Live fluorescence imaging reveals early recruitment of emerin, LBR, RanBP2, and Nup153 to reforming functional nuclear envelopes

2000 ◽  
Vol 113 (5) ◽  
pp. 779-794 ◽  
Author(s):  
T. Haraguchi ◽  
T. Koujin ◽  
T. Hayakawa ◽  
T. Kaneda ◽  
C. Tsutsumi ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Membrane ◽  
Nuclear Import ◽  
Nuclear Pore ◽  
Simultaneous Observation ◽  
Lamin B ◽  
Nuclear Membranes ◽  
Lamin B Receptor ◽  
The Times ◽  
Early Recruitment

We determined the times when the nuclear membrane, nuclear pore complex (NPC) components, and nuclear import function were recovered during telophase in living HeLa cells. Simultaneous observation of fluorescently-labeled NLS-bearing proteins, lamin B receptor (LBR)-GFP, and Hoechst33342-stained chromosomes revealed that nuclear membranes reassembled around chromosomes by 5 minutes after the onset of anaphase (early telophase) whereas nuclear import function was recovered later, at 8 minutes. GFP-tagged emerin also accumulated on chromosomes 5 minutes after the onset of anaphase. Interestingly, emerin and LBR initially accumulated at distinct, separate locations, but then became uniform 8 minutes after the onset of anaphase, concurrent with the recovery of nuclear import function. We further determined the timing of NPC assembly by immunofluorescence staining of cells fixed at precise times after the onset of anaphase. Taken together, these results showed that emerin, LBR, and several NPC components (RanBP2, Nup153, p62), but not Tpr, reconstitute around chromosomes very early in telophase prior to the recovery of nuclear import activity.

scholarly journals Induction of a Massive Endoplasmic Reticulum and Perinuclear Space Expansion by Expression of Lamin B Receptor Mutants and the Related Sterol Reductases TM7SF2 and DHCR7

2010 ◽  
Vol 21 (2) ◽  
pp. 354-368 ◽  
Author(s):  
Monika Zwerger ◽  
Thorsten Kolb ◽  
Karsten Richter ◽  
Iakowos Karakesisoglou ◽  
Harald Herrmann
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Cell Lines ◽  
Nuclear Membrane ◽  
Cultured Cells ◽  
Membrane Separation ◽  
Reductase Activity ◽  
Nuclear Pore ◽  
Lamin B ◽  
Lamin B Receptor

Lamin B receptor (LBR) is an inner nuclear membrane protein involved in tethering the nuclear lamina and the underlying chromatin to the nuclear envelope. In addition, LBR exhibits sterol reductase activity. Mutations in the LBR gene cause two different human diseases: Pelger-Huët anomaly and Greenberg skeletal dysplasia, a severe chrondrodystrophy causing embryonic death. Our study aimed at investigating the effect of five LBR disease mutants on human cultured cells. Three of the tested LBR mutants caused a massive compaction of chromatin coincidental with the formation of a large nucleus-associated vacuole (NAV) in several human cultured cell lines. Live cell imaging and electron microscopy revealed that this structure was generated by the separation of the inner and outer nuclear membrane. During NAV formation, nuclear pore complexes and components of the linker of nucleoskeleton and cytoskeleton complex were lost in areas of membrane separation. Concomitantly, a large number of smaller vacuoles formed throughout the cytoplasm. Notably, forced expression of the two structurally related sterol reductases transmembrane 7 superfamily member 2 and 7-dehydrocholesterol reductase caused, even in their wild-type form, a comparable phenotype in susceptible cell lines. Hence, LBR mutant variants and sterol reductases can severely interfere with the regular organization of the nuclear envelope and the endoplasmic reticulum.

scholarly journals Stepwise reassembly of the nuclear envelope at the end of mitosis

1993 ◽  
Vol 122 (2) ◽  
pp. 295-306 ◽  
Author(s):  
N Chaudhary ◽  
JC Courvalin
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Nuclear Pore ◽  
Membrane Domains ◽  
Lamin B ◽  
Inner Nuclear Membrane ◽  
Domain Specific ◽  
Lamin B Receptor ◽  
Different Populations ◽  
Pore Complexes

