The evolutionary conserved proteins CEP90, FOPNL, and OFD1 recruit centriolar distal appendage proteins to initiate their assembly.

In metazoa, cilia assembly is a cellular process that starts with centriole to basal body maturation, migration to the cell surface, and docking to the plasma membrane. Basal body docking involves the interaction of both the distal end of the basal body and the transition fibers/distal appendages, with the plasma membrane. Mutations in numerous genes involved in basal body docking and transition zone assembly are associated with the most severe ciliopathies, highlighting the importance of these events in cilium biogenesis. In this context, the ciliate Paramecium has been widely used as a model system to study basal body and cilia assembly. However, despite the evolutionary conservation of cilia assembly events across phyla, whether the same molecular players are functionally conserved, is not fully known. Here, we demonstrated that CEP90, FOPNL, and OFD1 are evolutionary conserved proteins crucial for ciliogenesis. Using ultrastructure expansion microscopy, we unveiled that these proteins localize at the distal end of both centrioles/basal bodies in Paramecium and mammalian cells. Moreover, we found that these proteins are recruited early during centriole duplication on the external surface of the procentriole. Functional analysis performed both in Paramecium and mammalian cells demonstrate the requirement of these proteins for distal appendage assembly and basal body docking. Finally, we show that mammalian centrioles require another component, Moonraker (MNR), to recruit OFD1, FOPNL, and CEP90, which will then recruit the distal appendage proteins CEP83, CEP89, and CEP164. Altogether, we propose that this OFD1, FOPNL, and CEP90 functional module is required to determine in mammalian cells the future position of distal appendage proteins.

TTC12 Loss-of-Function Mutations Cause Primary Ciliary Dyskinesia and Unveil Distinct Dynein Assembly Mechanisms in Motile Cilia Versus Flagella.

Cilia and flagella are evolutionarily conserved organelles whose motility relies on the outer and inner dynein arm complexes (ODAs and IDAs). Defects in ODAs and IDAs result in primary ciliary dyskinesia (PCD), a disease characterized by recurrent airway infections and male infertility. PCD mutations in assembly factors have been shown to cause a combined ODA-IDA defect, affecting both cilia and flagella. We identified four loss-of-function mutations in TTC12, which encodes a cytoplasmic protein, in four independent families in which affected individuals displayed a peculiar PCD phenotype characterized by the absence of ODAs and IDAs in sperm flagella, contrasting with the absence of only IDAs in respiratory cilia. Analyses of both primary cells from individuals carrying TTC12 mutations and human differentiated airway cells invalidated for TTC12 by a CRISPR-Cas9 approach revealed an IDA defect restricted to a subset of single-headed IDAs that are different in flagella and cilia, whereas TTC12 depletion in the ciliate Paramecium tetraurelia recapitulated the sperm phenotype. Overall, our study, which identifies TTC12 as a gene involved in PCD, unveils distinct dynein assembly mechanisms in human motile cilia versus flagella.

MKS-NPHP module proteins control ciliary shedding at the transition zone.

Ciliary shedding occurs from unicellular organisms to metazoans. Although required during the cell cycle and during neurogenesis, the process remains poorly understood. In all cellular models, this phenomenon occurs distal to the transition zone (TZ), suggesting conserved molecular mechanisms. The TZ module proteins (Meckel Gruber syndrome [MKS]/Nephronophtysis [NPHP]/Centrosomal protein of 290 kDa [CEP290]/Retinitis pigmentosa GTPase regulator-Interacting Protein 1-Like Protein [RPGRIP1L]) are known to cooperate to establish TZ formation and function. To determine whether they control deciliation, we studied the function of 5 of them (Transmembrane protein 107 [TMEM107], Transmembrane protein 216 [TMEM216], CEP290, RPGRIP1L, and NPHP4) in Paramecium. All proteins are recruited to the TZ of growing cilia and localize with 9-fold symmetry at the level of the most distal part of the TZ. We demonstrate that depletion of the MKS2/TMEM216 and TMEM107 proteins induces constant deciliation of some cilia, while depletion of either NPHP4, CEP290, or RPGRIP1L prevents Ca2+/EtOH deciliation. Our results constitute the first evidence for a role of conserved TZ proteins in deciliation and open new directions for understanding motile cilia physiology.

