The cytoskeleton is a dynamic network of protein filaments

extending throughout the cytoplasm.

The cytoskeleton is critical for many essential cell functions :

cell division, cell movement, cell growth, differentiation ...

The main functions of the cytoskeleton are :

 § To provide a structural framework for the cell and

support the large volume of cytoplasm (especially in
animal cells that have no cell wall).

 § To serve as a scaffold that determines the position of

organelles.

 § To facilitate the internal transport of organelles or

other structures (including

chromosomes during cell division).

 § To allow cell movements (cell migration), cell

contraction, or changes in cell shape.

 The cytoskeleton is composed of proteins that assemble

through non-covalent interactions to form long filament
structures .
Three types of structures can distinguished based on
their diameter, protein component and subunit
arrangement :

 Ø MICROTUBULES (22-25 nm) hollow cylinders made of

Tubulin
 Ø INTERMEDIATE FILAMENTS (10 nm) show a rope-like
structure and are composed of fibrous proteins (keratin,
vimentin ...)

 Ø ACTIN FILAMENTS OR MICROFILAMENTS (5-9 nm)

two stranded helycal polymers of Actin

 a) MICROTUBULES radiate from the microtubule-

organizing center (MTOC) or
 Centrosome
 b) MICROFILAMENTS show a scattered
distribution in the cytosol, but are especially
concentrated just beneath the plasma membrane.
 c) INTERMEDIATE FILAMENTS extend accross the
cytosol forming a framework that attaches to the
plasma membrane and provides mechanical
strength to the cell.

 MICROTUBULES
 Cells contain 2 types of microtubule populations:
ØUNSTABLEMICROTUBULES,short-livedand
verydynamic.
 They form the Mitotic Spindle and mediate
changes in cell shape and organelle movements
 Ø STABLE MICROTUBULES, long- lived and non
dynamic Present in Cilia, Flagella and Centrioles
A microtubule is a polymer of globular tubulin subunits
arranged in a hollow cylindrical tube.
Each microtubule subunit is a
heterodimer of α - Tubulin and β-Tubulin.
The tubulin subunits are aligned end to end forming
protofilaments that pack side by side to create the wall
of the microtubule. Each microtubule contains 13
protofilaments .
Tubulins are very similar in all animal species and
together with Histones are the most evolutionary
conserved proteins.

13
Diameter: 25 nm
Length: variable (1- >100 μm)

MICROTUBULE DYNAMICS
Microtubules are dynamic structures that go through
continuous assembly and disassembly processes and
are constantly growing and shrinking . This behavior is
known as DYNAMIC INSTABILITY.
In addition to their structural polarity, microtubule
display GROWTH POLARITY because tubulin dimers are
added preferentially at one end, designated the (+)
end (fast growing end) , and are lost preferentially
from the other end, the (-) end (non-growing or slow
growing end). The (+) end is the one where the β-
tubulin monomers are exposed. The (-) end is the one
where the α-tubulin monomers are exposed.
In a cell the (-) ends of all microtubules are found
around a microtubule organizing center or MTOC (the
centrosome for example) and radiate from there to the
cell periphery.

MICROTUBULE ASSEMBLY AND

DISASSEMBLY
1- αβ-tubulin dimers bind head to tail to form short
protofilaments.

2- These proteofilaments associate laterally into more stable

curved sheets. Eventually sheets made up of 13
protofilaments wrap around forming a short hollow cylinder.
The microtubule then grows by the addition of new tubulin
dimers to the ends of the protofilaments.

3- The free tubulin dimers have GTP bound to both tubulin

monomers. However, shortly after the tubulin dimer is added
to the growing microtubule, GTP in the β- tubulin monomer
is hydrolized to GDP due to the intrinsic GTPase activity of
tubulin.
4- GTP hydrolysis weakens the binding affinity of tubulin
dimers for each other leading to disassembly or
depolimerization of the GDP-bound dimers.

MICROTUBULE ASSEMBLY AND

DISASSEMBLY
THE DYNAMIC INSTABILITY MODEL
 § Only microtubules with (+) ends associated with GTP-
tubulin are stable and can prime tubulin polymerization.
 § MTs with bound GDP-tubulin depolymerize more
rapidly and can disappear in 1 min.
 §At high concentrations of non-polymerized GTP-
tubulin, the rate of tubulin assembly is faster than the
rate of GTP hydrolysis. Therefore the MT grows.

