Effects of the central pair apparatus on microtubule sliding velocity in sea urchin sperm flagella.

To produce oscillatory bending movement in cilia and flagella, the activity of dynein arms must be regulated. The central-pair microtubules, located at the centre of the axoneme, are often thought to be involved in the regulation, but this has not been demonstrated definitively. In order to determine whether the central-pair apparatus are directly involved in the regulation of the dynein arm activity, we analyzed the movement of singlet microtubules that were brought into contact with dynein arms on bundles of doublets obtained by sliding disintegration of elastase-treated flagellar axonemes. An advantage of this new assay system was that we could distinguish the bundles that contained the central pair apparatus from those that did not, the former being clearly thicker than the latter. We found that microtubule sliding occurred along both the thinner and the thicker bundles, but its velocity differed between the two kinds of bundles in an ATP concentration dependent manner. At high ATP concentrations, such as 0.1 and 1 mM, the sliding velocity on the thinner bundles was significantly higher than that on the thicker bundles, while at lower ATP concentrations the sliding velocity did not change between the thinner and the thicker bundles. We observed similar bundle width-related differences in sliding velocity after removal of the outer arms. These results provide first evidence suggesting that the central pair and its associated structures may directly regulate the activity of the inner (and probably also the outer) arm dynein.     

The actin nucleator Cordon-bleu is required for development of motile cilia in zebrafish

The cordon-bleu (Cobl) gene is widely conserved in vertebrates, with developmentally regulated axial and epithelial expression in mouse and chick embryos. In vitro, Cobl can bind monomeric actin and nucleate formation of unbranched actin filaments, while in cultured cells it can modulate the actin cytoskeleton. However, an essential role for Cobl in vivo has yet to be determined. We have used zebrafish as a model to assess the requirements for Cobl in embryogenesis. We find that cobl shows enriched expression in ciliated epithelial tissues during zebrafish organogenesis. Cobl protein is enriched in the apical domain of ciliated cells, in close proximity to the apical actin cap. Reduction of Cobl by antisense morpholinos reveals an essential role in development of motile cilia in organs such as Kupffer's vesicle and the pronephros. In Kupffer's vesicle, the reduction in Cobl coincides with a reduction in the amount of apical F-actin. Thus, Cobl represents a molecular activity that couples developmental patterning signals with local intracellular cytoskeletal dynamics to support morphogenesis of motile cilia.     

Direct measurement of the velocity of the metachronal wave in beating cilia

Recently a computerized electro-optical method was developed which enables one to simultaneously measure the frequency and the wavelength of the metachronal waves in beating cilia. The method is based on measurement of scattered light from two areas at a given distance apart. The distance between measured areas can be varied from zero to hundreds of microns. The relative ease of the measurement and data analysis of this method enable one to create large statistical ensembles in order to obtain reliable averages. In this work we show that in addition to the previously mentioned parameters this system can measure directly the velocity of the metachronal wave. It was found that the average velocity in the tissue culture from frog's palate epithelium at room temperature is approximately 270 micron/sec, about twice the average particle velocity at the frog's palate.     

Orientation of the central pair complex during flagellar bend formation in Chlamydomonas

Thin section electron micrographs of rapidly fixed Chlamydomonas cells were used to establish a relationship between flagellar bends and orientation of the central pair microtubule complex. Using conditions that preserve flagellar waveforms during both forward swimming (asymmetric bends) and backward swimming (symmetric bends), we found that central pair orientation differs in bent regions and straight regions. During forward swimming, a plane through the two central pair microtubules is parallel to the bend plane throughout principal bends, in both effective stroke and recovery stroke phases of the beat cycle. In these curved segments, the C1 microtubule always faces the outer edge of the curve. This parallel orientation twists in straight regions both proximal and distal to bends. During backward swimming episodes induced by photoshock, when Chlamydomonas flagella beat with principal and reverse bends of similar magnitude, the central pair twists by 180 degrees between successive bends. These observations support a model in which central pair orientation in Chlamydomonas is linked to doublet-specific dynein activation, and bend propagation is linked to rotation of the central pair complex.     

