Chapter 3 How Did the Cilium Evolve?

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How Did the Cilium Evolve? Peter Satir,* David R. Mitchell,† and Ga´spa´r Je´kely‡ Contents 1. 2. 3. 4.

Introduction The Link Between Ciliary Evolution and Eukaryotic Divergence Hypotheses of the Origin of Cilia The Viral Hypothesis of Ciliary Origin 4.1. Statement of the hypothesis 4.2. What the viral hypothesis can explain 4.3. Unusual predictions as a consequence of the viral hypothesis 4.4. Objections to the viral hypothesis 5. The Autogenous Model for the Origin of Cilia 5.1. Autogenous origin of microtubule-organizing centers 5.2. Autogenous origin of ninefold symmetric basal bodies and the axoneme 6. Origin of Intraflagellar Transport and the Sensory Function of Cilia 7. The Evolution of Ciliary Motility Acknowledgments References

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Abstract The cilium is a characteristic organelle of eukaryotes constructed from over 600 proteins. Bacterial flagella are entirely different. 9 + 2 motile cilia evolved before the divergence of the last eukaryotic common ancestor (LECA). This chapter explores, compares, and contrasts two potential pathways of evolution: (1) via invasion of a centriolar-like virus and (2) via autogenous formation from a pre-existing microtubule-organizing center (MTOC). In either case, the intraflagellar transport (IFT) machinery that is nearly universally required for the assembly and maintenance of cilia derived from the evolving intracellular vesicular transport system. The sensory function of cilia evolved first and the ciliary axoneme evolved gradually with ciliary motility, an important selection mechanism, as one of the driving forces.

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Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, New York Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tu¨bingen, Germany

Current Topics in Developmental Biology, Volume 85 ISSN 0070-2153, DOI: 10.1016/S0070-2153(08)00803-X

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2008 Elsevier Inc. All rights reserved.

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1. Introduction In this chapter, we explore the evolution of one of the most characteristic organelles of the eukaryotic cell, the cilium. Cilia, sometimes called flagella in the literature, are found exclusively on eukaryotic cells, throughout the protista, in many plant phyla on gametes, and on somatic cells and/ or gametes in virtually every metazoan phylum. (Bacterial flagella are entirely different organelles.) In humans, motile cilia are present on nasal and tracheal lining cells, ependymal cells of the brain, and cells lining the oviduct. The sperm tail is a cilium with accessory elements. The sensory hair cells of the ear and semicircular canals possess a single ‘‘kinocilium’’ (the ‘‘stereocilia’’ of the ear and epididymis are microvilli, not cilia). Olfactory sense cells each possess multiple cilia, which may be immotile. During embryogenesis, motile cilia present at the node during gastrulation evidently initiate the determination of left–right body asymmetry. In addition, many cells in the body possess a single nonmotile cilium called a primary cilium; a primary cilium with extensive membrane modifications forms the outer segment of the ciliary photoreceptors of the vertebrate eye. A current review of the structure and function of motile and primary mammalian cilia is found in Satir and Christensen (2007). A series of combined genomic–proteomic studies on protistan (Chlamydomonas, Tetrahymena) motile cilia, invertebrate (primarily Caenorhabditis elegans) sensory cilia, and mammalian and human cilia reveal that cilia are comprised of upwards of 600 proteins (Avidor Reiss et al., 2004; Li et al., 2004; Ostrowski et al., 2002; Pazour et al., 2005; Scholey, 2003; Smith et al., 2005). Many or most of the genes related to cilia are missing in genomes of nonciliated organisms, such as Arabidopsis. When cilia are present, a remarkable portion of the nuclear genome, perhaps 3–5% of the typical genome, is devoted to this single organelle. The proteins produced by these genes are highly conserved. Three well-studied illustrative examples of proteins equally important in the single-celled alga Chlamydomonas reinhardtii and man are (1) axonemal dyneins, for example, OADa (the a-subunit of outer arm dynein) (Wilkes et al., 2008); (2) hydin (Lechtreck et al., 2008), a central pair projection; and (3) polaris or IFT88, a component of the intraflagellar transport (IFT) system (Pazour et al., 2000; Yoder et al., 2002). Proteins such as these form defined parts of complex structures in the cilium. As has been understood for over half a century, almost all motile cilia are built on a similar plan, a 200 nm diameter cytoskeletal bundle of nine peripheral doublet microtubules surrounding a central singlet pair, the socalled 9 + 2 axoneme, which grows from a basal body and is enclosed in an extension of the cell membrane called the ciliary membrane (Fig. 3.1). Theciliary membrane incorporates specialized receptors and channels and confers on all cilia a sensory function, the ability to respond to external molecular signals (Christensen et al., 2007). The basal body itself is almost universally an

