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Kinesin is a motor protein that moves along microtubules. It was first identified in the giant axon of the squid, where it carries membrane-enclosed organelles away from the neuronal cell body toward the axon terminal by walking toward the plus end of microtubules. Kinesin is similar structurally to myosin II in having two heavy chains and two light chains per active motor, two globular head motor domains, and an elongated coiled-coil responsible for heavy chain dimerization. Like myosin, kinesin is a member of a large protein superfamily, for which the motor domain is the only common element (Figure 16-55). The yeastSaccharomyces cerevisiae has six distinct kinesins. The nematode C. elegans has 16 kinesins, and humans have about 40.

There are at least ten families of kinesin-related proteins, or KRPs, in the kinesin superfamily. Most of them have the motor domain at the N-terminus of the heavy chain and walk toward the plus end of the microtubule. A particularly interesting family has the motor domain at the C-terminus and walks in the opposite direction, toward the minus end of the microtubule. Some KRP heavy chains lack a coiled-coil sequence and seem to function as monomers, analogous to myosin I. Some others are homodimers, and yet others are heterodimers. At least one KRP (BimC) can self-associate through the tail domain, forming a bipolar motor that slides oppositely oriented microtubules past one another, much as a myosin II thick filament does for actin filaments. Most kinesins carry a binding site in the tail for either a membrane-enclosed organelle or another microtubule. Many of the kinesin superfamily members have specific roles in mitotic and meiotic spindle formation and chromosome separation during cell division.

The dyneins are a family of minus-end-directed microtubule motors, but they are unrelated to the kinesin superfamily. They are composed of two or three heavy chains (that include the motor domain) and a large and variable number of associated light chains. The dynein family has two major branches (Figure 16-56). The most ancient branch contains thecytoplasmic dyneins, which are typically heavy-chain homodimers, with two large motor domains as heads. Cytoplasmic dyneins are probably found in all eucaryotic cells, and they are important for vesicle trafficking, as well as for localization of the Golgi apparatus near the center of the cell. Axonemal dyneins, the other large branch, include heterodimers and heterotrimers, with two or three motor-domain heads, respectively. They are highly specialized for the rapid and efficient sliding movements of microtubules that drive the beating of cilia and flagella (discussed later). A third, minor, branch shares greater sequence similarity with cytoplasmic than with axonemal dyneins but seems to be involved in the beating of cilia.

Dyneins are the largest of the known molecular motors, and they are also among the fastest: axonemal dyneins can move microtubules in a test tube at the remarkable rate of 14 μm/sec. In comparison, the fastest kinesins can move their microtubules at about 2–3 μm/sec.

The motor domain of myosins is substantially larger than that of kinesins, about 850 amino acids compared with about 350. The two classes of motor proteins track along different filaments and have different kinetic properties, and they have no identifiable amino acid sequence similarities. However, determination of the three-dimensional structure of the motor domains of myosin and kinesin has revealed that these two motor domains are built around nearly identical cores (Figure 16-57). The central force-generating element that the two types of motor proteins have in common includes the site of ATP binding and the machinery necessary to translate ATP hydrolysis into an allosteric conformational change. The differences in domain size and in the choice of track can be attributed to large loops extending outward from this central core. These loops include the actin-binding and microtubule-binding sites, respectively.

An important clue to how the central core is involved in force generation has come from the observation that the motor core also bears some structural resemblance to the nucleotidebinding site of the small GTPases of the Ras superfamily. As discussed in Chapter 3 (see Figure 3-74), these proteins exhibit distinct conformations in their GTP-bound (active) and GDP-bound (inactive) forms: mobile “switch” loops in the nucleotide-binding site are in close contact with the γ-phosphate in the GTP-bound state, but these loops swing out when the hydrolyzed γ-phosphate is released. Although the details of the movement are different for the two motor proteins, and ATP rather than GTP is hydrolyzed, the relatively small structural change in the active site—the presence or absence of a terminal phosphate—is similarly amplified to cause a rotation of a different part of the protein. In kinesin and myosin, a switch loop interacts extensively with those regions of the protein involved in microtubule and actin binding, respectively, allowing the structural transitions caused by the ATP hydrolysis cycle to be relayed to the polymer-binding interface. The relay of structural changes between the polymer-binding site and the nucleotide hydrolysis site seems to work in both directions, since theATPase activity of motor proteins is strongly activated by binding to their filament tracks.

