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1.
Nat Methods ; 16(12): 1247-1253, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31636459

RESUMO

Segmenting the nuclei of cells in microscopy images is often the first step in the quantitative analysis of imaging data for biological and biomedical applications. Many bioimage analysis tools can segment nuclei in images but need to be selected and configured for every experiment. The 2018 Data Science Bowl attracted 3,891 teams worldwide to make the first attempt to build a segmentation method that could be applied to any two-dimensional light microscopy image of stained nuclei across experiments, with no human interaction. Top participants in the challenge succeeded in this task, developing deep-learning-based models that identified cell nuclei across many image types and experimental conditions without the need to manually adjust segmentation parameters. This represents an important step toward configuration-free bioimage analysis software tools.


Assuntos
Núcleo Celular/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Ciência de Dados , Humanos , Microscopia de Fluorescência/métodos
2.
PLoS Biol ; 17(10): e3000145, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31589603

RESUMO

Male reproductive glands like the mammalian prostate and the paired Drosophila melanogaster accessory glands secrete seminal fluid components that enhance fecundity. In humans, the prostate, stimulated by environmentally regulated endocrine and local androgens, grows throughout adult life. We previously showed that in fly accessory glands, secondary cells (SCs) and their nuclei also grow in adults, a process enhanced by mating and controlled by bone morphogenetic protein (BMP) signalling. Here, we demonstrate that BMP-mediated SC growth is dependent on the receptor for the developmental steroid ecdysone, whose concentration is reported to reflect sociosexual experience in adults. BMP signalling appears to regulate ecdysone receptor (EcR) levels via one or more mechanisms involving the EcR's N terminus or the RNA sequence that encodes it. Nuclear growth in virgin males is dependent on ecdysone, some of which is synthesised in SCs. However, mating induces additional BMP-mediated nuclear growth via a cell type-specific form of hormone-independent EcR signalling, which drives genome endoreplication in a subset of adult SCs. Switching to hormone-independent endoreplication after mating allows growth and secretion to be hyperactivated independently of ecdysone levels in SCs, permitting more rapid replenishment of the accessory gland luminal contents. Our data suggest mechanistic parallels between this physiological, behaviour-induced signalling switch and altered pathological signalling associated with prostate cancer progression.


Assuntos
Proteínas Morfogenéticas Ósseas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Ecdisona/metabolismo , Genoma de Inseto , Proteínas de Insetos/genética , Receptores de Esteroides/genética , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Copulação/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/metabolismo , Masculino , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Esteroides/antagonistas & inibidores , Receptores de Esteroides/metabolismo , Transdução de Sinais
3.
Nat Cell Biol ; 21(10): 1248-1260, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31576060

RESUMO

While nuclear lamina abnormalities are hallmarks of human diseases, their interplay with epigenetic regulators and precise epigenetic landscape remain poorly understood. Here, we show that loss of the lysine acetyltransferase MOF or its associated NSL-complex members KANSL2 or KANSL3 leads to a stochastic accumulation of nuclear abnormalities with genomic instability patterns including chromothripsis. SILAC-based MOF and KANSL2 acetylomes identified lamin A/C as an acetylation target of MOF. HDAC inhibition or acetylation-mimicking lamin A derivatives rescue nuclear abnormalities observed in MOF-deficient cells. Mechanistically, loss of lamin A/C acetylation resulted in its increased solubility, defective phosphorylation dynamics and impaired nuclear mechanostability. We found that nuclear abnormalities include EZH2-dependent histone H3 Lys 27 trimethylation and loss of nascent transcription. We term this altered epigenetic landscape "heterochromatin enrichment in nuclear abnormalities" (HENA). Collectively, the NSL-complex-dependent lamin A/C acetylation provides a mechanism that maintains nuclear architecture and genome integrity.