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Figure 3.1 Architecture of the motile 9 + 2 cilium and the basal body. (A) Longitudinal section through one Chlamydomonas reinhardtii flagellum (a second flagellum is out of the plane of section, although its basal body is visible). Abbreviations: CP, central pair; DM, doublet microtubule; MEM, plasma membrane; CW, cell wall; BB, basal bodies; TZ, transition zone. Inset, cross section through an isolated axoneme shows the location of radial spokes (RSP), outer arm dyneins (OAD), and inner arm dyneins (IAD). (B) and (C) Longitudinal and cross sections of a field of oviduct cilia (courtesy Dirksen and Satir, 1972, unpublished micrograph). In these cilia, the basal foot (arrowhead) points in the direction of the effective stroke. A multirowed ciliary necklace (arrow) is present in the transition zone near the IFT loading area, above which the cilium projects from the cell and the central pair of the 9 + 2 axoneme originates. Each axoneme is surrounded by the ciliary membrane, in which special receptors and channels become concentrated. The diameter of the cilium (0.25 mm) provides a scale for the micrographs.

arrangement of nine triplet microtubules, essentially identical to a centriole that has become attached to the cell membrane and formed a specialized structure called the ciliary necklace (Gilula and Satir, 1972). The cilium grows beyond the necklace with each of the nine triplet microtubules reduced to an axonemal doublet. In the transition zone, the central pair arises. Growth to lengths often greater than 10 mm occurs, again almost universally, by a process discovered more recently that has been named intraflagellar transport (Blacque et al., 2008; Rosenbaum and Witman, 2002; Scholey, 2003). The motile cilium is a nanomachine powered by molecular motors, the axonemal dyneins attached to one edge of each axonemal doublet. The ATPase activity of the OADs and IADs (inner arm dyneins) produces sliding between the doublets, powers the axoneme, and causes ciliary beat. The form and frequency of beat are modulated in a highly complex manner by second messengers generated via ciliary membrane channels or coupled receptors and by actions of the radial spoke–central pair proteins. The radial spokes (Fig. 3.1) extend from each doublet microtubule to specific protein

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projections surrounding the central singlet microtubules (Smith and Yang, 2004). Orthologous proteins of different organisms form the identical structures and perform the same functions in corresponding axonemes. Moreover, mutations in these proteins lead to structural and motility defects in Chlamydomonas and, because of the corresponding defects in human cilia, to ciliopathies in man (Badano et al., 2006; Li et al., 2004; Satir and Christensen, 2008). As examples of human ciliopathies, defects in OADs lead to primary ciliary dyskinesia (PCD), defects in hydin lead to hydrocephalus and defects in IFT88 (polaris) lead to autosomal recessive polycystic kidney disease (PKD). Specialized sensory cilia like the photoreceptor outer segment and primary cilia also possess an axoneme, a ciliary membrane, a ciliary necklace and they grow above a basal body by IFT. However, although axonemal structure is largely preserved, these cilia are often nonmotile because the dynein arms, the radial spokes, and proteins involved in dynein regulation are missing. In most cases, the central microtubules are also missing and the axonemes are referred to as 9 + 0. Examples abound in vertebrates, as such 9 + 0 ‘‘primary’’ cilia are found on most cell types. In contrast to motile function, the presence or absence of sensory function, regulated adhesion, and surface motility are less easily detected. These functions may be retained by most motile as well as nonmotile cilia.

2. The Link Between Ciliary Evolution and Eukaryotic Divergence Conservation of primary sequence, protein composition, structure, and function throughout eukaryotic history as recorded in current phyletic divergence suggests a unitary origin of cilia at or very near the beginning of eukaryotic cell evolution. Certainly, the presence of cilia can be traced back to the last eukaryotic common ancestor (LECA) through analysis of gene/ protein sequences and studies of ultrastructure in existing organisms. These determinations have been made possible only by the extensive recent advances in our overall understanding of eukaryotic phylogeny (e.g., Brinkmann and Philippe, 2007; Rodriguez-Ezpeleta et al., 2007; Yoon et al., 2008). Using such phylogenetic approaches, it has been possible to confidently state, regardless of specific branch points and groupings, that all of the major identified branches of existing eukaryotes include organisms with nearly indistinguishable 9 + 2 motile cilia, and therefore such cilia evolved prior to the divergence from the LECA (Cavalier-Smith, 2002; Mitchell, 2004). Although the possibility remains that an organism with a more primitive motile apparatus will be definitively identified as a member of a new phyletic clade, not closely related to any of the presently

