In Vivo Dynamics of Nuclear Pore Complexes in Yeast

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.

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Reticulon-like REEP4 at the inner nuclear membrane promotes nuclear pore complex formation

The Journal of Cell Biology ◽

10.1083/jcb.202101049 ◽

2021 ◽

Vol 221 (2) ◽

Author(s):

Banafsheh Golchoubian ◽

Andreas Brunner ◽

Helena Bragulat-Teixidor ◽

Annett Neuner ◽

Busra A. Akarlar ◽

...

Keyword(s):

Nuclear Envelope ◽

Nuclear Pore Complex ◽

Nuclear Membrane ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Inner Nuclear Membrane ◽

Pore Complexes ◽

Late Stages ◽

Protein Complex Assembly

Nuclear pore complexes (NPCs) are channels within the nuclear envelope that mediate nucleocytoplasmic transport. NPCs form within the closed nuclear envelope during interphase or assemble concomitantly with nuclear envelope reformation in late stages of mitosis. Both interphase and mitotic NPC biogenesis require coordination of protein complex assembly and membrane deformation. During early stages of mitotic NPC assembly, a seed for new NPCs is established on chromatin, yet the factors connecting the NPC seed to the membrane of the forming nuclear envelope are unknown. Here, we report that the reticulon homology domain protein REEP4 not only localizes to high-curvature membrane of the cytoplasmic endoplasmic reticulum but is also recruited to the inner nuclear membrane by the NPC biogenesis factor ELYS. This ELYS-recruited pool of REEP4 promotes NPC assembly and appears to be particularly important for NPC formation during mitosis. These findings suggest a role for REEP4 in coordinating nuclear envelope reformation with mitotic NPC biogenesis.

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Dissection of the NUP107 nuclear pore subcomplex reveals a novel interaction with spindle assembly checkpoint protein MAD1 in Caenorhabditis elegans

Molecular Biology of the Cell ◽

10.1091/mbc.e11-11-0927 ◽

2012 ◽

Vol 23 (5) ◽

pp. 930-944 ◽

Cited By ~ 31

Author(s):

Eduardo Ródenas ◽

Cristina González-Aguilera ◽

Cristina Ayuso ◽

Peter Askjaer

Keyword(s):

Caenorhabditis Elegans ◽

Nuclear Envelope ◽

Molecular Mechanisms ◽

Spindle Assembly Checkpoint ◽

Protein Import ◽

Spindle Assembly ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Pore Complexes

Nuclear pore complexes consist of several subcomplexes. The NUP107 complex is important for nucleocytoplasmic transport, nuclear envelope assembly, and kinetochore function. However, the underlying molecular mechanisms and the roles of individual complex members remain elusive. We report the first description of a genetic disruption of NUP107 in a metazoan. Caenorhabditis elegans NUP107/npp-5 mutants display temperature-dependent lethality. Surprisingly, NPP-5 is dispensable for incorporation of most nucleoporins into nuclear pores and for nuclear protein import. In contrast, NPP-5 is essential for proper kinetochore localization of NUP133/NPP-15, another NUP107 complex member, whereas recruitment of NUP96/NPP-10C and ELYS/MEL-28 is NPP-5 independent. We found that kinetochore protein NUF2/HIM-10 and Aurora B/AIR-2 kinase are less abundant on mitotic chromatin upon NPP-5 depletion. npp-5 mutants are hypersensitive to anoxia, suggesting that the spindle assembly checkpoint (SAC) is compromised. Indeed, NPP-5 interacts genetically and physically with SAC protein MAD1/MDF-1, whose nuclear envelope accumulation requires NPP-5. Thus our results strengthen the emerging connection between nuclear pore proteins and chromosome segregation.

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

eLife ◽

10.7554/elife.45284 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 37

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.

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

eLife ◽

10.7554/elife.49774 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 8

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.

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Nucleocytoplasmic transport of macromolecules

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.61.2.193-211.1997 ◽

1997 ◽

Vol 61 (2) ◽

pp. 193-211

Author(s):

A H Corbett ◽

P A Silver

Keyword(s):

Nuclear Envelope ◽

Nuclear Transport ◽

Bound State ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Yeast Saccharomyces Cerevisiae ◽

