scholarly journals Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells

2001 ◽  
Vol 154 (1) ◽  
pp. 71-84 ◽  
Author(s):  
Nathalie Daigle ◽  
Joël Beaudouin ◽  
Lisa Hartnell ◽  
Gabriela Imreh ◽  
Einar Hallberg ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Nuclear Lamina ◽  
Nuclear Pore ◽  
Global Changes ◽  
Nuclear Pore Complexes ◽  
Annulate Lamellae ◽  
Lamin B1 ◽  
Green Fluorescent ◽  
Pore Complexes

The nuclear pore complex (NPC) and its relationship to the nuclear envelope (NE) was characterized in living cells using POM121–green fluorescent protein (GFP) and GFP-Nup153, and GFP–lamin B1. No independent movement of single pore complexes was found within the plane of the NE in interphase. Only large arrays of NPCs moved slowly and synchronously during global changes in nuclear shape, strongly suggesting mechanical connections which form an NPC network. The nuclear lamina exhibited identical movements. NPC turnover measured by fluorescence recovery after photobleaching of POM121 was less than once per cell cycle. Nup153 association with NPCs was dynamic and turnover of this nucleoporin was three orders of magnitude faster. Overexpression of both nucleoporins induced the formation of annulate lamellae (AL) in the endoplasmic reticulum (ER). Turnover of AL pore complexes was much higher than in the NE (once every 2.5 min). During mitosis, POM121 and Nup153 were completely dispersed and mobile in the ER (POM121) or cytosol (Nup153) in metaphase, and rapidly redistributed to an immobilized pool around chromatin in late anaphase. Assembly and immobilization of both nucleoporins occurred before detectable recruitment of lamin B1, which is thus unlikely to mediate initiation of NPC assembly at the end of mitosis.

scholarly journals Concentration-dependent lamin assembly and its roles in the localization of other nuclear proteins

2014 ◽  
Vol 25 (8) ◽  
pp. 1287-1297 ◽  
Author(s):  
Yuxuan Guo ◽  
Youngjo Kim ◽  
Takeshi Shimi ◽  
Robert D. Goldman ◽  
Yixian Zheng
Keyword(s):  
Nuclear Lamina ◽  
Nuclear Pore ◽  
Lamin A ◽  
Nuclear Pore Complexes ◽  
Developmental Defects ◽  
Protein Levels ◽  
Lamin B1 ◽  
Mouse Cells ◽  
Pore Complexes ◽  
And Function

The nuclear lamina (NL) consists of lamin polymers and proteins that bind to the polymers. Disruption of NL proteins such as lamin and emerin leads to developmental defects and human diseases. However, the expression of multiple lamins, including lamin-A/C, lamin-B1, and lamin-B2, in mammals has made it difficult to study the assembly and function of the NL. Consequently, it has been unclear whether different lamins depend on one another for proper NL assembly and which NL functions are shared by all lamins or are specific to one lamin. Using mouse cells deleted of all or different combinations of lamins, we demonstrate that the assembly of each lamin into the NL depends primarily on the lamin concentration present in the nucleus. When expressed at sufficiently high levels, each lamin alone can assemble into an evenly organized NL, which is in turn sufficient to ensure the even distribution of the nuclear pore complexes. By contrast, only lamin-A can ensure the localization of emerin within the NL. Thus, when investigating the role of the NL in development and disease, it is critical to determine the protein levels of relevant lamins and the intricate shared or specific lamin functions in the tissue of interest.

scholarly journals Lamin B1 protein is required for dendrite development in primary mouse cortical neurons

2016 ◽  
Vol 27 (1) ◽  
pp. 35-47 ◽  
Author(s):  
Caterina Giacomini ◽  
Sameehan Mahajani ◽  
Roberta Ruffilli ◽  
Roberto Marotta ◽  
Laura Gasparini
Keyword(s):  
Cortical Neurons ◽  
Nuclear Lamina ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Loss Of Function ◽  
Lamin B1 ◽  
Dendrite Development ◽  
Primary Mouse ◽  
Nuclear Shuttling ◽  
Pore Complexes

