RESUMEN
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
Centrioles and basal bodies are MT based structures that present a highly conserved ninefold symmetry. Centrioles can be found at the core of the centrosome where they participate in PCM recruitment and organization, contributing to cytoplasmic MT nucleation. Basal bodies are normally located closely to the plasma membrane where they are responsible for axoneme assembly to form structures such as cilia or flagella. While it is well accepted that these organelles have important roles in cell and tissue organization, their contribution to certain phases of animal development is still not entirely established. Here we review the role of centrosomes and cilia in Drosophila melanogaster and briefly discuss the implications of these findings to other model organisms.
Asunto(s)
Centriolos/fisiología , Drosophila melanogaster/fisiología , Animales , Centriolos/ultraestructura , Cilios/fisiología , Cilios/ultraestructura , Drosophila melanogaster/ultraestructuraRESUMEN
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
Ploidy variations such as genome doubling are frequent in human tumors and have been associated with genetic instability favoring tumor progression. How polyploid cells deal with increased centrosome numbers and DNA content remains unknown. Using Drosophila neuroblasts and human cancer cells to study mitotic spindle assembly in polyploid cells, we found that most polyploid cells divide in a multipolar manner. We show that even if an initial centrosome clustering step can occur at mitotic entry, the establishment of kinetochore-microtubule attachments leads to spatial chromosome configurations, whereby the final coalescence of supernumerary poles into a bipolar array is inhibited. Using in silico approaches and various spindle and DNA perturbations, we show that chromosomes act as a physical barrier blocking spindle pole coalescence and bipolarity. Importantly, microtubule stabilization suppressed multipolarity by improving both centrosome clustering and pole coalescence. This work identifies inhibitors of bipolar division in polyploid cells and provides a rationale to understand chromosome instability typical of polyploid cancer cells.
Asunto(s)
Centrosoma/metabolismo , Poliploidía , Huso Acromático/metabolismo , Animales , Células Cultivadas , Drosophila , Femenino , Células HEK293 , Humanos , Huso Acromático/genéticaRESUMEN
Defects in mitotic spindle orientation (MSO) disrupt the organization of stem cell niches impacting tissue morphogenesis and homeostasis. Mutations in centrosome genes reduce MSO fidelity, leading to tissue dysplasia and causing several diseases such as microcephaly, dwarfism, and cancer. Whether these mutations perturb spindle orientation solely by affecting astral microtubule nucleation or whether centrosome proteins have more direct functions in regulating MSO is unknown. To investigate this question, we analyzed the consequences of deregulating Plk4 (the master centriole duplication kinase) activity in Drosophila asymmetrically dividing neural stem cells. We found that Plk4 functions upstream of MSO control, orchestrating centriole symmetry breaking and consequently centrosome positioning. Mechanistically, we show that Plk4 acts through Spd2 phosphorylation, which induces centriole release from the apical cortex. Overall, this work not only reveals a role for Plk4 in regulating centrosome function but also links the centrosome biogenesis machinery with the MSO apparatus.
Asunto(s)
Proteínas Cdh1/metabolismo , Centriolos/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células-Madre Neurales/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Huso Acromático/fisiología , Animales , Proteínas Cdh1/genética , Ciclo Celular , Células Cultivadas , Centrosoma/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Masculino , Células-Madre Neurales/citología , Fosforilación , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Centrosomes act as the main microtubule-organizing centre of animal cells and play critical roles in the cell, such as mitotic spindle organization, cell polarity, and motility. They are composed of two barrel-shaped structures, the centrioles, surrounded by the pericentriolar matrix. In mammalian cells, the two centrioles differ structurally due to generational difference, the oldest one bearing appendages which allow the transient docking of the centriole at the plasma membrane in order to grow a primary cilium. Centrosome components are highly conserved throughout evolution and several pathologies have been associated with centrosomal defects. The understanding of such a complex organelle has therefore been a challenge for many researchers and has led to the development of centrosomal purification procedures to assess molecular composition, biological function, and structural organization of centrosomes. In this paper, we detail a step-by-step procedure to generate high yield of purified centrosome obtained from various mammalian cell lines.
