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Cilia and flagella are eukaryotic machineries for cell motility propelled by the propagation
of bending waves. The internal cytoskeletal structures, called axonemes, are constructed
from 9+2 microtubules with axonemal dyneins and regulatory structures such as the
central apparatus and radial spokes 1]. These structures are well conserved in all eukaryotes except those that have lost
them during evolution. Ciliary and flagellar bend propagations are generated by propagation
of sliding of doublet microtubules by axonemal dyneins 2]-7]. The propulsive forces generated by bend propagation of cilia and flagella are considered
an adaptation for efficient movements by generating fluid flow in microenvironments
with low Reynolds numbers 8].

Motility of cilia and flagella is modulated by several extracellular stimuli to enable
directed and harmonious movement of cells and tissues. Ca²⁺ is an important factor for these modulations. Here, I first introduce the diversified
roles of Ca²⁺ in ciliary and flagellar motility over several eukaryotes and then focus on the Ca²⁺ sensors that directly regulate the motile machinery, the axonemes. In addition, I
present a phylogenetic analysis of Ca²⁺ sensors, demonstrating the evolution of Ca²⁺ sensors and proposing a pathway of eukaryotic evolution.

Cilia and flagella change their motility in response to Ca²⁺

Cilia and flagella respond to extracellular stimuli and change their motility. Ca²⁺ is a well-known intracellular regulator for modulation of ciliary and flagellar movements.
These modulations range across diverse modes, including (1) changes in ciliary or
flagellar waveforms, (2) rotation or reversal of the direction of ciliary or flagellar
bending, (3) arrest of beating, and (4) increasing of beat frequency (Figure 1).

Figure 1. Schematic drawings of various Ca²⁺-dependent changes in wave propagation of cilia and flagella and the direction of
locomotion and water flow in several organisms and tissues. Red dots in Ciona sperm and Chlamydomonas flagella indicate acrosomes and mating structure (fertilization tubules), respectively.
Black and gray arrows represent the direction of wave propagation and cell locomotion,
respectively.

Changes in ciliary or flagellar waveforms

Sperm swim with the tip of the head (acrosome) ahead of the direction of movement
for fertilization of the egg. Sperm of the ascidian Ciona intestinalis dramatically increase flagellar asymmetry in response to increases in intracellular
Ca²⁺ concentration caused by a chemoattractant from the egg 9],10]. This change enables the sperm to make turns and move forward towards the egg.

The unicellular alga Chlamydomonas reinhardtii has two flagella and usually swims in breast stroke fashion with the flagella located
anterior to the cell body. A structure for mating is formed between the two flagella
at fertilization 11],12]. When exposed to intense light, Chlamydomonas stops its motility and then moves in the reverse direction with conversion of flagella
into a symmetrical waveform 13]. Analysis with a demembranated cell model suggests that the conversion of flagellar
waveform from asymmetric to symmetric is caused by an increase in Ca²⁺ concentration. The increase of intracellular Ca²⁺ appears to be performed by Ca²⁺ influx through a voltage-dependent channel CAV2 14]. Similar flagellar response to Ca²⁺ is observed in the prasinophyte Spermatozopsis similis15].

Rotation or reversal of the direction of ciliary or flagellar bending

In Paramecium, Ca²⁺ causes reversal of the beating plane of cilia 16]-18]. Extracellular stimuli such as mechanical collision induce membrane depolarization
and subsequent Ca²⁺ influx, resulting in ciliary reversal and backward swimming. It is considered that
ciliary reversal in Paramecium cilia is caused by the rotation of the central pair in the axoneme 19],20]. Rotation through 180° causes complete reversal of the beating plane of the cilia.
In the case of Ctenophora, the ciliary comb plate also shows ciliary reversal in a
Ca²⁺-dependent manner 21]. However, these comb plate cilia perform reversal of the beating plane without rotation
of the central axoneme pair 22].

Trypanosoma propagate flagellar waves both from the base to tip and the tip to base 23],24]. Demembranated cell models demonstrate that the direction of flagellar bend propagation
reverses when the cell is demembranated by glycerol or detergent and is reactivated
by ATP at low concentrations of Ca²⁺ in the trypanosomatid Crithidia oncopelti25].

Sperm in some insects and snails reverse the direction of bend propagation in a Ca²⁺-dependent manner 26]-30]. For example, in the sperm of the gastropod Strombus luhuanus, the reversal of bend propagation appears to be involved in sperm release from the
sperm storage site in the female genital tract 30].

Arrest of beating

Epithelial cilia of marine invertebrates show ciliary arrest in response to Ca²⁺. Spontaneous arrest of mussel gill cilia is caused by membrane depolarization, depending
on calcium ions 31],32]. Most of the gill cilia in demembranated cell models show arrest of beating at 10^?4 M Ca²⁺33]. Ciliary arrest in Ciona stigmatal cells also depends on the presence of external Ca²⁺34]. Cilia of sea urchin embryos or larvae undergo a series of changes in the beating
pattern. Spontaneous ciliary arrest is observed in early stages of development; in
later stages, cilia show spontaneous reversal or arrest and increase in beat frequency.
In many cases, these changes are accelerated by the presence of the Ca²⁺ ionophore A23187 in seawater 35].

Increase in beat frequency

Ca²⁺ induces increased beat frequency in airway cilia in mammals 36]-38] and in oviductal cilia 39], without alteration of beating direction. Increase in beat frequency is also observed
in the Triton-extracted Paramecium model and is inhibited by a calmodulin (CaM) antagonist 18]. However, sperm flagella show no significant increase in beat frequency due to Ca²⁺ in sea urchin 2] or Ciona (Mizuno and Inaba, unpublished observation), although a demembranated model of sea
urchin sperm flagella changed to an asymmetric waveform on stimulation with Ca²⁺, and showed quiescence at Ca²⁺ concentration 10^?4 M 40].

The effects of Ca²⁺ on ciliary and flagellar motility appear diverse among organisms, but the roles of
Ca²⁺ in the regulation can be classified into two parts. One is a signaling pathway upstream
of the modulation of the axonemes. Influx of Ca²⁺ is an important trigger for the modulation of ciliary and flagellar motility. Several
Ca²⁺ channels and Ca²⁺-binding enzymes, such as protein kinases and phosphatases, have been reported to
be localized and functional in the ciliary/flagellar plasma membrane and the ciliary/flagellar
matrix 6],7]. The other is the direct modulation of axonemal movements. Ca²⁺-binding proteins such as calaxin, dynein light chain 4 (LC4), CaM, and centrin are
bound to the substructures of the axonemes and directly modulate dyneins or their
regulatory elements, the radial spokes, and central apparatus. In this paper, I focus
on the Ca²⁺ sensors that directly act on the outer arm dynein in the axonemes.

