This would be the first of several podcasts on the cytoskeleton and this
first podcast will give you a quick overview of the cytoskeleton and talk
about microtubules. Now your learning objectives, you should be able to
recognize components of the cytoskeleton on electron micrographs and you should
be able to explain the structure and function of the cytoskeleton as it
relates to specialized histology, so for example as it would relate to the
histology muscle cells to neurons or to how we relate to stages of the cell
cycle that is comparing the cytoskeleton in an interphase cell versus a cell that's
going through division. The cytoskeleton is part of the non-membranous organelle
systems of cells and we can talk about several components of the cytoskeleton,
we'll talk about some general principles and some functions. So the components of
the cytoskeleton be microtubules, these are hollow tubes that are composed of
tubulin molecules, centrioles which appear in cylindrical structures at the
centrosome, the so called microtubule organizing center and then there are two
classes of filaments that are involved in the cytoskeleton. There are
microfilaments, a good example would be actin these are helical polymers, and
then there are intermediate filaments two prime examples would be keratins and
vimentins, these are rope or cable like fibers. If we look at just some general
principles of the cytoskeleton, we can say that the cytoskeleton consists of
networks of proteins, the assembly of the cytoskeleton requires polymerization, the
cytoskeleton by in large consists of very dynamic structures at least from
the perspective of microtubules and microfilaments. These are very dynamic
structures. The cytoskeleton supports cytoplasmic activities so things that
might relate to cell signaling in metabolic activity might relate to the
cytoskeleton and the cytoskeleton also supports movement so that would be the
cell moving, cell surface appendages moving, and the cytoskeleton also
provides the machinery for organelle movement and localization within the
cell. The
functions have been likened by some people to saying that the cytoskeleton
might be the bones of the cell and the muscles of the cell. These images they're
just pretty pictures but they're of the same cell, probably a fibroblast, and then
just stained with different antibodies to highlight the intermediate filaments
in this case the antibody is the vimentin, that's shown on the left an antibody
to microtubules shown in the middle and then an antibody to actin so called
microfilaments shown on the far right. Now let's just talk about microtubules
for the remainder of this podcast, microtubules are non-branching rigid
hollow tubes that can rapidly disassemble in one location and
reassemble in another location and here in an electron micrograph of the mitotic
spindle, you can see microtubules, they're highlighted by the arrows on
this image and you can see profiles of chromosomes. Here microtubules are shown
in the axon of a neuron. Microtubules can be found in interphase cells, they'd be
found at the centrosome and they'd be radiating out towards the cortical
cytoskeleton that's shown in this cartoon. Microtubules are also found in
dividing cells as pericentriolar material and in the spindle apparatus.
That's shown here and microtubules are in ciliated cells, in the basal body, the
microtubule organizing center of the microtubules that are going up into the
cilia. Here we're showing microtubules visualized by fluorescence microscopy,
you have an interphase cell here, the microtubules are shown as long fibers,
they're filling the cytoplasm. This happens to be from a cultured cell that's
stained with a fluorescent anti-tubulin antibody, the insert shows a mitotic cell
that's stained with the same anti tubulin antibody and it doesn't take
much imagination to look at how completely the microtubule cytoskeleton
has rearranged from an interface cell for us as a cell that's in cell
division. Microtubules are elongated polymers and they essentially consist of
equal molar ratios of alpha tubulin and beta tubulin. They're rigid cylinders.
They may be 20 to 25 nanometers in diameter. They consist of 13 circularly
arranged globular dimeric tubular molecules that are called proto
filaments and these make up the wall of a microtubule, so in this diagram on the
bottom this would be a proto filament that I'm tracing like that, another
protofilaments like this, and there would be 13 of these protofilaments arranged in a circle
around this hollow tube. The wall is about five nanometers thick. The
molecular weight of the alpha and beta tubulin dimer might be something on the
order of a hundred and ten kilo Dalton's. Microtubules are very highly
dynamic structures, all you have to think about is the mitotic or meiotic spindle
to understand that. Microtubules can be permanent though if you think about the
structure of cilia and flagella, the axinine is essentially composed of
microtubules and those are permanent structures. This cartoon just shows the
microtubules, here would be the protofilaments like this so display just
like that the protofilaments. Here you can see them in an electron micrograph
in cross-section and then in a longitudinal section like this. I'm going
to talk in just one second about the plus end and minus end of microtubules.
