RESUMO
Understanding nuclear architecture is indispensable for understanding the cell-type-dependent orchestration of active and silent genes and other nuclear functions, such as RNA splicing, DNA replication and repair. Yet, while it is now generally agreed that chromosomes in the cell nucleus are organized as chromosome territories, present models of chromosome territory architecture differ widely with respect to the possible functional implications of dynamic changes of this architecture during the cell cycle and terminal cell differentiation.
Assuntos
Estruturas Cromossômicas/genética , Modelos Genéticos , Animais , Montagem e Desmontagem da Cromatina , Humanos , Modelos Moleculares , Conformação MolecularRESUMO
The precise localization of transcribed DNA and resulting RNA is an important aspect of the functional architecture of the nucleus. To this end we have developed a novel in situ hybridization approach in combination with immunoelectron microscopy, using sense and anti-sense RNA probes that are derived from total cellular or cytoplasmic poly(A+) RNA. This new technology is much more gentle than classical in situ hybridization using DNA probes and shows excellent preservation of nuclear structure. Carried out on ultrathin sections of fixed and resin-embedded COS-7 cells, it revealed at high resolution the localization of the genes that code for the cellular mRNAs. Quantitative analysis shows that most transcribed DNA is concentrated in the perichromatin region, i.e. the interface between subchromosomal compact chromatin domains and the interchromatin space essentially devoid of DNA. The RNA that is produced is found mainly in the perichromatin region and the interchromatin space. These results imply that in the mammalian nucleus the chromatin fiber is folded so that active genes are predominantly present in the perichromatin region, which is the most prominent site of transcription.
Assuntos
Núcleo Celular/química , Cromatina/química , DNA/análise , Transcrição Gênica , Animais , Células COS , Chlorocebus aethiops , Hibridização In Situ/métodos , RNA/biossíntese , Sondas RNARESUMO
BACKGROUND INFORMATION: Sphingomyelin is one of the major phospholipids in the cell nucleus. However, its intranuclear distribution with regard to different functional nuclear domains as well as its possible involvement in the nuclear functional architecture remains to be elucidated. RESULTS: We carried out an ultrastructural cytochemical study of the intranuclear distribution of SM (sphingomyelin) using an in situ binding assay of neutral SMase (sphingomyelinase) conjugated to colloidal gold particles. The enzymatic labelling was carried out on ultrathin sections of different mammalian cells prepared by means of various fixation and resin-embedding protocols. Transmission electron microscopic analysis revealed preferential localization of SM within the PR (perichromatin region), a functionally important nucleoplasmic domain containing sites of pre-mRNA synthesis and processing. In the nucleolus, SM is mostly associated with the dense fibrillar component containing transcriptionally active ribosomal genes. Microinjection of enzymatically active SMase into living cells resulted in a rapid degradation of intranuclear structure. CONCLUSIONS: Our observations, supported by biochemical data, provide evidence for the involvement of SM in important nuclear functions. They bring additional information pointing out the PR as an essential functional nuclear domain. Furthermore, they suggest a role for SM in the internal nuclear architecture.
Assuntos
Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Cromatina/metabolismo , Cromatina/ultraestrutura , Esfingomielinas/metabolismo , Animais , Camundongos , Microscopia Eletrônica de Transmissão , Ratos , Transcrição GênicaRESUMO
The nuclear architecture is considered an important contributor to genome function. Although the fine structural features of the cell nucleus have been investigated extensively by means of ultrastructural cytochemistry, mainly on ultrathin sections in two dimensions (2D), there was a of lack routine methods for a rapid reconstruction of three-dimensional (3D) distribution of different structural constituents throughout the nuclear volume. We have now filled this gap by the application of a novel approach associating a pre-embedding selective visualization of nuclear components with a method making use of ultramicrotomy combined with scanning electron microscopy (microtome serial block face scanning electron microscopy--'3View'). We have been able to apply this method to the study of DNA distribution within the nuclear volume and reconstruction of 3D chromatin arrangement in nuclei of rat hepatocytes and endothelial cells. Our observations demonstrate that while chromatin appears to occupy the interior of nuclei rather sparsely on 2D images, once reconstructed in 3D from a series of sequential 2D images it gives the impression of considerably filling the nuclear volume. However, quantitative evaluation of the nuclear volume occupied by DNA in the above two types of nuclei leaves a significant part to the interchromatin space (66.2% for hepatic cells and 41.7% for endothelial cells, including nuclear space occupied by nucleoli). Detailed analysis of the reconstructed nuclei reveals a high degree of superposition of chromatin domains, giving rise to a false impression that they fill a much larger part of the nuclear volume than they really do. Our results show the importance of the contribution of such reconstruction techniques to our understanding of the nuclear architecture.
