Cilia and flagella are evolutionarily homologous cell organelles, with cilium being a generic term for either a motile or nonmotile axoneme-containing protrusion and a flagellum being a motile cilium used for locomotion (Mitchell, 2007
; Satir et al.
). Recent studies have shown that most cells have one or more cilia that mediate sensory perception and fluid homeostasis necessary for normal development and physiology of living systems (Baker and Beales, 2009
; Mirzadeh et al.
; Vogel et al.
; Wilson et al.
). Ciliary dysfunction results in a variety of ciliopathic diseases, including many that are caused by abnormal motility of motile cilia (Baker and Beales, 2009
). During evolution, cilium motility-dependent functions have expanded beyond swimming, food gathering, and parasitism in unicellular organisms to many more complex fluid-driven cellular processes in vertebrates, including humans. For example, the nodal cilia generate a directional flow that provides positional cues to the surrounding cells, leading to left–right body asymmetry in the developing embryos. Disruption of the nodal flow leads to situs inversus
with mispositioning and malformation of internal organs (Okada et al.
). The motile cilia of ependymal cells generate a flow of cerebrospinal fluid that plays roles in neuronal migration to the olfactory lobes (Sawamoto et al.
). Disruption of the cerebrospinal fluid flow affects the neuronal migration and also causes fluid retention and hydrocephalus (Sawamoto et al., 2006
; Lechtreck et al., 2008
; Wilson et al., 2010
). In the reproductive tract, abnormal motility of motile cilia may cause ectopic pregnancy (Lyons et al.
). Abnormal motility of the airway cilia leads to inefficient mucociliary clearance and respiratory problems (Braiman and Priel, 2008
). To accomplish these diverse physiological roles, motile cilia and flagella exhibit a large repertoire of waveforms and are capable of versatile motility changes in response to the external environment (Gibbons, 1981
A well-known mechanism for eliciting motility changes is alteration of intraflagellar Ca2+
concentrations (Kaupp et al.
; King, 2010
). This has been shown for ciliary reversal in Paramecium
(Naito and Kaneko, 1972
), reversal of flagellar bending chirality in various sperm (Ishijima and Hamaguchi, 1993
; Kaupp et al.
), sperm hyperactivation and chemotaxis (Publicover et al.
), and waveform conversion from the asymmetric ciliary form to the symmetric flagellar form in the green alga Chlamydomonas reinhardtii
(Schmidt and Eckert, 1976;
Bessen et al.
). Aside from inducing alternative waveforms, intraflagellar calcium can also reverse the flagellar wave propagation direction along the longitudinal axis of the axoneme (Sugrue et al.
; Ishijima et al.
). Whereas flagellar waves of most protozoa and spermatozoa start at the base and propagate toward the tip, some species, such as the Tephritid fly and the marine invertebrates Myzostomum
, have flagella that can propagate waves in both axial directions (Baccetti et al.
; Ishijima et al.
). Trypanosomes are unicellular parasites that use their singular flagella for movement in host tissues. Trypanosome flagella are unique in that they routinely propagate tip-to-base waves, which generate a flagellum-tip-leading, forward movement (Walker, 1961
; Sugrue et al.
; Hill, 2003
). However, in response to lights or obstacles, trypanosome flagella initiate short bursts of base-to-tip waves that generate a backward movement. Flagellar waveform conversion or reversal of wave direction provides a means of backing up, reorienting the cell, and avoiding obstacles in physiological settings.
The axoneme of nearly all motile cilia and flagella has the canonical “9+2” structure (Porter and Sale, 2000
; Heuser et al.
). The cylindrical wall of the axoneme consists of nine outer doublet microtubules (ODs) that enclose a central pair (CP) of singlet microtubules in the center, extending from the basal body to the distal tip. Serving as interconnecting structures between the ODs and the CP are radial spokes, which are anchored along the lengths of each of the nine ODs and project toward the CP. The radial spokes are known to interact with regulatory proteins located on the CP and to relay signals back to the dynein motors that are anchored on the ODs (Smith, 2002b
; Smith and Yang, 2004
; Mitchell, 2004
). The isolated demembranated axonemes of C. reinhardtii
and Crithidia oncopelti
have been shown to execute reversible waveform conversion and wave direction reversal, respectively, when reactivated in solutions of varying free Ca2+
concentrations (Bessen et al.