Vigorous beating of Chlamydomonas axonemes lacking central pair/radial spoke structures in the presence of salts and organic compounds

Flagella of Chlamydomonas mutants lacking the central pair of microtubules or radial spokes do not beat; however, axonemes isolated from these mutants were found to display vigorous bending movements in the presence of ATP and various salts, sugars, alcohols, and other organic compounds. For example, about 15% of the total axonemes isolated from pf18, a mutant lacking the central pair, displayed beating in the presence of 10 mM MgSO(4) and 0.2 mM ATP at about 22 Hz, while none beat with the same concentration of ATP and < or = 5 mM or > or = 25 mM MgSO(4). The beat frequency and waveform of beating pf18 axonemes were similar to those of wild type axonemes beating under the same conditions. Similarly, 10-50% of the axonemes beat in the presence of 0.5 M sucrose, 2.0 M glycerol, or 1.7 M[10% (v/v)] ethanol. The appearance of motility did not correlate with the change in axonemal ATPase; however, these substances at those concentrations commonly increased the amplitude of nanometer-scale oscillation (hyper-oscillation) in pf18 axonemes, as well as the extent of ATP-induced sliding disintegration of protease-treated axonemes. Axonemes of double mutants lacking both the central pair and various subspecies of inner-arm dynein also beat at increased MgSO(4) concentrations, but axonemes lacking outer-arm dynein in addition to the central pair did not beat. These and other observations suggest that small molecules perturb the regulation of microtubule sliding through some change in water activity or osmotic stress. Axonemes must have an intrinsic ability to beat without the central pair/radial spokes under a variety of non-physiological solution conditions, as long as the outer dynein arms are present. Apparently, the major function of the central pair/radial spoke structures is to restore this activity under physiological conditions.     

Substructure of inner dynein arms, radial spokes, and the central pair/projection complex of cilia and flagella

The substructure of the components of the axoneme interior--the inner dynein arms, the radial spokes, and the central pair/projection complex--was analyzed for Chlamydomonas. Tetrahymena, Strongelocentrotus, and Mnemiopsis using the quick-freeze, deep-etch technique. The inner arms are shown to resemble the outer arms in overall molecular organization, but they are disposed differently on the microtubule and have two distinct morphologies--dyads with two heads and triads with three. The dyads associate with spokes S3 and S2; the triads associate with S1. The spokes form a three-start right-handed helix with a 288-nm rise; the central pair makes a shallow left-handed twist. The spoke heads are shown to be made up of four major subunits; two bind to the spoke shaft and two bind to a pair of central-sheath projections.     

CFAP54 is required for proper ciliary motility and assembly of the central pair apparatus in mice

Motile cilia and flagella play critical roles in fluid clearance and cell motility, and dysfunction commonly results in the pediatric syndrome primary ciliary dyskinesia (PCD). CFAP221, also known as PCDP1, is required for ciliary and flagellar function in mice and Chlamydomonas reinhardtii, where it localizes to the C1d projection of the central microtubule apparatus and functions in a complex that regulates flagellar motility in a calcium-dependent manner. We demonstrate that the genes encoding the mouse homologues of the other C. reinhardtii C1d complex members are primarily expressed in motile ciliated tissues, suggesting a conserved function in mammalian motile cilia. The requirement for one of these C1d complex members, CFAP54, was identified in a mouse line with a gene-trapped allele. Homozygous mice have PCD characterized by hydrocephalus, male infertility, and mucus accumulation. The infertility results from defects in spermatogenesis. Motile cilia have a structural defect in the C1d projection, indicating that the C1d assembly mechanism requires CFAP54. This structural defect results in decreased ciliary beat frequency and perturbed cilia-driven flow. This study identifies a critical role for CFAP54 in proper assembly and function of mammalian cilia and flagella and establishes the gene-trapped allele as a new model of PCD.     

IP3 signaling is required for cilia formation and left-right body axis determination in Xenopus embryos

Vertebrate left–right (LR) body axis is manifested as an asymmetrical alignment of the internal organs such as the heart and the gut. It has been proposed that the process of LR determination commonly involves a cilia-driven leftward flow in the mammalian node and its equivalents (Kupffer’s vesicle in zebrafish and the gastrocoel roof plate in Xenopus). Recently, it was reported that Ca²⁺ flux regulates Kupffer’s vesicle development and is required for LR determination. As a basis of Ca²⁺ flux in many cell types, inositol 1,4,5-trisphosphate (IP₃) receptor-mediated calcium release from the endoplasmic reticulum (ER) plays important roles. However, its involvement in LR determination is poorly understood. We investigated the role of IP₃ signaling in LR determination in Xenopus embryos. Microinjection of an IP₃ receptor-function blocking antibody that can inhibit IP₃ calcium channel activity randomized the LR axis in terms of left-sided Pitx2 expression and organ laterality. In addition, an IP₃ sponge that could inhibit IP₃ signaling by binding IP₃ more strongly than the IP₃ receptor impaired LR determination. Examination of the gastrocoel roof plate revealed that the number of cilia was significantly reduced by IP₃ signal blocking. These results provide evidence that IP₃ signaling is involved in LR asymmetry formation in vertebrates.     

