more transcription which in turn leads to yet more ComK molecules
00:05:11.00 that lead to this on state in which large levels of ComK accumulate in the cell.
00:05:17.14 The key point is that this positive feedback loop has a threshold.
00:05:22.28 You can think of it as being poised on a knife edge.
00:05:26.10 And under the right conditions the cells have just less than a threshold
00:05:31.17 amount of ComK molecules in them and if, due to noise,
00:05:37.18 there are fluctuations in the amount of ComK from cell to cell then
00:05:43.21 some cells will have a bit more ComK than other cells.
00:05:47.18 Those cells that have a bit more have reached the threshold
00:05:51.13 and get the positive feedback loop going. Those that are below
00:05:55.20 the threshold can't get the positive feedback going.
00:05:59.08 And what makes this switch a bi-stable switch
00:06:03.10 is that multiple ComK molecules bind to the promoter in a cooperative fashion
00:06:11.05 by interacting with each other. This makes the switch highly sensitive
00:06:16.07 to small fluctuations in the level of ComK molecules.
00:06:20.24 So, when the amount of ComK in a cell is just below the threshold,
00:06:25.13 most cells will not activate ComK, but a few cells will have,
00:06:31.06 by noise driven processes, accumulated enough ComK molecules
00:06:36.19 to activate the positive feedback loop and get it going
00:06:39.27 and go into the competence on state.
00:06:42.12 Why does B. subtilis do this?
00:06:44.24 Well, we don't know for sure but, obviously, entering a state in which you're not
00:06:49.29 growing puts you at a disadvantage. But by deploying, stochastically, some cells
00:06:56.15 that are on the prowl for new genetic information, then B. subtilis is always
00:07:02.03 preparing itself for unexpected changes in its environment
00:07:06.07 when new kinds of genetic information may be important.
00:07:11.16 So, we can think of this as an example of bet hedging.
00:07:15.09 That is, B. subtilis is hedging its bets by deploying, stochastically,
00:07:20.02 a small proportion of cells that enter a non-growing state temporarily
00:07:25.05 so that should circumstances change
00:07:29.18 and should the right genetic sequences appear
00:07:32.14 then those cells will be at an advantage.
00:07:34.17 Remember, that evolution selects for the genome and not the individual.
00:07:39.00 So, deploying two kinds of cells in the population
00:07:42.02 can be advantageous to the genome
00:07:44.16 even if itâ€™s not advantageous to the individual.
00:07:47.19 Let me come to my second example, motility versus chaining.
00:07:52.26 This is a phase contrast micrograph that depicts B. subtilis cells as
00:07:58.04 we've traditionally seen them over many decades of research with this organism.
00:08:05.16 And if you look closely you can see there are two kinds of cells here.
00:08:09.03 There are long chains of cells that have completed cell division
00:08:12.01 but haven't separated from each other and there are also singlets and doublets.
00:08:17.17 The singlets and doublets, it turns out, are motile cells,
00:08:20.17 where as the long chains are non-motile cells. They're sessile cells, if you will.
00:08:26.03 Well, we saw this image for many, many years but didn't pay much attention to it.
00:08:33.01 But over time it emerged that a single transcription factor
00:08:36.07 called sigmaD is responsible for the production of enzymes that
00:08:42.29 degrade the cell wall material between newly divided cells,
00:08:46.25 enabling them to separate and also for the production of the machinery
00:08:51.18 that's responsible for motility.
00:08:55.16 So, with that in mind we revisited this field of exponential phase cells
00:09:02.08 but this time using cells that were tagged with green fluorescence protein reporter
00:09:07.21 gene fused to a promoter under the control sigmaD.
00:09:12.17 And at the same time we stained the cells with a red membrane dye
00:09:16.22 so that we could see the division septa.
00:09:18.25 And now, all of a sudden, we get a radically different view of
00:09:23.11 the field of cells that's very illuminating.
00:09:26.08 As you can see there are cells in two states.
00:09:30.04 There are sigmaD on cells that are doublets or singlets
00:09:34.28 and these are the motile cells. They undergo cell division
00:09:38.26 and then the products of cell division can separate from each other.
00:09:43.02 And then there are the sigmaD off cells which we see as long
00:09:47.06 red membrane staining cells. You can see the division septa in these cells.
00:09:51.19 The cells have divided but the daughter cells haven't separated from each other.
00:09:55.22 These cells are off for sigmaD and they're non-motile.
00:10:00.13 Why would B. subtilis do this?
00:10:02.03 Well, of course we don't know but itâ€™s attractive to imagine
00:10:05.26 itâ€™s another example of bet hedging.
