Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 19 de 19
Filtrar
1.
Nat Immunol ; 23(11): 1644-1652, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36271145

RESUMEN

Interleukin-17A (IL-17A) is a key mediator of protective immunity to yeast and bacterial infections but also drives the pathogenesis of several autoimmune diseases, such as psoriasis or psoriatic arthritis. Here we show that the tetra-transmembrane protein CMTM4 is a subunit of the IL-17 receptor (IL-17R). CMTM4 constitutively associated with IL-17R subunit C to mediate its stability, glycosylation and plasma membrane localization. Both mouse and human cell lines deficient in CMTM4 were largely unresponsive to IL-17A, due to their inability to assemble the IL-17R signaling complex. Accordingly, CMTM4-deficient mice had a severe defect in the recruitment of immune cells following IL-17A administration and were largely resistant to experimental psoriasis, but not to experimental autoimmune encephalomyelitis. Collectively, our data identified CMTM4 as an essential component of IL-17R and a potential therapeutic target for treating IL-17-mediated autoimmune diseases.


Asunto(s)
Artritis Psoriásica , Encefalomielitis Autoinmune Experimental , Psoriasis , Humanos , Ratones , Animales , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo , Interleucina-17/metabolismo , Encefalomielitis Autoinmune Experimental/genética , Proteínas con Dominio MARVEL/genética
2.
EMBO Rep ; 22(2): e50785, 2021 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-33426789

RESUMEN

Bardet-Biedl Syndrome (BBS) is a pleiotropic genetic disease caused by the dysfunction of primary cilia. The immune system of patients with ciliopathies has not been investigated. However, there are multiple indications that the impairment of the processes typically associated with cilia may have influence on the hematopoietic compartment and immunity. In this study, we analyze clinical data of BBS patients and corresponding mouse models carrying mutations in Bbs4 or Bbs18. We find that BBS patients have a higher prevalence of certain autoimmune diseases. Both BBS patients and animal models have altered red blood cell and platelet compartments, as well as elevated white blood cell levels. Some of the hematopoietic system alterations are associated with BBS-induced obesity. Moreover, we observe that the development and homeostasis of B cells in mice is regulated by the transport complex BBSome, whose dysfunction is a common cause of BBS. The BBSome limits canonical WNT signaling and increases CXCL12 levels in bone marrow stromal cells. Taken together, our study reveals a connection between a ciliopathy and dysregulated immune and hematopoietic systems.


Asunto(s)
Enfermedades Autoinmunes , Síndrome de Bardet-Biedl , Hematopoyesis , Animales , Síndrome de Bardet-Biedl/complicaciones , Síndrome de Bardet-Biedl/genética , Cilios , Modelos Animales de Enfermedad , Hematopoyesis/genética , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/genética , Mutación
3.
EMBO J ; 37(14)2018 07 13.
Artículo en Inglés | MEDLINE | ID: mdl-29752423

RESUMEN

Virtual memory T cells are foreign antigen-inexperienced T cells that have acquired memory-like phenotype and constitute 10-20% of all peripheral CD8+ T cells in mice. Their origin, biological roles, and relationship to naïve and foreign antigen-experienced memory T cells are incompletely understood. By analyzing T-cell receptor repertoires and using retrogenic monoclonal T-cell populations, we demonstrate that the virtual memory T-cell formation is a so far unappreciated cell fate decision checkpoint. We describe two molecular mechanisms driving the formation of virtual memory T cells. First, virtual memory T cells originate exclusively from strongly self-reactive T cells. Second, the stoichiometry of the CD8 interaction with Lck regulates the size of the virtual memory T-cell compartment via modulating the self-reactivity of individual T cells. Although virtual memory T cells descend from the highly self-reactive clones and acquire a partial memory program, they are not more potent in inducing experimental autoimmune diabetes than naïve T cells. These data underline the importance of the variable level of self-reactivity in polyclonal T cells for the generation of functional T-cell diversity.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Diferenciación Celular , Memoria Inmunológica , Receptores de Antígenos de Linfocitos T/análisis , Animales , Homeostasis , Ratones
4.
J Biol Chem ; 295(42): 14279-14290, 2020 10 16.
Artículo en Inglés | MEDLINE | ID: mdl-32759308

