Immunoelectron microscopic demonstration of S-100b protein-like in centriole, cilia, and basal body

We report here the ultrastructural localization of S-100b protein-like immunoreactivity in the centriole, cilia, and basal body. Duodenum and trachea of guinea pigs and rats were fixed and immunostained by the protein A-gold method. All centrioles, cilia, and basal bodies observed showed clear S-100b protein-like immunoreactivity. Specific colloidal gold particles were located over the microtubules in these cell organelles. However, other microtubules scattered throughout the cytoplasm were devoid of immunoreactivity. Although the functional significance of S-100b protein-like immunoreactivity in the centriole, cilia, and basal bodies remains to be elucidated, the present results introduce new perspectives into the investigation of localization and function of S-100 proteins.     

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.     

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.     

Sas-4 proteins are required during basal body duplication in Paramecium

Centrioles and basal bodies are structurally related organelles composed of nine microtubule (MT) triplets. Studies performed in Caenorhabditis elegans embryos have shown that centriole duplication takes place in sequential way, in which different proteins are recruited in a specific order to assemble a procentriole. ZYG-1 initiates centriole duplication by triggering the recruitment of a complex of SAS-5 and SAS-6, which then recruits the final player, SAS-4, to allow the incorporation of MT singlets. It is thought that a similar mechanism (that also involves additional proteins) is present in other animal cells, but it remains to be investigated whether the same players and their ascribed functions are conserved during basal body duplication in cells that exclusively contain basal bodies. To investigate this question, we have used the multiciliated protist Paramecium tetraurelia. Here we show that in the absence of PtSas4, two types of defects in basal body duplication can be identified. In the majority of cases, the germinative disk and cartwheel, the first structures assembled during duplication, are not detected. In addition, if daughter basal bodies were formed, they invariably had defects in MT recruitment. Our results suggest that PtSas4 has a broader function than its animal orthologues.     

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.     

Double life of centrioles: CP110 in the spotlight

Centrioles lead an important double life: they can give rise to the centrosome or convert to basal bodies and template cilia. Little is known about the control of centriole fate. Spektor and colleagues have now identified a centriolar complex, composed of CP110 and CEP97, which inhibits centriole to basal body conversion, preventing cilia formation. This work paves the way to understanding centriole and cilia biogenesis, which are two processes misregulated in human diseases, such as cancer and polycystic kidney disease.     

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.     

The UNI1 and UNI2 Genes Function in the Transition of Triplet to Doublet Microtubules between the Centriole and Cilium in Chlamydomonas

One fundamental role of the centriole in eukaryotic cells is to nucleate the growth of cilia. The unicellular alga Chlamydomonas reinhardtii provides a simple genetic system to study the role of the centriole in ciliogenesis. Wild-type cells are biflagellate, but “uni” mutations result in failure of some centrioles (basal bodies) to assemble cilia (flagella). Serial transverse sections through basal bodies in uni1 and uni2 single and double mutant cells revealed a previously undescribed defect in the transition of triplet microtubules to doublet microtubules, a defect correlated with failure to assemble flagella. Phosphorylation of the Uni2 protein is reduced in uni1 mutant cells. Immunogold electron microscopy showed that the Uni2 protein localizes at the distal end of the basal body where microtubule transition occurs. These results provide the first mechanistic insights into the function of UNI1 and UNI2 genes in the pathway mediating assembly of doublet microtubules in the axoneme from triplet microtubules in the basal body template.     

Cilia on bovine mammary epithelium: ultrastructural observations

Ultrastructural examination of bovine mammary tissues revealed the presence of 9+0 or primary cilia protruding from surfaces of alveolar epithelial and myoepithelial cells. Cilia of epithelial cells protruded approximately 1200 nm into lumina of alveoli and arose from a basal body centriole, the associated centriole of the diplosome, and an accessory rootlet system. Cilia on epithelial cells were more frequently observed than cilia on myoepithelial cells. Occasional cilia made contact with macrophages in the alveolar lumen. The structures were more commonly found in tissues from nonlactating cows, and most were observed in the ventral portion of the mammary gland.     

