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Hi, everyone.
So let's just quickly review one of the critical experiment
that we talked at the very end of last lecture.
This is the Rothman experiment, where they wanted
to understand whether fusion occurs between two populations
of vesicles--
in this case, a cis-Golgi vesicle and a
medial-Golgi vesicle.
And they did that by generating two vesicle
populations.
And in a wild-type vesicle population, the enzymes that
catalyze this specific glycosylation reaction exist.
And so it's expressed in the localizing these vesicles.
And in the mutant vesicles, this is derived from the
E-strain that specifically lack that enzyme.
A protein here, inconveniently named the
G-protein, is expressed.
So only when the two vesicles fuse, then this particular
G-protein can be glycosylated.
And therefore, they can detect this using a chemical assay.
So therefore, the assay is that in order
for this to be detected--
the signal to be detected --the two population vesicles
has to fuse.
And it turned out that, if you just add the two population
vesicles, the normal vesicles and the mutant vesicles, they
actually don't yield the glycosylation of the
substrate, suggesting that just having these two
population vesicles is not enough.
They don't spontaneously fuse with each other.
However, when the cytosol is added, then the fusion occurs.
And that argues that the cytosols are critical, and
there's stuff that is contained in the cytosol that
drives this reaction to go forward.
And from this assay, they then went on and purified factors
that are in the cytosol that supports
these fusion reactions.
And they isolated two proteins.
One is called NSF, and the other one is called SNAP.
And now we know that NSF and SNAP are the two proteins that
are required to break up the assaults, the snares that are
already associated.
The snares are the proteins that bind to each other very
tightly and then drag the target membrane and the
recipient compartment membrane together very close so that
they can fuse with each other.
And in order to break up those snares, you need ATPase.
This is the--
these two proteins uses the energy of ATP to unwind, to
dissociate those snare complexes.
And we've come across a lot of acronyms, and this is
certainly not all of them, but I found this, sometimes it
helps to understand where the acronyms come from.
It helps you to remember it.
Like COP1 is Coat Protein 1.
This you will probably remember, but others it
doesn't really help you.
ARF is the small G-protein that we talked about that's--
it's critical for budding off vesicles from the Golgi.
And its name comes from ADP ribosylation factor.
It came from a history of how this protein was discovered
that we won't get into.
This was before this was realized as a small G-protein.
But other small G-proteins, like Rabs is Ras-related
protein, the B family.
It doesn't really help you.
Snares are a SNAP receptor.
What is SNAP?
SNAP is soluble NSF attachment protein.
NSF is NEM-sensitive fusion protein.
So it doesn't really help you.
I mean, you can start guessing how they
got discovered, right?
I mean actually, the snares were discovered as a protein
that binds to SNAP, right?
SNAP and NSF are the two proteins that are required to
dissociate snares.
Of course, they have to bind to snares first.
It tells you something about how they were discovered, but
you have to remember some of them, the
critical ones, I guess.
OK, so I think by now I have spent a lot of time here in
this, in my section of Bio 42 going over a critical event in
the cell, which is how to distribute proteins.
Cell makes a lot of proteins.
They send the proteins strategically
to different places.
And so we've systematically gone through the different
signals that is embedded in the genetic information and
then is read out by cells.
And then that results in different
destinations of proteins.
And I've given you the big picture.
What are the most important things.
Always remember there are exceptions, but we're going to
call it a day here.
So now we're going to move on to a very
exciting topic, cell skeleton.
What are cell skeletons?
Cell skeletons are proteins inside the cells that gives
the cell its shape, and it helps cells to move around.
And it also helps to move organelles and
vesicles inside the cell.
So its name, skeleton, probably comes from one of its
obvious phenotypes that has been
studied at the very beginning.
It comes from--
determines the shape.
It gives the cell its rigid--
many of these are rigid structures, so it sticks out
and gives cell a shape.
And you will realize that when we go through these couple of
lectures, you'll find that a lot of these structures are
not only rigid, but also dynamic.
So when the cytoskeletons change, the cells move around.
So if you keep adding the bricks on one side of the
cell, it actually will push the cells
to move in one direction.
And they also serve as highways.
These are filaments, so they are long protein polymers, and
they can serve as roads for trafficking.
OK?
So these are the three functions, and there are three
major types of protein filaments or cell
skeletons in cells.
The best understood ones are the actin and the
microtubules.
Sandwiched in between is something called
intermediate filaments.
So these are filaments.
These are like ropes, OK?
And initially, this designation is made by looking
at how thick or how big the filaments are.
The microfilaments or actin filaments are the smallest
filaments among the three.
And the microtubules, as you will hear from the next
lecture, are much larger structures.
It's like a hollow tube formed by polymers.
And so this is small, that's big, and then there's a bunch
of filaments that fit falling between the
size, and they're called--
essentially called intermediate filaments.
So we're going to first talk about the function of the
actin, so this lecture is essentially exclusively on
actin And then we'll talk about
microtubules in the next lecture.
Actin has multiple functions, and we're first--
the best understood example of actin's function is in
striated muscle cells.
And the first part of this is going to
be on skeletal muscles.
And skeletal muscles control voluntary
actions, like limb movement.
So in our body view--
many of you are going to go to medical school --you'll learn
that there are three types of muscles.
There are the skeletal muscles, which are the muscles
that are attached to the bones.
And the muscles usually attach across a joint.
So the tendon of your biceps is attached to your forearm
bones, and then the other tendon is anchored on here.
And then when the muscle shortens, it will cause a flex
of your forearm, OK?
And the other two type of muscles are cardiac muscles,
which is what makes your heart, and the smooth muscles,
which are found in blood vessels and also your GI
tract-- gastrointestinal tract.
And these skeletal muscle is only one that gives rise to
voluntary actions.
That means if I want to flex my forearm, I can do that.
And cardiac muscles and smooth muscles don't do that.