§ At low concentrations of non-polymerized GTP-tubulin, the

rate of tubulin assembly decreases and the GTP hydrolysis
rate is higher. An unstable GDP cap is formed, the
protofilaments spring apart and tubulin subunits are
released. The MT shortens.
MICROTUBULE ASSEMBLY AND
DISASSEMBLY
α-tubulinànon-hydrolyzable GTP β-tubulinàGTP is
hydrolyzed to GDP
Figure 16-16c Molecular Biology of the Cell (© Garland Science 2008)

Both ends of the microtubule have the ability to grow,

but they will do it at very different rates. Tubulin
polimerization rate at the (+) end is 2-3 times higher
than at
the (-) end.
The concentration of free tubulin dimers determines
whether a microtubule will grow or shrink.
The Critical Tubulin Concentration (Cc) is the
concentration at which microtubule length is constant.
If the tubulin dimer concentration is higher than Cc,
polymerization will be favored and the microtubule
will grow.
If the tubulin dimer concentration is lower than Cc,
depolymerization will be favored and the microtubule
will shrink.
The concentration of tubulin in the cytoplasm depends
on the rate of synthesis and degradation but varies also
with polymerization or depolymerization events.
(-) (+)

[Tubulin] = Critical Concentration (Cc)

(-) (+)

[Tubulin] > Cc Polymerization

(-) (+)

[Tubulin] < Cc Depolymerization

MICROTUBULE-ASSOCIATED
PROTEINS (MAPs)
They bind specifically to microtubules and modulate their
stability of and their association with other cell structures.
There are two types of MAPs :

ØPROTEINS THAT STABILIZE MICROTUBULES : they bind to

the negatively charged C- terminal part of Tubulin and
stabilize the outer wall of a microtubule.

They can increase the growth rate of microtubules or

suppress microtubule catastrophe. They are cell-specific.
Some are present in neurons : MAP1A, MAP1B, TAU (in axons
and dendrites) and MAP2 (only present in dendrites). Others
are not present in neurons : MAP4.

Ø PROTEINS THAT DESTABILIZE MICROTUBULES : they may

sever intact cytosolic microtubules through an ATP-
dependent process by breaking-up internal bonds between
tubulin dimers (Katanin ) or they may promote disassembly
of tubulin dimers at the (+) end (Catastrophin)
MICROTUBULE-ASSOCIATED
PROTEINS (MAP)
The binding of MAPs to Microtubules is inhibited by
PHOSPHORYLATION . Therefore, phosphorylation of
MAPs favors microtubule disassembly.
Hyperphosphorylation can promote disassembly in
disorders like alzheimer ́s disease.
+ MAPs
VESICULAR TRANSPORT THROUGH
MICROTUBULES
Vesicles can be transported by MOTOR PROTEINS like
Kinesin and Dinein moving along microtubules . The
movement can be towards the (+) end (anterograde)
or towards the (-) end (retrograde) of microtubules.
§ Kinesins mediate anterograde transport § Dyneins
mediate retrograde transport
The type of receptors present on the vesicle surface
will determine in what direction it will be transported.
Vesicle transport is particularly important in neurons
where molecules synthesized in the cell body or even
mitochondria must be delivered to the axon terminal.
Axonal transport occurs in both directions (up and
down the axon) , is fast (up to 400 mm/day), and relies
on kinesin, dinein and microtubules.
http://www.youtube.com/watch?v=kOeJwQ0OXc4&feature=related

FUNCTIONS OF UNSTABLE
MICROTUBULES
1. MAINTAIN THE CELL SHAPE
2. CELLULAR TRANSPORT. Axonal transport
The oriented microtubules in the axon serve as
tracks for the directional transport
3. MITOTIC SPINDLE FORMATION, which will
distribute chromosomes between daughter
cells when the cell enters mitosis.

