Hello.  I'm Julie Theriot.
I'm a professor at Stanford University.
And for the second part of my
iBioSeminars presentation today,
I'd like to delve into the details
of the mechanics and dynamics
of rapid cell motility.
Now, to get us started here,
we see two images of a particular kind of
very rapidly moving cell
that comes from the skin of fish, called a keratocyte.
Over on the left, we have a fixed cell
that's been labeled for filamentous actin,
so you can see the distribution
of the actin cytoskeleton throughout the cell.
On the right, we have a video image
of the same kind of cell moving
in the same direction
that the fixed cell was at the time that it was
frozen with formaldehyde.
And what I'd like to talk about particularly
is the way that all of the different molecular machines
that have to operate in the context
of a moving cell
are able to coordinate with one another
in order to get this incredibly smooth and elegant
gliding motion
that you see in rapidly moving cells.
So, focusing a little bit on the details
of these molecular machines...
these are things that have actually been intensively studied
by biochemists and cell biologists
for many decades,
and at this point we're pretty familiar
with many of the proteins that are necessary
and, to some extent even maybe sufficient,
for generating the forces and dynamics
that we see associated with cell motility.
And in general, for large-scale cell biological processes
like motility,
the proteins within the cell
that are responsible for that kind of behavior
arrange themselves into nanomachines
where a number of different proteins will work together.
Now, one nanomachine that's very important
in cell motility
is the assembly of branched actin structures
at the leading edge of a motile cell,
and in the first part of this presentation
I talked in some detail
about how these different components have been identified
and how they're thought to work together.
But this particular little nanomachine
of growing actin filaments
pushing against the membrane r
eally only operates
in this very front part of the cell,
just a few microns back from the plasma membrane
at the very leading edge.
Another nanomachine that's also been quite intensively studied
and is very important for motility
is shown here.
This is the adhesion that actually binds the cell to the substrate,
enabling it to generate traction
so that it can move itself forward,
and here too a lot of the protein components have been identified,
and in this particularly beautiful example
from Clare Waterman's lab
even the spatial orientation of all the different components
have been very carefully measured
with respect to one another.
But this particular nanomachine
that makes this well-organized adhesion
really operates only in, again,
a very small zone right at the back of the cell.
And in order to understand
the overall process of cell motility...
when the cell is moving forward,
it's incredible how well all of these things
seem to be coordinated with one another.
The cell looks like it's
gliding over the substrate without changing shape,
even though it's got to be assembling actin filaments like mad
at the leading edge in order to push that membrane forward,
and then it's got to be building and then disassembling
the adhesions at the rear.
All of these things have to be coordinated
to happen at the same pace,
so that the left side of the cell
moves at the same rate as the right side of the cell
so it can go straight,
so that the front extends
at exactly the same rate that the back retracts
so that it can appear to move forward
without changing its size.
So it's a really fundamental and interesting question, I think,
about how all of these different nanomachines
can coordinate with one another
over the entire span of the cell,
which is tens of microns across,
so many of orders of magnitude larger
than, certainly, the individual proteins
that make up the machine,
but even any of these individual assemblies on their own.
Now, in order to address these kinds of questions
about how you get large-scale coordination
in moving cells,
we've taken a lot of advantage
of these fish skin cells,
so I'd just like to give you a bit of background
about where they come from
and how we isolate them.
It turns out that pretty much all fish
and many other amphibians
have a bilayered epidermis,
and the basal layer of that epidermis
is made up of these cells
that seem to be specialized
for very rapid wound healing.
So in the context of a fish,
when there are scales
coming out of the flank of the fish,
the epidermis actually wraps around the scale,
so if you go in with a pair of blunt forceps
and pluck a scale off of a fish
and then put it in culture,
the scale comes along with just a little bit of skin
right along the edge.
Now, the fish is not happy about this,
but it can grow a new scale,
and actually turning over the scales
is part of its normal skin regenerative process,
so it doesn't cause any significant damage.
