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Micronuclei are a hallmark of cancer and several other human disorders. Recently, micronuclei were implicated in chromothripsis, a series of massive genomic rearrangements that may drive tumor evolution and progression. Here, we show that Aurora B kinase mediates a surveillance mechanism that integrates error correction during anaphase with spatial control of nuclear envelope reassembly to prevent micronuclei formation. Using high-resolution live-cell imaging of human cancer and non-cancer cells, we uncover that anaphase lagging chromosomes are more frequent than previously anticipated, yet they rarely form micronuclei. Micronuclei formation from anaphase lagging chromosomes is prevented by a midzone-based Aurora B phosphorylation gradient that stabilizes kinetochore-microtubule attachments and assists spindle forces required for anaphase error correction while delaying nuclear envelope reassembly on lagging chromosomes, independently of microtubule density. We propose that a midzone-based Aurora B phosphorylation gradient actively monitors and corrects frequent chromosome segregation errors to prevent micronuclei formation during human cell division., Competing Interests: Declaration of interests B.O. declares that he is a consultant specialist at Volastra Therapeutics., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Small GTPases of the Rho family are binary molecular switches that regulate a variety of processes including cell migration and oriented cell divisions. Known Cdc42 effectors include proteins involved in cytoskeletal remodeling and kinase-dependent transcription induction, but none are involved in the maintenance of nuclear envelope integrity or ER morphology. Maintenance of nuclear envelope integrity requires the EndoSomal Complexes Required for Transport (ESCRT) proteins, but how they are regulated in this process remains unknown. Here, we show by live-cell imaging a novel Cdc42 localization with ESCRT proteins at sites of nuclear envelope and ER fission and, by genetic analysis of cdc42 mutant yeast, uncover a unique Cdc42 function in regulation of ESCRT proteins at the nuclear envelope and sites of ER tubule fission. Our findings implicate Cdc42 in nuclear envelope sealing and ER remodeling, where it regulates ESCRT disassembly to maintain nuclear envelope integrity and proper ER architecture., (© 2020 Lu and Drubin.)

Mitosis has been traditionally considered a metabolically inactive phase. We have previously shown, however, that extensive alterations in lipids occur as the cells traverse mitosis, including increased de novo fatty acid (FA) and phosphatidylcholine (PtdCho) synthesis and decreased lysophospholipid content. Given the diverse structural and functional properties of these lipids, we sought to study their metabolic fate and their importance for cell cycle completion. Here we show that FA and PtdCho synthesized at the mitotic exit are destined to the nuclear envelope. Importantly, FA and PtdCho synthesis, but not the decrease in lysophospholipid content, are necessary for cell cycle completion beyond G 2 /M. Moreover, the presence of alternative pathways for PtdCho synthesis renders the cells less sensitive to its inhibition than to the impairment of FA synthesis. FA synthesis, thus, represents a cell cycle-related metabolic vulnerability that could be exploited for combined chemotherapy. We explored the combination of fatty acid synthase (FASN) inhibition with agents that act at different phases of the cell cycle. Our results show that the effect of FASN inhibition may be enhanced under some drug combinations.

The integrity of the nuclear membranes coupled to the selective barrier of nuclear pore complexes (NPCs) are essential for the segregation of nucleoplasm and cytoplasm. Mechanical membrane disruption or perturbation to NPC assembly triggers an ESCRT-dependent surveillance system that seals nuclear pores: how these pores are sensed and sealed is ill defined. Using a budding yeast model, we show that the ESCRT Chm7 and the integral inner nuclear membrane (INM) protein Heh1 are spatially segregated by nuclear transport, with Chm7 being actively exported by Xpo1/Crm1. Thus, the exposure of the INM triggers surveillance with Heh1 locally activating Chm7. Sites of Chm7 hyperactivation show fenestrated sheets at the INM and potential membrane delivery at sites of nuclear envelope herniation. Our data suggest that perturbation to the nuclear envelope barrier would lead to local nuclear membrane remodeling to promote membrane sealing. Our findings have implications for disease mechanisms linked to NPC assembly and nuclear envelope integrity., Competing Interests: DT, MA, SB, PR, MB, CL No competing interests declared, (© 2019, Thaller et al.)

As a dividing cell exits mitosis and daughter cells enter interphase, many proteins must be dephosphorylated. The protein phosphatase 2A (PP2A) with its B55 regulatory subunit plays a crucial role in this transition, but the identity of its substrates and how their dephosphorylation promotes mitotic exit are largely unknown. We conducted a maternal-effect screen in Drosophila melanogaster to identify genes that function with PP2A-B55/Tws in the cell cycle. We found that eggs that receive reduced levels of Tws and of components of the nuclear envelope (NE) often fail development, concomitant with NE defects following meiosis and in syncytial mitoses. Our mechanistic studies using Drosophila cells indicate that PP2A-Tws promotes nuclear envelope reformation (NER) during mitotic exit by dephosphorylating BAF and suggests that PP2A-Tws targets additional NE components, including Lamin and Nup107. This work establishes Drosophila as a powerful model to further dissect the molecular mechanisms of NER and suggests additional roles of PP2A-Tws in the completion of meiosis and mitosis., (© 2018 Mehsen et al.)

In metazoans, the nuclear envelope (NE) breakdown (NEBD) occurs during "open" mitosis and meiosis. In the fission yeast Schizosaccharomyces pombe, the mitosis and the first meiotic division (MI) are "closed," during which the NE is maintained. Intriguingly, during the second meiotic division (MII), the NE is also maintained, but nuclear and cytoplasmic molecules are mixed similarly to open mitosis, a phenomenon of unknown biological significance called "virtual" NEBD (vNEBD). Here, we show that importin-α-dependent nucleocytoplasmic transport regulates spindle disassembly late in anaphase B at MI, as previously reported for mitosis. At MII, however, spindle dissolution is triggered by vNEBD early in anaphase B, a mechanism that short-circuits the nucleocytoplasmic transport system. We demonstrate that the sequential action of these two spindle disassembly systems regulates the spatiotemporal order and ploidy of the meiotic products., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Self-assembly on target membranes is one of the important properties of all dynamin family proteins. Drp6, a dynaminrelated protein in Tetrahymena, controls nuclear remodelling and undergoes cycles of assembly/disassembly on the nuclear envelope. To elucidate the mechanism of Drp6 function, we have characterized its biochemical and biophysical properties using size exclusion chromatography, chemical cross-linking and electron microscopy. The results demonstrate that Drp6 readily forms high-molecular-weight self-assembled structures as determined by size exclusion chromatography and chemical cross-linking. Negative stain electron microscopy revealed that Drp6 assembles into rings and spirals at physiological ionic strength. We have also shown that the recombinant Drp6 expressed in bacteria is catalytically active and its GTPase activity is not enhanced by low salt. These results suggest that, in contrast to dynamins but similar to MxA, Drp6 self-assembles in the absence of membrane templates, and its GTPase activity is not affected by ionic strength of the buffer. We discuss the self-assembly structure of Drp6 and explain the basis for lack of membrane-stimulated GTPase activity.