It's kind of hard to--
if you want to contract your guts, it's kind of hard to do.
You can probably try to make it do it, but not on a very
fast time scale.
And we're actually going to talk about why this is.
It has everything to do with what are the cytoskeleton
structures found in these muscles and how differently
they're organized.
OK, so then, what are muscles?
So skeletal muscles are made of muscle fibers.
It's kind of confusing why some are called muscle fibers.
Others are called myofibrials.
So muscle fibers is a cell, OK?
It's a large cylindrical cell.
And so just remember it's--
you can just remember it's muscle cells.
Of course, tissues are made of cells.
But these muscle cells are long and cylindrical in shape.
And the one thing that is special thing about these
muscle cells is that they are multinucleated, meaning that
they contain many nucleus, muscle nucleus.
OK, and that's formed by fusion events during
development.
So during development, there are--
fusion-competent cells will come to fuse with the
[? founder ?]
cells.
And after many fusion events, the mature muscle forms with
many nuclei.
But the focus of today's discussion is this structure
here that is found inside in the
cytoplasm of muscle fibers.
And these are called myofibrials.
And myofibrials are fibers.
They're made of cytosolic proteins.
They're made of cell skeleton proteins, as you will hear.
They are very long, and you can see that within each of
the muscle fiber, muscle cells, they are packed with
these myofibrials.
And the myofibrials are the contraction apparatus, because
if you purify myofibrials, isolate them from the muscle
cells, now you've lost all the nucleus and other stuff, and
all you need is myofibrials.
And if you add ATP and calcium, this
myofibrial will contract.
So this is the contraction apparatus.
And myofibrials contain two different kinds of protein
filaments, thin filaments and thick filaments.
And the thin filaments are made of a major
protein called actin.
And the actins are polymers.
They are made of single monomeric structures.
The monomers weighs about 43 kilodaltons, and they assemble
into a fiber in a head-to-tail fashion.
And therefore, so if you look at each of the individual
actin monomers, this is not--
actin doesn't come as a globe.
It's an asymmetric structure that has a
tail and has a head.
And when--
the assembly process dictates that only the head and tail
interactions are allowed, and therefore you don't get
something like head-to-head assembly.
This is the only way to make actin filaments.
So as a result of this polarity, the two ends of the
actin filaments are different from each other, right?
This end consists of the tail of the monomer, and that end
consists of the head of the monomer, if you wish.
So therefore, these two ends have different properties, and
sometimes they're called plus end and minus end.
Other times, just to confuse you, they're called barbed end
and pointed end.
But that just means that these two ends are different.
And we're going to talk about why these polarized structures
are very critical for its function.
Oh, and then the actin is the major component of the thin
filaments, but there are also other
proteins that are present.
But actin is the bulk of the stuff.
The structure of actin has been solved, so you can see
why-- understand why.
This is the monomer, and the assembly occurs this way, so
that the monomers pile up into these filaments.
And you can see the plus end and the minus end, they are
very different from each other, from 3-dimensional
structures, as you can tell.
Here you've got a bunch of beta strand, and then where
the alpha helixes turn.
And over here, you've got a couple of alpha helix
structures.
So they're different, OK?
So don't have to really remember the structure.
OK, so that's the thin filaments.
And the other major component of myofibrials is called the
thick filament.
This is a much larger protein.
It's made of six polypeptides, and it sums up to over half a
million Dalton.
And it comes with two heavy chains, two light chains, and
two regulatory light chains, so that's an essential light
chain and a regulatory light chain.
And the thick filaments, the myosin, the heavy chain, forms
a dimer and then it has a head structure and
then the tail structure.
And the tail structure of the two heavy chains intertwined
with each other, forming this filament structure, gives rise
to the filament.
And the head domain is ATPase that binds through ATP and has
the intrinsic ability to hydrolyze ATP.
And this is also the part that binds to actin in forming the
cross bridge, forming the link between actin and myosin.
And the two light chains are right here in the neck region.
And I'll show you in detail that this is a fascinating
molecule because it can generate movement.
And the movement is generated by hydrolyzing the ATP and
somehow converting that chemical energy into movement
right around the neck region.
So through this conformational change, if you wish, that is
driven by the ATP cycle, it generates movement.
We'll delve into that a lot more in the next slide.
So it's a molecular machine that converts chemical energy
into physical displacement, OK?
But first, let's look at it in a more subcellular resolution,
and then we'll delve into the molecular details.
So the head binds to the actin and hydrolyzes it, and the
tail is the coil region, and it forms these filaments.
And then there's a higher order organization as well, so
each of the myosin, which contains the six polypeptides,
get assembled.
And they're assembled in the skeletal muscle.
They're assembled in this bipolar filament.
So the tails, or there's multiple ones that are coming
together, and then from the other side, there are also
filaments formation.
So at the end, there's a line in the middle that anchors
this bipolar filament, and it becomes much clearer here.
So one of the ways that people have studied the skeletal
muscle, this is back in the days, in the early days, in
the '60s, when electron microscopy becomes available.
And muscle is the most beautiful structure that you
can imagine for electron microscopy students, because
when you do this, the myofibrials essentially repeat
very stereotyped structures, and that gives a lot of power
for the scientists who do the experiment.
Here, essentially, in the EM, what you're seeing is that
there are alternating light bands and dark bands,
and they are units.
And then this is from--
then the scientists give them names.
This is called the Z disc or the Z line.
And here is an M line.
M line means the middle, and then eventually goes to here.
It's a mirror structure, and you encounter another Z line.
And then the myofibrial essentially is a repeat.
Many of these from Z line to Z line, these units repeated
against each other again and again.
OK, and this unit was named--
was called a sarcomere.
The sarcomere, essentially, is the unit.
It's the elementary contractile apparatus.
When the muscles contract, the sarcomere shortens, and all
the sarcomeres shorten by the same extent.
