Biochemistry of Metabolism:
Cell Biology
Microtubule Motors
Copyright © 2000-2006 by Joyce J. Diwan.
All rights reserved.
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
tail domains
light chains
stalk
domain
N-terminal heavy
chain motor
domains (heads)
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
tail domains
light chains
stalk
domain
N-terminal heavy
chain motor
domains (heads)
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
tail domains
light chains
stalk
domain
N-terminal heavy
chain motor
domains (heads)
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.
microtubule
Cargo
proteins
bound by
kinesins are
diverse.
scaffolding
protein
kinesin
cargo
vesicle
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.
microtubule
scaffolding
In absence
protein
cargo
of cargo, the
vesicle
kinesin heavy
kinesin
chain stalk
receptor
folds at hinge
regions,
bringing
inactive kinesin
heavy chain
tail domains
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
microtubule
kinesin
cargo
vesicle
receptor
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.
Different
members of
the kinesin
protein
family vary
in structure.
C-terminal
tail domains
light chains
stalk
domain
N-terminal heavy
chain motor
domains (heads)
hinge
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
ADP
sequence homology,
to that of myosin.
Kinesin & myosin
heads both have
Kinesin heavy chain
nucleotide binding
head & neck domains
ADP
domains similar to
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 coil.
Kinesin heavy chain
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.
Structure of the motor
domain of monomeric
kinesin KIF1A with a
bound ATP analog,
complexed to a
microtubule has been
determined by high
resolution cryo-EM
(PDB 1IA0).
KIF1A head domain
with bound
Mg++-ATP
a-tubulin-GTP
b-tubulin-GDP-taxol
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
ADP
to a microtubule when
it has bound ATP.
 Myosin dissociates
Kinesin heavy chain
from actin upon
head & neck domains
ADP
binding ATP.
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.
View an animation emphasizing the cycle of ATP
binding, hydrolysis & product dissociation during
processive movement of kinesin along a microtubule.
Additional animations based on atomic resolution
structures of kinesin and tubulin:
 animation from a web site of the Mandelkow lab, MaxPlanck Unit for Structural Molecular Biology, Hamburg.
 animation from a web site of the Milligan lab, Scripps
Research Institute, La Jolla.
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.
()
nerve cell body
kinesin
(+)
axon
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.
Kinesins &
myosins may
cooperate in
vesicle transport.

nerve cell body
axon with
microtubules

axon ending
with actin
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
astral
of microtubules,
microtubule
perhaps by inducing
polar
chromosomal
curvature at ends of
microtubule
microtubule
protofilaments.
• 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
subunit flow during kinetochore
is treadmilling in
treadmilling

kinetochore
() ()
microtubules:

• Tubulin subunits  tubulin dimers
  released during
 
flow toward the
 metaphase or

tubulin dimers added
poles, as dimers
anaphase
during metaphase or
are added at plus
released in anaphase
ends & removed
at minus ends.
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 spindle
poles separate.



BimC
dynein
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 enddirected motor proteins.
nucleus
They were first studied in
cilia & flagella.
Many cytoplasmic dyneins
have now been discovered.

microtubules
Cytoplasmic dyneins mediate ATP-dependent retrograde
movements of vesicles and organelles along microtubules
toward the centrosome (MTOC-microtubule organizing
center).
head
domain that
interacts with
microtubule
stalk
Dynein is large &
complex.
Cytoplasmic dynein has
a MW exceeding 106.
Dynein
(approximate
structure)
motor
domain
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
motor domain is a narrow
stalk that ends in a small
globular domain.
head
domain that
interacts with
microtubule
stalk
It is this domain at the end
of the stalk that interacts
Dynein
with microtubules.
(approximate
structure)
motor
domain
The stalk may help avoid
steric interference when
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.
 Each heavy chain motor domain of dynein includes
6 repeats of an ATPase of the AAA gene family.
AAA ATPases typically form a wheel-like structure with
6 ATPase domains.
Diagrams: website  Berkeley; website  Imperial Coll.
 High resolution EM with image averaging indicates a
heptameric wheel-like structure of the dynein motor
domain, with the 6 AAA domains plus an additional
C-terminal domain.
One of the AAA domains is postulated to be the
functional ATPase that drives movement.
A stalk (assumed to be the microtubule-binding segment)
protrudes out from between 2 of 6 AAA domains.
Diagrams: website  U. Conn.; website  UCSF.
At a Univ. Leeds website:
 animated model of the dynein power stroke
 animation based on electron microscope images of a
flagellar dynein.
Dynactin is a large complex that mediates binding of
dynein to membranes or other cargo.
Dynein may bind to some cargo proteins directly via its
light chains, but interactions with cargo are often mediated
by dynactin.
Dynactin structure: diagram on webpage of T. Schroer.
Constituents of dynactin are listed on the next slide.
 Glued, an elongated 150 kDa dimeric protein, includes:
• 2 microtubule-binding globular heads at distal end.
Binding of glued to microtubules may increase
processivity of dynein movement on microtubules.
• domains of glued that bind dyneins & kinesins.
Coordination of bidirectional transport along
microtubules may involve regulated binding to
dynactin of plus & minus end-directed motors.
 Arp1, an actin-related protein, forms a short filament
(rod) of constant length (8-10 subunits).
Arp1 rod is capped at one end by actin-capping protein
CapZ, & at the other end by proteins unique to dynactin.
 Dynamitin, with another small protein, links the Arp1
rod to glued.
Dynein is often found in the cell cortex.
The Arp1 rod of dynactin binds to spectrin, an actinbinding 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
associated with golgi
membranes, which also
have a spectrin network
on their surface.
nucleus

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
microtubule
chromosomal
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.
Cilium
Cilia & flagella
 Bounded by plasma membrane.
plasma
membrane
 Basal body: a single centriole
cylinder at the base of each cilium
or flagellum. Electron micrograph
axoneme
(article by 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
An axoneme includes:
Nine doublet
microtubules around
the periphery.
The A tubule of each
doublet has attached
Cilium
cross section
dynein arms.
B
AA
B
radial
spoke
nexin
link
dynein
arm
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
AB
domains of A tubule dynein arms
+
along adjacent B tubules, toward
the minus end. This causes sliding
of microtubule doublets.
Minus ends are anchored in the
basal body, & flexible links
dynein
arms
between doublets limit sliding.
The result is bending of the cilium.
Evidence for this mechanism:
Sliding of ¯
microtubule
 ATP is required for bending.
doublets
 Inactivating dynein mutations
eliminate ciliary bending.
AB
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
toward the ciliary/flagellar tip.
Cytosolic dyneins transport the
particles with associated proteins
(including kinesins after they
discharge their cargo) along
axonemal microtubules back
toward the cytosol.
plasma
membrane
axoneme
basal body
(centriole)
cytosol
Video and diagram of intraflagellar transport at a website
of the Rosenbaum lab at Yale University.
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.