Although the cytoskeletal motor proteins and GTP-binding proteins both use structural changes in their nucleoside-triphosphate-binding sites to produce cyclic interactions with a partner protein, the motor proteins have a further requirement: each cycle of binding and release must propel them forward in a single direction along a filament to a newbinding site on the filament. For such unidirectional motion, a motor protein must use the energy derived from ATP binding and hydrolysis to force a large movement in part of the protein molecule. For myosin, each step of the movement along actin is generated by the swinging of an 8.5-nm-long α helix, or lever arm (see Figure 16-57), which is structurally stabilized by the binding of light chains. At the base of this lever arm next to the head, there is a piston-like helix that connects movements at the ATP-binding cleft in the head to small rotations of the so-called converter domain. A small change at this point can swing the helix like a long lever, causing the far end of the helix to move by about 5.0 nm. These changes in the conformation of the myosin are coupled to changes in its binding affinity for actin, allowing the myosin head to release its grip on the actin filament at one point and snatch hold of it again at another. The full mechanochemical cycle of nucleotide binding, nucleotide hydrolysis, and phosphate release (which causes the “power stroke”) produces a single step of movement (Figure 16-58). In the myosin VI subfamily of myosins, which move backward (toward the minus end of the actin filament), the converter domain probably lies in a different orientation, so that the same piston-like movement of the small helix causes the lever arm to rotate in the opposite direction.

In kinesin, instead of the rocking of a lever arm, the small movements of switch loops at the nucleotide–binding siteregulate the docking and undocking of the motor head domain to a long linker region that connects this motor head at one end to the coiled-coil dimerization domain at the other end. When the front (leading) kinesin head is bound to amicrotubule before the power stroke, its linker region is relatively unstructured. On the binding of ATP to this bound head, its linker region docks along the side of the head, which throws the second head forward to a position where it will be able to bind a new attachment site on the protofilament, 8 nm closer to the microtubule plus end than the binding site for the first head. The nucleotide hydrolysis cycles in the two heads are closely coordinated, so that this cycle of linker docking and undocking can allow the two-headed motor to move in a hand-over-hand (or head-over-head) stepwise manner (Figure 16-59A).

The coiled-coildomain seems both to coordinate the mechanochemical cycles of the two heads (motor domains) of thekinesin dimer and to determine its directionality of movement. Recall that whereas most members of the kinesin superfamily, with their motor domains at the N-terminus, move toward the plus end of the microtubule, a few superfamily members have their motor domains at the C-terminus and move toward the minus end. Since the motor domains of these two types of kinesins are essentially identical, how can they move in opposite directions? The answer seems to lie in the way in which the heads are connected. In high-resolution images of forward-walking and backward-walking members of the kinesin superfamily bound to microtubules, the heads that are attached to the microtubule are essentially indistinguishable, but the second, unattached heads are oriented very differently. This difference in tilt apparently biases the next binding site for the second head, and thereby determines the directionality of motor movement (Figure 16-60).

Although both myosin and kinesin undergo analogous mechanochemical cycles, the exact nature of the coupling between the mechanical and chemical cycles is different in the two cases (see Figure 16-60). For example, myosin without any nucleotide is tightly bound to its actin track, in a so-called “rigor” state, and it is released from this track by the association of ATP. In contrast, kinesin forms a rigor-like tight association with a microtubule when ATP is bound to the kinesin, and it is hydrolysis of ATP that promotes release of the motor from its track.

Thus, cytoskeletal motor proteins work in a manner highly analogous to GTP-binding proteins, except that in motor proteins the small protein conformational changes (a few tenths of a nanometer) associated with nucleotide hydrolysis are amplified by special protein domains—the lever arm in the case of myosin and the linker in the case of kinesin—to generate large-scale (several nanometers) conformational changes that move the motor proteins stepwise along their filament tracks. The analogy between the GTPases and the cytoskeletal motor proteins has recently been extended by the observation that one of the GTP-binding proteins—the bacterial elongation factorG—translates the chemical energy of GTP hydrolysis into directional movement of the mRNAmolecule on the ribosome.

The motor proteins in the myosin and kinesin superfamilies exhibit a remarkable diversity of motile properties, well beyond their choice of different polymer tracks. Most strikingly, a single dimer of conventional kinesin moves in a highly processive fashion, traveling for hundreds of ATPase cycles along a microtubule without dissociating. Skeletal muscle myosin II, in contrast, cannot move processively and makes just one or a few steps along an actin filament before letting go. These differences are critical for the motors’ various biological roles. A small number of kinesin molecules must be able to transport a mitochondrion all the way down a nerve cellaxon, and therefore require a high level of processivity. Skeletal muscle myosin, in contrast, never operates as a single molecule but rather as part of a huge array of myosin II molecules. Here processivity would actually inhibit biological function, since efficient muscle contraction requires that each myosin head perform its power stroke and then quickly get out of the way, to avoid interfering with the actions of the other heads attached to the same actin filament.