And then the myofibrial, as a result, shortens.
And that's when the balls move or the muscles contract.
OK, let's take a closer look why do these light bands and
dark bands really translate into the thick filaments and
the thin filaments that we've talked about.
So the Z disc--
also the sarcomere in humans is about two
micrometers in length.
Two microns.
Bacteria is about one micron, OK, the larger bacteria.
So this is a very tiny structure.
So a myofibrial is a repeat of many of these hundreds and
thousands of these sarcomeres.
OK, let's see how it works.
So the thin filament covers the region of the light bands,
but also goes into the dark bands, OK?
And the thick filament is only found in the dark bands.
So we said that the myosins assemble into this bipolar
fashion, and you can see that they are anchored--
the thick filament is anchored by the M line.
And the thin filament is attached to the Z disc.
OK, so when the muscles contract--
the sarcomeres contract, the length of the thin filaments
and the thick filaments don't change.
It's not like the filaments are breaking down or getting
added, more things are getting added.
But it's the sliding action between the thin and the thick
filaments that shortens the sarcomere.
OK, if you imagine that these thin and the thick filaments
interdigitate, and they go into each other, and the more
overlap they have, the shorter the sarcomere structure is.
So in the relaxed state, when the muscle is not contracting,
they're barely, let's say, overlapping with each other,
these two filaments.
But then when the contraction occurs, the sliding motion
results in more dramatic overlap between the two
filaments, and that causes the shortening of the sarcomere.
Right here, let's say if the M line is immobile, the thick
filaments are anchored, and the thin filament is sliding
towards the M line, and then you can see that the
sarcomere is short.
Is that clear?
This is kind of important.
OK, and of course, initially this was not--
these are cartoons.
Initially, when people work doing these studies, they were
looking at the light bands and the dark bands.
And you can imagine that when the contractions occurred,
which band is becoming shorter?
Anyone?
Light band.
Great.
OK.
I think you guys got it.
So that means--
so we want to understand the molecular mechanism of
contraction.
So we know that from these initial
studies that the thin--
that it's the sliding of the thin filaments against the
thick filaments that generates this motion.
So how does this gliding occur?
OK, what is the mechanism of this gliding?
And we know that the thin filament is made of actin, and
the thick filament is made of myosin.
So how do actin and myosin interaction generate this
sliding motion?
That's the critical--
this is the most important slide for this lecture, so pay
attention now, if you haven't been.
So and not just because this, but also because this is a
fascinating phenomenon.
As we said that, you've heard of a lot of biological
reactions, enzymes reactions.
You add enzymes, you add a substrate.
The enzyme does something to the substrate.
You create another two, some substrates, OK?
It's a product of a reaction.
That's OK.
We've heard many examples of that, but this is different.
This is way cooler than that, OK?
So essentially, this is generating
movement, OK, right?
I mean, so when the most primitive organism starts back
in the days, it's a bunch of enzymes doing their thing.
But now all of a sudden, the organism needs to move, OK?
And this is how movement is generated, how chemical energy
is transformed in a form of physical displacement of a
macromolecule.
So that is fascinating stuff.
So it's--
so first of all, it's a cycle.
So we could start in any of the steps within the cycle.
It doesn't matter.
But let's just say we start from this stage here.
So we said about the myosin, the myosin head.
Remember, the heavy chain has these two heads, and the head
has the ability to bind ATP and then also to bind actin.
So here, the polymer is the actin polymer.
And then this is the thick filament, out of which one of
the myosin head is sticking out, and it
binds to the actin.
And this state is called Rigor state.
Rigor is the Latin word for stiffness, so this interaction
is pretty tight.
It's rigid, and they're just stuck with each other.
So this is state number one.
Start with the actin and the myosin interact with each
other, and in this case, there's no nucleotide that
binds to the head of the myosin.
And then the ATP comes in, binds to the myosin head, and
this causes the link between the myosin and the actin to
become much looser.
And then this causes the head to dissociate from the actin
filament, OK?
And then the magic occurs.
The ATP is hydrolyzed.
The ATP is hydrolyzed because the myosin heavy chain, the
head is ATPase.
It cleaves the chemical bond, and this ATP hydrolysis causes
a conformational change around the neck region of the myosin.
Now the myosin head looks different than before.
So in the state number one, the myosin head is bent.
So then the actin-- the actin is here, so when the ATP
binds, this link gets looser.
And then when the ATP is hydrolyzed, the neck sticks
forward, OK?
And then it sticks forward, and it binds to another
monomer on the actin filament.
And this is not the same one, but it's the next one on the
filament that it grabs onto.
So initially, it comes here, and then the conformational
change allows it to reach for the next actin monomer, OK?
And then the other consequence of ATP hydrolysis is that now
it's no longer ATP, right?
It's ADP and the inorganic phosphate.
And the inorganic phosphate first comes off.
And when this comes off, this interaction between the myosin
head and the next actin monomer becomes
much tighter, OK?
And the next step in the--
when the binding becomes much stronger, it's called the
cocked state.
It's like a gun being cocked.
And then ADP dissociates.
The ADP dissociation causes the myosin head--
the heavy chain head --to return to this nucleotide-free
state, right?
Because now the inorganic phosphate is gone.
The ADP is gone, so it's a nucleotide-free state.
And the nucleotide-free state goes back to here.
So the head needs to be bent again, right?
So and that generates the motion.
OK, and this is called the power stroke.
Now it has gone back to this stage with
one difference, right?
The one difference is that, instead of binding to this
actin monomer, it is now bound to the next
one along the polymer.
So this is, again, it cycles through this process again, so
then it continues to grab the next actin filament--
the next actin polymer on the--
monomer --on the polymer.
And therefore, it's basically dragging, tugging the actin
towards one direction.
OK, so and you can see here that the key is it's a cycle.
