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1.
Annu Rev Cell Dev Biol ; 33: 23-49, 2017 10 06.
Artículo en Inglés | MEDLINE | ID: mdl-28813178

RESUMEN

The centriole is a beautiful microtubule-based organelle that is critical for the proper execution of many fundamental cellular processes, including polarity, motility, and division. Centriole biogenesis, the making of this miniature architectural wonder, has emerged as an exemplary model to dissect the mechanisms governing the assembly of a eukaryotic organelle. Centriole biogenesis relies on a set of core proteins whose contributions to the assembly process have begun to be elucidated. Here, we review current knowledge regarding the mechanisms by which these core characters function in an orderly fashion to assemble the centriole. In particular, we discuss how having the correct proteins at the right place and at the right time is critical to first scaffold, then initiate, and finally execute the centriole assembly process, thus underscoring fundamental principles governing organelle biogenesis.


Asunto(s)
Centriolos/metabolismo , Biogénesis de Organelos , Animales , Humanos , Microtúbulos/metabolismo , Modelos Biológicos
2.
EMBO J ; 42(24): e115076, 2023 Dec 11.
Artículo en Inglés | MEDLINE | ID: mdl-37987153

RESUMEN

In most metazoans, centrioles are lost during oogenesis, ensuring that the zygote is endowed with the correct number of two centrioles, which are paternally contributed. How centriole architecture is dismantled during oogenesis is not understood. Here, we analyze with unprecedent detail the ultrastructural and molecular changes during oogenesis centriole elimination in Caenorhabditis elegans. Centriole elimination begins with loss of the so-called central tube and organelle widening, followed by microtubule disassembly. The resulting cluster of centriolar proteins then disappears gradually, usually moving in a microtubule- and dynein-dependent manner to the plasma membrane. Our analysis indicates that neither Polo-like kinases nor the PCM, which modulate oogenesis centriole elimination in Drosophila, do so in C. elegans. Furthermore, we demonstrate that the central tube protein SAS-1 normally departs initially from the organelle, which loses integrity earlier in sas-1 mutants. Overall, our work provides novel mechanistic insights regarding the fundamental process of oogenesis centriole elimination.


Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Centriolos/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Microtúbulos/metabolismo , Drosophila/metabolismo , Oogénesis , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo
3.
Cell ; 144(3): 364-75, 2011 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-21277013

RESUMEN

The centriole, and the related basal body, is an ancient organelle characterized by a universal 9-fold radial symmetry and is critical for generating cilia, flagella, and centrosomes. The mechanisms directing centriole formation are incompletely understood and represent a fundamental open question in biology. Here, we demonstrate that the centriolar protein SAS-6 forms rod-shaped homodimers that interact through their N-terminal domains to form oligomers. We establish that such oligomerization is essential for centriole formation in C. elegans and human cells. We further generate a structural model of the related protein Bld12p from C. reinhardtii, in which nine homodimers assemble into a ring from which nine coiled-coil rods radiate outward. Moreover, we demonstrate that recombinant Bld12p self-assembles into structures akin to the central hub of the cartwheel, which serves as a scaffold for centriole formation. Overall, our findings establish a structural basis for the universal 9-fold symmetry of centrioles.


Asunto(s)
Caenorhabditis elegans/citología , Centriolos/química , Centriolos/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis/química , Caenorhabditis/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Multimerización de Proteína , Proteínas Recombinantes/metabolismo , Alineación de Secuencia
4.
PLoS Biol ; 20(9): e3001784, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-36107993