RESUMEN

Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of primary cilia. More than half of BBS patients carry mutations in one of eight genes encoding for subunits of a protein complex, the BBSome, which mediates trafficking of ciliary cargoes. In this study, we elucidated the mechanisms of the BBSome assembly in living cells and how this process is spatially regulated. We generated a large library of human cell lines deficient in a particular BBSome subunit and expressing another subunit tagged with a fluorescent protein. We analyzed these cell lines utilizing biochemical assays, conventional and expansion microscopy, and quantitative fluorescence microscopy techniques: fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. Our data revealed that the BBSome formation is a sequential process. We show that the pre-BBSome is nucleated by BBS4 and assembled at pericentriolar satellites, followed by the translocation of the BBSome into the ciliary base mediated by BBS1. Our results provide a framework for elucidating how BBS-causative mutations interfere with the biogenesis of the BBSome.


Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/metabolismo , Síndrome de Bardet-Biedl/patología , Sistemas CRISPR-Cas/genética , Línea Celular , Cilios/metabolismo , Citoplasma/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Edición Génica , Humanos , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/genética , Mutación , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo
5.
J Cell Sci ; 132(16)2019 08 29.
Artículo en Inglés | MEDLINE | ID: mdl-31331965

RESUMEN

The Arf GTPase controls formation of the COPI vesicle coat. Recent structural models of COPI revealed the positioning of two Arf1 molecules in contrasting molecular environments. Each of these pockets for Arf1 is expected to also accommodate an Arf GTPase-activating protein (ArfGAP). Structural evidence and protein interactions observed between isolated domains indirectly suggest that each niche preferentially recruits one of the two ArfGAPs known to affect COPI, i.e. Gcs1/ArfGAP1 and Glo3/ArfGAP2/3, although only partial structures are available. The functional role of the unique non-catalytic domain of either ArfGAP has not been integrated into the current COPI structural model. Here, we delineate key differences in the consequences of triggering GTP hydrolysis through the activity of one versus the other ArfGAP. We demonstrate that Glo3/ArfGAP2/3 specifically triggers Arf1 GTP hydrolysis impinging on the stability of the COPI coat. We show that the Snf1 kinase complex, the yeast homologue of AMP-activated protein kinase (AMPK), phosphorylates the region of Glo3 that is crucial for this effect and, thereby, regulates its function in the COPI-vesicle cycle. Our results revise the model of ArfGAP function in the molecular context of COPI.This article has an associated First Person interview with the first author of the paper.


Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Proteína Coat de Complejo I/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/genética , Proteína Coat de Complejo I/genética , Proteínas Activadoras de GTPasa/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
6.
Chromosome Res ; 28(3-4): 355-368, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32939606

RESUMEN

Expansion microscopy (ExM) is a method to magnify physically a specimen with preserved ultrastructure. It has the potential to explore structural features beyond the diffraction limit of light. The procedure has been successfully used for different animal species, from isolated macromolecular complexes through cells to tissue slices. Expansion of plant-derived samples is still at the beginning, and little is known, whether the chromatin ultrastructure becomes altered by physical expansion. In this study, we expanded isolated barley nuclei and compared whether ExM can provide a structural view of chromatin comparable with super-resolution microscopy. Different fixation and denaturation/digestion conditions were tested to maintain the chromatin ultrastructure. We achieved up to ~4.2-times physically expanded nuclei corresponding to a maximal resolution of ~50-60 nm when imaged by wild-field (WF) microscopy. By applying structured illumination microscopy (SIM, super-resolution) doubling the WF resolution, the chromatin structures were observed at a resolution of ~25-35 nm. WF microscopy showed a preserved nucleus shape and nucleoli. Moreover, we were able to detect chromatin domains, invisible in unexpanded nuclei. However, by applying SIM, we observed that the preservation of the chromatin ultrastructure after the expansion was not complete and that the majority of the tested conditions failed to keep the ultrastructure. Nevertheless, using expanded nuclei, we localized successfully centromere repeats by fluorescence in situ hybridization (FISH) and the centromere-specific histone H3 variant CENH3 by indirect immunolabelling. However, although these repeats and proteins were localized at the correct position within the nuclei (indicating a Rabl orientation), their ultrastructural arrangement was impaired.


