Distribution of calcium-independent phospholipase A<Subscript>2</Subscript> (iPLA<Subscript>2</Subscript>) in monkey brain

The present study was carried out to elucidate the distribution of calcium-independent phospholipase A₂ (iPLA₂) in the normal monkey brain. iPLA₂ immunoreactivity was observed in structures derived from the telencephalon, including the cerebral neocortex, amygdala, hippocampus, caudate nucleus, putamen, and nucleus accumbens, whereas structures derived from the diencephalon, including the thalamus, hypothalamus and globus pallidus were lightly labeled. The midbrain, vestibular, trigeminal and inferior olivary nuclei, and the cerebellar cortex were densely labeled. Immunoreactivity was observed on the nuclear envelope of neurons, and dendrites and axon terminals at electron microscopy. Western blot analysis showed higher levels of iPLA₂ protein in the cytosolic, than the nuclear fraction, but little or no protein in the membrane fraction. Similarly, subcellular fractionation studies of iPLA₂ activity in rat brain cortical cell cultures showed greater enzymatic activity in the cytosolic, than the nuclear fraction, and the least activity in non-nuclear membranes. The association of iPLA₂ with the nuclear envelope suggests a role of the enzyme in nuclear signaling, such as during neuronal proliferation and differentiation or death. In addition, the localization of iPLA₂ in dendrites and axon terminals suggests a role of the enzyme in neuronal signaling.     

Cytosolic stress granules relieve the ubiquitin‐proteasome system in the nuclear compartment

The role of cytosolic stress granules in the integrated stress response has remained largely enigmatic. Here, we studied the functionality of the ubiquitin‐proteasome system (UPS) in cells that were unable to form stress granules. Surprisingly, the inability of cells to form cytosolic stress granules had primarily a negative impact on the functionality of the nuclear UPS. While defective ribosome products (DRiPs) accumulated at stress granules in thermally stressed control cells, they localized to nucleoli in stress granule‐deficient cells. The nuclear localization of DRiPs was accompanied by redistribution and enhanced degradation of SUMOylated proteins. Depletion of the SUMO‐targeted ubiquitin ligase RNF4, which targets SUMOylated misfolded proteins for proteasomal degradation, largely restored the functionality of the UPS in the nuclear compartment in stress granule‐deficient cells. Stress granule‐deficient cells showed an increase in the formation of mutant ataxin‐1 nuclear inclusions when exposed to thermal stress. Our data reveal that stress granules play an important role in the sequestration of cytosolic misfolded proteins, thereby preventing these proteins from accumulating in the nucleus, where they would otherwise infringe nuclear proteostasis.     

N‐methyl‐N‐nitrosourea induces retinal degeneration in the rat via the inhibition of NF‐κB activation

Retinitis pigmentosa (RP) is a group of inherited neurodegenerative diseases characterized by the loss of photoreceptor cells through apoptosis. N‐methyl‐N‐nitrosourea (MNU) is an alkylating toxicant that induces photoreceptor cell death resembling hereditary RP. This study aimed to investigate the role of nuclear factor κB (NF‐κB) in MNU‐induced photoreceptor degeneration. Adult rats received a single intraperitoneal injection of MNU (60 mg/kg bodyweight). Hematoxylin and eosin staining demonstrated progressive outer nuclear layer (ONL) loss after MNU treatment. Transmission electron microscopy revealed nuclear pyknosis, chromatin margination in the photoreceptors, increased secondary lysosomes, and lobulated retinal‐pigmented epithelial cells in MNU‐treated rats. Numerous photoreceptor cells in the ONL showed positive TUNEL staining and apoptosis rate peaked at 24 hours. Enhanced depth imaging spectral‐domain optical coherence tomography showed ONL thinning and decreased choroid thickness. Electroretinograms showed decreased A wave amplitude that predominated in scotopic conditions. Western blot analysis showed that nuclear IκBα level increased, whereas nuclear NF‐κB p65 decreased significantly in the retinas of MNU‐treated rats. These findings indicate that MNU leads to selective photoreceptor degradation, and this is associated with the inhibition of NF‐κB activation.     

