Identification of Microtubule Motor Proteins

Kinesin and dynein, the prototypes of microtubule motor proteins, move along microtubules in opposite directions—kinesin toward the plus end and dynein toward the minus end (Figure 11.45). The first of these microtubule motor proteins to be identified was dynein, which was isolated by Ian Gibbons in 1965. The purification of this form of dynein (called axonemal dynein) was facilitated because it is a highly abundant protein in cilia, just as the abundance of myosin facilitated its isolation from muscle cells. The identification of other microtubule-based motors, however, was more problematic because the proteins responsible for processes such as chromosome movement and organelle transport are present at comparatively low concentrations in the cytoplasm. Isolation of these proteins therefore depended on the development of new experimental methods to detect the activity of molecular motors in cell-free systems.

Figure 11.45

Microtubule motor proteins. Kinesin and dynein move in opposite directions along microtubules, toward the plus and minus ends, respectively. Kinesin consists of two heavy chains, wound around each other in a coiled-coil structure, and two light chains. (more...)

The development of in vitro assays for cytoplasmic motor proteins was based on the use of video-enhanced microscopy, developed by Robert Allen and Shinya Inoué in the early 1980s, to study the movement of membrane vesicles and organelles along microtubules in squid axons. In this method, a video camera is used to increase the contrast of images obtained with the light microscope, substantially improving the detection of small objects and allowing the movement of organelles to be followed in living cells. Using this approach, Allen, Scott Brady, and Ray Lasek demonstrated that organelle movements also took place in a cell-free system in which the plasma membrane had been removed and a cytoplasmic extract had been spread on a glass slide. These observations led to the development of an in vitro reconstructed system, which provided an assay capable of detecting cellular proteins responsible for organelle movement. In 1985 Brady, as well as Ronald Vale, Thomas Reese, and Michael Sheetz, capitalized on these developments to identify kinesin as a novel microtubule motor protein, present in both squid axons and bovine brain.

Further studies demonstrated that kinesin translocates along microtubules in only a single direction—toward the plus end. Because the plus ends of microtubules in axons are all oriented away from the cell body (see Figure 11.44), the movement of kinesin in this direction transports vesicles and organelles away from the cell body, toward the tip of the axon. Within intact axons, however, vesicles and organelles also had been observed to move back toward the cell body, implying that a different motor protein might be responsible for movement along microtubules in the opposite direction—toward the minus end. Consistent with this prediction, further experiments showed that a protein previously identified as the microtubule-associated protein MAP-1C was in fact a motor protein that moved along microtubules in the minus end direction. Subsequent analysis demonstrated that MAP-1C is related to the dynein isolated from cilia (axonemal dynein), so MAP-1C is now referred to as cytoplasmic dynein.

Kinesin is a molecule of approximately 380 kd, consisting of two heavy chains (120 kd each) and two light chains (64 kd each) (see Figure 11.45). The heavy chains have long α-helical regions that wind around each other in a coiled-coil structure. The amino-terminal globular head domains of the heavy chains are the motor domains of the molecule: They bind to both microtubules and ATP, the hydrolysis of which provides the energy required for movement. Although the motor domain of kinesin (approximately 340 amino acids) is much smaller than that of myosin (about 850 amino acids), X-ray crystallography indicates that the kinesin and myosin motor domains are structurally similar, suggesting that kinesin and myosin evolved from a common ancestor. The tail portion of the kinesin molecule consists of the light chains in association with the carboxy-terminal domains of the heavy chains. This portion of kinesin is responsible for binding to other cell components (such as membrane vesicles and organelles) that are transported along microtubules by the action of kinesin motors.

Dynein is an extremely large molecule (up to 2000 kd), which consists of two or three heavy chains (each about 500 kd) complexed with a variable number of light and intermediate polypeptides, which range from 14 to 120 kd (see Figure 11.45). As in kinesin, the heavy chains form globular ATP-binding motor domains that are responsible for movement along microtubules. The basal portion of the molecule, including the light and intermediate chains, is thought to bind to other subcellular structures, such as organelles and vesicles.

Like the myosins, both kinesin and dynein define families of related motor proteins. Following the initial isolation of kinesin in 1985, a variety of kinesin-related proteins have been identified. Eighteen different kinesins are encoded in the genome of C. elegans, and it is thought that there may be as many as 100 different members of the kinesin family in humans. Some members of the kinesin family, like kinesin itself, move along microtubules in the plus end direction (see Figure 11.45). Other members of the kinesin family, however, move in the opposite direction, toward the minus end. Different members of the kinesin family vary in the sequences of their carboxy-terminal tails and are responsible for the movements of different types of “cargo,” including vesicles, organelles, and chromosomes, along microtubules. There are also several types of axonemal dynein, as well as multiple cytoplasmic dyneins. All members of the dynein family move toward the minus ends of microtubules, but different cytoplasmic dyneins may transport different cargoes.