Lamin B1, a key component of the nuclear lamina, plays an important role in brain development and function. A duplication of the human lamin B1 ( LMNB1) gene has been linked to adult-onset autosomal dominant leukodystrophy, and mouse and human loss-of-function mutations in lamin B1 are susceptibility factors for neural tube defects. In the mouse, experimental ablation of endogenous lamin B1 (Lmnb1) severely impairs embryonic corticogenesis. Here we report that in primary mouse cortical neurons, LMNB1 overexpression reduces axonal outgrowth, whereas deficiency of endogenous Lmnb1 results in aberrant dendritic development. In the absence of Lmnb1, both the length and complexity of dendrites are reduced, and their growth is unresponsive to KCl stimulation. This defective dendritic outgrowth stems from impaired ERK signaling. In Lmnb1-null neurons, ERK is correctly phosphorylated, but phospho-ERK fails to translocate to the nucleus, possibly due to delocalization of nuclear pore complexes (NPCs) at the nuclear envelope. Taken together, these data highlight a previously unrecognized role of lamin B1 in dendrite development of mouse cortical neurons through regulation of nuclear shuttling of specific signaling molecules and NPC distribution.

scholarly journals Nucleus–Vacuole Junctions in Saccharomyces cerevisiae Are Formed Through the Direct Interaction of Vac8p with Nvj1p

2000 ◽  
Vol 11 (7) ◽  
pp. 2445-2457 ◽  
Author(s):  
Xiaozhou Pan ◽  
Paul Roberts ◽  
Yan Chen ◽  
Erik Kvam ◽  
Natalyia Shulga ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Fluorescent Protein ◽  
Close Contact ◽  
Direct Interaction ◽  
Integral Membrane Protein ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Importin Α ◽  
Green Fluorescent ◽  
Pore Complexes

Vac8p is a vacuolar membrane protein that is required for efficient vacuole inheritance and fusion, cytosol-to-vacuole targeting, and sporulation. By analogy to other armadillo domain proteins, including β-catenin and importin α, we hypothesize that Vac8p docks various factors at the vacuole membrane. Two-hybrid and copurfication assays demonstrated that Vac8p does form complexes with multiple binding partners, including Apg13p, Vab2p, and Nvj1p. Here we describe the surprising role of Vac8p-Nvj1p complexes in the formation of nucleus–vacuole (NV) junctions. Nvj1p is an integral membrane protein of the nuclear envelope and interacts with Vac8p in the cytosol through its C-terminal 40–60 amino acids (aa). Nvj1p green fluorescent protein (GFP) concentrated in small patches or rafts at sites of close contact between the nucleus and one or more vacuoles. Previously, we showed that Vac8p-GFP concentrated in intervacuole rafts, where is it likely to facilitate vacuole-vacuole fusion, and in “orphan” rafts at the edges of vacuole clusters. Orphan rafts of Vac8p red-sifted GFP (YFP) colocalize at sites of NV junctions with Nvj1p blue-sifted GFP (CFP). GFP-tagged nuclear pore complexes (NPCs) were excluded from NV junctions. In vac8-Δ cells, Nvj1p-GFP generally failed to concentrate into rafts and, instead, encircled the nucleus. NV junctions were absent in both nvj1-Δ andvac8-Δ cells. Overexpression of Nvj1p caused the profound proliferation of NV junctions. We conclude that Vac8p and Nvj1p are necessary components of a novel interorganelle junction apparatus.

scholarly journals A Novel Fluorescence-based Genetic Strategy Identifies Mutants ofSaccharomyces cerevisiaeDefective for Nuclear Pore Complex Assembly

1998 ◽  
Vol 9 (9) ◽  
pp. 2439-2461 ◽  
Author(s):  
Mirella Bucci ◽  
Susan R. Wente
Keyword(s):  
Fluorescent Protein ◽  
Nuclear Pore ◽  
Temperature Sensitive ◽  
Nuclear Pore Complexes ◽  
Specific Subset ◽  
Powerful Approach ◽  
Sensitive Allele ◽  
Green Fluorescent ◽  
Pore Complexes ◽  
Carboxy Terminal

