Cell Nuclear Envelope Research Articles

Molecular docking of brazilin and its analogs to barrier-to-autointegration factor 1 (BAF1).

The tetracyclic phenolic compound brazilin, derived from the wood of Caesalpinia sappan, has been shown to bind to the chromatin protein BAF1 (barrier-to-autointegration factor 1), a protein essential to maintain integrity of the nuclear envelope in cells. BAF1 plays a role in cancer development. Using molecular docking, we have located the binding site for brazilin on the surface of the BAF1 monomer and compared its binding to that of four analogs. The oxidized product brazilein (ΔE=-57.7 kcal/mol) exhibits a higher affinity for BAF1 compared to the reduced form brazilin (ΔE=-38.2 kcal/mol). Incorporation of a 4-hydroxyl substituent on the indenochromene unit affords hematoxylin and hematein. In silico analysis predicts that the oxidized form hematein (ΔE=-66.2 kcal/mol) displays a higher affinity for BAF1 than the reduced form hematoxylin (ΔE=-42.2 kcal/mol). In contrast, the atypical bis-lactone product brazilide A cannot form good complexes with BAF1. The analysis points to the formation of more stable BAF1 complexes with the oxidized molecules compared to the reduced ones, but the position of the binding site on the protein cavity is different for brazilin/hematoxylin compared to brazilein/hematein. Our study may be useful to guide the design of BAF1 ligands.

PUL21 regulation of pUs3 kinase activity influences the nature of nuclear envelope deformation by the HSV-2 nuclear egress complex.

It is well established that the herpesvirus nuclear egress complex (NEC) has an intrinsic ability to deform membranes. During viral infection, the membrane-deformation activity of the NEC must be precisely regulated to ensure efficient nuclear egress of capsids. One viral protein known to regulate herpes simplex virus type 2 (HSV-2) NEC activity is the tegument protein pUL21. Cells infected with an HSV-2 mutant lacking pUL21 (ΔUL21) produced a slower migrating species of the viral serine/threonine kinase pUs3 that was shown to be a hyperphosphorylated form of the enzyme. Investigation of the pUs3 substrate profile in ΔUL21-infected cells revealed a prominent band with a molecular weight consistent with that of the NEC components pUL31 and pUL34. Phosphatase sensitivity and retarded mobility in phos-tag SDS-PAGE confirmed that both pUL31 and pUL34 were hyperphosphorylated by pUs3 in the absence of pUL21. To gain insight into the consequences of increased phosphorylation of NEC components, the architecture of the nuclear envelope in cells producing the HSV-2 NEC in the presence or absence of pUs3 was examined. In cells with robust NEC production, invaginations of the inner nuclear membrane were observed that contained budded vesicles of uniform size. By contrast, nuclear envelope deformations protruding outwards from the nucleus, were observed when pUs3 was included in transfections with the HSV-2 NEC. Finally, when pUL21 was included in transfections with the HSV-2 NEC and pUs3, decreased phosphorylation of NEC components was observed in comparison to transfections lacking pUL21. These results demonstrate that pUL21 influences the phosphorylation status of pUs3 and the HSV-2 NEC and that this has consequences for the architecture of the nuclear envelope.

Cryotherapy mediates histopathological and microstructural changes during the treatment of skin and subcutaneous tumors in dogs

The therapeutic effects of cryotherapy on skin and subcutaneous tumors in dogs were retrospectively studied in 20 dogs with 37 tumor lesions, of which 30 were benign and seven were malignant. Our results showed that during follow-up, 94.5% of lesions were completely exfoliated, without relapse or metastasis (mean time = 245.7 days). To investigate the effects of cryotherapy, we compared histopathological observations and microstructural changes in healthy tissues and tumor tissues, before and after cryotherapy. After cryotherapy, both normal skin and tumor tissue exhibited edema and hyperemia, with inflammatory cell infiltration. The cell nuclei exhibited pyknosis, disintegration and necrosis, and tight junctions were decreased in size. Cell morphology was varied, along with fragmented cell nuclear envelopes, crenulated nuclei and indistinct and necrotic intracellular organelles. Vacuoles were apparent in the cytoplasm and intercellular desmosomes were absent. These observations suggested that cryosurgery inhibited skin and subcutaneous tumors via cold-induced injury to cells, and cellular microenvironment changes induced by apoptosis. The results suggested that cryosurgery prevented skin and subcutaneous tumors via cold-induced injury to cells, and cellular microenvironment changes induced by apoptosis. We believe these data will provide general cryotherapy guidance to scientists and veterinary surgeons.

