RESUMEN
Centrioles are the core constituent of centrosomes, microtubule-organizing centers involved in directing mitotic spindle assembly and chromosome segregation in animal cells. In sexually reproducing species, centrioles degenerate during oogenesis and female meiosis is usually acentrosomal. Centrioles are retained during male meiosis and, in most species, are reintroduced with the sperm during fertilization, restoring centriole numbers in embryos. In contrast, the presence, origin, and function of centrioles in parthenogenetic species is unknown. We found that centrioles are maternally inherited in two species of asexual parthenogenetic nematodes and identified two different strategies for maternal inheritance evolved in the two species. In Rhabditophanes diutinus, centrioles organize the poles of the meiotic spindle and are inherited by both the polar body and embryo. In Disploscapter pachys, the two pairs of centrioles remain close together and are inherited by the embryo only. Our results suggest that maternally-inherited centrioles organize the embryonic spindle poles and act as a symmetry-breaking cue to induce embryo polarization. Thus, in these parthenogenetic nematodes, centrioles are maternally-inherited and functionally replace their sperm-inherited counterparts in sexually reproducing species.
Asunto(s)
Centriolos , Herencia Materna , Partenogénesis , Animales , Partenogénesis/genética , Femenino , Centriolos/metabolismo , Centriolos/genética , Masculino , Herencia Materna/genética , Meiosis/genética , Huso Acromático/metabolismo , Nematodos/genética , Rhabditoidea/genética , Rhabditoidea/fisiología , Espermatozoides/metabolismo , Cuerpos Polares/metabolismo , Embrión no MamíferoRESUMEN
Cilia are cellular projections that perform sensory and motile functions in eukaryotic cells. A defining feature of cilia is that they are evolutionarily ancient, yet not universally conserved. In this study, we have used the resulting presence and absence pattern in the genomes of diverse eukaryotes to identify a set of 386 human genes associated with cilium assembly or motility. Comprehensive tissue-specific RNAi in Drosophila and mutant analysis in C. elegans revealed signature ciliary defects for 70-80% of novel genes, a percentage similar to that for known genes within the cluster. Further characterization identified different phenotypic classes, including a set of genes related to the cartwheel component Bld10/CEP135 and two highly conserved regulators of cilium biogenesis. We propose this dataset defines the core set of genes required for cilium assembly and motility across eukaryotes and presents a valuable resource for future studies of cilium biology and associated disorders.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Humanos , Caenorhabditis elegans/genética , Filogenia , Cilios/genética , Proteínas de Drosophila/genéticaRESUMEN
[This corrects the article DOI: 10.1371/journal.pgen.1010150.].
RESUMEN
Proximity-dependent labeling approaches such as BioID have been a great boon to studies of protein-protein interactions in the context of cytoskeletal structures such as centrosomes which are poorly amenable to traditional biochemical approaches like immunoprecipitation and tandem affinity purification. Yet, these methods have so far not been applied extensively to invertebrate experimental models such as C. elegans given the long labeling times required for the original promiscuous biotin ligase variant BirA*. Here, we show that the recently developed variant TurboID successfully probes the interactomes of both stably associated (SPD-5) and dynamically localized (PLK-1) centrosomal components. We further develop an indirect proximity labeling method employing a GFP nanobody-TurboID fusion, which allows the identification of protein interactors in a tissue-specific manner in the context of the whole animal. Critically, this approach utilizes available endogenous GFP fusions, avoiding the need to generate multiple additional strains for each target protein and the potential complications associated with overexpressing the protein from transgenes. Using this method, we identify homologs of two highly conserved centriolar components, Cep97 and BLD10/Cep135, which are present in various somatic tissues of the worm. Surprisingly, neither protein is expressed in early embryos, likely explaining why these proteins have escaped attention until now. Our work expands the experimental repertoire for C. elegans and opens the door for further studies of tissue-specific variation in centrosome architecture.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Biotinilación , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Centriolos , Centrosoma , Proteínas Serina-Treonina QuinasasRESUMEN