But in the meantime, in culture,
now we have this scale
with a little bit of skin that's just wrapped around the tip of it,
and the cells at the edge
of that little bit of tissue
essentially think that the surface of the fish
has been wounded,
and so their response
is to try to start crawling outward
to close that gap,
and so you can see here,
both at the bottom and at the top of this particular scale,
these big clumps of cells
that start coming off first as epithelial sheets
and then eventually break up to make
all these little individual cells that seem to go
buzzing around more or less on their own.
Now, here we can look at that same process
at higher magnification,
here at the edge of a sheet
as if first starts coming off,
and then here, again,
this is what the isolated cells look like
once they break away from that epithelium.
And I hope you can appreciate
from looking at these movies
why these cells are such a spectacular model system
for studying the mechanics of cell motility.
They move extremely fast.
They are among the fastest animal cells that are known.
And they also have this very characteristic,
stereotyped geometry,
which is very well illustrated by the cell over here.
It has a very large, flat,
broad lamellipodium, which is its motile organ,
and then it carries all the rest of its organelles,
its nucleus, its Golgi apparatus,
even all its microtubules,
it carries in this little package of a cell body
that it just keeps right behind it.
The movement is very fast,
the movement is very persistent,
the movement is essentially unidirectional
- that is, they don't really have a very strong tendency
to change.
So they're essentially moving at steady state.
The fact that the movement is so regular
and is so stereotyped
makes it very good for biophysical analyses
of the kind that I'm going to be delving into today.
This shows you a little bit more detail
about how the cytoskeleton is organized in these particular cells,
and in this beautiful
structured illumination micrograph
taken by my student Sunny Lou,
you can see the distribution of actin filaments, shown in green,
myosin-II filaments, that is, contractile myosin, shown in red,
and we're going to come back to the role of myosin
in this coordination quite a lot.
And then, also, you can see labeled in blue
the focal adhesions
that are adhering the cell to its substrate.
And diagrammatically,
shown down here looking at the cell from the top,
you can see the actin filament branched network
is primarily oriented towards the front of the cell
and then in the back of the cell,
down here at the bottom,
you can see the filaments have become rearranged
to form these parallel bundles
that are being organized by the myosin.
Now, if we take a cross-section
through one of these cells and look at it sideways,
it looks like a baseball cap,
where the lamellipodium is very, very thin and flat,
only about 200 nanometers from top to bottom,
and the the cell body can rise up several microns high.
As these cells move forward,
they follow the same general steps
of actin-based cell motility
that are shared by many other motile animal cells
and also a large number of
eukaryotic unicellular organisms,
such as amoeba.
Overall, the first thing that has to happen
is the cell has to establish polarity,
that is, it has to distinguish its front from its back.
And then it has to be able extend the leading edge,
and in this cell,
as in many other motile cells,
the force for that extension
is thought to be driven by actin polymerization itself.
As it's extending the new leading edge,
it needs to form new adhesions to its substrate,
and at the same time be able to contract its rear,
to bring the cell body forward,
and then retract and disassemble
the adhesions that are at the back.
One of the fun things about keratocytes
is it's actually possible to demonstrate in these cells
that all of the components necessary
for that whole cycle of motility
are contained only in the lamellipodium
- you don't actually need any contributions from the cell body.
And that was first proved in this really classic
1984 experiment by Ursula Euteneuer and Manfred Schliwa,
where they sliced little bits
off of the lamellipodium of a keratocyte,
leaving the cell body behind,
and were able to see that those small fragments
of the keratocyte lamellipodium
were able to continue to translocate on their own,
and move at just about the same speed
and just about as persistently
as the whole cell was when it was intact.
And this movie shows a modern reenactment of that experiment
that was done by my student Erin Barnhart.
Here you see a fragment that's been
isolated away from its cell body
that's nothing but lamellipodium,
with all of these dynamic cytoskeletal structures inside of it.
And when the movie plays you can see
it's crawling along very nicely,
it's got a clear front and a clear rear.
It's about to crawl over a little piece of schmutz on the coverslip
that actually is going to separate
this crawling lamellipodial fragment into two bits.
The membrane connection between them resolves
and they're both able to crawl off on their own,
until they eventually get sliced into some unit
that's too small to movie.
