Hi.
I am Christine Jacobs-Wagner.
I am the director of the Microbial Sciences Institute
at Yale University,
and I am also a professor of molecular, cellular,
and developmental biology,
and an investigator of the Howard Hughes Medical Institute.
In the first presentation,
I told you a little bit about
the cell biology of bacteria
and how important it is for cellular function
and cellular behavior.
In this presentation,
I'd like to tell you a little bit about research ongoing in my lab
and, more specifically,
I would like to tell you about a particular project
that relates to DNA segregation,
so, how bacteria are able to segregate the chromosome
and achieve active transport.
Now, broadly speaking,
what my lab is really interested in
is understanding how bacterial cells self-replicate.
So, as you probably know,
and it's illustrated in this movie,
bacterial cells,
if they are put in the right growth condition,
they can proliferate at a remarkable pace,
something that often we would like to control.
And so my lab is really interested
in understanding the molecular and physical mechanisms
by which bacteria multiply at this fantastic pace.
And so we're... to do this,
we're addressing a lot of questions,
such as, how do bacterial cells grow?
How do they divide?
How do they couple these two processes
to ensure that they're going to achieve
an optimal shape and size for function?
How do they replicate their DNA?
How do their segregate their DNA?
How do they coordinate cell cycle events
with cell division and growth?
We also want to understand how they regulate gene expression
to maximize self-replication.
But today, because of time constraints,
I would like to focus on one particular question,
which is, how do bacterial cells partition their cellular content
to ensure that each daughter cell
is going to get its fair share of cellular components
so that it can continue the self-replication process?
Now, actually, bacteria...
because, as i said in my first presentation,
they are very small relative to eukaryotic cells.
So, this is how the bacteria...
so, I drew here how most bacteria would look like
relative to an animal's cells.
So, they're very tiny,
and this is actually an advantage
because it means that diffusion,
which is a passive process,
is going to be an effective means for molecular transport
inside the cell.
So, diffusion, which is driven by thermal fluctuations,
is going to work well in bacterial cells because they are small,
and diffusion works well over short distances.
So, this is illustrated in this simulation
that we made for you.
So, just consider the cellular component
that is in 100 copies,
and all 100 copies are localized in the middle of the cells,
so just imagine a protein with 100 copies.
Now, as I start the simulation,
in a very little amount of time,
simply due to Brownian motion, so, passive diffusion,
you're going to have a quick distribution of those
100 copies of the cellular component,
and random distribution here, or, you know,
dispersion of this component
is just going to be good enough for partitioning,
for equal partitioning between the daughter cells.
So, if the cell division, here,
happens in the middle,
each daughter cell is going to get its fair share
of about half of the copies.
And so, diffusion alone is going to be an effective means
for equal partitioning of many cellular components in bacterial cells.
So, that works well if the cellular component
is in many copies,
but it you have very few copies
of that cellular component...
so, there is many components in the cytoplasm
that are in few copies...
now, random distribution is not going to work as well.
So, here we have the same simulation
but, now, instead of having 100 copies,
we have only 2 copies,
and as you can imagine, depending when division is going to happen,
there is actually a chance that
one of the daughter cells is going to get both copies
and one daughter cell will get zero copies.
And you can do a very simple calculation,
which is what we did here,
to show that random distribution...
random distribution is going to,
for components that have...
that are in copy number of 10 or less,
there's going to be a significant probability,
and this probability is going to increase
as the copy number decreases,
the probability that one of the daughter cells
is not going to any copies of that component.
And so, that means that bacterial cells
had to evolve systems
to ensure the partitioning of cellular components
that are in low counts.
And a very important and versatile system
that bacteria have evolved
is the Par system,
which we call also the ParABS.
And so, ParABS systems
are important for chromosome segregation.
They've been shown also to be important
for plasmid partition...
so, some low-copy plasmids
encode their own ParABS system
that is important for the partitioning
such that each daughter cell is going to get
an equal number of these plasmids.