Outer arm dynein is essential for Ca²⁺-mediated changes of ciliary movement

The extent of flagellar or ciliary bending correlates with the velocity of microtubule
sliding 41],42]. The flagellar waveform is composed of a bend with a larger angle (principal bend)
and an opposite bend with a smaller angle (reverse bend) 2]. Formation of bends and propagation are achieved by local microtubule sliding, for
which dyneins are considered to be locally activated on one side to bend the axoneme,
while those on the other side are inactive 2],43].

The central apparatus (CP) – along with the radial spokes (RS) – plays an important
role in flagellar motility as revealed by the paralysis of Chlamydomonas CP mutants 44],45]. The CP is involved in determining the bending plane, demonstrated by the helical
movement with 9+0 axonemal structures of eel and Asian horseshoe crab sperm 46],47], and the loss of planar bend movement and the development of helical movement after
treatment of a sperm model by antibodies against radial spokes 48]. The activation of specific axonemal dyneins by CP/RS is thought to enable mutual
sliding of microtubules across the axoneme, resulting in planar bend propagation 49]-51]. Studies on Chlamydomonas flagella have shown that signals from the central apparatus activate specific dyneins
for local bending 45],52]. As previously reported, the f (I1) inner arm dynein is regulated by phosphorylation/dephosphorylation
of a 138 kDa intermediate chain (IC) through a kinase/phosphatase system present in
the RS and CP 53],54].

Axonemes have two dynein motors with different properties: outer arm dynein and inner
arm dynein. Subunits of the outer arm dynein have been well studied in Chlamydomonas and in the sperm of Ciona and sea urchins 7],55]-59]. They have two or three motor subunits (heavy chains) in the sperm or Chlamydomonas, respectively. Other subunits, including intermediate chains and light chains, are
involved in the assembly and regulation of dyneins. Several studies with Chlamydomonas mutants and outer arm extracted sea urchin sperm indicate that outer and inner arm
dyneins are involved in the elevation of microtubule sliding velocity (increasing
beat frequency) and formation and propagation of flagellar bending, respectively 3],4].

Much experimental evidence demonstrates that outer arm dynein is essential for Ca²⁺-dependent modulation of ciliary motility. The conversion of flagellar wavelength
from symmetric to asymmetric is transiently observed during chemotaxis of the sperm
to the egg 9],60],61]. This is caused by Ca²⁺-dependent regulation of outer arm dynein (see below). Lack of outer arm dynein in
the human sperm causes low swimming velocity, loss of circular movement with an asymmetric
waveform, and low efficiency of penetration into the egg coat 62],63].

Chlamydomonas changes swimming direction in response to light. There are two types of response:
a photophobic reaction to very strong light, photoshock, and a positively or negatively
directed movement towards a light source, phototaxis. Both photoshock and phototaxis
depend on changes in intracellular Ca²⁺. Reactivated Chlamydomonas axonemes show an asymmetric beat pattern at Ca²⁺ concentrations below 10^?6 M, become quiescent at 10^?5 M, and then resume beating with a symmetric waveform at 10^?4 M 64]. This waveform conversion does not occur in mutants lacking dynein outer arms 58],59],65]. In contrast, phototaxis is caused by different responses of the cis- and trans-flagellum.
The cis- and trans- flagellar axonemes of demembranated Chlamydomonas cell models differentially respond to Ca²⁺ concentration in the range 10^?8 M to 10^?6 M 57]. Studies using axonemal dynein mutants indicate that phototaxis requires the inner,
but not the outer, row of dynein arms 58],59].

Specific knockdown of outer arm dynein LC1 in Trypanosoma brucei results in the loss of tip to base propulsive propagation of the flagellar wave 66] that is usually observed in normal forward swimming. A similar phenotype is obtained
when LC2 is knocked down 67]. The tip to base propagation is Ca²⁺-dependent, and the base to tip propagation is only observed in demembranated models
when demembranated and reactivated in the presence of EGTA 25]. RNAi knockdown of LC1 in the planarian Schmidtea mediterranea demonstrated that outer arm dynein is essential for the increase of beat frequency
and coordination of cilia to produce ciliary oscillation with metachronal waves 68].

Calaxin is the calcium sensor of outer arm dynein necessary for chemotactic turns
of the sperm with asymmetric waveforms

Changes in ciliary and flagellar motility by Ca²⁺ are mediated by Ca²⁺-binding proteins. The most common motif for Ca²⁺ binding is the EF hand. It is a helix-loop-helix structural motif of 12 residues
(+X)x(+Y)x(+Z)x(?Y)x(?X)xx(?Z) for metal coordination, where +X, +Y, +Z and ?X, ?Y,
?Z are the vertices of an octahedron 69]-71]. The EF hand family contains the CTER, CRP, and S100 subfamilies. These three show
mutual congruence to one another within a subfamily. There are many other subfamilies
containing EF hands with no strong congruence to one another (Table 1) 72]. Both CTER and CRP basically contain four EF hands, at least one of which lacks the
capacity to bind Ca²⁺ in CRP and does not match the consensus sequence in a PROSITE search (Figure 2A). CTER subfamily proteins, such as CaM, centrin, and troponin C, have dumbbell shape
structures with two globular lobes connected by an eight-turn ?-helix, whereas CRP,
such as recoverin and NCS-1 (frequenin), have a globular structure without the long
?-helix link (Figure 2B) 73].

Table 1. Classification of EF-hand Proteins

Figure 2. Structures of EF-hand Ca²⁺-binding proteins. (A) Domain structures of Ciona and Chlamydomonas Ca²⁺-sensors, drawn based on SMART searches (http://smart.embl-heidelberg.de/). The length of each protein and the positions of EF hand motifs are scaled below.
(B) Molecular models of ligand-unbound Ciona centrin and NCS-1, built using SWISS-MODEL (http://swissmodel.expasy.org) 175]. Templates used are 1tnx.1 (skeletal muscle troponin) and 2d8n.1 (human recoverin)
for Ciona centrin and NCS-1, respectively.

