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Sample records for pore complexes harbor

  1. Nuclear pores. Architecture of the nuclear pore complex coat.

    PubMed

    Stuwe, Tobias; Correia, Ana R; Lin, Daniel H; Paduch, Marcin; Lu, Vincent T; Kossiakoff, Anthony A; Hoelz, André

    2015-03-01

    The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. Despite half a century of structural characterization, the architecture of the NPC remains unknown. Here we present the crystal structure of a reconstituted ~400-kilodalton coat nucleoporin complex (CNC) from Saccharomyces cerevisiae at a 7.4 angstrom resolution. The crystal structure revealed a curved Y-shaped architecture and the molecular details of the coat nucleoporin interactions forming the central "triskelion" of the Y. A structural comparison of the yeast CNC with an electron microscopy reconstruction of its human counterpart suggested the evolutionary conservation of the elucidated architecture. Moreover, 32 copies of the CNC crystal structure docked readily into a cryoelectron tomographic reconstruction of the fully assembled human NPC, thereby accounting for ~16 megadalton of its mass. PMID:25745173

  2. Viral Subversion of the Nuclear Pore Complex

    PubMed Central

    Le Sage, Valerie; Mouland, Andrew J.

    2013-01-01

    The nuclear pore complex (NPC) acts as a selective barrier between the nucleus and the cytoplasm and is responsible for mediating communication by regulating the transport of RNA and proteins. Numerous viral pathogens have evolved different mechanisms to hijack the NPC in order to regulate trafficking of viral proteins, genomes and even capsids into and out of the nucleus thus promoting virus replication. The present review examines the different strategies and the specific nucleoporins utilized during viral infections as a means of promoting their life cycle and inhibiting host viral defenses. PMID:23959328

  3. Physical modelling of the nuclear pore complex

    PubMed Central

    Fassati, Ariberto; Ford, Ian J.; Hoogenboom, Bart W.

    2013-01-01

    Physically interesting behaviour can arise when soft matter is confined to nanoscale dimensions. A highly relevant biological example of such a phenomenon is the Nuclear Pore Complex (NPC) found perforating the nuclear envelope of eukaryotic cells. In the central conduit of the NPC, of ∼30–60 nm diameter, a disordered network of proteins regulates all macromolecular transport between the nucleus and the cytoplasm. In spite of a wealth of experimental data, the selectivity barrier of the NPC has yet to be explained fully. Experimental and theoretical approaches are complicated by the disordered and heterogeneous nature of the NPC conduit. Modelling approaches have focused on the behaviour of the partially unfolded protein domains in the confined geometry of the NPC conduit, and have demonstrated that within the range of parameters thought relevant for the NPC, widely varying behaviour can be observed. In this review, we summarise recent efforts to physically model the NPC barrier and function. We illustrate how attempts to understand NPC barrier function have employed many different modelling techniques, each of which have contributed to our understanding of the NPC.

  4. Purification of the Vertebrate Nuclear Pore Complex by Biochemical Criteria

    PubMed Central

    Miller, Brian R.; Forbes, Douglass J.

    2015-01-01

    The nuclear pore is a large and complex biological machine, mediating all signal-directed transport between the nucleus and the cytoplasm. The vertebrate pore has a mass of ~120 million daltons or 30 times the size of a ribosome. The large size of the pore, coupled to its tight integration in the nuclear lamina, has hampered the isolation of pore complexes from vertebrate sources. We have now developed a strategy for the purification of nuclear pores from in vitro assembled annulate lamellae (AL), a cytoplasmic mimic of the nuclear envelope that lacks a lamina, nuclear matrix, and chromatin-associated proteins. We find that purified pore complexes from annulate lamellae contain every nuclear pore protein tested. In addition, immunoblotting reveals the presence of soluble transport receptors and factors known to play important roles in the transport of macromolecules through the pore. While transport factors such as Ran and NTF2 show only transient interaction with the pores, a number of soluble transport receptors, including importin β, show a tight association with the purified pores. In summary, we report that we have purified the vertebrate pore by biochemical criteria; silver staining reveals ~40–50 distinct protein bands. PMID:11208084

  5. Gating Immunity and Death at the Nuclear Pore Complex.

    PubMed

    Dasso, Mary; Fontoura, Beatriz M A

    2016-09-01

    The nuclear pore complex is the primary conduit for nuclear import and export of molecules. In this issue, Gu et al. uncover a novel mechanism in which immune signaling and programmed cell death require nuclear pore rearrangement and release of sequestered cyclin-dependent kinase inhibitors to elicit immunity and death. PMID:27610561

  6. In situ structural analysis of the human nuclear pore complex.

    PubMed

    von Appen, Alexander; Kosinski, Jan; Sparks, Lenore; Ori, Alessandro; DiGuilio, Amanda L; Vollmer, Benjamin; Mackmull, Marie-Therese; Banterle, Niccolo; Parca, Luca; Kastritis, Panagiotis; Buczak, Katarzyna; Mosalaganti, Shyamal; Hagen, Wim; Andres-Pons, Amparo; Lemke, Edward A; Bork, Peer; Antonin, Wolfram; Glavy, Joseph S; Bui, Khanh Huy; Beck, Martin

    2015-10-01

    Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ~60 nm in diameter. The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human nuclear pore complex to date. Our data suggest previously unknown protein interfaces across Y complexes and to inner-ring complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block--although compositionally identical--engage in different local sets of interactions and conformations. PMID:26416747

  7. The Nuclear Pore Complex as a Flexible and Dynamic Gate.

    PubMed

    Knockenhauer, Kevin E; Schwartz, Thomas U

    2016-03-10

    Nuclear pore complexes (NPCs) perforate the nuclear envelope and serve as the primary transport gates for molecular exchange between nucleus and cytoplasm. Stripping the megadalton complex down to its most essential organizational elements, one can divide the NPC into scaffold components and the disordered elements attached to them that generate a selective barrier between compartments. These structural elements exhibit flexibility, which may hold a clue in understanding NPC assembly and function. Here we review the current status of NPC research with a focus on the functional implications of its structural and compositional heterogeneity. PMID:26967283

  8. Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells

    PubMed Central

    Raghunayakula, Sarita; Subramonian, Divya; Dasso, Mary; Kumar, Rita; Zhang, Xiang-Dong

    2015-01-01

    Annulate lamellae are cytoplasmic organelles containing stacked sheets of membranes embedded with pore complexes. These cytoplasmic pore complexes at annulate lamellae are morphologically similar to nuclear pore complexes at the nuclear envelope. Although annulate lamellae has been observed in nearly all types of cells, their biological functions are still largely unknown. Here we show that SUMO1-modification of the Ran GTPase-activating protein RanGAP1 not only target RanGAP1 to its known sites at nuclear pore complexes but also to annulate lamellae pore complexes through interactions with the Ran-binding protein RanBP2 and the SUMO-conjugating enzyme Ubc9 in mammalian cells. Furthermore, upregulation of annulate lamellae, which decreases the number of nuclear pore complexes and concurrently increases that of annulate lamellae pore complexes, causes a redistribution of nuclear transport receptors including importin α/β and the exportin CRM1 from nuclear pore complexes to annulate lamellae pore complexes and also reduces the rates of nuclear import and export. Moreover, our results reveal that importin α/β-mediated import complexes initially accumulate at annulate lamellae pore complexes upon the activation of nuclear import and subsequently disassociate for nuclear import through nuclear pore complexes in cells with upregulation of annulate lamellae. Lastly, CRM1-mediated export complexes are concentrated at both nuclear pore complexes and annulate lamellae pore complexes when the disassembly of these export complexes is inhibited by transient expression of a Ran GTPase mutant arrested in its GTP-bound form, suggesting that RanGAP1/RanBP2-activated RanGTP hydrolysis at these pore complexes is required for the dissociation of the export complexes. Hence, our findings provide a foundation for further investigation of how upregulation of annulate lamellae decreases the rates of nuclear transport and also for elucidation of the biological significance of the

  9. Mitogen activated protein kinase at the nuclear pore complex

    PubMed Central

    Faustino, Randolph S; Maddaford, Thane G; Pierce, Grant N

    2011-01-01

    Abstract Mitogen activated protein (MAP) kinases control eukaryotic proliferation, and import of kinases into the nucleus through the nuclear pore complex (NPC) can influence gene expression to affect cellular growth, cell viability and homeostatic function. The NPC is a critical regulatory checkpoint for nucleocytoplasmic traffic that regulates gene expression and cell growth, and MAP kinases may be physically associated with the NPC to modulate transport. In the present study, highly enriched NPC fractions were isolated and investigated for associated kinases and/or activity. Endogenous kinase activity was identified within the NPC fraction, which phosphorylated a 30 kD nuclear pore protein. Phosphomodification of this nucleoporin, here termed Nup30, was inhibited by apigenin and PD-98059, two MAP kinase antagonists as well as with SB-202190, a pharmacological blocker of p38. Furthermore, high throughput profiling of enriched NPCs revealed constitutive presence of all members of the MAP kinase family, extracellular regulated kinases (ERK), p38 and Jun N-terminal kinase. The NPC thus contains a spectrum of associated MAP kinases that suggests an intimate role for ERK and p38 in regulation of nuclear pore function. PMID:20497490

  10. The Structure Inventory of the Nuclear Pore Complex.

    PubMed

    Schwartz, Thomas U

    2016-05-22

    The nuclear pore complex (NPC) is the principal gateway for molecular exchange between nucleus and cytoplasm across the nuclear envelope. Due to its sheer size of estimated 50-112MDa and its complex buildup from about 500-1000 individual proteins, it is a difficult object to study for structural biologists. Here, I review the extensive ensemble of high-resolution structures of the building blocks of the NPC. Concurrent with the increase in size and complexity, these latest, large structures and assemblies can now be used as the basis for hybrid approaches, primarily in combination with cryo-electron microscopic analysis, generating the first structure-based assembly models of the NPC. Going forward, the structures will be critically important for a detailed analysis of the NPC, including function, evolution, and assembly. PMID:27016207

  11. In situ structural analysis of the human nuclear pore complex

    PubMed Central

    Ori, Alessandro; DiGuilio, Amanda L.; Vollmer, Benjamin; Mackmull, Marie-Therese; Banterle, Niccolo; Parca, Luca; Kastritis, Panagiotis; Buczak, Katarzyna; Mosalaganti, Shyamal; Hagen, Wim; Andres-Pons, Amparo; Lemke, Edward A.; Bork, Peer; Antonin, Wolfram; Glavy, Joseph S.; Bui, Khanh Huy; Beck, Martin

    2016-01-01

    Summary Nuclear pore complexes (NPCs) are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Elucidating their 110 MDa structure imposes a formidable challenge and requires in situ structural biology approaches. Fifteen out of about thirty nucleoporins (Nups) are structured and form the Y- and inner ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ∼60 nm in diameter 1. The scaffold is decorated with transport channel Nups that often contain phenylalanine (FG)-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y-complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here, we combined cryo electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modeling to generate the most comprehensive architectural model of the NPC to date. Our data suggest previously unknown protein interfaces across Y-complexes and to inner ring complex members. We demonstrate that the higher eukaryotic transport channel Nup358 (RanBP2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport channel Nups. We conclude that, similarly to coated vesicles, multiple copies of the same structural building block - although compositionally identical - engage in different local sets of interactions and conformations. PMID:26416747

  12. Architecture of the fungal nuclear pore inner ring complex.

    PubMed

    Stuwe, Tobias; Bley, Christopher J; Thierbach, Karsten; Petrovic, Stefan; Schilbach, Sandra; Mayo, Daniel J; Perriches, Thibaud; Rundlet, Emily J; Jeon, Young E; Collins, Leslie N; Huber, Ferdinand M; Lin, Daniel H; Paduch, Marcin; Koide, Akiko; Lu, Vincent; Fischer, Jessica; Hurt, Ed; Koide, Shohei; Kossiakoff, Anthony A; Hoelz, André

    2015-10-01

    The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. We present the reconstitution and interdisciplinary analyses of the ~425-kilodalton inner ring complex (IRC), which forms the central transport channel and diffusion barrier of the NPC, revealing its interaction network and equimolar stoichiometry. The Nsp1•Nup49•Nup57 channel nucleoporin heterotrimer (CNT) attaches to the IRC solely through the adaptor nucleoporin Nic96. The CNT•Nic96 structure reveals that Nic96 functions as an assembly sensor that recognizes the three-dimensional architecture of the CNT, thereby mediating the incorporation of a defined CNT state into the NPC. We propose that the IRC adopts a relatively rigid scaffold that recruits the CNT to primarily form the diffusion barrier of the NPC, rather than enabling channel dilation. PMID:26316600

  13. Architecture of the nuclear pore inner ring complex

    PubMed Central

    Stuwe, Tobias; Bley, Christopher J.; Thierbach, Karsten; Petrovic, Stefan; Schilbach, Sandra; Mayo, Daniel J.; Perriches, Thibaud; Rundlet, Emily J.; Jeon, Young E.; Collins, Leslie N.; Huber, Ferdinand M.; Lin, Daniel H.; Paduch, Marcin; Koide, Akiko; Lu, Vincent; Fischer, Jessica; Hurt, Ed; Koide, Shohei; Kossiakoff, Anthony A.; Hoelz, André

    2016-01-01

    The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. We present the reconstitution and interdisciplinary analyses of the ~425-kDa inner ring complex (IRC), which forms the central transport channel and diffusion barrier of the NPC, revealing its interaction network and equimolar stoichiometry. The Nsp1•Nup49•Nup57 channel nucleoporin hetero-trimer (CNT) attaches to the IRC solely through the adaptor nucleoporin Nic96. The CNT•Nic96 structure reveals that Nic96 functions as an assembly sensor that recognizes the three dimensional architecture of the CNT, thereby mediating the incorporation of a defined CNT state into the NPC. We propose that the IRC adopts a relatively rigid scaffold that recruits the CNT to primarily form the diffusion barrier of the NPC, rather than enabling channel dilation. PMID:26316600

  14. Energetics of Transport through the Nuclear Pore Complex.

    PubMed

    Ghavami, Ali; van der Giessen, Erik; Onck, Patrick R

    2016-01-01

    Molecular transport across the nuclear envelope in eukaryotic cells is solely controlled by the nuclear pore complex (NPC). The NPC provides two types of nucleocytoplasmic transport: passive diffusion of small molecules and active chaperon-mediated translocation of large molecules. It has been shown that the interaction between intrinsically disordered proteins that line the central channel of the NPC and the transporting cargoes is the determining factor, but the exact mechanism of transport is yet unknown. Here, we use coarse-grained molecular dynamics simulations to quantify the energy barrier that has to be overcome for molecules to pass through the NPC. We focus on two aspects of transport. First, the passive transport of model cargo molecules with different sizes is studied and the size selectivity feature of the NPC is investigated. Our results show that the transport probability of cargoes is significantly reduced when they are larger than ∼5 nm in diameter. Secondly, we show that incorporating hydrophobic binding spots on the surface of the cargo effectively decreases the energy barrier of the pore. Finally, a simple transport model is proposed which characterizes the energy barrier of the NPC as a function of diameter and hydrophobicity of the transporting particles. PMID:26894898

  15. Assembly of nuclear pore complexes mediated by major vault protein.

    PubMed

    Vollmar, Friederike; Hacker, Christian; Zahedi, René-Peiman; Sickmann, Albert; Ewald, Andrea; Scheer, Ulrich; Dabauvalle, Marie-Christine

    2009-03-15

    During interphase growth of eukaryotic cells, nuclear pore complexes (NPCs) are continuously incorporated into the intact nuclear envelope (NE) by mechanisms that are largely unknown. De novo formation of NPCs involves local fusion events between the inner and outer nuclear membrane, formation of a transcisternal membranous channel of defined diameter and the coordinated assembly of hundreds of nucleoporins into the characteristic NPC structure. Here we have used a cell-free system based on Xenopus egg extract, which allows the experimental separation of nuclear-membrane assembly and NPC formation. Nuclei surrounded by a closed double nuclear membrane, but devoid of NPCs, were first reconstituted from chromatin and a specific membrane fraction. Insertion of NPCs into the preformed pore-free nuclei required cytosol containing soluble nucleoporins or nucleoporin subcomplexes and, quite unexpectedly, major vault protein (MVP). MVP is the main component of vaults, which are ubiquitous barrel-shaped particles of enigmatic function. Our results implicate MVP, and thus also vaults, in NPC biogenesis and provide a functional explanation for the association of a fraction of vaults with the NE and specifically with NPCs in intact cells. PMID:19240118

  16. Structure and gating of the nuclear pore complex

    PubMed Central

    Eibauer, Matthias; Pellanda, Mauro; Turgay, Yagmur; Dubrovsky, Anna; Wild, Annik; Medalia, Ohad

    2015-01-01

    Nuclear pore complexes (NPCs) perforate the nuclear envelope and allow the exchange of macromolecules between the nucleus and the cytoplasm. To acquire a deeper understanding of this transport mechanism, we analyse the structure of the NPC scaffold and permeability barrier, by reconstructing the Xenopus laevis oocyte NPC from native nuclear envelopes up to 20 Å resolution by cryo-electron tomography in conjunction with subtomogram averaging. In addition to resolving individual protein domains of the NPC constituents, we propose a model for the architecture of the molecular gate at its central channel. Furthermore, we compare and contrast this native NPC structure to one that exhibits reduced transport activity and unveil the spatial properties of the NPC gate. PMID:26112706

  17. Quantifying Nucleoporin Stoichiometry Inside Single Nuclear Pore Complexes In vivo

    PubMed Central

    Mi, Lan; Goryaynov, Alexander; Lindquist, Andre; Rexach, Michael; Yang, Weidong

    2015-01-01

    The nuclear pore complex (NPC) is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring-scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. NPCs are composed of ~30 different nucleoporins (Nups), averaged at 8, 16 or 32 copies per NPC. This estimate has not been confirmed for individual NPCs in living cells due to the inherent difficulty of counting proteins inside single supramolecular complexes. Here we used single-molecule SPEED microscopy to directly count the copy-number of twenty-four different Nups within individual NPCs of live yeast, and found agreement as well as significant deviation from previous estimates. As expected, we counted 8 copies of four peripheral Nups and 16 copies of fourteen scaffold Nups. Unexpectedly, we counted a maximum of 16 copies of Nsp1 and Nic96, rather than 32 as previously estimated; and found only 10–15 copies of six other Nups, rather than 8 or 16 copies as expected. This in situ molecular-counting technology can test structure-function models of NPCs and other supramolecular structures in cells. PMID:25797490

  18. Regulation of mRNA Trafficking by Nuclear Pore Complexes

    PubMed Central

    Bonnet, Amandine; Palancade, Benoit

    2014-01-01

    Over the last two decades, multiple studies have explored the mechanisms governing mRNA export out of the nucleus, a crucial step in eukaryotic gene expression. During transcription and processing, mRNAs are assembled into messenger ribonucleoparticles (mRNPs). mRNPs are then exported through nuclear pore complexes (NPCs), which are large multiprotein assemblies made of several copies of a limited number of nucleoporins. A considerable effort has been put into the dissection of mRNA export through NPCs at both cellular and molecular levels, revealing the conserved contributions of a subset of nucleoporins in this process, from yeast to vertebrates. Several reports have also demonstrated the ability of NPCs to sort out properly-processed mRNPs for entry into the nuclear export pathway. Importantly, changes in mRNA export have been associated with post-translational modifications of nucleoporins or changes in NPC composition, depending on cell cycle progression, development or exposure to stress. How NPC modifications also impact on cellular mRNA export in disease situations, notably upon viral infection, is discussed. PMID:25184662

  19. The Yeast Nuclear Pore Complex and Transport Through It

    PubMed Central

    Aitchison, John D.; Rout, Michael P.

    2012-01-01

    Exchange of macromolecules between the nucleus and cytoplasm is a key regulatory event in the expression of a cell’s genome. This exchange requires a dedicated transport system: (1) nuclear pore complexes (NPCs), embedded in the nuclear envelope and composed of proteins termed nucleoporins (or “Nups”), and (2) nuclear transport factors that recognize the cargoes to be transported and ferry them across the NPCs. This transport is regulated at multiple levels, and the NPC itself also plays a key regulatory role in gene expression by influencing nuclear architecture and acting as a point of control for various nuclear processes. Here we summarize how the yeast Saccharomyces has been used extensively as a model system to understand the fundamental and highly conserved features of this transport system, revealing the structure and function of the NPC; the NPC’s role in the regulation of gene expression; and the interactions of transport factors with their cargoes, regulatory factors, and specific nucleoporins. PMID:22419078

  20. Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution.

    PubMed

    Löschberger, Anna; Franke, Christian; Krohne, Georg; van de Linde, Sebastian; Sauer, Markus

    2014-10-15

    Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of nuclear pore complexes in isolated Xenopus laevis oocyte nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the nuclear pore complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of <20 nm on electron micrographs. The correlative images demonstrate quantitative molecular labeling and localization of nuclear pore complex proteins by standard immunocytochemistry with primary and secondary antibodies and reveal that the nuclear pore complex is composed of eight gp210 (also known as NUP210) protein homodimers. In addition, we find subpopulations of nuclear pore complexes with ninefold symmetry, which are found occasionally among the more typical eightfold symmetrical structures. PMID:25146397

  1. Phenylbutyrate increases pyruvate dehydrogenase complex activity in cells harboring a variety of defects

    PubMed Central

    Ferriero, Rosa; Boutron, Audrey; Brivet, Michele; Kerr, Douglas; Morava, Eva; Rodenburg, Richard J; Bonafé, Luisa; Baumgartner, Matthias R; Anikster, Yair; Braverman, Nancy E; Brunetti-Pierri, Nicola

    2014-01-01

    Objective Deficiency of pyruvate dehydrogenase complex (PDHC) is the most common genetic disorder leading to lactic acidosis. PDHC deficiency is genetically heterogenous and most patients have defects in the X-linked E1-α gene but defects in the other components of the complex encoded by PDHB, PDHX, DLAT, DLD genes or in the regulatory enzyme encoded by PDP1 have also been found. Phenylbutyrate enhances PDHC enzymatic activity in vitro and in vivo by increasing the proportion of unphosphorylated enzyme through inhibition of pyruvate dehydrogenase kinases and thus, has potential for therapy of patients with PDHC deficiency. In the present study, we investigated response to phenylbutyrate of multiple cell lines harboring all known gene defects resulting in PDHC deficiency. Methods Fibroblasts of patients with PDHC deficiency were studied for their enzyme activity at baseline and following phenylbutyrate incubation. Drug responses were correlated with genotypes and protein levels by Western blotting. Results Large deletions affecting PDHA1 that result in lack of detectable protein were unresponsive to phenylbutyrate, whereas increased PDHC activity was detected in most fibroblasts harboring PDHA1 missense mutations. Mutations affecting the R349-α residue were directed to proteasome degradation and were consistently unresponsive to short-time drug incubation but longer incubation resulted in increased levels of enzyme activity and protein that may be due to an additional effect of phenylbutyrate as a molecular chaperone. Interpretation PDHC enzyme activity was enhanced by phenylbutyrate in cells harboring missense mutations in PDHB, PDHX, DLAT, DLD, and PDP1 genes. In the prospect of a clinical trial, the results of this study may allow prediction of in vivo response in patients with PDHC deficiency harboring a wide spectrum of molecular defects. PMID:25356417

  2. Nuclear pore complex assembly studied with a biochemical assay for annulate lamellae formation.

    PubMed

    Meier, E; Miller, B R; Forbes, D J

    1995-06-01

    Formation of the nuclear pore is an intricate process involving membrane fusion and the ordered assembly of up to 1,000 pore proteins. As such, the study of pore assembly is not a simple one. Interestingly, annulate lamellae, a cytoplasmic organelle consisting of stacks of flattened membrane cisternae perforated by numerous pore complexes, have been found to form spontaneously in a reconstitution system derived from Xenopus egg extracts, as determined by electron microscopy (Dabauvalle et al., 1991). In this work, a biochemical assay for annulate lamellae (AL) formation was developed and used to study the mechanism of AL assembly in general and the assembly of individual nucleoporins into pore complexes in particular. Upon incubation of Xenopus egg cytosol and membrane vesicles, the nucleoporins nup58, nup60, nup97, nup153, and nup200 initially present in a disassembled form in the cytosol became associated with membranes and were pelletable. The association was time and temperature dependent and could be measured by immunoblotting. Thin-section electron microscopy as well as negative staining confirmed that annulate lamellae were forming coincident with the incorporation of pore proteins into membranes. Homogenization and subsequent flotation of the membrane fraction allowed us to separate a population of dense membranes, containing the integral membrane pore protein gp210 and all other nucleoporins tested, from the bulk of cellular membranes. Electron microscopy indicated that annulate lamellae were enriched in this dense, pore protein-containing fraction. GTP gamma S prevented incorporation of the soluble pore proteins into membranes. To address whether AL form in the absence of N-acetylglucosaminylated pore proteins, AL assembly was carried out in WGA-sepharose-depleted cytosol. Under these conditions, annulate lamellae formed but were altered in appearance. When the membrane fraction containing this altered AL was homogenized and subjected to flotation, the

  3. The membrane attack complex, perforin and cholesterol-dependent cytolysin superfamily of pore-forming proteins.

    PubMed

    Lukoyanova, Natalya; Hoogenboom, Bart W; Saibil, Helen R

    2016-06-01

    The membrane attack complex and perforin proteins (MACPFs) and bacterial cholesterol-dependent cytolysins (CDCs) are two branches of a large and diverse superfamily of pore-forming proteins that function in immunity and pathogenesis. During pore formation, soluble monomers assemble into large transmembrane pores through conformational transitions that involve extrusion and refolding of two α-helical regions into transmembrane β-hairpins. These transitions entail a dramatic refolding of the protein structure, and the resulting assemblies create large holes in cellular membranes, but they do not use any external source of energy. Structures of the membrane-bound assemblies are required to mechanistically understand and modulate these processes. In this Commentary, we discuss recent advances in the understanding of assembly mechanisms and molecular details of the conformational changes that occur during MACPF and CDC pore formation. PMID:27179071

  4. More Than a Pore: Ion Channel Signaling Complexes

    PubMed Central

    Fakler, Bernd; Kaczmarek, Leonard K.; Isom, Lori L.

    2014-01-01

    Voltage- and ligand-gated ion channels form the molecular basis of cellular excitability. With >400 members and accounting for ∼1.5% of the human genome, ion channels are some of the most well studied of all proteins in heterologous expression systems. Yet, ion channels often exhibit unexpected properties in vivo because of their interaction with a variety of signaling/scaffolding proteins. Such interactions can influence the function and localization of ion channels, as well as their coupling to intracellular second messengers and pathways, thus increasing the signaling potential of these ion channels in neurons. Moreover, functions have been ascribed to ion channels that are largely independent of their ion-conducting roles. Molecular and functional dissection of the ion channel proteome/interactome has yielded new insights into the composition of ion channel complexes and how their dysregulation leads to human disease. PMID:25392484

  5. Structural basis for assembly and function of the Nup82 complex in the nuclear pore scaffold

    PubMed Central

    Gaik, Monika; Flemming, Dirk; von Appen, Alexander; Kastritis, Panagiotis; Mücke, Norbert; Fischer, Jessica; Stelter, Philipp; Ori, Alessandro; Bui, Khanh Huy; Baßler, Jochen; Barbar, Elisar

    2015-01-01

    Nuclear pore complexes (NPCs) are huge assemblies formed from ∼30 different nucleoporins, typically organized in subcomplexes. One module, the conserved Nup82 complex at the cytoplasmic face of NPCs, is crucial to terminate mRNA export. To gain insight into the structure, assembly, and function of the cytoplasmic pore filaments, we reconstituted in yeast the Nup82–Nup159–Nsp1–Dyn2 complex, which was suitable for biochemical, biophysical, and electron microscopy analyses. Our integrative approach revealed that the yeast Nup82 complex forms an unusual asymmetric structure with a dimeric array of subunits. Based on all these data, we developed a three-dimensional structural model of the Nup82 complex that depicts how this module might be anchored to the NPC scaffold and concomitantly can interact with the soluble nucleocytoplasmic transport machinery. PMID:25646085

  6. An assay for clogging the ciliary pore complex distinguishes mechanisms of cytosolic and membrane protein entry

    PubMed Central

    Takao, Daisuke; Dishinger, John F; Kee, H Lynn; Pinskey, Justine M; Allen, Ben L; Verhey, Kristen J

    2014-01-01

    Summary As a cellular organelle, the cilium contains a unique protein composition [1, 2]. Entry of both membrane [3–5] and cytosolic components [6–8] is tightly regulated by gating mechanisms at the cilium base, however, the mechanistic details of ciliary gating are largely unknown. We previously proposed that entry of cytosolic components is regulated by mechanisms similar to those of nuclear transport and is dependent on nucleoporins (NUPs) which comprise a ciliary pore complex (CPC) [6, 9]. To investigate ciliary gating mechanisms, we developed a system to clog the pore by inhibiting NUP function via forced dimerization. We targeted NUP62, a component of the central channel of the nuclear pore complex (NPC) [10], for forced dimerization by tagging it with the homodimerizing Fv domain. As proof of principle, we show that forced dimerization of NUP62-Fv attenuated active transport of bovine serum albumin into the nuclear compartment and of the kinesin-2 motor KIF17 into the ciliary compartment. Using the pore clogging technique, we find that forced dimerization of NUP62 attenuated the gated entry of cytosolic proteins but did not affect entry of membrane proteins or diffusional entry of small cytosolic proteins. We propose a model in which active transport of cytosolic proteins into both nuclear and ciliary compartments requires functional NUPs of the central pore whereas lateral entry of membrane proteins utilizes a different mechanism that is likely specific to each organelle’s limiting membrane. PMID:25264252

  7. Disentangling the Complex Pore-Scale Dispersion Process in Natural Porous Media by Means of DNS

    NASA Astrophysics Data System (ADS)

    Medellin-Azuara, J.; Howitt, R. E.; MacEwan, D.; Lund, J. R.

    2014-12-01

    Over the recent past, important advances have been made in the area of micro-CT scanning of natural porous media. The acquired pore-space geometries enable detailed investigations of flow and transport via pore-scale direct numerical simulation (DNS). In this work, we utilize pore-scale DNS to investigate single-phase pore-scale dispersion. We focus on data stemming from beadpacks, Bentheimer sandstone, and Ketton and Estaillades carbonates. Our DNS results clearly show the transition from ballistic dispersion to the asymptotic Fickian regime (see figure a) at decimeter or meter scale depending on the medium type. We outline a universal Lagrangian model for tracer dispersion that is based on a low-dimensional parametrization of the complex three-dimensional motion of tracer particles (see figure b). We relate the process parameters to certain pore-geometry characteristics such as the tortuosity. Our model accurately captures the wide range of flow and transport dynamics observed in the samples considered. We establish the accuracy of the model by validating its limiting dispersion behavior, the resulting velocity statistics, and also, most challenging, snapshots of tracer plumes at travel times encompassing both ballistic and Fickian behavior.

