Algal Flagella

Susan K Dutcher, University of Colorado, Boulder, Colorado, USA

Flagella are extremely complex machines that must assemble over 250 components and coordinate their function. The molecular motors dynein and kinesin are found in the flagella and are believed to be necessary for the generation of a waveform.

Secondary article
Article Contents
. Introduction . Chlamydomonas, a Model Green Alga . Flagellar Waveforms . Microtubule Axoneme . Flagellar Assembly

Introduction
The green alga Chlamydomonas reinhardtii is a model system for the study of eukaryotic agella. The agella can use either of two waveforms for movement in a liquid environment. The choice of agellar waveform is inuenced by the intensity of the light. When examined by electron microscopy, the agella contain an axoneme with a ring of nine doublet microtubules with a complement of attached structures. These structures include the outer and inner dynein arms, the radial spokes, and the central pair of microtubules and its projections. Two-dimensional gel electrophoresis of isolated axonemes reveals that the axoneme is composed of over 250 polypeptides. Most of the polypeptides are in substiochiometric amounts compared to a and b tubulin, which make up 70% of the protein in an axoneme. A large collection of mutant strains that are lacking various substructures within the axoneme has suggested roles for each of these structures. The role of the central pair of microtubule projections and the radial spokes is to activate the dynein arms, which are needed to generate either a breast stroke-like waveform or the sinusoidal waveform. These two dierent waveforms move the cells forward or backwards, respectively. The outer dynein arms are needed to provide increased power to the waveform. The inner dynein arms are needed to generate the waveform and to respond to environmental signals. The assembly of the agellar axoneme occurs at the distal end, but requires the presence of a basal body at the proximal end. The transport of the components for building the axonemes requires several molecular motors. A heterotrimeric kinesin is required for transporting intraagellar transport (IFT) particles and their cargo toward the tip and a cytoplasmic dynein is required for returning the IFT particles without their cargo to the proximal end of the agella.

. Summary

swimming. The agella are also necessary for the initiation of mating between the two genetically dened matingtypes. Recognition of the opposite mating-type is mediated by glycoproteins in the agellar membranes. Chlamydomonas can be propagated in the laboratory as either haploid or diploid cultures. Haploid cells are used extensively for the isolation of mutations that aect the assembly and function of the agella. Mutations in over 135 loci have been isolated and characterized that have defects in agellar assembly or function. The phenotypes of these strains range from nonmotile cells with paralysed agella, to slowly swimming cells, to cells with agella that are longer or shorter than wild-type agella, to cells with no agella or too many agella. The characterization of these strains is described below. They provide a framework for thinking about how the dierent structures in this complex machine function together to produce a waveform.

Flagellar Waveforms
A agellum is about 0.25 mm in diameter and can move in two dierent ways. The agella can move with a whiplike motion or with a sinusoidal motion. The whiplike motion or breaststroke motion can be broken into two motions. The power stroke begins with the agellum fully extended and it moves against the liquid to lie next to the cell body. The recovery stroke involves an unfolding of the agellum and this minimizes the viscous drag through the liquid surroundings (Figure 1a). This waveform is often referred to as the ciliary waveform as it is used by the lateral cilia of the clam gill. The waveform results in the cell moving forward with the agella leading the way. In a wild-type Chlamydomonas cell, the beat frequency is generally about 60 Hz and the swimming velocity is about 150 mm s 2 1. A sinusoidal waveform is also observed. A wave is propagated along the agella (Figure 1d). This type of waveform is referred to as the agellar waveform and is similar to the waveform used by both vertebrate and invertebrate sperm tails. This motion results in the backward motion of a Chlamydomonas cell with the agella trailing. The controlled sliding of adjacent microtubules in the axoneme generates the waveform. Sliding is generated by
1

Chlamydomonas, a Model Green Alga

Chlamydomonas is a unicellular green alga that has two agella at its anterior end. In nature, it can be found in a wide variety of habitats from tropical seas to polar ice caps. The cell uses its two agella for moving on solid strata by gliding and for moving in a liquid environment by

