Biochemistry of Metabolism: Cell Biology

Microtubule Motors

Contents of this page:
Classes of microtubule motors
Kinesins
Dyneins
Cilia & Flagella

Note: Page numbers for this topic refer to the textbook Molecular Biology of the Cell by Alberts et al. (A). For additional information see the page on microtubules.

Two main classes of microtubule motor proteins carry out ATP-dependent movement along microtubules (A. p. 952-953):

• Kinesins are a large family of motor proteins, most of which walk along microtubules toward the plus end, away from the centrosome (MTOC).
• 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 and hydrolyzed, and products are released.

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 (conventional type) has a structure somewhat analogous to but distinct from that of myosin. There are 2 copies each of a heavy chain and a light chain. Each heavy chain includes a globular ATP-binding motor domain at the N-terminus. Stalk domains of the heavy chains interact in an a-helical coiled coil that extends from the heavy chain neck to the tail. The coiled coil is interrupted by a few hinge regions that give flexibility to the otherwise stiff stalk domain.

N-termini of the two light chains associate with the two heavy chains near the tail. The diagram above is over simplified. Light chains at the N-terminus include a series of hydrophobic heptad repeats that are predicted to interact with similar repeats in heavy chains near the tail region, in a 4-helix coiled coil. 

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 TPR repeats are involved in binding of kinesins to cargo. C terminal domains of heavy chains may also participate in binding some kinesins to cargo.

Cargo proteins bound by kinesins are diverse.

• Some organelle membranes contain transmembrane receptor proteins that bind kinesins. Kinectin is an endoplasmic reticulum 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 and fusion, are known to bind particular kinesins.

In the absence of cargo, the kinesin heavy chain stalk folds at hinge regions, bringing 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. 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 (A p. 952).

• In contrast to conventional kinesin I, a few kinesins have their motor domain located in the interior of the heavy chain sequence or at the C-terminus, instead of at the N-terminus. Those with the 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 (see A p. 957).
• One class of kinesin (KIF1) has a heavy chain that lacks the coiled coil domain and 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.

Kinesin's globular motor domain exhibits structural similarity, but little sequence homology, to that of myosin.  Kinesin and myosin heads both have nucleotide binding domains similar to that of the GTP-binding protein Ras. Positions of most b-strands and a-helices in their motor domains are equivalent. However kinesin has short connecting loops where the larger myosin head has longer stretches of amino acids.

Explore at right a dimer of kinesin heavy chain head and neck domains.   In vitro experiments have used digital video with differential interference microscopy to record ATP-dependent movements 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

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. Movement of the two-headed kinesin is processive, meaning that it takes many steps without dissociating from a microtubule. A hand over hand reaction cycle involving the two heads has been proposed. Myosin V, which transports vesicles along actin filaments, also exhibits processive movement.

The kinesin I reaction cycle differs from that of myosin II in that each kinesin motor domain binds tightly to a microtubule when it has bound ATP, while myosin dissociates from actin upon binding ATP. For details see A. p. 956. View at right an animation emphasizing the cycle of ATP binding, hydrolysis, and product dissociation during processive movement of kinesin along a microtubule.

of kinesin movement

Processivity such as observed for kinesin 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. (See article by Sablin & Fletterick.)

Kinesin has been observed 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.

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.

Various members of the kinesin family of proteins have diverse roles. Conventional kinesin has a role in movement of vesicles and lysosomes, from the vicinity of the golgi apparatus near the centrosome (MTOC - microtubule organizing center, which is usually adjacent to the cell nucleus), toward the plus ends of microtubules in the cell periphery.

Microtubule array in an interphase cell

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 along microtubules from near the centrosome in the cell body to axon endings. Such transport away from the centrosome, toward the plus ends of microtubules, is referred to as anterograde transport.

Kinesins and myosins may cooperate in vesicle transport. Vesicles in extruded nerve axoplasm were found to attach to and move along both microtubules and actin filaments. Kinesins and myosin V are both associated with precursors of synaptic vesicles. 

• Kinesin transports vesicles along microtubules within 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. 

During metaphase of mitosis, there is treadmilling in microtubules connecting kinetochores to the poles. Tubulin subunits flow toward the poles, as heterodimers are added at plus ends and removed at minus ends. During anaphase A, chromosomes move to the spindle poles, as microtubules linking kinetochores to the poles shorten. This involves:  dissociation of tubulin dimers at the kinetochore. continued dissociation of tubulin dimers at the poles in some cells.