Assuntos
Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , Células Endoteliais/ultraestrutura , Hepatócitos/ultraestrutura , Microscopia Eletrônica de Transmissão , Animais , RatosRESUMO
We have developed a novel approach for in situ labeling and detection of nucleic acids in cultured cells. It is based on in vivo incorporation of chlorouridine (ClU) or iododeoxyuridine (IdU) into Chinese hamster ovary cells with the aim of labeling RNA and DNA, respectively. The halogenated nucleotides are immunolabeled on ultrathin sections with anti-bromodeoxyuridine (BrdU) monoclonal antibodies that specifically react with either IdU or ClU. Furthermore, we combined ClU and IdU incubation to label simultaneously RNA and DNA in the same cell. Both were visualized by means of anti-BrdU antibodies exhibiting strong affinity for one of the two halogenated epitopes. Confocal imaging of interphase nuclei and electron microscopic analysis showed evidence of a partial colocalization of newly synthesized DNA and RNA inside the cell nucleus. RNase and DNase digestion of ultrathin sections after formaldehyde fixation and acrylic resin embedding confirmed the specificity of incorporation. Consequently, this method allows us to differentially label DNA and RNA on the same section. Using short pulses with the precursors, we could show that newly synthesized DNA and RNA both preferentially occur within the perichromatin region at the border of condensed chromatin domains.
Assuntos
DNA/análise , Desoxiuridina/análogos & derivados , Idoxuridina , RNA/análise , Animais , Anticorpos , Bromodesoxiuridina/imunologia , Células CHO , Cricetinae , Cricetulus , Reações Cruzadas , Microscopia de Fluorescência/métodos , Microscopia Imunoeletrônica/métodosRESUMO
Ribonucleoprotein (RNP) containing structural constituents in hepatocyte nuclei of adult, old and adult, vitamin E-deficient rats were investigated to assess the effect of aging and increased oxidative stress on nuclear functions. Fibrillar centres (FCs), dense fibrillar (DFC) and granular (GC) components of nucleoli as well as perichromatin granules (PGs) in the nucleoplasm were preferentially evidenced by the ethylenediaminetetracetic acid (EDTA) method and measured by computer-assisted morphometric procedures. FCs size and the percentage of nucleolar surface occupied by FCs significantly decreased during aging and vitamin E-deficiency. The percentage of nucleolar surface occupied by GC and DFC remained unchanged in adult and old rats, but in vitamin E-deficient animals GC increased and DFC decreased significantly. PG density significantly changed in aging and vitamin E-deficiency. Functionally, FCs, DFC and GC constitute sites of transcription and processing of ribosomal RNA while PGs are involved in intranuclear storage and transport of messenger RNA. Thus, the present structural changes during aging and vitamin E-deficiency correlate with a decay of nuclear responsiveness to cellular metabolic needs. Considering the antioxidant action of alpha-tocopherol, our data lend further support to the importance of free radical production and control in the aging process.
Assuntos
Envelhecimento/genética , Envelhecimento/metabolismo , RNA/metabolismo , Deficiência de Vitamina E/genética , Deficiência de Vitamina E/metabolismo , Animais , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Nucléolo Celular/ultraestrutura , Feminino , Hepatócitos/ultraestrutura , Microscopia Eletrônica , Estresse Oxidativo , RNA/ultraestrutura , Ratos , Ratos WistarRESUMO
Fibrillar centers (FCs), dense fibrillar (DFC) and granular (GC) components in nucleoli, and perichromatin granules (PGs) in nucleoplasm were measured by morphometry. FC size and their nucleolar surface fraction significantly decreased in aging and vitamin E deficiency. The GC and DFC nucleolar fraction was unchanged in adult and old rats, but in vitamin E-deficient animals GC increased and DFC decreased significantly. PG density significantly increased in aging and decreased in vitamin E deficiency. The quantitative evaluation of immunolabeled transcription and splicing factors revealed that polymerase II and SC-35 significantly decreased in old and vitamin E-deficient versus adult animals. Fibrillarin and snRNPs did not change between adult and old rats, but were significantly lower in vitamin E-deficient rats. These data document altered RNA pathways in aging and vitamin E deficiency. Considering the antioxidant role of vitamin E, they lend further support to the importance of free radical production and control in the aging process.
