Temperature-induced changes in nuclear pore complex frequencies nuclear envelope surface areas and nuclear volumes in light-synchronized Euglena

1981 ◽  
Vol 59 (10) ◽  
pp. 802-809 ◽  
Author(s):  
John S. Greenwood ◽  
John N. A. Lott

An autotrophic culture of Euglena, synchronized using a day: night (D:N), 14:10-h cycle, was subjected to a 21.5 → 31.5 °C temperature shift and then to a reversed shift in temperature after three D:N cycles at 31.5 °C. Nuclear pore complex (NPC) number per square micrometre and nuclear surface area and volume determinations were made on G1 cells at various intervals. Cells sampled immediately prior to the 21.5 → 31.5 °C shift had a mean value of 37.68 NPC∙μm−2 nuclear envelope surface area, 30.40 NPCs/μm2 after three D:N cycles at.31.5 °C and 39.98 NPCs/μm2 after three D:N cycles at the resumed culture temperature of 21.5 °C. Thus temperature changes affect NPC numbers per square micrometre and these changes are reversible.Mean nuclear surface area was 125.76 μm2 immediately prior to the 21.5 → 31.5 °C shift, and decreased over two D:N cycles at 31.5 °C to 101.30 μm2 by the end of the third D:N cycle. Nuclear envelope surface area, one and two D:N cycles after the 31.5 → 21.5 °C shift, was approximately equal that prior to the 21.5 → 31.5 °C shift. After the third D:N cycle, however, nuclear surface area had increased to 173.05 μm2. The changes in nuclear surface area resulted in large differences in the estimates of the total number of NPCs per nucleus. Euglena immediately prior to the 21.5 → 31.5 °C temperature shift had 4739 NPCs/nucleus; immediately prior to the 31.5 → 21.5 °C shift had 3079 NPCs/nucleus; and had 6919 NPCs/nucleus at 21.5 °C and three D:N cycles after the 31.5 → 21.5 °C shift. Estimates of the number of NPCs per cubic micrometre of nuclear volume were almost identical between these samples.

1987 ◽  
Vol 104 (5) ◽  
pp. 1143-1156 ◽  
Author(s):  
C M Snow ◽  
A Senior ◽  
L Gerace

Using monoclonal antibodies we identified a group of eight polypeptides of rat liver nuclear envelopes that have common epitopes. Most or all of these proteins are structurally distinct, as shown by tryptic peptide mapping and analysis with polyclonal antibodies. While these polypeptides are relatively tightly bound to nuclear membranes, only one is an integral membrane protein. The eight antigens cofractionate with the nuclear pore complex under various conditions of ionic strength and detergent. It can be seen by immunofluorescence microscopy that the monoclonal antibodies reacting with these antigens stain the nuclear surface of interphase cells in a finely punctate pattern. When the nuclear envelope is disassembled and subsequently reformed during mitosis, the proteins are reversibly dispersed throughout the cytoplasm in the form of minute foci. By EM immunogold localization on isolated nuclear envelopes, the monoclonal antibodies label exclusively the nuclear pore complex, at both its nucleoplasmic and cytoplasmic margins. Considered together, our biochemical and localization data indicate that the eight nuclear envelope polypeptides are pore complex components. As shown in the accompanying paper (Holt, G. D., C. M. Snow, A. Senior, R. S. Haltiwanger, L. Gerace, and G. W. Hart, J. Cell Biol., 104:1157-1164) these eight polypeptides contain a novel form of glycosylation, O-linked N-acetylglucosamine. The relative abundance and disposition of these O-linked glycoproteins in the pore complex are consistent with their having a role in nucleocytoplasmic transport.


2011 ◽  
Vol 192 (5) ◽  
pp. 855-871 ◽  
Author(s):  
Stéphanie Bolhy ◽  
Imène Bouhlel ◽  
Elisa Dultz ◽  
Tania Nayak ◽  
Michela Zuccolo ◽  
...  

Centrosomes are closely associated with the nuclear envelope (NE) throughout the cell cycle and this association is maintained in prophase when they separate to establish the future mitotic spindle. At this stage, the kinetochore constituents CENP-F, NudE, NudEL, dynein, and dynactin accumulate at the NE. We demonstrate here that the N-terminal domain of the nuclear pore complex (NPC) protein Nup133, although largely dispensable for NPC assembly, is required for efficient anchoring of the dynein/dynactin complex to the NE in prophase. Nup133 exerts this function through an interaction network via CENP-F and NudE/EL. We show that this molecular chain is critical for maintaining centrosome association with the NE at mitotic entry and contributes to this process without interfering with the previously described RanBP2–BICD2-dependent pathway of centrosome anchoring. Finally, our study reveals that tethering of centrosomes to the nuclear surface at the G2/M transition contributes, along with other cellular mechanisms, to early stages of bipolar spindle assembly.