Localization of transient receptor potential ion channels in primary and motile cilia of the female murine reproductive organs

We have examined the subcellular localization of transient receptor potential (TRP) ion channels and the potential sensory role of cilia in murine female reproductive organs using confocal laser scanning microscopy analysis on ovary and oviduct tissue sections as well as on primary cultures of follicular granulosa cells. We show that the Ca2+ permeable cation channel, polycystin-2, as well as polycystin-1, a receptor that forms a functional protein complex with polycystin 2, distinctively localize to primary cilia emerging from granulosa cells of antral follicles in vivo and in vitro. Both polycystins are localized to motile oviduct cilia and this localization is greatly increased upon ovulatory gonadotropic stimulation. Further, the Ca2+ permeable cation channel, TRP vaniloid 4 (TRPV4), localizes to a sub-population of motile cilia on the epithelial cells of the ampulla and isthmus with high intensity in proximal invaginations of the epithelial folds. These observations are the first to demonstrate ciliary localization of TRP ion channels and their possible receptor function in the female reproductive organs. We suggest that polycystins 1 and 2 play an important role in granulosa cell differentiation and in development and maturation of ovarian follicles. In the oviduct both TRPV4 and polycystins could be important in relaying physiochemical changes in the oviduct upon ovulation.     

Early stages of zebrafish eye formation require the coordinated activity of Wnt11, Fz5, and the Wnt/beta-catenin pathway

During regional patterning of the anterior neural plate, a medially positioned domain of cells is specified to adopt retinal identity. These eye field cells remain coherent as they undergo morphogenetic events distinct from other prospective forebrain domains. We show that two branches of the Wnt signaling pathway coordinate cell fate determination with cell behavior during eye field formation. Wnt/beta-catenin signaling antagonizes eye specification through the activity of Wnt8b and Fz8a. In contrast, Wnt11 and Fz5 promote eye field development, at least in part, through local antagonism of Wnt/beta-catenin signaling. Additionally, Wnt11 regulates the behavior of eye field cells, promoting their cohesion. Together, these results allow us to postulate a model in which Wnt11 and Fz5 signaling promotes early eye development through the coordinated antagonism of signals that suppress retinal identity and promotion of coherence of eye field cells.     

A Highlights from MBoC Selection: Regulation of Chlamydomonas flagella and ependymal cell motile cilia by ceramide-mediated translocation of GSK3

Cilia are important organelles formed by cell membrane protrusions; however, little is known about their regulation by membrane lipids. We characterize a novel activation mechanism for glycogen synthase kinase-3 (GSK3) by the sphingolipids phytoceramide and ceramide that is critical for ciliogenesis in Chlamydomonas and murine ependymal cells, respectively. We show for the first time that Chlamydomonas expresses serine palmitoyl transferase (SPT), the first enzyme in (phyto)ceramide biosynthesis. Inhibition of SPT in Chlamydomonas by myriocin led to loss of flagella and reduced tubulin acetylation, which was prevented by supplementation with the precursor dihydrosphingosine. Immunocytochemistry showed that (phyto)ceramide was colocalized with phospho–Tyr-216-GSK3 (pYGSK3) at the base and tip of Chlamydomonas flagella and motile cilia in ependymal cells. The (phyto)ceramide distribution was consistent with that of a bifunctional ceramide analogue UV cross-linked and visualized by click-chemistry–mediated fluorescent labeling. Ceramide depletion, by myriocin or neutral sphingomyelinase deficiency (fro/fro mouse), led to GSK3 dephosphorylation and defective flagella and cilia. Motile cilia were rescued and pYGSK3 localization restored by incubation of fro/fro ependymal cells with exogenous C24:1 ceramide, which directly bound to pYGSK3. Our findings suggest that (phyto)ceramide-mediated translocation of pYGSK into flagella and cilia is an evolutionarily conserved mechanism fundamental to the regulation of ciliogenesis.     

