Hi.
My name is Michele LeRoux
and I did my graduate work at the University of Washington in Seattle.
And I'm going to tell you today about work I did in graduate school
studying how bacteria interact with one another
and how they survive out in the environment
and in our bodies.
So, I'm going to start out by telling you that
I really love yogurt.
It's one of my favorite foods,
I eat it almost every day,
I even make my own yogurt now,
but early on in graduate school
I was looking at one of those little yogurt containers
where it says, "Contains live and active cultures,"
and I wondered whether that was really true.
Would I really see bacteria in there?
So, I took a little bit of yogurt
and I put it on a slide
and I went and looked at it under the microscope.
And this is the picture I took.
Look at all those bacteria
-- there's long rods, there's short rods,
there's these little coccoid bacteria
that are forming these chains --
and actually different types of yogurt
had slightly different bacterial groups in them
that gave them different tastes,
so this was really my first exciting glimpse
into this invisible world of bacteria
that are all around us.
They live in these communities
and they make delicious things for us to eat.
So this got me really excited about the idea of
studying how bacteria live in these communities together.
So, of course, bacteria are not just in our yogurt;
they're out in the environment,
everywhere you look, basically,
associated with plants,
in the soil, in the water,
there's bacteria in the ocean.
Every teaspoon of sea water
has about five million bacteria in it,
so think about that the next time you
accidentally swallow a little ocean water.
That's a lot of bacteria that you've just taken into your body.
And then, as I mentioned before,
there's bacteria in our foods.
Cheese is another type of food
that we require bacteria to make for us,
and of course there's bacteria in our bodies,
in our guts there's billions of bacteria,
there's bacteria in our mouths and our skin,
and a lot of them perform
really important functions for us.
They help us digest our food
and they help our immune systems work properly as well.
But of course, bacteria can also cause
really, really devastating infections,
and that's actually usually what we've been studying them for
in the past.
So, the organism that I'm going to tell you about today
is this organism called Pseudomonas aeruginosa.
And this is an organism that's
actually mostly found out in the environment.
It lives in the soil, it lives in the water,
and we come into contact with it all the time.
It lives all over the place.
So, mostly, it doesn't really cause us a lot of problems...
but occasionally Pseudomonas can get into
a particular human environment
and it can really wreak havoc.
So, this is a little bit like a termite.
You know, a termite,
mostly, it lives out in nature,
it can build some really cool mounds,
it will digest cellulose in wood
and it plays a really important role in the ecosystem,
but if a termite gets into someone's home,
into the wooden framework,
and starts chewing up that wood,
it can be really hard to get rid of
and it can bring the house down.
So, it's the same with Pseudomonas.
You know, mostly we don't worry about it,
but if Pseudomonas gets into the wrong human environment,
it can be really devastating.
So, what environments are those?
A lot of might have loved ones, family members,
that are in nursing homes
or are confined to wheelchairs,
and people like this are often
prone to getting bedsores or chronic wounds.
These are also common in people with type II diabetes.
And these are wounds that just cannot heal;
they just stay open wounds for,
sometimes, years.
And they can get infected with bacteria.
So, especially if Pseudomonas gets in there,
it can be really, really bad for these people.
It can get deep into the tissues
and it can be impossible to eradicate
and sometimes leads to amputations or even death.
And we don't quite understand
why it's so difficult to treat Pseudomonas infections
when they get into these chronic wound environments.
Another environment that Pseudomonas really thrives in
are the lungs of patients with the genetic disease
cystic fibrosis.
So, these people have
a mutation that causes them to have
really thick mucus in their lungs,
and this makes an ideal environment for this bacterium.
In fact, 80% or more of people with cystic fibrosis
will acquire Pseudomonas at some point in their lifetimes,
and once they do it's
impossible to eradicate it most of the time.
And it will go on to lead to the
decline of their lung function and ultimately their deaths.
So we really, really need to understand better
how Pseudomonas is causing these problems
and how we can treat these infections with Pseudomonas.
So, what I decided to do was
to kind of step back from thinking about Pseudomonas
interacting with humans, directly,
and think more about,
what does Pseudomonas need to survive?
What is motivating a lot of its behavior?
How has it evolved over the years?
So what I decided to do was
think about putting myself in the shoes of this bacterium...
well, of course, not exactly in its shoes,
because it's a bacterium, it's a single-cell organism,
it doesn't wear shoes...
but thinking about what it needs to survive.
