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.     

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.     

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 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.     

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.     

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.     

Scoring a backstage pass: mechanisms of ciliogenesis and ciliary access

Cilia are conserved, microtubule-based cell surface projections that emanate from basal bodies, membrane-docked centrioles. The beating of motile cilia and flagella enables cells to swim and epithelia to displace fluids. In contrast, most primary cilia do not beat but instead detect environmental or intercellular stimuli. Inborn defects in both kinds of cilia cause human ciliopathies, diseases with diverse manifestations such as heterotaxia and kidney cysts. These diseases are caused by defects in ciliogenesis or ciliary function. The signaling functions of cilia require regulation of ciliary composition, which depends on the control of protein traffic into and out of cilia.     

Cyclin O (Ccno) functions during deuterosome-mediated centriole amplification of multiciliated cells

Mucociliary clearance and fluid transport along epithelial surfaces are carried out by multiciliated cells (MCCs). Recently, human mutations in Cyclin O (CCNO) were linked to severe airway disease. Here, we show that Ccno expression is restricted to MCCs and the genetic deletion of Ccno in mouse leads to reduced numbers of multiple motile cilia and characteristic phenotypes of MCC dysfunction including severe hydrocephalus and mucociliary clearance deficits. Reduced cilia numbers are caused by compromised generation of centrioles at deuterosomes, which serve as major amplification platform for centrioles in MCCs. Ccno-deficient MCCs fail to sufficiently generate deuterosomes, and only reduced numbers of fully functional centrioles that undergo maturation to ciliary basal bodies are formed. Collectively, this study implicates CCNO as first known regulator of deuterosome formation and function for the amplification of centrioles in MCCs.     

Drosophila chibby is required for basal body formation and ciliogenesis but not for Wg signaling

Centriole-to-basal body conversion, a complex process essential for ciliogenesis, involves the progressive addition of specific proteins to centrioles. CHIBBY (CBY) is a coiled-coil domain protein first described as interacting with β-catenin and involved in Wg-Int (WNT) signaling. We found that, in Drosophila melanogaster, CBY was exclusively expressed in cells that require functional basal bodies, i.e., sensory neurons and male germ cells. CBY was associated with the basal body transition zone (TZ) in these two cell types. Inactivation of cby led to defects in sensory transduction and in spermatogenesis. Loss of CBY resulted in altered ciliary trafficking into neuronal cilia, irregular deposition of proteins on spermatocyte basal bodies, and, consequently, distorted axonemal assembly. Importantly, cby(1/1) flies did not show Wingless signaling defects. Hence, CBY is essential for normal basal body structure and function in Drosophila, potentially through effects on the TZ. The function of CBY in WNT signaling in vertebrates has either been acquired during vertebrate evolution or lost in Drosophila.     

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.     

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.     

Comparative genomics identifies a flagellar and basal body proteome that includes the BBS5 human disease gene

Cilia and flagella are microtubule-based structures nucleated by modified centrioles termed basal bodies. These biochemically complex organelles have more than 250 and 150 polypeptides, respectively. To identify the proteins involved in ciliary and basal body biogenesis and function, we undertook a comparative genomics approach that subtracted the nonflagellated proteome of Arabidopsis from the shared proteome of the ciliated/flagellated organisms Chlamydomonas and human. We identified 688 genes that are present exclusively in organisms with flagella and basal bodies and validated these data through a series of in silico, in vitro, and in vivo studies. We then applied this resource to the study of human ciliation disorders and have identified BBS5, a novel gene for Bardet-Biedl syndrome. We show that this novel protein localizes to basal bodies in mouse and C. elegans, is under the regulatory control of daf-19, and is necessary for the generation of both cilia and flagella.     

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.     

Life without centrioles: cilia in the spotlight

Centrioles are critical cellular components that form the architectural core of both centrosomes and basal bodies, the nucleating structures of cilia. New work, including a study in this issue (), highlights the unexpected finding that lack of centrioles does not impede development in the fruit fly. Rather, flies reach maturity but then die because their sensory neurons lack cilia.     

Cilia in the accessory hyperstriatum of the domestic fowl

Summary Numerous neurons and glia in the accessory hyperstriatum of the domestic fowl contain a cilium that is attached to a basal body. The accessory centriole is in the vicinity of the basal body and in some instances a connection between the two centrioles is noted. Cross-striated rootlets are associated with the basal body and the accessory centriole, however, some rootlets are found distant to centrioles. Cross sections of cilia show that most accessory hyperstriatal cilia have an 8+1 fiber pattern. Several proposed functional roles of neuronal cilia are discussed.This investigation was supported by a research grant from the National Institute of Neurological Diseases and Stroke (5 RO 1 NSO 7557-02) awarded to Norma Jean Adamo.     

