Branchial Cilia and Sperm Flagella Recruit Distinct Axonemal Components

Eukaryotic cilia and flagella have highly conserved 9 + 2 structures. They are functionally diverged to play cell-type-specific roles even in a multicellular organism. Although their structural components are therefore believed to be common, few studies have investigated the molecular diversity of the protein components of the cilia and flagella in a single organism. Here we carried out a proteomic analysis and compared protein components between branchial cilia and sperm flagella in a marine invertebrate chordate, Ciona intestinalis. Distinct feature of protein recruitment in branchial cilia and sperm flagella has been clarified; (1) Isoforms of α- and β-tubulins as well as those of actins are distinctly used in branchial cilia or sperm flagella. (2) Structural components, such as dynein docking complex, tektins and an outer dense fiber protein, are used differently by the cilia and flagella. (3) Sperm flagella are specialized for the cAMP- and Ca²⁺-dependent regulation of outer arm dynein and for energy metabolism by glycolytic enzymes. Our present study clearly demonstrates that flagellar or ciliary proteins are properly recruited according to their function and stability, despite their apparent structural resemblance and conservation.     

Intraflagellar transport particles participate directly in cilium-generated signaling in Chlamydomonas

Primary cilia are widely used for signal transduction during development and in homeostasis and are assembled and maintained by intraflagellar transport (IFT). Here, we have dissected the role of IFT in signaling within the flagella (structural and functional counterparts of cilia) of the biflagellated green alga Chlamydomonas. Using a conditional IFT mutant enables us to deplete the IFT machinery from intact, existing flagella. We identify a cGMP-dependent protein kinase (CrPKG) within flagella as the substrate of a protein tyrosine kinase activated by flagellar adhesion during fertilization. We demonstrate that flagellar adhesion stimulates association of CrPKG with a new flagellar compartment. Moreover, formation of the compartment requires IFT, and IFT particles themselves are part of the compartment. Our results lead to a model in which the IFT machinery is required not only for assembling cilia and flagella but also for organizing a signaling pathway within the organelles during cilium-generated signaling.     

Pix Proteins and the Evolution of Centrioles

We have made a wide phylogenetic survey of Pix proteins, which are constituents of vertebrate centrioles in most eukaryotes. We have also surveyed the presence and structure of flagella or cilia and centrioles in these organisms, as far as is possible from published information. We find that Pix proteins are present in a vast range of eukaryotes, but not all. Where centrioles are absent so are Pix proteins. If one considers the maintenance of Pix proteins over evolutionary time scales, our analysis would suggest that their key function is to make cilia and flagella, and the same is true of centrioles. Moreover, this survey raises the possibility that Pix proteins are only maintained to make cilia and flagella that undulate, and even then only when they are constructed by transporting ciliary constituents up the cilium using the intraflagellar transport (IFT) system. We also find that Pix proteins have become generally divergent within Ecdysozoa and between this group and other taxa. This correlates with a simplification of centrioles within Ecdysozoa and a loss or divergence of cilia/flagella. Thus Pix proteins act as a weathervane to indicate changes in centriole function, whose core activity is to make cilia and flagella.     

Centrosomes and cilia in human disease

Centrioles are microtubule-derived structures that are essential for the formation of centrosomes, cilia and flagella. The centrosome is the major microtubule organiser in animal cells, participating in a variety of processes, from cell polarisation to cell division, whereas cilia and flagella contribute to several mechanisms in eukaryotic cells, from motility to sensing. Although it was suggested more than a century ago that these microtubule-derived structures are involved in human disease, the molecular bases of this association have only recently been discovered. Surprisingly, there is very little overlap between the genes affected in the different diseases, suggesting that there are tissue-specific requirements for these microtubule-derived structures. Knowledge of these requirements and disease mechanisms has opened new avenues for therapeutical strategies. Here, we give an overview of recent developments in this field, focusing on cancer, diseases of brain development and ciliopathies.     

Mechanisms of mammalian ciliary motility: Insights from primary ciliary dyskinesia genetics

Motile cilia and flagella are organelles that, historically, have been poorly understood and inadequately investigated. However, cilia play critical roles in fluid clearance in the respiratory system and the brain, and flagella are required for sperm motility. Genetic studies involving human patients and mouse models of primary ciliary dyskinesia over the last decade have uncovered a number of important ciliary proteins and have begun to elucidate the mechanisms underlying ciliary motility. When combined with genetic, biochemical, and cell biological studies in Chlamydomonas reinhardtii, these mammalian genetic analyses begin to reveal the mechanisms by which ciliary motility is regulated.     