The key is that the ATP is being
hydrolyzed by the ATPase.
And there's energy that is generated by this, and the
energy is transformed into a shape, a change of shape in
the myosin heavy chain.
And this change of shape is then coupled onto the
interaction cycle between the myosin and the actin.
And this concerted movement essentially generates the
shortening of the muscle fiber.
OK.
We have a video that will make this a little easier to
remember, but I didn't like this one, actually.
This was from a few years ago, and I think the timing of some
of the events were not accurate.
So I found another one this morning, and I'm
going to use this one.
OK, so now this is essentially--
oops.
It's kind of bright here.
Maybe I can turn it off.
Double?
No, that's not right.
Overhead is probably the better one.
OK, so here in--
this is the state that we started with, right?
So this is the Rigor state when the myosin heavy chain is
bound tightly to the actin monomer, and the heavy chain
is in a nucleotide-free state, and the ATP started to bind.
And then that loosens this interaction, and the ATP is
hydrolyzed.
And this chemical reaction is converted into a
conformational change of this head.
And now it reaches out into extended form.
And then the second consequence of the hydrolysis
is that now it allows the--
I guess in this movie, this is step one.
It allows the binding onto the next monomer.
And this dissociation of the inorganic phosphate tightens
this interaction.
Now it's in a cocked state.
Then the ADP dissociates, and this generates--
this is the power stroke.
And so it pulls the filament to this direction.
And then now you need the ATP to, again, loosen the
interaction.
The cross bridge detachment occurs in this case.
And what's in the background here is the calcium ions,
which we'll talk next.
And you can see that it hydrolyzes the ATP, and that
causes the conformational change.
And now it can start binding again.
And then you can also see that--
OK, this is getting too fancy.
You are welcome to watch this.
I can't talk fast enough to explain all the events that
occurs there.
OK, I'll give you the link.
So that was actually a quite nice video someone made.
OK, so let's think about this a little bit, right?
So we said that this occurs in the skeletal muscles.
Skeletal muscles generate voluntary movement.
The voluntary movements are pretty accurate and fast.
Go ahead.
Two questions.
First of all, in the video only one of the heads binded.
What does the second head do?
Sorry.
OK, so the question is that in the video, there's--
only one of the head binds.
What's the other one?
So the other one is shaded.
It means that that's the other conformation.
So that is only illustrating one of the heads.
So there's two possible conformations.
One is the bent, and the other one is the more
extended form, OK?
But it doesn't mean that only one head binds the one time.
So, as you can probably see that here in the thick
filaments, there are many heads.
It's made of many myosin chains.
So if only one head binds, and one end let go of it, it'll
spring back, right?
So in this case, you should imagine there are many hands
that grabs onto the rope, and one hand
probably needs to do this.
But then in the meantime, there are many other hands
that are grabbing onto the same rope.
OK, what's the second question?
Secondly, it seems like this mechanism is only useful for
[INAUDIBLE]?
How do you make the muscle go back to normal?
Oh how do you go back to-- the muscles go back to normal?
Great question.
We're just going to be talking about that, yes.
OK, so the question is, OK, so now within this cycle, the ATP
binding is critical.
And the ATP hydrolysis gives the power, OK?
But the ATP is not what is being regulated
in this cell, right?
The ATP level in the cell is kind of constant.
If you have it in your breakfast, it's all going to
be pretty good, since ATP is always saturated there.
OK, so what regulates the contractility?
What makes the muscles contract?
Calcium?
Calcium, yes.
Calcium ions.
Excellent.
OK.
So the muscles, all the skeletal muscles are
innervated by neurons.
They're really--
they're the soldiers.
The neurons are actually the commanders, right?
So the neurons regulate the muscles by
regulating their calcium.
So first of all, the calcium is required for the
contraction, but why?
So in low calcium, actin--
this actin filament is not naked.
It's actually decorated by a set of proteins.
And one of the proteins is called tropomyosin.
Tropomyosin is these long strings.
This tropomyosin just wraps around the actin so that it
just covers all the binding site between the heavy chain
and the actin.
OK, so basically, it blocks the interaction between the
heavy chain and the actin.
And you saw from that video that the actin
actually twists around.
It's like you can imagine.
It's not like what the cartoon draws, like a single profile,
a head-to-tail assembly.
It's actually--
it assembles.
It has two binding sites on each monomer, so it actually
is sort of like two filaments that are
wrapping around each other.
And the tropomyosin just goes in and blocks every single
myosin binding site.
And the troponin is a three--
it's a trimer.
It has three subunits, and one of the
subunits binds to calcium.
And one of the subunits binds to tropomyosin.
And in low calcium, the tropomyosin binds to actin and
blocks the access between the myosin to actin.
But at high calcium, calcium comes in and binds to the
troponin C subunit, and this causes a movement of the
tropomyosin, and therefore exposing the binding site of
the myosin heavy chain.
So the calcium rushes in, and the calcium--
and the tropomyosin moves the tropomyosin.
The troponin moves the tropomyosin, and then the
interaction can occur.
OK, so now it becomes-- so the calcium is the one that
actually dictates when the muscles are going
to contract or relax.
So that means the calcium level has to be regulated
incredibly precisely.
Like when you want the muscles to contract, the calcium level
goes up, but then immediately when you want the muscles to
relax, the calcium level has to go down.
So how does this occur?
Just show you an image of a movie of a muscle.
This is actually from C. elegans.
It is a nematode, and the round structure in the middle
here is the throat of the worms.
And worms don't have teeth, so they have to chew their food,
and this is the organ that chews their food.
And they eat bacteria.
And these muscles, there's a ball of muscle that contracts,
and it grinds the bacteria, breaking them, and suck out
the nutrients from the bacteria.
OK, so you can see that here.
What you're seeing is a fluorescence indicator.
