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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.

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Molecular Cell Biology. 4th edition.

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Section 19.4Cilia and Flagella: Structure and Movement

Swimming is the major form of movement exhibited by sperm and by many protozoans. Some cells are propelled at velocities approaching 1 mm/s by the beating of cilia and flagella, flexible membrane extensions of the cell. Cilia and flagella range in length from a few microns to more than 2 mm in the case of some insect sperm flagella.

Although cilia and flagella are the same, they were given different names before their structures were studied. Typically, cells possess one or two long flagella, whereas ciliated cells have many short cilia. For example, the mammalian spermatozoon has a single flagellum, the unicellular green alga Chlamydomonas has two flagella, and the unicellular protozoan Paramecium is covered with a few thousand cilia, which are used both to move and to bring in food particles. In mammals, many epithelial cells are ciliated in order to sweep materials across the tissue surface. For instance, huge numbers of cilia (more than 107/mm2) cover the surfaces of mammalian respiratory passages (the nose, pharynx, and trachea), where they dislodge and expel particulate matter that collects in the mucus secretions of these tissues.

Ciliary and flagellar beating is characterized by a series of bends, originating at the base of the structure and propagated toward the tip. High-speed strobe microscopy allows the waveform of the beat to be seen (Figure 19-27). Beating can be planar or three-dimensional; like waves that you have studied in physics, it can be described by its amplitude, wavelength, and frequency. The bends push against the surrounding fluid, propelling the cell forward or moving the fluid across a fixed epithelium.

Figure 19-27. Flagellar motions in sperm and Chlamydomonas.

Figure 19-27

Flagellar motions in sperm and Chlamydomonas. 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 (more...)

All Eukaryotic Cilia and Flagella Contain Bundles of Doublet Microtubules

Virtually all eukaryotic cilia and flagella are remarkably similar in their organization, possessing a central bundle of microtubules, called the axoneme, in which nine outer doublet microtubules surround a central pair of singlet microtubules (Figure 19-28). This characteristic “9 + 2” arrangement of microtubules is seen when the axoneme is viewed in cross section with the electron microscope. As shown in Figure 19-3, each doublet microtubule consists of A and B tubules, or subfibers: the A tubule is a complete microtubule with 13 protofilaments, while the B tubule contains 10 protofilaments. The bundle of microtubules comprising the axoneme is surrounded by the plasma membrane. Regardless of the organism or cell type, the axoneme is about 0.25 μm in diameter, but it varies greatly in length, from a few microns to more than 2 mm.

Figure 19-28. Structure of ciliary and flagellar axonemes.

Figure 19-28

Structure of ciliary and flagellar axonemes. (a) Cross-sectional diagram of a typical flagellum showing its major structures. The dynein arms and radial spokes with attached heads occur only (more...)

At its point of attachment to the cell, the axoneme connects with the basal body (Figure 19-29). Like centrioles, basal bodies are cylindrical structures, about 0.4 μm long and 0.2 μm wide, which contain nine triplet microtubules. Each triplet contains one complete 13-protofilament microtubule, the A tubule, fused to the incomplete B tubule, which in turn is fused to the incomplete C tubule (see Figure 19-3). The A and B tubules of basal bodies continue into the axonemal shaft, whereas the C tubule terminates within the transition zone between the basal body and the shaft. The two central tubules in a flagellum or a cilium also end in the transition zone, above the basal body. As we will see later, the basal body plays an important role in initiating the growth of the axoneme.

Figure 19-29. Electron micrograph of the basal regions of the two flagella in Chlamydomonas reinhardtii.

Figure 19-29

Electron micrograph of the basal regions of the two flagella in Chlamydomonas reinhardtii. The bundles of microtubules and some fibers connecting them are visible in the flagella (FL). (more...)

Within the axoneme, the two central singlet and nine outer doublet microtubules are continuous for the entire length of the structure. Doublet microtubules, which represent a specialized polymer of tubulin, are found only in the axoneme. Permanently attached to the A tubule of each doublet microtubule is an inner and an outer row of dynein arms (see Figure 19-28a). These dyneins reach out to the B tubule of the neighboring doublet. The junction between A and B tubules of one doublet is probably strengthened by the protein tektin, a highly α-helical protein that is similar in structure to intermediate-filament proteins. Each tektin filament, which is 2 nm in diameter and approximately 48 nm long, runs longitudinally along the wall of the outer doublet where the A tubule is joined to the B tubule.

