RESUMEN
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.
Asunto(s)
Centriolos/metabolismo , Cilios/ultraestructura , Paramecium/metabolismo , Animales , Membrana Celular , Centriolos/química , Cilios/metabolismo , Mamíferos , Paramecium/química , Paramecium/citologíaRESUMEN
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.
Asunto(s)
Cilios/patología , Trastornos de la Motilidad Ciliar/etiología , Dineínas/metabolismo , Flagelos/patología , Mutación , Proteínas/genética , Cola del Espermatozoide/patología , Adulto , Axonema , Niño , Cilios/metabolismo , Trastornos de la Motilidad Ciliar/patología , Dineínas/genética , Femenino , Flagelos/metabolismo , Homocigoto , Humanos , Infertilidad Masculina/etiología , Infertilidad Masculina/patología , Masculino , Persona de Mediana Edad , Linaje , Fenotipo , Motilidad Espermática , Cola del Espermatozoide/metabolismo , Adulto JovenRESUMEN
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.
Asunto(s)
Cilios/metabolismo , Paramecium tetraurelia/citología , Proteínas Protozoarias/metabolismo , Proliferación Celular , Cilios/fisiología , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Expresión Génica , Fusión de Membrana/genética , Paramecium tetraurelia/genética , Dominios Proteicos , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Interferencia de ARNRESUMEN
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.
Asunto(s)
Dineínas Axonemales/metabolismo , Trastornos de la Motilidad Ciliar/genética , Proteínas del Citoesqueleto/genética , Flagelos/metabolismo , Mutación/genética , Proteínas Nucleares/genética , Alelos , Secuencia de Aminoácidos , Dineínas Axonemales/ultraestructura , Secuencia de Bases , Transporte Biológico , Diferenciación Celular/genética , Chlamydomonas/metabolismo , Secuencia Conservada/genética , Flagelos/ultraestructura , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Proteínas Nucleares/química , Paramecium/metabolismo , Paramecium/ultraestructura , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Dineínas Axonemales/genética , Cilios/genética , Dineínas/genética , Mutación/genética , Situs Inversus/genética , Adolescente , Secuencia de Aminoácidos , Niño , Preescolar , Trastornos de la Motilidad Ciliar/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , Sistema Respiratorio/patología , Alineación de SecuenciaRESUMEN
The intraflagellar transport IFT57 protein is essential for ciliary growth and maintenance. Also known as HIPPI, human IFT57 can be translocated to the nucleus via a molecular partner of the Huntingtin, Hip1, inducing gene expression changes. In Paramecium tetraurelia, we identified four IFT57 genes forming two subfamilies IFT57A/B and IFT57C/D arising from whole genome duplications. The depletion of proteins of the two subfamilies induced ciliary defects and IFT57A and IFT57C localized in basal bodies and cilia. We observed that IFT57A, but not IFT57C, is also present in the macronucleus and able to traffic toward the developing anlage during autogamy. Analysis of chimeric IFT57A-IFT57C-GFP-tagged proteins allowed us to identify a region of IFT57A necessary for nuclear localization. We studied the localization of the unique IFT57 protein of Paramecium caudatum, a species, which diverged from P. tetraurelia before the whole genome duplications. The P. caudatumIFT57C protein was excluded from the nucleus. We also analyzed whether the overexpression of IFT57A in Paramecium could affect gene transcription as the human protein does in HeLa cells. The expression of some genes was indeed affected by overexpression of IFT57A, but the set of affected genes poorly overlaps the set of genes affected in human cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Macronúcleo/fisiología , Familia de Multigenes/genética , Paramecium tetraurelia/fisiología , Proteínas Protozoarias/genética , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Genes Protozoarios/genética , Macronúcleo/genética , Paramecium tetraurelia/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
Membrana Celular/metabolismo , Centrosoma/metabolismo , Secuencia Conservada , Paramecium/citología , Paramecium/metabolismo , Proteínas Protozoarias/metabolismo , Cilios/metabolismo , Cilios/ultraestructura , Genes Protozoarios , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Paramecium/genética , Paramecium/ultraestructura , Transporte de Proteínas , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The individual role of the outer dynein arm light chains in the molecular mechanisms of ciliary movements in response to second messengers, such as Ca(2+) and cyclic nucleotides, is unclear. We examined the role of the gene termed the outer dynein arm light chain 1 (LC1) gene of Paramecium tetraurelia (ODAL1), a homologue of the outer dynein arm LC1 gene of Chlamydomonas reinhardtii, in ciliary movements by RNA interference (RNAi) using a feeding method. The ODAL1-silenced (ODAL1-RNAi) cells swam slowly, and their swimming velocity did not increase in response to membrane-hyperpolarizing stimuli. Ciliary movements on the cortical sheets of ODAL1-RNAi cells revealed that the ciliary beat frequency was significantly lower than that of control cells in the presence of ≥ 1 mM Mg(2+)-ATP. In addition, the ciliary orientation of ODAL1-RNAi cells did not change in response to cyclic AMP (cAMP). A 29-kDa protein phosphorylated in a cAMP-dependent manner in the control cells disappeared in the axoneme of ODAL1-RNAi cells. These results indicate that ODAL1 is essential for controlling the ciliary response by cAMP-dependent phosphorylation.