Characterization of an Importin α/β‐recognized nuclear localization signal in β‐dystroglycan

β‐dystroglycan (β‐DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of β‐DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of β‐DG, characterizing a functional nuclear localization signal (NLS) in the β‐DG cytoplasmic domain, within amino acids 776–782. The NLS either alone or in the context of the whole β‐DG protein was able to target the heterologous GFP protein to the nucleus, with site‐directed mutagenesis indicating that amino acids R⁷⁷⁹ and K⁷⁸⁰ are critical for NLS functionality. The nuclear transport molecules Importin (Imp)α and Impβ bound with high affinity to the NLS of β‐DG and were found to be essential for NLS‐dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of β‐DG may result in cytoplasmic retention, with Y⁸⁹² playing a key role. β‐DG thus follows a conventional Impα/β‐dependent nuclear import pathway, with important implications for its potential function in the nucleus. J. Cell. Biochem. 110: 706–717, 2010. © 2010 Wiley‐Liss, Inc.     

Lamin C and chromatin organization in <Emphasis Type="Italic">Drosophila</Emphasis>

Drosophila lamin C (LamC) is a developmentally regulated component of the nuclear lamina. The lamC gene is situated in the fifth intron of the essential gene tout velu (ttv). We carried out genetic analysis of lamC during development. Phenotypic analyses of RNAi-mediated downregulation of lamC expression as well as targeted misexpression of lamin C suggest a role for lamC in cell survival. Of particular interest in the context of laminopathies is the caspase-dependent apoptosis induced by the overexpression of lamin C. Interestingly, misexpression of lamin C in the central nervous system, where it is not normally expressed, did not affect organization of the nuclear lamina. lamC mutant alleles suppressed position effect variegation normally displayed at near-centromeric and telomeric regions. Further, both downregulation and misexpression of lamin C affected the distribution of heterochromatin protein 1. Our results suggest that Drosophila lamC has a tissue-specific role during development and is required for chromatin organization.     

Follower fish of the goldspotted eel <Emphasis Type="Italic">Myrichthys ocellatus</Emphasis> with a review on anguilliform fish as nuclear species

In a nuclear-follower fish foraging association, the follower benefits from food uncovered or flushed out when the nuclear fish disturbs the bottom, while nuclear species generally do not seem to be benefiting. Among nuclear species, eels (anguilliform fish) are known to be one of the most represented groups. Here we investigated the frequency and time duration of foraging associations among the goldspotted eel Myrichthys ocellatus and reef fish in a subtropical marginal reef. In addition, we reviewed nuclear eel species and their followers described in the literature. From a total of 211 goldspotted eels observed, seven follower species were recorded in 19% of the samples. The average time of the following associations per species ranged from 40 to 190 s. Four species were reported for the first time as M. ocellatus followers (Bodianus rufus, B. pulchellus, Stephanolepis hispidus, and Serranus baldwini) and three of them have never been reported in the literature as eel followers (B. pulchellus, S. hispidus, and S. baldwini). The literature describes 13 eel species acting as nuclear for 66 fish species, represented mainly by groupers and sea basses. The size of the eel was not correlated with the size of its follower and neither with the number of described follower species. The nuclear role of eels is likely to be an important component of the trophic ecology of small and medium-sized macrocarnivore fish.     

Dynamics of nuclear matrix proteome during embryonic development in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Embryonic development is a complex and dynamic process that involves spatiotemporal expression of genes in a highly coordinated manner. Multiple levels of nuclear architecture maintain the fidelity of gene expression programme. One of the components of nuclear architecture, which is believed to play an important role in regulation of gene expression, is the nuclear matrix (NuMat). Many studies over the past few years have tried to analyse the components of this non-chromatin scaffolding of the nucleus and have provided evidences of its structural and functional complexity. However, the relationship of NuMat with the process of embryonic development still remains poorly understood. Here, we report a comparative analysis of the NuMat proteomes of early and late stage Drosophila melanogaster embryos and show that 65% of the NuMat proteome is dynamic during development. Our study establishes links between the dynamics of nuclear architecture and embryonic development and provides tools to further understand the process such as cellular differentiation in the context of higher-order nuclear organization.     

A transmission electron microscopy investigation: the membrane complex in spermatogenesis of <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis>

The transforming characteristics of the membrane complex in spermatogenesis of Fenneropenaeus chinensis have been studied by using transmission electron microscopy. Two types of membrane complex have been investigated based on their sources: one originating from nucleus and the other from cytoplasm. The first one, consisted of annular structures, monolayer membrane blebs, and double or multi-lamellar membrane vesicles, emerges in the primary spermatocyte, then diffuses with the nuclear membrane and finally enters the cytoplasm. This type of membrane complex seems to play an important role in the materials transfusion from nucleus to cytoplasm, and it mainly exists inside the primary spermatocyte with some inside the secondary spermatocyte. The latter, originated from cytoplasm, is formed during the anaphase of spermiogenesis. It also exists in mature sperm, locating at both sides of the nucleus under the acrosomal cap. This type of membrane complex mainly comprises rings of convoluted membrane pouches, together with mitochondria, annular lamina bodies, fragments of endoplasmic reticulum, nuclear membrane and some nuclear particles. It releases vesicles and particles into the acrosomal area during the formation of the perforatorium, suggesting a combined function of the endoplasmic reticulum, mitochondria and Golgi’s mechanism.     

