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In some cases, a vesicle must traverse microtubule-poor but microfilament-rich regions in the cell. For example, during endocytosis, vesicles from the actin-rich plasma membrane are carried inward, whereas during secretion, vesicles derived from the endoplasmic reticulum and Golgi are moved outward. The results of several complementary experiments imply that microtubule and microfilament motor proteins bind to the same vesicles and cooperate in their transport. One piece of evidence was obtained from microscopy of vesicle movements in extruded cytoplasm from a squid giant axon. As observed many times before, vesicles traveled along microtubule tracks; surprisingly, movement continued at the periphery of the extruded cytoplasm through a region containing microfilaments but no microtubules. Subsequent experiments demonstrated that a given vesicle could move on a microtubule or a microfilament. Thus at least two motor proteins, myosin and either kinesin or cytosolic dynein, must be bound to the same vesicle (Figure 20-24). The discovery that a given vesicle can travel along both cytoskeletal systems suggests that, in a neuron, synaptic vesicles are transported at a fast rate by kinesin in the microtubule-rich axon and then travel through the actin-rich cortex at the nerve terminal on a myosin motor.

▲ FIGURE 20-24 Cooperation of myosin and kinesin at the cell cortex. Microtubules approach the actin-rich cell membrane. Consequently, some cargoes are transported to the cell periphery by kinesin motor proteins on microtubules but complete the journey on microfilaments under the power of myosin motor proteins.

Eukaryotic Cilia and Flagella Contain a Core of Doublet Microtubules Studded with Axonemal Dyneins

Cilia and flagella are flexible membrane extensions that project from certain cells. They range in length from a few

(a) Plasma membrane Nexin Central pair

(a) Plasma membrane Nexin Central pair

Doublet microtubule

50 |im

▲ EXPERIMENTAL FIGURE 20-26 Freeze-etching reveals structure of axonemal dynein. Electron micrograph of freeze-etched outer-arm dynein from Tetrahymena cilia and an artist's interpretation of the structure. The base contains several intermediate and light chains. Attached to the common base are three heavy chains each composed of a long stem, large globular head domain and small globular domain, and short stalk connecting the globular domains. Microtubules bind to the tip of the stalk. All axonemal dyneins are thought to have the general structure shown here, although some outer-arm dyneins contain two heavy chains, and inner-arm dyneins contain one or two heavy chains. [Electron micrograph from U. W. Goodenough and J. E. Heuser, 1984, J. Mol. Biol. 18:1083.]

Doublet microtubule

▲ FIGURE 20-25 Structure of an axoneme.

(a) Cross-sectional diagram of a typical flagellum showing its major structures. The dynein arms and radial spokes with attached heads surround a central pair of singlet microtubules.

(b) Micrograph of a transverse section through an isolated demembranated cilium. [See U. W. Goodenough and J. E. Heuser, 1985, J. Cell Biol. 100:2008. Part (b) courtesy of L. Tilney.]

micrometers to more than 2 mm for some insect sperm flagella. Virtually all eukaryotic cilia and flagella possess a central bundle of microtubules, called the axoneme, which consists of nine doublet microtubules surrounding a central pair of singlet mi-crotubules (Figure 20-25). This characteristic "9 + 2 " arrangement of microtubules is seen in cross section with the electron microscope. Each doublet microtubule consists of A and B tubules. The (+) end of axonemal microtubules is at the distal end of the axoneme. At its point of attachment to the cell, the axoneme connects with the basal body. Containing nine triplet microtubules (see Figure 20-4), the basal body plays an important role in initiating the growth of the axoneme.

The axoneme is held together by three sets of protein cross-links (see Figure 20-25a). The central pair of singlet microtubules is connected by periodic bridges, like rungs on a ladder, and is surrounded by a fibrous structure termed the inner sheath. A second set of linkers, composed of the protein nexin, joins adjacent outer doublet microtubules. Radial spokes, which radiate from the central singlets to each A tubule of the outer doublets, are proposed to regulate dynein.

