Basal Body Assembly in Ciliates: The Power of Numbers

Centrioles perform the dual functions of organizing both centrosomes and cilia. The biogenesis of nascent centrioles is an essential cellular event that is tightly coupled to the cell cycle so that each cell contains only two or four centrioles at any given point in the cell cycle. The assembly of centrioles and their analogs, basal bodies, is well characterized at the ultrastructural level whereby structural modules are built into a functional organelle. Genetic studies in model organisms combined with proteomic, bioinformatic and identifying ciliary disease gene orthologs have revealed a wealth of molecules requiring further analysis to determine their roles in centriole duplication, assembly and function. Nonetheless, at this stage, our understanding of how molecular components interact to build new centrioles and basal bodies is limited. The ciliates, Tetrahymena and Paramecium , historically have been the subject of cytological and genetic study of basal bodies. Recent advances in the ciliate genetic and molecular toolkit have placed these model organisms in a favorable position to study the molecular mechanisms of centriole and basal body assembly.     

Elucidation of basal body and centriole functions in Chlamydomonas reinhardtii

In eukaryotic cells, basal bodies and their structural equivalents, centrioles, play essential roles. They are needed for the assembly of flagella or cilia as well as for cell division. Chlamydomonas reinhardtii provides an excellent model organism for the study of the basal body and centrioles. Genes for two new members of the tubulin superfamily are needed for basal body/centriole duplication. In addition, other genes that play roles in the duplication and segregation of basal bodies are discussed.     

Replication of basal bodies and centrioles.

During the past year, studies on the centrioles and basal bodies of animal and algal cells, and the spindle pole bodies of yeast and other fungi, have added significantly to our knowledge of how these cell organelles form and how they function in initiating microtubule assembly throughout the cell cycle. Most of these studies have used antibodies to identify proteins within and around these organelles and, in some cases, to disrupt their ability to nucleate microtubules. Genetic methods have been used to identify specific proteins, including a new member of the tubulin superfamily, involved in the function and replication of spindle pole bodies and centrioles.     

Structural inheritance in Paramecium: ultrastructural evidence for basal body and associated rootlets polarity transmission through binary fission

Abstract One main difference between basal bodies and centrioles resides in the expression of their polarity: centrioles display a structural nine‐fold radial symmetry, whereas basal bodies express a circumferential polarity, thanks to their asymmetric set of rootlets. The origin of this polarity during organelle duplication still remains under debate: is it intrinsic to the nine‐fold structure itself (i.e. the nine microtubular triplets are not equivalent) or imposed by its immediate environment at time of assembly? We have reinvestigated this problem using the Ciliate Paramecium, in which the pattern of basal body duplication is well known. In this cell, all basal bodies produced within ciliary rows appear immediately anterior to parental ones. Observations on cells fixed with the tannic acid protocol suggest that, to be competent for basal body assembly, parental basal bodies have to be individually associated with a complete set of rootlets (monokinetid structure). During pro‐basal body assembly, full microtubular triplets were detected according to a random circumferential sequence; during the whole process, the new basal body and its associated rootlets maintained structural relations with the parental monokinetid structure by way of specific links. These results strongly suggest that basal body and associated rootlets (kinetid) polarity is driven by its immediate environment and provide a basis for the structural heredity property observed by Sonneborn some decades ago.     

Polarities of the centriolar structure: morphogenetic consequences.

Centrioles and basal bodies are two versions of the same conserved eukaryotic organelle and share two remarkable properties: nine-fold symmetry of their microtubular shaft and capacity to generate a new organelle in a fixed geometrical relationship to the mother organelle. It can thus be postulated that what is true for basal bodies is likely to be true also for centrioles. While the functions of centrioles are difficult to dissect, the functions of basal bodies are easier to approach. Over more than two decades, studies on protists have led to the notion that ciliary and flagellar basal bodies display polarities, not only a proximo-distal polarity, like in centrioles, but also a circumferential polarity accorded to the polarities of the cell and of its cytoskeleton. The major cytological and genetical data, mainly of Chlamydomonas, Paramecium and Tetrahymena, which support the notion that the microtubule triplets of basal bodies are non-equivalent, are reviewed. The morphogenetic implications of this circumferential anisotropy, perpetuated through the process of basal body duplication itself, are discussed. The question is raised of the possibility that centrioles also display a circumferential polarity, like basal bodies, and whether at least certain of their functions depend on such asymmetries.     

Centrioles want to move out and make cilia

Cilia formation in mammalian cells requires basal bodies that are either derived from centrioles that transition from their cytoplasmic role in centrosome organization or that form en masse in multiciliated cells. Several recent studies have begun to uncover the links between centriole duplication and their transformation to basal bodies.     