The nuclear envelope (NE) is a unique topological structure formed by lipid membranes (Inner and Outer Membrane: IM and OM) interrupted by open channels (Nuclear Pore complexes). Besides its well-established structural role in providing a physical separation between the genome and the cytoplasm and regulating the exchanges between the two cellular compartments, it has become quite evident in recent years that the NE also represents a hub for localized signal transduction. Mechanical, stress, or mitogen signals reach the nucleus and trigger the activation of several pathways, many effectors of which are processed at the NE. Therefore, the concept of the NE acting just as a barrier needs to be expanded to embrace all the dynamic processes that are indeed associated with it. In this context, dynamic protein association and turnover coupled to reversible post-translational modifications of NE components can provide important clues on how this integrated cellular machinery functions as a whole. Reversible protein phosphorylation is the most used mechanism to control protein dynamics and association in cells. Keys to the reversibility of the system are protein phosphatases and the regulation of their activity in space and time. As the NE is clearly becoming an interesting compartment for the control and transduction of several signalling pathways, in this review we will focus on the role of Protein Phosphatases at the NE since the significance of this class of proteins in this context has been little explored., (© 2018 The Author(s).)

Kinetochores (KTs) are large multiprotein complexes that constitute the interface between centromeric chromatin and the mitotic spindle during chromosome segregation. In spite of their essential role, little is known about how centromeres and KTs are assembled and how their precise stoichiometry is regulated. In this study, we show that the nuclear pore basket component Alm1 is required to maintain both the proteasome and its anchor, Cut8, at the nuclear envelope, which in turn regulates proteostasis of certain inner KT components. Consistently, alm1 -deleted cells show increased levels of KT proteins, including CENP-C Cnp3 , spindle assembly checkpoint activation, and chromosome segregation defects. Our data demonstrate a novel function of the nucleoporin Alm1 in proteasome localization required for KT homeostasis., (© 2017 Salas-Pino et al.)

Interleukin-1 mediates inflammation and stress response through nuclear activity of p38α. Although IL-1 receptor is not degraded, p38α activation is transient. IL-1 also causes cell migration and EMT by modulating cell-cell junctions. Although molecules involved in p38 activation are known, mechanism of the transient nuclear response and its basal activity remains unknown. By mathematical modeling of IL1/p38 signaling network, we show that IL-1 induces robust p38α activation both in the nucleus and in the cytoplasm/membrane. While nuclear response consists of an acute phase, membrane response resembles a step change. Following stimulation, p38α activity returns to a basal level in absence of receptor degradation. While nuclear pulse is controlled by MKP1 through a negative feedback to pp38, its basal activity is controlled by both TAB1 and MKP1 through a positive feedback loop. Our model provides insight into the mechanism of p38α activation, reason for its transient nuclear response, and explanation of the basal activity of MKK3/6 and p38α, which has been experimentally observed by other groups.

Phosphatidylcholine (PC) is the main constituent of mammalian cell membranes. Consequently, preservation of membrane PC content and composition - PC homeostasis - is crucial to maintain cellular life. PC biosynthetic pathway is generally controlled by CTP:phosphocholine cytidylyltransferase (CCT), which is considered the rate-limiting enzyme. CCTα is an amphitropic protein, whose enzymatic activity is commonly associated with endoplasmic reticulum redistribution. However, most of the enzyme is located inside the nuclei. Here, we demonstrate that CCTα is the most abundant isoform in renal collecting duct cells, and its redistribution is dependent on endogenous prostaglandins. Previously we have demonstrated that PC synthesis was inhibited by indomethacin (Indo) treatment, and this effect was reverted by exogenous PGD(2). In this work we found that Indo induced CCTα distribution into intranuclear Lamin A/C foci. Exogenous PGD(2) reverted this effect by inducing CCTα redistribution to nuclear envelope, suggesting that PGD(2) maintains PC synthesis by CCTα mobilization. Interestingly, we found that the effect of PGD(2) was dependent on ERK1/2 activation. In conclusion, our previous observations and the present results lead us to suggest that papillary cells possess the ability to maintain their structural integrity through the synthesis of their own survival molecule, PGD(2), by modulating CCTα intracellular location., (Copyright © 2016. Published by Elsevier B.V.)

The reversible association of CTP:phosphocholine cytidylyltransferase α (CCTα) with membranes regulates the synthesis of phosphatidylcholine (PC) by the CDP-choline (Kennedy) pathway. Based on results with insect CCT homologues, translocation of nuclear CCTα onto cytoplasmic lipid droplets (LDs) is proposed to stimulate the synthesis of PC that is required for LD biogenesis and triacylglycerol (TAG) storage. We examined whether this regulatory mechanism applied to LD biogenesis in mammalian cells. During 3T3-L1 and human preadipocyte differentiation, CCTα expression and PC synthesis was induced. In 3T3-L1 cells, CCTα translocated from the nucleoplasm to the nuclear envelope and cytosol but did not associate with LDs. The enzyme also remained in the nucleus during human adipocyte differentiation. RNAi silencing in 3T3-L1 cells showed that CCTα regulated LD size but did not affect TAG storage or adipogenesis. LD biogenesis in nonadipocyte cell lines treated with oleate also promoted CCTα translocation to the nuclear envelope and/or cytoplasm but not LDs. In rat intestinal epithelial cells, CCTα silencing increased LD size, but LD number and TAG deposition were decreased due to oleate-induced cytotoxicity. We conclude that CCTα increases PC synthesis for LD biogenesis by translocation to the nuclear envelope and not cytoplasmic LDs., (© 2015 Aitchison et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Phosphatidylinositol 4,5-bisphosphate hydrolysis at the plasma membrane by phospholipase C is one of the major hormone regulated intracellular signaling systems. The system generates the diffusible second messenger IP3 and the membrane bound messenger diacylglycerol. Spatial regulation of this system has been thought to be through specific subcellular distributions of the IP3 receptor or PKC. As is becoming increasingly apparent, receptor-stimulated signaling systems are also found at intracellular membranes. As discussed in this issue, G protein-coupled receptors have been identified at the nuclear envelope implying intracellular localization of the signaling systems that respond to G protein-coupled receptors. Here, we discuss the evidence for the existence of PLC signals that regulate nuclear processes, as well as the evidence for nuclear and nuclear envelope localization of PLC signaling components, and their implications for cardiac physiology and disease.

The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression. The outer nuclear membrane is continuous with the endoplasmic reticulum and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by endoplasmic-reticulum-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc7 (refs 2, 3). However, little is known about protein quality control at the INM. Here we describe a protein degradation pathway at the INM in yeast (Saccharomyces cerevisiae) mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi3 (ref. 4). We report that the Asi complex functions together with the ubiquitin-conjugating enzymes Ubc6 and Ubc7 to degrade soluble and integral membrane proteins. Genetic evidence suggests that the Asi ubiquitin ligase defines a pathway distinct from, but complementary to, ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquitin ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalized integral membrane proteins, thus acting to maintain and safeguard the identity of the INM.

The ubiquitin (Ub)-conjugating enzyme, Ubc13, has been known to be involved in error-free DNA damage tolerance (or post-replication repair) via catalyzing Lys63-linked polyubiquitin chains formation together with a Ubc variant. However, its functions remain largely unknown in plant species, especially in monocotyledons. In this study, we cloned a Ub-conjugating enzyme, OsUbc13, that shares the conserved domain of Ubc with AtUBC13B in Oryza sativa L., which encodes a protein of 153 amino acids; the deduced sequence shares high similarities with other homologs. Real-time quantitative polymerase chain reaction (PCR) indicated that OsUbc13 transcripts could be detected in all tissues examined, and the expression level was higher in palea, pistil, stamen, and leaf, and lower in root, stem, and lemma; the expression of OsUbc13 was induced by low temperature, methylmethane sulfate (MMS), and H(2)O(2), but repressed by mannitol, abscisic acid (ABA), and NaCl. OsUbc13 was probably localized in the plasma and nuclear membranes. About 20 proteins, which are responsible for the positive yeast two-hybrid interaction of OsUbc13, were identified. These include the confirmed OsVDAC (correlated with apoptosis), OsMADS1 (important for development of floral organs), OsB22EL8 (related to reactive oxygen species (ROS) scavenging and DNA protection), and OsCROC-1 (required for formation of Lys63 polyubiquitylation and error-free DNA damage tolerance). The molecular characterization provides a foundation for the functional study of OsUbc13.