Many studies showed that CaM is an important Ca²⁺ sensor for the regulation of ciliary and flagellar movements 74],75]. Although CaM was a strong candidate to be the Ca²⁺-dependent regulator for outer arm dynein in the sperm, several experiments suggest
the presence of Ca²⁺-binding proteins other than CaM. Unlike the light chain 4 (LC4) in Chlamydomonas, the outer dynein could not be isolated from sperm flagella in association with any
Ca²⁺-binding proteins. Moreover, conversion into an asymmetric flagellar waveform is achieved
at high concentrations of Ca²⁺ in the sea urchin sperm model demembranated by Triton X-100 in the presence of millimolar
Ca²⁺2],40]. In this condition, CaM is extracted from the axonemes. These reactivated sperm models
called ‘potentially symmetric’ sperm show symmetric waveforms at low concentrations
of Ca²⁺ but become asymmetric when Ca²⁺ is increased in the reactivation medium. The asymmetric flagellar waveform is seen
only in the presence of high concentrations of ATP 40], which induces motility with high beat frequency and therefore implies a role of
outer arm dynein.

Ca²⁺-dependent conversion of flagellar waveform is essential for sperm chemotaxis 9],10],60],76]-79] and rheotaxis 80], response of sea urchin sperm to mechanical stimuli 81], self-nonself recognition of sperm 82], hyperactivation 83],84], and release from the epithelium of sperm storage sites 85],86]. In the ascidian Ciona intestinalis, correlation between the increase in intracellular Ca²⁺ concentration and conversion of flagellar asymmetry is clearly observed 9]. Ciona sperm show rather planar wave propagation in seawater with a slight asymmetric flagellar
waveform, resulting in a circular trajectory. The reception of the gradient of chemoattractant
(sperm activating and attracting factor; SAAF) from the egg 87] induced a transient increase in intracellular Ca²⁺ concentration. Flagellar axonemes respond to the change and temporarily form and
propagate an asymmetric waveform, resulting in a turning movement towards the egg
9].

A previous study found a Ca²⁺-binding protein that is expressed in Ciona testis during the course of extensive descriptions of axonemal proteins 88]. It turned out that this protein is an axonemal protein localized at the outer arm
dynein, named Ca²⁺-binding axonemal protein calaxin 89]. Calaxin is grouped into one of the CRP EF hand protein families, the neuronal calcium
sensor (NCS) protein family, which is expressed in retinal photoreceptors or neurons
and neuroendocrine cells 90],91]. A phylogenetic analysis shows that calaxin is a new type of NCS protein in the axoneme;
other proteins, such as CaM and centrin, are all grouped into different phylogenetic
clades (Figure 3A).

Figure 3. Calaxin is an opisthokont-specific Ca²⁺sensor. (A) A phylogenetic tree of Ca²⁺-binding proteins in the ascidian Ciona intestinalis. Proteins were aligned by CLUSTALW, and the tree was constructed by MEGA5. Ciona parvalbumin-like protein (XP_002129217) was used as the outgroup. The value shown
on each branch represents the number of times that a node was supported in 1,000 bootstrap
pseudo-replications. Accession numbers or NCBI reference sequence numbers of the sequence
resources are as follows: calmodulin (AB076905), calaxin (AB079059), centrin (XP_004227465),
troponin C (XP_002129347), NCS-1 (XP_002126443), hippocalcin (XP_002124848), KChIP
(XP_004226075), calcineurin B subunit (CNB) (XP_002130765). (B) Multiple alignment of calaxin in opisthokont species. Asterisks, colons, or dots
indicate identical residues in all sequences in the alignment, conserved substitutions,
or semi-conserved substitutions, respectively. The amino acid residues identical to
Ciona calaxin or to calaxin in other organisms are in red or blue, respectively. The sources
of amino acid sequences are as follows: human calaxin (NP_078869), mouse calaxin (NP_080045),
Ciona calaxin (AB079059), oyster calaxin (EKC38288), sponge calaxin (XP_003383675), and
chytrid fungus calaxin (XP_006677085).

Calaxin has three Ca²⁺-binding EF hand motifs (amino acids 62 to 90, 98 to 126, and 151 to 166 in Ciona calaxin) 10],89]. Ca²⁺-binding to these sites was directly demonstrated by isothermal titration calorimetry
(ITC), showing a three-site sequential binding model 10]. Two of the three EF hand motifs exhibited endothermic binding and the other exothermic
binding. Ca²⁺-dependent hydrophobic interactions are suggested from positive enthalpy in ITC, as
in the case of Ca²⁺ binding to calmodulin 92]. Several investigations demonstrate membrane-associated roles of NCS in the modulation
of neurotransmitter release, biosynthesis of polyphosphoinositides, and in the direct
regulation of ion channels 93],94]. In fact, the N-termini of NCS proteins are myristoylated and become exposed outside
the protein molecules by binding of Ca²⁺, allowing them to associate with membranes. The consensus sequence for myristoylation,
N-terminal GXXXSX 94], is found in mammalian NCS-1 and calcineurin B. However, it is not present in calaxin
or its mammalian orthologs 89], suggesting that the N-terminal is not myristoylated and that calaxin does not have
the Ca²⁺-myristoyl switch property of NCS. Immunohistochemical observations indicate that
calaxin is located on the outer arm dyneins along the axoneme of sperm flagella 89]. Calaxin is also distributed in the cilia of ciliated tissues, such as the branchial
basket and endostyle 84]. Far western blotting shows that calaxin binds to ?-tubulin in the absence of Ca²⁺ and to the ? heavy chain (ortholog of the Chlamydomonas ? heavy chain) of the outer arm dynein 89]^a. Calaxin binds to the N-terminal stem region, as revealed by far-western blotting
against UV-cleaved fragments of the ? heavy chain (Mizuno and Inaba, unpublished data).
Although two IQ consensus motifs for binding CaM-like proteins are located within
the stem domain of the Chlamydomonas ? heavy chain 95], there is no such motif in the corresponding region of the Ciona ? heavy chain.

Ciona sperm shows a unique turning movement associated with a flagellar change to asymmetric
waveforms, followed by straight-ahead movement towards the chemoattractant SAAF 87]. In the presence of an NCS inhibitor, repaglinide, the sperm do not exhibit this
unique turning movement, showing less-effective chemotaxis 10]. Repaglinide-treated sperm can transiently form asymmetric flagellar waveforms in
the gradient of chemoattractant. However, they do not sustain the asymmetric waveform
and rapidly return to a symmetric form, resulting in less chemotactic behavior. The
flagellar waveforms of sperm demembranated with 0.04% Triton X-100 become more asymmetric
when reactivated at 10^?6 M Ca²⁺. Repaglinide attenuates propagation of asymmetric waveforms, but not the relatively
symmetric waveforms seen at low concentrations of Ca²⁺. Calaxin directly suppresses the velocity of microtubule sliding by outer arm dynein
at high Ca²⁺ concentrations. Repaglinide and anti-calaxin antibody cancel the suppression of microtubule
translocation at high concentrations of Ca²⁺. All of these data demonstrate that calaxin plays an essential role in the propagation
of asymmetric flagellar bending by suppression of dynein-driven microtubule sliding
at high concentration of Ca²⁺10]. Calaxin appears evenly located to every doublet microtubule 89]. Then, how does calaxin work to propagate an asymmetric planar waveform, in which
dyneins on the two sides of axonemes mainly participate in microtubule sliding? Although
there has not been any experimental evidence to elucidate this question, the function
of calaxin might be regulated through a mechanical feedback, such as thrust from flagellar
bending, or through a biochemical mechanism, such as protein phosphorylation and dephosphorylation.