Assuntos
Núcleo Celular/ultraestrutura , Histona Acetiltransferases/metabolismo , Lamina Tipo A/metabolismo , Proteínas Nucleares/metabolismo , Acetilação , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Epigênese Genética , Fibroblastos , Heterocromatina , Histona Acetiltransferases/genética , Histonas/genética , Histonas/metabolismo , Lamina Tipo A/química , Lamina Tipo A/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética
4.
PLoS Biol ; 17(10): e3000509, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31613895

RESUMO

The Hippo signalling pathway restricts cell proliferation in animal tissues by inhibiting Yes-associated protein (YAP or YAP1) and Transcriptional Activator with a PDZ domain (TAZ or WW-domain-containing transcriptional activator [WWTR1]), coactivators of the Scalloped (Sd or TEAD) DNA-binding transcription factor. Drosophila has a single YAP/TAZ homolog named Yorkie (Yki) that is regulated by Hippo pathway signalling in response to epithelial polarity and tissue mechanics during development. Here, we show that Yki translocates to the nucleus to drive Sd-mediated cell proliferation in the ovarian follicle cell epithelium in response to mechanical stretching caused by the growth of the germline. Importantly, mechanically induced Yki nuclear localisation also requires nutritionally induced insulin/insulin-like growth factor 1 (IGF-1) signalling (IIS) via phosphatidyl inositol-3-kinase (PI3K), phosphoinositide-dependent kinase 1 (PDK1 or PDPK1), and protein kinase B (Akt or PKB) in the follicular epithelium. We find similar results in the developing Drosophila wing, where Yki becomes nuclear in the mechanically stretched cells of the wing pouch during larval feeding, which induces IIS, but translocates to the cytoplasm upon cessation of feeding in the third instar stage. Inactivating Akt prevents nuclear Yki localisation in the wing disc, while ectopic activation of the insulin receptor, PI3K, or Akt/PKB is sufficient to maintain nuclear Yki in mechanically stimulated cells of the wing pouch even after feeding ceases. Finally, IIS also promotes YAP nuclear localisation in response to mechanical cues in mammalian skin epithelia. Thus, the Hippo pathway has a physiological function as an integrator of epithelial cell polarity, tissue mechanics, and nutritional cues to control cell proliferation and tissue growth in both Drosophila and mammals.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinase/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transativadores/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Animais , Fenômenos Biomecânicos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Polaridade Celular , Proliferação de Células , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Células Epiteliais/citologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Larva/citologia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Mecanotransdução Celular , Camundongos , Proteínas Nucleares/metabolismo , Folículo Ovariano/citologia , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Transporte Proteico , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transativadores/metabolismo , Asas de Animais/citologia , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismo
5.
Nucleic Acids Res ; 47(18): e108, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31562528

RESUMO

The integrity of the chromatin structure is essential to every process occurring within eukaryotic nuclei. However, there are no reliable tools to decipher the molecular composition of metaphase chromosomes. Here, we have applied infrared nanospectroscopy (AFM-IR) to demonstrate molecular difference between eu- and heterochromatin and generate infrared maps of single metaphase chromosomes revealing detailed information on their molecular composition, with nanometric lateral spatial resolution. AFM-IR coupled with principal component analysis has confirmed that chromosome areas containing euchromatin and heterochromatin are distinguishable based on differences in the degree of methylation. AFM-IR distribution of eu- and heterochromatin was compared to standard fluorescent staining. We demonstrate the ability of our methodology to locate spatially the presence of anticancer drug sites in metaphase chromosomes and cellular nuclei. We show that the anticancer 'rule breaker' platinum compound [Pt[N(p-HC6F4)CH2]2py2] preferentially binds to heterochromatin, forming localized discrete foci due to condensation of DNA interacting with the drug. Given the importance of DNA methylation in the development of nearly all types of cancer, there is potential for infrared nanospectroscopy to be used to detect gene expression/suppression sites in the whole genome and to become an early screening tool for malignancy.


