RESUMO
A universal feature of metazoan sexual development is the generation of oocyte P granules that withhold certain mRNA species from translation to provide coding potential for proteins during early post-fertilization development. Stabilisation of translationally quiescent mRNA pools in female Plasmodium gametocytes depends on the RNA helicase DOZI, but the molecular machinery involved in the silencing of transcripts in these protozoans is unknown. Using affinity purification coupled with mass-spectrometric analysis we identify a messenger ribonucleoprotein (mRNP) from Plasmodium berghei gametocytes defined by DOZI and the Sm-like factor CITH (homolog of worm CAR-I and fly Trailer Hitch). This mRNP includes 16 major factors, including proteins with homologies to components of metazoan P granules and archaeal proteins. Containing translationally silent transcripts, this mRNP integrates eIF4E and poly(A)-binding protein but excludes P body RNA degradation factors and translation-initiation promoting eIF4G. Gene deletion mutants of 2 core components of this mRNP (DOZI and CITH) are fertilization-competent, but zygotes fail to develop into ookinetes in a female gametocyte-mutant fashion. Through RNA-immunoprecipitation and global expression profiling of CITH-KO mutants we highlight CITH as a crucial repressor of maternally supplied mRNAs. Our data define Plasmodium P granules as an ancient mRNP whose protein core has remained evolutionarily conserved from single-cell organisms to germ cells of multi-cellular animals and stores translationally silent mRNAs that are critical for early post-fertilization development during the initial stages of mosquito infection. Therefore, translational repression may offer avenues as a target for the generation of transmission blocking strategies and contribute to limiting the spread of malaria.
Assuntos
Regulação da Expressão Gênica/fisiologia , Plasmodium berghei/fisiologia , Proteínas de Protozoários/fisiologia , Interferência de RNA/fisiologia , Animais , Southern Blotting , Western Blotting , Feminino , Citometria de Fluxo , Expressão Gênica , Perfilação da Expressão Gênica , Células Germinativas , Imunoprecipitação , Filogenia , RNA Mensageiro/genética , Ribonucleoproteínas/fisiologia , Desenvolvimento Sexual , ZigotoRESUMO
BACKGROUND: In cytokinesis, when the cleavage furrow has been formed, the two centrioles in each daughter cell separate. It has been suggested that the centrioles facilitate and regulate cytokinesis to some extent. It has been postulated that termination of cytokinesis (abscission) depends on the migration of a centriole to the intercellular bridge and then back to the cell center. To investigate the involvement of centrioles in cytokinesis, we monitored the movements of centrioles in three mammalian epithelial cell lines, HeLa, MCF 10A, and the p53-deficient mouse mammary tumor cell line KP-7.7, by time-lapse imaging. Centrin1-EGFP and alpha-Tubulin-mCherry were co-expressed in the cells to visualize respectively the centrioles and microtubules. RESULTS: Here we report that separated centrioles that migrate from the cell pole are very mobile during cytokinesis and their movements can be characterized as 1) along the nuclear envelope, 2) irregular, and 3) along microtubules forming the spindle axis. Centriole movement towards the intercellular bridge was only seen occasionally and was highly cell-line dependent. CONCLUSIONS: These findings show that centrioles are highly mobile during cytokinesis and suggest that the repositioning of a centriole to the intercellular bridge is not essential for controlling abscission. We suggest that centriole movements are microtubule dependent and that abscission is more dependent on other mechanisms than positioning of centrioles.