BLASTP searches for Ciona calaxin in the genomes of Chlamydomonas reinhardtii and Paramecium tetraurelia hit hypothetical proteins CHLREDRAFT_119565 (XP_001696107) (E = 4e^?13) and XP_001433234 (E = 2e^?15), respectively. Both hypothetical proteins show a best match with calcineurin subunit
B type 1-like protein (CBL-1), not calaxin, in the Ciona genome. LC4 is a Ca²⁺-binding subunit of outer arm dynein first identified in Chlamydomonas96]. It shows sequence similarity to CaM and CaM-related proteins such as centrin/caltractin
and troponin C. Ca²⁺-binding assays demonstrate that LC4 has at least one functional Ca²⁺-binding site. LC4 is isolated in association with the ? heavy chain of outer arm
dynein. These properties suggest functions of LC4 analogous to those of calaxin, although
the proteins are phylogenetically distinct from each other.

Calaxin is an opisthokont-innovated calcium sensor in cilia and flagella

The current view of eukaryote phylogeny includes its basal division into unikonts
(Opisthokonts and Amoebozoa) and bikonts (Archaeplastida, Hacrobia, Stramenopiles,
Alveolates, Rhizaria, and Excavata), based on the concept of eukaryotic cells with
a single flagellum or two flagella, respectively. Opisthokonts are groups shown to
propel cells by a posterior flagellum 97]-99]. Homologs of calaxin were searched in available genome databases. Calaxin homologs
were not found in any bikont species, such as Archaeplastida (Chlamydomonas) or Stramenopiles (ciliates, dinoflagellates, and blown algae). Calaxin homologs
were only found, and were well conserved, in species of the opisthokont supergroup,
including Homo sapiens, Mus musculus, Ciona intestinalis, Strongylocentrotus purpuratus, Amphimedon queenslandica, Drosophila melanogaster, Monosiga brevicollis, and Crassostrea gigas. The opisthokont organisms that lack motile cilia or flagella throughout their life
cycles, such as C. elegans, Vericrustaceans (except Notostraca and Thecostraca), yeast, and higher fungi show
no calaxin gene in their genomes, although these organisms have genes for other NCSs
such as NCS-1 (frequenin). The chytrid fungus Batrachochytrium dendrobatidis, grouped into the opisthokonta with metazoa, contains a calaxin gene (XP_006677085)
in its genome. The calaxin of B. dendrobatidis shares 38% amino acid identity with Ciona calaxin (Figure 3B). Because of insufficient genome information, the presence of calaxin in Amoebozoa
has not been elucidated. BLASTP searches show that calaxin is not present in either
the aflagellate amoebozoan Dictyostelium discoideum or the flagellated amoebozoid Breviata anathema which lacks outer arm dynein 100]. However, one of the well-investigated genera in the Amoebozoa, Physarum polycephalum, has a flagellated period in its life cycle. Because it possesses an axoneme of 9+2
structure with outer arm dynein 101]-103], it is possible that calaxin could be present in Amoebozoa and could be a unikont-innovated
protein.

A previous study identified proteins with a unique combination of domains: an intermediate
chain of outer arm dynein, thioredoxin domain and nucleoside diphosphate kinase domain
(TNDK-IC, 104],105]) and a radial spoke protein CMUB116 (IQ motif and ubiquitin domain 106]). These proteins are also opisthokont-specific proteins, suggesting that a critical
evolutionary event occurred during specification of axonemes in the opisthokont lineage.

Mirror-image relationship between calaxin and LC4

Knowledge of the molecular components of axonemal dyneins and the molecular mechanism
of ciliary and flagellar motility has been accumulated mostly from metazoan sperm
and certain protists such as Chlamydomonas. In the present study, an attempt has been made to biochemically compare the outer
arm dynein and its Ca²⁺ sensor between Ciona sperm flagella and Chlamydomonas flagella and to correlate their functions in the regulation of motility.

The outer arm dynein of Ciona sperm flagella consists of two heavy chains and represents a two-headed structure,
but that of Chlamydomonas flagella consists of three heavy chains with a three-headed structure. Each of the
two heavy chains of sperm outer arm dynein is known to have distinct properties 107]-110]. Sea urchin ? heavy chain (an ortholog of Ciona ? and Chlamydomonas ? heavy chains) mediates structural and rigor binding to the microtubules 110]. In vitro motility assays indicate that the absence of the Chlamydomonas ? heavy chain increases both microtubule gliding and ATPase activity 111], suggesting that the ? heavy chain suppresses the activities of outer arm dynein.

Ciona calaxin and Chlamydomonas LC4 bind to Ciona ? and Chlamydomonas ? heavy chains, respectively 89],112]. However, Ca²⁺ dependency of the binding is reversed between Ciona and Chlamydomonas (Figure 4). Calaxin binds to intermediate chain 2 (IC2) and ? tubulin in the absence of Ca²⁺ but becomes associated with the ? heavy chain at higher concentrations of Ca²⁺89]. The binding of calaxin to the heavy chain results in the suppression of microtubule-gliding
activity by outer arm dynein 10]. In the case of Chlamydomonas, LC4 is bound to the ? heavy chain in the absence of Ca²⁺ but becomes newly tethered to IC1 (an ortholog of Ciona IC2) in the presence of Ca²⁺95],112]. Although the effect of Ca²⁺ binding to LC4 on dynein-driven microtubule sliding has not been examined in Chlamydomonas, the binding of Ca²⁺ to LC4 induces activation of ATPase activity of the outer arm dynein in the mutant
lacking the ? heavy chain 112]. A model has been proposed for Ca²⁺-dependent regulation of the ? heavy chain; in the absence of Ca²⁺, LC4 is tightly bound to the ? HC, resulting in inefficient formation of a rigor
bond with microtubules. In the presence of high Ca²⁺, Ca²⁺-bound LC4 detaches from the IQ region of the ? heavy chain and becomes attached to
IC1, resulting in a structural change of the N-terminal stem domain and the activation
of motor activity 95].

