Fine Structure and Function of Pharynx Cilia in Glossobalanus minutus Kowalewsky (Enteropneusta)

Two kinds of cilia have been observed in the pharynx of Glossobalanus minutus Kowalewsky. From the present study, a ciliary specialization can be found in order to move a determinate substance, i.e. mucus or water. Mucus-moving cilia (type I cilia) have a single basal centriole and poorly developed ciliary rootlets. Their tips are rounded, bearing an inner, asymmetrical cap attached to some tubules. Water-moving cilia (type II cilia) are exclusively located at lateral epithelia of branchial bars, giving rise to the water current through the gills. They have two basal centrioles, proximal and distal, and a complex system of ciliary rootlets made up of a principal rootlet, a secondary or accessory rootlet and a 'fan' rootlet. The tips of type II cilia have a long process with some tubules inside. All basal structures are precisely orientated in order to assure a good coordination of ciliary beat. The possible functional significance of ciliary substructure is also discussed. From these observations a model for mucus and water currents through gill slits is postulated.     

C-NAP1 and rootletin restrain DNA damage-induced centriole splitting and facilitate ciliogenesis

Cilia are found on most human cells and exist as motile cilia or non-motile primary cilia. Primary cilia play sensory roles in transducing various extracellular signals, and defective ciliary functions are involved in a wide range of human diseases. Centrosomes are the principal microtubule-organizing centers of animal cells and contain two centrioles. We observed that DNA damage causes centriole splitting in non-transformed human cells, with isolated centrioles carrying the mother centriole markers CEP170 and ninein but not kizuna or cenexin. Loss of centriole cohesion through siRNA depletion of C-NAP1 or rootletin increased radiation-induced centriole splitting, with C-NAP1-depleted isolated centrioles losing mother markers. As the mother centriole forms the basal body in primary cilia, we tested whether centriole splitting affected ciliogenesis. While irradiated cells formed apparently normal primary cilia, most cilia arose from centriolar clusters, not from isolated centrioles. Furthermore, C-NAP1 or rootletin knockdown reduced primary cilium formation. Therefore, the centriole cohesion apparatus at the proximal end of centrioles may provide a target that can affect primary cilium formation as part of the DNA damage response.     

C-NAP1 and rootletin restrain DNA damage-induced centriole splitting and facilitate ciliogenesis

Cilia are found on most human cells and exist as motile cilia or non-motile primary cilia. Primary cilia play sensory roles in transducing various extracellular signals, and defective ciliary functions are involved in a wide range of human diseases. Centrosomes are the principal microtubule-organizing centers of animal cells and contain two centrioles. We observed that DNA damage causes centriole splitting in non-transformed human cells, with isolated centrioles carrying the mother centriole markers CEP170 and ninein but not kizuna or cenexin. Loss of centriole cohesion through siRNA depletion of C-NAP1 or rootletin increased radiation-induced centriole splitting, with C-NAP1-depleted isolated centrioles losing mother markers. As the mother centriole forms the basal body in primary cilia, we tested whether centriole splitting affected ciliogenesis. While irradiated cells formed apparently normal primary cilia, most cilia arose from centriolar clusters, not from isolated centrioles. Furthermore, C-NAP1 or rootletin knockdown reduced primary cilium formation. Therefore, the centriole cohesion apparatus at the proximal end of centrioles may provide a target that can affect primary cilium formation as part of the DNA damage response.     

Centriole maturation requires regulated Plk1 activity during two consecutive cell cycles

Newly formed centrioles in cycling cells undergo a maturation process that is almost two cell cycles long before they become competent to function as microtubule-organizing centers and basal bodies. As a result, each cell contains three generations of centrioles, only one of which is able to form cilia. It is not known how this long and complex process is regulated. We show that controlled Plk1 activity is required for gradual biochemical and structural maturation of the centrioles and timely appendage assembly. Inhibition of Plk1 impeded accumulation of appendage proteins and appendage formation. Unscheduled Plk1 activity, either in cycling or interphase-arrested cells, accelerated centriole maturation and appendage and cilia formation on the nascent centrioles, erasing the age difference between centrioles in one cell. These findings provide a new understanding of how the centriole cycle is regulated and how proper cilia and centrosome numbers are maintained in the cells.     

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.     