So, Pseudomonas needs, just like us,
food to eat.
It needs a place to live, it needs a home.
And it will fight to the death
to be able to make sure
that it maintains these resources.
And, actually, usually what it's fighting
are other bacteria that it's competing with
for its food and its home.
So this is where it's really helpful to think about
how long bacteria have been around
and what's really been driving their evolution.
So, the Earth first evolved
around four and a half, give or take, billion years ago,
and not too long after that, in evolutionary terms,
there's evidence of the first bacterial fossils.
Sometime later,
we have evidence of the first bacterial ecosystem
-- so, these are where groups of bacteria live together
and interact with one another,
so this is where bacterial interactions
would have first started evolving.
Now, eukaryotes didn't show up on the scene
until much, much later,
and of course humans have really only been around
for the blink of an eye in terms of evolutionary time.
So, this green bar
represents the amount of time that bacteria
have been interacting with other bacteria.
That green bar, that you can barely almost see over there,
that I'm showing you down here,
is how long that bacteria have had
to interact with humans.
So, it's a much, much smaller amount of time,
and yet that's what we usually end up studying in the lab.
So I thought, well,
there's probably a lot to be learned about bacterial behavior
if we focus a little bit more on
bacterial interactions with one another,
and that might even inform
how we think about their interactions with humans.
So, what I decided to do was
to start by focusing on one particular bacterial weapon
that it uses to compete with other organisms,
and that is this secretion system
called the type six secretion system.
You can think of it as a syringe
that injects toxins from one cell into another,
and this pathway was actually
not discovered too long ago,
and the lab that I did my graduate work in, the Mougous lab,
were one of the first to show that
this pathway actually
injects toxins from bacteria into other bacteria.
So this is kind of what this might look like.
If you have this green cell,
let's say this is the Pseudomonas cell,
it has these little red dots that are representing toxins,
it will literally inject them into the cell next to it,
which will cause...
these toxins will cause this other cell to die.
And if we zoom in on this a little bit,
you can see this syringe, here,
I'm depicting as kind of a tube,
but it's actually a molecular machine
made up of a bunch of different proteins
that are able to push proteins
-- toxins --
from one cell into another cell.
And these toxins have all kinds of different functions,
we're still discovering new ones all the time,
but, for example,
they might degrade the cell wall of the recipient bacterium,
which would cause it to burst,
the might attack the membrane of the cell,
and some of them even go after molecules
that are required for survival,
such as RNA or DNA or energy molecules.
And all of these things will cause the cell to die.
So, the next thing I want to do is show you
what type six looks like in action.
But before I can do that,
I have to explain to you what to look for.
So, how do we watch cells dying in the lab?
Well, what I like to do is
I have cells that are expressing
a colored protein, such as green fluorescent protein
-- they're making a lot of it,
so they appear green when you look at them --
but if that cell were to get
a rupture in its membrane or its cell wall,
what would happen is that the green protein
will flood out of the cell,
and then after just a second or two
that cell will appear dark.
So you're literally just going to see a green cell disappear.
But really what's happening is that cell is bursting open
and spewing out its guts into the surrounding area.
And so just keep that in mind:
when you see a cell go dark, really it's exploding in a...
actually a pretty violent way.
So, let me show you what I'm talking about.
So, here we have Pseudomonas aeruginosa
and it's labeled in a red color,
and we have a competitor organism
that's labeled green.
So, you're going to be looking for these green cells
to just disappear.
So, to make it a little bit easier to identify those,
I had the software that I use to analyze this data
outline them in a white outline
so you can find them more easily with your eye.
So, just one note about this competitor organism:
this is an organism that's called
Burkholderia thailandensis.
This is another bacterium that lives in the soil
that would potentially interact with Pseudomonas
in the environment.
You don't have to remember the name
-- from now on, I'm just going to refer to it as the competitor --
but one thing you should know about it is
it also has a type six secretion system,
and that will become relevant later on in my talk.
So, these systems are actually found in a lot of different bacteria,
about 30% of bacteria that we've looked at so far
have these secretion systems,
so this is a common weapon that bacteria
employ to fight one another with.
Alright, so let's see what this system can do.
I'm going to show you this movie
of these two populations growing together.