It's a genetically encoded fluorescence indicator, but
when the indicator become red, that means the calcium--
it's a calcium indicator --the calcium is high.
And you can track that.
If you look carefully, before each of the contractions, the
calcium level goes up.
So it's just--
this has been demonstrated in many systems in mammalian
muscle cells.
But this is just a little bit more cool because it's an
intact organism.
But anyway, so we now know that the calcium drives this
contraction to occur.
So but again, thanks, Juajita.
That means the calcium level has to be regulated both real
precisely temporally and spatially.
OK, so I said that the muscle is a huge cell.
You have to basically flood the cells at one moment with a
lot of calcium.
But then, next thing you know, you have to clear up all the
calcium in the cytosol.
How does the occur?
It's very difficult.
This is a large cell, and you have to do it in a very
precise fashion.
I know that many of you can type 150
characters per minute.
Or if you play music, you're probably--
your fingers can move even faster than that.
So that means all these reactions have to occur on a
millisecond scale.
So how does this occur?
It turned out that the resting calcium levels in all cells--
the vast majority of cells --are very low.
Anyone knows what is the calcium level in your blood?
Ten to the -7?
Ten to the -7?
No, that's way too low.
So in your blood, it's about--
it's about a couple millimolars.
So if you look at the actual cellular solutions, like your
saline solutions that people do IV with, it's a couple of--
two millimolars, OK?
But inside the cell, across the plasma membrane inside the
cell, in the cytosol, the resting calcium concentrations
of hundreds of nanomolars, so that's 10 to the fifth.
There's a 10 to the fifth gradient between the
extracellular to the intracellular calcium.
And in the ER, the ER is a calcium store.
In the ER, there's a couple of millimolars just like
extracellular.
So there's a millimolar range of calcium.
So there's a huge calcium gradient
between internal storage--
this is the ER --or the extracellular solutions, and
between them and the cytosol.
So there's a huge gradient.
That means if you--
and then there are calcium channels that are on the
membrane, on the ER membrane.
There are calcium channels on the plasma membrane.
So if you open the gates, then the calcium just flood the
cytosol, OK?
There's a huge gradient, and therefore it achieves a very
rapid increase, rise of the calcium in the cytosol.
And in the skeletal muscle, there is even more of these
very specialized programs to make sure that this occurs.
And if you look at the myofibrials, and we talked
about the myofibrials being the contract.
It's made of thick filament and the thin filament.
And there's also this sarcoplasmic reticulum, which
is a specialized form of endoplasmic reticulum.
It is the endoplasmic reticulum of muscles, but it
forms this regular shape.
So it basically penetrates into all the myofibrials,
surrounding them and forming these tubes.
And these SR structures, they contain a lot of calcium.
Their calcium levels are in the levels of millimolars.
And they're also connected to a T-tube.
The T-tube, essentially, is the
extension of a plasma membrane.
OK, so there are these T-tube SR complexes that surround the
myofibrials.
So when the nerve activates the muscles, the action
potential comes into the nerve terminals.
The nerve releases acetylcholine, and that
activates the postsynaptic membrane, which
is the muscle membranes.
And by the conduction of--
so the action potential then triggers the depolarization of
the muscle membrane, and that very rapidly propagates onto
the SR, the sarcoplasmic reticulum.
And the SRs get depolarized, and they open up
their calcium channels.
The calcium floods the whole cytoplasm of the muscles, to
ensure that each of the myofibrials are getting the
calcium they want, they need.
And then there are also very potent calcium pumps on the
SRs that very quickly, as soon as the nerve impulse is gone,
take up all the calcium that has gone into the cytosol.
And then the resting--
the muscles relax, because the calcium is pumped away.
OK, so this rapid release of calcium causes simultaneous
contraction of all the myofibrials, therefore causing
the muscles to shorten.
So this actually explains the interesting phenomenon called
rigor mortis, which happens right after death.
So essentially, animal or people, shortly after we die,
we become stiff, OK?
This is a fantastic dinner conversation subject again.
So anyone wants to take a stab at why when you die, when
people die, they become stiff?
You lose control of calcium gradients?
Why do you lose control of calcium?
Because these transport proteins across the membranes
stop working.
There are no nerve impulses.
There are no nerve impulses, but also importantly, these
calcium pumps on the SR are ATPase.
They burn ATP, and in the expense of that, pump calcium.
So when you lose--
when people die, their ATP stops to be generated, and
then because they're not breathing anymore.
And then this calcium goes up, OK?
And you raised your hand.
I was just going to say, no more ATP.
No more ATP.
Great.
OK.
And so this is one effect.
So one of the reasons is that the calcium floods the muscle,
and then they have the capacity of contracting.
Any other reasons?
Yes.
ATP can't bind to myosin causing it to release from the
[INAUDIBLE].
Awesome.
Yeah, that's the other reason.
So two reasons.
One is that the calcium rises, so you can imagine the calcium
rises, so then it can bind to troponin, drag away the
tropomyosin, and that exposes the binding site.
But also because there's no ATP.
So the cross bridge cycle, or the actin-myosin contracting
cycle, it's stuck in this rigor state, which is
nucleotide-free.
So in the human, this condition starts to develop a
couple of hours after death, and then so it reaches a peak
at 12 hours, and then slowly it'll go away, because by that
time, decomposition starts.
And then the myosin head is getting degraded, and
therefore there's no stiffness anymore.
OK, so that's what we want to talk about on the actin-myosin
cycle in the context of skeletal muscle contraction.
And I think the key to that part of the lecture is to
understand how a biochemical reaction can be converted into
physical movement and how the cycle works, OK?
And the smooth muscles in our blood vessels and
gastrointestinal tracts are different.
So they work differently.
So there's no myofibrials.
There's no repeated sarcomere structures.
But the movement is also generated with the same cycle,
with the same actin-myosin cycle.
The actin organization is different, and that accounts
for the difference between the two
different types of muscles.