Permanently attached periodically along the length of the A tubule of each doublet microtubule are inner-arm and outer-arm dyneins (see Figure 20-25a). These axonemal dyneins are complex multimers of heavy chains, intermediate chains, and light chains. When isolated axonemal dyneins are slightly denatured and spread out on an electron microscope grid, they are seen as a bouquet of two or three "blossoms" connected to a common base (Figure 20-26). Each blossom consists of a large globular head domain attached to a small globular domain through a short stalk; a stem connects one or more blossoms to a common base. The base is thought to attach a dynein to the A tubule, whereas the globular domains project outward toward the B tubule of the neighboring doublet.

A single dynein heavy chain, which forms each stalk, head, and stem is enormous, approximately 4500 amino acids in length with a molecular weight exceeding 540,000. At least eight or nine different heavy chains have been identified, each capable of hydrolyzing ATP. On the basis of sequence comparisons with the ATP-binding sites in other proteins, the ATP-binding site of axonemal dynein is predicted to lie in the globular head domain of the heavy chain, with the microtubule-binding site being at the tip of the stalk. Inner-arm dyneins are either one- or two-headed structures, containing one or two heavy chains. Outer-arm dyneins contain two heavy chains (e.g., in a sea urchin sperm flagellum) or three heavy chains (e.g., in Tetrahymena cilia and Chla-mydomonas flagella).

The intermediate and light chains in axonemal dynein are thought to form the base region. These chains help mediate the attachment of the dynein arm to the A tubule and may also participate in regulating dynein activity. The base proteins of axonemal dyneins are thus analogous to those composing the dynactin complexes associated with cytosolic dynein.

Ciliary and Flagellar Beating Are Produced by Controlled Sliding of Outer Doublet Microtubules

Ciliary and flagellar beating is characterized by a series of bends, originating at the base of the structure and propagated toward the tip (Figure 20-27). The bends push against the surrounding fluid, propelling the cell forward or moving the fluid across a fixed epithelium. A bend results from the sliding of adjacent doublet microtubules past one another. Because active sliding occurs all along the axoneme, bends can be propagated without damping. Findings from microscopic studies with isolated axonemes from which the cross-linkage proteins (e.g., nexin) are removed have shown that doublet microtubules slide past one another in the presence of ATP but no bending occurs (Figure 20-28a). Thus the

► EXPERIMENTAL FIGURE 20-27 Video microscopy shows flagellar movements that propel sperm and Chlamydomonas forward. In both cases, the cells are moving to the left. (a) In the typical sperm flagellum, successive waves of bending originate at the base and are propagated out toward the tip; these waves push against the water and propel the cell forward. Captured in this multiple-exposure sequence, a bend at the base of the sperm in the first (top) frame has moved distally halfway along the flagellum by the last frame. A pair of gold beads on the flagellum are seen to slide apart as the bend moves through their region. (b) Beating of the two flagella on Chlamydomonas occurs in two stages, called the effective stroke (top three frames) and the recovery stroke (remaining frames). The effective stroke pulls the organism through the water. During the recovery stroke, a different wave of bending moves outward from the bases of the flagella, pushing the flagella along the surface of the cell until they reach the position to initiate another effective stroke. Beating commonly occurs 5-10 times per second. [Part (a) from C. Brokaw, 1991, J. Cell Biol. 114(6): cover photograph; courtesy of C. Brokaw. Part (b) courtesy of S. Goldstein.]

ATP-dependent movement of doublet microtubules must be restricted by cross-linking proteins in order for sliding to be converted into the bending of an axoneme.

On the basis of the polarity and direction of sliding of the doublet microtubules and the properties of axonemal dyneins, the small head domains of the dynein arms on the A tubule of one doublet are thought to "walk" along the adjacent doublet's B tubule toward its base, the (—) end (Figure 20-28b). The force producing active sliding requires ATP and probably entails a conformational change in the head and stem that translocates the stalk. Successive binding and hydrolysis of ATP causes the dynein stalks to successively release from and attach to the adjacent doublet. Although this general model is most likely correct, many important details such as the mechanism of force transduction by dynein are still unknown.

Dynein arms

Dynein arms

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