And hopefully what you can see is that
these green cells, the ones that are outlined in white,
are just disappearing from view,
and they're just popping as the cells die.
So, how do we know that this is because of the type six system?
Well, what I did next is I took the same competitor cells,
but then I grew them with a Pseudomonas strain
that did not have this type six secretion system anymore
-- I inactivated it by deleting one of the key proteins.
And now what you can see is
the Pseudomonas cells are growing with this competitor,
and I'm playing both movies at the same time
so you can appreciate the difference.
So, now you see that
there's actually not a lot of these green cells
that are popping anymore,
because the Pseudomonas has lost its weapon.
And so, especially by the end of the movie,
you can really appreciate how many more green cells
there are in this movie,
where the Pseudomonas doesn't have its weapon,
compared to the one where it does.
So, this is what Pseudomonas can do:
it can basically wipe out most of these competitor cells.
But the question that I really had going into this was,
what is this system itself doing?
When does it get turned on?
When does Pseudomonas know that
it's time to mount this weapon
and fight against a competitor?
So let me show you one of the ways
that we can look at that,
and that's by labeling one component of the system itself
with a green fluorescent protein.
So, now we're not labeling the whole cell,
but you can see these little dots forming in these cells,
and each time you see one of those dots,
that's actually the system coming together
and we think it corresponds to
when it fires these toxins out of the cell.
So, what I've shown you here are just Pseudomonas growing by themselves,
so they don't really have anyone to attack,
and I've activated the system artificially
so we have something to look at,
to study in the lab,
because when you just look at the cells normally,
there's not much going on.
This kind of makes sense.
These cells don't want to be
making all of this protein
and firing it all out of the cell for no reason.
That uses up a lot of their resources
and, remember, I told you,
you know, bacteria are trying to survive,
they don't have that many resources.
So how do they know
when is a good time to turn this system on
so that they can fight
and actually have it mean something to them?
So, what I decided to do
was take kind of a similar approach.
I labeled one of the proteins of the type six apparatus,
and then what I did is
I mixed Pseudomonas with this competitor
and looked to see what happened to the system.
So, the first movie, here,
that you can see over there,
is Pseudomonas just growing by itself.
The movie on the other side is Pseudomonas
that's growing with this competitor.
Now, in this case, I've labeled the competitor with red
-- I'm going to switch colors back and forth a little bit,
we had to do that for some technical reasons,
but I'll always make sure to tell you which population is which.
And so I'm going to play these two movies simultaneously
so you can appreciate the differences
and look to see what happens with the green protein.
So, you see some dots forming
and then you see the cells get green.
And, hopefully, what you can appreciate
is that the cells growing with the competitor
are a lot greener than the cells that didn't have...
that were just growing by themselves.
And so what does this mean?
What this means is that
the Pseudomonas cells, in the presence of a competitor,
are actually making more type six protein,
and not only that, they're actually assembling it
more frequently into those foci,
those dots that you see,
which means that they're firing it more frequently.
So, this makes kind of...
it makes a lot of sense, actually, when you think about it.
Pseudomonas somehow realizes that it's near a competitor
and it makes more of this weapon to fight it with.
The next thing, the next experiment I did,
the result was a little bit more puzzling.
So, the first two movies I'm showing you here
are the same ones I just showed you,
Pseudomonas by itself,
Pseudomonas with a competitor,
but this last movie is Pseudomonas
growing with the same competitor,
but now, remember that I told you that competitor
also has a type six secretion system?...
I've deleted the type six secretion system
from the competitor.
So now let's look to see what happens.
So, the first two movies you see the same thing,
you see the cells with the competitor
get really bright,
but what you see here is the cells
growing with the competitor that doesn't have
its own type six system...
they don't turn on.
They look just like Pseudomonas growing by itself.
And we can see this in a graph form as well,
so we quantified this data.
And we're looking here at the levels of type six protein over time,
and you can see that when Pseudomonas grows by itself,
you know, you see a little bit of an increase,
but when it grows with a competitor
that increase is much more,
it's making much more of those type six proteins.
Now, when the competitor doesn't have
its own type six secretion system,
Pseudomonas doesn't turn its on either.
So, this was really puzzling.
How is this working?
How does Pseudomonas know the competitor is there,
first of all,
then how does it know that it has
this pathway as well?
So, this is a little bit of a silly example,
but let's just imagine for a second that
Pseudomonas is kind of like a...
these Pseudomonas cells are kind of like a fleet of spaceships.