The ParABS systems have been also important
for the partitioning,
the segregation of other cellular components
including enzymatic microcompartments
and cytoplasmic chemotaxis clusters.
What I'm going to talk about today
is the ParABS system
that is important for chromosome segregation,
and the model that we use is Caulobacter crescentus.
So, what is nice about the ParABS system
is that it's only made of three components.
One is the DNA sequence and the other two are proteins.
So, the DNA sequence is called parS,
and so parS has been shown to be very conserved,
to be found on many bacterial genomes,
and it's typically located close to the origin of replication.
ParB is a protein, it's a DNA binding protein,
that's going to bind to the parS sequence
and form what we call the partition complex.
And then ParA,
this is a Walker Type-A ATPase
that is going to be important for the translocation
of the partition complex.
So, since we are looking at this
in Caulobacter crescentus,
I just want to remind you that Caulobacter
is basically a typical bacterium in the sense that
it has one circular chromosome,
it has an origin of replication here,
and the parS sequence in Caulobacter,
just as in many bacteria,
is actually located close to the origin of replication.
As I mentioned in my first presentation,
the chromosome in bacterial cells
has to be compacted about a dozen times
to fit into the bacterial cells,
and so this is more the way we think
it would look like inside the Caulobacter crescentus cells.
So, the DNA, which I hope you can see,
is those blue strands
that are packed into the bacterial cells.
But, as I mentioned in my first presentation,
the chromosome is folded in a spatially organized fashion,
such that each gene is going to be located
at a specific location inside the cells,
and such that, in Caulobacter,
the origin of replication is always found
at one end of the cells,
which we call the old pole,
to distinguish it from the new pole at the opposite end,
which is called new pole because
it was made by the last division.
Since the parS region
is close to the origin of replication,
it means that it's also going to be found at that location,
at the old cell pole.
And, as I will show you in a minute,
this parS region is the first region
that's going to be translocated...
after duplication, translocated to the opposite end of the cells
in the process of chromosome segregation.
So now, to explain what happens
during chromosome segregation,
I just want to show you
some more rudimentary schematics.
So now, in this schematic, for the sake of simplicity,
I drew the chromosome just as this grey line
and, again, the parS sequence
is located at the old pole,
and since ParB is going to bind to this parS sequence
it's going to form the partition complex
that is shown here in green.
It's actually tethered to the end of the cells there,
because ParB is going to interact, physically interact,
with another protein called PopZ,
which is located at that end of the cells.
So, PopZ is a protein that multimerizes
and forms a matrix at the end of the cells,
and it's sticky, it's going to bind to ParB
and thereby tether the chromosome
at that end of the cell.
So, this is the rest of the Caulobacter cell cycle,
and Caulobacter has a very interesting cell cycle
by having, you know, polar appendages like stalks.
It's going to have, also, a flagellum here,
and it's going to divide asymmetrically.
These are all very interesting features that we study.
However, today, those are not important
because today I'm just going to talk about
chromosome segregation,
which happens in all bacteria
and it's not specific to a cell cycle.
Alright, so, let's go back here.
So, at this stage, now,
DNA replication occurs.
And since the parS sequence
is close to the origin of replication,
it means that it's going to be one of the first regions
that's going to be duplicated,
and therefore this is going to create two partition complexes.
One is going to stay at one end of the cells.
The other,
which is the first step of chromosome segregation,
the other is going to be actively transported
to the other side of the cells,
and this is going to be happen
in a ParA-dependent fashion.
And this step, so,
this active motion from one end of the cells
to the other,
is essential for viability.
Okay, so this is essentially happening
while replication is still ongoing.
So, in the bacteria, unlike in eukaryotic cells,
duplication and segregation
occur in the same time.
So, as the partition complex,
one of the them is migrating to the opposite end,
when it reaches the end it's going to tether,
it's going to be tethered
because it's going to now interact with the growing PopZ matrix,
and now DNA replication is going to keep on going
and the chromosome is going to unzip,
such that each of the daughter cells
is going to inherit a chromosome following division.
Okay, so the partition complex, what is it made of?