00:10:09.04 Imagine that B. subtilis is in a particular niche where there are nutrients.
00:10:13.04 The sessile chains of cells can stay put and exploit the existing niche.
00:10:21.07 But the motile cells, the sigmaD on cells, we can think of those
00:10:25.11 as nomadic cells that wander off to look few niches.
00:10:29.28 B. subtilis doesn't know what the future holds in store and so its immediate niche
00:10:34.14 may run out of nutrients or exhibit other adverse environmental factors.
00:10:41.04 So, by deploying some cells to be motile and some cells to be sessile
00:10:46.06 the bacterium can hedge its bets. Some cells stay put
00:10:50.21 and exploit existing circumstances whereas other cells swim off
00:10:56.00 looking for new niches in anticipation of the possibility that the original
00:11:02.15 niche may become exhausted.
00:11:06.12 My third example is called eating versus being eaten.
00:11:11.13 So, as we saw in the first part of my presentation, when starved for nutrients
00:11:16.29 B. subtilis enters the pathway to sporulate.
00:11:20.26 So just as competence represents a distinct state,
00:11:24.00 sporulation represents a specialized developmental state.
00:11:28.13 Entry into sporulation is governed by a master regulatory protein called Spo0A.
00:11:35.01 Sporulation is a complex process that takes multiple hours.
00:11:40.17 It takes time and it takes energy,
00:11:43.16 and it culminates in the formation of a dormant cell type,
00:11:47.28 the spore that can remain dormant for many years.
00:11:50.22 So I think itâ€™s easy to imagine that the decision to sporulate
00:11:54.11 is not one that B. subtilis wants to take lightly.
00:11:58.01 Entry into this pathway is governed by Spo0A
00:12:01.18 which sits at the top of this regulatory sequence.
00:12:06.04 It becomes activated under conditions in which nutrients become limiting.
00:12:12.28 Well, there is a crucial window of time near the start of sporulation
00:12:17.25 when the cells can change their minds, so to speak.
00:12:22.17 Initially, when Spo0A become active but before the hallmark process
00:12:28.16 of asymmetric division takes place, if nutrients reappear, sporulation is arrested
00:12:34.16 and the cells can even start growing again.
00:12:37.21 But once the cells cross the Rubicon, so to speak, of asymmetric division
00:12:42.25 now, they're committed to making a spore,
00:12:44.18 even if lots of nutrients appear at later stages.
00:12:48.18 So there's this window of time up until asymmetric division takes place
00:12:54.05 when the process is reversible,
00:12:56.14 and then it becomes irreversible later on.
00:12:59.15 So hold that thought in mind when I tell you what I
00:13:02.29 think at this point will not come as a surprise, that Spo0A
00:13:06.21 is itself subject to a bi-stable switch.
00:13:11.01 That is, when cells are limited for nutrients, only some of the cells become on for Spo0A
00:13:18.02 whereas others remain off for Spo0A.
00:13:22.01 And once again, we can visualize this by using a green fluorescence protein gene
00:13:27.07 fusion to a promoter under the control Spo0A.
00:13:31.10 And as you can see there are two kinds of cells:
00:13:33.19 those in which Spo0A is off and those in which Spo0A is on.
00:13:38.20 Only some of the cells are on for Spo0A.
00:13:41.24 Well, why have a bi-stable switch? Why is Spo0A subject to a bi-stable switch?
00:13:49.00 Well we believe that the answer has to do with a phenomenon
00:13:52.18 that we refer to as cannibalism.
00:13:55.22 When the cells are deprived of nutrients
00:13:59.16 and Spo0A becomes activated, the Spo0A on cells produce and export
00:14:05.15 a toxin, a peptide toxin that kills their sibling cells, the cells that are off for Spo0A.
00:14:13.07 The producing cells, the on cells are immune to the toxin
00:14:16.20 but the non-sporulating cells are killed by it. They lyse and liberate their nutrients.
00:14:22.23 Now, remember, Spo0A activation is triggered by nutrient limitation in the first place.
00:14:28.19 So, if some of the cells are lysing and liberating nutrients
00:14:30.26 that will have the affect of impeding Spo0A activation
00:14:36.08 and that will arrest sporulation or perhaps even reverse it.
00:14:39.17 So, cannibalism is a process for slowing down sporulation.
00:14:44.26 Let me illustrate that for you with this single agar plate experiment.
00:14:52.08 So the left side of the slide shows a streak of wild type cells
00:14:58.08 and the right hand portion of the slide shows a cannibalism mutant.
00:15:05.00 When cells start to sporulate, the colonies become white and opaque
00:15:09.27 and so we can see that with the naked eye.
00:15:12.28 And as you can see at the early time when this photograph was taken
00:15:16.26 the wild type cells have only just begun to sporulate.
00:15:21.13 The colonies are not yet white and opaque.
00:15:23.11 But the mutant cells are filled with spores.