RESUMEN

Uncovering organizing principles of organelle assembly is a fundamental pursuit in the life sciences. Caenorhabditis elegans was key in identifying evolutionary conserved components governing assembly of the centriole organelle. However, localizing these components with high precision has been hampered by the minute size of the worm centriole, thus impeding understanding of underlying assembly mechanisms. Here, we used Ultrastructure Expansion coupled with STimulated Emission Depletion (U-Ex-STED) microscopy, as well as electron microscopy (EM) and electron tomography (ET), to decipher the molecular architecture of the worm centriole. Achieving an effective lateral resolution of approximately 14 nm, we localize centriolar and PeriCentriolar Material (PCM) components in a comprehensive manner with utmost spatial precision. We found that all 12 components analysed exhibit a ring-like distribution with distinct diameters and often with a 9-fold radial symmetry. Moreover, we uncovered that the procentriole assembles at a location on the centriole margin where SPD-2 and ZYG-1 also accumulate. Moreover, SAS-6 and SAS-5 were found to be present in the nascent procentriole, with SAS-4 and microtubules recruited thereafter. We registered U-Ex-STED and EM data using the radial array of microtubules, thus allowing us to map each centriolar and PCM protein to a specific ultrastructural compartment. Importantly, we discovered that SAS-6 and SAS-4 exhibit a radial symmetry that is offset relative to microtubules, leading to a chiral centriole ensemble. Furthermore, we established that the centriole is surrounded by a region from which ribosomes are excluded and to which SAS-7 localizes. Overall, our work uncovers the molecular architecture of the C. elegans centriole in unprecedented detail and establishes a comprehensive framework for understanding mechanisms of organelle biogenesis and function.


Asunto(s)
Proteínas de Caenorhabditis elegans , Centriolos , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Centrosoma/metabolismo , Proteínas Quinasas/metabolismo
5.
Dev Biol ; 502: 68-76, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37414202

RESUMEN

Centrioles, together with the surrounding peri-centriolar material (PCM), constitute the centrosome, a major microtubule-organizing center of animal cells. Despite being critical in many cells for signaling, motility and division, centrioles can be eliminated in some systems, including in the vast majority of differentiating cells during embryogenesis in Caenorhabditis elegans. Whether the cells retaining centrioles in the resulting L1 larvae do so because they lack an activity that eliminates centrioles in the other cells is not known. Moreover, the extent to which centrioles and PCM remain present in later stages of worm development, when all cells but those of the germ line are terminally differentiated, is not known. Here, by fusing cells that lack centrioles with cells that retain them, we established that L1 larvae do not possess a diffusible elimination activity sufficient to remove centrioles. Moreover, analyzing PCM core proteins in L1 larval cells that retain centrioles, we found that some such proteins, but not all, are present as well. Furthermore, we uncovered that foci of centriolar proteins remain present in specific terminally differentiated cells of adult hermaphrodites and males, in particular in the somatic gonad. Correlating the time at which cells were born with the fate of their centrioles revealed that it is not cell age, but instead cell fate, that determines whether and when centrioles are eliminated. Overall, our work maps the localization of centriolar and PCM core proteins in the post-embryonic C. elegans lineage, thereby providing an essential blueprint for uncovering mechanisms modulating their presence and function.


Asunto(s)
Proteínas de Caenorhabditis elegans , Centriolos , Animales , Masculino , Centriolos/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Centrosoma/metabolismo , Centro Organizador de los Microtúbulos/metabolismo
6.
EMBO J ; 39(22): e106249, 2020 11 16.
Artículo en Inglés | MEDLINE | ID: mdl-32954505

RESUMEN

Centrioles are polarized microtubule-based organelles that seed the formation of cilia, and which assemble from a cartwheel containing stacked ring oligomers of SAS-6 proteins. A cryo-tomography map of centrioles from the termite flagellate Trichonympha spp. was obtained previously, but higher resolution analysis is likely to reveal novel features. Using sub-tomogram averaging (STA) in T. spp. and Trichonympha agilis, we delineate the architecture of centriolar microtubules, pinhead, and A-C linker. Moreover, we report ~25 Å resolution maps of the central cartwheel, revealing notably polarized cartwheel inner densities (CID). Furthermore, STA of centrioles from the distant flagellate Teranympha mirabilis uncovers similar cartwheel architecture and a distinct filamentous CID. Fitting the CrSAS-6 crystal structure into the flagellate maps and analyzing cartwheels generated in vitro indicate that SAS-6 rings can directly stack onto one another in two alternating configurations: with a slight rotational offset and in register. Overall, improved STA maps in three flagellates enabled us to unravel novel architectural features, including of centriole polarity and cartwheel stacking, thus setting the stage for an accelerated elucidation of underlying assembly mechanisms.