Molecular characterization of centriole assembly in ciliated epithelial cells

Ciliated epithelial cells have the unique ability to generate hundreds of centrioles during differentiation. We used centrosomal proteins as molecular markers in cultured mouse tracheal epithelial cells to understand this process. Most centrosomal proteins were up-regulated early in ciliogenesis, initially appearing in cytoplasmic foci and then incorporated into centrioles. Three candidate proteins were further characterized. The centrosomal component SAS-6 localized to basal bodies and the proximal region of the ciliary axoneme, and depletion of SAS-6 prevented centriole assembly. The intraflagellar transport component polaris localized to nascent centrioles before incorporation into cilia, and depletion of polaris blocked axoneme formation. The centriolar satellite component PCM-1 colocalized with centrosomal components in cytoplasmic granules surrounding nascent centrioles. Interfering with PCM-1 reduced the amount of centrosomal proteins at basal bodies but did not prevent centriole assembly. This system will help determine the mechanism of centriole formation in mammalian cells and how the limitation on centriole duplication is overcome in ciliated epithelial cells.     

Rootletin interacts with C-Nap1 and may function as a physical linker between the pair of centrioles/basal bodies in cells

Rootletin, a major structural component of the ciliary rootlet, is located at the basal bodies and centrosomes in ciliated and nonciliated cells, respectively. Here we investigated its potential role in the linkage of basal bodies/centrioles and the mechanism involved in such linkages. We show that rootletin interacts with C-Nap1, a protein restricted at the ends of centrioles and functioning in centrosome cohesion in interphase cells. Their interaction in vivo is supported by their colocalization at the basal bodies/centrioles and coordinated association with the centrioles during the cell cycle. Ultrastructural examinations demonstrate that rootletin fibers connect the basal bodies in ciliated cells and are present both at the ends of and in between the pair of centrioles in nonciliated cells. The latter finding stands in contrast with C-Nap1, which is present only at the ends of the centrioles. Transient expression of C-Nap1 fragments dissociated rootletin fibers from the centrioles, resulting in centrosome separation in interphase. Overexpression of rootletin in cells caused multinucleation, micronucleation, and irregularity of nuclear shape and size, indicative of defects in chromosome separation. These data suggest that rootletin may function as a physical linker between the pair of basal bodies/centrioles by binding to C-Nap1.     

Proteomic analysis of isolated chlamydomonas centrioles reveals orthologs of ciliary-disease genes

BACKGROUND: The centriole is one of the most enigmatic organelles in the cell. Centrioles are cylindrical, microtubule-based barrels found in the core of the centrosome. Centrioles also act as basal bodies during interphase to nucleate the assembly of cilia and flagella. There are currently only a handful of known centriole proteins. RESULTS: We used mass-spectrometry-based MudPIT (multidimensional protein identification technology) to identify the protein composition of basal bodies (centrioles) isolated from the green alga Chlamydomonas reinhardtii. This analysis detected the majority of known centriole proteins, including centrin, epsilon tubulin, and the cartwheel protein BLD10p. By combining proteomic data with information about gene expression and comparative genomics, we identified 45 cross-validated centriole candidate proteins in two classes. Members of the first class of proteins (BUG1-BUG27) are encoded by genes whose expression correlates with flagellar assembly and which therefore may play a role in ciliogenesis-related functions of basal bodies. Members of the second class (POC1-POC18) are implicated by comparative-genomics and -proteomics studies to be conserved components of the centriole. We confirmed centriolar localization for the human homologs of four candidate proteins. Three of the cross-validated centriole candidate proteins are encoded by orthologs of genes (OFD1, NPHP-4, and PACRG) implicated in mammalian ciliary function and disease, suggesting that oral-facial-digital syndrome and nephronophthisis may involve a dysfunction of centrioles and/or basal bodies. CONCLUSIONS: By analyzing isolated Chlamydomonas basal bodies, we have been able to obtain the first reported proteomic analysis of the centriole.     

Visualization of centrioles and basal bodies by fluorescent staining with nonimmune rabbit sera

Several sera from nonimmunized rabbits have been found which stain centrioles and basal bodies by indirect immunofluorescence in a wide variety of cell types. So far, approximately 10% of the rabbit sera that we have examined gave strong positive staining of centrioles and basal bodies. Cytoplasmic networks, mitotic spindles, and ciliary axonemes, however, remain unstained. This specific fluorescent staining of centrioles and basal bodies could not be abolished by absorption of sera with purified brain tubulin. This technique is superior to previous methods for the visualization of basal bodies and centrioles at the light microscopic level and should be useful for rapid and convenient detection of these organelles in large populations of cells.     

