Cytoskeleton

Providing structural support to the cell,

the cytoskeleton also functions in cell
motility and regulation
 Structural Support
• Mechanical support
– Maintains shape
• Fibers act like a geodesic dome to
stabilize and balance opposing forces
• Provides anchorage for organelles
• Dynamic
– Dismantles in one spot and reassembles in
another to change cell shape
• Introduction
• The cytoskeleton is a network of fibers
extending throughout the cytoplasm.
• The cytoskeleton
organizes the
structures and
activities of
the cell.
• The cytoskeleton also plays a major role in
cell motility.
– This involves both changes in cell location and
limited movements of parts of the cell.
• The cytoskeleton 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.

Fig. 7.21a
• Motor molecules also carry vesicles or
organelles to various destinations along
“monorails’ provided by the cytoskeleton.
• Interactions of motor proteins and the
cytoskeleton circulates materials within a
cell via streaming.
• Recently, evidence is accumulating that the
cytoskeleton may
transmit mechanical
signals that rearrange
the nucleoli and
other structures.
Fig. 7.21b
• There are three main types of fibers in the
cytoskeleton: microtubules,
microfilaments, and intermediate
filaments.
• Microtubules, the thickest fibers, are hollow rods
about 25 microns in diameter.
– Microtubule fibers are constructed of the
globular protein, tubulin, and they grow or
shrink as more tubulin molecules are added or
removed.
• They move chromosomes during cell division.
• Another function is
as tracks that guide
motor proteins
carrying organelles
to their destination.

Fig. 7.21b
• In many cells, microtubules grow out from a
centrosome near the nucleus.
– These microtubules resist compression to the
cell.
• In animal cells, the centrosome has a pair of
centrioles, each with nine triplets of microtubules
arranged in a ring.

• During cell division the

centrioles replicate.

Fig. 7.22
• Microtubules are the central structural
supports in cilia and flagella.
– Both can move unicellular and small
multicellular organisms by propelling water
past the organism.
– If these structures are anchored in a large
structure, they move fluid over a surface.
• For example, cilia sweep mucus carrying trapped
debris from the lungs.

Fig. 7.2
• Cilia usually occur in large numbers on the
cell surface.
– They are about 0.25 microns in diameter and
2-20 microns long.
• There are usually just one or a few flagella
per cell.
– Flagella are the same width as cilia, but 10-200
microns long.
• A flagellum has an undulatory movement.
– Force is generated parallel to the flagellum’s
axis.

Fig. 7.23a
• Cilia move more like oars with alternating
power and recovery strokes.
– They generate force perpendicular to the cilia’s
axis.

Fig. 7.23b
• In spite of their differences, both cilia and
flagella have the same ultrastructure.
– Both have a core of microtubules sheathed by
the plasma membrane.
– Nine doublets of microtubules arranged around
a pair at the center, the “9 + 2” pattern.
– Flexible “wheels” of proteins connect outer
doublets to each other and to the core.
– The outer doublets are also connected by
motor proteins.
– The cilium or flagellum is anchored in the cell
by a basal body, whose structure is identical
to a centriole.
Fig. 7.24
• The bending of cilia and flagella is driven by
the arms of a motor protein, dynein.
– Addition to dynein of a phosphate group from
ATP and its removal causes conformation
changes in the protein.
– Dynein arms alternately
grab, move, and release
the outer microtubules.
– Protein cross-links limit
sliding and the force is
expressed as bending.

Fig. 7.25
• Microfilaments, the thinnest class of the
cytoskeletal fibers, are solid rods of the
globular protein actin.
– An actin microfilament consists of a twisted
double chain of actin subunits.
• Microfilaments are designed to resist
tension.
• With other proteins, they form a three-
dimensional network just inside the plasma
membrane.
Fig. 7.26 The shape of the
microvilli in this intestinal cell
are supported by microfilaments,
anchored to a network of
intermediate filaments.
• In muscle cells, thousands of actin filaments are
arranged parallel to one another.
• Thicker filaments, composed of a motor protein,
myosin, interdigitate with the thinner actin fibers.
– Myosin molecules walk along the actin filament,
pulling stacks of actin fibers together and
shortening
the cell.

Fig. 7.21a
• In other cells, these actin-myosin aggregates are less
organized but still cause localized contraction.
– A contracting belt of microfilaments divides the
cytoplasm of animals cells during cell division.
– Localized contraction also drives amoeboid movement.
• Pseudopodia, cellular extensions, extend and
contract through the reversible assembly and
contraction of actin subunits into microfilaments.