1996 ◽  
Vol 109 (7) ◽  
pp. 1813-1824 ◽  
Author(s):  
A. Ewald ◽  
U. Kossner ◽  
U. Scheer ◽  
M.C. Dabauvalle

Pore complexes are not confined to the nuclear envelope but can also be found in the cytoplasm of numerous cell types in the form of annulate lamellae (AL). We have induced formation of AL by exposure of rat cells (line RV) to sublethal doses of the antimitotic drug vinblastine sulfate, and compared the distribution of several nuclear pore complex proteins (nucleoporins) in the nuclear envelope and AL by immunocytochemistry, cytochemical lectin binding studies and immunoblot analyses of nuclear and AL-enriched fractions. All the antibodies used yielded punctate nuclear surface staining in immunofluorescence microscopy which is characteristic for nuclear pore complex components. When we applied antibodies against the nucleoporin p62, AL were visualized as numerous cytoplasmic dot-like structures. Immunogold electron microscopy confirmed the correspondence of the cytoplasmic bodies with stacks of AL. Antibodies to constituents of the cytoplasmic (nup180) and nucleoplasmic (nup153) filaments extending from both sides of nuclear pore complexes also stained the AL, indicating that pore complexes are intrinsically asymmetric assemblies independent of their specific intracellular topology. By contrast, AL were negative with five different antibodies against the transmembrane nuclear pore glycoprotein gp210 and the lectin concanavalin A (ConA) known to bind to the oligosaccharide side chains of gp210. Similarly, there was no staining of the AL with antibodies to the other nuclear pore membrane protein so far known in higher eukaryotes, POM121. Immunoblot analyses confirmed the presence of p62, nup180 and nup153 in both the nuclear and AL fractions and the absence of gp210 and POM121 from AL. Our results do not support the generally held view that gp210 and POM121 function in anchoring the pore complex scaffold to the pore membrane. Rather, they point to a role for these proteins in transport processes through the nuclear pore complexes. Since AL are not involved in nucleocytoplasmic transport processes they may lack components of the transport machinery.


Nucleus ◽  
2013 ◽  
Vol 4 (2) ◽  
pp. 105-114 ◽  
Author(s):  
Michaela Clever ◽  
Yasuhiro Mimura ◽  
Tomoko Funakoshi ◽  
Naoko Imamoto

2009 ◽  
Vol 20 (2) ◽  
pp. 616-630 ◽  
Author(s):  
Hui-Lin Liu ◽  
Colin P.C. De Souza ◽  
Aysha H. Osmani ◽  
Stephen A. Osmani

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


1992 ◽  
Vol 119 (6) ◽  
pp. 1441-1449 ◽  
Author(s):  
R W Wozniak ◽  
G Blobel

The glycoprotein gp210 is located in the "pore membrane," a specialized domain of the nuclear envelope to which the nuclear pore complex (NPC) is anchored. gp210 contains a large cisternal domain, a single transmembrane segment (TM), and a COOH-terminal, 58-amino acid residue cytoplasmic tail (CT) (Wozniak, R. W., E. Bartnik, and G. Blobel. 1989. J. Cell Biol. 108:2083-2092; Greber, U. F., A. Senior, and L. Gerace. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:1495-1502). To locate determinants for sorting of gp210 to the pore membrane, we constructed various cDNAs coding for wild-type, mutant, and chimeric gp210, and monitored localization of the expressed protein in 3T3 cells by immunofluorescence microscopy using appropriate antibodies. The large cisternal domain of gp210 (95% of its mass) did not reveal any sorting determinants. Surprisingly, the TM of gp210 is sufficient for sorting to the pore membrane. The CT also contains a sorting determinant, but it is weaker than that of the TM. We propose specific lateral association of the transmembrane helices of two proteins to yield either a gp210 homodimer or a heterodimer of gp210 and another protein. The cytoplasmically oriented tails of these dimers may bind cooperatively to the adjacent NPCs. In addition, we demonstrate that gp210 co-localizes with cytoplasmically dispersed nucleoporins, suggesting a cytoplasmic association of these components.


2020 ◽  
Author(s):  
Julie Jacquemyn ◽  
Joyce Foroozandeh ◽  
Katlijn Vints ◽  
Jef Swerts ◽  
Patrik Verstreken ◽  
...  