Microtubules polymerize in an end-to-end manner sometimes called the head to tail
or the plus end to minus end manner so we can talk about the plus end here and the
minus end here, now this does not relate to charge in the least bit
it just is to indicate a head and a tail region and now as this diagram shows
alpha tubulin of one dimer will bind to beta tubulin of the next dimer and this
will occur in a repeating fashion and they'll be longitudinal contacts that
link dimers together into the linear structure,
the so-called protofilament. Microtubules initially grow from gamma
tubulin rings that are within the microtubule organizing center. These
gamma tubulin rings within the microtubule organizing center would
equal the nucleation site for alpha and beta tubulin dimers. These dimers are
added to the gamma tubulin ring and polymerization of the dimers requires
GTP and magnesium and that's what's shown in this diagram, the tubulin dimers
bound to GTP like this. Here the tubulin dimers bound to GDP, the growing end of
the microtubules is always the beta tubulin that the plus end of the proto
filament and these can extend towards the cell periphery, the non-growing end
is always at the minus end and that's always embedded or it's usually embedded
within the microtubule organizing center. Polymerization and depolymerization is
usually in a steady state and in an invitro condition. This polymerization and
steady state can be altered with temperature in living cells micro
tubular associate proteins can change the rate of polymerization and
depolymerization. This just gives you an example of microtubule assembly and
disassembly so here we have nucleation at the gamma tubulin rings that's shown
here in the cartoon, notice the centriole pair that's clustered in this nucleation
center or this microtubule organizing center, elongation of the microtubules
add the alpha and beta dimers on these gamma tubulin so they're going to grow
out with the plus end extending out towards the cell periphery an addition
of tubulin dimers occurs at this plus end so the microtubules get longer at the
plus end. Interestingly though microtubules exhibit a dynamic
instability, as the tubules shrink you also remove
dimers from the plus end. Now microtubule stabilization occurs as
there are association of microtubular associated proteins at the plus end.
Those proteins block the dynamic instability of microtubules and so we'll
make the tubulin a more stable structure. The important thing to understand is
that the natural state for microtubules is they want to polymerize and
depolymerize very rapidly, thus the sense of dynamic instability. This instability
must be stabilized by microtubular associated proteins to lead to permanent
structures. We've hinted at this in an earlier podcast organelle translocation
within cells can occur along roadways or railroad tracks if you want of
microtubules and this cartoon is just showing that and I think we've already
touched on the fact that transport along these microtubules is powered by micro
tubular motor proteins and in an axon we talk about things going from the cell
body towards the axon terminal so called movement in an anterior grade direction
or we talk about movement going from the axon terminal back to the cell body in a
retrograde direction. Whether something moves in an anterograde or retrograde
direction depends on the specificity of microtubular associated motor proteins.
There are two major motor proteins one being kinesin, the other being dynein.
Kinesin has a specificity of moving cargo towards the plus end of the
microtubules, dynein has the specificity of moving cargo towards the minus end of
the microtubules. Both of these microtubule motors have conserved
globular head regions that have ATPase activity and then the tail region binds
cargo and the tail region can be highly variable which would suggest that the
cargo would be variable, so the issue would be what type of cargo can be
transported with these microtubular motor proteins
and as you might imagine the cargo might be organelles like endocytic
vessels, lysosomes, mitochondria, the cargo could be other microtubules, the cargo
could also be chromosomes. Let's talk very briefly about cilia and flagella.
Cilia and flagella have a nine plus two arrangement of microtubules. Here is the
electron micrograph of a ciliary axoneme. So if we're looking at a cilia,
there'd be a plasma membrane that's surrounding this axoneme, so if you
look at for example these pseudo stratified columnar epithelium you can
see the cilia on the surface while the plasma membrane is coming out around
each cilia, around the axoneme just as the plasma membrane is coming out around
the sperm tail in the flagella. You may remember the structure of the axoneme 9
outer microtubule doublets. One member of the doublet is a complete tubulin with
13 protofilaments, the other member of the doublet is actually a partial
tubule in that it shares some of the protofilaments with its partner. We
talked about dynein arms, there's an inner and an outer dynein arm and there
are these linking proteins in the middle like this and then there's a central
doublet that has a structure of 13 protofilaments individually around each
of the tubules so you have this 9 plus 2 arrangement of the ciliary axoneme. If
you look at ciliary movement, it's all about dynein, the dynein arms,
dynein moves the microtubules in the minus direction so here if you just had
isolated microtubules in an invitro condition, these microtubules, if you
had dynein in the mix, would just walk past each other moving towards the minus
direction. If you go back and look at the structure of the ciliary axoneme, you've
got these various linking proteins along these microtubules. These linking
proteins allow the axoneme to bend because the microtubules are going to
slide past each other with this tiny moving towards the minus direction but
they can only slide as far as these link proteins allow them to
slide so they can't totally slide off of each other so they're gonna just bend.
This image shows the distribution of kinesin motor proteins on a mitotic
spindle. It's basically a confocal immunofluorescent image of mammary
epithelial cells and it's a particular cell that was captured at anaphase and
you can see the pair of centrioles at each spindle pole so here's one pair and
the pair at the opposite spindle pole. These have been stained with the
monoclonal antibody two proteins that associate with centrioles in the
microtubule organizing center and it was tagged with a molecule that will fluoresce
green. The red is an antibody to a mitotic kinesin, the mitotic kinesin is
called Eg5. You don't have to remember that but it's stained red because the
antibody was labeled with a fluorescent probe probably rotamine that will
fluoresce back red. So you can see this mitotic kinesin motor on these
kinetochore microtubules. Kinetochores are fluorescent white because kinetochore
proteins were stained with an antibody to the proteins that was labeled with a
white fluorescent probe. The sister chromatids are just reflecting
autofluorescence at the DNA fluorescent blue.