The Resorption of Cilia in Cyathodinium piriforme*

SYNOPSIS. The fine structure of the cilium, kinetosome, kinetodesmal fiber, and basal microtubules has been described in Cyathodinium piriforme. The ciliary axoneme is encased in an electron-dense jacket termed the axonemal jacket. This jacket surrounds the axoneme and is found midway between the axoneme and the ciliary membrane when viewed in cross section. Before division or reorganization the cilia are withdrawn into the cell. Intact cilia surrounded by their jackets are found in the cytoplasm during the early phases of retraction. Degradation of the axonemal microtubules precedes the dissolution of the axonemal jacket. Profiles of the jackets are observed after the microtubules have been resorbed. The cilia appear to detach from the kinetosomes. Barren kinetosomes are seen below the cell surface frequently with kinetodesmal fibers still attached. Whether all or some of these barren kinetosomes contribute to the formation of the new ciliary anlage cannot be ascertained.     

Evolution and persistence of the cilium

The origin of cilia, a fundamental eukaryotic organelle, not present in prokaryotes, poses many problems, including the origins of motility and sensory function, the origins of nine-fold symmetry, of basal bodies, and of transport and selective mechanisms involved in ciliogenesis. We propose the basis of ciliary origin to be a self-assembly RNA enveloped virus that contains unique tubulin and tektin precursors. The virus becomes the centriole and basal body, which would account for the self-assembly and self-replicative properties of these organelles, in contrast to previous proposals of spirochaete origin or endogenous differentiation, which do not readily account for the centriole or its properties. The viral envelope evolves into a sensory bud. The host cell supplies the transport machinery and molecular motors to construct the axoneme. Polymerization of cytoplasmic microtubules in the 9+0 axoneme completes the 9+2 pattern.     

Mutations in Hydin impair ciliary motility in mice

Chlamydomonas reinhardtii hydin is a central pair protein required for flagellar motility, and mice with Hydin defects develop lethal hydrocephalus. To determine if defects in Hydin cause hydrocephalus through a mechanism involving cilia, we compared the morphology, ultrastructure, and activity of cilia in wild-type and hydin mutant mice strains. The length and density of cilia in the brains of mutant animals is normal. The ciliary axoneme is normal with respect to the 9 + 2 microtubules, dynein arms, and radial spokes but one of the two central microtubules lacks a specific projection. The hydin mutant cilia are unable to bend normally, ciliary beat frequency is reduced, and the cilia tend to stall. As a result, these cilia are incapable of generating fluid flow. Similar defects are observed for cilia in trachea. We conclude that hydrocephalus in hydin mutants is caused by a central pair defect impairing ciliary motility and fluid transport in the brain.     

Kinesin-13 regulates the quantity and quality of tubulin inside cilia

Kinesin-13, an end depolymerizer of cytoplasmic and spindle microtubules, also affects the length of cilia. However, in different models, depletion of kinesin-13 either lengthens or shortens cilia, and therefore the exact function of kinesin-13 in cilia remains unclear. We generated null mutations of all kinesin-13 paralogues in the ciliate Tetrahymena. One of the paralogues, Kin13Ap, localizes to the nuclei and is essential for nuclear divisions. The remaining two paralogues, Kin13Bp and Kin13Cp, localize to the cell body and inside assembling cilia. Loss of both Kin13Bp and Kin13Cp resulted in slow cell multiplication and motility, overgrowth of cell body microtubules, shortening of cilia, and synthetic lethality with either paclitaxel or a deletion of MEC-17/ATAT1, the α-tubulin acetyltransferase. The mutant cilia assembled slowly and contained abnormal tubulin, characterized by altered posttranslational modifications and hypersensitivity to paclitaxel. The mutant cilia beat slowly and axonemes showed reduced velocity of microtubule sliding. Thus kinesin-13 positively regulates the axoneme length, influences the properties of ciliary tubulin, and likely indirectly, through its effects on the axonemal microtubules, affects the ciliary dynein-dependent motility.     

Presence of a Ciliary Patch in Preoral Epithelium of Sea Urchin Plutei

Removal of the hyaline layer from sea urchin embryos at the pluteus stage discloses a densely ciliated region in the preoral area of the ectodermal epithelium. In four-armed plutei, this ciliary path is located between the anterolateral arms and in eight-armed plutei it becomes surrounded by preoral and anterolateral arms. The area of the patch and the number of cilia increase with age. This patch is covered by cilia of unusual morphology and orientation. There are more than two cilia per cell which are coiled together several times around a small cone at the apical end of the cell. These coiled cilia run parallel to the surface of the cell but do not extend beyond the hyaline layer. The ciliary axoneme consists of a "9+2" microtubular structure, but no outer or inner dynein arms are observed. Although the cells with coiled cilia are present in a cluster constituting a part of the epithelium, they have axons that project from their basal (inner) ends. The structural characteistics of the ciliary patch suggest that it possesses a sensory function.     