SUV39h‐ and A‐type lamin‐dependent telomere nuclear rearrangement

Telomeres are specialized chromatin structures that are situated at the end of linear chromosomes and play an important role in cell senescence and immortalization. Here, we investigated whether changes in histone signature influence the nuclear arrangement and positioning of telomeres. Analysis of mouse embryonic fibroblasts revealed that telomeres were organized into specific clusters that partially associated with centromeric clusters. This nuclear arrangement was influenced by deficiency of the histone methyltransferase SUV39h, LMNA deficiency, and the histone deacetylase inhibitor Trichostatin A (TSA). Similarly, nuclear radial distributions of telomeric clusters were preferentially influenced by TSA, which caused relocation of telomeres closer to the nuclear center. Telomeres also co‐localized with promyelocytic leukemia bodies (PML). This association was increased by SUV39h deficiency and decreased by LMNA deficiency. These differences could be explained by differing levels of the telomerase subunit, TERT, in SUV39h‐ and LMNA‐deficient fibroblasts. Taken together, our data show that SUV39h and A‐type lamins likely play a key role in telomere maintenance and telomere nuclear architecture. J. Cell. Biochem. 109: 915–926, 2010. © 2010 Wiley‐Liss, Inc.     

Characterization of cellular DGK-θ

In this report we have summarized the data regarding the regulation of DGK-θ by two phospholipids: PtdSer and PtdOH. Our previous data has shown that stimulation of quiescent fibroblasts with a potent mitogen (α-thrombin) leads to an increase in nuclear localized DGK-θ ([Bregoli et al., 2001] and [Bregoli et al., 2002]), as has been seen in neuronal cells ([Tabellini et al., 2003] and [Tabellini et al., 2004]). Furthermore, these previous studies demonstrated that DGK-θ is actually localized to the nuclear matrix ([Bregoli et al., 2002] and [Tabellini et al., 2003]). As we have also previously shown that PtdSer and PtdOH modulate DGK-θ activity ([Tu-Sekine et al., 2006] and [Tu-Sekine et al., 2007]), we examined the phospholipid composition of the nuclear matrix as well as the phospholipid composition of the intact nuclei and non-nuclear membranes. This analysis has revealed that there are phospholipids in the “matrix” of nuclei and the composition of these lipids largely resembles those of the other membranes. The physical form and localization of the matrix-associated lipids has not been established, though the nearly identical percent composition of the non-nuclear membranes and the nuclear matrix lend credence to the hypothesis that at least some of the internal nuclear lipid is derived from invaginations of the cellular membranes through the nuclear interior known as nuclear tubules ([Fricker et al., 1997] and [Lee et al., 2006]).An important finding resulting from this analysis is that the nuclear envelope is enriched in PtdSer relative to the nuclear matrix and to cellular membranes, and that over-expression of DGK-θ reduces both the PtdSer and PtdEth levels of the nuclear envelope. While the mechanism behind these fluctuations is unknown, the data are consistent with a DGK-regulated phosphatidylcholine-dependent phosphatidylserine synthase (PSS-1) (EC 2.7.8.8) activity at the nuclear envelope. PtdSer synthesis in mammalian cells proceeds by headgroup exchange with PtdCho or PtdEth, catalyzed by PSS-1 or PSS-2, respectively. Interestingly, the decrease in both PtdSer and PtdEth at the nuclear membrane mirrors effects observed in the CHO-K1 mutant cell lines M.6.1.1 and PSA-3, which have been shown to be deficient in PSS-1 activity (Vance and Steenbergen, 2005). While there is very little information on the regulation of mammalian PSS enzymes, phosphorylation has been shown to regulate serine exchange activities in rat brain (Kanfer et al., 1988), and we have shown that nuclear DGK-θ regulates the exit of PKC-α from the nucleus (see regulation of PKC-α). To our knowledge, there is currently no reported study of nuclear phosphatidylyserine synthases.One other notable fluctuation in cellular lipids from DGK-θ expression is an increase in plasmalogen-PtdEth in both the NNM and nuclear matrix. While the majority of studies conducted on plasmalogen synthesis agree in that there is an almost complete dependence on the classical CDP-ethanolamine pathway to provide the headgroup for precursor plasmalogen synthesized in the peroxisome, there are several studies utilizing radiolabeled serine that report approximately 20–30% of cellular plasmalogen-PtdEth can be derived from PtdSer in at least some cell models ([Yorek et al., 1985] and [Xu et al., 1991]). Whether nuclear PtdSer-derived PtdEth a precursor for the membrane plasmalogen-PtdEth present in DGK-θ expressing cells is not evident from the data presented here, though one potential mechanism for an increase in flux through this pathway could be an increase in PtdSer-derived PtdEth. At this juncture we can only speculate on the cause of the lipid perturbations, and further work is required to support our hypothesis that DGK-θ impacts PtdSer and plasmalogen metabolism.The physiological role for nuclear DGK-θ has remained a mystery. It has long been recognized that an obvious role for DGKs is to modulate cellular levels of its DAG substrate thereby modulating DAG-sensitive proteins like such as many PKCs (Merida et al., 2008). Our data support this notion as suppression of DGK-θ, either by expression of RhoA or a dominant-negative construct of DGK-θ, leads to a sustained increase in nuclear PKC-α (Fig. 2).In addition to the regulation by PtdSer and PtdOH, we report here that the regulation of nuclear DGK-θ by RhoA is modulated by a Class I PI 3-kinase. Furthermore, we have examined the effect of various inhibitors on the activity of DGK-θ in lysates over-expressing this isoform. This is important as many studies often use pharmacologic inhibitors to determine the role of selected enzymes in signaling pathways. While this approach is useful for comparative analyses, there are no data pertaining to the effect of these inhibitors on DGK-θ. We have found that one well-established PI-PLC inhibitor, U73122, but not its inactive analog U73343, competitively inhibits this isoform with respect to its diacylglycerol (DAG) substrate. These data indicate that studies designed to examine the role of PI-PLC in modulating DGK-θ activity using this inhibitor should be interpreted with caution.     

