Cilia and flagella are related microtubule-based and membrane-enveloped extensions of the plasma membrane that project from many protozoan and most animal cells. Abundant motile cilia are found on the surfaces of specific epithelia, such as those that line the trachea, where they beat in an orchestrated wavelike fashion to move fluids. Animal-cell flagella, which are longer than cilia but have a very similar structure, can propel a cell, such as a sperm, through liquid. Cilia and flagella contain many different microtubule-based motors: axonemal dyneins are responsible for the beating of flagella and cilia, whereas kinesin-2 and cytoplasmic dynein are responsible for flagellum and cilium assembly and turnover.
Eukaryotic Cilia and Flagella Contain Long Doublet Microtubules Bridged by Dynein Motors
Cilia and flagella range in length from a few micrometers to more than 2 mm for some insect sperm flagella. They possess a central bundle of microtubules, called the axoneme, which consists of a so-called 9 + 2 arrangement of nine doublet microtubules surrounding a central pair of singlet, yet ultrastructurally distinct, microtubules (Figure 1a, b). Each of the nine outer doublets consists of an A microtubule with 13 protofilaments and a B microtubule with 10 proto filaments. All the microtubules in cilia and flagella have the same polarity: the (+) ends are located at the distal tip. At its point of attachment in the cell, the axoneme connects with the basal body, a complicated structure containing nine triplet microtubules (Figure 1a).
Fig1. Structural organization of cilia and flagella. (a) Cilia and flagella are assembled from a basal body, a structure built around nine linked triplet microtubules. Continuous with the A and B microtubules of the basal body are the A and B tubules of the axoneme—the membrane-enveloped core of the cilium or flagellum. Between the basal body and axoneme is the transitional zone. The diagram and accompanying transverse sections of the basal body, transitional zone, and axoneme show their intricate structures. (b) A transverse section of a cilium, to show the identity of the structures. See S. K. Dutcher, 2001, Curr. Opin. Cell Biol. 13:49–54. [Micrographs republished with permission of Elsevier, from Dutcher, S. K., “The tubulin fraternity: alpha to eta,” Curr. Opin. Cell Bio., 2001, 13(1):49-54.]
The axoneme is held together by three sets of protein cross-links (Figure 1b). The two central singlet micro tubules are connected to each other by periodic bridges, like rungs on a ladder. A second set of linkers, composed of the protein nexin, joins adjacent outer doublet microtubules to each other. Radial spokes project from each A tubule of the outer doublets toward the central pair.
The major motor protein present in cilia and flagella is axonemal dynein, a large, multisubunit protein related to cytoplasmic dynein. Two rows of axonemal dynein motors are attached periodically down the length of each A tubule of the outer doublet microtubules; these motors are called the inner-arm and outer-arm dyneins (see Figure1b). It is the interaction of these dynein motors with the B tubule in the ad jacent doublet that brings about ciliary and flagellar bending.
Ciliary and Flagellar Beating Are Produced by Controlled Sliding of Outer Doublet Microtubules
Cilia and flagella are motile structures because activation of the axonemal dynein motors causes them to bend. A close examination of their movement using video microscopy reveals that a bend starts at the base of a cilium or flagellum and then propagates along the structure (Figure 2). A clue to how this occurs came from studies of isolated axonemes. In these classic experiments, axonemes were gently treated with a protease that cleaves only the nexin linkers. When ATP was added to the treated axonemes, the doublet microtubules slid past one another as dynein, attached to the A tubule of one doublet, “walked” down the B tubule of the adjacent doublet (Figure 3b, c). In an axoneme with intact nexin linkers, the action of dynein induces flagellar bending because the microtubule doublets are connected to one another (Figure 3a).
Fig2. 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 surrounding fluid and propel the cell forward. Captured in this multiple-exposure sequence, a bend at the base of the flagellum in the first (top) frame has moved distally halfway along the flagellum by the last frame. A pair of gold beads attached to 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) ©1991 C. Brokaw et al., The Journal of Cell Biology, 114:1201–1215. doi: 10.1083/jcb.114.6.1201. Part (b) courtesy of Stuart Goldstein.]