The iris diaphragm model of centriole and basal body formation

This paper suggests that the formation and structure of the microtubular skeleton of centrioles and basal bodies can be derived from the following simple geometric principle. A closed ring of nine microtubular initiation sites defines (1) a template for the packing of 18 additional microtubular initiation sites, and (2) the shape of nine rigid arms. Upon swivelling of each arm around a point located four initiation sites away on the initial ring, the array unfolds in a manner similar to the opening of an iris diaphragm. As a consequence, the curved shape of the microtubular triplet blades arises together with the clockwise rotational sense of the slanted blades of the centriole or basal body. The final diameter of the centriole (basal body) self-adjusts. Furthermore, the pitch of the triplet blades, the taper of centrioles and basal bodies, and the change of slant of the blades towards the distal end can be derived. In addition, the model points to a method of replication of pro-centrioles (pro-basal bodies). The hypothesis was tested by the fitting of electron microscopical cross sections of centrioles of 3T3 cells to the geometric shapes predicted by the model.     

Tubulin superfamily: giving birth to triplets

Two new studies show that e tubulin is required for centriole/basal body duplication in both Chlamydomonas and Paramecium, adding to the list of new tubulin family members specifically involved in forming the centriole triplet microtubules. The function of these triplets, and the precise role of e tubulin in triplet formation, remains unclear.     

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.     

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.     

The ultrastructure of primary cilia in the endocrine and excretory duct cells of the pancreas of mice and rats

A primary cilium was frequently observed in the endocrine alpha, beta and delta cells, as well as in the excretory duct cells of the pancreas of normal mice and rats. The characteristic components of the cilium including the basal body, axoneme (shaft), and terminal part were clearly recognizable. The basal body or distal centriole surrounded by Golgi vesicles was perpendicularly oriented to the proximal centriole, and a dense striated band was seen filling the gap between them. The microtubules of the basal body consisted of nine peripheral triplets exhibiting a 9 + 0 pattern, an appearance similar to that of the proximal centriole. Rootlets, basal feet and alar sheets associated with the basal body were occasionally seen. The axoneme usually consisted of a 9 + 0 pattern of microtubule doublets, but other irregular patterns of 7 + 2, 7 + 3, and 8 + 1 were also seen. The microtubules in the terminal part of the cilium became fewer in number and had no peculiar arrangement. The cilium of the endocrine cells always projected into the intercellular canaliculus and was covered by the ciliary sheath, and occasionally, double cilia were visualized in the vicinity of beta cells. In the excretory duct cells, the cilium showed similar features, but it was slightly longer and always projected into the dense secretory content of duct lumen. On the other hand, no primary cilium was ever observed in the acinar cells of mouse and rat pancreas. In conclusion, the present study describes the morphology of primary cilia and its associated components in the endocrine and excretory duct cells of the pancreas of mice and rats. The findings suggest that the primary cilium should be considered as a constant intracellular organelle though its function and significance remain speculative.     

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.     

The PCM–basal body/primary cilium coalition

The centrosome is an organelle that acts as a microtubule-organizing center (MTOC) throughout the cell cycle. Within the centrosome are often two components that each have an ability to organize microtubule structures: the centriole that has the potential to function as a basal body and nucleate a cilium or a flagellum and a mass of protein material that in the presence of a centriole is commonly referred to as the pericentriolar material (PCM) that organizes cytoplasmic and spindle microtubule arrays. One characteristic of a large variety of cells is the ability to express a non-motile primary cilium. It is now appreciated that the function of the primary cilium is integral to a variety of essential cell functions and defects affecting this structure underlie a variety of human disease. While the function of the primary cilium and manner in which a basal body organizes a primary cilium has received extensive attention there is now a need to explore the inter-relationship between the PCM and the basal body/primary cilium. It is this latter topic that is the focus of this review where we show that the PCM is integrated with the centriole to form a coalition that is essential for both the expression and function of the primary cilium as well as the organization and function of the cellular environment that surrounds it.     

