110 research outputs found
Structural and functional characterization of the p62 complex, a subcomplex of the nuclear pore complex
No full textThe nuclear pore complex (NPC) is a highly conserved eukaryotic protein complex, which perforates
the nuclear envelope and regulates nucleocytoplasmic transport of cargos between the cytoplasm and nucleus. The structure and function of NPCs were examined in recent years by different molecular and structural biology techniques, such as immunoprecipitation, RNA interference, or electron- and fluorescence microscopy, allowing deeper insights into the molecular mechanisms underlying nucleocytoplasmic
transport. In this introduction, I will highlight recent developments in understanding the organization of four subcomplexes of the central region of the NPC, namely, the p62 complex, the Nup93 complex, the Nup107-160 complex, and the Nup155 complex as well as their impact on nucleocytoplasmic transport. In addition, I will discuss the role of these subcomplexes in cell cycle regulation and their impact
on human diseases. Furthermore, the molecular interactions between different transport receptors, cargos, and components of the NPC are described.
The nuclear pore complex (NPC) is the only known gateway for exchange of macromolecules between the cytoplasm and nucleus of eukaryotic cells. One key compound of the NPC is the p62 subcomplex,
which consists of the nucleoporins p62, p54, and p58/p45 and is supposed to be involved in nuclear protein import and export. In this study we show the localization of different domains of the p62 complex by immuno-electron microscopy using isolated nuclei from Xenopus oocytes. To determine the exact position of the p62 complex, we examined the localization of the C- and N-terminal domains of p62 by immunolabeling using domain-specific antibodies against p62. In addition, we expressed epitope-
tagged versions of p62, p54, and p58 in Xenopus oocytes and localized the domains with antibodies against the tags. This first systematic analysis of the domain topology of the p62 complex within the NPC revealed that the p62 complex is anchored to the cytoplasmic face of the NPC most likely by the coiled-coil domains of the three nucleoporins. Furthermore, we found the phenylalanine-glycine (FG)-repeat domain of p62, but not of p58 and p54, to be mobile and flexible nature.
Nuclear pore complexes (NPCs) are large protein complexes, which are embedded in the nuclear envelope (NE) and control the traffic of proteins and RNAs between the nucleus and the cytoplasm in a signal-dependent manner. Transport receptors bind cargos and interact particularly with certain nucleoporins.
Phenylalanine-glycine (FG) repeat motifs were found in about a third of the nucleoporins, functioning as a major docking site for soluble transport receptors. The p62 complex, which contains several FG repeat domains, was previously described to be involved in protein import and to interact directly with soluble transport receptors. To examine the localization of this docking site and to find out how antibodies against single components of the p62 complex influence nucleocytoplasmic transport, we performed in vitro transport assays using nucleoplasmin-GFP as cargo. This cargo was used in transport assays with digitonin-permeabilized HeLa cells, which were treated with antibodies against the p62 complex components, or was directly conjugated to colloidal gold in ultrastructural transport assays, using isolated nuclei from Xenopus oocytes. Our data suggest that antibodies against the components of the p62 complex inhibit or reduce transport of cargos through the NPC. The ultratructural transport studies revealed that the second docking site for cargo/receptor complexes is masked when p62 complex antibodies are used in the transport assay.
During the past few years, evidence accumulated that distinct nuclear pore complex proteins do not only function in the nucleocytoplasmic transport, but also in the regulation of other cellular processes, such as mitosis. To study the function of the p62-complex (i.e. p62, p54, and p58) in a cellular context, we depleted its components from HeLa cells by RNA interference, which led to an arrest in cell growth and an increase of apoptotic cells as analyzed by fluorescence activated cell sorting. In vitro transport studies of p62- and p54-depleted HeLa cells further showed that the depletion of these p62 complex components had a significant inhibitory effect on nuclear protein import. Taken together, these results indicate that the p62 complex is not only critical for mediating nuclear protein import, but also for cell growth and cell division.
To determine the location of different domains of the p62 complex within the 3D structure of the NPC, we localized the different FG-repeat and coiled-coil domains of this subcomplex with immuno-EM. Previous immuno-EM showed controversial results concerning the anchoring sites and the localization of the p62 complex within the 3D structure of the NPC. Our new data revealed that the p62 complex is anchored with its coiled-coil domains to the cytoplasmic side of the NPC and that its FG-repeat domains show differences in their flexibility and their distribution within the 3D architecture of the NPC. Thus, the FG-repeat domain of the nucleoporin p62 could be found at the cytoplasmic as well as at the nuclear
side of the NPC, whereas the FG-repeat domains of p54 and p58 were restricted to the cytoplasmic side. This might be due to different anchoring sites of the distint FG-repeat domains, the varied length of the FG-repeat domains of the complex, and differences in the biophysical behavior of the FG-repeat domains.
Recently, a complementary technique to immuno-EM was established to observe FG-repeat domains
in vitro by AFM. Lim et al. examined the biophysical behavior of the FG-repeat domain of Nup153, immobilized to a gold dot, by AFM simulating nuclear import by adding importin β.and RanGTP (Lim, Fahrenkrog et al. 2007). The immobilized FG-repeat domains formed a polymer brush with a certain height, which collapsed after addition of importin β. This collapse could be reversed after addition of RanGTP, demonstrating a new biophysical principle for nucleocytoplasmic transport. The AFM-studies were attended by immuno-EM-studies with isolated nuclei of Xenopus oocytes, which showed that, after addition of importin β, the FG-repeat domains of Nup153 collapse to their anchoring sites at the nuclear basket.
It would be interesting to use the same experimental approach equally for the different FG-repeat domains of the p62 complex in order to examine if the FG-repeat domains can form polymer brushes similar to the ones formed by the FG-repeat domains of Nup153. Beyond that, it would be worth to uncover
the anchoring sites of the FG-repeat domains of the p62 complex by immuno-EM after addition of importin β (Lim, Fahrenkrog et al. 2007). In the near future, progress in EM tomography will facilitate the localization of antibodies against certain nucleoporins without the use of gold particles, which would reveal the exact binding sites of the antibodies to structural elements of the NPC
Hoelz, Jacob (Death, 1873-11-07)
No full textAddress: 53 MulberryAge at death: 43Pg 235/1873/147/M W M/Germany/Dr. Thornton/Hust/Carthage Rd.Original record filed in drawer labeled 'HOBSON-HOFFMAN'
Premiere
No full textDie 1968 gegründete Zeitschrift "Kritische Justiz" stand anfangs am äußerst linken Rand der juristischen Publikationen. Auch der Rezensent, der in einer Ausgabe des Jahrgangs 1970 die Neuauflage der Lebenserinnerungen des KommunistenMax Hoelz "Vom 'Weißen Kreuz' zur roten Fahne" besprach, machte aus seinem dezidiert linken Standpunkt kein Geheimnis: die Neuauflage sei "wichtiges Lehrmaterial für die gegenwärtigen Klassenkämpfe" (Kritische Justiz 1970, 363–365). ..
Electrical and electromagnetic methods for submarine massive sulfide exploration: a case study of the Palinuro Seamount, Tyrrhenian Sea
No full textIncludes bibliographical references.2018 Spring.Recent years have seen increasing interest in exploring for mineral resources on the seafloor. I examine the capabilities of two geophysical methods in exploring for and characterizing seafloor massive sulfide (SMS) deposits: the electromagnetic (EM) and the self-potential (SP) methods. Working with a team from the Helmholtz Center for Ocean Research Kiel (GEOMAR) in Germany, I carried out a first test of a new marine EM configuration consisting of a towed-loop inductive source transmitter and remote ocean-bottom electric dipole receivers. The system was tested at the Palinuro Seamount, offshore Italy, where shallowly buried massive sulfides had previously been recovered from drill cores. Data from the first test of this EM configuration were collected using a horizontal loop transmitter, and analysis of the data found higher apparent conductivities when the transmitter was in proximity to the zone of known mineralization, suggesting that the buried massive sulfides were detected by the system. I also carried out 3D forward modeling which suggests that this configuration is sensitive to a shallowly buried conductive target of dimensions consistent with the drilling zone at a remote receiver up to ~100 m away from the transmitter when the target is located in line between the transmitter and receiver. I used both 1D and 3D forward modeling to compare the sensitivity of a horizontal loop transmitter vs a vertical loop transmitter in the marine EM configuration. The horizontal loop was found to be more sensitive than the vertical loop to the thickness of the target, which is advantageous in attempting to characterize the depth extent of mineralization. The vertical loop is more sensitive than the horizontal loop to a resistive target, such as a gas hydrate deposit. The vertical loop is also less affected by changes in the transmitter towing depth caused by the bathymetry of the field area. In addition to EM, I investigated the SP method in exploring for SMS deposits. A test of a marine SP system consisting of two perpendicular electrode pairs towed above the seafloor was carried out at the Palinuro study area. To my knowledge this was the first test of a marine SP system at a buried SMS site. The SP data showed elevated electric field strengths on the order of 1 - 3 mV/m over the zone of known mineralization, demonstrating that a shallowly buried massive sulfide occurrence can be detected by a marine SP system. My results show that both the marine SP and EM methods have applications in the exploration for SMS occurrences. Furthermore, data collected with the EM and SP systems both suggest that the mineralization at Palinuro extends southward of the drilling zone by ~40 m, which demonstrates that these methods are not only useful in detecting SMS deposits, but in characterizing their size and geometry as well
O Mundo é Um Moinho: Sacrifício E Cotidiano Em Mário De Andrade
No full textMário de Andrade's prolific and diverse activity in the press is one of the most recursive features of his life and work. Focusing on the modernist leader's role as a music critic in the press, this article argues that the idea of "sacrifice", inseparable from the fulfillment of an intellectual destiny, is central to articulate the different dimensions of his trajectory and work. It also contends that the question of "sacrifice" is what puts the duality between permanence and transience of his work into perspective. Finally, this article emphasizes that the sociological relevance of this idea is tied to the absence, in the Brazilian modernist context, of an autonomous intellectual field in which the different actors' positions, as well as in its internal and external relationships, were exclusively regulated by a particular habitus.19725128
As décadas do silêncio: a representação dos imigrantes no Brasil entre as duas guerras mundiais
No full textNucleoporins interacting with MX2 are located at the cytoplasmic face of the nuclear pore.
No full textSchematic diagram indicating the structure and organization of the nuclear pore complex as described in Hoelz et al [22]. Nucleoporins interacting with MX2 (identified by Y-2-H screening) are shown in red. Nucleoporins required for MX2 activity (defined as those for which specific depletion elicited >50% reduction in MX2 dependent inhibition of HIV-1, in either HeLa or U87-MG cells) are shown in bold. Nucleoporins that interact with MX2 and are required for MX2 inhibition are shown in red and bold.</p
Molecular basis for protection of ribosomal protein L4 from cellular degradation
No full textEukaryotic ribosome biogenesis requires the nuclear import of ∼80 nascent ribosomal proteins and the elimination of excess amounts by the cellular degradation machinery. Assembly chaperones recognize nascent unassembled ribosomal proteins and transport them together with karyopherins to their nuclear destination. We report the crystal structure of ribosomal protein L4 (RpL4) bound to its dedicated assembly chaperone of L4 (Acl4), revealing extensive interactions sequestering 70 exposed residues of the extended RpL4 loop. The observed molecular recognition fundamentally differs from canonical promiscuous chaperone–substrate interactions. We demonstrate that the eukaryote-specific RpL4 extension harbours overlapping binding sites for Acl4 and the nuclear transport factor Kap104, facilitating its continuous protection from the cellular degradation machinery. Thus, Acl4 serves a dual function to facilitate nuclear import and simultaneously protect unassembled RpL4 from the cellular degradation machinery
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