So, with this system
we have favorable geometry
-- it's very, very simple, very reproducible from one cell to another --
and we also have a fairly simple self-contained system
where we know that it's only the
components of the lamellipodium
that are necessary for persisten motility.
So, from analyzing the behavior of these cells
over many years,
my group has been able to identify and specifically
measure the contributions of all of the
different force-generating elements
that help the cell to move,
and those are all illustrated here.
At the leading edge, we have actin polymerization,
that's shown in red,
which pushes the membrane outward,
and that polymerization is actually opposed
by tension in the plane of the membrane.
And that tension serves
both to act as the barrier
that the growth of the actin filament pushes against,
and also, in fact,
helps to coordinate the motion
over the entire surface of the cell,
as we'll see in a little bit more detail.
Now, there's also adhesions that have to contribute,
and those adhesions are assembled in the front
and then disassembled in the back.
And then there's contractile forces that are driven by myosin,
primarily acting at the back.
Because that myosin contraction is happening
at the back and squeezing the cytoskeleton inward,
that actually creates a forward
hydrodynamic fluid flow
that squirts fluid through the meshwork of the lamellipodium
to deliver components up to the front of the leading edge.
Now, as I said, we've been able to measure
the quantitative contributions of each of these different forces
within the context of this very simple kind of motile cell,
and what I'd like to do over the next few minutes
is share with you a couple of highlights of things
that we've learned
that are somewhat surprising in retrospect
as to how this coordination is able to work
over such a large scale.
So, in order to make these kind of measurements,
we've had to develop methods
both for measuring behaviors of the cells very precisely,
and also methods for perturbing the behaviors of the cells
so that we could understand what aspects
were dependent on what other aspects.
So, one example of a kind of measurement that we can make,
shown here in a movie from Cyrus Wilson,
is tracking of the overall motion of the actin network
using a technique called speckle microscopy
that was originally developed by Clare Waterman.
And in this technique,
the cells are electroporated with a small amount
of a fluorescent dye that binds to the actin filaments,
but a sufficiently small amount that
rather than labeling the whole cell uniformly
you instead see this little speckly, textured pattern.
And then if we take the movies
as the come off the microscope,
which is what you see up top,
and then convert them into a different frame of reference,
where instead of looking at the cell in the lab frame of reference,
we now translate everything
as if the cell had a GoPro camera attached to its head,
and we're looking just right down at the cell itself
from its own point of view.
Then you can see, now, quite a bit more detail in terms of...
both in phase contrast and then with this fluorescent speckle microscopy,
how everything inside the cell is moving.
So, looking at the fluorescent speckles,
I hope you can now appreciate
that the whole actin network is sort of raining downwards
from the front towards the back of the cell
in the cell's frame of reference,
and is also being gathered inwards on the side,
down at the back here,
where the myosin is able to contract it.
Now, once we can do that frame of reference shift
and look at things from the cell's point of view,
Cyrus Wilson was able to work together with
Gaudenz Danuser and people in his lab,
including Lin Ji,
to develop quantitative methods for
measuring the flow of all of this material
in the lamellipodium very precisely,
and was able to map, overall,
how the motion of these particles
depend on the location inside of the cell.
So, here from the lab frame of reference,
what you can see is that the motion of the particles
with respect to the substrate is actually very little,
that is, the actin is actually pretty stationary
with respect to the glass that the cell is crawling over,
except at the very back where you see this massive
inward sweeping driven by myosin.
However, if you look from the cell's point of view,
you see there's a low of flux,
a lot of turnover of constantly treadmilling actin network,
where it's assembling at the front
and then disassembling under the cell body.
So, in order to try to understand this process
of assembly and disassembly a little bit better,
we also wanted to be able to
manipulate the behaviors of the cells,
to perturb them so that we could look at
how they responded to changes in their environments.
And one kind of perturbation
that was actually very informative
for understanding how these things couple together
was worked out by Erin Barnhart,
specifically where she was able to
change the degree of adhesivity,
or the degree of stickiness,
of the substrate that the cells were crawling over.
And what she found is that when cells
are on a sort of moderately sticky substrate,
they're able to move exactly the same way
that they would on glass
or in fact on the surface of an aminal.