Asunto(s)
Cilios/metabolismo , AMP Cíclico/metabolismo , Dineínas/metabolismo , Locomoción , Paramecium tetraurelia/metabolismo , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Axonema/genética , Axonema/metabolismo , Cloruro de Calcio/farmacología , Cilios/efectos de los fármacos , Técnicas de Cultivo , Dineínas/genética , Electroforesis en Gel de Poliacrilamida , Genes Protozoarios , Datos de Secuencia Molecular , Paramecium tetraurelia/genética , Fenotipo , Fosforilación , Proteínas Protozoarias/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The duplication of entire genomes has long been recognized as having great potential for evolutionary novelties, but the mechanisms underlying their resolution through gene loss are poorly understood. Here we show that in the unicellular eukaryote Paramecium tetraurelia, a ciliate, most of the nearly 40,000 genes arose through at least three successive whole-genome duplications. Phylogenetic analysis indicates that the most recent duplication coincides with an explosion of speciation events that gave rise to the P. aurelia complex of 15 sibling species. We observed that gene loss occurs over a long timescale, not as an initial massive event. Genes from the same metabolic pathway or protein complex have common patterns of gene loss, and highly expressed genes are over-retained after all duplications. The conclusion of this analysis is that many genes are maintained after whole-genome duplication not because of functional innovation but because of gene dosage constraints.
Asunto(s)
Evolución Molecular , Duplicación de Gen , Genoma de Protozoos/genética , Genómica , Paramecium tetraurelia/genética , Animales , Células Eucariotas/metabolismo , Genes Duplicados/genética , Genes Protozoarios/genética , Datos de Secuencia Molecular , FilogeniaRESUMEN
Observations in various organisms1-3 suggest that the transfer of mitochondrial DNA sequences to the nucleus has occurred in the course of evolution. Wright and Cummings have reported4, on the basis of hybridization experiments, that there is such a transposition in the fungus Podospora anserina during senescence5 (arrest of vegetative growth) which is accompanied by amplification of specific mitochondrial sequences6-9. They suggested that this transposition could explain senescence, and, as it could be regularly observed under laboratory conditions, senescence in Podospora could constitute a model system to study the molecular mechanisms of the transfer of DNA sequences from mitochondria to the nucleus. We report here experiments similar to those of Wright and Cummings4 but include additional critical controls. Our results do not confirm the data of Wright and Cummings; the only positive signals we observed after hybridization of mitochondrial DNA sequences on nuclear DNAs can be attributed to contaminating DNA from mitochondria or plasmids.
RESUMEN
Centrioles are characterized by a nine-fold arrangement of microtubule triplets held together by an inner protein scaffold. These structurally robust organelles experience strenuous cellular processes such as cell division or ciliary beating while performing their function. However, the molecular mechanisms underlying the stability of microtubule triplets, as well as centriole architectural integrity remain poorly understood. Here, using ultrastructure expansion microscopy for nanoscale protein mapping, we reveal that POC16 and its human homolog WDR90 are components of the microtubule wall along the central core region of the centriole. We further found that WDR90 is an evolutionary microtubule associated protein. Finally, we demonstrate that WDR90 depletion impairs the localization of inner scaffold components, leading to centriole structural abnormalities in human cells. Altogether, this work highlights that WDR90 is an evolutionary conserved molecular player participating in centriole architecture integrity.
Cells are made up of compartments called organelles that perform specific roles. A cylindrical organelle called the centriole is important for a number of cellular processes, ranging from cell division to movement and signaling. Each centriole contains nine blades made up of protein filaments called microtubules, which link together to form a cylinder. This well-known structure can be found in a variety of different species. Yet, it is unclear how centrioles are able to maintain this stable architecture whilst carrying out their various different cell roles. In early 2020, a group of researchers discovered a scaffold protein at the center of centrioles that helps keep the microtubule blades stable. Further investigation suggested that another protein called WDR90 may also help centrioles sustain their cylindrical shape. However, the exact role of this protein was poorly understood. To determine the role of WDR90, Steib et al. including many of the researchers involved in the 2020 study used a method called Ultrastructure Expansion Microscopy to precisely locate the WDR90 protein in centrioles. This revealed that WDR90 is located on the microtubule wall of centrioles in green algae and human cells grown in the lab. Further experiments showed that the protein binds directly to microtubules and that removing WDR90 from human cells causes centrioles to lose their scaffold proteins and develop structural defects. This investigation provides fundamental insights into the structure and stability of centrioles. It shows that single proteins are key components in supporting the structural integrity of organelles and shaping their overall architecture. Furthermore, these findings demonstrate how ultrastructure expansion microscopy can be used to determine the role of individual proteins within a complex structure.
