Hello, my name is Bob Goldman
and I'd like to speak to you today about intermediate filaments.
Intermediate filaments are found both as complex networks
in the cytoplasm and in the nucleus of vertebrate cells.
The naming of intermediate filaments may be of interest
to some of you as the name
is derived from the fact that they are midway in size
between microfilaments and microtubules,
so they have a diameter of 10 nanometers (nm).
So they were originally called 10-nm filaments
but now they're called intermediate filaments.
The intermediate protein family is complex
and in fact is encoded by one of the largest families of genes
in the entire human genome.
Typically, intermediate filaments have a
highly, structurally conserved central rod domain, here seen mainly in red,
and there are five major types of intermediate filaments.
The first four, here,
types I - IV, are found in the cytoplasm,
so those are the cytoskeletal intermediate filaments.
The characterization of filaments, structurally, is
based on their highly,
as I mentioned highly conserved central rod domain,
which is separated by these linker groups
called L1, L12, and L2.
And these separate the central rod into segments 1A, 1B, 2A, and 2B,
which are all highly alpha helical in nature.
The keratins, which are the first two types,
both the acidic keratins and basic keratins,
are complex because they form heterodimers
as the basic building block.
And these heterodimers then interact to form, ultimately, 10-nm filaments.
They're presently known to be over 50 keratin genes
in the human genome.
In the case of Type III intermediate filaments,
such as vimentin, desmin, peripherin,
and glial fibrillary acidic protein,
these form homopolymers.
And in fact,
vimentin will be the major topic for today
because it's the simplest type of intermediate filament protein,
having only one protein chain
which self-assembles into 10-nm filaments.
And then the Type IV intermediate filaments,
the last type in the cytoplasm,
consist of neurofilaments,
which are actually three protein chains
forming a complex mainly found in the nervous system
-- nerve cells of the brain and the spinal cord
and the peripheral nervous system --
and a protein called nestin,
which is expressed mainly during early development,
synemin, and alpha-internexin,
which are other forms of intermediate filament proteins
which also form complex polymers.
And finally, there's the Type V intermediate filaments,
which are the nuclear lamins,
which we will discuss in the second part,
or in the second lecture.
Within cells,
there's a remarkable complexity of intermediate filament networks.
Almost every cell type that one looks at has a different,
not only intermediate filament composition,
but also intermediate filament network formation.
You can see on the left a vimentin filament network
in endothelial cells with a
ring of filaments around the nucleus.
And then on the right you can see keratin
in an epithelial cell and you can see this
remarkably complex network radiating
all the way from the center of the cell,
where it surrounds the nucleus as a cage,
all the way to the cell surface.
Now, the functions of cytoskeletal intermediate filaments
are numerous and we won't have time to discuss all of them today,
but we'll discuss a few of them.
But let me summarize by saying they're
very important in the mechanical integrity of cells,
they're very important in the shape of cells,
they're involved in both shape determination and maintenance,
they're very involved in cytoskeletal crosstalk
and stability,
that is they frequently bridge between microtubules
and actin filaments,
they're involved in signal transduction,
adhesion -- both cell-cell adhesion
and cell-extracellular matrix adhesion,
they're involved in cell motility,
and they're involved in the positioning
and tethering of organelles within the cytoplasm.
So let me just briefly go over the
structural features of intermediate filaments
and how they're built in the cell,
how they're assembled.
So the typical intermediate filament
is made of a dimer,
so two protein chains,
for instance in the case of vimentin,
since it's a homopolymer, it makes homodimers
and two dimers interact to form a
head-to-tail assemblies
which are in exact parallel and in register.
So the central rod domains actually line up
precisely so that they form a coiled-coil
and then we have the tail at one end
and the head domain, or the N-terminus and the C-terminus,
at the opposite ends of the dimer.
And then we can see,
this is just a blowup of this type of schematic,
and you can see that so-called Helix 1 and Helix 2
are separated by this region in the center
called the Linker 12 (L12) region.
And then we have the C-terminus,
which is relatively unstructured
and then the N-terminus at the other end
which is also relatively unstructured
in the case of cytoskeletal intermediate filaments.
During polymerization of,
in this case, human vimentin,
or any form of vimentin
because it's a highly conserved protein,
we can see in the far left panel
the formation of dimers.
And the two globular regions at one of the dimer
actually are the C-terminal domains.
And if you then go the next step in assembly,
and this is all done in vitro using bacterially expressed protein,
you assemble what are called Unit Length Filaments (ULF).
These are actually dimers
which interact with each other
and there are 8 of them,
so there are 16 dimers which form a 32-protein chain
Unit Length Filament.