Asunto(s)
Centriolos/ultraestructura , Microscopía por Crioelectrón/métodos , Tomografía/métodos , Adhesión Celular , Cilios/ultraestructura , Microtúbulos/ultraestructura , Parabasalidea/citología
7.
Nat Rev Mol Cell Biol ; 13(7): 425-35, 2012 Jun 13.
Artículo en Inglés | MEDLINE | ID: mdl-22691849

RESUMEN

The centriole is an evolutionarily conserved macromolecular structure that is crucial for the formation of flagella, cilia and centrosomes. The ultrastructure of the centriole was first characterized decades ago with the advent of electron microscopy, revealing a striking ninefold radial arrangement of microtubules. However, it is only recently that the molecular mechanisms governing centriole assembly have begun to emerge, including the elucidation of the crucial role of spindle assembly abnormal 6 (SAS-6) proteins in imparting the ninefold symmetry. These advances have brought the field to an exciting era in which architecture meets function.


Asunto(s)
Centriolos/fisiología , Microtúbulos/metabolismo , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Centrosoma/fisiología , Cilios/metabolismo , Flagelos/metabolismo , Humanos , Microscopía Electrónica/métodos , Conformación Molecular , Conformación Proteica
8.
BMC Bioinformatics ; 24(1): 120, 2023 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-36977999

RESUMEN

BACKGROUND: High-throughput and selective detection of organelles in immunofluorescence images is an important but demanding task in cell biology. The centriole organelle is critical for fundamental cellular processes, and its accurate detection is key for analysing centriole function in health and disease. Centriole detection in human tissue culture cells has been achieved typically by manual determination of organelle number per cell. However, manual cell scoring of centrioles has a low throughput and is not reproducible. Published semi-automated methods tally the centrosome surrounding centrioles and not centrioles themselves. Furthermore, such methods rely on hard-coded parameters or require a multichannel input for cross-correlation. Therefore, there is a need for developing an efficient and versatile pipeline for the automatic detection of centrioles in single channel immunofluorescence datasets. RESULTS: We developed a deep-learning pipeline termed CenFind that automatically scores cells for centriole numbers in immunofluorescence images of human cells. CenFind relies on the multi-scale convolution neural network SpotNet, which allows the accurate detection of sparse and minute foci in high resolution images. We built a dataset using different experimental settings and used it to train the model and evaluate existing detection methods. The resulting average F1-score achieved by CenFind is > 90% across the test set, demonstrating the robustness of the pipeline. Moreover, using the StarDist-based nucleus detector, we link the centrioles and procentrioles detected with CenFind to the cell containing them, overall enabling automatic scoring of centriole numbers per cell. CONCLUSIONS: Efficient, accurate, channel-intrinsic and reproducible detection of centrioles is an important unmet need in the field. Existing methods are either not discriminative enough or focus on a fixed multi-channel input. To fill this methodological gap, we developed CenFind, a command line interface pipeline that automates cell scoring of centrioles, thereby enabling channel-intrinsic, accurate and reproducible detection across experimental modalities. Moreover, the modular nature of CenFind enables its integration in other pipelines. Overall, we anticipate CenFind to prove critical for accelerating discoveries in the field.


Asunto(s)
Aprendizaje Profundo , Microscopía , Humanos , Centriolos/metabolismo , Centrosoma/metabolismo
9.
Nat Methods ; 17(7): 726-733, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32572233

RESUMEN

Super-resolution microscopies have become an established tool in biological research. However, imaging throughput remains a main bottleneck in acquiring large datasets required for quantitative biology. Here we describe multifocal flat illumination for field-independent imaging (mfFIFI). By integrating mfFIFI into an instant structured illumination microscope (iSIM), we extend the field of view (FOV) to >100 × 100 µm2 while maintaining high-speed, multicolor, volumetric imaging at double the diffraction-limited resolution. We further extend the effective FOV by stitching adjacent images for fast live-cell super-resolution imaging of dozens of cells. Finally, we combine our flat-fielded iSIM with ultrastructure expansion microscopy to collect three-dimensional (3D) images of hundreds of centrioles in human cells, or thousands of purified Chlamydomonas reinhardtii centrioles, per hour at an effective resolution of ~35 nm. Classification and particle averaging of these large datasets enables 3D mapping of posttranslational modifications of centriolar microtubules, revealing differences in their coverage and positioning.