Ultrastructure of paired coniform 9 + 0 sensory cilia: a new type in the organ of Bellonci of the marine amphipod Gammarus setosus

Summary The structure of modified 9 + 0 cilia in the organ of Bellonci was studied in Gammarus setosus from late embryonic development to adult after routine fixation, fixation with lanthanum treatment, and prefixation with ethylene diamine tetraacetic acid and sodium dodecyl sulphate. The cilia are distinct from known sensory cilia in that they occur in pairs and lack centrioles. The basal bodies are at right angles to each other. The basal body cylinders consist of dense microtubule doublets and have 3 regions: the basal cartwheel, the middle pinwheel and the distal transitional. The pinwheel, which has 9 fins of dense material attached to the doublets, is differentiated into a spiral attachment of the ciliary roots whose periodicity is 70 nm. The scanning electron microscope shows the roots as beaded, tapering ribbons. The coniform outer segments give rise to tubules, each with 1 or 2 single or double microtubules in its core. The tubules are in contact with extracellular chains of calcium granules inside the organ. A bend in the axoneme brings the paired outer segments together. Lamellar bodies develop from the ciliary tubules in embryos and juveniles, but not in adults, except after exposure to lanthanum.     

Development of the Nervous System of Sea Urchin Embryos: Formation of Ciliary Bands and the Appearance of Two Types of Ectoneural Cells in the Pluteus

An ultrastructural study of the larval integument of the sea urchin, Hemicentrotus pulcherrimus , was conducted with special emphasis on the development of the nervous system in relation to the formation of ciliary bands. In the integument of 4-armed pluteus larvae, cells associated with the ciliary band, which have 200 nm-thick projections at their apices, and cells in the squamous epithelium, which have a cilium and long, fine radiating processes in the apical region, were observed. Both cell types have axons at their basal ends that form nerve bundles beneath the ciliary bands, where the axons make contact with ectodermal effector cells with motile cilia. The cilia and other apical projections of these ectoneural cells run parallel to the surface of the cells, and are under the hyaline layer. The axoneme of the cilium has a typical "9 + 2" microtubular arrangement, but generally has no dynein arms. These ectoneural cells are more frequent on the oral surface than on the antioral surface.     

THE CILIARY NECKLACE : A Ciliary Membrane Specialization

Cilia, primarily of the lamellibranch gill (Elliptio and Mytilus), have been examined in freeze-etch replicas. Without etching, cross fractures rarely reveal the 9 + 2 pattern, although suggestions of ninefold symmetry are present. In etched preparations, longitudinal fractures through the matrix show a triplet spoke alignment corresponding to the spoke periodicity seen in thin sections. Dynein rows can be visualized along the peripheral microtubules in some preparations. Fracture faces of the ciliary membrane are smooth with few membrane particles, except in the regions adjacent to the basal plate. In the transition region below the plate, a unique particle arrangement, the ciliary necklace, is found. In the Elliptio gill, on fracture face A the necklace is comprised of three well-defined rows or strands of membrane particles that encircle the ciliary shaft. The rows are scalloped and each scallop corresponds to a peripheral doublet microtubule. In thin sections at the level of these particles, a series of champagne-glass structures link the microtubular doublets to the ciliary membrane. The ciliary necklace and this "membrane-microtubule" complex may be involved in energy transduction or the timing of ciliary beat. Comparative studies show that these features are present in all somatic cilia examined including those of the ameboflagellate Tetramitus, sea urchin embryos, rat trachea, and nonmotile cilia of cultured chick embryo fibroblasts. The number of necklace strands differs with each species. The necklace has not been found in rat or sea urchin sperm.     

Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 null Tetrahymena cells reveals functional MIPs

The core structure of motile cilia and flagella, the axoneme, is built from a stable population of doublet microtubules. This unique stability is brought about, at least in part, by a network of microtubule inner proteins (MIPs) that are bound to the luminal side of the microtubule walls. Rib72A and Rib72B were identified as MIPs in the motile cilia of the protist Tetrahymena thermophila. Loss of these proteins leads to ciliary defects and loss of additional MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost in RIB72A/B knockout Tetrahymena axonemes. We identified a number of candidate MIPs and pursued one, Fap115, for functional characterization. We find that loss of Fap115 results in disrupted cell swimming and aberrant ciliary beating. Cryo-electron tomography reveals that Fap115 localizes to MIP6a in the A-tubule of the doublet microtubules. Overall, our results highlight the complex relationship between MIPs, ciliary structure, and ciliary function.     