There are two reasons for the high degree of processivity of kinesin movement. The first is that the mechanochemical cycles of the two motor heads in a kinesin dimer are coordinated with each other, so that one kinesin head does not let go until the other is poised to bind. This coordination allows the motor protein to operate in a hand-over-hand fashion, never allowing the organelle cargo to diffuse away from the microtubule track. There is no apparent coordination between the myosin heads in a myosin II dimer. The second reason for the high processivity of kinesin movement is that kinesin spends a relatively large fraction of its ATPase cycle tightly bound to the microtubule. For both kinesin and myosin, the conformational change that produces the force-generating working stroke must occur while the motor protein is tightly bound to its polymer, and the recovery stroke in preparation for the next step must occur while the motor is unbound. But as we have seen in Figure 16-59, myosin spends only about 5% of its ATPase cycle in the tightly bound state and is unbound the rest of the time.

What myosin loses in processivity it gains in speed; in an array in which many motor heads are interacting with the sameactin filament, a set of linked myosins can move their filament a total distance equivalent to 20 steps during a single cycle time, while kinesins can move only two. Thus, myosins can typically drive filament sliding much more rapidly than kinesins, even though they hydrolyze ATP at comparable rates and take molecular steps of comparable length.

Within each motor protein class, movement speeds vary widely, from about 0.2 to 60 μm/sec for myosins, and from about 0.02 to 2 μm/sec for kinesins. These differences arise from a fine-tuning of the mechanochemical cycle. The number of steps that an individual motor molecule can take in a given time, and thereby the velocity, can be increased by either increasing the motor protein’s intrinsic ATPase rate or decreasing the proportion of cycle time spent bound to the filament track. Moreover, the size of each step can be changed by either changing the length of the lever arm (for example, the lever arm of myosin V is about three times longer than the lever arm of myosin II) or the angle through which the helix swings (Figure 16-61). Each of these parameters varies slightly among different members of the myosin and kinesin families, corresponding to slightly different protein sequences and structures. It is assumed that the behavior of each motor protein, whose function is determined by the identity of the cargo attached through its tail-domain, has been fine-tuned during evolution for speed and processivity according to the specific needs of the cell.

A major function of cytoskeletal motors in interphase cells is the transport and positioning of membrane-enclosed organelles. Kinesin was originally identified as the protein responsible for fast axonal transport, the rapid movement of mitochondria, secretory vesicle precursors, and various synapse components down the microtubule highways of theaxon to the distant nerve terminals. Although organelles in most cells need not cover such long distances, their polarized transport is equally necessary. A typical microtubule array in an interphase cell is oriented with the minus ends near the center of the cell at the centrosome, and the plus ends extending to the cell periphery. Thus, centripetal movements of organelles toward the cell center require the action of minus-end-directed motor proteins such as cytoplasmic dynein, whereas centrifugal movements toward the periphery require plus-end-directed motors such as kinesins.

The role of microtubules and microtubule motors in the behavior of intracellular membranes is best exemplified by the part they play in organizing the endoplasmic reticulum (ER) and the Golgi apparatus. The network of ER membranetubules aligns with microtubules and extends almost to the edge of the cell, whereas the Golgi apparatus is located near the centrosome. When cells are treated with a drug that depolymerizes microtubules, such as colchicine or nocodazole, the ER collapses to the center of the cell, while the Golgi apparatus fragments and disperses throughout the cytoplasm(Figure 16-62). In vitro, kinesins can tether ER-derived membranes to preformed microtubule tracks, and walk toward the microtubule plus ends, dragging the ER membranes out into tubular protrusions and forming a membranous web very much like the ER in cells. Likewise, the outward movement of ER tubules toward the cell periphery is associated with microtubule growth in living cells. Conversely, dyneins are required for positioning the Golgi apparatus near the cell center, moving Golgi vesicles along microtubule tracks toward minus ends at the centrosome.

The different tails and their associated light chains on specific motor proteins allow the motors to attach to their appropriate organelle cargo. For example, there is evidence for membrane-associated motor receptors, sorted to specific membrane-enclosed compartments, that interact directly or indirectly with the tails of the appropriate kinesin family members. One of these receptors seems to be the amyloid precursor protein, APP, which binds directly to a light chainon the tail of kinesin-I and is proposed to be a transmembrane motor proteinreceptormolecule in nerve-cell axons. It is the abnormal processing of this protein that gives rise to Alzheimer’s disease, as discussed in Chapter 15.