The actins are attached to the plasma membrane, OK?
And there are--
these yellow sites are the attachment sites, and you can
see that they're still bridged by the myosin.
And then when the actin-myosin interact with each other, it's
the same actin-myosin cycle.
And they also generate--
that generates the sliding motion between the myosin and
the actin that causes the whole cell too shorten.
The calcium control is also different, as we said that the
skeleton muscle has to be controlled at a very high
temporal resolution, but the smooth muscles don't.
And they usually generate a slow but sustained
contraction.
When you're nervous, your blood pressures increase.
The blood vessels will contract, and that's not
controlled in a millisecond.
It's like a mood thing, so you have a much longer-- this
happens in a matter of minutes,
and it is more sustained.
So there's no specialized sarcoplasmic reticulum
structures, and there's also no troponin.
So then how does the calcium get in to regulate the
contraction?
The contraction also requires calcium.
So it turned out that calcium uses a different protein,
called calmodulin, to activate the contractile apparatus.
And the calcium calmodulin then binds to a kinase called
myosin light chain kinase, and the myosin light chain kinase
phosphorylates the myosin light chain.
The light chain are these two smaller peptides that are
sitting right next to-- right on the neck
of the heavy chain.
And once the light chain is phosphorylated, then the heavy
chain can bind to the actin and generate movement.
So experiments that demonstrate the importance of
this came from blocking or sufficiency experiments, so
you can inject an antibody to the myosin light chain kinase,
and that shuts off the contraction.
And if you just inject the phosphorylated--
or the kinase alone, it will increase the contractility.
So again, calcium serves as a switch.
So when calcium is high, it allows the--
it phosphorylates the light chain, and it allows the
binding between the heavy chain and the light chain.
And it turned out that calcium not only is the signal for
actin-myosin cycles.
It is not only the critical control or the signal system
to control the contraction, it also is the signal to control
many other biological activities.
You know, when the oocytes are fertilized, the sperms
penetrate into the oocyte, and it sets a calcium wave.
And that wave walks other entry of other sperms.
And there are many, many examples of calcium being a
major what's called "second messenger" in the cells.
But calcium is a very small molecule, right?
It's a small ion, so how does it actually generate
conformational change in cells?
So that's a fascinating question that has attracted
many researchers.
And calmodulin is one of the very important pluripotent or
pluritropic calcium adapters.
The calcium binds to these adapters, such as calmodulin.
And calmodulin has two forms.
It has an extended form that looks like a dumbbell, with
four calcium binding sites, two on this
head, two on that head.
And when calcium binds, this dumbbell moves, and generates
a much larger conformational change.
And this movement then can be coupled to different proteins.
So you can imagine that it becomes much easier, when you
have such a large conformational change, to do
something to proteins.
So essentially, if you look at the calmodulin, this appears
in all cells.
It started way before metazoan mammalians.
You can find calmodulin in the East.
And so that means that evolution has come up with the
solution from an early point and decided to keep it as a
pretty efficient way of transforming the information
that is carried out by calcium to a lot of the proteins, a
lot of the processes, that involve
changes in other proteins.
So this conformational change is a critical step in reading
out different calcium scenarios.
So that's the--
we talked about the skeletal muscles.
We talked about the smooth muscles.
And then, even in non-muscle cells, the actin-myosin
interaction do play important roles.
So the muscles have to contract to generate movement
but there are also movement in non-muscle cells.
For example, during cell division, the cytoplasm--
so this cell division is a process when the nuclei or the
genetic material is duplicated, and then the
cytokinesis needs to kick in to make two cells
out of the one cell.
In this process, the cytoplasm in the middle of the two
cells, in the division plane, becomes less and less.
This is caused by a constriction of the membrane
right around this plane.
And this constriction is mediated by actin-myosin
interactions.
And we know that because if you inject the myosin
antibody, it blocks the cytokinesis event.
So here I just want to show you a couple
of movies for this.
And here you see the cells dividing.
And this is to make the point that when they
divide, they round up.
But then, the cytoplasm between the two cells becomes
smaller and smaller, due to this contractile ring.
And on the right is a fluorescence image where the
cell is expressing GFP myosin II.
And here you can see in the start of the division, the
myosin is everywhere.
But then you can see that at certain stages when the
contractile ring is being formed, you see this myosin is
concentrated in the bridge between the two cells.
It is the force that constricts the cytoplasm--
the plasma membrane at that place, at
that contractile ring.
And it turned out that this is very similar to the, actually,
to the muscle myosin and actin.
So the power is generated by the same actin-myosin cycle.
It burns ATP and then translates into movements,
cellular movement.
And these movements add up to shorten the distance between
these filaments.
And calcium regulation--
this is true with the myosin light chain kinase, and that's
very similar to the smooth muscle.
And the actin are gliding against the myosin head.
OK, so that's quite similar.
And so nature has really used this actin-myosin cycle in
different places to generate movement.
And so in non-muscle cells, the actin-myosin can generate
movement and then cause the cells to constrict and divide.
And also, the second function is that it also serves as a
highway, if you wish, or a rope that the other myosins
can bind to vesicles and then to transport the vesicles and
organelles.
So these--
here the myosin that is used in these reactions are not the
same as the muscle myosin.
And we said the muscle myosin has this long tail.
And the long tails assemble into coiled structures, and
that really is what tethers the myosin to the right place,
immobilizes them, and therefore glides the actin
across them.
These myosins that are used for vesicle transport have
short tails.
They don't form polymers, and the short tails bind to the
cargo vesicles.
And the head, however, is very similar to the muscle myosin.
So again, the head actually interacts with the actin and
then generates these repeated cycles of movement.
And that drags the-- since the tail is not anchored, the tail
is binding to the vesicles, and this causes the net
movement of the vesicles towards one end of the actin.