Nuclear pore complexes (NPCs) are large proteinaceous portals for exchanging macromolecules between the nucleus and the cytoplasm. Revealing how this transport apparatus is assembled will be critical for understanding the nuclear transport mechanism. To address this issue and to identify factors that regulate NPC formation and dynamics, a novel fluorescence-based strategy was used. This approach is based on the functional tagging of NPC proteins with the green fluorescent protein (GFP), and the hypothesis that NPC assembly mutants will have distinct GFP-NPC signals as compared with wild-type (wt) cells. By fluorescence-activated cell sorting for cells with low GFP signal from a population of mutagenized cells expressing GFP-Nup49p, three complementation groups were identified: two correspond to mutantnup120 and gle2 alleles that result in clusters of NPCs. Interestingly, a third group was a novel temperature-sensitive allele of nup57. The lowered GFP-Nup49p incorporation in the nup57-E17 cells resulted in a decreased fluorescence level, which was due in part to a sharply diminished interaction between the carboxy-terminal truncated nup57pE17and wt Nup49p. Interestingly, thenup57-E17 mutant also affected the incorporation of a specific subset of other nucleoporins into the NPC. Decreased levels of NPC-associated Nsp1p and Nup116p were observed. In contrast, the localizations of Nic96p, Nup82p, Nup159p, Nup145p, and Pom152p were not markedly diminished. Coincidentally, nuclear import capacity was inhibited. Taken together, the identification of such mutants with specific perturbations of NPC structure validates this fluorescence-based strategy as a powerful approach for providing insight into the mechanism of NPC biogenesis.

scholarly journals Esc1, a Nuclear Periphery Protein Required for Sir4-Based Plasmid Anchoring and Partitioning

2002 ◽  
Vol 22 (23) ◽  
pp. 8292-8301 ◽  
Author(s):  
Erik D. Andrulis ◽  
David C. Zappulla ◽  
Athar Ansari ◽  
Severine Perrod ◽  
Catherine V. Laiosa ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Nuclear Periphery ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Uncharacterized Protein ◽  
Telomeric Silencing ◽  
Green Fluorescent ◽  
Pore Complexes ◽  
Redundant Pathway

ABSTRACT A targeted silencing screen was performed to identify yeast proteins that, when tethered to a telomere, suppress a telomeric silencing defect caused by truncation of Rap1. A previously uncharacterized protein, Esc1 (establishes silent chromatin), was recovered, in addition to well-characterized proteins Rap1, Sir1, and Rad7. Telomeric silencing was slightly decreased in Δesc1 mutants, but silencing of the HM loci was unaffected. On the other hand, targeted silencing by various tethered proteins was greatly weakened in Δesc1 mutants. Two-hybrid analysis revealed that Esc1 and Sir4 interact via a 34-amino-acid portion of Esc1 (residues 1440 to 1473) and a carboxyl-terminal domain of Sir4 known as PAD4 (residues 950 to 1262). When tethered to DNA, this Sir4 domain confers efficient partitioning to otherwise unstable plasmids and blocks the ability of bound DNA segments to rotate freely in vivo. Here, both phenomena were shown to require ESC1. Sir protein-mediated partitioning of a telomere-based plasmid also required ESC1. Fluorescence microscopy of cells expressing green fluorescent protein (GFP)-Esc1 showed that the protein localized to the nuclear periphery, a region of the nucleus known to be functionally important for silencing. GFP-Esc1 localization, however, was not entirely coincident with telomeres, the nucleolus, or nuclear pore complexes. Our data suggest that Esc1 is a component of a redundant pathway that functions to localize silencing complexes to the nuclear periphery.

scholarly journals Reorganization of Nuclear Pore Complexes and the Lamina in Late-Stage Parvovirus Infection