The nuclear envelope consists of three distinct membrane domains: the outer membrane with the bound ribosomes, the inner membrane with the bound lamina, and the pore membrane with the bound pore complexes. Using biochemical and morphological methods, we observed that the nuclear membranes of HeLa cells undergoing mitosis are disassembled in a domain-specific manner, i.e., integral membrane proteins representing the inner nuclear membrane (the lamin B receptor) and the nuclear pore membrane (gp210) are segregated into different populations of mitotic vesicles. At the completion of mitosis, the inner nuclear membrane-derived vesicles associate with chromatin first, beginning in anaphase, whereas the pore membranes and the lamina assemble later, during telophase and cytokinesis. Our data suggest that the ordered reassembly of the nuclear envelope is triggered by the early attachment of inner nuclear membrane-derived vesicles to the chromatin.

scholarly journals Localization of Pom121 to the inner nuclear membrane is required for an early step of interphase nuclear pore complex assembly

2011 ◽  
Vol 22 (7) ◽  
pp. 1058-1069 ◽  
Author(s):  
Tomoko Funakoshi ◽  
Michaela Clever ◽  
Ai Watanabe ◽  
Naoko Imamoto
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Membrane ◽  
Nuclear Pore ◽  
Functional Domain ◽  
Early Step ◽  
Nuclear Localization Signals ◽  
Inner Nuclear Membrane ◽  
Lamin B Receptor ◽  
Importin Α ◽  
A Cell

The nuclear pore complex (NPC) is a large protein assembly that mediates molecular trafficking between the cytoplasm and the nucleus. NPCs assemble twice during the cell cycle in metazoans: postmitosis and during interphase. In this study, using small interfering RNA (siRNA) in conjunction with a cell fusion–based NPC assembly assay, we demonstrated that pore membrane protein (Pom)121, a vertebrate-specific integral membrane nucleoporin, is indispensable for an early step in interphase NPC assembly. Functional domain analysis of Pom121 showed that its nuclear localization signals, which bind to importin β via importin α and likely function with RanGTP, play an essential role in targeting Pom121 to the interphase NPC. Furthermore, a region of Pom121 that interacts with the inner nuclear membrane (INM) and lamin B receptor was found to be crucial for its NPC targeting. Based on these findings and on evidence that Pom121 localizes at the INM in the absence of a complete NPC structure, we propose that the nuclear migration of Pom121 and its subsequent interaction with INM proteins are required to initiate interphase NPC assembly. Our data also suggest, for the first time, the importance of the INM as a seeding site for “prepores” during interphase NPC assembly.

Nuclear envelope disassembly in mitotic extract requires functional nuclear pores and a nuclear lamina

1998 ◽  
Vol 111 (9) ◽  
pp. 1293-1303 ◽  
Author(s):  
P. Collas
Keyword(s):  
Nuclear Envelope ◽  
Sea Urchin ◽  
Nuclear Membrane ◽  
Critical Role ◽  
Nuclear Lamina ◽  
Nuclear Pore ◽  
Nuclear Pores ◽  
Lamin B ◽  
Nuclear Membranes

Using sea urchin embryonic and in-vitro-assembled nuclei incubated in sea urchin mitotic extract, I provide evidence for a requirement for functional nuclear pores and a nuclear lamina for nuclear envelope disassembly in vitro. In interphase gastrula nuclei, lamin B interacts with p56, an integral protein of inner nuclear membrane cross-reacting with antibodies to human lamin B receptor. Incubation of gastrula nuclei in mitotic cytosol containing an ATP-generating system rapidly induces hyperphosphorylation of p56 and lamin B. Subsequently, p56-lamin B interactions are weakened and the two proteins segregate into distinct nuclear envelope-derived vesicles upon disassembly of nuclear membranes and of the lamina. Nuclear disassembly is accompanied by chromatin condensation. Blocking nuclear pore function with wheat germ agglutinin or antibodies to nucleoporins prevents p56 and lamin B hyperphosphorylation, nuclear membrane breakdown and lamina solubilization. These events are not rescued by permeabilization of nuclear membranes to molecules of 150, 000 Mr with lysolecithin. In-vitro-assembled nuclei containing nuclear membranes with functional pores but no lamina do not disassemble in mitotic cytosol in spite of p56 hyperphosphorylation. Nuclear import of soluble lamin B and reformation of a lamina in interphase extract restores nuclear disassembly in mitotic cytosol. The data indicate a role for functional nuclear pores in nuclear disassembly in vitro. They show that p56 hyperphosphorylation is not sufficient for nuclear membrane disassembly in mitotic cytosol and argue that the nuclear lamina plays a critical role in nuclear disassembly at mitosis.