The primary cilium as a multiple cellular signaling scaffold in development and disease

Primary cilia, single hair-like appendage on the surface of the most mammalian cells, were once considered to be vestigial cellular organelles for a past century because of their tiny structure and unknown function. Although they lack ancestral motility function of cilia or flagella, they share common ground with multiciliated motile cilia and flagella on internal structure such as microtubule based nine outer doublets nucleated from the base of mother centrioles called basal body. Making cilia, ciliogenesis, in cells depends on the cell cycle stage due to reuse of centrioles for cell division forming mitotic spindle pole (M phase) and assembling cilia from basal body (starting G1 phase and maintaining most of interphase). Ciliary assembly required two conflicting processes such as assembly and disassembly and balance between these two processes determines the length of cilia. Both process required highly conserved transport system to supply needed substance to grow tip of cilia and bring ciliary turnover product back to the base of cilia using motor protein, kinesin and dynein, and transport protein complex, IFT particles. Disruption of ciliary structure or function causes multiple human disorder called ciliopathies affecting disease of diverse ciliated tissues ranging from eye, kidney, respiratory tract and brain. Recent explosion of research on the primary cilia and their involvement on animal development and disease attracts scientific interest on how extensively the function of cilia related to specific cell physiology and signaling pathway. In this review, I introduce general features of primary cilia and recent progress in understanding of the ciliary length control and signaling pathways transduced through primary cilia in vertebrates. [BMB Reports 2012; 45(8): 427-432].     

Centrin2 regulates CP110 removal in primary cilium formation

Primary cilia are antenna-like sensory microtubule structures that extend from basal bodies, plasma membrane–docked mother centrioles. Cellular quiescence potentiates ciliogenesis, but the regulation of basal body formation is not fully understood. We used reverse genetics to test the role of the small calcium-binding protein, centrin2, in ciliogenesis. Primary cilia arise in most cell types but have not been described in lymphocytes. We show here that serum starvation of transformed, cultured B and T cells caused primary ciliogenesis. Efficient ciliogenesis in chicken DT40 B lymphocytes required centrin2. We disrupted CETN2 in human retinal pigmented epithelial cells, and despite having intact centrioles, they were unable to make cilia upon serum starvation, showing abnormal localization of distal appendage proteins and failing to remove the ciliation inhibitor CP110. Knockdown of CP110 rescued ciliation in CETN2-deficient cells. Thus, centrin2 regulates primary ciliogenesis through controlling CP110 levels.     

Development of a multiciliated cell

Multiciliated cells (MCC) are evolutionary conserved, highly specialized cell types that contain dozens to hundreds of motile cilia that they use to propel fluid directionally. To template these cilia, each MCC produces between 30 and 500 basal bodies via a process termed centriole amplification. Much progress has been made in recent years in understanding the pathways involved in MCC fate determination, differentiation, and ciliogenesis. Recent studies using mammalian cell culture systems, mice, Xenopus, and other model organisms have started to uncover the mechanisms involved in centriole and cilia biogenesis. Yet, how MCC progenitor cells regulate the precise number of centrioles and cilia during their differentiation remains largely unknown. In this review, we will examine recent findings that address this fundamental question.     

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.     

RTTN Mutations Link Primary Cilia Function to Organization of the Human Cerebral Cortex

Polymicrogyria is a malformation of the developing cerebral cortex caused by abnormal organization and characterized by many small gyri and fusion of the outer molecular layer. We have identified autosomal-recessive mutations in RTTN, encoding Rotatin, in individuals with bilateral diffuse polymicrogyria from two separate families. Rotatin determines early embryonic axial rotation, as well as anteroposterior and dorsoventral patterning in the mouse. Human Rotatin has recently been identified as a centrosome-associated protein. The Drosophila melanogaster homolog of Rotatin, Ana3, is needed for structural integrity of centrioles and basal bodies and maintenance of sensory neurons. We show that Rotatin colocalizes with the basal bodies at the primary cilium. Cultured fibroblasts from affected individuals have structural abnormalities of the cilia and exhibit downregulation of BMP4, WNT5A, and WNT2B, which are key regulators of cortical patterning and are expressed at the cortical hem, the cortex-organizing center that gives rise to Cajal-Retzius (CR) neurons. Interestingly, we have shown that in mouse embryos, Rotatin colocalizes with CR neurons at the subpial marginal zone. Knockdown experiments in human fibroblasts and neural stem cells confirm a role for RTTN in cilia structure and function. RTTN mutations therefore link aberrant ciliary function to abnormal development and organization of the cortex in human individuals.