Conserved patterns of nuclear compartmentalization are not observed in the chordate Oikopleura.

Background Information: Recent results from a limited number of eukaryotic model organisms suggest that major principles governing spatial organization of the genome in functionally distinct nuclear compartments are conserved through evolution.

Results: We examined the in situ spatial organization of major nuclear components and nuclear patterns of gene loci with strictly defined expression patterns in endocycling cells of the transparent urochordate Oikopleura dioica, a complex metazoan with a very compact genome. Endocycling cells with different functions and similar DNA content displayed distinct topologies of nuclear components.

Trend towards varying combinatorial centromere association in morphologically identical clusters in Purkinje neurons.

Neurons with similar morphology and neurotransmitter content located at a specific brain region may be part of the same or functionally separate networks. To address the question whether morphologically similar neurons have similar structural architecture at the chromosomal level, we studied Purkinje neurons in the cerebellum. Previous studies have shown that in Purkinje neurons centromeres of several chromosomes form clusters and that the number and size of these clusters remain stable in the adult brain.

Cell-type specific proximity of centromeric domains of one homologue each of chromosomes 2 and 11 in nuclei of cerebellar Purkinje neurons.

In Purkinje neurons of the mouse cerebellum, the centromeres of several chromosomes are placed in close proximity to form a distinct pattern of clusters and exhibit reproducible spatial redistributions during development. In granule neurons, an adjacent cell type in the cerebellum, the pattern, size, and number of centromeric aggregations are different from those of Purkinje neurons. The present work was undertaken to test the hypothesis that the same chromosomes form part of one aggregate in a cell-type-specific manner.

A survey of genetic diversity and reproductive biology of Puya raimondii (Bromeliaceae), the endangered queen of the Andes.

Puya raimondii Harms is an outstanding giant rosette bromeliad found solely around 4000 m above sea level in the Andes. It flowers at the end of an 80 - 100-year or even longer life cycle and yields an enormous (4 - 6 m tall) spike composed of from 15,000 to 20,000 flowers. It is endemic and currently endangered, with populations distributed from Peru to the north of Bolivia.

Organotypic slices in vitro: repeated, same-cell, high-resolution tracking of nuclear and cytoplasmic fluorescent signals in live, transfected cerebellar neurons by confocal microscopy.

The culture of organotypic slices for the purposes of tracking dynamic cellular events within the same live cell at high resolution, as a function of development in vitro has not been previously reported. The present study was undertaken to define the conditions most suitable for both the in vitro organotypic development of Purkinje neurons in cerebellar slices of neonatal mice, and the repeated visualization of nuclear signals within such cells. Slices of cerebella were maintained on 25 mm diameter, collagen-coated Anodisc membranes, placed in six-well plates and raised to the air-medium interface by use of glass fibre filter supports.

Intranuclear relocation of the Plc beta3 sequence in cerebellar purkinje neurons: temporal association with de novo expression during development.

Interphase nuclei exhibit a cell type-specific topology of chromatin domains. This topology has been proposed to be established at a specific developmental stage and to be associated, in turn, with cell type-specific gene expression. Using murine, cerebellar Purkinje neurons, we have shown previously that the number and the extent of clustering as well as the spatial, intranuclear distribution of centromeric domains change as a function of postnatal development.

Association of DNAse sensitive chromatin domains with the nuclear periphery in 3T3 cells in vitro.

DNAse sensitive chromatin, putative transcriptionally competent sequences, exists either as pan-nuclear speckles in cells with nuclei which exhibit a flat geometry, or as a shell apposed to the nuclear envelope in cells with spheroidal nuclei. To test the hypothesis that DNAse sensitive chromatin is similarly associated with the nuclear periphery in cell types with a very flat geometry such as 3T3 fibroblasts, cells were subjected to hypotonic expansion to change their nuclei from a flat ellipsoid to a spheriod. This was based on the assumption that such a spatial association is not resolvable due to the interdigitation at the nuclear midplane of DNAse sensitive chromatin associated with the upper and lower nuclear surfaces.

Nuclear topology of murine, cerebellar Purkinje neurons: changes as a function of development.

The interphase nucleus is a structurally ordered, three-dimensional structure, in which specific chromatin domains occupy distinct spatial positions that can, in turn, be modified with changes in cell function. A fundamental goal in developmental neurobiology is the identification of mechanisms that dictate the orderly expression of genes in a cell-specific manner. Given that different neuronal populations feature a characteristic spatial topology of centromeric sequences, the positioning of specific DNA sequences may constitute such a mechanism.