Structure and function of vertebrate cilia, towards a new taxonomy

In this review, we propose a new classification of vertebrate cilia/flagella and discuss the evolution and prototype of cilia. Cilia/flagella are evolutionarily well-conserved membranous organelles in eukaryotes and serve a variety of functions, including motility and sensation. Vertebrate cilia have been traditionally classified into conventional motile cilia and sensory primary cilia. However, an avalanche of emerging evidence on the variations of cilia has made it almost impossible to classify them in a simple dichotomic manner. For example, conventional motile cilia are also involved in the sensation of bitter taste to facilitate the beating of cilia as a defense system of the respiratory system. On the other hand, the primary cilium, often regarded as a non-motile sensory organelle, has been revealed to be motile in vertebrate embryonic nodes, where they play a crucial role in the determination of left-right asymmetry of the body. Moreover, choroid plexus epithelial cells in the cerebral ventricular system exhibit multiple primary cilia on a single cell. Considering these lines of evidence on the diversity of cilia, we believe the classification of cilia should be based on their structure and function, and include more detailed criteria. Another intriguing issue is how in the evolution of cilia, their function and morphology are combined. For example, has motility been acquired from originally sensory cilia, or vice versa? Alternatively, were they originally hybrid in nature? These questions are inseparable from the classification of cilia per se. We would like to address these conundrums in this review article, principally from the standpoint of differentiation of the animal cell.     

Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella

Intraflagellar transport (IFT) is a rapid movement of multi-subunit protein particles along flagellar microtubules and is required for assembly and maintenance of eukaryotic flagella. We cloned and sequenced a Chlamydomonas cDNA encoding the IFT88 subunit of the IFT particle and identified a Chlamydomonas insertional mutant that is missing this gene. The phenotype of this mutant is normal except for the complete absence of flagella. IFT88 is homologous to mouse and human genes called Tg737. Mice with defects in Tg737 die shortly after birth from polycystic kidney disease. We show that the primary cilia in the kidney of Tg737 mutant mice are shorter than normal. This indicates that IFT is important for primary cilia assembly in mammals. It is likely that primary cilia have an important function in the kidney and that defects in their assembly can lead to polycystic kidney disease.     

A protein methylation pathway in Chlamydomonas flagella is active during flagellar resorption

During intraflagellar transport (IFT), the regulation of motor proteins, the loading and unloading of cargo and the turnover of flagellar proteins all occur at the flagellar tip. To begin an analysis of the protein composition of the flagellar tip, we used difference gel electrophoresis to compare long versus short (i.e., regenerating) flagella. The concentration of tip proteins should be higher relative to that of tubulin (which is constant per unit length of the flagellum) in short compared with long flagella. One protein we have identified is the cobalamin-independent form of methionine synthase (MetE). Antibodies to MetE label flagella in a punctate pattern reminiscent of IFT particle staining, and immunoblot analysis reveals that the amount of MetE in flagella is low in full-length flagella, increased in regenerating flagella, and highest in resorbing flagella. Four methylated proteins have been identified in resorbing flagella, using antibodies specific for asymmetrically dimethylated arginine residues. These proteins are found almost exclusively in the axonemal fraction, and the methylated forms of these proteins are essentially absent in full-length and regenerating flagella. Because most cells resorb cilia/flagella before cell division, these data indicate a link between flagellar protein methylation and progression through the cell cycle.     

Cell motility: deaf Drosophila keep the beat

Intraflagellar transport is involved in the assembly of cilia and eukaryotic flagella. Two recent studies have shown that defects in intraflagellar transport prevent assembly of sensory cilia in Drosophila, leaving the fly deaf and uncoordinated. Surprisingly, the mutant sperm flagella have normal structure and motility.     

Ciliary motility: the components and cytoplasmic preassembly mechanisms of the axonemal dyneins

Motile cilia and flagella are organelles, which function in cell motility and in the transport of fluids over the surface of cells. Motility defects often result in a rare human disease, primary ciliary dyskinesia (PCD). Cell motility depends on axonemal dynein, a molecular motor that drives the beating of cilia and flagella. The dyneins are composed of multiple subunits, which are thought to be preassembled in the cytoplasm before they are transported into cilia and flagella. Axonemal dyneins have been extensively studied in Chlamydomonas. In addition, analyses of human PCDs over the past decade, together with studies in other model animals, have identified the conserved components required for dynein assembly. Recently also, the first cytoplasmic component of dynein assembly, kintoun (ktu), was elucidated through the analysis of a medaka mutant in combination with human genetics and cell biology and biochemical studies of Chlamydomonas. The components of dynein and the proteins involved in its cytoplasmic assembly process are discussed.     