Fig3. Ciliary and flagellar bending is mediated by axonemal dynein. (a) Axonemal dynein attached to an A tubule of an outer doublet pulls on the B tubule of the adjacent doublet trying to move toward the (−) end. Because the adjacent tubules are tethered by nexin, the force generated by dynein bends the cilium or flagellum. (b) Experimental evidence for the model in (a). When the nexin linkers are cleaved with a protease and ATP added to induce dynein activity, the microtubule doublets slide past one another. (c) Electron micro graph of two doublet microtubules in a protease-treated axoneme incubated with ATP. In the absence of cross-linking proteins, doublet microtubules slide excessively. The dynein arms can be seen projecting from A tubules and interacting with B tubules of the left microtubule doublet. [Part (c) courtesy of P. Satir.]
How specific subsets of dynein are activated and how a wave of activation is propagated down the axoneme are not yet fully understood, but post-translational modifications of tubulin may play a role. post-translational modifications of tubulin subunits can affect the interactions between microtubules and motor proteins. The B tubules of the outer axoneme doublets are often polyglutamylated, and this modification strongly affects the interaction of the inner-arm dynein with the B tubule. Because inner-arm dynein motors mainly affect the waveform of the ciliary beat, it is this aspect of ciliary function that is compromised in mutants unable to undergo polyglutamylation.
Intraflagellar Transport Moves Material Up and Down Cilia and Flagella
Although axonemal dynein is involved in bending cilia and flagella, another type of motility has been observed in these structures more recently. Careful examination of flagella on the biflagellate green alga Chlamydomonas reinhardtii revealed cytoplasmic particles moving at a constant speed of about 2.5 μm/s toward the tip of a flagellum (anterograde movement) and other particles moving at about 4 μm/s from the tip to the base (retrograde movement). This movement, known as intraflagellar transport (IFT), occurs in both cilia and flagella. Light and electron microscopy revealed that the particles move between the outer doublet microtubules and the plasma mem brane (Figure 4). Analysis of algal mutants demonstrated that the anterograde movement is powered by kinesin-2 and the retrograde movement by cytoplasmic dynein.
Fig4. Intraflagellar transport. (a) Particles are transport ed between the plasma membrane and the outer doublet microtubules. Transport of the particles to the tip is dependent on kinesin-2, whereas transport toward the base is mediated by cytoplasmic dynein. (b) This thin-section electron micrograph shows IFT particles in a section of a Chlamydomonas flagellum. [Part (b) reprinted by permission from Macmillan Publishers Ltd: Rosenbaum J.L., and Witman, G.B., “Intraflagellar transport,” Nature Reviews Molecular Cell Biology, 2002, 3, 813-825.]
The anterograde and retrograde IFT particles transported in Chlamydomonas flagella have been isolated and their composition determined. They consist of two distinct protein complexes, called IFT complex A and IFT complex B. By analyzing the phenotypes of cells having mutations affecting these complexes, it has been found that complex B is necessary for anterograde IFT, whereas complex A is important for retrograde IFT. Despite this segregation of function, both complexes are transported in both directions. All the components of IFT particles have homologs in organisms containing cilia, such as nematodes, fruit flies, mice, and humans, but these particles are absent from the genomes of yeasts and plants that lack cilia, suggesting that they are specific to IFT.
What is the function of IFT? Because all the microtubules in a flagellum have their growing (+) ends at the tip, that is the site where new tubulin subunits and flagellar structural proteins are added. Moreover, even in cells with flagella of uniform length, the microtubules are turning over, with assembly and disassembly occurring at the flagellar tip. In cells defective for kinesin-2, flagella shrink, suggesting that IFT transports new material to the tip for growth. Since IFT is a continually occur ring process, what happens to the kinesin-2 molecules when they get to the tip, and where do the dynein motors come from to transport the particles retrogradely? Remarkably, dynein is carried to the tip as cargo on the anterograde-moving particles, powered by kinesin-2, and then kinesin-2 becomes cargo as the particles are transported back to the base by dynein.
Primary Cilia Are Sensory Organelles on Interphase Cells
Many vertebrate cells bear a solitary nonmotile cilium known as the primary cilium. The primary cilium is a stable structure that is resistant to drugs such as colchicine that disassemble most microtubules; after colchicine treatment, the only remaining microtubules are found in the centrioles and the primary cilium. Moreover, the tubulin in the primary cilium is highly acetylated, so using antibodies that specifically recognize acetylated α-tubulin readily identifies the single primary cilium on each interphase cell (Figure 5a).