Assuntos
Centríolos/metabolismo , Células Epiteliais/metabolismo , Microtúbulos/metabolismo , Animais , Biomarcadores/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Centríolos/ultraestrutura , Proteínas Cromossômicas não Histona/metabolismo , Citocinese , Células Epiteliais/ultraestrutura , Células HeLa , Humanos , Mamíferos , Camundongos , Microscopia , Microtúbulos/ultraestrutura , Tubulina (Proteína)/metabolismoRESUMO
Trimethylation of histone H3 lysine 9 and the subsequent binding of heterochromatin protein 1 (HP1) mediate the formation and maintenance of pericentromeric heterochromatin. Trimethylation of H3K9 is governed by the histone methyltransferase SUV39H1. Recent studies of HP1 dynamics revealed that HP1 is not a stable component of heterochromatin but is highly mobile (Cheutin, T., A.J. McNairn, T. Jenuwein, D.M. Gilbert, P.B. Singh, and T. Misteli. 2003. Science. 299:721-725; Festenstein, R., S.N. Pagakis, K. Hiragami, D. Lyon, A. Verreault, B. Sekkali, and D. Kioussis. 2003. Science. 299:719-721). Because the mechanism by which SUV39H1 is recruited to and interacts with heterochromatin is unknown, we studied the dynamic properties of SUV39H1 in living cells by using fluorescence recovery after photobleaching and fluorescence resonance energy transfer. Our results show that a substantial population of SUV39H1 is immobile at pericentromeric heterochromatin, suggesting that, in addition to its catalytic activity, SUV39H1 may also play a structural role at pericentromeric regions. Analysis of SUV39H1 deletion mutants indicated that the SET domain mediates this stable binding. Furthermore, our data suggest that the recruitment of SUV39H1 to heterochromatin is at least partly independent from that of HP1 and that HP1 transiently interacts with SUV39H1 at heterochromatin.
Assuntos
Heterocromatina/metabolismo , Metiltransferases/química , Metiltransferases/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Catálise/efeitos dos fármacos , Linhagem Celular Tumoral , Centrômero/metabolismo , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Metilação de DNA/efeitos dos fármacos , Recuperação de Fluorescência Após Fotodegradação , Transferência Ressonante de Energia de Fluorescência , Inibidores de Histona Desacetilases , Humanos , Ácidos Hidroxâmicos/farmacologia , Proteínas Luminescentes/metabolismo , Camundongos , Proteínas Mutantes/metabolismo , Células NIH 3T3 , Fenótipo , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Senescence associated-beta-galactosidase (SA-beta-gal) activity is a widely used marker for cellular senenescence. SA-beta-gal activity is routinely detected cytochemically, manually discriminating negative from positive cells. This method is time-consuming, subjective and therefore prone to operator-error. We aimed to optimize a flow cytometric method described by other workers using endothelial cells to better differentiate between populations of fibroblasts in degrees of SA-beta-gal activity. Skin fibroblasts were isolated from young (mean age +/- SD: 25.5 +/- 1.8) and very old (age 90.2 +/- 0.3) subjects. Different pH modulators were tested for toxicity. To induce stress-induced senescence, fibroblasts were exposed to rotenone. Senescence was assessed measuring SA-beta-gal activity by cytochemistry (X-gal) and by flow cytometry (C(12)FDG). The pH modulator Bafilomycin A1 (Baf A1) was found to be least toxic for fibroblasts and to differentiate best between nonstressed and stressed fibroblast populations. Under nonstressed conditions, fibroblasts from very old subjects showed higher SA-beta-gal activity than fibroblasts from young subjects. This difference was found for both the flow cytometric and cytochemical methods (P = 0.013 and P = 0.056 respectively). Under stress-induced conditions the flow cytometric method but not the cytochemical method revealed significant higher SA-beta-gal activity in fibroblasts from very old compared to young subjects (P = 0.004 and P = 0.635 respectively). We found the modified flow cytometric method measuring SA-beta-gal activity superior in discriminating between degrees of senescence in different populations of fibroblasts.