Asunto(s)
Cromatina/ultraestructura , Microscopía/métodos , Núcleo Celular/ultraestructura , Técnica del Anticuerpo Fluorescente , Hordeum/genética , Hibridación Fluorescente in Situ , Microscopía/normas
7.
Hum Mutat ; 40(11): 2068-2087, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31283077

RESUMEN

Bardet-Biedl syndrome (BBS) is a recessive genetic disease causing multiple organ anomalies. Most patients carry mutations in genes encoding for the subunits of the BBSome, an octameric ciliary transport complex, or accessory proteins involved in the BBSome assembly or function. BBS proteins have been extensively studied using in vitro, cellular, and animal models. However, the molecular functions of particular BBS proteins and the etiology of the BBS symptoms are still largely elusive. In this study, we applied a meta-analysis approach to study the genotype-phenotype association in humans using our database of all reported BBS patients. The analysis revealed that the identity of the causative gene and the character of the mutation partially predict the clinical outcome of the disease. Besides their potential use for clinical prognosis, our analysis revealed functional differences of particular BBS genes in humans. Core BBSome subunits BBS2, BBS7, and BBS9 manifest as more critical for the function and development of kidneys than peripheral subunits BBS1, BBS4, and BBS8/TTC8, suggesting that incomplete BBSome retains residual function at least in the kidney.


Asunto(s)
Síndrome de Bardet-Biedl/diagnóstico , Síndrome de Bardet-Biedl/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Fenotipo , Factores de Ribosilacion-ADP/genética , Disfunción Cognitiva/genética , Anomalías Congénitas/genética , Estudios de Asociación Genética/métodos , Humanos , Riñón/anomalías , Enfermedades Renales/congénito , Enfermedades Renales/genética , Mutación , Penetrancia , Proteínas/genética
8.
EMBO Rep ; 17(2): 202-19, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26742961

RESUMEN

The trans-Golgi network (TGN) is the main secretory pathway sorting station, where cargoes are packed into appropriate transport vesicles targeted to specific destinations. Exomer is a cargo adaptor necessary for direct transport of a subset of cargoes from the TGN to the plasma membrane in yeast. Here, we show that unlike classical adaptor complexes, exomer is not recruited en bloc to the TGN, but rather assembles through a stepwise pathway, in which first the scaffold protein Chs5 and then the cargo-binding units, the ChAPs, are recruited. Although all ChAPs are able to assemble functional exomer complexes, they do so with different efficiencies. The mutual relationship between ChAPs varies from cooperation to competition depending on their expression levels and affinities to Chs5 allowing an optimized and efficient cargo transport. The multifactorial assembly pathway results in an exquisitely fine-tuned adaptor complex, enabling the cell to quickly respond and adapt to changes such as stress.


Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Quitina Sintasa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Vesículas Secretoras/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Quitina Sintasa/genética , Unión Proteica , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Red trans-Golgi/metabolismo
9.
Gastroenterology ; 144(2): 381-391, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23142137

RESUMEN

BACKGROUND & AIMS: The Wnt signaling pathway is required for maintenance of the intestinal epithelia; blocking this pathway reduces the proliferative capacity of the intestinal stem cells. However, aberrant Wnt signaling leads to intestinal cancer. We investigated the roles of the Wnt pathway in homeostasis of the intestinal epithelium and during malignant transformation in human cells and mice. METHODS: We performed chromatin immunoprecipitation (ChIP) with DNA microarray analysis (ChIP-on-chip) to identify genes regulated by Wnt signaling in human colorectal cancer cells Colo320, DLD1, LS174T, and SW480. Formation of intestinal tumor was induced in C57BL/6J mice using azoxymethane and dextran sulfate. Intestinal tissues from these mice, as well as Apc(+/Min) and Apc(CKO/CKO)/Lgr5-EGFP-IRES-CreERT2 mice, were analyzed by immunohistochemistry and in situ hybridization. RESULTS: We identified promoter regions of 960 genes that interacted with the Wnt pathway nuclear effector T-cell factor 4 in 4 different human colorectal cancer-derived cell lines; 18 of these promoters were present in all chromatin precipitates. Wnt signaling up-regulated a member of the tumor necrosis factor receptor superfamily called TROY. Levels of TROY messenger RNA were increased in human cells with deficiencies in the adenomatous polyposis coli (APC) gene and in cells stimulated with the Wnt3a ligand. Expression of Troy was significantly up-regulated in neoplastic tissues from mice during intestinal tumorigenesis. Lineage tracing experiments revealed that Troy is produced specifically by fast-cycling intestinal stem cells. TROY associated with a unique marker of these cells, leucine-rich repeat-containing G-protein coupled receptor (LGR) 5. In organoids established from the intestinal crypts, Troy suppressed signaling mediated by R-spondin, a Wnt agonist. CONCLUSIONS: TROY is up-regulated in human colorectal cancer cell lines and in intestinal tumors in mice. It functions as a negative modulator of the Wnt pathway in LGR5-positive stem cells.