Parvoviruses are serious pathogens but also serve as platforms for gene therapy or for using their lytic activity in experimental cancer treatment. Despite of their growing importance during the last decade little is known on how the viral genome is transported into the nucleus of the infected cell, which is crucial for replication. As nucleic acids are not karyophilic per se nuclear import must be driven by proteins attached to the viral genome. In turn, presence and conformation of these proteins depend upon the entry pathway of the virus into the cell. This review focuses on the trafficking of the parvoviral genome from the cellular periphery to nucleus. Despite of the uncertainties in knowledge about the entry pathway we show that parvoviruses developed a unique strategy to pass the nuclear envelope by hijacking enzymes involved in mitosis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Disassembly of the nuclear lamina is essential in mitosis and apoptosis requiring multiple coordinated enzymatic activities in nucleus and cytoplasm. Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases. Here we used the ability of parvoviruses to induce nuclear membrane breakdown to understand the triggers of key mitotic enzymes. Nuclear envelope disintegration was shown upon infection, microinjection but also upon their application to permeabilized cells. The latter technique also showed that nuclear envelope disintegration was independent upon soluble cytoplasmic factors. Using time-lapse microscopy, we observed that nuclear disassembly exhibited mitosis-like kinetics and occurred suddenly, implying a catastrophic event irrespective of cell- or type of parvovirus used. Analyzing the order of the processes allowed us to propose a model starting with direct binding of parvoviruses to distinct proteins of the nuclear pore causing structural rearrangement of the parvoviruses. The resulting exposure of domains comprising amphipathic helices was required for nuclear envelope disintegration, which comprised disruption of inner and outer nuclear membrane as shown by electron microscopy. Consistent with Ca⁺⁺ efflux from the lumen between inner and outer nuclear membrane we found that Ca⁺⁺ was essential for nuclear disassembly by activating PKC. PKC activation then triggered activation of cdk-2, which became further activated by caspase-3. Collectively our study shows a unique interaction of a virus with the nuclear envelope, provides evidence that a nuclear pool of executing enzymes is sufficient for nuclear disassembly in quiescent cells, and demonstrates that nuclear disassembly can be uncoupled from initial phases of mitosis.

Lipin-1 catalyzes the formation of diacylglycerol from phosphatidic acid. Lipin-1 mutations cause lipodystrophy in mice and acute myopathy in humans. It is heavily phosphorylated, and the yeast ortholog Pah1p becomes membrane-associated and active upon dephosphorylation by the Nem1p-Spo7p membrane complex. A mammalian ortholog of Nem1p is the C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1, formerly "dullard"), but its Spo7p-like partner is unknown, and the need for its existence is debated. Here, we identify the metazoan ortholog of Spo7p, TMEM188, renamed nuclear envelope phosphatase 1-regulatory subunit 1 (NEP1-R1). CTDNEP1 and NEP1-R1 together complement a nem1Δspo7Δ strain to block endoplasmic reticulum proliferation and restore triacylglycerol levels and lipid droplet number. The two human orthologs are in a complex in cells, and the amount of CTDNEP1 is increased in the presence of NEP1-R1. In the Caenorhabditis elegans embryo, expression of nematode CTDNEP1 and NEP1-R1, as well as lipin-1, is required for normal nuclear membrane breakdown after zygote formation. The expression pattern of NEP1-R1 and CTDNEP1 in human and mouse tissues closely mirrors that of lipin-1. CTDNEP1 can dephosphorylate lipins-1a, -1b, and -2 in human cells only in the presence of NEP1-R1. The nuclear fraction of lipin-1b is increased when CTDNEP1 and NEP1-R1 are co-expressed. Therefore, NEP1-R1 is functionally conserved from yeast to humans and functions in the lipin activation pathway.

Protein tyrosine phosphatase (PTP)1B is an abundant non-transmembrane enzyme that plays a major role in regulating insulin and leptin signaling. Recently, we reported that PTP1B is inhibited by sumoylation, and that sumoylated PTP1B accumulates in a perinuclear distribution, consistent with its known localization in the endoplasmic reticulum (ER) and the contiguous outer nuclear membrane. Here, we report that, in addition to its localization at the ER, PTP1B also is found at the inner nuclear membrane, where it is heavily sumoylated. We also find that PTP1B interacts with emerin, an inner nuclear membrane protein that is known to be tyrosine phosphorylated, and that PTP1B expression levels are inversely correlated with tyrosine phosphorylation levels of emerin. PTP1B sumoylation greatly increases as cells approach mitosis, corresponding to the stage where tyrosine phosphorylation of emerin is maximal. In addition, expression of a non-sumoylatable mutant of PTP1B greatly reduced levels of emerin tyrosine phosphorylation. These results suggest that PTP1B regulates the tyrosine phosphorylation of a key inner nuclear membrane protein in a sumoylation- and cell-cycle-dependent manner.

The nuclear envelope (NE) breaks down and reforms during each mitotic cycle. A similar process happens to the sperm NE following fertilisation. The formation of the NE in both these circumstances involves endoplasmic reticulum membranes enveloping the chromatin, but PLCγ-dependent membrane fusion events are also essential. Here we demonstrate the activation of PLCγ by a Src family kinase (SFK1) during NE assembly. We show by time-resolved FRET for the first time the direct in vivo interaction and temporal regulation of PLCγ and SFK1 in sea urchins. As a prerequisite for protein activation, there is a rapid phosphorylation of PLCγ on its Y783 residue in response to GTP in vitro. This phosphorylation is dependent upon SFK activity; thus Y783 phosphorylation and NE assembly are susceptible to SFK inhibition. Y783 phosphorylation is also observed on the surface of the male pronucleus (MPN) in vivo during NE formation. Together the corroborative in vivo and in vitro data demonstrate the phosphorylation and activation of PLCγ by SFK1 during NE assembly. We discuss the potential generality of such a mechanism.

Background: Protein mono-ADP-ribosylation is a reversible post-translational modification that modulates the function of target proteins. The enzymes that catalyze this reaction in mammalian cells are either bacterial pathogenic toxins or endogenous cellular ADP-ribosyltransferases. The latter include members of three different families of proteins: the well characterized arginine-specific ecto-enzymes ARTCs, two sirtuins and, more recently, novel members of the poly(ADP-ribose) polymerase (PARP/ARTD) family that have been suggested to act as cellular mono-ADP-ribosyltransferases. Here, we report on the characterisation of human ARTD15, the only known ARTD family member with a putative C-terminal transmembrane domain., Methodology/principal Findings: Immunofluorescence and electron microscopy were performed to characterise the sub-cellular localisation of ARTD15, which was found to be associated with membranes of the nuclear envelope and endoplasmic reticulum. The orientation of ARTD15 was determined using protease protection assay, and is shown to be a tail-anchored protein with a cytosolic catalytic domain. Importantly, by combining immunoprecipitation with mass spectrometry and using cell lysates from cells over-expressing FLAG-ARTD15, we have identified karyopherin-ß1, a component of the nuclear trafficking machinery, as a molecular partner of ARTD15. Finally, we demonstrate that ARTD15 is a mono-ADP-ribosyltransferase able to induce the ADP-ribosylation of karyopherin-ß1, thus defining the first substrate for this enzyme., Conclusions/significance: Our data reveal that ARTD15 is a novel ADP-ribosyltransferase enzyme with a new intracellular location. Finally, the identification of karyopherin-ß1 as a target of ARTD15-mediated ADP-ribosylation, hints at a novel regulatory mechanism of karyopherin-ß1 functions.