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Algal Flagella

(a)

(b)

(c)

(d)

Figure 1 Tracing of flagellar waveforms. (a) Wild-type cells. (b) oda mutant cells that are lacking outer dynein arms. There is no obvious difference between these mutant flagella and wild-type flagella. (c) ida mutant cells that are lacking the I1 or f dynein arms. The amplitude of the initial waveform is reduced. This results in a less effective waveform. (d) Sinusoidal waveform from backward swimming wild-type cells.

the hydrolysis of adenosine triphosphate (ATP) by the dynein arms (see below) and is converted into bending to create the waveform. The key to understanding the generation of specic waveforms will be the understanding of the control of the activity of the dynein arms and their hydrolysis of ATP. There must be radial control of the arms around the axoneme as well as control of the arms along the length of the axoneme. If all of the dynein arms were active at one time, the agella would be in rigor (paralysed). One tool for studying the activity of dynein arms is the use of an in vitro assay in isolated axonemes. The rate of sliding of adjacent microtubules can be measured in isolated agella in the presence of ATP as an indication of the activity of the dynein arms. In Chlamydomonas, one mechanism that allows for radial control of the dynein arms is based on the observation that the central pair of microtubules rotate within the axoneme. The type of waveform is controlled by the light intensity in the environment. When the light is intense, Chlamydomonas cells use the sinusoidal waveform and move backwards, perhaps as a means to escape the bright light. Under most light conditions, the cells will swim forward and toward the light using the breaststroke motion.

Radial spokes Doublet microtubules Inner dynein arms

Outer dynein arms B-tubule projections Interdoublet bridge

C2 central microtubule C1 central microtubule Dynein regulatory complex

Figure 2 The structures found in a cross-section of a wild-type Chlamydomonas axoneme, seen from base to tip or from tip to base. The A and B microtubules of the nine outer tubule microtubules are shown with their protofilaments indicated as circles. The radial spokes are attached to the A tubule and point to the central pair of microtubules and their associated projections. The outer dynein arms appear as three balloons attached to the outer circumference and the inner dynein arms appear as two balloons attached to the inner circumference. The dynein regulatory complex is a small structure located between the radial spokes and the dynein arms. The B-tubule projections are found inside the B tubule and are only found in the proximal one-third of the axoneme.

Microtubule Axoneme
The backbone of the agellum is composed of nine doublet microtubules known as the axoneme (Figure 2). These microtubules are similar in their composition to cytoplasmic microtubules in that they are made of polymers of a and b tubulin. But they dier from cytoplasmic microtubules in their doublet structure as well as in their
2

increased stability to depolymerization. The doublet microtubules consist of one complete microtubule with 13 protolaments known as the A tubule and an incomplete microtubule with 11 protolaments known as the B tubule. The structures described below are attached to the microtubule lattice with a periodicity related to a b heterodimer of 8 nm. In the centre of the agellar axoneme are two singlet microtubules. These microtubules are similar in their structure and their stability to the cytoplasmic microtubules. The axoneme is continuous with the microtubules of the basal body. Eukaryotic basal bodies are also composed of a and b tubulin, but have triplet microtubules. The C tubule ends in a transition zone between the basal body and the agellar axoneme. Mutant cells that lack d tubulin are unable to assemble the C tubule. These mutant cells show an increased number of cells that lack two agella and they

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Algal Flagella

have defects in cytokinesis. These phenotypes suggest that the C tubule is needed for ecient agellar assembly and organizing cytoplasmic cytoskeletal elements, but the role of the C tubule is still in question.

