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Hello, my name is Richard Losick and I'm a professor at Harvard University.
The title of my presentation is "Developmental biology of a simple organism."
We generally think of biological development in the context of complex multicellular organisms.
But, even the most primitive kinds of cells can also exhibit dramatic processes
of cellular differentiation and morphogenesis. I'm going to tell you one such example,
with the spore forming bacterium Bacillus subtilis.
Let me introduce Bacillus subtilis to you in the way it was introduced
to the academic community in 1877 by its discoverer Ferdinand Cohn.
Ferdinand Cohn published his findings in the Biology of Plants and he drew what he saw
in this marvelous plate that you see here. And what's apparent in the plate
is that he saw long chains of cells that had tiny ovoid bodies in them
which he recognized as bacterial spores. So this marked the discovery
of spore formation by a bacterium.
You should also know that this benign bacterium Bacillus subtilis has an evil cousin.
It's the anthrax causing pathogen Bacillus anthracis which was discovered
by Robert Koch at about the same time as Cohn. And Cohn and Koch
agreed to publish their findings back to back in the Biology of Plants and, in fact, this cartoon
of drawings contains contributions of Koch for Bacillus anthracis
as well as for Cohn for Bacillus subtilis.
I've divided my presentation into three parts. The first part concerns the process,
the overall process by which B. subtilis makes a spore.
The second part is new research on multicellularity. We traditionally have thought
of Bacillus subtilis and many other bacteria as being solitary creatures
that go about their business as individual cells, but now we've come to appreciate that
Bacillus subtilis makes elaborate multicellular communities and that
spore formation takes place in these communities.
The last part is the topic of stochasticity and cell fate.
It's generally believed that cell fate decisions
in developmental biology are highly determined.
And indeed they are. But increasingly we've begun to see examples in which
decisions about cell fate are made in a stochastic fashion
and that will be the topic of my last presentation
which will have four examples from Bacillus subtilis.
OK, so to begin this part of my talk:
Bacillus subtilis makes a spore. How does it do it?
So there are three main topics: The first is that spore formation involves two cells.
So this is really a tale of two cells. You'll see that two cells collaborate in making a single spore.
Having created two cells, each cell has its own fate,
follows its own distinct program of gene expression,
and this is governed by a series of transcription factors that act in a cell specific fashion.
The final part of my talk concerns how the two cells talk to each other.
The two cells are not completely independent, even though they follow
their own distinct programs of gene expression, but rather they talk back and forth
at each stage of development to keep the two processes in coordination with each other.
Here in cartoon form are the principle stages of spore formation.
Spore formation is triggered by nutrient limitation and that results in the cells entering a pathway
that involves the formation of two different cells. At the very start we have a single cell,
and I'll refer to that as the pre-divisional sporangium.
Then that pre-divisional sporangium undergoes
a conspicuously asymmetric process of cell division in which a division septum is
formed near an extreme pole of the cell. That divides the developing cell into
two cells; a forespore cell, the smaller cell, and a larger mother cell.
The forespore is destined to become the spore,
whereas the mother cell nurtures the developing spore
and eventually liberates the mature spore. At this early stage of development,
the forespore and the mother cell lie side by side. But later in development,
in a remarkable cell biological process, the mother cell swallows up the forespore,
in a process that resembles phagocytosis in higher cells,
fully engulfing the forespore and pinching
it off as a free cell in the mother cell cytoplasm to create a cell within a cell.
So that inner cell will become the spore and the outer mother cell nurtures the spore and
then eventually will liberate the spore by lysing.
Here are fluorescence micrographs of cells at these various stages of sporulation.
The cells have been stained with a membrane dye
to highlight the features that I've been speaking about.
So, at the stage of asymmetric division, you can see that a polar septum is formed
at an extreme polar position in the sporangium. Next, the mother cell membrane
starts to migrate around the forespore, eventually fully engulfing it,
and then pinching it off as a free cell in the sporangium.
So, how does this process of asymmetric division take place?
Well, bacteria divide by means of a tubulin-like protein called FtsZ
which forms a cytokinetic ring known as the Z-ring.
Higher cells rely on actin, bacteria rely on tubulin.
Here is a fluorescence micrograph showing the Z-ring,
which has been tagged with the green fluorescence protein.
It's in the center of the cells and it's at the future site of cell division in a vegetatively growing cell.
So, when cells are growing, a Z-ring forms in the middle
and it's then converted into a division septum
to give rise to two equal sized daughter cells. But when cells enter the pathway to sporulate,
what happens is that two Z-rings form. One near each pole of the sporangium.
And then only one of these two Z-rings gets converted into a division septum
whereas the other one is disassembled.