Asunto(s)
Centriolos , Proteínas del Citoesqueleto , Microtúbulos , Animales , Bovinos , Línea Celular , Células Cultivadas , Centriolos/metabolismo , Centriolos/ultraestructura , Chlamydomonas , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/ultraestructura , Humanos , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/ultraestructuraRESUMEN
BACKGROUND: Ciliary or flagellar basal bodies and centrioles share the same architecture and remarkable property of duplicating once per cell cycle. Duplication is known to proceed by budding of the daugther organelle close to and at right angles to the mother structure, but the molecular basis of this geometry remains unknown. Among the handful of proteins implicated in basal-body/centriole duplication, centrins seem required in all eukaryotes tested, but their mode of action is not clear. We have investigated centrin function in Paramecium, whose cortical organization allows detection of any spatial or temporal alteration in the pattern of basal-body duplication. RESULTS: We have characterized two pairs of genes, PtCEN2a and PtCEN2b as well as PtCEN3a and PtCEN3b, orthologs of HsCEN2 and HsCEN3, respectively. GFP tags revealed different localization for the two pairs of gene products, at basal bodies or on basal-body-associated filamentous arrays, respectively. Centrin depletion induced by RNAi caused mislocalization of the neoformed basal bodies: abnormal site of budding (PtCen2ap) or absence of separation between mother and daughter organelles (PtCen3ap). Over successive divisions, new basal bodies continued to be assembled, but internalization of the mispositionned basal bodies led to a progressive decrease in the number of cortical basal bodies. CONCLUSIONS: Our observations show that centrins (1) are required to define the site and polarities of duplication and to sever the mother-daughter links and (2) play no triggering or instrumental role in assembly. Our data underscore the biological importance of the geometry of the duplication process.
Asunto(s)
Proteínas de Unión al Calcio/deficiencia , Centriolos/metabolismo , Proteínas Cromosómicas no Histona/deficiencia , Flagelos/metabolismo , Paramecium/metabolismo , Filogenia , Secuencia de Aminoácidos , Animales , Proteínas de Unión al Calcio/genética , División Celular/fisiología , Proteínas Cromosómicas no Histona/genética , Clonación Molecular , Análisis por Conglomerados , Proteínas Fluorescentes Verdes/metabolismo , Immunoblotting , Microscopía Electrónica , Microscopía Fluorescente , Datos de Secuencia Molecular , Paramecium/ultraestructura , Interferencia de ARN , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
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.
RESUMEN
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.
RESUMEN
BACKGROUND: New generation technologies in cell and molecular biology generate large amounts of data hard to exploit for individual proteins. This is particularly true for ciliary and centrosomal research. Cildb is a multi-species knowledgebase gathering high throughput studies, which allows advanced searches to identify proteins involved in centrosome, basal body or cilia biogenesis, composition and function. Combined to localization of genetic diseases on human chromosomes given by OMIM links, candidate ciliopathy proteins can be compiled through Cildb searches. METHODS: Othology between recent versions of the whole proteomes was computed using Inparanoid and ciliary high throughput studies were remapped on these recent versions. RESULTS: Due to constant evolution of the ciliary and centrosomal field, Cildb has been recently upgraded twice, with new species whole proteomes and new ciliary studies, and the latter version displays a novel BioMart interface, much more intuitive than the previous ones. CONCLUSIONS: This already popular database is designed now for easier use and is up to date in regard to high throughput ciliary studies.
RESUMEN
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.