  8. Surveillance of nuclear pore complex assembly by ESCRT-III/Vps4

    PubMed Central

    Webster, Brant M.; Colombi, Paolo; Jäger, Jens; Lusk, C. Patrick

    2014-01-01

    SUMMARY The maintenance of nuclear compartmentalization by the nuclear envelope and nuclear pore complexes (NPCs) is essential for cell function; loss of compartmentalization is associated with cancers, laminopathies and aging. We uncovered a pathway that surveils NPC assembly intermediates to promote the formation of functional NPCs. Surveillance is mediated by Heh2, a member of the LEM (Lap2-emerin-MAN1) family of integral inner nuclear membrane proteins, which binds to an early NPC assembly intermediate, but not to mature NPCs. Heh2 recruits the Endosomal Sorting Complex Required for Transport (ESCRT) – III subunit Snf7 and the AAA-ATPase Vps4 to destabilize and clear defective NPC assembly intermediates. When surveillance or clearance is compromised, malformed NPCs accumulate in a Storage of Improperly assembled Nuclear Pore Complexes compartment, or SINC. The SINC is retained in old mothers to prevent loss of daughter lifespan, highlighting a continuum of mechanisms to ensure nuclear compartmentalization. PMID:25303532

  9. The TOM core complex: the general protein import pore of the outer membrane of mitochondria.

    PubMed

    Ahting, U; Thun, C; Hegerl, R; Typke, D; Nargang, F E; Neupert, W; Nussberger, S

    1999-11-29

    Translocation of nuclear-encoded preproteins across the outer membrane of mitochondria is mediated by the multicomponent transmembrane TOM complex. We have isolated the TOM core complex of Neurospora crassa by removing the receptors Tom70 and Tom20 from the isolated TOM holo complex by treatment with the detergent dodecyl maltoside. It consists of Tom40, Tom22, and the small Tom components, Tom6 and Tom7. This core complex was also purified directly from mitochondria after solubilization with dodecyl maltoside. The TOM core complex has the characteristics of the general insertion pore; it contains high-conductance channels and binds preprotein in a targeting sequence-dependent manner. It forms a double ring structure that, in contrast to the holo complex, lacks the third density seen in the latter particles. Three-dimensional reconstruction by electron tomography exhibits two open pores traversing the complex with a diameter of approximately 2.1 nm and a height of approximately 7 nm. Tom40 is the key structural element of the TOM core complex. PMID:10579717

  10. The nuclear pore complex--structure and function at a glance.

    PubMed

    Kabachinski, Greg; Schwartz, Thomas U

    2015-02-01

    Nuclear pore complexes (NPCs) are indispensable for cell function and are at the center of several human diseases. NPCs provide access to the nucleus and regulate the transport of proteins and RNA across the nuclear envelope. They are aqueous channels generated from a complex network of evolutionarily conserved proteins known as nucleporins. In this Cell Science at a Glance article and the accompanying poster, we discuss how transport between the nucleoplasm and the cytoplasm is regulated, what we currently know about the structure of individual nucleoporins and the assembled NPC, and how the cell regulates assembly and disassembly of such a massive structure. Our aim is to provide a general overview on what we currently know about the nuclear pore and point out directions of research this area is heading to. PMID:26046137

  11. The Molecular Basis for Ca2+ Signalling by NAADP: Two-Pore Channels in a Complex?

    PubMed Central

    Marchant, Jonathan S.; Lin-Moshier, Yaping; Walseth, Timothy F.; Patel, Sandip

    2014-01-01

    NAADP is a potent Ca2+ mobilizing messenger in a variety of cells but its molecular mechanism of action is incompletely understood. Accumulating evidence indicates that the poorly characterized two-pore channels (TPCs) in animals are NAADP sensitive Ca2+-permeable channels. TPCs localize to the endo-lysosomal system but are functionally coupled to the better characterized endoplasmic reticulum Ca2+ channels to generate physiologically relevant complex Ca2+ signals. Whether TPCs directly bind NAADP is not clear. Here we discuss the idea based on recent studies that TPCs are the pore-forming subunits of a protein complex that includes tightly associated, low molecular weight NAADP-binding proteins. PMID:25309835

  12. Single molecule atomic force microscopy of aerolysin pore complexes reveals unexpected star-shaped topography.

    PubMed

    He, Jianfeng; Wang, Jiabin; Hu, Jun; Sun, Jielin; Czajkowsky, Daniel Mark; Shao, Zhifeng

    2016-04-01

    Aerolysin is the paradigmatic member of a large family of toxins that convert from a water-soluble monomer/dimer into a membrane-spanning oligomeric pore. While there is x-ray crystallographic data of its water-soluble conformation, the most recent structural model of the membrane-inserted pore is based primarily on data of water-soluble tetradecamers of mutant protein, together with computational modeling ultimately performed in vacuum. Here we examine this pore model with atomic force microscopy (AFM) of membrane-associated wild-type complexes and all-atom molecular dynamics (MD) simulations in water. In striking contrast to a disc-shaped cap region predicted by the present model, the AFM images reveal a star-shaped complex, with a central ring surrounded by seven radial projections. Further, the MD simulations suggest that the locations of the receptor-binding (D1) domains in the present model are not correct. However, a modified model in which the D1 domains, rather than localized at fixed positions, adopt a wide range of configurations through fluctuations of an intervening linker is compatible with existing data. Thus our work not only demonstrates the importance of directly resolving such complexes in their native environment but also points to a dynamic receptor binding region, which may be critical for toxin assembly on the cell surface. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26537438

  13. Zero-Mode Waveguide detection of biomolecules transport through artificial nanopores and nuclear pore complexes

    NASA Astrophysics Data System (ADS)

    Auger, Thomas; Auvray, Loic; Montel, Fabien

    We have developed a novel single molecule optical observation method using a custom Zero-Mode Waveguide setup to study the translocation of biopolymers through artificial and biological nanopores. Our work focuses on two aspects. First we monitored the flow driven injection of DNA molecules through solid state nanopores and showed that DNA starts translocating over a flow threshold independent of the pore radius, the DNA concentration and length. We demonstrate that the translocation is controlled by an energy barrier as proposed by the de Gennes - Brochard suction model. The height of the energy barrier can be modulated by functionalizing the nanopores with PEG-Thiols. More recently we adapted our setup to the study of transport through the nuclear pore complex (NPC) using extracted nuclear membranes from Xenopus Laevis oocytes. We aim at probing the conformation of unstructured proteins - the FG-Nucleoporins - crowding the central channel of the NPC by monitoring the free diffusion of small Dextran molecules (3kDa). We have been able to estimate the radius of the central pore of the NPC. We want to study the effects of transporter molecules, which have a high affinity for the FG-Nups, on the central pore size and correlate it to the conformation of FG-Nups.

  14. Pore-controlled formation of 0D metal complexes in anionic 3D metal-organic frameworks

    SciTech Connect

    Zhang, MW; Bosch, M; Zhou, HC

    2015-01-01

    The host-guest chemistry between a series of anionic MOFs and their trapped counterions was investigated by single crystal XRD. The PCN-514 series contains crystallographically identifiable metal complexes trapped in the pores, where their formation is controlled by the size and shape of the MOF pores. A change in the structure and pore size of PCN-518 indicates that the existence of guest molecules may reciprocally affect the formation of host MOFs.

  15. Structure Determination of the Nuclear Pore Complex with Three-Dimensional Cryo electron Microscopy.

    PubMed

    von Appen, Alexander; Beck, Martin

    2016-05-22

    Determining the structure of the nuclear pore complex (NPC) imposes an enormous challenge due to its size, intricate composition and membrane-embedded nature. In vertebrates, about 1000 protein building blocks assemble into a 110-MDa complex that fuses the inner and outer membranes of a cell's nucleus. Here, we review the recent progress in understanding the in situ architecture of the NPC with a specific focus on approaches using three-dimensional cryo electron microscopy. We discuss technological benefits and limitations and give an outlook toward obtaining a high-resolution structure of the NPC. PMID:26791760

  16. Size-dependent leak of soluble and membrane proteins through the yeast nuclear pore complex

    PubMed Central

    Popken, Petra; Ghavami, Ali; Onck, Patrick R.; Poolman, Bert; Veenhoff, Liesbeth M.

    2015-01-01

    Nuclear pore complexes (NPCs) allow selective import and export while forming a barrier for untargeted proteins. Using fluorescence microscopy, we measured in vivo the permeability of the Saccharomyces cerevisiae NPC for multidomain proteins of different sizes and found that soluble proteins of 150 kDa and membrane proteins with an extralumenal domain of 90 kDa were still partly localized in the nucleus on a time scale of hours. The NPCs thus form only a weak barrier for the majority of yeast proteins, given their monomeric size. Using FGΔ-mutant strains, we showed that specific combinations of Nups, especially with Nup100, but not the total mass of FG-nups per pore, were important for forming the barrier. Models of the disordered phase of wild-type and mutant NPCs were generated using a one bead per amino acid molecular dynamics model. The permeability measurements correlated with the density predictions from coarse-grained molecular dynamics simulations in the center of the NPC. The combined in vivo and computational approach provides a framework for elucidating the structural and functional properties of the permeability barrier of nuclear pore complexes. PMID:25631821

  17. Components of the Arabidopsis nuclear pore complex play multiple diverse roles in control of plant growth

    PubMed Central

    Parry, Geraint

    2014-01-01

    The nuclear pore complex (NPC) is a multisubunit protein conglomerate that facilitates movement of RNA and protein between the nucleus and cytoplasm. Relatively little is known regarding the influence of the Arabidopsis NPC on growth and development. Seedling development, flowering time, nuclear morphology, mRNA accumulation, and gene expression changes in Arabidopsis nucleoporin mutants were investigated. Nuclear export of mRNA is differentially affected in plants with defects in nucleoporins that lie in different NPC subcomplexes. This study reveals differences in the manner by which nucleoporins alter molecular and plant growth phenotypes, suggesting that nuclear pore subcomplexes play distinct roles in nuclear transport and reveal a possible feedback relationship between the expression of genes involved in nuclear transport. PMID:25165147

  18. Structural Evidence for Common Ancestry of the Nuclear Pore Complex and Vesicle Coats

    SciTech Connect

    Brohawn, S.; Leksa, N; Spear, E; Rajashankar, K; Schwartz, T

    2008-01-01

    Nuclear pore complexes (NPCs) facilitate nucleocytoplasmic transport. These massive assemblies comprise an eightfold symmetric scaffold of architectural proteins and central-channel phenylalanine-glycine-repeat proteins forming the transport barrier. We determined the nucleoporin 85 (Nup85)bulletSeh1 structure, a module in the heptameric Nup84 complex, at 3.5 angstroms resolution. Structural, biochemical, and genetic analyses position the Nup84 complex in two peripheral NPC rings. We establish a conserved tripartite element, the ancestral coatomer element ACE1, that reoccurs in several nucleoporins and vesicle coat proteins, providing structural evidence of coevolution from a common ancestor. We identified interactions that define the organization of the Nup84 complex on the basis of comparison with vesicle coats and confirmed the sites by mutagenesis. We propose that the NPC scaffold, like vesicle coats, is composed of polygons with vertices and edges forming a membrane-proximal lattice that provides docking sites for additional nucleoporins.

  19. Nanoscale stiffness topography reveals structure and mechanics of the transport barrier in intact nuclear pore complexes

    PubMed Central

    Labokha, Aksana A.; Osmanović, Dino; Liashkovich, Ivan; Orlova, Elena V.; Ford, Ian J.; Charras, Guillaume; Fassati, Ariberto; Hoogenboom, Bart W.

    2014-01-01

    The nuclear pore complex (NPC) is the gate for transport between the cell nucleus and the cytoplasm. Small molecules cross the NPC by passive diffusion, but molecules larger than ~5 nm must bind to nuclear transport receptors to overcome a selective barrier within the NPC1. Whilst the structure and shape of the cytoplasmic ring of the NPC are relatively well characterized2-5, the selective barrier is situated deep within the central channel of the NPC and depends critically on unstructured nuclear pore proteins5,6, and is therefore not well understood. Here, we show that stiffness topography7 with sharp atomic force microscopy tips can generate nanoscale cross sections of the NPC. The cross sections reveal two distinct structures, a cytoplasmic ring and a central plug structure, which are consistent with the three-dimensional NPC structure derived from electron microscopy2-5. The central plug persists after reactivation of the transport cycle and resultant cargo release, indicating that the plug is an intrinsic part of the NPC barrier. Added nuclear transport receptors accumulate on the intact transport barrier and lead to a homogenization of the barrier stiffness. The observed nanomechanical properties in the NPC indicate the presence of a cohesive barrier to transport, and are quantitatively consistent with the presence of a central condensate of nuclear pore proteins in the NPC channel. PMID:25420031

  20. Reorganization of Nuclear Pore Complexes and the Lamina in Late-Stage Parvovirus Infection.

    PubMed

    Mäntylä, Elina; Niskanen, Einari A; Ihalainen, Teemu O; Vihinen-Ranta, Maija

    2015-11-01

    Canine parvovirus (CPV) infection induces reorganization of nuclear structures. Our studies indicated that late-stage infection induces accumulation of nuclear pore complexes (NPCs) and lamin B1 concomitantly with a decrease of lamin A/C levels on the apical side of the nucleus. Newly formed CPV capsids are located in close proximity to NPCs on the apical side. These results suggest that parvoviruses cause apical enrichment of NPCs and reorganization of nuclear lamina, presumably to facilitate the late-stage infection. PMID:26311881

  1. The transmembrane nucleoporin NDC1 is required for targeting of ALADIN to nuclear pore complexes

    SciTech Connect

    Yamazumi, Yusuke; Kamiya, Atsushi; Nishida, Ayumu; Nishihara, Ayako; Iemura, Shun-ichiro; Natsume, Tohru; Akiyama, Tetsu

    2009-11-06

    NDC1 is a transmembrane nucleoporin that is required for NPC assembly and nucleocytoplasmic transport. We show here that NDC1 directly interacts with the nucleoporin ALADIN, mutations of which are responsible for triple-A syndrome, and that this interaction is required for targeting of ALADIN to nuclear pore complexes (NPCs). Furthermore, we show that NDC1 is required for selective nuclear import. Our findings suggest that NDC1-mediated localization of ALADIN to NPCs is essential for selective nuclear protein import, and that abrogation of the interaction between ALADIN and NDC1 may be important for the development of triple-A syndrome.

  2. Reorganization of Nuclear Pore Complexes and the Lamina in Late-Stage Parvovirus Infection

    PubMed Central

    Mäntylä, Elina; Niskanen, Einari A.; Ihalainen, Teemu O.

    2015-01-01

    Canine parvovirus (CPV) infection induces reorganization of nuclear structures. Our studies indicated that late-stage infection induces accumulation of nuclear pore complexes (NPCs) and lamin B1 concomitantly with a decrease of lamin A/C levels on the apical side of the nucleus. Newly formed CPV capsids are located in close proximity to NPCs on the apical side. These results suggest that parvoviruses cause apical enrichment of NPCs and reorganization of nuclear lamina, presumably to facilitate the late-stage infection. PMID:26311881

  3. Complex resistivity spectra in relation to multiscale pore geometry in carbonates and mixed-siliciclastic rocks

    NASA Astrophysics Data System (ADS)

    Norbisrath, Jan Henrik

    Carbonate rocks are known to have complex and heterogeneous pore structures, which result from their biogenic origin and strong affinity for diagenetic processes that change their pore structure after burial. The combination of sheer endless variations of precursor biogenic material, depositional environments, and diagenetic effects results in rocks that are interesting to study but intricate to understand. Many schemes to categorize the diversity of carbonate rocks are in use today; most are based on the macropore structure and qualitative thin-section analysis. Many studies, however, acknowledge that micropores have a significant influence on the macroscopic petrophysical rock properties, which are essential to determine reservoir quality. Micropores are, by definition, smaller than the thickness of a thin-section (< 30 microm) and hence cannot be quantified with conventional methods. For their analysis, scanning electron microscopy (SEM) is the logical next step. The challenge is that mechanical polishing methods produce excessive surface roughness at micron scale; the resulting surfaces are not suited for quantification of micropores. Advances in broad-ion-beam (BIB) milling enable preparation of nanometer-precision 2D sections that are suited for quantitative analysis with the SEM. To accomplish the objective of accurate quantification of carbonate micropores, part one of this dissertation employs the BIB-SEM technique on a variety of carbonate rock samples and finds four major carbonate microporosity types: (1) small intercrystalline, (2) large inter-crystalline, (3) intercement, and (4) micromoldic. Each microporosity type shows a distinct capacity to conduct electrical charge, which largely controls the magnitude and range of cementation factors (m) in rocks with such microporosity type. The BIB-SEM method is also used on a dataset of mixed carbonate-siliciclastic (mudrock) samples with high kerogen and pyrite content. Results show that the nanopore

  4. Nuclear pore complex proteins mark the implantation window in human endometrium

    PubMed Central

    Guffanti, Elisa; Kittur, Nupur; Brodt, Z. Nilly; Polotsky, Alex J.; Kuokkanen, Satu M.; Heller, Debra S.; Young, Steven L.; Santoro, Nanette; Meier, U. Thomas

    2009-01-01

    Summary Nucleolar channel systems (NCSs) are membranous organelles appearing transiently in the epithelial cell nuclei of postovulatory human endometrium. Their characterization and use as markers for a healthy receptive endometrium have been limited because they are only identifiable by electron microscopy. Here we describe the light microscopic detection of NCSs using immunofluorescence. Specifically, the monoclonal nuclear pore complex antibody 414 shows that NCSs are present in about half of all human endometrial epithelial cells but not in any other cell type, tissue or species. Most nuclei contain only a single NCS of uniform 1 μm diameter indicating a tightly controlled organelle. The composition of NCSs is as unique as their structure; they contain only a subset each of the proteins of nuclear pore complexes, inner nuclear membrane, nuclear lamina and endoplasmic reticulum. Validation of our robust NCS detection method on 95 endometrial biopsies defines a 6-day window, days 19-24 (±1) of an idealized 28 day cycle, wherein NCSs occur. Therefore, NCSs precede and overlap with the implantation window and serve as potential markers of uterine receptivity. The immunodetection assay, combined with the hitherto underappreciated prevalence of NCSs, now enables simple screening and further molecular and functional dissection. PMID:18505792

  5. Polymer brushes infiltrated by nanoparticles and applications to the nuclear pore complex

    NASA Astrophysics Data System (ADS)

    Opferman, Michael G.

    Systems of grafted polymers in the presence of additives are useful in a variety of contexts including industrial applications, solar cells, organic electronics, drug delivery, and nucleocytoplasmic transport. In this thesis, we will consider the morphologies that polymer brushes attain when exposed to a solution of additives (which we generically term "nanoparticles"), particularly when those nanparticles interact attractively with the polymers. We find that nanoparticles of this type can have a dramatic effect on the height of the polymer chains above the grafting surface, and they can induce highly non-uniform morphologies, including ones in which a dense layer of nanoparticles and monomers forms near the grafting surface. We consider especially the relevance of the system to several experiments performed on biopolymers in the nuclear pore complex when they interact attractively with transport factors that regulate nucleocytoplasmic transport. We find that, although these experiments appear to give inconsistent results, the inconsistencies can be reconciled through two simple models: the Alexander-de Gennes polymer brush, and the Milner-Witten-Cates polymer brush. Our findings should contribute to the understanding of the nuclear pore complex in that experiments can be better understood in the context of their relevant control parameters.

  6. Structure–function mapping of a heptameric module in the nuclear pore complex

    PubMed Central

    Fernandez-Martinez, Javier; Phillips, Jeremy; Sekedat, Matthew D.; Diaz-Avalos, Ruben; Velazquez-Muriel, Javier; Franke, Josef D.; Williams, Rosemary; Stokes, David L.; Chait, Brian T.

    2012-01-01

    The nuclear pore complex (NPC) is a multiprotein assembly that serves as the sole mediator of nucleocytoplasmic exchange in eukaryotic cells. In this paper, we use an integrative approach to determine the structure of an essential component of the yeast NPC, the ∼600-kD heptameric Nup84 complex, to a precision of ∼1.5 nm. The configuration of the subunit structures was determined by satisfaction of spatial restraints derived from a diverse set of negative-stain electron microscopy and protein domain–mapping data. Phenotypic data were mapped onto the complex, allowing us to identify regions that stabilize the NPC’s interaction with the nuclear envelope membrane and connect the complex to the rest of the NPC. Our data allow us to suggest how the Nup84 complex is assembled into the NPC and propose a scenario for the evolution of the Nup84 complex through a series of gene duplication and loss events. This work demonstrates that integrative approaches based on low-resolution data of sufficient quality can generate functionally informative structures at intermediate resolution. PMID:22331846

  7. Posttranslational marks control architectural and functional plasticity of the nuclear pore complex basket.

    PubMed

    Niño, Carlos A; Guet, David; Gay, Alexandre; Brutus, Sergine; Jourquin, Frédéric; Mendiratta, Shweta; Salamero, Jean; Géli, Vincent; Dargemont, Catherine

    2016-01-18

    The nuclear pore complex (NPC) serves as both the unique gate between the nucleus and the cytoplasm and a major platform that coordinates nucleocytoplasmic exchanges, gene expression, and genome integrity. To understand how the NPC integrates these functional constraints, we dissected here the posttranslational modifications of the nuclear basket protein Nup60 and analyzed how they intervene to control the plasticity of the NPC. Combined approaches highlight the role of monoubiquitylation in regulating the association dynamics of Nup60 and its partner, Nup2, with the NPC through an interaction with Nup84, a component of the Y complex. Although major nuclear transport routes are not regulated by Nup60 modifications, monoubiquitylation of Nup60 is stimulated upon genotoxic stress and regulates the DNA-damage response and telomere repair. Together, these data reveal an original mechanism contributing to the plasticity of the NPC at a molecular-organization and functional level. PMID:26783300

  8. Posttranslational marks control architectural and functional plasticity of the nuclear pore complex basket

    PubMed Central

    Niño, Carlos A.; Guet, David; Gay, Alexandre; Brutus, Sergine; Jourquin, Frédéric; Mendiratta, Shweta; Salamero, Jean; Géli, Vincent

    2016-01-01

    The nuclear pore complex (NPC) serves as both the unique gate between the nucleus and the cytoplasm and a major platform that coordinates nucleocytoplasmic exchanges, gene expression, and genome integrity. To understand how the NPC integrates these functional constraints, we dissected here the posttranslational modifications of the nuclear basket protein Nup60 and analyzed how they intervene to control the plasticity of the NPC. Combined approaches highlight the role of monoubiquitylation in regulating the association dynamics of Nup60 and its partner, Nup2, with the NPC through an interaction with Nup84, a component of the Y complex. Although major nuclear transport routes are not regulated by Nup60 modifications, monoubiquitylation of Nup60 is stimulated upon genotoxic stress and regulates the DNA-damage response and telomere repair. Together, these data reveal an original mechanism contributing to the plasticity of the NPC at a molecular-organization and functional level. PMID:26783300

  9. Influence of pore fluid chemistry on the complex conductivity and induced polarization responses of Berea sandstone

    NASA Astrophysics Data System (ADS)

    Lesmes, David P.; Frye, Kevin M.

    2001-01-01

    The spectral induced-polarization (IP) response of rocks and soils is a complex function of pore solution chemistry, sample microgeometry, and surface chemical properties. We measure the complex conductivity and the time domain IP responses of Berea sandstone as a function of pore fluid ionic strength and pH. Complex conductivity is measured over the frequency range 10-3 to 106 Hz, and chargeability is computed using a time window of 0.16 to 1.74 s. The field IP parameters: phase, percent frequency effect, and chargeability are functions of both the surface and bulk electrical properties of the sample and are observed to decrease with increasing solution conductivity. Dividing these parameters by the sample resistivity yields normalized IP parameters (quadrature conductivity, metal factor, normalized chargeability) that are proportional to the imaginary component of the complex surface conductivity. Normalized IP parameters increase with ionic strength up to concentrations of 10-1 M NaCl and show a reduced response at pH 3, the point of zero charge for quartz-dominated systems. For concentrations >10-1 M NaCl, the normalized parameters decrease with increasing concentration. This decrease in surface polarization may indicate a decrease in the effective mobility of polarizing charges at high solution concentration. Our data indicate that normalized IP parameters are directly related to the physiochemical parameters that control the surface conductivity responses of rocks and soils. Normalization of IP measurements in environmental investigations should increase the effectiveness of IP surveys, especially in high-conductivity environments.

  10. Comparative Genomic Evidence for a Complete Nuclear Pore Complex in the Last Eukaryotic Common Ancestor

    PubMed Central

    Neumann, Nadja; Lundin, Daniel; Poole, Anthony M.

    2010-01-01

    Background The Nuclear Pore Complex (NPC) facilitates molecular trafficking between nucleus and cytoplasm and is an integral feature of the eukaryote cell. It exhibits eight-fold rotational symmetry and is comprised of approximately 30 nucleoporins (Nups) in different stoichiometries. Nups are broadly conserved between yeast, vertebrates and plants, but few have been identified among other major eukaryotic groups. Methodology/Principal Findings We screened for Nups across 60 eukaryote genomes and report that 19 Nups (spanning all major protein subcomplexes) are found in all eukaryote supergroups represented in our study (Opisthokonts, Amoebozoa, Viridiplantae, Chromalveolates and Excavates). Based on parsimony, between 23 and 26 of 31 Nups can be placed in LECA. Notably, they include central components of the anchoring system (Ndc1 and Gp210) indicating that the anchoring system did not evolve by convergence, as has previously been suggested. These results significantly extend earlier results and, importantly, unambiguously place a fully-fledged NPC in LECA. We also test the proposal that transmembrane Pom proteins in vertebrates and yeasts may account for their variant forms of mitosis (open mitoses in vertebrates, closed among yeasts). The distribution of homologues of vertebrate Pom121 and yeast Pom152 is not consistent with this suggestion, but the distribution of fungal Pom34 fits a scenario wherein it was integral to the evolution of closed mitosis in ascomycetes. We also report an updated screen for vesicle coating complexes, which share a common evolutionary origin with Nups, and can be traced back to LECA. Surprisingly, we find only three supergroup-level differences (one gain and two losses) between the constituents of COPI, COPII and Clathrin complexes. Conclusions/Significance Our results indicate that all major protein subcomplexes in the Nuclear Pore Complex are traceable to the Last Eukaryotic Common Ancestor (LECA). In contrast to previous screens

  11. Nuclear Pore Complex Protein Sequences Determine Overall Copolymer Brush Structure and Function?

    NASA Astrophysics Data System (ADS)

    Ando, David; Kim, Yongwoon; Zandi, Roya; Colvin, Michael; Rexach, Michael; Gopinathan, Ajay

    2015-03-01

    Disordered proteins are an interesting class of unfolded protein biopolymers which are functionally versatile. Their sequences are unconstrained by a sequence-structure relationship, and allow for a wide range of chemical and physical polymer properties. The Nuclear Pore Complex (NPC) contains over one hundred of such proteins (FG nups), which collectively function to regulate the exchange of all materials between the nucleus and cytoplasm. We perform coarse grained simulations of both individual FG nups and grafted rings of nups mimicking the in vivo geometry of the NPC, supplemented with polymer brush modeling. Our results indicate that different regions or ``blocks'' of an individual FG nup can have distinctly different forms of disorder, and that this property appears to be a conserved feature across eukarya. Furthermore, this block structure at the individual protein level is critical to the formation of a unique higher-order polymer brush architecture. Because the interactions between FG nups may be modulated by certain forms of transport factors, our results indicate that transitions between brush morphologies could play an important role in regulating transport across the NPC, suggesting novel forms of gated transport across membrane pores with wide biomimetic applicability.

  12. Molecular modeling of zinc paddlewheel molecular complexes and the pores of a flexible metal organic framework.

    PubMed

    Alzahrani, Khalid A H; Deeth, Robert J

    2016-04-01

    A new all-atom first-principles force field (FF) is constructed for the bimetallic, four-bladed zinc paddlewheel (ZPW) motif. Zinc-ligand interactions are described via Morse functions and the angular geometry at the metal centers is modeled with a pure ligand-ligand repulsion term. The ZPW-FF is principally based on 15 DFT-optimized model systems of general formula ZnPR.nL, where ZnP is the base Zn2(O2CR)4 unit, R = H, CH3 or CF3, L = NH3 or pyridine, and n = 0, 1 or 2. It correctly generates the distorted tetrahedral coordination of the uncapped [Zn2(O2CR)4] species in their ground states as well as giving reasonable structures and energies for the higher symmetry D4h transition state conformations. The zinc-ligand Morse function reference distance, r 0 , is further refined against 30 complexes located in the Cambridge Structural Database and this FF is applied to pore models of the flexible metal-organic framework (MOF) [Zn(bdc)2(dabco)]n (bdc = 1,4-benzendicarboxylate; dabco = 1,4-diazabicyclo(2.2.2)octane). A single pore model reproduces the unit cell of the evacuated MOF system while a 3×3 grid model is necessary to provide good agreement with the observed pronounced structural changes upon adsorption of either dimethylformamide or benzene. PMID:26979608

  13. From hypothesis to mechanism: uncovering nuclear pore complex links to gene expression.

    PubMed

    Burns, Laura T; Wente, Susan R

    2014-06-01

    The gene gating hypothesis put forth by Blobel in 1985 was an alluring proposal outlining functions for the nuclear pore complex (NPC) in transcription and nuclear architecture. Over the past several decades, collective studies have unveiled a full catalog of nucleoporins (Nups) that comprise the NPC, structural arrangements of Nups in the nuclear pore, and mechanisms of nucleocytoplasmic transport. With this foundation, investigations of the gene gating hypothesis have now become possible. Studies of several model organisms provide credence for Nup functions in transcription, mRNA export, and genome organization. Surprisingly, Nups are not only involved in transcriptional events that occur at the nuclear periphery, but there are also novel roles for dynamic Nups within the nucleoplasmic compartment. Several tenants of the original gene gating hypothesis have yet to be addressed. Knowledge of whether the NPC impacts the organization of the genome to control subsets of genes is limited, and the cooperating molecular machinery or specific genomic anchoring sequences are not fully resolved. This minireview summarizes the current evidence for gene gating in Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and mammalian model systems. These examples highlight new and unpredicted mechanisms for Nup impacts on transcription and questions that are left to be explored. PMID:24615017

  14. Effect of pore sizes on catalytic activities of arenetricarbonyl metal complexes constructed within Zr-based MOFs.

    PubMed

    Saito, Masakazu; Toyao, Takashi; Ueda, Kozo; Kamegawa, Takashi; Horiuchi, Yu; Matsuoka, Masaya

    2013-07-14

    Arenetricarbonyl metal complexes ([-phM(CO)3-] and [-biphM(CO)3-]; ph = phenylene, biph = biphenylene, M = Mo, Cr) constructed within Zr-based MOFs act as highly active and selective catalysts for epoxidation of cyclooctene. Catalytic activities of these complexes are enhanced with increasing the pore sizes of Zr-based MOFs. PMID:23694976

  15. Single-Molecule Imaging to Characterize the Transport Mechanism of the Nuclear Pore Complex.

    PubMed

    Jeremy, Grace; Stevens, James; Lowe, Alan R

    2016-01-01

    In the eukaryotic cell, a large macromolecular channel, known as the Nuclear Pore Complex (NPC), mediates all molecular transport between the nucleus and cytoplasm. In recent years, single-molecule fluorescence (SMF) imaging has emerged as a powerful tool to study the molecular mechanism of transport through the NPC. More recently, techniques such as single-molecule localization microscopy (SMLM) have enabled the spatial and temporal distribution of cargos, transport receptors and even structural components of the NPC to be determined with nanometre accuracy. In this protocol, we describe a method to study the position and/or motion of individual molecules transiting through the NPC with high spatial and temporal precision. PMID:27283299

  16. Modern tools to study nuclear pore complexes and nucleocytoplasmic transport in Caenorhabditis elegans.

    PubMed

    Askjaer, Peter; Galy, Vincent; Meister, Peter

    2014-01-01

    The nematode Caenorhabditis elegans is characterized by many features that make it highly attractive to study nuclear pore complexes (NPCs) and nucleocytoplasmic transport. NPC composition and structure are highly conserved in nematodes and being amenable to a variety of genetic manipulations, key aspects of nuclear envelope dynamics can be observed in great details during breakdown, reassembly, and interphase. In this chapter, we provide an overview of some of the most relevant modern techniques that allow researchers unfamiliar with C. elegans to embark on studies of nucleoporins in an intact organism through its development from zygote to aging adult. We focus on methods relevant to generate loss-of-function phenotypes and their analysis by advanced microscopy. Extensive references to available reagents, such as mutants, transgenic strains, and antibodies are equally useful to scientists with or without prior C. elegans or nucleoporin experience. PMID:24857735

  17. Toward the atomic structure of the nuclear pore complex: when top down meets bottom up.

    PubMed

    Hoelz, André; Glavy, Joseph S; Beck, Martin

    2016-07-01

    Elucidating the structure of the nuclear pore complex (NPC) is a prerequisite for understanding the molecular mechanism of nucleocytoplasmic transport. However, owing to its sheer size and flexibility, the NPC is unapproachable by classical structure determination techniques and requires a joint effort of complementary methods. Whereas bottom-up approaches rely on biochemical interaction studies and crystal-structure determination of NPC components, top-down approaches attempt to determine the structure of the intact NPC in situ. Recently, both approaches have converged, thereby bridging the resolution gap from the higher-order scaffold structure to near-atomic resolution and opening the door for structure-guided experimental interrogations of NPC function. PMID:27273515

  18. Super-resolution 3D tomography of interactions and competition in the nuclear pore complex.

    PubMed

    Ma, Jiong; Goryaynov, Alexander; Yang, Weidong

    2016-03-01

    A selective barrier formed by intrinsically disordered Phe-Gly (FG) nucleoporins (Nups) allows transport receptor (TR)-facilitated translocation of signal-dependent cargos through the nuclear pore complexes (NPCs) of eukaryotic cells. However, the configuration of the FG-Nup barrier and its interactions with multiple TRs in native NPCs remain obscure. Here, we mapped the interaction sites of various TRs or FG segments within the FG-Nup barrier by using high-speed super-resolution microscopy and used these sites to reconstruct the three-dimensional tomography of the native barrier in the NPC. We found that each TR possesses a unique interaction zone within the FG-Nup barrier and that two major TRs, importin β1 and Crm1, outcompete other TRs in binding FG Nups. Moreover, TRs may alter the tomography of the FG-Nup barrier and affect one another's pathways under circumstances of heavy competition. PMID:26878241

  19. Diffusion in an elastic medium: A model for macromolecule transport across the nuclear pore complex

    NASA Astrophysics Data System (ADS)

    Chakrabarti, Rajarshi; Debnath, Ananya; Sebastian, K. L.

    2014-06-01

    Nuclear pore complexes (NPCs) are very selective filters that sit on the membrane of the nucleus and monitor the transport between the cytoplasm and the nucleoplasm. For the central plug of NPC two models have been suggested in the literature. The first suggests that the plug is a reversible hydrogel while the other suggests that it is a polymer brush. Here we propose a model for the transport of a protein through the plug, which is general enough to cover both the models. The protein stretches the plug and creates a local deformation, which together with the protein, we refer to as the bubble. We start with the free energy for creation of the bubble and consider its motion within the plug. The relevant coordinate is the center of the bubble which executes random walk. We find that for faster relaxation of the gel, the diffusion of the bubble is greater.

  20. Simple biophysics underpins collective conformations of the intrinsically disordered proteins of the Nuclear Pore Complex.

    PubMed

    Vovk, Andrei; Gu, Chad; Opferman, Michael G; Kapinos, Larisa E; Lim, Roderick Yh; Coalson, Rob D; Jasnow, David; Zilman, Anton

    2016-01-01

    Nuclear Pore Complexes (NPCs) are key cellular transporter that control nucleocytoplasmic transport in eukaryotic cells, but its transport mechanism is still not understood. The centerpiece of NPC transport is the assembly of intrinsically disordered polypeptides, known as FG nucleoporins, lining its passageway. Their conformations and collective dynamics during transport are difficult to assess in vivo. In vitro investigations provide partially conflicting results, lending support to different models of transport, which invoke various conformational transitions of the FG nucleoporins induced by the cargo-carrying transport proteins. We show that the spatial organization of FG nucleoporin assemblies with the transport proteins can be understood within a first principles biophysical model with a minimal number of key physical variables, such as the average protein interaction strengths and spatial densities. These results address some of the outstanding controversies and suggest how molecularly divergent NPCs in different species can perform essentially the same function. PMID:27198189

  1. Respiratory complex I dysfunction due to mitochondrial DNA mutations shifts the voltage threshold for opening of the permeability transition pore toward resting levels.