Assuntos
Fibroblastos/metabolismo , Citometria de Fluxo/métodos , beta-Galactosidase/metabolismo , Adulto , Fatores Etários , Idoso de 80 Anos ou mais , Envelhecimento , Senescência Celular , Inibidores Enzimáticos/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Imuno-Histoquímica/métodos , Lisossomos/metabolismo , Macrolídeos/farmacologia , Modelos Biológicos , Rotenona/farmacologia , Pele/metabolismoRESUMO
Many of the protein factors that play a role in nuclear export of mRNAs have been identified, but still little is known about how mRNAs are transported through the cell nucleus and which nuclear compartments are involved in mRNA transport. Using fluorescent 2'O-methyl oligoribonucleotide probes, we investigated the mobility of poly(A)+ RNA in the nucleoplasm and in nuclear speckles of U2OS cells. Quantitative analysis of diffusion using photobleaching techniques revealed that the majority of poly(A)+ RNA move throughout the nucleus, including in and out of speckles (also called SC-35 domains), which are enriched for splicing factors. Interestingly, in the presence of the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, the association of poly(A)+ RNA with speckles remained dynamic. Our results show that RNA movement is energy dependent and that the proportion of nuclear poly(A)+ RNA that resides in speckles is a dynamic population that transiently interacts with speckles independent of the transcriptional status of the cell. Rather than the poly(A)+ RNA within speckles serving a stable structural role, our findings support the suggestion of a more active role of these regions in nuclear RNA metabolism and/or transport.
Assuntos
Núcleo Celular/metabolismo , Corantes Fluorescentes/metabolismo , RNA Mensageiro/metabolismo , Transcrição Gênica , Transporte Ativo do Núcleo Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Desoxiadenosinas/metabolismo , Diclororribofuranosilbenzimidazol/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Humanos , Proteínas Nucleares/metabolismo , Inibidores da Síntese de Ácido Nucleico/metabolismo , Oligorribonucleotídeos/química , Oligorribonucleotídeos/metabolismo , Proteína II de Ligação a Poli(A)/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Rodaminas/química , Rodaminas/metabolismo , Fatores de Processamento de Serina-ArgininaRESUMO
The cell nucleus is highly organized with chromosomes occupying discrete, partially overlapping territories, and proteins that localize to specific nuclear compartments. This spatial organization of the nucleus is considered to be dynamic in response to environmental and cellular conditions to support changes in transcriptional programs. Chromatin, however, is relatively immobile when analyzed in living cells and shows a constrained Brownian type of movement. A possible explanation for this relative immobility is that chromatin interacts with a nuclear matrix structure and/or with nuclear compartments. Here, we explore the use of photoactivatable GFP fused to histone H4 as a potential tool to analyze the mobility of chromatin at various nuclear compartments. Selective photoactivation of photoactivatable-GFP at defined nuclear regions was achieved by two-photon excitation with 820 nm light. Nuclear speckles, which are considered storage sites of splicing factors, were visualized by coexpression of a fluorescent protein fused to splicing factor SF2/ASF. The results reveal a constrained chromatin motion, which is not affected by transcriptional inhibition, and suggests an intimate interaction of chromatin with speckles.
Assuntos
Cromatina/fisiologia , Proteínas de Fluorescência Verde/análise , Histonas/análise , Transporte Biológico/genética , Compartimento Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Histonas/metabolismo , Humanos , Espaço Intranuclear/fisiologia , Espaço Intranuclear/ultraestrutura , Microscopia ConfocalRESUMO
To better understand the impact of changes in nuclear architecture on nuclear functions, it is essential to quantitatively elucidate the three-dimensional organization of nuclear components using image processing tools. We have developed a novel image segmentation method, which involves a contrast enhancement and a subsequent thresholding step. In addition, we have developed a new segmentation method of the nuclear volume using the fluorescent background signal of a probe. After segmentation of the nucleus, a first-order normalization is performed on the signal positions of the component of interest to correct for the shape of the nucleus. This method allowed us to compare various signal positions within a single nucleus, and also on pooled data obtained from multiple nuclei, which may vary in size and shape. The algorithms have been tested by analyzing the spatial localization of nuclear bodies in relation to the nuclear center. Next, we used this new tool to study the change in the spatial distribution of nuclear components in cells before and after caspase-8 activation, which leads to cell death. Compared to the morphological TopHat method, this method gives similar but significantly faster results. A clear shift in the radial distribution of centromeres has been found, while the radial distribution of telomeres was changed much less. In addition, we have used this new tool to follow changes in the spatial distribution of two nuclear components in the same nucleus during activation of apoptosis. We show that after caspase-8 activation, when centromeres shift to a peripheral localization, the spatial distribution of PML-NBs does not change while that of centromeres did. We propose that the use of this new image segmentation method will contribute to a better understanding of the 3D spatial organization of the cell nucleus.