The axoneme is held together by three sets of protein cross-links (see Figure 19-28a). The central pair of singlet microtubules are connected by periodic bridges, like rungs on a ladder, and are surrounded by a fibrous structure termed the inner sheath. A second set of linkers, composed of the protein nexin, joins adjacent outer doublet microtubules. Spaced every 86 nm along the axoneme, nexin is proposed to be part of a dynein regulatory complex. Radial spokes, which radiate from the central singlets to each A tubule of the outer doublets, form the third linkage system.

The biflagellated, unicellular alga Chlamydomonas reinhardtii has proved especially amenable to biochemical and genetic studies on the function, structure, and assembly of flagella. A population of cells, shorn of their flagella by mechanical or chemical methods, provide flagella in good purity and high yield, and the deflagellated cells quickly regenerate new flagella. Analysis of the sheared flagella by two-dimensional gel electrophoresis reveals approximately 250 discrete polypeptides, in addition to α- and β-tubulin. The functions of these polypeptides have been assessed by analysis of flagella from Chlamydomonas mutants that are nonmotile or otherwise defective in flagellar function. Some nonmotile mutants, for example, lack an entire substructure, such as the radial spokes or central-pair microtubules. Many mutants that are missing a particular flagellar substructure also have been found to lack certain specific proteins, thus permitting these proteins to be assigned to a specific substructure and associated with specific genes. Such studies have identified 17 polypeptides that are components of the radial spokes and spoke heads. The components of the inner and outer dynein arms, the central-pair microtubules, and other axonemal structures have been similarly identified.

Although the 9 + 2 pattern is the fundamental pattern of virtually all cilia and flagella, the axonemes of certain protozoans and some insect sperm show some interesting variations. The simplest such axoneme, containing three doublet microtubules and no central singlets (3 + 0) is found in Daplius, a parasitic protozoan. Its flagellum beats slowly (1.5 beats/s) in a helical pattern. Other axonemes consist of 6 + 0 or 9 + 0 arrangements of microtubules. These atypical cilia and flagella, which are all motile, show that the central pair of singlet microtubules is not necessary for axonemal beating and that fewer than nine outer doublets can sustain motility, but at a lower frequency.

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

Having examined the complex structure of cilia and flagella, we now discuss how the various components contribute to their characteristic motions. Cilia and flagella, from which the plasma membrane has been removed by nonionic detergents, can beat when ATP is added; this in vitro movement can be indistinguishable from that observed in living cells. Thus the forces that generate movement must reside within the axoneme and are not located in the plasma membrane or elsewhere in the cell body.

As in the movement of muscle during contraction, the basis for axonemal movement is the sliding of protein filaments relative to one another. In cilia and flagella, the filaments are the doublet microtubules, all of which are arranged with their (+) end at the outer tip of the axoneme. Axonemal bending is produced by forces that cause sliding between pairs of doublet microtubules. The active sliding occurs all along the axoneme, so that the resulting bends can be propagated without damping.

Sliding was seen in an activation-type experiment. Demembranated axonemes were briefly treated with proteolytic enzymes such as trypsin or elastase to digest the structural linkages and the radial spokes. Upon addition of ATP, the digested axonemes telescoped apart, but no bending was observed (Figure 19-30). The sliding was often nearly complete, so that the resulting structure was greater than five times longer than the original length of the axoneme. Clearly then, the ATP-dependent movement of outer doublets must be restricted by cross-linkage proteins in order for sliding to be converted into bending of an axoneme.

Figure 19-30. Electron micrograph of two doublet microtubules in a protease-treated axoneme incubated with ATP.

Figure 19-30

Electron micrograph of two doublet microtubules in a protease-treated axoneme incubated with ATP. In the absence of cross-linking proteins, which are removed by protease, excessive sliding (more...)