    PubMed

    Porcelli, Anna Maria; Angelin, Alessia; Ghelli, Anna; Mariani, Elisa; Martinuzzi, Andrea; Carelli, Valerio; Petronilli, Valeria; Bernardi, Paolo; Rugolo, Michela

    2009-01-23

    We have studied mitochondrial bioenergetics in HL180 cells (a cybrid line harboring the T14484C/ND6 and G14279A/ND6 mtDNA mutations of Leber hereditary optic neuropathy, leading to an approximately 50% decrease of ATP synthesis) and XTC.UC1 cells (derived from a thyroid oncocytoma bearing a disruptive frameshift mutation in MT-ND1, which impairs complex I assembly). The addition of rotenone to HL180 cells and of antimycin A to XTC.UC1 cells caused fast mitochondrial membrane depolarization that was prevented by treatment with cyclosporin A, intracellular Ca2+ chelators, and antioxidant. Both cell lines also displayed an anomalous response to oligomycin, with rapid onset of depolarization that was prevented by cyclosporin A and by overexpression of Bcl-2. These findings indicate that depolarization by respiratory chain inhibitors and oligomycin was due to opening of the mitochondrial permeability transition pore (PTP). A shift of the threshold voltage for PTP opening close to the resting potential may therefore be the underlying cause facilitating cell death in diseases affecting complex I activity. This study provides a unifying reading frame for previous observations on mitochondrial dysfunction, bioenergetic defects, and Ca2+ deregulation in mitochondrial diseases. Therapeutic strategies aimed at normalizing the PTP voltage threshold may be instrumental in ameliorating the course of complex I-dependent mitochondrial diseases. PMID:19047048

  2. Interactome Mapping Reveals the Evolutionary History of the Nuclear Pore Complex

    PubMed Central

    Obado, Samson O.; Brillantes, Marc; Uryu, Kunihiro; Zhang, Wenzhu; Ketaren, Natalia E.; Chait, Brian T.; Field, Mark C.; Rout, Michael P.

    2016-01-01

    The nuclear pore complex (NPC) is responsible for nucleocytoplasmic transport and constitutes a hub for control of gene expression. The components of NPCs from several eukaryotic lineages have been determined, but only the yeast and vertebrate NPCs have been extensively characterized at the quaternary level. Significantly, recent evidence indicates that compositional similarity does not necessarily correspond to homologous architecture between NPCs from different taxa. To address this, we describe the interactome of the trypanosome NPC, a representative, highly divergent eukaryote. We identify numerous new NPC components and report an exhaustive interactome, allowing assignment of trypanosome nucleoporins to discrete NPC substructures. Remarkably, despite retaining similar protein composition, there are exceptional architectural dissimilarities between opisthokont (yeast and vertebrates) and excavate (trypanosomes) NPCs. Whilst elements of the inner core are conserved, numerous peripheral structures are highly divergent, perhaps reflecting requirements to interface with divergent nuclear and cytoplasmic functions. Moreover, the trypanosome NPC has almost complete nucleocytoplasmic symmetry, in contrast to the opisthokont NPC; this may reflect divergence in RNA export processes at the NPC cytoplasmic face, as we find evidence supporting Ran-dependent mRNA export in trypanosomes, similar to protein transport. We propose a model of stepwise acquisition of nucleocytoplasmic mechanistic complexity and demonstrate that detailed dissection of macromolecular complexes provides fuller understanding of evolutionary processes. PMID:26891179

  3. Interactome Mapping Reveals the Evolutionary History of the Nuclear Pore Complex.

    PubMed

    Obado, Samson O; Brillantes, Marc; Uryu, Kunihiro; Zhang, Wenzhu; Ketaren, Natalia E; Chait, Brian T; Field, Mark C; Rout, Michael P

    2016-02-01

    The nuclear pore complex (NPC) is responsible for nucleocytoplasmic transport and constitutes a hub for control of gene expression. The components of NPCs from several eukaryotic lineages have been determined, but only the yeast and vertebrate NPCs have been extensively characterized at the quaternary level. Significantly, recent evidence indicates that compositional similarity does not necessarily correspond to homologous architecture between NPCs from different taxa. To address this, we describe the interactome of the trypanosome NPC, a representative, highly divergent eukaryote. We identify numerous new NPC components and report an exhaustive interactome, allowing assignment of trypanosome nucleoporins to discrete NPC substructures. Remarkably, despite retaining similar protein composition, there are exceptional architectural dissimilarities between opisthokont (yeast and vertebrates) and excavate (trypanosomes) NPCs. Whilst elements of the inner core are conserved, numerous peripheral structures are highly divergent, perhaps reflecting requirements to interface with divergent nuclear and cytoplasmic functions. Moreover, the trypanosome NPC has almost complete nucleocytoplasmic symmetry, in contrast to the opisthokont NPC; this may reflect divergence in RNA export processes at the NPC cytoplasmic face, as we find evidence supporting Ran-dependent mRNA export in trypanosomes, similar to protein transport. We propose a model of stepwise acquisition of nucleocytoplasmic mechanistic complexity and demonstrate that detailed dissection of macromolecular complexes provides fuller understanding of evolutionary processes. PMID:26891179

  4. Systematic analysis of barrier-forming FG hydrogels from Xenopus nuclear pore complexes.

    PubMed

    Labokha, Aksana A; Gradmann, Sabine; Frey, Steffen; Hülsmann, Bastian B; Urlaub, Henning; Baldus, Marc; Görlich, Dirk

    2013-01-23

    Nuclear pore complexes (NPCs) control the traffic between cell nucleus and cytoplasm. While facilitating translocation of nuclear transport receptors (NTRs) and NTR·cargo complexes, they suppress passive passage of macromolecules 30 kDa. Previously, we reconstituted the NPC barrier as hydrogels comprising S. cerevisiae FG domains. We now studied FG domains from 10 Xenopus nucleoporins and found that all of them form hydrogels. Related domains with low FG motif density also substantially contribute to the NPC's hydrogel mass. We characterized all these hydrogels and observed the strictest sieving effect for the Nup98-derived hydrogel. It fully blocks entry of GFP-sized inert objects, permits facilitated entry of the small NTR NTF2, but arrests importin β-type NTRs at its surface. O-GlcNAc modification of the Nup98 FG domain prevented this arrest and allowed also large NTR·cargo complexes to enter. Solid-state NMR spectroscopy revealed that the O-GlcNAc-modified Nup98 gel lacks amyloid-like β-structures that dominate the rigid regions in the S. cerevisiae Nsp1 FG hydrogel. This suggests that FG hydrogels can assemble through different structural principles and yet acquire the same NPC-like permeability. PMID:23202855

  5. Systematic analysis of barrier-forming FG hydrogels from Xenopus nuclear pore complexes

    PubMed Central

    Labokha, Aksana A; Gradmann, Sabine; Frey, Steffen; Hülsmann, Bastian B; Urlaub, Henning; Baldus, Marc; Görlich, Dirk

    2013-01-01

    Nuclear pore complexes (NPCs) control the traffic between cell nucleus and cytoplasm. While facilitating translocation of nuclear transport receptors (NTRs) and NTR·cargo complexes, they suppress passive passage of macromolecules ⩾30 kDa. Previously, we reconstituted the NPC barrier as hydrogels comprising S. cerevisiae FG domains. We now studied FG domains from 10 Xenopus nucleoporins and found that all of them form hydrogels. Related domains with low FG motif density also substantially contribute to the NPC's hydrogel mass. We characterized all these hydrogels and observed the strictest sieving effect for the Nup98-derived hydrogel. It fully blocks entry of GFP-sized inert objects, permits facilitated entry of the small NTR NTF2, but arrests importin β-type NTRs at its surface. O-GlcNAc modification of the Nup98 FG domain prevented this arrest and allowed also large NTR·cargo complexes to enter. Solid-state NMR spectroscopy revealed that the O-GlcNAc-modified Nup98 gel lacks amyloid-like β-structures that dominate the rigid regions in the S. cerevisiae Nsp1 FG hydrogel. This suggests that FG hydrogels can assemble through different structural principles and yet acquire the same NPC-like permeability. PMID:23202855

  6. An evaluation of factors influencing pore pressure in accretionary complexes: Implications for taper angle and wedge mechanics

    USGS Publications Warehouse

    Saffer, D.M.; Bekins, B.A.

    2006-01-01

    At many subduction zones, accretionary complexes form as sediment is off-scraped from the subducting plate. Mechanical models that treat accretionary complexes as critically tapered wedges of sediment demonstrate that pore pressure controls their taper angle by modifying basal and internal shear strength. Here, we combine a numerical model of groundwater flow with critical taper theory to quantify the effects of sediment and de??collement permeability, sediment thickness, sediment partitioning between accretion and underthrusting, and plate convergence rate on steady state pore pressure. Our results show that pore pressure in accretionary wedges can be viewed as a dynamically maintained response to factors which drive pore pressure (source terms) and those that limit flow (permeability and drainage path length). We find that sediment permeability and incoming sediment thickness are the most important factors, whereas fault permeability and the partitioning of sediment have a small effect. For our base case model scenario, as sediment permeability is increased, pore pressure decreases from near-lithostatic to hydrostatic values and allows stable taper angles to increase from ??? 2.5?? to 8??-12.5??. With increased sediment thickness in our models (from 100 to 8000 m), increased pore pressure drives a decrease in stable taper angle from 8.4??-12.5?? to 15?? to <4??) with increased sediment thickness (from <1 to 7 km). One key implication is that hydrologic properties may strongly influence the strength of the crust in a wide range of geologic settings. Copyright 2006 by the American Geophysical Union.

  7. Porosity-Tortuosity Relations in Complex Porous Media Using Pore-Scale Numerical Simulation

    NASA Astrophysics Data System (ADS)

    Xiao, F.; Yin, X.

    2015-12-01

    Diffusive transport is an important mechanism of mass transfer in gas or liquid phases in porous media. The continuous presence of solids in porous media causes the diffusion paths to deviate from straight lines, and this behavior is descried as the tortuosity of porous media. In this contribution, the porosity-tortuosity relations were studied using a random walk particle tracking (RWPT) code for three-dimensional complex porous medium geometries. The RWPT code was massively parallelized and has been tested on Titan (Cray XK7) at the Oak Ridge National Laboratory and on Mira (IBM Blue Gene/Q) at the Argonne National Laboratory with up to 16,384 cores. The porous medium geometries were synthetically created based on Voronoi tessellations, and include homogeneous fibrous/tubular/granular geometries as well as heterogeneous (vuggy) tubular/granular geometries. Simulation results show that the homogenous granular geometry is the least tortuous, the homogeneous tubular geometry is intermediately tortuous, and the homogeneous fibrous geometry is the most tortuous. For homogeneous granular and tubular geometries, as porosity increases, the difference between the tortuosity of tubular geometry and that of granular geometry decreases. The vuggy porosity increases the tortuosity, due to the fact that mass "particles" trapped in a vug cannot exit the vug except along specific directions where the vug has narrow openings. The size of vugs relative to the size of inter-vug pores also affects tortuosity: the larger the vug size relative to the inter-vug pores, the higher the tortuosity, because in smaller vugs (with the same vuggy porosity and total porosity) the chance is higher for mass "particles" to diffuse into the exiting cylindrical pores. To test the effect of vuggy porosity and tortuosity on permeability, three vuggy geometries were set up with the same vuggy porosity, but different vug sizes. It is found, interestingly, that they have the same dimensionless

  8. Characterization of nuclear pore complex components in fission yeast Schizosaccharomyces pombe

    PubMed Central

    Asakawa, Haruhiko; Yang, Hui-Ju; Yamamoto, Takaharu G; Ohtsuki, Chizuru; Chikashige, Yuji; Sakata-Sogawa, Kumiko; Tokunaga, Makio; Iwamoto, Masaaki; Hiraoka, Yasushi; Haraguchi, Tokuko

    2014-01-01

    The nuclear pore complex (NPC) is an enormous proteinaceous complex composed of multiple copies of about 30 different proteins called nucleoporins. In this study, we analyzed the composition of the NPC in the model organism Schizosaccharomyces pombe using strains in which individual nucleoporins were tagged with GFP. We identified 31 proteins as nucleoporins by their localization to the nuclear periphery. Gene disruption analysis in previous studies coupled with gene disruption analysis in the present study indicates that 15 of these nucleoporins are essential for vegetative cell growth and the other 16 nucleoporins are non-essential. Among the 16 non-essential nucleoporins, 11 are required for normal progression through meiosis and their disruption caused abnormal spore formation or poor spore viability. Based on fluorescence measurements of GFP-fused nucleoporins, we estimated the composition of the NPC in S. pombe and found that the organization of the S. pombe NPC is largely similar to that of other organisms; a single NPC was estimated as being 45.8–47.8 MDa in size. We also used fluorescence measurements of single NPCs and quantitative western blotting to analyze the composition of the Nup107-Nup160 subcomplex, which plays an indispensable role in NPC organization and function. Our analysis revealed low amounts of Nup107 and Nup131 and high amounts of Nup132 in the Nup107-Nup160 subcomplex, suggesting that the composition of this complex in S. pombe may differ from that in S. cerevisiae and humans. Comparative analysis of NPCs in various organisms will lead to a comprehensive understanding of the functional architecture of the NPC. PMID:24637836

  9. Slide-and-exchange mechanism for rapid and selective transport through the nuclear pore complex.

    PubMed

    Raveh, Barak; Karp, Jerome M; Sparks, Samuel; Dutta, Kaushik; Rout, Michael P; Sali, Andrej; Cowburn, David

    2016-05-01

    Nucleocytoplasmic transport is mediated by the interaction of transport factors (TFs) with disordered phenylalanine-glycine (FG) repeats that fill the central channel of the nuclear pore complex (NPC). However, the mechanism by which TFs rapidly diffuse through multiple FG repeats without compromising NPC selectivity is not yet fully understood. In this study, we build on our recent NMR investigations showing that FG repeats are highly dynamic, flexible, and rapidly exchanging among TF interaction sites. We use unbiased long timescale all-atom simulations on the Anton supercomputer, combined with extensive enhanced sampling simulations and NMR experiments, to characterize the thermodynamic and kinetic properties of FG repeats and their interaction with a model transport factor. Both the simulations and experimental data indicate that FG repeats are highly dynamic random coils, lack intrachain interactions, and exhibit significant entropically driven resistance to spatial confinement. We show that the FG motifs reversibly slide in and out of multiple TF interaction sites, transitioning rapidly between a strongly interacting state and a weakly interacting state, rather than undergoing a much slower transition between strongly interacting and completely noninteracting (unbound) states. In the weakly interacting state, FG motifs can be more easily displaced by other competing FG motifs, providing a simple mechanism for rapid exchange of TF/FG motif contacts during transport. This slide-and-exchange mechanism highlights the direct role of the disorder within FG repeats in nucleocytoplasmic transport, and resolves the apparent conflict between the selectivity and speed of transport. PMID:27091992

  10. Simple biophysics underpins collective conformations of the intrinsically disordered proteins of the Nuclear Pore Complex

    PubMed Central

    Vovk, Andrei; Gu, Chad; Opferman, Michael G; Kapinos, Larisa E; Lim, Roderick YH; Coalson, Rob D; Jasnow, David; Zilman, Anton

    2016-01-01

    Nuclear Pore Complexes (NPCs) are key cellular transporter that control nucleocytoplasmic transport in eukaryotic cells, but its transport mechanism is still not understood. The centerpiece of NPC transport is the assembly of intrinsically disordered polypeptides, known as FG nucleoporins, lining its passageway. Their conformations and collective dynamics during transport are difficult to assess in vivo. In vitro investigations provide partially conflicting results, lending support to different models of transport, which invoke various conformational transitions of the FG nucleoporins induced by the cargo-carrying transport proteins. We show that the spatial organization of FG nucleoporin assemblies with the transport proteins can be understood within a first principles biophysical model with a minimal number of key physical variables, such as the average protein interaction strengths and spatial densities. These results address some of the outstanding controversies and suggest how molecularly divergent NPCs in different species can perform essentially the same function. DOI: http://dx.doi.org/10.7554/eLife.10785.001 PMID:27198189

  11. Physical modeling of the conformation of the unfolded proteins of the Nuclear Pore Complex

    NASA Astrophysics Data System (ADS)

    Zilman, Anton; Opferman, Michael; Coalson, Rob; Jasnow, David

    2013-03-01

    Nuclear Pore Complex (NPC) is a biological ``nano-machine'' that controls the macromolecular transport between the cell nucleus and the cytoplasm. NPC functions without direct input of metabolic energy and without transitions of the gate from a ``closed'' to an ``open'' state during transport. The key and unique aspect of transport is the interaction of the transported molecules with the unfolded, natively unstructured proteins that cover the lumen of the NPC. Recently, the NPC inspired creation of artificial bio-mimetic for nano-technology applications. Although several models have been proposed, it is still not clear how the passage of the transport factors is coupled to the conformational dynamics of the unfolded proteins within the NPC. Morphology changes in assemblies of the unfolded proteins induced by the transport factors have been investigated experimentally in vitro. I will present a coarse-grained theoretical and simulation framework that mimics the interactions of unfolded proteins with nano-sized transport factors. The simple physical model predicts morphology changes that explain the recent puzzling experimental results and suggests possible new modes of transport through the NPC. It also provides insights into the physics of the behavior of unfolded proteins.

  12. Plant nuclear pore complex proteins are modified by novel oligosaccharides with terminal N-acetylglucosamine.

    PubMed Central

    Heese-Peck, A; Cole, R N; Borkhsenious, O N; Hart, G W; Raikhel, N V

    1995-01-01

    Only a few nuclear pore complex (NPC) proteins, mainly in vertebrates and yeast but none in plants, have been well characterized. As an initial step to identify plant NPC proteins, we examined whether NPC proteins from tobacco are modified by N-acetylglucosamine (GlcNAc). Using wheat germ agglutinin, a lectin that binds specifically to GlcNAc in plants, specific labeling was often found associated with or adjacent to NPCs. Nuclear proteins containing GlcNAc can be partially extracted by 0.5 M salt, as shown by a wheat germ agglutinin blot assay, and at least eight extracted proteins were modified by terminal GlcNAc, as determined by in vitro galactosyltransferase assays. Sugar analysis indicated that the plant glycans with terminal GlcNAc differ from the single O-linked GlcNAc of vertebrate NPC proteins in that they consist of oligosaccharides that are larger in size than five GlcNAc residues. Most of these appear to be bound to proteins via a hydroxyl group. This novel oligosaccharide modification may convey properties to the plant NPC that are different from those of vertebrate NPCs. PMID:8589629

  13. SUMO-Dependent Relocalization of Eroded Telomeres to Nuclear Pore Complexes Controls Telomere Recombination.

    PubMed

    Churikov, Dmitri; Charifi, Ferose; Eckert-Boulet, Nadine; Silva, Sonia; Simon, Marie-Noelle; Lisby, Michael; Géli, Vincent

    2016-05-10

    In budding yeast, inactivation of telomerase and ensuing telomere erosion cause relocalization of telomeres to nuclear pore complexes (NPCs). However, neither the mechanism of such relocalization nor its significance are understood. We report that proteins bound to eroded telomeres are recognized by the SUMO (small ubiquitin-like modifier)-targeted ubiquitin ligase (STUbL) Slx5-Slx8 and become increasingly SUMOylated. Recruitment of Slx5-Slx8 to eroded telomeres facilitates telomere relocalization to NPCs and type II telomere recombination, a counterpart of mammalian alternative lengthening of telomeres (ALT). Moreover, artificial tethering of a telomere to a NPC promotes type II telomere recombination but cannot bypass the lack of Slx5-Slx8 in this process. Together, our results indicate that SUMOylation positively contributes to telomere relocalization to the NPC, where poly-SUMOylated proteins that accumulated over time have to be removed. We propose that STUbL-dependent relocalization of telomeres to NPCs constitutes a pathway in which excessively SUMOylated proteins are removed from "congested" intermediates to ensure unconventional recombination. PMID:27134164

  14. P granules extend the nuclear pore complex environment in the C. elegans germ line

    PubMed Central

    Updike, Dustin L.; Hachey, Stephanie J.; Kreher, Jeremy

    2011-01-01

    The immortal and totipotent properties of the germ line depend on determinants within the germ plasm. A common characteristic of germ plasm across phyla is the presence of germ granules, including P granules in Caenorhabditis elegans, which are typically associated with the nuclear periphery. In C. elegans, nuclear pore complex (NPC)–like FG repeat domains are found in the VASA-related P-granule proteins GLH-1, GLH-2, and GLH-4 and other P-granule components. We demonstrate that P granules, like NPCs, are held together by weak hydrophobic interactions and establish a size-exclusion barrier. Our analysis of intestine-expressed proteins revealed that GLH-1 and its FG domain are not sufficient to form granules, but require factors like PGL-1 to nucleate the localized concentration of GLH proteins. GLH-1 is necessary but not sufficient for the perinuclear location of granules in the intestine. Our results suggest that P granules extend the NPC environment in the germ line and provide insights into the roles of the PGL and GLH family proteins. PMID:21402789

  15. Global motions of the nuclear pore complex: insights from elastic network models.

    PubMed

    Lezon, Timothy R; Sali, Andrej; Bahar, Ivet

    2009-09-01

    The nuclear pore complex (NPC) is the gate to the nucleus. Recent determination of the configuration of proteins in the yeast NPC at approximately 5 nm resolution permits us to study the NPC global dynamics using coarse-grained structural models. We investigate these large-scale motions by using an extended elastic network model (ENM) formalism applied to several coarse-grained representations of the NPC. Two types of collective motions (global modes) are predicted by the ENMs to be intrinsically favored by the NPC architecture: global bending and extension/contraction from circular to elliptical shapes. These motions are shown to be robust against tested variations in the representation of the NPC, and are largely captured by a simple model of a toroid with axially varying mass density. We demonstrate that spoke multiplicity significantly affects the accessible number of symmetric low-energy modes of motion; the NPC-like toroidal structures composed of 8 spokes have access to highly cooperative symmetric motions that are inaccessible to toroids composed of 7 or 9 spokes. The analysis reveals modes of motion that may facilitate macromolecular transport through the NPC, consistent with previous experimental observations. PMID:19730674

  16. Effect of charge, hydrophobicity, and sequence of nucleoporins on the translocation of model particles through the nuclear pore complex.

    PubMed

    Tagliazucchi, Mario; Peleg, Orit; Kröger, Martin; Rabin, Yitzhak; Szleifer, Igal

    2013-02-26

    The molecular structure of the yeast nuclear pore complex (NPC) and the translocation of model particles have been studied with a molecular theory that accounts for the geometry of the pore and the sequence and anchoring position of the unfolded domains of the nucleoporin proteins (the FG-Nups), which control selective transport through the pore. The theory explicitly models the electrostatic, hydrophobic, steric, conformational, and acid-base properties of the FG-Nups. The electrostatic potential within the pore, which arises from the specific charge distribution of the FG-Nups, is predicted to be negative close to pore walls and positive along the pore axis. The positive electrostatic potential facilitates the translocation of negatively charged particles, and the free energy barrier for translocation decreases for increasing particle hydrophobicity. These results agree with the experimental observation that transport receptors that form complexes with hydrophilic/neutral or positively charged proteins to transport them through the NPC are both hydrophobic and strongly negatively charged. The molecular theory shows that the effects of electrostatic and hydrophobic interactions on the translocating potential are cooperative and nonequivalent due to the interaction-dependent reorganization of the FG-Nups in the presence of the translocating particle. The combination of electrostatic and hydrophobic interactions can give rise to complex translocation potentials displaying a combination of wells and barriers, in contrast to the simple barrier potential observed for a hydrophilic/neutral translocating particle. This work demonstrates the importance of explicitly considering the amino acid sequence and hydrophobic, electrostatic, and steric interactions in understanding the translocation through the NPC. PMID:23404701

  17. The chromosome 2p21 region harbors a complex genetic architecture for association with risk for renal cell carcinoma

    PubMed Central

    Han, Summer S.; Yeager, Meredith; Moore, Lee E.; Wei, Ming-Hui; Pfeiffer, Ruth; Toure, Ousmane; Purdue, Mark P.; Johansson, Mattias; Scelo, Ghislaine; Chung, Charles C.; Gaborieau, Valerie; Zaridze, David; Schwartz, Kendra; Szeszenia-Dabrowska, Neonilia; Davis, Faith; Bencko, Vladimir; Colt, Joanne S.; Janout, Vladimir; Matveev, Vsevolod; Foretova, Lenka; Mates, Dana; Navratilova, M.; Boffetta, Paolo; Berg, Christine D.; Grubb, Robert L.; Stevens, Victoria L.; Thun, Michael J.; Diver, W. Ryan; Gapstur, Susan M.; Albanes, Demetrius; Weinstein, Stephanie J.; Virtamo, Jarmo; Burdett, Laurie; Brisuda, Antonin; McKay, James D.; Fraumeni, Joseph F.; Chatterjee, Nilanjan; Rosenberg, Philip S.; Rothman, Nathaniel; Brennan, Paul; Chow, Wong-Ho; Tucker, Margaret A.; Chanock, Stephen J.; Toro, Jorge R.

    2012-01-01

    In follow-up of a recent genome-wide association study (GWAS) that identified a locus in chromosome 2p21 associated with risk for renal cell carcinoma (RCC), we conducted a fine mapping analysis of a 120 kb region that includes EPAS1. We genotyped 59 tagged common single-nucleotide polymorphisms (SNPs) in 2278 RCC and 3719 controls of European background and observed a novel signal for rs9679290 [P = 5.75 × 10−8, per-allele odds ratio (OR) = 1.27, 95% confidence interval (CI): 1.17–1.39]. Imputation of common SNPs surrounding rs9679290 using HapMap 3 and 1000 Genomes data yielded two additional signals, rs4953346 (P = 4.09 × 10−14) and rs12617313 (P = 7.48 × 10−12), both highly correlated with rs9679290 (r2 > 0.95), but interestingly not correlated with the two SNPs reported in the GWAS: rs11894252 and rs7579899 (r2 < 0.1 with rs9679290). Genotype analysis of rs12617313 confirmed an association with RCC risk (P = 1.72 × 10−9, per-allele OR = 1.28, 95% CI: 1.18–1.39) In conclusion, we report that chromosome 2p21 harbors a complex genetic architecture for common RCC risk variants. PMID:22113997

  18. Multi-scale characterization of pore evolution in a combustion metamorphic complex, Hatrurim basin, Israel: Combining (ultra) small-angle neutron scattering and image analysis

    NASA Astrophysics Data System (ADS)

    Wang, Hsiu-Wen; Anovitz, Lawrence M.; Burg, Avihu; Cole, David R.; Allard, Lawrence F.; Jackson, Andrew J.; Stack, Andrew G.; Rother, Gernot

    2013-11-01

    Backscattered scanning electron micrograph and ultra small- and small-angle neutron scattering data have been combined to provide statistically meaningful data on the pore/grain structure and pore evolution of combustion metamorphic complexes from the Hatrurim basin, Israel. Three processes, anti-sintering roughening, alteration of protolith (dehydration, decarbonation, and oxidation) and crystallization of high-temperature minerals, occurred simultaneously, leading to significant changes in observed pore/grain structures. Pore structures in the protoliths, and in low- and high-grade metamorphic rocks show surface (Ds) and mass (Dm) pore fractal geometries with gradual increases in both Ds and Dm values as a function of metamorphic grade. This suggests that increases in pore volume and formation of less branching pore networks are accompanied by a roughening of pore/grain interfaces. Additionally, pore evolution during combustion metamorphism is also characterized by reduced contributions from small-scale pores to the cumulative porosity in the high-grade rocks. At high temperatures, small-scale pores may be preferentially closed by the formation of high-temperature minerals, producing a rougher morphology with increasing temperature. Alternatively, large-scale pores may develop at the expense of small-scale pores. These observations (pore fractal geometry and cumulative porosity) indicate that the evolution of pore/grain structures is correlated with the growth of high-temperature phases and is a consequence of the energy balance between pore/grain surface energy and energy arising from heterogeneous phase contacts. The apparent pore volume density further suggests that the localized time/temperature development of the high-grade Hatrurim rocks is not simply an extension of that of the low-grade rocks. The former likely represents the "hot spots (burning foci)" in the overall metamorphic terrain while the latter may represent contact aureoles.

  19. Multi-scale characterization of pore evolution in a combustion metamorphic complex, Hatrurim basin, Israel: Combining (ultra) small-angle neutron scattering and image analysis

    SciTech Connect

    Wang, Hsiu-Wen; Anovitz, Lawrence {Larry} M; Burg, Avihu; Cole, David; Allard Jr, Lawrence Frederick; Jackson, Andrew J; Stack, Andrew G; Rother, Gernot; Ciarlette, Diane D

    2013-01-01

    Backscattered scanning electron micrograph and ultra small- and small-angle neutron scattering data have been combined to provide statistically meaningful data on the pore/grain structure and pore evolution of combustion metamorphic complexes from the Hatrurim basin, Israel. Three processes, anti-sintering roughening, alteration of protolith (dehydration, decarbonation, and oxidation) and crystallization of high-temperature minerals, occurred simultaneously, leading to significant changes in observed pore/grain structures. Pore structures in the protoliths, and in lowand high-grade metamorphic rocks show surface (Ds) and mass (Dm) pore fractal geometries with gradual increases in both Ds and Dm values as a function of metamorphic grade. This suggests that increases in pore volume and formation of less branching pore networks are accompanied by a roughening of pore/grain interfaces. Additionally, pore evolution during combustion metamorphism is also characterized by reduced contributions from small-scale pores to the cumulative porosity in the high-grade rocks. At high temperatures, small-scale pores may be preferentially closed by the formation of high-temperature minerals, producing a rougher morphology with increasing temperature. Alternatively, large-scale pores may develop at the expense of small-scale pores. These observations (pore fractal geometry and cumulative porosity) indicate that the evolution of pore/grain structures is correlated with the growth of high-temperature phases and is a consequence of the energy balance between pore/grain surface energy and energy arising from heterogeneous phase contacts. The apparent pore volume density further suggests that the localized time/temperature development of the high-grade Hatrurim rocks is not simply an extension of that of the low-grade rocks. The former likely represents the "hot spots (burning foci)" in the overall metamorphic terrain while the latter may represent contact aureoles.

  20. A characterization of the coupled evolution of grain fabric and pore space using complex networks: Pore connectivity and optimized flows in the presence of shear bands

    NASA Astrophysics Data System (ADS)

    Russell, Scott; Walker, David M.; Tordesillas, Antoinette

    2016-03-01

    A framework for the multiscale characterization of the coupled evolution of the solid grain fabric and its associated pore space in dense granular media is developed. In this framework, a pseudo-dual graph transformation of the grain contact network produces a graph of pores which can be readily interpreted as a pore space network. Survivability, a new metric succinctly summarizing the connectivity of the solid grain and pore space networks, measures material robustness. The size distribution and the connectivity of pores can be characterized quantitatively through various network properties. Assortativity characterizes the pore space with respect to the parity of the number of particles enclosing the pore. Multiscale clusters of odd parity versus even parity contact cycles alternate spatially along the shear band: these represent, respectively, local jamming and unjamming regions that continually switch positions in time throughout the failure regime. Optimal paths, established using network shortest paths in favor of large pores, provide clues on preferential paths for interstitial matter transport. In systems with higher rolling resistance at contacts, less tortuous shortest paths thread through larger pores in shear bands. Notably the structural patterns uncovered in the pore space suggest that more robust models of interstitial pore flow through deforming granular systems require a proper consideration of the evolution of in situ shear band and fracture patterns - not just globally, but also inside these localized failure zones.

  1. Direct numerical simulation of supercritical gas flow in complex nanoporous media: Elucidating the relationship between permeability and pore space geometry

    NASA Astrophysics Data System (ADS)

    Landry, C. J.; Prodanovic, M.; Eichhubl, P.

    2015-12-01

    Mudrocks and shales are currently a significant source of natural gas and understanding the basic transport properties of these formations is critical to predicting long-term production, however, the nanoporous nature of mudrocks presents a unique challenge. Mudrock pores are predominantly in the range of 1-100 nm, and within this size range the flow of gas at reservoir conditions will fall within the slip-flow and early transition-flow regime (0.001 < Kn < 1.0). Therefore, flow-rates will significantly deviate from Navier-Stokes predictions. Currently, the study of slip-flows is mostly limited to simple tube and channel geometries, but the geometry of mudrock pores is often sponge-like (organic matter) and/or platy (clays). Here we present a local effective viscosity lattice Boltzmann model (LEV-LBM) constructed for flow simulation in the slip- and early-transition flow regimes, adapted here for complex geometries. At the macroscopic scale the LEV-LBM is parameterized with local effective viscosities at each node to capture the variance of the mean free path of gas molecules in a bounded system. The LEV-LBM is first validated in simple tube geometries, where excellent agreement with linearized Boltzmann solutions is found for Knudsen numbers up to 1.0. The LEV-LBM is then employed to quantify the length effect on the apparent permeability of tubes, which suggests pore network modeling of flow in the slip and early-transition regime will result in overestimation unless the length effect is considered. Furthermore, the LEV-LBM is used to evaluate the predictive value of commonly measured pore geometry characteristics such as porosity, pore size distribution, and specific solid surface area for the calculation of permeability. We show that bundle of tubes models grossly overestimate apparent permeability, as well as underestimate the increase in apparent permeability with decreasing pressure as a result of excluding topology and pore shape from calculations.