The Formation, Structure, Distribution and Frequency of Nuclear Pores

1971 ◽  
Vol 29 ◽  
pp. 518-519
Author(s):  
G. G. Maul
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Membrane ◽  
Dynamic Structure ◽  
Nuclear Pore ◽  
Nuclear Pores ◽  
Filter Function ◽  
Etching Technique ◽  
Selective Filter ◽  
Membranous Part

The chromatin of eukaryotic cells is separated from the cytoplasm by a double membrane. One obvious structural specialization of the nuclear membrane is the presence of pores which have been implicated to facilitate the selective nucleocytoplasmic exchange of a variety of large molecules. Thus, the function of nuclear pores has mainly been regarded to be a passive one. Non-membranous diaphragms, radiating fibers, central rings, and other pore-associated structures were thought to play a role in the selective filter function of the nuclear pore complex. Evidence will be presented that suggests that the nuclear pore is a dynamic structure which is non-randomly distributed and can be formed during interphase, and that a close relationship exists between chromatin and the membranous part of the nuclear pore complex.Octagonality of the nuclear pore complex has been confirmed by a variety of techniques. Using the freeze-etching technique, it was possible to show that the membranous part of the pore complex has an eight-sided outline in human melanoma cells in vitro. Fibers which traverse the pore proper at its corners are continuous and indistinguishable from chromatin at the nucleoplasmic side, as seen in conventionally fixed and sectioned material. Chromatin can be seen in octagonal outline if serial sections are analyzed which are parallel but do not include nuclear membranes (Fig. 1). It is concluded that the shape of the pore rim is due to fibrous material traversing the pore, and may not have any functional significance. In many pores one can recognize a central ring with eight fibers radiating to the corners of the pore rim. Such a structural arrangement is also found to connect eight ribosomes at the nuclear membrane.

Lamin B receptor: role on chromatin structure, cellular senescence and possibly aging

2020 ◽  
Vol 477 (14) ◽  
pp. 2715-2720
Author(s):  
Susana Castro-Obregón
Keyword(s):  
Cellular Senescence ◽  
Nuclear Membrane ◽  
Chromatin Structure ◽  
Cholesterol Synthesis ◽  
Reductase Activity ◽  
Chimeric Protein ◽  
Nuclear Lamina ◽  
Lamin B ◽  
Inner Nuclear Membrane ◽  
Lamin B Receptor

The nuclear envelope is composed by an outer nuclear membrane and an inner nuclear membrane, which is underlain by the nuclear lamina that provides the nucleus with mechanical strength for maintaining structure and regulates chromatin organization for modulating gene expression and silencing. A layer of heterochromatin is beneath the nuclear lamina, attached by inner nuclear membrane integral proteins such as Lamin B receptor (LBR). LBR is a chimeric protein, having also a sterol reductase activity with which it contributes to cholesterol synthesis. Lukasova et al. showed that when DNA is damaged by ɣ-radiation in cancer cells, LBR is lost causing chromatin structure changes and promoting cellular senescence. Cellular senescence is characterized by terminal cell cycle arrest and the expression and secretion of various growth factors, cytokines, metalloproteinases, etc., collectively known as senescence-associated secretory phenotype (SASP) that cause chronic inflammation and tumor progression when they persist in the tissue. Therefore, it is fundamental to understand the molecular basis for senescence establishment, maintenance and the regulation of SASP. The work of Lukasova et al. contributed to our understanding of cellular senescence establishment and provided the basis that lead to the further discovery that chromatin changes caused by LBR reduction induce an up-regulated expression of SASP factors. LBR dysfunction has relevance in several diseases and possibly in physiological aging. The potential bifunctional role of LBR on cellular senescence establishment, namely its role in chromatin structure together with its enzymatic activity contributing to cholesterol synthesis, provide a new target to develop potential anti-aging therapies.