Figure 4. Mirror image in the function of outer arm dynein Ca²⁺sensors betweenCionaandChlamydomonas.Ciona calaxin binds to the ?-heavy chain, suppresses microtubule-sliding and induces propagation
of an asymmetric waveform at high concentration of Ca²⁺. In contrast, Chlamydomonas LC4 binds to the ?-heavy chain, becomes tethered to IC1 and induces propagation of
a symmetric waveform at high concentration of Ca²⁺. Direct evidence for the activation of microtubule-sliding by Chlamydomonas outer arm dynein has not been obtained.

These mirror-image relationships in the effect of Ca²⁺ on the regulation of outer arm dynein in Ciona and Chlamydomonas are likely to connect with the difference in the changes of flagellar waveforms (Table 2). At high concentrations of intracellular Ca²⁺, Ciona sperm show asymmetric waveforms whereas Chlamydomonas flagella become symmetric. The molecular mechanisms of Ca²⁺-dependent regulation of outer arm dynein appear quite similar to each other, but
the response to Ca²⁺ in the conversion of flagellar waveforms is completely reversed. This implies the
possibility of an evolutionary event in the functional diversification of cilia and
flagella at the onset of eukaryotic radiation.

Table 2. Comparison of Ca²⁺-dependent regulation of outer arm dynein betweenCionasperm flagella andChlamydomonasflagella

It is unlikely that ciliary response in waveform conversion depends on the extracellular
Ca²⁺ concentration in the environment (such as in seawater or freshwater). For example,
sperm of freshwater fish show asymmetric waveforms depending on an increase in intracellular
Ca²⁺ concentration 113],114]. The marine alga Pyramimonas parkae shows waveform conversion similar to Chlamydomonas reinhardtii115], although the relationship between the conversion and intracellular Ca²⁺ concentration has not been elucidated. An interesting experiment was the examination
of the relationship between intracellular Ca²⁺ concentration and flagellar waveform in the prasinophyte algae Pterosperma and Cymbomonas, both of which show conversion of flagellar waveforms similar to metazoan sperm:
symmetric flagellar waveforms in normal swimming and asymmetric waveforms when they
change swimming direction 115]. Anterior flagella of Stramenopiles bear hair-like structures called mastigonemes
116]. These organisms or their gametes normally swim with the anterior flagellum ahead.
The flagella show symmetric wave propagation from base to tip, but the direction of
propulsive force changes because of the reversal of water current by mastigonemes
117]. They change swimming direction in phototactic behavior by altering the flagellar
waveform or the orientation of the anterior or posterior flagellum 118], but the relationship between waveform change and intracellular Ca²⁺ is unclear.

Usage of distinct Ca²⁺ sensors in unikont and bikont supergroups

A phylogenetic analysis of Ciona calaxin, CaM, centrin, NCS, calcineurin B-subunit (CN-B), Chlamydomonas LC4, and a Ca²⁺-binding subunit of outer arm dynein docking complex 3 (DC3) 119],120] using available genome information resulted in distinct distribution of calaxin and
LC4/DC3 in the opisthokont and bikont supergroups, respectively (Figure 5). Chlamydomonas LC4 and its orthologs were grouped into a clade different from that of calaxin but
were more closely related to calaxin than were CaM or centrin. BLASTP searches of
Chlamydomonas LC4 against genomes of bikonts resulted in finding orthologs in flagellated species
including ciliates, dinoflagellates, diatoms, brown algae, haptophytes, and cryptophytes.
Exceptions are seen in organisms lacking outer arm dynein such as angiosperm, moss,
and fern 121]. BLASTP searches of Chlamydomonas LC4 against these species resulted in best hits to CaM. Search of Chlamydomonas LC4 in the genomes of opisthokonts failed to find any homologs in this supergroup.
For example, the protein most homologous to LC4 in Ciona intestinalis was CaM (E = 3e^?22).

Figure 5. Phylogenetic analysis of Ca²⁺-binding proteins. Proteins were aligned by CLUSTALW, and the tree was constructed by MEGA5. Ciona parvalbumin-like protein (XP_002129217) was used as the outgroup. The value shown
on each branch represents the number of times that a node was supported in 1,000 bootstrap
pseudo-replications. Sequences were obtained from the organisms Ciona (Ciona intestinalis), human (Homo sapiens), fungus (Batrachochytrium dendrobatidis), Naegleria (Naegleria gruberi), Euglena (Euglena gracilis), Trypanosoma (Trypanosoma cruzi or T. brucei), Giardia (Giardia intestinalis or G. lamblia), Trichomonas (Trichomonas vaginalis), Chlamydomonas (Chlamydomonas reinhardtii), Paramecium (Paramecium tetraurelia), and Ectocarpus (Ectocarpus siliculosus). The sources of amino acid sequences are as follows: Ciona calmodulin (AB076905), Ciona calaxin (AB079059), Ciona centrin (XP_004227465), Ciona NCS-1 (XP_002126443), Ciona CNB (XP_002130765); human CaM (CAA36839), human calaxin (NP_078869), human NCS1 (NP_055101),
human CNB (NP_000936), human centrin (NP_004057); chytrid fungus calaxin (XP_006677085),
chytrid fungus CaM (XP_006678916), chytrid fungus centrin (XP_006682970), chytrid
fungus NCS1 (XP_006675998), chytrid fungus CNB (XP_006677028); Naegleria CaM (XP_002683533), Naegleria centrin (XP_002678269); Trypanosoma CaM (XP_805243), Trypanosoma centrin (XP_805423), Trypanosoma calflagin (Q26680); Euglena CaM (P11118), Euglena centrin (AGS09408); Giardia CaM (XP_001705820), Giardia centrin (XP_001707577), Giardia LC4 (XP_001705117); Trichomonas CaM (XP_001326924), Trichomonas centrin (CAB55607), Trichomonas CNB (XP_002680632); Paramecium CaM (XP_001448363), Paramecium LC4 (XP_001442002), Paramecium centrin (XP_001347281), Paramecium DC3 (XP_001444482); Ectocarpus LC4 (CBN80105), Ectocarpus CaM (CBN74265), Ectocarpus centrin (CBN79657), Ectocarpus DC3 (CBJ30770). The protein sequences with specific accession numbers were obtained
from DDBJ/EMBL/GenBank, or from genome browsers with the following URLs: Chlamydomonashttp://genome.jgi-psf.org/Chlre4/Chlre4.home.html; Parameciumhttp://paramecium.cgm.cnrs-gif.fr; Naegleriahttp://genome.jgi-psf.org/Naegr1/Naegr1.home.html; Trichomonashttp://trichdb.org; and Trypanosomahttps://www.sanger.ac.uk/resources/downloads/protozoa/trypanosoma-brucei.html.