  2. Structures of the autoproteolytic domain from the Saccharomyces cerevisiae nuclear pore complex component, Nup145

    SciTech Connect

    Sampathkumar, Parthasarathy; Ozyurt, Sinem A.; Do, Johnny; Bain, Kevin T.; Dickey, Mark; Rodgers, Logan A.; Gheyi, Tarun; Sali, Andrej; Kim, Seung Joong; Phillips, Jeremy; Pieper, Ursula; Fernandez-Martinez, Javier; Franke, Josef D.; Martel, Anne; Tsuruta, Hiro; Atwell, Shane; Thompson, Devon A.; Emtage, J. Spencer; Wasserman, Stephen R.; Rout, Michael P.; Sauder, J. Michael; Burley, Stephen K.

    2012-04-30

    Nuclear pore complexes (NPCs) are large, octagonally symmetric dynamic macromolecular assemblies responsible for exchange of proteins and RNAs between the nucleus and cytoplasm. NPCs are made up of at least 456 polypeptides from {approx}30 distinct nucleoporins. Several of these components, sharing similar structural motifs, form stable subcomplexes that form a coaxial structure containing two outer rings (the nuclear and cytoplasmic rings), two inner rings, and a membrane ring. The yeast (Saccharomyces cerevisiae) Nup145 and its human counterpart are unique among the nucleoporins, in that they undergo autoproteolysis to generate functionally distinct proteins. The human counterpart of Nup145 is expressed as two alternatively spliced mRNA transcripts. The larger 190 kDa precursor undergoes post-translational autoproteolysis at the Phe863-Ser864 peptide bond yielding the 92 kDa Nup98 and the 96 kDa Nup96. The smaller 98 kDa precursor is also autoproteolysed at an analogous site giving 92 kDa Nup98-N and a 6 kDa C-terminal fragment, which may form a noncovalent complex. The yeast Nup145 precursor [Fig. 1(A)] contains twelve repeats of a 'GLFG' peptide motif (FG repeats) at its N-terminus, an internal autoproteolytic domain (a region of high conservation with the homologous yeast nucleoporins Nup110 and Nup116, neither of which undergo autoproteolysis), followed by the C-terminal domain. Various forms of the FG repeats are present in nearly half of all nucleoporins; they form intrinsically disordered regions implicated in gating mechanisms that control passage of macromolecules through NPCs. Nup145 undergoes autoproteolysis at the Phe605-Ser606 peptide bond to generate two functionally distinct proteins, Nup145N and Nup145C. Subsequently, Nup145C associates with six other proteins to form the heptameric Y-complex, a component of the outer rings of the NPC. Nup145N, on the other hand, can shuttle between the NPC and the nuclear interior. It has been suggested that Nup

  3. Structure of a pore-blocking toxin in complex with a eukaryotic voltage-dependent K+ channel

    PubMed Central

    Banerjee, Anirban; Lee, Alice; Campbell, Ernest; MacKinnon, Roderick

    2013-01-01

    Pore-blocking toxins inhibit voltage-dependent K+ channels (Kv channels) by plugging the ion-conduction pathway. We have solved the crystal structure of paddle chimera, a Kv channel in complex with charybdotoxin (CTX), a pore-blocking toxin. The toxin binds to the extracellular pore entryway without producing discernable alteration of the selectivity filter structure and is oriented to project its Lys27 into the pore. The most extracellular K+ binding site (S1) is devoid of K+ electron-density when wild-type CTX is bound, but K+ density is present to some extent in a Lys27Met mutant. In crystals with Cs+ replacing K+, S1 electron-density is present even in the presence of Lys27, a finding compatible with the differential effects of Cs+ vs K+ on CTX affinity for the channel. Together, these results show that CTX binds to a K+ channel in a lock and key manner and interacts directly with conducting ions inside the selectivity filter. DOI: http://dx.doi.org/10.7554/eLife.00594.001 PMID:23705070

  4. [Pearl Harbor.

    ERIC Educational Resources Information Center

    Johnson, Jennifer, Ed.

    1992-01-01

    This issue of "Loblolly Magazine" was written in observance of the 50th anniversary of the U.S. entrance into World War II. The publication features interviews conducted by East Texas high school students with Clarence Otterman, one of the few survivors of the crew of the USS Arizona, which was bombed during the attack on Pearl Harbor, and with a…

  5. Saccharomyces cerevisiae Porin Pore Forms Complexes with Mitochondrial Outer Membrane Proteins Om14p and Om45p

    PubMed Central

    Lauffer, Susann; Mäbert, Katrin; Czupalla, Cornelia; Pursche, Theresia; Hoflack, Bernard; Rödel, Gerhard; Krause-Buchholz, Udo

    2012-01-01

    Numerous transport processes occur between the two mitochondrial (mt) membranes due to the diverse functions and metabolic processes of the mt organelle. The metabolite and ion transport through the mt outer membrane (OM) is widely assumed to be mediated by the porin pore, whereas in the mt inner membrane (IM) specific carriers are responsible for transport processes. Here, we provide evidence by means of Blue Native (BN)-PAGE analysis, co-immunoprecipitation, and tandem affinity purification that the two mt OM proteins Om14p and Om45p associate with the porin pore. Porin molecules seem to assemble independently to build the core unit. A subpopulation of these core units interacts with Om14p and Om45p. With preparative tandem affinity purification followed by MS analysis, we could identify interaction partners of this OM complex, which are mainly localized within the mt IM and function as carriers for diverse molecules. We propose a model for the role of the two OM proteins in addressing the porin pore to bind to specific channels in the mt IM to facilitate transport of metabolites. PMID:22461620

  6. Importin-β modulates the permeability of the nuclear pore complex in a Ran-dependent manner

    PubMed Central

    Lowe, Alan R; Tang, Jeffrey H; Yassif, Jaime; Graf, Michael; Huang, William YC; Groves, Jay T; Weis, Karsten; Liphardt, Jan T

    2015-01-01

    Soluble karyopherins of the importin-β (impβ) family use RanGTP to transport cargos directionally through the nuclear pore complex (NPC). Whether impβ or RanGTP regulate the permeability of the NPC itself has been unknown. In this study, we identify a stable pool of impβ at the NPC. A subpopulation of this pool is rapidly turned-over by RanGTP, likely at Nup153. Impβ, but not transportin-1 (TRN1), alters the pore's permeability in a Ran-dependent manner, suggesting that impβ is a functional component of the NPC. Upon reduction of Nup153 levels, inert cargos more readily equilibrate across the NPC yet active transport is impaired. When purified impβ or TRN1 are mixed with Nup153 in vitro, higher-order, multivalent complexes form. RanGTP dissolves the impβ•Nup153 complexes but not those of TRN1•Nup153. We propose that impβ and Nup153 interact at the NPC's nuclear face to form a Ran-regulated mesh that modulates NPC permeability. DOI: http://dx.doi.org/10.7554/eLife.04052.001 PMID:25748139

  7. Nuclear pore complex remodeling by p75NTR cleavage controls TGF-β signaling and astrocyte functions

    PubMed Central

    Schachtrup, Christian; Ryu, Jae Kyu; Mammadzada, Könül; Khan, Abdullah S.; Carlton, Peter M.; Perez, Alex; Christian, Frank; Le Moan, Natacha; Vagena, Eirini; Baeza-Raja, Bernat; Rafalski, Victoria; Chan, Justin P.; Nitschke, Roland; Houslay, Miles D.; Ellisman, Mark H.; Wyss-Coray, Tony; Palop, Jorge J.; Akassoglou, Katerina

    2016-01-01

    Astrocytes play critical roles in neuronal activity and inhibition of regeneration. Here we show that the cleaved p75 neurotrophin receptor (p75NTR) is a component of the nuclear pore complex (NPC) required for glial scar formation and reduced gamma oscillations in mice via regulation of TGF-β signaling. The cleaved p75NTR interacts with nucleoporins to promote Smad2 nucleocytoplasmic shuttling. Thus, NPC remodeling by regulated intramembrane cleavage of p75NTR controls astrocyte-neuronal communication in response to profibrotic factors. PMID:26120963

  8. Extracellular ATP dissociates nonmuscle myosin from P2X(7) complex: this dissociation regulates P2X(7) pore formation.

    PubMed

    Gu, Ben J; Rathsam, Catherine; Stokes, Leanne; McGeachie, Andrew B; Wiley, James S

    2009-08-01

    The P2X(7) receptor is a ligand-gated cation channel that is highly expressed on monocyte-macrophages and that mediates the pro-inflammatory effects of extracellular ATP. Dilation of the P2X(7) channel and massive K(+) efflux follows initial channel opening, but the mechanism of secondary pore formation is unclear. The proteins associated with P2X(7) were isolated by using anti-P2X(7) monoclonal antibody-coated Dynabeads from both interferon-gamma plus LPS-stimulated monocytic THP-1 cells and P2X(7)-transfected HEK-293 cells. Two nonmuscle myosins, NMMHC-IIA and myosin Va, were found to associate with P2X(7) in THP-1 cells and HEK-293 cells, respectively. Activation of the P2X(7) receptor by ATP caused dissociation of P2X(7) from nonmuscle myosin in both cell types. The interaction of P2X(7) and NMMHC-IIA molecules was confirmed by fluorescent life time measurements and fluorescent resonance of energy transfer-based time-resolved flow cytometry assay. Reducing the expression of NMMHC-IIA or myosin Va by small interfering RNA or short hairpin RNA led to a significant increase of P2X(7) pore function without any increase in surface expression or ion channel function of P2X(7) receptors. S-l-blebbistatin, a specific inhibitor of NMMHC-IIA ATPase, inhibited both ATP-induced ethidium uptake and ATP-induced dissociation of P2X(7)-NMMHC-IIA complex. In both cell types nonmuscle myosin closely interacts with P2X(7) and is dissociated from the complex by extracellular ATP. Dissociation of this anchoring protein may be required for the transition of P2X(7) channel to a pore. PMID:19494237

  9. High-Resolution Scanning Electron Microscopy and Immuno-Gold Labeling of the Nuclear Lamina and Nuclear Pore Complex.

    PubMed

    Goldberg, Martin W

    2016-01-01

    Scanning electron microscopy (SEM) is a technique used to image surfaces. Field emission SEMs (feSEMs) can resolve structures that are ~0.5-1.5 nm apart. FeSEM, therefore is a useful technique for imaging molecular structures that exist at surfaces such as membranes. The nuclear envelope consists of four membrane surfaces, all of which may be accessible for imaging. Imaging of the cytoplasmic face of the outer membrane gives information about ribosomes and cytoskeletal attachments, as well as details of the cytoplasmic peripheral components of the nuclear pore complex, and is the most easily accessed surface. The nucleoplasmic face of the inner membrane is easily accessible in some cells, such as amphibian oocytes, giving valuable details about the organization of the nuclear lamina and how it interacts with the nuclear pore complexes. The luminal faces of both membranes are difficult to access, but may be exposed by various fracturing techniques. Protocols are presented here for the preparation, labeling, and feSEM imaging of Xenopus laevis oocyte nuclear envelopes. PMID:27147058

  10. Mislocalization of prelamin A Tyr646Phe mutant to the nuclear pore complex in human embryonic kidney 293 cells

    SciTech Connect

    Pan, Yong; Garg, Abhimanyu; Agarwal, Anil K. . E-mail: anil.agarwal@utsouthwestern.edu

    2007-03-30

    Mature lamin A is formed after post-translational processing of prelamin A, which includes prenylation and carboxymethylation of cysteine 661 in the CaaX motif, followed by two proteolytic cleavages by zinc metalloprotease (ZMPSTE24). We expressed several prelamin A mutants, C661S (defective in prenylation), Y646F (designed to undergo prenylation but not second proteolytic cleavage), double mutant, Y646F/C661S and Y646X (mature lamin A), and the wild-type construct in human embryonic kidney (HEK-293) cells. Only the Y646F mutant co-localized with nuclear pore complex proteins, including Nup53 and Nup98, whereas the other mutants localized to the nuclear envelope rim. The cells expressing Y646F mutant also revealed abnormal nuclear morphology which was partially rescued with the farnesyl transferase inhibitors. These data suggest that the unprenylated prelamin A is not toxic to the cells. The toxicity of prenylated prelamin A may be due to its association and/or accumulation at the nuclear pore complex which could be partially reversed by farnesyl transferase inhibitors.

  11. The Nuclear Pore Complex Function of Sec13 Protein Is Required for Cell Survival during Retinal Development*

    PubMed Central

    Niu, Xubo; Hong, Jian; Zheng, Xiaofeng; Melville, David B.; Knapik, Ela W.; Meng, Anming; Peng, Jinrong

    2014-01-01

    Sec13 is a dual function protein, being a core component of both the COPII coat, which mediates protein trafficking from the endoplasmic reticulum to the Golgi apparatus, and the nuclear pore complex (NPC), which facilitates nucleo-cytoplasmic traffic. Here, we present a genetic model to differentiate the roles of these two functions of Sec13 in vivo. We report that sec13sq198 mutant embryos develop small eyes that exhibit disrupted retinal lamination and that the mutant retina contains an excessive number of apoptotic cells. Surprisingly, we found that loss of COPII function by oligonucleotide-mediated gene knockdown of sec31a and sec31b or brefeldin A treatment did not disrupt retinal lamination, although it did result in digestive organ defects similar to those seen in sec13sq198, suggesting that the digestive organ defects observed in sec13sq198 are due to loss of COPII function, whereas the retinal lamination defects are due to loss of the NPC function. We showed that the retinal cells of sec13sq198 failed to form proper nuclear pores, leading to a nuclear accumulation of total mRNA and abnormal activation of the p53-dependent apoptosis pathway, causing the retinal defect in sec13sq198. Furthermore, we found that a mutant lacking Nup107, a key NPC-specific component, phenocopied the retinal lamination phenotype as observed in sec13sq198. Our results demonstrate a requirement for the nuclear pore function of Sec13 in development of the retina and provide the first genetic evidence to differentiate the contributions of the NPC and the COPII functions of Sec13 during organogenesis. PMID:24627485

  12. Nucleoporin's Like Charge Regions Are Major Regulators of FG Coverage and Dynamics Inside the Nuclear Pore Complex

    PubMed Central

    Peyro, Mohaddeseh; Soheilypour, Mohammad; Ghavami, Ali; Mofrad, Mohammad R. K.

    2015-01-01

    Nucleocytoplasmic transport has been the subject of a large body of research in the past few decades. Recently, the focus of investigations in this field has shifted from studies of the overall function of the nuclear pore complex (NPC) to the examination of the role of different domains of phenylalanine-glycine nucleoporin (FG Nup) sequences on the NPC function. In our recent bioinformatics study, we showed that FG Nups have some evolutionarily conserved sequence-based features that might govern their physical behavior inside the NPC. We proposed the ‘like charge regions’ (LCRs), sequences of charged residues with only one type of charge, as one of the features that play a significant role in the formation of FG network inside the central channel. In this study, we further explore the role of LCRs in the distribution of FG Nups, using a recently developed coarse-grained molecular dynamics model. Our results demonstrate how LCRs affect the formation of two transport pathways. While some FG Nups locate their FG network at the center of the NPC forming a homogeneous meshwork of FG repeats, other FG Nups cover the space adjacent to the NPC wall. LCRs in the former group, i.e. FG Nups that form an FG domain at the center, tend to regulate the size of the highly dense, doughnut-shaped FG meshwork and leave a small low FG density area at the center of the pore for passive diffusion. On the other hand, LCRs in the latter group of FG Nups enable them to maximize their interactions and cover a larger space inside the NPC to increase its capability to transport numerous cargos at the same time. Finally, a new viewpoint is proposed that reconciles different models for the nuclear pore selective barrier function. PMID:26658558

  13. SU-E-J-61: Electrodynamics and Nano-Scale Fluid Dynamics in Protein Localization of Nuclear Pore Complexes

    SciTech Connect

    Cunningham, J; Gatenby, R

    2014-06-01

    Purpose: To develop a simulation to catalyze a reevaluation of common assumptions about 3 dimensional diffusive processes and help cell biologists gain a more nuanced, intuitive understanding of the true physical hurdles of protein signaling cascades. Furthermore, to discuss the possibility of intracellular electrodynamics as a critical, unrecognized component of cellular biology and protein dynamics that is necessary for optimal information flow from the cell membrane to the nucleus. Methods: The Unity 3D gaming physics engine was used to build an accurate virtual scale model of the cytoplasm within a few hundred nanometers of the nuclear membrane. A cloud of simulated pERK proteins is controlled by the physics simulation, where diffusion is based on experimentally measured values and the electrodynamics are based on theoretical nano-fluid dynamics. The trajectories of pERK within the cytoplasm and through the 1250 nuclear pores on the nuclear surface is recorded and analyzed. Results: The simulation quickly demonstrates that pERKs moving solely by diffusion will rarely locate and come within capture distance of a nuclear pore. The addition of intracellular electrodynamics between charges on the nuclear pore complexes and on pERKs increases the number of successful translocations by allowing the electro-physical attractive effects to draw in pERKs from the cytoplasm. The effects of changes in intracellular shielding ion concentrations allowed for estimation of the “capture radius” under varying conditions. Conclusion: The simulation allows a shift in perspective that is paramount in attempting to communicate the scale and dynamics of intracellular protein cascade mechanics. This work has allowed researchers to more fully understand the parameters involved in intracellular electrodynamics, such as shielding anion concentration and protein charge. As these effects are still far below the spatial resolution of currently available measurement technology this

  14. Nucleoporin's Like Charge Regions Are Major Regulators of FG Coverage and Dynamics Inside the Nuclear Pore Complex.

    PubMed

    Peyro, Mohaddeseh; Soheilypour, Mohammad; Ghavami, Ali; Mofrad, Mohammad R K

    2015-01-01

    Nucleocytoplasmic transport has been the subject of a large body of research in the past few decades. Recently, the focus of investigations in this field has shifted from studies of the overall function of the nuclear pore complex (NPC) to the examination of the role of different domains of phenylalanine-glycine nucleoporin (FG Nup) sequences on the NPC function. In our recent bioinformatics study, we showed that FG Nups have some evolutionarily conserved sequence-based features that might govern their physical behavior inside the NPC. We proposed the 'like charge regions' (LCRs), sequences of charged residues with only one type of charge, as one of the features that play a significant role in the formation of FG network inside the central channel. In this study, we further explore the role of LCRs in the distribution of FG Nups, using a recently developed coarse-grained molecular dynamics model. Our results demonstrate how LCRs affect the formation of two transport pathways. While some FG Nups locate their FG network at the center of the NPC forming a homogeneous meshwork of FG repeats, other FG Nups cover the space adjacent to the NPC wall. LCRs in the former group, i.e. FG Nups that form an FG domain at the center, tend to regulate the size of the highly dense, doughnut-shaped FG meshwork and leave a small low FG density area at the center of the pore for passive diffusion. On the other hand, LCRs in the latter group of FG Nups enable them to maximize their interactions and cover a larger space inside the NPC to increase its capability to transport numerous cargos at the same time. Finally, a new viewpoint is proposed that reconciles different models for the nuclear pore selective barrier function. PMID:26658558

  15. Model Inspired by Nuclear Pore Complex Suggests Possible Roles for Nuclear Transport Receptors in Determining Its Structure

    PubMed Central

    Osmanović, Dino; Ford, Ian J.; Hoogenboom, Bart W.

    2013-01-01

    Nuclear transport receptors (NTRs) mediate nucleocytoplasmic transport via their affinity for unstructured proteins (polymers) in the nuclear pore complex (NPC). Here, we have modeled the effect of NTRs on polymeric structure in the nanopore confinement of the NPC central conduit. The model explicitly takes into account inter- and intramolecular interactions, as well as the finite size of the NTRs (∼20% of the NPC channel diameter). It reproduces various proposed scenarios for the channel structure, ranging from a central polymer condensate (selective phase) to brushlike polymer arrangements localized at the channel wall (virtual gate, reduction of dimensionality), with the transport receptors lining the polymer surface. In addition, it predicts a new structure in which NTRs become an integral part of the transport barrier by forming a cross-linked network with the unstructured proteins stretching across the pore. The model provides specific and distinctive predictions for the equilibrium spatial distributions of NTRs for these different scenarios that can be experimentally verified by, e.g., superresolution fluorescence microscopy. Moreover, it suggests mechanisms by which globular macromolecules (colloidal particles) can cause polymer-coated nanopores to switch between open and closed configurations, a possible explanation of the biological function of the NPC, and suggests potential technological applications for filtration and single-molecule sensing. PMID:24359750

  16. Spatiotemporal dynamics of the nuclear pore complex transport barrier resolved by high-speed atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Sakiyama, Yusuke; Mazur, Adam; Kapinos, Larisa E.; Lim, Roderick Y. H.

    2016-08-01

    Nuclear pore complexes (NPCs) are biological nanomachines that mediate the bidirectional traffic of macromolecules between the cytoplasm and nucleus in eukaryotic cells. This process involves numerous intrinsically disordered, barrier-forming proteins known as phenylalanine-glycine nucleoporins (FG Nups) that are tethered inside each pore. The selective barrier mechanism has so far remained unresolved because the FG Nups have eluded direct structural analysis within NPCs. Here, high-speed atomic force microscopy is used to visualize the nanoscopic spatiotemporal dynamics of FG Nups inside Xenopus laevis oocyte NPCs at timescales of ∼100 ms. Our results show that the cytoplasmic orifice is circumscribed by highly flexible, dynamically fluctuating FG Nups that rapidly elongate and retract, consistent with the diffusive motion of tethered polypeptide chains. On this basis, intermingling FG Nups exhibit transient entanglements in the central channel, but do not cohere into a tightly crosslinked meshwork. Therefore, the basic functional form of the NPC barrier is comprised of highly dynamic FG Nups that manifest as a central plug or transporter when averaged in space and time.

  17. Structural basis for binding the TREX2 complex to nuclear pores, GAL1 localisation and mRNA export.

    PubMed

    Jani, Divyang; Valkov, Eugene; Stewart, Murray

    2014-06-01

    The conserved Sac3:Thp1:Sem1:Sus1:Cdc31 (TREX2) complex binds to nuclear pore complexes (NPCs) and, in addition to integrating mRNA nuclear export with preceding steps in the gene expression pathway, facilitates re-positioning of highly regulated actively transcribing genes (such as GAL1) to NPCs. Although TREX2 is thought to bind NPC protein Nup1, defining the precise role of this interaction has been frustrated by the complex pleiotropic phenotype exhibited by nup1Δ strains. To provide a structural framework for understanding the binding of TREX2 to NPCs and its function in the gene expression pathway, we have determined the structure of the Nup1:TREX2 interaction interface and used this information to engineer a Sac3 variant that impairs NPC binding while not compromising TREX2 assembly. This variant inhibited the NPC association of both de-repressed and activated GAL1 and also produced mRNA export and growth defects. These results indicate that the TREX2:Nup1 interaction facilitates the efficient nuclear export of bulk mRNA together with the re-positioning of GAL1 to NPCs that is required for transcriptional control that is mediated by removal of SUMO from repressors by NPC-bound Ulp1. PMID:24705649

  18. The Three Fungal Transmembrane Nuclear Pore Complex Proteins of Aspergillus nidulans Are Dispensable in the Presence of an Intact An-Nup84-120 Complex

    PubMed Central

    Liu, Hui-Lin; De Souza, Colin P.C.; Osmani, Aysha H.

    2009-01-01

    In Aspergillus nidulans nuclear pore complexes (NPCs) undergo partial mitotic disassembly such that 12 NPC proteins (Nups) form a core structure anchored across the nuclear envelope (NE). To investigate how the NPC core is maintained, we affinity purified the major core An-Nup84-120 complex and identified two new fungal Nups, An-Nup37 and An-ELYS, previously thought to be vertebrate specific. During mitosis the An-Nup84-120 complex locates to the NE and spindle pole bodies but, unlike vertebrate cells, does not concentrate at kinetochores. We find that mutants lacking individual An-Nup84-120 components are sensitive to the membrane destabilizer benzyl alcohol (BA) and high temperature. Although such mutants display no defects in mitotic spindle formation, they undergo mitotic specific disassembly of the NPC core and transient aggregation of the mitotic NE, suggesting the An-Nup84-120 complex might function with membrane. Supporting this, we show cells devoid of all known fungal transmembrane Nups (An-Ndc1, An-Pom152, and An-Pom34) are viable but that An-ndc1 deletion combined with deletion of individual An-Nup84-120 components is either lethal or causes sensitivity to treatments expected to destabilize membrane. Therefore, the An-Nup84-120 complex performs roles, perhaps at the NPC membrane as proposed previously, that become essential without the An-Ndc1 transmembrane Nup. PMID:19019988

  19. On the complex structural diffusion of proton holes in nanoconfined alkaline solutions within slit pores

    PubMed Central

    Muñoz-Santiburcio, Daniel; Marx, Dominik

    2016-01-01

    The hydroxide anion OH−(aq) in homogeneous bulk water, that is, the solvated proton hole, is known to feature peculiar properties compared with excess protons solvated therein. In this work, it is disclosed that nanoconfinement of such alkaline aqueous solutions strongly affects the key structural and dynamical properties of OH−(aq) compared with the bulk limit. The combined effect of the preferred hypercoordinated solvation pattern of OH−(aq), its preferred perpendicular orientation relative to the confining surfaces, the pronounced layering of nanoconfined water and the topology of the hydrogen bond network required for proton hole transfer lead to major changes of the charge transport mechanism, in such a way that the proton hole migration mechanism depends exquisitely on the width of the confined space that hosts the water film. Moreover, the anionic Zundel complex, which is of transient nature in homogeneous bulk solutions, can be dynamically trapped as a shallow intermediate species by suitable nanoconfinement conditions. PMID:27550616

  20. On the complex structural diffusion of proton holes in nanoconfined alkaline solutions within slit pores.

    PubMed

    Muñoz-Santiburcio, Daniel; Marx, Dominik

    2016-01-01

    The hydroxide anion OH(-)(aq) in homogeneous bulk water, that is, the solvated proton hole, is known to feature peculiar properties compared with excess protons solvated therein. In this work, it is disclosed that nanoconfinement of such alkaline aqueous solutions strongly affects the key structural and dynamical properties of OH(-)(aq) compared with the bulk limit. The combined effect of the preferred hypercoordinated solvation pattern of OH(-)(aq), its preferred perpendicular orientation relative to the confining surfaces, the pronounced layering of nanoconfined water and the topology of the hydrogen bond network required for proton hole transfer lead to major changes of the charge transport mechanism, in such a way that the proton hole migration mechanism depends exquisitely on the width of the confined space that hosts the water film. Moreover, the anionic Zundel complex, which is of transient nature in homogeneous bulk solutions, can be dynamically trapped as a shallow intermediate species by suitable nanoconfinement conditions. PMID:27550616

  1. Molecular Basis for the Anchoring of Proto-Oncoprotein Nup98 to the Cytoplasmic Face of the Nuclear Pore Complex

    PubMed Central

    Stuwe, Tobias T.; von Borzyskowski, Lennart Schada; Davenport, Andrew M.; Hoelz, André

    2014-01-01

    The cytoplasmic filament nucleoporins of the nuclear pore complex (NPC) are critically involved in nuclear export and remodeling of mRNA ribonucleoprotein particles and are associated with various human malignancies. Here, we report the crystal structure of the Nup98 C-terminal autoproteolytic domain, frequently missing from leukemogenic forms of the protein, in complex with the N-terminal domain of Nup82 and the C-terminal tail fragment of Nup159. The Nup82 β propeller serves as a non-cooperative binding platform for both binding partners. Interaction of Nup98 with Nup82 occurs through a reciprocal exchange of loop structures. Strikingly, the same Nup98 groove promiscuously interacts with Nup82 and Nup96 in a mutually excusive fashion. Simultaneous disruption of both Nup82 interactions in yeast causes severe defects in mRNA export, while the severing of a single interaction is tolerated. Thus, the cytoplasmic filament network of the NPC is robust, consistent with its essential function in nucleocytoplasmic transport. PMID:22480613

  2. Nuclear pore complex evolution: a trypanosome Mlp analogue functions in chromosomal segregation but lacks transcriptional barrier activity

    PubMed Central

    Holden, Jennifer M.; Koreny, Ludek; Obado, Samson; Ratushny, Alexander V.; Chen, Wei-Ming; Chiang, Jung-Hsien; Kelly, Steven; Chait, Brian T.; Aitchison, John D.; Rout, Michael P.; Field, Mark C.

    2014-01-01

    The nuclear pore complex (NPC) has dual roles in nucleocytoplasmic transport and chromatin organization. In many eukaryotes the coiled-coil Mlp/Tpr proteins of the NPC nuclear basket have specific functions in interactions with chromatin and defining specialized regions of active transcription, whereas Mlp2 associates with the mitotic spindle/NPC in a cell cycle–dependent manner. We previously identified two putative Mlp-related proteins in African trypanosomes, TbNup110 and TbNup92, the latter of which associates with the spindle. We now provide evidence for independent ancestry for TbNup92/TbNup110 and Mlp/Tpr proteins. However, TbNup92 is required for correct chromosome segregation, with knockout cells exhibiting microaneuploidy and lowered fidelity of telomere segregation. Further, TbNup92 is intimately associated with the mitotic spindle and spindle anchor site but apparently has minimal roles in control of gene transcription, indicating that TbNup92 lacks major barrier activity. TbNup92 therefore acts as a functional analogue of Mlp/Tpr proteins, and, together with the lamina analogue NUP-1, represents a cohort of novel proteins operating at the nuclear periphery of trypanosomes, uncovering complex evolutionary trajectories for the NPC and nuclear lamina. PMID:24600046

  3. Biallelic Mutations in Nuclear Pore Complex Subunit NUP107 Cause Early-Childhood-Onset Steroid-Resistant Nephrotic Syndrome

    PubMed Central

    Miyake, Noriko; Tsukaguchi, Hiroyasu; Koshimizu, Eriko; Shono, Akemi; Matsunaga, Satoko; Shiina, Masaaki; Mimura, Yasuhiro; Imamura, Shintaro; Hirose, Tomonori; Okudela, Koji; Nozu, Kandai; Akioka, Yuko; Hattori, Motoshi; Yoshikawa, Norishige; Kitamura, Akiko; Cheong, Hae Il; Kagami, Shoji; Yamashita, Michiaki; Fujita, Atsushi; Miyatake, Satoko; Tsurusaki, Yoshinori; Nakashima, Mitsuko; Saitsu, Hirotomo; Ohashi, Kenichi; Imamoto, Naoko; Ryo, Akihide; Ogata, Kazuhiro; Iijima, Kazumoto; Matsumoto, Naomichi

    2015-01-01

    The nuclear pore complex (NPC) is a huge protein complex embedded in the nuclear envelope. It has central functions in nucleocytoplasmic transport, nuclear framework, and gene regulation. Nucleoporin 107 kDa (NUP107) is a component of the NPC central scaffold and is an essential protein in all eukaryotic cells. Here, we report on biallelic NUP107 mutations in nine affected individuals who are from five unrelated families and show early-onset steroid-resistant nephrotic syndrome (SRNS). These individuals have pathologically focal segmental glomerulosclerosis, a condition that leads to end-stage renal disease with high frequency. NUP107 is ubiquitously expressed, including in glomerular podocytes. Three of four NUP107 mutations detected in the affected individuals hamper NUP107 binding to NUP133 (nucleoporin 133 kDa) and NUP107 incorporation into NPCs in vitro. Zebrafish with nup107 knockdown generated by morpholino oligonucleotides displayed hypoplastic glomerulus structures and abnormal podocyte foot processes, thereby mimicking the pathological changes seen in the kidneys of the SRNS individuals with NUP107 mutations. Considering the unique properties of the podocyte (highly differentiated foot-process architecture and slit membrane and the inability to regenerate), we propose a “podocyte-injury model” as the pathomechanism for SRNS due to biallelic NUP107 mutations. PMID:26411495

  4. A clonal complex 12 methicillin-resistant Staphylococcus aureus strain, West Australian MRSA-59, harbors a novel pseudo-SCCmec element.