Assuntos
Cromossomos/ultraestrutura , DNA/ultraestrutura , Metáfase/genética , Espectrofotometria Infravermelho/métodos , Animais , Núcleo Celular/ultraestrutura , Eucromatina/ultraestrutura , Heterocromatina/ultraestrutura , Humanos , Interfase/genética
6.
Int J Mol Sci ; 20(18)2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31509978

RESUMO

Tumorous metastasis is a difficult challenge to resolve for researchers and for clinicians. Targeted delivery of antitumor drugs towards tumor cells' nuclei can be a practical approach to resolving this issue. This work describes an efficient nuclear-targeting delivery system prepared from trans-activating transcriptional activator (TAT) peptide-functionalized graphene nanocarriers. The TAT peptide, originally observed in a human immunodeficiency virus 1 (HIV-1), was incorporated with graphene via an edge-functionalized ball-milling method developed by the author's research group. High tumor-targeting capability of the resulting nanocarrier was realized by the strong affinity between TAT and the nuclei of cancer cells, along with the enhanced permeability and retention (EPR) effect of two-dimensional graphene nanosheets. Subsequently, a common antitumor drug, mitomycin C (MMC), was covalently linked to the TAT-functionalized graphene (TG) to form a nuclear-targeted nanodrug MMC-TG. The presence of nanomaterials inside the nuclei of ocular choroidal melanoma (OCM-1) cells was shown using transmission electron microscopy (TEM) and confocal laser scanning microscopy. In vitro results from a Transwell co-culture system showed that most of the MMC-TG nanodrugs were delivered in a targeted manner to the tumorous OCM-1 cells, while a very small amount of MMC-TG was delivered in a non-targeted manner to normal human retinal pigment epithelial (ARPE-19) cells. TEM results further confirmed that apoptosis of OCM-1 cells was started from the lysis of nuclear substances, followed by the disappearance of nuclear membrane and cytoplasm. This suggests that the as-synthesized MMC-TG is a promising nuclear-target nanodrugfor resolution of tumorous metastasis issues at the headstream.


Assuntos
Neoplasias da Coroide/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Grafite/química , Melanoma/tratamento farmacológico , Mitomicina/administração & dosagem , Peptídeos/química , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/química , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Neoplasias da Coroide/metabolismo , Neoplasias da Coroide/patologia , Portadores de Fármacos/química , Humanos , Melanoma/metabolismo , Melanoma/patologia , Microscopia Eletrônica de Transmissão , Mitomicina/química , Nanoestruturas/administração & dosagem , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química
7.
Gene ; 719: 144074, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31446094

RESUMO

Kinesin-14 motor es-kifc1 is highly expressed in the male reproductive system of the Chinese mitten crab Eriocheir sinensis (E. sinensis). In addition to acrosomal formation, es-KIFC1 also tightly surrounds the nucleus and its specific mechanism remains unknown. During spermatogenesis, sperm nucleus dents into a cup-shaped structure with several radial arms and completed the nuclear decondensation. In this study, the spatial expression pattern of es-KIFC1 indicates a potential function in nuclear formation with the nuclear localization sequence (NLS) on N-terminal domain which is crucial for the translocation of es-KIFC1 into the nucleus. The Motor domain is associated with microtubule modulation and the Golgi vesicles positioning. Furthermore, the expression level of es-KIFC1 is not only related to the seasonal variation of crustacean development, but also associates with mature sperm storage. The double strand RNA (dsRNA) mediated RNA interference manifests that the cup-shaped sperm nucleus is remarkably malformed and even separates the chromatin throughout the nuclei at the last stage of spermiogenesis. Besides, the sperm nucleus almost disperses its structure and separates the chromatin into several segments throughout the nucleus showing an asymmetrical performance without cytoskeleton. In summary, these results indicate the importance of es-KIFC1 in microtubule positioning and the maintenance of the mature sperm nuclei.


Assuntos
Proteínas de Artrópodes/genética , Braquiúros/fisiologia , Núcleo Celular/metabolismo , Cinesina/genética , Espermatogênese/genética , Animais , Proteínas de Artrópodes/metabolismo , Núcleo Celular/ultraestrutura , Citoesqueleto/metabolismo , Cinesina/metabolismo , Masculino , Microtúbulos/metabolismo , Transporte Proteico , RNA de Cadeia Dupla/genética , Espermatozoides/ultraestrutura
8.
Zygote ; 27(4): 232-240, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31397243