In animal cells, the functions of the microtubule cytoskeleton are coordinated by centriole-based centrosomes via γ-tubulin complexes embedded in the pericentriolar material or PCM.1 PCM assembly has been best studied in the context of mitosis, where centriolar SPD-2 recruits PLK-1, which in turn phosphorylates key scaffolding components like SPD-5 and CNN to promote expansion of the PCM polymer.2-4 To what extent these mechanisms apply to centrosomes in interphase or in differentiated cells remains unclear.5 Here, we examine a novel type of centrosome found at the ciliary base of C. elegans sensory neurons, which we show plays important roles in neuronal morphogenesis, cellular trafficking, and ciliogenesis. These centrosomes display similar dynamic behavior to canonical, mitotic centrosomes, with a stable PCM scaffold and dynamically localized client proteins. Unusually, however, they are not organized by centrioles, which degenerate early in terminal differentiation.6 Yet, PCM not only persists but continues to grow with key scaffolding proteins including SPD-5 expressed under control of the RFX transcription factor DAF-19. This assembly occurs in the absence of the mitotic regulators SPD-2, AIR-1 and PLK-1, but requires tethering by PCMD-1, a protein which also plays a role in the initial, interphase recruitment of PCM in early embryos.7 These results argue for distinct mechanisms for mitotic and non-mitotic PCM assembly, with only the former requiring PLK-1 phosphorylation to drive rapid expansion of the scaffold polymer.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Aurora Quinasa A , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Centriolos , Centrosoma , Humanos , Mitosis , Polímeros , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Centrioles are highly elaborate microtubule-based structures responsible for the formation of centrosomes and cilia. Despite considerable variation across species and tissues within any given tissue, their size is essentially constant [1, 2]. While the diameter of the centriole cylinder is set by the dimensions of the inner scaffolding structure of the cartwheel [3], how centriole length is set so precisely and stably maintained over many cell divisions is not well understood. Cep97 and CP110 are conserved proteins that localize to the distal end of centrioles and have been reported to limit centriole elongation in vertebrates [4, 5]. Here, we examine Cep97 function in Drosophila melanogaster. We show that Cep97 is essential for formation of full-length centrioles in multiple tissues of the fly. We further identify the microtubule deacetylase Sirt2 as a Cep97 interactor. Deletion of Sirt2 likewise affects centriole size. Interestingly, so does deletion of the acetylase Atat1, indicating that loss of stabilizing acetyl marks impairs centriole integrity. Cep97 and CP110 were originally identified as inhibitors of cilia formation in vertebrate cultured cells [6], and loss of CP110 is a widely used marker of basal body maturation. In contrast, in Drosophila, Cep97 appears to be only transiently removed from basal bodies and loss of Cep97 strongly impairs ciliogenesis. Collectively, our results support a model whereby Cep97 functions as part of a protective cap that acts together with the microtubule acetylation machinery to maintain centriole stability, essential for proper function in cilium biogenesis.
Asunto(s)
Centriolos/fisiología , Centrosoma , Cilios , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Proteínas Asociadas a Microtúbulos/fisiología , Morfogénesis/genética , Animales , Cuerpos Basales/metabolismo , Células Cultivadas , Centrosoma/metabolismo , Cilios/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Morfogénesis/fisiologíaRESUMEN
Woolly mammoths were among the most abundant cold-adapted species during the Pleistocene. Their once-large populations went extinct in two waves, an end-Pleistocene extinction of continental populations followed by the mid-Holocene extinction of relict populations on St. Paul Island â¼5,600 years ago and Wrangel Island â¼4,000 years ago. Wrangel Island mammoths experienced an episode of rapid demographic decline coincident with their isolation, leading to a small population, reduced genetic diversity, and the fixation of putatively deleterious alleles, but the functional consequences of these processes are unclear. Here, we show that a Wrangel Island mammoth genome had many putative deleterious mutations that are predicted to cause diverse behavioral and developmental defects. Resurrection and functional characterization of several genes from the Wrangel Island mammoth carrying putatively deleterious substitutions identified both loss and gain of function mutations in genes associated with developmental defects (HYLS1), oligozoospermia and reduced male fertility (NKD1), diabetes (NEUROG3), and the ability to detect floral scents (OR5A1). These data suggest that at least one Wrangel Island mammoth may have suffered adverse consequences from reduced population size and isolation.