Farnesylated prelamin A accumulates when the final endoproteolytic maturation of the protein fails to occur and causes a dysmorphic nuclear phenotype; however, the morphology and mechanisms of biogenesis of these changes remain unclear. We show here that acute prelamin A accumulation after reduction in the activity of the ZMPSTE24 endoprotease by short interfering RNA knockdown, results in the generation of a complex nucleoplasmic reticulum that depends for its formation on the enzyme CTP:phosphocholine-cytidylyltransferase-α (CCT-α, also known as choline-phosphate cytidylyltransferase A). This structure can form during interphase, confirming that it is independent of mitosis and therefore not a consequence of disordered nuclear envelope assembly. Serial-section dual-axis electron tomography reveals that these invaginations can take two forms: one in which the inner nuclear membrane infolds alone with an inter membrane space interior, and the other in which an invagination of both nuclear membranes occurs, enclosing a cytoplasmic core. Both types of invagination can co-exist in one nucleus and both are frequently studded with nuclear pore complexes (NPC), which reduces NPC abundance on the nuclear surface.

Myotonic dystrophy 1 (DM1) is a multisystemic disease caused by a triplet nucleotide repeat expansion in the 3' untranslated region of the gene coding for myotonic dystrophy protein kinase (DMPK). DMPK is a nuclear envelope (NE) protein that promotes myogenic gene expression in skeletal myoblasts. Muscular dystrophy research has revealed the NE to be a key determinant of nuclear structure, gene regulation, and muscle function. To investigate the role of DMPK in NE stability, we analyzed DMPK expression in epithelial and myoblast cells. We found that DMPK localizes to the NE and coimmunoprecipitates with Lamin-A/C. Overexpression of DMPK in HeLa cells or C2C12 myoblasts disrupts Lamin-A/C and Lamin-B1 localization and causes nuclear fragmentation. Depletion of DMPK also disrupts NE lamina, showing that DMPK is required for NE stability. Our data demonstrate for the first time that DMPK is a critical component of the NE. These novel findings suggest that reduced DMPK may contribute to NE instability, a common mechanism of skeletal muscle wasting in muscular dystrophies.

The aim of this study was to determine the capacity of some progesterone derivatives, to inhibit the conversion of labeled androstenedione ([(3)H] 4-dione) to [(3)H]dihydrotestosterone ([(3)H]DHT) in prostate nuclear membrane fractions, where the 5α-reductase activity is present. The enzyme 5α-reductase catalyzes the 5α-reduction of 4-dione whereas the 17β-hydroxysteroid dehydrogenase catalyzes the transformation of 4-dione to testosterone or 5α-dione to dihydrotestosterone (DHT). Moreover, we also investigated the role of unlabeled 5α-dione in these pathways. In order to determine the inhibitory effect of different concentrations of the progesterone derivatives in the conversion of [(3)H] 4-dione to [(3)H]DHT, homogenates of human prostate were incubated with [(3)H] 4-dione, NADPH and increasing concentrations of non-labeled 5α-dione. The incubating mixture was extracted and purified using thin layer chromatography. The fraction of the chromatogram corresponding to the standard of DHT was separated and the radioactivity determined. The results showed that the presence of [(3)H] 4-dione plus unlabelled 5α-dione produced similar levels of DHT as compared to [(3)H] 4-dione. On the other hand, the results indicated that 17α-hydroxypregn-4-ene-3,20-dione 5 and 4-bromo-17α-hydroxypregn-4-ene-3,20-dione 7b, were the most potent steroids to inhibit the conversion of [(3)H] 4-dione to [(3)H]DHT, showing IC(50) values of 2 and 1.6 nM, respectively.

ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a β-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase β-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS-PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was also characterized by SELDI-TOF mass spectrometry before and after deglycosylation. This allowed us to determine that the carbohydrate moiety of BetaM has molecular mass 5.9kDa and consists of short high-mannose type N-glycans. The results of direct analysis of the purified native eutherian BetaM protein provide first insights into structural properties underlying its entirely new evolutionarily acquired functions., (Copyright © 2011 Elsevier Inc. All rights reserved.)

TorsinA is a membrane-associated enzyme in the endoplasmic reticulum (ER) lumen that is mutated in DYT1 dystonia. How it remains in the ER has been unclear. We report that a hydrophobic N-terminal domain (NTD) directs static retention of torsinA within the ER by excluding it from ER exit sites, as has been previously reported for short transmembrane domains (TMDs). We show that despite the NTD's physicochemical similarity to TMDs, it does not traverse the membrane, defining torsinA as a lumenal monotopic membrane protein and requiring a new paradigm to explain retention. ER retention and membrane association are perturbed by a subset of nonconservative mutations to the NTD, suggesting that a helical structure with defined orientation in the membrane is required. TorsinA preferentially enriches in ER sheets, as might be expected for a lumenal monotopic membrane protein. We propose that the principle of membrane-based protein sorting extends to monotopic membrane proteins, and identify other proteins including the monotopic lumenal enzyme cyclooxygenase 1 (prostaglandin H synthase 1) that share this mechanism of retention with torsinA.

Budding yeast telomeres are reversibly bound at the nuclear envelope through two partially redundant pathways that involve the Sir2/3/4 silencing complex and the Yku70/80 heterodimer. To better understand how this is regulated, we studied the role of SUMOylation in telomere anchoring. We find that the PIAS-like SUMO E3 ligase Siz2 sumoylates both Yku70/80 and Sir4 in vivo and promotes telomere anchoring to the nuclear envelope. Remarkably, loss of Siz2 also provokes telomere extension in a telomerase-dependent manner that is epistatic with loss of the helicase Pif1. Consistent with our previously documented role for telomerase in anchorage, normal telomere anchoring in siz2 Δ is restored by PIF1 deletion. By live-cell imaging of a critically short telomere, we show that telomeres shift away from the nuclear envelope when elongating. We propose that SUMO-dependent association with the nuclear periphery restrains bound telomerase, whereas active elongation correlates with telomere release.

Nuclear fragmentation is a common feature in many neurodegenerative diseases, including Alzheimer's disease (AD). In this study, we show that nuclear lamina dispersion is an early and irreversible trigger for cell death initiated by deregulated Cdk5, rather than a consequence of apoptosis. Cyclin-dependent kinase 5 (Cdk5) activity is significantly increased in AD and contributes to all three hallmarks: neurotoxic amyloid-β (Aβ), neurofibrillary tangles (NFT), and extensive cell death. Using Aβ and glutamate as the neurotoxic stimuli, we show that deregulated Cdk5 induces nuclear lamina dispersion by direct phosphorylation of lamin A and lamin B1 in neuronal cells and primary cortical neurons. Phosphorylation-resistant mutants of lamins confer resistance to nuclear dispersion and cell death on neurotoxic stimulation, highlighting this as a major mechanism for neuronal death. Rapid alteration of lamin localization pattern and nuclear membrane change are further supported by in vivo data using an AD mouse model. After p25 induction, the pattern of lamin localization was significantly altered, preceding neuronal death, suggesting that it is an early pathological event in p25-inducible transgenic mice. Importantly, lamin dispersion is coupled with Cdk5 nuclear localization, which is highly neurotoxic. Inhibition of nuclear dispersion rescues neuronal cells from cell death, underscoring the significance of this event to Cdk5-mediated neurotoxicity.