When they're put on substrates that were less sticky,
so, ones that were more slippery,
you can see the cells actually change shape
- they become a little bit rounder
and you can see the accumulation of these characteristic
pleats in their lamellipodium,
where the inward flow of the actin
is now actually faster than the motion of the cell,
so it's really spinning its wheels
because it can't quite get a grip on its surface.
Now, most interestingly, I think,
when they're put on high adhesion substrates,
their behavior changes very dramatically,
and instead of now having this steady state motion
where they glide forward uniformly,
they now do this completely crazy thing
of putting out small bits of lamellipodium
that seem to sweep sideways.
And we're in the process of trying to figure out
how all of these different things work,
but I hope you can appreciate that even this
very, very simple motile cell
that seems like, you know, sort of the stripped down,
minimalized, like, soapbox derby version of a motile cell,
even this is able to extremely
expand its behaviors depending on cues
that it's getting from the environment,
in this particular case,
mechanical cues in the form of the stickiness of the substrate.
So, the several examples
that I want to tell you about
mostly have to do with surprising roles for myosin.
Now, myosin, of course,
we're mostly familiar with in the context of skeletal muscle,
where it's able to contract sarcomeres
by sliding stable arrays of actin filaments
relative to one another.
Myosin in non-muscle cells,
myosin II in non-muscle cells,
also acts as a contractile protein,
and its assembly is regulated,
so the monomeric state of the myosin
in non-muscle cells
is folded up on itself,
and then when it receives an appropriate signal,
the phosphorylation of the
regulatory light chains on myosin
enables it to extend outwards
so that it can then assemble into bipolar thick filaments
that are much more similar to the organization inside of muscle.
And so we look at the myosin in keratocytes...
what you can see is there's very little myosin at the front,
where the actin is actively polymerizing,
and instead there's actually quite a lot of myosin
right at the back,
and in particular it's in these very bright spots
right on either side of the cell body.
Okay, so bearing all that in mind,
now let's go back to this question of assembly and disassembly.
One of the things we're able, now, to measure,
that we can track the motion of the actin
and know where all these other elements are located,
is Cyrus was able to actually figure out
how to make a map of,
quantitatively,
how much assembly and disassembly of the actin cytoskeleton
takes place over the context of the whole cell.
And what he found was that the assembly
is very much biased towards the leading edge,
specifically right in the middle of the front of the leading edge,
which is very much what we'd expect,
but the disassembly, unexpectedly,
was found in these two very intense spots
right on either side of the cell body.
And Cyrus recognized that
those locations were actually
very similar to the locations where we found myosin.
Now, we can also look at the distribution
of mysoin II in these cells.
Here, using a cell that's been transfected
with myosin light chain carrying YFP.
And now, in the cell frame of reference,
you can actually see the motion of these little speckles,
which now are mini-filaments of myosin,
that is, bipolar filaments that have been assembled.
And what you can see is they seem to
stick onto the actin network
and then they rain backwards
towards the back of the cell body,
essentially riding on the actin network
until you get right all the way to the back,
where they then start forming these contractile cables,
pulling in the actin network
and making these bundles that go from one side to the other.
Now, it's suggestive that the spatial distribution of myosin II in these cells
is exactly the same as the foci of disassembly,
and we can also inhibit disassembly of actin in these cells,
for example, by inhibiting the motor activity of myosin.
So, we hypothesized that
the myosin itself is actually contributing
to the disassembly of the actin cytoskeleton
by buckling and breaking and ripping apart the actin filaments
using, directly, its force-generating capabilities.
And one of the strongest pieces of evidence
in favor of that hypothesis
is this very nice experiment done by Mark Tsuchida,
where instead of using moving living cells,
he used extracted cytoskeletons.
So, if you take a keratocyte
as it's crawling across a substrate
and then sneak up on it with a little bit of detergent,
you can get the membrane to dissociate,
leaving behind only the insoluble parts of the cytoskeleton,
so, the assembled actin filaments,
whatever actin binding proteins are bound to them,
but having now gotten rid of all soluble components,
including actin monomers, ATP, everything else.
