The Cytoskeleton
Intricate network of protein filaments that extends throughout the cytoplasm
Filamentous architecture helps support large volume of cytoplasm in animal cells,
cytoskeletal elements found in bacteria but cytoskeleton most prominent in
structurally complex eukaryotic cell
The many roles of the cytoskeleton…
- Mitosis: segregation of chromosomes into daughter cells, cytokinesis
- Cell movement (mammalian cells, gametes – sperm cells, WBCs)
- Organises internal structure of cell, hold organelles in place (Golgi, nucleus,
mitochondria)
*Golgi disperses into fragments, ER collapses around nucleus as it is continuous with
nuclear envelope
- Determine cell shape (epithelial cells, neurons with long dendrites)
*In general, cells lose their shape and form round balls when microtubules are
depolymerised
- Intracellular transportation system – movement of membrane vesicles and proteins
Cytoskeleton composed of three major types of structural proteins
Subunit Nucleotid Diameter Molecular Form of Motor
e binding of weight polymer Proteins
filament
Actin ATP/ADP 6-7 nm 42 kDa 2 Myosins
Actin
monomer stranded
filaments
helix
Tubulin GTP/GDP 24-25 nm 50 kDa Hollow Kinesins
Microtubules dimer tube: 13 Dyneins
filaments
Depends None 8-10 nm 40-210 Coiled coil None
Intermediat
on cell kDa
e filaments
type
*Microtubules and actin filaments are dynamic polymers (polymerise and depolymerise
rapidly in cells), IFs are not
*Microtubules and actin filaments are polarised protein polymers (each end of polymer
has different behaviour), IFs are not
,Microtubule/Actin Disrupting Agents
Actin-binding
Cytochalasins Cap+ ends of filaments
(fungual metabolites)
Inhibit actin polymerisation
Latrunculins Bind to actin monomers
(red sea sponge toxin)
Phalloidins
(Amantina mushroom)
Stabilise actin filaments Bind to polymerised actin
Jasplakinolide
(sea sponge toxin)
Tubulin-binding
Nocadazole Binds to tubulin dimer
Inhibits tubulin polymerisation
(benzimidazole derivative)
Taxol Binds to microtubules
Stabilises microtubules
(ark of Pacific yew)
Microtubules
Function of microtubules depend upon two “contradictory” features: can act as
stuff structural elements which can easily disassemble
Tubular structure and large diameter make them relatively stiff and allow them to
resist compression
Dynamic nature is very important for function – allows rapid reorganisation of
microtubules when necessary
May also have a role in allowing cells to navigate/change direction
Fibroblast: moves within body and changes directions – microtubules arranged in
starlike pattern, radiating outward from single point in nucleus (these are short-lived,
lasting only a fraction of the time it takes for the cell to move)
Neuron: large parallel microtubules with many vesicles which carry material to and from
synapse (stable tubules, essential for cell structure), growth cones have unstable
microtubules to help form synapses with other neurons
,Structure
Tubulin: a heterodimer made up of α-
and β-tubulin (both proteins share 40%
sequence identity, always associated
with each other)
Each molecule of α- and β-tubulin binds
a molecule of GTP (only the β-tubulin
bound GTP is exposed to exchange with
nucleotide in solution
Microtubules are protein polymers composed of thousands of tubulin subunits
organised into a hollow tube
Typically made up of 13 linear chains of subunits that run parallel to one another –
each linear chain = protofilament; 1 microtubule formed of 11-15 protofilaments,
majority have 13
Each heterodimer forms extensive noncovalent bonds with its neighbours – bonds
form longitudinally and laterally, linking adjacent protofilaments (lateral bonds
make the microtubule strong)
Heterodimers have same orientation within microtubule – allows two ends to be
structurally different and can behave differently
Polarity – inherent directionality throughout microtubules allows them to act as
directional tracks for molecular motor proteins (essential for organisation within
the cell)
Microtubule Organisation and Nucleation in Cells
Pure tubulin + buffer + GTP @ 37oC
initiates polymerisation in vitro
(formation of microtubules detected by
light scattering)
Spontaneous nucleation is a rare
limiting-step in polymerisation – a nuclei
are only stable if the grow faster than
they polymerise
There is a critical concentration of
subunits in solution – one that doesn’t
change no matter what the starting concentration is
, In cells, the MTOC functions to nucleate
microtubules as spontaneous nucleation is very
slow – MTOCs often remain associated with the
minus ends of microtubules they nucleate and
can dictate position + orientation
Most common MTOC in animal cells =
centrosome, composed of a pair of centrioles
surrounded by the pericentriolar material
Centrioles (organised at right angles to one
another in centre of centrosome) composed of triplet microtubules, 9 arranged
symmetrically to form walls of barrel
Centrioles contain α-,β-,ε-,δ-tubulin and the pericentriolar material is composed of
100 different types of proteins arranged in a 3D lattice structure
γ-tubulin and several other proteins of the pericentriolar material found in the
γ-tubulin ring complex (γTuRC)
This complex binds to α- and β- tubulins and are responsible for nucleating
microtubules (mechanism not yet clear?)