So, this is shown here.
So, as I mentioned, the parS region
is close to the origin of replication,
which is not shown in this schematic,
but in Caulobacter there's two parS sequences,
which are shown in red.
ParB is going to bind to those parS sequences
and, as it binds specifically
and recognizes those sequences,
now the protein, ParB protein,
is going to be able to spread,
some people say that it will polymerize on the DNA,
simply because of those two parS sequences,
when it binds to it,
this is going to result in spreading of the protein
on adjacent sequences,
regardless of what the sequence composition is.
Now, we can estimate the number of ParB proteins
that are associated with this DNA sequence,
and so, by doing quantitative western blotting,
we know the number,
the average number of ParB protein inside the cells,
and by doing quantitative fluorescence microscopy
we could establish that there was
about 80% of the ParB protein inside the cells
that is associated with the DNA,
the parS sequence,
and so that is roughly 580 molecules of ParB
that is associated to the DNA...
that is associated...
that is part of the partition complex.
This is important,
because it means that you're going to have a very strong,
very high local concentration of ParB
at the partition complex.
This is going become important later on.
Comparatively,
we have only about 140 molecules
of diffusing ParB,
so that's just 20% of the pool,
and that's going to result in a very low concentration of ParB
in the cytoplasm.
We estimate it to be under 1 micromolar,
where it's like over 500 micromolar
at the partition complex.
Okay, so we can track the partition complex
as it's duplicated and translocated.
So, because there are so many ParB proteins
binding to the DNA region,
it appears as a bright focus by fluorescence microscopy
if you label ParB protein with a fluorescent protein.
So, I'm going to start the movie.
We're going to have DNA replication appearing.
That generates a second partition complex that's going...
one of the them stays at the pole.
The other one that is free is going to migrate
to the opposite end of the cells,
and the movie is going to loop a few times
so that you get to see it more than once.
So, we can quantify this motion.
This is actually shown here.
So, we're looking at the partition complex
around the cells.
Over time, you see that, after duplication,
one is going to stay at one of the cells.
The other is going to be translocated
in a ParA-dependent fashion,
and then is going to get tethered
to the opposite end of the cells.
What we can do also is that we can visualize
both ParB and ParA in the same cell,
and we can do this by tagging them
to fluorescent proteins that have a different color,
and so this is what we did in this experiment.
So now, what you are basically seeing
is ParA is in red
and you also see, in green,
the trace of the partition complex,
which is rich in ParB.
So, this is actually a kymograph
where we are looking at the ParA signals
along the cell length over time.
And, just to help you,
here we have the schematic.
So, in the beginning, which is shown here,
the ParA signal is basically across the cells,
except where the partition complex is.
As I'm going to show you in the next slide,
it's actually forming a gradient.
But then, when the partition complex is duplicated,
something amazing happens,
and that is that when the partition complex
gets close to the edge of the ParA signal, shown in red,
now the ParA signal is going to shrink,
and this is going to be correlated
with the motion of the partition complex.
And this correlation,
these correlated dynamics between ParA and ParB,
which was first shown in Vibrio cholerae
for chromosome segregation in that organism,
has also been shown to be true for plasmid
and other ParABS systems.
Okay, so let's see this in a different way.
So, again, here you're looking inside the cells,
this is an epifluorescence image.
In red it's ParA,
in green is the partition complex.
As you can see, there are two of them,
one has just duplicated.
And now, if you look at it,
even if you look at it by super-resolution microscopy,
you find that the ParA signals
across the width of the cells is fairly uniform,
but across the length of the cells
it's actually forming a gradient, which is shown here.
So, here you see, along the cell position,
you see ParB in green
and you see ParA in red.
You can see that it forms this shallow gradient.
So, this is looking at the beginning,
following DNA replication,
but if we follow this over time
you can see here that the gradient, the ParA gradient in red,
is getting steeper and steeper
as the partition complex, shown in green,
is basically moving along,
as if it was walking across...
along, following the ParA gradient.
So, how does this happen?