Shape analysis of 3D nanoscale reconstructions of brain cell nuclear envelopes by implicit and explicit parametric representations

Shape analysis of cell nuclei is becoming increasingly important in biology and medicine. Recent results have identified that large variability in shape and size of nuclei has an important impact on many biological processes. Current analysis techniques involve automatic methods for detection and segmentation of histology and microscopy images, but are mostly performed in 2D. Methods for 3D shape analysis, made possible by emerging acquisition methods capable to provide nanometric-scale 3D reconstructions, are still at an early stage, and often assume a simple spherical shape. We introduce here a framework for analyzing 3D nanoscale reconstructions of nuclei of brain cells (mostly neurons), obtained by semiautomatic segmentation of electron micrographs. Our method considers two parametric representations: the first one customizes the implicit hyperquadricsformulation and it is particularly suited for convex shapes, while the latter considers a spherical harmonics decomposition of the explicit radial representation. Point clouds of nuclear envelopes, extracted from image data, are fitted to the parameterized models which are then used for performing statistical analysis and shape comparisons. We report on the analysis of a collection of 121 nuclei of brain cells obtained from the somatosensory cortex of a juvenile rat.

Lentiviral Vpx induces alteration of mammalian cell nuclear envelope integrity

Vpx, a virion-associated protein of Human Immunodeficiency Virus 2 (HIV-2) and Simian Immunodeficiency Virus (SIV) counteracts host restriction factor SAMDH1 for efficient viral DNA synthesis in the cytoplasm and mediates subsequent nuclear translocation of the viral genome. Vpx was found to be indispensable in the viral infection of terminally differentiated target cells and macaques infected with virions carrying truncated Vpx showed delayed pathogenesis, suggesting multiple roles of Vpx at different steps in the virus life cycle. The current study demonstrates a novel function of SIVsmPBj1.9 Vpx on the integrity of the nuclear envelope in HeLa cells. Results from the Super-Resolution Structured Illumination Microscopy (SR-SIM) analysis showed that Vpx puncta alter HeLa cell nuclear envelope assembly. Furthermore, three-dimensional (3D) SIM analysis of such regions suggests that Vpx is primed in a specific way to disrupt the nuclear envelope integrity. The nuclear incursion of cytoplasmic proteins through Vpx mediated ruptured nuclear envelope regions suggest that these events might play a critical role in the nuclear entry of otherwise cytoplasmically sequestered molecules and theirby may be assisting Vpx functions including the transport of viral genome into the nucleus, which is critical for the establishment of virus infection and pathogenesis.

70 - Reorganization of the nuclear lamina and cytoskeleton of human mesenchymal stem cells on a dendrimer-immobilized surface

Human mesenchymal stem cells (hMSCs) have great potential as a cell source for tissue engineering and regenerative therapy due to its capacity to differentiate into many cell types. Differentiation of mesenchymal stem cells is regulated by microenvironmental cues which play an important role in directing stem cell behavior. In this study, we investigated the cytoskeleton-nucleus reorganization of hMSCs cultured on the dendrimer-immobilized surface. Time-lapse observation of hMSCs on a fifth generation (G5) of dendrimer-immobilized surface exhibits active migration behavior and repetitive morphological changes through stretching and contracting, resulting cell aggregates formation. However, hMSCs cultured on polystyrene (PS) surface shows high attachment to the substrate with spreading, flattened and elongated shape of cells also performed passive migration behavior. Fluorescent staining of F-actin and nucleoskeletal protein, lamin A/C, found significant presence of immature stress fibers in the aggregated cells on the G5 surface. In addition, the nuclear envelopes of cells on the G5 surface were wrinkled and relaxed, whereas, on PS surface, the nuclei were flattened by stress fibers and appear stiff and smooth. The observation indicated that the G5 surface permits the regulation of nuclear skeleton structure reorganization associated with alteration in cellular morphology and migratory behaviors. It was also found that cardiomyocyte-specific marker, cardiac Troponin T expression, was, in particular, promoted on the G5 surface, thus supporting the hypothesis that cytoskeleton-nucleus reorganization during active migration induces cardiomyogenic lineage commitment in hMSCs. These results suggest that active migration driven by dendrimer surface could regulate cytoskeleton-nucleus reorganization which possibly initiate mechanotransduction inside cells and directed hMSCs towards cardiomyogenic lineage commitment.