RESUMO

Brilliant cresyl blue (BCB) vital labelling is a powerful method for analyzing the quality of porcine cumulus-oocyte complexes. Our aim was to investigate the correlation between the selection of porcine oocytes using BCB labelling and selected intranuclear characteristics of porcine oocytes and parthenotes. Moreover, BCB labelling was correlated with the diameter of the oocyte and the developmental potential of the parthenotes. The following methods were used: BCB labelling, measurement of the diameter of the oocyte, parthenogenetic activation, immunocytochemistry, transmission electron microscopy, enucleation and relative protein concentration (RPC) analysis. We determined that the diameter of the oocytes in the BCB-positive (BCB+) group was significantly larger than in the BCB-negative (BCB-) group. Immediately after oocyte selection according to BCB labelling, we found significant difference in chromatin configuration between the analyzed groups. BCB+ oocytes were significantly better at maturation than BCB- oocytes. BCB+ embryos were significantly more competent at cleaving and in their ability to reach the blastocyst stage than BCB- embryos. Ultrastructural analyses showed that the formation of active nucleoli in the BCB+ group started at the 8-cell stage. Conversely, most BCB- embryos at the 8-cell and 16-cell stages were fragmented. No statistically significant difference in RPC in nucleolus precursor bodies (NPBs) between BCB+ and BCB- oocytes was found. We can conclude that BCB labelling could be suitable for assessing the quality of porcine oocytes. Moreover, the evaluation of RPC indicates that the quantitative content of proteins in NPB is already established in growing oocytes.


Assuntos
Blastocisto/química , Núcleo Celular/química , Embrião de Mamíferos/química , Oócitos/química , Oxazinas/química , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Núcleo Celular/ultraestrutura , Tamanho Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/ultraestrutura , Feminino , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Proteínas Nucleares/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Reprodutibilidade dos Testes , Coloração e Rotulagem/métodos , Suínos
9.
Comput Math Methods Med ; 2019: 3072498, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31308855

RESUMO

The leukocyte nucleus quick segmentation is one of the key techniques in leukocyte real-time online scanning of human blood smear. We propose a quick leukocyte nucleus segmentation method based on the component difference in RGB color space. By analyzing the captured microscopic images of the peripheral blood smears from the autoscanning microscope, it is found that the difference values between B component and G component (B - G values) in the regions of the leukocyte nuclei and the platelets are much bigger than those in the other regions, even in the regions including the stains. So, the B - G values can segment the leukocyte nuclei and the platelets with an appropriate empirical threshold because the platelets are much smaller than the leukocyte nuclei, so the leukocyte nuclei can be segmented by size filtering. Also, only an 8 bit subtraction operation is performed for the B - G values, and it can improve the leukocyte nucleus segmentation speed significantly. Experimental results show that the proposed method performs well for the five types of leukocyte segmentation with a quick speed. It is very suitable for the real-time peripheral blood smear autoscanning test application. In addition, the five types of leukocytes can be counted accurately.


Assuntos
Núcleo Celular/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Leucócitos/citologia , Contagem de Linfócitos , Reconhecimento Automatizado de Padrão , Algoritmos , Cor , Simulação por Computador , Humanos , Microscopia , Linguagens de Programação
10.
Nucleic Acids Res ; 47(18): e109, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31340014

RESUMO

Fluorescence in situ hybridization (FISH) can be used for the intracellular detection of DNA or RNA molecules. The detection of DNA sequences by DNA FISH requires the denaturation of the DNA double helix to allow the hybridization of the fluorescent probe with DNA in a single stranded form. These hybridization conditions require high temperature and low pH that can damage RNA, and therefore RNA is not typically detectable by DNA FISH. In contrast, RNA FISH does not require a denaturation step since RNA is single stranded, and therefore DNA molecules are not detectable by RNA FISH. Hence, DNA FISH and RNA FISH are mutually exclusive. In this study, we show that plasmid DNA transiently transfected into cells is readily detectable in the cytoplasm by RNA FISH without need for denaturation, shortly after transfection and for several hours. The plasmids, however, are usually not detectable in the nucleus except when the plasmids are efficiently directed into the nucleus, which may imply a more open packaging state for DNA after transfection. This detection of plasmid DNA in the cytoplasm has implications for RNA FISH experiments and opens a window to study conditions when DNA is present in the cytoplasm.