And these ULFs,
which are approximately 50 nm in length,
actually interact with each other in tandem
to make longer and longer filaments.
There's two interacting at the lower left side
of this slide during the assembly process.
And then finally they go,
once you get long linkages of ULF,
you then go through a radial compaction,
which you can see in the last panel here.
And then,
it can be seen even in a more schematic way
in this next slide,
where you have Unit Length Filaments
interacting with each other,
linking in tandem,
to form longer and longer
intermediate filaments,
which then go through a super compaction
to form mature intermediate filaments.
The other important point about filament structure
is that it's apolar because the dimers,
which are in parallel in a register
with respect to the two protein chains,
interact in a head-to-tail fashion
which is called longitudinal assembly of dimers,
and they also interact laterally
in an oppositely-oriented and staggered way
to that you wind up getting tetramers
with opposite polarity.
And building up this structure into a 10-nm filament
gives you what's called an apolar filament
because it doesn't have a plus end
and a minus end like microtubules do,
and like actin.
So, now I'd like to go on just to tell you
how we prepare intermediate filament proteins from cells.
And, essentially, if you look on the left panel
it just shows you a blowup of a region of a cell
which was fixed and stained with antibody.
It's a fibroblast
and you can see the complex vimentin network.
In the middle panel, this cell has been extracted
with a solution containing high salt,
detergent,
and DNase I.
The DNase I is used to degrade chromatin,
it also helps to remove actin
from the preparation,
and there are no microtubules in these preparations.
And if you look on the next panel, you'll see that,
at higher solution
-- higher magnification in the EM,
in the electron microscope --
that in fact, these are intermediate filaments
and there's virtually no other structure
in the former cytoplasm of these cells.
And on the right panel, you can see the major band,
which is vimentin, and just above that
there are the nuclear lamins, which we'll speak about later
or in the next talk.
And finally, at the top of the gel
there's several proteins which are associated
with intermediate filaments such as nestin,
in this case,
and also a protein called plectin.
Because intermediate filaments
are so easy to isolate,
and in fact because they are pelleted
at low speed and almost virtually all of the protein
(intermediate filament protein)
in the cells like fibroblasts can be pelleted
at low speed and virtually no
protein is detectable in the supernatant,
for many years intermediate filaments were thought
to be very stable polymers
inside of cells.
Another property that led to their,
or leads to the,
I think, misconception that they are very stable,
is the fact that in an old paper
by Paul Janmey many years ago,
he demonstrated that under
conditions of stress
and strain in vitro,
in fact intermediate filaments
remained undamaged as you can see in the panel
on the left,
whereas microtubules and actin filaments
ruptured at relatively low shear stress
in this preparation.
On the right side, you can see a drawing
from a recent article in the literature
which shows that intermediate filaments,
in addition to being
undamaged by high shear,
can actually be stretched
up to three-and-a-half times
their normal length.
And once they become stretched to this extent,
they become very difficult to break.
So in fact this is an atomic force microscope
depicting what happens to an intermediate filament
when you stretch it,
and basically this is called,
their properties are known as
strain hardening properties.
And, for these two reasons,
both the fact that they're very extensible,
they can become very rigid when stretched,
and that they seem to be very insoluble
in biochemical preparations,
they were thought for many years
to be less dynamic that their cytoskeletal counterparts.
So let me go on and tell you a little bit
about the dynamic properties of intermediate filaments.
So, when we turn to live cells,
we realize that intermediate filaments
are very dynamic in vivo.
And in fact if you do a photobleaching experiment
where you look at recovery
of photobleaching with FRAP analysis,
you can see on the left side
a cell that was microinjected with
rhodamine-conjugated vimentin
and then in the next panel (B),
immediately afterwards that fiber
was bleached, photobleached.
And then in C D and E
you can see the photobleach recovery coming back.
And if you analyze in the bottom panel,
the recovery of fluorescence,
you'll find that it's equal all along the length of the fiber
as it recovers, which indicates that, in fact,
the filaments are not polar,
that they can actually exchange subunits
all along their length.
Another aspect of intermediate filament dynamics
can be seen very dramatically
when cells are attaining a shape in culture.
So, if you take trypsinized cells,
which are rounded,
placed in suspension,
and then place them down
onto a glass coverslip or slide,
and then watch them during the spreading process
by fixing and staining at different times
up to three or four hours,
you will in the top slide,
a lot of very small spots or particles,
and these are enriched in vimentin.
These cells were fixed and stained
with vimentin antibody for immunofluorescence.