What is important for us is to know the biochemistry
of the system.
So, the question is,
what are the biochemical properties?
And here, I'm going to summarize a lot of work
by many labs over the years, including ours,
with this biochemical model,
which was supported by in vitro studies
and also mutagenesis studies.
So, what is shown here is ParA.
ParA, in the presence...
in vitro, if you purify ParA
and you put it in the presence of ATP,
it's going to form dimers,
and then when it becomes a dimer,
now it's going to have an affinity for the DNA,
any unspecific sequence.
So now, the ParA dimer
is going to bind to the DNA,
and it is in this dimeric form
that now it's going to have a strong affinity
for ParB.
So now, it's going to bind to ParB,
but now, after it binds to ParB,
ParB is going to stimulate its ATPase activity.
So, in the absence of ParB,
there is very little ATPase activity,
I'll show you in the next slide,
but what is important is that,
when it gets in contact with ParB,
now this is going to stimulate ATP hydrolysis,
releasing the complex
between ParB and the ParA dimer,
because now the ParA dimer
is going to dissolve into monomer,
and then through nucleotide exchange
you can repeat the cycle.
Now, what is important on this slide
is that, yes,
it's going to bind to ParB and ParB
is going to stimulate ATP hydrolysis,
but ParB can do this only if there is a high concentration of ParB.
And so, this is shown in this biochemical experiment.
So, this is in vitro,
so you have purified ParB,
you have purified ParA,
you add DNA and ATP to your reaction.
Now, if you just have
no ParB at all
then what we're looking at here is the ATPase activity,
so, we're looking at the turnover of ATP
by ATP hydrolysis.
Without ParB,
you have very little amount of ParA ATPase activity.
You do have a little bit, but it's not relevant.
It's not high enough to be relevant for the reaction,
for the translocation process.
But if you increase the concentration of ParB
you see a really nice stimulation
of ParA ATPase activity.
But this...
you get only a significant or relevant
ParA ATPase activity
at very high concentrations of ParB.
So, you need over 100 micromolar of ParB
to get the nice stimulation of ParA ATPase activity.
And this is why it's only the partition complex,
which is really rich in ParB,
where you have hundreds of molecules of ParB,
that is able to stimulate ParA ATPase activity,
whereas the diffusing ParB,
whose concentration we estimate to be under 1 micromolar,
well, this is too low to have any effect.
So, it means that, again,
it's only the partition complex,
the ParB-rich partition complex,
that's going to bind to the ParA-ATP dimer
and that's going to stimulate ParA ATPase activity.
Alright, so this is how we think it looks like inside the cells.
So, you have DNA in blue
and you have the two partition complexes.
One is going to stay at one pole,
it's tethered there by the PopZ matrix,
and one is going to migrate along,
somehow, along this ParA gradient.
ParA, which I'm showing you in red here,
is basically to represent ParA-ATP dimer,
because there the concentration of ATP inside of cells
is high enough that ParA is going to bind ATP,
bind to the DNA,
and is going to do some away from the partition complex,
forming this gradient.
We know, by quantitative western blotting,
and we know also by single-molecule counting,
that there are about 90 dimers of ParA,
so this is what we put in this drawing.
So then, we wondered,
is it possible that the way the partition complex,
in green here,
is migrating to the opposite end of the cells
in a ParA-dependent fashion
is because it's simply diffusing,
interacting with some of those ParA-ATP dimers
along the way,
and this perhaps is resulting
in kind of a guided diffusion.
So, to wonder if that is true,
then the first thing that we wonder
is whether the partition complex,
when it moves to the opposite side of the cells,
does it do it in a linear fashion
or does it do it in a kind of zigzag-y fashion
because it has to interact with
so many ParA-ATP dimer through diffusion.
And, what's consistent with diffusion,
and we have other data that support that as well
that I won't have time to show, is that,
indeed, if we track, at very high temporal and spatial resolution,
the partition complex,
you see here that this is one of the cells, this is the other,
that it kind of follows this kind of zigzag-y fashion
and that there is really two phases.