Asunto(s)
Evolución Molecular , Mamuts/genética , Mutación , Animales , GenomaRESUMEN
Centrosomes, the predominant sites of microtubule nucleation and anchorage, coordinate spindle assembly and cell division in animal cells. At the onset of mitosis, centrioles accumulate microtubule-organizing pericentriolar material (PCM) in a process termed centrosome maturation. To what extent centrosome maturation depends on the continued activity of mitotic regulators or the presence of centrioles has hitherto been unclear. Using the C. elegans early embryo, we show that PCM expansion requires the Polo-like kinase PLK-1 and CEP192 (SPD-2 in C. elegans), but not its upstream regulator Aurora A (AIR-1), while maintenance of the PCM polymer depends exclusively on PLK-1. SPD-2 and PLK-1 are highly concentrated at centrioles. Unexpectedly, laser microsurgery reveals that while centrioles are required for PCM recruitment and centrosome structural integrity they are dispensable for PCM maintenance. We propose a model whereby centrioles promote centrosome maturation by recruiting PLK-1, but subsequent maintenance occurs via PLK-1 acting directly within the PCM.
Asunto(s)
Centriolos/metabolismo , Mitosis , Animales , Aurora Quinasa A/genética , Aurora Quinasa A/metabolismo , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Drosophila melanogaster , Células HeLa , Humanos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Meiotic chromosome movement is important for the pairwise alignment of homologous chromosomes, which is required for correct chromosome segregation. Movement is driven by cytoplasmic forces, transmitted to chromosome ends by nuclear membrane-spanning proteins. In animal cells, lamins form a prominent scaffold at the nuclear periphery, yet the role lamins play in meiotic chromosome movement is unclear. We show that chromosome movement correlates with reduced lamin association with the nuclear rim, which requires lamin phosphorylation at sites analogous to those that open lamina network crosslinks in mitosis. Failure to remodel the lamina results in delayed meiotic entry, altered chromatin organization, unpaired or interlocked chromosomes, and slowed chromosome movement. The remodeling kinases are delivered to lamins via chromosome ends coupled to the nuclear envelope, potentially enabling crosstalk between the lamina and chromosomal events. Thus, opening the lamina network plays a role in modulating contacts between chromosomes and the nuclear periphery during meiosis.
Asunto(s)
Animales Modificados Genéticamente/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Segregación Cromosómica , Cromosomas/genética , Profase Meiótica I/genética , Lámina Nuclear/patología , Animales , Animales Modificados Genéticamente/crecimiento & desarrollo , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/genética , Núcleo Celular/genética , Núcleo Celular/patología , Emparejamiento Cromosómico , Citoplasma , Regulación de la Expresión Génica , Membrana Nuclear/genética , Membrana Nuclear/patología , Lámina Nuclear/genética , FosforilaciónRESUMEN
Muscle development requires the coordinated activities of specific protein folding and degradation factors. UFD-2, a U-box ubiquitin ligase, has been reported to play a central role in this orchestra regulating the myosin chaperone UNC-45. Here, we apply an integrative in vitro and in vivo approach to delineate the substrate-targeting mechanism of UFD-2 and elucidate its distinct mechanistic features as an E3/E4 enzyme. Using Caenorhabditis elegans as model system, we demonstrate that UFD-2 is not regulating the protein levels of UNC-45 in muscle cells, but rather shows the characteristic properties of a bona fide E3 ligase involved in protein quality control. Our data demonstrate that UFD-2 preferentially targets unfolded protein segments. Moreover, the UNC-45 chaperone can serve as an adaptor protein of UFD-2 to poly-ubiquitinate unfolded myosin, pointing to a possible role of the UFD-2/UNC-45 pair in maintaining proteostasis in muscle cells.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Chaperonas Moleculares/metabolismo , Células Musculares/metabolismo , Miosinas/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Caenorhabditis elegans , Proteostasis , Ubiquitina/metabolismo , Ubiquitinación , Respuesta de Proteína DesplegadaRESUMEN
Cilia are cellular projections that assemble on centriole-derived basal bodies. While cilia assembly is absolutely dependent on centrioles, it is not known to what extent they contribute to downstream events. The nematode C. elegans provides a unique opportunity to address this question, as centrioles do not persist at the base of mature cilia. Using fluorescence microscopy and electron tomography, we find that centrioles degenerate early during ciliogenesis. The transition zone and axoneme are not completely formed at this time, indicating that cilia maturation does not depend on intact centrioles. The hydrolethalus syndrome protein HYLS-1 is the only centriolar protein known to remain at the base of mature cilia and is required for intraflagellar transport trafficking. Surprisingly, targeted degradation of HYLS-1 after initiation of ciliogenesis does not affect ciliary structures. Taken together, our results indicate that while centrioles are essential to initiate cilia formation, they are dispensable for cilia maturation and maintenance.