In the middle slide you can see,
in fact, that as the cell spreads,
small filaments or short filaments,
which we called squiggles,
form and then these squiggles appear
to link end-to-end as we saw in vitro
to form longer and longer filaments
until the fully formed mature network
is formed in spread cells.
Movies of this reveal how dynamic this process in fact is.
This is a later stage of spreading,
but if you watch the particles in the movie
and the short filaments or squiggles,
you'll see that they're very dynamic:
they're moving bidirectionally
towards the edge of the cell.
And they can both move
towards the nucleus and away from the nucleus
as you can see.
Now, the types of movements
and the speeds can be very fast,
up to 1.5-2 micrometers per second (um/s).
This indicated,
of course that they were probably moving
along structures like microtubules.
And in fact, those vimentin particles,
especially moving at high rates of speed,
can be colocalized
when you fix and stain using double immunofluorescence.
And you see here microtubules
in the middle in red and
vimentin on the left in green,
during early spreading.
And then
in the overlay on the right, on the upper set of pictures,
you will see that there's
a very nice overlap between the
vimentin particles and microtubules.
And in fact, if you look on the bottom two panels,
you'll see that most of the particles
colocalized with the molecular motor kinesin,
which of course is a microtubule-based motor.
And without giving you all the data,
I just want to summarize by saying
intermediate filament particles move bidirectionally
because in fact they're associated
with both dynein and kinesin.
So they can move both
towards the plus end and the minus end of microtubules,
and they move at relatively high rates of speed.
They seem to slow down when they form filaments,
and they continue moving until filaments meet each other,
short filaments, meet each other,
and then become longer filaments.
So mature IFs seem to be made
from particles, squiggles,
linking up to form long IF,
and depending on microtubules-based motors,
and in fact, there's evidence for microfilament-based
(or actin-based) motors
participating in this process.
Now that I've demonstrated that intermediate filaments
are dynamic,
let me go on to say that intermediate filaments
are also very important in cell shape.
And this is no better depicted than in the
Epithelial to Mesenchymal Transitions (EMT)
that take place both in early development
and in the conversion
of tumor cells to metastatic tumor cells,
or benign tumor cells to metastatic tumor cells.
And these changes in cell shape
are always accompanied
by an upregulation in vimentin expression.
Typically, epithelial cells, which you see on the left,
express keratin,
and when they go through the epithelial-mesenchymal transition
they begin to express, and
eventually vimentin is expressed to very, very high levels.
So vimentin expression is a hallmark
of the cytoskeletal changes that take place during this EMT.
On the next slide, we can show that vimentin
actually can induce changes
in epithelial cell shapes very rapidly.
So if we look at the top panel,
we can see in MCF7 cells,
which is a human breast epithelial cell,
which is round, there's a single isolated cell,
and then in the middle panel it's a cell
just observed two hours after it was microinjected
with bacterially-expressed vimentin
and then fixed and stained.
So in a very short period of time,
this cell changes its shape,
just due to the expression,
in the form of protein injection
into the cytoplasm of an MCF7 cell.
Eventually that cell will recover its rounded shape,
because in fact vimentin expression
can't be sustained.
On the bottom panel,
we can see vimentin on the left
and the result of silencing vimentin expression
using shRNA.
And you can see the cells that are silenced
by the green color,
which is using GFP as a marker in this case.
And you can see the cells that are not transfected, in fact,
are normally shaped,
like a mesenchymal cell,
and the ones that are transfected and silenced,
you can see have changed their overall shape
to an epithelial cell.
So it looks like both the reverse of the EMT,
the mesenchymal to epithelial transition,
and the EMT itself,
can take place
just by controlling vimentin expression.
So cell shape seems to be very dependent
in this case on the intermediate filament composition of cells.
I should mention that microtubules and actin
are not affected in these experiments.
Now I just want to mention that
not only do you get shape changes in epithelial cells
when you express vimentin in them,
but vimentin also increases epithelial cell motility.
So here we see in the upper left panel
vimentin being expressed in an epithelial cell colony,
this is an MCF7 colony
which you can see in phase contrast below,
and this is just a few hours after transfection,
you begin to see expression of vimentin
as you can see in green.
And 24 hours later,
you can still vimentin networks in these epithelial cells,
but now all of the cells
in the lower right panel
have moved away from the colony's edge.
So this tells us that vimentin expression
will alter the cell-cell adhesive properties,
the cell-substrate adhesive properties,
change the shape,
and cause the cells to migrate
away from the colony,
again showing the importance of vimentin in motility.
And finally,
I just want to emphasize
the requirement for vimentin
in motility
by looking at,
on the left, are normal vimentin-containing fibroblasts.