Asunto(s)
Microscopía Fluorescente/métodos , Animales , Células COS , Centriolos/ultraestructura , Chlorocebus aethiops , Humanos , Imagenología Tridimensional , Iluminación , Microtúbulos/ultraestructura
10.
J Chem Phys ; 158(8): 085102, 2023 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-36859084

RESUMEN

The Spindle Assembly Abnormal Protein 6 (SAS-6) forms dimers, which then self-assemble into rings that are critical for the nine-fold symmetry of the centriole organelle. It has recently been shown experimentally that the self-assembly of SAS-6 rings is strongly facilitated on a surface, shifting the reaction equilibrium by four orders of magnitude compared to the bulk. Moreover, a fraction of non-canonical symmetries (i.e., different from nine) was observed. In order to understand which aspects of the system are relevant to ensure efficient self-assembly and selection of the nine-fold symmetry, we have performed Brownian dynamics computer simulation with patchy particles and then compared our results with the experimental ones. Adsorption onto the surface was simulated by a grand canonical Monte Carlo procedure and random sequential adsorption kinetics. Furthermore, self-assembly was described by Langevin equations with hydrodynamic mobility matrices. We find that as long as the interaction energies are weak, the assembly kinetics can be described well by coagulation-fragmentation equations in the reaction-limited approximation. By contrast, larger interaction energies lead to kinetic trapping and diffusion-limited assembly. We find that the selection of nine-fold symmetry requires a small value for the angular interaction range. These predictions are confirmed by the experimentally observed reaction constant and angle fluctuations. Overall, our simulations suggest that the SAS-6 system works at the crossover between a relatively weak binding energy that avoids kinetic trapping and a small angular range that favors the nine-fold symmetry.

11.
PLoS Genet ; 15(2): e1007905, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30735500

RESUMEN

RNA interference (RNAi) related pathways are essential for germline development and fertility in metazoa and can contribute to inter- and trans-generational inheritance. In the nematode Caenorhabditis elegans, environmental double-stranded RNA provided by feeding can lead to heritable changes in phenotype and gene expression. Notably, transmission efficiency differs between the male and female germline, yet the underlying mechanisms remain elusive. Here we use high-throughput sequencing of dissected gonads to quantify sex-specific endogenous piRNAs, miRNAs and siRNAs in the C. elegans germline and the somatic gonad. We identify genes with exceptionally high levels of secondary 22G RNAs that are associated with low mRNA expression, a signature compatible with silencing. We further demonstrate that contrary to the hermaphrodite germline, the male germline, but not male soma, is resistant to environmental RNAi triggers provided by feeding, in line with previous work. This sex-difference in silencing efficacy is associated with lower levels of gonadal RNAi amplification products. Moreover, this tissue- and sex-specific RNAi resistance is regulated by the germline, since mutant males with a feminized germline are RNAi sensitive. This study provides important sex- and tissue-specific expression data of miRNA, piRNA and siRNA as well as mechanistic insights into sex-differences of gene regulation in response to environmental cues.


Asunto(s)
ARN Interferente Pequeño/genética , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Femenino , Regulación de la Expresión Génica/genética , Células Germinativas/fisiología , Gónadas/fisiología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Masculino , MicroARNs/genética , Interferencia de ARN/fisiología , ARN Bicatenario/genética , ARN Mensajero/genética , Caracteres Sexuales
12.
J Cell Sci ; 132(4)2019 02 20.
Artículo en Inglés | MEDLINE | ID: mdl-30787112