Asunto(s)
Cuerpos Basales/fisiología , Caenorhabditis elegans/fisiología , Centriolos/fisiología , Neurogénesis , Células Receptoras Sensoriales/fisiología , Animales , Animales Modificados Genéticamente , Axonema/fisiología , Cuerpos Basales/metabolismo , Cuerpos Basales/ultraestructura , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestructura , Proteínas de Caenorhabditis elegans/metabolismo , Centriolos/metabolismo , Centriolos/ultraestructura , Cilios/fisiología , Tomografía con Microscopio Electrónico , Microscopía Fluorescente , Microscopía por Video , Proteolisis , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/ultraestructura , Factores de Tiempo , Imagen de Lapso de TiempoRESUMEN
Microtubule-organizing centers (MTOCs) nucleate microtubules that can grow autonomously in any direction. To generate bundles of parallel microtubules originating from a single MTOC, the growth of multiple microtubules needs to coordinated, but the underlying mechanism is unknown. Here, we show that a conserved two-component system consisting of the plus-end tracker EB1 and the minus-end-directed molecular motor Kinesin-14 is sufficient to promote parallel microtubule growth. The underlying mechanism relies on the ability of Kinesin-14 to guide growing plus ends along existing microtubules. The generality of this finding is supported by yeast, Drosophila, and human EB1/Kinesin-14 pairs. We demonstrate that plus-end guiding involves a directional switch of the motor due to a force applied via a growing microtubule end. The described mechanism can account for the generation of parallel microtubule networks required for a broad range of cellular functions such as spindle assembly or cell polarization.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , ADN Helicasas/metabolismo , Cinesinas/metabolismo , Proteínas de Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Proteínas Oncogénicas/metabolismo , ARN Helicasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Animales , Drosophila melanogaster , Humanos , Fenómenos MecánicosRESUMEN
Transition fibres (TFs), together with the transition zone (TZ), are basal ciliary structures thought to be crucial for cilium biogenesis and function by acting as a ciliary gate to regulate selective protein entry and exit. Here we demonstrate that the centriolar and basal body protein HYLS-1, the C. elegans orthologue of hydrolethalus syndrome protein 1, is required for TF formation, TZ organization and ciliary gating. Loss of HYLS-1 compromises the docking and entry of intraflagellar transport (IFT) particles, ciliary gating for both membrane and soluble proteins, and axoneme assembly. Additional depletion of the TF component DYF-19 in hyls-1 mutants further exacerbates TZ anomalies and completely abrogates ciliogenesis. Our data support an important role for HYLS-1 and TFs in establishment of the ciliary gate and underline the importance of selective protein entry for cilia assembly.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Cilios/metabolismo , Deformidades Congénitas de la Mano/metabolismo , Cardiopatías Congénitas/metabolismo , Hidrocefalia/metabolismo , Animales , Caenorhabditis elegans , Cilios/ultraestructura , Flagelos/metabolismo , Eliminación de Gen , Modelos Biológicos , OrganogénesisRESUMEN
Primary microcephaly (MCPH) associated proteins CDK5RAP2, CEP152, WDR62 and CEP63 colocalize at the centrosome. We found that they interact to promote centriole duplication and form a hierarchy in which each is required to localize another to the centrosome, with CDK5RAP2 at the apex, and CEP152, WDR62 and CEP63 at sequentially lower positions. MCPH proteins interact with distinct centriolar satellite proteins; CDK5RAP2 interacts with SPAG5 and CEP72, CEP152 with CEP131, WDR62 with MOONRAKER, and CEP63 with CEP90 and CCDC14. These satellite proteins localize their cognate MCPH interactors to centrosomes and also promote centriole duplication. Consistent with a role for satellites in microcephaly, homozygous mutations in one satellite gene, CEP90, may cause MCPH. The satellite proteins, with the exception of CCDC14, and MCPH proteins promote centriole duplication by recruiting CDK2 to the centrosome. Thus, centriolar satellites build a MCPH complex critical for human neurodevelopment that promotes CDK2 centrosomal localization and centriole duplication.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Línea Celular , Humanos , Multimerización de ProteínaRESUMEN
In addition to organizing centrosomes, centrioles have an evolutionarily conserved role as basal bodies in the formation of cilia. The discovery of a range of human genetic disorders linked to cilia dysfunction has led to a resurgence of interest in this cellular organelle. The nematode Caenorhabditis elegans possesses several unique features (highly stereotypical morphology, dispensability of cilia for organismal viability and fertility) that make it an attractive model to study cilia assembly and function. However, both the adult worm and the embryo present particular challenges for electron microscopy (EM), which remains the gold standard for high-resolution morphological studies. Here, we present a step-by-step guide for the ultrastructural analysis of C. elegans cilia, including optimized protocols for standard chemical fixation as well as high-pressure freezing/freeze substitution and further processing for serial-section transmission electron microscopy and electron tomography.