And so the myosin always moves-- the head always moves
towards the plus end of the actin.
Remember, the actin, because it's an asymmetric structure,
one end is different from the other end, OK?
And let's just briefly compare these.
How could--
are these similar events or different events?
So there are similarities between these two muscles and
the vessicular transport.
So the similar event is that the myosin is always
interacting with the actin, and then it always steps
towards the plus end of the filament, right?
So in this case, this stepping towards the plus end resulted
in the cargo vesicles to move towards the
plus end of the actin.
But over here, it's the same way.
So the actin, the thin filament is organized so that
the plus end is at the Z line, and the minus end is towards
the M line, the middle of the sarcomere.
And when the myosin head moves, it always wants to move
to the plus end.
It always reaches over to grab-- towards the plus end.
And here, because the myosin is actually anchored, and it
wants to move to the plus end, that's why it always drags the
minus end towards it.
Is that clear?
If you--
so OK.
So it always--
you imagine this head is always extending towards the
plus end, right?
And then when it contracts--
because the actin is movable, but the myosin is anchored.
But when it contracts, it pulls the actin towards--
pulls the minus end of the actin towards the M line, or
the middle, towards the heavy chain.
How about that?
Is that clear?
So it's the same plus-end preference of this myosin head
that generates both the cargo movement towards the plus end
or, in a different scenario, in the skeletal muscle, it
generates this gliding motion that shortens the sarcomere.
It's the same mechanism.
So on to the last topic of today, which is that in
non-muscle cells, the actin can generate movement, even in
the absence of myosin.
So, so far all we've talked about is that you have to see
the myosin heavy chain.
The head interacting with the actin is how the ATP cycle
drives that.
But now I'm going to show you that the actin filament has
been co-opted to do other things that you don't even
need myosin, OK?
So the movement is generated by the differences in
properties between the minus end and the plus end.
And we talked about how, because of the asymmetry of
the monomer, one end of the filament is different from the
other end of the filament.
And before I go into the details, I have to tell you
that the actin is also an ATP binding protein.
How confusing, right?
Myosin is an ATP binding protein.
Actin is also an ATP binding protein.
So it's an ATP binding protein, and it can also
hydrolyze ATP to make ADP.
And there are exchange factors that helps the ATP to replace
the ATP to form ATP form of actin.
Before I continue to go further to confuse you, let's
just establish the fact that in the skeletal muscles, the
actins are very stable.
They are stabilized by other proteins that
we didn't talk about.
And so don't start thinking that OK, what happens if the
actin starts to break down and all dynamic, and how do the
myosins grab onto them, right?
So in the muscles, this actin is very stable.
Just think that as a static rope that the
myosin has to pull on.
But in non-muscle cells, the actin is not so stable.
It's very dynamic.
We'll show you examples of that.
So OK, let's imagine this.
So in the non-muscle cells, about 50% of the actin exists
in filaments, and the other 50% exists in unpolymerized--
this is called globular actin, G actin.
The filamentous actin is called F actin.
So that means there's exchange, constant exchange,
between the G actin and the F actin.
The actin monomer on the filaments continues to fall
off, giving rise to the G actin.
The G actin continues to polymerize, giving
rise to F actin, OK?
And because of the differences in the minus end and the plus
end, the plus end always has ATP actin.
So the ATP actin is more likely to join the plus end.
And once it joined the plus end, because it has intrinsic
ATPase activity, it'll slowly hydrolyze the ATP.
And therefore, the longer you stay here, you're more likely
to become ADP.
So on the plus end, you will always see a few or a bunch of
monomers with ATP.
And towards the minus end, it becomes ADP dominant, OK?
And on the minus end, the monomers are more
likely to fall off.
It doesn't mean that it doesn't get added here.
There's also addition here.
The G actin also comes--
are added at the minus end.
But the reaction favors the falling-off process.
On the plus end, the reaction favors the adding-on side of
the reaction.
So you can imagine that this is also dictated by how much G
actin it is in the cytosol.
If you have a lot of G actins in the cytosol, then there
will be more, I guess, added on to it than
falling off, right?
So it's a biochemical reaction with a K.
So that means--
so because of this property, there is more likely to be
addition at the plus end and more likely to be falling off
on the minus end.
You can find a concentration of the G actin where the
number of monomers added onto the plus end is exactly the
same as the monomers that have fallen off from the minus end.
If there is a concentration of G actin like that, then there
will be continuous adding here, continuous losing here,
but that the net length of the G actin stays the same.
So this is called treadmilling.
We're going to talk about-- yes, this is treadmilling.
At that concentration, the length of the
polymer is not changing.
The filament is not changing but it's like you're running
on the treadmill, right?
The belt is continuously disappearing on the end, and
then it reappears on the front.
So in this state, the actin filament is dynamic, although
if you watch it in cells, their
length might be constant.
OK, so why are we going to even talk about this?
Let me show you a movie first.
So this is a movie of a type of cell called keratinocytes.
These are from fish, and they're in their skin.
And these are professional migrators.
And you can see that not only does it migrate, but it
migrates with a huge leading edge.
And you can [? light ?] these cells and ask what is this
leading edge made of?
And people have done electromagnetic scanning
electron microscopy, and the leading edge is just filled
with filaments.
And these are actin filaments.
You don't find a whole lot of myosin here, but it's the
actin filaments.
And then take a closer look at these actin filaments, and
you'll find that these are not only actin filaments, but
there are also branched filaments.
You see a lot of these angles that are
branched, the filaments.
We're going to talk about more--
there's stereotyped branching of these filaments.
And the idea is that it's the polymerization, or the
movement of the actin, that drives, that pushes on the
leading edge, and that locomotes the cells.
So for cells to move around, there has to
be two things occurring.
One is that there has to be new adhesion sites, or there'd
be a force in the leading edge that pushes the cells to go
forward, right?