Assuntos
Citoplasma/ultraestrutura , DNA/ultraestrutura , Hibridização in Situ Fluorescente/métodos , RNA/química , Núcleo Celular/ultraestrutura , DNA/isolamento & purificação , Corantes Fluorescentes/química , Hibridização de Ácido Nucleico , Plasmídeos/genética , Sequências Repetitivas de Ácido Nucleico
11.
Int J Mol Sci ; 20(14)2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31340471

RESUMO

The side effects induced by nanoparticle exposure at a cellular level are one of the priority research topics due to the steady increase in the use of nanoparticles (NPs). Recently, the focus on cellular morphology and mechanical behavior is gaining relevance in order to fully understand the cytotoxic mechanisms. In this regard, we have evaluated the morphomechanical alteration in human breast adenocarcinoma cell line (MCF-7) exposed to TiO2NPs at two different concentrations (25 and 50 µg/mL) and two time points (24 and 48 h). By using confocal and atomic force microscopy, we demonstrated that TiO2NP exposure induces significant alterations in cellular membrane elasticity, due to actin proteins rearrangement in cytoskeleton, as calculated in correspondence to nuclear and cytoplasmic compartments. In this work, we have emphasized the alteration in mechanical properties of the cellular membrane, induced by nanoparticle exposure.


Assuntos
Núcleo Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citotoxinas/toxicidade , Nanopartículas/toxicidade , Titânio/toxicidade , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/ultraestrutura , Fenômenos Biomecânicos , Membrana Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , Citosol/ultraestrutura , Citotoxinas/química , Elasticidade/efeitos dos fármacos , Humanos , Células MCF-7 , Microscopia de Força Atômica , Nanopartículas/ultraestrutura , Titânio/química
12.
Mol Cell ; 74(6): 1110-1122, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31226276

RESUMO

During embryogenesis, precise gene transcription in space and time requires that distal enhancers and promoters communicate by physical proximity within gene regulatory landscapes. To achieve this, regulatory landscapes fold in nuclear space, creating complex 3D structures that influence enhancer-promoter communication and gene expression and that, when disrupted, can cause disease. Here, we provide an overview of how enhancers and promoters construct regulatory landscapes and how multiple scales of 3D chromatin structure sculpt their communication. We focus on emerging views of what enhancer-promoter contacts and chromatin domains physically represent and how two antagonistic fundamental forces-loop extrusion and homotypic attraction-likely form them. We also examine how these same forces spatially separate regulatory landscapes by functional state, thereby creating higher-order compartments that reconfigure during development to enable proper enhancer-promoter communication.


Assuntos
Cromatina/ultraestrutura , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Regiões Promotoras Genéticas , Transcrição Genética , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Cromatina/metabolismo , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Células Eucarióticas/metabolismo , Células Eucarióticas/ultraestrutura , Humanos , Conformação Molecular
13.
Gigascience ; 8(6)2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31247098

RESUMO

BACKGROUND: Cells are powered by a large set of macromolecular complexes, which work together in a crowded environment. The in situ mechanisms of these complexes are unclear because their 3D distribution, organization, and interactions are largely unknown. Electron cryotomography (cryo-ET) can address these knowledge gaps because it produces cryotomograms-3D images that reveal biological structure at ∼4-nm resolution. Cryo-ET uses no fixation, dehydration, staining, or plastic embedment, so cellular features are visualized in a life-like, frozen-hydrated state. To study chromatin and mitotic machinery in situ, we subjected yeast cells to genetic and chemical perturbations, cryosectioned them, and then imaged the cells by cryo-ET. FINDINGS: Here we share >1,000 cryo-ET raw datasets of cryosectioned budding yeast Saccharomyces cerevisiaecollected as part of previously published studies. These data will be valuable to cell biologists who are interested in the nanoscale organization of yeasts and of eukaryotic cells in general. All the unpublished tilt series and a subset of corresponding cryotomograms have been deposited in the EMPIAR resource for the community to use freely. To improve tilt series discoverability, we have uploaded metadata and preliminary notes to publicly accessible Google Sheets, EMPIAR, and GigaDB. CONCLUSIONS: Cellular cryo-ET data can be mined to obtain new cell-biological, structural, and 3D statistical insights in situ. These data contain structures not visible in traditional electron-microscopy data. Template matching and subtomogram averaging of known macromolecular complexes can reveal their 3D distributions and low-resolution structures. Furthermore, these data can serve as testbeds for high-throughput image-analysis pipelines, as training sets for feature-recognition software, for feasibility analysis when planning new structural-cell-biology projects, and as practice data for students.