Fig. 7.21b
• In plant cells (and others), actin-myosin
interactions and sol-gel transformations
drive cytoplasmic streaming.
– This creates a circular flow of cytoplasm in the
cell.
– This speeds the distribution of materials within
the cell.

Fig. 7.21c
• Intermediate filaments,
intermediate in size at 8 -
12 nanometers, are
specialized for bearing
tension.
– Intermediate filaments
are built from a diverse
class of subunits from a
family of proteins called
keratins.
• Intermediate filaments are
more permanent fixtures of
the cytoskeleton than are
the other two classes.
• They reinforce cell shape
and fix organelle location. Fig. 7.26
Two main families of microtubule motor proteins
carry out ATP-dependent movement along
microtubules:
1. Kinesins are a large family of motor proteins,
most of which walk along microtubules toward
the plus end, away from the centrosome
(MTOC).
2. Dyneins walk along microtubules toward the
minus end (toward the centrosome).
In each case there is postulated to be a reaction
cycle similar (but not identical) to that of myosin.
Motility arises from conformational changes in the
motor domain as ATP is bound & hydrolyzed, and
products released.
 Kinesins
Kinesins are a large family of proteins with diverse
structures. Mammalian cells have at least 40
different kinesin genes.
The best studied is referred to as conventional
kinesin, kinesin I, or simply kinesin.
Some are referred to as kinesin-related proteins
(KRPs).
Kinesin I has a structure analogous to but distinct
from that of myosin.
There are 2 copies each of a heavy chain and a light
chain.
 C-terminal N-terminal heavy
tail domains stalk chain motor
domain domains (heads)

light chains hinge

Kinesin I
Each heavy chain of kinesin I includes a globular
ATP-binding motor domain at the N-terminus.
Stalk domains of heavy chains interact in an a-
helical coiled coil that extends from heavy chain
neck to tail.
The coiled coil is interrupted by a few hinge regions
that give flexibility to the otherwise stiff stalk domain.
 C-terminal N-terminal heavy
tail domains stalk chain motor
domain domains (heads)

light chains hinge

Kinesin I

N-termini of the 2 light chains associate with the 2 heavy

chains near the tail. The diagram above is over simplified.
Light chains at the N-terminus include a series of hydrophobic
heptad repeats predicted to interact with similar repeats in the
heavy chains near the tail region, in a 4-helix coiled coil.
 C-terminal N-terminal heavy
tail domains stalk chain motor
domain domains (heads)

light chains hinge

Kinesin I
C-terminal tail domains of kinesin light chains include several
"tetratrico peptide repeats" (TPRs). The 34 amino acid TPRs
mediate protein-protein interactions.
Kinesin light chain TPRs are involved in binding of kinesins to
cargo.
C terminal domains of heavy chains may also participate in
binding some kinesins to cargo.
Cargo scaffolding

microtubule
proteins protein
cargo
bound by vesicle
kinesins are kinesin
diverse.
receptor

Some organelle membranes contain transmembrane

receptor proteins that bind kinesins. Kinectin is an ER
membrane receptor for kinesin-I.
Scaffolding proteins, first identified as being involved in
assembling signal protein complexes, mediate binding of
kinesin light chains to some cargo proteins or receptors.
Some membrane-associated Rab GTPases, that provide
specificity for vesicle transport & fusion, are known to bind
particular kinesins.
In absence scaffolding
of cargo, the protein
cargo
kinesin heavy vesicle
chain stalk kinesin
folds at hinge receptor
regions,
microtubule
bringing
heavy chain
tail domains inactive kinesin

into contact with the motor domains.

In this folded over state kinesin exhibits decreased ATPase
activity and diminished binding to microtubules.
This may prevent wasteful hydrolysis of ATP by kinesin when
it is not transporting cargo.
 scaffolding
protein
cargo
vesicle
kinesin
receptor
microtubule

inactive kinesin

Unfolding of kinesin into its more extended

active conformation is promoted by:
 phosphorylation of kinesin light chains,
catalyzed by a specific kinase, or
 binding of cargo.
 C-terminal N-terminal heavy
tail domains stalk chain motor
Different domains (heads)
members of domain
the kinesin
protein family
vary in light chains hinge
structure.
Kinesin I
In contrast to conventional kinesin I, a few kinesins have their
motor domain in the interior of the heavy chain sequence or
at the C-terminus, instead of at the N-terminus.
 Those with their motor domain at the C-terminus, e.g., Ncd
(KIFC2), move in the opposite direction along microtubules
(toward the minus end) than is typical for kinesins.
  microtubule 