Organelle Transport and Intracellular Organization

One of the major roles of microtubules is to transport membrane vesicles and organelles through the cytoplasm of eukaryotic cells. As already discussed, such cytoplasmic organelle transport is particularly evident in nerve cell axons, which may extend more than a meter in length. Ribosomes are present only in the cell body and dendrites, so proteins, membrane vesicles, and organelles (e.g., mitochondria) must be transported from the cell body to the axon. Via video-enhanced microscopy, the transport of membrane vesicles and organelles in both directions can be visualized along axon microtubules, where kinesin and dynein carry their cargoes to and from the tips of the axons, respectively. For example, secretory vesicles containing neurotransmitters are carried from the Golgi apparatus to the terminal branches of the axon by kinesin. In the reverse direction, cytoplasmic dynein transports endocytic vesicles from the axon back to the cell body.

Microtubules similarly transport membrane vesicles and organelles in other types of cells. Because microtubules are usually oriented with their minus end anchored in the centrosome and their plus end extending toward the cell periphery, different members of the kinesin and dynein families are thought to transport vesicles and organelles in opposite directions through the cytoplasm (Figure 11.46). Conventional kinesin and other plus end-directed members of the kinesin family carry their cargo toward the cell periphery, whereas cytoplasmic dyneins and minus end-directed members of the kinesin family transport materials toward the center of the cell. In addition to transporting membrane vesicles in the endocytic and secretory pathways, microtubules and associated motor proteins position membrane-enclosed organelles (such as the endoplasmic reticulum, Golgi apparatus, lysosomes, and mitochondria) within the cell. For example, the endoplasmic reticulum extends to the periphery of the cell in association with microtubules (Figure 11.47). Drugs that depolymerize microtubules cause the endoplasmic reticulum to retract toward the cell center, indicating that association with microtubules is required to maintain the endoplasmic reticulum in its extended state. This positioning of the endoplasmic reticulum appears to involve the action of kinesin (or possibly multiple members of the kinesin family), which pulls the endoplasmic reticulum along microtubules in the plus end direction, toward the cell periphery. Similarly, kinesin appears to play a key role in the positioning of lysosomes away from the center of the cell, and three different members of the kinesin family have been implicated in the movements of mitochondria.

Figure 11.46

Transport of vesicles along microtubules. Kinesin and other plus end-directed members of the kinesin family transport vesicles and organelles in the direction of microtubule plus ends, which extend toward the cell periphery. In contrast, dynein and minus (more...)

Figure 11.47

Association of the endoplasmic reticulum with microtubules. Fluorescence microscopy of the endoplasmic reticulum (A) and microtubules (B) in an epithelial cell. The endoplasmic reticulum is stained with a fluorescent dye and microtubules with an antibody (more...)

Conversely, cytoplasmic dynein is thought to play a role in positioning the Golgi apparatus. The Golgi apparatus is located in the center of the cell, near the centrosome. If microtubules are disrupted, either by a drug or when the cell enters mitosis, the Golgi breaks up into small vesicles that disperse throughout the cytoplasm. When the microtubules re-form, the Golgi apparatus also reassembles, with the Golgi vesicles apparently being transported to the center of the cell (toward the minus end of microtubules) by cytoplasmic dynein. Movement along microtubules is thus responsible not only for vesicle transport, but also for establishing the positions of membrane-enclosed organelles within the cytoplasm of eukaryotic cells.

Separation of Mitotic Chromosomes

As discussed earlier in this chapter, microtubules reorganize at the beginning of mitosis to form the mitotic spindle, which plays a central role in cell division by distributing the duplicated chromosomes to daughter nuclei. This critical distribution of the genetic material takes place during anaphase of mitosis, when sister chromatids separate and move to opposite poles of the spindle. Chromosome movement proceeds by two distinct mechanisms, referred to as anaphase A and anaphase B, which involve different types of spindle microtubules and associated motor proteins.

Anaphase A consists of the movement of chromosomes toward the spindle poles along the kinetochore microtubules, which shorten as chromosome movement proceeds (Figure 11.48). This type of chromosome movement appears to be driven principally by kinetochore-associated motor proteins that translocate chromosomes along the spindle microtubules in the minus end direction, toward the centrosomes. Cytoplasmic dynein is associated with kinetochores and may play a role in poleward chromosome movement, as may minus end-directed members of the kinesin family. The action of these kinetochore motor proteins is coupled to disassembly and shortening of the kinetochore microtubules, which may be mediated by some members of the kinesin family that act as microtubule-destabilizing enzymes.

Figure 11.48

Anaphase A chromosome movement. Chromosomes move toward the spindle poles along the kinetochore microtubules. Chromosome movement is thought to be driven by minus end-directed motor proteins associated with the kinetochore. The action of these motor proteins (more...)