Long-lost relatives reappear: identification of new members of the tubulin superfamily

The identification and analysis of new members of the tubulin superfamily has advanced the belief that these tubulins play important roles in the duplication and assembly of centrioles and basal bodies. This idea is supported by their distribution in organisms with centrioles containing triplet microtubules and by recent functional analysis of the new tubulins. delta- and epsilon-tubulin are found in most organisms that assemble triplet microtubules. delta-tubulin is needed for maintaining triplet microtubules in Chlamydomonas and Paramecium. epsilon-tubulin is needed for centriole and basal body duplication and is an essential gene in Chlamydomonas. The distribution of eta-tubulin is more limited and has been found in only four organisms to date. Phylogenetic analysis suggests that it is most closely related to delta-tubulin, which suggests that delta- and eta-tubulin could have overlapping functions.     

Centriole maturation and transformation to basal body

Centrioles and basal bodies are fascinating and mysterious organelles. They interconvert and seem to be crucial for a wide range of crucial cellular processes. However, intense research over the last years suggested that centrioles/basal bodies are essential mainly for the generation of cilia. Although a neglected organelle over a long time, interest in the primary cilia was recently rekindled by the notion that they are affected in a number of human diseases. Cilia formation is an intricate process that starts with the transformation of centrioles to basal bodies and their docking to the apical plasma membrane. Disturbance of basal body formation thus might cause ciliopathies. This review focuses on the formation of basal bodies in mammalian cells with an emphasis on basal bodies sprouting a primary cilium.     

Cilia-lacking respiratory cells in ciliary aplasia

This report describes the ultrastructural alterations observed in the nasal and bronchial mucosa of an 11-yr-old male suffering from immotile cilia syndrome (ICS). The morphological features observed in this patient are consistent with a ciliary aplasia. In fact, ciliated cells appeared to be replaced by columnar cells lacking cilia and basal bodies, and bearing on their surface cilium-like projections without any internal axonemal structure. In spite of the absence of basal bodies, centrioles, and kinocilia, these cells unexpectedly showed mature striated roots and centriolar precursor material scattered throughout the apical cytoplasm. These data suggest that control over basal body assembly is distinct from control over striated root formation. The presence of the above-reported structures in cells otherwise presenting many morphological features of normal ciliated cells is discussed on the basis of current knowledge of respiratory cilia biogenesis.     

Bld10p, a novel protein essential for basal body assembly in Chlamydomonas: localization to the cartwheel, the first ninefold symmetrical structure appearing during assembly

How centrioles and basal bodies assemble is a long-standing puzzle in cell biology. To address this problem, we analyzed a novel basal body-defective Chlamydomonas reinhardtii mutant isolated from a collection of flagella-less mutants. This mutant, bld10, displayed disorganized mitotic spindles and cytoplasmic microtubules, resulting in abnormal cell division and slow growth. Electron microscopic observation suggested that bld10 cells totally lack basal bodies. The product of the BLD10 gene (Bld10p) was found to be a novel coiled-coil protein of 170 kD. Immunoelectron microscopy localizes Bld10p to the cartwheel, a structure with ninefold rotational symmetry positioned near the proximal end of the basal bodies. Because the cartwheel forms the base from which the triplet microtubules elongate, we suggest that Bld10p plays an essential role in an early stage of basal body assembly. A viable mutant having such a severe basal body defect emphasizes the usefulness of Chlamydomonas in studying the mechanism of basal body/centriole assembly by using a variety of mutants.     

Cep120 is asymmetrically localized to the daughter centriole and is essential for centriole assembly

Centrioles form the core of the centrosome in animal cells and function as basal bodies that nucleate and anchor cilia at the plasma membrane. In this paper, we report that Cep120 (Ccdc100), a protein previously shown to be involved in maintaining the neural progenitor pool in mouse brain, is associated with centriole structure and function. Cep120 is up-regulated sevenfold during differentiation of mouse tracheal epithelial cells (MTECs) and localizes to basal bodies. Cep120 localizes preferentially to the daughter centriole in cycling cells, and this asymmetry between mother and daughter centrioles is relieved coincident with new centriole assembly. Photobleaching recovery analysis identifies two pools of Cep120, differing in their halftime at the centriole. We find that Cep120 is required for centriole duplication in cycling cells, centriole amplification in MTECs, and centriole overduplication in S phase-arrested cells. We propose that Cep120 is required for centriole assembly and that the observed defect in neuronal migration might derive from a defect in this process.     