Asunto(s)
Caenorhabditis elegans/ultraestructura , Cilios/ultraestructura , Animales , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Imagenología Tridimensional , Microscopía Electrónica de Transmisión , Microtomía , Adhesión en PlásticoRESUMEN
Cilia are cellular projections that perform sensory and motile functions. A key ciliary subdomain is the transition zone, which lies between basal body and axoneme. Previous work in Caenorhabditis elegans identified two ciliopathy-associated protein complexes or modules that direct assembly of transition zone Y-links. Here, we identify C. elegans CEP290 as a component of a third module required to form an inner scaffolding structure called the central cylinder. Co-inhibition of all three modules completely disrupted transition zone structure. Surprisingly, axoneme assembly was only mildly perturbed. However, dendrite extension by retrograde migration was strongly impaired, revealing an unexpected role for the transition zone in cell adhesion.
Asunto(s)
Axonema/metabolismo , Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/metabolismo , Adhesión Celular , Proteínas Asociadas a Microtúbulos/fisiología , Animales , Caenorhabditis elegans/citología , Microscopía por Video , Multimerización de ProteínaRESUMEN
Centrosome function in cell division requires their duplication, once, and only once, per cell cycle. Underlying centrosome duplication are alternating cycles of centriole assembly and separation. Work in vertebrates has implicated the cysteine protease separase in anaphase-coupled centriole separation (or disengagement) and identified this as a key step in licensing another round of assembly. Current models have separase cleaving a physical link between centrioles, potentially cohesin, that prevents reinitiation of centriole assembly unless disengaged. Here, we examine separase function in the C. elegans early embryo. We find that depletion impairs separation and consequently duplication of sperm-derived centrioles at the meiosis-mitosis transition. However, subsequent cycles proceed normally. Whereas mitotic centrioles separate in the context of cortical forces acting on a disassembling pericentriolar material, sperm centrioles are not associated with significant pericentriolar material or subject to strong forces. Increasing centrosomal microtubule nucleation restores sperm centriole separation and duplication in separase-depleted embryos, while forced pericentriolar material disassembly drives premature separation in mitosis. These results emphasize the critical role of cytoskeletal forces and the pericentriolar material in centriole separation. Separase contributes to separation where forces are limited, offering a potential explanation for results obtained in different experimental models.
Asunto(s)
Caenorhabditis elegans/genética , Centriolos/metabolismo , Embrión no Mamífero/metabolismo , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Ciclo Celular , División Celular , Embrión no Mamífero/embriología , Meiosis , Microscopía Fluorescente , Mitosis , Separasa/metabolismoRESUMEN
The centriole is a conserved microtubule-based organelle essential for both centrosome formation and cilium biogenesis. Five conserved proteins for centriole duplication have been identified. Two of them, SAS-5 and SAS-6, physically interact with each other and are codependent for their targeting to procentrioles. However, it remains unclear how these two proteins interact at the molecular level. Here, we demonstrate that the short SAS-5 C-terminal domain (residues 390-404) specifically binds to a narrow central region (residues 275-288) of the SAS-6 coiled coil. This was supported by the crystal structure of the SAS-6 coiled-coil domain (CCD), which, together with mutagenesis studies, indicated that the association is mediated by synergistic hydrophobic and electrostatic interactions. The crystal structure also shows a periodic charge pattern along the SAS-6 CCD, which gives rise to an anti-parallel tetramer. Overall, our findings establish the molecular basis of the specific interaction between SAS-5 and SAS-6, and suggest that both proteins individually adopt an oligomeric conformation that is disrupted upon the formation of the hetero-complex to facilitate the correct assembly of the nine-fold symmetric centriole.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Centriolos/química , Cristalografía por Rayos X , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
During meiotic prophase in fission yeast, the nucleus undergoes dramatic oscillatory movements. A newly identified structure, the radial microtubule organizing center (rMTOC), mediates these movements and shares some of the features of the pericentriolar material in higher eukaryotes.