Isolation and characterization of genes and/or proteins differentially expressed in ovaries are necessary for understanding ovarian development in the giant tiger shrimp (Penaeus monodon). In this study, the full-length cDNA of P. monodon mitogen-activating protein kinase 1 (PmMAPK1) was characterized. PmMAPK1 was 1,398 bp in length containing an open reading frame of 1,098 bp that corresponded to a polypeptide of 365 amino acids. PmMAPK1 was more abundantly expressed in ovaries than in testes of P. monodon. Quantitative real-time PCR revealed differential expression levels of PmMAPK1 mRNA during ovarian development of intact broodstock, where it peaked in early cortical rod (stage III) ovaries (P < 0.05) and slightly decreased afterwards (P > 0.05). Likewise, the expression level of PmMAPK1 in early cortical rod and mature (IV) ovaries was significantly greater than that in previtellogenic (I) and vitellogenic (II) ovaries of eyestalk-ablated broodstock (P < 0.05). The PmMAPK1 transcript was localized in ooplasm of previtellogenic oocytes. In intact broodstock, the expression of the PmMAPK1 protein was clearly increased from previtellogenic ovaries in subsequent stages of ovarian development (P < 0.05). In contrast, the level of ovarian PmMAPK1 protein was comparable during oogenesis in eyestalk-ablated broodstock (P > 0.05). The PmMAPK1 protein was localized in ooplasm of previtellogenic and vitellogenic oocytes. It was also detected around the nuclear membrane of early cortical rod oocytes in both intact and eyestalk-ablated broodstock. Results indicated that PmMAPK1 gene products seem to play functional roles in the development and maturation of oocytes/ovaries in P. monodon., (Copyright © 2011 Wiley-Liss, Inc.)

Recent studies have shown that inositol 1,4,5-trisphosphate 3-kinase isoform B (IP3KB) possesses important roles in the development of immune cells. IP3KB can be targeted to multiple cellular compartments, among them nuclear localization and binding in close proximity to the plasma membrane. The B isoform is the only IP3K that is almost ubiquitously expressed in mammalian cells. Detailed mechanisms of its targeting regulation will be important in understanding the role of Ins(1,4,5)P(3) phosphorylation on subcellular calcium signaling and compartment-specific initiation of pathways leading to regulatory active higher phosphorylated inositol phosphates. Here, we identified an exportin 1-dependent nuclear export signal ((134)LQRELQNVQV) and characterized the amino acids responsible for nuclear localization of IP3KB ((129)RKLR). These two targeting domains regulate the amount of nuclear IP3KB in cells. We also demonstrated that the localization of IP3KB at the plasma membrane is due to its binding to cortical actin structures. Intriguingly, all three of these targeting activities reside in one small polypeptide segment (amino acids 104-165), which acts as a multitargeting domain (MTD). Finally, a hitherto unknown subnuclear localization of IP3KB could be demonstrated in rapidly growing H1299 cells. IP3KB is specifically enriched at nuclear invaginations extending perpendicular between the apical and basal surface of the nucleus of these flat cells. Such nuclear invaginations are known to be involved in Ins(1,4,5)P(3)-mediated Ca(2+) signaling of the nucleus. Our findings indicate that IP3KB not only regulates cytoplasmic Ca(2+) signals by phosphorylation of subplasmalemmal and cytoplasmic Ins(1,4,5)P(3) but may also be involved in modulating nuclear Ca(2+) signals generated from these nuclear envelope invaginations.

The Saccharomyces cerevisiae PAH1-encoded phosphatidate phosphatase (PAP) catalyzes the penultimate step in the synthesis of triacylglycerol and plays a role in the transcriptional regulation of phospholipid synthesis genes. PAP is phosphorylated at multiple Ser and Thr residues and is dephosphorylated for in vivo function by the Nem1p-Spo7p protein phosphatase complex localized in the nuclear/endoplasmic reticulum membrane. In this work, we characterized seven previously identified phosphorylation sites of PAP that are within the Ser/Thr-Pro motif. When expressed on a low copy plasmid, wild type PAP could not complement the pah1Δ mutant in the absence of the Nem1p-Spo7p complex. However, phosphorylation-deficient PAP (PAP-7A) containing alanine substitutions for the seven phosphorylation sites bypassed the requirement of the phosphatase complex and complemented the pah1Δ nem1Δ mutant phenotypes, such as temperature sensitivity, nuclear/endoplasmic reticulum membrane expansion, decreased triacylglycerol synthesis, and derepression of INO1 expression. Subcellular fractionation coupled with immunoblot analysis showed that PAP-7A was highly enriched in the membrane fraction. In fluorescence spectroscopy analysis, the PAP-7A showed tighter association with phospholipid vesicles than wild type PAP. Using site-directed mutagenesis of PAP, we identified Ser(602), Thr(723), and Ser(744), which belong to the seven phosphorylation sites, as the sites phosphorylated by the CDC28 (CDK1)-encoded cyclin-dependent kinase. Compared with the dephosphorylation mimic of the seven phosphorylation sites, alanine substitution for Ser(602), Thr(723), and/or Ser(744) had a partial effect on circumventing the requirement for the Nem1p-Spo7p complex.

Inflammatory myofibroblastic tumor (IMT) is a mesenchymal neoplasm of intermediate biological potential, which may recur and rarely metastasize. Pathologic features do not correlate well with behavior. Approximately 50% of conventional IMTs harbor ALK gene rearrangement and overexpress ALK, most showing diffuse cytoplasmic staining. Rare IMTs with a distinct nuclear membrane or perinuclear pattern of ALK staining and epithelioid or round cell morphology have been reported. These cases pursued an aggressive clinical course, suggesting that such patterns may predict malignant behavior. We describe 11 cases of IMT with epithelioid morphology and a nuclear membrane or perinuclear pattern of immunostaining for ALK. Ten patients were male and 1 was female, ranging from 7 months to 63 years in age (median, 39 y). All tumors were intra-abdominal; most arose in the mesentery or omentum, measuring 8 to 26 cm (median, 15 cm). Six tumors were multifocal at presentation. The tumors were composed predominantly of sheets of round-to-epithelioid cells with vesicular nuclei, large nucleoli, and amphophilic-to-eosinophilic cytoplasm. In all cases, a minor spindle cell component was present. Nine tumors had abundant myxoid stroma. In 7 cases neutrophils were prominent and in 3 cases lymphocytes were prominent. Plasma cells were often absent. Median mitotic rate was 4/10 HPF; 6 tumors had necrosis. By immunohistochemistry, all tumors were positive for ALK, 9 tumors showing a nuclear membrane staining pattern and 2 tumors showing a cytoplasmic pattern with perinuclear accentuation. Other positive markers were desmin (10 of 11), focal smooth muscle actin (4 of 8), and CD30 (8 of 8). All tumors were negative for MYF4, caldesmon, keratins, EMA, and S-100. Fluorescence in situ hybridization was positive for ALK gene rearrangement in 9 cases, and in 3 cases tested, a RANBP2-ALK fusion was detected by reverse transcription polymerase chain reaction. Ten patients underwent surgical resection; 1 patient was inoperable. Follow-up was available for 8 patients and ranged from 3 to 40 months (median, 13 mo). All patients experienced rapid local recurrences; 4 patients had multiple recurrences. Eight patients were treated with postoperative chemotherapy; 2 patients received additional radiotherapy. Two patients also developed metastases (both patients developed metastases to the liver; 1 patient developed metastases to the lung and lymph nodes as well). Thus far, 5 patients died of disease, 2 patients are alive with disease, and 1 patient, treated with an experimental ALK inhibitor, has no evidence of disease. In summary, the epithelioid variant of IMT with nuclear membrane or perinuclear ALK is a distinctive intra-abdominal sarcoma with a predilection for male patients. Unlike conventional IMT, abundant myxoid stroma and prominent neutrophils are common. These tumors pursue an aggressive course with rapid local recurrences and are frequently fatal. We propose the designation "epithelioid inflammatory myofibroblastic sarcoma" to convey both the malignant behavior of these tumors and their close relationship with IMT.