Mutant torsinA in the heterozygous DYT1 state compromises HSV propagation in infected neurons and fibroblasts

Most cases of early onset torsion dystonia (DYT1) are caused by a 3-base pair deletion in one allele of the TOR1A gene causing loss of a glutamate in torsinA, a luminal protein in the nuclear envelope. This dominantly inherited neurologic disease has reduced penetrance and no other medical manifestations. It has been challenging to understand the neuronal abnormalities as cells and mouse models which are heterozygous (Het) for the mutant allele are quite similar to wild-type (WT) controls. Here we found that patient fibroblasts and mouse neurons Het for this mutation showed significant differences from WT cells in several parameters revealed by infection with herpes simplex virus type 1 (HSV) which replicates in the nucleus and egresses out through the nuclear envelope. Using a red fluorescent protein capsid to monitor HSV infection, patient fibroblasts showed decreased viral plaque formation as compared to controls. Mouse Het neurons had a decrease in cytoplasmic, but not nuclear HSV fluorescence, and reduced numbers of capsids entering axons as compared to infected WT neurons. These findings point to altered dynamics of the nuclear envelope in cells with the patient genotype, which can provide assays to screen for therapeutic agents that can normalize these cells.

Abstract 1887: Untethering heterochromatin: how loss of HP1a enhances cell invasion by altering nuclear envelope integrity

Abstract Malignant cell invasion is accompanied by complex changes in nuclear shape and organisation. The onset of invasion correlates with the loss of HP1a in many solid tumours including those of the thyroid, kidney, colon and breast, while in cancer cell lines HP1a suppresses invasive potential. In normal cells HP1a maintains the condensation of transcriptionally silent heterochromatin and sequesters it at the inner periphery of the nuclear envelope; an event critical for the maintenance of nuclear stability and envelope rigidity. This role for HP1a leads us to hypothesise that the un-tethering of heterochromatin from the nuclear periphery that would occur after loss of HP1 causes a remodelling of nuclear envelope that enhances cellular invasion. If true this would explain how the nucleus, the largest and most rigid cellular organelles, becomes malleable enough to squeeze through interstitial spaces in the surrounding tissue during metastatic invasion. In support of this hypothesis we observed a 44% increase in the invasive potential of the poorly invasive MCF7 breast cells when HP1a is knocked down. Lamin A staining and electron microscopy also confirmed that these HP1a knockdown cells had the expected alterations in nuclear morphology and loss of heterochromatin at the nuclear periphery. Furthermore the loss of HP1 was correlated with a decrease in the mechanical stability of the nuclei. The significant enrichment (p-val 6.7x10-8) of nuclear envelope proteins shown by RNAseq analysis of transcripts that are differentially expressed in the MCF7 versus HP1a knock-down cells, suggests this loss of nuclear envelope integrity is associated with alterations to its composition. The most striking change identified in the nuclear envelope of cells lacking HP1a was the loss of two Nesprin1 isoforms. While reintroduction of HP1a in highly invasive MDA-MB-231 cells shows the return of these isoforms to the nuclear envelope. As the Nesprin1 isoforms act as signalling scaffolds connecting to the cytoskeleton or reaching into the nucleus, they represent a means of modulating nuclear malleability. Together these findings suggest that when loss of HP1a increases invasive potential there is a corresponding disruption to the nuclear periphery that is a weakness of these cells that could be exploited. Citation Format: Tracy K. Hale, Sarah Bond, David Wheeler. Untethering heterochromatin: how loss of HP1a enhances cell invasion by altering nuclear envelope integrity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1887. doi:10.1158/1538-7445.AM2017-1887