The shape of the complex (arranged as a shallow helix) suggests that it serves as a
template for the formation of a microtubule (from the minus end)
Variations of centrosomes
Centrosomes reproduced themselves during each cycle of mitosis
Centrioles duplicate first, simultaneously with the DNA – each new centriole formed
at right angles to the two original centrioles
Each centrosome has one old and daughter centriole
Centrosome is a dynamic structure, changes size – once duplicated centrosomes
grow bigger as cells prepare for mitosis: high density of microtubules is needed to
build a mitotic spindle
Motile animal cells (eg. sperm cells) have specialised MTOC, the basal body – serves
as templates for assembly for axoneme
Not all cells use centrosomes to nucleate microtubules:
Fungi MTOC is the spindle pole body embedded into nuclear envelope
Plant cells lack any defined structure that acts as MTOC but have a number of
microtubule nucleating sites
Epithelial cells, neuron cells and muscle cells have microtubule arrays not attached
to the centrosome – suggests that smaller types of MTOC can be positions within
cell to created specialised arrangements
Eg. epithelial cells have nucleation sites near apical end of cell – plus ends from
apical end to the basal end of the cell
Intricate network of protein filaments that extends throughout the cytoplasm
Filamentous architecture helps support large volume of cytoplasm in animal cells,
cytoskeletal elements found in bacteria but cytoskeleton most prominent in
structurally complex eukaryotic cell
The many roles of the cytoskeleton…
- Mitosis: segregation of chromosomes into daughter cells, cytokinesis
- Cell movement (mammalian cells, gametes – sperm cells, WBCs)
- Organises internal structure of cell, hold organelles in place (Golgi, nucleus,
mitochondria)
*Golgi disperses into fragments, ER collapses around nucleus as it is continuous with
nuclear envelope
- Determine cell shape (epithelial cells, neurons with long dendrites)
*In general, cells lose their shape and form round balls when microtubules are
depolymerised
- Intracellular transportation system – movement of membrane vesicles and proteins
Cytoskeleton composed of three major types of structural proteins
Subunit Nucleotid Diameter Molecular Form of Motor
e binding of weight polymer Proteins
filament
Actin ATP/ADP 6-7 nm 42 kDa 2 Myosins
Actin
monomer stranded
filaments
helix
Tubulin GTP/GDP 24-25 nm 50 kDa Hollow Kinesins
Microtubules dimer tube: 13 Dyneins
filaments
Depends None 8-10 nm 40-210 Coiled coil None
Intermediat
on cell kDa
e filaments
type
*Microtubules and actin filaments are dynamic polymers (polymerise and depolymerise
rapidly in cells), IFs are not
*Microtubules and actin filaments are polarised protein polymers (each end of polymer
has different behaviour), IFs are not
,Microtubule/Actin Disrupting Agents
Actin-binding
Cytochalasins Cap+ ends of filaments
(fungual metabolites)
Inhibit actin polymerisation
Latrunculins Bind to actin monomers
(red sea sponge toxin)
Phalloidins
(Amantina mushroom)
Stabilise actin filaments Bind to polymerised actin
Jasplakinolide
(sea sponge toxin)
Tubulin-binding
Nocadazole Binds to tubulin dimer
Inhibits tubulin polymerisation
(benzimidazole derivative)
Taxol Binds to microtubules
Stabilises microtubules
(ark of Pacific yew)
Microtubules
Function of microtubules depend upon two “contradictory” features: can act as
stuff structural elements which can easily disassemble
Tubular structure and large diameter make them relatively stiff and allow them to
resist compression
Dynamic nature is very important for function – allows rapid reorganisation of
microtubules when necessary
May also have a role in allowing cells to navigate/change direction
Fibroblast: moves within body and changes directions – microtubules arranged in
starlike pattern, radiating outward from single point in nucleus (these are short-lived,