Asunto(s)
Centrosoma , Fraccionamiento Celular/métodos , Línea Celular , HumanosRESUMEN
Aneuploidy is associated with a variety of diseases such as cancer and microcephaly. Although many studies have addressed the consequences of a non-euploid genome in cells, little is known about their overall consequences in tissue and organism development. Here we use two different mutant conditions to address the consequences of aneuploidy during tissue development and homeostasis in Drosophila. We show that aneuploidy causes brain size reduction due to a decrease in the number of proliferative neural stem cells (NSCs), but not through apoptosis. Instead, aneuploid NSCs present an extended G1 phase, which leads to cell cycle exit and premature differentiation. Moreover, we show that this response to aneuploidy is also present in adult intestinal stem cells but not in the wing disc. Our work highlights a neural and intestine stem cell-specific response to aneuploidy, which prevents their proliferation and expansion.
Asunto(s)
Aneuploidia , Encéfalo/embriología , Diferenciación Celular/genética , Fase G1/genética , Intestinos/citología , Células-Madre Neurales/metabolismo , Animales , Encéfalo/metabolismo , Proliferación Celular/genética , Drosophila , Inmunohistoquímica , Células-Madre Neurales/citología , Tamaño de los Órganos , Células Madre/citología , Células Madre/metabolismo , Alas de Animales/citologíaRESUMEN
Centrosome amplification has severe consequences during development and is thought to contribute to a variety of diseases such as cancer and microcephaly. However, the adverse effects of centrosome amplification in epithelia are still not known. Here, we investigate the consequences of centrosome amplification in the Drosophila wing disc epithelium. We found that epithelial cells exhibit mechanisms of clustering but also inactivation of extra centrosomes. Importantly, these mechanisms are not fully efficient, and both aneuploidy and cell death can be detected. Epithelial cells with extra centrosomes generate tumors when transplanted into WT hosts and inhibition of cell death results in tissue over-growth and disorganization. Using SILAC-fly, we found that Moesin, a FERM domain protein, is specifically upregulated in wing discs with extra centrosomes. Moesin localizes to the centrosomes and mitotic spindle during mitosis, and we show that Moesin upregulation influences extra-centrosome behavior and robust bipolar spindle formation. This study provides a mechanistic explanation for the increased aneuploidy and transformation potential primed by centrosome amplification in epithelial tissues.
Asunto(s)
Centrosoma/metabolismo , Drosophila/metabolismo , Células Epiteliales/metabolismo , Proteínas de Microfilamentos/metabolismo , Huso Acromático/metabolismo , Regulación hacia Arriba , Aneuploidia , Animales , Muerte Celular , Células Epiteliales/citologíaRESUMEN
Cilia and flagella are organelles essential for motility and sensing of environmental stimuli. Depending on the cell type, cilia acquire a defined set of functions and, accordingly, are built with an appropriate length and molecular composition. Several ciliary proteins display a high degree of conservation throughout evolution and mutations in ciliary genes are associated with various diseases such as ciliopathies and infertility. Here, we describe the role of the highly conserved ciliary protein, Bug22, in Drosophila. Previous studies in unicellular organisms have shown that Bug22 is required for proper cilia function, but its exact role in ciliogenesis has not been investigated yet. Null Bug22 mutant flies display cilia-associated phenotypes and nervous system defects. Furthermore, sperm differentiation is blocked at the individualization stage, due to impaired migration of the individualization machinery. Tubulin post-translational modifications (PTMs) such as polyglycylation, polyglutamylation or acetylation, are determinants of microtubule (MT) functions and stability in centrioles, cilia and neurons. We found defects in the timely incorporation of polyglycylation in sperm axonemal MTs of Bug22 mutants. In addition, we found that depletion of human Bug22 in RPE1 cells resulted in the appearance of longer cilia and reduced axonemal polyglutamylation. Our work identifies Bug22 as a protein that plays a conserved role in the regulation of PTMs of the ciliary axoneme.
RESUMEN
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
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.