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well-defined clades, and thus displaying traits of eukaryotes that existed before the great divergence of most present day eukaryotes, this possibility is becoming increasingly remote as sequence coverage expands. Thus singlet, doublet, and triplet microtubules, and a-, b-, g-, d-, and e-tubulin, a central pair complex, radial spokes, at least five axonemal dynein complexes, and IFT complexes with their associated kinesin and dynein motors, all appear to have evolved prior to eukaryotic divergence. Further evolution in these organelles during the ca. 1 billion years that have passed since the LECA (Brinkmann and Philippe, 2007) has generated variations that are more apparent at the level of gene duplication than at the level of overall structure, a common example being the diversification of single-headed IAD heavy chains (Wickstead and Gull, 2007; Wilkes et al., 2008). However, diversification in the use of cilia appears to be largely the result of refinements of traits already common to these organelles prior to divergence from the LECA. These common uses include planar beating to generate fluid flow, ciliary membrane surface motility to produce particle transport (or gliding of single cells), sensory receptor display, and regulated adhesion, including adhesion-based signaling (Mitchell, 2007). Some of these traits have been lost when no longer needed, such as the loss of beating motility in organelles specialized for sensory functions.

3. Hypotheses of the Origin of Cilia Three hypotheses of the origin and subsequent evolution of the cilium have been proposed. The first, the symbiotic model, derives cilia from a spirochete bacterium (Margulis, 1981). However, no comparative genomic evidence has been found to support a spirochete ancestry of the ciliary proteome. Given this and the cell biological difficulties with such a symbiotic cell fusion model we and others think that this explanation is highly unlikely. The second hypothesis is autogenous origin of the centriole and cilium within the evolving eukaryotic cytoplasm (Cavalier-Smith, 1978; Je´kely and Arendt, 2006; Mitchell, 2004). There is little controversy that some aspects of the evolution of cilia are autogenous, in part because axonemes are built of microtubules mostly made of the same proteins as cytoplasmic microtubules, in part because of the commonality of molecular motors, and in part because of the relationship of IFT proteins to endocytic scaffold proteins and perhaps to nuclear pore proteins. This chapter will explore what these relationships are in detail and whether autogenous formation of the cilium can account for the whole of evolutionary history of the organelle. The third hypothesis, relatively new (Satir et al., 2007), assigns a supporting role to the developing eukaryotic cytoplasm. In this hypothesis, the cytoplasm plays host to an invading virus which becomes the primitive

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centriole. We begin by restating the main features of this sequence of ciliary evolution and the questions it poses. We then consider the pros and cons of this hypothesis, present an alternative autogenous scenario for centriole evolution, and discuss those features of ciliary evolution which are independent of the specific model of centriole origins.

4. The Viral Hypothesis of Ciliary Origin 4.1. Statement of the hypothesis The viral hypothesis (Fig. 3.2) suggests that the cilium evolves at a time after the invention of membrane trafficking in the protoeukaryotic cytoplasm. The sequence of events postulated is as follows. An RNA enveloped virus with ninefold symmetry invades the cell. The basal structure of the virus is the cartwheel, each spoke of which initiates a

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Figure 3.2 The viral hypothesis of ciliary evolution. (A) Membrane trafficking in the protoeukaryotic bacterial cytoplasm leads to endocytosis of cell organelles. An RNA enveloped virus with ninefold symmetry invades this cytoplasm. Cross section shows ninefold symmetry at the cartwheel. (B) The centriolar virus remains attached to the cell membrane and a sensory bulge capable of positional signaling develops. GPS function leads to restriction in organelle number and development of cell polarity. Cross section shows area of attachment at the ciliary necklace. (C) Elongation of the centriolar capsid, utilizing a- and b-tubulin, IFT and motor proteins of the host cell produces the 9 + 0 axoneme (cross section) in a sensory organelle. (D) Cytoplasmic dynein diversifies to give axonemal isoforms—arms between doublets in cross section— motility begins. (E) Misplaced cytoplasmic MTs give rise to the central pair leading to an increase in efficient motility in a stabilized 9 + 2 axoneme (cross section). Efficient signaling and motility in the 9 + 2 axoneme are advantageous for protistan survival and outcompete earlier forms. Images from Satir et al. (2007). Courtesy Cell Motil. Cytoskeleton.