Fig5. Many interphase cells contain a nonmotile primary cilium. (a) Fluorescence micrograph of mouse epithelial cells stained with antibodies to acetylated α-tubulin (green), which decorate the primary cilia; to pericentrin (magenta), which decorate the centrosome; and to ZO-1(red), which label the tight junctions that encircle each cell. (b) A diagram depicting how the presence of the primary cilium is tied to the centrioles, one of which serves as its basal body. (c) A diagram depicting a section through a nonmotile primary cilium, showing the lack of the central pair of microtubules and dynein arms typical of motile cilia and flagella. (d) Scanning electron micrographs of epithelial cells of a kidney collecting tubule from a wild-type mouse (left) and a mutant mouse defective in a component of the IFT particles. Arrows point to the primary cilia, which are short stubs in the mutant mouse. [(a) Republished with permission of John Wiley & Sons, from “A Cell-Based Screen for Inhibitors of Flagella-Driven Motility in Chlamydomonas Reveals a Novel Modulator of Ciliary Length and Retrograde Actin Flow,” Enge, B.D. et al., Cytoskeleton, 68(3). Copyright © 2011. (d) ©1991 Douglas J. Cole, from Pazour, G. et al., The Journal of Cell Biology. 151:709-718. doi: 10.1083/jcb.151.3.709.]
Terminally differentiated cells and dividing cells in interphase contain primary cilia. In the latter case, the presence of the primary cilium is tied to the duplication cycle of the centrioles, with the older, “mother” centriole functioning as the basal body for the assembly of the primary cilium (Figure5b).
The primary cilium is nonmotile because it lacks the central pair of microtubules and the dynein arms that are found in other cilia and in flagella (Figure 5c). Recent work has led to the discovery that the primary cilium is instead a sensory organelle, acting as the cell’s “antenna” by detecting extracellular signals. For example, the sense of smell is due to binding of odorants by receptors located in the primary cilia of olfactory sensory neurons in the nose. In another example, the rod and cone cells of the eye have a primary cilium with a greatly expanded tip to accommodate the proteins involved in photoreception. The retinal protein opsin moves through the primary cilium at about 2000 molecules per minute, transported by kinesin-2 as part of the IFT system. Defects in this transport cause retinal degeneration.
The primary cilium has a diffusion barrier at its base so that only the appropriate proteins can enter it: globular proteins of 10 kDa have ready access, but proteins above 40 kDa are excluded. Remarkably, transport through this barrier has similarities to transport into the nucleus through nuclear pores, and indeed, these two forms of transport have some components in common. Recall that import though nuclear pores requires a gradient of the Ran GTPase and involves binding of cargo proteins to importins. Transport of at least some proteins, such as kinesin-2, across the base and into a primary cilium requires both a gradient of the Ran GTPase and importins.
Defects in Primary Cilia Underlie Many Diseases
For many years, the existence and function of the primary cilium was ignored. However, this situation has changed dramatically over the last decade as it has become appreciated that defects in intraflagellar transport result in the loss of primary cilia in mice (Figure 5d), and as diseases have been traced to defects in primary cilia and IFT. One of the first clues came from the discovery that loss of a mammalian homolog of a Chlamydomonas IFT protein results in defects in the primary cilia and causes autosomal dominant polycystic kidney disease (ADPKD). It is believed that the primary cilia on the epithelial cells of the kidney collecting tubule act as mechanochemical sensors to measure the rate of fluid flow by the degree to which they are bent.
In another example, patients with Bardet-Biedl syn drome have retinal degeneration, polydactyly (from the Greek for “many fingers”), and obesity. The syndrome can be caused by mutations in any one of 14 genes and has been traced to defects in the function of primary cilia. Many of these genes encode subunits of the BBsome, an octameric complex that forms a coat with structural elements in common with COPI, COPII, and clathrin and that traffics membrane proteins to cilia. While defects in many of the BBsome’s components do not affect the structure of the primary cilium itself, they result in a lack of specific membrane receptors that would normally be delivered to primary cilia through the interaction of the BBsome with the IFT apparatus. For example, the polydactyly seen in patients with Bardet-Biedl syndrome is due to a loss of localized Hedgehog signaling in the primary cilium that is necessary for patterning during embryogenesis.