DC3 is also a CaM type of EF hand protein localized on the outer arm dynein docking
complex and shows redox-sensitive Ca²⁺-binding with a ratio of 1 mol Ca²⁺/mol protein 120]. However, it is unclear whether DC3 actually binds Ca²⁺ under physiological conditions because it also significantly binds Mg²⁺122]. Genes of DC3 homologs are present in Bikonta such as Stramenopiles (ciliates, brown
algae, and Plasmodium) and Cryptophytes but could not be found in the Ciona or human genomes. DC3 grouped into a clade closer than LC4 to CNB/calaxin/NCS (Figure 5). Intriguingly, BLASTP searches using recent genomic information on the chlorarachniophyte
Bigelowiella natans did not detect orthologs of Chlamydomonas LC4 or DC3. The protein with highest similarity was CaM (ID 54077), although ultrastructural
observation of the flagella clearly shows the presence of outer arm dynein 123]. LC4 was also absent from Plasmodium (Apicomplexa).

Both CN-B and NCS have been found in animals and fungi 124] but do not appear in plants. In plants, the CNB-like protein (CBL) family represents
a unique group of calcium sensors and plays a key role in intracellular Ca²⁺ signaling 124]. CNB-like proteins in plants are most closely related to CNB and NCS proteins in
animals and fungi (Figure 5). Proteins in Chlamydomonas (ID391130) and in Paramecium (GSPATP9660001) are grouped with CNB-like protein. Separation of these proteins from
the CNB group is supported by the bootstrap value (986/1,000).

Figure 6. Phylogenetic analysis of homologs of Ca²⁺sensor proteins in Excavata. Proteins (EF-hand proteins, length less than 350 amino acids) were searched against
genomes of each excavate by BLASTP and those with E-value e^?9 were aligned with Ciona or Chlamydomonas Ca²⁺-sensors by CLUSTALW. An unrooted tree was drawn by MEGA5. Branches of each Ca²⁺-sensor are highlighted by colors. The protein sequences (with accession numbers indicated)
were obtained from DDBJ/EMBL/GenBank, or from the genome browsers shown in the legend
of Figure 5.

The supergroup Excavata includes eight taxa 125]-128]. Phylogenetic analysis supports the monophyly of the Excavata 128] which consists of two major groups, Discoba and Metamonada. An additional organism,
Malawimonas, may also be included as a genus in the Excavata. Discoba includes four
phyla, Jakobida, Euglenozoa (for example, Euglena, Trypanosoma), Heterolobosea (for example, Naegleria), and Tsukubamonadida. The Metamonada includes amitochondriate flagellate Fornicata
(for example, Giardia), Parabasalids (for example, Trichomonas), and Preaxostyla 126]. Although Excavata are often considered the extant organisms closest to the ancient
eukaryotes, there are debates concerning their phylogenetic position.

Analysis of Ca²⁺ sensors in Excavata leads to an interesting point of view concerning the evolution
of Ca²⁺ sensor proteins (Figures 5, 6 and 7). First, both Giardia lamblia (XP_001705117) and Naegleria gruberi (ID 70962) contain clear orthologs of Chlamydomonas LC4 (Figure 5). Second, Naegleria has clear orthologs of NCS-1 and CNB (Figure 5). Third, several excavate species have multiple proteins with similarity to CNB,
NCS-1, LC4, or DC3 (Figure 6), although they could only be grouped into each Ca²⁺ sensor family with weak bootstrap support. Euglena has three DC3-like proteins. Naegleria has a LC4-like protein. Trypanosoma Tb10707970 is a CNB-like protein. Trichomonas has three NCS-1-like proteins. There are other proteins in Trichomonas, Naegleria, and Euglena that are similar to, but could not be grouped with, any ciliary Ca²⁺ sensors (Figures 6 and 7). These features of Ca²⁺ sensors or their homologs in Excavata suggest that duplication and divergence of
Ca²⁺ sensors occurred in this supergroup.

Figure 7. Distribution of Ca²⁺sensor proteins in eukaryotes. Based on the BLASTP search and the phylogenetic analyses in Figures 5 and 6, occurrence of each Ca²⁺ sensor in eukaryotic groups is summarized. Occurrence is indicated in the same colors
as used in Figures 5 and 6. Closed circles in a specific color represent an occurrence of homologs with weak
bootstrap support.

Figure 8. Structure of outer arm dynein and its Ca²⁺sensor across eukaryotic groups. (A) Schematic representation of the number of dynein heavy chains and the morphology
of outer arm dyneins observed by electron microscopy. Chlamydomonas outer arm dynein is composed of three heavy chains, ?, ?, and ?. Ciona outer arm dynein has two heavy chains homologous to the Chlamydomonas ? and ? chains. The ? and ? heavy chains in Ciona and the ? and ? heavy chains in sea urchin correspond to Chlamydomonas ? and ?, respectively. ODA, outer arm dynein; IDA, inner arm dynein; N-DRC, nexin
link/dynein regulatory complex. (B) Distribution of two-headed or three-headed outer arm dynein, and calaxin or LC4,
across eukaryotic groups. The occurrence of calaxin or LC4 is indicated in red or
blue, respectively, in the name of the group. A group name in black or gray indicates
the lack of both calaxin and LC4, or not enough genomic information, respectively.
The references for the EM images of the axonemes and the outer arm dynein are as follows:
Naegleria146]; Euglena176],177]; Trypanosoma66],67]; Giardia144]; Trichomonas147]: amoebozoan (Physarum) 101]-103]; choanoflagellate (Codosiga botrytis) 178]; chordate (Ciona intestinalis and human) 62],88]; echinoderm (sea urchin: Colobocentrotus atratus) 1],3]; platyhelminthes (Dugesia tigrina) 68],179]; arthropod (Exechia seriara) 180]; Mollusca (Crassostrea gigas) 181]; chytrid fungus (Rhizophlyctis) 182]; green alga (Chlamydomonas) 137]; diatom (Biddulphia levis) 183]; golden alga (Ochromonas) 116]; ciliate (Tetrahymena pyriformis) 184]; dinoflagellate (Wolszymkia micra) 185]; apicomplexan (Plasmodium) 141]; chlorarachnion (Bigelowiella natans) 123]; haptophyte (Chrysochromulina) 186]; and phytomyxean (Plasmodiophora brassicae) 140].