00:15:28.04 Now remember, cannibalism is a process for delaying sporulation.
00:15:32.12 So, therefore, in its absence sporulation is accelerated.
00:15:37.29 So we see rapid sporulation in a cannibalism mutant
00:15:42.20 and slow sporulation in the wild type.
00:15:45.03 Once again we can ask, "Why would B. subtilis do such a thing?"
00:15:50.17 And the appealing interpretation is, yet once again, itâ€™s hedging its bets.
00:15:55.27 Consider a population of B. subtilis cells that
00:15:59.18 experiences a drop in the availability of nutrients.
00:16:02.23 How does it know whether this decrease in nutrients is a simple
00:16:07.21 fluctuation, a temporary decrease in nutrients or the beginning of a famine?
00:16:13.07 If it willy-nilly committed itself to making a spore when nutrients were depleted
00:16:21.08 and went through this multi-hour, expensive process
00:16:24.12 and nutrients actually returned after the time of
00:16:28.21 commitment, well, it would put itself
00:16:30.25 at a disadvantage relative to other bacteria that would be simple waiting out
00:16:35.17 the period of low nutrients.
00:16:39.00 On the other hand, if itâ€™s in a period of prolonged starvation,
00:16:42.03 then going ahead to make spores makes good sense.
00:16:46.28 So, cannibalism is a way to stall for as long as possible
00:16:51.01 before crossing the Rubicon, before committing to spore formation
00:16:56.22 even at the expense of committing fratricide, killing and feeding on
00:17:01.07 genetically identical sibling cells.
00:17:05.19 Finally, I come to the example of individuality versus community.
00:17:11.19 This represents work done in collaboration with Roberto Kolter
00:17:16.21 and concerns the topic of multicellularity in biofilm formation
00:17:21.12 which was the subject of the second part of my presentation.
00:17:26.08 So, wild strains of B. subtilis can make architecturally complex communities.
00:17:31.24 In a standing culture, these communities form at the air liquid interface
00:17:37.22 in a structure known as the pellicle, that has an elaborate and distinctive architecture.
00:17:43.04 And on colonies, on solid medium, we also see an elaborate architecture
00:17:48.08 with thick veins and aerial structures.
00:17:51.25 The cells in these communities are held together
00:17:54.15 by an extracellular matrix, kind of a cement, that holds long chains
00:18:00.15 of cells together so that the architecture can be built.
00:18:05.08 This matrix consists of two components: a polysaccharide and a protein component
00:18:11.05 that are exported from the cells.
00:18:15.01 The matrix is subject to intricate regulation for its production.
00:18:20.02 And I've summarized, in a simplified form, the regulatory pathway
00:18:25.20 by which the matrix is produced.
00:18:29.12 But, most proximal to the genes for the matrix is
00:18:32.02 a repressor protein that holds them inactive.
00:18:35.20 The repressor protein is inactivated by another protein that we call an anti-repressor.
00:18:41.12 And finally, the anti-repressor is produced under the control
00:18:45.27 of our good friend Spo0A. And you will recall that Spo0A
00:18:50.14 is subject to a bi-stable switch.
00:18:54.18 Well, this predicts that if we look in cells that are about to form
00:18:59.15 a biofilm, we'll see the repressor being produced in all of the cells
00:19:04.27 but the anti-repressor, which is under the control Spo0A
00:19:08.15 will be produced in only a subset of the cells.
00:19:12.04 Let's look. First, I'll show you a field of cells that has
00:19:16.10 a green fluorescence protein gene fusion to the repressor gene.
00:19:20.08 And you can see, more or less, all of the cells are green.
00:19:25.06 All of them are producing repressor.
00:19:27.22 Now let's look at a comparable field of cells but this time
00:19:30.15 the green fluorescence protein gene is fused to the anti-repressor gene.
00:19:35.04 And now we get a radically different picture.
00:19:38.25 Only some of the cells, a minority of the cells are on for anti-repressor production.
00:19:45.18 That is, they're on for Spo0A and therefore, on for anti-repressor.
00:19:50.03 Hence, they're inactivating the repressor. Hence, these are the matrix producing cells.
00:19:54.18 And from this we conclude that some cells make matrix for the entire community.
00:20:00.02 This is a kind of altruism in which some cells are dedicated to making
00:20:05.12 matrix for the entire community of cells
00:20:10.13 and the other cells specializing in other directions.
00:20:17.05 OK, so, I've given you four examples from a bacterium,
00:20:20.21 a single bacterium in which cell choices are made in a stochastic manner.
00:20:26.15 But I don't want to leave you with the impression that stochasticity is unique to bacteria.
00:20:32.29 I'd like, in closing, to consider the case of the mouse olfactory neuron
00:20:37.25 and the eye of the fly which provide two examples of stochasticity
