Microtubule Motors

Copyright © 2000-2006 by Joyce J. Diwan. All rights reserved.

Biochemistry of Metabolism:Cell Biology

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.

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 -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.

stalk domain

N-terminal heavy chain motor

domains (heads)

Kinesin I

hinge light chains

C-terminal tail domains

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.

stalk domain

N-terminal heavy chain motor

domains (heads)

Kinesin I

hinge light chains

C-terminal tail domains

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.

stalk domain

N-terminal heavy chain motor

domains (heads)

Kinesin I

hinge light chains

C-terminal tail domains

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.

Cargo proteins bound by kinesins are diverse.

cargo vesicle

kinesin

mic

rotu

bule

scaffolding protein

receptor

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.

In absence of cargo, the kinesin heavy chain stalk folds at hinge regions, bringing heavy chain tail domains

cargo vesicle

kinesin

mic

rotu

bule

scaffolding protein

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.

cargo vesicle

kinesin m

icro

tubu

le

scaffolding protein

receptor

inactive kinesin

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.

stalk domain

N-terminal heavy chain motor

domains (heads)

Kinesin I

hinge light chains

C-terminal tail domains

Different members of the kinesin protein family vary in structure.

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.

microtubule

microtubule

BimC

that of the GTP-binding protein Ras.

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

Kinesin's globular motor domain exhibits structural similarity, but little sequence homology, to that of myosin.

Kinesin & myosin heads both have nucleotide binding domains similar to

Kinesin heavy chain head & neck domains

PDB 3KIN

ADP

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.

The neck domain of kinesin I is an -helical coiled coil.

Kinesin heavy chain head & neck domains

PDB 3KIN

ADP

ADP

The kinesin's microtubule-binding domain is positioned opposite the ATP-binding cleft, equivalent to the position of the actin-binding domain of myosin.

KIF1A head domain

with bound Mg++-ATP

-tubulin-GTP -tubulin-GDP-taxol

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).

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.

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.

(+) microtubule (-)

Kinesin transporting a vesicle along a 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.

(+) microtubule (-)

Kinesin transporting a vesicle along a microtubule

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 heavy chain head & neck domains

PDB 3KIN

ADP

ADP

Kinesin reaction cycle differs from myosin: Each kinesin motor

domain binds tightly to a microtubule when it has bound ATP.

Myosin dissociates from actin upon binding ATP.

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, Max-Planck Unit for Structural Molecular Biology, Hamburg.

animation from a web site of the Milligan lab, Scripps Research Institute, La Jolla.

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.

nucleus

MTOC

microtubules

Various members of the kinesin family have diverse roles.

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.

nerve cell body axon

ending

( ) (+) kinesin

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.

nerve cell body axon ending

with actin cytoskeleton

axon with microtubules Kinesins &

myosins may cooperate in vesicle transport.

• 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. 

Various kinesins function in mitosis.

Some kinesins promote shortening of microtubules, perhaps by inducing curvature at ends of protofilaments.

astral microtubule

chromosomal microtubule

polar microtubule

centrosome

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.

( ) ()

kinetochore subunit flow during treadmilling

tubulin dimers released during metaphase or anaphase

tubulin dimers added during metaphase or released in anaphase

In metaphase there is treadmilling in kinetochore microtubules: • Tubulin subunits

flow toward the poles, as dimers are added at plus ends & removed at minus ends.

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.

dynein

BimC

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

Anaphase B

During prophase and in anaphase B the mitotic spindle poles separate.

Cytoplasmic dyneins mediate ATP-dependent retrograde movements of vesicles and organelles along microtubules toward the centrosome (MTOC-microtubule organizing center).

nucleus

MTOC

microtubules

Dyneins are minus end-directed motor proteins.

They were first studied in cilia & flagella.

Many cytoplasmic dyneins have now been discovered.

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.

motor domain

stalk

head

Dynein (approximate structure)

domain that interacts with

microtubule

Dynein is large & complex.

Cytoplasmic dynein has a MW exceeding 106.

multiple dyneins interact with a microtubule.

The stalk is an intra-molecular coiled coil, formed by interaction of -helical segments on either side of the microtubule-binding segment in the primary sequence of the dynein heavy chain.

motor domain

stalk

head

Dynein (approximate structure)

domain that interacts with

microtubule

Extending out from each motor domain is a narrow stalk that ends in a small globular domain.

It is this domain at the end of the stalk that interacts with microtubules.

The stalk may help avoid steric interference when

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 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.

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.

nucleus

MTOC

microtubules

Dynein & dynactin are associated with golgi membranes, which also have a spectrin network on their surface.

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.

astral microtubule

chromosomal microtubule

polar microtubule

centrosome

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.

dynein

BimC

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

Anaphase B

• 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.

Cilia & flagella Bounded by plasma membrane. Basal body: a single centriole

cylinder at the base of each cilium or flagellum. Electron micrograph (article by J. Beisson & M. Wright).

Core axoneme: a complex of microtubules & associated proteins.

Some distinctions:

plasma membrane axoneme basal body (centriole)

Cilium

cytosol

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.

plasma membrane A B

radial spoke

nexin link

central sheath Cilium cross section

dynein arm

B A

An axoneme includes:

Nine doublet microtubules around the periphery.

The A tubule of each doublet has attached dynein arms.

Bending of a cilium involves ATP-dependent walking of motor 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 between doublets limit sliding. The result is bending of the cilium.

Evidence for this mechanism: ATP is required for bending. Inactivating dynein mutations

eliminate ciliary bending.

Sliding of microtubule doublets

+

¯

A B

A B

dynein arms

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.

ATP-dependent sliding of microtubule doublets, with radial spokes & nexin links cleaved.

Fewer microtubule doublets at the tip

of a bent cilium.

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.

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)

Cilium

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.