Ca²⁺ sensors appear to evolve with dynein heavy chains

As described above, Ciona and Chlamydomonas use distinct Ca²⁺ sensors for outer arm dynein. The molecular properties of these two proteins differ
from each other, and this might be related to the difference in Ca²⁺-dependent regulation of flagellar motility. BLAST searches using genomic information
from several organisms indicate that calaxin is an opisthokont-specific protein. Orthologs
of Chlamydomonas LC4 are distributed in Archaeplastida, Alveolata, Stramenopiles, Cryptophytes, Giardia and Naegleria, but not in Opisthokonta or the excavates Euglena and Trypanosoma.

Ca²⁺ sensors directly act on the motor subunits of outer arm dynein. The heavy chains
of outer arm dynein are phylogenetically classified into ODA? and ODA? families 129]. The ODA? family includes the Chlamydomonas ? heavy chain, the Ciona ? heavy chain, and the sea urchin ? heavy chain, all of which are located at the
innermost part of the outer arm 130],131]. The ODA? family includes the Chlamydomonas ? and ? heavy chains, the Ciona ? heavy chain, and the sea urchin ? heavy chain^a.

It is known that the number of heavy chains of outer arm dynein is two in metazoan
sperm but three in Chlamydomonas and ciliates 132]-136]; from the molecular structure of dynein, they are called two-headed and three-headed.
EM images of cross sections of the axonemes enable analysis of the number of heavy
chains of outer arm dynein (Figure 8A; 133]). The outer arm of a Chlamydomonas mutant lacking the ? heavy chain lacks the outermost part and appears similar to
the outer arm of sperm flagella 137],138], indicating that the outermost part corresponds to the ? heavy chain. Other observations
by transmission electron microscopy (TEM) 138] or cryo-electron tomography 130],131] indicate that the innermost part and the center part of the TEM image is composed
of the ? and ? heavy chain in Chlamydomonas, respectively. Following the idea of Mohri et al. 133], the number of heavy chains could be predicted from the morphology of outer arm dynein
observed by TEM (Figure 8A). I surveyed published TEM images of outer arm dyneins in several organisms. It
is intriguing to note that the number of dynein heads and the Ca²⁺ sensor used for regulation of outer arm dynein turn out to be well correlated (Figure 8B).

Figure 9. A possible model for the evolution of, and diversification in, the structures of outer
arm dynein and corresponding Ca²⁺sensors during eukaryotic evolution. The model is based on analyses of the structures of outer arm dynein (two-headed,
three-headed) and the types of Ca²⁺-sensor in each group of eukaryotes. It is assumed that the heavy chains and Ca²⁺-sensors of outer arm dynein of the last eukaryotic common ancestor (LECA) preceded
duplication, and that duplication and divergence of Ca²⁺-sensors occurred at an early stage of eukaryotic diversification. The model is arranged
so that the positions of eukaryotic groups match with widely accepted phylogenetic
relationships 128],158]. The number of cilia/flagella per cell is also indicated in parenthesis (brown letters).
Note that the numbers of cilia/flagella in Euglena and Trypanosoma are indicated as ‘1+,’ since these organisms are considered to have been biflagellates
but lost or largely degenerated one of the two flagella during evolution. In this
model, duplication of dynein heavy chain occurred at the root of the bikont lineage.
Duplication and divergence of Ca²⁺-sensors would have already occurred in the ancestral organisms that contained three-headed
dynein. An ancestral organism containing three-headed dynein might have recruited
LC4-like sensors or CNB/NCS-like sensors and then branched into the Metamonadan (Trichomonas + Giardia) and Discoban lineages. Loss of dynein heavy chains would have occurred in Giardia and the Euglenozoa. Red or blue asterisks represent duplication or loss of a dynein
heavy chain, respectively. Colored dots next to the two- or three-headed dyneins represent
Ca²⁺-sensors (red, calaxin; blue, LC4; magenta, DC3; green, NCS; cyan, CNB). In the lineage
of opisthokonts or Archaeplastida/Stramenopile/Alveolata, calaxin, LC4 or DC3 is demonstrated
to be bound to the dynein heavy chain, although it is not known whether Ca²⁺-sensors in Excavates or any of the hypothetical ancestors could bind to the dynein
or not.

It is believed that the two heavy chains of the ODA? family resulted from gene duplication
139], but the exact phylogenetic position of the duplication is not clear. The biflagellated
swarm cells in the amoebozoan Physarum possess 9+2-structured flagella. Cross sections of Physarum axonemes suggest that the outer arm dynein is two-headed 101]-103], like those in opisthokonts. However, the presence of calaxin and the number of heavy
chains in the outer arm dynein remain unclear because of the lack of a genome sequence.
Recent genome information reveals no gene similar to Chlamydomonas LC4 or DC3 in the chlorarachnion Bigelowiella natans. The number of heavy chains is possibly three judged from an EM image 123]. Another cercozoan, Plasmodiophora brassicae, apparently possesses three-headed outer arm dynein 140], but no genomic information is available. Ciliates, such as Paramecium and Tetrahymena, have three-headed outer arm dynein and a gene orthologous to Chlamydomonas LC4. However, another group of Alveolata, the Apicomplexa, shows a different feature;
the axonemes of Plasmodium berghei have normal 9+2 structure with three-headed outer arm dynein 141]. It is not clear whether P. berghei has LC4 since the genome sequence of this organism is not available. The gregarin
Lecudina tuzetae has a 6+0 structured axoneme, but the detailed structure of the outer arm dynein
is unclear from the available EM images 142].

Six species in the Excavata were available for prediction of the number of heavy chains
from EM images. First, the euglenozoan species Euglena, Leishmania, and Trypanosoma show a two-headed shape of outer arm dynein. The genome sequences reveal that neither
Euglena nor Trypanosoma have LC4. Second, Giardia has an LC4 homolog in the genome. EM images, however, are very close to those of
two-headed outer arm dynein 143],144]. This might be because Giardia lamblia is a fast-evolving parasitic species, leading to an error in phylogenetic analysis
due to long-branch attraction (LBA) 145]. Lastly, the outer arm dyneins of two excavate species, Naegleria gruberi and Trichomonas vaginalis, appear three-headed, although little TEM data with clear images of outer arm dynein
is available 146],147].

