scholarly journals Actin assembly ruptures the nuclear envelope by prying the lamina away from nuclear pores and nuclear membranes in starfish oocytes

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Natalia Wesolowska ◽  
Ivan Avilov ◽  
Pedro Machado ◽  
Celina Geiss ◽  
Hiroshi Kondo ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Super Resolution ◽  
Germinal Vesicle ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Actin Assembly ◽  
Nuclear Membranes ◽  
Pore Complexes ◽  
Distinct Membrane ◽  
Nuclear Rupture

The nucleus of oocytes (germinal vesicle) is unusually large and its nuclear envelope (NE) is densely packed with nuclear pore complexes (NPCs) that are stockpiled for embryonic development. We showed that breakdown of this specialized NE is mediated by an Arp2/3-nucleated F-actin ‘shell’ in starfish oocytes, in contrast to microtubule-driven tearing in mammalian fibroblasts. Here, we address the mechanism of F-actin-driven NE rupture by correlated live-cell, super-resolution and electron microscopy. We show that actin is nucleated within the lamina, sprouting filopodia-like spikes towards the nuclear membranes. These F-actin spikes protrude pore-free nuclear membranes, whereas the adjoining stretches of membrane accumulate NPCs that are associated with the still-intact lamina. Packed NPCs sort into a distinct membrane network, while breaks appear in ER-like, pore-free regions. We reveal a new function for actin-mediated membrane shaping in nuclear rupture that is likely to have implications in other contexts, such as nuclear rupture observed in cancer cells.

scholarly journals Rupture of nuclear envelope in starfish oocytes proceeds by F-actin-driven segregation of pore-dense and pore-free membranes

2018 ◽  
Author(s):  
Natalia Wesolowska ◽  
Pedro Machado ◽  
Ivan Avilov ◽  
Celina Geiss ◽  
Hiroshi Kondo ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Germinal Vesicle ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Detailed Mechanism ◽  
Nuclear Membranes ◽  
Vesicular Membrane ◽  
Pore Complexes

AbstractThe nucleus of oocytes, traditionally referred to as the germinal vesicle, is unusually large and its nuclear envelope (NE) is densely packed with nuclear pore complexes (NPCs) stockpiled for embryonic development. We have shown that breakdown of this specialized NE during meiosis of starfish oocytes is mediated by an Arp2/3-nucleated F-actin ‘shell’, in contrast to microtubule-driven tearing in somatic cells. The detailed mechanism of how the cytoskeletal forces disrupt the NE remains poorly understood in any system. Here, we address the mechanism of F-actin-driven NE rupture by using live-cell and correlated super-resolution light and electron microscopy. We show that actin is nucleated within the lamina and sprouts filopodia-like spikes towards the nuclear membranes forcing lamina and nuclear membranes apart. These F-actin spikes protrude pore-free nuclear membranes, whereas the adjoining membrane stretches accumulate packed NPCs associated with the still-intact lamin network. NPC conglomerates sort into a distinct tubular-vesicular membrane network, while breaks appear in pore-free, ER-like regions. Together, our work reveals a novel function for Arp2/3-mediated membrane shaping in NE rupture that is likely to have broad relevance in regulating NE dynamics in diverse other contexts such as nuclear rupture frequently observed in cancer cells.

scholarly journals In Vivo Dynamics of Nuclear Pore Complexes in Yeast

1997 ◽  
Vol 136 (6) ◽  
pp. 1185-1199 ◽  
Author(s):  
Mirella Bucci ◽  
Susan R. Wente
Keyword(s):  
Nuclear Envelope ◽  
Recipient Cell ◽  
Nucleocytoplasmic Transport ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Central Location ◽  
Nuclear Membranes ◽  
Mutant Strains ◽  
Pore Complexes