Inner dynein arms

Inner dynein arms are found along the inner circumference of the nine outer doublet microtubules. Unlike the outer dynein arms, there are at least seven biochemically distinct inner arm structures. However, the location of these seven dynein species within the axoneme is not known. It is possible that some of these arms are found only near the base of the agella and others are found at the tip. Despite this biochemical heterogeneity, electron microscopic analysis of the inner dynein arm region suggests that there is a repeating structure every 96 nm from the base to the tip (Mastronarde et al., 1992). Within this 96 nm repeat are ten densities (Figure 4). Three of these densities are part of the I1 or f dynein. This dynein arm has two dynein heavy chains, Ia and Ib, and four or ve other polypeptides. The absence of this arm results in an altered waveform (Figure 1c) and slowed swimming speeds. These strains have also lost the ability to respond to directional light signals. Two of the three densities observed by electron microscopy contain the globular heads of the dynein heavy chains and the third lobe contains the attachment sites to the tubulin lattice. The remaining six inner dynein arms that have been identied by biochemical analysis dier from the previous dynein arms in that they are likely to be single-headed dynein arms, which means that they have only a single dynein heavy chain associated with each arm. Biochemically, they are known as a, b, c, d, e and g. Mutations have been identied that aect the assembly of dyneins a, c, d and e and these mutant strains show altered waveforms that are similar to the waveform of the strains missing the I1 or f dynein. These altered waveforms result in slowed swimming speeds (Myster et al., 1997). Two low-molecular weight polypeptides, monomeric actin and a calcium-

Outer dynein arms

The outer dynein arms are found on the outer circumference of the nine outer doublet microtubules (Figures 2 and 3). They are placed every 24 nm along the B tubule and are homogeneous along the length of the agella. Outer dynein arms can be removed from the axoneme by treatment with high salt concentrations and can be biochemically puried. The puried structures contain at least 15 polypeptides. The outer dynein arm consists of three dynein heavy chains called a, b and g, which have molecular weights of greater than 500 000. These heavy chains assemble with two intermediate-molecular weight polypeptides of 69 and 78 kDa and at least 10 small (8 to 22 kDa polypeptides). Mutations in 13 dierent loci fail to assemble all or part of the outer dynein arms as monitored by electron microscopy and by biochemical analysis of salt-extracted mutant axonemes (Kamiya, 1988; Koutoulis et al., 1997). The failure to assemble the arms suggests that the gene products encoded by these loci are each needed to promote the assembly of the entire outer dynein arm structure. The spacing of the outer dynein arms along the microtubules requires the assembly of a docking complex, which is a bre that runs along the length of the microtubule and appears to be responsible for the placement of the arms every 24 nm. The role of the outer dynein arms is suggested by the phenotype of mutant stains that lack them. The agellar waveform is unaected, but the frequency of beating is reduced (Figure 1b). Thus, these structures are needed for increasing the power of the agellar stroke. These mutant strains move forward more slowly and the agellar beat is reduced by about one-half.

Radial spokes

19 8 8 20 11 69 14 14 78 16

22 18

22 18

2 1

5 4 10 8 3 9a 9b

19 16

Outer dynein arms

Figure 4 A diagram of the densities observed by electron microscopy of longitudinal sections of the Chlamydomonas axoneme. At the bottom are the homogeneous outer dynein arms and at the top are the two radial spokes that are present every 96 nm. Between these structures are the inner dynein arms and the dynein regulatory complex. Densities 1, 2 and 3 are the I1 or f dynein, densities 4, 5 and 6 are the I2 or a, c and d dyneins, density 9a is the e dynein, density 9b is the dynein regulatory complex. Densities 7 and 8 are not altered in any mutant strain and so the dynein composition is unknown. Density 10 is missing in an inner arm mutant that affects only a subset of the dynein arms on the nine doublet microtubules.

8 8 20 11 69 14 14 78

Figure 3 A diagram of an outer dynein arm with the arrangement of the different polypeptides within the outer dynein arm. The small spheres at the bottom of the figure represent the microtubule lattice.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Algal Flagella

binding protein known as centrin, are associated with these arms. The function of these proteins in the dynein arms is not known. Mutations that aect the other arms (b and g) have not been identied; this result may suggest that mutant cells lacking these arms have a subtler phenotype.