Mark was then able to label those extracted cytoskeletons with phalloidin
to see where the actin filaments were,
and then add back ATP
to those extracted cytoskeletons.
That added ATP was able to then
activate the myosins that were left behind,
so he could see if,
in this sort-of semi in vitro environment,
myosin activity could actually
drive destruction of the actin filament network.
And so that's what you're going to see in this movie
- this is an extracted cytoskeleton, now.
When the movie starts to play,
the ATP is going to be added, and you can see the network
just melted right in the back,
right where the myosin is located.
And you can also see that by comparing
this before and after shot,
where the blue shows the places where
the actin network disappeared
when the myosin was activated.
So, although we normally think of myosin
as actually contributing to contraction,
in this context, at least,
it seems like one of its more important functions
is destroying the actin network
when it gets to the back of the cell.
So, putting that together,
we came up with this idea for
myosin driving overall network treadmilling in the lamellipodium,
as illustrated here,
where initially, towards the front,
there's very little myosin in the network,
it's hard for the myosin mini-filaments
to diffuse through the network,
as it's actively assembling and
essentially pushing everything backwards,
but a few fo them get ahold of the filaments,
and then as they start contracting
they start rearranging the actin filaments
to form more parallel structures
that are of more favorable geometry for force generation by myosin.
After that goes on for a while,
by the time you get to the back of the cell,
the actin is now all in parallel bundles
rather than a dendritic network,
and the high concentration of myosin
that's able to accumulate there over time
is enough to rip that network apart.
So, overall, we think that's a major mechanism
for determining what the distance is
from the front of the cell to the back of the cell.
It's just determined by how much time it takes
for myosin to incorporate,
and for myosin to destroy the network.
Now, so far, I've been talking about keratocytes
as if they're all exactly identical,
and certainly that's one of the useful things about them
is that they're similar, but,
like any other organism,
if you look at the closely enough,
you'll see they actually have very interesting differences
from one another.
So, this shows a gallery of a whole bunch of different keratocytes
that were collected by Kinneret Keren and Zach Pincus,
showing that from even one scale
of a particular individual fish
you can have quite a lot of variation,
both in terms of the size of the individual cells,
and then also their shape.
So, some of them are quite round
and some of them are quite elongated
and almost canoe-shaped.
And to summarize a lot of work,
what we've been able to find is that
these cell-to-cell shape differences
are both persistent
-- so, if you follow a cell over time,
it keeps its shape --
and they're also cell-intrisic
-- so, if you disassmble all the actin cytoskeleton
and let it grow back,
it will grow back to exactly the same shape it was before.
And from quantitative analysis of those kinds of measurements,
what we found is that these shape differences
are essentially extremes of a continuous spectrum,
where some cells are very large and wide and smooth,
and these are the ones that are canoe-shaped,
and those are also the fastest moving cells.
And some of the other cells,
such as the ones over on the left side of this gallery,
and rounder, they tend to be smaller,
their leading edges look kind of rough,
and they also move kind of slow
and in a less persistent manner.
And so we call the wide, smooth cells,
we call those coherent cells,
and the smaller, narrower, rough cells,
we call decoherent cells.
But overall, we can find every behavior in between,
so we think the differences that we see among these shapes of the cells
basically just has to do with the
exact quantitative amount of all of these
force-generating elements they have present
within their cytoplasm
that balance each other in slightly different ways
to give overall cell shapes.
And overall, we can
quantitatively measure these variations in cell shape,
particularly identifying principle modes of shape variation,
and the mode I've been most frequently referring to
is this second mode,
where we go from the wide cells to
the rounder, more D-shaped cells.
And our modeling that we've done together with Alex Mogilner,
together with experimental work,
has suggested that really the variation in those shapes
is primarily due to
the back-and-forth force balance
between actin polymerization pushing on the membrane
and membrane tension restraining the actin polymerzation.
And the short version is that
cells that have very forceful actin polymerization
are able to assume this coherent, wide lamellipodium,
and cells that have weaker actin polymerization,
for whatever reason,
are the ones that end up in the rounder D-shape,
and also move slower.