STABLE MICROTUBULES

• CILIA AND FLAGELLA • CENTRIOLES

CILIA AND FLAGELLA
§ They are thin and flexible proyections of the cell.
§Cilia and Flagella have essentially the same structure
but cilia are short (few micrometers) and abundant
while flagella are long (e.g. more than 2 mm in an
insect sperm) and scarce.
§Function: locomotion of free cells (sperm) or
movement of fluids along the cell surface (respiratory
epithelia).
§Structure : 2 main regions
• Axoneme: central bundle of microtubules
• Basal body: point of attachment to the cell.
Figure 16-81b Molecular Biology of the Cell (© Garland Science 2008)

AXONEME
§ The axoneme is formed by 9 doublet MT surrounding a
central pair of singlet MT (9+2 arrangement).

 § Each doublet consists of A and B microtubules (A is

complete: 13 protofilaments; B is incomplete: 11
protofilaments) with the (+) end located at the distal
end of the axoneme.
 § The outer MT doublets are connected to the central
pair by radial spokes and to each

other by a protein called Nexin.

 § Permanently attached to each A tubule of the

doublet are inner-arm and outer-arm

dyneins, that drive the movement of cilia and flagella.

Figure 16-83b Molecular Biology of the Cell (© Garland Science 2008)

BASAL BODY
It is the growing point of cilia and flagella, where the (-)
end of axoneme microtubules are oriented.
It has the same structure as centrioles : 9 triplets of
microtubules tilted towards the central axis.
DIFFERENCES BETWEEN CILIA AND
FLAGELLA
1 Type of movement :
Cilia show a pendular or whiping movement, with an
effective stroke followed by

recovery stroke to initiate a new movement. They sweep

materials across tissues. Flagella show a waving movement
that propels cells forward.

2 Length: Cilia are shorter than flagella.

3 Number : Cilia are present in high numbers in each cell
while flagella are scarce (1 or 2)
FLAGELLA CILIA 106/μm2
CENTRIOLES
 § Two cylindrical structures located at the center of the
centrosome.
 § Exclusive of animal cells.
 § In interphase they are perpendicularly oriented and
constitute the DIPLOSOME.
 § Together with the pericentriolar (PC) matrix they
form the Centrosome
 § Each centriole is formed by 9 triplets of
microtubules, tilted towards the central axis of the
structure.
 § Each triplet is attached to the adjacent triplet by the
NEXIN protein.
 § Before entering mitosis cells must duplicate their
centrioles. Each centriole gives rise to

a new centriole so that once mitosis is complete each

daughter cell gets a diplosome.

 § They are involved in the formation of the mitotic

spindle but they are not essential (e.g. plants do not
have centrioles but form mitotic spindles).
C
C B
A
diplosome

THE CENTROSOME
§ The centrosome functions as a Microtubule Organizing
Center (MTOC) .

 § It is made up of pericentriolar material (PCM) and

sometimes, but not always, it contains 2 centrioles
oriented perpendicular to each other.
 § The (-) ends of cytosolic microtubules are immersed
in the pericentriolar matrix but they do not contact the
centrioles.
 § A gamma-tubulin ring (γTuRc) is found at the (-) end
of microtubules , but the (+) ends are free.
MICROFILAMENTS

MICROFILAMENTS
 § Microfilaments are the thinnest filaments in the
cytoskeleton (5-9 nm)
 § They are two stranded helical polymers of
ACTIN
 § Actin is the most abundant intracellular protein
in eukaryotes
  § Microfilaments show structural and growing
polarity : (+) end , fast growing, and (-) end, slow
growing.
 § The rate of polymerization and
depolymerization is very high.
 § They are usually shorter and more flexible than
microtubules.

Actin F-ADP
Actin G-ATP

ACTIN
 § Actin can be found as a free globular monomer
(G-ACTIN) or it can form fibrous polymers (F-
ACTIN).
 § Free Actin monomers are bound to ATP, but
ATP is hydrolyzed to ADP shortly after it is
incorporated into the filament.
 § Hydrolysis of ATP reduces the binding strength
between actin monomers and leads to
depolymerization.
G-Actin
F-Actin

POLYMERIZATION OF ACTIN
MICROFILAMENTS
 1. 1) NUCLEATION : formation of small aggregates of
Actin. It is the rate-limiting step in the formation of
an actin polymer.
2. 2) ELONGATION : the small initial nucleus
elongates by addition of new actin units to both
ends. The (+) end grows faster.
3. 3) HYDROLYSIS OF ACTIN-BOUND ATP AND
STABILIZATION : a steady state is reached at which
the rate of addition of new subunits to the
filament ends exactly balances the rate of subunit
dissociation. The filament length does not change.