    PubMed

    Monecke, Stefan; Coombs, Geoffrey W; Pearson, Julie; Hotzel, Helmut; Slickers, Peter; Ehricht, Ralf

    2015-11-01

    A West Australian methicillin-resistant Staphylococcus aureus strain (WA MRSA-59) was characterized by microarray and sequencing. Its pseudo-staphylococcal cassette chromosome mec (SCCmec) element comprised dcs, Q9XB68-dcs, mvaS-SCC, Q5HJW6, dru, ugpQ, ydeM, mecA-mecR-mecI, txbi mecI, tnp IS431, copA2-mco (copper resistance), ydhK, arsC-arsB-arsR (arsenic resistance), open reading frame PT43, and per-2. Recombinase genes, xylR (mecR2), and PSM-mec (phenol-soluble modulin) were absent. We suggest that mec complex A should be split into two subtypes. One harbors PSM-mec and xylR (mecR2). It is found in SCCmec types II, III, and VIII. The second subtype, described herein, is present in WA MRSA-59 and some coagulase-negative staphylococci. PMID:26349822

  5. A Clonal Complex 12 Methicillin-Resistant Staphylococcus aureus Strain, West Australian MRSA-59, Harbors a Novel Pseudo-SCCmec Element

    PubMed Central

    Coombs, Geoffrey W.; Pearson, Julie; Hotzel, Helmut; Slickers, Peter; Ehricht, Ralf

    2015-01-01

    A West Australian methicillin-resistant Staphylococcus aureus strain (WA MRSA-59) was characterized by microarray and sequencing. Its pseudo-staphylococcal cassette chromosome mec (SCCmec) element comprised dcs, Q9XB68-dcs, mvaS-SCC, Q5HJW6, dru, ugpQ, ydeM, mecA-mecR-mecI, txbi mecI, tnp IS431, copA2-mco (copper resistance), ydhK, arsC-arsB-arsR (arsenic resistance), open reading frame PT43, and per-2. Recombinase genes, xylR (mecR2), and PSM-mec (phenol-soluble modulin) were absent. We suggest that mec complex A should be split into two subtypes. One harbors PSM-mec and xylR (mecR2). It is found in SCCmec types II, III, and VIII. The second subtype, described herein, is present in WA MRSA-59 and some coagulase-negative staphylococci. PMID:26349822

  6. Yeast Integral Membrane Proteins Apq12, Brl1, and Brr6 Form a Complex Important for Regulation of Membrane Homeostasis and Nuclear Pore Complex Biogenesis

    PubMed Central

    Lone, Museer A.; Atkinson, Aaron E.; Hodge, Christine A.; Cottier, Stéphanie; Martínez-Montañés, Fernando; Maithel, Shelley; Mène-Saffrané, Laurent

    2015-01-01

    Proper functioning of intracellular membranes is critical for many cellular processes. A key feature of membranes is their ability to adapt to changes in environmental conditions by adjusting their composition so as to maintain constant biophysical properties, including fluidity and flexibility. Similar changes in the biophysical properties of membranes likely occur when intracellular processes, such as vesicle formation and fusion, require dramatic changes in membrane curvature. Similar modifications must also be made when nuclear pore complexes (NPCs) are constructed within the existing nuclear membrane, as occurs during interphase in all eukaryotes. Here we report on the role of the essential nuclear envelope/endoplasmic reticulum (NE/ER) protein Brl1 in regulating the membrane composition of the NE/ER. We show that Brl1 and two other proteins characterized previously—Brr6, which is closely related to Brl1, and Apq12—function together and are required for lipid homeostasis. All three transmembrane proteins are localized to the NE and can be coprecipitated. As has been shown for mutations affecting Brr6 and Apq12, mutations in Brl1 lead to defects in lipid metabolism, increased sensitivity to drugs that inhibit enzymes involved in lipid synthesis, and strong genetic interactions with mutations affecting lipid metabolism. Mutations affecting Brl1 or Brr6 or the absence of Apq12 leads to hyperfluid membranes, because mutant cells are hypersensitive to agents that increase membrane fluidity. We suggest that the defects in nuclear pore complex biogenesis and mRNA export seen in these mutants are consequences of defects in maintaining the biophysical properties of the NE. PMID:26432634

  7. Yeast Integral Membrane Proteins Apq12, Brl1, and Brr6 Form a Complex Important for Regulation of Membrane Homeostasis and Nuclear Pore Complex Biogenesis.

    PubMed

    Lone, Museer A; Atkinson, Aaron E; Hodge, Christine A; Cottier, Stéphanie; Martínez-Montañés, Fernando; Maithel, Shelley; Mène-Saffrané, Laurent; Cole, Charles N; Schneiter, Roger

    2015-12-01

    Proper functioning of intracellular membranes is critical for many cellular processes. A key feature of membranes is their ability to adapt to changes in environmental conditions by adjusting their composition so as to maintain constant biophysical properties, including fluidity and flexibility. Similar changes in the biophysical properties of membranes likely occur when intracellular processes, such as vesicle formation and fusion, require dramatic changes in membrane curvature. Similar modifications must also be made when nuclear pore complexes (NPCs) are constructed within the existing nuclear membrane, as occurs during interphase in all eukaryotes. Here we report on the role of the essential nuclear envelope/endoplasmic reticulum (NE/ER) protein Brl1 in regulating the membrane composition of the NE/ER. We show that Brl1 and two other proteins characterized previously-Brr6, which is closely related to Brl1, and Apq12-function together and are required for lipid homeostasis. All three transmembrane proteins are localized to the NE and can be coprecipitated. As has been shown for mutations affecting Brr6 and Apq12, mutations in Brl1 lead to defects in lipid metabolism, increased sensitivity to drugs that inhibit enzymes involved in lipid synthesis, and strong genetic interactions with mutations affecting lipid metabolism. Mutations affecting Brl1 or Brr6 or the absence of Apq12 leads to hyperfluid membranes, because mutant cells are hypersensitive to agents that increase membrane fluidity. We suggest that the defects in nuclear pore complex biogenesis and mRNA export seen in these mutants are consequences of defects in maintaining the biophysical properties of the NE. PMID:26432634

  8. GLE2, a Saccharomyces cerevisiae homologue of the Schizosaccharomyces pombe export factor RAE1, is required for nuclear pore complex structure and function.

    PubMed Central

    Murphy, R; Watkins, J L; Wente, S R

    1996-01-01

    To identify and characterize novel factors required for nuclear transport, a genetic screen was conducted in the yeast Saccharomyces cerevisiae. Mutations that were lethal in combination with a null allele of the gene encoding the nucleoporin Nup100p were isolated using a colony-sectoring assay. Three complementation groups of gle (for GLFG lethal) mutants were identified. In this report, the characterization of GLE2 is detailed. GLE2 encodes a 40.5-kDa polypeptide with striking similarity to that of Schizosaccharomyces pombe RAE1. In indirect immunofluorescence and nuclear pore complex fractionation experiments, Gle2p was associated with nuclear pore complexes. Mutated alleles of GLE2 displayed blockage of polyadenylated RNA export; however, nuclear protein import was not apparently diminished. Immunofluorescence and thin-section electron microscopic analysis revealed that the nuclear pore complex and nuclear envelope structure was grossly perturbed in gle2 mutants. Because the clusters of herniated pore complexes appeared subsequent to the export block, the structural perturbations were likely indirect consequences of the export phenotype. Interestingly, a two-hybrid interaction was detected between Gle2p and Srp1p, the nuclear localization signal receptor, as well as Rip1p, a nuclear export signal-interacting protein. We propose that Gle2p has a novel role in mediating nuclear transport. Images PMID:8970155

  9. Structure of the C-terminal domain of Saccharomyces cerevisiae Nup133, a component of the nuclear pore complex

    SciTech Connect

    Sampathkumar, Parthasarathy; Gheyi, Tarun; Miller, Stacy A.; Bain, Kevin T.; Dickey, Mark; Bonanno, Jeffrey B.; Kim, Seung Joong; Phillips, Jeremy; Pieper, Ursula; Fernandez-Martinez, Javier; Franke, Josef D.; Martel, Anne; Tsuruta, Hiro; Atwell, Shane; Thompson, Devon A.; Emtage, J. Spencer; Wasserman, Stephen R.; Rout, Michael P.; Sali, Andrej; Sauder, J. Michael; Burley, Stephen K.

    2012-10-23

    Nuclear pore complexes (NPCs), responsible for the nucleo-cytoplasmic exchange of proteins and nucleic acids, are dynamic macromolecular assemblies forming an eight-fold symmetric co-axial ring structure. Yeast (Saccharomyces cerevisiae) NPCs are made up of at least 456 polypeptide chains of {approx}30 distinct sequences. Many of these components (nucleoporins, Nups) share similar structural motifs and form stable subcomplexes. We have determined a high-resolution crystal structure of the C-terminal domain of yeast Nup133 (ScNup133), a component of the heptameric Nup84 subcomplex. Expression tests yielded ScNup133(944-1157) that produced crystals diffracting to 1.9{angstrom} resolution. ScNup133(944-1157) adopts essentially an all {alpha}-helical fold, with a short two stranded {beta}-sheet at the C-terminus. The 11 {alpha}-helices of ScNup133(944-1157) form a compact fold. In contrast, the previously determined structure of human Nup133(934-1156) bound to a fragment of human Nup107 has its constituent {alpha}-helices are arranged in two globular blocks. These differences may reflect structural divergence among homologous nucleoporins.

  10. Biased assembly of the nuclear pore complex is required for somatic and germline nuclear differentiation in Tetrahymena

    PubMed Central

    Iwamoto, Masaaki; Koujin, Takako; Osakada, Hiroko; Mori, Chie; Kojidani, Tomoko; Matsuda, Atsushi; Asakawa, Haruhiko; Hiraoka, Yasushi; Haraguchi, Tokuko

    2015-01-01

    Ciliates have two functionally distinct nuclei, a somatic macronucleus (MAC) and a germline micronucleus (MIC) that develop from daughter nuclei of the last postzygotic division (PZD) during the sexual process of conjugation. Understanding this nuclear dimorphism is a central issue in ciliate biology. We show, by live-cell imaging of Tetrahymena, that biased assembly of the nuclear pore complex (NPC) occurs immediately after the last PZD, which generates anterior-posterior polarized nuclei: MAC-specific NPCs assemble in anterior presumptive MACs but not in posterior presumptive MICs. MAC-specific NPC assembly in the anterior nuclei occurs much earlier than transport of Twi1p, which is required for MAC genome rearrangement. Correlative light-electron microscopy shows that addition of new nuclear envelope (NE) precursors occurs through the formation of domains of redundant NE, where the outer double membrane contains the newly assembled NPCs. Nocodazole inhibition of the second PZD results in assembly of MAC-specific NPCs in the division-failed zygotic nuclei, leading to failure of MIC differentiation. Our findings demonstrate that NPC type switching has a crucial role in the establishment of nuclear differentiation in ciliates. PMID:25788697

  11. Biased assembly of the nuclear pore complex is required for somatic and germline nuclear differentiation in Tetrahymena.

    PubMed

    Iwamoto, Masaaki; Koujin, Takako; Osakada, Hiroko; Mori, Chie; Kojidani, Tomoko; Matsuda, Atsushi; Asakawa, Haruhiko; Hiraoka, Yasushi; Haraguchi, Tokuko

    2015-05-01

    Ciliates have two functionally distinct nuclei, a somatic macronucleus (MAC) and a germline micronucleus (MIC) that develop from daughter nuclei of the last postzygotic division (PZD) during the sexual process of conjugation. Understanding this nuclear dimorphism is a central issue in ciliate biology. We show, by live-cell imaging of Tetrahymena, that biased assembly of the nuclear pore complex (NPC) occurs immediately after the last PZD, which generates anterior-posterior polarized nuclei: MAC-specific NPCs assemble in anterior presumptive MACs but not in posterior presumptive MICs. MAC-specific NPC assembly in the anterior nuclei occurs much earlier than transport of Twi1p, which is required for MAC genome rearrangement. Correlative light-electron microscopy shows that addition of new nuclear envelope (NE) precursors occurs through the formation of domains of redundant NE, where the outer double membrane contains the newly assembled NPCs. Nocodazole inhibition of the second PZD results in assembly of MAC-specific NPCs in the division-failed zygotic nuclei, leading to failure of MIC differentiation. Our findings demonstrate that NPC type switching has a crucial role in the establishment of nuclear differentiation in ciliates. PMID:25788697

  12. High-resolution imaging reveals new features of nuclear export of mRNA through the nuclear pore complexes.

    PubMed

    Kelich, Joseph M; Yang, Weidong

    2014-01-01

    The nuclear envelope (NE) of eukaryotic cells provides a physical barrier for messenger RNA (mRNA) and the associated proteins (mRNPs) traveling from sites of transcription in the nucleus to locations of translation processing in the cytoplasm. Nuclear pore complexes (NPCs) embedded in the NE serve as a dominant gateway for nuclear export of mRNA. However, the fundamental characterization of export dynamics of mRNPs through the NPC has been hindered by several technical limits. First, the size of NPC that is barely below the diffraction limit of conventional light microscopy requires a super-resolution microscopy imaging approach. Next, the fast transit of mRNPs through the NPC further demands a high temporal resolution by the imaging approach. Finally, the inherent three-dimensional (3D) movements of mRNPs through the NPC demand the method to provide a 3D mapping of both transport kinetics and transport pathways of mRNPs. This review will highlight the recently developed super-resolution imaging techniques advanced from 1D to 3D for nuclear export of mRNPs and summarize the new features in the dynamic nuclear export process of mRNPs revealed from these technical advances. PMID:25141104

  13. Atomic structure of the nuclear pore complex targeting domain of a Nup116 homologue from the yeast, Candida glabrata

    SciTech Connect

    Sampathkumar, Parthasarathy; Kim, Seung Joong; Manglicmot, Danalyn; Bain, Kevin T.; Gilmore, Jeremiah; Gheyi, Tarun; Phillips, Jeremy; Pieper, Ursula; Fernandez-Martinez, Javier; Franke, Josef D.; Matsui, Tsutomu; Tsuruta, Hiro; Atwell, Shane; Thompson, Devon A.; Emtage, J. Spencer; Wasserman, Stephen R.; Rout, Michael P.; Sali, Andrej; Sauder, J. Michael; Almo, Steven C.; Burley, Stephen K.

    2012-10-23

    The nuclear pore complex (NPC), embedded in the nuclear envelope, is a large, dynamic molecular assembly that facilitates exchange of macromolecules between the nucleus and the cytoplasm. The yeast NPC is an eightfold symmetric annular structure composed of {approx}456 polypeptide chains contributed by {approx}30 distinct proteins termed nucleoporins. Nup116, identified only in fungi, plays a central role in both protein import and mRNA export through the NPC. Nup116 is a modular protein with N-terminal 'FG' repeats containing a Gle2p-binding sequence motif and a NPC targeting domain at its C-terminus. We report the crystal structure of the NPC targeting domain of Candida glabrata Nup116, consisting of residues 882-1034 [CgNup116(882-1034)], at 1.94 {angstrom} resolution. The X-ray structure of CgNup116(882-1034) is consistent with the molecular envelope determined in solution by small-angle X-ray scattering. Structural similarities of CgNup116(882-1034) with homologous domains from Saccharomyces cerevisiae Nup116, S. cerevisiae Nup145N, and human Nup98 are discussed.

  14. Rapid evolution and complex structural organization in genomic regions harboring multiple prolamin genes in the polyploid wheat genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genes encoding wheat prolamins belong to complicated multi-gene families in the wheat genome. To understand the structural complexity of storage protein loci, we sequenced and analyzed orthologous regions containing both gliadin and LMW-glutenin genes from the A and B genomes of a tetraploid wheat ...

  15. Major histocompatibility complex harbors widespread genotypic variability of non-additive risk of rheumatoid arthritis including epistasis.

    PubMed

    Wei, Wen-Hua; Bowes, John; Plant, Darren; Viatte, Sebastien; Yarwood, Annie; Massey, Jonathan; Worthington, Jane; Eyre, Stephen

    2016-01-01

    Genotypic variability based genome-wide association studies (vGWASs) can identify potentially interacting loci without prior knowledge of the interacting factors. We report a two-stage approach to make vGWAS applicable to diseases: firstly using a mixed model approach to partition dichotomous phenotypes into additive risk and non-additive environmental residuals on the liability scale and secondly using the Levene's (Brown-Forsythe) test to assess equality of the residual variances across genotype groups per marker. We found widespread significant (P < 2.5e-05) vGWAS signals within the major histocompatibility complex (MHC) across all three study cohorts of rheumatoid arthritis. We further identified 10 epistatic interactions between the vGWAS signals independent of the MHC additive effects, each with a weak effect but jointly explained 1.9% of phenotypic variance. PTPN22 was also identified in the discovery cohort but replicated in only one independent cohort. Combining the three cohorts boosted power of vGWAS and additionally identified TYK2 and ANKRD55. Both PTPN22 and TYK2 had evidence of interactions reported elsewhere. We conclude that vGWAS can help discover interacting loci for complex diseases but require large samples to find additional signals. PMID:27109064

  16. Major histocompatibility complex harbors widespread genotypic variability of non-additive risk of rheumatoid arthritis including epistasis

    PubMed Central

    Wei, Wen-Hua; Bowes, John; Plant, Darren; Viatte, Sebastien; Yarwood, Annie; Massey, Jonathan; Worthington, Jane; Eyre, Stephen

    2016-01-01

    Genotypic variability based genome-wide association studies (vGWASs) can identify potentially interacting loci without prior knowledge of the interacting factors. We report a two-stage approach to make vGWAS applicable to diseases: firstly using a mixed model approach to partition dichotomous phenotypes into additive risk and non-additive environmental residuals on the liability scale and secondly using the Levene’s (Brown-Forsythe) test to assess equality of the residual variances across genotype groups per marker. We found widespread significant (P < 2.5e-05) vGWAS signals within the major histocompatibility complex (MHC) across all three study cohorts of rheumatoid arthritis. We further identified 10 epistatic interactions between the vGWAS signals independent of the MHC additive effects, each with a weak effect but jointly explained 1.9% of phenotypic variance. PTPN22 was also identified in the discovery cohort but replicated in only one independent cohort. Combining the three cohorts boosted power of vGWAS and additionally identified TYK2 and ANKRD55. Both PTPN22 and TYK2 had evidence of interactions reported elsewhere. We conclude that vGWAS can help discover interacting loci for complex diseases but require large samples to find additional signals. PMID:27109064

  17. Dominant-negative mutants of importin-beta block multiple pathways of import and export through the nuclear pore complex.

    PubMed Central

    Kutay, U; Izaurralde, E; Bischoff, F R; Mattaj, I W; Görlich, D

    1997-01-01

    Nuclear protein import proceeds through the nuclear pore complex (NPC). Importin-beta mediates translocation via direct interaction with NPC components and carries importin-alpha with the NLS substrate from the cytoplasm into the nucleus. The import reaction is terminated by the direct binding of nuclear RanGTP to importin-beta which dissociates the importin heterodimer. Here, we analyse the sites of interaction on importin-beta for its multiple partners. Ran and importin-alpha respectively require residues 1-364 and 331-876 of importin-beta for binding. Thus, RanGTP-mediated release of importin-alpha from importin-beta is likely to be an active displacement rather than due to simple competition between Ran and importin-alpha for a common binding site. Importin-beta has at least two non-overlapping sites of interaction with the NPC, which could potentially be used sequentially during translocation. Our data also suggest that termination of import involves a transient release of importin-beta from the NPC. Importin-beta fragments which bind to the NPC, but not to Ran, resist this release mechanism. As would be predicted from this, these importin-beta mutants are very efficient inhibitors of NLS-dependent protein import. Surprisingly, however, they also inhibit M9 signal-mediated nuclear import as well as nuclear export of mRNA, U snRNA, and the NES-containing Rev protein. This suggests that mediators of these various transport events share binding sites on the NPC and/or that mechanisms exist to coordinate translocation through the NPC via different nucleocytoplasmic transport pathways. PMID:9135132

  18. A laboratory study to estimate pore geometric parameters of sandstones using complex conductivity and nuclear magnetic resonance for permeability prediction

    NASA Astrophysics Data System (ADS)

    Osterman, Gordon; Keating, Kristina; Binley, Andrew; Slater, Lee

    2016-06-01

    We estimate parameters from the Katz and Thompson permeability model using laboratory complex electrical conductivity (CC) and nuclear magnetic resonance (NMR) data to build permeability models parameterized with geophysical measurements. We use the Katz and Thompson model based on the characteristic hydraulic length scale, determined from mercury injection capillary pressure estimates of pore throat size, and the intrinsic formation factor, determined from multisalinity conductivity measurements, for this purpose. Two new permeability models are tested, one based on CC data and another that incorporates CC and NMR data. From measurements made on forty-five sandstone cores collected from fifteen different formations, we evaluate how well the CC relaxation time and the NMR transverse relaxation times compare to the characteristic hydraulic length scale and how well the formation factor estimated from CC parameters compares to the intrinsic formation factor. We find: (1) the NMR transverse relaxation time models the characteristic hydraulic length scale more accurately than the CC relaxation time (R2 of 0.69 and 0.33 and normalized root mean square errors (NRMSE) of 0.16 and 0.21, respectively); (2) the CC estimated formation factor is well correlated with the intrinsic formation factor (NRMSE=0.23). We demonstrate that that permeability estimates from the joint-NMR-CC model (NRMSE=0.13) compare favorably to estimates from the Katz and Thompson model (NRMSE=0.074). This model advances the capability of the Katz and Thompson model by employing parameters measureable in the field giving it the potential to more accurately estimate permeability using geophysical measurements than are currently possible.

  19. Nucleoporin FG Domains Facilitate mRNP Remodeling at the Cytoplasmic Face of the Nuclear Pore Complex

    PubMed Central

    Adams, Rebecca L.; Terry, Laura J.; Wente, Susan R.

    2014-01-01

    Directional export of messenger RNA (mRNA) protein particles (mRNPs) through nuclear pore complexes (NPCs) requires multiple factors. In Saccharomyces cerevisiae, the NPC proteins Nup159 and Nup42 are asymmetrically localized to the cytoplasmic face and have distinct functional domains: a phenylalanine-glycine (FG) repeat domain that docks mRNP transport receptors and domains that bind the DEAD-box ATPase Dbp5 and its activating cofactor Gle1, respectively. We speculated that the Nup42 and Nup159 FG domains play a role in positioning mRNPs for the terminal mRNP-remodeling steps carried out by Dbp5. Here we find that deletion (Δ) of both the Nup42 and Nup159 FG domains results in a cold-sensitive poly(A)+ mRNA export defect. The nup42ΔFG nup159ΔFG mutant also has synthetic lethal genetic interactions with dbp5 and gle1 mutants. RNA cross-linking experiments further indicate that the nup42ΔFG nup159ΔFG mutant has a reduced capacity for mRNP remodeling during export. To further analyze the role of these FG domains, we replaced the Nup159 or Nup42 FG domains with FG domains from other Nups. These FG “swaps” demonstrate that only certain FG domains are functional at the NPC cytoplasmic face. Strikingly, fusing the Nup42 FG domain to the carboxy-terminus of Gle1 bypasses the need for the endogenous Nup42 FG domain, highlighting the importance of proximal positioning for these factors. We conclude that the Nup42 and Nup159 FG domains target the mRNP to Gle1 and Dbp5 for mRNP remodeling at the NPC. Moreover, these results provide key evidence that character and context play a direct role in FG domain function and mRNA export. PMID:24931410

  20. CHARLOTTE HARBOR IR, 2002

    EPA Science Inventory

    The 2002 Charlotte Harbor Implementation Review (IR) summarizes the progress and challenges ahead for the Charlotte Harbor National Estuary Program (CHNEP). The implementation review report requires seven components: Status of CCMP implementation (programmatic progress); Environm...

  1. Two conformational states of the membrane-associated Bacillus thuringiensis Cry4Ba {delta}-endotoxin complex revealed by electron crystallography: Implications for toxin-pore formation

    SciTech Connect

    Ounjai, Puey; Unger, Vinzenz M.; Sigworth, Fred J.; Angsuthanasombat, Chanan

    2007-10-05

    The insecticidal nature of Cry {delta}-endotoxins produced by Bacillus thuringiensis is generally believed to be caused by their ability to form lytic pores in the midgut cell membrane of susceptible insect larvae. Here we have analyzed membrane-associated structures of the 65-kDa dipteran-active Cry4Ba toxin by electron crystallography. The membrane-associated toxin complex was crystallized in the presence of DMPC via detergent dialysis. Depending upon the charge of the adsorbed surface, 2D crystals of the oligomeric toxin complex have been captured in two distinct conformations. The projection maps of those crystals have been generated at 17 A resolution. Both complexes appeared to be trimeric; as in one crystal form, its projection structure revealed a symmetrical pinwheel-like shape with virtually no depression in the middle of the complex. The other form revealed a propeller-like conformation displaying an obvious hole in the center region, presumably representing the toxin-induced pore. These crystallographic data thus demonstrate for the first time that the 65-kDa activated Cry4Ba toxin in association with lipid membranes could exist in at least two different trimeric conformations, conceivably implying the closed and open states of the pore.

  2. Complex interactions among residues within pore region determine the K+ dependence of a KAT1-type potassium channel AmKAT1.

    PubMed

    Yang, Guangzhe; Sentenac, Hervé; Véry, Anne-Aliénor; Su, Yanhua

    2015-08-01

    KAT1-type channels mediate K(+) influx into guard cells that enables stomatal opening. In this study, a KAT1-type channel AmKAT1 was cloned from the xerophyte Ammopiptanthus mongolicus. In contrast to most KAT1-type channels, its activation is strongly dependent on external K(+) concentration, so it can be used as a model to explore the mechanism for the K(+) -dependent gating of KAT1-type channels. Domain swapping between AmKAT1 and KAT1 reveals that the S5-pore-S6 region controls the K(+) dependence of AmKAT1, and residue substitutions show that multiple residues within the S5-Pore linker and Pore are involved in its K(+) -dependent gating. Importantly, complex interactions occur among these residues, and it is these interactions that determine its K(+) dependence. Finally, we analyzed the potential mechanism for the K(+) dependence of AmKAT1, which could originate from the requirement of K(+) occupancy in the selectivity filter to maintain its conductive conformation. These results provide new insights into the molecular basis of the K(+) -dependent gating of KAT1-type channels. PMID:26032087

  3. Biogenesis of porin of the outer mitochondrial membrane involves an import pathway via receptors and the general import pore of the TOM complex.

    PubMed

    Krimmer, T; Rapaport, D; Ryan, M T; Meisinger, C; Kassenbrock, C K; Blachly-Dyson, E; Forte, M; Douglas, M G; Neupert, W; Nargang, F E; Pfanner, N

    2001-01-22

    Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial. We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin. Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria. A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore. Using an assay established to monitor the assembly of in vitro-imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane [TOM] core complex). The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40. Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway. We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore. PMID:11266446

  4. Structural Characterization by Cross-linking Reveals the Detailed Architecture of a Coatomer-related Heptameric Module from the Nuclear Pore Complex*

    PubMed Central

    Shi, Yi; Fernandez-Martinez, Javier; Tjioe, Elina; Pellarin, Riccardo; Kim, Seung Joong; Williams, Rosemary; Schneidman-Duhovny, Dina; Sali, Andrej; Rout, Michael P.; Chait, Brian T.

    2014-01-01

    Most cellular processes are orchestrated by macromolecular complexes. However, structural elucidation of these endogenous complexes can be challenging because they frequently contain large numbers of proteins, are compositionally and morphologically heterogeneous, can be dynamic, and are often of low abundance in the cell. Here, we present a strategy for the structural characterization of such complexes that has at its center chemical cross-linking with mass spectrometric readout. In this strategy, we isolate the endogenous complexes using a highly optimized sample preparation protocol and generate a comprehensive, high-quality cross-linking dataset using two complementary cross-linking reagents. We then determine the structure of the complex using a refined integrative method that combines the cross-linking data with information generated from other sources, including electron microscopy, X-ray crystallography, and comparative protein structure modeling. We applied this integrative strategy to determine the structure of the native Nup84 complex, a stable hetero-heptameric assembly (∼600 kDa), 16 copies of which form the outer rings of the 50-MDa nuclear pore complex (NPC) in budding yeast. The unprecedented detail of the Nup84 complex structure reveals previously unseen features in its pentameric structural hub and provides information on the conformational flexibility of the assembly. These additional details further support and augment the protocoatomer hypothesis, which proposes an evolutionary relationship between vesicle coating complexes and the NPC, and indicates a conserved mechanism by which the NPC is anchored in the nuclear envelope. PMID:25161197

  5. Localization of Pom121 to the inner nuclear membrane is required for an early step of interphase nuclear pore complex assembly

    PubMed Central

    Funakoshi, Tomoko; Clever, Michaela; Watanabe, Ai; Imamoto, Naoko

    2011-01-01

    The nuclear pore complex (NPC) is a large protein assembly that mediates molecular trafficking between the cytoplasm and the nucleus. NPCs assemble twice during the cell cycle in metazoans: postmitosis and during interphase. In this study, using small interfering RNA (siRNA) in conjunction with a cell fusion–based NPC assembly assay, we demonstrated that pore membrane protein (Pom)121, a vertebrate-specific integral membrane nucleoporin, is indispensable for an early step in interphase NPC assembly. Functional domain analysis of Pom121 showed that its nuclear localization signals, which bind to importin β via importin α and likely function with RanGTP, play an essential role in targeting Pom121 to the interphase NPC. Furthermore, a region of Pom121 that interacts with the inner nuclear membrane (INM) and lamin B receptor was found to be crucial for its NPC targeting. Based on these findings and on evidence that Pom121 localizes at the INM in the absence of a complete NPC structure, we propose that the nuclear migration of Pom121 and its subsequent interaction with INM proteins are required to initiate interphase NPC assembly. Our data also suggest, for the first time, the importance of the INM as a seeding site for “prepores” during interphase NPC assembly. PMID:21289085

  6. Evaluation of sediment contamination in Pearl Harbor. Final report

    SciTech Connect

    Grovhoug, J.G.

    1992-06-01

    Pearl Harbor demonstrates remarkable resilience to natural and human-induced contaminant stresses. A review of more than fifty harbor-specific data sets reveals a complex contamination and recovery history. Siltation is a major contaminant pathway in Pearl Harbor. Dredging operations, which are necessary due to high siltation rates, reduce contaminant loading by periodically removing the upper harbor sediment layers. The response of test organisms during sediment toxicity and bioaccumulation studies showed negligible effects from sediment toxicity. The environmental quality at an offshore dredge disposal site for the harbor is not measurable affected. Urban runoff via storm drains and tributaries is an important nonpoint source of contaminant exposure to the Pearl Harbor ecosystem. Most contaminants experience extensive physical, chemical, and biological, modification after entering the harbor environment. Certain contaminants, including PCBs, petroleum hydrocarbons, and silver, were reported at sufficiently elevated sediment concentrations to warrant environmental concern in some harbor regions and may warrant further evaluation. The overall sediment quality in Pearl Harbor, however, is less degraded than that of many U.S. mainland coastal harbors. Further detailed study of the abundance and distribution of important marine resources in Pearl Harbor is recommended.

  7. BGLF4 Kinase Modulates the Structure and Transport Preference of the Nuclear Pore Complex To Facilitate Nuclear Import of Epstein-Barr Virus Lytic Proteins

    PubMed Central

    Chang, Chou-Wei; Lee, Chung-Pei; Su, Mei-Tzu; Tsai, Ching-Hwa

    2014-01-01

    ABSTRACT BGLF4 kinase, the only Ser/Thr protein kinase encoded by the Epstein-Barr virus (EBV) genome, phosphorylates multiple viral and cellular substrates to optimize the cellular environment for viral DNA replication and the nuclear egress of nucleocapsids. Previously, we found that nuclear targeting of BGLF4 is through direct interaction with the FG repeat-containing nucleoporins (FG-Nups) Nup62 and Nup153 independently of cytosolic transport factors. Here, we investigated the regulatory effects of BGLF4 on the structure and biological functions of the nuclear pore complex (NPC). In EBV-positive NA cells, the distribution of FG-Nups was modified during EBV reactivation. In transfected cells, BGLF4 changed the staining pattern of Nup62 and Nup153 in a kinase activity-dependent manner. Detection with anti-phospho-Ser/Thr-Pro MPM-2 antibody demonstrated that BGLF4 induced the phosphorylation of Nup62 and Nup153. The nuclear targeting of importin β was attenuated in the presence of BGLF4, leading to inhibition of canonical nuclear localization signal (NLS)-mediated nuclear import. An in vitro nuclear import assay revealed that BGLF4 induced the nuclear import of larger molecules. Notably, we found that BGLF4 promoted the nuclear import of several non-NLS-containing EBV proteins, including the viral DNA-replicating enzymes BSLF1, BBLF2/3, and BBLF4 and the major capsid protein (VCA), in cotransfected cells. The data presented here suggest that BGLF4 interferes with the normal functions of Nup62 and Nup153 and preferentially helps the nuclear import of viral proteins for viral DNA replication and assembly. In addition, the nuclear import-promoting activity was found in cells expressing the BGLF4 homologs of another two gammaherpesviruses but not those from alpha- and betaherpesviruses. IMPORTANCE During lytic replication, many EBV genome-encoded proteins need to be transported into the nucleus, not only for viral DNA replication but also for the assembly of

  8. The Nectin-4/Afadin Protein Complex and Intercellular Membrane Pores Contribute to Rapid Spread of Measles Virus in Primary Human Airway Epithelia

    PubMed Central

    Singh, Brajesh K.; Hornick, Andrew L.; Krishnamurthy, Sateesh; Locke, Anna C.; Mendoza, Crystal A.; Mateo, Mathieu; Miller-Hunt, Catherine L.; Cattaneo, Roberto

    2015-01-01

    ABSTRACT The discovery that measles virus (MV) uses the adherens junction protein nectin-4 as its epithelial receptor provides a new vantage point from which to characterize its rapid spread in the airway epithelium. We show here that in well-differentiated primary cultures of airway epithelial cells from human donors (HAE), MV infectious centers form rapidly and become larger than those of other respiratory pathogens: human respiratory syncytial virus, parainfluenza virus 5, and Sendai virus. While visible syncytia do not form after MV infection of HAE, the cytoplasm of an infected cell suddenly flows into an adjacent cell, as visualized through wild-type MV-expressed cytoplasmic green fluorescent protein (GFP). High-resolution video microscopy documents that GFP flows through openings that form on the lateral surfaces between columnar epithelial cells. To assess the relevance of the protein afadin, which connects nectin-4 to the actin cytoskeleton, we knocked down its mRNA. This resulted in more-limited infectious-center formation. We also generated a nectin-4 mutant without the afadin-binding site in its cytoplasmic tail. This mutant was less effective than wild-type human nectin-4 at promoting MV infection in primary cultures of porcine airway epithelia. Thus, in airway epithelial cells, MV spread requires the nectin-4/afadin complex and is based on cytoplasm transfer between columnar cells. Since the viral membrane fusion apparatus may open the passages that allow cytoplasm transfer, we refer to them as intercellular membrane pores. Virus-induced intercellular pores may contribute to extremely efficient measles contagion by promoting the rapid spread of the virus through the upper respiratory epithelium. IMPORTANCE Measles virus (MV), while targeted for eradication, still causes about 120,000 deaths per year worldwide. The recent reemergence of measles in insufficiently vaccinated populations in Europe and North America reminds us that measles is extremely

  9. The transient pore formed by homologous terminal complement complexes functions as a bidirectional route for the transport of autocrine and paracrine signals across human cell membranes.