C11orf70 Mutations Disrupting the Intraflagellar Transport-Dependent Assembly of Multiple Axonemal Dyneins Cause Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetically and phenotypically heterogeneous disorder characterized by destructive respiratory disease and laterality abnormalities due to randomized left-right body asymmetry. PCD is mostly caused by mutations affecting the core axoneme structure of motile cilia that is essential for movement. Genes that cause PCD when mutated include a group that encode proteins essential for the assembly of the ciliary dynein motors and the active transport process that delivers them from their cytoplasmic assembly site into the axoneme. We screened a cohort of affected individuals for disease-causing mutations using a targeted next generation sequencing panel and identified two unrelated families (three affected children) with mutations in the uncharacterized C11orf70 gene (official gene name CFAP300). The affected children share a consistent PCD phenotype from early life with laterality defects and immotile respiratory cilia displaying combined loss of inner and outer dynein arms (IDA+ODA). Phylogenetic analysis shows C11orf70 is highly conserved, distributed across species similarly to proteins involved in the intraflagellar transport (IFT)-dependant assembly of axonemal dyneins. Paramecium C11orf70 RNAi knockdown led to combined loss of ciliary IDA+ODA with reduced cilia beating and swim velocity. Tagged C11orf70 in Paramecium and Chlamydomonas localizes mainly in the cytoplasm with a small amount in the ciliary component. IFT139/TTC21B (IFT-A protein) and FLA10 (IFT kinesin) depletion experiments show that its transport within cilia is IFT dependent. During ciliogenesis, C11orf70 accumulates at the ciliary tips in a similar distribution to the IFT-B protein IFT46. In summary, C11orf70 is essential for assembly of dynein arms and C11orf70 mutations cause defective cilia motility and PCD.

Mutations in Outer Dynein Arm Heavy Chain DNAH9 Cause Motile Cilia Defects and Situs Inversus.

Motile cilia move body fluids and gametes and the beating of cilia lining the airway epithelial surfaces ensures that they are kept clear and protected from inhaled pathogens and consequent respiratory infections. Dynein motor proteins provide mechanical force for cilia beating. Dynein mutations are a common cause of primary ciliary dyskinesia (PCD), an inherited condition characterized by deficient mucociliary clearance and chronic respiratory disease coupled with laterality disturbances and subfertility. Using next-generation sequencing, we detected mutations in the ciliary outer dynein arm (ODA) heavy chain gene DNAH9 in individuals from PCD clinics with situs inversus and in one case male infertility. DNAH9 and its partner heavy chain DNAH5 localize to type 2 ODAs of the distal cilium and in DNAH9-mutated nasal respiratory epithelial cilia we found a loss of DNAH9/DNAH5-containing type 2 ODAs that was restricted to the distal cilia region. This confers a reduced beating frequency with a subtle beating pattern defect affecting the motility of the distal cilia portion. 3D electron tomography ultrastructural studies confirmed regional loss of ODAs from the distal cilium, manifesting as either loss of whole ODA or partial loss of ODA volume. Paramecium DNAH9 knockdown confirms an evolutionarily conserved function for DNAH9 in cilia motility and ODA stability. We find that DNAH9 is widely expressed in the airways, despite DNAH9 mutations appearing to confer symptoms restricted to the upper respiratory tract. In summary, DNAH9 mutations reduce cilia function but some respiratory mucociliary clearance potential may be retained, widening the PCD disease spectrum.

The conserved centrosomal protein FOR20 is required for assembly of the transition zone and basal body docking at the cell surface.

Within the FOP family of centrosomal proteins, the conserved FOR20 protein has been implicated in the control of primary cilium assembly in human cells. To ascertain its role in ciliogenesis, we have investigated the function of its ortholog, PtFOR20p, in the multiciliated unicellular organism Paramecium. Using combined functional and cytological analyses, we found that PtFOR20p specifically localises at basal bodies and is required to build the transition zone, a prerequisite to their maturation and docking at the cell surface and hence to ciliogenesis. We also found that PtCen2p (one of the two basal body specific centrins, an ortholog of HsCen2) is required to recruit PtFOR20p at the developing basal body and to control its length. By contrast, the other basal-body-specific centrin PtCen3p is not needed for assembly of the transition zone, but is required downstream, for basal body docking. Comparison of the structural defects induced by depletion of PtFOR20p, PtCen2p or PtCen3p, respectively, illustrates the dual role of the transition zone in the biogenesis of the basal body and in cilium assembly. The multiple potential roles of the transition zone during basal body biogenesis and the evolutionary conserved function of the FOP proteins in microtubule membrane interactions are discussed.