2015 ◽  
Vol 89 (22) ◽  
pp. 11706-11710 ◽  
Author(s):  
Elina Mäntylä ◽  
Einari A. Niskanen ◽  
Teemu O. Ihalainen ◽  
Maija Vihinen-Ranta
Keyword(s):  
Late Stage ◽  
Nuclear Lamina ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Lamin B1 ◽  
Apical Side ◽  
Close Proximity ◽  
Nuclear Structures ◽  
Pore Complexes ◽  
Stage Infection

Canine parvovirus (CPV) infection induces reorganization of nuclear structures. Our studies indicated that late-stage infection induces accumulation of nuclear pore complexes (NPCs) and lamin B1 concomitantly with a decrease of lamin A/C levels on the apical side of the nucleus. Newly formed CPV capsids are located in close proximity to NPCs on the apical side. These results suggest that parvoviruses cause apical enrichment of NPCs and reorganization of nuclear lamina, presumably to facilitate the late-stage infection.

scholarly journals Involvement of the Lamin Rod Domain in Heterotypic Lamin Interactions Important for Nuclear Organization

2001 ◽  
Vol 153 (3) ◽  
pp. 479-490 ◽  
Author(s):  
Eric C. Schirmer ◽  
Tinglu Guan ◽  
Larry Gerace
Keyword(s):  
Nuclear Envelope ◽  
Cultured Cells ◽  
Nuclear Lamina ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Binding Studies ◽  
Lamin B1 ◽  
Vitro Binding ◽  
Pore Complexes

The nuclear lamina is a meshwork of intermediate-type filament proteins (lamins) that lines the inner nuclear membrane. The lamina is proposed to be an important determinant of nuclear structure, but there has been little direct testing of this idea. To investigate lamina functions, we have characterized a novel lamin B1 mutant lacking the middle ∼4/5 of its α-helical rod domain. Though retaining only 10 heptads of the rod, this mutant assembles into intermediate filament-like structures in vitro. When expressed in cultured cells, it concentrates in patches at the nuclear envelope. Concurrently, endogenous lamins shift from a uniform to a patchy distribution and lose their complete colocalization, and nuclei become highly lobulated. In vitro binding studies suggest that the internal rod region is important for heterotypic associations of lamin B1, which in turn are required for proper organization of the lamina. Accompanying the changes in lamina structure induced by expression of the mutant, nuclear pore complexes and integral membrane proteins of the inner membrane cluster, principally at the patches of endogenous lamins. Considered together, these data indicate that lamins play a major role in organizing other proteins in the nuclear envelope and in determining nuclear shape.

scholarly journals Pml39, a Novel Protein of the Nuclear Periphery Required for Nuclear Retention of Improper Messenger Ribonucleoparticles

2005 ◽  
Vol 16 (11) ◽  
pp. 5258-5268 ◽  
Author(s):  
Benoît Palancade ◽  
Michela Zuccolo ◽  
Sophie Loeillet ◽  
Alain Nicolas ◽  
Valérie Doye
Keyword(s):  
Fluorescent Protein ◽  
Specific Interaction ◽  
Nuclear Periphery ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Retention ◽  
Reading Frame ◽  
Nuclear Domains ◽  
Green Fluorescent ◽  
Pore Complexes

Using a genetic screen, we have identified a previously uncharacterized Saccharomyces cerevisiae open reading frame (renamed PML39) that displays a specific interaction with nucleoporins of the Nup84 complex. Localization of a Pml39-green fluorescent protein (GFP) fusion and two-hybrid studies revealed that Pml39 is mainly docked to a subset of nuclear pore complexes opposite to the nucleolus through interactions with Mlp1 and Mlp2. The absence of Pml39 leads to a specific leakage of unspliced mRNAs that is not enhanced upon MLP1 deletion. In addition, overexpression of PML39-GFP induces a specific trapping of mRNAs transcribed from an intron-containing reporter and of the heterogenous nuclear ribonucleoprotein Nab2 within discrete nuclear domains. In a nup60Δ mutant, Pml39 is mislocalized together with Mlp1 and Mlp2 in intranuclear foci that also recruit Nab2. Moreover, pml39Δ partially rescues the thermosensitive phenotypes of messenger ribonucleoparticles (mRNPs) assembly mutants, indicating that PML39 deletion also bypasses the requirement for normally assembled mRNPs. Together, these data indicate that Pml39 is an upstream effector of the Mlps, involved in the retention of improper mRNPs in the nucleus before their export.