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triplet microtubule. The virus disassembles with the nucleic acid integrating into the evolving eukaryotic cell nucleus. The virus reassembles in a cytoplasmic viral factory. Many capsids (protocentrioles) form at once. Originally RNA is reincorporated into each protocentriole, but eventually some capsids do not incorporate all or even part of the viral genome. The maturing virus then attaches to a specialized exocytic vesicle, which attachment site eventually gives rise to the ciliary necklace. The virus then becomes encapsulated by the cell membrane when it exits the cell. A mutation in the encapsulation process occurs so that the centriolar capsid remains attached to the cell membrane with an expanded membrane bulge above it. As IFT proteins and motors evolve within the cell, specific receptors become incorporated into the membrane in this bulge, which then becomes capable of positional signaling. This signaling is used by the cell for spatial identification of cellular axes, which in many, but not all, cells leads to restriction in centriolar number and/or to development of cell polarity. A second mutation permitting elongation of the centriolar capsid, utilizing a- and b-tubulin, probably manufactured by the host cell, together with IFT motors and proteins, produces a 9 + 0 sensory cilium, where the original protocentriole is now a true basal body. Elongation mutants are common in some viruses (e.g., bacteriophage—Kellenberger, 1965).

4.2. What the viral hypothesis can explain The viral hypothesis offers relatively easy explanation of several remarkable features of ciliary morphogenesis: 1. In many cells, basal body (centriole) formation is not templated as a mother–daughter relationship, but is explosive and de novo, with many basal bodies, sometimes hundreds, arising at once in a cytoplasmic factory (reminiscent of a viral factory). Some examples are anarchic oral field formation in ciliates (Bell et al., 2008), gametogenesis in Gingko or Marsilia (Mizukami and Gall, 1966), and ciliogenesis in mammalian respiratory epithelia. In the ameboflagellate Naegleria and similar genera, normally only two to four cilia form, but ciliogenesis is de novo (Fulton and Walsh, 1980). 2. Centriole assembly is stereotypic self-assembly with microtubules polymerizing on a basal plate (the cartwheel) (Salisbury, 2008), length regulation, defined ninefold symmetry and a single enantiomorphic form, all reminiscent of viral assembly. 3. Basal bodies contain unique tubulins, tektins, and ribbon proteins not present in cytoplasmic microtubules, permitting the formation of stable triplet and doublet microtubules. Similarly, all direct attachment of

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centrioles to membranes or membrane vesicles that fuse with the cell membrane involves a ciliary necklace (Satir et al., 1976). 4. There may be specific centriolar or basal body RNAs with sequences reminiscent of retroviruses sometimes found within the organelle (Alliegro et al., 2006). Although the centriolar genome is now completely or largely nuclear and dispersed, during morphogenesis of the organelle centriolar genes must be turned on as cassettes in specific sequences. 5. Ciliary axonemal microtubules of the nine doublets are the only microtubules that grow directly from basal body (centriolar) microtubules. Other cytoplasmic microtubules including spindle microtubules arise from g-tubulin-containing foci within pericentriolar centrosomal material (Salisbury, 2008). These observations imply that only the ciliary doublet microtubules are templated by the centriole, and not vice versa. 6. Without a centriole, there are no cilia, but there are microtubules, microtubule-containing cell extensions such as axopodia, and mitosis. In a particularly informative experiment, Basto et al. (2006) ablated centrioles in Drosophila cells. These observations uncouple centriole and cilia evolution from cell division, suggesting that centriolar duplication connected to the cell cycle is a secondary event.

4.3. Unusual predictions as a consequence of the viral hypothesis Two specific predictions of the viral hypothesis as stated are that: 1. The sensory cilium originates before the motile cilium. 2. The first ciliated cells were neither unikonts nor bikonts, but rather multiciliated, as might be expected from an abortive viral budding process. These predictions are quite different from those made from the autogenous hypothesis and might be testable via extensive genomic analysis of primitive protists.

4.4. Objections to the viral hypothesis Several objections that may be raised to the viral hypothesis are now discussed: 1. The basal body would be an unusually large and complicated virus. This is correct, and it might imply that the host cell which the virus invaded was unusually large for its time also. The initial invader need not be exactly like the modern centriole, but if the hypothesis is correct, all the essential features, especially ninefold symmetry, and probably the unique proteins involved in genesis of the centriole, would be present.

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2. There are no viruses with ninefold symmetry. This also appears to be largely true, although an artificial virus with ninefold symmetry has been constructed (Martin-Benito et al., 2001), which suggests that viruses with such symmetry could potentially exist. Of course, finding such a modern day virus, especially if some of its proteins were centriole-related, would provide a strong demonstration in favor of the viral origin hypothesis. 3. There are organisms whose gametes have large numbers of doublet microtubules that do not originate from a centriole (Phillips, 1974); alternatively there are gametes with less than nine doublets and minimal centrioles (Goldstein and Schrevel, 1982). These organisms are not primitive and have no general significance for early evolution, but rather show that the selection pressure on gametes results in the fixation or divergence from the ancestral state. 4. It should be possible to find more evidence of the ancient retroviral sequences in centriolar genes and more viral RNA in extant centrioles than has been reported. Maybe so, but retroviral sequences are found throughout the eukaryotic genome and tracing lineages in this way may be futile. It seems likely that RNA could be found in other centrioles, but its presence could be confined to specific times in development of the organism or to specific stages of centriologenesis. 5. There is no sequence similarity between centriolar proteins and viral capsid proteins (G. Je´kely, unpublished data). This is true, but our sampling of viral diversity is still scarce, and such viruses may one day be found.