[Paramecium, a model organism to study ciliogenesis and ciliopathies]. / La paramécie, un organisme modèle pour étudier la ciliogenèse et les maladies ciliaires.

The cilium is a cell extension forming a distinct compartment of eukaryotic cell body with a complex and dynamic structure. This structure is highly conserved across species and ensures various functions as sensory and motility. In humans, ciliary dysfunction results in diseases (ciliopathies) that can affect all organs. Thanks to its complex ciliary structure, the unicellular and ciliated microorganism, Paramecium, constitutes a model of choice not only to study the structure, assembly and function of cilia but also to validate the specific role of mutations of genes linked to the ciliopathies.

TITLE: La paramécie, un organisme modèle pour étudier la ciliogenèse et les maladies ciliaires. ABSTRACT: Le cil est une extension présente à la surface de la quasi-totalité des cellules eucaryotes. Conservé au cours de l'évolution, il assure des fonctions sensorielles et/ou motiles. Chez l'homme, le dysfonctionnement ciliaire est à l'origine de différentes maladies regroupées sous le nom de ciliopathies. Grâce à sa ciliature complexe, la paramécie constitue un modèle de choix pour étudier non seulement la structure, l'assemblage et les fonctions des cils, mais aussi pour valider les mutations de gènes associées à ces ciliopathies.

Differentially expressed genes during the encystment-excystment cycle of the ciliate Sterkiella histriomuscorum.

Under unfavourable environmental conditions, many ciliates transform into resting cysts through a developmental process called encystment. Excystment is the reverse transformation of the resting cyst into a vegetative cell when favourable conditions are restored. In the oxytrichid Sterkiella histriomuscorum, the encystment - excystment (E-E) cycle involves extensive morphological changes since the whole cytoskeleton is disassembled during encystment. Assuming that these changes in cellular organization may be significantly reflected in the gene expression pattern, we used a "DNA macroarray" strategy to measure the transcript levels of 37 selected genes present at four distinct cellular stages (starved, encysted, excysting and vegetative cells). Differential expression was observed for 16 genes; four transcripts appeared to be markedly accumulated in a stage-specific manner. For most of the differentially expressed genes, the mRNA level was increased in cysts and excysting cells. When these mRNA are transcribed and when they are used, are still open questions. We showed that the copy number of the differentially expressed genes is the same in the macronuclei of cysts and vegetative cells ruling out a modulation of gene expression through a variation in the gene copy number upon encystment.

Basal body positioning and anchoring in the multiciliated cell Paramecium tetraurelia: roles of OFD1 and VFL3.

BACKGROUND: The development of a ciliary axoneme requires the correct docking of the basal body at cytoplasmic vesicles or plasma membrane. In the multiciliated cell Paramecium, three conserved proteins, FOR20, Centrin 2, and Centrin 3 participate in this process, FOR20 and Centrin 2 being involved in the assembly of the transition zone. We investigated the function of two other evolutionary conserved proteins, OFD1 and VFL3, likely involved in this process. RESULTS: In Paramecium tetraurelia, a single gene encodes OFD1, while four genes encode four isoforms of VFL3, grouped into two families, VFL3-A and VFL3-B. Depletion of OFD1 and the sole VFL3-A family impairs basal body docking. Loss of OFD1 yields a defective assembly of the basal body distal part. Like FOR20, OFD1 is recruited early during basal body assembly and localizes at the transition zone between axoneme and membrane at the level of the microtubule doublets. While the recruitment of OFD1 and Centrin 2 proceed independently, the localizations of OFD1 and FOR20 at the basal body are interdependent. In contrast, in VFL3-A depleted cells, the unanchored basal bodies harbor a fully organized distal part but display an abnormal distribution of their associated rootlets which mark their rotational asymmetry. VFL3-A, which is required for the recruitment of Centrin 3, is transiently present near the basal bodies at an early step of their duplication. VFL3-A localizes at the junction between the striated rootlet and the basal body. CONCLUSION: Our results demonstrate the conserved role of OFD1 in the anchoring mechanisms of motile cilia and establish its relations with FOR20 and Centrin 2. They support the hypothesis of its association with microtubule doublets. They suggest that the primary defect of VFL3 depletion is a loss of the rotational asymmetry of the basal body which specifies the sites of assembly of the appendages which guide the movement of basal bodies toward the cell surface. The localization of VFL3 outside of the basal body suggests that extrinsic factors could control this asymmetry.