Dynein regulatory complex

The dynein regulatory complex is localized to the same region as the inner dynein arms. The complex of six polypeptides does not contain a dynein heavy chain, but may exert its action by promoting the assembly of a subset of the inner dynein arms and perhaps regulating their activity by transmitting signals from the radial spokes to multiple dynein arms. Mutations that are missing subsets of these six polypeptides have a reducing velocity of swimming and altered waveforms as is observed in the mutant strains lacking dynein arms (Piperno et al., 1994). For this reason, it is hypothesized that the complex aects the inner dynein arms directly or indirectly through the radial spokes. However, the mechanism by which the dynein regulatory complex acts is unknown.

activation of radial spokes around the axoneme and their subsequent activation of the dynein arms. Another set of central-pair mutations result in the assembly of an aberrant microtubule. The pf15, pf18, pf19 and pf20 mutations result in a single large microtubule instead of a pair of microtubules. This result suggests that these gene products are needed for the specication or templating of the central microtubules. The template for the central pair of microtubules is not known, as they are not continuous with microtubules of the basal body. The presence of this unusual single microtubule results in paralysed agella. It is not known if these aberrant microtubules are capable of rotation or have all of the necessary projections to activate the radial spokes and dynein arms.

Radial spokes
Radial spokes are assembled twice every 96 nm along the axonemes. They consist of a stalk and a head. Mutations in ve genes result in the complete absence of the radial spoke and mutations in two genes result in the absence of the head (Deiner et al., 1990). These mutant strains have paralysed agella, which suggests that the radial spokes are needed to activate the dynein arms. Their radial position has raised the hypothesis that they are needed to activate dynein arms around the axoneme so that while dynein arms on one side of the axoneme are on, the arms on the other side are o. To support the role of radial spokes in vitro experiments have been employed. Experiments measuring the rate of sliding of adjacent microtubules in isolated axonemes suggest that the activity of dynein arms is reduced if radial spokes are missing. Increased rates of sliding can be reconstituted with the addition of radial spokes in vitro or by treatment with agents that block phosphorylation. The target for this regulation is a 138 kDa polypeptide of the I1 or f inner dynein arm (Habermacher and Sale, 1997). When the 138 kDa polypeptide is phosphorylated, the dynein arms are inactive and when it is dephosphorylated, the dynein arms are active.

Central pair of microtubules and their projections

The central pair of microtubules are two singlet microtubules that resemble the singlet microtubules found in the cytoplasm of eukaryotic cells. Along the length of the microtubules are at least six dierent types of projections. The absence of these projections results in paralysed agella. The projections on the central pair of microtubules may regulate or activate the dynein arms through their interaction with the radial spokes (see radial spokes below). The central pair of microtubules are dierent from one another and are known as the C1 and C2 microtubules (Mitchell and Sale, 1999). The assembly of the projections on the microtubules requires the action of several known gene products. The PF16 gene product, which contains armadillo repeats, is needed for the assembly of the C2 microtubule but not the C1 microtubule. The PF6 gene product is needed for the assembly of one of the projections on the C2 microtubule. Mutations of these two genes result in paralysis or near paralysis. The CPP1 gene product is required for the assembly of projections on the C1 microtubule. The mutant phenotype diers from pf6 and pf16 mutant strains in that cells remain motile (Smith and Lefebvre, 1996). Thus, the projections on the two microtubules appear to perform dierent functions. A kinesin-like protein (KLP1) is found on the C1 microtubule. It has been hypothesized that this motor protein may be responsible for the turning of the central pair complex within the axoneme. This turning of the central pair complex would allow the
4

B-tubule projection
Within the B tubules of outer doublets 1, 5 and 6 are beaklike projections. These projections are found only in the proximal one-third of the agella. Cells with a mutation in one of three dierent genes, called MBO, lack these projections. These cells lack the ability to generate the ciliary waveform. They are only able to generate the agellar waveform and so only move backwards. Flagellar axonemes isolated from these mutant cells are missing up to six polypeptides. Four of these six polypeptides are phosphorylated. In wild-type cells, agellar waveforms are initiated when cells are exposed to intense light signals. These light signals

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Algal Flagella

cause an inux of calcium ions into the cells. In vitro assays suggest that the phosphorylation of one of the B-tubule projection polypeptides is stimulated by calcium concentrations of greater than 10 2 6 mol L 2 1. This result suggests that the initiation of the agellar waveform could be inuenced by a calcium-stimulated kinase acting on the 95 kDa polypeptide of the B-tubule projection.