There is a slow motion
and it is followed by fast motion.
So then, we wanted to test,
well, is it possible that what happens is that
the partition complex is diffusing
and interacting with ParA-ATP dimers
and this is sufficient to reproduce
the translocation kinetics that we observe inside the cells?
So, we wanted to test that through simulation.
And so we built a model
that was absolutely constrained
by experimentally-derived parameters.
So, we measure the parameter inside of cells
and then we constrain our model
by the value that we obtain from experiments.
So, we know the diffusion coefficient
for the partition complex,
we know how many ParA-ATP dimers there are,
which are shown here in red,
we know how many ParB proteins there are,
we know that they can interact with multiple ParA-ATP dimers,
we know how the ParA gradient evolves
during the translocating process...
so, we can put all this in our model,
and then just what we do is a Brownian dynamics simulation,
when we track the translocation
of the partition complex over time.
So, this is an example of one simulation.
The partition complex is diffusing,
it's exhibiting Brownian motion,
interacting with ParA dimers
until ATP hydrolysis occurs,
we know the rate from our biochemical reaction,
that we have applied to the model.
Now, you see here that, in the experiments,
within 10-15 minutes,
translocation should be complete.
But in this case, in our simulations,
it's not complete.
It's not just complete,
it doesn't work just for that simulation...
we've run thousands of simulations
and then looked at the average behavior,
which is shown here.
The simulation is in blue,
the experiment, which is the average trajectory for the experiment,
2 in is black,
and you see in the black curve
that within 10 minutes
the partition complex has moved to the other side of the cell,
on average,
but this is not the case for our simulation.
So, it meant that something was missing
from our model.
What was missing is DNA dynamics.
Now, in our model,
the ParA-ATP dimer was static.
But the ParA-ATP dimer
is bound to the DNA, which we know is dynamic,
and this is shown here.
Here, what we have is one chromosomal region
that is fluorescently labeled
so that we can track it over time,
and as you can see here it's actually moving.
It's wiggling around,
and this has been known for quite some time.
And you can take many different chromosomal regions,
different regions of the chromosome
and you see the same behavior.
You can quantify this motion,
so you can obtain trajectories,
and you can analyze those trajectories,
which is what we've done here,
and we've found is that the chromosomal loci,
so the chromosome,
display elastic dynamics.
And this is shown here
because we see this harmonic potential
that implies elastic dynamics.
So, what it means is that chromosomal loci
are basically wiggling around,
but always coming back to an equilibrium point
as if they were on elastic.
So now, because ParA-ATP dimer is bound to the DNA,
it means that those ParA-ATP dimers
are going to experience the elastic dynamics
of the underlying DNA.
So, now we're going to add this dynamics
that we are measuring in experiments,
from the motion of chromosomal loci,
now we're going to incorporate it into our model.
So, this is exactly the same model,
the same parameters,
but the only difference is that now the ParA-ATP dimers,
which are shown in red here,
are going to wiggle around,
displaying the DNA dynamics that we observe in the experiment.
So, we have the same thing,
this is an example of a simulation.
In the beginning, we see a slow translocation,
but as it goes through the more dense region of ParA
you see an acceleration of the translocation.
Now, the ParA-ATP dimers
are not disappearing in our experiments.
We are just simply being redistributed...
as they interact with ParA they are being redistributed,
forming the evolution of the ParA gradient
that we see in the experiments.
So, this is in one experiment, and one simulation.
We did thousands of simulations,
thousands of simulations, we can average it,
and now we see that our model agrees very well
with the experiment.
So, in green is the result from the model,
where the only difference is that we incorporate
the DNA dynamics.
As you can see, it follows very nicely the experiment
shown in black.
Okay, so we called this model
the DNA-relay model,
because what we think is going on
is that the partition complex,
which diffuses,
when it's going to interact with the ParA-ATP dimer
that is moving, that is exhibiting this elastic dynamics,
when it interacts with it
it's going to experience the elastic dynamics,
bringing the partition complex to the equilibrium point
until hydrolysis occurs, being released,
and now it's going to be passed on to another DNA region.