Eukaryote evolution in view of outer arm dynein and its calcium sensors

The structure of the axoneme and the regulation of ciliary and flagellar motility
are basic aspects of all major eukaryotic groups and undoubtedly one of the ancestral
features of eukaryotes 148]-151]. There are three hypotheses for how cilia were acquired in the last eukaryotic common
ancestor (LECA): endosymbiosis of a Spirochete and an Archaebacterium 152], viral infection 153], and autogenous origin 153] (see reviews 149],154]). The latter hypothesis is widely accepted at present. During overall evolution of
cell motility, ciliary movement and amoeboid movement were selectively or cooperatively
used depending on the body plan of the organisms. In the most probable LECA unicellular
organism, both ciliary and amoeboid locomotion systems appear to have been used 151]. Ancient flagella are considered to be used for attachment to a substrate and to
pull the organism by gliding. It is possible that flagella then acquired regulatory
systems for directed, tactical, or avoiding movement with high speed, with the aid
of extracellular signaling molecules such Ca²⁺; examples of such regulated movement are reversal of bend propagation and altering
of flagellar waveforms (Figure 1). In this case, as much evidence indicates, Ca²⁺-dependent regulation of the outer arm dynein is thought to be critical. During diversification,
some organisms lost components of the axoneme. For example, loss of outer arm dynein
is probably due to the loss of a requirement for rapid and/or extensive reorientation
of the cell. Other organisms have lost motile flagella or cilia, probably due to disuse
of their motility, in, for example, reproduction. The former include the gregarin
Lecudina tuzetae, Breviata, fern, moss, eel, and insects like Acerentomon microrhinus, and the latter include nematodes, crustaceans, and angiosperms 154],155].

Taking into account the fact that cilia have been inherited through the major pathways
of eukaryotic evolution, I here propose a hypothesis for eukaryotic evolution based
on phylogenetic analyses of Ca²⁺ sensors and the number of dynein heads. The most evident feature is that the majority
of opisthokonts show two-headed outer arm dynein with the Ca²⁺ sensor calaxin, whereas the majority of bikonts (Archaeplastida, Stramenopiles, Alveolata,
and some (but not all) Excavata) have three-headed outer arm dynein with Chlamydomonas LC4-type Ca²⁺ sensors. Excavata robustly emerge between unikonts and Archaeplastida/Hacrobia/Stramenopiles/Alveolata/Rhizaria
and form a monophyletic supergroup 128]. Several phylogenetic analyses of diverse eukaryotes have led to the idea that the
eukaryotic root could be set at the base between unikonts and bikonts 156]-158], but this is still controversial 158]-162].

The Excavata is certainly a supergroup that could provide key clues to understanding
the evolution of dynein and its Ca²⁺ sensors and shed light on the origin of Ca²⁺-dependent regulation of cilia and flagella. A phylogenetic analysis in this study
showed that excavates had already evolved several Ca²⁺ sensors, including those with similarities to extant Ca²⁺ sensors. Based on the widely accepted relationship among excavate species 128],158], a possible pathway could be considered with respect to evolution of dynein structure
and Ca²⁺ sensors (Figure 9). This model is based on the hypothesis that the LECA had two-headed dynein and that
Ca²⁺ sensors were duplicated in the initial stage of eukaryotic evolution and became divergent
(and then possibly became functional) during evolution. Loss of dynein heavy chains
or Ca²⁺ sensors in Excavata, possibly by reduction of genomes in obligate parasites 143],163], is also taken into consideration.

The duplication of dynein heavy chains would have occurred at the root of the bikont
lineage (Figure 9). From the strong bootstrap supports (Figure 5), it appears that three-headed dynein might have recruited LC4 in the last common
ancestor of bikonts, which would be involved in the diversification in Metamonada
(Trichomonas and Giardia). Similarly, CNB/NCS-like Ca²⁺ sensor homologs must have existed in the last common eukaryotic ancestor. Another
route for Discoba diversification might have involved retentions of CNB/NCS-like Ca²⁺ sensors.

Excavates show a variety in the number of motile flagella per cell. For example, the
euglenoids Trypanosoma brucei and Euglena gracilis are biflagellate but one of the two flagella is highly reduced. There are two flagella
in Naegleria gruberi, five flagella in Trichomonas vaginalis, and eight flagella in Giardia lamblia (see Figure 9). It is worth pointing out that the excavate species bearing a single motile flagellum,
that is, Euglena and Trypanosoma, have two-headed dyneins; Giardia is the only excavate with two-headed dynein and multiple flagella (Figure 9). The only other eukaryotic group containing organisms (or cells) with a single motile
flagellum is the Opisthokonta.

The Amoebozoa, Physarum polycephalum and Breviata anathema, originally grouped into unikonts 125], bear two basal bodies. It has therefore been debated whether Amoebozoa and Opisthokonta
can be monophyletically grouped 157],164]. Physarum has one long and one short flagellum connected to two basal bodies, and Breviata anathema, a small amoeba-like cell, has a single flagellum from each of the two basal bodies.
The presence of two basal bodies is proposed as one of the characteristics of bikonts
165]. From TEM images of axonemes, Physarum appears to have two-headed outer arm dyneins (Figure 8), which is a common aspect of opisthokonts 133]. Breviata does not have outer arm dynein 100], meaning there is no evidence for its grouping based on the criterion of the structure
of outer arm dynein. It would be intriguing to search for calaxin (also TNDK-IC and
CMUB, see above and 166]) in organisms that have been under debate in terms of classification into bikonts
or unikonts.

New genes with novel functions are evolved by gene duplication 167]. Several models have been proposed for mechanisms of how new protein functions evolve
through gene duplication and divergence 168]. Recruitment of functional Ca²⁺ sensors seems particularly important in cilia and flagella because they participate
in gamete motility, essential for the success of reproduction in most organisms. For
Ca²⁺ sensors of outer arm dynein, the functions of calaxin and Chlamydomonas LC4 regulate the motor activity in flagella, but their response to Ca²⁺ concentration is different. The distribution of these Ca²⁺ sensors in extant species in eukaryotes is described in the present paper. Calaxin
and LC4 appear to be preserved in Opisthokonta and the majority of bikonts (Archaeplastida,
Stramenopiles, and Alveolata), respectively.

It is possible that these proteins became preserved after protein evolution by gene
duplication and divergence because of their specific functions in the interaction
with the cytoskeleton and the regulation of a molecular motor. The module-dominant
conservation, as seen in axonemes 166], is possibly because of the need for preservation of multiple proteins in this cytoskeletal
architecture. No biochemical evidence has been obtained for the localization or functions
of Ca²⁺ sensors, except Ciona calaxin and Chlamydomonas LC4. To learn whether evolution of proteins by gene duplication and divergence accompanies
or precedes innovation of protein function, it would be fascinating to examine the
interaction of an ancient calaxin with microtubules or dyneins.