RESUMEN

The centriole organelle consists of microtubules (MTs) that exhibit a striking 9-fold radial symmetry. Centrioles play fundamental roles across eukaryotes, notably in cell signaling, motility and division. In this Cell Science at a Glance article and accompanying poster, we cover the cellular life cycle of this organelle - from assembly to disappearance - focusing on human centrioles. The journey begins at the end of mitosis when centriole pairs disengage and the newly formed centrioles mature to begin a new duplication cycle. Selection of a single site of procentriole emergence through focusing of polo-like kinase 4 (PLK4) and the resulting assembly of spindle assembly abnormal protein 6 (SAS-6) into a cartwheel element are evoked next. Subsequently, we cover the recruitment of peripheral components that include the pinhead structure, MTs and the MT-connecting A-C linker. The function of centrioles in recruiting pericentriolar material (PCM) and in forming the template of the axoneme are then introduced, followed by a mention of circumstances in which centrioles form de novo or are eliminated.


Asunto(s)
Centriolos/ultraestructura , Microtúbulos/ultraestructura , Biogénesis de Organelos , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Embrión de Mamíferos , Células Eucariotas/metabolismo , Células Eucariotas/ultraestructura , Regulación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Marsileaceae/genética , Marsileaceae/metabolismo , Marsileaceae/ultraestructura , Ratones , Microtúbulos/metabolismo , Mitosis , Naegleria/genética , Naegleria/metabolismo , Naegleria/ultraestructura , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal
13.
Development ; 145(11)2018 05 30.
Artículo en Inglés | MEDLINE | ID: mdl-29724757

RESUMEN

Asymmetric division is crucial for embryonic development and stem cell lineages. In the one-cell Caenorhabditis elegans embryo, a contractile cortical actomyosin network contributes to asymmetric division by segregating partitioning-defective (PAR) proteins to discrete cortical domains. In the current study, we found that the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) localizes to polarized dynamic structures in C. elegans zygotes, distributing in a PAR-dependent manner along the anterior-posterior (A-P) embryonic axis. PIP2 cortical structures overlap with F-actin, and coincide with the actin regulators RHO-1 and CDC-42, as well as ECT-2. Particle image velocimetry analysis revealed that PIP2 and F-actin cortical movements are coupled, with PIP2 structures moving slightly ahead of F-actin. Importantly, we established that PIP2 cortical structure formation and movement is actin dependent. Moreover, we found that decreasing or increasing the level of PIP2 resulted in severe F-actin disorganization, revealing interdependence between these components. Furthermore, we determined that PIP2 and F-actin regulate the sizing of PAR cortical domains, including during the maintenance phase of polarization. Overall, our work establishes that a lipid membrane component, PIP2, modulates actin organization and cell polarity in C. elegans embryos.


Asunto(s)
Actinas/metabolismo , División Celular Asimétrica/fisiología , Caenorhabditis elegans/embriología , Polaridad Celular/fisiología , Lípidos de la Membrana/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animales , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Embrión no Mamífero/metabolismo , Proteínas de Unión al GTP/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Unión al GTP rho/metabolismo
14.
Nat Methods ; 15(10): 777-780, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30275574

RESUMEN

Single-particle reconstruction (SPR) from electron microscopy (EM) images is widely used in structural biology, but it lacks direct information on protein identity. To address this limitation, we developed a computational and analytical framework that reconstructs and coaligns multiple proteins from 2D super-resolution fluorescence images. To demonstrate our method, we generated multicolor 3D reconstructions of several proteins within the human centriole, which revealed their relative locations, dimensions and orientations.


Asunto(s)
Centriolos/ultraestructura , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Electrónica/instrumentación , Microscopía Electrónica/métodos , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Imagen Individual de Molécula/métodos , Algoritmos , Centriolos/metabolismo , Humanos
15.
Mol Biol Rep ; 48(7): 5459-5471, 2021 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-34304367