Centrin deficiency in Paramecium affects the geometry of basal-body duplication

BACKGROUND: Ciliary or flagellar basal bodies and centrioles share the same architecture and remarkable property of duplicating once per cell cycle. Duplication is known to proceed by budding of the daugther organelle close to and at right angles to the mother structure, but the molecular basis of this geometry remains unknown. Among the handful of proteins implicated in basal-body/centriole duplication, centrins seem required in all eukaryotes tested, but their mode of action is not clear. We have investigated centrin function in Paramecium, whose cortical organization allows detection of any spatial or temporal alteration in the pattern of basal-body duplication. RESULTS: We have characterized two pairs of genes, PtCEN2a and PtCEN2b as well as PtCEN3a and PtCEN3b, orthologs of HsCEN2 and HsCEN3, respectively. GFP tags revealed different localization for the two pairs of gene products, at basal bodies or on basal-body-associated filamentous arrays, respectively. Centrin depletion induced by RNAi caused mislocalization of the neoformed basal bodies: abnormal site of budding (PtCen2ap) or absence of separation between mother and daughter organelles (PtCen3ap). Over successive divisions, new basal bodies continued to be assembled, but internalization of the mispositionned basal bodies led to a progressive decrease in the number of cortical basal bodies. CONCLUSIONS: Our observations show that centrins (1) are required to define the site and polarities of duplication and to sever the mother-daughter links and (2) play no triggering or instrumental role in assembly. Our data underscore the biological importance of the geometry of the duplication process.     

Evidence for a functional role of RNA in centrioles.

Basal bodies, purified from Chlamydomonas and Tetrahymena, were exposed to various enzymatic treatments and then assayed for their ability to nucleate aster formation upon injection into eggs of Xenopus laevis. Untreated basal bodies injected into frog eggs act as centrioles and induce the formation of asters. The aster-inducing activity of basal bodies was eliminated by treatment with proteolytic enzymes and ribonucleases. Aster-inducing activity was not affected by DNAse and a number of other enzymes. The effect of proteolytic digestion on aster-inducing activity appeared to be directly correlated with the degree of structural damage to the basal body. Low concentrations of pancreatic ribonuclease A, ribonuclease T1, and S1 nuclease also completely abolished aster-inducing activity, although these enzymes had no effect on basal body structure. Ribonuclease-treated basal bodies remained capable of supporting microtubule elongation in vitro. Preliminary evidence indicates that basal bodies from Chlamydomonas and Tetrahymena contain about 5 x 10(-16) g of RNA which co-band with basal bodies and aster-inducing activity by equilibrium density gradient sedimentation. We conclude first, that centrioles contain RNA which is required for initiation of aster formation, and second, that the centriole activity or ability to assemble a mitotic aster is separable from the basal body activity, or ability to serve directly as a template for microtubule growth.     

Centriole and basal body formation during ciliogenesis revisited.

This review is concerned with the formation during ciliogenesis of centrioles and basal bodies, primarily in epithelial multi-ciliated cells from the developing vertebrate respiratory and reproductive tracts. During ciliated cell differentiation, in these as well as in other cell types, cilium formation is preceded by the formation of centrioles assembled from precursor structures having little resemblance to the mature organelle. The origin, composition and function of the centriole precursor structures in generating large numbers of centrioles in a short period of time during ciliogenesis is discussed. This review also focuses on the biochemistry of centrioles and basal bodies and on recent experimental evidence that DNA might be associated with these structures.     

Regulating the transition from centriole to basal body

The role of centrioles changes as a function of the cell cycle. Centrioles promote formation of spindle poles in mitosis and act as basal bodies to assemble primary cilia in interphase. Stringent regulations govern conversion between these two states. Although the molecular mechanisms have not been fully elucidated, recent findings have begun to shed light on pathways that regulate the conversion of centrioles to basal bodies and vice versa. Emerging studies also provide insights into how defects in the balance between centrosome and cilia function could promote ciliopathies and cancer.     

Drosophila Bld10 Is a Centriolar Protein That Regulates Centriole, Basal Body, and Motile Cilium Assembly

Cilia and flagella play multiple essential roles in animal development and cell physiology. Defective cilium assembly or motility represents the etiological basis for a growing number of human diseases. Therefore, how cilia and flagella assemble and the processes that drive motility are essential for understanding these diseases. Here we show that Drosophila Bld10, the ortholog of Chlamydomonas reinhardtii Bld10p and human Cep135, is a ubiquitous centriolar protein that also localizes to the spermatid basal body. Mutants that lack Bld10 assemble centrioles and form functional centrosomes, but centrioles and spermatid basal bodies are short in length. bld10 mutant flies are viable but male sterile, producing immotile sperm whose axonemes are deficient in the central pair of microtubules. These results show that Drosophila Bld10 is required for centriole and axoneme assembly to confer cilium motility.