AbstractTorsin ATPases of the endoplasmic reticulum (ER) and nuclear envelope (NE) lumen inhibit Lipin-mediated phosphatidate (PA) to diacylglycerol (DAG) conversion by an unknown mechanism. This excess PA metabolism is implicated in TOR1A/TorsinA diseases, but it is unclear whether it explains why Torsin concomitantly affects nuclear structure, lipid droplets (LD), organelle and cell growth. Here a fly miniscreen identified that Torsins affect these events via the NEP1R1-CTDNEP1 phosphatase complex. Further, Torsin homo-oligomerization rather than ATPase activity was key to function. NEP1R1-CTDNEP1 activates Lipin by dephosphorylation. We show that Torsin prevents CTDNEP1 from accumulating in the NE and excludes Lipin from the nucleus. Moreover, this repression of nuclear PA metabolism is required for interphase nuclear pore biogenesis. We conclude that Torsin is an upstream regulator of the NEP1R1-CTDNEP1/ Lipin pathway. This connects the ER/NE lumen with PA metabolism, and affects numerous cellular events including it has a previously unrecognized role in nuclear pore biogenesis.HighlightsNuclear envelope PA-DAG-TAG synthesis is independently regulated by Torsin and Torip/LAP1Torsin removes CTDNEP1 from the nuclear envelope and excludes Lipin from the nucleusExcess nuclear envelope NEP1R1-CTDNEP1/ Lipin activity impairs multiple aspects of NPC biogenesisNEP1R1-CTDNEP1/ Lipin inhibition prevents cellular defects associated with TOR1A and TOR1AIP1 / LAP1 disease


1993 ◽  
Vol 106 (1) ◽  
pp. 23-29 ◽  
Author(s):  
D.C. Chugani ◽  
L.H. Rome ◽  
N.L. Kedersha

Vaults are cytoplasmic ribonucleoprotein organelles that are highly conserved among diverse eukaryotic species. Their mass (12.9 MDa), diameter (26-35 nm) and shape (two halves, each with eightfold radial symmetry) have recently been determined and are similar to those ascribed to the central plug (or transporter) of the nuclear pore complex (NPC). The size and eightfold symmetry of the vault particle make it conducive to interacting physically in a complementary manner with NPCs. The present study demonstrates that vaults specifically associate with nuclei by both immunoblotting and immunofluorescence. Immunogold EM confirmed that vaults associate with the nuclear envelope in tissue sections and with NPCs of isolated nuclei.


1999 ◽  
Vol 77 (4) ◽  
pp. 321-329 ◽  
Author(s):  
Khaldon Bodoor ◽  
Sarah Shaikh ◽  
Paul Enarson ◽  
Sharmin Chowdhury ◽  
Davide Salina ◽  
...  

Nuclear pore complexes (NPCs) are extremely elaborate structures that mediate the bidirectional movement of macromolecules between the nucleus and cytoplasm. The current view of NPC organization features a massive symmetrical framework that is embedded in the double membranes of the nuclear envelope. It embraces a central channel of as yet ill-defined structure but which may accommodate particles with diameters up to 26 nm provided that they bear specific import/export signals. Attached to both faces of the central framework are peripheral structures, short cytoplasmic filaments, and a nuclear basket assembly, which interact with molecules transiting the NPC. The mechanisms of assembly and the nature of NPC structural intermediates are still poorly understood. However, mutagenesis and expression studies have revealed discrete sequences within certain NPC proteins that are necessary and sufficient for their appropriate targeting. In addition, some details are emerging from observations on cells undergoing mitosis where the nuclear envelope is disassembled and its components, including NPC subunits, are dispersed throughout the mitotic cytoplasm. At the end of mitosis, all of these components are reutilized to form nuclear envelopes in the two daughter cells. To date, it has been possible to define a time course of postmitotic assembly for a group of NPC components (CAN/Nup214, Nup153, POM121, p62 and Tpr) relative to the integral inner nuclear membrane protein LAP2 and the NPC membrane glycoprotein gp210. Nup153, a dynamic component of the nuclear basket, associates with chromatin towards the end of anaphase coincident with, although independent of, the inner nuclear membrane protein, LAP2. Assembly of the remaining proteins follows that of the nuclear membranes and occurs in the sequence POM121, p62, CAN/Nup214 and gp210/Tpr. Since p62 remains as a complex with three other NPC proteins (p58, p54, p45) during mitosis, and CAN/Nup214 maintains a similar interaction with its partner, Nup84, the relative timing of assembly of these additional four proteins may also be inferred. These observations suggest that there is a sequential association of NPC proteins with chromosomes during nuclear envelope reformation and the recruitment of at least eight of these precedes that of gp210. These findings support a model in which it is POM121 rather than gp210 that defines initial membrane-associated NPC assembly intermediates and which may therefore represent an essential component of the central framework of the NPC. Key words: nuclear pore complex, nucleoporin, mitosis, nuclear transport


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