Assembly and persistence of primary cilia in dividing Drosophila spermatocytes

Basal bodies are freed from cilia and transition into?centrioles to organize centrosomes in dividing cells. A mutually exclusive centriole/basal body existence during cell-cycle progression has become a widely accepted principle. Contrary to this view, we?show here that cilia assemble and persist through?two meiotic divisions in Drosophila spermatocytes. Remarkably, all four centrioles assemble primary cilia-centriole complexes that transit from the plasma membrane encased in a packet of membrane, recruit centrosomal material into microtubule-organizing centers, and persist at the spindle poles through division. Thus, spermatocyte centrioles organize centrosomes and cilia simultaneously at cell division. These findings challenge the prevailing view that cilia antagonize cell-cycle progression and raise the possibility that cilium retention at cell division may occur in diverse organisms and cell types.     

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.     

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.     

Centriole is the pivot co-ordinating dynamic signaling for cell proliferation and organization during early development in the vertebrates

Vertebrates have an elaborate and functionally segmented body. It evolves from a single cell by systematic cell proliferation but attains a complex body structure with exquisite precision. This development requires two cellular events: cell cycle and ciliogenesis. For these events, the dynamic molecular signaling is converged at the centriole. The cell cycle helps in cell proliferation and growth of the body and is a highly regulated and integrated process. Its errors cause malignancies and developmental disorders. The cells newly proliferated are organized during organogenesis. For a cellular organization, dedicated signaling hubs are developed in the cells, and most often cilia are utilized. The cilium is generated from one of the centrioles involved in cell proliferation. The developmental signaling pathways hosted in cilia are essential for the elaboration of the body plan. The cilium's compartmental seclusion is ideal for noise-free molecular signaling and is essential for the precision of the body layout. The dysfunctional centrioles and primary cilia distort the development of body layout that manifest as serious developmental disorders. Thus, centriole has a dual role in the growth and cellular organization. It organizes dynamically expressed molecules of cell cycle and ciliogenesis and plays a balancing act to generate new cells and organize them during development. A putative master molecule may regulate and co-ordinate the dynamic gene expression at the centrioles. The convergence of many critical signaling components at the centriole reiterates the idea that centriole is a major molecular workstation involved in elaborating the structural design and complexity in vertebrates.     

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.     

Mitochondrial origin of centrioles and cilia in some human endocrine neoplasms.

Fine structural investigation of surgically removed human pituitary and parathyroid adenomas, pheochromocytomas and bronchial carcinoids revealed a hitherto undetected sequence of events in the formation of centrioles and cilia indicating that mitochondria may serve as their progenitors. The first steps seem to be the disappearance of mitochondrial cristae and a polar accumulation of a fibrillar-granular material with a subsequent increase of electron density of the double mitochondrial membranes and deposition of more electron opaque substance within and around these procentriolar bodies. This process is followed by the disintegration of the double membranes and an asymmetrical division of the electron dense aggregate. The larger part seems to be the precursor of the primary centriole (basal body) whereas the smaller one that of the secondary centriole. Formation of centrioleand rudimentary cilium-like structures was disclosed within the unaltered mitochondrial membranes of oncocytic cells present in two pituitary adenomas and in one pheochromocytoma. Accumulation of procentriolar bodies and mature centrioles, noted in some tumors, may be due to a defect in the process of centriolo- and ciliogenesis. It is conceivable that the mitochondrial genome plays an important role in formation of centrioles and cilia.     

Centriole translocation and degeneration during ciliogenesis in Caenorhabditis elegans neurons

Neuronal cilia that are formed at the dendritic endings of sensory neurons are essential for sensory perception. However, it remains unclear how the centriole‐derived basal body is positioned to form a template for cilium formation. Using fluorescence time‐lapse microscopy, we show that the centriole translocates from the cell body to the dendrite tip in the Caenorhabditis elegans sensory neurons. The centriolar protein SAS‐5 interacts with the dynein light‐chain LC8 and conditional mutations of cytoplasmic dynein‐1 block centriole translocation and ciliogenesis. The components of the central tube are essential for the biogenesis of centrioles, which later drive ciliogenesis in the dendrite; however, the centriole loses these components at the late stage of centriole translocation and subsequently recruits transition zone and intraflagellar transport proteins. Together, our results provide a comprehensive model of ciliogenesis in sensory neurons and reveal the importance of the dynein‐dependent centriole translocation in this process.     

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.     