So that at the end of the process we have a single polar septum
that's created the two unequal size cells.
Now, immediately this raises interesting issues about how
each of these two cells acquire a chromosome.
The pre-divisional sporangium has two chromosomes and the challenge for the developing cell
is to ensure that the forespore and the mother cell each inherit a complete chromosome.
Sporulating B. subtilis goes about this process in a fascinating way
that differs from almost all other cell types that we know about.
Here is a cartoon that illustrates what the chromosomes look like in a growing cell.
They're in two masses referred to as nucleoids,
and each chromosome, of course, has an origin of replication
and the origins are at the outer edges of the two DNA masses.
When the cells enter the pathway to sporulate, those two DNA masses get remodeled into
a filament, known as the axial filament, that extends across the cell
from pole to pole with each of the two origins at the extreme opposite poles of the sporangium.
You can see this in this fluorescence micrograph, in which the DNA in this slide
is labeled in green and the outline of the cell in red.
In the growing cell you can see two distinct DNA masses
and in the cell that's begun to sporulate you can see that the DNA is
elongated and is extending all the way across the cell.
And the interpretive cartoon shows that the origins are at
the extreme opposite poles of this axial filament.
How does this happen? Well, this process of remodeling the chromosome into a filament and
anchoring it at the poles is mediated by two proteins.
They're called RacA and DivIVA. RacA binds to the DNA at multiple sites to collapse it
but especially around the origin to create a kind of Velcro that will stick it
to DivIVA, a protein which is anchored at the poles of the cell.
So you can see that in this cartoon. DivIVA is at opposite poles of the cell.
Here are the two DNA masses.
When RacA appears, it binds at diverse sites around the two chromosomes
to help collapse it, but forms a structure with many RacA molecules
at the origin regions. And as depicted in this cartoon, the chromosomes get stretched out
and adhere to the poles. Let me show that to you one more time. Here's the RacA protein,
and here's the process by which it gets pulled to opposite poles of the sporangium.
Now, finally, asymmetric division takes place.
But you'll immediately appreciate that when the division septum comes down,
because of its extreme polar placement,
only some of the chromosome destined to the forespore
will be in the little cell. So you can see this in these fluorescence micrographs.
Just after the division septum forms, only a little bit of DNA is in the forespore,
that which was trapped by virtue of being anchored at the pole. But then over time
the remainder of that chromosome gets pumped into the forespore compartment
until a complete chromosome is present in the forespore. This pumping
of a chromosome into the forespore is mediated by a molecular machine,
a protein called the DNA translocase,
which is located in the septum and uses energy from ATP
to pump the remaining portion of the chromosome into the forespore compartment.
So, most cells, or almost all cells that we know about separate their chromosomes prior
to cytokinesis. But, in sporulating cells, cytokinesis takes place before chromosome segregation.
We can visualize the DNA translocase that mediates this chromosome segregation by
tagging it with the green fluorescence protein shown in this cartoon.
So here's a sporangium, and you can see the polar septum
and this bright focus of green fluorescence
from the DNA translocase that's sitting in between
the mother cell and the forespore and is poised to pump DNA
across the septum into the small chamber
of the sporangium. So to review everything that I've said so far
in the very first stage of asymmetric division, the Z-protein is remodeled to form rings
at each pole of the sporangium. Then, one of these two Z-rings is converted into a division septum
and the other Z-ring is disassembled.
Next, chromosomes need to be segregated into the two cells.
And so while the Z-rings are forming, the two chromosomes are remodeled
into an axial filament by the RacA protein, which causes them to collapse
into elongated filament and anchors the origins at the poles,
where the DivIVA protein is present.
Then asymmetric division takes place, and the DNA translocase located in the division septum
pumps the remainder of the forespore chromosome into the small chamber of the sporangium.
So that when this process is complete we have two cells that lie side by side;
each has a complete chromosome. In the next stage of development,
the mother cell membrane migrates around the forespore
to fully engulf it and pinch it off as a free cell within a cell. So now the process of sporulation
is well underway and that inner cell will mature into a spore. This conversion of that inner cell
into a spore involves three principle, morphogenetic processes.
One, is the remodeling of the chromosome of the forespore into a doughnut-like structure,
in which state it's highly resistant to radiation. The second is the formation of a thick
layer of cell wall material called the cortex around the forespore.
And then a thick protein shell made up of
many different proteins that creates a protective shell
around the spore. This next cartoon illustrates these processes.
So you'll see the forespore chromosome being remodeled
into a doughnut. The white area is the cortex,
and the thick protein shell of coat proteins is created on the outside.