Dynein heavy chain isoforms and axonemal motility

The translocation of dynein along microtubules is the basis for a wide variety of essential cellular movements. Dynein was first discovered in the ciliary axoneme, where it causes the directed sliding between outer doublet microtubules that underlies ciliary bending. The initiation and propagation of ciliary bends are produced by a precisely located array of different dyneins containing eight or more different dynein heavy chain isoforms. The detailed clarification of the structural and functional diversity of axonemal dynein heavy chains will not only provide the key to understanding how cilia function, but also give insights applicable to the study of non-axonemal microtubule motors.     

The structure of the tips of mammalian respiratory cilia

Summary The ciliary crown and the relationship of the ciliary crown to the underlying axoneme were studied by electron microscopy in cilia from hamster and rat trachea and bronchioles, and rabbit trachea. The ciliary crown is a cluster of 4 to 6 fibrils 35 nm long protruding beyond the plasma membrane at the tips of the cilia. The fibrils are well preserved after tannic acidglutaraldehyde-osmium tetroxide fixation and have high contrast with a periodic density of 4.5 nm. They stain relatively weakly with phosphotungstic acid. The surface of the fibrils stains with ruthenium red.The microtubules of the axoneme end in a plate of electron dense amorphous material. A five layered disc occupies the space between the membrane and the amorphous plate at the tip of the axoneme. The plasma membrane can be dissolved with the detergent triton X-100 without loss of the ciliary crown. This indicates that the ciliary crown is composed of transmembranous filaments which are bound to the disc at the tip of the axoneme.Supported by U.S.P.H.S. Research Grant number HL-12650     

Tracheal motile cilia in mice require CAMSAP3 for the formation of central microtubule pair and coordinated beating

Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body (BB), which are linked by a “transition zone” (TZ). The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), a protein that can stabilize the minus-end of a microtubule, concentrates at multiple sites of the cilium–BB complex, including the upper region of the TZ or the axonemal basal plate (BP) where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the BP, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme and thereby supports the coordinated motion of multicilia in airway epithelial cells.     

Rpgrip1l controls ciliary gating by ensuring the proper amount of Cep290 at the vertebrate transition zone

A range of severe human diseases called ciliopathies is caused by the dysfunction of primary cilia. Primary cilia are cytoplasmic protrusions consisting of the basal body (BB), the axoneme, and the transition zone (TZ). The BB is a modified mother centriole from which the axoneme, the microtubule-based ciliary scaffold, is formed. At the proximal end of the axoneme, the TZ functions as the ciliary gate governing ciliary protein entry and exit. Since ciliopathies often develop due to mutations in genes encoding proteins that localize to the TZ, the understanding of the mechanisms underlying TZ function is of eminent importance. Here, we show that the ciliopathy protein Rpgrip1l governs ciliary gating by ensuring the proper amount of Cep290 at the vertebrate TZ. Further, we identified the flavonoid eupatilin as a potential agent to tackle ciliopathies caused by mutations in RPGRIP1L as it rescues ciliary gating in the absence of Rpgrip1l.     

CFAP53 regulates mammalian cilia-type motility patterns through differential localization and recruitment of axonemal dynein components

Motile cilia can beat with distinct patterns, but how motility variations are regulated remain obscure. Here, we have studied the role of the coiled-coil protein CFAP53 in the motility of different cilia-types in the mouse. While node (9+0) cilia of Cfap53 mutants were immotile, tracheal and ependymal (9+2) cilia retained motility, albeit with an altered beat pattern. In node cilia, CFAP53 mainly localized at the base (centriolar satellites), whereas it was also present along the entire axoneme in tracheal cilia. CFAP53 associated tightly with microtubules and interacted with axonemal dyneins and TTC25, a dynein docking complex component. TTC25 and outer dynein arms (ODAs) were lost from node cilia, but were largely maintained in tracheal cilia of Cfap53^-/- mice. Thus, CFAP53 at the base of node cilia facilitates axonemal transport of TTC25 and dyneins, while axonemal CFAP53 in 9+2 cilia stabilizes dynein binding to microtubules. Our study establishes how differential localization and function of CFAP53 contributes to the unique motion patterns of two important mammalian cilia-types.     