Assuntos
Envelhecimento , RNA/metabolismo , Deficiência de Vitamina E/patologia , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Feminino , Hepatócitos/metabolismo , Imuno-Histoquímica , Splicing de RNA , RNA Ribossômico/metabolismo , Ratos , Ratos Wistar , Fatores de Tempo , Transcrição GênicaRESUMO
In this review we describe major contributions of light and electron microscopic approaches to the present understanding of functional nuclear architecture. The large gap of knowledge, which must still be bridged from the molecular level to the level of higher order structure, is emphasized by differences of currently discussed models of nuclear architecture. Molecular biological tools represent new means for the multicolor visualization of various nuclear components in living cells. New achievements offer the possibility to surpass the resolution limit of conventional light microscopy down to the nanometer scale and require improved bioinformatics tools able to handle the analysis of large amounts of data. In combination with the much higher resolution of electron microscopic methods, including ultrastructural cytochemistry, correlative microscopy of the same cells in their living and fixed state is the approach of choice to combine the advantages of different techniques. This will make possible future analyses of cell type- and species-specific differences of nuclear architecture in more detail and to put different models to critical tests.
Assuntos
Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Microscopia/tendências , Animais , Núcleo Celular/química , Humanos , Modelos MolecularesRESUMO
Nuclear architecture has been investigated intensively by various electron microscopy (EM) methods. Most of these require chemical fixation of the sample, although cryofixation has also been used in combination with cryosubstitution and resin embedding. This approach allowed one to considerably increase the knowledge about the structural features of different nuclear domains and their involvement in nuclear functions. Cryoelectron microscopy of vitreous sections (CEMOVIS) has added a new dimension to the ultrastructural analysis of the cell nucleus, especially thanks to the possibility of observing the specimen in its hydrated state. In this way one can analyse, at high resolution, cellular structures as close as possible to their native state. In this chapter we describe in detail the different steps of the CEMOVIS method, which should allow an electron microscopist to perform cryosectioning and cryoelectron microscopy of vitrified biological material.
Assuntos
Núcleo Celular/ultraestrutura , Microscopia Crioeletrônica/métodos , Animais , Linhagem Celular Tumoral , Crioultramicrotomia , RatosRESUMO
Visualisation of RNA at an ultrastructural level represents a major approach to study organisation and function of the cell nucleus. In addition to methods allowing one to visualise a general distribution of RNA-containing structural constituents, in situ hybridisation (ISH) is a powerful tool for revealing specific RNA sequences or species. In this chapter we describe a method for detecting RNA by electron microscopic in situ hybridisation (EMISH) using anti-sense RNAs as probes. We first present the protocol for preparation of anti-sense RNA probes labeled with different markers, and then describe how such probes are applied to ultrathin sections by a method of ultrastructural ISH. The great advantage of this method is that it does not require denaturing either the specimen or the probe, thus allowing nuclear fine structure to be well preserved. The presence of the marker in the probe can be detected by immunoelectron microscopy using colloidal gold-conjugated antibodies, offering the possibility to evaluate the signal quantitatively. The method can also be combined with cytochemical techniques such as EDTA staining for preferential visualisation of ribonucleoprotein-containing nuclear structural components.
Assuntos
Hibridização In Situ , Microscopia Eletrônica/métodos , RNA/química , RNA/ultraestrutura , Animais , Ácido Edético/química , HumanosRESUMO
Nucleotide excision repair (NER) is an evolutionary conserved DNA repair system that is essential for the removal of UV-induced DNA damage. In this study we investigated how NER is compartmentalized in the interphase nucleus of human cells at the ultrastructural level by using electron microscopy in combination with immunogold labeling. We analyzed the role of two nuclear compartments: condensed chromatin domains and the perichromatin region. The latter contains transcriptionally active and partly decondensed chromatin at the surface of condensed chromatin domains. We studied the distribution of the damage-recognition protein XPC and of XPA, which is a central component of the chromatin-associated NER complex. Both XPC and XPA rapidly accumulate in the perichromatin region after UV irradiation, whereas only XPC is also moderately enriched in condensed chromatin domains. These observations suggest that DNA damage is detected by XPC throughout condensed chromatin domains, whereas DNA-repair complexes seem preferentially assembled in the perichromatin region. We propose that UV-damaged DNA inside condensed chromatin domains is relocated to the perichromatin region, similar to what has been shown for DNA replication. In support of this, we provide evidence that UV-damaged chromatin domains undergo expansion, which might facilitate the translocation process. Our results offer novel insight into the dynamic spatial organization of DNA repair in the human cell nucleus.