Asterless is required for centriole length control and sperm development

Centrioles are the foundation of two organelles, centrosomes and cilia. Centriole numbers and functions are tightly controlled, and mutations in centriole proteins are linked to a variety of diseases, including microcephaly. Loss of the centriole protein Asterless (Asl), the Drosophila melanogaster orthologue of Cep152, prevents centriole duplication, which has limited the study of its nonduplication functions. Here, we identify populations of cells with Asl-free centrioles in developing Drosophila tissues, allowing us to assess its duplication-independent function. We show a role for Asl in controlling centriole length in germline and somatic tissue, functioning via the centriole protein Cep97. We also find that Asl is not essential for pericentriolar material recruitment or centrosome function in organizing mitotic spindles. Lastly, we show that Asl is required for proper basal body function and spermatid axoneme formation. Insights into the role of Asl/Cep152 beyond centriole duplication could help shed light on how Cep152 mutations lead to the development of microcephaly.     

Epsilon-tubulin is an essential component of the centriole

Centrioles and basal bodies are cylinders composed of nine triplet microtubule blades that play essential roles in the centrosome and in flagellar assembly. Chlamydomonas cells with the bld2-1 mutation fail to assemble doublet and triplet microtubules and have defects in cleavage furrow placement and meiosis. Using positional cloning, we have walked 720 kb and identified a 13.2-kb fragment that contains epsilon-tubulin and rescues the Bld2 defects. The bld2-1 allele has a premature stop codon and intragenic revertants replace the stop codon with glutamine, glutamate, or lysine. Polyclonal antibodies to epsilon-tubulin show peripheral labeling of full-length basal bodies and centrioles. Thus, epsilon-tubulin is encoded by the BLD2 allele and epsilon-tubulin plays a role in basal body/centriole morphogenesis.     

Basal body duplication in Paramecium requires γ-tubulin

First discovered in the fungus Aspergillus nidulans[1], γ-tubulin is a ubiquitous component of microtubule organizing centres [2]. In centrosomes, γ-tubulin has been immunolocalized at the pericentriolar material, suggesting a role in cytoplasmic microtubule nucleation [3], as well as within the centriole core itself [4]. Although its function in the nucleation of the mitotic spindle and of cytoplasmic interphasic microtubules has been demonstrated in vitro[5], [6] and in vivo[7], [8], [9], the hypothesis that γ-tubulin could intervene in centriole assembly has never been experimentally addressed because the mitotic arrest caused by the inactivation of γ-tubulin in vivo precludes any further phenotypic analysis of putative centriole defects. The issue can be addressed in the ciliate Paramecium, which is characterized by numerous basal bodies that are similar to centrioles but the biogenesis of which is not tightly coupled to the nuclear division cycle. We demonstrate that the inactivation of the Paramecium γ-tubulin genes leads to inhibition of basal body duplication.     

Effects of nuclease and protease digestion on the ultrastructure of Paramecium basal bodies

The action of deoxyribonuclease, ribonuclease, perchloric acid, and pronase on the fine structure of basal bodies of sectioned Paramecium was observed as part of a more extensive autoradiographic electron microscope analysis directed toward the problem of basal body DNA. DNase was found to have no detectable effect on basal body fine structure. Pronase first solubilized the linkers and C tubules of the triplets, then attacked the protein portion of the axosome, a localized portion of the ciliary axoneme adjacent to the distal end of the basal body, the rim fiber, and newly described lumen spiral complex. Prolonged pronase treatment disrupted the remaining microtubular elements, basal body plates, and cartwheel. RNase removed material from the axosome and the lumen complex, a conspicuous structure occupying the central portion of the basal body and consisting of a twisted or looped 90-A diam fiber or, more probably, pair of fibers, in association with large, dense granules. The apparent removal of both RNA and protein from this basal body structure by either of the two corresponding enzymes suggests an unusual organization of the two components. Observations from this and other laboratories suggest that the basal body RNA is single stranded. Its function is unknown but alternatives are discussed.