Now, if that idea is true,
then it should be the case that we can
take an individual cell
and somehow increase or decrease
its overall rate of actin polymerization,
and have that individual cell
change across this entire shape spectrum.
And so that experiment actually was done by Greg Allen,
where the method he chose to change the rate of actin polymerization in the cell
was simply to lower and raise the temperature.
So, here we're going to watch a movie
of a cell and as it moves along
you can see it's fairly slow,
it's got this more sort-of D-shaped pattern,
and Greg is first going to start
dropping the temperature,
and as the temperature drops
you can see the lamellipodium gets rounder and rounder,
and the cell is moving slower and slower.
And at this point,
when we get down to just about 7 degrees Celsius,
he's now going to start raising the temperature,
and as the temperature comes back up
you can see the cell not only goes faster and faster,
but it also assumes a wider shape.
And so following cells like this quantitatively
using a variety of different metrics,
what we were able to find is that, in fact,
individual cells can explore
this entire range of behavior
that we see in the context of cell-to-cell variation,
and it's all consistent with the idea that
the primary determinant of the shape of the cell
as well as the speed of the cell
is simply how fast the actin is polymerizing.
Now, another really fun thing about keratocytes
is they have the ability to sense and respond to electric fields,
and this is something they actually have in common
with many other motile cells.
Pretty much any motile cell
that you put in a DC electric field
will choose either the anode or the cathode
and will head in one direction.
This was first described for keratocytes
by Cooper and Schliwa back in 1986,
and Greg was able to replicate this
using a setup that he built in our lab
to look at motion of individual cells
as the electric field was switched from one direction to the other.
So, in this movie, we see a cell
that's moving along in an electric field
that is oriented in this direction
-- you can see the orientation of the electric field
and also the magnitude listed over here --
and you can see the cell is following that line.
Now, when the label turned red,
that was when the field was flipped around,
and you can see the cell has turned
and is now heading back in the other direction.
And now, once again, the orientation of the field is flipped,
the cell flips back around,
and now heads back in the direction that it has been told to go.
So, the cells obviously...
even though I've been emphasizing
how good they are at balancing forces
across the front of the cell and between the front and the back of the cell,
they are able to also initiate imbalances in their forces
so that they're able to turn.
So, Greg Allen looked a little bit more deeply into the mechanism
of how they turn
and he found a couple of interesting things.
So, for example,
if we look at a single turning cell
-- in this case we're looking at it both in the lab frame of reference,
like it looks on the microscope,
and then in the cell frame of reference,
where we've repositioned everything
to see things in the cell's point of view --
you can see there is a physical asymmetry
in a turning cell,
where the part that's on the inside of the curve,
that is, the part that's going slower,
has this very round shape that we call decoherent,
and it's characteristic of slow motion.
And on the outside of the turn, the part that's going faster,
has a much more elongated, coherent shape
that we associate with fast motion.
So, this variation that we see,
both at the population level
and in individual cells as the temperature
is raised and lowered,
can actually also happen even within the context of an individual cell,
where one side can end up being much faster
than the other side.
Now, there's a number of different things
that contribute to this asymmetry,
but at this point you won't be surprised
that one of the major things that contributes
is the left-right distribution of myosin.
So, I showed you before that the myosin
accumulates in these two spots on either side of the cell body,
and those two spots aren't always necessarily equal in size.
So, this is an example of a cell
where there's a relatively low amount of myosin
in the spot on the left side,
and a much higher amount of myosin in the spot on the right side.
And looking in the movie,
you can see the consequences of that,
here with the myosin labeled:
the side that has more myosin is moving faster
and is therefore sweeping around the outside part of the turn.
Now, there's of course other elements
that also contribute to this turning
-- there are differences in adhesion,
there are differences in traction force,
there are differences in rates of actin polymerization --
but they all seem connected to one another,
and specifically connected to one another
through the mechanism of myosin action.
So, to summarize what we think is going on here,
we think as the cell begins to turn
the actin network flow starts to flow...
instead of just flowing straight back to the back of the cell,
starts to flow at a slight angle.
Because the myosin is carried along
on that flowing actin network,
the myosin then accumulates
at the outside corner of the cell.