For dynein, attachment to membranes is known to be mediated by a large macromolecular assembly. Cytoplasmic dynein is itself a huge proteincomplex, and it requires association with a second large protein complex called dynactinto translocate organelles effectively. The dynactin complex includes a short actinlike filament that is made of the actin-related protein Arp1 (distinct from Arp2 and Arp3, the components of the ARP complex involved in the nucleation of conventional actin filaments). Membranes of the Golgi apparatus are coated with the proteins ankyrin and spectrin, which have been proposed to associate with the Arp1 filament in the dynactin complex to form a planar cytoskeletal array reminiscent of the erythrocyte membranecytoskeleton (see Figure 10-31). The spectrin array probably gives structural stability to the Golgi membrane, and—via the Arp1 filament—it may mediate the regulatable attachment of dynein to the organelle (Figure 16-63).

Motor proteins also have a significant role in organelle transport along actin filaments. The first myosin shown to mediate organelle motility was myosin V, a two-headed myosin with a large step size (see Figure 16-61). In mice, mutations in the myosin V gene result in a “dilute” phenotype, in which fur color looks faded. In mice (and humans),membrane-enclosed pigment granules, called melanosomes, are synthesized in cells called melanocytes beneath the skin surface. These melanosomes move out to the ends of dendritic processes in the melanocytes, from where they are delivered to the overlying keratinocytes that form the skin and fur. Myosin V is associated with the surface of melanosomes, and it is able to mediate their actin-based movement in a test tube (Figure 16-64). In dilute mutant mice, the melanosomes are not delivered to the keratinocytes efficiently, and pigmentation is defective. Other myosins, including myosin I, are associated with endosomes and a variety of other organelles.

Motor Protein Function Can Be Regulated

The cell can regulate the activity of motor proteins, allowing it to change either the positioning of its membrane-enclosed organelles or its whole-cell movements. One of the most dramatic examples is provided by fish melanocytes. These giant cells, which are responsible for rapid changes in skin coloration in several species of fish, contain large pigment granules that can alter their location in response to neuronal or hormonal stimulation (Figure 16-65). These pigment granules aggregate or disperse by moving along an extensive network of microtubules. The minus ends of these microtubules are nucleated by the centrosome and are located in the center of the cell, while the plus ends are distributed around the cell periphery. The tracking of individual pigment granules reveals that the inward movement is rapid and smooth, while the outward movement is jerky, with frequent backward steps (Figure 16-66). Both dynein and kinesinare associated with the pigment granules. The jerky outward movements apparently result from a tug-of-war between the two motor proteins, with the stronger kinesin winning out overall. When the kinesin light chains become phosphorylated after a hormonal stimulation that signals skin color change, kinesin is inactivated, leaving dynein free to drag the pigment granules rapidly toward the cell center, changing the fish’s color. In a similar way, the movement of other membrane organelles coated with particular motor proteins is controlled by a complex balance of competing signals that regulate both motor protein attachment and activity.

Myosin activity can also be regulated by phosphorylation. In nonmuscle cells, myosin II can be phosphorylated on a variety of sites on both heavy and light chains, affecting both motor activity and thick filament assembly. The myosin II can exist in two different conformational states in such cells, an extended state that is capable of forming bipolar filaments, and a bent state in which the tail domain apparently interacts with the motor head. Phosphorylation of the regulatory light chain by the calcium-dependent myosin light-chain kinase (MLCK) causes the myosin II to preferentially assume the extended state, which promotes its assembly into a bipolar filament and leads to cell contraction (Figure 16-67). Myosin light-chain phosphorylation is an indirect target of activated Rho, the small GTPasediscussed previously whose activation causes a reorganization of the actincytoskeleton into contractile stress fibers. The MLCK is also activated during mitosis, causing myosin II to assemble into the contractile ring that is responsible for dividing the mitotic cell into two. Regulation of other members of the myosin superfamily is not as well understood, but control of these myosins is also likely to involve site-specific phosphorylations.

Summary

Motor proteins use the energy of ATP hydrolysis to move along microtubules or actin filaments. They mediate the sliding of filaments relative to one another and the transport of membrane-enclosed organelles along filament tracks. All known motor proteins that move on actin filaments are members of the myosin superfamily. The motor proteins that move on microtubules are members of either the kinesin superfamily or the dynein family. The myosin and kinesin superfamilies are diverse, with about 40 genes encoding each type of protein in humans. The only structural element shared among all members of each superfamily is the motor “head” domain. These heads can be attached to a wide variety of “tails,” which attach to different types of cargo and enable the various family members to perform different functions in the cell. Although myosin and kinesin walk along different tracks and use different mechanisms to produce force and movement by ATP hydrolysis, they share a common structural core, suggesting that they are derived from a common ancestor.

Two types of specialized motility structures in eucaryotic cells consist of highly ordered arrays of motor proteins that move on stabilized filament tracks. The myosin-actin system of the sarcomere powers the contraction of various types of muscle, including skeletal, smooth, and cardiac muscle. The dynein–microtubule system of the axoneme powers the beating of cilia and the undulations of flagella.