Listeria monocytogenes induces mast cell extracellular traps

Mast cells play an essential role in different immunological phenomena including allergy and infectious diseases. Several bacteria induce mast cell activation leading to degranulation and the production of several cytokines and chemokines. However, mast cells also have different microbicidal activities such as phagocytosis and the release of DNA with embedded granular proteins known as Mast Cell Extracellular Traps (MCETs). Although previous reports indicate that extracellular bacteria are able to induce MCETs little is known if intracellular bacteria can induce these structures. In this work, we evaluated MCETs induction by the intracellular bacteria Listeria monocytogenes. We found that mast cells released DNA after stimulation with L. monocytogenes, and this DNA was complexed to histone and tryptase. Before extracellular DNA release, L. monocytogenes induced modifications to the mast cell nuclear envelope and DNA was detected outside the nucleus. L. monocytogenes stimulated mast cells to produce significant amounts of reactive oxygen species (ROS) and blocking NADPH oxidase diminished DNA release by mast cells. Finally, MCETs showed antimicrobial activity against L. monocytogenes that was partially blocked when β-hexosaminidase activity was inhibited. These results show that L. monocytogenes induces mast cells to produce microbicidal MCETs, suggesting a role for mast cells in containing infection beyond the induction of inflammation.

Nuclear-localized cyclic nucleotide-gated channels mediate symbiotic calcium oscillations.

Nuclear-associated Ca(2+) oscillations mediate plant responses to beneficial microbial partners--namely, nitrogen-fixing rhizobial bacteria that colonize roots of legumes and arbuscular mycorrhizal fungi that colonize roots of the majority of plant species. A potassium-permeable channel is known to be required for symbiotic Ca(2+) oscillations, but the calcium channels themselves have been unknown until now. We show that three cyclic nucleotide-gated channels in Medicago truncatula are required for nuclear Ca(2+) oscillations and subsequent symbiotic responses. These cyclic nucleotide-gated channels are located at the nuclear envelope and are permeable to Ca(2+) We demonstrate that the cyclic nucleotide-gated channels form a complex with the postassium-permeable channel, which modulates nuclear Ca(2+) release. These channels, like their counterparts in animal cells, might regulate multiple nuclear Ca(2+) responses to developmental and environmental conditions.

Calcium signals the making of symbiosis

Plant Science Plant cell nuclei respond to signals from symbiotic nitrogenfixing rhizobial bacteria or arbuscular mycorrhizal fungi with oscillating Ca2+ release. Charpentier et al. identified a trio of responsible Ca2+ channels in a legume. These channels contain nuclear localization signals and are expressed in root cell nuclear envelopes. The channels function early in the establishment of symbiosis to produce oscillations in Ca2+ release from nuclear stores. Science , this issue p. [1102][1] [1]: /lookup/doi/10.1126/science.aae0109

Visualizing the Spatial Relationship of the Genome with the Nuclear Envelope Using Fluorescence In Situ Hybridization.

The genome has a special relationship with the nuclear envelope in cells. Much of the genome is anchored at the nuclear periphery, tethered by chromatin binding proteins such nuclear lamins and other integral membrane proteins. Even though there are global assays such as DAM-ID or ChIP to assess what parts of the genome are associated with the nuclear envelope, it is also essential to be able to visualize regions of the genome in order to reveal their individual relationships with nuclear structures in single cells. This is executed by fluorescence in situ hybridization (FISH) in 2-dimensional flattened nuclei (2D-FISH) or 3-dimensionally preserved cells (3D-FISH) in combination with indirect immunofluorescence to reveal structural proteins. This chapter explains the protocols for 2D- and 3D-FISH in combination with indirect immunofluorescence and discusses options for image capture and analysis. Due to the nuclear envelope proteins being part of the non-extractable nucleoskeleton, we also describe how to prepare DNA halos through salt extraction and how they can be used to study genome behavior and association when combined with 2D-FISH.

GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport.

The GGGGCC (G4C2) repeat expansion in a noncoding region of C9orf72 is the most common cause of sporadic and familial forms of amyotrophic lateral sclerosis and frontotemporal dementia. The basis for pathogenesis is unknown. To elucidate the consequences of G4C2 repeat expansion in a tractable genetic system, we generated transgenic fly lines expressing 8, 28 or 58 G4C2-repeat-containing transcripts that do not have a translation start site (AUG) but contain an open-reading frame for green fluorescent protein to detect repeat-associated non-AUG (RAN) translation. We show that these transgenic animals display dosage-dependent, repeat-length-dependent degeneration in neuronal tissues and RAN translation of dipeptide repeat (DPR) proteins, as observed in patients with C9orf72-related disease. This model was used in a large-scale, unbiased genetic screen, ultimately leading to the identification of 18 genetic modifiers that encode components of the nuclear pore complex (NPC), as well as the machinery that coordinates the export of nuclear RNA and the import of nuclear proteins. Consistent with these results, we found morphological abnormalities in the architecture of the nuclear envelope in cells expressing expanded G4C2 repeats in vitro and in vivo. Moreover, we identified a substantial defect in RNA export resulting in retention of RNA in the nuclei of Drosophila cells expressing expanded G4C2 repeats and also in mammalian cells, including aged induced pluripotent stem-cell-derived neurons from patients with C9orf72-related disease. These studies show that a primary consequence of G4C2 repeat expansion is the compromise of nucleocytoplasmic transport through the nuclear pore, revealing a novel mechanism of neurodegeneration.