In the middle,
these are fibroblasts
which have no vimentin,
in fact they don't express any intermediate filaments,
they're from the original vimentin knockout mouse.
And if you look, you can still see in those cells in the middle,
lots of activity around the surface,
these are surface ruffles
but no directional movement.
They can't move anywhere.
And you can see on the bottom
of each of these movies,
you can see the tracks made by the cells
over a 10-12 hour period.
In the right panel,
all of the cells are normal,
expect for this one at the tip of my finger,
which in fact is a cell
that is expressing a dominant-negative
mutant of vimentin
which you can see at the top.
It's basically a small piece of vimentin
which contains the initiation site
of the coiled domain plus the N-terminus,
and this acts to disassemble filaments in cells.
And you notice that one cell that's expressing
this mutant protein
is the only one in the field,
virtually, that doesn't move,
as you can see by the tracks below.
We then decided to look
at vimentin intermediate filaments in moving cells.
What do they look like?
So we just take moving fibroblasts,
in case it can be mouse fibroblasts
or human fibroblasts,
they look virtually indistinguishable,
and you can see in the top left the typical
fan-shaped moving fibroblast.
You can see, only now looking at vimentin,
you can see vimentin particles
are present in the lamellipod region
and at the leading edge the lamellipodium contains,
in the upper right panel,
you'll see lots of closely packed vimentin particles.
But filaments only seem to be forming,
or assembling,
in the rear region of the lamellipodium.
But the protein is certain there
in a disassembled state.
Below, in the top panel,
the top three images,
you can see a serum starved fibroblast
with vimentin filaments radiating all the way out to the edge.
And then it's very easy to induce ruffling in such cells,
as has been done for many years,
in many laboratories,
just by adding serum back.
So you take serum deprived cells
-- the cells were starved for 48-72 hours --
vimentin typically goes all the way to the surface,
the cells don't move, they're very quiet,
but as soon as you add,
actually within 10-15 minutes
after you add serum back,
on the bottom panel you can see the rapid formation
of lamellipodia and you can see rapidly,
that the same configuration that we see in normal moving cells,
we can now see in these
serum add-back experiments,
where you see vimentin particles
enriched in the leading edge
and then sort of a gradient of filament formation
back towards the nucleus.
Then we asked the question:
does the disassembly and retraction
of vimentin intermediate filaments
following serum addition involve phosphorylation?
And to do this, we used a phospho-specific antibody
which we received from the Inagaki lab in Japan.
And you can see in the top three images on the left,
vimentin on the left is
stained with vimentin antibody,
in the middle it's an antibody
against phospho-serine 38 (pSer-38),
which is in the N-terminal domain of vimentin.
And on the right is an overlay,
which of course just gives you the same pattern.
On the bottom, we can see a cell which was
fixed and stained
within a very short period of time,
within a couple of minutes after we added serum back.
And you can see now in the middle panel,
vimentin is on the left,
the middle panel shows what the Ser38 phospho-specific antibody looks like,
and you can see now
that the entire cell lights up,
is fluorescent (all these are indirect immunofluorescence images),
on the right is the overlay,
but a blowup of the region seen in the first panel,
panel D on the left,
is seen over on the right side here
where you can see that actually it looks like
the vimentin is retracted back from the surface
and that it's left behind particles and short filaments.
And here we can see,
in an SDS-acrylamide gel
and a western blot,
that in fact phosphorylation,
within one minute after adding serum,
goes up very significantly: many, many fold.
If you look at the zero time versus one minute,
you'll see the vimentin is rapidly phosphorylated;
in fact it only takes seconds
to become hyperphosphorylated under these conditions.
So this suggests that vimentin phosphorylation
is involved
in the local disassembly
and retraction of filaments
away from the cell surface in these regions.
And I should mention that these are the regions
which seem to initiate lamellipodia formation.
We also used another trick
to look at vimentin phosphorylation,
and that is using a form
of the small GTPase signaling factor called Rac1,
which is a member of the Rho family of GTPases.
It's very important,
we know from the actin literature,
that this particular signaling molecule
is very important in reorganizing actin
in the lamellipodial region.
And to do these experiments we used the photoactivatable form of Rac1,
provided to us by Klaus Hahn
from the University of North Carolina.
And this photoactivable form of Rac1
will become active
only when you shine 458 nm light on it,
and when we expose cells
which are expressing this
to global irradiation,
that is we just irradiate the whole cell
or a whole culture dish,
you can see that in fact
the intermediate filaments seem to retract
back away from the cell surface
under these conditions,
as in fact lamellipodia form.