ACTIN FILAMENT
TREADMILLING
 § ATP-actin monomers are added rapidly to the (+) end
of the microfilament and the ATP is hydrolyzed to ADP
after polymerization. The ADP-actin is less tightly bound
and can dissociate from the (-) end.
  § This cycle is called TREADMILLING. It creates a flow of
actin monomers from the (+) end to the (-) end of the
filament.
 § Treadmilling illustrates the dynamic behavior of actin
filaments.

ACTIN-BINDING PROTEINS
The formation and stability of actin filaments
in the cytosol is controlled by different actin-
binding proteins
Ø PROTEINS THAT PROMOTE
POLYMERIZATION: PROFILIN Ø PROTEINS
THAT PREVENT POLYMERIZATION:
THYMOSIN
Ø SEVERING PROTEINS : GELSOLIN AND
COFILIN Ø CAPPING PROTEINS : CapZ AND
TROPOMODULIN

REGULATION OF POLYMERIZATION-
DEPOLYMERIZATION
Actin polymerization is regulated by proteins that bind
free actin monomers (G- actin). These proteins either
promote or inhibit actin polymerization.
PROTEINS THAT PROMOTE POLYMERIZATION:
PROFILIN : it forms a complex with G-actin-ATP that
contributes to monomer aggregation at the (+) end. It
is a nucleotide-exchange factor.
PROTEINS THAT INHIBIT POLYMERIZATION:
THYMOSIN : it binds to G-actin-ATP, sequesters it and
prevents its binding to the (+) end of the filament.

SEVERING PROTEINS
Another group of proteins control the length of actin
filaments by breaking them into shorter fragments .
These proteins stabilize a conformational change in the
actin subunit to which they bind and create a small gap
between neighboring subunits and, eventually, a
break .
These proteins are GELSOLIN and COFILIN.
After breaking a filament , the severing protein remains
bound at the (+) end of one of the resulting fragments,
where it prevents the addition or exchange of actin
subunits (FILAMENT CAPPING).
They bind to actin filament ends and stabilize them .
§ CapZ: binds to the (+) ends of actin filaments and
prevents addition or loss of
actin subunits at that end .
§ TROPOMODULIN: Caps the (-) ends of actin filaments
CapZ

An actin filament that is capped at both ends is

effectively stabilized, undergoing neither addition nor
loss of subunits. Such capped filaments are needed in
places where the cytoskeleton organization does not
change, as in a muscle sarcomere or in the erythrocyte
membrane.

DIFFERENT ACTIN ARRAYS IN A CELL

ORGANIZATION OF ACTIN
FILAMENTS
Actin filaments filaments are assembled to form two
types of stable structures :
 ACTIN BUNDLES
 ACTIN NETWORKS
ORGANIZATION OF ACTIN
FILAMENTS
Actin filaments filaments are assembled to form two
types of stable structures :
 ACTIN BUNDLES
 ACTIN NETWORKS
Both structures require ACTIN CROSS-LINKING
PROTEINS to link one filament to another in
different ways. The filament pattern created by
this proteins is determined by their size and shape.
 Actin-Bundling proteins : FIMBRIN, α-ACTININ
 Network-forming proteins : FILAMIN

ACCESSORY PROTEINS IN ACTIN CYTOSKELETON

MICROFILAMENT FUNCTIONS
1- DEFINING AND CHANGING THE CELL SHAPE :
For example : changes in platelet shape during blood
clotting.
Resting platelets have a Following activation by clotting
biconcave, disk-like shape agents platelets extend

numerous FILOPODIA (thin cellular projections)

Finally, platelets spread out and form LAMELLIPODIA (sheet-

like extensions)

These changes in morphology are the result of complex

rearrangements in the actin cytoskeleton that is cross-
linked to the plasma membrane.

MICROFILAMENT FUNCTIONS
2- FACILITATING CELL MOTILITY (CELL CRAWLING)
AND MUSCLE CONTRACTION:
1) Extension of the cell membrane and formation of a
lamellipodium.

2) Polymerization of actin filaments and further protrusion of

the lamellipodium.

3) Formation of focal adhesions in the lamellipodium and

adhesion to the substratum.

4) Stable contacts with the underlying surface prevent the

membrane from retracting. There is a cytosolic flux forward.