Abnormal nuclear envelope in the cerebellar Purkinje cells and impaired motor learning in DYT11 myoclonus-dystonia mouse models

Myoclonus-dystonia (M-D) is a movement disorder characterized by myoclonic jerks with dystonia. DYT11 M-D is caused by mutations in SGCE which codes for ɛ-sarcoglycan. SGCE is maternally imprinted and paternally expressed. Abnormal nuclear envelope has been reported in mouse models of DYT1 generalized torsion dystonia. However, it is not known whether similar alterations occur in DYT11 M-D. We developed a mouse model of DYT11 M-D using paternally inherited Sgce heterozygous knockout ( Sgce KO) mice and reported that they had myoclonus and motor coordination and learning deficits in the beam-walking test. However, the specific brain regions that contribute to these phenotypes have not been identified. Since ɛ-sarcoglycan is highly expressed in the cerebellar Purkinje cells, here we examined the nuclear envelope in these cells using a transmission electron microscope and found that they are abnormal in Sgce KO mice. Our results put DYT11 M-D in a growing family of nuclear envelopathies. To analyze the effect of loss of ɛ-sarcoglycan function in the cerebellar Purkinje cells, we produced paternally inherited cerebellar Purkinje cell-specific Sgce conditional knockout ( Sgce pKO) mice. Sgce pKO mice showed motor learning deficits, while they did not show abnormal nuclear envelope in the cerebellar Purkinje cells, robust motor deficits, or myoclonus. The results suggest that ɛ-sarcoglycan in the cerebellar Purkinje cells contributes to the motor learning, while loss of ɛ-sarcoglycan in other brain regions may contribute to nuclear envelope abnormality, myoclonus and motor coordination deficits.

Nesprins, but not sun proteins, switch isoforms at the nuclear envelope during muscle development

Nesprins are a family of nuclear transmembrane proteins anchored via Sun proteins to the nuclear membrane. Analysis of nesprins during human muscle development revealed an increase in nesprin-1-giant during early myogenesis in vitro. During the transition from immature to mature muscle fibres in vivo, nesprin-2 partly replaced nesprin-1 at the nuclear envelope and short nesprin isoforms became dominant. Sun1 and Sun2 proteins remained unchanged during this fibre maturation. In emerin-negative skin fibroblasts, nesprin-2-giant was relocated from the nuclear envelope to the cytoplasm, not to the endoplasmic reticulum, while nesprin-1 remained at the nuclear envelope. In emerin-negative keratinocytes lacking nesprin-1, nesprin-2 remained at the nuclear envelope. HeLa cell nuclear envelopes lacked nesprin-1, which was the dominant form in myoblasts, while a novel 130-kD nesprin-2 isoform dominated Ntera-2 cells. The results suggest the possibility of isoform-specific and tissue-specific roles for nesprins in nuclear positioning.

Focal distortion of the nuclear envelope by huntingtin aggregates revealed by lamin immunostaining

Huntington’s disease is an autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine repeat tract in the huntingtin protein. Polyglutamine-expanded huntingtin forms intranuclear as well as perinuclear inclusion bodies. Perinuclear aggregates formed by polyglutamine-expanded proteins are associated with a characteristic indentation of the nuclear envelope. We examined the nuclear envelope in cells containing huntingtin aggregates using immunostaining for lamin B1, a major component of the nuclear lamina. Laser confocal microscopy analysis revealed that huntingtin aggregates in a juxtanuclear position were associated with a clear focal distortion in the nuclear envelope in cells transfected with polyglutamine-expanded huntingtin. Lamin B1 distribution was not altered by aggregates of polyglutamine-expanded ataxin-1, that are exclusively intranuclear. Thus lamin immunocytochemistry demonstrates clearly the depression of the nuclear envelope resulting from the formation of perinuclear aggregates by polyglutamine-expanded huntingtin. Lamin immunocytochemistry would be of value to monitor the state of the nuclear envelope in experimental paradigms aimed at establishing the significance of perinuclear aggregates of pathogenic proteins.