5. The Autogenous Model for the Origin of Cilia 5.1. Autogenous origin of microtubule-organizing centers An alternative to the viral origin of basal bodies and cilia is an autogenous model. In this model, the basal body structure evolved from pre-existing cellular components via the process of gene duplication and divergence and the final ninefold symmetry is the result of a coevolutionary process to optimize cilium-based motility. According to this model, basal body structure and axonemal structure tightly coevolved from the very beginning, and therefore neither structure was templated on the other one. There is strong

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indication that the symmetry and structure of the basal body and the axoneme are tightly linked and if the axoneme is lost, the centriole is lost completely (e.g., flowering plants). The starting point according to this model was a cell already possessing a microtubule cytoskeleton, which regulated cell shape and served as a scaffold for the motility of intracellular membranes. (In the viral hypothesis, this is the host cell that is invaded by the centriolar virus.) The microtubule cytoskeleton could have been unpolarized initially. Basal body and cilium evolution started when the cell first acquired polarity by evolving a primitive microtubule-organizing center (MTOC) that served as the major site to nucleate microtubules (Fig. 3.3). Such MTOCs that also form the ends of the mitotic spindle must have evolved very early as structures needed for microtubule organization and cell polarity determination. In the autogenous model, the evolution of a cilium is assumed to have occurred through modification of a single MTOC to support assembly of a specialized set of microtubules that became the axoneme (Fig. 3.3). According to this model the need to anchor the axoneme drove evolution directly from a simpler MTOC that supported a simpler protoaxoneme into (eventually) a centriole–basal body system that serves dual purposes in axoneme assembly and in microtubule nucleation during mitosis. This dual-purpose MTOC evolved into the centrosome during animal evolution. This association of centrioles with a mitotic organizing function has been secondarily lost in multiciliated cells, including both protists such as Tetrahymena and vertebrate cells such as those of ciliated airway epithelia, where tens or hundreds of centrioles dock with the cell membrane to become the basal bodies of cilia, separated from sites of spindle assembly. Plausible scenarios exist for evolution of an axoneme from a unique organizing center that only later developed an association with the mitotic MTOC, but analysis of the evolution of centrin, a protein essential for spindle pole body duplication in yeasts and also important for basal body/centriole replication (Loncarek et al., 2008; Salisbury, 2007), provides support to the concept that basal bodies evolved linearly through modification of an ancestral mitotic MTOC (Bornens and Azimzadeh, 2007). A MTOC could have originated due to the self-organizational capacity of the microtubule system and associated molecular motors. One can conceive a scenario where the minus end of microtubules became focused by the action of dimeric minus end-directed motors (Surrey et al., 2001). Such foci could have nucleated further microtubules either on the locally concentrated microtubules (self-nucleation) or on an emerging MTOC material that could have accumulated at the foci as cargo carried by molecular motors. Self-nucleation and transport could have allowed the emergence of a robust MTOC via positive feedback loops (i.e., more localized microtubule nucleation leading to more localized transport of MTOC material). During further evolution, these MTOC became independent and were maintained also in the absence of microtubules. MTOCs evolved

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Figure 3.3 The autogenous hypothesis of the evolution of motile ciliary functions and IFT from coated vesicle transport. (A) The evolving eukaryotic cell possesses microtubules emanating from an organizing center (MTOC) and receptors randomly distributed on the cell surface. (B) Sensory receptor capping and directional transport lead to MT elongation and bundling. A specialized membrane patch (red) appears at the ends of elongated MT. (C) The bundled MTs form the primitive basal body (centriole) and a primitive axoneme. Vesicle transport motors evolve into IFT kinesin and dynein (green). The axoneme uses IFT motors for gliding motility, promoting selection of the polarized MT bundle/protoaxoneme. (D) Axonemal dynein isoforms evolve (red) that permit bending movement of the axoneme, which drives selection of doublet microtubules and of a centriole with membrane anchors. The 9 + 2 pattern became fixed at a later time based on its superior regulation of bending parameters. (E) The proto-IFT complex in a protoeukaryote mediated transport of transmembrane proteins (endocytosis and/or recycling) at a specialized region of the plasma membrane (shown in red). (F) Microtubule growth exposed this membrane domain to the environment. Transmembrane proteins were transported in vesicles, proteins of the developing axoneme arrived by diffusion. Nuclear pore complexes also diverged from a protocoatomer complex. (G) Bidirectional IFT in modern cilia with kinesin-II as the anterograde and dynein 1b/2 as the retrograde motor. The motors are thought to have evolved from the ones that moved coated vesicles. IFT cargo can be either transmembrane or cytoplasmic. During the subsequent stages of cilia evolution basal body structure coevolved with axonemal structure. (H), (I), and (J) Organization of coated vesicle complexes, nuclear pore complexes, and the IFT complex. Adapted from Mitchell (2004) and Je´kely and Arendt (2006).