Vertebrate primary cilia: a sensory part of centrosomal complex in tissue cells,but a “sleeping beauty” in cultured cells?

Primary cilium development along with other components of the centrosome in mammalian cells was analysed ultrastructurally and by immunofluorescent staining with anti-acetylated tubulin antibodies. We categorized two types of primary cilia, nascent cilia that are about 1microm long located inside the cytoplasm, and true primary cilia that are several microm long and protrude from the plasma membrane. The primary cilium is invariably associated with the older centriole of each diplosome, having appendages at the distal end and pericentriolar satellites with cytoplasmic microtubules emanating from them. Only one cilium per cell is formed normally through G(0), S and G(2)phases. However, in some mouse embryo fibroblasts with two mature centrioles, bicilates were seen. Primary cilia were not observed in cultured cells where the mature centriole had no satellites and appendages (Chinese hamster kidney cells, line 237, some clones of l-fibroblasts). In contrast to primary cilia, striated rootlets were found around active and non-active centrioles with the same frequency. In proliferating cultured cells, a primary cilium can be formed several hours after mitosis, in fibroblasts 2-4 h after cell division and in PK cells only during the S-phase. In interphase cells, formation of the primary cilium can be stimulated by the action of metabolic inhibitors and by reversed depolymerization of cytoplasmic microtubules with cold or colcemid treatments. In mouse renal epithelial cells in situ, the centrosome was located near the cell surface and mature centrioles in 80% of the cells had primary cilium protruding into the duct lumen. After cells were explanted and subcultured, the centrosome comes closer to the nucleus and the primary cilium was depolymerized or reduced. Later primary cilia appeared in cells that form islets on the coverslip. However, the centrosome in cultured ciliated cells was always located near the cell nucleus and primary cilium never formed a characteristic distal bulb. A sequence of the developmental stages of the primary cilium is proposed and discussed. We also conclude that functioning primary cilium does not necessarily operate in culture cells, which might explain some of the contradictory data on cell ciliation in vitro reported in the literature.     

The ultrastructure of primary cilia in quiescent 3T3 cells

Electron microscopy was used to investigate primary cilia in quiescent 3T3 cells. As in the case of primary cilia of other cell types, their basal centriole was found to be a focal point of numerous cytoplasmic microtubules which terminate at the basal feet. There are also intermediate filaments which appear to converge at the basal centriole. Cross-striated fibers of microtubular diameter, reminiscent of striated rootlets of ordinary cilia, appear associated with the proximal end of the basal centriole. Usually less than nine cross-banded basal feet surround the basal centriole in a well-defined plane perpendicular to the centriolar axis. The ciliary shaft was found to be entirely enclosed in the cytoplasm of fully flattened cells. In rounded cells, it could be found extending to the outside of the cell. Periodic striations along the entire shaft were observed after preparing the cells in a special way. The tip of the shaft showed an electron-dense specialization. Several unusual forms of primary cilia were observed which were reminiscent of olfactory flagella or retinal rods.Using tubulin antibody for indirect immunofluorescence, a fluorescent rod is visible in the cells [18] which we demonstrate is identical with the primary cilium.     

Centrosome behavior in the exposure of tissue culture cells to energy metabolism inhibitors

The organization of the centrosome in PK cells has been analysed according to several parameters: the presence of primary cilium, the number of pericentriolar satellites, the presence of striated rootlets, the distance between two centrioles and their orientation as regards the substrate plane. 2,4-Dinitrophenol (DNP), DNP with deoxyglucose (DOG), sodium azide cause the increase of frequency of occurrence of primary cilia and the growth of mean number of satellites per active centriole. The distribution of active and inactive centrioles in control cells is described by a histogram corresponding to a histogram of accidental distribution. Under the action of DNP or DNP with DOG, but not of sodium azide a part of active centrioles settled down perpendicularly to the substrate plane increases. The orientation of inactive centrioles under all the treatments used doesn't practically change.     