Summary
Algal agella have served as a model for the understanding of eukaryotic agella. Through the combined use of genetic and biochemical analyses, the polypeptide composition of the axoneme has been studied. It is a complex structure in which over 250 polypeptides must assemble at precise positions. It involves two molecular motors that are needed to transport the preassembled structures to the site of assembly at the tip. Flagella generate two types of waveforms that require the action of large protein complexes known as dynein arms. There is great heterogeneity in these arms. The role of the dierent dyneins is only now being elucidated. The outer dynein arms are needed for added power while the inner arms are needed for the type of waveform. Why the cells need seven dierent inner dynein arms is not known. The activity of the inner dynein arms can be inuenced by environmental signals that are likely to act through dierent intracellular calcium concentrations. Increased intracellular calcium results in a agellar waveform and backward swimming. Cells can also orient toward a directional light and the intracellular calcium level inuences this behaviour. Further genetic, biochemical and ultrastructural analyses will help to reveal the regulation of dyneins and agellar beating.

Flagellar Assembly
The assembly of the agella requires the presence of a basal body. Basal bodies, like agella, are microtubule-based structures. The basal body has triplet microtubules that become doublet microtubules at the distal end. Flagella in Chlamydomonas are about 12 mm in length and it is unlikely that diusion is sucient to transport all of the proteins needed for the assembly of the agella. Transport is an active process that requires two dierent molecular motors and the assembly of rafts or intraagellar particles. Using dierential interference contrast optics, the movement of these particles was observed. They move toward the tip of agella at 2 mm s 2 1 (anterograde movement) and move toward the base of the agella at 3.5 mm s 2 1 (retrograde movement) (Kozminski et al., 1995). The anterograde movement requires the presence of FLA10p, a member of the heterotrimeric kinesin-like protein family. The retrograde movement to move particles from the tip to the base requires the presence of a cytoplasmic dynein. The particles, when examined by electron microscopy, are large raft-like structures (  400 nm in length). These rafts have also been observed by electron microscopy in C. elegans and in vertebrate rod cells of the retina. Biochemical analysis has suggested that the rafts are composed of at least 20 polypeptides. The rafts are hypothesized to carry agellar polypeptides to the tip for assembly. Several lines of evidence suggest that the structures are preassembled in the cytoplasm and that single polypeptides are not the cargo for the transport machinery. When several of the components of the IFT particles were subjected to microsequencing, the sequences revealed signicant similarities to genes in C. elegans and in the human, mouse, and rat Expressed Sequence Tag databases. Mutations in several of the homologous C. elegans genes have defects in assembly of sensory cilia. Mice that are homozygous for a deletion mutation in KIF3B, a homologue of FLA10, show a lethal phenotype, but early embryos show a defect in the establishment of leftright asymmetry. Early nodal cells in wild-type embryos have a single, motile cilium that is hypothesized to move a signalling molecule in the extraembryonic uid toward the left side of the embryo, which will initiate the cascade of events involved in the establishment of leftright asymmetry. In these homozygous mutant mice, these cilia do not assemble and bilateral symmetry is not broken.