While much is known about the role of nuclear pore complexes (NPCs) in nucleocytoplasmic transport, the mechanism of NPC assembly into pores formed through the double lipid bilayer of the nuclear envelope is not well defined. To investigate the dynamics of NPCs, we developed a live-cell assay in the yeast Saccharomyces cerevisiae. The nucleoporin Nup49p was fused to the green fluorescent protein (GFP) of Aequorea victoria and expressed in nup49 null haploid yeast cells. When the GFP–Nup49p donor cell was mated with a recipient cell harboring only unlabeled Nup49p, the nuclei fused as a consequence of the normal mating process. By monitoring the distribution of the GFP–Nup49p, we could assess whether NPCs were able to move from the donor section of the nuclear envelope to that of the recipient nucleus. We observed that fluorescent NPCs moved and encircled the entire nucleus within 25 min after fusion. When assays were done in mutant kar1-1 strains, where nuclear fusion does not occur, GFP–Nup49p appearance in the recipient nucleus occurred at a very slow rate, presumably due to new NPC biogenesis or to exchange of GFP– Nup49p into existing recipient NPCs. Interestingly, in a number of existing mutant strains, NPCs are clustered together at permissive growth temperatures. This has been explained with two different hypotheses: by movement of NPCs through the double nuclear membranes with subsequent clustering at a central location; or, alternatively, by assembly of all NPCs at a central location (such as the spindle pole body) with NPCs in mutant cells unable to move away from this point. Using the GFP–Nup49p system with a mutant in the NPCassociated factor Gle2p that exhibits formation of NPC clusters only at 37°C, it was possible to distinguish between these two models for NPC dynamics. GFP– Nup49p-labeled NPCs, assembled at 23°C, moved into clusters when the cells were shifted to growth at 37°C. These results indicate that NPCs can move through the double nuclear membranes and, moreover, can do so to form NPC clusters in mutant strains. Such clusters may result by releasing NPCs from a nuclear tether, or by disappearance of a protein that normally prevents pore aggregation. This system represents a novel approach for identifying regulators of NPC assembly and movement in the future.

scholarly journals An ESCRT-LEM protein surveillance system is poised to directly monitor the nuclear envelope and nuclear transport system

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
David J Thaller ◽  
Matteo Allegretti ◽  
Sapan Borah ◽  
Paolo Ronchi ◽  
Martin Beck ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Surveillance System ◽  
Nuclear Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Membranes ◽  
Disease Mechanisms ◽  
Pore Complexes ◽  
Membrane Sealing

The integrity of the nuclear membranes coupled to the selective barrier of nuclear pore complexes (NPCs) are essential for the segregation of nucleoplasm and cytoplasm. Mechanical membrane disruption or perturbation to NPC assembly triggers an ESCRT-dependent surveillance system that seals nuclear pores: how these pores are sensed and sealed is ill defined. Using a budding yeast model, we show that the ESCRT Chm7 and the integral inner nuclear membrane (INM) protein Heh1 are spatially segregated by nuclear transport, with Chm7 being actively exported by Xpo1/Crm1. Thus, the exposure of the INM triggers surveillance with Heh1 locally activating Chm7. Sites of Chm7 hyperactivation show fenestrated sheets at the INM and potential membrane delivery at sites of nuclear envelope herniation. Our data suggest that perturbation to the nuclear envelope barrier would lead to local nuclear membrane remodeling to promote membrane sealing. Our findings have implications for disease mechanisms linked to NPC assembly and nuclear envelope integrity.

scholarly journals Subcellular analyses of planarian meiosis implicates a novel, double-membraned vesiculation process in nuclear envelope breakdown

2019 ◽  
Author(s):  
Longhua Guo ◽  
Fengli Guo ◽  
Shasha Zhang ◽  
Kexi Yi ◽  
Melainia McClain ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
De Novo ◽  
Germinal Vesicle ◽  
Protein Composition ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Cell Nuclei ◽  
Germinal Vesicle Breakdown ◽  
Evolutionary Origins ◽  
Pore Complexes