RESUMEN

BACKGROUND: The Canadian prairie ecosystem presents a rich source of natural products from plants that are subjected to herbivory by grazing mammals. This type of ecological competition may contribute to the production of natural products of interest in cell biology and medical research. We provide the first biological description of the sesquiterpene lactone, pulchelloid A, which we isolated from the prairie plant, Gaillardia aristata (Asteraceae) and report that it inhibits mitosis in human cells. METHODS AND RESULTS: We found that G. aristata (Blanket flower) extracts were cytotoxic to human cell lines and used phenotypic assays to characterize the bioactivity of extracts. Before dying, cells were characterized by a rounded morphology, phospho-histone H3 signals, mitotic spindles, and active Cdk1. By biology-guided fractionation of Gaillardia extracts, we isolated a sesquiterpene lactone named pulchelloid A. We used immunofluorescence microscopy and observed that cells treated with pulchelloid A have phospho-histone H3 positive chromosomes and a mitotic spindle, confirming that they were in mitosis. Treated cells arrest with an unusual phenotype; they enter a prolonged mitotic arrest in which the spindles become multipolar and the chromosomes acquire histone γH2AX foci, a hallmark of damaged DNA. CONCLUSIONS: We propose that pulchelloid A, a natural product present in the prairie plant Gaillardia aristata, delays cells in mitosis. There is a growing body of evidence that a small number of members of the sesquiterpene lactone chemical family may target proteins that regulate mitosis.


Asunto(s)
Asteraceae/química , Extractos Vegetales/química , Huso Acromático/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular , Células HT29 , Humanos , Mitosis/efectos de los fármacos , Extractos Vegetales/farmacología , Hojas de la Planta/genética
16.
Nat Rev Mol Cell Biol ; 9(5): 355-66, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18431399

RESUMEN

Asymmetric cell division is fundamental for generating diversity in multicellular organisms. The mechanisms that govern asymmetric cell division are increasingly well understood, owing notably to studies that were conducted in Drosophila melanogaster and Caenorhabditis elegans. Lessons learned from these two model organisms also apply to cells that divide asymmetrically in other metazoans, such as self-renewing stem cells in mammals.


Asunto(s)
Caenorhabditis elegans/citología , División Celular/fisiología , Drosophila melanogaster/citología , Animales , Proteínas de Ciclo Celular/metabolismo , Polaridad Celular , Centrosoma/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Hormonas Juveniles/genética , Hormonas Juveniles/metabolismo , Pliegue de Proteína , Receptores Notch/genética , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Huso Acromático/metabolismo , Huso Acromático/ultraestructura , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo
17.
Bioessays ; 40(4): e1700241, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29508910

RESUMEN

The cartwheel is a striking structure critical for building the centriole, a microtubule-based organelle fundamental for organizing centrosomes, cilia, and flagella. Over the last 50 years, the cartwheel has been described in many systems using electron microscopy, but the molecular nature of its constituent building blocks and their assembly mechanisms have long remained mysterious. Here, we review discoveries that led to the current understanding of cartwheel structure, assembly, and function. We focus on the key role of SAS-6 protein self-organization, both for building the signature ring-like structure with hub and spokes, as well as for their vertical stacking. The resemblance of assembly intermediates in vitro and in vivo leads us to propose a novel registration step in cartwheel biogenesis, whereby stacked SAS-6-containing rings are put in register through interactions with peripheral elements anchored to microtubules. We conclude by evoking some avenues for further uncovering cartwheel and centriole assembly mechanisms.


Asunto(s)
Centriolos/metabolismo , Orgánulos/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centriolos/genética , Centriolos/ultraestructura , Cilios/genética , Cilios/metabolismo , Cilios/ultraestructura , Microscopía Electrónica , Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Orgánulos/genética , Orgánulos/ultraestructura
18.
Dev Biol ; 434(2): 221-230, 2018 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-29307730