Characterization of a 65 kDa NIF in the nuclear matrix of the monocot Allium cepa that interacts with nuclear spectrin‐like proteins

Plant cells have a well organized nucleus and nuclear matrix, but lack orthologues of the main structural components of the metazoan nuclear matrix. Although data is limited, most plant nuclear structural proteins are coiled‐coil proteins, such as the NIFs (nuclear intermediate filaments) in Pisum sativum that cross‐react with anti‐intermediate filament and anti‐lamin antibodies, form filaments 6–12 nm in diameter in vitro, and may play the role of lamins. We have investigated the conservation and features of NIFs in a monocot species, Allium cepa, and compared them with onion lamin‐like proteins. Polyclonal antisera against the pea 65 kDa NIF were used in 1D and 2D Western blots, ICM (imunofluorescence confocal microscopy) and IEM (immunoelectron microscopy). Their presence in the nuclear matrix was analysed by differential extraction of nuclei, and their association with structural spectrin‐like proteins by co‐immunoprecipitation and co‐localization in ICM. NIF is a conserved structural component of the nucleus and its matrix in monocots with M_r and pI values similar to those of pea 65 kDa NIF, which localized to the nuclear envelope, perichromatin domains and foci, and to the nuclear matrix, interacting directly with structural nuclear spectrin‐like proteins. Its similarities with some of the proteins described as onion lamin‐like proteins suggest that they are highly related or perhaps the same proteins.     

Histone chaperone CAF‐1 promotes HIV‐1 latency by leading the formation of phase‐separated suppressive nuclear bodies

HIV‐1 latency is a major obstacle to achieving a functional cure for AIDS. Reactivation of HIV‐1‐infected cells followed by their elimination via immune surveillance is one proposed strategy for eradicating the viral reservoir. However, current latency‐reversing agents (LRAs) show high toxicity and low efficiency, and new targets are needed to develop more promising LRAs. Here, we found that the histone chaperone CAF‐1 (chromatin assembly factor 1) is enriched on the HIV‐1 long terminal repeat (LTR) and forms nuclear bodies with liquid–liquid phase separation (LLPS) properties. CAF‐1 recruits epigenetic modifiers and histone chaperones to the nuclear bodies to establish and maintain HIV‐1 latency in different latency models and primary CD4⁺ T cells. Three disordered regions of the CHAF1A subunit are important for phase‐separated CAF‐1 nuclear body formation and play a key role in maintaining HIV‐1 latency. Disruption of phase‐separated CAF‐1 bodies could be a potential strategy to reactivate latent HIV‐1.