1001 model organisms to study cilia and flagella

Most mammalian cell types have the potential to assemble at least one cilium. Immotile cilia participate in numerous sensing processes, while motile cilia are involved in cell motility and movement of extracellular fluid. The functional importance of cilia and flagella is highlighted by the growing list of diseases due to cilia defects. These ciliopathies are marked by an amazing diversity of clinical manifestations and an often complex genetic aetiology. To understand these pathologies, a precise comprehension of the biology of cilia and flagella is required. These organelles are remarkably well conserved throughout eukaryotic evolution. In this review, we describe the strengths of various model organisms to decipher diverse aspects of cilia and flagella biology: molecular composition, mode of assembly, sensing and motility mechanisms and functions. Pioneering studies carried out in the green alga Chlamydomonas established the link between cilia and several genetic diseases. Moreover, multicellular organisms such as mouse, zebrafish, Xenopus, Caenorhabditis elegans or Drosophila, and protists such as Paramecium, Tetrahymena and Trypanosoma or Leishmania each bring specific advantages to the study of cilium biology. For example, the function of genes involved in primary ciliary dyskinesia (due to defects in ciliary motility) can be efficiently assessed in trypanosomes.     

The mouse ortholog of EFHC1 implicated in juvenile myoclonic epilepsy is an axonemal protein widely conserved among organisms with motile cilia and flagella

The gene product of EFHC1 recently implicated in juvenile myoclonic epilepsy (JME) was found to be a homolog of Chlamydomonas axonemal protein Rib72, whose homologs are present in a wide variety of organisms that have motile cilia and flagella. Western blot analyses and immunofluorescence localization of the mouse ortholog mRib72-1/Efhc1 indicated that it is indeed abundantly present in sperm flagella and tracheal cilia but only in a small amount in the brain. It is not present in immotile primary cilia. These observations raise the possibility that malfunction of motile cilia is involved in the development of JME.     

Intraflagellar transport is required in Drosophila to differentiate sensory cilia but not sperm

BACKGROUND: Intraflagellar transport (IFT) uses kinesin II to carry a multiprotein particle to the tips of eukaryotic cilia and flagella and a nonaxonemal dynein to return it to the cell body. IFT particle proteins and motors are conserved in ciliated eukaryotes, and IFT-deficient mutants in algae, nematodes, and mammals fail to extend or maintain cilia and flagella, including sensory cilia. In Drosophila, the only ciliated cells are sensory neurons and sperm. no mechanoreceptor potential (nomp) mutations have been isolated that affect the differentiation and function of ciliated sense organs. The nompB gene is here shown to encode an IFT protein. Its mutant phenotypes reveal the consequences of an IFT defect in an insect. RESULTS: Mechanosensory and olfactory neurons in nompB mutants have missing or defective cilia. nompB encodes the Drosophila homolog of the IFT complex B protein IFT88/Polaris/OSM-5. nompB is expressed in the ciliated sensory neurons, and a functional, tagged NOMPB protein is located in sensory cilia and around basal bodies. Surprisingly, nompB mutant males produce normally elongated, motile sperm. Neuronally restricted expression and male germline mosaic experiments show that nompB-deficient sperm are fully functional in transfer, competition, and fertilization. CONCLUSIONS: NOMPB, the Drosophila homolog of IFT88, is required for the assembly of sensory cilia but not for the extension or function of the sperm flagellum. Assembly of this extremely long axoneme is therefore independent of IFT.     

Cilia and ciliopathies: From Chlamydomonas and beyond

The biological function of motile cilia/flagella has long been recognized. The non-motile primary cilium, once regarded as a vestigial organelle, however, has been found recently to play unexpected roles in mammalian physiology and development. Defects in cilia have profound impact on human health. Diseases related to cilia, collectively called ciliopathies include male infertility, primary cilia dyskinesia, renal cyst formation, blindness, polydactyly, obesity, hypertension, and even mental retardation. Our current understanding of cilia and ciliopathies has been fueled by basic research employing various model organisms including Chlamydomonas, a unicellular green alga. This review article provides a general introduction to the cell biology of cilia and an overview of various cilia-related diseases.     