So, basically, the partition complex
is going to move from one DNA region to another
using ParA-ATP dimer
as a transient tether.
And so, this is what we tried to illustrate here
in this animation.
So, in this animation,
before I start the movie I'll just tell you what you're going to see...
so, here you have the partition complex,
which is rich in ParB,
and then you have ParA-ATP dimers
that are bound to the DNA.
Now, the DNA starts to form those plectoneme loops,
which is shown here,
and those loops are basically going to exhibit
elastic dynamics.
And then, as I start the movie,
the partition complex is basically diffusing...
then it's going to encounter
a ParA-ATP dimer.
That's basically, because of the elastic dynamics,
it's going to bring it to the equilibrium point
until ATP hydrolysis occurs
and now it's going to be released,
and now it's going to interact
with one or more ParA-ATP dimers.
That's going to bring it to the next DNA region.
And the reason we think that it works,
as shown in our simulations,
is because the partition complex comes from one side,
relative to the ParA-ATP dimers,
that is going to be...
that the probability of being in the stretched configuration
to bring it to the equilibrium point.
And so, together with XVIVO,
we created an animation
that illustrates what we think happens from the top.
Caulobacter crescentus
has a circular chromosome
that is condensed into a spatially organized 3-D structure
inside the cell.
The origin of replication and two nearby parS sequences
are located at one end of the cell called the old cell pole.
The DNA protein ParB
recognizes parS
and spreads onto flanking DNA.
This makes the so-called partition complex.
Replication creates a second partition complex
that diffuses away.
Inside the cell,
the DNA is highly dynamic,
and elastic force returns each DNA locus
to its equilibrium position.
ParA, another protein critical for chromosome segregation,
binds ATP
and forms dimers that associate with the dynamic DNA,
away from the partition complex.
When the diffusing partition complex
interacts with one or more DNA-bound ParA dimers,
it experiences the elastic force
from the underlying DNA.
ParB stimulates ParA-ATP hydrolysis,
leading to the release of ParA monomers
from the partition complex and the DNA.
Repeated rounds of association and disassociation
between ParA and ParB
effectively transport the partition complex
towards the new cell pole.  I hope you have enjoyed the animation.
So, in conclusion,
the data suggest that the cell
is using a DNA-relay mechanism
in which the cells are harnessing
the elastic dynamics of the DNA
to achieve directed motion,
so, in other words,
the chromosome is playing a mechanical function.
So, of course, now we have
a million questions to address, too many to list,
but some of the first questions
that we're interested in addressing
is, how is the ParA-ATP dimer gradient achieved,
and is it really important
for achieving this directed motion?
Another important question is,
how general is this DNA-relay mechanism?
We think that it might be general,
that it might be important for chromosome segregation,
but that it also might be important
for other processes that exploit a ParABS system.
The reason why we think this
is because the elastic dynamics
of the DNA that we observe in Caulobacter crescentus
are also true in other bacterial cells, such as E. coli,
and it has also been shown
that they are actually in eukaryotic cells,
where chromatin displays elastic dynamics in nuclei.
So, it is likely that those elastic dynamics
of the chromosome
are universal properties of DNA that is in a confined space.
Furthermore, every time it was tested,
it was shown that the DNA binding activity of ParA
is conserved,
which would be important for this DNA-relay mechanism.
And so, to end,
I would just like to acknowledge the work
that was done by... the experimental work was done
by Hoong Chuin Lim and Whitman Schofield,
whereas the Brownian dynamics simulation
was done by Ivan Surovtsev
and Bruno Beltran in my lab.
In addition, I would like to thank
all of the members, past and current, of my lab
for their really nice contributions over the years,
and in particular I would like to thank Brad Parry,
who was the graduate student that made the two first simulations
that I showed in the beginning of the talk.
And, of course, I would like to give a big thanks
to the National Institute of Health
and the Howard Hughes Medical Institute
for their generous support over the years.
And I would like also to thank you for your attention.