    PubMed Central

    Acosta, J. A.; Benzaquen, L. R.; Goldstein, D. J.; Tosteson, M. T.; Halperin, J. A.

    1996-01-01

    BACKGROUND: We have previously shown that the membrane attack complex (MAC) of complement stimulates cell proliferation and that insertion of homologous MAC into the membranes of endothelial cells results in the release of potent mitogens, including basic fibroblast growth factor (bFGF). The mechanism of secretion of bFGF and other polypeptides devoid of signal peptides, such as interleukin 1 (IL-1) is still an open problem in cell biology. We have hypothesized that the homologous MAC pore itself could constitute a transient route for the diffusion of biologically active macromolecules in and out of the target cells. MATERIALS AND METHODS: Human red blood cell ghosts and artificial lipid vesicles were loaded with labeled growth factors, cytokines and IgG, and exposed to homologous MAC. The release of the 125I-macromolecules was followed as a function of time. The incorporation of labeled polypeptides and fluorescent dextran (MW: 10,000) was measured in MAC-impacted human red blood cells and human umbilical endothelial cells (HUVEC), respectively. RESULTS: Homologous MAC insertion into HUVEC resulted in the massive uptake of 10-kD dextran and induced the release of bFGF, in the absence of any measurable lysis. Red blood cell ghosts preloaded with bFGF, IL-1 beta, and the alpha-chain of interferon-gamma (IFN-gamma) released the polypeptides upon MAC insertion, but they did not release preloaded IgG. MAC-impacted ghosts took up radioactive IFN-gamma from the extracellular medium. Vesicles loaded with IL-I released the polypeptide when exposed to MAC. CONCLUSIONS: The homologous MAC pore in its nonlytic form allows for the export of cytosolic proteins devoid of signal peptides that are not secreted through the classical endoplasmic reticulum/Golgi exocytotic pathways. Our results suggest that the release, and perhaps the uptake, of biologically active macromolecules through the homologous MAC pore is a novel biological function of the complement system in mammals

  10. Effect of the ostreolysin A/pleurotolysin B pore-forming complex on intracellular Ca2+ activity in the vascular smooth muscle cell line A10.

    PubMed

    Vrecl, Milka; Babnik, Monika; Sepčić, Kristina; Žužek, Monika C; Maček, Peter; Diacci, Uroš; Frangež, Robert

    2015-12-01

    Ostreolysin A/pleurotolysin B (OlyA/PlyB) is a binary pore-forming protein complex that produces a rapid cardiorespiratory arrest. Increased tonus of the coronary vascular wall produced by OlyA/PlyB may lead to ischemia, arrhythmias, the hypoxic injury of cardiomyocytes and cardiotoxicity. We evaluated the effects of OlyA/PlyB in cultured vascular smooth muscle A10 cells. Fluorometric measurements using the Ca(2+) indicator Fluo-4 AM and Fura-2 AM revealed that nanomolar concentrations of OlyA/PlyB increased the intracellular Ca(2+) activity [Ca(2+)]i in A10 cells. This effect was absent in a Ca(2+)-free medium, indicating that OlyA/PlyB-induced [Ca(2+)]i increase was dependent on Ca(2+) influx into cells. The increase in [Ca(2+)]i by OlyA/PlyB was partially prevented by: i) the calcium channel blockers verapamil and La(3+), ii) the inhibitor of the sodium-calcium exchanger (NCX) benzamil, and iii) the iso-osmotic replacement of NaCl by sucrose. The pre-treatment of cells with the Ca(2+)-ATPase inhibitor thapsigargin reduced the [Ca(2+)]i increase evoked by OlyA/PlyB, whereas the plasma membrane depolarization with high K(+) in the medium did not prevent OlyA/PlyB-induced [Ca(2+)]i. In summary, our data could suggest that the OlyA/PlyB-induced increase in [Ca(2+)]i is due to an influx of Ca(2+) through a variety of co-existing plasma membrane Ca(2+)-permeable channels, Ca(2+) entry through non-selective ion permeable pores formed de novo by OlyA/PlyB in the plasma membrane and calcium-induced intracellular Ca(2+) release, altogether leading to disturbed Ca(2+) homeostasis in A10 cells. PMID:26320834

  11. SUMO modification through rapamycin-mediated heterodimerization reveals a dual role for Ubc9 in targeting RanGAP1 to nuclear pore complexes

    SciTech Connect

    Zhu Shanshan; Zhang Hong; Matunis, Michael J. . E-mail: mmatunis@jhsph.edu

    2006-04-15

    SUMOs (small ubiquitin-related modifiers) are eukaryotic proteins that are covalently conjugated to other proteins and thereby regulate a wide range of important cellular processes. The molecular mechanisms by which SUMO modification influences the functions of most target proteins and cellular processes, however, remain poorly defined. A major obstacle to investigating the effects of SUMO modification is the availability of a system for selectively inducing the modification or demodification of an individual protein. To address this problem, we have developed a procedure using the rapamycin heterodimerizer system. This procedure involves co-expression of rapamycin-binding domain fusion proteins of SUMO and candidate SUMO substrates in living cells. Treating cells with rapamycin induces a tight association between SUMO and a single SUMO substrate, thereby allowing specific downstream effects to be analyzed. Using RanGAP1 as a model SUMO substrate, the heterodimerizer system was used to investigate the molecular mechanism by which SUMO modification targets RanGAP1 from the cytoplasm to nuclear pore complexes (NPCs). Our results revealed a dual role for Ubc9 in targeting RanGAP1 to NPCs: In addition to conjugating SUMO-1 to RanGAP1, Ubc9 is also required to form a stable ternary complex with SUMO-1 modified RanGAP1 and Nup358. As illustrated by our studies, the rapamycin heterodimerizer system represents a novel tool for studying the molecular effects of SUMO modification.

  12. Novel in situ multiharmonic EQCM-D approach to characterize complex carbon pore architectures for capacitive deionization of brackish water

    NASA Astrophysics Data System (ADS)

    Shpigel, Netanel; Levi, Mikhael D.; Sigalov, Sergey; Aurbach, Doron; Daikhin, Leonid; Presser, Volker

    2016-03-01

    Multiharmonic analysis by electrochemical quartz-crystal microbalance with dissipation monitoring (EQCM-D) is introduced as an excellent tool for quantitative studying electrosorption of ions from aqueous solution in mesoporous (BP-880) or mixed micro-mesoporous (BP-2000) carbon electrodes. Finding the optimal conditions for gravimetric analysis of the ionic content in the charged carbon electrodes, we propose a novel approach to modeling the charge-dependent gravimetric characteristics by incorporation of Gouy-Chapman-Stern electric double layer model for ions electrosorption into meso- and micro-mesoporous carbon electrodes. All three parameters of the gravimetric equation evaluated by fitting it to the experimental mass changes curves were validated using supplementary nitrogen gas sorption analysis and complementing atomic force microscopy. Important overlap between gravimetric EQCM-D analysis of the ionic content of porous carbon electrodes and the classical capacitive deionization models has been established. The necessity and usefulness of non-gravimetric EQCM-D characterizations of complex carbon architectures, providing insight into their unique viscoelastic behavior and porous structure changes, have been discussed in detail.

  13. Novel in situ multiharmonic EQCM-D approach to characterize complex carbon pore architectures for capacitive deionization of brackish water.

    PubMed

    Shpigel, Netanel; Levi, Mikhael D; Sigalov, Sergey; Aurbach, Doron; Daikhin, Leonid; Presser, Volker

    2016-03-23

    Multiharmonic analysis by electrochemical quartz-crystal microbalance with dissipation monitoring (EQCM-D) is introduced as an excellent tool for quantitative studying electrosorption of ions from aqueous solution in mesoporous (BP-880) or mixed micro-mesoporous (BP-2000) carbon electrodes. Finding the optimal conditions for gravimetric analysis of the ionic content in the charged carbon electrodes, we propose a novel approach to modeling the charge-dependent gravimetric characteristics by incorporation of Gouy-Chapman-Stern electric double layer model for ions electrosorption into meso- and micro-mesoporous carbon electrodes. All three parameters of the gravimetric equation evaluated by fitting it to the experimental mass changes curves were validated using supplementary nitrogen gas sorption analysis and complementing atomic force microscopy. Important overlap between gravimetric EQCM-D analysis of the ionic content of porous carbon electrodes and the classical capacitive deionization models has been established. The necessity and usefulness of non-gravimetric EQCM-D characterizations of complex carbon architectures, providing insight into their unique viscoelastic behavior and porous structure changes, have been discussed in detail. PMID:26902741

  14. Photonic hybrid crystals constructed from in situ host-guest nanoconfinement of a light-emitting complex in metal-organic framework pores

    NASA Astrophysics Data System (ADS)

    Chaudhari, Abhijeet K.; Ryder, Matthew R.; Tan, Jin-Chong

    2016-03-01

    We report the concept underpinning the facile nanoconfinement of a bulky luminous guest molecule in the pores of a metal-organic framework (MOF) host, which yields a hybrid host ⊃ guest nanomaterial with tunable opto-electronic characteristics and enhanced photostability. Utilizing an in situ host-guest confinement strategy enabled by molecular self-assembly, we show that the highly emitting ZnQ [Zn-(bis-8-hydroxyquinoline)] guest complexes could be rapidly encapsulated within the sodalite nanocages of zeolitic imidazolate framework (ZIF-8) host crystals. The nature of optical and electronic transitions phenomena of the guest-encapsulated ZIF-8 ⊃ ZnQ has been elucidated by means of fluorescence and absorption spectroscopy measurements, and substantiated further via theoretical molecular orbital calculations revealing the plausible host-guest charge transfer mechanism involved. Evidence suggests that its photophysical properties are not only strongly determined by the host-guest co-operative bonding interactions within the environment of the confined MOF nanocage, but also can be engineered to manipulate its emission color chromaticity or to shield light-sensitive emitting guests against rapid photochemical degradation.We report the concept underpinning the facile nanoconfinement of a bulky luminous guest molecule in the pores of a metal-organic framework (MOF) host, which yields a hybrid host ⊃ guest nanomaterial with tunable opto-electronic characteristics and enhanced photostability. Utilizing an in situ host-guest confinement strategy enabled by molecular self-assembly, we show that the highly emitting ZnQ [Zn-(bis-8-hydroxyquinoline)] guest complexes could be rapidly encapsulated within the sodalite nanocages of zeolitic imidazolate framework (ZIF-8) host crystals. The nature of optical and electronic transitions phenomena of the guest-encapsulated ZIF-8 ⊃ ZnQ has been elucidated by means of fluorescence and absorption spectroscopy measurements, and

  15. Port and Harbor Security

    SciTech Connect

    Saito, T; Guthmuller, H; DeWeert, M

    2004-12-15

    Port and Harbor Security is a daunting task to which optics and photonics offers significant solutions. We are pleased to report that the 2005 Defense and Security Symposium (DSS, Orlando, FL) will include reports on active and passive photonic systems operating from both airborne and subsurface platforms. In addition to imaging techniques, there are various photonic applications, such as total internal reflection fluorescence (TIRF), which can be used to ''sniff'' for traces of explosives or contaminants in marine. These non-imaging technologies are beyond the scope of this article, but will also be represented at DSS 2005. We encourage colleagues to join our technical group to help us to make our ports and harbors safer and more secure.

  16. B-type nuclear lamin and the nuclear pore complex Nup107-160 influences maintenance of the spindle envelope required for cytokinesis in Drosophila male meiosis.

    PubMed

    Hayashi, Daisuke; Tanabe, Karin; Katsube, Hiroka; Inoue, Yoshihiro H

    2016-01-01

    In higher eukaryotes, nuclear envelope (NE) disassembly allows chromatin to condense and spindle microtubules to access kinetochores. The nuclear lamina, which strengthens the NE, is composed of a polymer meshwork made of A- and B-type lamins. We found that the B-type lamin (Lam) is not fully disassembled and continues to localize along the spindle envelope structure during Drosophila male meiosis I, while the A-type lamin (LamC) is completely dispersed throughout the cytoplasm. Among the nuclear pore complex proteins, Nup107 co-localized with Lam during this meiotic division. Surprisingly, Lam depletion resulted in a higher frequency of cytokinesis failure in male meiosis. We also observed the similar meiotic phenotype in Nup107-depleted cells. Abnormal localization of Lam was found in the Nup-depleted cells at premeiotic and meiotic stages. The central spindle microtubules became abnormal and recruitment of a contractile ring component to the cleavage sites was disrupted in Lam-depleted cells and Nup107-depleted cells. Therefore, we speculate that both proteins are required for a reinforcement of the spindle envelope, which supports the formation of central spindle microtubules essential for cytokinesis in Drosophila male meiosis. PMID:27402967

  17. Nup2 requires a highly divergent partner, NupA, to fulfill functions at nuclear pore complexes and the mitotic chromatin region

    PubMed Central

    Markossian, Sarine; Suresh, Subbulakshmi; Osmani, Aysha H.; Osmani, Stephen A.

    2015-01-01

    Chromatin and nuclear pore complexes (NPCs) undergo dramatic changes during mitosis, which in vertebrates and Aspergillus nidulans involves movement of Nup2 from NPCs to the chromatin region to fulfill unknown functions. This transition is shown to require the Cdk1 mitotic kinase and be promoted prematurely by ectopic expression of the NIMA kinase. Nup2 localizes with a copurifying partner termed NupA, a highly divergent yet essential NPC protein. NupA and Nup2 locate throughout the chromatin region during prophase but during anaphase move to surround segregating DNA. NupA function is shown to involve targeting Nup2 to its interphase and mitotic locations. Deletion of either Nup2 or NupA causes identical mitotic defects that initiate a spindle assembly checkpoint (SAC)–dependent mitotic delay and also cause defects in karyokinesis. These mitotic problems are not caused by overall defects in mitotic NPC disassembly–reassembly or general nuclear import. However, without Nup2 or NupA, although the SAC protein Mad1 locates to its mitotic locations, it fails to locate to NPCs normally in G1 after mitosis. Collectively the study provides new insight into the roles of Nup2 and NupA during mitosis and in a surveillance mechanism that regulates nucleokinesis when mitotic defects occur after SAC fulfillment. PMID:25540430

  18. Curcumin Pretreatment Prevents Potassium Dichromate-Induced Hepatotoxicity, Oxidative Stress, Decreased Respiratory Complex I Activity, and Membrane Permeability Transition Pore Opening

    PubMed Central

    García-Niño, Wylly Ramsés; Tapia, Edilia; Zazueta, Cecilia; Zatarain-Barrón, Zyanya Lucía; Hernández-Pando, Rogelio; Vega-García, Claudia Cecilia; Pedraza-Chaverrí, José

    2013-01-01

    Curcumin is a polyphenol derived from turmeric with recognized antioxidant properties. Hexavalent chromium is an environmental toxic and carcinogen compound that induces oxidative stress. The objective of this study was to evaluate the potential protective effect of curcumin on the hepatic damage generated by potassium dichromate (K2Cr2O7) in rats. Animals were pretreated daily by 9-10 days with curcumin (400 mg/kg b.w.) before the injection of a single intraperitoneal of K2Cr2O7 (15 mg/kg b.w.). Groups of animals were sacrificed 24 and 48 h later. K2Cr2O7-induced damage to the liver was evident by histological alterations and increase in the liver weight and in the activity of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and alkaline phosphatase in plasma. In addition, K2Cr2O7 induced oxidative damage in liver and isolated mitochondria, which was evident by the increase in the content of malondialdehyde and protein carbonyl and decrease in the glutathione content and in the activity of several antioxidant enzymes. Moreover, K2Cr2O7 induced decrease in mitochondrial oxygen consumption, in the activity of respiratory complex I, and permeability transition pore opening. All the above-mentioned alterations were prevented by curcumin pretreatment. The beneficial effects of curcumin against K2Cr2O7-induced liver oxidative damage were associated with prevention of mitochondrial dysfunction. PMID:23956771

  19. B-type nuclear lamin and the nuclear pore complex Nup107-160 influences maintenance of the spindle envelope required for cytokinesis in Drosophila male meiosis

    PubMed Central

    Hayashi, Daisuke; Tanabe, Karin; Katsube, Hiroka

    2016-01-01

    ABSTRACT In higher eukaryotes, nuclear envelope (NE) disassembly allows chromatin to condense and spindle microtubules to access kinetochores. The nuclear lamina, which strengthens the NE, is composed of a polymer meshwork made of A- and B-type lamins. We found that the B-type lamin (Lam) is not fully disassembled and continues to localize along the spindle envelope structure during Drosophila male meiosis I, while the A-type lamin (LamC) is completely dispersed throughout the cytoplasm. Among the nuclear pore complex proteins, Nup107 co-localized with Lam during this meiotic division. Surprisingly, Lam depletion resulted in a higher frequency of cytokinesis failure in male meiosis. We also observed the similar meiotic phenotype in Nup107-depleted cells. Abnormal localization of Lam was found in the Nup-depleted cells at premeiotic and meiotic stages. The central spindle microtubules became abnormal and recruitment of a contractile ring component to the cleavage sites was disrupted in Lam-depleted cells and Nup107-depleted cells. Therefore, we speculate that both proteins are required for a reinforcement of the spindle envelope, which supports the formation of central spindle microtubules essential for cytokinesis in Drosophila male meiosis. PMID:27402967

  20. Contaminant levels in Herring (Larus argentatus) and Great Black-backed Gull (Larus marinus) eggs from colonies in the New York harbor complex between 2012 and 2013

    PubMed Central

    Elbin, Susan

    2014-01-01

    Birds living in coastal areas are exposed to severe storms and tidal flooding during the nesting season, but also to contaminants that move up the food chain from the water column and sediment to their prey items. We examine metals in Herring Gull (Larus argentatus) and Great Black-backed Gull (Larus marinus) eggs collected from the New York/New Jersey harbor estuary in 2012 and in 2013 to determine if there were significant yearly differences in metal levels. We test the null hypothesis that there were no significant yearly differences in metal levels. We investigate whether there were consistent differences in metals from 2012 to 2013 that might suggest a storm-related effect because Superstorm Sandy landed in New Jersey in October 2012 with high winds and extensive flooding, and view this research as exploratory. Except for arsenic, there were significant inter-year variations in the mean levels for all colonies combined for Herring Gull, and for lead, mercury and selenium for Great Black-backed Gulls. All metal levels in 2013 were less than in 2012, except for lead. These differences were present for individual colonies as well. Metal levels varied significantly among islands for Herring Gulls in both years (except for cadmium in 2013). No one colony had the highest levels of all metals for Herring Gulls. A long term data set on mercury levels in Herring Gulls indicated that the differences between 2012 and 2013 were greater than usual. Several different factors could account for these differences, and these are discussed. PMID:25471353

  1. The nuclear pore complex protein ALADIN is anchored via NDC1 but not via POM121 and GP210 in the nuclear envelope

    SciTech Connect

    Kind, Barbara; Koehler, Katrin; Lorenz, Mike; Huebner, Angela

    2009-12-11

    The nuclear pore complex (NPC) consists of {approx}30 different proteins and provides the only sites for macromolecular transport between cytoplasm and nucleus. ALADIN was discovered as a new member of the NPC. Mutations in ALADIN are known to cause triple A syndrome, a rare autosomal recessive disorder characterized by adrenal insufficiency, alacrima, and achalasia. The function and exact location of the nucleoporin ALADIN within the NPC multiprotein complex is still unclear. Using a siRNA-based approach we downregulated the three known membrane integrated nucleoporins NDC1, GP210, and POM121 in stably expressing GFP-ALADIN HeLa cells. We identified NDC1 but not GP210 and POM121 as the main anchor of ALADIN within the NPC. Solely the depletion of NDC1 caused mislocalization of ALADIN. Vice versa, the depletion of ALADIN led also to disappearance of NDC1 at the NPC. However, the downregulation of two further membrane-integral nucleoporins GP210 and POM121 had no effect on ALADIN localization. Furthermore, we could show a direct association of NDC1 and ALADIN in NPCs by fluorescence resonance energy transfer (FRET) measurements. Based on our findings we conclude that ALADIN is anchored in the nuclear envelope via NDC1 and that this interaction gets lost, if ALADIN is mutated. The loss of integration of ALADIN in the NPC is a main pathogenetic aspect for the development of the triple A syndrome and suggests that the interaction between ALADIN and NDC1 may be involved in the pathogenesis of the disease.

  2. Poring over two-pore channel pore mutants

    PubMed Central

    Penny, Christopher J.; Patel, Sandip

    2016-01-01

    Two-pore channels are members of the voltage-gated ion channel superfamily. They localise to the endolysosomal system and are likely targets for the Ca2+ mobilising messenger NAADP. In this brief review, we relate mutagenesis of the TPC pore to a recently published homology model and discuss how pore mutants are informing us of TPC function. Molecular physiology of these ubiquitous proteins is thus emerging. PMID:27226934

  3. Tide- and wind-driven flushing of Boston Harbor, Massachusetts

    USGS Publications Warehouse

    Signell, Richard

    1992-01-01

    The flushing of Boston Harbor, a shallow, tidally dominated embayment with little fresh water input, is investigated using a depth-averaged model. The modeled tidal currents exhibit strong spatial variability and ebb/flood asymmetry due to complex topography and coastline geometry and were verified by shipboard acoustic Doppler current profiler measurements. At the inlets to the harbor, the asymmetry between flood and ebb gives rise to a net exchange of water, which acts over successive tidal cycles to flush the harbor. The flushing is examined by tracking water that starts out in Boston Harbor for 40 M2 tidal cycles. The tidal flushing is very efficient at mixing water in the vicinity of the inlets over several tidal cycles, but efficiency decreases with time as ``tidal mixing regions'' form on either side of the harbor inlets. When wind forcing is included, the wind-driven currents act to flush the tidal mixing regions, giving rise to more efficient flushing. The exception is when the wind is from the southwest, which confines the jet-like ebb flow from the harbor and therefore reduces the flushing efficiency. In general, flushing is shown to be a two-step process: (1) rapid exchange due to tides over a large region in the vicinity of the harbor inlets and (2) flushing of this region by wind-driven flow. The model also demonstrates that flushing is not uniform over the entire harbor but occurs rapidly in the deep tidal channels and slowly in the regions of weak tidal currents around the harbor periphery. Although the depth-averaged approach to flushing is appropriate over most of the harbor due to the harbor's shallow depth and broad depth distribution, the lack of bathymetric variability and the presence of locally important density driven currents in the Boston Inner Harbor indicates that flushing of this localized area must be approached with a three-dimensional model.

  4. Effect of the ostreolysin A/pleurotolysin B pore-forming complex on neuroblastoma cell morphology and intracellular Ca²⁺ activity.

    PubMed

    Vrecl, Milka; Babnik, Monika; Diacci, Uroš; Benoit, Evelyne; Frangež, Robert

    2015-04-01

    Ostreolysin A (OlyA) and pleurotolysin B (PlyB), isolated from edible oyster mushrooms, form a cytolytic complex (OlyA/PlyB) in membrane cells that causes respiratory arrest. This study evaluated the mechanisms underlying cytotoxic OlyA/PlyB activity in neuroblastoma NG108-15 cells. Confocal microscopy with morphometric analysis revealed that OlyA/PlyB increased the 3-dimensional projected area of differentiated cells. Iso-osmotic replacement of NaCl by sucrose or Na-isethionate prevented the cellular swelling. This suggests that formation of cellular edema requires the presence of Na(+) and/or Cl(-) in the extracellular space and may be related to an influx of Na(+) and/or a shift in Cl(-), which induce a marked influx of water that is ultimately responsible for cellular swelling. In addition, extracellular Ca(2+) moderately contributed to the swelling because benzamil (10 µM), a 3Na(+)/Ca(2+) exchange (NCX) inhibitor, and Ca(2+)-free medium partially prevented this response. Fluorometric measurements revealed that OlyA/PlyB, at approximately 15-fold higher concentrations, increased the intracellular Ca(2+) activity [Ca(2+)]i. This increase was dependent on the presence of Na(+) and Ca(2+) in the external medium and was sensitive to benzamil. It is thus likely that a switch in the NCX mode, associated with the de novo formation of non-selective ion pores by OlyA/PlyB in cellular plasma membranes, plays an important role in this effect. Overall, OlyA/PlyB affects neuroblastoma cell morphology and Ca(2+) homeostasis to influence the toxin-induced respiratory arrest. PMID:25556216

  5. Isolation and characterization of a proteinaceous subnuclear fraction composed of nuclear matrix, peripheral lamina, and nuclear pore complexes from embryos of Drosophila melanogaster.

    PubMed

    Fisher, P A; Berrios, M; Blobel, G

    1982-03-01

    Morphologically intact nuclei have been prepared from embryos of Drosophila melanogaster by a simple and rapid procedure. These nuclei have been further treated with high concentrations of DNase I and RNase A followed by sequential extraction with 2% Triton X-100 and 1 M NaCl to produce a structurally and biochemically distinct preparation designated Drosophila subnuclear fraction I (DSNF-I). As seen by phase-contrast microscopy, DSNF-I is composed of material which closely resembles unfractionated nuclei; residual internal nuclear structures including nucleolar remnants are clearly visible. By transmission electron microscopy, nuclear lamina, pore complexes, and a nuclear matrix are similarly identified. Biochemically, DSNF-I is composed almost entirely of protein (greater than 93%). SDS PAGE analysis reveals several major polypeptides; species at 174,000, 74,000, and 42,000 predominate. A polypeptide coincident with the Coomassie Blue-stainable 174-kdalton band has been shown by a novel technique of lectin affinity labeling to be a glycoprotein; a glycoprotein of similar or identical molecular weight has been found to be a component of nuclear envelope fractions isolated from the livers of rats, guinea pigs, opossums, and chickens. Antisera against several of the polypeptides in DSNF-I have been obtained from rabbits, and all of them show only little or no cross-reactivity with Drosophila cytoplasmic fractions. Initial results of immunocytochemical studies, while failing to positively localize either the 174- or 16-kdalton polypeptides, demonstrate a nuclear localization of the 74-kdalton antigen in all of several interphase cell types obtained from both Drosophila embryos and third-instar larvae. PMID:6177701

  6. Pore Velocity Estimation Uncertainties

    NASA Astrophysics Data System (ADS)

    Devary, J. L.; Doctor, P. G.

    1982-08-01

    Geostatistical data analysis techniques were used to stochastically model the spatial variability of groundwater pore velocity in a potential waste repository site. Kriging algorithms were applied to Hanford Reservation data to estimate hydraulic conductivities, hydraulic head gradients, and pore velocities. A first-order Taylor series expansion for pore velocity was used to statistically combine hydraulic conductivity, hydraulic head gradient, and effective porosity surfaces and uncertainties to characterize the pore velocity uncertainty. Use of these techniques permits the estimation of pore velocity uncertainties when pore velocity measurements do not exist. Large pore velocity estimation uncertainties were found to be located in the region where the hydraulic head gradient relative uncertainty was maximal.

  7. Measuring kinetic drivers of pneumolysin pore structure.

    PubMed

    Gilbert, Robert J C; Sonnen, Andreas F-P

    2016-05-01

    Most membrane attack complex-perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins are thought to form pores in target membranes by assembling into pre-pore oligomers before undergoing a pre-pore to pore transition. Assembly during pore formation is into both full rings of subunits and incomplete rings (arcs). The balance between arcs and full rings is determined by a mechanism dependent on protein concentration in which arc pores arise due to kinetic trapping of the pre-pore forms by the depletion of free protein subunits during oligomerization. Here we describe the use of a kinetic assay to study pore formation in red blood cells by the MACPF/CDC pneumolysin from Streptococcus pneumoniae. We show that cell lysis displays two kinds of dependence on protein concentration. At lower concentrations, it is dependent on the pre-pore to pore transition of arc oligomers, which we show to be a cooperative process. At higher concentrations, it is dependent on the amount of pneumolysin bound to the membrane and reflects the affinity of the protein for its receptor, cholesterol. A lag occurs before cell lysis begins; this is dependent on oligomerization of pneumolysin. Kinetic dissection of cell lysis by pneumolysin demonstrates the capacity of MACPF/CDCs to generate pore-forming oligomeric structures of variable size with, most likely, different functional roles in biology. PMID:26906727

  8. Laser powder-bed fusion additive manufacturing: Physics of complex melt flow and formation mechanisms of pores, spatter, and denudation zones

    DOE PAGESBeta

    Khairallah, Saad A.; Anderson, Andrew T.; Rubenchik, Alexander; King, Wayne E.

    2016-02-23

    Our study demonstrates the significant effect of the recoil pressure and Marangoni convection in laser powder bed fusion (L-PBF) of 316L stainless steel. A three-dimensional high fidelity powder-scale model reveals how the strong dynamical melt flow generates pore defects, material spattering (sparking), and denudation zones. The melt track is divided into three sections: a topological depression, a transition and a tail region, each being the location of specific physical effects. The inclusion of laser ray-tracing energy deposition in the powder-scale model improves over traditional volumetric energy deposition. It enables partial particle melting, which impacts pore defects in the denudation zone.more » Different pore formation mechanisms are observed at the edge of a scan track, at the melt pool bottom (during collapse of the pool depression), and at the end of the melt track (during laser power ramp down). Finally, we discuss remedies to these undesirable pores are discussed. The results are validated against the experiments and the sensitivity to laser absorptivity.« less

  9. MRSA Clonal Complex 22 Strains Harboring Toxic Shock Syndrome Toxin (TSST-1) Are Endemic in the Primary Hospital in Gaza, Palestine

    PubMed Central

    Chen, Liang; Abdelateef, Nahed; Elamreen, Farid Abu; Ginocchio, Christine C.; Pierard, Denis; Becker, Karsten; Kreiswirth, Barry N.

    2015-01-01

    Background Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen in both community and healthcare-related settings worldwide. Current knowledge regarding the epidemiology of S. aureus and MRSA in Gaza is based on a single community-based carriage study. Here we describe a cross-sectional analysis of 215 clinical isolates collected from Al-Shifa Hospital in Gaza during 2008 and 2012. Methods All isolates were characterized by spa typing, SCCmec typing, and detection of genes encoding Panton-Valentine leukocidin (PVL) and toxic shock syndrome toxin (TSST-1). Representative genotypes were also subjected to multilocus sequence typing (MLST). Antibiotic susceptibility testing was performed using VITEK2 and MicroScan. Results MRSA represented 56.3% of all S. aureus strains, and increased in frequency from 2008 (54.8%) to 2012 (58.4%). Aside from beta-lactams, resistance was observed to tetracycline, erythromycin, clindamycin, gentamicin, and fluoroquinolones. Molecular typing identified 35 spa types representing 17 MLST clonal complexes (CC), with spa 998 (Ridom t223, CC22) and spa 70 (Ridom t044, CC80) being the most prevalent. SCCmec types I, III, IV, V and VI were identified among MRSA isolates, while type II was not detected. PVL genes (lukF/S-PV) were detected in 40.0% of all isolates, while the TSST-1 gene (tst) was detected in 27.4% of all isolates, with surprisingly high frequency within CC22 (70.4%). Both PVL and TSST-1 genes were found in several isolates from 2012. Conclusions Molecular typing of clinical isolates from Gaza hospitals revealed unusually high prevalence of TSST-1 genes among CC22 MRSA, which is noteworthy given a recent community study describing widespread carriage of a CC22 MRSA clone known as the ‘Gaza strain’. While the latter did not address TSST-1, tst-positive spa 998 (Ridom t223) has been detected in several neighboring countries, and described as endemic in an Italian NICU, suggesting international spread of a

  10. Safe harbor: protecting ports with shipboard fuel cells.

    PubMed

    Taylor, David A

    2006-04-01

    With five of the largest harbors in the United States, California is beginning to take steps to manage the large amounts of pollution generated by these bustling centers of transport and commerce. One option for reducing diesel emissions is the use of fuel cells, which run cleaner than diesel and other internal combustion engines. Other technologies being explored by harbor officials are diesel-electric hybrid and gas turbine locomotives for moving freight within port complexes. PMID:16581531

  11. Incomplete pneumolysin oligomers form membrane pores.

    PubMed

    Sonnen, Andreas F-P; Plitzko, Jürgen M; Gilbert, Robert J C

    2014-01-01

    Pneumolysin is a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming proteins that are produced as water-soluble monomers or dimers, bind to target membranes and oligomerize into large ring-shaped assemblies comprising approximately 40 subunits and approximately 30 nm across. This pre-pore assembly then refolds to punch a large hole in the lipid bilayer. However, in addition to forming large pores, pneumolysin and other CDCs form smaller lesions characterized by low electrical conductance. Owing to the observation of arc-like (rather than full-ring) oligomers by electron microscopy, it has been hypothesized that smaller oligomers explain smaller functional pores. To investigate whether this is the case, we performed cryo-electron tomography of pneumolysin oligomers on model lipid membranes. We then used sub-tomogram classification and averaging to determine representative membrane-bound low-resolution structures and identified pre-pores versus pores by the presence of membrane within the oligomeric curve. We found pre-pore and pore forms of both complete (ring) and incomplete (arc) oligomers and conclude that arc-shaped oligomeric assemblies of pneumolysin can form pores. As the CDCs are evolutionarily related to the membrane attack complex/perforin family of proteins, which also form variably sized pores, our findings are of relevance to that class of proteins as well. PMID:24759615

  12. Pore-Scale Modeling of Pore Structure Effects on P-Wave Scattering Attenuation in Dry Rocks

    PubMed Central

    Li, Tianyang; Qiu, Hao; Wang, Feifei

    2015-01-01

    Underground rocks usually have complex pore system with a variety of pore types and a wide range of pore size. The effects of pore structure on elastic wave attenuation cannot be neglected. We investigated the pore structure effects on P-wave scattering attenuation in dry rocks by pore-scale modeling based on the wave theory and the similarity principle. Our modeling results indicate that pore size, pore shape (such as aspect ratio), and pore density are important factors influencing P-wave scattering attenuation in porous rocks, and can explain the variation of scattering attenuation at the same porosity. From the perspective of scattering attenuation, porous rocks can safely suit to the long wavelength assumption when the ratio of wavelength to pore size is larger than 15. Under the long wavelength condition, the scattering attenuation coefficient increases as a power function as the pore density increases, and it increases exponentially with the increase in aspect ratio. For a certain porosity, rocks with smaller aspect ratio and/or larger pore size have stronger scattering attenuation. When the pore aspect ratio is larger than 0.5, the variation of scattering attenuation at the same porosity is dominantly caused by pore size and almost independent of the pore aspect ratio. These results lay a foundation for pore structure inversion from elastic wave responses in porous rocks. PMID:25961729

  13. Pore-scale modeling of pore structure effects on P-wave scattering attenuation in dry rocks.

    PubMed

    Wang, Zizhen; Wang, Ruihe; Li, Tianyang; Qiu, Hao; Wang, Feifei

    2015-01-01

    Underground rocks usually have complex pore system with a variety of pore types and a wide range of pore size. The effects of pore structure on elastic wave attenuation cannot be neglected. We investigated the pore structure effects on P-wave scattering attenuation in dry rocks by pore-scale modeling based on the wave theory and the similarity principle. Our modeling results indicate that pore size, pore shape (such as aspect ratio), and pore density are important factors influencing P-wave scattering attenuation in porous rocks, and can explain the variation of scattering attenuation at the same porosity. From the perspective of scattering attenuation, porous rocks can safely suit to the long wavelength assumption when the ratio of wavelength to pore size is larger than 15. Under the long wavelength condition, the scattering attenuation coefficient increases as a power function as the pore density increases, and it increases exponentially with the increase in aspect ratio. For a certain porosity, rocks with smaller aspect ratio and/or larger pore size have stronger scattering attenuation. When the pore aspect ratio is larger than 0.5, the variation of scattering attenuation at the same porosity is dominantly caused by pore size and almost independent of the pore aspect ratio. These results lay a foundation for pore structure inversion from elastic wave responses in porous rocks. PMID:25961729

  14. Environment-Dependent Distribution of the Sediment nifH-Harboring Microbiota in the Northern South China Sea

    PubMed Central

    Yang, Jinying; Li, Jing; Luan, Xiwu; Zhang, Yunbo; Gu, Guizhou; Xue, Rongrong; Zong, Mingyue; Klotz, Martin G.

