scholarly journals Dissecting Torsin/cofactor function at the nuclear envelope: a genetic study

2016 ◽  
Vol 27 (25) ◽  
pp. 3964-3971 ◽  
Author(s):  
Ethan Laudermilch ◽  
Pei-Ling Tsai ◽  
Morven Graham ◽  
Elizabeth Turner ◽  
Chenguang Zhao ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Structural Analysis ◽  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Compositional Analysis ◽  
Immunogold Labeling ◽  
Nuclear Pore ◽  
Functional Link ◽  
Experimental Platform ◽  
Bleb Formation

The human genome encodes four Torsin ATPases, the functions of which are poorly understood. In this study, we use CRISPR/Cas9 engineering to delete all four Torsin ATPases individually and in combination. Using nuclear envelope (NE) blebbing as a phenotypic measure, we establish a direct correlation between the number of inactivated Torsin alleles and the occurrence of omega-shaped herniations within the lumen of the NE. A similar, although not identical, redundancy is observed for LAP1 and LULL1, which serve as regulatory cofactors for a subset of Torsin ATPases. Unexpectedly, deletion of Tor2A in a TorA/B/3A-deficient background results in a stark increase of bleb formation, even though Tor2A does not respond to LAP1/LULL1 stimulation. The robustness of the observed phenotype in Torsin-deficient cells enables a structural analysis via electron microscopy tomography and a compositional analysis via immunogold labeling. Ubiquitin and nucleoporins were identified as distinctively localizing components of the omega-shaped bleb structure. These findings suggest a functional link between the Torsin/cofactor system and NE/nuclear pore complex biogenesis or homeostasis and establish a Torsin-deficient cell line as a valuable experimental platform with which to decipher Torsin function.

scholarly journals The role of the integral membrane nucleoporins Ndc1p and Pom152p in nuclear pore complex assembly and function

2006 ◽  
Vol 173 (3) ◽  
pp. 361-371 ◽  
Author(s):  
Alexis S. Madrid ◽  
Joel Mancuso ◽  
W. Zacheus Cande ◽  
Karsten Weis
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Nuclear Envelope ◽  
Lipid Bilayers ◽  
Nuclear Pore Complex ◽  
Transmembrane Proteins ◽  
Nuclear Pore ◽  
Large Channel ◽  
And Function

The nuclear pore complex (NPC) is a large channel that spans the two lipid bilayers of the nuclear envelope and mediates transport events between the cytoplasm and the nucleus. Only a few NPC components are transmembrane proteins, and the role of these proteins in NPC function and assembly remains poorly understood. We investigate the function of the three integral membrane nucleoporins, which are Ndc1p, Pom152p, and Pom34p, in NPC assembly and transport in Saccharomyces cerevisiae. We find that Ndc1p is important for the correct localization of nuclear transport cargoes and of components of the NPC. However, the role of Ndc1p in NPC assembly is partially redundant with Pom152p, as cells lacking both of these proteins show enhanced NPC disruption. Electron microscopy studies reveal that the absence of Ndc1p and Pom152p results in aberrant pores that have enlarged diameters and lack proteinaceous material, leading to an increased diffusion between the cytoplasm and the nucleus.

scholarly journals Torsin ATPases are required to complete nuclear pore complex biogenesis in interphase

2019 ◽  
Author(s):  
Anthony J. Rampello ◽  
Ethan Laudermilch ◽  
Nidhi Vishnoi ◽  
Sarah M. Prohet ◽  
Lin Shao ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Nuclear Pore ◽  
Ubiquitin Conjugation ◽  
Bleb Formation ◽  
Kinetics Of