The ciliary basal apparatus is adapted to the structure and mechanics of the epithelium

The basal apparatuses which anchor the gill cilia in Branchiostoma lanceolatum (Pallas) and the actinopharynx cilia in Calliactis parasitica (Couch) are similar in structure. In C. parasitica the pharynx epithelium and the basal apparatuses are flexible. The basal apparatuses, however, bend in only one direction. This mechanism may permit epithelial flexibility whilst maintaining a similar basal orientation between cilia. In B. lanceolatum the ciliated gill epithelia are mechanically stable but the epithelial surfaces are curved. The basal apparatuses may correct for this curvature, with short rootlets between the distal centrioles (basal bodies) and the cell membranes, so that their cilia also share a common orientation. A common basal orientation between cilia is important for their coordination. The degree of coordination depends upon the function of the cilia; water-propelling cilia are more precisely coordinated than mucus-propelling cilia. Much of the structural diversity of ciliary basal apparatuses in Metazoa may be due to variation in the demands of anchoring functionally different cilia to epithelia which have different structural and mechanical properties.     

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.     

Flagellar basal apparatus and its utility in phylogenetic analyses of the porifera

A broadly based comparative study was initiated to assess components of the flagellar basal apparatus as a character set in phylogenetic analyses of poriferans. The flagellated (monociliated) epidermal cells of sponge larvae were selected for study. Taken together, they create a field of locomotory cells analogous to a multiciliated surface. Larvae of six species in four orders of the Demospongiae were examined by transmission electron microscopy. Results are compared with findings taken from the literature on larvae of five additional species of demosponges and four species of calcareans. Data were assembled on six components of the basal apparatus: (1) basal body, (2) basal foot, (3) accessory centriole, (4) transverse cytoskeletal system, (5) longitudinal cytoskeletal system, and (6) association with Golgi body. Where evidence permits assessment, all have Type II basal bodies. Basal feet are diverse and are subdivided into three categories based on structural complexity. The most anatomically intricate (Type III) is found only in larvae of Mycale spp. Accessory centrioles are present or absent depending on the species, but their occurrence is without overall taxonomic pattern. When present, accessory centrioles are oriented perpendicularly to the long axis of the basal body, but as ascertained from relationship to the anterior-posterior axis of the larvae they are without consistent orientation with regard to the plane of effective beat of the flagellum. Transverse and longitudinal cytoskeletal systems are also diverse among larvae. The existence of cross-striated rootlets is convincingly established only in larvae of calcareans, and such rootlets are present in larvae of all four calcareans studied to date. Three apparently new rootlet structures are described: lateral arms of the transverse cytoskeletal system from larvae of Aplysilla sp. and Haliclona tubifera; laminar sheets of the longitudinal system from larvae of Aplysilla sp. and M. cecilia; and paraxial rootlet in larvae of H. tubifera. A robust similarity in structure of the basal appartus is observed among the three species of halichondrids reported here for the first time. In comparison with the flagellar basal apparatus found in adults, those of larvae are more complex and more diverse. Review of studies on adult sponges that include information on the basal apparatus reveals the absence of a longitudinal rootlet system in all cases. Additionally, there exists a high degree of concordance between properties of the basal apparatus in the one sclerosponge and the one hexactinellid studied to date. These basal apparatus are also the simplest in construction of those found in sponges. Conversely, the basal apparatus of demosponges are varied. Although consistent presentation of the basal apparatus is evident in certain taxa, any discernable systematic pattern in their overall configuration remains obscure. Finally, we conclude that the flagellar basal apparatus of sponges is more similar to that found in choanoflagellates than it is to that observed in eumetazoans. © 1995 Wiley-Liss, Inc.     

A molecular marker for centriole maturation in the mammalian cell cycle

The centriole pair in animals shows duplication and structural maturation at specific cell cycle points. In G1, a cell has two centrioles. One of the centrioles is mature and was generated at least two cell cycles ago. The other centriole was produced in the previous cell cycle and is immature. Both centrioles then nucleate one procentriole each which subsequently elongate to full-length centrioles, usually in S or G2 phase. However, the point in the cell cycle at which maturation of the immature centriole occurs is open to question. Furthermore, the molecular events underlying this process are entirely unknown. Here, using monoclonal and polyclonal antibody approaches, we describe for the first time a molecular marker which localizes exclusively to one centriole of the centriolar pair and provides biochemical evidence that the two centrioles are different. Moreover, this 96-kD protein, which we name Cenexin (derived from the Latin, senex for "old man," and Cenexin for centriole) defines very precisely the mature centriole of a pair and is acquired by the immature centriole at the G2/M transition in prophase. Thus the acquisition of Cenexin marks the functional maturation of the centriole and may indicate a change in centriolar potential such as its ability to act as a basal body for axoneme development or as a congregating site for microtubule-organizing material.