Dynamics of structure-function relationships in interphase nuclei.

The interphase nucleus is a topologically ordered, three-dimensional structure. While it remains unclear whether this structural organization also represents compartmentalization of function, the presence of the latter would likely be reflected in the spatial coupling of molecular factors involved in related events. This review summarizes morphological evidence, derived from in situ experiments, which indicates the existence of compartmentalization of both chromatin and non-chromatin components in the interphase nucleus.

Kindling-induced neurogenesis in the dentate gyrus of the rat.

Kindling, a form of neuronal plasticity produced by repeated low intensity electrical brain stimulation, leads to epileptic seizures. To address possible causes of this phenomenon, we have prepared amygdala-kindled animals and measured neurogenesis, by bromodeoxyuridine incorporation. Early, when focal seizures were present, there was no evidence of a change in the rate of hippocampal neurogenesis.

A specific conformation of the territory of chromosome 17 locates ERBB-2 sequences to a DNase-hypersensitive domain at the nuclear periphery.

Chromatin in interphase nuclei exhibits a topology that is associated with the transcriptional state of cells. We examined the spatial, intranuclear distribution of chromosome 17 and the ERBB-2 (HER2/neu) sequence thereon, relative to that of DNase-hypersensitive chromatin (DHC), in breast tumour cells exhibiting different levels of expression of ERBB-2. These sequences were specifically associated with the nuclear periphery, within a band of DHC.

Changes in morphology and spatial position of coiled bodies during NGF-induced neuronal differentiation of PC12 cells.

Interphase nuclei are organized into structural and functional domains. The coiled body, a nuclear organelle of unknown function, exhibits cell type-specific changes in number and morphology. Its association with nucleoli and with small nuclear ribonucleo-proteins (snRNPs) indicates that it functions in RNA processing.

Spatial repositioning of centromeric domains during regrowth of axons in nuclei of murine dorsal root ganglion neurons in vitro.

Specific chromatin domains within interphase nuclei are organized in cell type specific distributions and are rearranged in association with changes in cell function. Axotomy leads to changes in gene expression. Dorsal root ganglion (DRG) neurons in vitro are a model for axotomy because they are detached from their axons in preparation for the culturing procedure.

Transposition of DNase hypersensitive chromatin to the nuclear periphery coincides temporally with nerve growth factor-induced up-regulation of gene expression in PC12 cells.

To test the hypothesis that the nonrandom organization of the contents of interphase nuclei represents a compartmentalization of function, we examined the relative, spatial relationship of small nuclear ribonucleoproteins (snRNPs) and of DNase I hypersensitive chromatin (DHC) in rat pheochromocytoma cells. In controls, DHC and snRNPs colocalized as pan-nuclear speckles. During nerve growth factor-induced differentiation, both snRNPs and DHC migrated to the nuclear periphery with the migration of DHC preceding that of snRNPs, resulting in their transient separation.

Dopamine secretion by PC12 cells microencapsulated in a hydroxyethyl methacrylate--methyl methacrylate copolymer.

A rat pheochromocytoma cell line (PC12) was encapsulated in a water-insoluble hydroxyethyl methacrylate-methyl methacrylate copolymer by interfacial precipitation from a polyethylene glycol 200 solution into phosphate-buffered saline. The resulting capsules (660 +/- 44 microns in diameter; 84 +/- 27 microns wall thickness) contained viable PC12 cells in a spheroidal arrangement, much like tumour spheroids, the latter grown on surfaces unsuitable for cell attachment. In these spheroids, the viable cells formed a band approximately 100 microns thick, surrounding an inner core of necrotic cells.

Organization of centromeric domains in hepatocyte nuclei: rearrangement associated with de novo activation of the vitellogenin gene family in Xenopus laevis.

The existence of a function-dependent, nonrandom organization of chromatin domains within interphase nuclei is supported by evidence which suggests that specific chromatin domains undergo spatial rearrangement under conditions which alter gene expression. Exposure to estrogen of male Xenopus laevis hepatocytes in vitro results in de novo activation of vitellogenin mRNA production and vitellogenin protein synthesis and provides an ideal model to study the association between chromatin organization and changes in gene expression. In a test of the hypothesis that the de novo induction of vitellogenesis in male X.