During prophase as well as in anaphase B the mitotic spindle poles separate. BimC, which forms bipolar complexes (see above), 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. For more information on kinesins, see the Kinesin Home Page.

Dyneins are minus end-directed motor proteins.

• Dyneins were first studied in cilia & flagella.
• Many cytoplasmic dyneins have now been discovered. Cytoplasmic dyneins mediate ATP-dependent retrograde movements of vesicles and organelles along microtubules toward the centrosome (MTOC - microtubule organizing center) (diagram in A p. 980).

Dynein is large and complex. Cytoplasmic dynein has a molecular weight exceeding one million. Electron micrographs are shown in A. p. 953.

Dynein includes 2 or 3 heavy chains. Each is about 4600 amino acid residues long and includes a globular motor domain. There are also multiple intermediate and light chains. In addition, dynein 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. It is this domain at the end of the stalk that interacts with microtubules. 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 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. For diagrams and additional information on AAA ATPases, see:      website of the Kurian lab at Berkeley, &      website of the Macromolecular Structure & Function Research Group, Imperial College London.

High resolution electron microscopy with image averaging indicates a heptameric wheel-like structure of the dynein motor domain, with the six 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 two of the six AAA domains.
Diagrams of dynein structure may be found at:
      website of S. King at University of Connecticut Health Center, &
      website of the Vale Lab at UCSF.

An animated model of the dynein power stroke, and an animation based on electron microscope images of a flagellar dynein, are found on a University of Leeds website.

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 includes:

• Glued, an elongated 150 kDa dimeric protein, has at its distal end two microtubule-binding globular heads. Binding of glued to microtubules may increase processivity of dynein movement along microtubules.
  Other domains of glued bind to dyneins as well as kinesins. Coordination of bidirectional transport along microtubules may involve regulated binding to dynactin of plus and minus end-directed motors.
• Arp1, an actin-related protein forms a short filament (rod) of constant length (8-10 subunits), capped at one end by the actin-capping protein CapZ, and at the other end by proteins unique to dynactin.
• Dynamitin, with another small protein, links the Arp1 rod to glued.

See diagrams in A. p. 959, and on a webpage of Trina A. Schroer at Johns Hopkins University.

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 the dynactin complex anchors dynein to the plasma membrane.

Early and late endocytic vesicles have associated dynein and dynactin, which may (with myosin VI) be responsible for moving these vesicles inward from the cell surface.

Interaction of cortical dynein with astral microtubules is considered essential to orientation of the mitotic spindle and separation of poles during mitosis. 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. See also diagram above and diagram in A. p. 1049.
 

Cilia & flagella Cilia and flagella are bounded by the plasma membrane. A basal body, which is a single centriole cylinder, is at the base of each cilium or flagellum. See electron micrograph in an article by J. Beisson & M. Wright (requires subscription to Current Opinion in Cell Biology). Cilia and flagella have a core axoneme, a complex of microtubules and associated proteins. Diagrams in A p. 966-968. Some distinctions between cilia and flagella: Flagella are usually 1 or 2 per cell. They tend to have a rotary or sinusoidal movement. they may have additional structures outside of the core axoneme. Cilia are usually many per cell. They tend to have a whip-like movement.

The axoneme of cilia or flagella includes: Nine doublet microtubules around the periphery. The A tubule of each doublet has attached dynein arms. 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 adjacent B tubules, toward the minus end. This causes the sliding of microtubule doublets. Minus ends are anchored in the basal body, and flexible links between doublets (radial spokes and nexin links) limit sliding. The result is bending of the cilium. Several lines of evidence support this mechanism: ATP is required for bending. Inactivating dynein mutations eliminate ciliary bending

If isolated axonemes, with their membrane removed, are treated with mild protease, the radial spokes and nexin links are degraded. ATP addition then causes the microtubule doublets to slide apart. If a bent cilium is examined in cross-section by electron microscopy, 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 and 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 and plasma membrane proteins are transported to and from the tip of a cilium or flagellum. This intraflagellar transport is important for formation and maintenance of cilia and 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.
• A video of intraflagellar transport may be viewed at a website maintained by 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 and flagella lead to polycystic kidney disease and retinal degeneration in mammals. A non-motile primary cilia in kidney epithelial cells fails to develop in this disease. Development of retinal rod and cone photoreceptors 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 that are 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.

Copyright © 1999-2006 by Joyce J. Diwan. All rights reserved.

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