Development of the ciliary complex and microtubules in the cells of rat subcommissural organ

Summary The fine structure of the rat subcommissural organs from the late stages of gestation through the postnatal to the adult stages was studied with the electron microscope. Emphasis in this report is placed on the development of the cilium with its affiliated structures. With the progress of cytodifferentiation centrioles originally located in the Golgi region migrate to the cell apex, where each then serves as a basal body to form a cilium which has a 9+2 organization of substructures. Thus, each of the mature subcommissural cells is provided with two cilia of motile type. Satellites first appear on one side of the basal body at about 17 fetal days, rapidly increase in number with age, and finally surround the basal body, forming an elaborate latticework. In the perinatal period microtubules progressively increase in number in the distal cytoplasm, which concurrently elongates and forms a prominent projection in the brain ventricle. Closely associated with the microtubules are large clusters of dense granular masses with an undefined border, which bear a close resemblance to the dense masses appearing in the differentiating cells of respiratory epithelium and having been generally assumed to be the precursor substance for centriole replication. However, the mature subcommissural cells contain no centrioles other than the preexisting pair, each of which has organized a cilium. The dense masses in the subcommissural cells are presumed to be involved in the formation of the cytoplasmic microtubules instead of new centrioles.Work supported by the National Science Council, the Republic of China, and by the China Medical Board of New York, Inc. A preliminary report was presented at the 6th International Congress for Electron Microscopy, Kyoto, 1966 (Lin, H.-S., andI-1. Chen: Satellites of the ciliary basal body and microtubules in the cells of the rat subcommissural organ. In: Electron Microscopy (R. Ueda, ed.) Vol. II, 461–462. Tokyo: Maruzen Co., Ltd. 1966).     

Ultrastructure of cilia and flagella - back to the future!

Eukaryotic cilia and flagella perform motility and sensory functions which are essential for cell survival in protozoans, and to organism development and homoeostasis in metazoans. Their ultrastructure has been studied from the early beginnings of electron microscopy, and these studies continue to contribute to much of our understanding about ciliary biology. In the light of the progress made in the visualization of cellular structures over the last decade, we revisit the ultrastructure of cilia and flagella. We briefly describe the typical features of a 9+2 axoneme before focusing extensively on the transition zone, the ciliary necklace, the singlet zone, the ciliary cap and the ciliary crown. We discuss how the singlet zone is linked to sensory and/or motile function, the contribution of the ciliary crown to ovocyte and mucosal propulsion, and the relationship between the ciliary cap and microtubule growth and shortening, and its relation to ciliary beat. We further examine the involvement of the transition zone/the ciliary necklace in axonemal stabilization, autotomy and as a diffusion barrier.     

Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms

Seven monoclonal antibodies raised against tubulin from the axonemes of sea urchin sperm flagella recognize an acetylated form of alpha-tubulin present in the axoneme of a variety of organisms. The antigen was not detected among soluble, cytoplasmic alpha-tubulin isoforms from a variety of cells. The specificity of the antibodies was determined by in vitro acetylation of sea urchin and Chlamydomonas cytoplasmic tubulins in crude extracts. Of all the acetylated polypeptides in the extracts, only alpha-tubulin became antigenic. Among Chlamydomonas tubulin isoforms, the antibodies recognize only the axonemal alpha-tubulin isoform acetylated in vivo on the epsilon-amino group of lysine(s) (L'Hernault, S.W., and J.L. Rosenbaum, 1985, Biochemistry, 24:473-478). The antibodies do not recognize unmodified axonemal alpha-tubulin, unassembled alpha-tubulin present in a flagellar matrix-plus-membrane fraction, or soluble, cytoplasmic alpha-tubulin from Chlamydomonas cell bodies. The antigen was found in protein fractions that contained axonemal microtubules from a variety of sources, including cilia from sea urchin blastulae and Tetrahymena, sperm and testis from Drosophila, and human sperm. In contrast, the antigen was not detected in preparations of soluble, cytoplasmic tubulin, which would not have contained tubulin from stable microtubule arrays such as centrioles, from unfertilized sea urchin eggs, Drosophila embryos, and HeLa cells. Although the acetylated alpha-tubulin recognized by the antibodies is present in axonemes from a variety of sources and may be necessary for axoneme formation, it is not found exclusively in any one subset of morphologically distinct axonemal microtubules. The antigen was found in similar proportions in fractions from sea urchin sperm axonemes enriched for central pair or outer doublet B or outer doublet A microtubules. Therefore the acetylation of alpha-tubulin does not provide the mechanism that specifies the structure of any one class of axonemal microtubules. Preliminary evidence indicates that acetylated alpha-tubulin is not restricted to the axoneme. The antibodies described in this report may allow us to deduce the role of tubulin acetylation in the structure and function of microtubules in vivo.     