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the ability to self-assemble either in a templated manner on a mother MTOC or independently. Mechanisms to coordinate MTOC duplication and cell division also evolved. At this stage g-tubulin diverged from cytoplasmic tubulin (following gene duplication) and specialized to nucleate microtubules in the MTOC. By regulating the microtubule skeleton and cell polarity, these MTOCs also became indispensable for the positioning of the nucleus and for cell division. The MTOC had no ninefold symmetry at this stage, and was similar to the pericentriolar material, being able to nucleate microtubules in all directions. During subsequent stages the MTOC acquired polarity, triplet microtubules, and the ability to nucleate the developing axonemal microtubules from one end only (Fig. 3.3C). This origin of centriolar complexity from a simpler MTOC is of course not necessary in the viral hypothesis. However, other examples of microtubules forming complex symmetrical structures are seen in the suctorian tentacle, where a sevenfold microtubule array develops (Tucker, 1974) and the heliozoan axopodium, where large 12-fold symmetric double-spiral helical microtubule arrays form (Tilney and Porter, 1965).

5.2. Autogenous origin of ninefold symmetric basal bodies and the axoneme Formation of triplet microtubules, from which axonemal doublets arise, requires both d- and e-tubulin (Dutcher, 2003), which are present in all organisms with motile cilia and flagella. In contrast to the viral hypothesis which assumes that these proteins were brought into the protoeukaryotic cell with the centriolar virus, the autogenous hypothesis assumes that these tubulin forms arose via gene duplication and divergence of pre-existing tubulin forms, most likely g-tubulin, within the nuclear genome (Dutcher, 2001). The forces that led to the evolution of these more complex microtubule structures are likely linked to the asymmetry provided by a doublet, which provides differentiation into a cargo-binding surface for permanent dynein attachment (the A-tubule), and a track for dynein-based movement (the B-tubule). Further development into a triplet may have more to do with the anchoring of basal bodies to withstand the forces of axonemal beating, since basal bodies in C. elegans, which lacks motile cilia, have only doublet microtubules (Dutcher, 2003), as do the basal bodies of the 9 + 0 sperm axonemes of a termite (Baccetti and Dallai, 1978), whose sperm are described as being ‘‘feebly motile.’’ Recent study of mutations that alter basal body structure and function in Chlamydomonas have revealed for the first time at least part of the basis of the ninefold symmetry of basal bodies. Two conserved gene products, BLD10 and SAS-6 in Chlamydomonas (homologs of previously identified metazoan centriolar proteins CEP135 and SAS-6) are both located in the cartwheel found at the proximal end of basal bodies and centrioles. BLD10