Asunto(s)
Transformación Celular Neoplásica/genética , Regulación Neoplásica de la Expresión Génica , Mucosa Intestinal/metabolismo , Células Madre Neoplásicas/metabolismo , Receptores Acoplados a Proteínas G/fisiología , Receptores del Factor de Necrosis Tumoral/fisiología , Vía de Señalización Wnt/fisiología , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Humanos , Inmunohistoquímica , Hibridación in Situ , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales , Células Madre Neoplásicas/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Neoplásico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
10.
Cells ; 12(22)2023 11 20.
Artículo en Inglés | MEDLINE | ID: mdl-37998397

RESUMEN

Bardet-Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate.


Asunto(s)
Síndrome de Bardet-Biedl , Humanos , Ratones , Animales , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/metabolismo , Síndrome de Bardet-Biedl/patología , Ratones Noqueados , Proteínas/metabolismo , Cilios/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo
11.
STAR Protoc ; 3(1): 101199, 2022 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-35257113

RESUMEN

Primary cilia are hair-like sensory organelles protruding from the surface of most human cells. As cilia are dynamic, several aspects of their biology can only be revealed by real-time analysis in living cells. Here we describe the generation of primary cilia reporter cell lines. Furthermore, we provide a detailed protocol of how to use the reporter cell lines for live-cell imaging microscopy analysis of primary cilia to study their growth as well as intraciliary transport. For complete details on the use and execution of this protocol, please refer to Bernatik et al. (2020) and Pejskova et al. (2020).


Asunto(s)
Cilios , Procesamiento de Imagen Asistido por Computador , Línea Celular , Cilios/metabolismo , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía/métodos
12.
Front Immunol ; 13: 1009198, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36275704

RESUMEN

Mature T cells are selected for recognizing self-antigens with low to intermediate affinity in the thymus. Recently, the relative differences in self-reactivity among individual T-cell clones were appreciated as important factors regulating their fate and immune response, but the role of self-reactivity in T-cell biology is incompletely understood. We addressed the role of self-reactivity in T-cell diversity by generating an atlas of mouse peripheral CD8+ T cells, which revealed two unconventional populations of antigen-inexperienced T cells. In the next step, we examined the steady-state phenotype of monoclonal T cells with various levels of self-reactivity. Highly self-reactive clones preferentially differentiate into antigen-inexperienced memory-like cells, but do not form a population expressing type I interferon-induced genes, showing that these two subsets have unrelated origins. The functional comparison of naïve monoclonal CD8+ T cells specific to the identical model antigen did not show any correlation between the level of self-reactivity and the magnitude of the immune response.


Asunto(s)
Linfocitos T CD8-positivos , Interferón Tipo I , Ratones , Animales , Timo , Células Clonales , Autoantígenos
13.
Hum Mol Genet ; 18(11): 2014-23, 2009 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-19293337