00:20:43.10 in complex metazoans.
00:20:46.16 So, the mouse devotes a great deal of its genetic material to the process of smell.
00:20:55.04 Fully 4% of its genes encode receptors, membrane receptors for odorants.
00:21:03.06 There are about 2000 such genes in the chromosomes of the mouse,
00:21:08.04 in the diploid mouse and...but itâ€™s the case that any given neuron
00:21:15.09 must express only a single receptor.
00:21:20.08 Otherwise, the mouse would be confused as to what odor it was sensing.
00:21:24.00 So, how does this work?
00:21:30.00 Here, in a cartoon form, is depicted a neuron that's expressing
00:21:36.06 a particular odorant receptor depicted in red.
00:21:40.18 Itâ€™s on and all of the other 999 odorant receptor genes on one haploid set
00:21:49.10 and the homolog on the homologous chromosome are off.
00:21:54.11 This cell expresses only one out of 2000 genes.
00:21:57.25 Now, how does this work?
00:21:59.07 Well, you could imagine, I suppose, a very complicated regulatory network
00:22:05.08 that was special for every neuron that ensured that only one
00:22:09.23 out of 2000 genes was turned on.
00:22:11.24 But, that would be so complicated itâ€™s hard even to imagine how it would work.
00:22:16.14 Instead, the mouse has evolved a very elegant strategy.
00:22:21.17 It turns on, in any given neuron a single receptor gene stochastically.
00:22:26.10 Each neuron throws a roll of the dice to decide which receptor gene to turn on
00:22:34.09 and then, by mechanisms that are not yet fully clear, all other genes in that neuron,
00:22:40.07 all other odorant receptor genes, are prevented from being expressed.
00:22:45.26 My last example, concerns the eye of the fly.
00:22:49.26 The eye of the fly is a compound eye. Flies don't have the simple eyes
00:22:55.02 that we have. They have many eyes as do other insects.
00:22:58.08 These compound eyes consist of many clusters of light sensitive cells
00:23:04.06 called ommatidia. Each of these ommatidia
00:23:07.12 can produce either of two color sensitive rhodopsins called rh5 or rh6.
00:23:15.13 So the eye is a field of many ommatidia and each of these ommatidia
00:23:21.07 switch on either the blue rh5 or the green rh6.
00:23:28.06 And they do so, they make this choice stochastically.
00:23:32.15 This is the work of Claude Desplan and I illustrate it to you
00:23:37.08 with this marvelous image from Desplan in which
00:23:40.24 you'll see a field of ommatidia in which some cells are producing the green rhodopsin
00:23:48.08 and others are producing the blue rhodopsin.
00:23:52.05 And if you stare at this image for a while you'll see that there's no consistent pattern.
00:23:56.25 It's stochastic. Itâ€™s not a simple flip of coin.
00:24:00.29 Itâ€™s not fifty-fifty. Itâ€™s a biased stochastic switch.
00:24:04.12 Itâ€™s biased in favor of the green choice in a ratio of about 70 to 30,
00:24:10.16 but any individual ommatidium is making its choice randomly, stochastically.
00:24:20.14 Such that, on average, the average decision is a ratio 70 to 30.
00:24:25.15 And if you look closely at this you'll see no consistent pattern
00:24:29.04 and if you look a the other eye of the fly you would see a different pattern.
00:24:32.21 If you looked at other flies you would see yet other patterns.
00:24:35.18 So the choice is stochastic.
00:24:38.21 So, in conclusion, we can say that nature does, indeed, know how to
00:24:44.20 make deterministic decisions, but, in contrast to Einstein's view of the universe,
00:24:50.07 she also knows how to leave certain decisions to a roll of the dice.
00:24:55.19 Thank you very much.

Videos in this Talk

Part 1: Spore Formation in B. Subtilis
Audience:
- Student
- Researcher
- Educators of H. School / Intro Undergrad
- Educators of Adv. Undergrad / Grad
Part 2: New Research on Multicellularity
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad
Part 3: Stochasticity and Cell Fate
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad

Speaker: Richard Losick

Total Duration: 1:12:13

Recorded: April 2008

Educator Resources for this Talk

All Talks in Microbiology

Talk Overview

How do simple cells differentiate, assemble into communities, and cope with change? Losick’s seminar addresses these questions in the spore-forming bacterium Bacillus subtilis.

Part I is an overview of how B. subtilis makes a spore.

Part II presents research on the capacity of B. subtilis cells to form architecturally complex communities.

Part III presents research showing that B. subtilis uses a bet hedging strategy for coping with uncertainty.

Speaker Bio

Richard Losick

Richard Losick is a Howard Hughes Medical Institute Professor, the Maria Moors Cabot Professor of Biology, and a Harvard College Professor. He received his B.A. from Princeton University in 1965 and his Ph.D. from MIT in 1969. He was elected to the Harvard Society of Fellows as a Junior Fellow in 1969, and he joined… Continue Reading