Interaction between the movement protein of barley yellow dwarf virus and the cell nuclear envelope: Role of a putative amphiphilic α‐helix at the N‐terminus of the movement protein

The open reading frame 4 (ORF 4) gene product of barley yellow dwarf virus (BYDV) may act as a movement protein (MP) by assisting the transport of viral genomic RNA across the nuclear envelope (NE) of host plant cells. To investigate interactions between BYDV MP and the NE, wild-type and mutant open reading frame (ORF 4)-green fluorescent protein (GFP) fusion cistrons were expressed in insect cells. A fusion protein expressed by the wild-type ORF 4-GFP cistron associated with the NE and caused protrusions from its surface. The fusion protein expressed by the mutant ORF 4-GFP cistron lacked a putative amphiphilic alpha-helix at its N-terminus and although associating with the NE, showed decreased levels of protrusions. A peptide homologue of this putative alpha-helix induced an increase of 7 degrees C in the phase transition temperature of dimyrystoyl phosphatidylserine (DMPS) membranes, accompanied by a decrease in membrane fluidity, but exhibited no significant interaction with either dimyristoyl phosphatidylcholine (DMPC) or dimyristoyl phosphatidylethanolamine (DMPE) membranes. These results strongly support the view that BYDV MP may interact with the NE to help transport viral genomic RNA into the nuclear compartment. This function of BYDV MP appears to involve protrusions on the surface of the NE and may require the presence of an N-terminal amphiphilic alpha-helix, which is speculated to destabilize membranes, thereby assisting the entry of BYDV-GAV into the nuclear compartment.

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. In situ nick translation was used to visualize the distribution of DNAse sensitive chromatin as a function of nuclear geometry. Both unexpanded and expanded cells exhibit DNAse sensitive chromatin as a dome at the apical side of the nucleus, i.e., that aspect of the cell facing the culture medium. The results argue for a polarized association of DNAse sensitive chromatin with the nuclear envelope and indicate that the nuclear periphery may function as a compartment for the spatial coupling of transcription and nucleo-cytoplasmic transport.Key words: nuclear organization, DNAse sensitive chromatin, hypotonic expansion, 3T3 cells.

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. In situ nick translation was used to visualize the distribution of DNAse sensitive chromatin as a function of nuclear geometry. Both unexpanded and expanded cells exhibit DNAse sensitive chromatin as a dome at the apical side of the nucleus, i.e., that aspect of the cell facing the culture medium. The results argue for a polarized association of DNAse sensitive chromatin with the nuclear envelope and indicate that the nuclear periphery may function as a compartment for the spatial coupling of transcription and nucleo-cytoplasmic transport.

Distribution of apoptosis-like cells in sea urchin early embryogenesis

It has been reported that the number of cells per embryo increases from the cleavage stage to the pluteus stage. Also, it has been reported that the number of cells per embryo from the early gastrula stage to the mid-gastrula stage increases very slightly (Mizoguchi, 1999). A detailed analysis of cell proliferation during this period would thus seem to be necessary.On the other hand, Roccheri et al. (1997) reported spontaneous apoptosis at the early pluteus stage, especially in the regions of arm and intestine. However, it is unknown whether apoptosis occurs before the pluteus stage.Using the Tumor, Neuro and/or Cardio TACS in situ apoptosis detection kit (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling: TUNEL method, Trevigen, USA), the Comet assay kit (Trevigen, USA) and DAPI (4,6-diamidino-2-phenylinodole dihydrochloride) staining, we investigated the distribution of apoptosis-like cells in sea urchins during early embryogenesis to clarify the relationship between cell death and morphogenesis.The embryos of the sea urchin Hemicentrotus pulcherrimus were used in the present study. Three methods of detection of apoptosis signals in the embryos were performed: (1) defects in chromosomes and punctured nuclear envelopes of cells were detected by DAPI staining; (2) apoptosis-like cells were stained a brown colour by the TUNEL method; (3) some clear tails which detected the fragmentation of DNA were found by the Comet assay. These signals indicate apoptosis-like cells.