That myosin is able to contract faster,
pull in that side of the
back of the wing of the lamellipodium
and help the cell sort of flip around.
At the same time, because the myosin
is depolymerizing the actin filaments,
it's generating a gradient of G-actin,
such that there's more actin available for polymerization
on that same side of the cell
where you have more myosin
and where you have faster motion.
And all of these things, we think,
are able to actually feed back on one another
in a positive sense,
such that once a cell starts making one of these turns
it actually is able to continue to make that turn
in a persistent way
actually for quite a long time,
until it's then forced to turn in another direction.
So, so far what I've shown you
is that in the context of these very simple
steady-state moving cells,
myosin in the back of the cell
is actually doing a tremendous number of exciting things
that help the cell move overall and that help coordinate the front and the back.
It's helping to disassemble the network
and it's also specifically contributing to
left-right aymmetries that help the cell to turn.
Now, the keratocytes are fairly unusual cells
-- they're unusual in their appearance,
they're unusual in the steadiness of their motion --
and so it became very natural then for us to ask
whether similar mechanisms might be at play
in more complicated cells
that are doing more complicated tasks.
And one of the very interesting cells that's been well-studied
in the context of motility
is the neutrophil, the human neutrophil,
a white blood cell,
whose job is to go after and engulf the
bacteria that are invading the human body.
And you can actually isolate neutrophils
from your own blood
and watch them crawl around and eat things
-- it's really very gratifying --
but also we have a call line,
a neutrophil-like cell line,
that is able to behave much like a neutrophil
but that we can also transform
and look at protein distributions
in moving versions of the cell.
So, Tony Tsai in the lab
decided that it was time
to actually break out of the keratocyte mold
and start looking at motion in more complicated kinds of cells,
including neutrophils,
and just to show you how dramatic
the behavior of these cells is,
this is one of these HL60 cells
that's been put in a chamber with some Candida albicans,
which is a pathogenic yeast,
and what you see as the movie loops
is the neutrophil starts off over on the right side, here,
and then runs across
to this little pile of yeast
and is able to actually phagocytose and engulf them.
So it really is a very
neutrophil-behaving tissue culture cell.
Now, looking at the shapes,
they're obviously much more complicated than the keratocytes,
and putting in labels for the actin,
which is shown here in green,
the myosin, shown in red,
and then DAPI to stain the nucleus in blue,
what you can see is that the shapes are not only
much more variable than keratocytes
but also much, much more dynamic.
All of the cytoskeletal elements
are drastically rearranging themselves
over periods of just a few seconds
as the cell is crawling around.
So, although this makes it a more interesting question,
I think,
to figure out what is going on in terms of
the mechanics and dynamics of this behavior,
it's also a much more challenging problem
as far as quantitative analysis goes.
So, Tony so far has been able to work out
a number of quantitative techniques
to be able to break down this complex motion
so we can actually watch changes over time.
So, for example, he can track the edges
of one of these moving neutrophils
and then go back and calculate,
for the cell as it's moving,
how much of the cell is extending in every time frame,
in this case, every couple of seconds,
how much is retracting,
calculate the overall area of the cell
as well as the extension of its leading edge,
and the amount of retraction of its body.
At the same time, we can look at labeled proteins inside the cell,
and obviously one of the ones we're most interested in looking at
is myosin,
and look at the overall fluorescence intensity distribution
and see how that changes as the cell moves around.
And what you'll probably be able to see
is that the myosin localization itself is also very dynamic.
It's often in the back of the cell,
sometimes in these bright spots,
but then those bright spots will disassemble,
the myosin will become more uniform
or will move to a different location within the cell.
And tracking all those things quantitatively over time,
what Tony was able to find
was that when a cell was speeding up,
that the accumulation of the myosin
in response to that change in cell behavior
happens later, happens about 12 or 15 seconds
after the initial movement of the leading edge.
So, whereas in the keratocytes
it seems like the actin and the myosin
were always in perfect balance
so that the cells were always gliding forward,
in the neutrophils the story is a little bit different
- it seems like the actin is calling the initial shots
and the myosin is then reacting.