Centrin diversity and basal body patterning across evolution: new insights from Paramecium.

First discovered in unicellular eukaryotes, centrins play crucial roles in basal body duplication and anchoring mechanisms. While the evolutionary status of the founding members of the family, Centrin2/Vfl2 and Centrin3/cdc31 has long been investigated, the evolutionary origin of other members of the family has received less attention. Using a phylogeny of ciliate centrins, we identify two other centrin families, the ciliary centrins and the centrins present in the contractile filaments (ICL centrins). In this paper, we carry on the functional analysis of still not well-known centrins, the ICL1e subfamily identified in Paramecium, and show their requirement for correct basal body anchoring through interactions with Centrin2 and Centrin3. Using Paramecium as well as a eukaryote-wide sampling of centrins from completely sequenced genomes, we revisited the evolutionary story of centrins. Their phylogeny shows that the centrins associated with the ciliate contractile filaments are widespread in eukaryotic lineages and could be as ancient as Centrin2 and Centrin3.

Paramecium tetraurelia basal body structure.

Paramecium is a free-living unicellular organism, easy to cultivate, featuring ca. 4000 motile cilia emanating from longitudinal rows of basal bodies anchored in the plasma membrane. The basal body circumferential polarity is marked by the asymmetrical organization of its associated appendages. The complex basal body plus its associated rootlets forms the kinetid. Kinetids are precisely oriented within a row in correlation with the cell polarity. Basal bodies also display a proximo-distal polarity with microtubule triplets at their proximal ends, surrounding a permanent cartwheel, and microtubule doublets at the transition zone located between the basal body and the cilium. Basal bodies remain anchored at the cell surface during the whole cell cycle. On the opposite to metazoan, there is no centriolar stage and new basal bodies develop anteriorly and at right angle from the base of the docked ones. Ciliogenesis follows a specific temporal pattern during the cell cycle and both unciliated and ciliated docked basal bodies can be observed in the same cell. The transition zone is particularly well organized with three distinct plates and a maturation of its structure is observed during the growth of the cilium. Transcriptomic and proteomic analyses have been performed in different organisms including Paramecium to understand the ciliogenesis process. The data have incremented a multi-organism database, dedicated to proteins involved in the biogenesis, composition and function of centrosomes, basal bodies or cilia. Thanks to its thousands of basal bodies and the well-known choreography of their duplication during the cell cycle, Paramecium has allowed pioneer studies focusing on the structural and functional processes underlying basal body duplication. Proteins involved in basal body anchoring are sequentially recruited to assemble the transition zone thus indicating that the anchoring process parallels the structural differentiation of the transition zone. This feature offers an opportunity to dissect spatio-temporally the mechanisms involved in the basal body anchoring process and transition zone formation.

Ciliary heterogeneity within a single cell: the Paramecium model.

Paramecium is a single cell able to divide in its morphologically differentiated stage that has many cilia anchored at its cell surface. Many thousands of cilia are thus assembled in a short period of time during division to duplicate the cell pattern while the cell continues swimming. Most, but not all, of these sensory cilia are motile and involved in two main functions: prey capture and cell locomotion. These cilia display heterogeneity, both in their length and their biochemical properties. Thanks to these properties, as well as to the availability of many postgenomic tools and the possibility to follow the regrowth of cilia after deciliation, Paramecium offers a nice opportunity to study the assembly of the cilia, as well as the genesis of their diversity within a single cell. In this paper, after a brief survey of Paramecium morphology and cilia properties, we describe the tools and the protocols currently used for immunofluorescence, transmission electron microscopy, and ultrastructural immunocytochemistry to analyze cilia, with special recommendations to overcome the problem raised by cilium diversity.

Localization of centrins in the hypotrich ciliate Paraurostyla weissei.