RESUMEN

Genome stability relies notably on the integrity of centrosomes and on the mitotic spindle they organize. Structural and numerical centrosome aberrations are frequently observed in human cancer, and there is increasing evidence that centrosome amplification can promote tumorigenesis. Here, we use C. elegans seam cells as a model system to analyze centrosome homeostasis in the context of a stereotyped stem like lineage. We found that overexpression of the Plk4-related kinase ZYG-1 leads to the formation of one supernumerary centriolar focus per parental centriole during the cell cycle that leads to the sole symmetric division in the seam lineage. In the following cell cycle, such supernumerary foci function as microtubule organizing centers, but do not cluster during mitosis, resulting in the formation of a multipolar spindle and then aneuploid daughter cells. Intriguingly, we found also that supernumerary centriolar foci do not assemble in the asymmetric cell divisions that precedes or that follows the symmetric seam cell division, despite the similar presence of GFP::ZYG-1. Furthermore, we established that supernumerary centrioles form earlier during development in animals depleted of the heterochronic gene lin-14, in which the symmetric division is precocious. Conversely, supernumerary centrioles are essentially not observed in animals depleted of lin-28, in which the symmetric division is lacking. These findings lead us to conclude that ZYG-1 promotes limited centriole amplification solely during the symmetric division in the C. elegans seam lineage.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , División Celular , Centriolos/metabolismo , Inestabilidad Genómica , Proteínas Quinasas/metabolismo , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Centriolos/genética , Proteínas Quinasas/genética
19.
EMBO J ; 33(16): 1815-30, 2014 Aug 18.
Artículo en Inglés | MEDLINE | ID: mdl-24996901

RESUMEN

The positioning and the elongation of the mitotic spindle must be carefully regulated. In human cells, the evolutionary conserved proteins LGN/Gαi1-3 anchor the coiled-coil protein NuMA and dynein to the cell cortex during metaphase, thus ensuring proper spindle positioning. The mechanisms governing cortical localization of NuMA and dynein during anaphase remain more elusive. Here, we report that LGN/Gαi1-3 are dispensable for NuMA-dependent cortical dynein enrichment during anaphase. We further establish that NuMA is excluded from the equatorial region of the cell cortex in a manner that depends on the centralspindlin components CYK4 and MKLP1. Importantly, we reveal that NuMA can directly associate with PtdInsP (PIP) and PtdInsP2 (PIP2) phosphoinositides in vitro. Furthermore, chemical or enzymatic depletion of PIP/PIP2 prevents NuMA cortical localization during mitosis, and conversely, increasing PIP2 levels augments mitotic cortical NuMA. Overall, our study uncovers a novel function for plasma membrane phospholipids in governing cortical NuMA distribution and thus the proper execution of mitosis.


Asunto(s)
Antígenos Nucleares/metabolismo , Membrana Celular/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Fosfatidilinositoles/metabolismo , Huso Acromático/metabolismo , Anafase , Antígenos Nucleares/genética , Proteínas de Ciclo Celular , Dineínas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metafase , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis , Proteínas Asociadas a Matriz Nuclear/genética , Estructura Terciaria de Proteína
20.
J Cell Sci ; 129(15): 3015-25, 2016 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-27335426

RESUMEN

Accurate spindle positioning is essential for error-free cell division. The one-cell Caenorhabditis elegans embryo has proven instrumental for dissecting mechanisms governing spindle positioning. Despite important progress, how the cortical forces that act on astral microtubules to properly position the spindle are modulated is incompletely understood. Here, we report that the PP6 phosphatase PPH-6 and its associated subunit SAPS-1, which positively regulate pulling forces acting on spindle poles, associate with the Aurora A kinase AIR-1 in C. elegans embryos. We show that acute inactivation of AIR-1 during mitosis results in excess pulling forces on astral microtubules. Furthermore, we uncover that AIR-1 acts downstream of PPH-6-SAPS-1 in modulating spindle positioning, and that PPH-6-SAPS-1 negatively regulates AIR-1 localization at the cell cortex. Moreover, we show that Aurora A and the PP6 phosphatase subunit PPP6C are also necessary for spindle positioning in human cells. There, Aurora A is needed for the cortical localization of NuMA and dynein during mitosis. Overall, our work demonstrates that Aurora A kinases and PP6 phosphatases have an ancient function in modulating spindle positioning, thus contributing to faithful cell division.


Asunto(s)
Aurora Quinasa A/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Animales , Antígenos Nucleares/metabolismo , Fenómenos Biomecánicos , Proteína Quinasa CDC2 , Proteínas de Ciclo Celular/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Biológicos , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Unión Proteica , Huso Acromático/metabolismo
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