So, as the cell initially extends,
it then activates the accumulation of myosin at the rear
to pull the back together.
So, instead of gliding,
it's doing more of an inchworm motion.
How does this affect cell turning?
Well, the same way that Tony was able to come up with
quantitative metrics for the localization of myosin,
he was also able to come up with
quantitative descriptions for the orientation change of the cell
and then the left-right asymmetry of the myosin
with respect to its immediate orientation.
And you can already see the answer, actually,
quite dramatically,
with this maximal intensity projection,
where this is just a low magnification movie
of a cell that's undergoing a sinusoidal path,
and we're looking now only at the myosin
that's accumulating at its rear,
and what you can see is that the myosin
always accumulates on the outside of the turn,
and when it changes its direction,
the myosin then changes which side of the cell it's on.
So, this is actually very reminiscent of the keratocytes,
where we saw, again,
the myosin on the outside of the turn,
except in the case of neutrophils, again,
there's a little bit of a time lag
between when the turning initially starts
versus when the myosin accumulates on the outside of the turn.
So, here too it looks like
the actin is calling the shots in terms of direction,
the flow direction of the actin
that's changing
then gathers the myosin on the outside of the turn,
that then causes disassembly of that cytoskeleton
in a way that enables the neutrophil
to essentially swing its tail around
so that the whole cell is now oriented in the proper direction.
So, overall, comparing these two stories...
if you look at a movie of a keratocyte versus a neutrophil,
they seem like they're behaving rather differently,
but what we understand when we dissect
the mechanics and the dynamics of this behavior
is that they're strikingly similar.
In particular, I've shown you
recent data that myosin accumulates at the cell rear
due to this actin network retrograde transport
and mediates actin network disassembly
in a way that is then able to coordinate
not only front-rear motion of the cell
but also give you asymmetries
that can lead to turning.
And both of those things seem to happen
in very similar ways
in both the keratocytes and the neutrophils.
Now, one last little hint I want to leave you with is,
as I showed you before with this movie,
we can force keratocytes
to behave more like neutrophils
in terms of changing their shape all the time
if we simply change the environment,
and particularly if we put them on
very, very sticky substrates.
So you might wonder, can we do the flipside;
can we make a neutrophil behave more like a keratocyte,
into something that will have a steady-state motion
where everything is happening at the same rate?
Well, it turns out when you take a neutrophil
and you confine it to a very narrow channel
and then watch it move over time,
these guys now are moving at steady-state.
The speed is an absolute constant,
the distribution of myosin is constant,
it's always found at the rear,
and they'll continue to move like this
for many tens of minutes
without any obvious changes
in terms of their overall shape or overall behavior.
And taking this same movie and now making a kymograph of it,
where the time is moving from top to bottom
and each one of these slices
is an individual frame of this movie,
you can see, really, how constant this speed is over time.
So, not only can we force keratocytes
to become more crazy
and change their direction like neutrophils,
we can also force neutrophils
to behave more in a steady-state like keratocytes.
And moving forward, I think the combination
of our ability to both measure and manipulate
these different kinds of motile cells
will help us to understand
the general principles that govern motility
for all animal cells
that use actin polymerization to drive their movement.
So, as an overall summary,
I think the main point here is that
actin and myosin have to cooperate
in order to make cells move,
not just in order to generate force,
but also just in order to do things
like steer them and determine their shape.
And we found that myosin plays actually
several very unexpected roles at the rear of cells,
not just contributing to contraction,
as we might have expected,
but also contributing specifically to actin network turnover
and to asymmetries that lead to cell turning.
And overall, we've been very surprised
by how similar the mechanics are
between fish skin keratocytes and human neutrophils.
So, obviously, there have been
a lot of very talented people
who have contributed to the work that I just described,
and I've listed here the many members of my group
who have contributed to different aspects of cell motility projects
over the last 15 years or so,
and also our wonderful collaborators.
And I'd particularly like to mention this in this context
our very productive long-term collaboration
with Alex Mogilner,
who has done a lot of the quantitative physical modeling
that has driven the thought processes behind our experiments.
Thank you.