So, they're out in space,
they're trying to figure out
which other space ships are attacking them,
which ones are just kind of flying around and not bothering them.
So, let's say...
so, what I've just told you is that
if detects another spaceship that also has this weapon,
it's going to activate its weapon
and fire at it.
So, you can kind of think...
if you think about, you know, a cell,
how is it detecting that this other thing is there?
These are single-celled organisms,
they don't have eyes or ears
or the senses that we have,
that we can see what's going on around us.
So, we wondered, well,
maybe it's somehow sensing this
syringe puncturing it on the outside of its cell
or maybe there's some other way it has of
sensing some things going on around
only when these cells have a type six secretion system.
So, to distinguish between those two possibilities,
what I next decided to do
was to look to see if Pseudomonas
needs to be touching this other organism.
So, the syringe can only attack Pseudomonas
if the cells are directly contacting each other.
So, this is data from that same movie I showed you before,
where we looked at the type six secretion system turning on,
but now we're just looking at one of the fields
at the very end of the movie,
and instead of looking at in green,
I've colored it so that you can appreciate
the differences in expression a little bit better.
So, if it's red,
that means that there's more of the type six protein,
and the bluer it is,
the less there is of it.
And what I've also done is
I've highlighted which cells are contacting the competitor
and which are not contacting the competitor.
So, you can't see the competitor the way I'm displaying it here,
you'll just have to take my word for it,
but the cells up at the top marked with the "C",
those are cells that are directly touching
this competitor organism,
whereas the "NC" cells are not touching,
they're just only touching other Pseudomonas cells.
And what you can see is that
there's really not a huge difference,
they're all able to turn on their type six secretion system,
and in this particular image
it looks like maybe the ones not touching it
are even turning it on a little higher,
but let me show you what this looks like in a graph form.
So, here we're again looking at type six levels
on the y axis
and we're looking at time...
over time, how they turn on.
So, when the competitor
doesn't have a type six secretion system,
you don't see that much going on.
When it has a type six system,
now you see Pseudomonas is turning on,
this is exactly what I showed you before,
but the cells I'm showing you right here on this graph
are the cells that were directly touching the competitor.
Now let's graph the cells that aren't touching competitor.
You can see that they look pretty much the same.
So it doesn't seem to matter
if it's contacting this other organism or not.
So, this was a little puzzling.
How... how does it detect this at a distance if it's not...
the membrane isn't getting punctured,
it's not somehow feeling this other organism there, directly?
This was one of those moments
where I had to really kind of step back from all of this
and think hard about,
what is going on with Pseudomonas?
What is it experiencing in these interactions
where this competitor has a type six secretion system?
Up to this point in my PhD, actually,
I had really only been thinking about
Pseudomonas attacking other cells.
I hadn't really stopped to think about,
well, what if the other cells are maybe also
attacking Pseudomonas?
It wasn't happening so much that it was really striking,
because I would have noticed all my cells dying
if that was the case,
but I decided to take a look and see
if there was a difference in cell death of Pseudomonas
when the competitor had or did not have
its type six secretion system.
So, that's what this experiment is showing,
and so you can see here that
that's exactly what I saw.
There's more Pseudomonas cells dying
when the competitor has its type six secretion system
compared with when it doesn't
-- then, we basically don't see any cells dying.
So that's kind of interesting...
so, this competitor is also killing Pseudomonas.
So, the next clue is solving this puzzle
actually came in looking at the timing of all of these events unfolded.
So, here we're looking at three parameters.
The first one I'm showing you is
the cell death of Pseudomonas,
so, the same thing I just showed you
but now you can see how it happens over time,
and what you can see is that
the Pseudomonas cells start dying
almost immediately after you put them in contact
with the Burkholderia cell.
You start to see, even at 15 minutes,
there's already been some cell death.
The next parameter I looked at was
the increase in type six secretion system levels,
so that's just, you know,
plotted on top of the other one,
so you can see that type six levels start to go up,
but only after some of the Pseudomonas
have already started dying.
There's a little bit of a delay there.
Now, the last parameter we looked at
was the death of the competitor organism,
and that's shown here in these white squares.
So, here, this also kind of makes sense,
because you only start to see competitor dying
after some of the Pseudomonas cells
have turned on their type six secretion systems.