Centrins are ubiquitous cytoskeletal proteins that are generally associated with the centrosome and form large cytoskeletal networks in protists. To obtain more data on the respective role of different centrin proteins, we studied their distribution and behavior in one ciliate species, Paraurostyla weissei, using specific antibodies. In this species, only two major proteins of 21 and 24 kDa corresponding to centrins, were identified by 1D and 2D electrophoresis. Immunofluorescence analysis showed that these two proteins displayed non-overlapping localization in the interphase cell and during morphogenesis. Both centrin proteins localize on the fibrous network linking the oral basal bodies in the interphase cell and in the form of marginal dots, which correspond to the proximal ends of the striated rootlets; the 21 kDa centrin was also detected within the basal bodies, whereas the 24 kDa centrin allowed identifying new structures, the frontal dashes. During morphogenesis, the 21 kDa centrin locates at the basal bodies, while the 24 kDa centrin is detected along the striated rootlets and in close association with the basal bodies pairs. These data are discussed in terms of the potential roles of the two centrins in different cellular functions.

Epiplasmins and epiplasm in paramecium: the building of a submembraneous cytoskeleton.

In ciliates, basal bodies and associated appendages are bound to a submembrane cytoskeleton. In Paramecium, this cytoskeleton takes the form of a thin dense layer, the epiplasm, segmented into regular territories, the units where basal bodies are inserted. Epiplasmins, the main component of the epiplasm, constitute a large family of 51 proteins distributed in 5 phylogenetic groups, each characterized by a specific molecular design. By GFP-tagging, we analyzed their differential localisation and role in epiplasm building and demonstrated that: 1) The epiplasmins display a low turnover, in agreement with the maintenance of an epiplasm layer throughout the cell cycle; 2) Regionalisation of proteins from different groups allows us to define rim, core, ring and basal body epiplasmins in the interphase cell; 3) Their dynamics allows definition of early and late epiplasmins, detected early versus late in the duplication process of the units. Epiplasmins from each group exhibit a specific combination of properties. Core and rim epiplasmins are required to build a unit; ring and basal body epiplasmins seem more dispensable, suggesting that they are not required for basal body docking. We propose a model of epiplasm unit assembly highlighting its implication in structural heredity in agreement with the evolutionary history of epiplasmins.

Maintaining cell polarity through vegetative cell pattern dedifferentiation: cytoskeleton and morphogenesis in the hypotrich ciliate Sterkiella histriomuscorum.

The morphological differentiation of ciliates is achieved through the development of a submembraneous cytoskeleton in which the cilia are anchored. In most hypotrich ciliates, this cytoskeleton is mainly constructed of microtubules. In these species, cells pass through vegetative cell pattern dedifferentiated stages during their biological cycle. In order to investigate the behaviour of the cytoskeleton during these stages, we analysed the reorganization of the cytoskeleton during the sexual cycle of Sterkiella histriomuscorum by microscopy. Sterkiella exconjugants transiently dedifferentiate to form zygocysts devoid of ciliature and infraciliature. Immunofluorescence images obtained with antibodies directed against pericentrosomal material and tubulin showed that the cells resorb their ciliature and basal bodies, but retain their submembraneous microtubular cytoskeleton during the whole process and that the body plan is maintained through vegetative cell pattern dedifferentiation: the cell polarity remains printed on the cell surface by the microtubular cytoskeleton which in turn could mark the sites of basal body assembly during zygocyst morphogenesis. The results are discussed in terms of mechanisms of cell patterning.

The sperm roller: a modified testicular duct linked to giant sperm transport within the male reproductive tract.

The male reproductive tracts of Drosophila display considerable variation in the relative size of their components, notably of the testes, but there are few structural differences between species. Here we report a remarkable coiled structure separating the testes from the seminal vesicles in the giant sperm species Drosophila bifurca. This evolutionary novelty, known as the 'sperm roller', seems to be an exaggeration in the size of the testicular duct as revealed by light and electron microscopic observations. It consists of a tubular monocellular epithelium lying on the basal laminae and muscle and conjunctive cells. The lumen of the roller contains crypts. The apical membrane of the epithelial cells presents numerous long microvilli protruding into the lumen. The sperm roller structure is probably involved in managing sperm during their transit through the male genital tract, because sperm are seen in bundles at the base of the testis, whereas they are singly rolled up by the time they enter the seminal vesicles. The hypercoiling of the individual spermatozoon within the roller probably occurs as the result of an osmotic process produced by features of the epithelial wall and the dramatically increased exchange surface. This is the first report of a specialized device of this type in Drosophila or, more generally, in insects.