scholarly journals The Entire Nup107-160 Complex, Including Three New Members, Is Targeted as One Entity to Kinetochores in Mitosis

2004 ◽  
Vol 15 (7) ◽  
pp. 3333-3344 ◽  
Author(s):  
Isabelle Loïodice ◽  
Annabelle Alves ◽  
Gwénaël Rabut ◽  
Megan van Overbeek ◽  
Jan Ellenberg ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
New Members ◽  
Multiple Copies ◽  
Bidirectional Transport ◽  
Higher Eukaryotes ◽  
Green Fluorescent ◽  
Pore Complexes ◽  
Transport Of Macromolecules

In eukaryotes, bidirectional transport of macromolecules between the cytoplasm and the nucleus occurs through elaborate supramolecular structures embedded in the nuclear envelope, the nuclear pore complexes (NPCs). NPCs are composed of multiple copies of ∼30 different proteins termed nucleoporins, of which several can be biochemically isolated as subcomplexes. One such building block of the NPC, termed the Nup107-160 complex in vertebrates, was so far demonstrated to be composed of six different nucleoporins. Here, we identify three WD (Trp-Asp)-repeat nucleoporins as new members of this complex, two of which, Nup37 and Nup43, are specific to higher eukaryotes. The third new member Seh1 is more loosely associated with the Nup107-160 complex biochemically, but its depletion by RNA interference leads to phenotypes similar to knock down of other constituents of this complex. By combining green fluorescent protein-tagged nucleoporins and specific antibodies, we show that all the constituents of this complex, including Nup37, Nup43, Seh1, and Sec13, are targeted to kinetochores from prophase to anaphase of mitosis. Together, our results indicate that the entire Nup107-160 complex, which comprises nearly one-third of the so-far identified nucleoporins, specifically localizes to kinetochores in mitosis.

scholarly journals The nuclear pore complex: mediator of translocation between nucleus and cytoplasm

2000 ◽  
Vol 113 (10) ◽  
pp. 1651-1659 ◽  
Author(s):  
T.D. Allen ◽  
J.M. Cronshaw ◽  
S. Bagley ◽  
E. Kiseleva ◽  
M.W. Goldberg
Keyword(s):  
Nuclear Export ◽  
Mammalian Cells ◽  
Complex Structure ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Complete Breakdown ◽  
Import And Export ◽  
Complex Structural ◽  
Pore Complexes ◽  
Giant Nuclei

The enclosure of nuclear contents in eukaryotes means that cells require sites in the boundary that mediate exchange of material between nucleus and cytoplasm. These sites, termed nuclear pore complexes (NPCs), number 100–200 in yeast, a few thousand in mammalian cells and approximately 50 million in the giant nuclei of amphibian oocytes. NPCs are large (125 MDa) macromolecular complexes that comprise 50–100 different proteins in vertebrates. In spite of their size and complex structure, NPCs undergo complete breakdown and reformation at cell division. Transport through NPCs can be rapid (estimated at several hundred molecules/pore/second) and accommodates both passive diffusion of relatively small molecules, and active transport of complexes up to several megadaltons in molecular mass. Each pore can facilitate both import and export. The two processes apparently involve multiple pathways for different cargoes, and their transport signals, transport receptors and adapters, and the molecules (and their regulators) that underpin the transport mechanisms. Over the past few years there has been an increasing interest in the pore complex: structural studies have been followed by elucidation of the biochemical aspects of nuclear import, and subsequent investigations into nuclear export. The current challenge is to understand the interactions between the structural elements of the pore complex and the mechanisms that drive the physical processes of translocation through it.

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