The Cytoskeleton

The cytoskeleton is an aggregate

structure formed of 3 kinds of
cytoplasmic filamentous proteins:
microfilaments (actins), intermediate
filaments and microtubules
(tubulins)
 I. Microfilaments (Actin filaments)

• Positioning of proteins and organelles

• Cell shape – all cells
• Shape changes
– Ameboid locomotion
– Neuronal growth cones
– Muscle contraction
 Microfilaments: visualized in a kidney cell with fluorescently-
labeled phalloidin (a poisonous mushroom toxin that stabilizes
filaments and inhibits processes that depend on actin filament
turnover)
Actin filaments have polarity, a fact that is relevant
for filament growth and interaction with other
cellular elements

+ or ‘barbed’ ends

- or ‘pointed’ ends
 Assembly of microfilaments

Monomers of G (globular) actin polymerize to form long

filaments, the F (filamentous) actin. The addition of
actin monomers to the ends of the filament occurs at
different rates: addition to the + end, also called the
barbed end occurs 5-10x faster than to the minus or
pointed end. Polymerization requires hydrolysis of an
ATP.
 Reversible polymerization of actin:
Treadmilling of actin monomers
(In some cells, remodeling of actin filaments is responsible
for half of the ATP hydrolysis in the cell)
Growth of a microfilament requires an initial nucleation by an actin
trimer, and can stop if the rate constant for adding monomers at
the + end and one for removing them at the – end approach the
same value – at that point, treadmilling occurs.
Some intracellular pathogens such as the bacteria Listeria and Shigella and the
vaccinia virus usurp the host cell’s mechanism of assembling actin networks
and propel themselves through the cytoplasm with actin “tails”.
Association of actin filaments with the membrane
• A network of actin
filaments and other
cytoskeletal proteins
underlies the plasma
membrane and
determines cell
shape:example, rbc:

• Actin bundles also

attach to the plasma
membrane to anchor
cell-cell and cell-
substratum contacts,
as shown here:
Microvilli in an intestinal epithelial cell
• Each of the ~ 1000
microvilli contain several
dozen microfilaments with
their + ends facing
outward; they are tightly
packed together by actin-
bundling proteins and are
connected to the inner
surface of the plasma
membrane by lateral
crosslinks composed of
calmodulin and myosin 1.
They project into the
dense actin network within
the cell.
Structure of microvilli
• Microvilli are relatively
stable projections of
the plasma
membrane that are
supported by
microfilaments.
Microvilli increase the
surface area of cells
such as those lining
the intestine.
 The actin cytoskeleton is
1) linked to the plasma membrane and
2) able to change to promote cell movement
• In this picture of a resting,
attached fibroblast, the long
filaments are revealed by
phalloidin staining, but when a
fibroblast begins to move, the
filaments break down into short
filament fragments and G actin
and filaments are reassembled
in the cell’s extensions.
Actin filament turnover and monomer recycling is
regulated by accessory proteins
Actin exists in cells as bundles and networks:
actin-binding proteins crosslink the filaments
 Extrinsic signals can regulate the
actin cytoskeleton
• Phosphoinositol bis phosphate (PIP2) binds
profilin, preventing it from interacting with actin.
• Phospholipase C hydrolyzes PIP2 to IP3,
releasing profilin, which promotes filament
growth
• Gelsolin and cofilin are actin-severing proteins –
they are inhibited by PIP2 - when they are active,
they stimulate formation of new filaments by
increasing the number of + ends available
Several g-protein coupled pathways control
cell shape and behavior
• The Rho protein family includes the small
monomeric G proteins Cdc 42, Rac and Rho
• The Cdc42 pathway activates rapid actin
polymerization and bundling, causing cells to put
out filopodia
• The Rac pathway uncaps + ends of filaments,
stimulating filament elongation, causing cells to
form lamellipodia and membrane ruffles
• The Rho pathway stimulates filament bundling,
causing cells to form stress fibers that interact
with focal contact points with the extracellular
matrix
Fibroblast cell shape changes resulting
from single g-protein signals

Dynamic interaction of these signals is necessary for complex

cellular behavior, as seen in the next slide of cell crawling.
Dynamics of actin in a crawling cell: note the
polarization of the microfilaments
 II. Intermediate filaments
• Formed by a large, heterogeneous group of
proteins
At least 4 major filament types
– Keratin - epithelial cells
– Neurofilaments – neurons
– Vimentin-containing filaments – fibroblasts, glial cells,
muscle cells
– Nuclear lamina – all nucleated cells
• No energy is required for filament assembly
• Filaments are not polarized
 Intermediate filaments in kidney cell (left, green) and
cultured epithelial cell (right, orange; desmosomes are
green)
Intermediate filaments: basic structure & assembly
Genetic diseases of skin and nervous system

• Mutant keratin genes are responsible for some

human skin diseases – some lead to
susceptibility to skin damage and death due to
the trauma of birth, others impact the
organization and stability of skin.
• Abnormalities of neurofilaments underlie the loss
of motor control in Lou Gehrig’s disease (ALS)
and the motor neuron disease that afflicts the
physicist Stephen Hawking.
 III. Microtubules
• Microtubules are polymers of tubulin
• Microtubules are dynamically unstable – they
can be assembled and disassembled rapidly
• Tubules are organized by the centrosome
complex
• Functions:
– Vesicle and organelle transport
– Cilia and flagella
– Mitotic spindles
Microtubules in kidney cell
 III. Microtubules: α,β,γ tubulin
• Microtubules are stiff, cylindrical polymers of α
and β tubulin.
• The polymers project from the centrosome,
where the γ tubulin is present that is necessary
for the “nucleation” of the polymers.
• There is a + end projecting into the cytoplasm
and a – end that often remains anchored in the
centrosome. (cilia and flagella are a special
case, as we will see).
Formation of microtubules
 Regulation of growth and dissolution by subunit
supply: high and low concentrations of GTP-tubulin
Compared with the microtubules of a normal fibroblast (A), the cell
in (B) is regrowing microtubules from the centromere after
treatment with colcemid blocked assembly of microtubules
 Chemicals that affect microtubules: drugs
that inhibit mitosis by having opposite effects
• Colchicine (Colcemid) binds to tubulin monomers, inhibiting their assembly.
• Vinblastine and vincristine are used in cancer chemotherapy because they
cause tubulin to aggregate.
• Taxol binds to tubules and stabilizes them, making it an anticancer drug (for
breast and ovarian cancers) because it inhibits cell division.
 Like microfilaments, microtubules in the cytoplasm exhibit
dynamic instability, including treadmilling and motility that results
from the pattern of growth, such as growing towards and then
moving chromosomes in mitosis.
• Microtubules interacts with motor proteins.
– In cilia and flagella motor proteins pull
components of the cytoskeleton past each
other.
– This is also true in muscle cells.
• Motor molecules also carry vesicles or
organelles to various destinations along
“monorails’ provided by microtubules.
• Interactions of motor proteins and the
cytoskeleton circulates materials within a
cell via streaming.
 Putting them all
together: microfilaments,
intermediate filaments
and microtubules in
kidney cells
 Microtubules,
intermediate
filaments and
the actin
filaments (gray)
are all linked by
a protein called
plectin
(orange, with
gold particles
on the strands)