This, then, matures into a spore, a golf ball-like spore. The mother cell, having done her job
lyses and liberates the mature spore, which can remain inert
for many years, but on a moment's notice, when
good conditions return, it can crack open like an egg, and give rise to a cell
that can resume vegetative growth and binary fission.
OK, so we've seen now how during sporulation, two cells are formed.
And the key point is that each of these two cells needs to follow its own distinct program
of gene expression. These two cells have their own pathways of cellular differentiation,
the forespore and the mother cell.
These pathways of differentiation are driven by transcription factors
that act in a cell specific manner and that's the topic I want to talk about now.
So, in this cartoon I've indicated the five principle
transcription factors that drive the process of sporulation.
The first one is known as Spo0A and it's the master regulator for sporulation.
It's the protein which becomes activated in response to nutrient limitation
and causes the cell to enter this pathway and causes all the events
that I've been talking about to unfold, including asymmetric division.
After asymmetric division takes place,
then a transcription factor appears that's called sigmaF.
SigmaF is a member of a family of regulatory proteins
in bacteria known as RNA polymerase sigma factors that work by
binding to RNA polymerase and directing it to particular kinds of promoter...
promoter sequences in the chromosome.
The first of these sigma factors, sigmaF, becomes activated in the forespore compartment.
Then, a sigma factor called sigmaE gets activated in the mother cell compartment.
Then, after engulfment takes place, sigmaF gets replaced in the forespore
by a transcription factor called sigmaG.
And then lastly, in the mother cell, the final transcription factor
in the developmental program, sigmaK, appears.
That these transcription factors act in a cell specific manner
can be seen in the inset on the right.
Here I show you single sporangia that harbor a fusion of the gene for the
green fluorescence protein joined to a promoter under the control of sigmaE
in the top example or a promoter controlled by sigmaG in the bottom example.
You can see in the upper case fluorescence is restricted to the mother cell,
the compartment in which sigmaE is active.
Whereas in the bottom example, we have the opposite pattern.
The fluorescence is accumulated in the forespore compartment
where sigmaG is active.
So each of these four sigma factors act in a cell specific fashion.
And each one of them has presented a puzzle as to the molecular mechanisms
that cause it to become activated in a specific cell type.
We've, over the years, helped to unravel these mysteries
and figure out how these four transcription factors are activated.
And let me tell you just one story about what we know about the activation of sigmaF.
So, sigmaF as you've seen is activated in the forespore
but it's actually synthesized and present in the pre-divisional sporangium.
It's not active in the pre-divisional sporangium because it's held inactive
in the pre-divisional sporangium by an antagonistic protein called AB.
AB is a so-called anti-sigma factor that binds to sigmaF and holds it in an inert state.
AB holds sigmaF inert in the pre-divisional cell and also
after asymmetric division in the mother cell.
But in the forespore, sigmaF manages to escape from AB and
becomes active in directing gene expression.
How does it escape?
Well, its escape is mediated by another protein that we call AA.
AA is an anti-anti-sigma factor that reacts with a complex of AB and sigmaF
to discharge free and active sigmaF.
AA itself is regulated by phosphorylation. It's a phospho-protein and
in its phosphorylated state it is inactive and in its dephosphorylated state
it's active and capable of triggering the activation of sigmaF.
This conversion from the phospho-form to the dephospho-form is mediated by
a phosphatase called E. So E converts AA-phosphate to AA.
And then AA reacts with AB-sigmaF to discharge
free and active sigmaF in the forespore compartment.
How does this happen just in the forespore?
Well, we don't fully know the answer to this question.
But undoubtedly, an important clue is the discovery that the E phosphatase
is itself situated in the septum that divides the two cells from each other.
So here we've tagged the E phosphatase with the green fluorescence protein.
You can see that in the left fluorescence panel.
And in the right fluorescence panel we've stained the sporangium membranes with
a red membrane dye. And you can see that the E protein is located right in the septum.
Somehow, it acts preferentially or exclusively on the forespore side of the septum
to cause sigma F to be activated in that compartment and not the mother cell compartment.
So let me bring everything I've said up until now together;
AA, AB, activation of sigmaF, using a field of cells
in which each cell harbors a fusion of the gene
for the green fluorescence protein to the promoter
to a promoter under the control of sigmaF.
So as this movie begins you can see that all of the cells are dark;
they have no fluorescence. Then asymmetric division takes place.
And then sigmaF becomes activated and you can see the bright green foci appearing
massively in the cells in this field as they begin to sporulate.
Then those bright green foci get converted into opaque phase bright bodies
as sporulation is completed. Let's look at this one last time.
Here's the field of cells, then massively, green fluorescence appears
near one end of each of those sporangia.