How do cilia organize signalling cascades?

Cilia and flagella are closely related centriole-nucleated protrusions of the cell with roles in motility and signal transduction. Two of the best-studied signalling pathways organized by cilia are the transduction cascade for the morphogen Hedgehog in vertebrates and the mating pathway that initiates gamete fusion in the unicellular green alga Chlamydomonas reinhardtii. What is the role of cilia in these signalling transduction cascades? In both Hedgehog and mating pathways, all signalling intermediates have been found to localize to cilia, and, for some signalling factors, ciliary localization is regulated by pathway activation. Given a concentration factor of three orders of magnitude provided by translocating a protein into the cilium, the compartment model proposes that cilia act as miniaturized reaction tubes bringing signalling factors and processing enzymes in close proximity. On the other hand, the scaffolding model views the intraflagellar transport machinery, whose primary function is to build cilia and flagella, as a molecular scaffold for the mating transduction cascade at the flagellar membrane. While these models may coexist, it is hoped that a precise understanding of the mechanisms that govern signalling inside cilia will provide a satisfying answer to the question ‘how do cilia organize signalling?’. This review covers the evidence supporting each model of signalling and outlines future directions that may address which model applies in given biological settings.     

Rotation and translocation of microtubules in vitro induced by dyneins from Tetrahymena cilia

Dynein, the force-generating enzyme that powers the movement of cilia and flagella, has been characterized biochemically, but no simple system has been available for examining its motile properties. Here we describe a quantitative in vitro motility assay in which dynein adsorbed onto a glass surface induces linear translocation of purified bovine microtubules. Using this assay, we show that both 22S and 14S dyneins from Tetrahymena cilia induce movement but have distinct motile properties. A unique property of 14S dynein, which has not been described for other motility proteins, is its ability to generate torque that causes microtubules to rotate during forward translocation. In the axoneme, 14S dynein-induced torque may induce rotation of central-pair microtubules and may play an important role in generating three-dimensional ciliary beating patterns.     

Primary ciliary dyskinesia in mice lacking the novel ciliary protein Pcdp1

Primary ciliary dyskinesia (PCD) results from ciliary dysfunction and is commonly characterized by sinusitis, male infertility, hydrocephalus, and situs inversus. Mice homozygous for the nm1054 mutation develop phenotypes associated with PCD. On certain genetic backgrounds, homozygous mutants die perinatally from severe hydrocephalus, while mice on other backgrounds have an accumulation of mucus in the sinus cavity and male infertility. Mutant sperm lack mature flagella, while respiratory epithelial cilia are present but beat at a slower frequency than wild-type cilia. Transgenic rescue demonstrates that the PCD in nm1054 mutants results from the loss of a single gene encoding the novel primary ciliary dyskinesia protein 1 (Pcdp1). The Pcdp1 gene is expressed in spermatogenic cells and motile ciliated epithelial cells. Immunohistochemistry shows that Pcdp1 protein localizes to sperm flagella and the cilia of respiratory epithelial cells and brain ependymal cells in both mice and humans. This study demonstrates that Pcdp1 plays an important role in ciliary and flagellar biogenesis and motility, making the nm1054 mutant a useful model for studying the molecular genetics and pathogenesis of PCD.     

Serological similarity of flagellar and mitotic microtubules

An antiserum to flagellar axonemes from sperm of Arbacia punctulata contains antibodies which react both with intact flagellar outer fibers and with purified tubulin from the outer fibers. Immunodiffusion tests indicate the presence of similar antigenic determinants on outer-fiber tubulins from sperm flagella of five species of sea urchins and a sand dollar, but not a starfish. The antibodies also react with extracts containing tubulins from different classes of microtubules, including central-pair fibers and both A- and B-subfibers from outer fibers of sperm flagella, an extract from unfertilized eggs, mitotic apparatuses from first cleavage embryos, and cilia from later embryos. Though most tubulins tested share similar antigenic determinants, some clear differences have been detected, even, in Pseudoboletia indiana, between the outer-fiber tubulins of sperm flagella and blastular cilia. Though tubulins are "actin-like" proteins, antitubulin serum does not react with actin from sea urchin lantern muscle. On the basis of these observations, we suggest that various echinoid microtubules are built of similar, but not identical, tubulins.