Ultrastructural localization of filamentous actin within neuronal interphase nuclei in situ.

Previous biochemical studies utilizing isolated nuclei and nuclear matrices have shown actin to be a constituent of the interphase nucleus. In addition, recent ultrastructural work has shown the presence of actin and myosin within nuclei of interphase cells in situ. It was unclear, however, whether this intranuclear actin is present in the unpolymerized globular actin or the filamentous (F)-actin form.

Cytochemical localization of actin and myosin aggregates in interphase nuclei in situ.

Biochemical and ultrastructural studies on isolated nuclear compartments have previously shown actin and myosin to be constituents of interphase nuclei. In the present work, immunocytochemistry, in conjunction with confocal microscopy and ultrastructural immunogold techniques, shows that interphase nuclei of intact dorsal root ganglion neurons and of PC12 cells contain actin and myosin. Nuclear actin was observed to be distributed throughout the nucleoplasm occurring as distinct aggregates.

Distribution of snRNPs, splicing factor SC-35 and actin in interphase nuclei: immunocytochemical evidence for differential distribution during changes in functional states.

Small nuclear ribonucleoproteins (snRNPs) play an integral role in the processing of pre-mRNA in eukaryotic nuclei. snRNPs often occur in a speckled intranuclear distribution, together with the non-snRNP splicing factor SC-35. snRNPs have also been shown to be associated with actin in the nuclear matrix, suggesting that both actin and snRNPs may be involved in the processing and transport of transcripts.

Diethylstilbestrol alters the morphology and calcium levels of growth cones of PC12 cells in vitro.

Diethylstilbestrol (DES) is a synthetic estrogen with carcinogenic properties. DES is known to alter cytoskeletal components, including the organization of actin stress fibres in C6 rat glioma cells. In a test of the hypothesis that DES disrupts actin filaments of growth cones in neuron-like cells, DES-induced changes in filopodial lengths were quantified in rat pheochromocytoma (PC12) cells in vitro.

Spatial rearrangement and enhanced clustering of kinetochores in interphase nuclei of dorsal root ganglion neurons in vitro: association with nucleolar fusion.

Interphase nuclei of several cell types display distinct, nonrandom arrangements of specific chromatin domains. It has been suggested that this arrangement is associated with the functional commitment of the cell and results from compartmentalization of specific DNA sequences to transcriptionally competent sites. In a test of the hypothesis that such topological organization is established during cellular differentiation, the spatial distribution of centromeres was determined, in dorsal root ganglion neurons in vitro, using immunocytochemistry in conjunction with fluorescence microscopy, confocal laser microscopy, and ultrastructural immunogold techniques.

Morphological assessment of hepatoma cells (HepG2) microencapsulated in a HEMA-MMA copolymer with and without Matrigel.

Hepatoma cells (HepG2), an anchorage-dependent cell line, were microencapsulated in a HEMA-MMA polyacrylate membrane to which the cells do not adhere. This environment was altered by the coencapsulation of Matrigel, a reconstituted extracellular matrix derived from the Engelbreth-Holm-Swarm (EHS) mouse tumor basement membrane, to provide sites for cell attachment. The effect on the cells of these two capsule microenvironments during a 2-week in vitro culture period was assessed by examining the spatial arrangement, morphology, and viability of the cells using light microscopy and scanning electron microscopy (SEM).

Nuclear membrane modifications in polytene nuclei of Drosophila melanogaster: serial reconstruction and cytochemistry.

The nuclear envelope of polytene nuclei of salivary glands of Drosophila melanogaster displays modifications consisting of nuclear envelope invaginations (NEI) and evaginations (NEE). Ultrastructural analyses combined with three-dimensional reconstruction and cytochemistry show that NEI are bounded by a single membrane and that they may arise as invaginations of the inner nuclear membrane. NEI extend deeply into the nucleus.

Rearrangement of centromeric satellite DNA in hippocampal neurons exhibiting long-term potentiation.

In situ hybridization in conjunction with three-dimensional reconstruction was used to examine the topology of satellite DNA (sDNA) sequences in hippocampal CA1 neurons. In slices fixed immediately after preparation, 4-5 signals/nucleus were detected in CA1, CA3 and dentate neurons. 70-80% of 154 neurons examined in these 3 areas displayed all signals at the nuclear periphery.