AbstractNuclear envelope herniations (blebs) containing FG-nucleoporins and ubiquitin are the phenotypic hallmark of Torsin ATPase manipulation. Both the dynamics of blebbing and the connection to nuclear pore biogenesis remain poorly understood. We employ a proteomics-based approach to identify MLF2 as a luminal component of the bleb. Using an MLF2-based live cell imaging platform, we demonstrate that NE blebbing occurs rapidly and synchronously immediately after nuclear envelope reformation during mitosis. Bleb formation is independent of ubiquitin conjugation within the bleb, but strictly dependent on POM121, a transmembrane nucleoporin essential for interphase nuclear pore biogenesis. Nup358, a late marker for interphase nuclear pore complex (NPC) biogenesis, is underrepresented relative to FG nucleoporins in nuclear envelopes of Torsin-deficient cells. The kinetics of bleb formation, its dependence on POM121, and a reduction of mature NPCs in Torsin deficient cells lead us to conclude that the hallmark phenotype of Torsin manipulation represents the accumulation of stalled NPC assembly intermediates.

scholarly journals Torsin ATPase deficiency leads to defects in nuclear pore biogenesis and sequestration of MLF2

2020 ◽  
Vol 219 (6) ◽  
Author(s):  
Anthony J. Rampello ◽  
Ethan Laudermilch ◽  
Nidhi Vishnoi ◽  
Sarah M. Prophet ◽  
Lin Shao ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Myeloid Leukemia ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Nuclear Pore ◽  
Ubiquitin Conjugation ◽  
Bleb Formation ◽  
Kinetics Of

Nuclear envelope herniations (blebs) containing FG-nucleoporins and ubiquitin are the phenotypic hallmark of Torsin ATPase manipulation. Both the dynamics of blebbing and the connection to nuclear pore biogenesis remain poorly understood. We employ a proteomics-based approach to identify myeloid leukemia factor 2 (MLF2) as a luminal component of the bleb. Using an MLF2-based live-cell imaging platform, we demonstrate that nuclear envelope blebbing occurs rapidly and synchronously immediately after nuclear envelope reformation during mitosis. Bleb formation is independent of ubiquitin conjugation within the bleb, but strictly dependent on POM121, a transmembrane nucleoporin essential for interphase nuclear pore biogenesis. Nup358, a late marker for interphase nuclear pore complex (NPC) biogenesis, is underrepresented relative to FG-nucleoporins in nuclear envelopes of Torsin-deficient cells. The kinetics of bleb formation, its dependence on POM121, and a reduction of mature NPCs in Torsin-deficient cells lead us to conclude that the hallmark phenotype of Torsin manipulation represents aberrant NPC intermediates.

scholarly journals Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy

1993 ◽  
Vol 122 (1) ◽  
pp. 1-19 ◽  
Author(s):  
CW Akey ◽  
M Radermacher
Keyword(s):  
Electron Microscopy ◽  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Three Dimensional ◽  
Nucleocytoplasmic Transport ◽  
Domain Model ◽  
Nuclear Pore ◽  
Transport Channel ◽  
Macromolecular Transport ◽  
Cryo Electron Microscopy

The nuclear pore complex spans the nuclear envelope and functions as a macromolecular transporter in the ATP-dependent process of nucleocytoplasmic transport. In this report, we present three dimensional (3D) structures for both membrane-associated and detergent-extracted Xenopus NPCs, imaged in frozen buffers by cryo-electron microscopy. A comparison of the differing configurations present in the 3D maps suggests that the spokes may possess an intrinsic conformational flexibility. When combined with recent data from a 3D map of negatively stained NPCs (Hinshaw, J. E., B. O. Carragher, and R. A. Milligan. 1992. Cell. 69:1133-1141), these observations suggest a minimal domain model for the spoke-ring complex which may account for the observed plasticity of this assembly. Moreover, lumenal domains in adjacent spokes are interconnected by radial arm dimers, forming a lumenal ring that may be responsible for anchoring the NPC within the nuclear envelope pore. Importantly, the NPC transporter is visualized as a centrally tapered cylinder that spans the entire width of the NPC, in a direction normal to the nuclear envelope. The central positioning, tripartite structure, and hollow nature of the transporter suggests that it may form a macromolecular transport channel, with a globular gating domain at each end. Finally, the packing of the transporter within the spokes creates a set of eight internal channels that may be responsible, in part, for the diffusion of ions and small molecules across the nuclear envelope.