Mutations in the nuclear envelope protein lamin A or in its processing protease ZMPSTE24 cause human accelerated aging syndromes, including Hutchinson-Gilford progeria syndrome. Similarly, Zmpste24-deficient mice accumulate unprocessed prelamin A and develop multiple progeroid symptoms, thus representing a valuable animal model for the study of these syndromes. Zmpste24-deficient mice also show marked transcriptional alterations associated with chromatin disorganization, but the molecular links between both processes are unknown. We report herein that Zmpste24-deficient mice show a hypermethylation of rDNA that reduces the transcription of ribosomal genes, being this reduction reversible upon treatment with DNA methyltransferase inhibitors. This alteration has been previously described during physiological aging in rodents, suggesting its potential role in the development of the progeroid phenotypes. We also show that Zmpste24-deficient mice present global hypoacetylation of histones H2B and H4. By using a combination of RNA sequencing and chromatin immunoprecipitation assays, we demonstrate that these histone modifications are associated with changes in the expression of several genes involved in the control of cell proliferation and metabolic processes, which may contribute to the plethora of progeroid symptoms exhibited by Zmpste24-deficient mice. The identification of these altered genes may help to clarify the molecular mechanisms underlying aging and progeroid syndromes as well as to define new targets for the treatment of these dramatic diseases., (© 2010 The Authors. Aging Cell © 2010 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.)

Many inducible genes in yeast are targeted to the nuclear pore complex when active. We find that the peripheral localization of the INO1 and GAL1 genes is regulated through the cell cycle. Active INO1 and GAL1 localized at the nuclear periphery during G1, became nucleoplasmic during S-phase, and then returned to the nuclear periphery during G2/M. Loss of peripheral targeting followed the initiation of DNA replication and was lost in cells lacking a cyclin-dependent kinase (Cdk) inhibitor. Furthermore, the Cdk1 kinase and two Cdk phosphorylation sites in the nucleoporin Nup1 were required for peripheral targeting of INO1 and GAL1. Introduction of aspartic acid residues in place of either of these two sites in Nup1 bypassed the requirement for Cdk1 and resulted in targeting of INO1 and GAL1 to the nuclear periphery during S-phase. Thus, phosphorylation of a nuclear pore component by cyclin dependent kinase controls the localization of active genes to the nuclear periphery through the cell cycle.

Akt is involved in the regulation of diverse cellular functions such as cell proliferation, energy metabolism, and apoptosis. Although three Akt isoforms are known, the function of each isoform is poorly understood. To gain a better understanding of each Akt isoform, we examined the subcellular localization and expression of each isoform in transformed and nontransformed cells. Akt1 was localized in the cytoplasm, which is in agreement with the currently accepted model that cytoplasmic Akt is translocated and activated at the inner leaflet of the plasma membrane. Interestingly, HEK-293 and HEK-293T cells contained Akt1 in the nucleus and cytoplasm, respectively, suggesting that SV40 T-antigen plays a crucial role in the cytoplasmic localization and activation of Akt1 in HEK-293T. Akt2 was colocalized with the mitochondria, while Akt3 was localized in both the nucleus and nuclear membrane. The subcellular localization of the Akt isoforms was not substantially altered in response to ionizing radiation or EGF. Furthermore, the ablation of one Akt isoform by small interfering RNA (siRNA) did not alter the subcellular location of the remaining isoforms, suggesting that the major function of one isoform is not compensated for by other isoforms. Together, our data support the notion that Akt2 and Akt3 are regulated at the mitochondrial and nuclear membranes, respectively. The mitochondrial localization of Akt2 raises the possibility that this isoform may be involved in both glucose-based energy metabolism and suppression of apoptosis, two Akt functions previously identified with anti-pan-Akt antibodies.

Previous reports indicated the presence of both gangliosides and sialidase in the nuclear envelope (NE) of primary neurons and the NG108-15 neural cell line. GM1, one of the major gangliosides of this membrane, was shown to be tightly associated with a sodium-calcium exchanger in the inner membrane of the NE and to potentiate exchanger activity. GD1a was the other major ganglioside detected in the NE and, like GM1, occurs in both inner and outer membranes. A subsequent report indicated the presence of sialidase activity in the NE without specification as to which of the two membranes express it. The present study was undertaken to determine the nature and locus of this activity within the NE of two cell lines: NG108-15 and SH-SY5Y. Western blot analysis of the separated membranes revealed occurrence of Neu3 in the inner membrane and Neu1 in the outer membrane of the NE. Moreover, sialidase activity at both sites was shown capable of catalyzing conversion of endogenous GD1a to GM1.

The variations of the intracellular localization of the individual protein kinase C (PKC) isoforms are related with their different biological functions. In this study, we have investigated the precise intracellular translocation of endogenous PKCalpha and PKCepsilon in PMA-stimulated normal and tumoral lactotroph cells by using confocal and immunogold electron microscopy, which was correlated with the rate of cell proliferation of both pituitary cell phenotypes. The present results showed that the short phorbol ester incubation stimulated the proliferation of normal and tumoral lactotroph cells, as determined by the measurement of the BrdU-labelling index. The translocation of PKCalpha to plasma and nuclear membranes induced by PMA was more marked than that observed for PKCepsilon in normal and tumoral lactotroph cells. Our results showed that PKCs translocation to the plasma and nuclear membranes varied from isozyme to isozyme emphasizing that PKCalpha could be related with the mitogenic stimulus exerted by phorbol ester. These data support the notion that specific PKC isozymes may exert spatially defined effects by virtue of their directed translocation to distinct intracellular sites.

Trm1 is a tRNA specific m(2)(2)G methyltransferase shared by nuclei and mitochondria in Saccharomyces cerevisiae. In nuclei, Trm1 is peripherally associated with the inner nuclear membrane (INM). We investigated the mechanism delivering/tethering Trm1 to the INM. Analyses of mutations of the Ran pathway and nuclear pore components showed that Trm1 accesses the nucleoplasm via the classical nuclear import pathway. We identified a Trm1 cis-acting sequence sufficient to target passenger proteins to the INM. Detailed mutagenesis of this region uncovered specific amino acids necessary for authentic Trm1 to locate at the INM. The INM information is contained within a sequence of less than 20 amino acids, defining the first motif for addressing a peripheral protein to this important subnuclear location. The combined studies provide a multi-step process to direct Trm1 to the INM: (i) translation in the cytoplasm; (ii) Ran-dependent import into the nucleoplasm; and (iii) redistribution from the nucleoplasm to the INM via the INM motif. Furthermore, we demonstrate that the Trm1 mitochondrial targeting and nuclear localization signals are in competition with each other, as Trm1 becomes mitochondrial if prevented from entering the nucleus.