    2013-01-01

    The South China Sea (SCS), the largest marginal sea in the Western Pacific Ocean, is a huge oligotrophic water body with very limited influx of nitrogenous nutrients. This suggests that sediment microbial N2 fixation plays an important role in the production of bioavailable nitrogen. To test the molecular underpinning of this hypothesis, the diversity, abundance, biogeographical distribution, and community structure of the sediment diazotrophic microbiota were investigated at 12 sampling sites, including estuarine, coastal, offshore, deep-sea, and methane hydrate reservoirs or their prospective areas by targeting nifH and some other functional biomarker genes. Diverse and novel nifH sequences were obtained, significantly extending the evolutionary complexity of extant nifH genes. Statistical analyses indicate that sediment in situ temperature is the most significant environmental factor influencing the abundance, community structure, and spatial distribution of the sediment nifH-harboring microbial assemblages in the northern SCS (nSCS). The significantly positive correlation of the sediment pore water NH4+ concentration with the nifH gene abundance suggests that the nSCS sediment nifH-harboring microbiota is active in N2 fixation and NH4+ production. Several other environmental factors, including sediment pore water PO43− concentration, sediment organic carbon, nitrogen and phosphorus levels, etc., are also important in influencing the community structure, spatial distribution, or abundance of the nifH-harboring microbial assemblages. We also confirmed that the nifH genes encoded by archaeal diazotrophs in the ANME-2c subgroup occur exclusively in the deep-sea methane seep areas, providing for the possibility to develop ANME-2c nifH genes as a diagnostic tool for deep-sea methane hydrate reservoir discovery. PMID:23064334

  15. Narrow 'safe harbors' may create tough choices for hospitals.

    PubMed

    Hudson, T

    1991-10-01

    The release of the "safe harbors" regulations by the Department of Health and Human Services has left a variety of important issues unresolved. Health care attorneys and their clients are now poring over the new regulations, trying to determine, among other things, how much risk tolerance to accept in relation to the new restrictions, and what level and types of monitoring the federal government will apply. Ultimately, say many experts, the regulations could result in a strong trailoff in joint venture volume. PMID:1894272

  16. 33 CFR 207.480 - Lake Huron, Mich.; Harbor of refuge, Harbor Beach; use and navigation.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 3 2014-07-01 2014-07-01 false Lake Huron, Mich.; Harbor of refuge, Harbor Beach; use and navigation. 207.480 Section 207.480 Navigation and Navigable Waters CORPS... Huron, Mich.; Harbor of refuge, Harbor Beach; use and navigation. (a) All boats, barges, and...

  17. 33 CFR 207.480 - Lake Huron, Mich.; Harbor of refuge, Harbor Beach; use and navigation.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 3 2012-07-01 2012-07-01 false Lake Huron, Mich.; Harbor of refuge, Harbor Beach; use and navigation. 207.480 Section 207.480 Navigation and Navigable Waters CORPS... Huron, Mich.; Harbor of refuge, Harbor Beach; use and navigation. (a) All boats, barges, and...

  18. 33 CFR 207.480 - Lake Huron, Mich.; Harbor of refuge, Harbor Beach; use and navigation.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 3 2013-07-01 2013-07-01 false Lake Huron, Mich.; Harbor of refuge, Harbor Beach; use and navigation. 207.480 Section 207.480 Navigation and Navigable Waters CORPS... Huron, Mich.; Harbor of refuge, Harbor Beach; use and navigation. (a) All boats, barges, and...

  19. 33 CFR 207.480 - Lake Huron, Mich.; Harbor of refuge, Harbor Beach; use and navigation.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 3 2010-07-01 2010-07-01 false Lake Huron, Mich.; Harbor of refuge, Harbor Beach; use and navigation. 207.480 Section 207.480 Navigation and Navigable Waters CORPS... Huron, Mich.; Harbor of refuge, Harbor Beach; use and navigation. (a) All boats, barges, and...

  20. 33 CFR 207.480 - Lake Huron, Mich.; Harbor of refuge, Harbor Beach; use and navigation.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 3 2011-07-01 2011-07-01 false Lake Huron, Mich.; Harbor of refuge, Harbor Beach; use and navigation. 207.480 Section 207.480 Navigation and Navigable Waters CORPS... Huron, Mich.; Harbor of refuge, Harbor Beach; use and navigation. (a) All boats, barges, and...

  1. X-ray microtomography application in pore space reservoir rock.

    PubMed

    Oliveira, M F S; Lima, I; Borghi, L; Lopes, R T

    2012-07-01

    Characterization of porosity in carbonate rocks is important in the oil and gas industry since a major hydrocarbons field is formed by this lithology and they have a complex media porous. In this context, this research presents a study of the pore space in limestones rocks by x-ray microtomography. Total porosity, type of porosity and pore size distribution were evaluated from 3D high resolution images. Results show that carbonate rocks has a complex pore space system with different pores types at the same facies. PMID:22264795

  2. Visualization of enzyme activities inside earthworm pores

    NASA Astrophysics Data System (ADS)

    Hoang, Duyen; Razavi, Bahar S.

    2015-04-01

    In extremely dynamic microhabitats as bio-pores made by earthworm, the in situ enzyme activities are assumed as a footprint of complex biotic interactions. Our study focused on the effect of earthworm on the enzyme activities inside bio-pores and visualizing the differences between bio-pores and earthworm-free soil by zymography technique (Spohn and Kuzyakov, 2013). For the first time, we aimed at quantitative imaging of enzyme activities in bio-pores. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). After two weeks when bio-pore systems were formed by earthworms, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine-aminopeptidase, and phosphatase. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. However, the differences in activity of cellobiohydrolase and leucine aminopeptidase between bio-pore and bulk soil were less pronounced. This demonstrated an applicability of zymography approach to monitor and to distinguish the in situ activity of hydrolytic enzymes in soil biopores.

  3. Nup358/RanBP2 Attaches to the Nuclear Pore Complex via Association with Nup88 and Nup214/CAN and Plays a Supporting Role in CRM1-Mediated Nuclear Protein Export

    PubMed Central

    Bernad, Rafael; van der Velde, Hella; Fornerod, Maarten; Pickersgill, Helen

    2004-01-01

    Nuclear pore complexes (NPCs) traverse the nuclear envelope (NE), providing a channel through which nucleocytoplasmic transport occurs. Nup358/RanBP2, Nup214/CAN, and Nup88 are components of the cytoplasmic face of the NPC. Here we show that Nup88 localizes midway between Nup358 and Nup214 and physically interacts with them. RNA interference of either Nup88 or Nup214 in human cells caused a strong reduction of Nup358 at the NE. Nup88 and Nup214 showed an interdependence at the NPC and were not affected by the absence of Nup358. These data indicate that Nup88 and Nup214 mediate the attachment of Nup358 to the NPC. We show that localization of the export receptor CRM1 at the cytoplasmic face of the NE is Nup358 dependent and represents its empty state. Also, removal of Nup358 causes a distinct reduction in nuclear export signal-dependent nuclear export. We propose that Nup358 provides both a platform for rapid disassembly of CRM1 export complexes and a binding site for empty CRM1 recycling into the nucleus. PMID:14993277

  4. The Arabidopsis Nuclear Pore and Nuclear Envelope

    PubMed Central

    Meier, Iris; Brkljacic, Jelena

    2010-01-01

    The nuclear envelope is a double membrane structure that separates the eukaryotic cytoplasm from the nucleoplasm. The nuclear pores embedded in the nuclear envelope are the sole gateways for macromolecular trafficking in and out of the nucleus. The nuclear pore complexes assembled at the nuclear pores are large protein conglomerates composed of multiple units of about 30 different nucleoporins. Proteins and RNAs traffic through the nuclear pore complexes, enabled by the interacting activities of nuclear transport receptors, nucleoporins, and elements of the Ran GTPase cycle. In addition to directional and possibly selective protein and RNA nuclear import and export, the nuclear pore gains increasing prominence as a spatial organizer of cellular processes, such as sumoylation and desumoylation. Individual nucleoporins and whole nuclear pore subcomplexes traffic to specific mitotic locations and have mitotic functions, for example at the kinetochores, in spindle assembly, and in conjunction with the checkpoints. Mutants of nucleoporin genes and genes of nuclear transport components lead to a wide array of defects from human diseases to compromised plant defense responses. The nuclear envelope acts as a repository of calcium, and its inner membrane is populated by functionally unique proteins connected to both chromatin and—through the nuclear envelope lumen—the cytoplasmic cytoskeleton. Plant nuclear pore and nuclear envelope research—predominantly focusing on Arabidopsis as a model—is discovering both similarities and surprisingly unique aspects compared to the more mature model systems. This chapter gives an overview of our current knowledge in the field and of exciting areas awaiting further exploration. PMID:22303264

  5. Soil pore structure and substrate C mineralization

    NASA Astrophysics Data System (ADS)

    Sleutel, Steven; Maenhout, Peter; Vanhoorebeke, Luc; Cnudde, Veerle; De Neve, Stefaan

    2014-05-01

    Our aim was to investigate the complex interactions between soil pore structure, soil biota and decomposition of added OM substrates. We report on a lab incubation experiment in which CO2 respiration from soil cores was monitored (headspace GC analysis) and an X-ray CT approach yielded soil pore size distributions. Such combined use of X-ray CT with soil incubation studies was obstructed, until now, by many practical constraints such as CT-volume quality, limited resolution, scanning time and complex soil pore network quantification, which have largely been overcome in this study. We incubated a sandy loam soil (with application of ground grass or sawdust) in 18 small aluminium rings (Ø 1 cm, h 1 cm). Bulk density was adjusted to 1.1 or 1.3 Mg m-3 (compaction) and 6 rings were filled at a coarser Coarse Sand:Fine Sand:Silt+Clay ratio. While compaction induced a strong reduction in the cumulative C mineralization for both grass and sawdust substrates, artificial change to a coarser soil texture only reduced net C mineralization from the added sawdust. There thus appears to be a strong interaction effect between soil pore structure and substrate type on substrate decomposition. Correlation coefficients between the C mineralization rates and volumes of 7 pore size classes (from the X-ray CT data) also showed an increasing positive correlation with increasing pore size. Since any particulate organic matter initially present in the soil was removed prior to the experiment (sieving, ashing the >53µm fraction and recombining with the <53µm fraction), the added OM can be localized by means of X-ray CT. Through on-going image analysis the surrounding porosity of the added grass or sawdust particles is being quantified to further study the interaction between the soil pore structure and substrate decomposition.

  6. The pore space scramble

    NASA Astrophysics Data System (ADS)

    Gormally, Alexandra; Bentham, Michelle; Vermeylen, Saskia; Markusson, Nils

    2015-04-01

    Climate change and energy security continue to be the context of the transition to a secure, affordable and low carbon energy future, both in the UK and beyond. This is reflected in for example, binding climate policy targets at the EU level, the introduction of renewable energy targets, and has also led to an increasing interest in Carbon Capture and Storage (CCS) technology with its potential to help mitigate against the effects of CO2 emissions from fossil fuel burning. The UK has proposed a three phase strategy to integrate CCS into its energy system in the long term focussing on off-shore subsurface storage (DECC, 2014). The potential of CCS therefore, raises a number of challenging questions and issues surrounding the long-term storage of CO2 captured and injected into underground spaces and, alongside other novel uses of the subsurface, contributes to opening a new field for discussion on the governance of the subsurface. Such 'novel' uses of the subsurface have lead to it becoming an increasingly contested space in terms of its governance, with issues emerging around the role of ownership, liability and property rights of subsurface pore space. For instance, questions over the legal ownership of pore space have arisen with ambiguity over the legal standpoint of the surface owner and those wanting to utilise the pore space for gas storage, and suggestions of whether there are depths at which legal 'ownership' becomes obsolete (Barton, 2014). Here we propose to discuss this 'pore space scramble' and provide examples of the competing trajectories of different stakeholders, particularly in the off-shore context given its priority in the UK. We also propose to highlight the current ambiguity around property law of pore space in the UK with reference to approaches currently taken in different national contexts. Ultimately we delineate contrasting models of governance to illustrate the choices we face and consider the ethics of these models for the common good

  7. EGO-1, a Putative RNA-Directed RNA Polymerase, Promotes Germline Proliferation in Parallel With GLP-1/Notch Signaling and Regulates the Spatial Organization of Nuclear Pore Complexes and Germline P Granules in Caenorhabditis elegans

    PubMed Central

    Vought, Valarie E.; Ohmachi, Mitsue; Lee, Min-Ho; Maine, Eleanor M.

    2005-01-01

    Caenorhabditis elegans EGO-1, a putative cellular RNA-directed RNA polymerase, promotes several aspects of germline development, including proliferation, meiosis, and gametogenesis, and ensures a robust response to RNA interference. In C. elegans, GLP-1/Notch signaling from the somatic gonad maintains a population of proliferating germ cells, while entry of germ cells into meiosis is triggered by the GLD-1 and GLD-2 pathways. GLP-1 signaling prevents germ cells from entering meiosis by inhibiting GLD-1 and GLD-2 activity. We originally identified the ego-1 gene on the basis of a genetic interaction with glp-1. Here, we investigate the role of ego-1 in germline proliferation. Our data indicate that EGO-1 does not positively regulate GLP-1 protein levels or GLP-1 signaling activity. Moreover, GLP-1 signaling does not positively regulate EGO-1 activity. EGO-1 does not inhibit expression of GLD-1 protein in the distal germline. Instead, EGO-1 acts in parallel with GLP-1 signaling to influence the proliferation vs. meiosis fate choice. Moreover, EGO-1 and GLD-1 act in parallel to ensure germline health. Finally, the size and distribution of nuclear pore complexes and perinuclear P granules are altered in the absence of EGO-1, effects that disrupt germ cell biology per se and probably limit germline growth. PMID:15911573

  8. EGO-1, a putative RNA-directed RNA polymerase, promotes germline proliferation in parallel with GLP-1/notch signaling and regulates the spatial organization of nuclear pore complexes and germline P granules in Caenorhabditis elegans.

    PubMed

    Vought, Valarie E; Ohmachi, Mitsue; Lee, Min-Ho; Maine, Eleanor M

    2005-07-01

    Caenorhabditis elegans EGO-1, a putative cellular RNA-directed RNA polymerase, promotes several aspects of germline development, including proliferation, meiosis, and gametogenesis, and ensures a robust response to RNA interference. In C. elegans, GLP-1/Notch signaling from the somatic gonad maintains a population of proliferating germ cells, while entry of germ cells into meiosis is triggered by the GLD-1 and GLD-2 pathways. GLP-1 signaling prevents germ cells from entering meiosis by inhibiting GLD-1 and GLD-2 activity. We originally identified the ego-1 gene on the basis of a genetic interaction with glp-1. Here, we investigate the role of ego-1 in germline proliferation. Our data indicate that EGO-1 does not positively regulate GLP-1 protein levels or GLP-1 signaling activity. Moreover, GLP-1 signaling does not positively regulate EGO-1 activity. EGO-1 does not inhibit expression of GLD-1 protein in the distal germline. Instead, EGO-1 acts in parallel with GLP-1 signaling to influence the proliferation vs. meiosis fate choice. Moreover, EGO-1 and GLD-1 act in parallel to ensure germline health. Finally, the size and distribution of nuclear pore complexes and perinuclear P granules are altered in the absence of EGO-1, effects that disrupt germ cell biology per se and probably limit germline growth. PMID:15911573

  9. Modelling multiphase dynamics during infiltration using a pore network model

    NASA Astrophysics Data System (ADS)

    Tzavaras, Jannis; Arns, Ji-Youns; Max, Koehne; Hans-Joerg, Vogel

    2013-04-01

    We present an implementation of water infiltration into a pore network model where the local water pressures is continuously updated during the transient process. The network geometry is designed to represent structured soil which is different from simple granular porous media in some respect: Pores are more elongated and less isometric and the pore size distribution is much wider and structured hierarchically. To reproduce these properties, the classical concept of pore-bodies and throats is replaced by direct measurements of pore topology and the pores below the minimal pore size of the network model are represented by a continuous network of water saturated micro pores. The latter ensures that the water phase is always continuous which affects the propagation of the water potential during infiltration. The network model is based on cylindrical pores and considers capillary and gravitational forces. The propagation of interfaces is calculated for each time step by repeatedly solving the complete set of linear equation arising from Kirchhoff's law based on mass balance at each node of the network. This is done using the public domain package ITPack. The successive overrelaxation (SOR) and the Jacobi conjugate gradient (JCG) method proved to be more robust and faster than other solvers tested for the complex topology. The model accounts for entrapped air which is assumed to be incompressible. We present first results demonstrating the impact of external forcing (i.e infiltration rate) and pore topology on the dynamics of water-gas interfaces, the volume of entrapped air and hysteresis.

  10. 33 CFR 100.109 - Winter Harbor Lobster Boat Race, Winter Harbor, ME.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Winter Harbor Lobster Boat Race, Winter Harbor, ME. 100.109 Section 100.109 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF... Lobster Boat Race, Winter Harbor, ME. (a) Regulated area. The regulated area includes all waters of...

  11. Magnetic-resonance pore imaging of nonsymmetric microscopic pore shapes

    NASA Astrophysics Data System (ADS)

    Hertel, Stefan Andreas; Wang, Xindi; Hosking, Peter; Simpson, M. Cather; Hunter, Mark; Galvosas, Petrik

    2015-07-01

    Imaging of the microstructure of porous media such as biological tissue or porous solids is of high interest in health science and technology, engineering and material science. Magnetic resonance pore imaging (MRPI) is a recent technique based on nuclear magnetic resonance (NMR) which allows us to acquire images of the average pore shape in a given sample. Here we provide details on the experimental design, challenges, and requirements of MRPI, including its calibration procedures. Utilizing a laser-machined phantom sample, we present images of microscopic pores with a hemiequilateral triangular shape even in the presence of NMR relaxation effects at the pore walls. We therefore show that MRPI is applicable to porous samples without a priori knowledge about their pore shape and symmetry. Furthermore, we introduce "MRPI mapping," which combines MRPI with conventional magnetic resonance imaging (MRI). This enables one to resolve microscopic pore sizes and shapes spatially, thus expanding the application of MRPI to samples with heterogeneous distributions of pores.

  12. HIV-1 remodels the nuclear pore complex

    PubMed Central

    Monette, Anne; Panté, Nelly

    2011-01-01

    Human immunodeficiency virus type 1 (HIV-1) commandeers host cell proteins and machineries for its replication. Our earlier work showed that HIV-1 induced the cytoplasmic retention of nucleocytoplasmic shuttling and ribonucleic acid (RNA)–binding proteins. This retention is dependent on nuclear export of the viral genomic RNA and on changes in the localization and expression level of the nucleoporin (Nup) p62 (Nup62). To further characterize the extent of perturbation induced by HIV-1, we performed proteomics analyses of nuclear envelopes (NEs) isolated from infected T cells. Infection induced extensive changes in the composition of the NE and its associated proteins, including a remarkable decrease in the abundance of Nups. Immunogold electron microscopy revealed the translocation of Nups into the cytoplasm. Nup62 was identified as a component of purified virus, and small interfering RNA depletion studies revealed an important role for this Nup in virus gene expression and infectivity. This detailed analysis highlights the profound effects on NE composition induced by HIV-1 infection, providing further evidence of the magnitude of viral control over the cell biology of its host. PMID:21576391

  13. 33 CFR 117.603 - Manchester Harbor.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Manchester Harbor. 117.603... DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Massachusetts § 117.603 Manchester Harbor. The Massachusetts Bay Transportation Authority Bridge at mile 1.0 in Manchester, shall operate as follows: (a)...

  14. 33 CFR 117.1061 - Tacoma Harbor.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false Tacoma Harbor. 117.1061 Section 117.1061 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Washington § 117.1061 Tacoma Harbor. (a) When...

  15. 33 CFR 117.1061 - Tacoma Harbor.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 false Tacoma Harbor. 117.1061 Section 117.1061 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Washington § 117.1061 Tacoma Harbor. (a) When...

  16. 33 CFR 117.1061 - Tacoma Harbor.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false Tacoma Harbor. 117.1061 Section 117.1061 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Washington § 117.1061 Tacoma Harbor. (a) When...

  17. 33 CFR 117.1061 - Tacoma Harbor.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Tacoma Harbor. 117.1061 Section 117.1061 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Washington § 117.1061 Tacoma Harbor. (a) When...

  18. 33 CFR 117.1061 - Tacoma Harbor.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false Tacoma Harbor. 117.1061 Section 117.1061 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Washington § 117.1061 Tacoma Harbor. (a) When...

  19. 31 CFR 212.10 - Safe harbor.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 31 Money and Finance:Treasury 2 2011-07-01 2011-07-01 false Safe harbor. 212.10 Section 212.10... PAYMENTS § 212.10 Safe harbor. (a) Protection during examination and pending review. A financial... failing to honor a garnishment order, for account activity during: (1) The two business days following...

  20. 31 CFR 212.10 - Safe harbor.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 31 Money and Finance: Treasury 2 2014-07-01 2014-07-01 false Safe harbor. 212.10 Section 212.10... PAYMENTS § 212.10 Safe harbor. (a) Protection during examination and pending review. A financial... failing to honor a garnishment order, for account activity during: (1) The two business days following...

  1. 33 CFR 117.811 - Tonawanda Harbor.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false Tonawanda Harbor. 117.811 Section 117.811 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements New York § 117.811 Tonawanda Harbor. The draw of...

  2. 33 CFR 117.811 - Tonawanda Harbor.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Tonawanda Harbor. 117.811 Section 117.811 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements New York § 117.811 Tonawanda Harbor. The draw of...

  3. Soils, Pores, and NMR

    NASA Astrophysics Data System (ADS)

    Pohlmeier, Andreas; Haber-Pohlmeier, Sabina; Haber, Agnes; Sucre, Oscar; Stingaciu, Laura; Stapf, Siegfried; Blümich, Bernhard

    2010-05-01

    Within Cluster A, Partial Project A1, the pore space exploration by means of Nuclear Magnetic Resonance (NMR) plays a central role. NMR is especially convenient since it probes directly the state and dynamics of the substance of interest: water. First, NMR is applied as relaxometry, where the degree of saturation but also the pore geometry controls the NMR signature of natural porous systems. Examples are presented where soil samples from the Selhausen, Merzenhausen (silt loams), and Kaldenkirchen (sandy loam) test sites are investigated by means of Fast Field Cycling Relaxometry at different degrees of saturation. From the change of the relaxation time distributions with decreasing water content and by comparison with conventional water retention curves we conclude that the fraction of immobile water is characterized by T1 < 5 ms. Moreover, the dependence of the relaxation rate on magnetic field strength allows the identification of 2D diffusion at the interfaces as the mechanism which governs the relaxation process (Pohlmeier et al. 2009). T2 relaxation curves are frequently measured for the rapid characterization of soils by means of the CPMG echo train. Basically, they contain the same information about the pore systems like T1 curves, since mostly the overall relaxation is dominated by surface relaxivity and the surface/volume ratio of the pores. However, one must be aware that T2 relaxation is additionally affected by diffusion in internal gradients, and this can be overcome by using sufficiently short echo times and low magnetic fields (Stingaciu et al. 2009). Second, the logic continuation of conventional relaxation measurements is the 2-dimensional experiment, where prior to the final detection of the CPMG echo train an encoding period is applied. This can be T1-encoding by an inversion pulse, or T2 encoding by a sequence of 90 and 180° pulses. During the following evolution time the separately encoded signals can mix and this reveals information about

  4. P2X7R large pore is partially blocked by pore forming proteins antagonists in astrocytes.

    PubMed

    Faria, Robson X; Reis, Ricardo A M; Ferreira, Leonardo G B; Cezar-de-Mello, Paula F T; Moraes, Milton O

    2016-06-01

    The ATP-gated P2X7R (P2X7R) is a channel, which is involved in events, such as inflammation, cell death, and pain. The most intriguing event concerning P2X7R functions is the phenomenon of pore dilation. Once P2X7R is activated, the permeability of the plasma membrane becomes higher, leading to the permeation of 1000 Da-weight solutes. The mechanisms involved in this process remain unclear. Nevertheless, this event is not exclusively through P2X7R, as other proteins may form large pores in the plasma membrane. Recent evidence concerning pore formation reveals putative P2X7R and other pores-associated protein complexes, revealing cross-interactive pharmacological and biophysical issues. In this work, we showed results that corroborated with cross-interactive aspects with P2X7R and pores in astrocytes. These cells expressed most of the pores, including P2X7R. We discovered that different pore types open with peculiar characteristics, as both anionic and cationic charged solutes permeate the plasma membrane, following P2X7R activation. Moreover, we showed that both synergic and additive relationships are found within P2X7, cationic, and anionic large pores. Therefore, our data suggest that other protein-related pores are assembled following the formation of P2X7R pore. PMID:26830892

  5. A smart ROV solution for ship hull and harbor inspection

    NASA Astrophysics Data System (ADS)

    Reed, Scott; Wood, Jon; Vazquez, Jose; Mignotte, Pierre-Yves; Privat, Benjamin

    2010-04-01

    Hull and harbor infrastructure inspections are frequently performed manually and involve quite a bit of risk and human and monetary resources. In any kind of threat and resource constrained environment, this involves unacceptable levels of risk and cost. Modern Remotely Operated Vehicles are highly refined machines that provide features and capabilities previously unavailable. Operations once carried out by divers can now be carried out more quickly, efficiently and safely by smart enabled ROVs. ROVs are rapidly deployable and capable of continuous, reliable operations in adverse conditions. They also provide a stable platform on which multiple sensors may be mounted and utilized to meet the harbor inspection problem. Automated Control software provides ROV's and their pilots with the capability to inspect complex, constrained environments such as those found in a harbor region. This application and the user interface allow the ROV to automatically conduct complex maneuvers relative to the area being inspected and relieves the training requirements and work load for the pilot, allowing he or she to focus on the primary task of survey, inspection and looking for possible threats (such as IEDs, Limpet Mines, signs of sabotage, etc). Real-time sensor processing tools can be integrated into the smart ROV solution to assist the operator. Automatic Target Recognition (ATR) algorithms are used to search through the sensor data collected by the ROV in real time. These algorithms provide immediate feedback on possible threats and notify the operator of regions that may require manual verification. Sensor data (sonar or video) is also mosaiced, providing the operator with real-time situational awareness and a coverage map of the hull or seafloor. Detected objects may also be placed in the context of the large scale characteristics of the hull (or bottom or pilings) and localized. Within the complex areas such as the harbor pier pilings and the running gear of the ship, real

  6. Modeling tidal exchange and dispersion in Boston Harbor

    USGS Publications Warehouse

    Signell, Richard P.; Butman, Bradford

    1992-01-01

    Tidal dispersion and the horizontal exchange of water between Boston Harbor and the surrounding ocean are examined with a high-resolution (200 m) depth-averaged numerical model. The strongly varying bathymetry and coastline geometry of the harbor generate complex spatial patterns in the modeled tidal currents which are verified by shipboard acoustic Doppler surveys. Lagrangian exchange experiments demonstrate that tidal currents rapidly exchange and mix material near the inlets of the harbor due to asymmetry in the ebb/flood response. This tidal mixing zone extends roughly a tidal excursion from the inlets and plays an important role in the overall flushing of the harbor. Because the tides can only efficiently mix material in this limited region, however, harbor flushing must be considered a two step process: rapid exchange in the tidal mixing zone, followed by flushing of the tidal mixing zone by nontidal residual currents. Estimates of embayment flushing based on tidal calculations alone therefore can significantly overestimate the flushing time that would be expected under typical environmental conditions. Particle-release simulations from point sources also demonstrate that while the tides efficiently exchange material in the vicinity of the inlets, the exact nature of dispersion from point sources is extremely sensitive to the timing and location of the release, and the distribution of particles is streaky and patchlike. This suggests that high-resolution modeling of dispersion from point sources in these regions must be performed explicitly and cannot be parameterized as a plume with Gaussian-spreading in a larger scale flow field.

  7. Assembly and stability of nisin-lipid II pores.

    PubMed

    Hasper, Hester Emilie; de Kruijff, Ben; Breukink, Eefjan

    2004-09-14

    The peptide antibiotic nisin was the first reported example of an antibiotic that kills bacteria via targeted pore formation. The specific target of nisin is Lipid II, an essential intermediate in the bacterial cell-wall synthesis. High-affinity binding of the antibiotic to Lipid II is followed by rapid permeabilization of the membrane. Here, we investigated the assembly and stability of nisin-Lipid II pore complexes by means of pyrene fluorescence and circular dichroism. We demonstrated that nisin uses all available Lipid II molecules in the membrane to form pore complexes. The pore complexes have a uniform structure and consist of 8 nisin and 4 Lipid II molecules. Moreover, the pores displayed a remarkable stability, because they were able to resist the solubilization of the membrane environment by mild detergents. Similar experiments with [N20P/M21P]nisin showed that the hinge region is essential for the assembly into stable pore complexes. The new insights were used to propose a refined model for nisin pore formation. PMID:15350143

  8. Pore dynamics in lipid membranes

    NASA Astrophysics Data System (ADS)

    Gozen, I.; Dommersnes, P.

    2014-09-01

    Transient circular pores can open in plasma membrane of cells due to mechanical stress, and failure to repair such pores lead to cell death. Similar pores in the form of defects also exist among smectic membranes, such as in myelin sheaths or mitochondrial membranes. The formation and growth of membrane defects are associated with diseases, for example multiple sclerosis. A deeper understanding of membrane pore dynamics can provide a more refined picture of membrane integrity-related disease development, and possibly also treatment options and strategies. Pore dynamics is also of great importance regarding healthcare applications such as drug delivery, gene or as recently been implied, cancer therapy. The dynamics of pores significantly differ in stacks which are confined in 2D compared to those in cells or vesicles. In this short review, we will summarize the dynamics of different types of pores that can be observed in biological membranes, which include circular transient, fusion and hemi-fusion pores. We will dedicate a section to floral and fractal pores which were discovered a few years ago and have highly peculiar characteristics. Finally, we will discuss the repair mechanisms of large area pores in conjunction with the current cell membrane repair hypotheses.

