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.     

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.     

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.     

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.     

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.     

Basal body/centriole assembly and continuity

The long-standing interest in centrioles and basal bodies stems from the evolutionary conservation of their structural design and from their dual mode of assembly (templated versus de novo), revealed by electron microscopic studies nearly four decades ago and unique for a subcellular organelle. Molecular dissection of the assembly pathway during the past few years has recently progressed, essentially through direct and reverse genetic approaches. These studies revealed essential roles for centrins and the gamma-, delta-, epsilon - and eta-tubulins in assembly or as specific signals for centriole duplication. Identification of further components of basal bodies and centrioles might help to unravel the two assembly pathways and their regulation.     

Organelle positioning and cell polarity

In spite of conspicuous differences in their polarized architecture, swimming unicellular eukaryotes and migrating cells from metazoa display a conserved hierarchical interlocking of the main cellular compartments, in which the microtubule network has a dominant role. A microtubule array can organize the distribution of endomembranes owing to a cell-wide and polarized extension around a unique nucleus-associated structure. The nucleus-associated structure in animal cells contains a highly conserved organelle, the centriole or basal body. This organelle has a defined polarity that can be transmitted to the cell. Its conservative mode of duplication seems to be a core mechanism for the transmission of polarities through cell division.     

Cep70 and Cep131 contribute to ciliogenesis in zebrafish embryos

Background  

The centrosome is the cell's microtubule organising centre, an organelle with important roles in cell division, migration and polarity. However, cells can divide and flies can, for a large part of development, develop without them. Many centrosome proteins have been identified but the roles of most are still poorly understood. The centrioles of the centrosome are similar to the basal bodies of cilia, hair-like extensions of many cells that have important roles in cell signalling and development. In a number of human diseases, such Bardet-Biedl syndrome, centrosome/cilium proteins are mutated, leading to polycystic kidney disease, situs inversus, and neurological problems, amongst other symptoms.     

Kif3a regulates planar polarization of auditory hair cells through both ciliary and non-ciliary mechanisms

Auditory hair cells represent one of the most prominent examples of epithelial planar polarity. In the auditory sensory epithelium, planar polarity of individual hair cells is defined by their V-shaped hair bundle, the mechanotransduction organelle located on the apical surface. At the tissue level, all hair cells display uniform planar polarity across the epithelium. Although it is known that tissue planar polarity is controlled by non-canonical Wnt/planar cell polarity (PCP) signaling, the hair cell-intrinsic polarity machinery that establishes the V-shape of the hair bundle is poorly understood. Here, we show that the microtubule motor subunit Kif3a regulates hair cell polarization through both ciliary and non-ciliary mechanisms. Disruption of Kif3a in the inner ear led to absence of the kinocilium, a shortened cochlear duct and flattened hair bundle morphology. Moreover, basal bodies are mispositioned along both the apicobasal and planar polarity axes of mutant hair cells, and hair bundle orientation was uncoupled from the basal body position. We show that a non-ciliary function of Kif3a regulates localized cortical activity of p21-activated kinases (PAK), which in turn controls basal body positioning in hair cells. Our results demonstrate that Kif3a-PAK signaling coordinates planar polarization of the hair bundle and the basal body in hair cells, and establish Kif3a as a key component of the hair cell-intrinsic polarity machinery, which acts in concert with the tissue polarity pathway.     

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.     

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.     

Effect of alteration in the global body plan on the deployment of morphogenesis-related protein epitopes labeled by the monoclonal antibody 12G9 in Tetrahymena thermophila

We have employed monoclonal antibodies to reinvestigate the janus mutants of the ciliate Tetrahymena thermophila, which cause reversal of circumferential polarity on the dorsal surface of the cell. This reversal brings about frequent ectopic expression of ventral cortical landmarks, such as a "secondary" oral apparatus, on the dorsal surface. The principal antibody employed, FXXXIX-12G9, immunolabels both transient cortical structures not directly associated with basal bodies (the fission line and the postoral meridional filament) and more permanent structures (apical band and oral crescent) that are associated with basal bodies. 12G9-immunolabeling of janus cells has revealed additional phenotypes, including disorder of ciliary rows. Further, this labeling has shown that the postoral meridional filament is often expressed and the apical band is frequently interrupted on the mid-dorsal surface of janus cells irrespective of whether or not these cells express a "secondary" oral apparatus. Of the permanent structures revealed by 12G9 immunofluorescence, modifications of the oral crescent (OC) are associated with prior modifications in the development of basal body-containing structures in the secondary oral apparatus. The formation of the apical band (AB) is also commonly abnormal in janus cells; analysis of specific abnormalities shows that the AB depends both on its initiation at a specific site near the anterior basal body of apical basal body couplets and on the normal location of these couplets just posterior to the fission line. We also have uncovered an intriguing difference in the reactivity of apical-band filaments to the 12G9 antibody in the two non-allelic janus mutants (janA1 and janC2) that we have investigated. Taken together, our observations indicate that the formation of new cellular structures at division depends both upon pre-existing cytoskeletal structures and upon the positional information provided by large-scale cellular polarities.     

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.     

Remodeling Epithelial Cell Organization: Transitions Between Front–Rear and Apical–Basal Polarity

Polarized epithelial cells have a distinctive apical–basal axis of polarity for vectorial transport of ions and solutes across the epithelium. In contrast, migratory mesenchymal cells have a front–rear axis of polarity. During development, mesenchymal cells convert to epithelia by coalescing into aggregates that undergo epithelial differentiation. Signaling networks and protein complexes comprising Rho family GTPases, polarity complexes (Crumbs, PAR, and Scribble), and their downstream effectors, including the cytoskeleton and the endocytic and exocytic vesicle trafficking pathways, together regulate the distributions of plasma membrane and cytoskeletal proteins between front–rear and apical–basal polarity. The challenge is to understand how these regulators and effectors are adapted to regulate symmetry breaking processes that generate cell polarities that are specialized for different cellular activities and functions.     

Immunocytochemical localization of myosin during ciliogenesis of quail oviduct

Myosin has been localized during ciliogenesis of quail oviduct by immunocytochemistry (immunofluorescence, immunoperoxidase, immunogold labeling) using a previously characterized monoclonal antibody. In ovariectomized quail oviduct many undifferentiated epithelial cells present a primary cilium arising from one of the diplosome centrioles. Myosin is associated with material located between the two centrioles. In contrast, in estrogen-stimulated quail oviduct, the material preceding the procentioles is never labeled. Basal bodies become labeled just before their migration toward the apical plasma membrane. During the anchoring phase, the labeling is mainly associated with the basal feet. In mature ciliated cells, myosin appears associated with an apical network embedding the basal bodies. This network is connected to a myosin-rich belt associated with the apical junctional complex which differentiates at the beginning of centriologenesis. The association of myosin with migrating basal bodies suggests that myosin could be involved in basal body movements.     

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.     

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.     

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.