And this is accompanied, again,
by a huge increase in the phosphorylation of Ser38.
So normally, you grow the cells in the dark
and when you shine light on them
they activate this Rac1.
And Rac1, it turns out, binds to PAK,
and PAK is a kinase
which is known to phosphorylation Ser38
in the vimentin head domain.
On the upper left,
we see the result of
a local irradiation experiment
with Klaus Hahn's PA-Rac1.
This cell, if you look beginning
now, was serum starved
and then we photoactivated
that small region
which you see in the movie.
But the important thing here
is to look at the edge of the cell,
and you'll see that when vimentin pulls back,
now,
the ruffle in fact forms.
So vimentin seems to disassemble
and retract from the cell surface.
And you can see this
even in regions which have very thick cables
of vimentin near the cell surface.
In the second upper panel,
you can see actually it looks like a steel cable
unwinding
after photoactivation of Rac1 in this small region.
And as this unwinds,
you can see the formation of short filament
and squiggles which seem to spin off
from this cable,
and it's only then that we really start to see
active lamellipodia forming.
So, in both cases,
it looks like vimentin actually
is stabilizing the cell surface,
and only when you disassemble and dismantle it,
presumably by phosphorylation,
and disassembly, and retraction away from the cell surface,
do you get lamellipods forming.
Now we did one other series of experiments
to show the importance of vimentin.
And that is using a peptide
called the 2B2 vimentin peptide.
2B2 is a mimetic peptide
which is derived
from the 2B region of the central rod domain.
And with our colleagues
Harald Herrmann and Ueli Aebi, we discovered that,
in fact, if you expose vimentin filaments,
on the left side panel A,
to this peptide
and leave it for a relatively short period of time
in vitro (this is an in vitro experiment),
you disassemble intermediate filaments
into these short pieces
which in fact are unit length filaments.
So using this 2B2 peptide,
we carried out some microinjection experiments.
In the top panels are just a repeat
of what the peptide does to intermediate filaments:
disassembles them
into small pieces
which in fact are unit length filaments.
And if you look at cells following injection of this peptide,
you see on the lower left figure D,
a cell which was injected
which had been grown in the absence of serum,
you can see that, in fact,
after injection the filament network
is retracted back towards the nucleus
and there are many particles
and short filaments in the region
between the nucleus and the cell surface.
And in fact, you also induce lamellipodia to form,
as seen here by one of the leading edge markers,
Arp2/3, which is an actin-associated protein.
And here we see a control cell
which was injected with scrambled peptide,
and you can see there's no retraction
back of the network, there's no disassembly.
On the next slide, I just want to show you a movie
of a cell which was injected with a
relatively high concentration, 2 micrograms/mL,
of the 2B2 vimentin mimetic peptide
and the filaments retracted.
This was a GFP-tagged vimentin-expressing cell.
And you can see that the filaments were retracted back towards the nucleus.
And then we,
in the absence of serum,
we get basically uncontrolled ruffling all over the surface.
And this cell cannot move anywhere, it just sits there.
So this fits nicely with our vimentin-null cells,
where we saw ruffling all over the surface.
In this case,
all we did was to inject the 2B2 peptide
in the absence of serum.
And then we did another experiment
using very low concentration,
0.5 micrograms/mL in the injection buffer,
and this cell was injected
just to the left side of the nucleus,
and this cell was in low serum
(1.5% serum)
and it was not moving at all
and there was no obvious ruffling.
And we injected just to the left of the nucleus.
And when you do this,
you see at the point of injection, very close to that region,
we induced a ruffle.
And if you look carefully,
you can see that this cell even begins to migrate
in the direction of the ruffle,
up towards the left of the screen.
So in summary, these observations tell us
that vimentin intermediate filament assembly
and disassembly are required
for the proper mesenchymal/fibroblast-type cell locomotion.
The regional and local disassembly of intermediate filaments
into their structural building blocks,
both the particle and squiggles,
modulates the formation of lamellipodia.
And conversely,
the regional and local assembly of intermediate filament networks
appears to be involved in inhibiting the formation of lamellipodia,
and what this means to us is that
it actually acts as a brake or a stabilizer
to the cell surface.
Intermediate filament disassembly and assembly appears,
in turn, to be regulated by kinases and phosphatases,
many of which are involved in signal transduction.
I'd like to acknowledge and thank
all of the members of my own laboratory
who have contributed in recent years to these studies,
and our collaborators,
both here and overseas,
and especially to our granting agencies,
the National Institute of General Medical Sciences,
the National Cancer Institute,
and Hannah's Hope Foundation,
for supporting our work.
Thank you.