Anaphase B refers to the separation of the spindle poles themselves (Figure 11.49). Spindle-pole separation is accompanied by elongation of the polar microtubules and is similar to the initial separation of duplicated centrosomes to form the spindle poles at the beginning of mitosis (see Figure 11.43). During anaphase B the overlapping polar microtubules slide against one another, pushing the spindle poles apart. This type of movement has been found to result from the action of several plus end-directed members of the kinesin family, which crosslink polar microtubules and move them toward the plus end of their overlapping microtubule—away from the opposite spindle pole. In addition, the spindle poles may be pulled apart by the astral microtubules. The mechanism responsible for this type of movement has not been established, but it could result from the action of cytoplasmic dynein anchored to the cell cortex or another structure in the cytoplasm. The translocation of such an anchored dynein motor along astral microtubules in the minus end direction would have the effect of pulling the spindle poles apart, toward the periphery of the cell. Alternatively, a motor protein associated with the spindle poles could move along astral microtubules in the plus end direction, which would also pull the spindle poles toward the cell periphery.

Figure 11.49

Spindle pole separation in anaphase B. The separation of spindle poles results from two types of movement. First, overlapping polar microtubules slide past each other to push the spindle poles apart, probably as a result of the action of plus end-directed (more...)

Cilia and Flagella

Cilia and flagella are microtubule-based projections of the plasma membrane that are responsible for movement of a variety of eukaryotic cells. Many bacteria also have flagella, but these prokaryotic flagella are quite different from those of eukaryotes. Bacterial flagella (which are not discussed further here) are protein filaments projecting from the cell surface, rather than projections of the plasma membrane supported by microtubules.

Eukaryotic cilia and flagella are very similar structures, each with a diameter of approximately 0.25 μm (Figure 11.50). Many cells are covered by numerous cilia, which are about 10 μm in length. Cilia beat in a coordinated back-and-forth motion, which either moves the cell through fluid or moves fluid over the surface of the cell. For example, the cilia of some protozoans (such as Paramecium) are responsible both for cell motility and for sweeping food organisms over the cell surface and into the oral cavity. In animals, an important function of cilia is to move fluid or mucus over the surface of epithelial cell sheets. A good example is provided by the ciliated cells lining the respiratory tract, which clear mucus and dust from the respiratory passages. Flagella differ from cilia in their length (they can be as long as 200 μm) and in their wavelike pattern of beating. Cells usually have only one or two flagella, which are responsible for the locomotion of a variety of protozoans and of sperm.

Figure 11.50

Examples of cilia and flagella. (A) Scanning electron micrograph showing numerous cilia covering the surface of Paramecium. (B) Scanning electron micrograph of ciliated epithelial cells lining the surface of a trachea. (C) Multiple-flash photograph (500 (more...)

The fundamental structure of both cilia and flagella is the axoneme, which is composed of microtubules and their associated proteins (Figure 11.51). The microtubules are arranged in a characteristic “9 + 2” pattern in which a central pair of microtubules is surrounded by nine outer microtubule doublets. The two fused microtubules of each outer doublet are distinct: One (called the A tubule) is a complete microtubule consisting of 13 protofilaments; the other (the B tubule) is incomplete, containing only 10 or 11 protofilaments fused to the A tubule. The outer microtubule doublets are connected to the central pair by radial spokes and to each other by links of a protein called nexin. In addition, two arms of dynein are attached to each A tubule, and it is the motor activity of these axonemal dyneins that drives the beating of cilia and flagella.

Figure 11.51

Structure of the axoneme of cilia and flagella. (A) Computer-enhanced electron micrograph of a cross section of the axoneme of a rat sperm flagellum. (B) Schematic cross section of an axoneme. The nine outer doublets consist of one complete (A) and one (more...)

The minus ends of the microtubules of cilia and flagella are anchored in a basal body, which is similar in structure to a centriole and contains nine triplets of microtubules (Figure 11.52). Centrioles were discussed earlier as components of the centrosome, in which their function is uncertain. Basal bodies, however, play a clear role in organization of the axoneme microtubules. Namely, each of the outer microtubule doublets of the axoneme is formed by extension of two of the microtubules present in the triplets of the basal body. Basal bodies thus serve to initiate the growth of axonemal microtubules, as well as anchoring cilia and flagella to the surface of the cell.

Figure 11.52

Electron micrographs of basal bodies. (A) A longitudinal view of cilia anchored in basal bodies. (B) A cross section of basal bodies. Each basal body consists of nine triplets of microtubules. (A, Conly L. Reider/Biological Photo Service; B, W. L. Dentler, (more...)

The movements of cilia and flagella result from the sliding of outer microtubule doublets relative to one another, powered by the motor activity of axonemal dynein (Figure 11.53). The dynein bases bind to the A tubules while the dynein head groups bind to the B tubules of adjacent doublets. Movement of the dynein head group in the minus end direction then causes the A tubule of one doublet to slide toward the basal end of the adjacent B tubule. Because the microtubule doublets in an axoneme are connected by nexin links, the sliding of one doublet along another causes them to bend, forming the basis of the beating movements of cilia and flagella. It is apparent, however, that the activities of dynein molecules in different regions of the axoneme must be carefully regulated to produce the coordinated beating of cilia and the wavelike oscillations of flagella—a process about which little is currently understood.

Figure 11.53

Movement of microtubules in cilia and flagella. The bases of dynein arms are attached to A tubules, and the motor head groups interact with the B tubules of adjacent doublets. Movement of the dynein head groups in the minus end direction (toward the base (more...)

Box

Key Experiment: The Isolation of Kinesin.