Assuntos
Bases de Dados Factuais , Saccharomyces cerevisiae/ultraestrutura , Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , Imagem Tridimensional
14.
PLoS Pathog ; 15(6): e1007827, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31181119

RESUMO

P10 is a small, abundant baculovirus protein that accumulates to high levels in the very late stages of the infection cycle. It is associated with a number of intracellular structures and implicated in diverse processes from occlusion body maturation to nuclear stability and lysis. However, studies have also shown that it is non-essential for virus replication, at least in cell culture. Here, we describe the use of serial block-face scanning electron microscopy to achieve high-resolution 3D characterisation of P10 structures within Trichoplusia ni TN-368 cells infected with Autographa californica multiple nucleopolyhedrovirus. This has enabled unparalleled visualisation of P10 and determined the independent formation of dynamic perinuclear and nuclear vermiform fibrous structures. Our 3D data confirm the sequence of ultrastructural changes that create a perinuclear cage from thin angular fibrils within the cytoplasm. Over the course of infection in cultured cells, the cage remodels to form a large polarised P10 mass and we suggest that these changes are critical for nuclear lysis to release occlusion bodies. In contrast, nuclear P10 forms a discrete vermiform structure that was observed in close spatial association with both electron dense spacers and occlusion bodies; supporting a previously suggested role for P10 and electron dense spacers in the maturation of occlusion bodies. We also demonstrate that P10 hyper-expression is critical for function. Decreasing levels of p10 expression, achieved by manipulation of promoter length, correlated with reduced P10 production, a lack of formation of P10 structures and a concomitant decrease in nuclear lysis.


Assuntos
Núcleo Celular/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Núcleo Celular/virologia , Mariposas , /genética , Domínios Proteicos , Proteínas Virais/química , Proteínas Virais/genética
15.
PLoS Genet ; 15(6): e1008061, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31170156

RESUMO

The nuclear pore complex (NPC) forms a gateway for nucleocytoplasmic transport. The outer ring protein complex of the NPC (the Nup107-160 subcomplex in humans) is a key component for building the NPC. Nup107-160 subcomplexes are believed to be symmetrically localized on the nuclear and cytoplasmic sides of the NPC. However, in S. pombe immunoelectron and fluorescence microscopic analyses revealed that the homologous components of the human Nup107-160 subcomplex had an asymmetrical localization: constituent proteins spNup132 and spNup107 were present only on the nuclear side (designated the spNup132 subcomplex), while spNup131, spNup120, spNup85, spNup96, spNup37, spEly5 and spSeh1 were localized only on the cytoplasmic side (designated the spNup120 subcomplex), suggesting the complex was split into two pieces at the interface between spNup96 and spNup107. This contrasts with the symmetrical localization reported in other organisms. Fusion of spNup96 (cytoplasmic localization) with spNup107 (nuclear localization) caused cytoplasmic relocalization of spNup107. In this strain, half of the spNup132 proteins, which interact with spNup107, changed their localization to the cytoplasmic side of the NPC, leading to defects in mitotic and meiotic progression similar to an spNup132 deletion strain. These observations suggest the asymmetrical localization of the outer ring spNup132 and spNup120 subcomplexes of the NPC is necessary for normal cell cycle progression in fission yeast.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/genética , Poro Nuclear/genética , Proteínas de Schizosaccharomyces pombe/genética , Transporte Ativo do Núcleo Celular/genética , Ciclo Celular/genética , Divisão Celular/genética , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Citoplasma/genética , Citoplasma/ultraestrutura , Humanos , Meiose/genética , Microscopia de Fluorescência , Membrana Nuclear/genética , Poro Nuclear/ultraestrutura , Ligação Proteica/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética
16.
Chemistry ; 25(41): 9592-9596, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31111975