RESUMEN

The AD29 mutation in HPRP31 belongs to a series of mutations that were initially linked with the autosomal dominant disorder retinitis pigmentosa (RP) type 11. The HPRP31 gene encodes the hPrp31 protein that specifically associates with spliceosomal small nuclear ribonucleoprotein particles (snRNPs). Despite intensive research, it is still unclear how the AD29 (Ala216Pro) mutation causes RP. In this study, we report that the expression of this mutant protein affects cell proliferation and alters the structure of nuclear Cajal bodies that are connected with snRNP metabolism. Interestingly, these effects can be reversed by the over-expression of the hPrp6 protein, a binding partner of hPrp31. Although Ala216 is not contained within the U4 or U5 snRNP interacting domains, we present several lines of evidence that demonstrate that the association between the AD29 mutant and snRNPs in the cell nucleus is significantly reduced. Finally, we show that the stability of the AD29 mutant is severely affected resulting in its rapid degradation. Taken together, our results indicate that the Ala216Pro mutation destabilizes the hPrp31 protein structure in turn reducing its interaction with snRNP binding partners and leading to its rapid degradation. These findings significantly impact our understanding of the molecular mechanisms underlying RP and suggest that the insufficiency of the functional hPrp31 protein combined with the potential cytotoxicity associated with the expression the AD29 mutant are at least partially causative of the RP phenotype.


Asunto(s)
Proteínas del Ojo/química , Proteínas del Ojo/genética , Mutación Missense , Retinitis Pigmentosa/genética , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Empalmosomas/metabolismo , Cuerpos Enrollados/genética , Cuerpos Enrollados/metabolismo , Proteínas del Ojo/metabolismo , Células HeLa , Humanos , Unión Proteica , Estabilidad Proteica , Retinitis Pigmentosa/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/genética , Empalmosomas/genética
14.
RNA ; 15(11): 2063-71, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19767419

RESUMEN

Expression of the nascent RNA transcript is regulated by its interaction with a number of proteins. The misregulation of such interactions can often result in impaired cellular functions that can lead to cancer and a number of diseases. Thus, our understanding of RNA-protein interactions within the cellular context is essential for the development of novel diagnostic and therapeutic tools. While there are many in vitro methods that analyze RNA-protein interactions in vivo approaches are scarce. Here we established a method based on fluorescence resonance energy transfer (FRET), which we term RNA-binding mediated FRET (RB-FRET), which determines RNA-protein interaction inside cells and tested it on hnRNP H protein binding to its cognate RNA. Using two different approaches, we provide evidence that RB-FRET is sensitive enough to detect specific RNA-protein interactions in the cell, providing a powerful tool to study spatial and temporal localization of specific RNA-protein complexes.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas de Unión al ARN/metabolismo , ARN/análisis , Secuencia de Bases , Sitios de Unión , Vectores Genéticos/genética , Células HeLa , Humanos , Unión Proteica , ARN/metabolismo , Proteínas de Unión al ARN/genética , Especificidad por Sustrato
15.
Methods Cell Biol ; 161: 197-216, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33478690

RESUMEN

The resolution achieved by conventional light microscopy is limited by light diffraction. This obstacle can be overcome either by optical super-resolution techniques or by the recently developed method to physically expand specimens, called expansion microscopy (ExM). The method utilizes polymer chemistry and the ability of a swellable polyelectrolyte hydrogel to absorb water, and thus to expand its size. The procedure was successfully applied to different species and tissue samples, mostly from the animal kingdom. Physically expanded nuclei and chromosomes in combination with specific protein labeling and super-resolution microscopy may provide new insight into the ultrastructure, dynamics, and function of plant chromatin. Here we provide a detailed protocol to expand isolated plant nuclei and visualize proteins by indirect immunolabeling. With the focus on chromatin structure, we expanded isolated barley nuclei from root tips and visualized the centromere-specific histone H3 variant CENH3. The achieved physical expansion of ~4.2 times allowed the detection of DAPI-labeled chromatin structures already by conventional wild-field (WF) microscopy with a maximal resolution of ~50-60nm. By applying structured illumination microscopy (SIM), doubling the WF resolution, chromatin structures at a resolution of ~25-35nm were observed. However, a certain distortion of the centromeric chromatin ultrastructure became obvious.