Assuntos
Núcleo Celular/metabolismo , Dano ao DNA , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , DNA , Linhagem Celular , DNA/metabolismo , DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismoRESUMO
The perichromatin region has emerged as an important functional domain of the interphase nucleus. Major nuclear functions, such as DNA replication and transcription, as well as different RNA processing factors, occur within this domain. In this review, we summarize in situ observations regarding chromatin structure analysed by transmission electron microscopy and compare results to data obtained by other methods. In particular, we address the functional architecture of the perichromatin region and the way chromatin may be folded within this nucleoplasmic domain.
Assuntos
Compartimento Celular/fisiologia , Núcleo Celular/química , Núcleo Celular/fisiologia , Cromatina/química , Animais , Núcleo Celular/ultraestrutura , Cromossomos/química , Cromossomos/ultraestrutura , Humanos , Microscopia Eletrônica/métodos , Dobramento de Proteína , RNA/síntese química , RNA/químicaRESUMO
In spite of strong evidence that the nucleus is a highly organized organelle, a consensus on basic principles of the global nuclear architecture has not so far been achieved. The chromosome territory-interchromatin compartment (CT-IC) model postulates an IC which expands between chromatin domains both in the interior and the periphery of CT. Other models, however, dispute the existence of the IC and claim that numerous chromatin loops expand between and within CTs. The present study was undertaken to resolve these conflicting views. (1) We demonstrate that most chromatin exists in the form of higher-order chromatin domains with a compaction level at least 10 times above the level of extended 30 nm chromatin fibers. A similar compaction level was obtained in a detailed analysis of a particularly gene-dense chromosome region on HSA 11, which often expanded from its CT as a finger-like chromatin protrusion. (2) We further applied an approach which allows the experimental manipulation of both chromatin condensation and the width of IC channels in a fully reversible manner. These experiments, together with electron microscopic observations, demonstrate the existence of the IC as a dynamic, structurally distinct nuclear compartment, which is functionally linked with the chromatin compartment.
Assuntos
Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , Animais , Células CHO , Permeabilidade da Membrana Celular , Cromossomos/ultraestrutura , Cricetinae , DNA/biossíntese , Células HeLa , Humanos , Microscopia Eletrônica de Transmissão , Modelos Genéticos , RNA/biossíntese , RNA Polimerase II/metabolismoRESUMO
BACKGROUND INFORMATION: NPY (neuropeptide Y) may have an effect on the properties of vascular endothelial cells such as pro-angiogenic effects and potentiation of noradrenaline-induced vasoconstriction. In HUVEC (human umbilical-vein endothelial cells), immunoreactive neuropeptide Y has been detected, but NPY synthesis, storage and secretion have not been studied. The aim of the present study was to establish NPY expression, storage and cellular transducing effects in HUVEC. RESULTS: HUVEC contain 0.19 fmol of NPY/microg of protein and 0.46 fmol of pro-NPY/microg of protein, as measured by ELISA. RT (reverse transcriptase)-PCR confirmed the expression of NPY in HUVEC. Immunofluorescence revealed the presence of NPY in small punctate structures, with a fluorescence pattern different from that observed for von Willebrand factor, indicating distinct storage compartments. Double labelling for NPY and Rab3A demonstrated similar granular patterns, with at least partial co-localization. Electron microscopy showed NPY immunoreactivity in vesicle-like cytoplasmic structures, of a fine fibrillar texture, as well as in mitochondria and in the nucleus. A similar general distribution pattern was also obtained for Rab3A. Y1 and Y2 receptors were expressed in HUVEC as assessed by RT-PCR, and they were functional since NPY induced a 42 nM intracellular calcium increase within 100 s, representing 22% of the histamine-induced response. In contrast with histamine, NPY did not induce acute von Willebrand factor secretion. CONCLUSIONS: HUVEC produce, store and respond to NPY, suggesting an autocrine regulatory role for NPY in the endothelium.