AbstractThe cell nuclei of Ophisthokonts, the eukaryotic supergroup defined by fungi and metazoans, is remarkable in the constancy of both their double-membraned structure and protein composition. Such remarkable structural conservation underscores common and ancient evolutionary origins. Yet, the dynamics of disassembly and reassembly displayed by Ophisthokont nuclei vary extensively. Besides closed mitosis in fungi and open mitosis in some animals, little is known about the evolution of nuclear envelope break down (NEBD) during cell division. Here, we uncovered a novel form of NEBD in primary oocytes of the flatworm Schmidtea mediterranea. From zygotene to metaphase II, both nuclear envelope (NE) and peripheral endoplasmic reticulum (ER) expand notably in size, likely involving de novo membrane synthesis. 3-D electron microscopy reconstructions demonstrated that the NE transforms itself into numerous double-membraned vesicles similar in membrane architecture to NE doublets in mammalian oocytes after germinal vesicle breakdown. The vesicles are devoid of nuclear pore complexes and DNA, yet are loaded with nuclear proteins, including a planarian homologue of PIWI, a protein essential for the maintenance of stem cells in this and other organisms. Our data contribute a new model to the canonical view of NE dynamics and support that NEBD is an evolutionarily adaptable trait in multicellular organisms.

scholarly journals ON THE ATTACHMENT OF THE NUCLEAR PORE COMPLEX

1974 ◽  
Vol 62 (3) ◽  
pp. 746-754 ◽  
Author(s):  
Robert Peter Aaronson ◽  
Günter Blobel
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Structural Integrity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Complete Removal ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Membranes ◽  
Peripheral Heterochromatin ◽  
Pore Complexes

Electron microscope examination of isolated rat liver nuclei after treatment with the detergent Triton X-100 revealed the complete removal of both the inner and outer membranes of the nuclear envelope. The envelope-denuded nuclei did not show any change in either shape or internal ultrastructure. Most strikingly, the nuclear pore complexes, which in untreated nuclei appear to be integral components of the nuclear envelope, were retained in their characteristic location at the distal ends of the channels leading through the peripheral heterochromatin. Determination of the chemical composition of detergent-treated nuclei showed that over 95% of the nuclear phospholipid was solubilized, thus corroborating the morphological absence of nuclear membranes. Furthermore, detergent treatment also solubilized approximately 10% of the nuclear protein. Analysis of the solubilized protein by polyacrylamide gel electrophoresis in the presence of SDS indicated that these proteins belong to a few specific classes which presumably represent the major polypeptides of the nuclear membranes. The total absence of the nuclear envelope on both morphological and biochemical grounds supports the idea that the nuclear pore complex does not require the membranes either for attachment to the nucleus or for maintenance of its own structural integrity.

Evolutionary conservation of ‘Basket’ structure in nuclear pore complexes, and their possible role in mRNA export

1994 ◽  
Vol 52 ◽  
pp. 12-13 ◽  
Author(s):  
T. D. Allen ◽  
M. W. Goldberg ◽  
C. Pelling ◽  
I. Solovei
Keyword(s):  
Nuclear Envelope ◽  
Germinal Vesicle ◽  
Evolutionary Conservation ◽  
Nuclear Pore ◽  
Macromolecular Complex ◽  
Nuclear Pore Complexes ◽  
Animal Kingdom ◽  
Nuclear Interior ◽  
Cytoplasmic Filaments ◽  
Pore Complexes

The nuclear pore complex (NPC) is a macromolecular complex which provides transport through the nuclear envelope. Each NPC has a diameter of around 120 nms, and extends outwards into the cytoplasm by some 20 nms via eight cytoplasmic filaments of granular substructure. On the nucleoplasmic face, NPC ‘cages’ or ‘baskets’ project some 60 nms into the nuclear interior where they terminate in an inner basket ring that supports a further structure, the nuclear envelope lattice (NEL), a network of filaments. Both the NEL and NPC baskets have been visualised to date only in amphibian germinal vesicle nuclear envelopes. All current models of NPC structure are based on data from amphibian oocytes, mainly due to the convenience of isolating clean and well preserved nuclear envelopes. It is important therefore to determine how widely these structures are conserved in other groups throughout the animal kingdom, as a basis for the overall evolutionary conservation of NPC structure.

scholarly journals POM121 and Sun1 play a role in early steps of interphase NPC assembly

2011 ◽  
Vol 194 (1) ◽  
pp. 27-37 ◽  
Author(s):  
Jessica A. Talamas ◽  
Martin W. Hetzer
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Molecular Mechanisms ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Outer Nuclear Membrane ◽  
Nuclear Membranes ◽  
Distinct Cell ◽  
Pore Complexes