  9. Cilia and Nuclear Pore Proteins: Pore No More?

    PubMed

    Obado, Samson O; Rout, Michael P

    2016-09-12

    Nuclear pore proteins at the base of cilia were thought to regulate transport into cilia. In this issue of Developmental Cell, Del Viso et al. (2016) challenge this view, showing instead that pore proteins localize to ciliary basal bodies and that their perturbation leads to congenital heart disease. PMID:27623377

  10. Open–closed switching of synthetic tubular pores

    PubMed Central

    Kim, Yongju; Kang, Jiheong; Shen, Bowen; Wang, Yanqiu; He, Ying; Lee, Myongsoo

    2015-01-01

    While encouraging progress has been made on switchable nanopores to mimic biological channels and pores, it remains a great challenge to realize long tubular pores with a dynamic open–closed motion. Here we report μm-long, dynamic tubular pores that undergo rapid switching between open and closed states in response to a thermal signal in water. The tubular walls consist of laterally associated primary fibrils stacked from disc-shaped molecules in which the discs readily tilt by means of thermally regulated dehydration of the oligoether chains placed on the wall surfaces. Notably, this pore switching mediates a controlled water-pumping catalytic action for the dehydrative cyclization of adenosine monophosphate to produce metabolically active cyclic adenosine monophosphate. We believe that our work may allow the creation of a variety of dynamic pore structures with complex functions arising from open–closed motion. PMID:26456695

  11. Silicon Pore Optics Technology

    NASA Astrophysics Data System (ADS)

    Beijersbergen, Marco; Collon, M. J.; Günther, R.; Partapsing, R.; Ackermann, M.; Olde Riekerink, M.; Cooper-Jensen, C.; Christensen, F.; Freyberg, M.; Krumrey, M.; Erhard, M.; van Baren, C.; Wallace, K.; Bavdaz, M.

    2009-01-01

    Silicon pore optics have been developed over the last years to enable future astrophysical X-ray telescopes and have now become a candidate mirror technology for the IXO mission. Scientific requirements demand an angular resolution better than 5” and a large effective area of several square meters at photon energies of 1 keV. This paper discusses the performance of the latest generation of these novel light, stiff and modular X-ray optics, based on ribbed plates made from commercial high grade 12” silicon wafers. Stacks with several tens of silicon plates have been assembled in the course of an ESA technology development program, by bending the plates into an accurate shape and directly bonding them on top of each other. Several mirror modules, using two stacks each, have been aligned and integrated to form the conical approximation of a Wolter-I design. This paper presents the status of the technology, addresses and discusses a number of activities in the ongoing ESA technology development and shows the latest results of full area measurements at the long-beamline MPE X-ray test facility (PANTER) and the PTB beam line at the BESSY electron storage ring in Berlin.

  12. Assembly of the Bak Apoptotic Pore

    PubMed Central

    Ma, Stephen; Hockings, Colin; Anwari, Khatira; Kratina, Tobias; Fennell, Stephanie; Lazarou, Michael; Ryan, Michael T.; Kluck, Ruth M.; Dewson, Grant

    2013-01-01

    Bak and Bax are the essential effectors of the intrinsic pathway of apoptosis. Following an apoptotic stimulus, both undergo significant changes in conformation that facilitates their self-association to form pores in the mitochondrial outer membrane. However, the molecular structures of Bak and Bax oligomeric pores remain elusive. To characterize how Bak forms pores during apoptosis, we investigated its oligomerization under native conditions using blue native PAGE. We report that, in a healthy cell, inactive Bak is either monomeric or in a large complex involving VDAC2. Following an apoptotic stimulus, activated Bak forms BH3:groove homodimers that represent the basic stable oligomeric unit. These dimers multimerize to higher-order oligomers via a labile interface independent of both the BH3 domain and groove. Linkage of the α6:α6 interface is sufficient to stabilize higher-order Bak oligomers on native PAGE, suggesting an important role in the Bak oligomeric pore. Mutagenesis of the α6 helix disrupted apoptotic function because a chimera of Bak with the α6 derived from Bcl-2 could be activated by truncated Bid (tBid) and could form BH3:groove homodimers but could not form high molecular weight oligomers or mediate cell death. An α6 peptide could block Bak function but did so upstream of dimerization, potentially implicating α6 as a site for activation by BH3-only proteins. Our examination of native Bak oligomers indicates that the Bak apoptotic pore forms by the multimerization of BH3:groove homodimers and reveals that Bak α6 is not only important for Bak oligomerization and function but may also be involved in how Bak is activated by BH3-only proteins. PMID:23893415

  13. 33 CFR 110.30 - Boston Harbor, Mass.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Boston Harbor, Mass. 110.30... ANCHORAGE REGULATIONS Special Anchorage Areas § 110.30 Boston Harbor, Mass. (a) Vicinity of South Boston... the local Harbor Master, Hull, Mass. (m) Hingham Harbor Area 1. Beginning at position latitude...

  14. 33 CFR 80.1122 - Channel Islands Harbor, CA.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false Channel Islands Harbor, CA. 80... INTERNATIONAL NAVIGATION RULES COLREGS DEMARCATION LINES Pacific Coast § 80.1122 Channel Islands Harbor, CA. (a) A line drawn from Channel Islands Harbor South Jetty Light 2 to Channel Islands Harbor...

  15. 33 CFR 80.1122 - Channel Islands Harbor, CA.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 false Channel Islands Harbor, CA. 80... INTERNATIONAL NAVIGATION RULES COLREGS DEMARCATION LINES Pacific Coast § 80.1122 Channel Islands Harbor, CA. (a) A line drawn from Channel Islands Harbor South Jetty Light 2 to Channel Islands Harbor...

  16. 33 CFR 80.1122 - Channel Islands Harbor, CA.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false Channel Islands Harbor, CA. 80... INTERNATIONAL NAVIGATION RULES COLREGS DEMARCATION LINES Pacific Coast § 80.1122 Channel Islands Harbor, CA. (a) A line drawn from Channel Islands Harbor South Jetty Light 2 to Channel Islands Harbor...

  17. 33 CFR 80.1122 - Channel Islands Harbor, CA.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Channel Islands Harbor, CA. 80... INTERNATIONAL NAVIGATION RULES COLREGS DEMARCATION LINES Pacific Coast § 80.1122 Channel Islands Harbor, CA. (a) A line drawn from Channel Islands Harbor South Jetty Light 2 to Channel Islands Harbor...

  18. 33 CFR 80.1122 - Channel Islands Harbor, CA.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false Channel Islands Harbor, CA. 80... INTERNATIONAL NAVIGATION RULES COLREGS DEMARCATION LINES Pacific Coast § 80.1122 Channel Islands Harbor, CA. (a) A line drawn from Channel Islands Harbor South Jetty Light 2 to Channel Islands Harbor...

  19. 33 CFR 110.30 - Boston Harbor, Mass.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false Boston Harbor, Mass. 110.30... ANCHORAGE REGULATIONS Special Anchorage Areas § 110.30 Boston Harbor, Mass. (a) Vicinity of South Boston... the local Harbor Master, Hull, Mass. (m) Hingham Harbor Area 1. Beginning at position latitude...

  20. 33 CFR 110.30 - Boston Harbor, Mass.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false Boston Harbor, Mass. 110.30... ANCHORAGE REGULATIONS Special Anchorage Areas § 110.30 Boston Harbor, Mass. (a) Vicinity of South Boston... the local Harbor Master, Hull, Mass. (m) Hingham Harbor Area 1. Beginning at position latitude...

  1. 33 CFR 110.30 - Boston Harbor, Mass.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false Boston Harbor, Mass. 110.30... ANCHORAGE REGULATIONS Special Anchorage Areas § 110.30 Boston Harbor, Mass. (a) Vicinity of South Boston... the local Harbor Master, Hull, Mass. (m) Hingham Harbor Area 1. Beginning at position latitude...

  2. 33 CFR 110.30 - Boston Harbor, Mass.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 false Boston Harbor, Mass. 110.30... ANCHORAGE REGULATIONS Special Anchorage Areas § 110.30 Boston Harbor, Mass. (a) Vicinity of South Boston... the local Harbor Master, Hull, Mass. (m) Hingham Harbor Area 1. Beginning at position latitude...

  3. 33 CFR 80.1140 - Pillar Point Harbor, CA.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Pillar Point Harbor, CA. 80.1140... NAVIGATION RULES COLREGS DEMARCATION LINES Pacific Coast § 80.1140 Pillar Point Harbor, CA. A line drawn from Pillar Point Harbor Light 6 to Pillar Point Harbor Entrance Light....

  4. 33 CFR 80.1140 - Pillar Point Harbor, CA.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false Pillar Point Harbor, CA. 80.1140... NAVIGATION RULES COLREGS DEMARCATION LINES Pacific Coast § 80.1140 Pillar Point Harbor, CA. A line drawn from Pillar Point Harbor Light 6 to Pillar Point Harbor Entrance Light....

  5. Managing Free-energy Barriers in Nuclear Pore Transport

    PubMed Central

    Nielsen, Brian; Jeppesen, Claus

    2006-01-01

    The Nuclear Pore Complexes (NPC) facilitate highly selective gateways for transport of macromolecules across the Nuclear Envelope (NE). Based on the current accumulated knowledge of the architecture of NPC we have established a minimal physical model of the pore and the transport mechanism. The barrier properties of the NPC model are analyzed by the recently established Wang–Landau Monte Carlo computer simulation technique and the transport properties are extracted by employing Kramers’ theory of reaction rates. We show that our physical model can account for a range of characteristics observed for nuclear pore transport. PMID:19669451

  6. 78 FR 54392 - Security Zone, Baltimore Harbor, Baltimore's Inner Harbor; Baltimore, MD

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-04

    ...The Coast Guard is establishing a temporary security zone encompassing certain waters of Baltimore Harbor, Baltimore's Inner Harbor, at Baltimore, Maryland. This action is necessary to safeguard persons and property, and prevent terrorist acts or incidents. This rule prohibits vessels and people from entering the security zone and requires vessels and persons in the security zone to depart the......

  7. 33 CFR 165.904 - Lake Michigan at Chicago Harbor & Burnham Park Harbor-Safety and Security Zone.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... & Burnham Park Harbor-Safety and Security Zone. 165.904 Section 165.904 Navigation and Navigable Waters... Guard District § 165.904 Lake Michigan at Chicago Harbor & Burnham Park Harbor—Safety and Security Zone... waters including Burnham Park Harbor and the southern part of Chicago Harbor, Lake Michigan, bounded...

  8. 33 CFR 165.904 - Lake Michigan at Chicago Harbor & Burnham Park Harbor-Safety and Security Zone.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... & Burnham Park Harbor-Safety and Security Zone. 165.904 Section 165.904 Navigation and Navigable Waters... Guard District § 165.904 Lake Michigan at Chicago Harbor & Burnham Park Harbor—Safety and Security Zone... waters including Burnham Park Harbor and the southern part of Chicago Harbor, Lake Michigan, bounded...

  9. Triggered pore-forming agents

    DOEpatents

    Bayley, Hagan; Walker, Barbara J.; Chang, Chung-yu; Niblack, Brett; Panchal, Rekha

    1998-01-01

    An inactive pore-forming agent which is activated to lytic function by a condition such as pH, light, heat, reducing potential, or metal ion concentration, or substance such as a protease, at the surface of a cell.

  10. Biomimetic collagen scaffolds with anisotropic pore architecture.

    PubMed

    Davidenko, N; Gibb, T; Schuster, C; Best, S M; Campbell, J J; Watson, C J; Cameron, R E

    2012-02-01

    Sponge-like matrices with a specific three-dimensional structural design resembling the actual extracellular matrix of a particular tissue show significant potential for the regeneration and repair of a broad range of damaged anisotropic tissues. The manipulation of the structure of collagen scaffolds using a freeze-drying technique was explored in this work as an intrinsically biocompatible way of tailoring the inner architecture of the scaffold. The research focused on the influence of temperature gradients, imposed during the phase of crystallisation of collagen suspensions, upon the degree of anisotropy in the microstructures of the scaffolds produced. Moulding technology was employed to achieve differences in heat transfer rates during the freezing processes. For this purpose various moulds with different configurations were developed with a view to producing uniaxial and multi-directional temperature gradients across the sample during this process. Scanning electron microscopy analysis of different cross-sections (longitudinal and horizontal) of scaffolds revealed that highly aligned matrices with axially directed pore architectures were obtained where single unidirectional temperature gradients were induced. Altering the freezing conditions by the introduction of multiple temperature gradients allowed collagen scaffolds to be produced with complex pore orientations, and anisotropy in pore size and alignment. PMID:22005330

  11. New Bedford Harbor Long Term Monitoring Program

    EPA Science Inventory

    New Bedford Harbor (NBH), located in southeastern Massachusetts, was designated as a Superfund site in 1983 due to unacceptably high levels of sediment contamination by polychlorinated biphenyls (PCBs). Based on human health and environmental concerns, the decision was made to d...

  12. 16 CFR 312.10 - Safe harbors.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 16 Commercial Practices 1 2010-01-01 2010-01-01 false Safe harbors. 312.10 Section 312.10 Commercial Practices FEDERAL TRADE COMMISSION REGULATIONS UNDER SPECIFIC ACTS OF CONGRESS CHILDREN'S ONLINE..., issued by representatives of the marketing or online industries, or by other persons, that, after...

  13. Genetics Home Reference: Floating-Harbor syndrome

    MedlinePlus

    ... Arpin S, Afenjar A, Dubern B, Toutain A, Cabrol S, Héron D. Floating-Harbor Syndrome: report on a case ... G, Whiteford ML, Quaio CR, Gomy I, Bertola DR, Albrecht B, Platzer K, McGillivray G, Zou R, ...

  14. HARBOR ISLAND REMEDIAL INVESTIGATION, MARINE SEDIMENT

    EPA Science Inventory

    The data set contains marine sediment data from a remedial investigation of Harbor Island, a National Priority List (NPL) Superfund site in Washington State. Both surface and subsurface marine sediments were collected. A station data set contain sampling station location and desc...

  15. Sediment bioaccumulation testing: Manistique Harbor sediments

    EPA Science Inventory

    Manistique Harbor AOC public meeting and availability session on August 28th in Manistique, MI. This meeting/session is organized by GLNPO; they are EPA's lead on AOC restoration efforts. The goal of the meeting is to engage with the community with all the work that has been d...

  16. The perforin pore facilitates the delivery of cationic cargos.

    PubMed

    Stewart, Sarah E; Kondos, Stephanie C; Matthews, Antony Y; D'Angelo, Michael E; Dunstone, Michelle A; Whisstock, James C; Trapani, Joseph A; Bird, Phillip I

    2014-03-28

    Cytotoxic lymphocytes eliminate virally infected or neoplastic cells through the action of cytotoxic proteases (granzymes). The pore-forming protein perforin is essential for delivery of granzymes into the cytoplasm of target cells; however the mechanism of this delivery is incompletely understood. Perforin contains a membrane attack complex/perforin (MACPF) domain and oligomerizes to form an aqueous pore in the plasma membrane; therefore the simplest (and best supported) model suggests that granzymes passively diffuse through the perforin pore into the cytoplasm of the target cell. Here we demonstrate that perforin preferentially delivers cationic molecules while anionic and neutral cargoes are delivered inefficiently. Furthermore, another distantly related pore-forming MACPF protein, pleurotolysin (from the oyster mushroom), also favors the delivery of cationic molecules, and efficiently delivers human granzyme B. We propose that this facilitated diffusion is due to conserved features of oligomerized MACPF proteins, which may include an anionic lumen. PMID:24558045

  17. Membrane pore formation at protein-lipid interfaces.

    PubMed

    Gilbert, Robert J C; Dalla Serra, Mauro; Froelich, Christopher J; Wallace, Mark I; Anderluh, Gregor

    2014-11-01

    Pore-forming proteins (PFPs) interact with lipid bilayers to compromise membrane integrity. Many PFPs function by inserting a ring of oligomerized subunits into the bilayer to form a protein-lined hydrophilic channel. However, mounting evidence suggests that PFPs can also generate 'proteolipidic' pores by contributing to the fusion of inner and outer bilayer leaflets to form a toroidal structure. We discuss here toroidal pore formation by peptides including melittin, protegrin, and Alzheimer's Aβ1-41, as well as by PFPs from several evolutionarily unrelated families: the colicin/Bcl-2 grouping including the pro-apoptotic protein Bax, actinoporins derived from sea anemones, and the membrane attack complex-perforin/cholesterol dependent cytolysin (MACPF/CDC) set of proteins. We also explore how the structure and biological role of toroidal pores might be investigated further. PMID:25440714

  18. Role of the synaptobrevin C terminus in fusion pore formation

    PubMed Central

    Ngatchou, Annita N.; Kisler, Kassandra; Fang, Qinghua; Walter, Alexander M.; Zhao, Ying; Bruns, Dieter; Sørensen, Jakob B.; Lindau, Manfred

    2010-01-01

    Neurotransmitter release is mediated by the SNARE proteins synaptobrevin II (sybII, also known as VAMP2), syntaxin, and SNAP-25, generating a force transfer to the membranes and inducing fusion pore formation. However, the molecular mechanism by which this force leads to opening of a fusion pore remains elusive. Here we show that the ability of sybII to support exocytosis is inhibited by addition of one or two residues to the sybII C terminus depending on their energy of transfer from water to the membrane interface, following a Boltzmann distribution. These results suggest that following stimulation, the SNARE complex pulls the C terminus of sybII deeper into the vesicle membrane. We propose that this movement disrupts the vesicular membrane continuity leading to fusion pore formation. In contrast to current models, the experiments suggest that fusion pore formation begins with molecular rearrangements at the intravesicular membrane leaflet and not between the apposed cytoplasmic leaflets. PMID:20937897

  19. Gas Hydrate and Pore Pressure

    NASA Astrophysics Data System (ADS)

    Tinivella, Umberta; Giustiniani, Michela

    2014-05-01

    Many efforts have been devoted to quantify excess pore pressures related to gas hydrate dissociation in marine sediments below the BSR using several approaches. Dissociation of gas hydrates in proximity of the BSR, in response to a change in the physical environment (i.e., temperature and/or pressure regime), can liberate excess gas incrising the local pore fluid pressure in the sediment, so decreasing the effective normal stress. So, gas hydrate dissociation may lead to excess pore pressure resulting in sediment deformation or failure, such as submarine landslides, sediment slumping, pockmarks and mud volcanoes, soft-sediment deformation and giant hummocks. Moreover, excess pore pressure may be the result of gas hydrate dissociation due to continuous sedimentation, tectonic uplift, sea level fall, heating or inhibitor injection. In order to detect the presence of the overpressure below the BSR, we propose two approachs. The fist approach models the BSR depth versus pore pressure; in fact, if the free gas below the BSR is in overpressure condition, the base of the gas hydrate stability is deeper with respect to the hydrostatic case. This effect causes a discrepancy between seismic and theoretical BSR depths. The second approach models the velocities versus gas hydrate and free gas concentrations and pore pressure, considering the approximation of the Biot theory in case of low frequency, i.e. seismic frequency. Knowing the P and S seismic velocity from seismic data analysis, it is possibile to jointly estimate the gas hydrate and free gas concentrations and the pore pressure regime. Alternatively, if the S-wave velocity is not availbale (due to lack of OBS/OBC data), an AVO analysis can be performed in order to extract information about Poisson ratio. Our modeling suggests that the areas characterized by shallow waters (i.e., areas in which human infrastructures, such as pipelines, are present) are significantly affected by the presence of overpressure condition

  20. Can ash clog soil pores?

    NASA Astrophysics Data System (ADS)

    Stoof, Cathelijne; Stoof, Cathelijne; Gevaert, Anouk; Gevaert, Anouk; Baver, Christine; Baver, Christine; Hassanpour, Bahareh; Hassanpour, Bahareh; Morales, Veronica; Morales, Veronica; Zhang, Wei; Zhang, Wei; Martin, Deborah; Martin, Deborah; Steenhuis, Tammo; Steenhuis, Tammo

    2015-04-01

    Wildfire can greatly increase a landscape's vulnerability to flooding and erosion events, and ash is thought to play a large role in controlling runoff and erosion processes after wildfire. Although ash can store rainfall and thereby reduce runoff and erosion for a limited period after wildfires, it has also been hypothesized to clog soil pores and reduce infiltration. Several researchers have attributed the commonly observed increase in runoff and erosion after fire to the potential pore-clogging effect of ash. Evidence is however incomplete, as to date, research has solely focused on identifying the presence of ash in the soil, with the actual flow processes associated with the infiltration and pore-clogging of ash remaining a major unknown. In several laboratory experiments, we tested the hypothesis that ash causes pore clogging to the point that infiltration is hampered and ponding occurs. We first visualized and quantified pore-scale infiltration of water and ash in sand of a range of textures and at various infiltration rates, using a digital bright field microscope capturing both photo and video. While these visualization experiments confirm field and lab observation of ash washing into soil pores, we did not observe any clogging of pores, and have not been able to create conditions for which this does occur. Additional electrochemical analysis and measurement of saturated hydraulic conductivity indicate that pore clogging by ash is not plausible. Electrochemical analysis showed that ash and sand are both negatively charged, showing that attachment of ash to sand and any resulting clogging is unlikely. Ash also had quite high saturated conductivity, and systems where ash was mixed in or lying on top of sand had similarly high hydraulic conductivity. Based on these various experiments, we cannot confirm the hypothesis that pore clogging by ash contributes to the frequently observed increase in post-fire runoff, at least for the medium to coarse sands

  1. Pore translocation of polymer chains with physical knots

    NASA Astrophysics Data System (ADS)

    Suma, Antonio; Rosa, Angelo; Micheletti, Cristian

    The driven traslocation of knotted chains through narrow pores has important implications for single-molecule manipulation contexts. Its complex phenomenology is, however, still largely unexplored, both as a function of knot complexity and the magnitude of the driving, translocating force. We accordingly report on a systematic theoretical and computational investigation of both aspects. In particular we consider the case of flexible chains accommodating a large repertoire of knots that are driven through pores too narrow to allow for their passage. We show that the observed rich translocation phenomenology can be rationalised in a transparent mechanical framework that can further be used for predictive purposes.

  2. 76 FR 8653 - Drawbridge Operation Regulation; Gulf Intracoastal Waterway, New Orleans Harbor, Inner Harbor...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-02-15

    ... Seeber/Claiborne Avenue) vertical lift bridge across the Inner Harbor Navigational Canal, mile 0.9, (Gulf... deviation from the published regulation for the SR 39 (Judge Seeber/Claiborne Avenue) vertical lift...

  3. Sediment quality assessment studies in Boston Harbor, Massachusetts

    SciTech Connect

    Carr, R.S.; Chapman, D.C.; Biedenbach, J.M.; Long, E.R.; Thursby, G.; MacDonald, D.D.

    1995-12-31

    As part of NOAA`s National Status and Trends program, a bioeffects assessment study was conducted in the vicinity of Boston Harbor, Massachusetts. Surficial sediment samples were collected at 55 sites and subsamples were tested for toxicity using (1) the 10-day whole sediment test with Ampelisca abdita, (2) the sea urchin (Arbacia punctulata) fertilization and embryological development assays with sediment pore water, and (3) Microtox{trademark} assay with organic sediment extracts. Eleven percent of the samples were significantly toxic in the amphipod test, only 4% were toxic in the sea urchin fertilization test whereas all of the samples were highly toxic in the sea urchin embryological development assay; the Microtox assay determined 56% of the organic sediment extracts to be significantly toxic. Sediment chemical analyses for metals, AVS/SEM, PAHs, PCBs, and pesticides were performed on 30 of the 55 samples. Twenty-seven of the 30 samples exceeded at least one probable effects level (PEL) value. For the 20 samples that exceeded 5 or more PELS, the concordance between the predicted and observed toxicity was 20% for the amphipod test, 60% for the Microtox test, and 100% for the sea urchin embryological development assay. There were no significant correlations among the different toxicity tests or between the tests and the contaminant concentrations in the bulk sediment. Possible explanations for the apparent lack of correlation between the sediment chemistry and the toxicity tests will be discussed.

  4. Geostatistical Modeling of Pore Velocity

    SciTech Connect

    Devary, J.L.; Doctor, P.G.

    1981-06-01

    A significant part of evaluating a geologic formation as a nuclear waste repository involves the modeling of contaminant transport in the surrounding media in the event the repository is breached. The commonly used contaminant transport models are deterministic. However, the spatial variability of hydrologic field parameters introduces uncertainties into contaminant transport predictions. This paper discusses the application of geostatistical techniques to the modeling of spatially varying hydrologic field parameters required as input to contaminant transport analyses. Kriging estimation techniques were applied to Hanford Reservation field data to calculate hydraulic conductivity and the ground-water potential gradients. These quantities were statistically combined to estimate the groundwater pore velocity and to characterize the pore velocity estimation error. Combining geostatistical modeling techniques with product error propagation techniques results in an effective stochastic characterization of groundwater pore velocity, a hydrologic parameter required for contaminant transport analyses.

  5. 8. NEW YORK HARBOR MODEL. VIEW FACING DOWN EAST RIVER ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    8. NEW YORK HARBOR MODEL. VIEW FACING DOWN EAST RIVER TO NEW YORK HARBOR, WITH LOWER MANHATTAN ISLAND AT RIGHT. - Waterways Experiment Station, Hydraulics Laboratory, Halls Ferry Road, 2 miles south of I-20, Vicksburg, Warren County, MS

  6. 7. NEW YORK HARBOR MODEL. VIEW FACING DOWN NEW YORK ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    7. NEW YORK HARBOR MODEL. VIEW FACING DOWN NEW YORK HARBOR, WITH LOWER MANHATTAN ISLAND AT LEFT. - Waterways Experiment Station, Hydraulics Laboratory, Halls Ferry Road, 2 miles south of I-20, Vicksburg, Warren County, MS

  7. Restricted Transport in Small Pores

    PubMed Central

    Anderson, John L.; Quinn, John A.

    1974-01-01

    The basic hydrodynamic equations governing transport in submicron pores are reexamined. Conditions necessary for a simplified, one-dimensional treatment of the diffusion/convection process are established. Steric restrictions and Brownian motion are incorporated directly into the resulting model. Currently available fluid mechanical results are used to evaluate an upper limit on hindered diffusion; this limit is valid for small particle-to-pore ratios. Extensions of the analysis are shown to depend on numerical solutions of the related hydrodynamic problem, that of asymmetrical particle motion in a bounded fluid. PMID:4813157

  8. Decadal Changes In Benthic Community Measures In New York Harbor

    EPA Science Inventory

    Monitoring in New York Harbor, NY, as part of the Regional Environmental Monitoring and Assessment Program has spanned a decade, and includes habitat and water quality measures and sediment contaminant levels from four sub-basins (Upper NY Harbor, Lower NY Harbor, Newark Bay, and...

  9. 33 CFR 110.130 - Bar Harbor, Maine.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Bar Harbor, Maine. 110.130... ANCHORAGE REGULATIONS Anchorage Grounds § 110.130 Bar Harbor, Maine. (a) Anchorage grounds. (1) Anchorage “A” is that portion of Frenchman Bay, Bar Harbor, ME enclosed by a rhumb line connecting the...

  10. 33 CFR 110.130 - Bar Harbor, Maine.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false Bar Harbor, Maine. 110.130... ANCHORAGE REGULATIONS Anchorage Grounds § 110.130 Bar Harbor, Maine. (a) Anchorage grounds. (1) Anchorage “A” is that portion of Frenchman Bay, Bar Harbor, ME enclosed by a rhumb line connecting the...

  11. 33 CFR 110.130 - Bar Harbor, Maine.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false Bar Harbor, Maine. 110.130... ANCHORAGE REGULATIONS Anchorage Grounds § 110.130 Bar Harbor, Maine. (a) Anchorage grounds. (1) Anchorage “A” is that portion of Frenchman Bay, Bar Harbor, ME enclosed by a rhumb line connecting the...

  12. 33 CFR 110.130 - Bar Harbor, Maine.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 false Bar Harbor, Maine. 110.130... ANCHORAGE REGULATIONS Anchorage Grounds § 110.130 Bar Harbor, Maine. (a) Anchorage grounds. (1) Anchorage “A” is that portion of Frenchman Bay, Bar Harbor, ME enclosed by a rhumb line connecting the...

  13. 33 CFR 110.130 - Bar Harbor, Maine.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false Bar Harbor, Maine. 110.130... ANCHORAGE REGULATIONS Anchorage Grounds § 110.130 Bar Harbor, Maine. (a) Anchorage grounds. (1) Anchorage “A” is that portion of Frenchman Bay, Bar Harbor, ME enclosed by a rhumb line connecting the...

  14. 33 CFR 110.205 - Chicago Harbor, Ill.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false Chicago Harbor, Ill. 110.205... ANCHORAGE REGULATIONS Anchorage Grounds § 110.205 Chicago Harbor, Ill. (a) The anchorage grounds—(1...) Anchorage D, Chicago Harbor Lock South. Beginning at a point 35.5 feet South (16 feet South of the...

  15. 26 CFR 1.401(k)-3 - Safe harbor requirements.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... fail to be satisfied merely because safe harbor matching contributions are made on both elective... contribution requirement of this paragraph (c) will not fail to be satisfied merely because safe harbor... paragraph (c) will not fail to be satisfied merely because the plan provides that safe harbor...

  16. 49 CFR 578.7 - Criminal safe harbor provision.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 7 2011-10-01 2011-10-01 false Criminal safe harbor provision. 578.7 Section 578... Criminal safe harbor provision. (a) Scope. This section sets forth the requirements regarding the reasonable time and the manner of correction for a person seeking safe harbor protection from...

  17. 26 CFR 1.401(m)-3 - Safe harbor requirements.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... plan will not fail to satisfy the safe harbor matching contribution requirements of this section merely... rule. A plan that provides for safe harbor matching contributions will not fail to satisfy the... 26 Internal Revenue 5 2013-04-01 2013-04-01 false Safe harbor requirements. 1.401(m)-3 Section...

  18. 49 CFR 578.7 - Criminal safe harbor provision.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 7 2012-10-01 2012-10-01 false Criminal safe harbor provision. 578.7 Section 578... Criminal safe harbor provision. (a) Scope. This section sets forth the requirements regarding the reasonable time and the manner of correction for a person seeking safe harbor protection from...

  19. 7. Photocopy of c 1837 map of Cleveland Harbor with ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    7. Photocopy of c 1837 map of Cleveland Harbor with two plans for additonal harbor. This is the first map to show the Cleveland Breakwater. Original in the Corps' files, Buffalo District. - Cleveland Breakwater at Cleveland Harbor, Cleveland, Cuyahoga County, OH

  20. 33 CFR 80.1136 - Moss Landing Harbor, CA.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false Moss Landing Harbor, CA. 80.1136... NAVIGATION RULES COLREGS DEMARCATION LINES Pacific Coast § 80.1136 Moss Landing Harbor, CA. A line drawn from the seaward extremity of the pier located 0.3 mile south of Moss Landing Harbor Entrance to...

  1. 33 CFR 80.1136 - Moss Landing Harbor, CA.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false Moss Landing Harbor, CA. 80.1136... NAVIGATION RULES COLREGS DEMARCATION LINES Pacific Coast § 80.1136 Moss Landing Harbor, CA. A line drawn from the seaward extremity of the pier located 0.3 mile south of Moss Landing Harbor Entrance to...

  2. 33 CFR 80.1136 - Moss Landing Harbor, CA.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 false Moss Landing Harbor, CA. 80.1136... NAVIGATION RULES COLREGS DEMARCATION LINES Pacific Coast § 80.1136 Moss Landing Harbor, CA. A line drawn from the seaward extremity of the pier located 0.3 mile south of Moss Landing Harbor Entrance to...

  3. 33 CFR 80.1136 - Moss Landing Harbor, CA.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Moss Landing Harbor, CA. 80.1136... NAVIGATION RULES COLREGS DEMARCATION LINES Pacific Coast § 80.1136 Moss Landing Harbor, CA. A line drawn from the seaward extremity of the pier located 0.3 mile south of Moss Landing Harbor Entrance to...

  4. 33 CFR 80.1136 - Moss Landing Harbor, CA.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false Moss Landing Harbor, CA. 80.1136... NAVIGATION RULES COLREGS DEMARCATION LINES Pacific Coast § 80.1136 Moss Landing Harbor, CA. A line drawn from the seaward extremity of the pier located 0.3 mile south of Moss Landing Harbor Entrance to...

  5. 33 CFR 110.95 - Newport Bay Harbor, Calif.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Beach Harbor Ordinance No. 543 for recreational and small craft of such size and alignment as permitted.... Fore and aft moorings will be allowed in this area conforming to the City of Newport Beach Harbor... moorings will be allowed in this area conforming to the City of Newport Beach Harbor Ordinance No. 543...

  6. 33 CFR 110.95 - Newport Bay Harbor, Calif.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Beach Harbor Ordinance No. 543 for recreational and small craft of such size and alignment as permitted.... Fore and aft moorings will be allowed in this area conforming to the City of Newport Beach Harbor... moorings will be allowed in this area conforming to the City of Newport Beach Harbor Ordinance No. 543...

  7. 33 CFR 117.272 - Boot Key Harbor.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Boot Key Harbor. 117.272 Section... DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Florida § 117.272 Boot Key Harbor. The draw of the Boot Key Harbor drawbridge, mile 0.13, between Marathon and Boot Key, will open as necessary on...

  8. 33 CFR 117.272 - Boot Key Harbor.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 false Boot Key Harbor. 117.272 Section... DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Florida § 117.272 Boot Key Harbor. The draw of the Boot Key Harbor drawbridge, mile 0.13, between Marathon and Boot Key, will open as necessary on...

  9. 33 CFR 117.272 - Boot Key Harbor.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false Boot Key Harbor. 117.272 Section... DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Florida § 117.272 Boot Key Harbor. The draw of the Boot Key Harbor drawbridge, mile 0.13, between Marathon and Boot Key, will open as necessary on...

  10. 33 CFR 117.272 - Boot Key Harbor.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false Boot Key Harbor. 117.272 Section... DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Florida § 117.272 Boot Key Harbor. The draw of the Boot Key Harbor drawbridge, mile 0.13, between Marathon and Boot Key, will open as necessary on...