Assuntos
Endotélio Vascular/metabolismo , Neuropeptídeo Y/biossíntese , Veias Umbilicais/citologia , Cálcio/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Meios de Cultura Livres de Soro/farmacologia , AMP Cíclico/metabolismo , Ensaio de Imunoadsorção Enzimática , Coloide de Ouro/farmacologia , Histamina/metabolismo , Humanos , Imuno-Histoquímica , Cinética , Microscopia Eletrônica , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Mitocôndrias/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteína rab3A de Ligação ao GTP/metabolismo , Fator de von Willebrand/metabolismoRESUMO
Ultrastructural cytochemistry has been, for many years now, a major tool for investigating structure-function relationships in the cell nucleus. It has been essential in approaching the roles which different nuclear structural constituents can play in nuclear functions. This article briefly summarises transmission electron microscopic studies aimed at characterising in situ nuclear architectural domains and their involvement in main nuclear functions, such as DNA replication, hnRNA transcription and pre-mRNA processing. It discusses the importance of ultrastructural cytochemistry in high resolution analyses of intranuclear distribution of chromatin domains and their topological relationships with other structural interphase nuclear constituents. It puts forward the central role of the perichromatin region as a functional nuclear domain. Finally, it attempts to critically evaluate some future applications of ultrastructural investigations of the nucleus and stresses the importance of combining them with light microscopic analyses of living cells.
Assuntos
Núcleo Celular/ultraestrutura , Histocitoquímica , Animais , Núcleo Celular/genética , Núcleo Celular/fisiologia , Cromatina/genética , Cromatina/fisiologia , Cromatina/ultraestrutura , Corpos Enovelados/metabolismo , Corpos Enovelados/ultraestrutura , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Replicação do DNA/fisiologia , Humanos , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
The model of in situ DNA replication provided by immunofluorescence and confocal imaging is compared with observations obtained by electron microscopic studies. Discrepancies between both types of observations call into question the replication focus as a persistent nuclear structure and as a replication entity where DNA replication takes place. Most electron microscopic analyses reveal that replication sites are confined to dispersed chromatin areas at the periphery of condensed chromatin, and the distribution of replication factors exhibits the same localization pattern. Moreover, rapid migration of newly synthesized DNA from the replication sites towards the interior of condensed chromatin regions obviously takes place during S-phase. It implies modifications of replication domains, hardly detectable by fluorescence microscopy. The confrontation of different observations carried out at light microscopic or electron microscopic levels of resolution lead to a conclusion that a combination of in vivo fluorescence analysis with a subsequent ultrastructural investigation performed on the same cells will represent an optimal approach in future studies of nuclear functions in situ.
Assuntos
Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , Replicação do DNA , Replicon/genética , Fase S/fisiologia , Animais , Núcleo Celular/genética , Cromatina/genética , Humanos , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Replicon/fisiologiaRESUMO
The cell nucleus is a highly compartmentalized structure. In this review we describe controversial views on higher order chromatin organization from the level of higher order chromatin domains built up from folded chromatin fibers to the level of chromosome territories and the interchromatin compartment (IC), which harbors non-chromatin nuclear domains, such as interchromatin granule clusters (IGCs visualized in the electron microscope) or splicing factor-containing speckles (visualized by fluorescence microscopy). Emphasis is laid on the definition and functional importance of a nuclear compartment located at the periphery of chromatin domains in direct contact with the IC, termed the perichromatin region (PR). Ongoing experiments to elucidate the topological relationships between PR and IC have provided new insights into the functional interplay between transcription and splicing. As an example, we discuss the structure and nuclear topology of perichromatin fibrils (FPs) contained in the PR and their functional interplay with IGCs/speckles. In addition we discuss the advantages and drawbacks of experimental approaches currently used to study nuclear architecture and function in fixed and living cells.
Assuntos
Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , Animais , Transporte Biológico , Núcleo Celular/fisiologia , Cromatina/fisiologia , Posicionamento Cromossômico/genética , Posicionamento Cromossômico/fisiologia , Cromossomos/fisiologia , Cromossomos/ultraestrutura , Replicação do DNA/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Hibridização in Situ Fluorescente/métodos , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Fluorescência/métodos , Splicing de RNA/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica/genéticaRESUMO
Human Polycomb group (PcG) proteins are involved in cell-type-dependent epigenetic gene silencing in an evolutionarily conserved manner. We have analysed the subnuclear localisation of these regulatory proteins in two different human cell lines and in rat liver tissue by means of light and electron immunomicroscopy using specific antibodies. We find that the PcG proteins HPC2, HPH1, BMI1 and RING1 are highly concentrated in the perichromatin compartment, situated at the surface of condensed chromatin domains. This compartment was demonstrated earlier to be the nuclear site where most pre-mRNA synthesis takes place. Interestingly, these PcG proteins are virtually absent from the interior of condensed chromatin areas. The present observations therefore show that transcriptionally active and PcG-silenced loci occur within the same spatially limited nuclear domain. Our novel high-resolution data strongly support the idea that epigenetic PcG-mediated gene silencing is a local event, rather than affecting large chromatin domains. In addition to being associated with the perichromatin region, PcG proteins also occur in the interchromatin space. Implications of these observations for higher order chromatin structure and for the mechanisms of PcG-mediated gene silencing are discussed.