Nuclear pore complexes (NPCs) assemble at the end of mitosis during nuclear envelope (NE) reformation and into an intact NE as cells progress through interphase. Although recent studies have shown that NPC formation occurs by two different molecular mechanisms at two distinct cell cycle stages, little is known about the molecular players that mediate the fusion of the outer and inner nuclear membranes to form pores. In this paper, we provide evidence that the transmembrane nucleoporin (Nup), POM121, but not the Nup107–160 complex, is present at new pore assembly sites at a time that coincides with inner nuclear membrane (INM) and outer nuclear membrane (ONM) fusion. Overexpression of POM121 resulted in juxtaposition of the INM and ONM. Additionally, Sun1, an INM protein that is known to interact with the cytoskeleton, was specifically required for interphase assembly and localized with POM121 at forming pores. We propose a model in which POM121 and Sun1 interact transiently to promote early steps of interphase NPC assembly.

Nuclear Envelope Dynamics During Mitosis

1988 ◽  
Vol 46 ◽  
pp. 224-225
Author(s):  
Brian Burke
Keyword(s):  
Nuclear Envelope ◽  
Membrane Vesicles ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Animal Cells ◽  
Interphase Chromosome ◽  
Lamin B ◽  
Chromosome Architecture ◽  
Complex Membrane ◽  
Pore Complexes

The nuclear envelope is a complex membrane structure that forms the boundary of the nuclear compartment in eukaryotes. It regulates the passage of macromolecules between the two compartments and may be important for organizing interphase chromosome architecture. In interphase animal cells it forms a remarkably stable structure consisting of a double membrane ouerlying a protein meshwork or lamina and penetrated by nuclear pore complexes. The latter form the channels for nucleocytoplasmic exchange of macromolecules, At the onset of mitosis, however, it rapidly disassembles, the membranes fragment to yield small vesicles and the lamina, which is composed of predominantly three polypeptides, lamins R, B and C (MW approx. 74, 68 and 65 kDa respectiuely), breaks down. Lamins B and C are dispersed as monomers throughout the mitotic cytoplasm, while lamin B remains associated with the nuclear membrane vesicles.

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.

scholarly journals O-GlcNAc modification of nuclear pore complexes accelerates bi-directional transport

2020 ◽  
Author(s):  
Tae Yeon Yoo ◽  
Timothy J Mitchison
Keyword(s):  
Nuclear Import ◽  
Nuclear Transport ◽  
Super Resolution ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Permeability Barrier ◽  
Nuclear Pore Complexes ◽  
Facilitated Diffusion ◽  
Import And Export ◽  
Pore Complexes

AbstractMacromolecular transport across the nuclear envelope depends on facilitated diffusion through nuclear pore complexes (NPCs). The interior of NPCs contains a permeability barrier made of phenylalanine-glycine (FG) repeat domains that selectively facilitates the permeation of cargoes bound to nuclear transport receptors (NTRs). FG repeats in NPC are a major site of O-linked N-acetylglucosamine (O-GlcNAc) modification, but the functional role of this modification in nucleocytoplasmic transport is unclear. We developed high-throughput assays based on optogenetic probes to quantify the kinetics of nuclear import and export in living human cells. We found that increasing O-GlcNAc modification of the NPC accelerated NTR-facilitated nucleocytoplasmic transport of proteins in both directions, and decreasing modification slowed transport. Super-resolution imaging revealed strong enrichment of O-GlcNAc at the FG-repeat barrier. O-GlcNAc modification also accelerated passive permeation of a small, inert protein through NPCs. We conclude that O-GlcNAc modification accelerates nucleocytoplasmic transport by enhancing the non-specific permeability the FG-repeat barrier, perhaps by steric inhibition of interactions between FG repeats.SummaryNuclear pore complexes mediate nuclear transport and are highly modified with O-linked N-acetylglucosamine (O-GlcNAc) on FG repeat domains. Using a new quantitative live-cell imaging assay, Yoo and Mitchison demonstrate acceleration of nuclear import and export by O-GlcNAc modification.

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