And then in the tail, the membrane
also has to be recycled.
You're letting go of whatever adhesion that you have
established with the substrate, right?
So it's establishing the new sites and pushing in the
leading edge of the cell and also retracting the rear end
of the cell.
So then that leading edge, the pushing event,
is caused by actin.
It's caused by the dynamic behavior of actin.
OK, let's see how this occurs.
And this is kind of cool.
This is a different type of cells, and this cell is
transfected with GFP actin.
So it tells you where all the actin is.
I want you to focus on this edge.
So it turned out that this is a migrating cell.
I'm going to play the movies right now, and then you'll see
that this is the leading edge.
This is the edge that is getting pushed
forward by the actin.
But I want you to make some observations.
And what do you see?
So you can see that at this moment, there is a band of
actin that exists in the leading edge.
I want you to focus on this band and tell me what you see.
OK, you see this edge is moving.
And OK, this is rewinding and replaying the movie again.
So tell me.
You can see that there's a band of actin, and then
there's some actin low concentration zone.
So what do you see here?
What do you see?
There's a feature of this actin
band that says something.
Can anyone?
Just take a brave guess.
So imagine, OK?
I said that it's the actin that is being added to the
filaments that pushes the leading edge forward, right?
So if we continue to add actin, what would you expect?
So imagine this band has many of the actin filaments that
are oriented in some way.
And you continue to add actin to this fiber.
What should you expect?
Longer?
Longer.
OK.
You would expect the actin filaments to become longer.
OK.
And do you--
let me replay this, OK?
And then so the key observation here that people
made was that, although the cells continue to move, this
actin band, the net length of the actin, did not change.
It's always the actin with a fixed length that is at the
tip, right?
It only makes sense, right?
If you continue to add, then the actin is going to be
across the entire cell, right?
So in some way, it has to be cycles of things.
So you're adding and then breaking at the same time, OK?
So this makes it--
next interesting question is where is actin added?
You could imagine that the actin could be added at the
rear end of the zone, that it's literally pushing.
Or it could be added at the front end of the zone.
We talked about treadmilling.
You have maybe adding and subtracting at the same time,
and so you can probably add always on one end, and then
subtracting on the other end.
So the next set of experiments really tried
to get at this question.
Where is actin added?
Any intuitive guesses?
On the other edge?
On the other edge?
You mean on the leading edge, or--
--the outer.
The outer rim?
Any other?
Well, the only other would be on the other side.
Your intuition is correct.
This is actually not so intuitive to many people.
OK, so how do you demonstrate that, right?
The problem is that if you go back to this movie, and here
we're labeling actin.
And the actin is--
the whole filament is decorated by actin, right?
So you can't see if one end is falling off and the other one
is being added.
You can't distinguish which way is being active, because
you're visualizing the entire actin all the time.
So there are a couple of experiments that
really get at this.
I think this is the first time that we're
going encounter this.
And we're going to go back to these experiments again in the
cell cycle, cell division lecture, but
this is a very useful.
It's called--
this is fluorescence recovery after photo bleaching.
Essentially, this is the same actin zone that I showed you
in the previous movie.
And you come in with a laser, beam of laser, and you control
exactly where this laser is scanned.
And you just bleach all the fluorescence in this part.
When you bleach a fluorescence protein, it makes it--
makes it dark.
It makes it no longer fluorescent.
And then this fluorescence is not going to come back in many
minutes, so you basically have bleached that fluorescence
fusion protein.
But you don't damage the protein.
The protein is still there.
The actin is still doing its thing, but then it's just
become invisible.
So now when you generate a dark zone, and over time, the
dark zone is filled by fluorescence that comes from
the addition of new actins, right?
So there are lots of G actins that are outside of this
bleached zone, let's say, from here.
OK, and then these actins are being added.
So now you can ask the question, where is the
fluorescence coming back first, or where does the
fluorescence come back next?
Right?
This is the bleached zone, and you can see from between these
images, the fluorescence is coming back first on the
leading edge, as she suspected.
And that argues that the G actin in being added at the
front edge, or at the leading edge.
So this is one way of doing it.
The next way of doing it is called fluorescence speckle
microscopy.
Essentially what it does is that, if you have a polymer
structure, and that is labeled entirely, in its entirety, by
GFP, you can't distinguish which one.
You can't track, even though the actin is moving this way.
Let's say this is the leading edge.
You can't see this movement because the
whole filament is actin.
So but if you can find a way to only label parts of the
filaments with fluorescence, then you can potentially see a
movement, OK?
So this is how.
The way that people have done this is to basically give the
cells a low concentration of GFP actin mixed with a lot of
unlabeled actin.
So then only part of the filament has GFP or has
fluorescence in it.
And this is called speckle--
fluorescence speckle microscopy.
And I just want to show you one example.
It's really cool when you do this.
So this is again in the leading edge, and this edge is
the leading edge.
And here you see a lot of speckles, because the actin is
being labeled fluorescently, but at a low concentration.
So now, let's play the movie so you can see that's the
leading edge.
I don't know if you can--
there are ways to quantify this, but you get a sense that
the flow is from the leading edge back towards the center
of the cell.
OK, so that means that you're adding new things, and you can
see the flow backwards.
OK so just show you one more.
This is very simple.
We know that the actin is also critical for determining the
shape of the cells, so you add a drug called cytochalasin.
This is a toxin from certain types of fungi that blocks
polymerization on the plus end so the actin cannot be added
efficiently on the plus end anymore.
And the cell will collapse, going from a fanned out shape
like this to this shape.
And the last movie that I'll show you today is these
keratinocytes that--
during migration, if you add latrunculin, you'll see that
the movement is stopped.
And the cell eventually will also lose their
shape or round up.
So that means that if you block the actin polymerization
process, the cell will fail to be pushed--
continue to push forward, and the migration stops.
OK.
Thank you.