The induction of cyclooxygenase-2 (COX-2) in tissue macrophages (MØ) increases prostaglandin E(2) (PGE(2)) release, potentially down-regulating granulomatous inflammation. In response to Mycobacteria, local MØ express COX-2, which is either nuclear envelope (NE)-associated or NE-dissociated. Persistent mycobacterial pulmonary inflammation is characterized by alveolar MØ expressing NE-dissociated (inactive) COX-2 without release of PGE(2). In this study, we examined COX-2 in alveolar MØ after intranasal exposure to heat-killed Mycobacterium bovis BCG (HK-BCG). After administration, whole lungs of C57Bl/6 mice were lavaged with saline; COX-2 expression and PGE(2) release by alveolar MØ and tumor necrosis factor (TNF)-alpha and nitric oxide levels in the lung lavage were monitored. Normal alveolar MØ had undetectable levels of COX-2 on Western blots. However, 1 day after intranasal administration, almost all alveolar MØ had phagocytosed HK-BCG and expressed NE-dissociated COX-2 without any increase in the release of PGE(2). At 28 days after intranasal administration, 68% of alveolar MØ still contained both BCG and the NE-dissociated form of COX-2. NE-associated (active) COX-2 was not observed in alveolar MØ. In contrast, 7 days after intraperitoneal injection of HK-BCG, peritoneal MØ containing HK-BCG were no longer detected. At 28 days after intranasal administration, TNF-alpha and nitrite levels in the lung lavage fluid were significantly higher than those in controls. Our results indicate that mycobacterial pulmonary inflammation is associated with suppressed PGE(2) production by alveolar MØ, with expression of COX-2 dissociated from the NE.

The double lipid bilayer of the nuclear envelope (NE) remains intact during closed mitosis. In the fission yeast Schizosaccharomyces pombe, the intranuclear mitotic spindle has envelope-embedded spindle pole bodies (SPB) at its ends. As the spindle elongates and the nucleus divides symmetrically, nuclear volume remains constant but nuclear area rapidly increases by 26%. When Ran-GTPase function is compromised in S. pombe, nuclear division is strikingly asymmetrical and the newly synthesized SPB is preferentially associated with the smaller nucleus, indicative of a Ran-dependent SPB defect that interferes with symmetrical nuclear division. A second defect, which specifically influences the NE, results in breakage of the NE upon spindle elongation. This defect, but not asymmetric nuclear division, is partially rescued by slowing spindle elongation, stimulating endoplasmic reticulum (ER) proliferation or changing conformation of the ER membrane. We propose that redistribution of lipid within the ER-NE network is crucial for mitosis-specific NE changes in both open and closed mitosis.

In addition to suppressing cholesterol synthesis and uptake, oxysterols also activate glycerophospholipid and SM (sphingomyelin) synthesis, possibly to buffer cells from excess sterol accumulation. In the present study, we investigated the effects of oxysterols on the CDP-choline pathway for PtdCho (phosphatidylcholine) synthesis using wild-type and sterol-resistant CHO (Chinese-hamster ovary) cells expressing a mutant of SCAP [SREBP (sterol-regulatory-element-binding protein) cleavage-activating protein] (CHO-SCAP D443N). [(3)H]Choline-labelling experiments showed that 25OH (25-hydroxycholesterol), 22OH (22-hydroxycholesterol) and 27OH (27-hydroxycholesterol) increased PtdCho synthesis in CHO cells as a result of CCTalpha (CTP:phosphocholine cytidylyltransferase alpha) translocation and activation at the NE (nuclear envelope). These oxysterols also activate PtdCho synthesis in J774 macrophages. in vitro, CCTalpha activity was stimulated 2- to 2.5-fold by liposomes containing 5 mol% 25OH, 22OH or 27OH. Inclusion of up to 5 mol% cholesterol did not further activate CCTalpha. 25OH activated CCTalpha in CHO-SCAP D443N cells leading to a transient increase in PtdCho synthesis and accumulation of CDP-choline. CCTalpha translocation to the NE and intranuclear tubules in CHO-SCAP D443N cells was complete after 1 h exposure to 25OH compared with only partial translocation by 4-6 h in CHO-Mock cells. These enhanced responses in CHO-D443N cells were sterol-dependent since depletion with cyclodextrin or lovastatin resulted in reduced sensitivity to 25OH. However, the lack of effect of cholesterol on in vitro CCT activity indicates an indirect relationship or involvement of other sterols or oxysterol. We conclude that translocation and activation of CCTalpha at nuclear membranes by side-chain hydroxylated sterols are regulated by the cholesterol status of the cell.

The link between nucleocytoplasmic transport and apoptosis remains controversial. Nucleocytoplasmic exchange of molecules seems indeed essential for the initiation and execution of the apoptotic programme; but inhibition of nuclear transport factors may also represent a powerful apoptotic trigger. The GTPase Ran (together with its partners), first discovered to be essential in nucleocytoplasmic transport, has multiple key functions in cell biology, and particularly in spindle assembly, kinetochore function and nuclear envelope assembly. Among the Ran partners studied, NTF2 appears to be solely involved in nucleocytoplasmic transport. Here, we localised Ran and several of its partners, RanBP1, CAS and NTF2, at the nuclear membrane in the trypanosomatid Leishmania major. Remarkably, these proteins fused to GFP decorated a perinuclear 'collar' of about 15 dots, colocalising at nuclear pore complexes with the homologue of nucleoporin Sec13. In the other trypanosomatid Trypanosoma brucei, RNAi knockdown of the expression of the corresponding genes resulted in an apoptosis-like phenomenon. These phenotypes show that Ran and its partners have a key function in trypanosomatids like they have in mammals. Our data, notably those about TbNTF2 RNAi, support the idea that active nucleocytoplasmic transport is not essential for the initiation and execution of apoptosis, and, rather, the impairment of this transport constitutes an intrinsic signal for triggering PCD.

Nuclear envelope (NE) and microsomal glucosa-6-phosphatase (G-6-Pase) activities were compared. Intact microsomes were unable to hydrolyze mannose-6-phosphate (M-6-P), on the other hand, intact NE hydrolyzes this substrate. Galactose-6-phosphate showed to be a good substrate for both NE and microsomal enzymes, with similar latency to that obtained with M-6-P using microsomes. In consequence, this substrate was used to measure the NE integrity. The kinetic parameters (Kii and Kis) of the intact NE G-6-Pase for the phlorizin inhibition using glucose-6-phosphate (G-6-P) and M-6-P as substrates, were very similar. The NE T1 transporter was more sensitive to amiloride than the microsomal T1. The microsomal system was more sensitive to N-ethylmalemide (NEM) than the NE and the latter was insensitive to anion transport inhibitors DIDS and SITS, which strongly affect the microsomal enzyme. The above results allowed to postulate the presence of a hexose-6-phosphate transporter in the NE which is able to carry G-6-P and M-6-P, and perhaps other hexose-6-phosphate which could be different from that present in microsomes or, if it is the same, its activity could by modified by the membrane system where it is included. The higher PPi hydrolysis activity of the intact NE G-6-Pase in comparison to the intact microsomal, suggests differences between the Pi/PPi transport (T2) of both systems. The lower sensitivity of the NE G-6-Pase to NEM suggests that the catalytic subunit of this system has some differences with the microsomal isoform.