scholarly journals Cryo-EM structure of the Inner Ring from Xenopus laevis Nuclear Pore Complex

2021 ◽  
Author(s):  
Gaoxingyu Huang ◽  
Xiechao Zhan ◽  
Chao Zeng ◽  
Ke Liang ◽  
Xuechen Zhu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Xenopus Laevis ◽  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Single Particle ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Shuttling ◽  
Cryo Electron Microscopy

Nuclear pore complex (NPC) mediates nucleocytoplasmic shuttling. Here we present single-particle cryo-electron microscopy (cryo-EM) structure of the inner ring (IR) subunit from Xenopus laevis NPC at an average resolution of 4.4 Å. The symmetric IR subunit comprises a cytoplasmic half and a nuclear half. A homo-dimer of Nup205 resides at the center of the IR subunit, flanked by two molecules of Nup188. Four molecules of Nup93 each places an extended helix into the axial groove of Nup205 or Nup188, together constituting the central scaffold. The channel nucleoporin heterotrimer (CNT) of Nup54/58/62 is anchored on the central scaffold. Six Nup155 molecules interact with the central scaffold and together with the NDC1-ALADIN hetero-dimers anchor the IR subunit to the nuclear envelope and to outer rings. The scarce inter-subunit contacts may allow sufficient latitude in conformation and diameter of the IR. Our structure of vertebrate IR reveals key insights that are functionally important.

scholarly journals Regulation and coordination of nuclear envelope and nuclear pore complex assembly

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

scholarly journals Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components.

1990 ◽  
Vol 110 (4) ◽  
pp. 883-894 ◽  
Author(s):  
R Reichelt ◽  
A Holzenburg ◽  
E L Buhle ◽  
M Jarnik ◽  
A Engel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Nuclear Envelope ◽  
Three Dimensional ◽  
Structural Features ◽  
Nuclear Pore ◽  
Xenopus Laevis Oocyte ◽  
Three Dimensional Model ◽  
Transmission Electron ◽  
Freeze Dried

Nuclear pore complexes (NPCs) prepared from Xenopus laevis oocyte nuclear envelopes were studied in "intact" form (i.e., unexposed to detergent) and after detergent treatment by a combination of conventional transmission electron microscopy (CTEM) and quantitative scanning transmission electron microscopy (STEM). In correlation-averaged CTEM pictures of negatively stained intact NPCs and of distinct NPC components (i.e., "rings," "spoke" complexes, and "plug-spoke" complexes), several fine structural features arranged with octagonal symmetry about a central axis could reproducibly be identified. STEM micrographs of unstained/freeze-dried intact NPCs as well as of their components yielded comparable but less distinct features. Mass determination by STEM revealed the following molecular masses: intact NPC with plug, 124 +/- 11 MD; intact NPC without plug, 112 +/- 11 MD; heavy ring, 32 +/- 5 MD; light ring, 21 +/- 4 MD; plug-spoke complex, 66 +/- 8 MD; and spoke complex, 52 +/- 3 MD. Based on these combined CTEM and STEM data, a three-dimensional model of the NPC exhibiting eightfold centrosymmetry about an axis perpendicular to the plane of the nuclear envelope but asymmetric along this axis is proposed. This structural polarity of the NPC across the nuclear envelope is in accord with its well-documented functional polarity facilitating mediated nucleocytoplasmic exchange of molecules and particles.

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

2009 ◽  
Vol 20 (2) ◽  
pp. 616-630 ◽  
Author(s):  
Hui-Lin Liu ◽  
Colin P.C. De Souza ◽  
Aysha H. Osmani ◽  
Stephen A. Osmani
Keyword(s):  
High Temperature ◽  
Nuclear Envelope ◽  
Aspergillus Nidulans ◽  
Nuclear Pore Complex ◽  
Mitotic Spindle ◽  
Spindle Pole ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Spindle Pole Bodies ◽  
Pore Complexes

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.

Sign in / Sign up

Export Citation Format

Share Document