Asunto(s)
Núcleo Celular , Centrómero , Cromatina , Plantas , Histonas/genética , Microscopía
16.
Curr Biol ; 30(15): 3045-3056.e7, 2020 08 03.
Artículo en Inglés | MEDLINE | ID: mdl-32589908

RESUMEN

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/fisiología
17.
Sci Rep ; 6: 29961, 2016 07 21.
Artículo en Inglés | MEDLINE | ID: mdl-27439481

RESUMEN

In search for the function of local chromatin environment on pre-mRNA processing we established a new tool, which allows for the modification of chromatin using a targeted approach. Using Transcription Activator-Like Effector domains fused to histone modifying enzymes (TALE-HME), we show locally restricted alteration of histone methylation modulates the splicing of target exons. We provide evidence that a local increase in H3K9 di- and trimethylation promotes inclusion of the target alternative exon, while demethylation by JMJD2D leads to exon skipping. We further demonstrate that H3K9me3 is localized on internal exons genome-wide suggesting a general role in splicing. Consistently, targeting of the H3K9 demethylase to a weak constitutive exon reduced co-transcriptional splicing. Together our data show H3K9 methylation within the gene body is a factor influencing recognition of both constitutive and alternative exons.


Asunto(s)
Exones/genética , Histonas/metabolismo , Lisina/metabolismo , Efectores Tipo Activadores de la Transcripción/metabolismo , Empalme Alternativo/genética , Cromatina/metabolismo , Fibronectinas/genética , Células HeLa , Humanos , Metilación , Transcripción Genética
18.
J Cell Biol ; 191(1): 75-86, 2010 Oct 04.
Artículo en Inglés | MEDLINE | ID: mdl-20921136

RESUMEN

Precursor messenger RNA (pre-mRNA) splicing is catalyzed by the spliceosome, a large ribonucleoprotein (RNP) complex composed of five small nuclear RNP particles (snRNPs) and additional proteins. Using live cell imaging of GFP-tagged snRNP components expressed at endogenous levels, we examined how the spliceosome assembles in vivo. A comprehensive analysis of snRNP dynamics in the cell nucleus enabled us to determine snRNP diffusion throughout the nucleoplasm as well as the interaction rates of individual snRNPs with pre-mRNA. Core components of the spliceosome, U2 and U5 snRNPs, associated with pre-mRNA for 15-30 s, indicating that splicing is accomplished within this time period. Additionally, binding of U1 and U4/U6 snRNPs with pre-mRNA occurred within seconds, indicating that the interaction of individual snRNPs with pre-mRNA is distinct. These results are consistent with the predictions of the step-wise model of spliceosome assembly and provide an estimate on the rate of splicing in human cells.


Asunto(s)
Precursores del ARN/metabolismo , Empalme del ARN/fisiología , ARN Mensajero/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Empalmosomas/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Células HeLa , Humanos , Cinética , Ribonucleoproteínas Nucleares Pequeñas/fisiología , Espectrometría de Fluorescencia
19.
Mol Biol Cell ; 19(6): 2534-43, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18367544

RESUMEN

The Cajal body (CB) is a nuclear structure closely associated with import and biogenesis of small nuclear ribonucleoprotein particles (snRNPs). Here, we tested whether CBs also contain mature snRNPs and whether CB integrity depends on the ongoing snRNP splicing cycle. Sm proteins tagged with photoactivatable and color-maturing variants of fluorescent proteins were used to monitor snRNP behavior in living cells over time; mature snRNPs accumulated in CBs, traveled from one CB to another, and they were not preferentially replaced by newly imported snRNPs. To test whether CB integrity depends on the snRNP splicing cycle, two human orthologues of yeast proteins involved in distinct steps in spliceosome disassembly after splicing, hPrp22 and hNtr1, were depleted by small interfering RNA treatment. Surprisingly, depletion of either protein led to the accumulation of U4/U6 snRNPs in CBs, suggesting that reassembly of the U4/U6.U5 tri-snRNP was delayed. Accordingly, a relative decrease in U5 snRNPs compared with U4/U6 snRNPs was observed in CBs, as well as in nuclear extracts of treated cells. Together, the data show that particular phases of the spliceosome cycle are compartmentalized in living cells, with reassembly of the tri-snRNP occurring in CBs.


Asunto(s)
Cuerpos Enrollados/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Empalmosomas/metabolismo , Biomarcadores/metabolismo , Proteínas Portadoras/metabolismo , Células HeLa , Humanos , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequeña U5/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA