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Sample records for envelope cytoplasmic tail

  1. Virion incorporation of envelope glycoproteins with long but not short cytoplasmic tails is blocked by specific, single amino acid substitutions in the human immunodeficiency virus type 1 matrix.

    PubMed Central

    Freed, E O; Martin, M A

    1995-01-01

    Incorporation of envelope glycoproteins into a budding retrovirus is an essential step in the formation of an infectious virus particle. By using site-directed mutagenesis, we identified specific amino acid residues in the matrix domain of the human immunodeficiency virus type 1 (HIV-1) Gag protein that are critical to the incorporation of HIV-1 envelope glycoproteins into virus particles. Pseudotyping analyses were used to demonstrate that two heterologous envelope glycoproteins with short cytoplasmic tails (the envelope of the amphotropic murine leukemia virus and a naturally truncated HIV-2 envelope) are efficiently incorporated into HIV-1 particles bearing the matrix mutations. Furthermore, deletion of the cytoplasmic tail of HIV-1 transmembrane envelope glycoprotein gp41 from 150 to 7 or 47 residues reversed the incorporation block imposed by the matrix mutations. These results suggest the existence of a specific functional interaction between the HIV-1 matrix and the gp41 cytoplasmic tail. PMID:7853546

  2. Infection of human and non-human cells by a highly fusogenic primary CD4-independent HIV-1 isolate with a truncated envelope cytoplasmic tail

    SciTech Connect

    Saha, Kunal . E-mail: sahak@pediatrics.ohio-state.edu; Yan Hui; Nelson, Julie A.E.; Zerhouni-Layachi, Bouchra

    2005-06-20

    Truncation of the envelope cytoplasmic tail has enabled FIV, SIV, and some laboratory HIV-1 strains to acquire broader cellular tropism and enhanced fusogenicity. Here we have characterized a primary CD4-independent HIV-1 isolate (92UG046-T8) with a truncated cytoplasmic tail that was able to infect and induce syncytia in primary lymphocytes from human, chimpanzee, and monkey, as well as CD4-negative cell lines from human and monkey. Increased syncytia were also noticeable with 293 cells expressing the cloned envelope from the 92UG046-T8 isolate suggesting envelope-mediated cellular fusion. Except pooled serum from HIV-1-infected individuals, monoclonal anti-envelope antibodies or antibodies/antagonists against CD4, CXCR4, and CCR5 were not able to prevent infection by the 92UG046-T8 isolate. This is the first report showing a primary HIV-1 variant with truncated cytoplasmic tail which is highly fusogenic and can infect a broad range of cells from human and non-human origins. In vivo evolution of similar HIV-1 mutants may have important implications in AIDS pathogenesis.

  3. Mutation of critical serine residues in HIV-1 matrix result in an envelope incorporation defect which can be rescued by truncation of the gp41 cytoplasmic tail

    SciTech Connect

    Bhatia, Ajay K.; Kaushik, Rajnish; Campbell, Nancy A.; Pontow, Suzanne E.; Ratner, Lee

    2009-02-05

    The human immunodeficiency virus type 1 (HIV-1) matrix (MA) domain is involved in both early and late events of the viral life cycle. Simultaneous mutation of critical serine residues in MA has been shown previously to dramatically reduce phosphorylation of MA. However, the role of phosphorylation in viral replication remains unclear. Viruses harboring serine to alanine substitutions at positions 9, 67, 72, and 77 are severely impaired in their ability to infect target cells. In addition, the serine mutant viruses are defective in their ability to fuse with target cell membranes. Interestingly, both the fusion defect and the infectivity defect can be rescued by truncation of the long cytoplasmic tail of gp41 envelope protein (gp41CT). Sucrose density gradient analysis also reveals that these mutant viruses have reduced levels of gp120 envelope protein incorporated into the virions as compared to wild type virus. Truncation of the gp41CT rescues the envelope incorporation defect. Here we propose a model in which mutation of specific serine residues prevents MA interaction with lipid rafts during HIV-1 assembly and thereby impairs recruitment of envelope to the sites of viral budding.

  4. The Envelope Cytoplasmic Tail of HIV-1 Subtype C Contributes to Poor Replication Capacity through Low Viral Infectivity and Cell-to-Cell Transmission.

    PubMed

    Santos da Silva, Eveline; Mulinge, Martin; Lemaire, Morgane; Masquelier, Cécile; Beraud, Cyprien; Rybicki, Arkadiusz; Servais, Jean-Yves; Iserentant, Gilles; Schmit, Jean-Claude; Seguin-Devaux, Carole; Perez Bercoff, Danielle

    2016-01-01

    The cytoplasmic tail (gp41CT) of the HIV-1 envelope (Env) mediates Env incorporation into virions and regulates Env intracellular trafficking. Little is known about the functional impact of variability in this domain. To address this issue, we compared the replication of recombinant virus pairs carrying the full Env (Env viruses) or the Env ectodomain fused to the gp41CT of NL4.3 (EnvEC viruses) (12 subtype C and 10 subtype B pairs) in primary CD4+ T-cells and monocyte-derived-macrophages (MDMs). In CD4+ T-cells, replication was as follows: B-EnvEC = B-Env>C-EnvEC>C-Env, indicating that the gp41CT of subtype C contributes to the low replicative capacity of this subtype. In MDMs, in contrast, replication capacity was comparable for all viruses regardless of subtype and of gp41CT. In CD4+ T-cells, viral entry, viral release and viral gene expression were similar. However, infectivity of free virions and cell-to-cell transmission of C-Env viruses released by CD4+ T-cells was lower, suggestive of lower Env incorporation into virions. Subtype C matrix only minimally rescued viral replication and failed to restore infectivity of free viruses and cell-to-cell transmission. Taken together, these results show that polymorphisms in the gp41CT contribute to viral replication capacity and suggest that the number of Env spikes per virion may vary across subtypes. These findings should be taken into consideration in the design of vaccines. PMID:27598717

  5. Critical Role of the Fusion Protein Cytoplasmic Tail Sequence in Parainfluenza Virus Assembly

    PubMed Central

    Stone, Raychel; Takimoto, Toru

    2013-01-01

    Interactions between viral glycoproteins, matrix protein and nucleocapsid sustain assembly of parainfluenza viruses at the plasma membrane. Although the protein interactions required for virion formation are considered to be highly specific, virions lacking envelope glycoprotein(s) can be produced, thus the molecular interactions driving viral assembly and production are still unclear. Sendai virus (SeV) and human parainfluenza virus type 1 (hPIV1) are highly similar in structure, however, the cytoplasmic tail sequences of the envelope glycoproteins (HN and F) are relatively less conserved. To unveil the specific role of the envelope glycoproteins in viral assembly, we created chimeric SeVs whose HN (rSeVhHN) or HN and F (rSeVh(HN+F)) were replaced with those of hPIV1. rSeVhHN grew as efficiently as wt SeV or hPIV1, suggesting that the sequence difference in HN does not have a significant impact on SeV replication and virion production. In sharp contrast, the growth of rSeVh(HN+F) was significantly impaired compared to rSeVhHN. rSeVh(HN+Fstail) which expresses a chimeric hPIV1 F with the SeV cytoplasmic tail sequence grew similar to wt SeV or rSeVhHN. Further analysis indicated that the F cytoplasmic tail plays a critical role in cell surface expression/accumulation of HN and F, as well as NP and M association at the plasma membrane. Trafficking of nucelocapsids in infected cells was not significantly affected by the origin of F, suggesting that F cytoplasmic tail is not involved in intracellular movement. These results demonstrate the role of the F cytoplasmic tail in accumulation of structural components at the plasma membrane assembly sites. PMID:23593451

  6. Cytoplasmic tail length influences fatty acid selection for acylation of viral glycoproteins.

    PubMed Central

    Veit, M; Reverey, H; Schmidt, M F

    1996-01-01

    We report remarkable differences in the fatty acid content of thioester-type acylated glycoproteins of enveloped viruses from mammalian cells. The E2 glycoprotein of Semliki Forest virus contains mainly palmitic acid like most other palmitoylated proteins analysed so far. However, the other glycoprotein (E1) of the same virus, as well as the HEF (haemagglutinin esterase fusion) glycoprotein of influenza C virus, are unique in this respect because they are acylated primarily with stearic acid. Comparative radiolabelling of uninfected cells with different fatty acids suggests that stearate may also be the prevailing fatty acid in some cellular acylproteins. To look for further differences between palmitoylated and stearoylated glycoproteins we characterized stearoylation in more detail. We identified the acylation site of HEF as a cysteine residue located at the boundary between the transmembrane region and the cytoplasmic tail. The attachment of stearate to HEF and E1 occurs post-translationally in a pre-Golgi compartment. Thus, stearoylated and palmitoylated proteins cannot be discriminated on the basis of the fatty acid linkage site or the intracellular compartment, where acylation occurs. However, stearoylated acylproteins contain a very short, positively charged cytoplasmic tail, whereas in palmitoylated proteins this molecular region is longer. Replacing the short cytoplasmic tail of stearoylated HEF with the long influenza A virus haemagglutinin (HA) tail in an HEF-HA chimera, and subsequent vaccinia T7 expression in CV-1 cells, yielded proteins with largely palmitic acid bound. The reverse chimera, HA-HEF with a short cytoplasmic tail was not fatty acylated at all during expression, indicating that conformational or topological constraints control fatty acid transfer. PMID:8761467

  7. Polybasic KKR Motif in the Cytoplasmic Tail of Nipah Virus Fusion Protein Modulates Membrane Fusion by Inside-Out Signaling▿

    PubMed Central

    Aguilar, Hector C.; Matreyek, Kenneth A.; Choi, Daniel Y.; Filone, Claire Marie; Young, Sophia; Lee, Benhur

    2007-01-01

    The cytoplasmic tails of the envelope proteins from multiple viruses are known to contain determinants that affect their fusogenic capacities. Here we report that specific residues in the cytoplasmic tail of the Nipah virus fusion protein (NiV-F) modulate its fusogenic activity. Truncation of the cytoplasmic tail of NiV-F greatly inhibited cell-cell fusion. Deletion and alanine scan analysis identified a tribasic KKR motif in the membrane-adjacent region as important for modulating cell-cell fusion. The K1A mutation increased fusion 5.5-fold, while the K2A and R3A mutations decreased fusion 3- to 5-fold. These results were corroborated in a reverse-pseudotyped viral entry assay, where receptor-pseudotyped reporter virus was used to infect cells expressing wild-type or mutant NiV envelope glycoproteins. Differential monoclonal antibody binding data indicated that hyper- or hypofusogenic mutations in the KKR motif affected the ectodomain conformation of NiV-F, which in turn resulted in faster or slower six-helix bundle formation, respectively. However, we also present evidence that the hypofusogenic phenotypes of the K2A and R3A mutants were effected via distinct mechanisms. Interestingly, the K2A mutant was also markedly excluded from lipid rafts, where ∼20% of wild-type F and the other mutants can be found. Finally, we found a strong negative correlation between the relative fusogenic capacities of these cytoplasmic-tail mutants and the avidities of NiV-F and NiV-G interactions (P = 0.007, r2 = 0.82). In toto, our data suggest that inside-out signaling by specific residues in the cytoplasmic tail of NiV-F can modulate its fusogenicity by multiple distinct mechanisms. PMID:17301148

  8. α integrin cytoplasmic tails have tissue-specific roles during C. elegans development

    PubMed Central

    MEIGHAN, CHRISTOPHER M.; SCHWARZBAUER, JEAN E.

    2015-01-01

    Integrin signaling impacts many developmental processes. The complexity of these signals increases when multiple, unique integrin heterodimers are expressed during a single developmental event. Since integrin heterodimers have different signaling capabilities, the signals originating at each integrin type must be separated in the cell. C. elegans have two integrin heterodimers, α INA-1/β PAT-3 and α PAT-2/β PAT-3, which are expressed individually or simultaneously, based on tissue type. We used chimeric α integrins to assess the role of α integrin cytoplasmic tails during development. Chimeric integrin ina-1 with the pat-2 cytoplasmic tail rescued lethality and maintained neuron fasciculation in an ina-1 mutant. Interestingly, the pat-2 tail was unable to completely restore distal tip cell migration and vulva morphogenesis. Chimeric integrin pat-2 with the ina-1 cytoplasmic tail had a limited ability to rescue a lethal mutation in pat-2, with survivors showing aberrant muscle organization, yet normal distal tip cell migration. In a wild type background, α integrin pat-2 with the ina-1 cytoplasmic tail had a dominant negative effect which induced muscle disorganization, cell migration defects and lethality. These results show the α integrin cytoplasmic tails impact unique cellular behaviors that vary by tissue type during development. PMID:25354452

  9. Arsenate arrests flagellar rotation in cytoplasm-free envelopes of bacteria.

    PubMed Central

    Margolin, Y; Barak, R; Eisenbach, M

    1994-01-01

    The effect of arsenate on flagellar rotation in cytoplasm-free flagellated envelopes of Escherichia coli and Salmonella typhimurium was investigated. Flagellar rotation ceased as soon as the envelopes were exposed to arsenate. Inclusion of phosphate intracellularly (but not extracellular) prevented the inhibition by arsenate. In a parallel experiment, the rotation was not affected by inclusion of an ATP trap (hexokinase and glucose) within the envelopes. It is concluded that arsenate affects the motor in a way other than reversible deenergization. This may be an irreversible damage to the cell or direct inhibition of the motor by arsenate. The latter possibility suggests that a process of phosphorylation or phosphate binding is involved in the motor function. PMID:8071237

  10. A structural basis for integrin activation by the cytoplasmic tail of the αIIb-subunit

    PubMed Central

    Vinogradova, Olga; Haas, Tom; Plow, Edward F.; Qin, Jun

    2000-01-01

    A key step in the activation of heterodimeric integrin adhesion receptors is the transmission of an agonist-induced cellular signal from the short α- and/or β-cytoplasmic tails to the extracellular domains of the receptor. The structural details of how the cytoplasmic tails mediate such an inside-out signaling process remain unclear. We report herein the NMR structures of a membrane-anchored cytoplasmic tail of the αIIb-subunit and of a mutant αIIb-cytoplasmic tail that renders platelet integrin αIIbβ3 constitutively active. The structure of the wild-type αIIb-cytoplasmic tail reveals a “closed” conformation where the highly conserved N-terminal membrane-proximal region forms an α-helix followed by a turn, and the acidic C-terminal loop interacts with the N-terminal helix. The structure of the active mutant is significantly different, having an “open” conformation where the interactions between the N-terminal helix and C-terminal region are abolished. Consistent with these structural differences, the two peptides differ in function: the wild-type peptide suppressed αIIbβ3 activation, whereas the mutant peptide did not. These results provide an atomic explanation for extensive biochemical/mutational data and support a conformation-based “on/off switch” model for integrin activation. PMID:10677482

  11. Alanine substitution of conserved residues in the cytoplasmic tail of herpes simplex virus gB can enhance or abolish cell fusion activity and viral entry

    SciTech Connect

    Ruel, Nancy . E-mail: n-ruel@northwestern.edu; Zago, Anna . E-mail: anna_zago@acgtinc.com; Spear, Patricia G. . E-mail: p-spear@northwestern.edu

    2006-03-01

    Herpes simplex virus (HSV) glycoprotein B (gB) is one of the four viral glycoproteins required for viral entry and cell fusion and is highly conserved among herpesviruses. Mutants of HSV type 2 gB were generated by substituting conserved residues in the cytoplasmic tail with alanine or by deleting 41 amino acids from the C-terminus. Some of the mutations abolished cell fusion activity and also prevented transport of gB to the cell surface, identifying residues in the gB cytoplasmic tail that are critical for intracellular transport of this glycoprotein. These mutations also prevented production of infectious virus, possibly because the mutant forms of gB were not transported to the site of envelopment. Other mutations, particularly the deletion, significantly enhanced cell fusion activity. These mutations, as well as others described previously, identify regions of the gB cytoplasmic domain that modulate cell fusion activity.

  12. Cytoplasmic tail domain of glycoprotein B is essential for HHV-6 infection.

    PubMed

    Mahmoud, Nora F; Jasirwan, Chyntia; Kanemoto, Satoshi; Wakata, Aika; Wang, Bochao; Hata, Yuuki; Nagamata, Satoshi; Kawabata, Akiko; Tang, Huamin; Mori, Yasuko

    2016-03-01

    Human herpesvirus 6 (HHV-6) glycoprotein B (gB) is an abundantly expressed viral glycoprotein required for viral entry and cell fusion, and is highly conserved among herpesviruses. The present study examined the function of HHV-6 gB cytoplasmic tail domain (CTD). A gB CTD deletion mutant was constructed which, in contrast to its revertant, could not be reconstituted. Moreover, deletion of gB cytoplasmic tail impaired the intracellular transport of gB protein to the trans-Golgi network (TGN). Taken together, these results suggest that gB CTD is critical for HHV-6 propagation and important for intracellular transportation. PMID:26802210

  13. Distinct roles of NR2A and NR2B cytoplasmic tails in long term potentiation

    PubMed Central

    Foster, Kelly A.; McLaughlin, Nathan; Edbauer, Dieter; Phillips, Marnie; Bolton, Andrew; Constantine-Paton, Martha; Sheng, Morgan

    2010-01-01

    NMDA receptors (NMDARs) are critical mediators of activity-dependent synaptic plasticity, but the differential roles of NR2A- versus NR2B-containing NMDARs have been controversial. Here, we investigate the roles of NR2A and NR2B in LTP in organotypic hippocampal slice cultures using RNAi and overexpression, to complement pharmacological approaches. In young slices, when NR2B is the predominant subunit expressed, LTP is blocked by the NR2B-selective antagonist Ro25-6981. As slices mature, and NR2A expression rises, activation of NR2B receptors became no longer necessary for LTP induction. LTP was blocked, however, by RNAi knockdown of NR2B, and this was rescued by coexpression of an RNAi-resistant NR2B (NR2B*) cDNA. Interestingly, a chimeric NR2B subunit in which the C-terminal cytoplasmic tail was replaced by that of NR2A failed to rescue LTP while the reverse chimera, NR2A channel with NR2B tail, was able to restore LTP. Thus expression of NR2B with its intact cytoplasmic tail is required for LTP induction, at an age when channel activity of NR2B-NMDARs is not required for LTP. Overexpression of wildtype NR2A failed to rescue LTP in neurons transfected with NR2B-RNAi construct, despite restoring NMDA-EPSC amplitude to a similar level as NR2B*. Surprisingly, an NR2A construct lacking its entire C-terminal cytoplasmic tail regained its ability to restore LTP. Together these data suggest that the NR2B subunit plays a critical role for LTP, presumably by recruiting relevant molecules important for LTP via its cytoplasmic tail. By contrast, NR2A is not essential for LTP and its cytoplasmic tail seems to carry inhibitory factors for LTP. PMID:20164351

  14. Case 35: an unusual haematological neoplasm characterized by cells with cytoplasmic tails.

    PubMed

    Kassam, Shireen; Rice, Alexandra; Morilla, Ricardo; Bain, Barbara J

    2007-06-01

    A middle aged Northern European man presented with a submental lymph node and was found on imaging to have generalized lymphadenopathy. There was bone marrow infiltration by small apparently lymphoid cells, many with cytoplasmic tails; these cells were CD56(+) and weakly CD4(+). A provisional diagnosis was confirmed by further immunophenotyping. PMID:17577785

  15. Importance of the short cytoplasmic domain of the feline immunodeficiency virus transmembrane glycoprotein for fusion activity and envelope glycoprotein incorporation into virions

    SciTech Connect

    Celma, Cristina C.P.; Paladino, Monica G.; Gonzalez, Silvia A.; Affranchino, Jose L.

    2007-09-30

    The mature form of the envelope (Env) glycoprotein of lentiviruses is a heterodimer composed of the surface (SU) and transmembrane (TM) subunits. Feline immunodeficiency virus (FIV) possesses a TM glycoprotein with a cytoplasmic tail of approximately 53 amino acids which is unusually short compared with that of the other lentiviral glycoproteins (more than 100 residues). To investigate the relevance of the FIV TM cytoplasmic domain to Env-mediated viral functions, we characterized the biological properties of a series of Env glycoproteins progressively shortened from the carboxyl terminus. All the mutant Env proteins were efficiently expressed in feline cells and processed into the SU and TM subunits. Deletion of 5 or 11 amino acids from the TM C-terminus did not significantly affect Env surface expression, fusogenic activity or Env incorporation into virions, whereas removal of 17 or 23 residues impaired Env-mediated cell-to-cell fusion. Further truncation of the FIV TM by 29 residues resulted in an Env glycoprotein that was poorly expressed at the cell surface, exhibited only 20% of the wild-type Env fusogenic capacity and was inefficiently incorporated into virions. Remarkably, deletion of the TM C-terminal 35 or 41 amino acids restored or even enhanced Env biological functions. Indeed, these mutant Env glycoproteins bearing cytoplasmic domains of 18 or 12 amino acids were found to be significantly more fusogenic than the wild-type Env and were efficiently incorporated into virions. Interestingly, truncation of the TM cytoplasmic domain to only 6 amino acids did not affect Env incorporation into virions but abrogated Env fusogenicity. Finally, removal of the entire TM cytoplasmic tail or deletion of as many as 6 amino acids into the membrane-spanning domain led to a complete loss of Env functions. Our results demonstrate that despite its relatively short length, the FIV TM cytoplasmic domain plays an important role in modulating Env-mediated viral functions.

  16. Mutants of the Rous sarcoma virus envelope glycoprotein that lack the transmembrane anchor and cytoplasmic domains: analysis of intracellular transport and assembly into virions.

    PubMed Central

    Perez, L G; Davis, G L; Hunter, E

    1987-01-01

    -infected cells contained normal levels of glycoprotein. The cytoplasmic tail of gp37 is thus not required for the assembly of envelope glycoproteins into virions. It is unlikely, therefore, that this region of gp37 interacts with viral core proteins during the selective incorporation of viral glycoproteins into the viral envelope. Images PMID:3041017

  17. A single amino acid change in the cytoplasmic domain of the simian immunodeficiency virus transmembrane molecule increases envelope glycoprotein expression on infected cells.

    PubMed Central

    LaBranche, C C; Sauter, M M; Haggarty, B S; Vance, P J; Romano, J; Hart, T K; Bugelski, P J; Marsh, M; Hoxie, J A

    1995-01-01

    We have described a virus termed CP-MAC, derived from the BK28 molecular clone of simian immunodeficiency virus, that was remarkable for its ability to infect Sup-T1 cells with rapid kinetics, cell fusion, and CD4 down-modulation (C. C. LaBranche, M. M. Sauter, B. S. Haggarty, P. J. Vance, J. Romano, T. K. Hart, P. J. Bugelski, and J. A. Hoxie, J. Virol. 68:5509-5522, 1994 [Erratum 68:7665-7667]). Compared with BK28, CP-MAC exhibited a number of changes in its envelope glycoproteins, including a highly stable association between the external (SU) and transmembrane (TM) molecules, a more rapid electrophoretic mobility of TM, and, of particular interest, a marked increase in the level of envelope protein expression on the surface of infected cells. These changes were shown to be associated with 11 coding mutations in the env gene (5 in SU and 6 in TM). In this report, we demonstrate that a single amino acid mutation of a Tyr to a Cys at position 723 (Y723C) in the TM cytoplasmic domain of CP-MAC is the principal determinant for the increased expression of envelope glycoproteins on the cell surface. When introduced into the env gene of BK28, the Y723C mutation produced up to a 25-fold increase in the levels of SU and TM on chronically infected cells, as determined by fluorescence-activated cell sorter analysis with monoclonal and polyclonal antibodies. A similar effect was observed when a Tyr-to-Cys change was introduced at the analogous position (amino acid 721) in the SIVmac239 molecular clone, which, unlike BK28 does not contain a premature stop codon in its TM cytoplasmic tail. Substituting other amino acids, including Ala, Ile, and Ser, at this position produced increases in surface envelope glycoproteins that were similar to that observed for the Cys substitution, while a Tyr-to-Phe mutation produced a smaller increase. These results could not be accounted for by differences in the kinetics or efficiency of envelope glycoprotein processing or by shedding of SU

  18. Cytoplasmic Tail Regulates the Intercellular Adhesion Function of the Epithelial Cell Adhesion Molecule

    PubMed Central

    Balzar, Maarten; Bakker, Hellen A. M.; Briaire-de-Bruijn, Inge H.; Fleuren, Gert Jan; Warnaar, Sven O.; Litvinov, Sergey V.

    1998-01-01

    Ep-CAM, an epithelium-specific cell-cell adhesion molecule (CAM) not structurally related to the major families of CAMs, contains a cytoplasmic domain of 26 amino acids. The chemical disruption of the actin microfilaments, but not of the microtubuli or intermediate filaments, affected the localization of Ep-CAM at the cell-cell boundaries, suggesting that the molecule interacts with the actin-based cytoskeleton. Mutated forms of Ep-CAM were generated with the cytoplasmic domain truncated at various lengths. All of the mutants were transported to the cell surface in the transfectants; however, the mutant lacking the complete cytoplasmic domain was not able to localize to the cell-cell boundaries, in contrast to mutants with partial deletions. Both the disruption of the actin microfilaments and a complete truncation of the cytoplasmic tail strongly affected the ability of Ep-CAM to mediate aggregation of L cells. The capability of direct aggregation was reduced for the partially truncated mutants but remained cytochalasin D sensitive. The tail truncation did not affect the ability of the transfectants to adhere to solid-phase-adsorbed Ep-CAM, suggesting that the ability to form stable adhesions and not the ligand specificity of the molecule was affected by the truncation. The formation of intercellular adhesions mediated by Ep-CAM induced a redistribution to the cell-cell boundaries of α-actinin, but not of vinculin, talin, filamin, spectrin, or catenins. Coprecipitation demonstrated direct association of Ep-CAM with α-actinin. Binding of α-actinin to purified mutated and wild-type Ep-CAMs and to peptides representing different domains of the cytoplasmic tail of Ep-CAM demonstrates two binding sites for α-actinin at positions 289 to 296 and 304 to 314 of the amino acid sequence. The results demonstrate that the cytoplasmic domain of Ep-CAM regulates the adhesion function of the molecule through interaction with the actin cytoskeleton via α-actinin. PMID:9671492

  19. Cytoplasmic tail regulates the intercellular adhesion function of the epithelial cell adhesion molecule.

    PubMed

    Balzar, M; Bakker, H A; Briaire-de-Bruijn, I H; Fleuren, G J; Warnaar, S O; Litvinov, S V

    1998-08-01

    Ep-CAM, an epithelium-specific cell-cell adhesion molecule (CAM) not structurally related to the major families of CAMs, contains a cytoplasmic domain of 26 amino acids. The chemical disruption of the actin microfilaments, but not of the microtubuli or intermediate filaments, affected the localization of Ep-CAM at the cell-cell boundaries, suggesting that the molecule interacts with the actin-based cytoskeleton. Mutated forms of Ep-CAM were generated with the cytoplasmic domain truncated at various lengths. All of the mutants were transported to the cell surface in the transfectants; however, the mutant lacking the complete cytoplasmic domain was not able to localize to the cell-cell boundaries, in contrast to mutants with partial deletions. Both the disruption of the actin microfilaments and a complete truncation of the cytoplasmic tail strongly affected the ability of Ep-CAM to mediate aggregation of L cells. The capability of direct aggregation was reduced for the partially truncated mutants but remained cytochalasin D sensitive. The tail truncation did not affect the ability of the transfectants to adhere to solid-phase-adsorbed Ep-CAM, suggesting that the ability to form stable adhesions and not the ligand specificity of the molecule was affected by the truncation. The formation of intercellular adhesions mediated by Ep-CAM induced a redistribution to the cell-cell boundaries of alpha-actinin, but not of vinculin, talin, filamin, spectrin, or catenins. Coprecipitation demonstrated direct association of Ep-CAM with alpha-actinin. Binding of alpha-actinin to purified mutated and wild-type Ep-CAMs and to peptides representing different domains of the cytoplasmic tail of Ep-CAM demonstrates two binding sites for alpha-actinin at positions 289 to 296 and 304 to 314 of the amino acid sequence. The results demonstrate that the cytoplasmic domain of Ep-CAM regulates the adhesion function of the molecule through interaction with the actin cytoskeleton via alpha

  20. Modulation of cell surface transport and lipid raft localization by the cytoplasmic tail of the influenza virus hemagglutinin.

    PubMed

    Scolari, Silvia; Imkeller, Katharina; Jolmes, Fabian; Veit, Michael; Herrmann, Andreas; Schwarzer, Roland

    2016-01-01

    Viral glycoproteins are highly variable in their primary structure, but on the other hand feature a high functional conservation to fulfil their versatile tasks during the pathogenic life cycle. Typically, all protein domains are optimized in that indispensable functions can be assigned to small conserved motifs or even individual amino acids. The cytoplasmic tail of many viral spike proteins, although of particular relevance for the virus biology, is often only insufficiently characterized. Hemagglutinin (HA), the receptor-binding protein of the influenza virus comprises a short cytoplasmic tail of 13 amino acids that exhibits three highly conserved palmitoylation sites. However, the particular importance of these modifications and the tail in general for intracellular trafficking and lateral membrane organization remains elusive. In this study, we generated HA core proteins consisting of transmembrane domain, cytoplasmic tail and a minor part of the ectodomain, tagged with a yellow fluorescent protein. Different mutation and truncation variants of these chimeric proteins were investigated using confocal microscopy, to characterize the role of cytoplasmic tail and palmitoylation for the intracellular trafficking to plasma membrane and Golgi apparatus. In addition, we assessed raft partitioning of the variants by Foerster resonance energy transfer with an established raft marker. We revealed a substantial influence of the cytoplasmic tail length on the intracellular distribution and surface exposure of the proteins. A complete removal of the tail hampers a physiological trafficking of the protein, whereas a partial truncation can be compensated by cytoplasmic palmitoylations. Plasma membrane raft partitioning on the other hand was found to imperatively require palmitoylations, and the cysteine at position 551 turned out to be of most relevance. Our data shed further light on the tight interconnection between cytoplasmic elements and intracellular trafficking and

  1. Membrane structure correlates to function of LLP2 on the cytoplasmic tail of HIV-1 gp41 protein.

    PubMed

    Boscia, Alexander L; Akabori, Kiyotaka; Benamram, Zachary; Michel, Jonathan A; Jablin, Michael S; Steckbeck, Jonathan D; Montelaro, Ronald C; Nagle, John F; Tristram-Nagle, Stephanie

    2013-08-01

    Mutation studies previously showed that the lentivirus lytic peptide (LLP2) sequence of the cytoplasmic C-terminal tail of the HIV-1 gp41 envelope protein inhibited viral-initiated T-cell death and T-cell syncytium formation, at which time in the HIV life cycle the gp41 protein is embedded in the T-cell membrane. In striking contrast, the mutants did not affect virion infectivity, during which time the gp41 protein is embedded in the HIV envelope membrane. To examine the role of LLP2/membrane interactions, we applied synchrotron x-radiation to determine structure of hydrated membranes. We focused on WT LLP2 peptide (+3 charge) and MX2 mutant (-1 charge) with membrane mimics for the T-cell and the HIV-1 membranes. To investigate the influence of electrostatics, cholesterol content, and peptide palmitoylation, we also studied three other LLP2 variants and HIV-1 mimics without negatively charged lipids or cholesterol as well as extracted HIV-1 lipids. All LLP2 peptides bound strongly to T-cell membrane mimics, as indicated by changes in membrane structure and bending. In contrast, none of the weakly bound LLP2 variants changed the HIV-1 membrane mimic structure or properties. This correlates well with, and provides a biophysical basis for, previously published results that reported lack of a mutant effect in HIV virion infectivity in contrast to an inhibitory effect in T-cell syncytium formation. It shows that interaction of LLP2 with the T-cell membrane modulates biological function. PMID:23931314

  2. The cleaved cytoplasmic tail of polycystin-1 regulates Src-dependent STAT3 activation.

    PubMed

    Talbot, Jeffrey J; Song, Xuewen; Wang, Xiaofang; Rinschen, Markus M; Doerr, Nicholas; LaRiviere, Wells B; Schermer, Bernhard; Pei, York P; Torres, Vicente E; Weimbs, Thomas

    2014-08-01

    Polycystin-1 (PC1) mutations result in proliferative renal cyst growth and progression to renal failure in autosomal dominant polycystic kidney disease (ADPKD). The transcription factor STAT3 (signal transducer and activator of transcription 3) was shown to be activated in cyst-lining cells in ADPKD and PKD mouse models and may drive renal cyst growth, but the mechanisms leading to persistent STAT3 activation are unknown. A proteolytic fragment of PC1 corresponding to the cytoplasmic tail, PC1-p30, is overexpressed in ADPKD. Here, we show that PC1-p30 interacts with the nonreceptor tyrosine kinase Src, resulting in Src-dependent activation of STAT3 by tyrosine phosphorylation. The PC1-p30-mediated activation of Src/STAT3 was independent of JAK family kinases and insensitive to the STAT3 inhibitor suppressor of cytokine signaling 3. Signaling by the EGF receptor (EGFR) or cAMP amplified the activation of Src/STAT3 by PC1-p30. Expression of PC1-p30 changed the cellular response to cAMP signaling. In the absence of PC1-p30, cAMP dampened EGFR- or IL-6-dependent activation of STAT3; in the presence of PC1-p30, cAMP amplified Src-dependent activation of STAT3. In the polycystic kidney (PCK) rat model, activation of STAT3 in renal cystic cells depended on vasopressin receptor 2 (V2R) signaling, which increased cAMP levels. Genetic inhibition of vasopressin expression or treatment with a pharmacologic V2R inhibitor strongly suppressed STAT3 activation and reduced renal cyst growth. These results suggest that PC1, via its cleaved cytoplasmic tail, integrates signaling inputs from EGFR and cAMP, resulting in Src-dependent activation of STAT3 and a proliferative response. PMID:24578126

  3. Cytoplasmic tail of IL-13Ralpha2 regulates IL-4 signal transduction.

    PubMed

    Andrews, Allison-Lynn; Nordgren, Ida Karin; Kirby, Isabelle; Holloway, John W; Holgate, Stephen T; Davies, Donna E; Tavassoli, Ali

    2009-08-01

    IL (interleukin)-4 and IL-13 are key cytokines in the pathogenesis of allergic inflammatory disease. IL-4 and IL-13 share many functional properties as a result of their utilization of a common receptor complex comprising IL-13Ralpha1 (IL-13 receptor alpha-chain 1) and IL-4Ralpha. The second IL-13R (IL-13 receptor) has been identified, namely IL-13Ralpha2. This has been thought to be a decoy receptor due to its short cytoplasmic tail and its high binding affinity for IL-13 but not IL-4. IL-13Ralpha2 exists on the cell membrane, intracellularly and in a soluble form. Recent reports revealed that membrane IL-13Ralpha2 may have some signalling capabilities, and a soluble form of IL-13Ralpha2 can be generated in the presence of environmental allergens such as DerP. Interestingly, IL-13Ralpha2 has also been shown to regulate both IL-13 and IL-4 response in primary airway cells, despite the fact that IL-13Ralpha2 does not bind IL-4. The regulator mechanism is still unclear but the physical association of IL-13Ralpha2 with IL-4Ralpha appears to be a key regulatory step. These results suggest that the cytoplasmic tail of IL-13Ralpha2 may interfere with the association or activation of signalling molecules, such as JAK1 (Janus kinase 1), on IL-4Ralpha and thus prevents downstream signal cascade. The receptor has more complicated functions than a simple decoy receptor. In this review, we discuss newly revealed functions of IL-13Ralpha2. PMID:19614610

  4. Skeletal Phenotype of Transgenic Mice Expressing the Beta1 Integrin Cytoplasmic Tail In Osteoblasts

    NASA Technical Reports Server (NTRS)

    Globus, R. K.; vanderMeulen, M. C. H.; Damsky, D.; Kim, J.-B.; Amblard, D.; Amblard, D.; Nishimura, Y.; Almeida, E.; Iwaniec, U. T.; Wronski, T. J.; Dalton, Bonnie (Technical Monitor)

    2002-01-01

    To define the physiologic role of beta1 integrin in bone formation and mechanical loading, transgenic mice were generated by expressing the cytoplasmic tall and transmembrane domain of Beta1 integrin under the control of the osteocalcin promoter. In cultured cells, this truncated fragment of Beta1 can act as a dominant negative. Previously, the matrix of calvariae was shown to be abnormal in transgenic (TG) compared to wildtype (WT) mice. In this study, we analyzed appendicular bone in TG and WT, male and female mice at 14, 35, 63, 90 and 365 days old (n=8-12/gp). To assess beta1 integrin function in mechanical loading, a pilot study using hindlimb unloading by tail suspension was performed. 35d old TG and WT females were hindlimb unloaded for 4 wks (n=3-5). Body mass, bone mineral content, histomorphometric (distal femur) and biomechanical parameters were analyzed. Statistical significance (P less than.05) was defined by ANOVA using the Tukey-Kramer post-hoc test. We confirmed transgene expression by immunoprecipitating then immunoblotting bone lysates using an antibody against the beta1 tail. Body masses of TG mice at 63, 90 and 365d old were greater (16-25%) than WT. Some TG female mice at 365d appeared obese; mean abdominal fat mass was 415% greater in TG than WT mice. Tibiae were longer (5-7%) in TG than WT mice at 63 and 90d. Tibial mineral mass of 35d males was 7% lower in TG than WT mice, but at 63d was 21% higher. The % osteoblast surface in 35d TG mice was 20% higher than WT, and at 63d was 17% lower, while % osteoclast surface did not differ. In 365d mice, cancellous bone volume (125%) and endocortical mineral apposition rate (40%) were greater in TG than WT males but not females. In WT mice, hindlimb unloading caused a reduction in mineral mass of tibiae (-20%) and lumbar vertebrae (-22%) relative to normally loaded controls. Surprisingly, hindlimb unloading also caused a relative reduction (-13%) in humerus mass. The effects of hindlimb unloading on

  5. Endocytosis via coated pits mediated by glycoprotein receptor in which the cytoplasmic tail is replaced by unrelated sequences.

    PubMed Central

    Verrey, F; Gilbert, T; Mellow, T; Proulx, G; Drickamer, K

    1990-01-01

    Rat 6 fibroblast cell lines expressing wild-type chicken liver glycoprotein receptor (CHL) or chimeric receptors with alternate cytoplasmic tails were produced to study the role of the cytoplasmic tail in mediating receptor localization in coated pits and endocytosis of ligand. Cells expressing CHL or cells expressing a hybrid receptor that contains the cytoplasmic tail of the asialoglycoprotein receptor display high-efficiency endocytosis of N-acetylglucosamine-conjugated bovine serum albumin in experiments designed to measure an initial internalization step, as well as in studies of continuous uptake and degradation. Substitution of the cytoplasmic tail by the equivalent domain of rat Na,K-ATPase beta subunit or by a stretch of Xenopus laevis globin beta chain does not abolish endocytosis but decreases the endocytosis rate constant from 15%-16%/min to 2.4% and 6.5%/min, respectively. Electron microscopy was used to visualize the glycoprotein binding sites at the surface of Rat 6 cells transfected with the various receptors. The percentage of receptors found in coated areas ranged from 32% for CHL to 9% for the Na,K-ATPase hybrid, indicating that clustering in coated pits correlates with efficiency of endocytosis. We concluded that replacement of the CHL cytoplasmic tail with unrelated sequences does not prevent, but decreases to varying extents, coated-pit localization and endocytosis efficiency. The construct with NH2-terminal globin tail lacks a signal for high-efficiency localization in coated pits but nevertheless is directed to the pits by an alternative mechanism. Images PMID:1963794

  6. Neutralization resistance of virological synapse-mediated HIV-1 Infection is regulated by the gp41 cytoplasmic tail.

    PubMed

    Durham, Natasha D; Yewdall, Alice W; Chen, Ping; Lee, Rebecca; Zony, Chati; Robinson, James E; Chen, Benjamin K

    2012-07-01

    Human immunodeficiency virus type 1 (HIV-1) infection can spread efficiently from infected to uninfected T cells through adhesive contacts called virological synapses (VSs). In this process, cell-surface envelope glycoprotein (Env) initiates adhesion and viral transfer into an uninfected recipient cell. Previous studies have found some HIV-1-neutralizing patient sera to be less effective at blocking VS-mediated infection than infection with cell-free virus. Here we employ sensitive flow cytometry-based infection assays to measure the inhibitory potency of HIV-1-neutralizing monoclonal antibodies (MAb) and HIV-1-neutralizing patient sera against cell-free and VS-mediated infection. To various degrees, anti-Env MAbs exhibited significantly higher 50% inhibitory concentration (IC(50)s) against VS-mediated infection than cell-free infection. Notably, the MAb 17b, which binds a CD4-induced (CD4i) epitope on gp120, displayed a 72-fold reduced efficacy against VS-mediated inocula compared to cell-free inocula. A mutant with truncation mutation in the gp41 cytoplasmic tail (CT) which is unable to modulate Env fusogenicity in response to virus particle maturation but which can still engage in cell-to-cell infection was tested for the ability to resist neutralizing antibodies. The ΔCT mutation increased cell surface staining by neutralizing antibodies, significantly enhanced neutralization of VS-mediated infection, and had reduced or no effect on cell-free infection, depending upon the antibody. Our results suggest that the gp41 CT regulates the exposure of key neutralizing epitopes during cell-to-cell infection and plays an important role in immune evasion. Vaccine strategies should consider immunogens that reflect Env conformations exposed on the infected cell surface to enhance protection against VS-mediated HIV-1 spread. PMID:22553332

  7. Integrin α1 Has a Long Helix, Extending from the Transmembrane Region to the Cytoplasmic Tail in Detergent Micelles

    PubMed Central

    Lai, Chaohua; Liu, Xiaoxi; Tian, Changlin; Wu, Fangming

    2013-01-01

    Integrin proteins are very important adhesion receptors that mediate cell-cell and cell-extracellular matrix interactions. They play essential roles in cell signaling and the regulation of cellular shape, motility, and the cell cycle. Here, the transmembrane and cytoplasmic (TMC) domains of integrin α1 and β1 were over-expressed and purified in detergent micelles. The structure and backbone relaxations of α1-TMC in LDAO micelles were determined and analyzed using solution NMR. A long helix, extending from the transmembrane region to the cytoplasmic tail, was observed in α1-TMC. Structural comparisons of α1-TMC with reported αIIb-TMC domains indicated different conformations in the transmembrane regions and cytoplasmic tails. An NMR titration experiment indicated weak interactions between α1-TMC and β1-TMC through several α1-TMC residues located at its N-terminal juxta-transmembrane region and C-terminal extended helix region. PMID:23646163

  8. Suppression of integrin activation by the membrane-distal sequence of the integrin alphaIIb cytoplasmic tail.

    PubMed Central

    Yamanouchi, Jun; Hato, Takaaki; Tamura, Tatsushiro; Fujita, Shigeru

    2004-01-01

    Integrin cytoplasmic tails regulate integrin activation including an increase in integrin affinity for ligands. Although there is ample evidence that the membrane-proximal regions of the alpha and beta tails interact with each other to maintain integrins in a low-affinity state, little is known about the role of the membrane-distal region of the alpha tail in regulation of integrin activation. We report a critical sequence for regulation of integrin activation in the membrane-distal region of the alphaIIb tail. Alanine substitution of the RPP residues in the alphaIIb tail rendered alphaIIbbeta3 constitutively active in a metabolic energy-dependent manner. Although an alphaIIb/alpha6Abeta3 chimaeric integrin, in which the alphaIIb tail was replaced by the alpha6A tail, was in an energy-dependent active state to bind soluble ligands, introduction of the RPP sequence into the alpha6A tail inhibited binding of an activation-dependent antibody PAC1. In alphaIIb/alpha6Abeta3, deleting the TSDA sequence from the alpha6A tail or single amino acid substitutions of the TSDA residues inhibited alphaIIb/alpha6Abeta3 activation and replacing the membrane-distal region of the alphaIIb tail with TSDA rendered alphaIIbbeta3 active, suggesting a stimulatory role of TSDA in energy-dependent integrin activation. However, adding TSDA to the alphaIIb tail containing the RPP sequence of the membrane-distal region failed to activate alphaIIbbeta3. These results suggest that the RPP sequence after the GFFKR motif of the alphaIIb tail suppresses energy-dependent alphaIIbbeta3 activation. These findings provide a molecular basis for the regulation of energy-dependent integrin activation by alpha subunit tails. PMID:14723599

  9. The Nectin-1α Transmembrane Domain, But Not The Cytoplasmic Tail, Influences Cell Fusion Induced by HSV-1 Glycoproteins

    PubMed Central

    Subramanian, Ravi P.; Dunn, Jennifer E.; Geraghty, Robert J.

    2006-01-01

    Nectin-1 is a receptor for herpes simplex virus (HSV), a member of the immunoglobulin superfamily, and a cellular adhesion molecule. To study domains of nectin-1α involved in cell fusion, we measured the ability of nectin-1α/nectin-2α chimeras, nectin-1α/CD4 chimeras, and transmembrane domain and cytoplasmic tail mutants of nectin-1α to promote cell fusion induced by HSV-1 glycoproteins. Our results demonstrate that only chimeras and mutants containing the entire V-like domain and a link to the plasma membrane conferred cell-fusion activity. The transmembrane domain and cytoplasmic tail of nectin-1 were not required for any viral receptor or cell adhesion function tested. Cellular cytoplasmic factors that bind to the nectin-1α cytoplasmic tail, therefore, did not influence virus entry or cell fusion. Interestingly, the efficiency of cell fusion was reduced when membrane spanning domains of nectin-1α and gD were replaced by glycosylphosphatidylinositol tethers, indicating that transmembrane domains may play a modulatory role in the gD/nectin-1α interaction in fusion. PMID:16005040

  10. The nectin-1{alpha} transmembrane domain, but not the cytoplasmic tail, influences cell fusion induced by HSV-1 glycoproteins

    SciTech Connect

    Subramanian, Ravi P.; Dunn, Jennifer E.; Geraghty, Robert J. . E-mail: rgeragh@uky.edu

    2005-09-01

    Nectin-1 is a receptor for herpes simplex virus (HSV), a member of the immunoglobulin superfamily, and a cellular adhesion molecule. To study domains of nectin-1{alpha} involved in cell fusion, we measured the ability of nectin-1{alpha}/nectin-2{alpha} chimeras, nectin-1{alpha}/CD4 chimeras, and transmembrane domain and cytoplasmic tail mutants of nectin-1{alpha} to promote cell fusion induced by HSV-1 glycoproteins. Our results demonstrate that only chimeras and mutants containing the entire V-like domain and a link to the plasma membrane conferred cell-fusion activity. The transmembrane domain and cytoplasmic tail of nectin-1 were not required for any viral receptor or cell adhesion function tested. Cellular cytoplasmic factors that bind to the nectin-1{alpha} cytoplasmic tail, therefore, did not influence virus entry or cell fusion. Interestingly, the efficiency of cell fusion was reduced when membrane-spanning domains of nectin-1{alpha} and gD were replaced by glycosylphosphatidylinositol tethers, indicating that transmembrane domains may play a modulatory role in the gD/nectin-1{alpha} interaction in fusion.

  11. Membrane-Mediated Regulation of the Intrinsically Disordered CD3ϵ Cytoplasmic Tail of the TCR

    PubMed Central

    López, Cesar A.; Sethi, Anurag; Goldstein, Byron; Wilson, Bridget S.; Gnanakaran, S.

    2015-01-01

    The regulation of T-cell-mediated immune responses depends on the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) on T-cell receptors. Although many details of the signaling cascades are well understood, the initial mechanism and regulation of ITAM phosphorylation remains unknown. We used molecular dynamics simulations to study the influence of different compositions of lipid bilayers on the membrane association of the CD3ϵ cytoplasmic tails of the T-cell receptors. Our results show that binding of CD3ϵ to membranes is modulated by both the presence of negatively charged lipids and the lipid order of the membrane. Free-energy calculations reveal that the protein-membrane interaction is favored by the presence of nearby basic residues and the ITAM tyrosines. Phosphorylation minimizes membrane association, rendering the ITAM motif more accessible to binding partners. In systems mimicking biological membranes, the CD3ϵ chain localization is modulated by different facilitator lipids (e.g., gangliosides or phosphoinositols), revealing a plausible regulatory effect on activation through the regulation of lipid composition in cell membranes. PMID:25992726

  12. Distinct Pathways Mediate the Sorting of Tail-Anchored Proteins to the Plastid Outer Envelope

    PubMed Central

    Dhanoa, Preetinder K.; Richardson, Lynn G. L.; Smith, Matthew D.; Gidda, Satinder K.; Henderson, Matthew P. A.; Andrews, David W.; Mullen, Robert T.

    2010-01-01

    Background Tail-anchored (TA) proteins are a distinct class of membrane proteins that are sorted post-translationally to various organelles and function in a number of important cellular processes, including redox reactions, vesicular trafficking and protein translocation. While the molecular targeting signals and pathways responsible for sorting TA proteins to their correct intracellular destinations in yeasts and mammals have begun to be characterized, relatively little is known about TA protein biogenesis in plant cells, especially for those sorted to the plastid outer envelope. Methodology/Principal Findings Here we investigated the biogenesis of three plastid TA proteins, including the 33-kDa and 34-kDa GTPases of the translocon at the outer envelope of chloroplasts (Toc33 and Toc34) and a novel 9-kDa protein of unknown function that we define here as an outer envelope TA protein (OEP9). Using a combination of in vivo and in vitro assays we show that OEP9 utilizes a different sorting pathway than that used by Toc33 and Toc34. For instance, while all three TA proteins interact with the cytosolic OEP chaperone/receptor, AKR2A, the plastid targeting information within OEP9 is distinct from that within Toc33 and Toc34. Toc33 and Toc34 also appear to differ from OEP9 in that their insertion is dependent on themselves and the unique lipid composition of the plastid outer envelope. By contrast, the insertion of OEP9 into the plastid outer envelope occurs in a proteinaceous-dependent, but Toc33/34-independent manner and membrane lipids appear to serve primarily to facilitate normal thermodynamic integration of this TA protein. Conclusions/Significance Collectively, the results provide evidence in support of at least two sorting pathways for plastid TA outer envelope proteins and shed light on not only the complex diversity of pathways involved in the targeting and insertion of proteins into plastids, but also the molecular mechanisms that underlie the delivery of TA

  13. Structures and Interaction Analyses of Integrin αMβ2 Cytoplasmic Tails*

    PubMed Central

    Chua, Geok-Lin; Tang, Xiao-Yan; Amalraj, Monalisa; Tan, Suet-Mien; Bhattacharjya, Surajit

    2011-01-01

    Integrins are heterodimeric (α and β subunits) signal transducer proteins involved in cell adhesions and migrations. The cytosolic tails of integrins are essential for transmitting bidirectional signaling and also implicated in maintaining the resting states of the receptors. In addition, cytosolic tails of integrins often undergo post-translation modifications like phosphorylation. However, the consequences of phosphorylation on the structures and interactions are not clear. The leukocyte-specific integrin αMβ2 is essential for myeloid cell adhesion, phagocytosis, and degranulation. In this work, we determined solution structures of the myristoylated cytosolic tail of αM and a Ser phosphorylated variant in dodecylphosphocholine micelles by NMR spectroscopy. Furthermore, the interactions between non-phosphorylated and phosphorylated αM tails with β2 tail were investigated by NMR and fluorescence resonance energy transfer (FRET). The three-dimensional structures of the 24-residue cytosolic tail of αM or phosphorylated αM are characterized by an N-terminal amphipathic helix and a loop at the C terminus. The residues at the loop are involved in packing interactions with the hydrophobic face of the helix. 15N-1H heteronuclear single quantum coherence experiments identified residues of αM and β2 tails that may be involved in the formation of a tail-tail heterocomplex. We further examined interactions between myristoylated β2 tail in dodecylphosphocholine micelles with dansylated αM tail peptides by FRET. These studies revealed enhanced interactions between αM or phosphorylated αM tails with β2 tail with Kd values ∼5.2 ± 0.6 and ∼4.4 ± 0.7 μm, respectively. Docked structures of tail-tail complexes delineated that the αM/β2 interface at the cytosolic region could be sustained by a network of polar interactions, ionic interactions, and/or hydrogen bonds. PMID:22052909

  14. Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of adhesion molecule CD44

    SciTech Connect

    Mori, Tomoyuki; Kitano, Ken; Terawaki, Shin-ichi; Maesaki, Ryoko; Hakoshima, Toshio

    2007-10-01

    The radixin FERM domain complexed with the CD44 cytoplasmic tail peptide has been crystallized. A diffraction data set from the complex was collected to 2.1 Å. CD44 is an important adhesion molecule that specifically binds hyaluronic acid and regulates cell–cell and cell–matrix interactions. Increasing evidence has indicated that CD44 is assembled in a regulated manner into the membrane–cytoskeletal junction, a process that is mediated by ERM (ezrin/radixin/moesin) proteins. Crystals of a complex between the radixin FERM domain and the C-terminal cytoplasmic region of CD44 have been obtained. The crystal of the radixin FERM domain bound to the CD44 cytoplasmic tail peptide belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 62.70, b = 66.18, c = 86.22 Å, and contain one complex in the crystallographic asymmetric unit. An intensity data set was collected to a resolution of 2.1 Å.

  15. The Plastid Outer Envelope – A Highly Dynamic Interface between Plastid and Cytoplasm

    PubMed Central

    Breuers, Frederique K. H.; Bräutigam, Andrea; Weber, Andreas P. M.

    2011-01-01

    Plastids are the defining organelles of all photosynthetic eukaryotes. They are the site of photosynthesis and of a large number of other essential metabolic pathways, such as fatty acid and amino acid biosyntheses, sulfur and nitrogen assimilation, and aromatic and terpenoid compound production, to mention only a few examples. The metabolism of plastids is heavily intertwined and connected with that of the surrounding cytosol, thus causing massive traffic of metabolic precursors, intermediates, and products. Two layers of biological membranes that are called the inner (IE) and the outer (OE) plastid envelope membranes bound the plastids of Archaeplastida. While the IE is generally accepted as the osmo-regulatory barrier between cytosol and stroma, the OE was considered to represent an unspecific molecular sieve, permeable for molecules of up to 10 kDa. However, after the discovery of small substrate specific pores in the OE, this view has come under scrutiny. In addition to controlling metabolic fluxes between plastid and cytosol, the OE is also crucial for protein import into the chloroplast. It contains the receptors and translocation channel of the TOC complex that is required for the canonical post-translational import of nuclear-encoded, plastid-targeted proteins. Further, the OE is a metabolically active compartment of the chloroplast, being involved in, e.g., fatty acid metabolism and membrane lipid production. Also, recent findings hint on the OE as a defense platform against several biotic and abiotic stress conditions, such as cold acclimation, freezing tolerance, and phosphate deprivation. Moreover, dynamic non-covalent interactions between the OE and the endomembrane system are thought to play important roles in lipid and non-canonical protein trafficking between plastid and endoplasmic reticulum. While proteomics and bioinformatics has provided us with comprehensive but still incomplete information on proteins localized in the plastid IE, the stroma

  16. Reevaluation of envelope profiles and cytoplasmic ultrastructure of mycobacteria processed by conventional embedding and freeze-substitution protocols.

    PubMed

    Paul, T R; Beveridge, T J

    1992-10-01

    The cell envelope architectures and cytoplasmic structures of Mycobacterium aurum CIPT 1210005, M. fortuitum, M. phlei 425, and M. thermoresistible ATCC 19527 were compared by conventional embedding and freeze-substitution methods. To ascertain the integrity of cells during each stage of the processing regimens, [1-14C]acetate was incorporated into the mycolic acids of mycobacterial walls, and the extraction of labeled mycolic acids was monitored by liquid scintillation counting. Radiolabeled mycolic acids were extracted by both processing methods; however, freeze-substitution resulted in the extraction of markedly less radiolabel. During conventional processing of cells, most of the radiolabel was extracted during the dehydration stage, whereas postsubstitution washes in acetone yielded the greatest loss of radiolabel during freeze-substitution. Conventional embedding frequently produced cells with condensed fibrous nucleoids and occasional mesosomes. Their cell walls were relatively thick (approximately 25 nm) but lacked substance. Freeze-substituted cells appeared more robust, with well-dispersed nucleoids and ribosomes. The walls of all species were much thinner than those of their conventionally processed counterparts, but these stained well, which was an indication of more wall substance; the fabric of these walls, in particular the plasma membrane, appeared highly condensed and tightly apposed to the peptidoglycan. Some species possessed a thick, irregular outer layer that was readily visualized in the absence of exogenous stabilizing agents by freeze-substitution. Since freeze-substituted mycobacteria retained a greater percentage of mycolic acids in their walls, and probably other labile wall and cytoplasmic constituents, we believe that freeze-substitution provides a more accurate image of structural organization in mycobacteria than that achieved by conventional procedures. PMID:1400203

  17. The B7-1 Cytoplasmic Tail Enhances Intracellular Transport and Mammalian Cell Surface Display of Chimeric Proteins in the Absence of a Linear ER Export Motif

    PubMed Central

    Lin, Yi-Chieh; Chen, Bing-Mae; Lu, Wei-Cheng; Su, Chien-I; Prijovich, Zeljko M.; Chung, Wen-Chuan; Wu, Pei-Yu; Chen, Kai-Chuan; Lee, I-Chiao; Juan, Ting-Yi; Roffler, Steve R.

    2013-01-01

    Membrane-tethered proteins (mammalian surface display) are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids) and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells. PMID:24073236

  18. The scaffolding protein IQGAP1 co-localizes with actin at the cytoplasmic face of the nuclear envelope: implications for cytoskeletal regulation

    PubMed Central

    Johnson, Michael A.

    2012-01-01

    IQGAP1 is an important cytoskeletal regulator, known to act at the plasma membrane to bundle and cap actin filaments, and to tether the cortical actin meshwork to microtubules via plus-end binding proteins. Here we describe the novel subcellular localization of IQGAP1 at the cytoplasmic face of the nuclear envelope, where it co-located with F-actin. The IQGAP1 and F-actin staining overlapped that of microtubules at the nuclear envelope, revealing a pattern strikingly similar to that observed at the plasma membrane. In detergent-extracted cells IQGAP1 was retained at cytoskeletal structures at the nuclear envelope. This finding has new implications for involvement of IQGAP1 in cell polarization and migration events and potentially in cell cycle-associated nuclear envelope assembly/disassembly. PMID:22964981

  19. A Novel Trafficking Signal within the HLA-C Cytoplasmic Tail Allows Regulated Expression Upon Differentiation of Macrophages

    PubMed Central

    Schaefer, Malinda R.; Williams, Maya; Kulpa, Deanna A.; Blakely, Pennelope K.; Yaffee, Anna Q.; Collins, Kathleen L.

    2008-01-01

    Major histocompatibility complex class I molecules (MHC-I) present peptides to cytotoxic T lymphocytes (CTLs). In addition, HLA-C allotypes are recognized by killer cell Ig-like receptors (KIR) found on natural killer (NK) cells and effector CTLs. Compared to other classical MHC-I allotypes, HLA-C has low cell surface expression and an altered intracellular trafficking pattern. We present evidence that this results from effects of both the extracellular domain and the cytoplasmic tail. Notably, we demonstrate that the cytoplasmic tail contains a dihydrophobic (LI) internalization and lysosomal targeting signal that is partially attenuated by an aspartic acid residue (DXSLI). In addition, we provide evidence that this signal is specifically inhibited by hypophosphorylation of the adjacent serine residue upon macrophage differentiation and that this allows high HLA-C expression in this cell type. We propose that tightly regulated HLA-C surface expression facilitates immune surveillance and allows HLA-C to serve a specialized role in macrophages. PMID:18523244

  20. Two Cytoplasmic Acylation Sites and an Adjacent Hydrophobic Residue, but No Other Conserved Amino Acids in the Cytoplasmic Tail of HA from Influenza A Virus Are Crucial for Virus Replication.

    PubMed

    Siche, Stefanie; Brett, Katharina; Möller, Lars; Kordyukova, Larisa V; Mintaev, Ramil R; Alexeevski, Andrei V; Veit, Michael

    2015-12-01

    Recruitment of the matrix protein M1 to the assembly site of the influenza virus is thought to be mediated by interactions with the cytoplasmic tail of hemagglutinin (HA). Based on a comprehensive sequence comparison of all sequences present in the database, we analyzed the effect of mutating conserved residues in the cytosol-facing part of the transmembrane region and cytoplasmic tail of HA (A/WSN/33 (H1N1) strain) on virus replication and morphology of virions. Removal of the two cytoplasmic acylation sites and substitution of a neighboring isoleucine by glutamine prevented rescue of infectious virions. In contrast, a conservative exchange of the same isoleucine, non-conservative exchanges of glycine and glutamine, deletion of the acylation site at the end of the transmembrane region and shifting it into the tail did not affect virus morphology and had only subtle effects on virus growth and on the incorporation of M1 and Ribo-Nucleoprotein Particles (RNPs). Thus, assuming that essential amino acids are conserved between HA subtypes we suggest that, besides the two cytoplasmic acylation sites (including adjacent hydrophobic residues), no other amino acids in the cytoplasmic tail of HA are indispensable for virus assembly and budding. PMID:26670246

  1. Two Cytoplasmic Acylation Sites and an Adjacent Hydrophobic Residue, but No Other Conserved Amino Acids in the Cytoplasmic Tail of HA from Influenza A Virus Are Crucial for Virus Replication

    PubMed Central

    Siche, Stefanie; Brett, Katharina; Möller, Lars; Kordyukova, Larisa V.; Mintaev, Ramil R.; Alexeevski, Andrei V.; Veit, Michael

    2015-01-01

    Recruitment of the matrix protein M1 to the assembly site of the influenza virus is thought to be mediated by interactions with the cytoplasmic tail of hemagglutinin (HA). Based on a comprehensive sequence comparison of all sequences present in the database, we analyzed the effect of mutating conserved residues in the cytosol-facing part of the transmembrane region and cytoplasmic tail of HA (A/WSN/33 (H1N1) strain) on virus replication and morphology of virions. Removal of the two cytoplasmic acylation sites and substitution of a neighboring isoleucine by glutamine prevented rescue of infectious virions. In contrast, a conservative exchange of the same isoleucine, non-conservative exchanges of glycine and glutamine, deletion of the acylation site at the end of the transmembrane region and shifting it into the tail did not affect virus morphology and had only subtle effects on virus growth and on the incorporation of M1 and Ribo-Nucleoprotein Particles (RNPs). Thus, assuming that essential amino acids are conserved between HA subtypes we suggest that, besides the two cytoplasmic acylation sites (including adjacent hydrophobic residues), no other amino acids in the cytoplasmic tail of HA are indispensable for virus assembly and budding. PMID:26670246

  2. Adaptation of Subtype A Human Immunodeficiency Virus Type 1 Envelope to Pig-Tailed Macaque Cells▿

    PubMed Central

    Humes, Daryl; Overbaugh, Julie

    2011-01-01

    The relevance of simian/human immunodeficiency virus (SHIV) infection of macaques to HIV-1 infection in humans depends on how closely SHIVs mimic HIV-1 transmission, pathogenesis, and diversity. Circulating HIV-1 strains are predominantly subtypes C and A and overwhelmingly require CCR5 for entry, yet most SHIVs incorporate CXCR4-using subtype B envelopes (Envs). While pathogenic subtype C-based SHIVs have been constructed, the subtype A-based SHIVs (SHIV-As) constructed to date have been unable to replicate in macaque cells. To understand the barriers to SHIV-A replication in macaque cells, HIVAQ23/SIVvif was constructed by engineering a CCR5-tropic subtype A provirus to express SIV vif, which counters the macaque APOBEC3G restriction. HIVAQ23/SIVvif replicated poorly in pig-tailed macaque (Ptm) lymphocytes, but viruses were adapted to Ptm lymphocytes. Two independent mutations in gp120, G312V (V3 loop) and A204E (C2 region), were identified that increased peak virus levels by >100-fold. Introduction of G312V and A204E to multiple subtype A Envs and substitution of G312 and A204 with other residues increased entry into Ptm cells by 10- to 100-fold. G312V and A204E Env variants continued to require CCR5 for entry but were up to 50- and 200-fold more sensitive to neutralization by IgG1b12 and soluble CD4 and had a 5- to 50-fold increase in their ability to utilize Ptm CD4 compared to their wild-type counterparts. These findings identify the inefficient use of Ptm CD4 as an unappreciated restriction to subtype A HIV-1 replication in Ptm cells and reveal amino acid changes to gp120 that can overcome this barrier. PMID:21325401

  3. Coupling of Human Immunodeficiency Virus Type 1 Fusion to Virion Maturation: a Novel Role of the gp41 Cytoplasmic Tail

    PubMed Central

    Wyma, Donald J.; Jiang, Jiyang; Shi, Jiong; Zhou, Jing; Lineberger, Janet E.; Miller, Michael D.; Aiken, Christopher

    2004-01-01

    Retrovirus particles are not infectious until they undergo proteolytic maturation to form a functional core. Here we report a link between human immunodeficiency virus type 1 (HIV-1) core maturation and the ability of the virus to fuse with target cells. Using a recently developed reporter assay of HIV-1 virus-cell fusion, we show that immature HIV-1 particles are 5- to 10-fold less active for fusion with target cells than are mature virions. The fusion of mature and immature virions was rendered equivalent by truncating the gp41 cytoplasmic domain or by pseudotyping viruses with the glycoprotein of vesicular stomatitis virus. An analysis of a panel of mutants containing mutated cleavage sites indicated that HIV-1 fusion competence is activated by the cleavage of Gag at any site between the MA and NC segments and not as an indirect consequence of an altered core structure. These results suggest a mechanism by which binding of the gp41 cytoplasmic tail to Gag within immature HIV-1 particles inhibits Env conformational changes on the surface of the virion that are required for membrane fusion. This “inside-out” regulation of HIV-1 fusion could play an important role in the virus life cycle by preventing the entry of immature, noninfectious particles. PMID:15016865

  4. Membrane fusion by the GTPase atlastin requires a conserved C-terminal cytoplasmic tail and dimerization through the middle domain

    PubMed Central

    Moss, Tyler J.; Andreazza, Camilla; Verma, Avani; Daga, Andrea; McNew, James A.

    2011-01-01

    The biogenesis and maintenance of the endoplasmic reticulum (ER) requires membrane fusion. ER homotypic fusion is driven by the large GTPase atlastin. Domain analysis of atlastin shows that a conserved region of the C-terminal cytoplasmic tail is absolutely required for fusion activity. Atlastin in adjacent membranes must associate to bring the ER membranes into molecular contact. Drosophila atlastin dimerizes in the presence of GTPγS but is monomeric with GDP or without nucleotide. Oligomerization requires the juxtamembrane middle domain three-helix bundle, as does efficient GTPase activity. A soluble version of the N-terminal cytoplasmic domain that contains the GTPase domain and the middle domain three-helix bundle serves as a potent, concentration-dependent inhibitor of membrane fusion both in vitro and in vivo. However, atlastin domains lacking the middle domain are without effect. GTP-dependent dimerization of atlastin generates an enzymatically active protein that drives membrane fusion after nucleotide hydrolysis and conformational reorganization. PMID:21690399

  5. Surface Exposure of the HIV-1 Env Cytoplasmic Tail LLP2 Domain during the Membrane Fusion Process

    PubMed Central

    Lu, Lu; Zhu, Yun; Huang, Jinghe; Chen, Xi; Yang, Hengwen; Jiang, Shibo; Chen, Ying-Hua

    2008-01-01

    HIV-1 gp41 cytoplasmic tail (CT) is highly conserved among HIV-1 isolates, particularly the region designated lentivirus lytic peptide (LLP1–2), which includes two α-helical domains LLP1 and LLP2. Although the gp41 CT is recognized as a modulator of viral fusogenicity, little is known about the regulatory mechanism of this region in the viral fusion process. Here we report that anti-LLP1–2 and anti-LLP2 antibodies (IgG) inhibited HIV-1 Env-mediated cell fusion and bound to the interface between effector and target cells at a suboptimal temperature (31.5 °C), which slows down the fusion process and prolongs the fusion intermediate state. This suggests that LLP1–2, especially the LLP2 region located inside the viral membrane, is transiently exposed on the membrane surface during the fusion process. Synthetic LLP2 peptide could bind to the gp41 six-helix bundle core with high binding affinity. These results suggest that the gp41 CT may interact with the gp41 core, via the surface-exposed LLP2 domain, to regulate Env-mediated membrane fusion. PMID:18408000

  6. The BRCA1-binding protein BRAP2 can act as a cytoplasmic retention factor for nuclear and nuclear envelope-localizing testicular proteins.

    PubMed

    Davies, Rebecca G; Wagstaff, Kylie M; McLaughlin, Eileen A; Loveland, Kate L; Jans, David A

    2013-12-01

    Regulation of nuclear protein import is central to many cellular processes such as development, with a key mechanism being factors that retain cargoes in the cytoplasm that normally localize in the nucleus. The breast cancer antigen BRCA1-binding protein BRAP2 has been reported as a novel negative regulator of nuclear import of various nuclear localization signal (NLS)-containing viral and cellular proteins, but although implicated in differentiation pathways and highly expressed in tissues including testis, the gamut of targets for BRAP2 action in a developmental context is unknown. As a first step towards defining the BRAP2 interactome, we performed a yeast-2-hybrid screen to identify binding partners of BRAP2 in human testis. Here we report characterization for the first time of three of these: the high mobility group (HMG)-box-domain-containing chromatin component HMG20A, nuclear mitotic apparatus protein NuMA1 and synaptic nuclear envelope protein SYNE2. Co-immunoprecipitation experiments indicate association of BRAP2 with HMG20A, NuMA1, and SYNE2 in testis, underlining the physiological relevance of the interactions, with immunohistochemistry showing that where BRAP2 is co-expressed with HMG20A and NuMA1, both are present in the cytoplasm, in contrast to their nuclear localization in other testicular cell types. Importantly, quantitative confocal microscopic analysis of cultured cells indicates that ectopic expression of BRAP2 inhibits nuclear localization of HMG20A and NuMA1, and prevents nuclear envelope accumulation of SYNE2, the first report of BRAP2 altering localization of a non-nuclear protein. These results imply for the first time that BRAP2 may have an important role in modulating subcellular localization during testicular development. PMID:23707952

  7. The Cytoplasmic Tail Domain of Influenza B Virus Hemagglutinin Is Important for Its Incorporation into Virions but Is Not Essential for Virus Replication in Cell Culture in the Presence of Compensatory Mutations

    PubMed Central

    Watanabe, Shinji

    2012-01-01

    Influenza B virus hemagglutinin (BHA) contains a predicted cytoplasmic tail of 10 amino acids that are highly conserved among influenza B viruses. To understand the role of this cytoplasmic tail in infectious virus production, we used reverse genetics to generate a recombinant influenza B virus lacking the BHA cytoplasmic tail domain. The resulting virus, designated BHATail−, had a titer approximately 5 log units lower than that of wild-type virus but grew normally when BHA was supplemented in trans by BHA-expressing cells. Although the levels of BHA cell surface expression were indistinguishable between truncated and wild-type BHA, the BHATail− virus produced particles containing dramatically less BHA. Moreover, removal of the cytoplasmic tail abrogated the association of BHA with Triton X-100-insoluble lipid rafts. Interestingly, long-term culture of a virus lacking the BHA cytoplasmic tail in Madin-Darby canine kidney (MDCK) cells yielded a mutant with infectivities somewhat similar to that of wild-type virus. Sequencing revealed that the mutant virus retained the original cytoplasmic tail deletion but acquired additional mutations in its BHA, neuraminidase (NA), and M1 proteins. Viral growth kinetic analysis showed that replication of BHA cytoplasmic tailless viruses could be improved by compensatory mutations in the NA and M1 proteins. These findings indicate that the cytoplasmic tail domain of BHA is important for efficient incorporation of BHA into virions and tight lipid raft association. They also demonstrate that the domain is not absolutely required for virus viability in cell culture in the presence of compensatory mutations. PMID:22896616

  8. Control of lipid organization and actin assembly during clathrin-mediated endocytosis by the cytoplasmic tail of the rhomboid protein Rbd2.

    PubMed

    Cortesio, Christa L; Lewellyn, Eric B; Drubin, David G

    2015-04-15

    Clathrin-mediated endocytosis (CME) is facilitated by a precisely regulated burst of actin assembly. PtdIns(4,5)P2 is an important signaling lipid with conserved roles in CME and actin assembly regulation. Rhomboid family multipass transmembrane proteins regulate diverse cellular processes; however, rhomboid-mediated CME regulation has not been described. We report that yeast lacking the rhomboid protein Rbd2 exhibit accelerated endocytic-site dynamics and premature actin assembly during CME through a PtdIns(4,5)P2-dependent mechanism. Combined genetic and biochemical studies showed that the cytoplasmic tail of Rbd2 binds directly to PtdIns(4,5)P2 and is sufficient for Rbd2's role in actin regulation. Analysis of an Rbd2 mutant with diminished PtdIns(4,5)P2-binding capacity indicates that this interaction is necessary for the temporal regulation of actin assembly during CME. The cytoplasmic tail of Rbd2 appears to modulate PtdIns(4,5)P2 distribution on the cell cortex. The syndapin-like F-BAR protein Bzz1 functions in a pathway with Rbd2 to control the timing of type 1 myosin recruitment and actin polymerization onset during CME. This work reveals that the previously unstudied rhomboid protein Rbd2 functions in vivo at the nexus of three highly conserved processes: lipid regulation, endocytic regulation, and cytoskeletal function. PMID:25694450

  9. Control of lipid organization and actin assembly during clathrin-mediated endocytosis by the cytoplasmic tail of the rhomboid protein Rbd2

    PubMed Central

    Cortesio, Christa L.; Lewellyn, Eric B.; Drubin, David G.

    2015-01-01

    Clathrin-mediated endocytosis (CME) is facilitated by a precisely regulated burst of actin assembly. PtdIns(4,5)P2 is an important signaling lipid with conserved roles in CME and actin assembly regulation. Rhomboid family multipass transmembrane proteins regulate diverse cellular processes; however, rhomboid-mediated CME regulation has not been described. We report that yeast lacking the rhomboid protein Rbd2 exhibit accelerated endocytic-site dynamics and premature actin assembly during CME through a PtdIns(4,5)P2-dependent mechanism. Combined genetic and biochemical studies showed that the cytoplasmic tail of Rbd2 binds directly to PtdIns(4,5)P2 and is sufficient for Rbd2's role in actin regulation. Analysis of an Rbd2 mutant with diminished PtdIns(4,5)P2-binding capacity indicates that this interaction is necessary for the temporal regulation of actin assembly during CME. The cytoplasmic tail of Rbd2 appears to modulate PtdIns(4,5)P2 distribution on the cell cortex. The syndapin-like F-BAR protein Bzz1 functions in a pathway with Rbd2 to control the timing of type 1 myosin recruitment and actin polymerization onset during CME. This work reveals that the previously unstudied rhomboid protein Rbd2 functions in vivo at the nexus of three highly conserved processes: lipid regulation, endocytic regulation, and cytoskeletal function. PMID:25694450

  10. The p55 tumour necrosis factor receptor TNFR1 contains a trans-Golgi network localization signal in the C-terminal region of its cytoplasmic tail.

    PubMed Central

    Storey, Helen; Stewart, Abigail; Vandenabeele, Peter; Luzio, J Paul

    2002-01-01

    It has been reported in some human cells that, in addition to a plasma membrane localization, members of the tumour necrosis factor receptor superfamily may be localized to the Golgi complex. We have shown by immunofluorescence and immunoelectron microscopy that the p55 tumour necrosis factor receptor, TNFR1, is principally localized to the trans-Golgi network in the human breast carcinoma cell line, MCF7. Chimaeras consisting of the extracellular and transmembrane domains of CD8 together with the cytoplasmic tail of TNFR1 were targeted to the trans-Golgi network in stably transfected rat fibroblastic cells. Deletions in the cytoplasmic tails of these chimaeras demonstrated the requirement for the C-terminal sequence of 23 amino acids for this targeting. The 23 amino acid sequence is mostly outside the death domain and contains both an acid patch and a dileucine motif. Interaction of this sequence with membrane traffic adaptor proteins may play an important role in controlling the responses of cells to tumour necrosis factor, since binding of signalling adaptor proteins has only been demonstrated for plasma membrane, and not Golgi-localized, TNFR1. PMID:11985495

  11. Novel localization of formin mDia2: importin β-mediated delivery to and retention at the cytoplasmic side of the nuclear envelope

    PubMed Central

    Shao, Xiaowei; Kawauchi, Keiko; Shivashankar, G. V.; Bershadsky, Alexander D.

    2015-01-01

    ABSTRACT The formin family proteins are important regulators of actin polymerization that are involved in many cellular processes. However, little is known about their specific cellular localizations. Here, we show that Diaphanous-related formin-3 (mDia2) localizes to the cytoplasmic side of the nuclear envelope. This localization of mDia2 to the nuclear rim required the presence of a nuclear localization signal (NLS) sequence at the mDia2 N-terminal. Consistent with this result, super-resolution images demonstrated that at the nuclear rim, mDia2 co-localized with the nuclear pore complexes and a nuclear transport receptor, importin β. Furthermore, an interaction between mDia2 and importin β was detected by immunoprecipitation, and silencing of importin β was shown to attenuate accumulation of mDia2 to the nuclear rim. We have shown previously that Ca2+ entry leads to the assembly of perinuclear actin rim in an inverted formin 2 (INF2) dependent manner. mDia2, however, was not involved in this process since abolishing its localization at the nuclear rim by silencing of importin β had no effect on actin assembly at the nuclear rim triggered by Ca2+ stimulation. PMID:26519515

  12. A retention signal necessary and sufficient for Golgi localization maps to the cytoplasmic tail of a Bunyaviridae (Uukuniemi virus) membrane glycoprotein.

    PubMed

    Andersson, A M; Melin, L; Bean, A; Pettersson, R F

    1997-06-01

    Members of the Bunyaviridae family mature by a budding process in the Golgi complex. The site of maturation is thought to be largely determined by the accumulation of the two spike glycoproteins, G1 and G2, in this organelle. Here we show that the signal for localizing the Uukuniemi virus (a phlebovirus) spike protein complex to the Golgi complex resides in the cytoplasmic tail of G1. We constructed chimeric proteins in which the ectodomain, transmembrane domain (TMD), and cytoplasmic tail (CT) of Uukuniemi virus G1 were exchanged with the corresponding domains of either vesicular stomatitis virus G protein (VSV G), chicken lysozyme, or CD4, all proteins readily transported to the plasma membrane. The chimeras were expressed in HeLa or BHK-21 cells by using either the T7 RNA polymerase-driven vaccinia virus system or the Semliki Forest virus system. The fate of the chimeric proteins was monitored by indirect immunofluorescence, and their localizations were compared by double labeling with markers specific for the Golgi complex. The results showed that the ectodomain and TMD (including the 10 flanking residues on either side of the membrane) of G1 played no apparent role in targeting chimeric proteins to the Golgi complex. Instead, all chimeras containing the CT of G1 were efficiently targeted to the Golgi complex and colocalized with mannosidase II, a Golgi-specific enzyme. Conversely, replacing the CT of G1 with that from VSV G resulted in the efficient transport of the chimeric protein to the cell surface. Progressive deletions of the G1 tail suggested that the Golgi retention signal maps to a region encompassing approximately residues 10 to 50, counting from the proposed border between the TMD and the tail. Both G1 and G2 were found to be acylated, as shown by incorporation of [3H]palmitate into the viral proteins. By mutational analyses of CD4-G1 chimeras, the sites for palmitylation were mapped to two closely spaced cysteine residues in the G1 tail. Changing

  13. Hepcidin induction by transgenic overexpression of Hfe does not require the Hfe cytoplasmic tail, but does require hemojuvelin

    PubMed Central

    Andrews, Nancy C.; Fleming, Mark D.

    2010-01-01

    Mutations in HFE cause the most common form of hereditary hemochromatosis (HH). We previously showed that liver-specific, transgenic overexpression of murine Hfe stimulates production of the iron regulatory hormone hepcidin. Here, we developed several additional transgenic mouse strains to further interrogate the structural basis of HFE function in the pathophysiology of HH. We hypothesized that the small, cytoplasmic domain of HFE might be necessary for HFE-mediated induction of hepcidin. We demonstrate that, like the full-length protein, overexpression of Hfe proteins lacking the cytoplasmic domain leads to hepcidin induction, iron deficiency and a hypochromic, microcytic anemia. However, high-level expression of a liver-specific Hfe transgene carrying the mouse equivalent of the common HFE C282Y human disease-causing mutation (murine C294Y) did not cause iron deficiency. Furthermore, hepcidin induction by transgenes encoding both WT Hfe and Hfe lacking its cytoplasmic domain is greatly attenuated in the absence of hemojuvelin (Hjv). Our observations indicate that the extracellular and transmembrane domains of Hfe are sufficient, and Hjv is essential, for Hfe-mediated induction of hepcidin expression. PMID:20837779

  14. The C-terminal tail of the gp41 transmembrane envelope glycoprotein of HIV-1 clades A, B, C, and D may exist in two conformations: an analysis of sequence, structure, and function

    SciTech Connect

    Hollier, Mark J.; Dimmock, Nigel J. . E-mail: n.j.dimmock@warwick.ac.uk

    2005-07-05

    In addition to the major ectodomain, the gp41 transmembrane glycoprotein of HIV-1 is now known to have a minor ectodomain that is part of the long C-terminal tail. Both ectodomains are highly antigenic, carry neutralizing and non-neutralizing epitopes, and are involved in virus-mediated fusion activity. However, data have so far been biologically based, and derived solely from T cell line-adapted (TCLA), B clade viruses. Here we have carried out sequence and theoretically based structural analyses of 357 gp41 C-terminal sequences of mainly primary isolates of HIV-1 clades A, B, C, and D. Data show that all these viruses have the potential to form a tail loop structure (the minor ectodomain) supported by three, {beta}-sheet, membrane-spanning domains (MSDs). This means that the first (N-terminal) tyrosine-based sorting signal of the gp41 tail is situated outside the cell membrane and is non-functional, and that gp41 that reaches the cell surface may be recycled back into the cytoplasm through the activity of the second tyrosine-sorting signal. However, we suggest that only a minority of cell-associated gp41 molecules - those destined for incorporation into virions - has 3 MSDs and the minor ectodomain. Most intracellular gp41 has the conventional single MSD, no minor ectodomain, a functional first tyrosine-based sorting signal, and in line with current thinking is degraded intracellularly. The gp41 structural diversity suggested here can be viewed as an evolutionary strategy to minimize HIV-1 envelope glycoprotein expression on the cell surface, and hence possible cytotoxicity and immune attack on the infected cell.

  15. Function of the cytoplasmic tail of human calcitonin receptor-like receptor in complex with receptor activity-modifying protein 2

    SciTech Connect

    Kuwasako, Kenji; Kitamura, Kazuo; Nagata, Sayaka; Hikosaka, Tomomi; Kato, Johji

    2010-02-12

    Receptor activity-modifying protein 2 (RAMP2) enables calcitonin receptor-like receptor (CRLR) to form an adrenomedullin (AM)-specific receptor. Here we investigated the function of the cytoplasmic C-terminal tail (C-tail) of human (h)CRLR by co-transfecting its C-terminal mutants into HEK-293 cells stably expressing hRAMP2. Deleting the C-tail from CRLR disrupted AM-evoked cAMP production or receptor internalization, but did not affect [{sup 125}I]AM binding. We found that CRLR residues 428-439 are required for AM-evoked cAMP production, though deleting this region had little effect on receptor internalization. Moreover, pretreatment with pertussis toxin (100 ng/mL) led to significant increases in AM-induced cAMP production via wild-type CRLR/RAMP2 complexes. This effect was canceled by deleting CRLR residues 454-457, suggesting Gi couples to this region. Flow cytometric analysis revealed that CRLR truncation mutants lacking residues in the Ser/Thr-rich region extending from Ser{sup 449} to Ser{sup 467} were unable to undergo AM-induced receptor internalization and, in contrast to the effect on wild-type CRLR, overexpression of GPCR kinases-2, -3 and -4 failed to promote internalization of CRLR mutants lacking residues 449-467. Thus, the hCRLR C-tail is crucial for AM-evoked cAMP production and internalization of the CRLR/RAMP2, while the receptor internalization is dependent on the aforementioned GPCR kinases, but not Gs coupling.

  16. Increased infectivity in human cells and resistance to antibody-mediated neutralization by truncation of the SIV gp41 cytoplasmic tail.

    PubMed

    Kuwata, Takeo; Kaori, Takaki; Enomoto, Ikumi; Yoshimura, Kazuhisa; Matsushita, Shuzo

    2013-01-01

    The role of antibodies in protecting the host from human immunodeficiency virus type 1 (HIV-1) infection is of considerable interest, particularly because the RV144 trial results suggest that antibodies contribute to protection. Although infection of non-human primates with simian immunodeficiency virus (SIV) is commonly used as an animal model of HIV-1 infection, the viral epitopes that elicit potent and broad neutralizing antibodies to SIV have not been identified. We isolated a monoclonal antibody (MAb) B404 that potently and broadly neutralizes various SIV strains. B404 targets a conformational epitope comprising the V3 and V4 loops of Env that intensely exposed when Env binds CD4. B404-resistant variants were obtained by passaging viruses in the presence of increasing concentration of B404 in PM1/CCR5 cells. Genetic analysis revealed that the Q733stop mutation, which truncates the cytoplasmic tail of gp41, was the first major substitution in Env during passage. The maximal inhibition by B404 and other MAbs were significantly decreased against a recombinant virus with a gp41 truncation compared with the parental SIVmac316. This indicates that the gp41 truncation was associated with resistance to antibody-mediated neutralization. The infectivities of the recombinant virus with the gp41 truncation were 7,900-, 1,000-, and 140-fold higher than those of SIVmac316 in PM1, PM1/CCR5, and TZM-bl cells, respectively. Immunoblotting analysis revealed that the gp41 truncation enhanced the incorporation of Env into virions. The effect of the gp41 truncation on infectivity was not obvious in the HSC-F macaque cell line, although the resistance of viruses harboring the gp41 truncation to neutralization was maintained. These results suggest that viruses with a truncated gp41 cytoplasmic tail were selected by increased infectivity in human cells and by acquiring resistance to neutralizing antibody. PMID:23717307

  17. A Cytoplasmic Tail Determinant in HIV-1 Vpu Mediates Targeting of Tetherin for Endosomal Degradation and Counteracts Interferon-Induced Restriction

    PubMed Central

    Kueck, Tonya; Neil, Stuart J. D.

    2012-01-01

    The HIV-1 accessory protein Vpu counteracts tetherin (BST-2/CD317) by preventing its incorporation into virions, reducing its surface expression, and ultimately promoting its degradation. Here we characterize a putative trafficking motif, EXXXLV, in the second alpha helix of the subtype-B Vpu cytoplasmic tail as being required for efficient tetherin antagonism. Mutation of this motif prevents ESCRT-dependent degradation of tetherin/Vpu complexes, tetherin cell surface downregulation, but not its physical interaction with Vpu. Importantly, this motif is required for efficient cell-free virion release from CD4+ T cells, particularly after their exposure to type-1 interferon, indicating that the ability to reduce surface tetherin levels and promote its degradation is important to counteract restriction under conditions that the virus likely encounters in vivo. Vpu EXXXLV mutants accumulate with tetherin at the cell surface and in endosomal compartments, but retain the ability to bind both β-TrCP2 and HRS, indicating that this motif is required for a post-binding trafficking event that commits tetherin for ESCRT-dependent degradation and prevents its transit to the plasma membrane and viral budding zones. We further found that while Vpu function is dependent on clathrin, and the entire second alpha helix of the Vpu tail can be functionally complemented by a clathrin adaptor binding peptide derived from HIV-1 Nef, none of the canonical clathrin adaptors nor retromer are required for this process. Finally we show that residual activity of Vpu EXXXLV mutants requires an intact endocytic motif in tetherin, suggesting that physical association of Vpu with tetherin during its recycling may be sufficient to compromise tetherin activity to some degree. PMID:22479182

  18. The putative invertebrate adaptive immune protein Litopenaeus vannamei Dscam (LvDscam) is the first reported Dscam to lack a transmembrane domain and cytoplasmic tail.

    PubMed

    Chou, Pin-Hsiang; Chang, Hao-Shuo; Chen, I-Tung; Lin, Han-You; Chen, Yi-Min; Yang, Huey-Lang; Wang, K C Han-Ching

    2009-12-01

    It has recently been suggested that Dscam (Down syndrome cell adhesion molecule), a member of the immunoglobulin superfamily (IgSF), plays an essential role in the alternative adaptive immune system of invertebrates. Here, we isolated and characterized the first shrimp Dscam from Litopenaeus vannamei. The LvDscam protein had an extracellular domain but lacked the expected transmembrane domain and cytoplasmic tail, both of which are found in all other members of the Dscam family (and may also be found in other L. vannamei Dscams that have not yet been isolated). In nervous tissue, expression levels of LvDscam were unexpectedly low. Phylogenetic analysis suggests that LvDscam is far from the Dscams found in other invertebrates. Nevertheless, the domain architecture of the extracellular region of LvDscam is similar to other invertebrate Dscams, and it exhibits the typical configuration of 10 immunoglobulin (Ig) domains, 6 fibronectin type 3 domains (FNIII) and one cell attachment sequence (RGD). Cloning and characterization of a total of 62 cDNAs from hemocytes collected from WSSV-free, WSSV-persistent and WSSV-acute-infected shrimp revealed 23 alternative amino acid sequences in the N-terminal of Ig2, 30 in the N-terminal of Ig3 and 13 in the Ig7 domain. This implies that LvDscam can potentially encode at least 8970 unique isoforms. Further analysis suggested that the LvDscam Ig2 and Ig3 regions are more functionally important than Ig7 in the shrimp's specific immune response against WSSV. We discuss how this tail-less, soluble Dscam can still play an active role in alternative adaptive immune response even while its axonal guidance functionality may be impaired. PMID:19635499

  19. The Fn14 cytoplasmic tail binds tumour-necrosis-factor-receptor-associated factors 1, 2, 3 and 5 and mediates nuclear factor-kappaB activation.

    PubMed Central

    Brown, Sharron A N; Richards, Christine M; Hanscom, Heather N; Feng, Sheau-Line Y; Winkles, Jeffrey A

    2003-01-01

    Fn14 is a growth-factor-inducible immediate-early-response gene encoding a 102-amino-acid type I transmembrane protein. The human Fn14 protein was recently identified as a cell-surface receptor for the tumour necrosis factor (TNF) superfamily member named TWEAK (TNF-like weak inducer of apoptosis). In the present paper, we report that the human TWEAK extracellular domain can also bind the murine Fn14 protein. Furthermore, site-specific mutagenesis and directed yeast two-hybrid interaction assays revealed that the TNFR-associated factor (TRAF) 1, 2, 3 and 5 adaptor molecules bind the murine Fn14 cytoplasmic tail at an overlapping, but non-identical, amino acid sequence motif. We also found that TWEAK treatment of quiescent NIH 3T3 cells stimulates inhibitory kappaBalpha phosphorylation and transcriptional activation of a nuclear factor-kappaB (NF-kappaB) enhancer/luciferase reporter construct. Fn14 overexpression in transiently transfected NIH 3T3 cells also promotes NF-kappaB activation, and this cellular response requires an intact TRAF binding site. These results indicate that Fn14 is a functional TWEAK receptor that can associate with four distinct TRAF family members and stimulate the NF-kappaB transcription factor signalling pathway. PMID:12529173

  20. Susceptibility to virus-cell fusion at the plasma membrane is reduced through expression of HIV gp41 cytoplasmic domains

    SciTech Connect

    Malinowsky, Katharina; Luksza, Julia; Dittmar, Matthias T.

    2008-06-20

    The cytoplasmic tail of the HIV transmembrane protein plays an important role in viral infection. In this study we analyzed the role of retroviral cytoplasmic tails in modulating the cytoskeleton and interfering with virus-cell fusion. HeLaP4 cells expressing different HIV cytoplasmic tail constructs showed reduced acetylated tubulin levels whereas the cytoplasmic tail of MLV did not alter microtubule stability indicating a unique function for the lentiviral cytoplasmic tail. The effect on tubulin is mediated through the membrane proximal region of the HIV cytoplasmic tail and was independent of membrane localization. Site-directed mutagenesis identified three motifs in the HIV-2 cytoplasmic tail required to effect the reduction in acetylated tubulin. Both the Yxx{phi} domain and amino acids 21 to 45 of the HIV-2 cytoplasmic tail need to be present to change the level of acetylated tubulin in transfected cells. T-cells stably expressing one HIV-2 cytoplasmic tail derived construct showed also a reduction in acetylated tubulin thus confirming the importance of this effect not only for HeLaP4 and 293T cells. Challenge experiments using transiently transfected HeLaP4 cells and T cells stably expressing an HIV cytoplasmic tail construct revealed both reduced virus-cell fusion and replication of HIV-1{sub NL4.3} compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and

  1. Keratin 1 Plays a Critical Role in Golgi Localization of Core 2 N-Acetylglucosaminyltransferase M via Interaction with Its Cytoplasmic Tail*

    PubMed Central

    Petrosyan, Armen; Ali, Mohamed F.; Cheng, Pi-Wan

    2015-01-01

    Core 2 N-acetylglucosaminyltransferase 2/M (C2GnT-M) synthesizes all three β6GlcNAc branch structures found in secreted mucins. Loss of C2GnT-M leads to development of colitis and colon cancer. Recently we have shown that C2GnT-M targets the Golgi at the Giantin site and is recycled by binding to non-muscle myosin IIA, a motor protein, via the cytoplasmic tail (CT). But how this enzyme is retained in the Golgi is not known. Proteomics analysis identifies keratin type II cytoskeletal 1 (KRT1) as a protein pulled down with anti-c-Myc antibody or C2GnT-M CT from the lysate of Panc1 cells expressing bC2GnT-M tagged with c-Myc. Yeast two-hybrid analysis shows that the rod domain of KRT1 interacts directly with the WKR6 motif in the C2GnT-M CT. Knockdown of KRT1 does not affect Golgi morphology but increases the interaction of C2GnT-M with non-muscle myosin IIA and its transportation to the endoplasmic reticulum, ubiquitination, and degradation. During Golgi recovery after brefeldin A treatment, C2GnT-M forms a complex with Giantin before KRT1, demonstrating CT-mediated sequential events of Golgi targeting and retention of C2GnT-M. In HeLa cells transiently expressing C2GnT-M-GFP, knockdown of KRT1 does not affect Golgi morphology but leaves C2GnT-M outside of the Golgi, resulting in the formation of sialyl-T antigen. Interaction of C2GnT-M and KRT1 was also detected in the goblet cells of human colon epithelial tissue and primary culture of colonic epithelial cells. The results indicate that glycosylation and thus the function of glycoconjugates can be regulated by a protein that helps retain a glycosyltransferase in the Golgi. PMID:25605727

  2. Prolonged activation of phospholipase D in Chinese hamster ovary cells expressing platelet-activating-factor receptor lacking cytoplasmic C-terminal tail.

    PubMed

    Liu, B; Nakashima, S; Adachi, T; Ito, Y; Takano, T; Shimizu, T; Nozawa, Y

    1997-10-01

    The mechanism and role of phospholipase D (PLD) activation by platelet-activating factor (PAF) were examined with Chinese hamster ovary cells stably expressing wild-type PAF receptor (WT-H cells) and truncated PAF receptor lacking the C-terminal cytoplasmic tail (D-H cells). Treatment of D-H cells with PAF resulted in the rapid formation of Ins(1,4,5)P3, which was followed by a sustained phase for more than 10 min. In these cells, PAF-induced PLD activation lasted for more than 20 min. In contrast, PLD activation in WT-H cells was transient. PAF stimulation caused the biphasic formation of 1,2-diacylglycerol (DG) in both types of cell. The first phase was rapid and transient, coinciding with the Ins(1,4,5)P3 peak. The second sustained phase of DG formation was attenuated by butanol, which produces phosphatidylbutanol at the expense of phosphatidic acid (PA) by transphosphatidylation activity of PLD, and by propranolol, a selective inhibitor for PA phosphohydrolase catalysing the conversion of PA into DG. The DG level returned nearly to basal at 20 min after PAF stimulation in WT-H cells, whereas in D-H cells the elevated DG level was sustained for more than 20 min. The profile of translocation of protein kinase Calpha (PKCalpha) to membrane was similar to that of DG formation. In WT-H cells, PKCalpha was transiently associated with membranes and then returned to the cytosol. However, in D-H cells PKCalpha was rapidly translocated to and remained in membranes for more than 20 min. Butanol suppressed this sustained translocation of PKCalpha. Furthermore the mRNA levels of c-fos and c-jun by PAF in WT-H cells were much lower than those in D-H cells. Propranolol and butanol at concentrations that inhibited the formation of DG suppressed the PAF-induced mRNA expression of c-fos and c-jun. Taken together, the prolonged PLD activation in D-H cells confirmed a primary role for phospholipase C/PKC in PLD activation by PAF. Furthermore the results obtained here suggest that

  3. The intermediate filament protein vimentin binds specifically to a recombinant integrin {alpha}2/{beta}1 cytoplasmic tail complex and co-localizes with native {alpha}2/{beta}1 in endothelial cell focal adhesions

    SciTech Connect

    Kreis, Stephanie; Schoenfeld, Hans-Joachim; Melchior, Chantal; Steiner, Beat; Kieffer, Nelly . E-mail: kieffer@cu.lu

    2005-04-15

    Integrin receptors are crucial players in cell adhesion and migration. Identification and characterization of cellular proteins that interact with their short {alpha} and {beta} cytoplasmic tails will help to elucidate the molecular mechanisms by which integrins mediate bi-directional signaling across the plasma membrane. Integrin {alpha}2{beta}1 is a major collagen receptor but to date, only few proteins have been shown to interact with the {alpha}2 cytoplasmic tail or with the {alpha}2{beta}1 complex. In order to identify novel binding partners of a {alpha}2{beta}1cytoplasmic domain complex, we have generated recombinant GST-fusion proteins, incorporating the leucine zipper heterodimerization cassettes of Jun and Fos. To ascertain proper functionality of the recombinant proteins, interaction with natural binding partners was tested. GST-{alpha}2 and GST-Jun {alpha}2 bound His-tagged calreticulin while GST-{beta}1 and GST-Fos {beta}1 proteins bound talin. In screening assays for novel binding partners, the immobilized GST-Jun {alpha}2/GST-Fos {beta}1 heterodimeric complex, but not the single subunits, interacted specifically with endothelial cell-derived vimentin. Vimentin, an abundant intermediate filament protein, has previously been shown to co-localize with {alpha}v{beta}3-positive focal contacts. Here, we provide evidence that this interaction also occurs with {alpha}2{beta}1-enriched focal adhesions and we further show that this association is lost after prolonged adhesion of endothelial cells to collagen.

  4. The Influenza M2 Cytoplasmic Tail Changes the Proton-Exchange Equilibria and the Backbone Conformation of the Transmembrane Histidine Residue to Facilitate Proton Conduction

    PubMed Central

    Liao, Shu Y.; Yang, Yu; Tietze, Daniel; Hong, Mei

    2015-01-01

    The influenza M2 protein forms an acid-activated tetrameric proton channel important for the virus lifecycle. Residue His37 in the transmembrane domain is responsible for channel activation and proton selectivity. While the structure and dynamics of His37 have been well studied in TM peptide constructs, it has not been investigated in the presence of the full cytoplasmic domain, which increases the proton conductivity by 2-fold compared to the TM peptide. We report here 13C and 15N chemical shifts of His37 in the cytoplasmic-containing M2(21-97), and show that cationic histidines are already present at neutral pH, in contrast to the TM peptide, indicating that the cytoplasmic domain shifts the protonation equilibria. Quantification of the imidazole 15N intensities yielded two resolved proton dissociation constants (pKa’s) of 7.1 and 5.4, which differ from the TM result but resemble the M2(18–60) result, suggesting cooperative proton binding. The average His37 pKa is higher for M2(21–97) than for the shorter constructs. We attribute this higher pKa to direct and indirect effects of the cytoplasmic domain, which is rich in acidic residues. 2D 13C-13C correlation spectra reveal seven His37 Cα-Cβ cross peaks at different pH, some of which are unique to the cytoplasmic-containing M2 and correspond to more ideal α-helical conformations. Based on the pH at which these chemical shifts appear and their sidechain structures, we assign these conformations to His37 in differently charged tetramers. Thus, the cytoplasmic domain facilitates proton conduction through the transmembrane pore by modifying the His37-water proton-exchange equilibria and the His37 backbone conformational distribution. PMID:25892574

  5. Identification of the phosphorylation sequence in the cytoplasmic tail of the varicella-zoster virus Fc receptor glycoprotein gpI.

    PubMed Central

    Yao, Z; Jackson, W; Grose, C

    1993-01-01

    Varicella-zoster virus (VZV) glycoprotein gpI, the homolog of herpes simplex virus gE, functions as a receptor for the Fc portion of immunoglobulin G. Like other cell surface receptors, this viral receptor is highly phosphorylated in cell culture. To identify the precise location of the cellular kinase-mediated phosphorylation, we generated a tailless deletion mutant and several point mutants which had altered serine and threonine residues within the cytoplasmic domain of gpI. The mutated and wild-type genes of gpI were transfected and expressed within a vaccinia virus-T7 polymerase transfection system in order to determine what effect these mutations had on the phosphorylation state of the protein in vivo and in vitro. Truncation of the cytoplasmic domain of gpI diminished the phosphorylation of gpI in vivo. Examination of the point mutants established that the major phosphorylation sequence of gpI was located between amino acids 593 and 598, a site which included four phosphorylatable serine and threonine residues. Phosphorylation analyses of the mutant and wild-type glycoproteins confirmed that gpI was a substrate for casein kinase II, with threonines 596 and 598 being critical residues. Although the mutant glycoproteins were phosphorylated by casein kinase I, protease V8 partial digestion profiles suggested that casein kinase II exerted the major effect. Thus, these mutagenesis studies demonstrated that the gpI cytoplasmic sequence Ser-Glu-Ser-Thr-Asp-Thr was phosphorylated in mammalian cells in the absence of any other herpesvirus products. Since the region defined by transfection was consistent with results obtained with in vitro phosphorylation by casein kinase II, we propose that VZV gpI is a physiologic substrate for casein kinase II. Immunofluorescence and pulse-chase experiments demonstrated that the mutant glycoproteins were processed and transported to the outer cell membrane. Images PMID:8392591

  6. Modified Cytoplasmic Ca2+ Sequestration Contributes to Spinal Cord Injury-Induced Augmentation of Nerve-Evoked Contractions in the Rat Tail Artery

    PubMed Central

    Al Dera, Hussain; Callaghan, Brid P.; Brock, James A.

    2014-01-01

    In rat tail artery (RTA), spinal cord injury (SCI) increases nerve-evoked contractions and the contribution of L-type Ca2+ channels to these responses. In RTAs from unoperated rats, these channels play a minor role in contractions and Bay K8644 (L-type channel agonist) mimics the effects of SCI. Here we investigated the mechanisms underlying the facilitatory actions of SCI and Bay K8644 on nerve-evoked contractions of RTAs and the hypothesis that Ca2+ entering via L-type Ca2+ channels is rapidly sequestered by the sarcoplasmic reticulum (SR) limiting its role in contraction. In situ electrochemical detection of noradrenaline was used to assess if Bay K8644 increased noradrenaline release. Perforated patch recordings were used to assess if SCI changed the Ca2+ current recorded in RTA myocytes. Wire myography was used to assess if SCI modified the effects of Bay K8644 and of interrupting SR Ca2+ uptake on nerve-evoked contractions. Bay K8644 did not change noradrenaline-induced oxidation currents. Neither the size nor gating of Ca2+ currents differed between myocytes from sham-operated (control) and SCI rats. Bay K8644 increased nerve-evoked contractions in RTAs from both control and SCI rats, but the magnitude of this effect was reduced by SCI. By contrast, depleting SR Ca2+ stores with ryanodine or cyclopiazonic acid selectively increased nerve-evoked contractions in control RTAs. Cyclopiazonic acid also selectively increased the blockade of these responses by nifedipine (L-type channel blocker) in control RTAs, whereas ryanodine increased the blockade produced by nifedipine in both groups of RTAs. These findings suggest that Ca2+ entering via L-type channels is normally rapidly sequestered limiting its access to the contractile mechanism. Furthermore, the findings suggest SCI reduces the role of this mechanism. PMID:25350563

  7. Cryo-EM structure of a native, fully glycosylated, cleaved HIV-1 envelope trimer.

    PubMed

    Lee, Jeong Hyun; Ozorowski, Gabriel; Ward, Andrew B

    2016-03-01

    The envelope glycoprotein trimer (Env) on the surface of HIV-1 recognizes CD4(+) T cells and mediates viral entry. During this process, Env undergoes substantial conformational rearrangements, making it difficult to study in its native state. Soluble stabilized trimers have provided valuable insights into the Env structure, but they lack the hydrophobic membrane proximal external region (MPER, an important target of broadly neutralizing antibodies), the transmembrane domain, and the cytoplasmic tail. Here we present (i) a cryogenic electron microscopy (cryo-EM) structure of a clade B virus Env, which lacks only the cytoplasmic tail and is stabilized by the broadly neutralizing antibody PGT151, at a resolution of 4.2 angstroms and (ii) a reconstruction of this form of Env in complex with PGT151 and MPER-targeting antibody 10E8 at a resolution of 8.8 angstroms. These structures provide new insights into the wild-type Env structure. PMID:26941313

  8. Forming a tough shell via an intracellular matrix and cellular junctions in the tail epidermis of Oikopleura dioica (Chordata: Tunicata: Appendicularia)

    NASA Astrophysics Data System (ADS)

    Nakashima, Keisuke; Nishino, Atsuo; Hirose, Euichi

    2011-08-01

    A postanal tail is a major synapomorphy of the phylum Chordata, which is composed of three subphyla: Vertebrata, Cephalochordata, and Tunicata (Urochordata). Among tunicates, appendicularians are the only group that retains the tail in the adult, and the adult tail functions in locomotion and feeding in combination with a cellulose-based house structure. Given the phylogenetic position of tunicates, the appendicularian adult tail may possess ancestral features of the chordate tail. We assess the ultrastructural development of the tail epidermis of the appendicularian Oikopleura dioica. The epidermis of the larval tail is enclosed by the larval envelope, which is a thin sheet similar to the outer tunic layer of ascidian larvae. The epidermis of the adult tail seems to bear no tunic-like cellulosic integuments, and the tail fin is a simple folding of the epidermis. Every epidermal cell, except for the triangular cells at the edge of the tail fin, has a conspicuous matrix layer of fibrous content in the apical cytoplasm without enclosing membranes. The epidermis of the larval tail does not have a fibrous matrix layer, suggesting the production of the layer during larval development and metamorphosis. Zonulae adhaerentes firmly bind the epidermal cells of the adult tail to one another, and the dense microfilaments lining the cell borders constitute a mechanical support for the cell membranes. The intracellular matrix, cell junctions, and cytoskeletons probably make the tail epidermis a tough, flexible shell supporting the active beating of the oikopleuran adult tail.

  9. Evidence for bacteriophage T7 tail extension during DNA injection

    PubMed Central

    Serwer, Philip; Wright, Elena T; Hakala, Kevin W; Weintraub, Susan T

    2008-01-01

    Background Electron micrographs of bacteriophage T7 reveal a tail shorter than needed to reach host cytoplasm during infection-initiating injection of a T7 DNA molecule through the tail and cell envelope. However, recent data indicate that internal T7 proteins are injected before the DNA molecule is injected. Thus, bacteriophage/host adsorption potentially causes internal proteins to become external and lengthen the tail for DNA injection. But, the proposed adsorption-induced tail lengthening has never been visualized. Findings In the present study, electron microscopy of particles in T7 lysates reveals a needle-like capsid extension that attaches partially emptied bacteriophage T7 capsids to non-capsid vesicles and sometimes enters an attached vesicle. This extension is 40–55 nm long, 1.7–2.4× longer than the T7 tail and likely to be the proposed lengthened tail. The extension is 8–11 nm in diameter, thinner than most of the tail, with an axial hole 3–4 nm in diameter. Though the bound vesicles are not identified by microscopy, these vesicles resemble the major vesicles in T7 lysates, found to be E. coli outer membrane vesicles by non-denaturing agarose gel electrophoresis, followed by mass spectrometry. Conclusion The observed lengthened tail is long enough to reach host cytoplasm during DNA injection. Its channel is wide enough to be a conduit for DNA injection and narrow enough to clamp DNA during a previously observed stalling/re-starting of injection. However, its outer diameter is too large to explain formation by passing of an intact assembly through any known capsid hole unless the hole is widened. PMID:18710489

  10. Identification of an epitope within the Bovine herpesvirus 1 glycoprotein E cytoplasmic tail and use of a monoclonal antibody directed against the epitope for the differentiation between vaccinated and infected animals.

    PubMed

    Chowdhury, Shafiqul I

    2016-07-01

    We constructed a recombinant bovine herpesvirus type 1 triple mutant virus (BoHV-1 tmv) that lacks UL49.5 residues 30-32 and 80-96, gE cytoplasmic tail (gE CT) residues 452-575 and the entire 435bp long Us9 ORF. To develop a gE CT-specific blocking ELISA test that is necessary to distinguish the BoHV-1 tmv vaccinated calves from the wild-type (wt) virus-infected calves, a mouse monoclonal antibody (mAb) 2H8F3 was generated by using the Escherichia coli expressed gE CT residues 452-575. Further, by performing a PEPSCAN analysis of 12 mer overlapping peptides spanning the entire gE CT, the epitope sequence recognized by the mAb2H8F3 was mapped within the gE CT residues 499SDDDGPASN507. A blocking ELISA test was then developed for detecting antibodies in wild-type BoHV-1 infected calves against the gE CT epitope specified by 499SDDDGPASN507. The assay is based on the use of HRP conjugated mAb2H8F3 and the E. coli expressed gE CT protein as an indicator antibody and a coating antigen, respectively. In this assay, serum from entire gE-deleted and BoHV-1 tmv-infected calves scored negative, whereas serum from calves infected with BoHV-1 wt scored positive. Therefore, the gE CT-ELISA, based on the mAb2H8F3 and E. coli expressed gE CT protein, is suitable for differentiating the wt virus-infected and BoHV-1 tmv-vaccinated cattle. PMID:26976821

  11. Membrane bound Indian clade C HIV-1 envelope antigen induces antibodies to diverse and conserved epitopes upon DNA prime/protein boost in rabbits.

    PubMed

    Rangasamy, Sneha Priya; Menon, Veena; Dhopeshwarkar, Priyanka; Pal, Ranajit; Vaniambadi, Kalyanaraman S; Mahalingam, Sundarasamy

    2016-05-01

    The partial success of RV144 human clinical trial demonstrated that ALVAC prime/envelope protein boost vaccine regimen may represent a promising strategy for the development of an effective HIV-1 vaccine. Our earlier study demonstrated that a trimeric HIV-1 envelope gp145 from an Indian clade C isolate elicited cross clade neutralizing antibodies primarily towards Tier 1 isolates. In the present study, we examined the immunogenicity of DNA prime/envelope protein boost vaccine in rabbits using gp160 DNA of the Indian clade C isolate with various cytoplasmic tail truncations and trimeric gp145 protein. Cytoplasmic tail mutants of gp160 exposed epitopes that reacted strongly with a number of broadly neutralizing human monoclonal antibodies against HIV-1. Overall, envelope specific titers were found to be similar in all rabbit groups with higher pseudovirus neutralization in protein only immunized rabbits. The complete linear epitope mapping of rabbit immune sera revealed strong binding to C1, C2, V3, C3 and C4 domains of gp145. Importantly, reactivity of gp41 ecto-domain peptides was observed in DNA prime/protein boost sera but not in the sera of rabbits immunized with protein alone. Moreover, membrane anchored but not soluble envelope encoding DNA immunization elicited antibodies against linear epitopes on the conserved gp41 ecto-domain. Together, these results suggest that priming with DNA encoding cytoplasmic domains of Env alters the quality of antibodies elicited following protein boost and hence may be utilized to generate protective immunity by HIV-1 vaccine. PMID:27032514

  12. A model for the coordinated stepping of cytoplasmic dynein.

    PubMed

    Zhao, X Y; Sun, W; Zhang, J P; Tala; Guo, W S

    2014-10-31

    Cytoplasmic dynein play an important role in transporting various intracellular cargos by coupling their ATP hydrolysis cycle with their conformational changes. Recent experimental results showed that the cytoplasmic dynein had a highly variable stepping pattern including "hand-over-hand", "inchworm" and "nonalternating-inchworm". Here, we developed a model to describe the coordinated stepping patterns of cytoplasmic dynein, based on its working cycle, construction and the interaction between its leading head and tailing head. The kinetic model showed how change in the distance between the two heads influences the rate of cytoplasmic dynein under different stepping patterns. Numerical simulations of the distribution of step size and striding rate are in good quantitative agreement with experimental observations. Hence, our coordinated stepping model for cytoplasmic dynein successfully explained its diverse stepping patterns as a molecular motor. The cooperative mechanism carried out by the two heads of cytoplasmic dynein shed light on the strategies adopted by the cytoplasmic dynein in executing various functions. PMID:25301561

  13. SAFEGUARDS ENVELOPE

    SciTech Connect

    Duc Cao; Richard Metcalf

    2010-07-01

    The Safeguards Envelope is a strategy to determine a set of specific operating parameters within which nuclear facilities may operate to maximize safeguards effectiveness without sacrificing safety or plant efficiency. This paper details advanced statistical techniques that will be applied to real plant process monitoring (PM) data from the Idaho Chemical Processing Plant (ICPP). In a simulation based on this data, multi-tank and multi-attribute correlations were tested against synthetic diversion scenarios. Kernel regression smoothing was used to fit a curve to the historical data, and multivariable, residual analysis and cumulative sum techniques set parameters for operating conditions. Diversion scenarios were created and tested, showing improved results when compared with a previous study utilizing only one-variable Z-testing. A brief analysis of the impact of the safeguards optimization on the rest of plant efficiency, criticality concerns, and overall requirements is presented.

  14. Floods from tailings dam failures.

    PubMed

    Rico, M; Benito, G; Díez-Herrero, A

    2008-06-15

    This paper compiles the available information on historic tailings dam failures with the purpose to establish simple correlations between tailings ponds geometric parameters (e.g., dam height, tailings volume) and the hydraulic characteristics of floods resulting from released tailings. Following the collapse of a mining waste dam, only a part of tailings and polluted water stored at the dam is released, and this outflow volume is difficult to estimate prior the incident. In this study, tailings' volume stored at the time of failure was shown to have a good correlation (r2=0.86) with the tailings outflow volume, and the volume of spilled tailings was correlated with its run-out distance (r2=0.57). An envelope curve was drawn encompassing the majority of data points indicating the potential maximum downstream distance affected by a tailings' spill. The application of the described regression equations for prediction purposes needs to be treated with caution and with support of on-site measurement and observations. However, they may provide a universal baseline approximation on tailing outflow characteristics (even if detailed dam information is unavailable), which is of a great importance for risk analysis purposes. PMID:18096316

  15. Co-evolution analysis to predict protein-protein interactions within influenza virus envelope.

    PubMed

    Mintaev, Ramil R; Alexeevski, Andrei V; Kordyukova, Larisa V

    2014-04-01

    Interactions between integral membrane proteins hemagglutinin (HA), neuraminidase (NA), M2 and membrane-associated matrix protein M1 of influenza A virus are thought to be crucial for assembly of functionally competent virions. We hypothesized that the amino acid residues located at the interface of two different proteins are under physical constraints and thus probably co-evolve. To predict co-evolving residue pairs, the EvFold ( http://evfold.org ) program searching the (nontransitive) Direct Information scores was applied for large samplings of amino acid sequences from Influenza Research Database ( http://www.fludb.org/ ). Having focused on the HA, NA, and M2 cytoplasmic tails as well as C-terminal domain of M1 (being the less conserved among the protein domains) we captured six pairs of correlated positions. Among them, there were one, two, and three position pairs for HA-M2, HA-M1, and M2-M1 protein pairs, respectively. As expected, no co-varying positions were found for NA-HA, NA-M1, and NA-M2 pairs obviously due to high conservation of the NA cytoplasmic tail. The sum of frequencies calculated for two major amino acid patterns observed in pairs of correlated positions was up to 0.99 meaning their high to extreme evolutionary sustainability. Based on the predictions a hypothetical model of pair-wise protein interactions within the viral envelope was proposed. PMID:24712535

  16. Biochemical evidence of a role for matrix trimerization in HIV-1 envelope glycoprotein incorporation.

    PubMed

    Tedbury, Philip R; Novikova, Mariia; Ablan, Sherimay D; Freed, Eric O

    2016-01-12

    The matrix (MA) domain of HIV Gag has important functions in directing the trafficking of Gag to sites of assembly and mediating the incorporation of the envelope glycoprotein (Env) into assembling particles. HIV-1 MA has been shown to form trimers in vitro; however, neither the presence nor the role of MA trimers has been documented in HIV-1 virions. We developed a cross-linking strategy to reveal MA trimers in virions of replication-competent HIV-1. By mutagenesis of trimer interface residues, we demonstrated a correlation between loss of MA trimerization and loss of Env incorporation. Additionally, we found that truncating the long cytoplasmic tail of Env restores incorporation of Env into MA trimer-defective particles, thus rescuing infectivity. We therefore propose a model whereby MA trimerization is required to form a lattice capable of accommodating the long cytoplasmic tail of HIV-1 Env; in the absence of MA trimerization, Env is sterically excluded from the assembling particle. These findings establish MA trimerization as an obligatory step in the assembly of infectious HIV-1 virions. As such, the MA trimer interface may represent a novel drug target for the development of antiretrovirals. PMID:26711999

  17. RhoA activation and actin reorganization involved in endothelial CAM-mediated endocytosis of anti-PECAM carriers: critical role for tyrosine 686 in the cytoplasmic tail of PECAM-1.

    PubMed

    Garnacho, Carmen; Shuvaev, Vladimir; Thomas, Anu; McKenna, Lindsay; Sun, Jing; Koval, Michael; Albelda, Steven; Muzykantov, Vladimir; Muro, Silvia

    2008-03-15

    Platelet-endothelial cell adhesion molecule-1 (PECAM-1), a transmembrane glycoprotein involved in leukocyte transmigration, represents a good target for endothelial drug delivery (eg, using antibody-directed nanocarriers, anti-PECAM/NCs). Although endothelial cells do not internalize PECAM antibodies, PECAM-1 engagement by multivalent anti-PECAM conjugates and nanocarriers causes endocytosis via a nonclassic CAM-mediated pathway. We found that endothelial uptake of multivalent anti-PECAM complexes is associated with PECAM-1 phosphorylation. Using model REN cells expressing a series of PECAM-1 deletion and point mutants, we found that the PECAM-1 cytoplasmic domain and, more precisely, PECAM-1 tyrosine 686, is critical in mediating RhoA activation and recruitment of EGFP-RhoA to anti-PECAM/NC binding sites at the plasmalemma, actin polymerization into phalloidin-positive stress fibers, and finally CAM endocytosis of anti-PECAM/NCs. Endothelial targeting and endocytosis of anti-PECAM/NCs were markedly efficient and did not compromise endothelial barrier function in vitro (determined by immunostaining of VE-cadherin and (125)I-albumin transport across endothelial monolayers) or in vivo (determined by electron microscopy imaging of pulmonary capillaries and (125)I-albumin transport from the blood into the lung tissue after intravenous injection of anti-PECAM/NCs in mice). These results reveal PECAM-1 signaling and interactions with the cytoskeleton, which are required for CAM-endocytosis, and may provide safe intra-endothelial drug delivery by anti-PECAM/NCs. PMID:18182571

  18. Chloroplast and Cytoplasmic Enzymes

    PubMed Central

    Anderson, Louise E.; Pacold, Ivan

    1972-01-01

    Several peaks of aldolase activity are found in the isoelectric focusing pattern of pea (Pisum sativum) leaf chloroplast extracts. One peak, separated by 0.5 pH unit from the major chloroplast aldolase peak, is found when cytoplasmic extracts are focused. The chloroplast and cytoplasmic enzymes have a pH 7.4 optimum with fructose 1,6-diphosphate. The Michaelis constant for fructose-1,6-diphosphate is 19 μM for the chloroplast, 21 μM for the cytoplasmic enzyme, and for sedoheptulose 1,7-diphosphate, 8 μM for the chloroplast enzyme, 18 μM for the cytoplasmic enzyme. Both enzymes are inhibited by d-glyceraldehyde 3-phosphate and by ribulose 1,5-diphosphate. The similarity in the catalytic properties of the isoenzymes suggests that both enzymes have an amphibolic role in carbon metabolism in the green leaf. PMID:16657968

  19. Immunogenicity of a novel engineered HIV-1 clade C synthetic consensus-based envelope DNA vaccine.

    PubMed

    Yan, Jian; Corbitt, Natasha; Pankhong, Panyupa; Shin, Thomas; Khan, Amir; Sardesai, Niranjan Y; Weiner, David B

    2011-09-22

    DNA vaccines require significant engineering in order to generate strong CTL responses in both non-human primates and humans. In this study, we designed a clade C env gene (EY3E1-C) to decrease the genetic distances of virus isolates within clade C and focus the induced T cell responses to conserved clade C epitopes. After generating a consensus sequence by analyzing full-length clade C env early transmitter sequences, several modifications were performed to increase the expression of the EY3E1-C, including codon/RNA optimization, addition of Kozak sequence and addition of an IgE leader sequence. We also shortened the V1 and V2 loops to approximate early transmitter isolate sequences and the cytoplasmic tail was truncated to prevent envelope recycling. When studied as a DNA vaccine in Balb/c mice, compared to a primary codon-optimized clade C envelope DNA vaccine (p96ZM651gp140-CD5), this novel construct is up to three times more potent in driving CTL responses. Importantly this construct not only induces stronger cross-reactive cellular responses within clade C, it also induces stronger immune responses against clade B and group M envelope peptide pools than p96ZM651gp140-CD5. Epitope mapping demonstrated that EY3E1-C was able to induce clade C envelope-specific immune responses against 15 peptide pools, clade B envelope-specific immune responses against 19 peptide pools and group M envelope-specific immune responses against 16 peptide pools out of 29, respectively, indicating that a significant increase in the breadth of induced immune responses. The analysis of antibody responses suggested that vaccination of pEY3E1-C could induce a clade C envelope-specific antibody response. The cellular immune responses of pEY3E1-C could be further enhanced when the DNA was delivered by using electroporation (EP). Thus, the synthetic engineered consensus EY3E1-C gene is capable of eliciting stronger and broader CTL responses than primary clade C envelopes. This finding

  20. Enhanced antagonism of BST-2 by a neurovirulent SIV envelope

    PubMed Central

    Matsuda, Kenta; Chen, Chia-Yen; Whitted, Sonya; Chertova, Elena; Roser, David J.; Wu, Fan; Plishka, Ronald J.; Ourmanov, Ilnour; Buckler-White, Alicia; Lifson, Jeffrey D.; Strebel, Klaus; Hirsch, Vanessa M.

    2016-01-01

    Current antiretroviral therapy (ART) is not sufficient to completely suppress disease progression in the CNS, as indicated by the rising incidence of HIV-1–associated neurocognitive disorders (HAND) among infected individuals on ART. It is not clear why some HIV-1–infected patients develop HAND, despite effective repression of viral replication in the circulation. SIV-infected nonhuman primate models are widely used to dissect the mechanisms of viral pathogenesis in the CNS. Here, we identified 4 amino acid substitutions in the cytoplasmic tail of viral envelope glycoprotein gp41 of the neurovirulent virus SIVsm804E that enhance replication in macrophages and associate with enhanced antagonism of the host restriction factor BM stromal cell antigen 2 (BST-2). Rhesus macaques were inoculated with a variant of the parental virus SIVsmE543-3 that had been engineered to contain the 4 amino acid substitutions present in gp41 of SIVsm804E. Compared with WT virus–infected controls, animals infected with mutant virus exhibited higher viral load in cerebrospinal fluid. Together, these results are consistent with a potential role for BST-2 in the CNS microenvironment and suggest that BST-2 antagonists may serve as a possible target for countermeasures against HAND. PMID:27159392

  1. Structural Characterization of the Crimean-Congo Hemorrhagic Fever Virus Gn Tail Provides Insight into Virus Assembly*

    PubMed Central

    Estrada, D. Fernando; De Guzman, Roberto N.

    2011-01-01

    The RNA virus that causes the Crimean Congo Hemorrhagic Fever (CCHF) is a tick-borne pathogen of the Nairovirus genus, family Bunyaviridae. Unlike many zoonotic viruses that are only passed between animals and humans, the CCHF virus can also be transmitted from human to human with an overall mortality rate approaching 30%. Currently, there are no atomic structures for any CCHF virus proteins or for any Nairovirus proteins. A critical component of the virus is the envelope Gn glycoprotein, which contains a C-terminal cytoplasmic tail. In other Bunyaviridae viruses, the Gn tail has been implicated in host-pathogen interaction and viral assembly. Here we report the NMR structure of the CCHF virus Gn cytoplasmic tail, residues 729–805. The structure contains a pair of tightly arranged dual ββα zinc fingers similar to those found in the Hantavirus genus, with which it shares about 12% sequence identity. Unlike Hantavirus zinc fingers, however, the CCHF virus zinc fingers bind viral RNA and contain contiguous clusters of conserved surface electrostatics. Our results provide insight into a likely role of the CCHF virus Gn zinc fingers in Nairovirus assembly. PMID:21507948

  2. An unresolved LINC in the nuclear envelope

    PubMed Central

    Torbati, Mehdi; Lele, Tanmay P; Agrawal, Ashutosh

    2016-01-01

    The nuclear envelope segregates the nucleoplasm from the cytoplasm and is a key feature of eukaryotic cells. Nuclear envelope architecture is comprised of two concentric membrane shells which fuse at multiple sites and yet maintain a uniform separation of 30–50 nm over the rest of the membrane. Studies have revealed the roles for numerous nuclear proteins in forming and maintaining the architecture of the nuclear envelope. However, there is a lack of consensus on the fundamental forces and physical mechanisms that establish the geometry. The objective of this review is to discuss recent findings in the context of membrane mechanics in an effort to define open questions and possible answers. PMID:27330571

  3. Cytoplasmic Z-RNA

    SciTech Connect

    Zarling, D.A.; Calhoun, C.J.; Hardin, C.C.; Zarling, A.H.

    1987-09-01

    Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgCs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Pre-or nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or single-stranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation.

  4. Protein diffusion in mammalian cell cytoplasm.

    PubMed

    Kühn, Thomas; Ihalainen, Teemu O; Hyväluoma, Jari; Dross, Nicolas; Willman, Sami F; Langowski, Jörg; Vihinen-Ranta, Maija; Timonen, Jussi

    2011-01-01

    We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS. PMID:21886771

  5. Equine Tetherin Blocks Retrovirus Release and Its Activity Is Antagonized by Equine Infectious Anemia Virus Envelope Protein

    PubMed Central

    Yin, Xin; Hu, Zhe; Gu, Qinyong; Wu, Xingliang; Zheng, Yong-Hui; Wei, Ping

    2014-01-01

    Human tetherin is a host restriction factor that inhibits replication of enveloped viruses by blocking viral release. Tetherin has an unusual topology that includes an N-terminal cytoplasmic tail, a single transmembrane domain, an extracellular domain, and a C-terminal glycosylphosphatidylinositol anchor. Tetherin is not well conserved across species, so it inhibits viral replication in a species-specific manner. Thus, studies of tetherin activities from different species provide an important tool for understanding its antiviral mechanism. Here, we report cloning of equine tetherin and characterization of its antiviral activity. Equine tetherin shares 53%, 40%, 36%, and 34% amino acid sequence identity with feline, human, simian, and murine tetherins, respectively. Like the feline tetherin, equine tetherin has a shorter N-terminal domain than human tetherin. Equine tetherin is localized on the cell surface and strongly blocks human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and equine infectious anemia virus (EIAV) release from virus-producing cells. The antiviral activity of equine tetherin is neutralized by EIAV envelope protein, but not by the HIV-1 accessory protein Vpu, which is a human tetherin antagonist, and EIAV envelope protein does not counteract human tetherin. These results shed new light on our understanding of the species-specific tetherin antiviral mechanism. PMID:24227834

  6. Tail Buffeting

    NASA Technical Reports Server (NTRS)

    Abdrashitov, G.

    1943-01-01

    An approximate theory of buffeting is here presented, based on the assumption of harmonic disturbing forces. Two cases of buffeting are considered: namely, for a tail angle of attack greater and less than the stalling angle, respectively. On the basis of the tests conducted and the results of foreign investigators, a general analysis is given of the nature of the forced vibrations the possible load limits on the tail, and the methods of elimination of buffeting.

  7. A Single Herpesvirus Protein Can Mediate Vesicle Formation in the Nuclear Envelope*

    PubMed Central

    Lorenz, Michael; Vollmer, Benjamin; Unsay, Joseph D.; Klupp, Barbara G.; García-Sáez, Ana J.; Mettenleiter, Thomas C.; Antonin, Wolfram

    2015-01-01

    Herpesviruses assemble capsids in the nucleus and egress by unconventional vesicle-mediated trafficking through the nuclear envelope. Capsids bud at the inner nuclear membrane into the nuclear envelope lumen. The resulting intralumenal vesicles fuse with the outer nuclear membrane, delivering the capsids to the cytoplasm. Two viral proteins are required for vesicle formation, the tail-anchored pUL34 and its soluble interactor, pUL31. Whether cellular proteins are involved is unclear. Using giant unilamellar vesicles, we show that pUL31 and pUL34 are sufficient for membrane budding and scission. pUL34 function can be bypassed by membrane tethering of pUL31, demonstrating that pUL34 is required for pUL31 membrane recruitment but not for membrane remodeling. pUL31 can inwardly deform membranes by oligomerizing on their inner surface to form buds that constrict to vesicles. Therefore, a single viral protein can mediate all events necessary for membrane budding and abscission. PMID:25605719

  8. A Mutation in the Human Immunodeficiency Virus Type 1 Gag Protein Destabilizes the Interaction of the Envelope Protein Subunits gp120 and gp41

    PubMed Central

    Davis, Melody R.; Jiang, Jiyang; Zhou, Jing; Freed, Eric O.; Aiken, Christopher

    2006-01-01

    The Gag protein of human immunodeficiency virus type 1 (HIV-1) associates with the envelope protein complex during virus assembly. The available evidence indicates that this interaction involves recognition of the gp41 cytoplasmic tail (CT) by the matrix protein (MA) region of Pr55Gag. Here we show that substitution of Asp for Leu at position 49 (L49D) in MA results in a specific reduction in particle-associated gp120 without affecting the levels of gp41. Mutant virions were markedly reduced in single-cycle infectivity despite a relatively modest defect in fusion with target cells. Studies with HIV-1 particles containing decreased levels of envelope proteins suggested that the L49D mutation also inhibits a postentry step in infection. Truncation of the gp41 tail, or pseudotyping by vesicular stomatitis virus glycoprotein, restored both the fusion and infectivity of L49D mutant virions to wild-type levels. Truncation of gp41 also resulted in equivalent levels of gp120 on particles with and without the MA mutation and enhanced the replication of the L49D mutant virus in T cells. The impaired fusion and infectivity of L49D mutant particles were also complemented by a single point mutation in the gp41 CT that disrupted the tyrosine-containing endocytic motif. Our results suggest that an altered interaction between the MA domain of Gag and the gp41 cytoplasmic tail leads to dissociation of gp120 from gp41 during HIV-1 particle assembly, thus resulting in impaired fusion and infectivity. PMID:16474147

  9. The cell envelope proteome of Aggregatibacter actinomycetemcomitans

    PubMed Central

    Smith, Kenneth P.; Fields, Julia G.; Voogt, Richard D.; Deng, Bin; Lam, Ying-Wai; Mintz, Keith P.

    2014-01-01

    Summary The cell envelope of Gram-negative bacteria serves a critical role in maintenance of cellular homeostasis, resistance to external stress, and host-pathogen interactions. Envelope protein composition is influenced by the physiological and environmental demands placed on the bacterium. In this study, we report a comprehensive compilation of cell envelope proteins from the periodontal and systemic pathogen Aggregatibacter actinomycetemcomitans VT1169, an afimbriated serotype b strain. The urea-extracted membrane proteins were identified by mass spectrometry-based shotgun proteomics. The membrane proteome, isolated from actively growing bacteria under normal laboratory conditions, included 648 proteins representing 28% of the predicted ORFs in the genome. Bioinformatic analyses were used to annotate and predict the cellular location and function of the proteins. Surface adhesins, porins, lipoproteins, numerous influx and efflux pumps, multiple sugar, amino acid and iron transporters, and components of the type I, II and V secretion systems were identified. Periplasmic space and cytoplasmic proteins with chaperone function were also identified. 107 proteins with unknown function were associated with the cell envelope. Orthologs of a subset of these uncharacterized proteins are present in other bacterial genomes, while others are found exclusively in A. actinomycetemcomitans. This knowledge will contribute to elucidating the role of cell envelope proteins in bacterial growth and survival in the oral cavity. PMID:25055881

  10. Enhanced Central Nervous System Transduction with Lentiviral Vectors Pseudotyped with RVG/HIV-1gp41 Chimeric Envelope Glycoproteins

    PubMed Central

    Trabalza, Antonio; Eleftheriadou, Ioanna; Sgourou, Argyro; Liao, Ting-Yi; Patsali, Petros; Lee, Heyne

    2014-01-01

    ABSTRACT To investigate the potential benefits which may arise from pseudotyping the HIV-1 lentiviral vector with its homologous gp41 envelope glycoprotein (GP) cytoplasmic tail (CT), we created chimeric RVG/HIV-1gp41 GPs composed of the extracellular and transmembrane sequences of RVG and either the full-length gp41 CT or C terminus gp41 truncations sequentially removing existing conserved motifs. Lentiviruses (LVs) pseudotyped with the chimeric GPs were evaluated in terms of particle release (physical titer), biological titers, infectivity, and in vivo central nervous system (CNS) transduction. We report here that LVs carrying shorter CTs expressed higher levels of envelope GP and showed a higher average infectivity than those bearing full-length GPs. Interestingly, complete removal of GP CT led to vectors with the highest transduction efficiency. Removal of all C-terminal gp41 CT conserved motifs, leaving just 17 amino acids (aa), appeared to preserve infectivity and resulted in a significantly increased physical titer. Furthermore, incorporation of these 17 aa in the RVG CT notably enhanced the physical titer. In vivo stereotaxic delivery of LV vectors exhibiting the best in vitro titers into rodent striatum facilitated efficient transduction of the CNS at the site of injection. A particular observation was the improved retrograde transduction of neurons in connected distal sites that resulted from the chimeric envelope R5 which included the “Kennedy” sequence (Ken) and lentivirus lytic peptide 2 (LLP2) conserved motifs in the CT, and although it did not exhibit a comparable high titer upon pseudotyping, it led to a significant increase in distal retrograde transduction of neurons. IMPORTANCE In this study, we have produced novel chimeric envelopes bearing the extracellular domain of rabies fused to the cytoplasmic tail (CT) of gp41 and pseudotyped lentiviral vectors with them. Here we report novel effects on the transduction efficiency and physical titer

  11. 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

  12. Wild-Type-Like Viral Replication Potential of Human Immunodeficiency Virus Type 1 Envelope Mutants Lacking Palmitoylation Signals

    PubMed Central

    Chan, Woan-Eng; Lin, Hui-Hua; Chen, Steve S.-L.

    2005-01-01

    Palmitoylation of the cytoplasmic domain of the human immunodeficiency type virus type 1 (HIV-1) envelope (Env) transmembrane protein, gp41, has been implicated in Env targeting to detergent-resistant lipid rafts, Env incorporation into the virus, and viral infectivity. In contrast, we provide evidence here to show that HIV-1 infectivity, Env targeting to lipid rafts, and Env incorporation into the virus are independent of cytoplasmic tail palmitoylation. The T-cell (T)-tropic HXB2-based virus, which utilizes CXCR4 as the entry coreceptor, carrying a Cys-to-Ser mutation at residue 764 or 837 or at both replicated with wild-type (WT) virus replication kinetics in CD4+ T cells. The properties of Env expression, precursor processing, cell surface expression, and Env incorporation of these three mutant viruses were normal compared to those of the WT virus. These three mutant Env proteins all effectively mediated one-cycle virus infection. When the Cys residues were replaced by Ala residues, all single and double mutants still retained the phenotypes of infectivity, Env incorporation, and lipid raft localization of the WT Env. When Cys-to-Ala substitutions were introduced into the macrophage (M)-tropic ConB virus, which utilizes CCR5 as the coreceptor, these mutations did not affect the replication potential, Env phenotypes, lipid raft targeting, or Env assembly into the virus of the WT Env. These T- and M-tropic mutants also productively replicated in human primary CD4+ T cells. Moreover, mutations at both Cys residues significantly reduced the level of palmitoylation of the Env. Our results together support the notion that palmitoylation of the cytoplasmic tail of the HIV-1 Env is not essential for the HIV-1 virus life cycle. PMID:15956582

  13. Cytoplasmic bacteriophage display system

    DOEpatents

    Studier, F.W.; Rosenberg, A.H.

    1998-06-16

    Disclosed are display vectors comprising DNA encoding a portion of a structural protein from a cytoplasmic bacteriophage, joined covalently to a protein or peptide of interest. Exemplified are display vectors wherein the structural protein is the T7 bacteriophage capsid protein. More specifically, in the exemplified display vectors the C-terminal amino acid residue of the portion of the capsid protein is joined to the N-terminal residue of the protein or peptide of interest. The portion of the T7 capsid protein exemplified comprises an N-terminal portion corresponding to form 10B of the T7 capsid protein. The display vectors are useful for high copy number display or lower copy number display (with larger fusion). Compositions of the type described herein are useful in connection with methods for producing a virus displaying a protein or peptide of interest. 1 fig.

  14. Cytoplasmic bacteriophage display system

    DOEpatents

    Studier, F. William; Rosenberg, Alan H.

    1998-06-16

    Disclosed are display vectors comprising DNA encoding a portion of a structural protein from a cytoplasmic bacteriophage, joined covalently to a protein or peptide of interest. Exemplified are display vectors wherein the structural protein is the T7 bacteriophage capsid protein. More specifically, in the exemplified display vectors the C-terminal amino acid residue of the portion of the capsid protein is joined to the N-terminal residue of the protein or peptide of interest. The portion of the T7 capsid protein exemplified comprises an N-terminal portion corresponding to form 10B of the T7 capsid protein. The display vectors are useful for high copy number display or lower copy number display (with larger fusion). Compositions of the type described herein are useful in connection with methods for producing a virus displaying a protein or peptide of interest.

  15. The study of burst pulses envelope in Ytterbium-doped fiber amplifier modulating by pulsed pump source

    NASA Astrophysics Data System (ADS)

    Wang, Lei; Yu, Haijuan; Zhang, Jingyuan; Qi, Yaoyao; Qiao, Zhongliang; Lin, Xuechun

    2016-02-01

    The characteristics of fiber amplifiers of burst pulses envelope during the propagation are comprehensively studied under the conditions of pulsed pump with low repetition rate. The variation of signal envelopes, pump power and upper-level population distribution are discussed in this paper. The impacts of linear varied and exponential varied pump to the raising edge and tailing edge of the output pulses' envelope, are mainly emphasized. Finally, the genetic algorithm is employed to reshape the pulses' envelopes.

  16. Nuclear Envelopes Properties and Physical Interactions with Nucleoplasm

    NASA Astrophysics Data System (ADS)

    Discher, Dennis; Dahl, Kris; Wilson, Kathy

    2004-03-01

    Given the stresses imposed on a cell and its organelles and the nuclear envelope's important role as a barrier between cytoplasm and nucleoplasm, we sought to measure and model mechanical properties of isolated nuclear envelopes. Xenopus laevis oocyte (XO) nuclei are primarily used since they have been widely studied in many fields as model systems for nuclear structure and function. We manipulate the nuclear envelope by both osmotic swelling and micromanipulation to determine an effective elastic modulus. We show the envelope properties are independent of the effects of the nucleoplasm. Micropipette aspiration of XO nuclei gives an effective elastic modulus of the nuclear envelope of 250 mN/m with similar results obtained from isotropic swelling of XO nuclear envelopes. The results suggest that these nuclear envelopes have relatively homogeneous properties and are highly elastic, sustaining strains of 50-100Square-net simulations and comparisons to polymer network models suggests that XO nuclear envelope physical properties are dominated by the lamin network. If applicable to nuclei in other cells, a "pre-compressed" state envisioned here would allow for significant shear flexibility, especially important for motile cells whose nuclei need to rapidly deform.

  17. Tegument Assembly and Secondary Envelopment of Alphaherpesviruses

    PubMed Central

    Owen, Danielle J.; Crump, Colin M.; Graham, Stephen C.

    2015-01-01

    Alphaherpesviruses like herpes simplex virus are large DNA viruses characterized by their ability to establish lifelong latent infection in neurons. As for all herpesviruses, alphaherpesvirus virions contain a protein-rich layer called “tegument” that links the DNA-containing capsid to the glycoprotein-studded membrane envelope. Tegument proteins mediate a diverse range of functions during the virus lifecycle, including modulation of the host-cell environment immediately after entry, transport of virus capsids to the nucleus during infection, and wrapping of cytoplasmic capsids with membranes (secondary envelopment) during virion assembly. Eleven tegument proteins that are conserved across alphaherpesviruses have been implicated in the formation of the tegument layer or in secondary envelopment. Tegument is assembled via a dense network of interactions between tegument proteins, with the redundancy of these interactions making it challenging to determine the precise function of any specific tegument protein. However, recent studies have made great headway in defining the interactions between tegument proteins, conserved across alphaherpesviruses, which facilitate tegument assembly and secondary envelopment. We summarize these recent advances and review what remains to be learned about the molecular interactions required to assemble mature alphaherpesvirus virions following the release of capsids from infected cell nuclei. PMID:26393641

  18. Closing a gap in the nuclear envelope.

    PubMed

    Vietri, Marina; Stenmark, Harald; Campsteijn, Coen

    2016-06-01

    The nuclear envelope (NE) ensures nucleo-cytoplasmic compartmentalization, with trafficking of macromolecules across this double membrane controlled by embedded nuclear pore complexes (NPCs). The NE and associated proteins are dismantled during open mitosis and reestablishment of this barrier during mitotic exit requires dynamic remodeling of endoplasmic reticulum (ER) membranes and coordination with NPC reformation, with NPC deposition continuing during subsequent interphase. In this review, we discuss recent progress in our understanding of NE reformation and nuclear pore complex generation, with special focus on work implicating the endosomal sorting complex required for transport (ESCRT) membrane remodeling machinery in these events. PMID:27016712

  19. The epididymis, cytoplasmic droplets and male fertility

    PubMed Central

    Cooper, Trevor G

    2011-01-01

    The potential of spermatozoa to become motile during post-testicular maturation, and the relationship between the cytoplasmic droplet and fertilizing capacity are reviewed. Post-testicular maturation of spermatozoa involves the autonomous induction of motility, which can occur in vivo in testes with occluded excurrent ducts and in vitro in testicular explants, and artefactual changes in morphology that appear to occur in the testis in vitro. Both modifications may reflect time-dependent oxidation of disulphide bonds of head and tail proteins. Regulatory volume decrease (RVD), which counters sperm swelling at ejaculation, is discussed in relation to loss of cytoplasmic droplets and consequences for fertility. It is postulated that: (i) fertile males possess spermatozoa with sufficient osmolytes to drive RVD at ejaculation, permitting the droplet to round up and pinch off without membrane rupture; and (ii) infertile males possess spermatozoa with insufficient osmolytes so that RVD is inadequate, the droplet swells and the resulting flagellar angulation prevents droplet loss. Droplet retention at ejaculation is a harbinger of infertility caused by failure of the spermatozoon to negotiate the uterotubal junction or mucous and reach the egg. In this hypothesis, the epididymis regulates fertility indirectly by the extent of osmolyte provision to spermatozoa, which influences RVD and therefore droplet loss. Man is an exception, because ejaculated human spermatozoa retain their droplets. This may reflect their short midpiece, approximating head length, permitting a swollen droplet to extend along the entire midpiece; this not only obviates droplet migration and flagellar angulation but also hampers droplet loss. PMID:21076437

  20. Stereopsis from contrast envelopes.

    PubMed

    Langley, K; Fleet, D J; Hibbard, P B

    1999-07-01

    We report two experiments concerning the site of the principal nonlinearity in second-order stereopsis. The first exploits the asymmetry in perceiving transparency with second-order stimuli found by Langley et al. (1998) (Proceedings of the Royal Society of London B, 265, 1837-1845) i.e. the product of a positive-valued contrast envelope and a mean-zero carrier grating can be seen transparently only when the disparities are consistent with the envelope appearing in front of the carrier. We measured the energy at the envelope frequencies that must be added in order to negate this asymmetry. We report that this amplitude can be predicted from the envelope sidebands and not from the magnitude of compressive pre-cortical nonlinearities measured by other researchers. In the second experiment, contrast threshold elevations were measured for the discrimination of envelope disparities following adaptation to sinusoidal gratings. It is reported that perception of the envelope's depth was affected most when the adapting grating was similar (in orientation and frequency) to the carrier, rather than to the contrast envelope. These results suggest that the principal nonlinearity in second-order stereopsis is cortical, occurring after orientation- and frequency-selective linear filtering. PMID:10367053

  1. The solar envelope

    NASA Technical Reports Server (NTRS)

    Burlaga, L. F.

    1971-01-01

    Processes which occur within the region between approximately 2 solar radii and 25 solar radii, which is called the solar envelope and the effect on the solar wind as seen at 1 AU are discussed. In the envelope the wind speed becomes supersonic and super-Alfvenic, the magnetic energy density is larger than the flow energy density, and the magnetic energy density is much larger than the thermal energy density. Large azimuthal gradients in the bulk speed are expected in the envelope, but the stream interactions near the outer edge of the envelope are probably relatively small. Cosmic ray observations suggest the presence of hydromagnetic waves in the envelope. The collisionless damping of such waves could heat protons out to approximately 25 solar radii and thereby cause an increase in V and T sub p consistent with the observed T sub p -V relation. A mechanism which couples protons and electrons would also heat and accelerate the wind. Alfven waves can accelerate the wind in the envelope without necessarily causing heating of protons; the Lorentz force might have a similar effect.

  2. Nuclear envelope-localized EGF family protein amphiregulin activates breast cancer cell migration in an EGF-like domain independent manner

    SciTech Connect

    Tanaka, Hisae; Nishioka, Yu; Yokoyama, Yuhki; Higashiyama, Shigeki; Matsuura, Nariaki; Matsuura, Shuji; Hieda, Miki

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer Nuclear envelope-localized proAREG activates cancer cell migration via its cytoplasmic domain. Black-Right-Pointing-Pointer The induction of cell migration does not require the EGF-like domain or EGR function. Black-Right-Pointing-Pointer Nuclear envelope-localized proAREG suppresses breast cancer cell growth without EGFR function. Black-Right-Pointing-Pointer This study revealed a novel function mediated by the intracellular domain of proAREG. -- Abstract: Amphiregulin (AREG), an EGF family protein, is synthesized as a type I transmembrane precursor (proAREG) and expressed on the cell surface with an extracellular EGF-like domain and an intracellular short cytoplasmic tail. The ectodomain shedding yields a soluble EGF receptor ligand (soluble AREG) which binds to EGF receptor (EGFR) and concomitantly induces migration of unshed proAREG from the plasma membrane to the nuclear envelope (NE). AREG is known to play a potential role in breast cancer and has been intensively investigated as an EGF receptor ligand, while the function of the NE-localized proAREG remains unknown. In this study we used a truncated mutant that mimics NE-localized proAREG without shedding stimuli to discriminate between the functions of NE-localized and plasma membrane-localized proAREG and demonstrate that NE-localized proAREG activates breast cancer cell migration, but suppresses cell growth. Moreover, the present study shows that induction of cell migration by NE-localized proAREG does not require the extracellular growth factor domain or EGF receptor function. Collectively these data demonstrate a novel function mediated by the intracellular domain of proAREG and suggest a significant role for NE-localized proAREG in driving human breast cancer progression.

  3. TssK Is a Trimeric Cytoplasmic Protein Interacting with Components of Both Phage-like and Membrane Anchoring Complexes of the Type VI Secretion System*

    PubMed Central

    Zoued, Abdelrahim; Durand, Eric; Bebeacua, Cecilia; Brunet, Yannick R.; Douzi, Badreddine; Cambillau, Christian; Cascales, Eric; Journet, Laure

    2013-01-01

    The Type VI secretion system (T6SS) is a macromolecular machine that mediates bacteria-host or bacteria-bacteria interactions. The T6SS core apparatus assembles from 13 proteins that form two sub-assemblies: a phage-like complex and a trans-envelope complex. The Hcp, VgrG, TssE, and TssB/C subunits are structurally and functionally related to components of the tail of contractile bacteriophages. This phage-like structure is thought to be anchored to the membrane by a trans-envelope complex composed of the TssJ, TssL, and TssM proteins. However, how the two sub-complexes are connected remains unknown. Here we identify TssK, a protein that establishes contacts with the two T6SS sub-complexes through direct interactions with TssL, Hcp, and TssC. TssK is a cytoplasmic protein assembling trimers that display a three-armed shape, as revealed by TEM and SAXS analyses. Fluorescence microscopy experiments further demonstrate the requirement of TssK for sheath assembly. Our results suggest a central role for TssK by linking both complexes during T6SS assembly. PMID:23921384

  4. Archaeal viruses at the cell envelope: entry and egress

    PubMed Central

    Quemin, Emmanuelle R. J.; Quax, Tessa E. F.

    2015-01-01

    The cell envelope represents the main line of host defense that viruses encounter on their way from one cell to another. The cytoplasmic membrane in general is a physical barrier that needs to be crossed both upon viral entry and exit. Therefore, viruses from the three domains of life employ a wide range of strategies for perforation of the cell membrane, each adapted to the cell surface environment of their host. Here, we review recent insights on entry and egress mechanisms of viruses infecting archaea. Due to the unique nature of the archaeal cell envelope, these particular viruses exhibit novel and unexpected mechanisms to traverse the cellular membrane. PMID:26097469

  5. FRACTIONAL CRYSTALLIZATION FEED ENVELOPE

    SciTech Connect

    HERTING DL

    2008-03-19

    Laboratory work was completed on a set of evaporation tests designed to establish a feed envelope for the fractional crystallization process. The feed envelope defines chemical concentration limits within which the process can be operated successfully. All 38 runs in the half-factorial design matrix were completed successfully, based on the qualitative definition of success. There is no feed composition likely to be derived from saltcake dissolution that would cause the fractional crystallization process to not meet acceptable performance requirements. However, some compositions clearly would provide more successful operation than other compositions.

  6. Retromer Regulates HIV-1 Envelope Glycoprotein Trafficking and Incorporation into Virions

    PubMed Central

    Groppelli, Elisabetta; Jolly, Clare

    2014-01-01

    The envelope glycoprotein (Env) of the Human Immunodeficiency Virus Type-1 (HIV-1) is a critical determinant of viral infectivity, tropism and is the main target for humoral immunity; however, little is known about the cellular machinery that directs Env trafficking and its incorporation into nascent virions. Here we identify the mammalian retromer complex as a novel and important cellular factor regulating Env trafficking. Retromer mediates endosomal sorting and is most closely associated with endosome-to-Golgi transport. Consistent with this function, inactivating retromer using RNAi targeting the cargo selective trimer complex inhibited retrograde trafficking of endocytosed Env to the Golgi. Notably, in HIV-1 infected cells, inactivating retromer modulated plasma membrane expression of Env, along with Env incorporation into virions and particle infectivity. Mutagenesis studies coupled with coimmunoprecipitations revealed that retromer-mediated trafficking requires the Env cytoplasmic tail that we show binds directly to retromer components Vps35 and Vps26. Taken together these results provide novel insight into regulation of HIV-1 Env trafficking and infectious HIV-1 morphogenesis and show for the first time a role for retromer in the late-steps of viral replication and assembly of a virus. PMID:25393110

  7. Targeting Nuclear Envelope Repair.

    PubMed

    2016-06-01

    Migrating cancer cells undergo repeated rupture of the protective nuclear envelope as they squeeze through small spaces in the surrounding tissue, compromising genomic integrity. Inhibiting both general DNA repair and the mechanism that seals these tears may enhance cell death and curb metastasis. PMID:27130435

  8. Jacketed lamp bulb envelope

    DOEpatents

    MacLennan, Donald A.; Turner, Brian P.; Gitsevich, Aleksandr; Bass, Gary K.; Dolan, James T.; Kipling, Kent; Kirkpatrick, Douglas A.; Leng, Yongzhang; Levin, Izrail; Roy, Robert J.; Shanks, Bruce; Smith, Malcolm; Trimble, William C.; Tsai, Peter

    2001-01-01

    A jacketed lamp bulb envelope includes a ceramic cup having an open end and a partially closed end, the partially closed end defining an aperture, a lamp bulb positioned inside the ceramic cup abutting the aperture, and a reflective ceramic material at least partially covering a portion of the bulb not abutting the aperture. The reflective ceramic material may substantially fill an interior volume of the ceramic cup not occupied by the bulb. The ceramic cup may include a structural feature for aiding in alignment of the jacketed lamp bulb envelope in a lamp. The ceramic cup may include an external flange about a periphery thereof. One example of a jacketed lamp bulb envelope includes a ceramic cup having an open end and a closed end, a ceramic washer covering the open end of the ceramic cup, the washer defining an aperture therethrough, a lamp bulb positioned inside the ceramic cup abutting the aperture, and a reflective ceramic material filling an interior volume of the ceramic cup not occupied by the bulb. A method of packing a jacketed lamp bulb envelope of the type comprising a ceramic cup with a lamp bulb disposed therein includes the steps of filling the ceramic cup with a flowable slurry of reflective material, and applying centrifugal force to the cup to pack the reflective material therein.

  9. COMMON ENVELOPE: ENTHALPY CONSIDERATION

    SciTech Connect

    Ivanova, N.; Chaichenets, S.

    2011-04-20

    In this Letter, we discuss a modification to the criterion for the common envelope (CE) event to result in envelope dispersion. We emphasize that the current energy criterion for the CE phase is not sufficient for an instability of the CE, nor for an ejection. However, in some cases, stellar envelopes undergo stationary mass outflows, which are likely to occur during the slow spiral-in stage of the CE event. We propose the condition for such outflows, in a manner similar to the currently standard {alpha}{sub CE}{lambda}-prescription but with an addition of P/{rho} term in the energy balance equation, accounting therefore for the enthalpy of the envelope rather than merely the gas internal energy. This produces a significant correction, which might help to dispense with an unphysically high value of energy efficiency parameter during the CE phase, currently required in the binary population synthesis studies to make the production of low-mass X-ray binaries with a black hole companion to match the observations.

  10. Cytoplasmic Domains and Voltage-Dependent Potassium Channel Gating

    PubMed Central

    Barros, Francisco; Domínguez, Pedro; de la Peña, Pilar

    2012-01-01

    The basic architecture of the voltage-dependent K+ channels (Kv channels) corresponds to a transmembrane protein core in which the permeation pore, the voltage-sensing components and the gating machinery (cytoplasmic facing gate and sensor–gate coupler) reside. Usually, large protein tails are attached to this core, hanging toward the inside of the cell. These cytoplasmic regions are essential for normal channel function and, due to their accessibility to the cytoplasmic environment, constitute obvious targets for cell-physiological control of channel behavior. Here we review the present knowledge about the molecular organization of these intracellular channel regions and their role in both setting and controlling Kv voltage-dependent gating properties. This includes the influence that they exert on Kv rapid/N-type inactivation and on activation/deactivation gating of Shaker-like and eag-type Kv channels. Some illustrative examples about the relevance of these cytoplasmic domains determining the possibilities for modulation of Kv channel gating by cellular components are also considered. PMID:22470342

  11. ESCRT III repairs nuclear envelope ruptures during cell migration to limit DNA damage and cell death.

    PubMed

    Raab, M; Gentili, M; de Belly, H; Thiam, H R; Vargas, P; Jimenez, A J; Lautenschlaeger, F; Voituriez, Raphaël; Lennon-Duménil, A M; Manel, N; Piel, M

    2016-04-15

    In eukaryotic cells, the nuclear envelope separates the genomic DNA from the cytoplasmic space and regulates protein trafficking between the two compartments. This barrier is only transiently dissolved during mitosis. Here, we found that it also opened at high frequency in migrating mammalian cells during interphase, which allowed nuclear proteins to leak out and cytoplasmic proteins to leak in. This transient opening was caused by nuclear deformation and was rapidly repaired in an ESCRT (endosomal sorting complexes required for transport)-dependent manner. DNA double-strand breaks coincided with nuclear envelope opening events. As a consequence, survival of cells migrating through confining environments depended on efficient nuclear envelope and DNA repair machineries. Nuclear envelope opening in migrating leukocytes could have potentially important consequences for normal and pathological immune responses. PMID:27013426

  12. STS-8 postal Stamp envelope

    NASA Technical Reports Server (NTRS)

    1983-01-01

    STS-8 postal Stamp envelope with Challenger insignia, USA eagle stamp, 25th NASA anniversary stamp. The envelope is stamped with various postmarks, one saying Kennedy Space Center, Fl., another saying 'Returned to earth, Edwards AFB, CA'.

  13. Tail biting in pigs.

    PubMed

    Schrøder-Petersen, D L; Simonsen, H B

    2001-11-01

    One of the costly and welfare-reducing problems in modern pig production is tail biting. Tail biting is an abnormal behaviour, characterized by one pig's dental manipulation of another pig's tail. Tail biting can be classified into two groups: the pre-injury stage, before any wound on the tail is present, and the injury stage, where the tail is wounded and bleeding. Tail biting in the injury stage will reduce welfare of the bitten pig and the possible spread of infection is a health as well as welfare problem. The pigs that become tail biters may also suffer, because they are frustrated due to living in a stressful environment. This frustration may result in an excessive motivation for biting the tails of pen mates. This review aims to summarize recent research and theories in relation to tail biting. PMID:11681870

  14. Restoration of flagellar clockwise rotation in bacterial envelopes by insertion of the chemotaxis protein CheY.

    PubMed Central

    Ravid, S; Matsumura, P; Eisenbach, M

    1986-01-01

    When cells of the bacterium Salmonella typhimurium are incubated with penicillin and lysed in a dilute buffer, flagellated cytoplasm-free envelopes are formed. When the envelopes are tethered to glass by their flagella and then energized, some of them spin. The direction of rotation of wild-type envelopes is exclusively counterclockwise (CCW). We perturbed this system by including in the lysis medium (and hence in the envelopes) the chemotaxis protein CheY. As a result, some of the envelopes rotated exclusively clockwise (CW). The fraction of envelopes that did so increased with the concentration of CheY; at a concentration of 48 microM (pH 8), all functional envelopes spun CW. The fraction also increased with the pH of the lysis medium in the range of 6.6-8.4. The results were the same in the presence or absence of intracellular Ca2+. Reconstituted envelopes failed to respond to chemotactic stimuli. None of them changed the direction of their rotation. However, when the intracellular pH was lowered to 6.6 or below, envelopes that spun CW stopped rotating, while envelopes that spun CCW continued to rotate. This phenomenon was reversible. We conclude that CheY per se, without any additional free cytoplasmic mediators, interacts with a switch at the base of the flagellum to cause CW rotation. PMID:3532103

  15. The Carboxyl Tail of Connexin32 Regulates Gap Junction Assembly in Human Prostate and Pancreatic Cancer Cells*

    PubMed Central

    Katoch, Parul; Mitra, Shalini; Ray, Anuttoma; Kelsey, Linda; Roberts, Brett J.; Wahl, James K.; Johnson, Keith R.; Mehta, Parmender P.

    2015-01-01

    Connexins, the constituent proteins of gap junctions, are transmembrane proteins. A connexin (Cx) traverses the membrane four times and has one intracellular and two extracellular loops with the amino and carboxyl termini facing the cytoplasm. The transmembrane and the extracellular loop domains are highly conserved among different Cxs, whereas the carboxyl termini, often called the cytoplasmic tails, are highly divergent. We have explored the role of the cytoplasmic tail of Cx32, a Cx expressed in polarized and differentiated cells, in regulating gap junction assembly. Our results demonstrate that compared with the full-length Cx32, the cytoplasmic tail-deleted Cx32 is assembled into small gap junctions in human pancreatic and prostatic cancer cells. Our results further document that the expression of the full-length Cx32 in cells, which express the tail-deleted Cx32, increases the size of gap junctions, whereas the expression of the tail-deleted Cx32 in cells, which express the full-length Cx32, has the opposite effect. Moreover, we show that the tail is required for the clustering of cell-cell channels and that in cells expressing the tail-deleted Cx32, the expression of cell surface-targeted cytoplasmic tail alone is sufficient to enhance the size of gap junctions. Our live-cell imaging data further demonstrate that gap junctions formed of the tail-deleted Cx32 are highly mobile compared with those formed of full-length Cx32. Our results suggest that the cytoplasmic tail of Cx32 is not required to initiate the assembly of gap junctions but for their subsequent growth and stability. Our findings suggest that the cytoplasmic tail of Cx32 may be involved in regulating the permeability of gap junctions by regulating their size. PMID:25548281

  16. Coilin Shuttles between the Nucleus and Cytoplasm In Xenopus Oocytes

    PubMed Central

    Bellini, Michel; Gall, Joseph G.

    1999-01-01

    Coiled bodies are discrete nuclear organelles often identified by the marker protein p80-coilin. Because coilin is not detected in the cytoplasm by immunofluorescence and Western blotting, it has been considered an exclusively nuclear protein. In the Xenopus germinal vesicle (GV), most coilin actually resides in the nucleoplasm, although it is highly concentrated in 50–100 coiled bodies. When affinity-purified anti-coilin antibodies were injected into the cytoplasm of oocytes, they could be detected in coiled bodies within 2–3 h. Coiled bodies were intensely labeled after 18 h, whereas other nuclear organelles remained negative. Because the nuclear envelope does not allow passive diffusion of immunoglobulins, this observation suggests that anti-coilin antibodies are imported into the nucleus as an antigen–antibody complex with coilin. Newly synthesized coilin is not required, because cycloheximide had no effect on nuclear import and subsequent targeting of the antibodies. Additional experiments with myc-tagged coilin and myc-tagged pyruvate kinase confirmed that coilin is a shuttling protein. The shuttling of Nopp140, NO38/B23, and nucleolin was easily demonstrated by the targeting of their respective antibodies to the nucleoli, whereas anti-SC35 did not enter the germinal vesicle. We suggest that coilin, perhaps in association with Nopp140, may function as part of a transport system between the cytoplasm and the coiled bodies. PMID:10512877

  17. Structure of Phage P22 Cell Envelope-Penetrating Needle

    SciTech Connect

    Olia,A.; Casjens, S.; Cingolani, G.

    2007-01-01

    Bacteriophage P22 infects Salmonella enterica by injecting its genetic material through the cell envelope. During infection, a specialized tail needle, gp26, is injected into the host, likely piercing a hole in the host cell envelope. The 2.1-Angstroms crystal structure of gp26 reveals a 240-Angstroms elongated protein fiber formed by two trimeric coiled-coil domains interrupted by a triple beta-helix. The N terminus of gp26 plugs the portal protein channel, retaining the genetic material inside the virion. The C-terminal tip of the fiber exposes beta-hairpins with hydrophobic tips similar to those seen in class II fusion peptides. The alpha-helical core connecting these two functionally polarized tips presents four trimerization octads with consensus sequence IXXLXXXV. The slender conformation of the gp26 fiber minimizes the surface exposed to solvent, which is consistent with the idea that gp26 traverses the cell envelope lipid bilayers.

  18. Structure of Phage P22 Cell Envelope-Penetrating Needle

    SciTech Connect

    Olia, A.S.; Casjens, S.; Cingolani, G.

    2009-06-02

    Bacteriophage P22 infects Salmonella enterica by injecting its genetic material through the cell envelope. During infection, a specialized tail needle, gp26, is injected into the host, likely piercing a hole in the host cell envelope. The 2.1-{angstrom} crystal structure of gp26 reveals a 240-{angstrom} elongated protein fiber formed by two trimeric coiled-coil domains interrupted by a triple {beta}-helix. The N terminus of gp26 plugs the portal protein channel, retaining the genetic material inside the virion. The C-terminal tip of the fiber exposes {beta}-hairpins with hydrophobic tips similar to those seen in class II fusion peptides. The {alpha}-helical core connecting these two functionally polarized tips presents four trimerization octads with consensus sequence IXXLXXXV. The slender conformation of the gp26 fiber minimizes the surface exposed to solvent, which is consistent with the idea that gp26 traverses the cell envelope lipid bilayers.

  19. Origin, development and ultrastructure of boar spermatozoa with folded tails and with two tails.

    PubMed

    Bonet, S; Briz, M; Fradera, A; Egozcue, J

    1992-04-01

    Spermatozoa from the three epididymal regions (head, body and tail) of healthy and sexually mature boars have been examined by light microscopy, and scanning and transmission electron microscopy. The origin, development and external and internal morphologies of aberrant spermatozoa with folded tails and spermatozoa with one or two heads and two fused tails have been established. A count carried out in each region of the epididymis indicated that significant differences (P less than 0.01) exist in the frequencies of each type of malformation and the epididymal region from which the spermatozoa come. Spermatozoa with folded tails at Jensen's ring originate in the cauda of the epididymis from immature spermatozoa that have not ejected the distal cytoplasmic droplet. The plasma membrane which covers the main piece is fused with the membranes of the midpiece, the connecting piece and the head. The fibrous sheath deforms the mitochondrial sheath and is placed between the plasma membrane and the postacrosomal dense lamina. Spermatozoa with one head and two fused tails originate in the epididymal body from spermatozoa with one head and two unfused tails coming from the cephalic region of the epididymis. Spermatozoa with two heads and two fused tails originate in the cephalic region of the epididymis by head-to-head agglutination of two spermatozoa and later fusion of their tails. The frequency of spermatozoa with two fused tails increases as they progress through the epididymal duct. Their tails, parallel in monocephalic spermatozoa and helicoid in bicephalic spermatozoa, have two complete axonemal axes. In their midpiece, the mitochondrial sheaths of the two axes are fused, producing an 8-shaped sheath. PMID:1522197

  20. Cap homeostasis is independent of poly(A) tail length

    PubMed Central

    Kiss, Daniel L.; Oman, Kenji M.; Dougherty, Julie A.; Mukherjee, Chandrama; Bundschuh, Ralf; Schoenberg, Daniel R.

    2016-01-01

    Cap homeostasis is a cyclical process of decapping and recapping that maintains the cap on a subset of the cytoplasmic transcriptome. Interfering with cytoplasmic capping results in the redistribution of target transcripts from polysomes to non-translating mRNPs, where they accumulate in an uncapped but nonetheless stable form. It is generally thought that decapping is preceded by shortening of the poly(A) tail to a length that can no longer support translation. Therefore recapped target transcripts would either have to undergo cytoplasmic polyadenylation or retain a reasonably long poly(A) tail if they are to return to the translating pool. In cells that are inhibited for cytoplasmic capping there is no change in the overall distribution of poly(A) lengths or in the elution profile of oligo(dT)-bound targets. Poly(A) tail lengths were similar for target mRNAs on polysomes or in non-translating mRNPs, and the presence of polyadenylated uncapped mRNA in mRNPs was confirmed by separation into capped and uncapped pools prior to assay. Finally, in silico analysis of cytoplasmic capping targets revealed significant correlations with genes encoding transcripts with uridylated or multiply modified 3′ ends, and genes possessing multiple 3′-untranslated regions (UTRs) generated by alternative cleavage and polyadenylation. PMID:26673707

  1. Model scattering envelopes of young stellar objects. II - Infalling envelopes

    NASA Technical Reports Server (NTRS)

    Whitney, Barbara A.; Hartmann, Lee

    1993-01-01

    We present scattered light images for models of young stellar objects surrounded by dusty envelopes. The envelopes are assumed to have finite angular momentum and are falling in steady flow onto a disk. The model envelopes include holes, such as might be created by energetic bipolar flows. We calculate images using the Monte Carlo method to follow the light scattered in the dusty envelope and circumstellar disk, assuming that the photons originate from the central source. Adopting typical interstellar medium dust opacities and expected mass infall rates for protostars of about 10 exp -6 solar mass/yr, we find that detectable amounts of optical radiation can escape from envelopes falling into a disk as small as about 10-100 AU, depending upon the viewing angle and the size of the bipolar flow cavity. We suggest that the extended optical and near-IR light observed around several young stars is scattered by dusty infalling envelopes rather than disks.

  2. Regulation of bacterial virulence gene expression by cell envelope stress responses

    PubMed Central

    Flores-Kim, Josué; Darwin, Andrew J

    2014-01-01

    The bacterial cytoplasm lies within a multilayered envelope that must be protected from internal and external hazards. This protection is provided by cell envelope stress responses (ESRs), which detect threats and reprogram gene expression to ensure survival. Pathogens frequently need these ESRs to survive inside the host, where their envelopes face dangerous environmental changes and attack from antimicrobial molecules. In addition, some virulence genes have become integrated into ESR regulons. This might be because these genes can protect the cell envelope from damage by host molecules, or it might help ESRs to reduce stress by moderating the assembly of virulence factors within the envelope. Alternatively, it could simply be a mechanism to coordinate the induction of virulence gene expression with entry into the host. Here, we briefly describe some of the bacterial ESRs, followed by examples where they control virulence gene expression in both Gram-negative and Gram-positive pathogens. PMID:25603429

  3. Refrigerated cryogenic envelope

    DOEpatents

    Loudon, John D.

    1976-11-16

    An elongated cryogenic envelope including an outer tube and an inner tube coaxially spaced within said inner tube so that the space therebetween forms a vacuum chamber for holding a vacuum. The inner and outer tubes are provided with means for expanding or contracting during thermal changes. A shield is located in the vacuum chamber intermediate the inner and outer tubes; and, a refrigeration tube for directing refrigeration to the shield is coiled about at least a portion of the inner tube within the vacuum chamber to permit the refrigeration tube to expand or contract along its length during thermal changes within said vacuum chamber.

  4. Cytoplasmic Control of Sense-Antisense mRNA Pairs.

    PubMed

    Sinturel, Flore; Navickas, Albertas; Wery, Maxime; Descrimes, Marc; Morillon, Antonin; Torchet, Claire; Benard, Lionel

    2015-09-22

    Transcriptome analyses have revealed that convergent gene transcription can produce many 3'-overlapping mRNAs in diverse organisms. Few studies have examined the fate of 3'-complementary mRNAs in double-stranded RNA-dependent nuclear phenomena, and nothing is known about the cytoplasmic destiny of 3'-overlapping messengers or their impact on gene expression. Here, we demonstrate that the complementary tails of 3'-overlapping mRNAs can interact in the cytoplasm and promote post-transcriptional regulatory events including no-go decay (NGD) in Saccharomyces cerevisiae. Genome-wide experiments confirm that these messenger-interacting mRNAs (mimRNAs) form RNA duplexes in wild-type cells and thus have potential roles in modulating the mRNA levels of their convergent gene pattern under different growth conditions. We show that the post-transcriptional fate of hundreds of mimRNAs is controlled by Xrn1, revealing the extent to which this conserved 5'-3' cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression. PMID:26344770

  5. Involvement of the Host Cell Nuclear Envelope Membranes in the Replication of Japanese Encephalitis Virus

    PubMed Central

    Zebovitz, E.; Leong, J. K. L.; Doughty, S. C.

    1974-01-01

    The distribution of viral ribonucleic acid (RNA) on various cell membrane fractions derived from a porcine kidney cell line infected with Japanese encephalitis virus was investigated. At 40 h postinfection, after virus growth had reached its peak, three viral RNAs, 45S, 27S, and 20S, were associated with the cytoplasmic membranes and intact nuclei. The amount of each RNA associated with the nucleus was two- to fivefold greater than that present with the cytoplasmic membranes. Treatment of washed infected nuclei with 1.0% Triton X-100, which removed the outer nuclear envelope membrane, also removed the viral RNA. When the nucleus was fractionated into nuclear envelope membranes and a large particle fraction which sedimented at 600 × g, nearly all of the viral RNA remained associated with the envelope membranes. The nuclear envelope membranes contained higher viral RNA polymerase activity than the cytoplasmic membranes derived from the same cells. These data suggest that major sites for Japanese encephalitis virus RNA synthesis may be localized on or in very close association with the nuclear envelope membranes. PMID:4842128

  6. Mutations at the C-terminus of the simian immunodeficiency virus envelope glycoprotein affect gp120-gp41 stability on virions

    SciTech Connect

    Affranchino, Jose L.; Gonzalez, Silvia A. . E-mail: sigonzal@ub.edu.ar

    2006-03-30

    The transmembrane (TM) subunit of the envelope (Env) glycoprotein of the simian immunodeficiency virus (SIV) contains an unusually long cytoplasmic domain of 164 amino acids. Previously, we identified domains in the SIV TM cytoplasmic tail that are necessary for Env incorporation into virions and viral infectivity. In this study, we investigated the relevance to Env function of the highly conserved sequence comprising the immediate C-terminal 19 residues of TM. To this end, small in-frame deletions as well as a premature stop codon mutation were introduced into the coding region for the SIV TM C-terminus. All the mutant Env glycoproteins were expressed, processed and transported to the cell surface in an essentially wild-type manner. Moreover, the ability of the mutant Env proteins to mediate cell-to-cell fusion was similar to or slightly lower than that of the wild-type Env. However, viruses expressing the mutant Env glycoproteins were found to be poorly infectious in single-cycle infectivity assays. Further characterization of the TM mutant viruses revealed that while exhibiting wild-type levels of the TM protein, they contained significantly lower levels of the Env surface (SU) subunit, which is consistent with increased SU shedding from virions after Env incorporation. This phenotype was independent of Gag processing, since genetic inactivation of the viral protease did not increase SU retention by the resulting immature particles. Our findings indicate that deletions at the C-terminus of the SIV Env promote the instability of the SU-TM association on the virion surface and point to an important role for the TM cytoplasmic domain in modulating Env structure.

  7. Envelope Glycoprotein Internalization Protects Human and Simian Immunodeficiency Virus-Infected Cells from Antibody-Dependent Cell-Mediated Cytotoxicity

    PubMed Central

    von Bredow, Benjamin; Arias, Juan F.; Heyer, Lisa N.; Gardner, Matthew R.; Farzan, Michael; Rakasz, Eva G.

    2015-01-01

    ABSTRACT The cytoplasmic tails of human and simian immunodeficiency virus (HIV and SIV, respectively) envelope glycoproteins contain a highly conserved, membrane-proximal endocytosis motif that prevents the accumulation of Env on the surface of infected cells prior to virus assembly. Using an assay designed to measure the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC), we show that substitutions in this motif increase the susceptibility of HIV-1- and SIV-infected cells to ADCC in a manner that directly correlates with elevated Env levels on the surface of virus-infected cells. In the case of HIV-1, this effect is additive with a deletion in vpu recently shown to enhance the susceptibility of HIV-1-infected cells to ADCC as a result of tetherin-mediated retention of budding virions on the cell surface. These results reveal a previously unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from antibody responses by regulating the amount of Env present on the cell surface. IMPORTANCE This study reveals an unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from elimination by Env-specific antibodies. Thus, strategies designed to interfere with this mechanism of Env internalization may improve the efficacy of antibody-based vaccines and antiretroviral therapies designed to enhance the immunological control of HIV-1 replication in chronically infected individuals. PMID:26269175

  8. Anisotropic charged core envelope star

    NASA Astrophysics Data System (ADS)

    Mafa Takisa, P.; Maharaj, S. D.

    2016-08-01

    We study a charged compact object with anisotropic pressures in a core envelope setting. The equation of state is quadratic in the core and linear in the envelope. There is smooth matching between the three regions: the core, envelope and the Reissner-Nordström exterior. We show that the presence of the electric field affects the masses, radii and compactification factors of stellar objects with values which are in agreement with previous studies. We investigate in particular the effect of electric field on the physical features of the pulsar PSR J1614-2230 in the core envelope model. The gravitational potentials and the matter variables are well behaved within the stellar object. We demonstrate that the radius of the core and the envelope can vary by changing the parameters in the speed of sound.

  9. Maturation-Dependent Human Immunodeficiency Virus Type 1 Particle Fusion Requires a Carboxyl-Terminal Region of the gp41 Cytoplasmic Tail▿

    PubMed Central

    Jiang, Jiyang; Aiken, Christopher

    2007-01-01

    Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), typically encode fusion glycoproteins with long cytoplasmic tails (CTs). We previously reported that immature HIV-1 particles are inhibited for fusion with target cells by a mechanism requiring the 152-amino-acid CT of gp41. The gp41 CT was also shown to mediate the detergent-resistant association of the HIV-1 envelope glycoprotein complex with immature HIV-1 particles, indicating that the gp41 CT forms a stable complex with Gag in immature virions. In the present study, we analyzed the effects of progressive truncations and point mutations in the gp41 CT on the fusion of mature and immature HIV-1 particles with target cells. We also determined the effects of these mutations on the detergent-resistant association of gp41 with immature HIV-1 particles. Removal of the C-terminal 28 amino acids relieved the dependence of HIV-1 fusion on maturation. However, a mutant Env protein lacking this region remained associated with immature HIV-1 particles treated with nonionic detergent. Further mutational analysis of the C-terminal region of gp41 revealed two specific sequences required for maturation-dependent HIV-1 fusion. Collectively, our results demonstrate that the extreme C terminus of gp41 plays a key role in coupling HIV-1 fusion competence to virion maturation. They further indicate that the stable association of gp41 with Gag in immature virions is not sufficient for inhibition of immature HIV-1 particle fusion. PMID:17609279

  10. Simulation on particle crushing of tailings material under high pressure

    NASA Astrophysics Data System (ADS)

    Liu, Hai-ming; Liu, Yi-ming; Yang, Chun-he; Cao, Jing

    2013-06-01

    With continuous increase of the high tailings dam, it has an important practical and theoretical significance to study the mechanical characteristics of the tailings material under high pressures. It is indicated that strength envelopes of the tailings material have a remarkable nonlinear characteristics through the triaxial test under high pressures. A further study stated that the particle crushing has a critical effect on the mechanical behavior of the tailings material. In order to quantitatively research its influence, the grain size distribution of the tailings material is analyzed for pre-and post-test and the particle crushing of the tailings material is measured. The particle flow code is employed to simulate and monitor the sample during testing. Firstly, a model which considers the particle crushing is built under the plane strain condition. Then, a series of biaxial numerical tests of the tailings specimen are simulated by using the model. It is found that the simulation result agrees with the triaxial test. Finally, a law between the particle crushing and strain of the tailings material under different confining pressures is obtained.

  11. Nucleocytoplasmic transport of nucleocapsid proteins of enveloped RNA viruses

    PubMed Central

    Wulan, Wahyu N.; Heydet, Deborah; Walker, Erin J.; Gahan, Michelle E.; Ghildyal, Reena

    2015-01-01

    Most viruses with non-segmented single stranded RNA genomes complete their life cycle in the cytoplasm of infected cells. However, despite undergoing replication in the cytoplasm, the structural proteins of some of these RNA viruses localize to the nucleus at specific times in the virus life cycle, primarily early in infection. Limited evidence suggests that this enhances successful viral replication by interfering with or inhibiting the host antiviral response. Nucleocapsid proteins of RNA viruses have a well-established, essential cytoplasmic role in virus replication and assembly. Intriguingly, nucleocapsid proteins of some RNA viruses also localize to the nucleus/nucleolus of infected cells. Their nuclear function is less well understood although significant advances have been made in recent years. This review will focus on the nucleocapsid protein of cytoplasmic enveloped RNA viruses, including their localization to the nucleus/nucleolus and function therein. A greater understanding of the nuclear localization of nucleocapsid proteins has the potential to enhance therapeutic strategies as it can be a target for the development of live-attenuated vaccines or antiviral drugs. PMID:26082769

  12. Isolation of Cytoplasmic Enzymes from Pollen 1

    PubMed Central

    Weeden, Norman F.; Gottlieb, Leslie D.

    1980-01-01

    The cytoplasmic isozyme of many cytoplasmic-organelle isozyme pairs, as well as other cytoplasmic enzymes in plants, can be readily obtained from pollen by soaking it in an appropriate buffer for 4 hours. Enzymes localized in subcellular organelles appear not to be released during the soaking period, although they are released if the pollen is crushed. The technique is a useful initial step in studies of subcellular localization of enzymes or for obtaining small quantities of cytoplasmic enzymes free of organellar contaminants. Images PMID:16661444

  13. Multifamily Envelope Leakage Model

    SciTech Connect

    Faakye, Omari; Griffiths, Dianne

    2015-05-08

    “The cost for blower testing is high, because it is labor intensive, and it may disrupt occupants in multiple units. This high cost and disruption deter program participants, and dissuade them from pursuing energy improvements that would trigger air leakage testing, such as improvements to the building envelope.” This statement found in a 2012 report by Heschong Mahone Group for several California interests emphasizes the importance of reducing the cost and complexity of blower testing in multifamily buildings. Energy efficiency opportunities are being bypassed. The cost of single blower testing is on the order of $300. The cost for guarded blower door testing—the more appropriate test for assessing energy savings opportunities—could easily be six times that, and that’s only if you have the equipment and simultaneous access to multiple apartments. Thus, the proper test is simply not performed. This research seeks to provide an algorithm for predicting the guarded blower door test result based upon a single, total blower door test.

  14. The Type VI Secretion TssEFGK-VgrG Phage-Like Baseplate Is Recruited to the TssJLM Membrane Complex via Multiple Contacts and Serves As Assembly Platform for Tail Tube/Sheath Polymerization

    PubMed Central

    Boyer, Frédéric; Douzi, Badreddine; Cascales, Eric

    2015-01-01

    The Type VI secretion system (T6SS) is a widespread weapon dedicated to the delivery of toxin proteins into eukaryotic and prokaryotic cells. The 13 T6SS subunits assemble a cytoplasmic contractile structure anchored to the cell envelope by a membrane-spanning complex. This structure is evolutionarily, structurally and functionally related to the tail of contractile bacteriophages. In bacteriophages, the tail assembles onto a protein complex, referred to as the baseplate, that not only serves as a platform during assembly of the tube and sheath, but also triggers the contraction of the sheath. Although progress has been made in understanding T6SS assembly and function, the composition of the T6SS baseplate remains mostly unknown. Here, we report that six T6SS proteins–TssA, TssE, TssF, TssG, TssK and VgrG–are required for proper assembly of the T6SS tail tube, and a complex between VgrG, TssE,-F and-G could be isolated. In addition, we demonstrate that TssF and TssG share limited sequence homologies with known phage components, and we report the interaction network between these subunits and other baseplate and tail components. In agreement with the baseplate being the assembly platform for the tail, fluorescence microscopy analyses of functional GFP-TssF and TssK-GFP fusion proteins show that these proteins assemble stable and static clusters on which the sheath polymerizes. Finally, we show that recruitment of the baseplate to the apparatus requires initial positioning of the membrane complex and contacts between TssG and the inner membrane TssM protein. PMID:26460929

  15. The kinase domain of mitochondrial PINK1 faces the cytoplasm

    PubMed Central

    Zhou, Chun; Huang, Yong; Shao, Yufang; May, Jessica; Prou, Delphine; Perier, Celine; Dauer, William; Schon, Eric A.; Przedborski, Serge

    2008-01-01

    Mutations in PTEN-induced putative kinase 1 (PINK1) are a cause of autosomal recessive familial Parkinson's disease (PD). Efforts in deducing the PINK1 signaling pathway have been hindered by controversy around its subcellular and submitochondrial localization and the authenticity of its reported substrates. We show here that this mitochondrial protein exhibits a topology in which the kinase domain faces the cytoplasm and the N-terminal tail is inside the mitochondria. Although deletion of the transmembrane domain disrupts this topology, common PD-linked PINK1 mutations do not. These results are critical in rectifying the location and orientation of PINK1 in mitochondria, and they should help decipher its normal physiological function and potential pathogenic role in PD. PMID:18687899

  16. Measurement of Cytoplasmic Streaming in Drosophila Melanogaster

    NASA Astrophysics Data System (ADS)

    Ganguly, Sujoy; Williams, Lucy; Palacios, Isabel; Goldstein, Raymond

    2010-11-01

    During stage 9 of Drosophila melanogastor oogenesis flow of the oocyte cytoplasm, driven by kinesin 1 motor protein is observed. This cytoplasmic streaming is analyzed by PIV in both wild type and kinesin light chain mutants, revealing striking statistical differences. Further measurements of the rheology of the oocyte allow for estimations of the mechanical energy needed to generate the observed flows.

  17. Functional analysis of the cytoplasmic domain of the integrin {alpha}1 subunit in endothelial cells.

    PubMed

    Abair, Tristin D; Bulus, Nada; Borza, Corina; Sundaramoorthy, Munirathinam; Zent, Roy; Pozzi, Ambra

    2008-10-15

    Integrin alpha1beta1, the major collagen type IV receptor, is expressed by endothelial cells and plays a role in both physiologic and pathologic angiogenesis. Because the molecular mechanisms whereby this collagen IV receptor mediates endothelial cell functions are poorly understood, truncation and point mutants of the integrin alpha1 subunit cytoplasmic tail (amino acids 1137-1151) were generated and expressed into alpha1-null endothelial cells. We show that alpha1-null endothelial cells expressing the alpha1 subunit, which lacks the entire cytoplasmic tail (mutant alpha1-1136) or expresses all the amino acids up to the highly conserved GFFKR motif (mutant alpha1-1143), have a similar phenotype to parental alpha1-null cells. Pro(1144) and Leu(1145) were shown to be necessary for alpha1beta1-mediated endothelial cell proliferation; Lys(1146) for adhesion, migration, and tubulogenesis and Lys(1147) for tubulogenesis. Integrin alpha1beta1-dependent endothelial cell proliferation is primarily mediated by ERK activation, whereas migration and tubulogenesis require both p38 MAPK and PI3K/Akt activation. Thus, distinct amino acids distal to the GFFKR motif of the alpha1 integrin cytoplasmic tail mediate activation of selective downstream signaling pathways and specific endothelial cell functions. PMID:18647959

  18. Cytoplasmic alkalization reduces calcium buffering in molluscan central neurons.

    PubMed

    Zucker, R S

    1981-11-23

    The effect of raised cytoplasmic pH (pHi) on intracellular concentration ([Ca2+]i) transients following calcium influx during membrane depolarization was studied in identified neurons in the abdominal ganglion of Aplysia californica. The pHi was monitored with pH-sensitive microelectrodes. Sea water containing 15 mM NH4Cl at pH 7.7 elevated pHi about 0.35 pH units from the normal level of 7.17. These cells have an estimated buffering power of about 60 mM/pH unit. Calcium influx was elicited by depolarizing pulses under voltage clamp and [Ca2+]i transients were monitored with the photoprotein aequorin or the metallochromic dye arsenazo III. Aequorin photo-emissions increased by 21--131% (mean, 48%) and arsenazo III absorbance changes accompanying depolarization increased by 9--33% (mean, 20%) after 30 min in NH4+, corresponding roughly to a 14% increase in [Ca2+]i transients. Calcium-dependent potassium tail currents following a depolarizing pulse were somewhat slower and 4--91% (mean, 38%) large in NH4+. The magnitude and time- and voltage-dependence of the membrane calcium conductance was studied using calcium tail currents following depolarizing pulses. The calcium current was unaffected by NH4+, so the enhanced [Ca2+]i transients must reflect reduced calcium buffering at high pHi. Either reduced cytoplasmic calcium binding or slowed active extrusion of calcium may be responsible for this effect. PMID:6271335

  19. Cytoplasmic Streaming - Skylab Student Experiment ED-63

    NASA Technical Reports Server (NTRS)

    1973-01-01

    This chart describes the Skylab student experiment (ED-63), Cytoplasmic Streaming, proposed by Cheryl A. Peitz of Arapahoe High School, Littleton, Colorado. Experiment ED-63 was to observe the effect of zero-gravity on cytoplasmic streaming in the aquatic plant named Elodea, commonly called water weed or water thyme. The phenomenon of cytoplasmic streaming is not well understood, but it is recognized as the circulation mechanism of the internal materials or cytoplasm of a cell. Cytoplasm is a gelatinous substance that has the ability to change its viscosity and flow, carrying various cell materials with it. The activity can be stimulated by sunlight or heat. In March 1972, NASA and the National Science Teachers Association selected 25 experiment proposals for flight on Skylab. Science advisors from the Marshall Space Flight Center aided and assisted the students in developing the proposals for flight on Skylab.

  20. The diagnostic pathology of the nuclear envelope in human cancers.

    PubMed

    Fischer, Andrew H

    2014-01-01

    Cancer is still diagnosed on the basis of altered tissue and cellular morphology. The criteria that pathologists use for diagnosis include many morphologically distinctive alterations in the nuclear envelope (NE). With the expectation that diagnostic NE changes will have biological relevance to cancer, a classification of the various types of NE structural changes into three groups is proposed. The first group predicts chromosomal instability. The changes in this group include pleomorphism of lamina size and shape, as if constraints to maintain a spherical shape were lost. Also characteristic of chromosomal instability are the presence of micronuclei, a specific structural feature likely related to the newly described physiology of chromothripsis. The second group is predicted to be functionally important during clonal evolution, because the NE changes in this group are conserved during the clonal evolution of genetically unstable tumors. Two examples of this group include increased ratio of nuclear volume to cytoplasmic volume and the relatively fragile nuclei of small-cell carcinomas. The third and most interesting group develops in a near-diploid, genetically stable background. Many of these (perhaps ultimately all) are directly related to the activation of particular oncogenes. The changes in this group so far include long inward folds of the NE and spherical invaginations of cytoplasm projecting partially into the nucleus ("intranuclear cytoplasmic inclusions"). This group is exemplified by papillary thyroid carcinoma in which RET and TRK tyrosine kinases, and probably B-Raf mutations, directly lead to diagnostic longitudinal folds of the lamina ("nuclear grooves") and intranuclear cytoplasmic inclusions. B-Raf activation may also be linked to intranuclear cytoplasmic inclusions in melanoma and to nuclear grooves in Langerhans cell histiocytosis. Nuclear grooves in granulosa cell tumor may be related to mutations in the FOXL2 oncogene. Uncovering the precise

  1. Nucleocapsid Protein from Fig Mosaic Virus Forms Cytoplasmic Agglomerates That Are Hauled by Endoplasmic Reticulum Streaming

    PubMed Central

    Ishikawa, Kazuya; Miura, Chihiro; Maejima, Kensaku; Komatsu, Ken; Hashimoto, Masayoshi; Tomomitsu, Tatsuya; Fukuoka, Misato; Yusa, Akira; Yamaji, Yasuyuki

    2014-01-01

    ABSTRACT Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of Fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while nonenveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant negative inhibition of the class XI myosins, XI-1, XI-2, and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localize in the cytoplasm but in close proximity to the ER membrane to form enveloped particles and that this causes passive movements of cytoplasmic NBs by ER streaming. IMPORTANCE Intracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors but have failed to provide evidence for the mechanism or biological significance of this motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly

  2. Deep cytoplasmic rearrangements in ventralized Xenopus embryos

    NASA Technical Reports Server (NTRS)

    Brown, E. E.; Denegre, J. M.; Danilchik, M. V.

    1993-01-01

    Following fertilization in Xenopus, dramatic rearrangements of the egg cytoplasm relocalize maternally synthesized egg components. During the first cell cycle the vegetal yolk mass rotates relative to the egg surface, toward the sperm entry point (SEP) (J. P. Vincent, G. F. Oster, and J. C. Gerhart, 1986, Dev. Biol. 113, 484-500), while concomitant deep cytoplasmic rearrangements occur in the animal hemisphere (M. V. Danilchik and J. M. Denegre, 1991, Development 111, 845-856). In this paper we examine the role of vegetal yolk mass rotation in producing the animal cytoplasmic rearrangements. We inhibited rotation by uv-irradiating embryos during the first cell cycle, a treatment that yields an extremely ventralized phenotype. Both uv-irradiated embryos and unirradiated control embryos show cytoplasmic rearrangements in the animal hemisphere during the first cell cycle. Cytoplasmic rearrangements on the SEP side of the embryo associated with the path of the sperm pronucleus, plus a swirl on the anti-SEP (dorsal) side, are seen, whether or not yolk mass rotation has occurred. This result suggests a role for the expanding sperm aster in directing animal hemisphere cytoplasmic movements. In unirradiated control embryos the anti-SEP (dorsal) swirl is larger than that in uv-irradiated embryos and often extends into the vegetal hemisphere, consistent with the animal cytoplasm having been pulled dorsally and vegetally by the sliding vegetal yolk mass. Thus the yolk mass rotation may normally enhance the dorsalward cytoplasmic movement, begun by the sperm aster, enough to induce normal axis formation. We extended our observations of unirradiated control and uv-irradiated embryos through early cleavages. The vegetal extent of the anti-SEP (dorsal) swirl pattern seen in control embryos persists through the early cleavage period, such that labeled animal cytoplasm extends deep into dorsal third-tier blastomeres at the 32-cell stage. Significantly, in uv-irradiated embryos

  3. Fumarate or a fumarate metabolite restores switching ability to rotating flagella of bacterial envelopes.

    PubMed Central

    Barak, R; Eisenbach, M

    1992-01-01

    Flagella of cytoplasm-free envelopes of Escherichia coli or Salmonella typhimurium can rotate in either the counterclockwise or clockwise direction, but they never switch from one direction of rotation to another. Exogenous fumarate, in the intracellular presence of the chemotaxis protein CheY, restored switching ability to envelopes, with a concomitant increase in clockwise rotation. An increase in clockwise rotation was also observed after fumarate was added to partially lysed cells of E. coli, but the proportion of switching cells remained unchanged. Images PMID:1729255

  4. Passage of heme-iron across the envelope of Staphylococcus aureus.

    PubMed

    Mazmanian, Sarkis K; Skaar, Eric P; Gaspar, Andrew H; Humayun, Munir; Gornicki, Piotr; Jelenska, Joanna; Joachmiak, Andrzej; Missiakas, Dominique M; Schneewind, Olaf

    2003-02-01

    The cell wall envelope of Gram-positive pathogens functions as a scaffold for the attachment of virulence factors and as a sieve that prevents diffusion of molecules. Here the isd genes (iron-regulated surface determinant) of Staphylococcus aureus were found to encode factors responsible for hemoglobin binding and passage of heme-iron to the cytoplasm, where it acts as an essential nutrient. Heme-iron passage required two sortases that tether Isd proteins to unique locations within the cell wall. Thus, Isd appears to act as an import apparatus that uses cell wall-anchored proteins to relay heme-iron across the bacterial envelope. PMID:12574635

  5. Macromolecular Transport between the Nucleus and the Cytoplasm: Advances in Mechanism and Emerging Links to Disease

    PubMed Central

    Tran, Elizabeth J.; King, Megan C.; Corbett, Anita H.

    2014-01-01

    Transport of macromolecules between the cytoplasm and the nucleus is critical for the function of all eukaryotic cells. Large macromolecular channels termed nuclear pore complexes that span the nuclear envelope mediate the bidirectional transport of cargoes between the nucleus and cytoplasm. However, the influence of macromolecular trafficking extends past the nuclear pore complex to transcription and RNA processing within the nucleus and signaling pathways that reach into the cytoplasm and beyond. At the Mechanisms of Nuclear Transport biennial meeting held from October 18-23, 2013 in Woods Hole, MA, researchers in the field met to report on their recent findings. The work presented highlighted significant advances in understanding nucleocytoplasmic trafficking including how transport receptors and cargoes pass through the nuclear pore complex, the many signaling pathways that impinge on transport pathways, interplay between the nuclear envelope, nuclear pore complexes, and transport pathways, and numerous links between transport pathways and human disease. The goal of this review is to highlight newly emerging themes in nuclear transport and underscore the major questions that are likely to be the focus of future research in the field. PMID:25116306

  6. The Tail of BPM

    NASA Astrophysics Data System (ADS)

    Kruba, Steve; Meyer, Jim

    Business process management suites (BPMS's) represent one of the fastest growing segments in the software industry as organizations automate their key business processes. As this market matures, it is interesting to compare it to Chris Anderson's 'Long Tail.' Although the 2004 "Long Tail" article in Wired magazine was primarily about the media and entertainment industries, it has since been applied (and perhaps misapplied) to other markets. Analysts describe a "Tail of BPM" market that is, perhaps, several times larger than the traditional BPMS product market. This paper will draw comparisons between the concepts in Anderson's article (and subsequent book) and the BPM solutions market.

  7. Review of Design Approaches Applicable to Dewatering Uranium Mill Tailings Disposal Pits

    SciTech Connect

    Gutknecht, P. J.; Gates, T. E.

    1982-03-01

    This report is a review of design approaches in the literature that may be applicable to uranium mill tailings drainage. Tailings dewatering is required in the deep mined-out pits used for wet tailings disposal. Agricultural drainage theory is reviewed because it is seen as the most applicable technology. It is concluded that the standard drain-pipe envelope design criteria should be easily adapted. The differences in dewatering objectives and physical characteristics between agricultural and tailings drainage systems prevent direct technology transfer with respect to drain spacing calculations. Recommendations for further research are based on the drainage features unique to uranium mill tailings. It is recommended that transient solutions be applied to describe liquid movement through saturated and partially saturated tailings. Modeling should be used to evaluate the benefits of drainage design approaches after careful consideration of potential construction problems.

  8. Cytoplasmic rearrangements associated with amphibian egg symmetrization

    NASA Technical Reports Server (NTRS)

    Malacinski, G. M.

    1984-01-01

    Cytoplasmic rearrangements which follow fertilization were mentioned in normal and inverted eggs. A set of yolk compartments was resolved by cytological analyses of both normally oriented and inverted eggs. Those compartments were characterized by their yolk platelet compositions and movement during egg inversion. It is found that during egg inversion the yolk compartments shift minor cytoplasmic compartments which line the egg cortex. Those yolk mass shifts occurred only after the inverted egg was activated. The direction of shift of the major yolk components, rather than the sperm entrance site, determines the dorsal/ventral polarity of the inverted egg. Among different spawnings the rate of shift varied. Eggs that displayed the fastest rate of shift exhibited the highest frequency of developmental abnormalities during organogenesis. Interpretation of novel observations on cytoplasmic organization provide criticism of some earlier models. A new density compartment model is presented as a coherent way to view the organization of the egg cytoplasm and the development of bilateral symmetry.

  9. Wagging tail vibration absorber

    NASA Technical Reports Server (NTRS)

    Barclay, R. G.; Humphrey, P. W.

    1969-01-01

    A 750-foot cantilever length of extendible-tape boom (very low stiffness) was considered as the main system to be damped. A number of tail lengths were tried from 20 feet to 80 feet after which 40 feet was investigated further as a desirable compromise between performance and practical lengths. A 40-foot damping tail produced a damping effect on the main boom for the first mode equivalent in decay rate to 3.1 percent of critical damping. In this case the spring-hinge and tail were tuned to the main boom first mode frequency and the hinge damping was set at 30 percent of critical based on the tail properties. With this same setting, damping of the second mode was .4 percent and the third mode .1 percent.

  10. Heat Recovery in Building Envelopes

    SciTech Connect

    Sherman, Max H.; Walker, Iain S.

    2001-01-01

    Infiltration has traditionally been assumed to contribute to the energy load of a building by an amount equal to the product of the infiltration flow rate and the enthalpy difference between inside and outside. Application of such a simple formula may produce an unreasonably high contribution because of heat recovery within the building envelope. Previous laboratory and simulation research has indicated that such heat transfer between the infiltrating air and walls may be substantial. In this study, Computational Fluid Dynamics was used to simulate sensible heat transfer in typical envelope constructions. The results show that the traditional method may over-predict the infiltration energy load by up to 95 percent at low leakage rates. A simplified physical model has been developed and used to predict the infiltration heat recovery based on the Peclet number of the flow and the fraction of the building envelope active in infiltration heat recovery.

  11. Envelope Inflation or Stellar Wind?

    NASA Astrophysics Data System (ADS)

    Ro, S.; Matzner, C. D.

    We an optically-thick, transonic, steady wind model for a H-free Wolf-Rayet star. A bifurcation is found across a critical mass loss rate Mb. Slower winds M < Mb extend by several hydrostatic stellar radii, reproduce features of envelope in ation from Petrovic et al. (2006) and Gräfener et al. (2012), and are energetically unbound. This work is of particular interest for extended envelopes and winds, radiative hydrodynamic instabilities (eg. wind stagnation, clumping, etc.), and NLTE atmospheric models.

  12. Carbon chemistry of circumstellar envelopes

    NASA Technical Reports Server (NTRS)

    Bieging, John H.

    1990-01-01

    The chemical composition of envelopes surrounding cool evolved stars, as determined from microwave spectroscopic observations, is reviewed. Emphasis is placed on recent observations with the new large mm-wavelength telescopes and interferometer arrays, and on new theoretical work, especially concerning ion-molecule chemistry of carbon-bearing in these envelopes. Thermal (as opposed to maser) emission lines are discussed. Much progress has been made in the past few years in the theoretical understanding of these objects. It is already clear, however, that observations with the new generation of mm-telescopes will require substantial improvements in the theoretical models to achieve a thorough understanding of the data now becoming available.

  13. Dopamine D2 receptor activates extracellular signal-regulated kinase through the specific region in the third cytoplasmic loop.

    PubMed

    Takeuchi, Yusuke; Fukunaga, Kohji

    2004-06-01

    To investigate whether the third cytoplasmic loop and the C-terminal cytoplasmic tail of dopamine D(2) receptor (D2R) are involved in extracellular signal-regulated kinase (ERK) activation and subsequent regulation of transcription factors, we established NG108-15 cells stably expressing D2LR and D2SR deleted 40 amino acid residues in the third cytoplasmic loop (NGD2LR-3rd-dele and NGD2SR-3rd-dele) or the C-terminal cytoplasmic tail (NGD2LR-C-dele and NGD2SR-C-dele) and evaluated these receptors' functions using luciferase reporter gene assay. Immunocytochemical studies showed similar intracellular distributions of D2LR-3rd-dele and D2SR-3rd-dele to D2LR and D2SR, respectively. Quinpirole-induced inhibition of forskolin-induced cyclic AMP responsive element (CRE) activation was not affected by the deletion of 40 amino acid residues. However, nuclear factor-kappa B (NF-kappaB) activation by D2R-3rd-dele was largely attenuated compared to that by D2R. Similarly, ERK or serum-responsive element (SRE) activation by quinpirole treatment was totally abolished in NGD2R-3rd-dele cells. Moreover, D2R-C-dele was diffusely distributed or clustered in the cell bodies and lost the receptor functions. Taken together, the 40 amino acid residues in the third cytoplasmic loop are essential for the ERK activation but not for inhibition of adenylyl cyclase through Gi/o proteins. In addition, the C-terminal cytoplasmic tail is essential for membrane association of D2Rs to elicit the receptor functions. PMID:15189353

  14. Dual Protein Localization to the Envelope and Thylakoid Membranes Within the Chloroplast.

    PubMed

    Klasek, Laura; Inoue, Kentaro

    2016-01-01

    The chloroplast houses various metabolic processes essential for plant viability. This organelle originated from an ancestral cyanobacterium via endosymbiosis and maintains the three membranes of its progenitor. Among them, the outer envelope membrane functions mainly in communication with cytoplasmic components while the inner envelope membrane houses selective transport of various metabolites and the biosynthesis of several compounds, including membrane lipids. These two envelope membranes also play essential roles in import of nuclear-encoded proteins and in organelle division. The third membrane, the internal membrane system known as the thylakoid, houses photosynthetic electron transport and chemiosmotic phosphorylation. The inner envelope and thylakoid membranes share similar lipid composition. Specific targeting pathways determine their defined proteomes and, thus, their distinct functions. Nonetheless, several proteins have been shown to exist in both the envelope and thylakoid membranes. These proteins include those that play roles in protein transport, tetrapyrrole biosynthesis, membrane dynamics, or transport of nucleotides or inorganic phosphate. In this review, we summarize the current knowledge about proteins localized to both the envelope and thylakoid membranes in the chloroplast, discussing their roles in each membrane and potential mechanisms of their dual localization. Addressing the unanswered questions about these dual-localized proteins should help advance our understanding of chloroplast development, protein transport, and metabolic regulation. PMID:26944623

  15. The mechanics of motility in dissociated cytoplasm.

    PubMed Central

    Dembo, M

    1986-01-01

    We stimulate the dynamical behavior of dissociated cytoplasm using the Reactive Flow Model (Dembo, M., and F. Harlow, 1986, Biophys. J., 50:109-121). We find that for the most part the predicted dynamical behavior of the cytoplasm is governed by three nondimensional numbers. Several other nondimensional parameters, the initial conditions, and boundary conditions are found to have lesser effects. Of the three major nondimensional parameters, one (D#) controls the percentage of ectoplasm, the second (C#) controls the sharpness of the endoplasm-ectoplasm boundary, and the third (R#) controls the topological complexity of the endoplasm-ectoplasm distribution. If R# is very small, then the cytoplasm contracts into a single uniform mass, and there is no bulk streaming. If R# is very large, then the cytoplasmic mass breaks up into a number of clumps scattered throughout the available volume. Between these clumps the solution undergoes turbulent or chaotic patterns of streaming. Intermediate values of R# can be found such that the mass of cytoplasm remains connected and yet undergoes coherent modes of motility similar to flares (Taylor, D.L., J.S. Condeelis, P.L. Moore, and R.D. Allen, 1973, J. Cell Biol., 59:378-394) and rosettes (Kuroda, K., 1979, Cell Motility: Molecules and Organization, 347-362). Images FIGURE 1 FIGURE 1B FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 FIGURE 8 FIGURE 9 PMID:3801576

  16. Nuclear envelope breakdown induced by herpes simplex virus type 1 involves the activity of viral fusion proteins

    SciTech Connect

    Maric, Martina; Haugo, Alison C.; Dauer, William; Johnson, David; Roller, Richard J.

    2014-07-15

    Herpesvirus infection reorganizes components of the nuclear lamina usually without loss of integrity of the nuclear membranes. We report that wild-type HSV infection can cause dissolution of the nuclear envelope in transformed mouse embryonic fibroblasts that do not express torsinA. Nuclear envelope breakdown is accompanied by an eight-fold inhibition of virus replication. Breakdown of the membrane is much more limited during infection with viruses that lack the gB and gH genes, suggesting that breakdown involves factors that promote fusion at the nuclear membrane. Nuclear envelope breakdown is also inhibited during infection with virus that does not express UL34, but is enhanced when the US3 gene is deleted, suggesting that envelope breakdown may be enhanced by nuclear lamina disruption. Nuclear envelope breakdown cannot compensate for deletion of the UL34 gene suggesting that mixing of nuclear and cytoplasmic contents is insufficient to bypass loss of the normal nuclear egress pathway. - Highlights: • We show that wild-type HSV can induce breakdown of the nuclear envelope in a specific cell system. • The viral fusion proteins gB and gH are required for induction of nuclear envelope breakdown. • Nuclear envelope breakdown cannot compensate for deletion of the HSV UL34 gene.

  17. Xenopus egg cytoplasm with intact actin.

    PubMed

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts. PMID:24630119

  18. Development of tissue-targeting hemagglutinating virus of Japan envelope vector for successful delivery of therapeutic gene to mouse skin.

    PubMed

    Kawachi, Masako; Tamai, Katsuto; Saga, Kotaro; Yamazaki, Takehiko; Fujita, Hiroshi; Shimbo, Takashi; Kikuchi, Yasushi; Nimura, Keisuke; Nishifuji, Koji; Amagai, Masayuki; Uitto, Jouni; Kaneda, Yasufumi

    2007-10-01

    We report a novel strategy for constructing a tissue-targeting hemagglutinating virus of Japan (HVJ; Sendai virus) envelope vector (HVJ-E), and its application in gene therapy of a mouse model of genetic skin disease. Chimeric genes encoding viral F protein and green fluorescent protein (GFP) were constructed on the basis of various deletion mutants. The product of one chimeric gene, containing signal peptide, transmembrane domain, and the cytoplasmic tail of F protein, was transported to the cell surface and incorporated into new viruses released from HVJ-infected LLC-MK2 cells. For tissue targeting, in the preceding construct GFP was replaced with single-chain antibody (scFv) against mouse desmoglein 3 (mDsg3), a desmosomal cadherin found in basal layer keratinocytes of the skin. HVJ encoding scFv-F chimeric protein bound to mDsg3-coated plates much more efficiently than did wild-type HVJ. When chimeric HVJ was injected into a skin blister of a mouse model of epidermolysis bullosa, in which defective expression of type VII collagen results in a failure to secure epidermis to the underlying dermis, viral F protein expression was detected in most of the basal keratinocytes. Furthermore, chimeric HVJ-E introduced type VII collagen expression more efficiently compared with wild-type HVJ in basal keratinocytes of type VII collagen-deficient mouse skin, resulting in efficient amelioration of the genetic defect. Thus, a novel tissue-targeting HVJ-E could be used to successfully target epidermal keratinocytes both in vitro and in vivo. PMID:17892442

  19. Consequences of Cytoplasmic Irradiation: Studies from Microbeam

    PubMed Central

    Zhou, Hongning; Hong, Mei; Chai, Yunfei; Hei, Tom K.

    2013-01-01

    The prevailing dogma for radiation biology is that genotoxic effects of ionizing radiation such as mutations and carcinogenesis are attributed mainly to direct damage to the nucleus. However, with the development of microbeam that can target precise positions inside the cells, accumulating evidences have shown that energy deposit by radiation in nuclear DNA is not required to trigger the damage, extra-nuclear or extra-cellular radiation could induce the similar biological effects as well. This review will summarize the biological responses after cytoplasm irradiated by microbeam, and the possible mechanisms involved in cytoplasmic irradiation. PMID:19346686

  20. How crowded is the prokaryotic cytoplasm?

    PubMed

    Spitzer, Jan; Poolman, Bert

    2013-07-11

    We consider biomacromolecular crowding within the cytoplasm of prokaryotic cells as a two-phase system of 'supercrowded' cytogel and 'dilute' cytosol; we simplify and quantify this model for a coccoid cell over a wide range of biomacromolecular crowding. The key result shows that the supercrowded cytogel extends the vectorial character of the plasma membrane deeper into the cytoplasm by about 20-70 nm. We discuss useful physiological insights that this model gives into the functioning of a prokaryotic cell on the micrometer scale. PMID:23735698

  1. Thermonuclear flash model for long X-ray tails from Aquila X-1

    NASA Technical Reports Server (NTRS)

    Fushiki, Ikko; Taam, Ronald E.; Woosley, S. E.; Lamb, D. Q.

    1992-01-01

    Attention is given to a thermonuclear flash model for long X-ray tails from the recurrent transient Aql X-1, in which the extended phase of nuclear burning is due to the fact that the envelope is out of thermal equilibrium. Only the first X-ray burst emitted by Aql X-1 during its transient outburst exhibits a long X-ray tail. The properties of subsequent bursts are distinguished by a lack of an X-ray tail reflecting the much smaller accumulated masses which result from the effects of thermal inertia in the neutron star envelope. The characteristics of the latter bursts are similar to those of typical X-ray bursters. For a neutron star characterized by a mass and radius of 1.4 solar mass and 9.1 km, respectively, the occurrence of the long X-ray tail requires that the mass of the accumulated layer be less than 10 exp 23 g and that the envelope temperatures of the neutron star be less than 1.5 x 10 exp 7. This interpretation is found to be consistent with the thermal relaxation of the neutron star envelope during the quiescent state of Aql X-1 and with the mass accretion rates inferred for the transient outburst itself.

  2. Analysis of Pseudomonas aeruginosa Cell Envelope Proteome by Capture of Surface-Exposed Proteins on Activated Magnetic Nanoparticles

    PubMed Central

    Vecchietti, Davide; Di Silvestre, Dario; Miriani, Matteo; Bonomi, Francesco; Marengo, Mauro; Bragonzi, Alessandra; Cova, Lara; Franceschi, Eleonora; Mauri, Pierluigi; Bertoni, Giovanni

    2012-01-01

    We report on specific magneto-capturing followed by Multidimensional Protein Identification Technology (MudPIT) for the analysis of surface-exposed proteins of intact cells of the bacterial opportunistic pathogen Pseudomonas aeruginosa. The magneto-separation of cell envelope fragments from the soluble cytoplasmic fraction allowed the MudPIT identification of the captured and neighboring proteins. Remarkably, we identified 63 proteins captured directly by nanoparticles and 67 proteins embedded in the cell envelope fragments. For a high number of proteins, our analysis strongly indicates either surface exposure or localization in an envelope district. The localization of most identified proteins was only predicted or totally unknown. This novel approach greatly improves the sensitivity and specificity of the previous methods, such as surface shaving with proteases that was also tested on P. aeruginosa. The magneto-capture procedure is simple, safe, and rapid, and appears to be well-suited for envelope studies in highly pathogenic bacteria. PMID:23226459

  3. Safeguards Envelope Progress FY08

    SciTech Connect

    Robert Bean; Richard Metcalf; Aaron Bevill

    2008-09-01

    The Safeguards Envelope Project met its milestones by creating a rudimentary safeguards envelope, proving the value of the approach on a small scale, and determining the most appropriate path forward. The Idaho Chemical Processing Plant’s large cache of reprocessing process monitoring data, dubbed UBER Data, was recovered and used in the analysis. A probabilistic Z test was used on a Markov Monte Carlo simulation of expected diversion data when compared with normal operating data. The data regarding a fully transient event in a tank was used to create a simple requirement, representative of a safeguards envelope, whose impact was a decrease in operating efficiency by 1.3% but an increase in material balance period of 26%. This approach is operator, state, and international safeguards friendly and should be applied to future reprocessing plants. Future requirements include tank-to-tank correlations in reprocessing facilities, detailed operations impact studies, simulation inclusion, automated optimization, advanced statistics analysis, and multi-attribute utility analysis.

  4. Managing 'tail liability'.

    PubMed

    Frese, Richard C; Weber, Ryan J

    2013-11-01

    To reduce and control their level of tail liability, hospitals should: Utilize a self-insurance vehicle; Consider combined limits between the hospital and physicians; Communicate any program changes to the actuary, underwriter, and auditor; Continue risk management and safety practices; Ensure credit is given to the organization's own medical malpractice program. PMID:24340649

  5. Nuclear envelope and genome interactions in cell fate

    PubMed Central

    Talamas, Jessica A.; Capelson, Maya

    2015-01-01

    The eukaryotic cell nucleus houses an organism’s genome and is the location within the cell where all signaling induced and development-driven gene expression programs are ultimately specified. The genome is enclosed and separated from the cytoplasm by the nuclear envelope (NE), a double-lipid membrane bilayer, which contains a large variety of trans-membrane and associated protein complexes. In recent years, research regarding multiple aspects of the cell nucleus points to a highly dynamic and coordinated concert of efforts between chromatin and the NE in regulation of gene expression. Details of how this concert is orchestrated and how it directs cell differentiation and disease are coming to light at a rapid pace. Here we review existing and emerging concepts of how interactions between the genome and the NE may contribute to tissue specific gene expression programs to determine cell fate. PMID:25852741

  6. Coronavirus envelope (E) protein remains at the site of assembly.

    PubMed

    Venkatagopalan, Pavithra; Daskalova, Sasha M; Lopez, Lisa A; Dolezal, Kelly A; Hogue, Brenda G

    2015-04-01

    Coronaviruses (CoVs) assemble at endoplasmic reticulum Golgi intermediate compartment (ERGIC) membranes and egress from cells in cargo vesicles. Only a few molecules of the envelope (E) protein are assembled into virions. The role of E in morphogenesis is not fully understood. The cellular localization and dynamics of mouse hepatitis CoV A59 (MHV) E protein were investigated to further understanding of its role during infection. E protein localized in the ERGIC and Golgi with the amino and carboxy termini in the lumen and cytoplasm, respectively. E protein does not traffic to the cell surface. MHV was genetically engineered with a tetracysteine tag at the carboxy end of E. Fluorescence recovery after photobleaching (FRAP) showed that E is mobile in ERGIC/Golgi membranes. Correlative light electron microscopy (CLEM) confirmed the presence of E in Golgi cisternae. The results provide strong support that E proteins carry out their function(s) at the site of budding/assembly. PMID:25726972

  7. Detection of cytoplasmic glycosylation associated with hydroxyproline.

    PubMed

    West, Christopher M; van der Wel, Hanke; Blader, Ira J

    2006-01-01

    A special class of glycosylation occurs on a proline residue of the cytoplasmic/nuclear protein Skp1 in the social amoeba Dictyostelium. For this glycosylation to occur, the proline must first be hydroxylated by the action of a soluble prolyl 4-hydroxylase acting on the protein. Cytoplasmic prolyl 4-hydroxylases are dioxygen-dependent enzymes that have low affinity for their O2 substrate and, therefore, have been implicated in O2-sensing in Dictyostelium, as well as in vertebrates and invertebrates. The sugar-hydroxyproline linkage has low abundance, is resistant to alkali cleavage and known glycosidases, and does not bind known lectins. However, initial screens for this modification can be made by assessing changes in electrophoretic mobility of candidate proteins after treatment of cells with prolyl hydroxylase inhibitors, and/or by metabolic labeling with [3H]sugar precursors. In addition, cytoplasmic hydroxylation/glycosylation can be assessed by assaying for cytoplasmic glycosyltransferases. Here we describe these methods and examples of their use in analyzing Skp1 glycosylation in Dictyostelium and the apicomplexan Toxoplasma gondii, the causative agent of toxoplasmosis in humans. PMID:17132515

  8. CNS Myelination Requires Cytoplasmic Dynein Function

    PubMed Central

    Yang, Michele L.; Shin, Jimann; Kearns, Christina A.; Langworthy, Melissa M.; Snell, Heather; Walker, Macie B.; Appel, Bruce

    2014-01-01

    Background Cytoplasmic dynein provides the main motor force for minus-end-directed transport of cargo on microtubules. Within the vertebrate central nervous system (CNS), proliferation, neuronal migration and retrograde axon transport are among the cellular functions known to require dynein. Accordingly, mutations of DYNC1H1, which encodes the heavy chain subunit of cytoplasmic dynein, have been linked to developmental brain malformations and axonal pathologies. Oligodendrocytes, the myelinating glial cell type of the CNS, migrate from their origins to their target axons and subsequently extend multiple long processes that ensheath axons with specialized insulating membrane. These processes are filled with microtubules, which facilitate molecular transport of myelin components. However, whether oligodendrocytes require cytoplasmic dynein to ensheath axons with myelin is not known. Results We identified a mutation of zebrafish dync1h1 in a forward genetic screen that caused a deficit of oligodendrocytes. Using in vivo imaging and gene expression analyses, we additionally found evidence that dync1h1 promotes axon ensheathment and myelin gene expression. Conclusions In addition to its well known roles in axon transport and neuronal migration, cytoplasmic dynein contributes to neural development by promoting myelination. PMID:25488883

  9. REAR PROFILE OF TAIL FROM SECOND LEVEL OF TAIL DOCK ...

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

    REAR PROFILE OF TAIL FROM SECOND LEVEL OF TAIL DOCK STAND, SHOWING AIRCRAFT NUMBER (319), HORIZONTAL STABILIZER, TAIL CONE AND COOLING CTS FOR THE AUXILIARY POWER UNIT (APU), MECHANIC PAUL RIDEOUT IS LOWERING THE BALANCE PANELS ON THE STABILIZERS FOR LUBRICATION AND INSPECTION. - Greater Buffalo International Airport, Maintenance Hangar, Buffalo, Erie County, NY

  10. Membrane anchoring domain of herpes simplex virus glycoprotein gB is sufficient for nuclear envelope localization.

    PubMed Central

    Gilbert, R; Ghosh, K; Rasile, L; Ghosh, H P

    1994-01-01

    We have used the glycoprotein gB of herpes simplex virus type 1 (gB-1), which buds from the inner nuclear membrane, as a model protein to study localization of membrane proteins in the nuclear envelope. To determine whether specific domains of gB-1 glycoprotein are involved in localization in the nuclear envelope, we have used deletion mutants of gB-1 protein as well as chimeric proteins constructed by replacing the domains of the cell surface glycoprotein G of vesicular stomatitis virus with the corresponding domains of gB. Mutant and chimeric proteins expressed in COS cells were localized by immunoelectron microscopy. A chimeric protein (gB-G) containing the ectodomain of gB and the transmembrane and cytoplasmic domains of G did not localize in the nuclear envelope. When the ectodomain of G was fused to the transmembrane and cytoplasmic domains of gB, however, the resulting chimeric protein (G-gB) was localized in the nuclear envelope. Substitution of the transmembrane domain of G with the 69 hydrophobic amino acids containing the membrane anchoring domain of gB allowed the hybrid protein (G-tmgB) to be localized in the nuclear envelope, suggesting that residues 721 to 795 of gB can promote retention of proteins in the nuclear envelope. Deletion mutations in the hydrophobic region further showed that a transmembrane segment of 21 hydrophobic amino acids, residues 774 to 795 of gB, was sufficient for localization in the nuclear envelope. Since wild-type gB and the mutant and chimeric proteins that were localized in the nuclear envelope were also retained in the endoplasmic reticulum, the membrane spanning segment of gB could also influence retention in the endoplasmic reticulum. Images PMID:8139012

  11. An integrated model for the nucleo-cytoplasmic transport of cytoplasmic poly(A)-binding proteins.

    PubMed

    Burgess, Hannah M; Gray, Nicola K

    2012-05-01

    Cytoplasmic poly(A)-binding proteins (PABPs) regulate mRNA stability and translation. Although predominantly localized in the cytoplasm, PABP proteins also cycle through the nucleus. Recent work has established that their steady-state localization can be altered by cellular stresses such as ultraviolet (UV) radiation, and infection by several viruses, resulting in nuclear accumulation of PABPs. Here, we present further evidence that their interaction with and release from mRNA and translation complexes are important in determining their sub-cellular distribution and propose an integrated model for regulated nucleo-cytoplasmic transport of PABPs. PMID:22896784

  12. The structure of common-envelope remnants

    NASA Astrophysics Data System (ADS)

    Hall, Philip D.

    2015-05-01

    We investigate the structure and evolution of the remnants of common-envelope evolution in binary star systems. In a common-envelope phase, two stars become engulfed in a gaseous envelope and, under the influence of drag forces, spiral to smaller separations. They may merge to form a single star or the envelope may be ejected to leave the stars in a shorter period orbit. This process explains the short orbital periods of many observed binary systems, such as cataclysmic variables and low-mass X-ray binary systems. Despite the importance of these systems, and of common-envelope evolution to their formation, it remains poorly understood. Specifically, we are unable to confidently predict the outcome of a common-envelope phase from the properties at its onset. After presenting a review of work on stellar evolution, binary systems, common-envelope evolution and the computer programs used, we describe the results of three computational projects on common-envelope evolution. Our work specifically relates to the methods and prescriptions which are used for predicting the outcome. We use the Cambridge stellar-evolution code STARS to produce detailed models of the structure and evolution of remnants of common-envelope evolution. We compare different assumptions about the uncertain end-of-common envelope structure and envelope mass of remnants which successfully eject their common envelopes. In the first project, we use detailed remnant models to investigate whether planetary nebulae are predicted after common-envelope phases initiated by low-mass red giants. We focus on the requirement that a remnant evolves rapidly enough to photoionize the nebula and compare the predictions for different ideas about the structure at the end of a common-envelope phase. We find that planetary nebulae are possible for some prescriptions for the end-of-common envelope structure. In our second contribution, we compute a large set of single-star models and fit new formulae to the core radii of

  13. Proteomic Response of Bacillus subtilis to Lantibiotics Reflects Differences in Interaction with the Cytoplasmic Membrane

    PubMed Central

    Wenzel, Michaela; Kohl, Bastian; Münch, Daniela; Raatschen, Nadja; Albada, H. Bauke; Hamoen, Leendert; Metzler-Nolte, Nils; Sahl, Hans-Georg

    2012-01-01

    Mersacidin, gallidermin, and nisin are lantibiotics, antimicrobial peptides containing lanthionine. They show potent antibacterial activity. All three interfere with cell wall biosynthesis by binding lipid II, but they display different levels of interaction with the cytoplasmic membrane. On one end of the spectrum, mersacidin interferes with cell wall biosynthesis by binding lipid II without integrating into bacterial membranes. On the other end of the spectrum, nisin readily integrates into membranes, where it forms large pores. It destroys the membrane potential and causes leakage of nutrients and ions. Gallidermin, in an intermediate position, also readily integrates into membranes. However, pore formation occurs only in some bacteria and depends on membrane composition. In this study, we investigated the impact of nisin, gallidermin, and mersacidin on cell wall integrity, membrane pore formation, and membrane depolarization in Bacillus subtilis. The impact of the lantibiotics on the cell envelope was correlated to the proteomic response they elicit in B. subtilis. By drawing on a proteomic response library, including other envelope-targeting antibiotics such as bacitracin, vancomycin, gramicidin S, or valinomycin, YtrE could be identified as the most reliable marker protein for interfering with membrane-bound steps of cell wall biosynthesis. NadE and PspA were identified as markers for antibiotics interacting with the cytoplasmic membrane. PMID:22926563

  14. Parvoviruses Cause Nuclear Envelope Breakdown by Activating Key Enzymes of Mitosis

    PubMed Central

    Porwal, Manvi; Cohen, Sarah; Snoussi, Kenza; Popa-Wagner, Ruth; Anderson, Fenja; Dugot-Senant, Nathalie; Wodrich, Harald; Dinsart, Christiane; Kleinschmidt, Jürgen A.; Panté, Nelly; Kann, Michael

    2013-01-01

    Disassembly of the nuclear lamina is essential in mitosis and apoptosis requiring multiple coordinated enzymatic activities in nucleus and cytoplasm. Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases. Here we used the ability of parvoviruses to induce nuclear membrane breakdown to understand the triggers of key mitotic enzymes. Nuclear envelope disintegration was shown upon infection, microinjection but also upon their application to permeabilized cells. The latter technique also showed that nuclear envelope disintegration was independent upon soluble cytoplasmic factors. Using time-lapse microscopy, we observed that nuclear disassembly exhibited mitosis-like kinetics and occurred suddenly, implying a catastrophic event irrespective of cell- or type of parvovirus used. Analyzing the order of the processes allowed us to propose a model starting with direct binding of parvoviruses to distinct proteins of the nuclear pore causing structural rearrangement of the parvoviruses. The resulting exposure of domains comprising amphipathic helices was required for nuclear envelope disintegration, which comprised disruption of inner and outer nuclear membrane as shown by electron microscopy. Consistent with Ca++ efflux from the lumen between inner and outer nuclear membrane we found that Ca++ was essential for nuclear disassembly by activating PKC. PKC activation then triggered activation of cdk-2, which became further activated by caspase-3. Collectively our study shows a unique interaction of a virus with the nuclear envelope, provides evidence that a nuclear pool of executing enzymes is sufficient for nuclear disassembly in quiescent cells, and demonstrates that nuclear disassembly can be uncoupled from initial phases of mitosis. PMID:24204256

  15. Arrest of cytoplasmic streaming induces algal proliferation in green paramecia.

    PubMed

    Takahashi, Toshiyuki; Shirai, Yohji; Kosaka, Toshikazu; Hosoya, Hiroshi

    2007-01-01

    A green ciliate Paramecium bursaria, bearing several hundreds of endosymbiotic algae, demonstrates rotational microtubule-based cytoplasmic streaming, in which cytoplasmic granules and endosymbiotic algae flow in a constant direction. However, its physiological significance is still unknown. We investigated physiological roles of cytoplasmic streaming in P. bursaria through host cell cycle using video-microscopy. Here, we found that cytoplasmic streaming was arrested in dividing green paramecia and the endosymbiotic algae proliferated only during the arrest of cytoplasmic streaming. Interestingly, arrest of cytoplasmic streaming with pressure or a microtubule drug also induced proliferation of endosymbiotic algae independently of host cell cycle. Thus, cytoplasmic streaming may control the algal proliferation in P. bursaria. Furthermore, confocal microscopic observation revealed that a division septum was formed in the constricted area of a dividing paramecium, producing arrest of cytoplasmic streaming. This is a first report to suggest that cytoplasmic streaming controls proliferation of eukaryotic cells. PMID:18159235

  16. Hybridization using cytoplasmic male sterility, cytoplasmic herbicide tolerance, and herbicide tolerance from nuclear genes

    SciTech Connect

    Beversdorf, W.D.; Erickson, L.R.; Grant, I.

    1987-04-14

    An improved process is described for producing a substantially homogeneous population of plants of a predetermined hybrid variety of crop which is capable of undergoing self-pollination and cross-pollination. The process comprises: growing in a first planting area a substantially random population of cytoplasmic male sterile plants which exhibit cytoplasmic herbicide tolerance to at least one Type A herbicide and exhibit tolerance to at least one Type B herbicide which is attributable solely to homozygous dominant nuclear genes and male fertile plants which are homozygous recessive maintainer plants for the cytoplasmic male sterile plants and which lack the cytoplasmic herbicide tolerance to at least one Type A herbicide and exhibit tolerance to at least one Type B herbicide attributable solely to the homozygous dominant nuclear genes.

  17. Processing of heparanase is mediated by syndecan-1 cytoplasmic domain and involves syntenin and α-actinin

    PubMed Central

    Shteingauz, Anna; Ilan, Neta; Vlodavsky, Israel

    2014-01-01

    Heparanase activity plays a decisive role in cell dissemination associated with cancer metastasis. Cellular uptake of heparanase is considered a pre-requisite for the delivery of latent 65 kDa heparanase to lysosomes and its subsequent proteolytic processing and activation into 8 and 50 kDa protein subunits by cathepsin L. Heparan sulfate proteoglycans, and particularly syndecan, are instrumental for heparanase uptake and activation, through a process that has been shown to occur independent of rafts. Nevertheless, the molecular mechanism underlying syndecan mediated internalization outside of rafts is unclear. Here, we examined the role of syndecan-1 cytoplasmic domain in heparanase processing, utilizing deletion constructs lacking the entire cytoplasmic domain (delta), the conserved (C1 or C2) or variable (V) regions. Heparanase processing was markedly increased following syndecan-1 over expression; In contrast, heparanase was retained at the cell membrane and its processing was impaired in cells over expressing syndecan-1 deleted for the entire cytoplasmic tail. We have next revealed that conserved domain 2 (C2) and variable (V) regions of syndecan-1 cytoplasmic tail mediate heparanase processing. Furthermore, we found that syntenin, known to interact with syndecan C2 domain, and α actinin are essential for heparanase processing. PMID:24788042

  18. Structural changes of envelope proteins during alphavirus fusion

    SciTech Connect

    Li, Long; Jose, Joyce; Xiang, Ye; Kuhn, Richard J.; Rossmann, Michael G.

    2010-12-08

    Alphaviruses are enveloped RNA viruses that have a diameter of about 700 {angstrom} and can be lethal human pathogens. Entry of virus into host cells by endocytosis is controlled by two envelope glycoproteins, E1 and E2. The E2-E1 heterodimers form 80 trimeric spikes on the icosahedral virus surface, 60 with quasi-three-fold symmetry and 20 coincident with the icosahedral three-fold axes arranged with T = 4 quasi-symmetry. The E1 glycoprotein has a hydrophobic fusion loop at one end and is responsible for membrane fusion. The E2 protein is responsible for receptor binding and protects the fusion loop at neutral pH. The lower pH in the endosome induces the virions to undergo an irreversible conformational change in which E2 and E1 dissociate and E1 forms homotrimers, triggering fusion of the viral membrane with the endosomal membrane and then releasing the viral genome into the cytoplasm. Here we report the structure of an alphavirus spike, crystallized at low pH, representing an intermediate in the fusion process and clarifying the maturation process. The trimer of E2-E1 in the crystal structure is similar to the spikes in the neutral pH virus except that the E2 middle region is disordered, exposing the fusion loop. The amino- and carboxy-terminal domains of E2 each form immunoglobulin-like folds, consistent with the receptor attachment properties of E2.

  19. Protein folding in the cell envelope of Escherichia coli.

    PubMed

    De Geyter, Jozefien; Tsirigotaki, Alexandra; Orfanoudaki, Georgia; Zorzini, Valentina; Economou, Anastassios; Karamanou, Spyridoula

    2016-01-01

    While the entire proteome is synthesized on cytoplasmic ribosomes, almost half associates with, localizes in or crosses the bacterial cell envelope. In Escherichia coli a variety of mechanisms are important for taking these polypeptides into or across the plasma membrane, maintaining them in soluble form, trafficking them to their correct cell envelope locations and then folding them into the right structures. The fidelity of these processes must be maintained under various environmental conditions including during stress; if this fails, proteases are called in to degrade mislocalized or aggregated proteins. Various soluble, diffusible chaperones (acting as holdases, foldases or pilotins) and folding catalysts are also utilized to restore proteostasis. These responses can be general, dealing with multiple polypeptides, with functional overlaps and operating within redundant networks. Other chaperones are specialized factors, dealing only with a few exported proteins. Several complex machineries have evolved to deal with binding to, integration in and crossing of the outer membrane. This complex protein network is responsible for fundamental cellular processes such as cell wall biogenesis; cell division; the export, uptake and degradation of molecules; and resistance against exogenous toxic factors. The underlying processes, contributing to our fundamental understanding of proteostasis, are a treasure trove for the development of novel antibiotics, biopharmaceuticals and vaccines. PMID:27573113

  20. Nuclear and cytoplasmic actin in dinoflagellates.

    PubMed

    Soyer-Gobillard, M O; Ausseil, J; Géraud, M L

    1996-01-01

    Experiments using monoclonal and polyclonal anti-actin antibodies allowed us to demonstrate the presence of F- or G-actin in original protists, dinoflagellates, either by biochemistry, immunofluorescence and in TEM. SDS-PAGE electrophoresis and immunoblottings made either from total or nuclear protein extracts revealed the presence of a 44-kDa band reacting with monoclonal anti-actin antibody in two species, Prorocentrum micans and Crypthecodinium cohnii, and thus demonstrated the presence of actin in nuclear and cytoplasmic fractions. After squash preparation of P micans cells, actin was identified within the nucleus and in some regions of the cytoplasm by immunofluorescence microscopy. Labelling of both the nucleolus and the centrosome region was evident together with amorphous nucleoplasmic material surrounding the chromosomes. The use of cryosections of intact P micans and C cohnii cells for immunofluorescence along with staining with DAPI to delineate the chromosomes themselves, yielded finer resolution of the intranuclear network labelling pattern and allowed us to complete our observations, in particular on the cytoplasmic labelling. In P micans, in addition to the centrosome region, the cytoplasmic channels passing through the nucleus in dividing cells are labelled. In C cohnii, the cortex, the centrosome region, the cytoplasmic channels, the region surrounding the nucleus, the filaments linking it to the cortex and the cleavage furrow are also labelled. In the nucleus of the two species, there is a prominent "weft' of fine actin filaments in the nucleoplasm forming a matrix of varying density around the persistent chromosomes. This actin matrix, of unknown function, is most conspicuous at the end of the S-phase of the cell cycle. Fluorescent derivatives of phalloidin, used as diagnostic cytochemical probes for polymeric actin (F-actin), gave similar results. Positive TEM immunolabelling of intranuclear actin confirms its presence in the nucleoplasm, in the

  1. Isolating The Building Thermal Envelope

    NASA Astrophysics Data System (ADS)

    Harrje, D. T.; Dutt, G. S.; Gadsby, K. J.

    1981-01-01

    The evaluation of the thermal integrity of building envelopes by infrared scanning tech-niques is often hampered in mild weather because temperature differentials across the envelope are small. Combining the infrared scanning with positive or negative building pressures, induced by a "blower door" or the building ventilation system, considerably extends the periods during which meaningful diagnostics can be conducted. Although missing or poorly installed insulation may lead to a substantial energy penalty, it is the search for air leakage sites that often has the largest potential for energy savings. Infrared inspection of the attic floor with air forced from the occupied space through ceiling by-passes, and inspecting the interior of the building when outside air is being sucked through the envelope reveals unexpected leakage sites. Portability of the diagnostic equipment is essential in these surveys which may include access into some tight spaces. A catalog of bypass heat losses that have been detected in residential housing using the combined infrared pressure differential technique is included to point out the wide variety of leakage sites which may compromise the benefits of thermal insulation and allow excessive air infiltration. Detection and suppression of such leaks should be key items in any building energy audit program. Where a calibrated blower door is used to pressurize or evacuate the house, the leakage rate can be quantified and an excessively tight house recognized. Houses that are too tight may be improved with a minimal energy penalty by forced ventilation,preferably with a heat recuperator and/or by providing combustion air directly to the furnace.

  2. The primary structure of rat brain (cytoplasmic) dynein heavy chain, a cytoplasmic motor enzyme.

    PubMed Central

    Zhang, Z; Tanaka, Y; Nonaka, S; Aizawa, H; Kawasaki, H; Nakata, T; Hirokawa, N

    1993-01-01

    Overlapping cDNA clones encoding the heavy chain of rat brain cytoplasmic dynein have been isolated. The isolated cDNA clones contain an open reading frame of 13,932 bp encoding 4644 aa (M(r), 532,213). The deduced protein sequence of the heavy chain of rat brain dynein shows significant similarity to sea urchin flagellar beta-dynein (27.0% identical) and to Dictyostelium cytoplasmic dynein (53.5% identical) throughout the entire sequence. The heavy chain of rat brain (cytoplasmic) dynein contains four putative nucleotide-binding consensus sequences [GX4GK(T/S)] in the central one-third region that are highly similar to those of sea urchin and Dictyostelium dyneins. The N-terminal one-third of the heavy chain of rat brain (cytoplasmic) dynein shows high similarity (43.8% identical) to that of Dictyostelium cytoplasmic dynein but poor similarity (19.4% identical) to that of sea urchin flagellar dynein. These results suggested that the C-terminal two-thirds of the dynein molecule is conserved and plays an essential role in microtubule-dependent motility activity, whereas the N-terminal regions are different between cytoplasmic and flagellar dyneins. Images Fig. 1 PMID:7690137

  3. Flexible Envelope Request Notation (FERN)

    NASA Technical Reports Server (NTRS)

    Zoch, David R.; Lavallee, David; Weinstein, Stuart

    1991-01-01

    The following topics are presented in view graph form and include the following: scheduling application; the motivation for the Flexible Envelope Request Notation (FERN); characteristics of FERN; types of information needed in requests; where information is stored in requests; FERN structures; generic requests; resource availability for pooled resources; expressive notation; temporal constraints; time formats; changes to FERN; sample FERN requests; the temporal relationship between two steps; maximum activity length to limit step delays; alternative requests; the temporal relationship between two activities; and idle resource usage between steps.

  4. The tail plane

    NASA Technical Reports Server (NTRS)

    Munk, Max M

    1923-01-01

    This report deals with the calculation of the equilibrium, statistical stability, and damping of the tail plane. The author has simplified the present theory of longitudinal stability for the particular purpose of obtaining one definite coefficient characteristics of the effect of the tail plane. This coefficient is obtained by substituting certain aerodynamic characteristics and some dimensions of the airplane in a comparatively simple mathematical expression. Care has been taken to confine all aerodynamical information necessary for the calculation of the coefficient to the well-known curves representing the qualities of the wing section. This is done by making use of the present results of modern aerodynamics. All formulas and relations necessary for the calculation are contained in the paper. They give in some cases only an approximation of the real values. An example of calculation is added in order to illustrate the application of the method. The coefficient indicates not only whether the effect of the tail plane is great enough, but also whether it is not too great. It appears that the designer has to avoid a certain critical length of the fuselage, which inevitably gives rise to periodical oscillations of the airplane. The discussion also shows the way and in what direction to carry out experimental work.

  5. Modelling Cometary Sodium Tails

    NASA Astrophysics Data System (ADS)

    Birkett, K. S.; Jones, G. H.; Coates, A. J.

    2013-12-01

    Neutral sodium is readily observed in cometary spectra and can be seen to form its own distinct tail at high activity comets. Solar radiation pressure accelerates the sodium atoms antisunward and, as strong sodium absorption lines are present in the solar spectrum, the magnitude of this force is dependent upon the Doppler shift of the incident solar radiation. Therefore the heliocentric velocity of the sodium atom directly determines its acceleration. This can produce unique effects, such as a stagnation region. Sodium is relatively easy to detect and so can potentially be used to trace mechanisms in the coma that are otherwise difficult to observe. The source of neutral sodium in the tail currently remains unknown. We have therefore developed a new, three dimensional Monte-Carlo model of neutral cometary sodium in order to facilitate testing of different source production functions. It includes weightings due to neutral sodium lifetime, variation of cometary sodium emission due to Fraunhofer absorption lines and solar flux variation with heliocentric distance. The Swings and Greenstein effects, which can have particularly dramatic effects in near-Sun comets, are also considered comprehensively. Preliminary results from this model are presented, focusing on a comparison of predictions of the neutral sodium tail of Comet C/2012 S1 (ISON) with initial observations.

  6. Secretion of Bacterial Lipoproteins: Through the Cytoplasmic Membrane, the Periplasm and Beyond

    PubMed Central

    Zückert, Wolfram R.

    2014-01-01

    Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., grampositive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep process that involves sequential modification of a cysteine residue and cleavage of the signal peptide by the signal II peptidase Lsp. In both monoderms and diderms, signal peptide processing is preceded by acylation with a diacylglycerol through preprolipoprotein diacylglycerol transferase (Lgt). In diderms but also some monoderms, lipoproteins are further modified with a third acyl chain through lipoprotein N-acyl transferase (Lnt). Fully modified lipoproteins that are destined to be anchored in the inner leaflet of the outer membrane (OM) are selected, transported and inserted by the Lol (lipoprotein outer membrane localization) pathway machinery, which consists of the inner-membrane (IM) ABC transporterlike LolCDE complex, the periplasmic LolA chaperone and the OM LolB lipoprotein receptor. Retention of lipoproteins in the cytoplasmic membrane results from Lol avoidance signals that were originally described as the “+2 rule”. Surface localization of lipoproteins in diderms is rare in most bacteria, with the exception of several spirochetal species. Type 2 (T2SS) and type 5 (T5SS) secretion systems are involved in secretion of specific surface lipoproteins of γ-proteobacteria. In the model spirochete Borrelia burgdorferi, surface lipoprotein secretion does not follow established sorting rules, but remains dependent on N-terminal peptide sequences. Secretion through the outer membrane requires maintenance of lipoproteins in a translocation-competent unfolded conformation

  7. Mechanism of Cytoplasmic mRNA Translation

    PubMed Central

    2015-01-01

    Protein synthesis is a fundamental process in gene expression that depends upon the abundance and accessibility of the mRNA transcript as well as the activity of many protein and RNA-protein complexes. Here we focus on the intricate mechanics of mRNA translation in the cytoplasm of higher plants. This chapter includes an inventory of the plant translational apparatus and a detailed review of the translational processes of initiation, elongation, and termination. The majority of mechanistic studies of cytoplasmic translation have been carried out in yeast and mammalian systems. The factors and mechanisms of translation are for the most part conserved across eukaryotes; however, some distinctions are known to exist in plants. A comprehensive understanding of the complex translational apparatus and its regulation in plants is warranted, as the modulation of protein production is critical to development, environmental plasticity and biomass yield in diverse ecosystems and agricultural settings. PMID:26019692

  8. PRA1 co-localizes with envelope but does not influence primate lentivirus production, infectivity or envelope incorporation.

    PubMed

    Blancou, Philippe; Evans, David T; Desrosiers, Ronald C

    2005-06-01

    The results of yeast and mammalian two-hybrid assays previously indicated complex formation between prenylated Rab acceptor 1 (PRA1) and the cytoplasmic domain of gp41 (gp41CD) for both the human and simian immunodeficiency viruses [Evans, D. T., Tilman, K. C. & Desrosiers, R. C. (2002). J Virol 76, 327-337]. The assembly and release of infectious virus particles was studied under conditions of PRA1 overexpression in a transient transfection assay or suppression by RNA interference. Although a clear pattern of co-localization of PRA1 and gp41 was observed, no changes in virion release, infectivity or envelope content were observed as a result of either PRA1 suppression or overexpression. These data show that PRA1 co-localizes with gp41 inside cells and they are consistent with a direct or indirect interaction between these proteins. However, variation in the levels of PRA1 expression did not influence virion production, infectivity or envelope incorporation under the conditions of these assays. PMID:15914857

  9. African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes.

    PubMed

    Hernáez, Bruno; Guerra, Milagros; Salas, María L; Andrés, Germán

    2016-04-01

    African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs. PMID:27110717

  10. African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes

    PubMed Central

    Hernáez, Bruno; Guerra, Milagros; Salas, María L.

    2016-01-01

    African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs. PMID:27110717

  11. Palmitoylation of the feline immunodeficiency virus envelope glycoprotein and its effect on fusion activity and envelope incorporation into virions

    SciTech Connect

    Gonzalez, Silvia A.; Paladino, Monica G.; Affranchino, Jose L.

    2012-06-20

    The feline immunodeficiency virus (FIV) envelope glycoprotein (Env) possesses a short cytoplasmic domain of 53 amino acids containing four highly conserved cysteines at Env positions 804, 811, 815 and 848. Since palmitoylation of transmembrane proteins occurs at or near the membrane anchor, we investigated whether cysteines 804, 811 and 815 are acylated and analyzed the relevance of these residues for Env functions. Replacement of cysteines 804, 811 and 815 individually or in combination by serine residues resulted in Env glycoproteins that were efficiently expressed and processed. However, mutations C804S and C811S reduced Env fusogenicity by 93% and 84%, respectively, compared with wild-type Env. By contrast, mutant C815S exhibited a fusogenic capacity representing 50% of the wild-type value. Remarkably, the double mutation C804S/C811S abrogated both Env fusion activity and Env incorporation into virions. Finally, by means of Click chemistry assays we demonstrated that the four FIV Env cytoplasmic cysteines are palmitoylated.

  12. Circumplanetary disc or circumplanetary envelope?

    NASA Astrophysics Data System (ADS)

    Szulágyi, J.; Masset, F.; Lega, E.; Crida, A.; Morbidelli, A.; Guillot, T.

    2016-08-01

    We present three-dimensional simulations with nested meshes of the dynamics of the gas around a Jupiter mass planet with the JUPITER and FARGOCA codes. We implemented a radiative transfer module into the JUPITER code to account for realistic heating and cooling of the gas. We focus on the circumplanetary gas flow, determining its characteristics at very high resolution (80 per cent of Jupiter's diameter). In our nominal simulation where the temperature evolves freely by the radiative module and reaches 13000 K at the planet, a circumplanetary envelope was formed filling the entire Roche lobe. Because of our equation of state is simplified and probably overestimates the temperature, we also performed simulations with limited maximal temperatures in the planet region (1000, 1500, and 2000 K). In these fixed temperature cases circumplanetary discs (CPDs) were formed. This suggests that the capability to form a CPD is not simply linked to the mass of the planet and its ability to open a gap. Instead, the gas temperature at the planet's location, which depends on its accretion history, plays also fundamental role. The CPDs in the simulations are hot and cooling very slowly, they have very steep temperature and density profiles, and are strongly sub-Keplerian. Moreover, the CPDs are fed by a strong vertical influx, which shocks on the CPD surfaces creating a hot and luminous shock-front. In contrast, the pressure supported circumplanetary envelope is characterized by internal convection and almost stalled rotation.

  13. Safeguards Envelope Progress FY10

    SciTech Connect

    Richard Metcalf

    2010-10-01

    The Safeguards Envelope is a strategy to determine a set of specific operating parameters within which nuclear facilities may operate to maximize safeguards effectiveness without sacrificing safety or plant efficiency. This paper details the additions to the advanced operating techniques that will be applied to real plant process monitoring (PM) data from the Idaho Chemical Processing Plant (ICPP). Research this year focused on combining disparate pieces of data together to maximize operating time with minimal downtime due to safeguards. A Chi-Square and Croiser's cumulative sum were both included as part of the new analysis. Because of a major issue with the original data, the implementation of the two new tests did not add to the existing set of tests, though limited one-variable optimization made a small increase in detection probability. Additional analysis was performed to determine if prior analysis would have caused a major security or safety operating envelope issue. It was determined that a safety issue would have resulted from the prior research, but that the security may have been increased under certain conditions.

  14. Nuclear envelope: positioning nuclei and organizing synapses

    PubMed Central

    Razafsky, David; Hodzic, Didier

    2015-01-01

    The nuclear envelope plays an essential role in nuclear positioning within cells and tissues. This review highlights advances in understanding the mechanisms of nuclear positioning during skeletal muscle and central nervous system development. New findings, particularly about Atype lamins and Nesprin1, may link nuclear envelope integrity to synaptic integrity. Thus synaptic defects, rather than nuclear mispositioning, may underlie human pathologies associated with mutations of nuclear envelope proteins. PMID:26079712

  15. Long-time tails of correlation and memory functions

    NASA Astrophysics Data System (ADS)

    Sawada, Isao

    2002-11-01

    We review the generalized Langevin equation, which is a transformation and reformulation of equation of motion, from the two viewpoints: the projection operator method developed by Mori and the recurrence relations method developed by Lee. The fluctuating forces acting on the Bloch electrons’ current are clarified the strongly colored quantum fluctuations with the spontaneous interband transitions leading to a long-time tail of 1/ t for the envelope of the memory function. The velocity autocorrelation functions in the coupled harmonic oscillator on the Bethe lattice have a long-time tail of 1/t t. The oscillation and the form of decay found in correlation functions affect transport coefficients given by the integrated intensity up to infinity. We also study the force-force correlation functions often used as an approximation to the memory function.

  16. Toxicity of gentamicin in red-tailed hawks.

    PubMed

    Bird, J E; Walser, M M; Duke, G E

    1983-07-01

    Gentamicin sulfate at dosage levels of 10 and 20 mg/kg of body weight was administered twice daily IV to red-tailed hawks. Clinical signs, water consumption, and changes in blood chemical values were monitored. Tissues were examined grossly and ultrastructurally, using light and electron microscopy. Clinical signs of weakness and apnea were attributed to gentamicin-induced neuromuscular blockade in the 20-mg/kg group. Serum values of aspartate transaminase, alanine transaminase, cholesterol, inorganic phosphorus, total protein, albumin, and uric acid increased in some birds. There was a decrease in periodic acid-Schiff staining of proximal tubular brush borders. Increased numbers of cytoplasmic lysosomes, many of which contained myelin figures, in renal epithelial cells were seen at the ultrastructural level. All birds given 20 mg/kg died. Both dosage levels were considered toxic in red-tailed hawks. PMID:6881667

  17. The geomagnetic tail

    SciTech Connect

    Birn, J. )

    1991-01-01

    A review is presented of the plasma sheet and lobe regions of the magnetotail, focusing principally on large-scale processes or microprocesses with some large-scale effects. Consideration is given to quiet and average structures, not necessarily related to activity phases, with quasi-steady convection aspects, and with the characteristics of dynamic phases including acceleration mechanisms and single particle aspects. Attention is given to various activity models, average and quiet time properties, properties and effects of magnetospheric convection, dynamics of the magnetotail, and the near tail, substorm current wedge.

  18. Connexin channel permeability to cytoplasmic molecules.

    PubMed

    Harris, Andrew L

    2007-01-01

    Connexin channels are known to be permeable to a variety of cytoplasmic molecules. The first observation of second messenger junctional permeability, made approximately 30 years ago, sparked broad interest in gap junction channels as mediators of intercellular molecular signaling. Since then, much has been learned about the diversity of connexin channels with regard to isoform diversity, tissue and developmental distribution, modes of channel regulation, assembly, expression, biochemical modification and permeability, all of which appear to be dynamically regulated. This information has expanded the potential roles of connexin channels in development, physiology and disease, and made their elucidation much more complex--30 years ago such an orchestra of junctional dynamics was unanticipated. Only recently, however, have investigators been able to directly address, in this more complex framework, the key issue: what specific biological molecules, second messengers and others, are able to permeate the various types of connexin channels, and how well? An important related issue, given the ever-growing list of connexin-related pathologies, is how these permeabilities are altered by disease-causing connexin mutations. Together, many studies show that a variety of cytoplasmic molecules can permeate the different types of connexin channels. A few studies reveal differences in permeation by different molecules through a particular type of connexin channel, and differences in permeation by a particular molecule through different types of connexin channels. This article describes and evaluates the various methods used to obtain these data, presents an annotated compilation of the results, and discusses the findings in the context of what can be inferred about mechanism of selectivity and potential relevance to signaling. The data strongly suggest that highly specific interactions take place between connexin pores and specific biological molecular permeants, and that those

  19. The cytoplasmic extension of the integrin β6 subunit regulates epithelial-to-mesenchymal transition.

    PubMed

    Lee, Carlin; Lee, Casey; Lee, Stacey; Siu, Amanda; Ramos, Daniel M

    2014-02-01

    Prognosis for oral cancer patients has not improved in over 60 years due to invasion and recurrence. To understand the invasive behavior of this tumor, we evaluated the role of the αvβ6 integrin. Invasive oral SCC cells express the αvβ6 integrin, which contains an 11-amino-acid extension on its β-subunit unique to the integrin family. We determined that this β6 cytoplasmic extension regulates the composition of the intermediate filament network and the organization of signaling structures called focal contacts. The auto-phosphorylation of FAK, which is localized to focal contacts, was also regulated by the β6-cytoplasmic tail, as were the transcription factors Notch and STAT3. Lastly, we also determined that activation of MAPK required the full-length β6 integrin. Together these results indicate that the signaling critical to epithelial-to-mesenchymal transition (EMT) is regulated by the β6 integrin cytoplasmic domain. PMID:24510996

  20. 14 CFR 23.333 - Flight envelope.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Flight envelope. 23.333 Section 23.333... STANDARDS: NORMAL, UTILITY, ACROBATIC, AND COMMUTER CATEGORY AIRPLANES Structure Flight Loads § 23.333 Flight envelope. (a) General. Compliance with the strength requirements of this subpart must be shown...

  1. Uranium mill tailings and radon

    SciTech Connect

    Hanchey, L A

    1981-04-01

    The major health hazard from uranium mill tailings is presumed to be respiratory cancer resulting from the inhalation of radon daughter products. A review of studies on inhalation of radon and its daughters indicates that the hazard from the tailings is extremely small. If the assumptions used in the studies are correct, one or two people per year in the United States may develop cancer as a result of radon exhaled from all the Uranium Mill Tailings Remedial Action program sites. The remedial action should reduce the hazard from the tailings by a factor of about 100.

  2. Uranium mill tailings and radon

    SciTech Connect

    Hanchey, L A

    1981-01-01

    The major health hazard from uranium mill tailings is presumed to be respiratory cancer resulting from the inhalation of radon daughter products. A review of studies on inhalation of radon and its daughters indicates that the hazard from the tailings is extremely small. If the assumptions used in the studies are correct, one or two people per year in the US may develop cancer as a result of radon exhaled from all the Uranium Mill Tailings Remedial Action Program sites. The remedial action should reduce the hazard from the tailings by a factor of about 100.

  3. Chemical ligation of the influenza M2 protein for solid-state NMR characterization of the cytoplasmic domain.

    PubMed

    Kwon, Byungsu; Tietze, Daniel; White, Paul B; Liao, Shu Y; Hong, Mei

    2015-07-01

    Solid-state NMR-based structure determination of membrane proteins and large protein complexes faces the challenge of limited spectral resolution when the proteins are uniformly (13)C-labeled. A strategy to meet this challenge is chemical ligation combined with site-specific or segmental labeling. While chemical ligation has been adopted in NMR studies of water-soluble proteins, it has not been demonstrated for membrane proteins. Here we show chemical ligation of the influenza M2 protein, which contains a transmembrane (TM) domain and two extra-membrane domains. The cytoplasmic domain, which contains an amphipathic helix (AH) and a cytoplasmic tail, is important for regulating virus assembly, virus budding, and the proton channel activity. A recent study of uniformly (13)C-labeled full-length M2 by spectral simulation suggested that the cytoplasmic tail is unstructured. To further test this hypothesis, we conducted native chemical ligation of the TM segment and part of the cytoplasmic domain. Solid-phase peptide synthesis of the two segments allowed several residues to be labeled in each segment. The post-AH cytoplasmic residues exhibit random-coil chemical shifts, low bond order parameters, and a surface-bound location, thus indicating that this domain is a dynamic random coil on the membrane surface. Interestingly, the protein spectra are similar between a model membrane and a virus-mimetic membrane, indicating that the structure and dynamics of the post-AH segment is insensitive to the lipid composition. This chemical ligation approach is generally applicable to medium-sized membrane proteins to provide site-specific structural constraints, which complement the information obtained from uniformly (13)C, (15)N-labeled proteins. PMID:25966817

  4. Chemical ligation of the influenza M2 protein for solid-state NMR characterization of the cytoplasmic domain

    PubMed Central

    Kwon, Byungsu; Tietze, Daniel; White, Paul B; Liao, Shu Y; Hong, Mei

    2015-01-01

    Solid-state NMR-based structure determination of membrane proteins and large protein complexes faces the challenge of limited spectral resolution when the proteins are uniformly 13C-labeled. A strategy to meet this challenge is chemical ligation combined with site-specific or segmental labeling. While chemical ligation has been adopted in NMR studies of water-soluble proteins, it has not been demonstrated for membrane proteins. Here we show chemical ligation of the influenza M2 protein, which contains a transmembrane (TM) domain and two extra-membrane domains. The cytoplasmic domain, which contains an amphipathic helix (AH) and a cytoplasmic tail, is important for regulating virus assembly, virus budding, and the proton channel activity. A recent study of uniformly 13C-labeled full-length M2 by spectral simulation suggested that the cytoplasmic tail is unstructured. To further test this hypothesis, we conducted native chemical ligation of the TM segment and part of the cytoplasmic domain. Solid-phase peptide synthesis of the two segments allowed several residues to be labeled in each segment. The post-AH cytoplasmic residues exhibit random-coil chemical shifts, low bond order parameters, and a surface-bound location, thus indicating that this domain is a dynamic random coil on the membrane surface. Interestingly, the protein spectra are similar between a model membrane and a virus-mimetic membrane, indicating that the structure and dynamics of the post-AH segment is insensitive to the lipid composition. This chemical ligation approach is generally applicable to medium-sized membrane proteins to provide site-specific structural constraints, which complement the information obtained from uniformly 13C, 15N-labeled proteins. PMID:25966817

  5. Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation

    PubMed Central

    Ansseau, Eugénie; Matteotti, Christel; Yip, Cassandre; Liu, Jian; Leroy, Baptiste; Hubeau, Céline; Gerbaux, Cécile; Cloet, Samuel; Wauters, Armelle; Zorbo, Sabrina; Meyer, Pierre; Pirson, Isabelle; Laoudj-Chenivesse, Dalila; Wattiez, Ruddy; Harper, Scott Q.; Belayew, Alexandra; Coppée, Frédérique

    2016-01-01

    recently shown to exit the nucleus via a novel mechanism of nuclear envelope budding. Following DUX4 or DUX4c overexpression in muscle cell cultures, we observed their association with similar nuclear buds. In conclusion, our study demonstrated unexpected interactions of DUX4/4c with cytoplasmic proteins playing major roles during muscle differentiation. Further investigations are on-going to evaluate whether these interactions play roles during muscle regeneration as previously suggested for DUX4c. PMID:26816005

  6. Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation.

    PubMed

    Ansseau, Eugénie; Eidahl, Jocelyn O; Lancelot, Céline; Tassin, Alexandra; Matteotti, Christel; Yip, Cassandre; Liu, Jian; Leroy, Baptiste; Hubeau, Céline; Gerbaux, Cécile; Cloet, Samuel; Wauters, Armelle; Zorbo, Sabrina; Meyer, Pierre; Pirson, Isabelle; Laoudj-Chenivesse, Dalila; Wattiez, Ruddy; Harper, Scott Q; Belayew, Alexandra; Coppée, Frédérique

    2016-01-01

    recently shown to exit the nucleus via a novel mechanism of nuclear envelope budding. Following DUX4 or DUX4c overexpression in muscle cell cultures, we observed their association with similar nuclear buds. In conclusion, our study demonstrated unexpected interactions of DUX4/4c with cytoplasmic proteins playing major roles during muscle differentiation. Further investigations are on-going to evaluate whether these interactions play roles during muscle regeneration as previously suggested for DUX4c. PMID:26816005

  7. 3. VIEW OF WEST TAILING DAM, LARGE TANK, AND TAILING, ...

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

    3. VIEW OF WEST TAILING DAM, LARGE TANK, AND TAILING, LOOKING NORTHEAST. A SIX-FOOT SCALE IS LOCATED AGAINST WALL ON LEFT. PURPOSE OF TANK IS UNKNOWN, BUT APPEARS TO HAVE FALLEN FROM ITS ORIGINAL LOCATION AT THE MILL SITE, UP AND TO THE RIGHT OF THIS VIEW. - Skidoo Mine, Park Route 38 (Skidoo Road), Death Valley Junction, Inyo County, CA

  8. PTEN functions by recruitment to cytoplasmic vesicles.

    PubMed

    Naguib, Adam; Bencze, Gyula; Cho, Hyejin; Zheng, Wu; Tocilj, Ante; Elkayam, Elad; Faehnle, Christopher R; Jaber, Nadia; Pratt, Christopher P; Chen, Muhan; Zong, Wei-Xing; Marks, Michael S; Joshua-Tor, Leemor; Pappin, Darryl J; Trotman, Lloyd C

    2015-04-16

    PTEN is proposed to function at the plasma membrane, where receptor tyrosine kinases are activated. However, the majority of PTEN is located throughout the cytoplasm. Here, we show that cytoplasmic PTEN is distributed along microtubules, tethered to vesicles via phosphatidylinositol 3-phosphate (PI(3)P), the signature lipid of endosomes. We demonstrate that the non-catalytic C2 domain of PTEN specifically binds PI(3)P through the CBR3 loop. Mutations render this loop incapable of PI(3)P binding and abrogate PTEN-mediated inhibition of PI 3-kinase/AKT signaling. This loss of function is rescued by fusion of the loop mutant PTEN to FYVE, the canonical PI(3)P binding domain, demonstrating the functional importance of targeting PTEN to endosomal membranes. Beyond revealing an upstream activation mechanism of PTEN, our data introduce the concept of PI 3-kinase signal activation on the vast plasma membrane that is contrasted by PTEN-mediated signal termination on the small, discrete surfaces of internalized vesicles. PMID:25866245

  9. Till disassembly do us part: a happy marriage of nuclear envelope and chromatin.

    PubMed

    Tsuchiya, Yuichi

    2008-02-01

    A characteristic feature of eukaryotic cells is the presence of nuclear envelope (NE) which separates genomic DNA from cytoplasm. NE is composed of inner nuclear membrane (INM), which interacts with chromatin, and outer nuclear membrane, which is connected to endoplasmic reticulum. Nuclear pore complexes are inserted into NE to form transport channels between nucleus and cytoplasm. In metazoan cells, an intermediate filament-based meshwork called as nuclear lamina exists between INM and chromatin. Sophisticated collaboration of these molecular machineries is necessary for the structure and functions of NE. Recent research advances have revealed that NE dynamically communicates with chromatin and cytoskeleton to control multiple nuclear functions. In this mini review, I briefly summarize the basic concepts and current topics of functional relationships between NE and chromatin. PMID:17999983

  10. Characterization of the VP39 envelope protein from Singapore grouper iridovirus.

    PubMed

    Zhang, Honglian; Zhou, Sheng; Xia, Liqun; Huang, Xiaohong; Huang, Youhua; Cao, Jianhao; Qin, Qiwei

    2015-12-01

    Singapore grouper iridovirus (SGIV) is a major pathogen that causes heavy economic losses to the grouper aquaculture industry in China and Southeast Asian countries. In the present study, a viral envelope protein, VP39, encoded by SGIV ORF39L, was identified and characterized. SGIV ORF39L was found in all sequenced iridoviruses and is now considered to be a core gene of the family Iridoviridae. ORF39L was classified as a late gene during in vitro infection using reverse transcription-polymerase chain reaction, western blotting, and a drug inhibition analysis. An indirect immunofluorescence assay revealed that the VP39 protein was confined to the cytoplasm, especially at viral assembly sites. Western blot and matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry analyses suggested that VP39 is an envelope protein. Immunogold electron microscopy further confirmed that VP39 is a viral envelope protein. Furthermore, a mouse anti-VP39 polyclonal antibody exhibited SGIV-neutralizing activity in vitro, suggesting that VP39 is involved in SGIV infection. Taken together, the current data suggest that VP39 represents a conserved envelope protein of iridoviruses that contributes to viral infection. PMID:26524136

  11. Telling tails: selective pressures acting on investment in lizard tails.

    PubMed

    Fleming, Patricia A; Valentine, Leonie E; Bateman, Philip W

    2013-01-01

    Caudal autotomy is a common defense mechanism in lizards, where the animal may lose part or all of its tail to escape entrapment. Lizards show an immense variety in the degree of investment in a tail (i.e., length) across species, with tails of some species up to three or four times body length (snout-vent length [SVL]). Additionally, body size and form also vary dramatically, including variation in leg development and robustness and length of the body and tail. Autotomy is therefore likely to have fundamentally different effects on the overall body form and function in different species, which may be reflected directly in the incidence of lost/regenerating tails within populations or, over a longer period, in terms of relative tail length for different species. We recorded data (literature, museum specimens, field data) for relative tail length (n=350 species) and the incidence of lost/regenerating tails (n=246 species). We compared these (taking phylogeny into account) with intrinsic factors that have been proposed to influence selective pressures acting on caudal autotomy, including body form (robustness, body length, leg development, and tail specialization) and ecology (foraging behavior, physical and temporal niches), in an attempt to identify patterns that might reflect adaptive responses to these different factors. More gracile species have relatively longer tails (all 350 spp., P < 0.001; also significant for five of the six families tested separately), as do longer (all species, P < 0.001; Iguanidae, P < 0.05; Lacertidae, P < 0.001; Scindidae, P < 0.001), climbing (all species, P < 0.05), and diurnal (all species, P < 0.01; Pygopodidae, P < 0.01) species; geckos without specialized tails (P < 0.05); or active-foraging skinks (P < 0.05). We also found some relationships with the data for caudal autotomy, with more lost/regenerating tails for nocturnal lizards (all 246 spp., P < 0.01; Scindidae, P < 0.05), larger skinks (P < 0.05), climbing geckos (P < 0

  12. The theoretical polarization of pure scattering axisymmetric circumstellar envelopes

    NASA Technical Reports Server (NTRS)

    Fox, G. K.

    1994-01-01

    The Sobolev approach to the scattering of starlight through a pure scattering circumstellar envelope is developed. The theoretical polarization due to electron scattering in Be star envelopes is calculated for two geometries (an equatorially enhanced envelope and a spheroidal envelope). Only the disk-type envelope is found to yield a maximum polarization consistent with the observed range for Be stars. A lower limit, analytical approximation to the theoretical polarization from a pure scattering envelope is obtained.

  13. Runaway tails in magnetized plasmas

    NASA Technical Reports Server (NTRS)

    Moghaddam-Taaheri, E.; Vlahos, L.; Rowland, H. L.; Papadopoulos, K.

    1985-01-01

    The evolution of a runaway tail driven by a dc electric field in a magnetized plasma is analyzed. Depending on the strength of the electric field and the ratio of plasma to gyrofrequency, there are three different regimes in the evolution of the tail. The tail can be (1) stable with electrons accelerated to large parallel velocities, (2) unstable to Cerenkov resonance because of the depletion of the bulk and the formation of a positive slope, (3) unstable to the anomalous Doppler resonance instability driven by the large velocity anisotropy in the tail. Once an instability is triggered (Cerenkov or anomalous Doppler resonance) the tail relaxes into an isotropic distribution. The role of a convection type loss term is also discussed.

  14. Safeguards Envelope Progress FY09

    SciTech Connect

    Richard Metcalf; Robert Bean

    2009-09-01

    The Safeguards Envelope is a strategy to determine a set of specific operating parameters which nuclear facilities may operate within to maximize safeguards effectiveness without sacrificing safety or plant efficiency. This paper details advanced statistical techniques will be applied to real plant process monitoring (PM) data from the Idaho Chemical Processing Plant (ICPP). As a result of the U.S. having no operating nuclear chemical reprocessing plants, there has been a strong interest in obtaining process monitoring data from the ICPP. The ICPP was shut down in 1996 and a recent effort has been made to retrieve the PM data from storage in a data mining effort. In a simulation based on this data, multi-tank and multi-attribute correlations were tested against synthetic diversion scenarios. Kernel regression smoothing was used to fit a curve to the historical data, and multivariable, residual analysis and cumulative sum techniques set parameters for operating conditions. Diversion scenarios were created and tested, showing improved results when compared with a previous study utilizing only one-variable Z- testing7.

  15. Personnel occupied woven envelope robot

    NASA Technical Reports Server (NTRS)

    Wessling, Francis; Teoh, William; Ziemke, M. Carl

    1988-01-01

    The Personnel Occupied Woven Envelope Robot (POWER) provides an alternative to extravehicular activity (EVA) of space suited astronauts and/or use of long slender manipulator arms such as are used in the Shuttle Remote Manipulator System. POWER provides the capability for a shirt sleeved astronaut to perform such work by entering a control pod through air locks at both ends of an inflated flexible bellows (access tunnel). The exoskeleton of the tunnel is a series of six degrees of freedom (Six-DOF) articulated links compressible to 1/6 of their fully extended length. The operator can maneuver the control pod to almost any location within about 50 m of the base attachment to the space station. POWER can be envisioned as a series of hollow Six-DOF manipulator segments or arms wherein each arm grasps the shoulder of the next arm. Inside the hollow arms ia a bellow-type access tunnel. The control pod is the fist of the series of linked hollow arms. The fingers of the fist are conventional manipulator arms under direct visual control of the nearby operator in the pod. The applications and progress to date of the POWER system is given.

  16. Loss of desmoplakin tail causes lethal acantholytic epidermolysis bullosa.

    PubMed

    Jonkman, Marcel F; Pasmooij, Anna M G; Pasmans, Suzanne G M A; van den Berg, Maarten P; Ter Horst, Henk J; Timmer, Albertus; Pas, Hendri H

    2005-10-01

    The cytoplasmic plaque protein desmoplakin (DP), which is located in desmosomes, plays a major role in epithelial and muscle cell adhesion by linking the transmembrane cadherins to the cytoplasmic intermediate filament network. Mutations of DP may cause striate palmoplantar keratoderma, arrhythmogenic right ventricular dysplasia, skin fragility/woolly hair syndrome, Naxos-like disease, and Carvajal syndrome. DP must be indispensable, because DP-/- mice are early abortive. Here, we report a patient with severe fragility of skin and mucous membranes caused by genetic truncation of the DP tail. The new phenotype is lethal in the neonatal period because of immense transcutaneous fluid loss. The phenotype also comprised universal alopecia, neonatal teeth, and nail loss. Histology showed suprabasal clefting and acantholysis throughout the spinous layer, mimicking pemphigus. Electron microscopy revealed disconnection of keratin intermediate filaments from desmosomes. Immunofluorescence staining of DP showed a distinct punctate intercellular pattern in the patient's skin. Protein analysis revealed expression of truncated DP polypeptides. Mutational analysis of the patient demonstrated compound heterozygosity for two DP mutations, 6079C-->T (R1934X) and 6370delTT, respectively. Aberrant mRNA transcripts that predict premature termination of translation with loss of the three intermediate filament-binding subdomains in the DP tail were detected by RT-PCR. The new dramatic phenotype, which we named "lethal acantholytic epidermolysis bullosa," underscores the paramount role of DP in epidermal integrity. PMID:16175511

  17. A New Family of Membrane Electron Transporters and Its Substrates, Including a New Cell Envelope Peroxiredoxin, Reveal a Broadened Reductive Capacity of the Oxidative Bacterial Cell Envelope

    PubMed Central

    Cho, Seung-Hyun; Parsonage, Derek; Thurston, Casey; Dutton, Rachel J.; Poole, Leslie B.; Collet, Jean-Francois; Beckwith, Jon

    2012-01-01

    ABSTRACT The Escherichia coli membrane protein DsbD functions as an electron hub that dispatches electrons received from the cytoplasmic thioredoxin system to periplasmic oxidoreductases involved in protein disulfide isomerization, cytochrome c biogenesis, and sulfenic acid reduction. Here, we describe a new class of DsbD proteins, named ScsB, whose members are found in proteobacteria and Chlamydia. ScsB has a domain organization similar to that of DsbD, but its amino-terminal domain differs significantly. In DsbD, this domain directly interacts with substrates to reduce them, which suggests that ScsB acts on a different array of substrates. Using Caulobacter crescentus as a model organism, we searched for the substrates of ScsB. We discovered that ScsB provides electrons to the first peroxide reduction pathway identified in the bacterial cell envelope. The reduction pathway comprises a thioredoxin-like protein, TlpA, and a peroxiredoxin, PprX. We show that PprX is a thiol-dependent peroxidase that efficiently reduces both hydrogen peroxide and organic peroxides. Moreover, we identified two additional proteins that depend on ScsB for reduction, a peroxiredoxin-like protein, PrxL, and a novel protein disulfide isomerase, ScsC. Altogether, our results reveal that the array of proteins involved in reductive pathways in the oxidative cell envelope is significantly broader than was previously thought. Moreover, the identification of a new periplasmic peroxiredoxin indicates that in some bacteria, it is important to directly scavenge peroxides in the cell envelope even before they reach the cytoplasm. PMID:22493033

  18. Mutations in the nuclear lamin proteins resulting in their aberrant assembly in the cytoplasm.

    PubMed Central

    Loewinger, L; McKeon, F

    1988-01-01

    We have constructed a series of mutations in the human A lamin cDNA to identify and alter the nuclear localization signal using an in vivo functional assay system. The nuclear localization signal in the lamin proteins has both structural and functional similarities with that of the SV40 large T-antigen. Mutations within this functional domain result in the assembly of cytoplasmic tubular structures, and the behavior of these mutants suggests a post-translational dimerization of the lamin proteins prior to their transport into the nucleus. In the course of this work other regions of the carboxy terminus of the A/C lamin proteins have been implicated in the proper assembly and structure of the nuclear envelope. Images PMID:3056713

  19. Resource envelope concepts for mission planning

    NASA Technical Reports Server (NTRS)

    Ibrahim, K. Y.; Weiler, J. D.; Tokaz, J. C.

    1991-01-01

    Seven proposed methods for creating resource envelopes for Space Station Freedom mission planning are detailed. Four reference science activity models are used to illustrate the effect of adding operational flexibility to mission timelines. For each method, a brief explanation is given along with graphs to illustrate the application of the envelopes to the power and crew resources. The benefits and costs of each method are analyzed in terms of resource utilization. In addition to the effect on individual activities, resource envelopes are analyzed at the experiment level.

  20. Flight investigation of the effect of tail boom strakes on helicopter directional control

    NASA Technical Reports Server (NTRS)

    Kelly, Henry L.; Crowell, Cynthia A.; Yenni, Kenneth R.; Lance, Michael B.

    1993-01-01

    A joint U.S. Army/NASA flight investigation was conducted utilizing a single-rotor helicopter to determine the effectiveness of horizontally mounted tail boom strakes on directional controllability and tail rotor power during low-speed, crosswind operating conditions. Three configurations were investigated: (1) baseline (strakes off), (2) single strake (strake at upper shoulder on port side of boom), and (3) double strake (upper strake plus a lower strake on same side of boom). The strakes were employed as a means to separate airflow over the tail boom and change fuselage yawing moments in a direction to improve the yaw control margin and reduce tail rotor power. Crosswind data were obtained in 5-knot increments of airspeed from 0 to 35 knots and in 30 deg increments of wind azimuth from 0 deg to 330 deg. At the most critical wind azimuth and airspeed in terms of tail rotor power, the strakes improved the pedal margin by 6 percent of total travel and reduced tail rotor power required by 17 percent. The increase in yaw control and reduction in tail rotor power offered by the strakes can expand the helicopter operating envelope in terms of gross weight and altitude capability. The strakes did not affect the flying qualities of the vehicle at airspeeds between 35 and 100 knots.

  1. Physical properties of cytoplasmic intermediate filaments.

    PubMed

    Block, Johanna; Schroeder, Viktor; Pawelzyk, Paul; Willenbacher, Norbert; Köster, Sarah

    2015-11-01

    Intermediate filaments (IFs) constitute a sophisticated filament system in the cytoplasm of eukaryotes. They form bundles and networks with adapted viscoelastic properties and are strongly interconnected with the other filament types, microfilaments and microtubules. IFs are cell type specific and apart from biochemical functions, they act as mechanical entities to provide stability and resilience to cells and tissues. We review the physical properties of these abundant structural proteins including both in vitro studies and cell experiments. IFs are hierarchical structures and their physical properties seem to a large part be encoded in the very specific architecture of the biopolymers. Thus, we begin our review by presenting the assembly mechanism, followed by the mechanical properties of individual filaments, network and structure formation due to electrostatic interactions, and eventually the mechanics of in vitro and cellular networks. This article is part of a Special Issue entitled: Mechanobiology. PMID:25975455

  2. Quantifying intermittent transport in cell cytoplasm

    NASA Astrophysics Data System (ADS)

    Lagache, Thibault; Holcman, David

    2008-03-01

    Active cellular transport is a fundamental mechanism for protein and vesicle delivery, cell cycle, and molecular degradation. Viruses can hijack the transport system and use it to reach the nucleus. Most transport processes consist of intermittent dynamics, where the motion of a particle, such as a virus, alternates between pure Brownian and directed movement along microtubules. In this Rapid Communication, we estimate the mean time for a particle to attach to a microtubule network. This computation leads to a coarse grained equation of the intermittent motion in radial and cylindrical geometries. Finally, by using the degradation activity inside the cytoplasm, we obtain refined asymptotic estimations for the probability and the mean time a virus reaches a small nuclear pore.

  3. Quantifying intermittent transport in cell cytoplasm.

    PubMed

    Lagache, Thibault; Holcman, David

    2008-03-01

    Active cellular transport is a fundamental mechanism for protein and vesicle delivery, cell cycle, and molecular degradation. Viruses can hijack the transport system and use it to reach the nucleus. Most transport processes consist of intermittent dynamics, where the motion of a particle, such as a virus, alternates between pure Brownian and directed movement along microtubules. In this Rapid Communication, we estimate the mean time for a particle to attach to a microtubule network. This computation leads to a coarse grained equation of the intermittent motion in radial and cylindrical geometries. Finally, by using the degradation activity inside the cytoplasm, we obtain refined asymptotic estimations for the probability and the mean time a virus reaches a small nuclear pore. PMID:18517320

  4. Non-ideal solution thermodynamics of cytoplasm.

    PubMed

    Ross-Rodriguez, Lisa U; Elliott, Janet A W; McGann, Locksley E

    2012-10-01

    Quantitative description of the non-ideal solution thermodynamics of the cytoplasm of a living mammalian cell is critically necessary in mathematical modeling of cryobiology and desiccation and other fields where the passive osmotic response of a cell plays a role. In the solution thermodynamics osmotic virial equation, the quadratic correction to the linear ideal, dilute solution theory is described by the second osmotic virial coefficient. Herein we report, for the first time, intracellular solution second osmotic virial coefficients for four cell types [TF-1 hematopoietic stem cells, human umbilical vein endothelial cells (HUVEC), porcine hepatocytes, and porcine chondrocytes] and further report second osmotic virial coefficients indistinguishable from zero (for the concentration range studied) for human hepatocytes and mouse oocytes. PMID:23840923

  5. RINGO/cdk1 and CPEB mediate poly(A) tail stabilization and translational regulation by ePAB

    PubMed Central

    Kim, Jong Heon; Richter, Joel D.

    2007-01-01

    One activity that controls mRNA translation in vertebrate oocytes, embryos, and neurons is cytoplasmic polyadenylation. In Xenopus oocytes, where much of the biochemistry of this process has been elucidated, nuclear pre-mRNAs containing a cytoplasmic polyadenylation element (CPE) in their 3′ untranslated regions (UTRs) have long poly(A) tails; once the RNAs are spliced and transported to the cytoplasm, the tails are shortened. Following the resumption of meiosis, the poly(A) tails are lengthened and translation ensues. CPEB is a sequence-specific RNA-binding protein that coordinates these events and does so by binding to the CPE as well as several factors including Gld2, a poly(A) polymerase, and PARN [poly(A)-specific ribonuclease], a deadenylase. Here, we show that ePAB, embryonic poly(A)-binding protein, transiently associates with the polyadenylation complex; it initially interacts with CPEB, but after polyadenylation, it binds the poly(A) tail. ePAB dissociation from CPEB is regulated by RINGO (Rapid Inducer of G2/M progression in Oocytes), a cyclin B1-like cofactor that activates cdk1, a protein kinase that phosphorylates CPEB. Subsequent ePAB binding to the poly(A) tail is necessary to protect the homopolymer from degradation by deadenylating enzymes. Poly(A)-bound ePAB also interacts with eIF4G, which instigates translation initiation of CPEB-bound mRNAs. PMID:17938241

  6. Inborn errors of cytoplasmic triglyceride metabolism.

    PubMed

    Wu, Jiang Wei; Yang, Hao; Wang, Shu Pei; Soni, Krishnakant G; Brunel-Guitton, Catherine; Mitchell, Grant A

    2015-01-01

    Triglyceride (TG) synthesis, storage, and degradation together constitute cytoplasmic TG metabolism (CTGM). CTGM is mostly studied in adipocytes, where starting from glycerol-3-phosphate and fatty acyl (FA)-coenzyme A (CoA), TGs are synthesized then stored in cytoplasmic lipid droplets. TG hydrolysis proceeds sequentially, producing FAs and glycerol. Several reactions of CTGM can be catalyzed by more than one enzyme, creating great potential for complex tissue-specific physiology. In adipose tissue, CTGM provides FA as a systemic energy source during fasting and is related to obesity. Inborn errors and mouse models have demonstrated the importance of CTGM for non-adipose tissues, including skeletal muscle, myocardium and liver, because steatosis and dysfunction can occur. We discuss known inborn errors of CTGM, including deficiencies of: AGPAT2 (a form of generalized lipodystrophy), LPIN1 (childhood rhabdomyolysis), LPIN2 (an inflammatory condition, Majeed syndrome, described elsewhere in this issue), DGAT1 (protein loosing enteropathy), perilipin 1 (partial lipodystrophy), CGI-58 (gene ABHD5, neutral lipid storage disease (NLSD) with ichthyosis and "Jordan's anomaly" of vacuolated polymorphonuclear leukocytes), adipose triglyceride lipase (ATGL, gene PNPLA2, NLSD with myopathy, cardiomyopathy and Jordan's anomaly), hormone-sensitive lipase (HSL, gene LIPE, hypertriglyceridemia, and insulin resistance). Two inborn errors of glycerol metabolism are known: glycerol kinase (GK, causing pseudohypertriglyceridemia) and glycerol-3-phosphate dehydrogenase (GPD1, childhood hepatic steatosis). Mouse models often resemble human phenotypes but may diverge markedly. Inborn errors have been described for less than one-third of CTGM enzymes, and new phenotypes may yet be identified. PMID:25300978

  7. Cytoplasmic location of α1A voltage-gated calcium channel C-terminal fragment (Cav2.1-CTF) aggregate is sufficient to cause cell death.

    PubMed

    Takahashi, Makoto; Obayashi, Masato; Ishiguro, Taro; Sato, Nozomu; Niimi, Yusuke; Ozaki, Kokoro; Mogushi, Kaoru; Mahmut, Yasen; Tanaka, Hiroshi; Tsuruta, Fuminori; Dolmetsch, Ricardo; Yamada, Mitsunori; Takahashi, Hitoshi; Kato, Takeo; Mori, Osamu; Eishi, Yoshinobu; Mizusawa, Hidehiro; Ishikawa, Kinya

    2013-01-01

    The human α1A voltage-dependent calcium channel (Cav2.1) is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C)-tail contains a small poly-glutamine (Q) tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6). A recent study has shown that a 75-kDa C-terminal fragment (CTF) containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (r)CTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12) cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range) than with Q13 (normal-length). Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB) and phosphorylated-CREB (p-CREB) in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei. PMID:23505410

  8. [Tail Plane Icing

    NASA Technical Reports Server (NTRS)

    1997-01-01

    The Aviation Safety Program initiated by NASA in 1997 has put greater emphasis in safety related research activities. Ice-contaminated-tailplane stall (ICTS) has been identified by the NASA Lewis Icing Technology Branch as an important activity for aircraft safety related research. The ICTS phenomenon is characterized as a sudden, often uncontrollable aircraft nose- down pitching moment, which occurs due to increased angle-of-attack of the horizontal tailplane resulting in tailplane stall. Typically, this phenomenon occurs when lowering the flaps during final approach while operating in or recently departing from icing conditions. Ice formation on the tailplane leading edge can reduce tailplane angle-of-attack range and cause flow separation resulting in a significant reduction or complete loss of aircraft pitch control. In 1993, the Federal Aviation Authority (FAA) and NASA embarked upon a four-year research program to address the problem of tailplane stall and to quantify the effect of tailplane ice accretion on aircraft performance and handling characteristics. The goals of this program, which was completed in March 1998, were to collect aerodynamic data for an aircraft tail with and without ice contamination and to develop analytical methods for predicting the effects of tailplane ice contamination. Extensive dry air and icing tunnel tests which resulted in a database of the aerodynamic effects associated with tailplane ice contamination. Although the FAA/NASA tailplane icing program generated some answers regarding ice-contaminated-tailplane stall (ICTS) phenomena, NASA researchers have found many open questions that warrant further investigation into ICTS. In addition, several aircraft manufacturers have expressed interest in a second research program to expand the database to other tail configurations and to develop experimental and computational methodologies for evaluating the ICTS phenomenon. In 1998, the icing branch at NASA Lewis initiated a second

  9. Time-resolved measurement technique for pulsed electron beam envelope basing on framing and streaking principle

    NASA Astrophysics Data System (ADS)

    Jiang, Xiao-Guo; Wang, Yuan; Yang, Zhi-Yong; Zhang, Huang; Wang, Yi

    2016-01-01

    The time-resolved electron beam envelope parameters, including cross sectional distribution and beam centroid position, are very important for the study of beam transmission characteristics in a magnetic field and for verifying the rationality of the magnetic field parameters employed. One kind of high time-resolved beam envelope measurement system has recently been developed, constituted of a high-speed framing camera and a streak camera. It can obtain three panoramic images of the beam and time continuous information along the given beam profile simultaneously. Recently obtained data has proved that several fast vibrations of the beam envelope along the diameter direction occur during the front and the tail parts of the electron beam. The vibration period is several nanoseconds. The effect of magnetic field on the electron beam is also observed and verified. Beam debugging experiments have proved that the existing beam transmission design is reasonable and viable. This beam envelope measurement system will establish a good foundation for beam physics research. Supported by National Natural Science Foundation of China (10675104, 11375162)

  10. Helicopter tail rotor noise analyses

    NASA Technical Reports Server (NTRS)

    George, A. R.; Chou, S. T.

    1986-01-01

    A study was made of helicopter tail rotor noise, particularly that due to interactions with the main rotor tip vortices, and with the fuselage separation mean wake. The tail rotor blade-main rotor tip vortex interaction is modelled as an airfoil of infinite span cutting through a moving vortex. The vortex and the geometry information required by the analyses are obtained through a free wake geometry analysis of the main rotor. The acoustic pressure-time histories for the tail rotor blade-vortex interactions are then calculated. These acoustic results are compared to tail rotor loading and thickness noise, and are found to be significant to the overall tail rotor noise generation. Under most helicopter operating conditions, large acoustic pressure fluctuations can be generated due to a series of skewed main rotor tip vortices passing through the tail rotor disk. The noise generation depends strongly upon the helicopter operating conditions and the location of the tail rotor relative to the main rotor.

  11. Personnel occupied woven envelope robot power

    NASA Technical Reports Server (NTRS)

    Wessling, F. C.

    1988-01-01

    The Personnel Occupied Woven Envelope Robot (POWER) concept has evolved over the course of the study. The goal of the project was the development of methods and algorithms for solid modeling for the flexible robot arm.

  12. Solar envelope concepts: moderate density building applications

    NASA Astrophysics Data System (ADS)

    Knowles, R. L.; Berry, R. D.

    1980-04-01

    The public policy mechanism for guaranteeing solar access is conceptualized as a solar zoning envelope that allows the largest possible building bulk on a land parcel without shadowing neighboring properties during specified times. Step-by-step methods for generating solar envelopes are described with extensive drawings, showing a variety of urban platting and lot configurations. Development and design possibilities are examined on a selected set of Los Angeles sites with typically diverse urban characteristics. Envelope attributes suitable for encouraging moderate-density commercial and residential building are examined in the context of two hypothetical but realistic development programs: one for speculative office buildings and one for condominium housing. Numerous illustrations of envelope forms and prototypical building designs are provided.

  13. Internal Sense of Direction: Sensing and Signaling from Cytoplasmic Chemoreceptors

    PubMed Central

    Collins, Kieran D.; Lacal, Jesus

    2014-01-01

    SUMMARY Chemoreceptors sense environmental signals and drive chemotactic responses in Bacteria and Archaea. There are two main classes of chemoreceptors: integral inner membrane and soluble cytoplasmic proteins. The latter were identified more recently than integral membrane chemoreceptors and have been studied much less thoroughly. These cytoplasmic chemoreceptors are the subject of this review. Our analysis determined that 14% of bacterial and 43% of archaeal chemoreceptors are cytoplasmic, based on currently sequenced genomes. Cytoplasmic chemoreceptors appear to share the same key structural features as integral membrane chemoreceptors, including the formations of homodimers, trimers of dimers, and 12-nm hexagonal arrays within the cell. Cytoplasmic chemoreceptors exhibit varied subcellular locations, with some localizing to the poles and others appearing both cytoplasmic and polar. Some cytoplasmic chemoreceptors adopt more exotic locations, including the formations of exclusively internal clusters or moving dynamic clusters that coalesce at points of contact with other cells. Cytoplasmic chemoreceptors presumably sense signals within the cytoplasm and bear diverse signal input domains that are mostly N terminal to the domain that defines chemoreceptors, the so-called MA domain. Similar to the case for transmembrane receptors, our analysis suggests that the most common signal input domain is the PAS (Per-Arnt-Sim) domain, but a variety of other N-terminal domains exist. It is also common, however, for cytoplasmic chemoreceptors to have C-terminal domains that may function for signal input. The most common of these is the recently identified chemoreceptor zinc binding (CZB) domain, found in 8% of all cytoplasmic chemoreceptors. The widespread nature and diverse signal input domains suggest that these chemoreceptors can monitor a variety of cytoplasmically based signals, most of which remain to be determined. PMID:25428939

  14. Structural basis for tubulin recognition by cytoplasmic linker protein 170 and its autoinhibition.

    PubMed

    Mishima, Masaki; Maesaki, Ryoko; Kasa, Miyuki; Watanabe, Takashi; Fukata, Masaki; Kaibuchi, Kozo; Hakoshima, Toshio

    2007-06-19

    Cytoplasmic linker protein 170 (CLIP-170) is a prototype of the plus end-tracking proteins that regulate microtubule dynamics, but it is obscure how CLIP-170 recognizes the microtubule plus end and contributes to polymerization rescue. Crystallographic, NMR, and mutation studies of two tandem cytoskeleton-associated protein glycine-rich (CAP-Gly) domains of CLIP-170, CAP-Gly-1 and CAP-Gly-2, revealed positively charged basic grooves of both CAP-Gly domains for tubulin binding, whereas the CAP-Gly-2 domain possesses a more basic groove and directly binds the EExEEY/F motif of the C-terminal acidic-tail ends of alpha-tubulin. Notably, the p150(Glued) CAP-Gly domain that is furnished with a less positively charged surface only weakly interacts with the alpha-tubulin acidic tail. Mutation studies showed that this acidic sextette motif is the minimum region for CAP-Gly binding. The C-terminal zinc knuckle domains of CLIP-170 bind the basic groove to inhibit the binding to the acidic tails. These results provide a structural basis for the proposed CLIP-170 copolymerization with tubulin on the microtubule plus end. CLIP-170 strongly binds the acidic tails of EB1 as well as those of alpha-tubulins, indicating that EB1 localized at the plus end contributes to CLIP-170 recruitment to the plus end. We suggest that CLIP-170 stimulates microtubule polymerization and/or nucleation by neutralizing the negative charges of tubulins with the highly positive charges of the CLIP-170 CAP-Gly domains. Once CLIP-170 binds microtubule, the released zinc knuckle domain may serve to recruit dynein to the plus end by interacting with p150(Glued) and LIS1. Thus, our structures provide the structural basis for the specific dynein loading on the microtubule plus end. PMID:17563362

  15. Survival of an Enveloped Virus on Toys.

    PubMed

    Bearden, Richard L; Casanova, Lisa M

    2016-08-01

    Children's toys may carry respiratory viruses. Inactivation of a lipid-enveloped bacteriophage, Φ6, was measured on a nonporous toy at indoor temperature and relative humidity (RH). Inactivation was approximately 2log10 after 24 hours at 60% RH and 6.8log10 at 10 hours at 40% RH. Enveloped viruses can potentially survive on toys long enough to result in exposures. PMID:27144972

  16. Creating a Lunar EVA Work Envelope

    NASA Technical Reports Server (NTRS)

    Griffin, Brand N.; Howard, Robert; Rajulu, Sudhakar; Smitherman, David

    2009-01-01

    A work envelope has been defined for weightless Extravehicular Activity (EVA) based on the Space Shuttle Extravehicular Mobility Unit (EMU), but there is no equivalent for planetary operations. The weightless work envelope is essential for planning all EVA tasks because it determines the location of removable parts, making sure they are within reach and visibility of the suited crew member. In addition, using the envelope positions the structural hard points for foot restraints that allow placing both hands on the job and provides a load path for reacting forces. EVA operations are always constrained by time. Tasks are carefully planned to ensure the crew has enough breathing oxygen, cooling water, and battery power. Planning first involves computers using a virtual work envelope to model tasks, next suited crew members in a simulated environment refine the tasks. For weightless operations, this process is well developed, but planetary EVA is different and no work envelope has been defined. The primary difference between weightless and planetary work envelopes is gravity. It influences anthropometry, horizontal and vertical mobility, and reaction load paths and introduces effort into doing "overhead" work. Additionally, the use of spacesuits other than the EMU, and their impacts on range of motion, must be taken into account. This paper presents the analysis leading to a concept for a planetary EVA work envelope with emphasis on lunar operations. There is some urgency in creating this concept because NASA has begun building and testing development hardware for the lunar surface, including rovers, habitats and cargo off-loading equipment. Just as with microgravity operations, a lunar EVA work envelope is needed to guide designers in the formative stages of the program with the objective of avoiding difficult and costly rework.

  17. Tail buffet alleviation of high performance twin tail aircraft using offset piezoceramic stack actuators and acceleration feedback control

    NASA Astrophysics Data System (ADS)

    Bayon de Noyer, Maxime P.

    In High Performance Twin-Tail Aircraft (HPTTA), tail buffet occurs during high angles of attack maneuvers. At high angles of attack, flow separates and vortices are convected by the geometry of the wing-fuselage interface toward the vertical tails. This phenomenon, along with the aeroelastic coupling of the tail structural assembly, results in vibrations that can shorten the fatigue life of the empennage assembly and limit the flight envelope due to the large amplitude of the fin vibrations. The main goal of this research was to develop an active buffet alleviation system for HPTTA using Offset Piezoceramic Stack Actuators (OPSA) in combination with Acceleration Feedback Control (AFC) theory. In order to complete this task, the research work was divided into three main areas. First, two new methods for the design of non-collocated AFC controller parameters were developed for pure active damping applications and for quadratic performance criterion minimization. Second, a new type of moment inducing actuator based on piezoceramic stacks, the OPSA, was developed to provide high control authority while satisfying high reliability and maintainability requirements. A modal model of the OPSA acting on a benchmark structure was developed to create a low frequency approximation of the actuator and to optimize its offset distance and its placement. Third, because of the non-availability of reliable models for the controlled structure and the buffet-induced loads, a control system design method, based solely on the use of experimental data, was developed. Finally, two sets of experiments were conducted to show the feasibility of controlling buffet-induced vibrations during high angle of attack operations of a HPTTA. The first experiment validated both the effectiveness and the robustness of the active buffet alleviation system on an aeroelastically scaled model in wind tunnel tests. The second experiment showed that the combination of OPSA and AFC could suppress vibrations in

  18. Mercury's Dynamic Magnetic Tail

    NASA Technical Reports Server (NTRS)

    Slavin, James A.

    2010-01-01

    The Mariner 10 and MESSENGER flybys of Mercury have revealed a magnetosphere that is likely the most responsive to upstream interplanetary conditions of any in the solar system. The source of the great dynamic variability observed during these brief passages is due to Mercury's proximity to the Sun and the inverse proportionality between reconnection rate and solar wind Alfven Mach number. However, this planet's lack of an ionosphere and its small physical dimensions also contribute to Mercury's very brief Dungey cycle, approx. 2 min, which governs the time scale for internal plasma circulation. Current observations and understanding of the structure and dynamics of Mercury's magnetotail are summarized and discussed. Special emphasis will be placed upon such questions as: 1) How much access does the solar wind have to this small magnetosphere as a function of upstream conditions? 2) What roles do heavy planetary ions play? 3) Do Earth-like substorms take place at Mercury? 4) How does Mercury's tail respond to extreme solar wind events such coronal mass ejections? Prospects for progress due to advances in the global magnetohydrodynamic and hybrid simulation modeling and the measurements to be taken by MESSENGER after it enters Mercury orbit on March 18, 2011 will be discussed.

  19. Genetic diversity of koala retroviral envelopes.

    PubMed

    Xu, Wenqin; Gorman, Kristen; Santiago, Jan Clement; Kluska, Kristen; Eiden, Maribeth V

    2015-03-01

    Genetic diversity, attributable to the low fidelity of reverse transcription, recombination and mutation, is an important feature of infectious retroviruses. Under selective pressure, such as that imposed by superinfection interference, gammaretroviruses commonly adapt their envelope proteins to use alternative receptors to overcome this entry block. The first characterized koala retroviruses KoRV subgroup A (KoRV-A) were remarkable in their absence of envelope genetic variability. Once it was determined that KoRV-A was present in all koalas in US zoos, regardless of their disease status, we sought to isolate a KoRV variant whose presence correlated with neoplastic malignancies. More than a decade after the identification of KoRV-A, we isolated a second subgroup of KoRV, KoRV-B from koalas with lymphomas. The envelope proteins of KoRV-A and KoRV-B are sufficiently divergent to confer the ability to bind and employ distinct receptors for infection. We have now obtained a number of additional KoRV envelope variants. In the present studies we report these variants, and show that they differ from KoRV-A and KoRV-B envelopes in their host range and superinfection interference properties. Thus, there appears to be considerable variation among KoRVs envelope genes suggesting genetic diversity is a factor following the KoRV-A infection process. PMID:25789509

  20. Cooling of neutron stars with diffusive envelopes

    NASA Astrophysics Data System (ADS)

    Beznogov, M. V.; Fortin, M.; Haensel, P.; Yakovlev, D. G.; Zdunik, J. L.

    2016-08-01

    We study the effects of heat blanketing envelopes of neutron stars on their cooling. To this aim, we perform cooling simulations using newly constructed models of the envelopes composed of binary ion mixtures (H-He, He-C, C-Fe) varying the mass of lighter ions (H, He or C) in the envelope. The results are compared with those calculated using the standard models of the envelopes which contain the layers of lighter (accreted) elements (H, He and C) on top of the Fe layer, varying the mass of accreted elements. The main effect is that the chemical composition of the envelopes influences their thermal conductivity and, hence, thermal insulation of the star. For illustration, we apply these results to estimate the internal temperature of the Vela pulsar and to study the cooling of neutron stars of ages of 105 - 106 yr at the photon cooling stage. The uncertainties of the cooling models associated with our poor knowledge of chemical composition of the heat insulating envelopes strongly complicate theoretical reconstruction of the internal structure of cooling neutron stars from observations of their thermal surface emission.

  1. The joke envelope: a neglected precursor of the psychic envelope concept in Freud's writing.

    PubMed

    Spero, Moshe Halevi

    2009-01-01

    The concepts of the primeval skin ego, psychic envelope, and related pre-ego containing and wrapping functions elaborated respectively by Esther Bick, Didier Anzieu, and Francis Tustin occupy an important position in contemporary psychoanalytic theory and clinical practice. The psychic envelope begins as a virtual mental protostructure ("proto" because it is not yet based on fully symbolized representations) that holds the budding mind together pending further developments. With maturity, the enveloping functions adopt symbolized, metaphoric form (for example, the aesthetic use of cloth, the analytic framework), but can regress to more concrete and pathological forms. The aforementioned authors based their ideas on a cluster of specific allusions to the idea of a psychic covering, barrier, or envelope in Freud's work. Yet they neglected one reference, hidden in Freud's analysis of the structure ofjokes and humor: the 'joke envelope"--die witzige Einkleidung. The present essay explores Freud's use of the term Einkleidung, including his intriguing idea that a joke requires three people whereas a dream does not and the fact that Freud nowhere speaks of a "dream envelope. "I take the "joke envelope" beyond its original context and posit a relationship between laughter and the early, normative traumas of breathing, crying, and loss, and the dawn of rhythmic envelopes that enable mentalization. Jokes and joking symbolically repeat the early rupture and rapture of breathing and self-other differentiation and the internalization of maternal containing and envelopment. PMID:20578439

  2. The RanGTP Pathway: From Nucleo-Cytoplasmic Transport to Spindle Assembly and Beyond

    PubMed Central

    Cavazza, Tommaso; Vernos, Isabelle

    2016-01-01

    The small GTPase Ran regulates the interaction of transport receptors with a number of cellular cargo proteins. The high affinity binding of the GTP-bound form of Ran to import receptors promotes cargo release, whereas its binding to export receptors stabilizes their interaction with the cargo. This basic mechanism linked to the asymmetric distribution of the two nucleotide-bound forms of Ran between the nucleus and the cytoplasm generates a switch like mechanism controlling nucleo-cytoplasmic transport. Since 1999, we have known that after nuclear envelope breakdown (NEBD) Ran and the above transport receptors also provide a local control over the activity of factors driving spindle assembly and regulating other aspects of cell division. The identification and functional characterization of RanGTP mitotic targets is providing novel insights into mechanisms essential for cell division. Here we review our current knowledge on the RanGTP system and its regulation and we focus on the recent advances made through the characterization of its mitotic targets. We then briefly review the novel functions of the pathway that were recently described. Altogether, the RanGTP system has moonlighting functions exerting a spatial control over protein interactions that drive specific functions depending on the cellular context. PMID:26793706

  3. The cytoplasmic domain of simian immunodeficiency virus transmembrane protein modulates infectivity.

    PubMed Central

    Chakrabarti, L; Emerman, M; Tiollais, P; Sonigo, P

    1989-01-01

    A striking characteristic of the simian immunodeficiency virus (SIV) and of the human immunodeficiency virus type 2 (HIV-2) is the presence of a nonsense mutation in the env gene resulting in the synthesis of a truncated transmembrane protein lacking the cytoplasmic domain. By mutagenesis of an infectious molecular clone of SIVmac142, we investigated the function of the cytoplasmic domain and the significance of the env nonsense mutation. When the nonsense codon (TAG) was replaced by a glutamine codon (CAG), the virus infected HUT78 cells with markedly delayed kinetics. This negative effect was counterselected in vitro as reversion of the slow phenotype frequently occurred. The sequencing of one revertant revealed the presence of a new stop codon three nucleotides 5' to the original mutation. Deletions or an additional nonsense mutation introduced 3' to the original stop codon did not modify SIV infectivity. In contrast, the same deletions or nonsense mutation introduced in the clone in which the stop codon was replaced by CAG abolished infectivity. These results indicated that the envelope domain located 3' to the stop codon is not necessary for in vitro replication. However, the presence of this domain in SIV transmembrane protein leads to a reduced infectivity. This negative effect might correspond to a function controlling the rate of spread of the virus during in vivo infection. Images PMID:2778881

  4. Insights into the Function of YciM, a Heat Shock Membrane Protein Required To Maintain Envelope Integrity in Escherichia coli

    PubMed Central

    Nicolaes, Valérie; El Hajjaji, Hayat; Davis, Rebecca M.; Van der Henst, Charles; Depuydt, Matthieu; Leverrier, Pauline; Aertsen, Abram; Haufroid, Vincent; Ollagnier de Choudens, Sandrine; De Bolle, Xavier; Ruiz, Natividad

    2013-01-01

    The cell envelope of Gram-negative bacteria is an essential organelle that is important for cell shape and protection from toxic compounds. Proteins involved in envelope biogenesis are therefore attractive targets for the design of new antibacterial agents. In a search for new envelope assembly factors, we screened a collection of Escherichia coli deletion mutants for sensitivity to detergents and hydrophobic antibiotics, a phenotype indicative of defects in the cell envelope. Strains lacking yciM were among the most sensitive strains of the mutant collection. Further characterization of yciM mutants revealed that they display a thermosensitive growth defect on low-osmolarity medium and that they have a significantly altered cell morphology. At elevated temperatures, yciM mutants form bulges containing cytoplasmic material and subsequently lyse. We also discovered that yciM genetically interacts with envC, a gene encoding a regulator of the activity of peptidoglycan amidases. Altogether, these results indicate that YciM is required for envelope integrity. Biochemical characterization of the protein showed that YciM is anchored to the inner membrane via its N terminus, the rest of the protein being exposed to the cytoplasm. Two CXXC motifs are present at the C terminus of YciM and serve to coordinate a redox-sensitive iron center of the rubredoxin type. Both the N-terminal membrane anchor and the C-terminal iron center of YciM are important for function. PMID:24187084

  5. Virtual Nuclear Envelope Breakdown and Its Regulators in Fission Yeast Meiosis.

    PubMed

    Asakawa, Haruhiko; Yang, Hui-Ju; Hiraoka, Yasushi; Haraguchi, Tokuko

    2016-01-01

    Ran, a small GTPase, is required for the spindle formation and nuclear envelope (NE) formation. After NE breakdown (NEBD) during mitosis in metazoan cells, the Ran-GTP gradient across the NE is lost and Ran-GTP becomes concentrated around chromatin, thus affecting the stability of microtubules and promoting the assembly of spindle microtubules and segregation of chromosomes. Mitosis in which chromosomes are segregated subsequent to NEBD is called "open mitosis." In contrast, many fungi undergo a process termed "closed mitosis" in which chromosome segregation and spindle formation occur without NEBD. Although the fission yeast Schizosaccharomyces pombe undergoes a closed mitosis, it exhibits a short period during meiosis (anaphase of the second meiosis; called "anaphase II") when nuclear and cytoplasmic proteins are mixed in the presence of intact NE and nuclear pore complexes (NPC). This "virtual" nuclear envelope breakdown (vNEBD) involves changes in the localization of RanGAP1, an activator of Ran-GTP hydrolysis. Recently, Nup132, a component of the structural core Nup107-160 subcomplex of the NPC, has been shown to be involved in the maintenance of the nuclear cytoplasmic barrier in yeast meiosis. In this review, we highlight the possible roles of RanGAP1 and Nup132 in vNEBD and discuss the biological significance of vNEBD in S. pombe meiosis. PMID:26870731

  6. Virtual Nuclear Envelope Breakdown and Its Regulators in Fission Yeast Meiosis

    PubMed Central

    Asakawa, Haruhiko; Yang, Hui-Ju; Hiraoka, Yasushi; Haraguchi, Tokuko

    2016-01-01

    Ran, a small GTPase, is required for the spindle formation and nuclear envelope (NE) formation. After NE breakdown (NEBD) during mitosis in metazoan cells, the Ran-GTP gradient across the NE is lost and Ran-GTP becomes concentrated around chromatin, thus affecting the stability of microtubules and promoting the assembly of spindle microtubules and segregation of chromosomes. Mitosis in which chromosomes are segregated subsequent to NEBD is called “open mitosis.” In contrast, many fungi undergo a process termed “closed mitosis” in which chromosome segregation and spindle formation occur without NEBD. Although the fission yeast Schizosaccharomyces pombe undergoes a closed mitosis, it exhibits a short period during meiosis (anaphase of the second meiosis; called “anaphase II”) when nuclear and cytoplasmic proteins are mixed in the presence of intact NE and nuclear pore complexes (NPC). This “virtual” nuclear envelope breakdown (vNEBD) involves changes in the localization of RanGAP1, an activator of Ran-GTP hydrolysis. Recently, Nup132, a component of the structural core Nup107-160 subcomplex of the NPC, has been shown to be involved in the maintenance of the nuclear cytoplasmic barrier in yeast meiosis. In this review, we highlight the possible roles of RanGAP1 and Nup132 in vNEBD and discuss the biological significance of vNEBD in S. pombe meiosis. PMID:26870731

  7. Two independent targeting signals in the cytoplasmic domain determine trans-Golgi network localization and endosomal trafficking of the proprotein convertase furin.

    PubMed Central

    Schäfer, W; Stroh, A; Berghöfer, S; Seiler, J; Vey, M; Kruse, M L; Kern, H F; Klenk, H D; Garten, W

    1995-01-01

    Furin, a subtilisin-like eukaryotic endoprotease, is responsible for proteolytic cleavage of cellular and viral proteins transported via the constitutive secretory pathway. Cleavage occurs at the C-terminus of basic amino acid sequences, such as R-X-K/R-R and R-X-X-R. Furin was found predominantly in the trans-Golgi network (TGN), but also in clathrin-coated vesicles dispatched from the TGN, on the plasma membrane as an integral membrane protein and in the medium as an anchorless enzyme. When furin was vectorially expressed in normal rat kidney (NRK) cells it accumulated in the TGN similarly to the endogenous glycoprotein TGN38, often used as a TGN marker protein. The signals determining TGN targeting of furin were investigated by mutational analysis of the cytoplasmic tail of furin and by using the hemagglutinin (HA) of fowl plague virus, a protein with cell surface destination, as a reporter molecule, in which membrane anchor and cytoplasmic tail were replaced by the respective domains of furin. The membrane-spanning domain of furin grafted to HA does not localize the chimeric molecule to the TGN, whereas the cytoplasmic domain does. Results obtained on furin mutants with substitutions and deletions of amino acids in the cytoplasmic tail indicate that wild-type furin is concentrated in the TGN by a mechanism involving two independent targeting signals, which consist of the acidic peptide CPSDSEEDEG783 and the tetrapeptide YKGL765. The acidic signal in the cytoplasmic domain of a HA-furin chimera is necessary and sufficient to localize the reporter molecule to the TGN, whereas YKGL is a determinant for targeting to the endosomes. The data support the concept that the acidic signal, which is the dominant one, retains furin in the TGN, whereas the YKGL motif acts as a retrieval signal for furin that has escaped to the cell surface. Images PMID:7781597

  8. In Vivo Dynamics of Drosophila Nuclear Envelope Components

    PubMed Central

    Katsani, Katerina R.; Karess, Roger E.; Dostatni, Nathalie

    2008-01-01

    Nuclear pore complexes (NPCs) are multisubunit protein entities embedded into the nuclear envelope (NE). Here, we examine the in vivo dynamics of the essential Drosophila nucleoporin Nup107 and several other NE-associated proteins during NE and NPCs disassembly and reassembly that take place within each mitosis. During both the rapid mitosis of syncytial embryos and the more conventional mitosis of larval neuroblasts, Nup107 is gradually released from the NE, but it remains partially confined to the nuclear (spindle) region up to late prometaphase, in contrast to nucleoporins detected by wheat germ agglutinin and lamins. We provide evidence that in all Drosophila cells, a structure derived from the NE persists throughout metaphase and early anaphase. Finally, we examined the dynamics of the spindle checkpoint proteins Mad2 and Mad1. During mitotic exit, Mad2 and Mad1 are actively imported back from the cytoplasm into the nucleus after the NE and NPCs have reformed, but they reassociate with the NE only later in G1, concomitantly with the recruitment of the basket nucleoporin Mtor (the Drosophila orthologue of vertebrate Tpr). Surprisingly, Drosophila Nup107 shows no evidence of localization to kinetochores, despite the demonstrated importance of this association in mammalian cells. PMID:18562695

  9. Coronavirus envelope (E) protein remains at the site of assembly

    SciTech Connect

    Venkatagopalan, Pavithra; Daskalova, Sasha M.; Lopez, Lisa A.; Dolezal, Kelly A.; Hogue, Brenda G.

    2015-04-15

    Coronaviruses (CoVs) assemble at endoplasmic reticulum Golgi intermediate compartment (ERGIC) membranes and egress from cells in cargo vesicles. Only a few molecules of the envelope (E) protein are assembled into virions. The role of E in morphogenesis is not fully understood. The cellular localization and dynamics of mouse hepatitis CoV A59 (MHV) E protein were investigated to further understanding of its role during infection. E protein localized in the ERGIC and Golgi with the amino and carboxy termini in the lumen and cytoplasm, respectively. E protein does not traffic to the cell surface. MHV was genetically engineered with a tetracysteine tag at the carboxy end of E. Fluorescence recovery after photobleaching (FRAP) showed that E is mobile in ERGIC/Golgi membranes. Correlative light electron microscopy (CLEM) confirmed the presence of E in Golgi cisternae. The results provide strong support that E proteins carry out their function(s) at the site of budding/assembly. - Highlights: • Mouse hepatitis coronavirus (MHV-CoV) E protein localizes in the ERGIC and Golgi. • MHV-CoV E does not transport to the cell surface. • MHV-CoV can be genetically engineered with a tetracysteine tag appended to E. • First FRAP and correlative light electron microscopy of a CoV E protein. • Live-cell imaging shows that E is mobile in ERGIC/Golgi membranes.

  10. Cytoplasmic Adenylation and Processing of Maternal RNA

    PubMed Central

    Slater, Isabel; Gillespie, David; Slater, D. W.

    1973-01-01

    Molecular hybridization between [3H]-poly(U) and unlabeled RNA prepared from sea urchin eggs and embryos has been used to contrast the subcellular localization as well as the size distribution of adenylylated maternal RNA preexisting in the unfertilized egg with that adenylylated as a function of fertilization. Evidence reported establishes that such preadenylylated genetic messages are predominantly located in the ovum's subribosomal fraction and that fertilization elicits a rapid reallocation of these latent transcripts into the zygote's ribosomal fraction. Examination of the size distribution of the adenylylated RNA further demonstrates that the unfertilized egg contains a substantial population of RNA transcripts of exceptionally high molecular weight that are used as primers for the 2-fold net synthesis of poly(A) that follows fertilization. The poly(A)-rich tracts are shown to be covalently bonded to RNA. Assessment of the poly(A) content of nuclear and cytoplasmic fractions suggests that the function of poly(A) is not confined to the transport of genetic messages from the nucleus. PMID:4510284