The nucleoplasmic reticulum (NR), a nuclear membrane network implicated in signaling and transport, is formed by the biosynthetic and membrane curvature-inducing properties of the rate-limiting enzyme in phosphatidylcholine synthesis, CTP:phosphocholine cytidylyltransferase (CCT) alpha. The NR is formed by invagination of the nuclear envelope and has an underlying lamina that may contribute to membrane tubule formation or stability. In this study we investigated the role of lamins A and B in NR formation in response to expression and activation of endogenous and fluorescent protein-tagged CCTalpha. Similarly to endogenous CCTalpha, CCT-green fluorescent protein (GFP) reversibly translocated to nuclear tubules projecting from the NE in response to oleate, a lipid promoter of CCT membrane binding. Coexpression and RNA interference experiments revealed that both CCTalpha and lamin A and B were necessary for NR proliferation. Expression of CCT-GFP mutants with compromised membrane-binding affinity produced fewer nuclear tubules, indicating that the membrane-binding function of CCTalpha promotes the expansion of the NR. Proliferation of atypical bundles of nuclear membrane tubules by a CCTalpha mutant that constitutively associated with membranes revealed that expansion of the double-bilayer NR requires the coordinated assembly of an underlying lamin scaffold and induction of membrane curvature by CCTalpha.

Over 25 years ago, it was observed that peritoneal macrophages (Mphi) isolated from mice given heat-killed Mycobacterium bovis bacillus Calmette-Guérin (HK-BCG) i.p. did not release PGE(2). However, when peritoneal Mphi from untreated mice are treated with HK-BCG in vitro, cyclooxygenase 2 (COX-2), a rate-limiting enzyme for PGE(2) biosynthesis, is expressed and the release of PGE(2) is increased. The present study of peritoneal Mphi obtained from C57BL/6 mice and treated either in vitro or in vivo with HK-BCG was undertaken to further characterize the cellular responses that result in suppression of PGE(2) release. The results indicate that Mphi treated with HK-BCG in vivo express constitutive COX-1 and inducible COX-2 that are catalytically inactive, are localized subcellularly in the cytoplasm, and are not associated with the nuclear envelope (NE). In contrast, Mphi treated in vitro express catalytically active COX-1 and COX-2 that are localized in the NE and diffusely in the cytoplasm. Thus, for local Mphi activated in vivo by HK-BCG, the results indicate that COX-1 and COX-2 dissociated from the NE are catalytically inactive, which accounts for the lack of PGE(2) production by local Mphi activated in vivo with HK-BCG. Our studies further indicate that the formation of catalytically inactive COX-2 is associated with in vivo phagocytosis of HK-BCG, and is not dependent on extracellular mediators produced by in vivo HK-BCG treatment. This attenuation of PGE(2) production may enhance Mphi-mediated innate and Th1-acquired immune responses against intracellular infections which are suppressed by PGE(2).

Sphingoid long-chain base 1-phosphates act as bioactive lipid molecules in eukaryotic cells. In budding yeast, long-chain base 1-phosphates are synthesized mainly by the long-chain base kinase Lcb4. We recently reported that, soon after yeast cells enter into the stationary phase, Lcb4 is rapidly degraded by being delivered to the vacuole in a palmitoylation- and phosphorylation-dependent manner. In this study, we investigated the complete trafficking pathway of Lcb4, from its synthesis to its degradation. After membrane anchoring by palmitoylation at the Golgi apparatus, Lcb4 is delivered to the plasma membrane (PM) through the late Sec pathway and then to the endoplasmic reticulum (ER). The yeast ER consists of a cortical network juxtaposed to the PM (cortical ER) with tubular connections to the nuclear envelope (nuclear ER). Remarkably, the localization of Lcb4 is restricted to the cortical ER. As the cells reach the stationary phase, G(1) cell cycle arrest initiates Lcb4 degradation and its delivery to the vacuole via the Golgi apparatus. The protein transport pathway from the PM to the ER found in this study has not been previously reported. We speculate that this novel pathway is mediated by the PM-ER contact.

Cyclooxygenase-2 (COX-2)-mediated prostaglandin E(2) (PGE(2)) biosynthesis by macrophages downregulates microbicidal activities in innate and acquired immune responses against intracellular bacteria. Previous studies in mice showed that intraperitoneal administration of heat-killed Mycobacterium bovis bacillus Calmette-Guérin (HK-BCG) resulted in induction of splenic PGE(2)-releasing macrophages in 7-14 days. In contrast, HK-BCG induced catalytically inactive COX-2 at relatively high levels in the macrophages within 1 day. In the present study, we found that COX-2 was localized subcellularly in the nuclear envelope (NE) 7 and 14 days after HK-BCG treatment, whereas COX-2 was dissociated from the NE 1 day after treatment. At 1 day after treatment, the majority of COX-2-positive macrophages had phagocytosed HK-BCG. In contrast, no intracellular HK-BCG was detected 7 and 14 days after treatment in COX-2-positive macrophages, where COX-2 was associated with the NE. However, when macrophages phagocytosed HK-BCG in vitro, all COX-2 was associated with the NE. Thus the administration of HK-BCG induces the biphasic COX-2 expression of an NE-dissociated catalytically inactive or an NE-associated catalytically active form in splenic macrophages. The catalytically inactive COX-2-positive macrophages develop microbicidal activities effectively, since they lack PGE(2) biosynthesis.

The mechanism underlying targeting of the nuclear membrane to chromatin at the end of mitosis was studied using an in vitro cell-free system comprising Xenopus egg membrane and cytosol fractions, and sperm chromatin. The mitotic phase membrane, which was separated from a mitotic phase extract of Xenopus eggs and could not bind to chromatin, became able to bind to chromatin on pretreatment with a synthetic phase cytosol fraction of Xenopus eggs. When the cytosol fraction was depleted of protein phosphatase 1 (PP1) with anti-Xenopus PP1gamma1 antibodies, this ability was lost. The addition of recombinant xPP1gamma1 to the PP1-depleted cytosol fraction restored the ability. These and other results suggested that dephosphorylation of mitotic phosphorylation sites on membranes by PP1 in the synthetic phase cytosol fraction promoted targeting of the membranes to chromatin. On the other hand, a fragment containing the chromatin-binding domain of lamin B receptor (LBR) but not emerin inhibited targeting of membrane vesicles. It was also shown that PP1 dephosphorylates a phosphate group(s) responsible for regulation of the binding of LBR to chromatin. A possible mechanism involving PP1 and LBR for the regulation of nuclear membrane targeting to chromatin was discussed.

Background: In the vascular system, elevated levels of reactive oxygen species (ROS) produce oxidative stress and predispose to the development of atherosclerosis. Therefore, it is important to understand the systems producing and those scavenging vascular ROS. Here, we analyzed the ROS-reducing capability of paraoxonase-2 (PON2) in different vascular cells and its involvement in the endoplasmic reticulum stress pathway known as the unfolded protein response., Methods and Results: Quantitative real-time polymerase chain reaction and Western blotting revealed that PON2 is equally expressed in vascular cells and appears in 2 distinct glycosylated isoforms. By determining intracellular ROS, we show that overexpression of PON2 markedly reduced ROS, whereas its knockdown increased ROS levels significantly. Using microscopic and biochemical methods, we found PON2 mainly in the nuclear membrane and endoplasmic reticulum. Furthermore, PON2 expression was induced at both the promoter and protein levels by endoplasmic reticulum stress pathway unfolded protein response. This pathway may promote both apoptotic and survival mechanisms. Functionally, PON2 reduced unfolded protein response-accompanying oxidative stress and unfolded protein response-derived caspase activation., Conclusions: We suggest that PON2 represents an endogenous defense mechanism against vascular oxidative stress and unfolded protein response-induced cell death, thereby contributing to the prevention of atherosclerosis.