Assuntos
Núcleo Celular/ultraestrutura , Imageamento Tridimensional/métodos , Células-Tronco Mesenquimais/ultraestrutura , Adulto , Apoptose , Caspase 8/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Centrômero/ultraestrutura , Corantes Fluorescentes/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Microscopia Confocal , Proteínas Nucleares/metabolismo , Telômero/ultraestruturaRESUMO
Little is known on how well senescence markers in vitro and in situ correlate within individual donors. We studied correlations between the same and different in vitro markers. Furthermore, we tested correlations between in vitro markers with in situ p16INK4a positivity.From 100 donors (20-91 years), cultured dermal fibroblasts were assessed for reactive oxygen species (ROS), telomere-associated foci (TAF), p16INK4a and senescence-associated ß-gal (SAß-gal), with/ without 0.6 µM rotenone for 3 days (short-term). In fibroblasts from 40 donors, telomere shortening, ROS and SAß-gal were additionally assessed, with/ without 20 nM rotenone for 7 weeks (long-term). In skin from 52 donors, the number of p16INK4a positive dermal cells was assessed in situ.More than half of the correlations of the same senescence markers in vitro between duplicate experiments and between short-term versus long-term experiments were significant. Half of the different senescence marker correlations were significant within the short-term and within the long-term experiments. The different senescence markers in vitro were not significantly correlated intra-individually with in situ p16INK4a positivity.In conclusion, the same and different senescence markers are frequently correlated significantly within and between in vitro experiments, but in vitro senescence markers are not correlated with p16INK4a positivity in situ.
Assuntos
Biomarcadores/metabolismo , Senescência Celular/genética , Genes p16/fisiologia , Telômero/genética , beta-Galactosidase/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/análise , Células Cultivadas , Senescência Celular/fisiologia , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Telômero/metabolismo , Adulto Jovem , beta-Galactosidase/análiseRESUMO
A combinatorial library of 1336 fluorescent styryl molecules was synthesized aiming to select dyes that are photostable, non-toxic, and specific for RNA molecules in living cells . These dyes are potentially important to the study of gene expression in live cells.
Assuntos
Sondas RNA/química , Sondas RNA/genética , RNA/genética , Animais , Corantes Fluorescentes/químicaRESUMO
Nucleic acids are typically detected in morphologically preserved fixed cells and tissues using in situ hybridization techniques. This review discusses a variety of established and more challenging fluorescence-based methods for the detection and tracking of DNA or RNA sequences in living cells. Over the past few years, various fluorescent in vivo labeling methods have been developed, and dedicated microscope and image analysis tools have been designed. These advances in technologies indicate that live-cell imaging of nucleic acids is likely to become a standard research tool for understanding genome organization and gene expression regulation in the near future. Recent live-cell imaging studies have already provided important insights into the dynamic behaviors of chromatin and RNAs in the cell.
Assuntos
Fenômenos Fisiológicos Celulares , DNA/metabolismo , Hibridização in Situ Fluorescente/métodos , Hibridização in Situ Fluorescente/tendências , Microscopia de Fluorescência/métodos , Microscopia de Fluorescência/tendências , RNA/metabolismo , Transporte Biológico , DNA/análise , Sondas de DNA , Corantes Fluorescentes , RNA/análise , Sondas RNARESUMO
Knowledge of how molecules interact in space and time is crucial for understanding cellular processes. A host of novel techniques have been developed for the visualisation of single target molecules in living cells, many based on fluorescence in situ hybridisation (FISH) or immunocytochemistry (IC). To extend the applicability of FISH to living cells, special backbone-modified probes and specific conformations (molecular beacons) have been designed. In the case of IC, conventional immunoreagents have been fine-tuned with respect to size and affinity or replaced with new protein scaffolds based on ankyrin repeat proteins. Other key advances include the use of proximity ligation to confirm vicinity binding and the use of quantum dots, which have proven potential for cellular labelling.