Dynein Arms Generate the Sliding Forces in Axonemes

Once it was clear that the doublet microtubules in axonemes slide past each other, researchers sought to identify the force-generating proteins responsible for this movement. The inner- and outer-arm dyneins, which bridge between the doublet microtubules, were the best candidates. The identity of dynein as the motor protein in axonemes is supported by various findings. For instance, cilia and flagella possess an active ATPase that is associated with the dynein arms. In addition, removal of outer-arm dyneins by treatment with high-salt solutions reduces the rate of ATP hydrolysis, microtubule sliding, and beat frequency of isolated axonemes by 50 percent. When the extracted outer-arm dyneins are added back to salt-stripped axonemes, both the ATPase activity and the beat frequency are restored, and electron microscopy reveals that the outer arms have reattached to the proper places.

Based on the polarity and direction of sliding of the doublet microtubules, we can propose a model in which the dynein arms on the A tubule of one doublet “walk” along the adjacent doublet’s B tubule toward its base, the (−) end (Figure 19-31). The force producing active sliding requires ATP and is caused by successive formation and breakage of cross-bridges between the dynein arm and the B tubule. Successive binding and hydrolysis of ATP causes the dynein arms to successively release from and attach to the adjacent doublet. Although this general model most likely is correct, many important details such as the mechanism of force transduction by dynein are still unknown.

Figure 19-31. Model for dynein-mediated sliding of axonemal outer doublet microtubules.

Figure 19-31

Model for dynein-mediated sliding of axonemal outer doublet microtubules. The dynein arms attached to the A subfiber of one microtubule walk along the B subfiber of the adjacent doublet toward (more...)

Axonemal Dyneins Are Multiheaded Motor Proteins

Axonemal dyneins are complex multimers of heavy chains, intermediate chains, and light chains. Isolated axonemal dyneins, when slightly denatured and spread out on an electron microscope grid, are seen as a bouquet of two or three “blossoms” (Figure 19-32a). Each blossom consists of a large globular domain attached to a small globular domain (the “head”) through a short stalk; another stalk connects one or more blossoms to a common base (Figure 19-32b). The base is thought to be the site where the dynein arm attaches to the A tubule, while the small globular heads bind to the adjacent B tubule (Figure 19-32c).

Figure 19-32. Structure of axonemal dynein.

Figure 19-32

Structure of axonemal dynein. (a) Electron micrograph of freeze-etched outer-arm dynein from Tetrahymena cilia reveal three globular “blossoms” connected by stems to a (more...)

Each globular head and its stalk is formed from a single dynein heavy chain. The dynein heavy chain is enormous, approximately 4,500 amino acids in length with a molecular weight exceeding 540,000. Each heavy chain is capable of hydrolyzing ATP, and on the basis of sequences commonly found at the ATP-binding sites in other proteins, the ATP-binding domain of axonemal dynein is predicted to lie in the globular head portion of the heavy chain. The intermediate and light chains, thought to form the base of the dynein arm, help mediate attachment of the dynein arm to the A tubule and may also participate in regulating dynein activity. These base proteins thus are analogous to the MBP complexes associated with cytosolic dynein.

Axonemes contain at least eight or nine different dynein heavy chains. All inner dynein arms are two-headed structures, containing two heavy chains. The outer dynein arms have two heavy chains (e.g., in a sea urchin sperm flagellum) or three heavy chains (e.g., in Chlamydomonas flagella).

Conversion of Microtubule Sliding into Axonemal Bending Depends on Inner-Arm Dyneins

As we saw earlier, flagellar and ciliary beating is characterized by the propagation of bends that originate from the base of the axoneme (see Figure 19-27). On the other hand, the active sliding of microtubules relative to each other is a linear phenomenon (see Figure 19-31). How, then, is microtubule sliding converted to bending of a cilium or flagellum?

A bend is formed between a region of sliding and a region that resists sliding. Bending is regulated by controlling the regions where dynein is active along and around the axoneme. A close examination of the axoneme cross-section reveals that the nine outer doublets and their dynein arms are arranged in a circle so that, when viewed from the base of the axoneme, the arms all point clockwise. Since the dynein arms walk in only one direction, toward the (−) end, and each doublet slides down only one of its two neighboring doublets, active sliding in one half of the axoneme produces bending toward one side and active sliding in the other half produces bending toward the opposite side (see Figure 19-27a). By regulating the timing and location in which dynein arms are active, the axoneme can propagate bends in both directions from base to tip.