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immunolocalizes to the outer ends of the cartwheel spokes, at the point where they interact with the inner-most microtubule (A-tubule) of each basal body triplet (Hiraki et al., 2007). Particularly interesting is the observation that C-terminal truncations of BLD10 can partially rescue the null mutant, but 25% of such cells have basal bodies with a perfect cylinder of only eight instead of the normal nine triplets. While the hub of the cartwheel often appeared normal in these BLD10 △C basal bodies, and cartwheels retained nine spokes, the spokes were shorter than normal and failed to interact properly with the triplet A-tubules. In bld12 mutant cells (lacking SAS-6), cartwheel spokes are present and retain their association with the A-tubule of each triplet, but the central hub of the cartwheel is missing and the number of triplets varies from 7 to 12, with only 70% retaining the normal number of 9 (Nakazawa et al., 2007). Importantly, the basal bodies with abnormal numbers of triplets retain their cylindrical shape. Immunolocalization of SAS-6 indicates that it is near the center of the cartwheel at about the spoke-hub junction, consistent with work on its homolog in C. elegans (Pelletier et al., 2006). Thus basal bodies have a built in propensity for radial symmetry, and the properties of only one or two proteins appear to stabilize that symmetry at ninefold. In the autogenous model, what then has driven the evolution of ninefold symmetry? One hint may come from examination of the flagellar axonemes that assemble onto the abnormal basal bodies of bld12 mutant cells. Axonemes with fewer than nine doublets retain dynein arms and radial spokes, but have a central lumen that lacks any central pair apparatus, presumably because the central pair complex could not fit in the remaining space. Those with 10 or 11 doublets retain the central pair, but radial spokes from only about half of the doublets can make contact with this central apparatus. Clearly the current geometry of radial spokes and central apparatus, once established, acted to fix the number of doublets in the axoneme. Some nonmotile insect spermatozoa that naturally lack radial spokes, dynein arms and a central apparatus have 12 or 14 doublets, which are assembled from basal bodies of 12 or 14 triplet microtubules (Baccetti et al., 1973). Naturally occurring nonmotile axonemes with 10 doublets have also been reported in sensory cilia of a parasitic nematode (Ross, 1967). Thus there is nothing inherent in the architecture of a basal body that requires ninefold symmetry. Is ninefold symmetry linked to the evolution of motility? Some of the more divergent insect spermatozoa that retain weak motility have large numbers of doublet microtubules arranged in spiral rows (Baccetti et al., 1974b). These lack radial spokes or a central apparatus, and have a single row of dyneins. A few organisms have evolved simplified axonemes with only six (Kuriyama et al., 2005) or three (Prensier et al., 1980) doublets, and also lack radial spokes and a central apparatus. Therefore, many geometries can support motility, and cylindrical basal bodies can exist that have 10-, 12-, or 14-fold symmetry, rather than ninefold symmetry.

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There is, however, a unique aspect of the motility of axonemes with ninefold symmetric basal bodies. Such motile axonemes come in three forms, the most common is the 9 + 2 variety with an asymmetric central apparatus built on a platform of two singlet microtubules. The 9 + 2 pattern is present on virtually all somatic cilia of eukaryotes and on most sperm tails as well. The other varieties are radially symmetric 9 + 0 axonemes, that completely lack a central apparatus and may or may not possess radial spokes (Gibbons et al., 1983), and in rare cases, axonemes that retain radial spokes but have a radially symmetric central apparatus built from three microtubules (Baccetti et al., 1970, 1974a) or from nonmicrotubular structures (Baccetti, 1986). The sperm tail flagella of such organisms with radially symmetric axonemes have invariably shown a simplified helical motility pattern, rather than the more complex planar or three-dimensional beat patterns typical of organelles that retain a 9 + 2 architecture. The planar motility supported by an asymmetric central apparatus may have provided an evolutionary advantage that drove fixation of this structural geometry (Mitchell, 2004). An interesting possibility of why fixation of a 9 + 2 pattern might be advantageous is suggested by an early speculation by Astbury and Beighton (1955), consistent with the discovery of central pair twisting or rotation in certain protists. They proposed that just ‘‘nine circles can be arranged in a ring to enclose two others, . . . to allow the latter to ‘roll around’ inside’’ touching each of the peripheral circles (the spoke heads?) in turn. Clearly an axoneme with other geometries could serve as well for the other functions common to these organelles (surface gliding motility and sensory receptor display), but only those with a 9 + 2 geometry survived to become the ancestor of all extant eukaryotes. Based on these arguments, and consistent with the autogenous origin of the centriole and cilium, ninefold symmetry was fixed late during the evolution of eukaryotes, but still prior to the divergence from our LECA. These considerations support the notion that ninefold symmetry is not the historical consequence of the invasion of a host cell with a virus with ninefold symmetry, but is the result of a long evolutionary process to optimize motility and the regulation and pattern of axonemal beating.

6. Origin of Intraflagellar Transport and the Sensory Function of Cilia The evolutionary history of centriolar and axonemal components suggests that protoeukaryotes must have already possessed a dynamic cytoskeleton when cilium evolution started (Cavalier-Smith, 2002). In addition, the history of IFT suggests that this protoeukaryotic cell also contained a secretory endomembrane system that was shaped by vesicle coat complexes and molecular motors ( Je´kely and Arendt, 2006) (Fig. 3.3). Based on these