And so, you know,
this all kind of fit together
and it led me to wonder if,
maybe, somehow,
Pseudomonas could tell when its siblings were getting killed,
and somehow that was sending a message
that maybe it's time to start
mobilizing this defensive weapon and fight back.
So, before I show you the outcome of that experiment,
I'm going to step back a little bit
and tell you a little bit more about
what we know about how this system gets turned on.
So, I told you we don't know what signal turns it on
or when it gets turned on,
but we do actually know, already,
a little bit about the proteins that are involved in the turning on.
So, bacteria, of course, as I said before,
don't have ears and eyes,
but they do have these proteins on the outside of their cells
that can detect changes in their environment,
such as specific molecules
-- it's almost like smelling, a little bit.
And so that's this little cup that's pictured here.
So, a protein like this has to
interact with a molecule outside of the cell
and then what it does is it sends a signal into the cell,
and in this case this particular one
will then turn on the type six secretion system.
However, we didn't know what the signal was
for this particular sensor,
we didn't know what molecule it detected
out in the environment.
So I started wondering,
what if there was something in a dead cell
that was released that this sensor could then detect
and send a signal into the surviving cells
and tell them to turn on their type six secretion systems.
So, this was something I could test directly.
Just to remind you, you know,
when a cell dies,
it spews out all of its stuff that's normally inside of the cell
that can't get to the outside of the cell
because of the cell membrane,
but when it dies all of that gets outside,
it's kind of like its guts are getting spewed out.
And we call this process lysis,
so this is a cell lysing.
So, I could take a bunch of Pseudomonas cells
and I could lyse them,
I could, you know, grind them up
and take all of their guts
and then I could add that to happy, living Pseudomonas cells
and see if this sensor protein could detect this.
So that's what I did in this next experiment.
So, here, we're looking at
the activation of this sensor protein,
and you can see that when you just have cells
and you don't add anything to them,
it doesn't get turned on.
Now, when I took this Pseudomonas guts, this lysate,
and added it to these cells,
you can see they get turned on really quickly.
Immediately, you start to see this sensor get activated.
And importantly, when we took the guts
of the competitor organism,
the one that it's killing normally,
you don't see any difference.
So this makes sense
-- it doesn't really care if it's killing the competitor,
that's the goal, right?
But if it's getting killed,
if some of its siblings are getting killed,
that's when it knows,
okay, now it's time to make type six
so we can fight back.
So, this was really exciting,
I mean, we finally figured out a signal,
an environmental cue that's turning on this system
and is triggering the activation
of the type six secretion system.
And we could basically think of these cell guts
as a type of danger signal.
You know, when something terrible is happening
in the rest of the colony,
the Pseudomonas cells say, okay, this is dangerous,
it's time to do something about it.
So, let's put this together
and look at it how it would look in a population of cells,
well, I'm just showing you four cells, for simplicity,
but the green cells represent the Pseudomonas.
So, a few of them get killed by this competitor organism,
they spew out their guts,
there's some danger signal in there
that activates the rest of their population
that's then able to defend itself by turning on
its type six secretion system.
So, there's one more exciting twist to this story
that I want to share with you.
So, I told you that this sensor protein,
we know it turns on the type six secretion system,
but the other thing that it does
is actually turns on a bunch of other proteins in the cell.
It turns on about 300 other proteins
and actually, a lot of them,
we have no idea what they do,
and we never really knew why this group of proteins
was all getting turned on together
by this particular sensor.
But now we know that the sensor
is responding to a danger signal,
so we thought, well,
it would make sense for the cell to want to turn on other things
that are helping it protect itself.
So, what would happen if we
inactivated this sensor
and Pseudomonas no longer had the ability to sense danger?
Would there be other ramifications of this?
So, that's what I did.
So, I'm going to show you three movies, now.
So, in this case
we're looking at Pseudomonas cells dying,
so these are the red cells,
and they're going to be outlined in white as they die,
so remember that's just when they disappear from view.
So, the first movie on that side of the screen,
you can see are just normal Pseudomonas cells
growing with its competitor.
In the middle, we have Pseudomonas cells
where I've inactivated their type six secretion system,
so they've lost that ability to fight
with the type six system.
And over here are cells where
I've taken away their ability to sense danger,
so they no longer have this...
the sensor doesn't work anymore.
So, let's see what happens.