BimC

 microtubule 

 One class of kinesins (KIF1) has a heavy chain that lacks the
coiled coil domain & is monomeric instead of dimeric.
 BimC, a kinesin related protein involved in mitosis, has a tail
domain that allows it to assemble into antiparallel dimers that
can mediate sliding of microtubules relative to one another.
This resembles the ability of myosin II to form bipolar filaments
that mediate sliding of actin filaments.
 PDB 3KIN
Kinesin's globular
motor domain exhibits
structural similarity, but
little sequence
homology, to that of
myosin. ADP
Kinesin & myosin
heads both have
nucleotide binding
domains similar to Kinesin heavy chain
head & neck domains ADP

that of the GTP-binding protein Ras.

Positions of most b-strands & a-helices in their motor
domains are equivalent. But kinesin has short connecting
loops where the larger myosin head has longer stretches of
amino acids.
 PDB 3KIN

ADP

The neck domain

of kinesin I is an
a-helical coiled Kinesin heavy chain
coil. head & neck domains ADP

Switch regions have been identified that change

conformation depending on what occupies the
nucleotide binding site.
These are equivalent to switch regions of myosin
and GTP-binding proteins.
 KIF1A head domain
Structure of the with bound
motor domain of Mg++-ATP
monomeric kinesin
KIF1A with a bound
ATP analog,
complexed to a
microtubule has
been determined by
high resolution cryo- a-tubulin-GTP b-tubulin-GDP-taxol
EM (PDB 1IA0).
The kinesin's microtubule-binding domain is
positioned opposite the ATP-binding cleft, equivalent
to the position of the actin-binding domain of myosin.
In vitro experiments have used digital video
with differential interference microscopy to
record:
 ATP-dependent movement of microtubules
along a surface coated with conventional
kinesin, and
 ATP-dependent kinesin-mediated movements
of vesicles along microtubules.
Videos may be viewed in a web site linked to the
Kinesin Home Page.
 Kinesin transporting a vesicle
along a microtubule

(+) microtubule (-)

Observations of conventional kinesin transporting

elongated particles have demonstrated that cargo
particles do not roll along the microtubule. Instead
kinesin walks along, maintaining the orientation of a
cargo particle.
 Kinesin transporting a vesicle
along a microtubule

(+) microtubule (-)

Movement of the 2-headed kinesin is processive, meaning

that it takes many steps without dissociating from a
microtubule. A hand over hand reaction cycle involving the 2
heads has been proposed.
Myosin V, which transports vesicles along actin filaments,
also exhibits processive movement.
 PDB 3KIN
Kinesin reaction cycle
differs from myosin:
 Each kinesin motor
domain binds tightly to
a microtubule when it ADP
has bound ATP.
 Myosin dissociates
from actin upon binding
ATP. Kinesin heavy chain
head & neck domains ADP
Kinesin processivity requires coordination between motor
domains.
Repositioning of the forward motor with its neck linker,
allowing it to bind ATP & attach more firmly to the microtubule,
is postulated to depend on the trailing motor hydrolyzing its
ATP & beginning to detach.
Kinesin has been found to limp along.
While each step length is 8 nm, the time it takes for
each sequential step alternates between short and
long.
It has been suggested that the irregular gait may
result from the coiled coil stalk being alternately
over and under-wound, as the two kinesin motor
domains go through their combined reaction cycle.
See diagram by S. Block & coworkers.
 MTOC

nucleus

Various members of the
kinesin family have
diverse roles.
microtubules

Conventional kinesin has a role in movement of

vesicles & lysosomes, from the vicinity of the golgi
apparatus near the centrosome (MTOC - adjacent to
the cell nucleus), toward the plus ends of
microtubules in the cell periphery.
 () kinesin (+)
axon
nerve cell body ending

Kinesin I was first isolated from brain tissue.