RESUMO

A genetically encoded fluorescent tag for live cell microscopy is presented. This tag is composed of previously published fluorogen-activating protein FAST and a novel fluorogenic derivative of green fluorescent protein (GFP)-like chromophore with red fluorescence. The reversible binding of the novel fluorogen and FAST is accompanied by three orders of magnitude increase in red fluorescence (580-650 nm). The proposed dye instantly stains target cellular proteins fused with FAST, washes out in a minute timescale, and exhibits higher photostability of the fluorescence signal in confocal and widefield microscopy, in contrast with previously published fluorogen:FAST complexes.


Assuntos
Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/química , Rodanina/análogos & derivados , Núcleo Celular/ultraestrutura , Fluorescência , Células HEK293 , Células HeLa , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Imagem Óptica
17.
Ann Anat ; 224: 117-123, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31117003

RESUMO

Specific ultrastructural anatomy of masticatory muscles is commonly referred to a general pattern assigned to striated muscles. Junctional feet consisting of calcium channels of the sarcoplasmic reticulum (i.e. the ryanodine receptors, RyRs) physically connected to the calcium channels of the t-tubules build triads within striated muscles. Functional RyRs were demonstrated in the nuclear envelopes of pancreas and of a skeletal muscle derived cell line, but not in muscle in situ. It was hypothesized that ryanodine receptors (RyRs) could also exist in the nuclear envelope in the masseter muscle, thus aiming at studying this by transmission electron microscopy. There were identified paired and consistent subsarcolemmal clusters of mitochondria, appearing as outpockets of the muscle fibers, usually flanking an endomysial microvessel. It was observed on grazing longitudinal cuts that the I-band-limited mitochondria were not strictly located in a single intermyofibrillar space but continued transversally over the I-band to the next intermyofibrillar space. It appeared that the I-band-limited transverse mitochondria participate with the column-forming mitochondria in building a rather incomplete mitochondrial reticulum of the masseter muscle. Subsarcolemmal nuclei presented nuclear envelope-associated RyRs. Moreover, t-tubules were contacting the nuclear envelope and they were seemingly filled from the perinuclear space. This could suggest that nucleoplasmic calcium could contribute to balance the cytosolic concentration via pre-built anatomical routes: (i) indirectly, via the RyRs of the nuclear envelope and (ii) directly via the communication of t-tubules and sarcoplasmic reticulum through the perinuclear space.


Assuntos
Cálcio/metabolismo , Músculo Masseter/metabolismo , Músculo Masseter/ultraestrutura , Animais , Núcleo Celular/ultraestrutura , Masculino , Microscopia Eletrônica de Transmissão , Microvasos/ultraestrutura , Mitocôndrias/ultraestrutura , Modelos Animais , Fibras Musculares Esqueléticas/ultraestrutura , Miofibrilas/ultraestrutura , Membrana Nuclear/ultraestrutura , Coelhos , Sarcolema/ultraestrutura , Sarcômeros/ultraestrutura
18.
Mol Cell ; 75(1): 26-38.e3, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31130364

RESUMO

Growth factor signaling is initiated at the plasma membrane and propagated through the cytoplasm for eventual relay to intracellular organelles such as lysosomes. The serine/threonine kinase mTOR participates in growth factor signaling as a component of two multi-subunit complexes, mTORC1 and mTORC2. mTORC1 associates with lysosomes, and its activity depends on the positioning of lysosomes within the cytoplasm, although there is no consensus regarding the exact effect of perinuclear versus peripheral distribution. mTORC2 and its substrate kinase AKT have a widespread distribution, but they are thought to act mainly at the plasma membrane. Using cell lines with knockout of components of the lysosome-positioning machinery, we show that perinuclear clustering of lysosomes delays reactivation of not only mTORC1, but also mTORC2 and AKT upon serum replenishment. These experiments demonstrate the existence of pools of mTORC2 and AKT that are sensitive to lysosome positioning.


