An anaphase surveillance mechanism prevents micronuclei formation from frequent chromosome segregation errors.

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.

Cdc42 GTPase regulates ESCRTs in nuclear envelope sealing and ER remodeling.

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.

De novo lipogenesis at the mitotic exit is used for nuclear envelope reassembly/expansion. Implications for combined chemotherapy.

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 G2/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.

An ESCRT-LEM protein surveillance system is poised to directly monitor the nuclear envelope and nuclear transport system.

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.

PP2A-B55 promotes nuclear envelope reformation after mitosis in Drosophila.

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.

Importin α and vNEBD Control Meiotic Spindle Disassembly in Fission Yeast.

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.

Tetrahymena dynamin-related protein 6 self-assembles independent of membrane association.

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.

Protein phosphatases at the nuclear envelope.

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.

The fission yeast nucleoporin Alm1 is required for proteasomal degradation of kinetochore components.

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-CCnp3, 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.

Model Predicts That MKP1 and TAB1 Regulate p38α Nuclear Pulse and Its Basal Activity through Positive and Negative Feedback Loops in Response to IL-1.

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.

Participation of prostaglandin D2 in the mobilization of the nuclear-localized CTP:phosphocholine cytidylyltransferase alpha in renal epithelial cells.

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.

Nuclear-localized CTP:phosphocholine cytidylyltransferase α regulates phosphatidylcholine synthesis required for lipid droplet biogenesis.

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.

Regulation of phosphatidylinositol-specific phospholipase C at the nuclear envelope in cardiac myocytes.

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.

Protein quality control at the inner nuclear membrane.

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.

Molecular identification and interaction assay of the gene (OsUbc13) encoding a ubiquitin-conjugating enzyme in rice.

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.

Interaction of parvoviruses with the nuclear envelope.

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.

Parvoviruses cause nuclear envelope breakdown by activating key enzymes of mitosis.

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.

Dynamics of PLCγ and Src family kinase 1 interactions during nuclear envelope formation revealed by FRET-FLIM.

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.

PARP16/ARTD15 is a novel endoplasmic-reticulum-associated mono-ADP-ribosyltransferase that interacts with, and modifies karyopherin-ß1.

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.

Sumoylated protein tyrosine phosphatase 1B localizes to the inner nuclear membrane and regulates the tyrosine phosphorylation of emerin.

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.