It is responsible for fast axonal flow, in which organelles
(e.g., mitochondria) and vesicles (e.g., precursors of synaptic
vesicles formed in the golgi) are carried from near the
centrosome in the cell body to axon endings.
Such transport away from the centrosome, toward the (+)
ends of microtubules, is called anterograde transport.
 axon with
Kinesins & 
 microtubules
myosins may
cooperate in axon ending
vesicle transport. with actin
nerve cell body
cytoskeleton
Vesicles in extruded nerve axoplasm were found to attach to
and move along both microtubules and actin filaments.
Kinesins & myosin V are both associated with precursors of
synaptic vesicles.
 Kinesin transports vesicles along microtubules in the
axon to the plus ends, where the axon ending begins.
 Axon endings instead have an extensive actin
cytoskeleton. Myosin V may take over to transport vesicles
along actin filaments to near the plasma membrane at the
synapse.
Various kinesins
function in mitosis. centrosome

Some kinesins
promote shortening
of microtubules, astral
perhaps by inducing microtubule
curvature at ends of
protofilaments. polar chromosomal
microtubule microtubule

• the catastrophe-promoting kinesin MCAK is in the

kinetochore, where plus ends of spindle microtubules
attach to chromosomes. Movie showing ATP-dependent
shortening of isolated microtubules by added MCAK (video
supplement #1, Helenius et al).
• the disassembly-promoting kinesin KLP10A is at minus
ends of spindle microtubules, at poles of the cell.
In metaphase there
is treadmilling in subunit flow during kinetochore
treadmilling 
kinetochore
microtubules: 
()  ()
• Tubulin subunits
flow toward the   tubulin dimers 
released during  
 
poles, as dimers  metaphase or 
are added at plus anaphase tubulin dimers added
ends & removed during metaphase or
at minus ends. released in anaphase

During anaphase A chromosomes move to spindle poles as

microtubules linking kinetochores to poles shorten by:
• dissociation of tubulin dimers at the kinetochore.
• continued dissociation of tubulin dimers at the poles in
some cells.
 Anaphase B

 
During prophase  
and in anaphase
B the mitotic BimC dynein
spindle poles
separate. BimC mediates sliding of polar microtubules;
dynein pulls asters to membrane; tubulin
dimers add at plus ends of polar microtubules.
BimC, which forms bipolar complexes, mediates sliding of
antiparallel spindle microtubules relative to one another.
BimC motor domains walk toward the plus ends of
overlapping polar microtubules, pushing the poles apart, as
tubulin heterodimers add to the plus ends.
 MTOC

Dyneins are minus end-

directed motor proteins. nucleus

They were first studied in
cilia & flagella.
Many cytoplasmic
dyneins have now been microtubules
discovered.
Cytoplasmic dyneins mediate ATP-dependent
retrograde movements of vesicles and organelles
along microtubules toward the centrosome (MTOC-
microtubule organizing center).
 domain that
interacts with
head microtubule

Dynein is large & stalk

complex.
Cytoplasmic dynein Dynein
has a MW exceeding (approximate motor
structure) domain
106.
Dynein includes 2 or 3 heavy chains. Each is about 4600
amino acid residues long & includes a globular motor
domain.
There are also multiple intermediate & light chains.
Dynein also requires large complexes of other proteins to
mediate binding to cargo such as membrane vesicles.
Extending out from each
domain that
motor domain is a narrow interacts with
stalk that ends in a small head microtubule
globular domain.
It is this domain at the end stalk
of the stalk that interacts
with microtubules.
The stalk may help avoid Dynein
steric interference when (approximate motor
structure) domain

multiple dyneins interact with a microtubule.

The stalk is an intra-molecular coiled coil, formed by
interaction of a-helical segments on either side of the
microtubule-binding segment in the primary sequence of the
dynein heavy chain.
Dynein is often found in the cell cortex.
The Arp1 rod of dynactin binds to spectrin, an
actin-binding protein of the cortical cytoskeleton.
Spectrin in turn binds to ankryn, which binds to
integral membrane proteins.
Thus dynactin anchors dynein to the plasma
membrane.
 MTOC

Dynein & dynactin are nucleus

associated with golgi 
membranes, which also
have a spectrin network on
their surface.
microtubules

Location of the golgi apparatus near the centrosome

(MTOC) is thought to be due to its being drawn along
microtubules toward their minus ends by dynein.
Early & late endocytic vesicles also have associated
dynein & dynactin, which may (with myosin VI) move these
vesicles inward from the cell surface.
 centrosome

astral
microtubule

polar chromosomal
microtubule microtubule
Metaphase of mitosis

Interaction of cortical dynein with astral

microtubules is considered essential to orientation
of the mitotic spindle and separation of poles
during mitosis.
 Anaphase B

 
 

BimC dynein

BimC mediates sliding of polar microtubules;

dynein pulls asters to membrane; tubulin
dimers add at plus ends of polar microtubules.