Assuntos
DNA/genética , DNA/metabolismo , Perfilação da Expressão Gênica/métodos , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Mapeamento de Interação de Proteínas/métodos , Análise de Sequência de DNA/métodos , Fenômenos Fisiológicos Celulares , DNA/análise , Sistemas de Liberação de Medicamentos/métodos , Biologia Molecular/métodosRESUMO
Expression of the cyclin-dependent kinase inhibitor gene p21 is induced after DNA damage and plays a role in cell survival. The exact mechanism of induction is not known, but enhancement of mRNA stability has recently been implicated as an important factor. To obtain further insight into the dynamics of p21 gene expression at the individual cell level, normal fibroblasts, GM1492 fibroblasts from a Bloom's syndrome patient, and U2OS osteosarcoma cells were UVC irradiated, fixed at different time points, and subjected to mRNA fluorescence in situ hybridization (FISH) and immunocytochemical staining. In mock-irradiated normal fibroblasts, a subfraction of cells revealed low levels of p21 mRNA synthesis. After UVC treatment, p21 transcripts accumulated over time in nuclear locations other than transcription foci. At 6 hr after irradiation, almost 50% of the cells displayed p21 mRNA in three different distribution patterns within the nuclei. The highest frequency of cells with cytoplasmic accumulation of p21 mRNA was seen at 17 hr after UVC treatment. We conclude that increased p21 gene transcription and possibly stabilization of newly synthesized p21 mRNA contribute to elevated levels of p21 protein after UVC irradiation. (J Histochem Cytochem 50:81-89, 2002)
Assuntos
Ciclinas/metabolismo , RNA Mensageiro/metabolismo , Raios Ultravioleta , Linhagem Celular , Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Citoplasma/metabolismo , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Humanos , Hibridização in Situ Fluorescente , Cinética , Microscopia de Fluorescência , RNA Mensageiro/biossíntese , Células Tumorais CultivadasRESUMO
In some myopathies, hypoxia can be the result of pathologic effects like muscle necrosis and abnormal blood flow. At the molecular level, the consequence of hypoxic conditions is not yet fully understood. Under stress conditions, many housekeeping gene mRNAs are translationally silenced, while translation of other mRNAs increases. Alterations to the pool of mRNAs available for translation lead to the formation of so-called stress granules containing both mRNAs and proteins. Stress granule formation and dynamics have been investigated using cells in culture, but have not yet been examined in vivo. In Drosophila embryonic muscles, we found that hypoxia induces the formation of sarcoplasmic granules containing the established stress granule markers RIN and dFMR1. Upon restoration of normoxia, the observed granules were decreased in size, indicating that their formation might be reversible. Employing photobleaching approaches, we found that a cytoplasmic reporter mRNA rapidly shuttles in and out of the granules. Hence, stress granules are highly dynamic complexes and not simple temporary storage sites. Although mRNA rapidly cycles through the granules, its movement throughout the muscle is, remarkably, spatially restricted by the presence of yet undefined myofiber domains. Our results suggest that in hypoxic muscles mRNA remains highly mobile; however, its movement throughout the muscle is restricted by certain boundaries. The development of this Drosophila hypoxia model makes it possible to study the formation and dynamics of stress granules and their associated mRNAs and proteins in a living organism.