Soluble Factors ◽

Pore Complexes ◽

Transport Of Macromolecules

Nucleocytoplasmic transport is a complex process that consists of the movement of numerous macromolecules back and forth across the nuclear envelope. All macromolecules that move in and out of the nucleus do so via nuclear pore complexes that form large proteinaceous channels in the nuclear envelope. In addition to nuclear pores, nuclear transport of macromolecules requires a number of soluble factors that are found both in the cytoplasm and in the nucleus. A combination of biochemical, genetic, and cell biological approaches have been used to identify and characterize the various components of the nuclear transport machinery. Recent studies have shown that both import to and export from the nucleus are mediated by signals found within the transport substrates. Several studies have demonstrated that these signals are recognized by soluble factors that target these substrates to the nuclear pore. Once substrates have been directed to the pore, most transport events depend on a cycle of GTP hydrolysis mediated by the small Ras-like GTPase, Ran, as well as other proteins that regulate the guanine nucleotide-bound state of Ran. Many of the essential factors have been identified, and the challenge that remains is to determine the exact mechanism by which transport occurs. This review attempts to present an integrated view of our current understanding of nuclear transport while highlighting the contributions that have been made through studies with genetic organisms such as the budding yeast, Saccharomyces cerevisiae.

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REEP4 is recruited to the inner nuclear membrane by ELYS and promotes nuclear pore complex formation

10.1101/2020.09.30.320333 ◽

2020 ◽

Cited By ~ 1

Author(s):

Banafsheh Golchoubian ◽

Andreas Brunner ◽

Helena Bragulat-Teixidor ◽

Busra A. Akarlar ◽

Nurhan Ozlu ◽

...

Keyword(s):

Nuclear Envelope ◽

Nuclear Pore Complex ◽

Nuclear Membrane ◽

Nucleocytoplasmic Transport ◽

Local Deformation ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Membrane Remodeling ◽

Inner Nuclear Membrane ◽

Pore Complexes

AbstractNuclear pore complexes (NPCs) are channels within the nuclear envelope that mediate nucleocytoplasmic transport. NPCs assemble either into the closed nuclear envelope during interphase or concomitantly with nuclear envelope reformation during anaphase. Both, interphase and post-mitotic NPC biogenesis require local deformation of membrane. Yet, the factors that control proper membrane remodeling for post-mitotic NPC assembly are unknown. Here, we report that the reticulon homology domain-protein REEP4 localizes not only to high-curvature membrane of the cytoplasmic endoplasmic reticulum (ER) but also to the inner nuclear membrane (INM). We show that REEP4 is recruited to the INM by the NPC biogenesis factor ELYS and promotes NPC assembly. REEP4 contributes mainly to anaphase NPC assembly, suggesting that REEP4 has an unexpected role in coordinating nuclear envelope reformation with post-mitotic NPC biogenesis.

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ON THE ATTACHMENT OF THE NUCLEAR PORE COMPLEX

The Journal of Cell Biology ◽

10.1083/jcb.62.3.746 ◽

1974 ◽

Vol 62 (3) ◽

pp. 746-754 ◽

Cited By ~ 102

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.

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Flexible Gates: Dynamic Topologies and Functions for FG Nucleoporins in Nucleocytoplasmic Transport

Eukaryotic Cell ◽

10.1128/ec.00225-09 ◽

2009 ◽

Vol 8 (12) ◽

pp. 1814-1827 ◽

Cited By ~ 157

Author(s):

Laura J. Terry ◽

Susan R. Wente

Keyword(s):

Nuclear Envelope ◽

Regulation Of Gene Expression ◽

Nucleocytoplasmic Transport ◽

Structural Features ◽

Nuclear Pore ◽

Permeability Barrier ◽

Nuclear Pore Complexes ◽

Cellular Functions ◽

Essential Components ◽

Pore Complexes

ABSTRACT The nuclear envelope is a physical barrier between the nucleus and cytoplasm and, as such, separates the mechanisms of transcription from translation. This compartmentalization of eukaryotic cells allows spatial regulation of gene expression; however, it also necessitates a mechanism for transport between the nucleus and cytoplasm. Macromolecular trafficking of protein and RNA occurs exclusively through nuclear pore complexes (NPCs), specialized channels spanning the nuclear envelope. A novel family of NPC proteins, the FG-nucleoporins (FG-Nups), coordinates and potentially regulates NPC translocation. The extensive repeats of phenylalanine-glycine (FG) in each FG-Nup directly bind to shuttling transport receptors moving through the NPC. In addition, FG-Nups are essential components of the nuclear permeability barrier. In this review, we discuss the structural features, cellular functions, and evolutionary conservation of the FG-Nups.

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

The Journal of Cell Biology ◽

10.1083/jcb.201012154 ◽

2011 ◽

Vol 194 (1) ◽

pp. 27-37 ◽

Cited By ~ 90

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.

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

10.1101/480434 ◽

2018 ◽

Cited By ~ 2

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.

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