Dynein, bound via dynactin to the plasma membrane/

cortical cytoskeleton, may generate force by
movement toward the centrosome along astral
microtubules, early in mitosis as well as during
anaphase B.
Cilia & flagella Cilium
 Bounded by plasma membrane. plasma
 Basal body: a single centriole membrane
cylinder at the base of each cilium or
flagellum. Electron micrograph (article by axoneme
J. Beisson & M. Wright).
 Core axoneme: a complex of
microtubules & associated proteins.
 Some distinctions: basal body
(centriole)
cytosol
• Flagella are usually 1 or 2 per cell.
They tend to have a rotary or sinusoidal movement.
There may be additional structures outside core axoneme.
• Cilia are usually many per cell.
They tend to have a whip-like movement.
 plasma
membrane B
AAB

An axoneme includes: radial

spoke
Nine doublet
microtubules around the nexin
periphery. link
The A tubule of each dynein
doublet has attached arm
dynein arms. Cilium
cross section central sheath

Two singlet central microtubules, surrounded by a sheath.

Nexin links & radial spokes. These provide elastic
connections between microtubule doublets and between the A
tubule of each doublet and the central sheath.
Bending of a cilium involves ATP-
dependent walking of motor domains
of A tubule dynein arms along AB
adjacent B tubules, toward the +
AB
minus end. This causes sliding of
microtubule doublets.
Minus ends are anchored in the
basal body, & flexible links between
doublets limit sliding. The result is
dynein
bending of the cilium. arms
Evidence for this mechanism:
 ATP is required for bending.
 Inactivating dynein mutations Sliding of ¯
eliminate ciliary bending. microtubule
doublets
 ATP-dependent sliding of
microtubule doublets, with radial
spokes & nexin links cleaved.

Fewer microtubule
doublets at the tip
of a bent cilium.

 If isolated axonemes, with their membrane removed, are

treated with mild protease, radial spokes & nexin links
are degraded. ATP addition then causes microtubule
doublets to slide apart.
 If a bent cilium is examined in cross-section by EM,
fewer than 9 doublet microtubules are seen at the tip.
Few mammalian cell types have motile cilia or
flagella, including some respiratory epithelial
cells and sperm cells.
Many mammalian cells have a single short non-
motile primary cilium.
The photoreceptor structure of each retinal rod
& cone cell develops from a non-motile cilium.
Intraflagellar transport:
In addition to their role in ciliary/flagellar movement,
microtubules of the axoneme provide pathways along which
cytosolic & plasma membrane proteins are transported to
& from the tip of a cilium or flagellum.
This intraflagellar transport is important for formation &
maintenance of cilia & flagella, which grow by addition of
subunits at the distal tip where plus ends of axonemal
microtubules are located.
Some axonemal precursor proteins are transported in
association with particles (rafts) that are large enough to be
visualized by differential interference contrast light
microscopy.
 Cilium
Kinesins transport the particles
along axonemal microtubules plasma
membrane
toward the ciliary/flagellar tip.
Cytosolic dyneins transport the
axoneme
particles with associated proteins
(including kinesins after they
discharge their cargo) along
axonemal microtubules back basal body
toward the cytosol. (centriole)
cytosol
A number of diseases have been attributed to
defects in transport along microtubules.
 Defects in protein subunits of particles (rafts)
that carry cargo proteins to the tips of cilia &
flagella lead to polycystic kidney disease &
retinal degeneration in mammals.
• A non-motile primary cilia in kidney epithelial
cells fails to develop in this disease.
• Development of retinal rod & cone
photoreceptors from non-motile cilia is also
impaired.
 Kinesin defects:
• Some neurodegenerative diseases are
associated with defects in kinesin-mediated
long distance transport of materials along
microtubules.
• Some types of cancer are associated with
abnormalities of kinesins involved in mitosis.
• Ciliary and flagellar defects can also arise
from deficiency of kinesins involved in
transport of cargo to the tips of cilia and
flagella.
 LIS1 protein is associated with dynein/dynactin
in the cell cortex.
• Genetic defects in LIS1 lead to the disease
lissencephaly, in which brain development is
severely impaired.
• In animals, overexpression or elimination of
LIS1 causes mitotic spindle abnormalities,
including altered spindle orientation in
polarized epithelial cells.
 Toxins & Drugs
Some toxins and drugs (all of which inhibit mitosis) affect
polymerization or depolymerization of tubulin:
 Taxol, an anti-cancer drug, stabilizes microtubules.
 Colchicine binds tubulin & blocks polymerization.
Microtubules depolymerize at high [colchicine].
 Vinblastine causes depolymerization and formation of
vinblastine-tubulin paracrystals.
 Nocodazole causes depolymerization of
microtubules.