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we can infer that the protoeukaryotic cell that started to evolve a cilium was already rather developed, with dynamic membranes and cytoskeleton, and thus the acquisition of cilia is a relatively late event in the history of eukaryote evolution. Below we will discuss what the evolutionary history of IFT has revealed about cilium evolution. IFT machinery is nearly universally required for the assembly and maintenance of cilia (Rosenbaum and Witman, 2002). IFT is characterized by a bidirectional, microtubule motor-driven transport process between the ciliary axoneme and the ciliary membrane (Kozminski et al., 1993). A large multiprotein complex, the IFT complex, serves as a platform or raft that carries both membrane and axonemal proteins along the length of cilia, using kinesin and dynein motor proteins. Transport is bidirectional, ensuring a constant turnover of the ciliary proteome, including the IFT particles and the associated motor proteins as well. The core proteins of IFT show structural similarity to components of vesicle coats (COPI and clathrin) (Avidor-Reiss et al., 2004; Je´kely and Arendt, 2006). These proteins all have unique domain architecture, with an N-terminal b-propeller and a C-terminal a-solenoid. Besides, distant sequence similarity can also be detected between these proteins using sensitive sequence similarity searches. The vesicle coat proteins and the IFT complex all evolved from a primordial membrane curving complex, the protocoatomer, which also gave rise to the nuclear pore complex in the protoeukaryotic cell as illustrated in Fig. 3.3D (Devos et al., 2004). This deep homology between vesicle coats and IFT core proteins suggests that IFT evolved from an intracellular bidirectional coated vesicle transport process (Fig. 3.3E–J), most likely one operating between the plasma membrane and the evolving Golgi complex ( Je´kely and Arendt, 2006). This evolutionary scenario is also supported by the similar mechanism of membrane recruitment of vesicle coats and the IFT complex. In both cases members of the Ras-like small GTPase family play a critical role, by binding and recruiting the complexes in their active, GTP-bound form (Omori et al., 2008). This scenario provides interesting insights into early cilium evolution. It is conceivable that the transport process that was the evolutionary precursor of IFT delivered membrane proteins to a specialized region of the plasma membrane, perhaps delineated by the ciliary necklace. This transport could have occurred along the polarized microtubule cytoskeleton that was organized by the primitive MTOC (see above). Such localized transport could have created a membrane domain with a distinct composition and could have allowed the cell to localize its membrane receptors in one membrane domain (Fig. 3.3). This could have functioned as a ‘‘sensory membrane patch’’ that later protruded when microtubules polymerized from the MTOC and pushed the membrane out. Alternatively, the membrane domain produced by the precursor of IFT might have been a

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preferred site for centriolar virus attachment and budding. According to these models, the sensory function of cilia evolved first and motility evolved later to increase exposure of sensory receptors to the environment.

7. The Evolution of Ciliary Motility Regardless of whether centrioles and basal bodies evolved from a virus or via an autogenous mechanism, it is widely accepted that the ciliary axoneme evolved gradually and that ciliary motility was one of the driving forces. Motility provides an important selection mechanism, since a motile single-celled organism is far better able to find nutrients and to avoid predators and unfavorable conditions. Since primitive eukaryotes likely had actin and myosin and a well-developed amoeboid motility, the motility provided by a microtubule-based axoneme must have had specific advantages that drove its continued evolution. Gliding motility may have evolved first, through simple regulated coupling of microtubule motor-dependent transport to membrane adhesion sites (Fig. 3.3C). Many single-celled organisms move by flagellar surface gliding (Bloodgood, 1988; Chantangsi et al., 2008; Tamm, 1967), which can transport cells at up to 30 mm/s (Saito et al., 2003). Such flagellar surface gliding might have had several selective advantages over amoeboid motility, including inherent polarity of the microtubule track, close association of the motile machinery with polarized surface receptors to aid chemotactic signaling, and a higher transport rate than could be achieved by amoeboid movement. Thus development of gliding motility, based on a polarized microtubule bundle, cytoplasmic dynein as a retrograde motor, and a vesicular trafficking kinesin as an anterograde motor to return dynein to the tip, might have been a selective force that drove formation of a simple axoneme. The addition of bending motility to the repertoire of this protocilium might first be driven by the ability of bending to create currents in the fluid environment, increasing both nutrient exchange and the rate of capture of suspended particulates, as is commonly seen among benthic protists today. To produce bending motility within the cilium, cytoplasmic dynein diversifies into axonemal dynein, becomes stabilized as an arm capable of producing sliding between adjacent axonemal microtubules (Fig. 3.3D) and ultimately drives selection for doublet microtubules. In the alternative viral hypothesis, axonemal doublets arose by lengthening of the viral capsid (Fig. 3.2C). The axonemal dyneins then diversify through selection for greater motile efficiency. In a further step, microtubules polymerize independently of the basal body within the axoneme, giving rise first to a symmetric central apparatus, and finally to the asymmetric central pair, which permits planar bending and provides a further increase in motility

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regulation. Efficient signaling and regulated motility provided by the 9 + 2 axoneme are so advantageous for protistan survival that other early eukaryote forms are outcompeted and disappear.

ACKNOWLEDGMENTS We thank Michael Cammer and Ann Holland for help in preparing this manuscript.

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