; Sugrue et al.
). This indicates that the mediators for waveform conversion and wave direction reversal are located on the axoneme. Many calcium-sensing proteins are integrated within the CP, radial spokes, and dynein motors (Smith, 2002a
; Smith and Yang, 2004
; Dymek and Smith, 2007
). Although less is known about calcium-sensing or responding proteins on the ODs, three C. reinhardtii mbo
(move backward only
) mutations result in the loss of six to eight OD-associated polypeptides, and these mutant flagella are unable to execute waveform conversion (Segal et al.
). The cloned mbo2
locus encodes a conserved coiled-coil protein on the ODs with yet undefined functions in other organisms (Tam and Lefebvre, 2002
The PKD2 family of calcium channels has been shown to function in motile as well as immotile cilia. Mutations in vertebrate PKD2
lead to renal cystic growth (Wu et al.
) and defects in left–right body asymmetry (Pennekamp et al.
) due to disruptions of fluid sensation by the kidney and nodal cilia, respectively. The mating defect of Caenorhabditis elegans Pkd2
is due to the loss of mechanosensation by the ciliated sensory neurons that mediate vulva location during mating (Barr and Sternberg, 1999
). A working model applicable to these situations is that mechanical depression/bending of cilia opens the PKD2 channels on the ciliary membranes, which results in calcium influx into the cilia; this eventually leads to signal transduction and physiological changes in the cell body. It is unclear whether this ciliary bending model applies to C. reinhardtii
where Pkd2 functions on flagella that are constantly beating (Gao et al
; Watnick et al
; Huang et al.
The C. reinhardtii
PKD2 (CrPKD2) is required for flagellar adhesion-mediated mating (Huang et al.
). RNAi knockdown of CrPKD2 does not interfere with flagellar adhesion between the (+) and (−) gametes, but does block downstream signaling, resulting in the reduction of gamete fusion and formation of diploid cells (Huang et al.
). Studies have shown that, in this pathway, CrPKD2 functions downstream of flagellar adhesion but upstream of a flagellar protein tyrosine kinase, which leads to downstream activation of the cGMP-dependent protein kinase CrPKG (Wang et al.
) and an adenylate cyclase that produces cAMP (Wang and Snell, 2003
). cAMP appears to be the critical second messenger because exogenously supplied cAMP is sufficient to induce gamete fusion, bypassing the steps of CrPKD2, CrPKG, and the adenylate cyclase. CrPKD2 is also present in flagella of vegetative cells, and so it is likely to have additional functions in other signaling pathways, possibly including control of flagellar motility (Huang et al.
Pkd2 is highly enriched on the sperm flagellum (Gao et al.
), and its expression in S2 Drosophila
tissue culture cells generates cation channel activities similar to those of mammalian PKD2 (Venglarik et al.
). As a normal reproductive process, Drosophila
sperm in the uterus travel into a long and narrow seminal receptacle (SR) tubule that has a closed end. Sperm are stored in the distal half of the SR for weeks, and during this period, they gradually exit out of the SR to fertilize the egg (Bloch Qazi and Wolfner, 2006
). Mutant sperm lacking wild-type Pkd2
show grossly normal motility but are unable to move efficiently into the SR (Watnick et al.
; Gao et al.
). This leads to a working model that Drosophila
sperm storage is induced by an SR entry signal that activates Pkd2, leading to calcium influx into the sperm and thereby producing the specific swimming behavior necessary for SR entry. Here, we report the identification of CG34110, another flagellar protein required for the Drosophila
sperm to enter the SR tubule. Loss-of-function CG34110
results in specific sperm motility defects that are essentially the same as those of the Pkd2
mutant. CG34110 has unequivocal orthologues in organisms possessing motile cilia and flagella, but is absent in organisms that do not have cilia and in C. elegans
, which does not have motile cilia. Biochemical analyses indicate that the C. reinhardtii
orthologue FAP50 (Pazour et al.
) is an OD-associated protein unaffected in all three mbo