Genetic studies of mutant Chlamydomonas with abnormal motility reveal that the inner- and outer-arm dyneins contribute differently to the waveform and beat frequency of an axoneme. For example, the absence of one set of inner arms affects the waveform of flagellar beating. In contrast, mutant flagella lacking outer arms have normal waveform but slower beat frequencies. Thus the outer-arm dyneins accelerate active sliding of the outer doublets but do not contribute to bending. In contrast, the inner-arm dyneins are responsible for producing the sliding forces that are converted to bending; this suggests that inner-arm dyneins are essential for bending.

Proteins Associated with Radial Spokes May Control Flagellar Beat

Several lines of evidence indicate that the radial spokes and central-pair microtubules play a critical role in controlling the bending of a flagellum. First, mutant flagella lacking radial spokes are paralyzed. Further, a dynein regulatory complex, located at the junction between the radial spokes and inner dynein arms, has recently been identified by genetic suppressor studies. One hypothesis is that phosphorylation of the inner dynein arm inactivates it, while dephosphorylation activates it to cause sliding between outer doublet microtubules. A bend is propagated when inner-arm dynein is inactivated in one region and activated in a neighboring region.

Axonemal Microtubules Are Dynamic and Stable

For axonemes to participate in movement, they must be stable structures anchored by at least one end. As noted already, a cilium or flagellum is anchored at its cytosolic end to a basal body. In addition to its anchoring role, the basal body serves as a nucleus for the assembly of flagellar microtubules. Recall that the basal body has nine triplet microtubules. The nine A and B tubules of these triplets appear to initiate assembly of the nine outer doublet microtubules of the cilium or flagellum by growing outward from the basal body during elongation of the axonemal shaft.

Studies on the assembly of flagella and cilia provided the first evidence that a microtubule elongates by incorporating tubulin subunits at its tip. This model was originally proved by autoradiography of cells that were regenerating their flagella in the presence of radioactive tubulin subunits (and other axonemal components). More recent studies using a recombinant Chlamydomonas cell have confirmed that addition of tubulin subunits and incorporation of other axonemal components occur at the distal end of a flagellum (Figure 19-33). In these experiments, a Chlamydomonas cell expressing an epitope-tagged tubulin subunit was mated with a wild-type cell whose flagella had been amputated. After mating, which involves fusion of the two cells and mixing of their cytoplasms, the diploid cell regenerated full-length flagella by incorporating the tagged tubulin subunits. Antibodies to the epitope tag showed that the recombinant tubulin was localized within the distal tips of the regenerated flagella. This pattern could arise only if elongation occurs at the tip and not the base.

Figure 19-33. Assembly of flagellar microtubules.

Figure 19-33

Assembly of flagellar microtubules. (a) Schematic diagram of an experiment in which two Chlamydomonas cells were mated. One cell has had its flagella amputated and is regenerating them, (more...)

SUMMARY

  •  Flagellar beating propels cells forward, and ciliary beating sweeps materials across tissues.
  •  Despite their different names, flagella and cilia have the same axoneme structure, including nine doublet microtubules arranged in a circle around two central singlet microtubules (see Figure 19-28).
  •  Walking of dynein arms extending from one doublet toward the (−) end of a neighboring doublet generates a sliding force in the axoneme (see Figure 19-31). This force is converted into a bend by regions that resist sliding.
  •  Axonemal dynein is larger and more complex than cytosolic dynein. Instead of the vesicle cargo transported by cytosolic dynein, axonemal dynein moves one microtubule across the surface of its neighboring microtubule (see Figure 19-32c).
  •  The (−) ends of the microtubules in a cilium or flagellum are anchored in the basal body and are extensions of microtubules located there. Elongation of cilia and flagella occurs by addition of αβ-tubulin subunits to the distal (+) end of axonemal microtubules.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, W. H. Freeman and Company.
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