Ciliary specializations in branchial stigmatal cells of protochordates

Tissues from the pharynx of five representative species of the protochordates (subphylum Tunicata, the three classes Ascidiacea, Thaliacea and Appendicularia, and subphylum Cephalochordata) were examined in both thin sections and freeze-fracture replicas. In all species, the stigmatal cilia of the branchial chamber are neatly arranged and move continuously to propel sea-water in a fixed direction for respiration and feeding of the organism. A number of specializations are found in the basal region of these cilia and are represented by: a) bridges connecting axonemal doublets numbers 5 and 6; b) dense fibrous material linking the doublet microtubules of the axoneme to the ciliary membrane, sometimes in the shape of longitudinal strands or as clusters of filaments; c) intramembrane particles (IMPs) associated with the P-face of the membrane, often arranged in clusters evenly aligned along the ciliary shaft in relation to the underlying axonemal doublets. Ciliary specializations are distributed along the plane of the effective stroke of the beat in both the ascidian Botryllus schlosseri and in the thaliacean Pyrosoma atlanticum and the amphioxus Branchiostoma lanceolatum, whereas in the thaliacean Doliolum nationalis and the appendicularian Oikopleura dioica a more uniform distribution of these specializations all around the basal portion of the cilia is observed. Whatever the disposition of the ciliary specializations in all the examined species, they are always present at the base of the water-propelling cilia. Some morphological evidence suggests that these specializations play a mechanical function in tethering the ciliary membrane to the axoneme. We propose that they help maintain the orientation of the cilia during beating, enhance their stiffness and improve their efficiency.     

Specific Features of Centriole Formation and Ciliogenesis in Ciliary Epithelium Cells of Respiratory Tracts in Patients with Kartagener Syndrome

An electron microscopic study of the ciliary epithelium of respiratory tracts was carried out in children (members of the same family) with Kartagener syndrome, which is a variant of ciliary dyskinesia. It was shown that in the case of both mobile cilia and ciliary dyskinesia in man, centrioles are formed during formation of the ciliary basal bodies predominantly de novo, involving deuterosomes. A wide spectrum of pathological changes was described in literature, such as the absence of dynein arms in the axoneme and disorganization of axoneme structure. In addition to these changes in the ciliary system, we found integration of several ciliary axonemes by the same plasma membrane, running of microtubules from the plasma membrane as bundles, different orientation of basal legs, etc.__________Translated from Ontogenez, Vol. 36, No. 3, 2005, pp. 190–198.Original Russian Text Copyright © 2005 by Domaratskii, Uvakina, Volkov, Onishchenko.     

Immune localization of calmodulin in the ciliated cells of hamster tracheal epithelium

Melachronous beating of cilia of epithelial surfaces of most respiratory airways moves the overlying mucous layer in a caudal direction. The molecular mechanisms controlling ciliary beat remain largely unknown. Calcium, an element in its cationic form, is ubiquitous in biological functions and its concentration is critical for ciliary beating. Calmodulin, a calcium-binding protein which regulates the activity of many enzymes and cellular processes, may regulate ciliary beating by controlling enzymes responsible for mechanochemical movement between adjacent peripheral microtubule doublets composing the ciliary axoneme. As a first step in describing a calmodulin-related controlling mechanism for ciliary beating, calmodulin was localized in the ciliated cells lining the respiratory tracts of hamsters by electron microscopy, using an indirect immunoperoxidase technique with anticalmodulin antibodies as the molecular probe. Thin-sections revealed calmodulin located on microtubules and dynein arms of the ciliary shaft, basal body, apical cytoskeletal microtubules, and plasma membranes in specimens fixed with 1 mM Ca+2. Specimens fixed with less Ca+2 (1 microM), Mn+2, Mg+2, and EGTA showed a diffuse pattern of calmodulin with loci of greatest densities on basal body microtubule triplets. Demembranated specimens showed a less specific localization on axonemal microtubules but only on cells fixed with Ca+2. Calmodulin, by binding calcium, may function in ciliary beating in the respiratory tract of mammals either directly or indirectly through its effects on the energy-producing enzymes and by control of Ca+2 flux through plasma membranes.