5) The cell “ tail” eventually detaches and retracts into the

cell body.

Cell contraction as well as muscle contraction involve Actin

and Myosin II
MICROFILAMENT FUNCTIONS
3- FORMATION OF THE CONTRACTILE RING AND
CYTOKINESIS:
Cytokinesis is the process that divides a cell in two daughter
cells following mitosis.

Each daughter cell must receive the same amount of

cytoplasm and organelles.
A CONTRACTILE RING consisting of Actin filaments and
Myosin II is assembled at the equator of the dividing cell. Its
contraction pulls the plasma membrane progressively inward
until it closes off and splits the cell in two.

Contractile ring (Actin and Myosin II)

Actin filaments
Cleavage furrow

Daughter cells

Myosin

STABLE MICROFILAMENT
STRUCTURES
ØMICROVILLI :
§Fingerlike extensions of the plasma membrane that
are particularly abundant in cells involved in
absorption such as the epithelial cells lining the
intestine. The purpose of these thin protrusions is to
increase the surface area available for absorption.
 § They do not possess intrinsic movement
capacity
 § They are smaller than cilia (1/10 or 1/20 of the
cilia size)
 § A single cell can have several thousand
microvilli.
ACTIN FILAMENTS

with associated proteins

MYOSIN I VILLIN FIMBRIN

INTERMEDIATE FILAMENTS
(IF)
3.3. INTERMEDIATE FILAMENTS

INTERMEDIATE FILAMENTS
 § Intermediate size (10 nm)
 § Present in all animal cells
  § Highly stable, felixible and resistant polymers
of fibrous proteins (there is NO polymerization and
depolymerization and no structural polarity.
 § They do not require ATP or GTP for assembly or
disassembly.
 § They are composed of different proteins
depending on the cell type :
 Epithelial cells : Keratins

 Neurones : neurofilament proteins

 Muscle cells : Desmin

 § Abundant in the cytosol of cells that are subject

to mechanical stress
FUNCTION: Intermediate Filaments DO NOT
contribute to cell motility. Their functional role is
to provide mechanical strength to the cell and to
dissipate tensile forces to avoid cell or tissue
damage. IF also provide support to the nuclear
membrane.

DIFFERENT TYPES OF INTERMEDIATE

FILAMENTS
INTERMEDIATE FILAMENT
STRUCTURE
All IF proteins have a central α-helical region flanked by two
globular N- and C-terminal domains.

The helical segments of two monomers interwind around

each other to form a coiled-coil dimer with both N- termini
on one side and both C- termini on the other side (PARALLEL
DIMER).

Two parallel dimers associate in an antiparallel orientation to

form a staggered TETRAMER .

Tetramers assemble end-to end to form PROTOFILAMENTS

Protofilaments associate laterally to form PROTOFIBRILS

Protofibrils wind around each other to form a ROPELIKE

FILAMENT
INTERMEDIATE FILAMENTS IN THE
NUCLEAR LAMINA
The NUCLEAR LAMINA is a fibrous meshwork that lines
the inner face of the nuclear envelope. It is composed
of Intermediate Filaments (made up of Lamins) and
other associated proteins.
LAMIN INTERMEDIATE FILAMENTS support and
strengthen the nuclear envelope.

DYNAMICS OF NUCLEAR
INTERMEDIATE FILAMENTS

During the initial stages of mitosis the nuclear

envelope breaks down and the nuclear lamina
intermediate filaments disassemble in a tightly
regulated process.
At the end of mitosis the nuclear envelope forms again
and the nuclear lamina re-assembles.

HUMAN DISORDERS ASSOCIATED WITH

INTERMEDIATE FILAMENTS
q Epidermolisis Bullosa Simplex :

Caused by mutations in several Keratin genes. As a result,

cells in the epidermis become fragile and easily damaged. In
affected individuals skin is less resistant to friction and
minor trauma and blisters easily.
Normal Keratin Mutant Keratin

q Amyotrophic Lateral Sclerosis (Lou Gehrig's syndrome) : It

is a Motor Neuron Disease that has been linked to alterations
in Neuron Intermediate Filaments and to accumulation and
abnormal assembly of neurofilaments
q Neurodegeneration
q Laminopathies : Progeria, Muscular Dystrophy