The diverse roles of the Nup93/Nic96 complex proteins – structural scaffolds of the nuclear pore complex with additional cellular functions

2014 ◽  
Vol 395 (5) ◽  
pp. 515-528 ◽  
Author(s):  
Benjamin Vollmer ◽  
Wolfram Antonin
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Membrane ◽  
Building Blocks ◽  
Transport Processes ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Cellular Functions ◽  
Complex Assembly ◽  
Essential Function ◽  
Pore Complexes

Abstract Nuclear pore complexes mediate the transport between the cell nucleoplasm and cytoplasm. These 125 MDa structures are among the largest assemblies found in eukaryotes, built from proteins organized in distinct subcomplexes that act as building blocks during nuclear pore complex biogenesis. In this review, we focus on one of these subcomplexes, the Nup93 complex in metazoa and its yeast counterpart, the Nic96 complex. We discuss its essential function in nuclear pore complex assembly as a linker between the nuclear membrane and the central part of the pore and its various roles in nuclear transport processes and beyond.

scholarly journals SARS-CoV-2 Orf6 hijacks Nup98 to block STAT nuclear import and antagonize interferon signaling

2020 ◽  
Vol 117 (45) ◽  
pp. 28344-28354 ◽  
Author(s):  
Lisa Miorin ◽  
Thomas Kehrer ◽  
Maria Teresa Sanchez-Aparicio ◽  
Ke Zhang ◽  
Phillip Cohen ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Nuclear Translocation ◽  
Nuclear Pore ◽  
Interferon Signaling ◽  
Antiviral Signaling ◽  
Pathogenic Viruses ◽  
Transcriptional Induction ◽  
Global Health Problem ◽  
Viral Accessory Protein

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that is a serious global health problem. Evasion of IFN-mediated antiviral signaling is a common defense strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation in order to impair transcriptional induction of IFN-stimulated genes (ISGs). Our results demonstrate that the viral accessory protein Orf6 exerts this anti-IFN activity. We found that SARS-CoV-2 Orf6 localizes at the nuclear pore complex (NPC) and directly interacts with Nup98-Rae1 via its C-terminal domain to impair docking of cargo-receptor (karyopherin/importin) complex and disrupt nuclear import. In addition, we show that a methionine-to-arginine substitution at residue 58 impairs Orf6 binding to the Nup98-Rae1 complex and abolishes its IFN antagonistic function. All together our data unravel a mechanism of viral antagonism in which a virus hijacks the Nup98-Rae1 complex to overcome the antiviral action of IFN.

The nuclear pore complex, nuclear transport, and apoptosisThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.

2006 ◽  
Vol 84 (3-4) ◽  
pp. 279-286 ◽  
Author(s):  
Birthe Fahrenkrog
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Morphological Changes ◽  
Nuclear Transport ◽  
Nuclear Pore ◽  
Simple Fact ◽  
Cell Death Program ◽  
A Cell ◽  
Cellular Compartments ◽  
Selection Of

The nuclear pore complex (NPC) is the sole gateway between the nucleus and the cytoplasm of interphase eukaryotic cells, and it mediates all trafficking between these 2 cellular compartments. As such, the NPC and nuclear transport play central roles in translocating death signals from the cell membrane to the nucleus where they initiate biochemical and morphological changes occurring during apoptosis. Recent findings suggest that the correlation between the NPC, nuclear transport, and apoptosis goes beyond the simple fact that NPCs mediate nuclear transport of key players involved in the cell death program. In this context, the accessibility of key regulators of apoptosis appears to be highly modulated by nuclear transport (e.g., impaired nuclear import might be an apoptotic trigger). In this review, recent findings concerning the unexpected tight link between NPCs, nuclear transport, and apoptosis will be presented and critically discussed.

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