Asunto(s)
Centriolos/genética , Cilios/metabolismo , Flagelos/metabolismo , Paramecium/ultraestructura , Proteínas Protozoarias/fisiología , Animales , Centriolos/ultraestructura , Flagelos/ultraestructura , Proteínas Fluorescentes Verdes/metabolismo , Paramecium/genética , Paramecium/metabolismo , Proteínas Protozoarias/metabolismo , Interferencia de ARNRESUMEN
In addition to their key role in the duplication of microtubule organising centres (MTOCs), centrins are major constituents of diverse MTOC-associated contractile arrays. A centrin partner, Sfi1p, has been characterised in yeast as a large protein carrying multiple centrin-binding sites, suggesting a model for centrin-mediated Ca2+-induced contractility and for the duplication of MTOCs. In vivo validation of this model has been obtained in Paramecium, which possesses an extended contractile array - the infraciliary lattice (ICL) - essentially composed of centrins and a huge Sfi1p-like protein, PtCenBP1p, which is essential for ICL assembly and contractility. The high molecular diversity revealed here by the proteomic analysis of the ICL, including ten subfamilies of centrins and two subfamilies of Sf1p-like proteins, led us to address the question of the functional redundancy, either between the centrin-binding proteins or between the centrin subfamilies. We show that all are essential for ICL biogenesis. The two centrin-binding protein subfamilies and nine of the centrin subfamilies are ICL specific and play a role in its molecular and supramolecular architecture. The tenth and most conserved centrin subfamily is present at three cortical locations (ICL, basal bodies and contractile vacuole pores) and might play a role in coordinating duplication and positioning of cortical organelles.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Familia de Multigenes , Paramecium tetraurelia/citología , Proteínas Protozoarias/metabolismo , Animales , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Proteínas Portadoras/clasificación , Proteínas Portadoras/metabolismo , Clonación Molecular , Evolución Molecular , Proteínas Fluorescentes Verdes/genética , Espectrometría de Masas , Microscopía Fluorescente , Datos de Secuencia Molecular , Paramecium tetraurelia/genética , Paramecium tetraurelia/fisiología , Filogenia , Unión Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
The previous characterization and structural analyses of Sfi1p, a Saccharomyces cerevisiae centrin-binding protein essential for spindle pole body duplication, have suggested molecular models to account for centrin-mediated, Ca2+-dependent contractility processes (S. Li, A. M. Sandercock, P. Conduit, C. V. Robinson, R. L. Williams, and J. V. Kilmartin, J. Cell Biol. 173:867-877, 2006). Such processes can be analyzed by using Paramecium tetraurelia, which harbors a large Ca2+ -dependent contractile cytoskeletal network, the infraciliary lattice (ICL). Previous biochemical and genetic studies have shown that the ICL is composed of diverse centrin isoforms and a high-molecular-mass centrin-associated protein, whose reduced size in the démaillé (dem1) mutant correlates with defective organization of the ICL. Using sequences derived from the high-molecular-mass protein to probe the Paramecium genome sequence, we characterized the PtCenBP1 gene, which encodes a 460-kDa protein. PtCenBP1p displays six almost perfect repeats of ca. 427 amino acids (aa) and harbors 89 potential centrin-binding sites with the consensus motif LLX11F/LX2WK/R, similar to the centrin-binding sites of ScSfi1p. The smaller (260-kDa) protein encoded by the dem1 mutant PtCenBP1 allele comprises only two repeats of 427 aa and 46 centrin-binding sites. By using RNA interference and green fluorescent protein fusion experiments, we showed that PtCenBP1p forms the backbone of the ICL and plays an essential role in its assembly and contractility. This study provides the first in vivo demonstration of the role of Sfi1p-like proteins in centrin-mediated Ca2+-dependent contractile processes.
Asunto(s)
Calcio/farmacología , Proteínas de Ciclo Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Paramecium tetraurelia/citología , Paramecium tetraurelia/efectos de los fármacos , Proteínas Protozoarias/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/química , Cilios/efectos de los fármacos , Cilios/ultraestructura , Proteínas Fluorescentes Verdes/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Paramecium tetraurelia/ultraestructura , Fenotipo , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Protozoarias/química , Interferencia de ARN , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/química , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
In Paramecium tetraurelia, the regulated secretory pathway of dense core granules called trichocysts can be altered by mutation and genetically studied. Seventeen nondischarge (ND) genes controlling exocytosis have already been identified by a genetic approach. The site of action of the studied mutations is one of the three compartments, the cytosol, trichocyst, or plasma membrane. The only ND genes cloned to date correspond to mutants affected in the cytosol or in the trichocyst compartment. In this work, we investigated a representative of the third compartment, the plasma membrane, by cloning the ND6 gene. This gene encodes a 1,925-amino-acid protein containing two domains homologous to the regulator of chromosome condensation 1 (RCC1). In parallel, 10 new alleles of the ND6 gene were isolated. Nine of the 12 available mutations mapped in the RCC1-like domains, showing their importance for the Nd6 protein (Nd6p) function. The RCC1 protein is well known for its guanine exchange factor activity towards the small GTPase Ran but also for its involvement in membrane fusion during nuclear envelope assembly. Other proteins with RCC1-like domains are also involved in intracellular membrane fusion, but none has been described yet as involved in exocytosis. The case of Nd6p is thus the first report of such a protein with a documented role in exocytosis.