So, you can see the normal cells are doing fine,
they're growing, the cells in the middle,
they're not doing quite so well,
but over here, when they don't have the ability to sense danger,
they are just getting decimated by this other organism.
Pretty much every Pseudomonas cell
that touches this competitor
is getting wiped out and, remember,
before it was the one that was winning.
So now it's just lost its ability to defend itself and to fight back,
in a much more dramatic way
than just when it's lost type six.
So what this tells us is that this pathway
is more general.
It's mobilizing a bunch of different things in the cell,
beyond the type six secretion system,
that are helping Pseudomonas defend itself.
So, let's just think back to the spaceships for a second.
So, what I've basically told you is that
if Pseudomonas, if one of its spaceships in its fleet
gets destroyed by a competitor and it blows apart,
the other spaceships can detect that debris flying through the air,
and they know that it's time to
not only get type six going,
but also to put up their defenses,
and we don't know what those other things are yet,
that's ongoing work,
but potentially they're changing the outsides of the cells,
their membranes,
making them more resistant to things like toxins or antibiotics.
And so this is a really exciting new area,
there's a lot of new pathways that we can probably discover here.
So, how does...
what does this look like on the cellular level?
This is similar to what I showed you before.
Pseudomonas, if it gets killed by a competitor
-- and I'll just point out,
this killing doesn't have to be from type six
from a competitor,
we showed that any kind of killing where the cells explode,
where they lyse,
will activate this pathway --
they will then turn on the type six system,
as well as a bunch of other factors that are also unknown,
and then they're able to defend themselves.
So, this opens up a lot of new, fun, exciting research possibilities.
There's all these new things that we now
have an idea of maybe what they're doing,
but we don't know how they work at all.
So, let's come back to what I started with,
with yogurt.
So, this was my first glimpse into this
invisible world of these alien creatures
that are living all around us,
so, now, hopefully I've convinced you that
these bacteria, even though they're kind of primitive,
we might call them,
and they're single-cellular,
they actually exhibit some really sophisticated behaviors,
almost like an immune system,
where they can detect danger and then they know to fight back,
which is something that cells in our body do.
They also have this interesting ability to work together.
It's not just each cell for itself;
when they detect that something bad has happened in the colony,
they're all ready to mobilize and get it together
so that they can survive as a whole.
So this is an interesting way that
these single-celled organisms
can almost behave a little bit like a multi-cellular group.
But now, finally, of course,
what does this mean for disease.
So, I started by telling you that
Pseudomonas causes really, really devastating infections
for both patients with cystic fibrosis
as well as people who have chronic wounds.
And what can we learn from what I've just told you,
these interactions with other bacteria,
why does that matter for humans?
Well, actually, a lot of these infections
are what you call poly-microbial,
which means that there's a lot of different bacteria
that make up these infections.
It's not always just Pseudomonas by itself,
and it's actually usually not.
So maybe Pseudomonas is really good at
fighting with other bacteria in our bodies
and that gives it a little bit of an edge.
That's something that would be really exciting to explore.
Another thing is that now we know that
when Pseudomonas is exposed to this danger signal
when some of the cells die,
it goes into this other program of fighting back.
So maybe our immune cells
are killing a few of the Pseudomonas cells
or some of the other bacteria in our bodies,
and then that activates the cells
to go into this danger mode,
and maybe that protects them against antibiotics,
for example.
So, most antibiotics actually are
derived from other bacteria
-- we've just kind of stolen their molecules and used them to fight bacteria ourselves --
so it would make sense for Pseudomonas
to fight back against some of these other antibiotics.
So, I mean, a lot of these things are kind of years in the future,
you know, it's going to take a lot of work to figure out
if these things are actually happening,
but I think by thinking a little bit more about it
from the perspective of the bacterium
and from its interactions with other bacteria,
we've learned a really important component of its behavior,
and this can help us think more carefully
about how we attack these organisms
when they get into our bodies.
So, I just want to acknowledge
a bunch of other people that were really involved in this project.
I did not do all this work by myself.
First and foremost is my graduate advisor,
Joseph Mougous,
whose lab I worked in when I did all of this work.
And then I've listed some of the lab members
that were directly involved in this project.
And I also need to acknowledge
our long-time collaborator, Paul Wiggins,
who was very involved with helping us
get the microscopy set up
as well as analyzing a lot of that data.
Thank you very much for listening.