Assuntos
Núcleo Celular/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Proteínas Proto-Oncogênicas c-akt/genética , Fatores de Ribosilação do ADP/deficiência , Fatores de Ribosilação do ADP/genética , Sistemas CRISPR-Cas , Núcleo Celular/ultraestrutura , Meios de Cultura Livres de Soro , Endossomos/metabolismo , Endossomos/ultraestrutura , Edição de Genes , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Cinesina/deficiência , Cinesina/genética , Lisossomos/ultraestrutura , Fatores de Transcrição MEF2/deficiência , Fatores de Transcrição MEF2/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
19.
Nucleic Acids Res ; 47(13): 6699-6713, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31127282

RESUMO

Numerous pieces of evidence support the complex, 3D spatial organization of the genome dictates gene expression. CTCF is essential to define topologically associated domain boundaries and to facilitate the formation of insulated chromatin loop structures. To understand CTCF's direct role in global transcriptional regulation, we integrated the miniAID-mClover3 cassette to the endogenous CTCF locus in a human pediatric B-ALL cell line, SEM, and an immortal erythroid precursor cell line, HUDEP-2, to allow for acute depletion of CTCF protein by the auxin-inducible degron system. In SEM cells, CTCF loss notably disrupted intra-TAD loops and TAD integrity in concurrence with a reduction in CTCF-binding affinity, while showing no perturbation to nuclear compartment integrity. Strikingly, the overall effect of CTCF's loss on transcription was minimal. Whole transcriptome analysis showed hundreds of genes differentially expressed in CTCF-depleted cells, among which MYC and a number of MYC target genes were specifically downregulated. Mechanically, acute depletion of CTCF disrupted the direct interaction between the MYC promoter and its distal enhancer cluster residing ∼1.8 Mb downstream. Notably, MYC expression was not profoundly affected upon CTCF loss in HUDEP-2 cells suggesting that CTCF could play a B-ALL cell line specific role in maintaining MYC expression.


Assuntos
Fator de Ligação a CCCTC/fisiologia , Cromatina/ultraestrutura , Elementos Facilitadores Genéticos/genética , Regulação Leucêmica da Expressão Gênica , Genes myc , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myc/biossíntese , Fator de Ligação a CCCTC/deficiência , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Cromatina/genética , Regulação para Baixo , Células Precursoras Eritroides/metabolismo , Técnicas de Introdução de Genes , Genes Reporter , Humanos , Conformação de Ácido Nucleico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Transcriptoma
20.
Sensors (Basel) ; 19(9)2019 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-31060334

RESUMO

[1-13C]pyruvate, the most widely used compound in dissolution-dynamic nuclear polarization (dDNP) magnetic resonance (MR), enables the visualization of lactate dehydrogenase (LDH) activity. This activity had been demonstrated in a wide variety of cancer models, ranging from cultured cells, to xenograft models, to human tumors in situ. Here we quantified the LDH activity in precision cut tumor slices (PCTS) of breast cancer xenografts. The Michigan Cancer Foundation-7 (MCF7) cell-line was chosen as a model for the luminal breast cancer type which is hormone responsive and is highly prevalent. The LDH activity, which was manifested as [1-13C]lactate production in the tumor slices, ranged between 3.8 and 6.1 nmole/nmole adenosine tri-phosphate (ATP) in 1 min (average 4.6 ± 1.0) on three different experimental set-ups consisting of arrested vs. continuous perfusion and non-selective and selective RF pulsation schemes and combinations thereof. This rate was converted to an expected LDH activity in a mass ranging between 3.3 and 5.2 µmole/g in 1 min, using the ATP level of these tumors. This indicated the likely utility of this approach in clinical dDNP of the human breast and may be useful as guidance for treatment response assessment in a large number of tumor types and therapies ex vivo.


Assuntos
Neoplasias da Mama/diagnóstico , Núcleo Celular/ultraestrutura , Lactato Desidrogenases/isolamento & purificação , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Núcleo Celular/química , Núcleo Celular/metabolismo , Polaridade Celular/efeitos dos fármacos , Liberação Controlada de Fármacos/efeitos dos fármacos , Feminino , Humanos , Lactato Desidrogenases/metabolismo , Imagem por Ressonância Magnética , Camundongos , Ácido Pirúvico/isolamento & purificação , Ácido Pirúvico/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
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