Asunto(s)
Citoesqueleto/metabolismo , Paramecium/citología , Paramecium/metabolismo , Proteínas Protozoarias/metabolismo , Ciclo Celular , Citoesqueleto/genética , Citoesqueleto/ultraestructura , Microscopía Electrónica , Paramecium/clasificación , Paramecium/crecimiento & desarrollo , Filogenia , Proteínas Protozoarias/genéticaRESUMEN
Basal bodies are tightly controlled not only for their time of duplication but also for their movements, which ensure proper division and morphogenesis. However, the mechanisms underlying these movements only begin to be explored. We describe here a novel basal body appendage in Paramecium, the anterior left filament (ALF), which develops transiently from the mother basal body before duplication and disassembles once the new basal body is docked at the surface. By comparing the ultrastructure of dividing wild type cells to that of cells defective in basal body duplication, either by depletion of conserved proteins required for basal body assembly, or by mutation, we showed 1) that assembly of the ALF requires PtCen3p, one of the two basal body specific centrins and 2) that absence of the ALF correlates with a failure of the newly assembled basal bodies to tilt up to their docking site at the surface. This correlation suggests that the function of the ALF consists in anchoring centrin-containing contractile fibers which pull up the new basal body toward its site of docking. The presence in T. thermophila of an ALF-like appendage suggests the conservation of an ancestral mechanism ensuring the coupling of basal body duplication and cell morphogenesis.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Orgánulos/metabolismo , Paramecium tetraurelia/metabolismo , Paramecium tetraurelia/ultraestructura , Animales , Proteínas de Unión al Calcio/genética , División Celular , Centriolos/genética , Centriolos/metabolismo , Centriolos/ultraestructura , Proteínas Cromosómicas no Histona/genética , Cilios/genética , Cilios/metabolismo , Cilios/ultraestructura , Orgánulos/genética , Orgánulos/ultraestructura , Paramecium tetraurelia/citología , Paramecium tetraurelia/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
BACKGROUND: Changes in genes coding for ciliary proteins contribute to complex human syndromes called ciliopathies, such as Bardet-Biedl Syndrome (BBS). We used the model organism Paramecium to focus on ciliary ion channels that affect the beat form and sensory function of motile cilia and evaluate the effects of perturbing BBS proteins on these channels. METHODS: We used immunoprecipitations and mass spectrometry to explore whether Paramecium proteins interact as in mammalian cells. We used RNA interference (RNAi) and swimming behavior assays to examine the effects of BBS depletion on ciliary ion channels that control ciliary beating. Combining RNA interference and epitope tagging, we examined the effects of BBS depletion of BBS 7, 8 and 9 on the location of three channels and a chemoreceptor in cilia. RESULTS: We found 10 orthologs of 8 BBS genes in P. tetraurelia. BBS1, 2, 4, 5, 7, 8 and 9 co-immunoprecipitate. While RNAi reduction of BBS 7 and 9 gene products caused loss and shortening of cilia, RNAi for all BBS genes except BBS2 affected patterns of ciliary motility that are governed by ciliary ion channels. Swimming behavior assays pointed to loss of ciliary K+ channel function. Combining RNAi and epitope tagged ciliary proteins we demonstrated that a calcium activated K+ channel was no longer located in the cilia upon depletion of BBS 7, 8 or 9, consistent with the cells' swimming behavior. The TRPP channel PKD2 was also lost from the cilia. In contrast, the ciliary voltage gated calcium channel was unaffected by BBS depletion, consistent with behavioral assays. The ciliary location of a chemoreceptor for folate was similarly unperturbed by the depletion of BBS 7, 8 or 9. CONCLUSIONS: The co-immunoprecipitation of BBS 1,2,4,5,7,8, and 9 suggests a complex of BBS proteins. RNAi for BBS 7, 8 or 9 gene products causes the selective loss of K+ and PKD2 channels from the cilia while the critical voltage gated calcium channel and a peripheral receptor protein remain undisturbed. These channels govern ciliary beating and sensory function. Importantly, in P. tetraurelia we can combine studies of ciliopathy protein function with behavior and location and control of ciliary channels.
RESUMEN
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.
Asunto(s)
Centriolos/metabolismo , Paramecium/citología , Proteínas Protozoarias/metabolismo , Animales , Centriolos/ultraestructura , Cilios/fisiología , Cilios/ultraestructura , Silenciador del Gen , Humanos , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Basal bodies which nucleate cilia and flagella, and centrioles which organize centrosomes share the same architecture characterized by the ninefold symmetry of their microtubular shaft. Among the conserved proteins involved in the biogenesis of the canonical 9-triplet centriolar structures, Sas-6 and Bld10 proteins have been shown to play central roles in the early steps of assembly and in establishment/stabilization of the ninefold symmetry. Using fluorescent tagged proteins and RNAi to study the localization and function of these two proteins in Paramecium, we focused on the early effects of their depletion, the consequences of their overexpression and their functional interdependence. We find that both genes are essential and their depletion affects cartwheel assembly and hence basal body duplication. We also show that, contrary to Sas6p, Bld10p is not directly responsible for the establishment of the ninefold symmetry, but is required not only for new basal body assembly and stability but also for Sas6p maintenance at mature basal bodies. Finally, ultrastructural analysis of cells overexpressing either protein revealed two types of early assembly intermediates, hub-like structures and generative discs, suggesting a conserved scaffolding process.