MNS1 is essential for spermiogenesis and motile ciliary functions in mice

During spermiogenesis, haploid round spermatids undergo dramatic cell differentiation and morphogenesis to give rise to mature spermatozoa for fertilization, including nuclear elongation, chromatin remodeling, acrosome formation, and development of flagella. The molecular mechanisms underlining these fundamental processes remain poorly understood. Here, we report that MNS1, a coiled-coil protein of unknown function, is essential for spermiogenesis. We find that MNS1 is expressed in the germ cells in the testes and localizes to sperm flagella in a detergent-resistant manner, indicating that it is an integral component of flagella. MNS1-deficient males are sterile, as they exhibit a sharp reduction in sperm production and the remnant sperm are immotile with abnormal short tails. In MNS1-deficient sperm flagella, the characteristic arrangement of "9+2" microtubules and outer dense fibers are completely disrupted. In addition, MNS1-deficient mice display situs inversus and hydrocephalus. MNS1-deficient tracheal motile cilia lack some outer dynein arms in the axoneme. Moreover, MNS1 monomers interact with each other and are able to form polymers in cultured somatic cells. These results demonstrate that MNS1 is essential for spermiogenesis, the assembly of sperm flagella, and motile ciliary functions.     

Epithelial sodium channels (ENaC) are uniformly distributed on motile cilia in the oviduct and the respiratory airways

Epithelial sodium channels (ENaCs) are located on the apical surface of cells and funnel Na⁺ ions from the lumen into the cell. ENaC function also regulates extracellular fluid volume as water flows across membranes accompanying Na⁺ ions to maintain osmolarity. To examine the sites of expression and intracellular localization of ENaC, we generated polyclonal antibodies against the extracellular domain of human α-ENaC subunit that we expressed in E. coli. Three-dimensional (3D) confocal microscopy of immunofluorescence using these antibodies for the first time revealed that ENaCs are uniformly distributed on the ciliary surface in all epithelial cells with motile cilia lining the bronchus in human lung and female reproductive tract, all along the fimbrial end of the fallopian tube, the ampulla and rare cells in the uterine glands. Quantitative analysis indicated that cilia increase cell surface area >70-fold and the amount of ENaC on cilia is >1,000-fold higher than on non-ciliated cell surface. These findings indicate that ENaC functions as a regulator of the osmolarity of the periciliary fluid bathing the cilia. In contrast to ENaC, cystic fibrosis transmembrane conductance regulator (CFTR) that channels chloride ions from the cytoplasm to the lumen is located mainly on the apical side, but not on cilia. The cilial localization of ENaC requires reevaluation of the mechanisms of action of CFTR and other modulators of ENaC function. ENaC on motile cilia should be essential for diverse functions of motile cilia, such as germ cell transport, fertilization, implantation, clearance of respiratory airways and cell migration.     

Subunits of the chaperonin CCT are associated with Tetrahymena microtubule structures and are involved in cilia biogenesis

The cytosolic chaperonin CCT is a heterooligomeric complex of about 900 kDa that mediates the folding of cytoskeletal proteins. We observed by indirect immunofluorescence that the Tetrahymena TpCCTalpha, TpCCTdelta, TpCCTepsilon, and TpCCTeta-subunits colocalize with tubulin in cilia, basal bodies, oral apparatus, and contractile vacuole pores. TpCCT-subunits localization was affected during reciliation. These findings combined with atomic force microscopy measurements in reciliating cells indicate that these proteins play a role during cilia biogenesis related to microtubule nucleation, tubulin transport, and/or axoneme assembly. The TpCCT-subunits were also found to be associated with cortex and cytoplasmic microtubules suggesting that they can act as microtubule-associated proteins. The TpCCTdelta being the only subunit found associated with the macronuclear envelope indicates that it has functions outside of the 900 kDa complex. Tetrahymena cytoplasm contains granular/globular-structures of TpCCT-subunits in close association with microtubule arrays. Studies of reciliation and with cycloheximide suggest that these structures may be sites of translation and folding. Combined biochemical techniques revealed that reciliation affects the oligomeric state of TpCCT-subunits being tubulin preferentially associated with smaller CCT oligomeric species in early stages of reciliation. Collectively, these findings indicate that the oligomeric state of CCT-subunits reflects the translation capacity of the cell and microtubules integrity.