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.