lasting only a fraction of the time it takes for the cell to move)
Neuron: large parallel microtubules with many vesicles which carry material to and from
synapse (stable tubules, essential for cell structure), growth cones have unstable
microtubules to help form synapses with other neurons
,Structure
Tubulin: a heterodimer made up of α-
and β-tubulin (both proteins share 40%
sequence identity, always associated
with each other)
Each molecule of α- and β-tubulin binds
a molecule of GTP (only the β-tubulin
bound GTP is exposed to exchange with
nucleotide in solution
Microtubules are protein polymers composed of thousands of tubulin subunits
organised into a hollow tube
Typically made up of 13 linear chains of subunits that run parallel to one another –
each linear chain = protofilament; 1 microtubule formed of 11-15 protofilaments,
majority have 13
Each heterodimer forms extensive noncovalent bonds with its neighbours – bonds
form longitudinally and laterally, linking adjacent protofilaments (lateral bonds
make the microtubule strong)
Heterodimers have same orientation within microtubule – allows two ends to be
structurally different and can behave differently
Polarity – inherent directionality throughout microtubules allows them to act as
directional tracks for molecular motor proteins (essential for organisation within
the cell)
Microtubule Organisation and Nucleation in Cells
Pure tubulin + buffer + GTP @ 37oC
initiates polymerisation in vitro
(formation of microtubules detected by
light scattering)
Spontaneous nucleation is a rare
limiting-step in polymerisation – a nuclei
are only stable if the grow faster than
they polymerise
There is a critical concentration of
subunits in solution – one that doesn’t
change no matter what the starting concentration is
, In cells, the MTOC functions to nucleate
microtubules as spontaneous nucleation is very
slow – MTOCs often remain associated with the
minus ends of microtubules they nucleate and
can dictate position + orientation
Most common MTOC in animal cells =
centrosome, composed of a pair of centrioles
surrounded by the pericentriolar material
Centrioles (organised at right angles to one
another in centre of centrosome) composed of triplet microtubules, 9 arranged
symmetrically to form walls of barrel
Centrioles contain α-,β-,ε-,δ-tubulin and the pericentriolar material is composed of
100 different types of proteins arranged in a 3D lattice structure
γ-tubulin and several other proteins of the pericentriolar material found in the
γ-tubulin ring complex (γTuRC)
This complex binds to α- and β- tubulins and are responsible for nucleating
microtubules (mechanism not yet clear?)
The shape of the complex (arranged as a shallow helix) suggests that it serves as a
template for the formation of a microtubule (from the minus end)
Variations of centrosomes
Centrosomes reproduced themselves during each cycle of mitosis
Centrioles duplicate first, simultaneously with the DNA – each new centriole formed
at right angles to the two original centrioles
Each centrosome has one old and daughter centriole
Centrosome is a dynamic structure, changes size – once duplicated centrosomes
grow bigger as cells prepare for mitosis: high density of microtubules is needed to
build a mitotic spindle
Motile animal cells (eg. sperm cells) have specialised MTOC, the basal body – serves
as templates for assembly for axoneme
Not all cells use centrosomes to nucleate microtubules:
Fungi MTOC is the spindle pole body embedded into nuclear envelope
Plant cells lack any defined structure that acts as MTOC but have a number of
microtubule nucleating sites
Epithelial cells, neuron cells and muscle cells have microtubule arrays not attached
to the centrosome – suggests that smaller types of MTOC can be positions within
cell to created specialised arrangements
Eg. epithelial cells have nucleation sites near apical end of cell – plus ends from
apical end to the basal end of the cell