Assuntos
Proteínas de Transporte , Compartimento Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , Células Eucarióticas/metabolismo , Inativação Gênica/fisiologia , Proteínas Repressoras/metabolismo , Animais , Diferenciação Celular/genética , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Cromatina/genética , Cromatina/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Eucarióticas/ultraestrutura , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Ligases , Microscopia Eletrônica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complexo Repressor Polycomb 1 , Proteínas do Grupo Polycomb , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Proteínas Repressoras/genética , Transcrição Gênica/genética , Células Tumorais Cultivadas , Ubiquitina-Proteína LigasesRESUMO
3D-FISH has become a major tool for studying the higher order chromatin organization in the cell nucleus. It is not clear, however, to what extent chromatin arrangement in the nucleus after fixation and 3D-FISH still reflects the order in living cells. To study this question, we compared higher order chromatin arrangements in living cells with those found after the 3D-FISH procedure. For in vivo studies we employed replication labeling of DNA with Cy3-conjugated nucleotides and/or chromatin labeling by GFP-tagged histone 2B. At the light microscope level, we compared the intranuclear distribution of H2B-GFP-tagged chromatin and the positions of replication-labeled chromatin domains in the same individual cells in vivo, after fixation with 4% paraformaldehyde, and after 3D-FISH. Light microscope data demonstrate a high degree of preservation of the spatial arrangement of approximately 1-Mb chromatin domains. Subsequent electron microscope investigations of chromatin structure showed strong alterations in the ultrastructure of the nucleus caused mainly by the heat denaturation step. Through this step chromatin acquires the appearance of a net with mesh size of 50-200 nm roughly corresponding to the average displacement of the chromatin domains observed at light microscope level. We conclude that 3D-FISH is a useful tool to study chromosome territory structure and arrangements down to the level of approximately 1-Mb chromatin domain positions. However, important ultrastructural details of the chromatin architecture are destroyed by the heat denaturation step, thus putting a limit to the usefulness of 3D-FISH analyses at nanometer scales.
Assuntos
Cromatina/ultraestrutura , Hibridização in Situ Fluorescente/métodos , Carbocianinas/química , Núcleo Celular/química , Núcleo Celular/ultraestrutura , Células Cultivadas , Replicação do DNA , Nucleotídeos de Desoxiuracil/química , Fibroblastos/ultraestrutura , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde , Células HeLa , Histonas/análise , Histonas/genética , Humanos , Imageamento Tridimensional/métodos , Indicadores e Reagentes/análise , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Microscopia Confocal , Proteínas Recombinantes de Fusão/análise , Células Tumorais CultivadasRESUMO
To define the human immunodeficiency virus type 1 (HIV-1) RNA maturation pathways, we analyzed the intracellular distribution of HIV-1 RNA and the viral regulatory proteins Rev and Tat in transfected COS cells and HIV-1-infected lymphoid C8166 cells by means of ultrastructural in situ hybridization using antisense RNA probes and immunoelectron microscopy. The intranuclear viral RNA occurs in ribonucleoprotein fibrils in the perichromatin and interchromatin regions. The simultaneous demonstration of Rev, Tat, Br-labeled RNA, and cellular proteins SC35 and CRM1 in such fibrils reveals the potential of Rev to associate with nascent HIV pre-mRNA and its splicing complex and transport machinery. In a rev-minus system, the env intron-containing, incompletely spliced viral RNAs are revealed only in the nucleus, indicating that Rev is required to initiate the transport to the cytoplasm. Moreover, env intron sequences frequently occur in the periphery of interchromatin granule clusters, while the probe containing the rev exon sequence does not associate with this nucleoplasmic domain. When cells were treated with the CRM1 inhibitor leptomycin B in the presence of Rev protein, the env intron containing HIV RNAs formed clusters throughout the nucleoplasm and accumulated at the nuclear pores. This suggests that Rev is necessary and probably also sufficient for the accumulation of incompletely spliced HIV RNAs at the nuclear pores while CRM1 is needed for translocation across the nuclear pore complex.