Assuntos
Grânulos Citoplasmáticos/genética , Drosophila/genética , Embrião não Mamífero/metabolismo , Hipóxia/genética , Músculo Esquelético/embriologia , RNA Mensageiro/genética , Animais , Grânulos Citoplasmáticos/metabolismo , Drosophila/metabolismo , Hipóxia/metabolismo , Músculo Esquelético/metabolismo , RNA Mensageiro/metabolismoRESUMO
Respiratory chain function becomes less efficient with age resulting in increased levels of damaging reactive oxygen species. We compared rotenone-exposed fibroblast strains from young and old subjects and from offspring of nonagenarian siblings and the partners of the offspring. Rotenone increased reactive oxygen species levels, inhibited growth rate, and increased telomere shortening (all p < .05). Non-stressed strains from young subjects showed lower reactive oxygen species levels (p = .031) and higher growth rates (p = .002) than strains from old subjects. Stressed strains from young subjects showed smaller increases in reactive oxygen species levels (p = .014) and larger decreases in growth rate (p < .001) than strains from old subjects. Telomere-shortening rates were not different between groups. Stress-induced decreases in growth rate were larger in strains from offspring than from partners (p = .05). Strains from young and old subjects are differentially affected by chronic inhibition of the respiratory chain. Changed growth rates in strains from offspring resemble those from strains from young subjects.
Assuntos
Envelhecimento/fisiologia , Transporte de Elétrons/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Rotenona/farmacologia , Fatores Etários , Idoso de 80 Anos ou mais , Biópsia , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Modelos Lineares , Masculino , Países Baixos , Espécies Reativas de Oxigênio/metabolismo , Encurtamento do Telômero , beta-Galactosidase/metabolismoRESUMO
The offspring of nonagenarian siblings suffer less from age related conditions and have a lower risk of mortality compared to their partners. Fibroblast strains derived from such offspring in middle age show different in vitro responses to stress, more stress-induced apoptosis and less senescence when compared to strains of their partners. Aiming to find differences in cellular metabolism in vitro between these fibroblast strains, cell culture supernatants collected at 24 hours and five days were analysed using (1)H nuclear magnetic resonance (NMR)-based metabolic footprinting. Between 24 hours and five days of incubation, supernatants of all fibroblast strains showed decreased levels of glucose, pyruvate, alanine-glutamine (ala-gln), valine, leucine, isoleucine, serine and lysine and increased levels of glutamine, alanine, lactate and pyroglutamic acid. Strains from offspring and their partners were compared using a partial least squares-discriminant analysis (PLS-DA) model based on the data of the five-day time point. The ala-gln and glucose consumption were higher for fibroblast strains derived from offspring when compared to strains of their partners. Also, production of glutamine, alanine, lactate and pyroglutamic acid was found to be higher for fibroblast strains derived from offspring. In conclusion, differences in NMR-based metabolic profiles of human cells in vitro reflect the propensity for human longevity of the subjects from whom these were derived.
Assuntos
Longevidade/fisiologia , Envelhecimento/metabolismo , Técnicas de Cultura de Células , Feminino , Glucose/metabolismo , Humanos , Lactatos/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Masculino , Metaboloma/fisiologia , Pessoa de Meia-Idade , Piruvatos/metabolismo , IrmãosRESUMO
Senescence is thought to play an important role in the progressive age-related decline in tissue integrity and concomitant diseases, but not much is known about the complex interplay between upstream regulators and downstream effectors. We profiled whole genome gene expression of non-stressed and rotenone-stressed human fibroblast strains from young and oldest old subjects, and measured senescence associated ß-gal activity. Microarray results identified gene sets involved in carbohydrate metabolism, Wnt/ß-catenin signaling, the cell cycle, glutamate signaling, RNA-processing and mitochondrial function as being differentially regulated with chronological age. The most significantly differentially regulated mRNA corresponded to the p16 gene. p16 was then investigated using qPCR, Western blotting and immunocytochemistry. In conclusion, we have identified cellular pathways that are differentially expressed between fibroblast strains from young and old subjects.
Assuntos
Envelhecimento/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/patologia , Células Cultivadas , Feminino , Fibroblastos/patologia , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de OligonucleotídeosRESUMO
The nuclear lamina is an intermediate filament network that provides a structural framework for the cell nucleus. Changes in lamina structure are found during changes in cell fate such as cell division or cell death and are associated with human diseases. An unbiased method that quantifies changes in lamina shape can provide information on cells undergoing changes in cellular functions. We have developed an image processing methodology that finds and quantifies the 3D structure of the nuclear lamina. We show that measurements on such images can be used for cell classification and provide information concerning protein spatial localization in this structure. To demonstrate the efficacy of this method, we compared the lamina of unmanipulated human mesenchymal stem cells (hMSCs) at passage 4 to cells activated for apoptosis. A statistically significant classification was found between the two populations.