And then those green foci become phase bright bodies that represent the maturing spores.
OK, let's now come to the last topic.
I may have left you with the impression up to now that after asymmetric division
the forespore and the mother cell each march to their own drummer,
independently follow their own independent programs of gene expression.
But nothing could be further from the truth,
because the two cells are having a conversation with each other
at each stage of development. They talk back and forth
to each other so as to coordinate the progress of development in one cell
to the progress of development in the other cell.
So, to begin, as we've seen sigmaF is activated in the forespore compartment.
But sigmaE does not appear until sigmaF is active.
SigmaF sends a signal across the membranes that separate the two cells
that leads to the activation of sigmaE in the mother cell.
Once sigmaE is activated in the mother cell, it in turn sends another signal
that leads to the activation of sigmaG in the forespore cell,
the now engulfed forespore compartment.
Once sigmaG is activated, it in turn sends a signal back to the mother cell
that allows the final transcription factor in this sequence, sigmaK,
to appear. So the two cells are talking to each other
in a two-way conversation: from forespore to mother cell,
to mother cell to forespore, to forespore to mother cell.
Let's listen in on one of these conversations
and see just how the language of one of these conversations between the two cells
the very last one, in which sigmaG tells the mother cell to activate sigmaK.
The way this works is as follows:
sigmaK is initially synthesized as an inactive pro-protein.
That is, the primary gene product has an N-terminal extension
of about 20 amino acids that renders pro-sigmaK inactive.
In order for it to be active a protease needs to chop off that N-terminal extension
to generate the mature and active form of the transcription factor.
You can see that in this Western blot experiment.
So this is an experiment in which all the proteins from the sporulating cell were
separated on a gel and then sigmaK and pro-sigmaK were visualized
with antibodies to the protein. And as you can see in a wild-type sporulating cell
most of the sigmaK is in the form of the mature and active protein
and relatively little in the larger pro-protein form.
This conversion of pro- to mature depends on the action of sigmaG.
And you can see this key point if we use a mutant of sigmaG.
When sigmaG is mutant there is no conversion and if you look at the Western blot
analysis on the far right of the mutant, now you can see that all of the protein
is in the pro-form and little or none is in the form of the mature sigma factor.
Somehow, the activation of sigmaK in one cell, depends on genetic events
taking place in the adjacent cell. How does this work?
Well, the protease is a membrane protein and it mediates the cleavage of the pro-sequence.
But initially it's held inactive by two other membrane proteins
that are inhibitory and together hold the protein in an inactive complex.
In order for the protease to become activated,
the protease needs to escape from this inhibition.
And that event is caused by a signaling protein
that's produced in the forespore compartment under the control of sigmaG.
So sigmaG turns on the gene for a signaling protein,
that signaling protein is secreted across the membrane
of the forespore where it interacts with a complex of proteins
that includes the protease and its inhibitory proteins
and reverses the inhibition so that now cleavage of
pro-sigmaK to the mature form of the transcription factor can take place.
This protease turns out to be especially interesting on two counts.
First, we infer that its active site is located in the membrane.
The blue bars represent the inferred catalytic regions
of the protease and it's inferred that the N-terminal extension on sigmaK
inserts into a cavity, in the membrane, created by the protease.
Well, this kind of membrane cleavage leading to gene... activation of gene expression
is a fore-runner of something that's widespread in biology
and is referred to as regulated inter-membrane proteolysis.
And interestingly, this very example of it in a bacterium
is conserved all the way up to mammals.
Mammals have a protease with homologous features, in particular the catalytic center,
to the bacterial protease. In the case of mammals,
the protease activates a transcription factor that's bound to the membrane
and cleavage by the protease releases it from the membrane
and so it can migrate to the nucleus and activate gene expression,
in this case, genes involved in cholesterol metabolism.
But, the overall principle is the same.
Two transcription factors are in inactive states that require proteolysis to be activated
so that they can turn on genes.
And the similar proteases, conserved over eons of evolution, mediate both processes.
So finally, let me point you in the direction of future research:
what I see as a principle challenge for the future.
We've talked about the cell biology of sporulation, and we've talked about
the orchestrated expression of genes under the control
of a series of cell-specific transcription factors.
These transcription factors are activating over 500 genes
in a cell-specific and temporal fashion. And it's the products of those genes
that mediate the morphogenesis that culminates in the spore.
So the final challenge is to understand how the myriad proteins
produced under the control of sigmaF, E, G and K mediate morphogenesis, drive
the assembly of the spore into this remarkable dormant structure that can resist
the ravages of time and insults of the environment in such a robust manner.
Thank you.