References
Deiner DR, Curry AM, Johnson KA, Williams BD, Lefebvre PA, Kindle KL and Rosenbaum JL (1990) Rescue of a paralyzed agella mutant of Chlamydomonas by transformation. Proceedings of the National Academy of Sciences of the USA 87: 57395743. Habermacher G and Sale WS (1997) Regulation of a agellar dynein by phosphorylation of a 138-kD inner dynein arm intermediate chain. Journal of Cell Biology 136: 167176. Kamiya R (1988) Mutations at twelve independent loci result in the absence of the outer dynein arms in Chlamydomonas reinhardtii. Journal of Cell Biology 107: 22532258. Koutoulis A, Pazour GJ, Wilkerson CG et al. (1997) The Chlamydomonas reinhardtii ODA3 gene encodes a protein of the outer dynein arm docking complex. Journal of Cell Biology 137: 10691080. Kozminski KG, Beech PL and Rosenbaum JL (1995) The Chlamydomonas kinesin-like protein Fla10 is involved in motility associated with the agellar membrane. Journal of Cell Biology 131: 15171527. Mastronarde DN, OToole ET, McDonald KL, McIntosh JR and Porter ME (1992) Arrangement of inner dynein arms in wild-type and mutant agella of Chlamydomonas. Journal of Cell Biology 118: 11451162. Mitchell DR and Sale WS (1999) Characterization of a Chlamydomonas insertional mutant that disrupts agellar central pair microtubuleassociated structures. Journal of Cell Biology 144: 293304. Myster SH, Knott JA, OToole E and Porter ME (1997) The Chlamydomonas DHC1 gene encodes a dynein heavy chain subunit required for the assembly of the I1 inner arm complex. Molecular Biology of the Cell 8: 607620. Piperno G, Mead K, LeDizet M and Moscatelli A (1994) Mutations in the dynein regulatory complex alter the ATP-insensitive binding sites for inner arm dyneins in Chlamydomonas axonemes. Journal of Cell Biology 125: 11091117.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Algal Flagella

Smith EF and Lefebvre PA (1996) PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas agella. Journal of Cell Biology 132: 359370.

Further Reading
Bernstein M, Beech PL, Katz SG and Rosenbaum JL (1994) A new kinesin-like protein (Klp1) localized to a single microtubule of the Chlamydomonas agellum. Journal of Cell Biology 125: 13131316. Cole DG, Diener DR, Himelblau AL et al. (1998) Chlamydomonas kinesin-ii-dependent intraagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons. Journal of Cell Biology 141: 9931008. Collet J, Spike CA, Lundquist EA, Shaw JE and Herman RK (1998) Analysis of osm-6, a gene that aects sensory cilium structure and sensory neuron function in Caenorhabditis elegans. Genetics 148: 187 200. Curry AM, Williams BD and Rosenbaum JL (1992) Sequence analysis reveals homology between two proteins of the agellar radial spoke. Molecular and Cell Biology 12: 39673977.

Dutcher SK (1995) Flagellar assembly in 250 easy-to-follow steps. Trends in Genetics 11: 398404. Harris EH (1989) The Chlamydomonas Sourcebook. A Comprehensive Guide to Biology and Laboratory Use. San Diego: Academic Press. King SJ and Dutcher SK (1997) Phosphoregulation of an inner dynein arm complex in Chlamydomonas reinhardtii is altered in phototactic mutant strains. Journal of Cell Biology 136: 177191. King SM, Barbarese E, Dillman JF 3rd et al. (1998) Cytoplasmic dynein contains a family of dierentially expressed light chains. Biochemistry 37: 1503315041. Lefebvre PA and Rosenbaum JL (1986) Regulation of the synthesis and assembly of ciliary and agellar proteins during regeneration. Annual Review of Cell Biology 2: 517546. Piperno G, Ramanis Z, Smith EF and Sale WS (1990) Three distinct inner dynein arms in Chlamydomonas agella: molecular composition and location in the axoneme. Journal of Cell Biology 110: 379389. Porter ME, Knott JA, Gardner LC, Mitchell DR and Dutcher SK (1994) Mutations in the SUP-PF-1 locus of Chlamydomonas reinhardtii identify a regulatory domain in the b-dynein heavy chain. Journal of Cell Biology 126: 14951507.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net