RESUMO
Increased aggregation of misfolded proteins is associated with aging, and characterizes a number of neurodegenerative disorders caused by homopolymeric amino acid expansion mutations. PABPN1 is an aggregation-prone nuclear protein. Natural aggregation of wild-type (WT) PABPN1 is not known to be disease-associated, but alanine-expanded PABPN1 (expPABPN1) accumulates in insoluble intranuclear inclusions in muscle of patients with oculopharyngeal muscular dystrophy (OPMD). We applied microscopic image quantification to study PABPN1 aggregation process in living cells. We identified transitional pre-inclusion foci and demonstrate that these structures significantly differ between WT- and expPABPN1-expressing cells, while inclusions of these proteins are indistinguishable. In addition to the immobile PABPN1 in inclusions, in the nucleoplasm of expPABPN1 expressing cells we also found a fraction of immobile proteins, representing pre-aggregated species. We found that pre-aggregated and pre-inclusion structures are reverted by a PABPN1 specific affinity binder while inclusion structures are not. Together our results demonstrate that the aggregation process of WT- and expPABPN1 differs in steps preceding inclusion formation, suggesting that pre-aggregated protein species could represent the cytotoxic structures.
Assuntos
Corpos de Inclusão Intranuclear/metabolismo , Proteína II de Ligação a Poli(A)/química , Multimerização Proteica , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Corpos de Inclusão Intranuclear/genética , Mutação , Proteína II de Ligação a Poli(A)/genética , Proteína II de Ligação a Poli(A)/metabolismo , Estrutura Quaternária de Proteína , Fatores de TempoRESUMO
Cellular senescence, an important factor in ageing phenotypes, can be induced by replicative exhaustion or by stress. We investigated the relation between maximum replicative capacity, telomere length, stress-induced cellular senescence, and apoptosis/cell death in human primary fibroblast strains obtained from nonagenarians of the Leiden 85-plus Study. Fibroblast strains were cultured until replicative senescence and stressed with rotenone at low passage. Telomere length, senescence-associated-ß-galactosidase activity, sub-G1 content, and Annexin-V/PI positivity were measured in nonstressed and stressed conditions. Fibroblast strains with a higher replicative capacity had longer telomeres (p = .054). In nonstressed conditions, replicative capacity was not associated with ß-gal activity (p = .07) and negatively with sub-G1 (p = .008). In rotenone-stressed conditions, replicative capacity was negatively associated with ß-gal activity (p = .034) and positively with sub-G1 (p = .07). Summarizing, fibroblast strains with a higher maximum replicative capacity have longer telomeres, are less prone to go into stress-induced cellular senescence, and more prone to die after stress.
Assuntos
Senescência Celular , Fibroblastos/fisiologia , Estresse Oxidativo , Idoso , Idoso de 80 Anos ou mais , Apoptose , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Masculino , Pele/citologia , TelômeroRESUMO
The analysis of the spatial-dynamic properties of DNA and RNA molecules in living cells will greatly extend our knowledge of genome organization and gene expression regulation in the cell nucleus. The development of hybridization methods allowing detection of specific endogenous DNA and RNA sequences in living cells has therefore been a challenge for many years. However, there are many technical issues that have proven so far to be difficult, or even impossible, to overcome. As a result, in most situations, the application of in vivo hybridization methods is currently limited to the visualization of highly repetitive DNA sequences or abundant RNA species. We describe a protocol that enables the visualization and tracking of telomeres in living cells by hybridization with a fluorescent peptide nucleic acid (PNA) probe. Furthermore, we describe a method that allows the detection of abundant endogenous RNAs in living cells by microinjecting fluorescently labeled complementary 2'-O-methyl RNA probes.